New Panorama of Animal Evolution: Proceedings of the XVIII International Congress of Zoology, Athens, Greece, September, 2000

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Author: A. Legakis | S. Sfenthourakis | R. Polymeni & M. Thessalou-Legaki (Editors)

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THE NEW PANORAMA OF ANIMAL EVOLUTION

Proceedings XVIII International Congress of Zoology XVIIIème Congrès International de Zoologie

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THE NEW PANORAMA OF

ANIMAL EVOLUTION Proceedings XVIII International Congress of Zoology XVIIIème Congrès International de Zoologie Organized by the Hellenic Zoological Society Athens, Greece 28.8.–2.9.2000 Edited by A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki

ISBN 954-642-164-2

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Opening Remarks

THE NEW PANORAMA OF ANIMAL EVOLUTION Proceedings XVIII International Congress of Zoology XVIIIème Congrés International de Zoologie Edited by A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki

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This book is published with the support of UNESCO

Opening Remarks

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THE NEW PANORAMA OF ANIMAL EVOLUTION Proceedings XVIII International Congress of Zoology XVIIIème Congrés International de Zoologie Organized by the Hellenic Zoological Society and held at the National and Kapodistrian University of Athens, Greece, from the 28th of August to the 2nd of September 2000

Edited by A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki

Sofia-Moscow 2003

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ORGANIZING COMMITTEE Francis Dov Por – Chairman (Israel) Rosa Polymeni – Secretary (Greece) Anastasios Legakis – Treasurer (Greece) Spyros Sfenthourakis (Greece) Maria Thessalou-Legaki (Greece) Chariton Chintiroglou (Greece) Maria Lazaridou (Greece) Drosos Koutsoumbas (Greece) Basil Chondropoulos (Greece) Stella Fraguedakis-Tsolis (Greece) INTERNATIONAL INITIATIVE COMMITTEE Francis Dov Por (Israel) Bruno Battaglia (Italy) Daniel R. Brooks (Canada) Edwin L. Cooper (USA) Vassili Kiortsis (Greece) Claude Levi (France) Paulo Nogueira Neto (Brasil) Stuart G. Poss (USA) Song Daxiang (China) Maher Houssain Khalifa (Egypt) PROCEEDINGS EDITORIAL COMMITTEE Anastasios Legakis (Greece) Spyros Sfenthourakis (Greece) Rosa Polymeni (Greece) Maria Thessalou-Legaki (Greece)

First published 2003 ISBN 954-642-164-2 © PENSOFT Publishers All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the copyright owner. Pensoft Publishers, Acad. G. Bonchev Str., Bl.6, 1113 Sofia, Bulgaria Fax: +359-2-70-45-08, e-mail: [email protected], www.pensoft.net Printed in Bulgaria, February 2003

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Contents Preface of the Editorial Committee .................................................................................... xi Chairman’s opening remarks .............................................................................................. xii Secretary’s opening remarks ............................................................................................... xv Invited lectures PIANKA E.R. A General Review of Zoological Trends During the 20th Century ......... 3 WILEY E.O. On Species and Speciation .............................................................................. 15 BUCKERIDGE J.St.J.S. Aristotle: Descriptor Animalium Princeps! ............................... 19 POR F.D. The Persistent Progression: a New View on Animal Evolution ..................... 27 The new paleontological panorama BERGSTRÖM J. Introduction: The new paleontological panorama ............................... 43 AHLBERG P.E. Fossils, developmental patterning and the origin of tetrapods .......... 45 CURRIE P.J. Feathered dinosaurs and the origin of birds ................................................ 55 FORTELIUS M. Evolution of Dental Capability in Western Eurasian Large Mammal Plant-Eaters 22-2 Million Years Ago: A Case for Environmental Forcing Mediated by Biotic Processes ................................................................................................................... 61 WALOSZEK D. Cambrian ‘Orsten’-type preserved Arthropods and the Phylogeny of Crustacea ............................................................................................................................. 69 BERGSTRÖM J. & HOU Xianguang. Cambrian arthropods: a lesson in convergent evolution .................................................................................................................................... 89 Molecular macroevolution MÜLLER W.E.G., MÜLLER I.M. The urmetazoa: Molecular biological studies with living fossils - Porifera ........................................................................................................... 99 The integrative approach in zoological evolution NYLIN S. Evolutionary dynamics of host plant range in the butterfly tribe Nymphalini (Insecta, Lepidoptera, Nymphalidae) ........................................................................... 107 Comparative Immunology of the animal kingdom COOPER E.L. Comparative Immunology of the Animal Kingdom ............................. 117 STOTZ H.U., AUGUSTIN R., KHALTURIN K., KUZNETSOV S., RINKEVICH B., SCHRÖDER J. & BOSCH T.C.G. Novel approaches for the analysis of immune reactions in Tunicate and Cnidarian model organisms ..................................................... 127

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KAUSCHKE E. & MOHRIG W. Does Functional Similarity of Certain Innate Immune Mechanisms of Invertebrates and Vertebrates Point to their Phylogenetic Relation? ............................................................................................................................................. 133 DE EGUILEOR M., GRIMALDI A., TETTAMANTI G., VALVASSORI R. & COOPER E.L. State of the art for the immune system in leeches ...................................................... 139 SALZET M., TASIEMSKI A., LEFEBVRE C. & COOPER E. Comparison of Molecular Neuroimmune Processes Between Leeches and Human .......................................... 147 PESTARINO M. Bidirectional communication between the immune and neuroendocrine systems: an evolutionary perspective ................................................................. 159 PARRINELLO N., CAMMARATA M., ARIZZA V., VAZZANA M. & COOPER E.L. How do cells of the invertebrate immune systems kill other cells? .................................. 167 ROCH P., MITTA G., VANDENBULCKE F., SALZET M., AUMELAS A., YANG Y.-S., CHAVNIEU A. & CALAS B. Originality of the Mytilus (Bivalve Mollusc) antibacterial peptides: structurally related to Insects but involved as in Mammals ............... 177 Evolution as reflected in embryonic development SHANKLAND M. Evolution of body axis segmentation in the bilaterian radiation ...... ............................................................................................................................................. 187 The role of parasitism in animal evolution MØLLER A.P. Behavioural, genetic and evolutionary interactions between cuckoos and their hosts .......................................................................................................................... 199 POULIN R. Phenotypic Manipulation and Parasite-Mediated Host Evolution ........ 205 MORAND S. Parasites and the evolution of host life history traits ............................. 213 CÔTÉ I.M. Parasites and the evolution of cleaning symbioses among fish ................ 219 COMBES C. Host behaviour: the first line of defense .................................................... 227 HUGOT J.P. TreeMap: an algorithm to maximize the number of codivergences when reconstructing the history of an associate and its host .............................................. 235 The Protozoa-Metazoa boundary SHIELDS G. & FOISSNER W. Diverse perspectives on the Protozoan – Metazoan transition ................................................................................................................................... 243 RIEGER R. The phenotypic transition from uni- to multicellular animals ................. 247 BRASIER M. From Famine to Feast: a context for the protozoan-metazoan transition .. ............................................................................................................................................. 259 DEWEL, R.A., DEWEL W.C. & MCKINNEY F.K. Origin and Diversification of the Metazoa: Superorganisms among the Ediacarans ............................................................... 269 HACKSTEIN J. The protozoan-metazoan boundary: a molecular biologist’s view ........ ............................................................................................................................................. 277

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BENGTSON S. Tracing metazoan roots in the fossil record .......................................... 289 Archaeozoology. Human-animal interactions as a tool for present and future WILKENS B. & DELUSSU F. Wild and domestic mammals in holocenic Sardinia ......... ............................................................................................................................................. 303 MARCINIAK A. People and animals in the early Neolithic in Central Europe. New approach to animal bones assemblages from farming settlements ......................... 309 Benchmark events and key figures in 20th century Zoology WINSTON J.E. Libbie Hyman and Invertebrate Zoology in the 20th Century ........... 321 BURKHARDT R.W. Konrad Lorenz, Niko Tinbergen, and the founding of ethology as a scientific discipline .......................................................................................................... 329 JAX K. From scientific natural history to ecosystem research: changing roles of the animal in the history of animal ecology ............................................................................. 337 DELSOL M., NOIROT C., GENERMONT J. & D’HONDT J.-L. Hommage à Pierre-Paul Grassé ................................................................................................................................. 345 SMOCOVITIS V.B. The Invisible Subject: Zoology and the Evolutionary Synthesis . 351 SCHRAM F.R. Our evolving understanding of biodiversity through history and its impact on the recognition of higher taxa of Metazoa ..................................................... 359 SCHMITT M. Willi Hennig and the Rise of Cladistics ................................................... 369 Diversity, endemism and conservation priorities in Madagascar LOURENÇO W.R. Diversity, Endemism and Conservation Priorities in Madagascar ... ............................................................................................................................................. 383 LOURENÇO W.R. The remarkable levels of diversity and endemicity in the scorpion fauna of Madagascar ....................................................................................................... 385 GANZHORN J.U., GOODMAN S.M., RAMANAMANJATO J.-B., RAKOTONDRAVONY D. & RAKOTOSAMIMANANA B. Biogeographic relations and life history characteristics of vertebrate communities in littoral forests of Madagascar .......... 393 ANDREONE F. The amphibians and reptiles of Madagascar: diversity, threats and conservation perspectives ..................................................................................................... 403 THALMANN U. An integrative approach to the study of diversity and regional endemism in lemurs (Primates, Mammalia) and their conservation ............................... 409 Comparative biology of sperm storage in vertebrates HAMLETT W.C., GREVEN H. & SCHINDLER J. Sperm Storage in the Class Chondrichthyes & Class Osteichthyes ............................................................................................ 421 SEVER D.M., RANIA L.C. & BRIZZI R. Sperm Storage in the Class Amphibia ........ 431

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SEVER D.M. & HAMLETT W.C. Sperm Storage in the Class Reptilia ........................ 439 BAKST M.R. Oviducal Sperm Storage in Turkeys (Meleagris Gallopavo): The Infundibulum as a secondary Sperm Storage Site, or is it? ......................................................... 447 SUAREZ S.S. Sperm Storage in the Class Mammalia .................................................... 451 Integrative approaches to phylogenetic relationships of arthropods SCHMIDT-RHAESA A. Integrative approaches to phylogenetic relationships of arthropods: Introduction to the Symposium .......................................................................... 461 KRISTENSEN R.M. Comparative Morphology: Do the ultrastructural investigations of Loricifera and Tardigrada support the clade Ecdysozoa? ......................................... 467 BUDD G.E. Arthropods as ecdysozoans: the fossil evidence ........................................ 479 SCHOLTZ G. Is the taxon Articulata obsolete? Arguments in favour of a close relationship between annelids and arthropods ........................................................................ 489 GAREY J.R. Ecdysozoa: the evidence for a close relationship between arthropods and nematodes ......................................................................................................................... 503 Zoological implications of the discovery of geothermally-driven communities GAILL F., ZBINDEN M. & PRADILLON F. Adaptations of hydrothermal vent organisms to their environment ............................................................................................... 513 The role of symbiosis in physiology and evolution NARDON P. & HEDDI A. The Role of Symbiosis in Physiology and Evolution ....... 521 BOURTZIS K. Wolbachia: Symbionts as Reproductive Parasites ................................... 523 HEDDI A. The weevil’s symbiocosm and its four intracellular genomes ................... 527 ISHIKAWA H. Characteristic features of the genome of an aphid endosymbiotic bacterium, Buchnera .................................................................................................................. 535 JEON K.W. Integration of bacterial endosymbionts in amoebae .................................. 541 Diversification and evolutionary Ecology MARTENS J. & PÄCKERT M. Disclosure of songbird diversity in the Palearctic/Oriental transition zone ............................................................................................................ 551 Ways for improving modern zoological education BUCKERIDGE J.St.J.S. Zoological Education in New Zealand: a 21st Century perspective ...................................................................................................................................... 561 AZARIAH J. A New Engine For a Holistic Zoology Education in the 21st Century ..... 569 POR F. D. The Crisis In Teaching Of Zoology: The Israeli Experience ......................... 575

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BUCKERIDGE J.St.J.S. Ways for improving modern zoological education: overview of the session ......................................................................................................................... 581 Coordinated development and use of collections databases POSS G. Coordinated Development and Use of Collections Databases ..................... 585 FORTUNATO H. Evolutionary Paleontology and Informatics: The Neogene Marine Biota of Tropical America (NMITA) Database ....................................................................... 591 LEGAKIS A. & EMBLOW C.S. The Register Of Collections Of European Marine Species: An Overview ............................................................................................................ 603 FROESE R. & REYES R.Jr. Use Them Or Lose Them: The Need to Make Collection Databases Publicly Available .................................................................................................. 611 WILEY E.O. & PETERSON A.T. Distributed Information Systems and Predictive Biogeography: Putting Natural History Collections to Work in the 21st Century ........... 619 The taxonomic impediment, in search of a remedy action POR F.D. Remedies for the Taxonomic Impediment in Zoology .................................. 627 CRESSWELL I.D. & BRIDGEWATER P.B. The Global Taxonomy Initiative (GTI) and the International Congress on Zoology – a perspective on the role of the Convention on Biological Diversity and UNESCO ............................................................................... 631 FAUCHALD K. Taxonomic impediment in the study of marine invertebrates ......... 637 POR F.D. A “Taxonomic Affidavit”. Why it is needed? .................................................. 643 The new International Code of Zoological Nomenclature and related issues MINELLI A. Zoological nomenclature after the publication of the Fourth Edition of the Code ................................................................................................................................... 649 HOWCROFT J.M.& THORNE M.J. Zoological Record – a bibliographic service and taxonomic resource ............................................................................................................... 659 GREUTER W. Biological nomenclature in the electronic era: chances, challenges, risks ............................................................................................................................................. 665 RIDE W.D.L. The International Code of Zoological Nomenclature, 4th Edition - What Next? .................................................................................................................................. 673 WYRWOLL T.W. Still Desiderata: Scientific Names for Domestic Animals and Their Feral Derivatives .............................................................................................................. 683 Special presentations TIAGO C.G. & HADEL V.F. Neotropical Biodiversity Conservation and Sustainable Use in São Paulo State (Brazil) - BIOTA/FAPESP - The Biodiversity Virtual Institute ...... ............................................................................................................................................. 701

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BUCKERIDGE J.St.J.S. & GORDON D. Species 2000 New Zealand: Outcomes of the February Symposium ............................................................................................................ 705 ´ PRÓSZY NSKI J. Large computer monographs in zoology - possibilities and perspective. Demonstration of a test case - “Salticidae (Araneae) of the World” ............... 711 BÃNÃRESCU P.M. New data on “satellite” fish species and their evolutionary significance ................................................................................................................................... 715 List of Participants .............................................................................................................. 725 Index ....................................................................................................................................... 737

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Preface of the Editorial Committee This volume contains the proceedings of the 18th International Congress of Zoology organized by the Hellenic Zoological Society and held at the National and Kapodistrian University of Athens, Greece from the 28th of August till the 2nd of September 2000. The congress is a continuation of the series of International Congresses of Zoology that were started in Paris in 1889. During the 5 days of the congress, 233 participants from 36 countries around the world attended 102 oral presentations that were divided into 3 opening lectures, 8 general symposia, 8 special symposia, 4 general discussions, 1 special mini symposium and 6 special presentations. 127 posters were also exhibited on the site of the congress. The proceedings editorial committee was established by the General Assembly of the congress, which also decided to hold the next International Congress of Zoology in Peking, China in 2004, expressing the willingness of the participants to continue this institution. This congress will be organized by the China Zoological Society and the Institute of Zoology of the Chinese Academy of Sciences. The General Assembly also established the International Congress of Zoology Committee, which will overlook the next congress and will also decide on the statutes of the International Congresses. This committee includes Dr. W. Bock (USA), Dr. J. Buckeridge (New Zealand), Dr. E. Cooper (USA), Dr. Daxiang Song (China), Dr. R. Polymeni (Greece) and Dr. M. Schmitt (Germany), with Dr. S. Poss (USA) as secretary. In this volume, we are happy to present you a number of very important contributions that can be considered as milestones for the study of Zoology in the beginning of the 21st century. The publication of this volume would not be possible without the generous assistance of UNESCO, which we warmly thank. We would also like to thank the colleagues who accepted the task of reviewing and providing valuable comments on the submitted papers, and also all the contributors to this volume. The Editorial Committee

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. xii-xiv, 2003

Opening Remarks Francis Dov Por, Chairman, Organizing Committee

Dear friend zoologists, It is with deep satisfaction and with a sentiment of humility that we are starting, in this olympic year, the International Congresses of Zoology of the New Era, in Athens where Aristotle, the father of Zoology taught. Zoology, the revolutionary science of Darwin, was among the very first disciplines to build an international organization. The Congresses started in 1889 on the initiative of the Société Zoologique de France on the occasion of the International Exposition in Paris. For decades to come, Milne-Edwards, Perrier, Caullery and Grassé were the Presidents of the Permanent Commission of the Congresses. Raphael Blanchard was the soul of the Congresses for more than 30 years. We are glad to have after 110 years, the representatives of the Société Zoologique de France here with us. Chers Mr. Daguzan, vice-président de la Société Zoologique, Mr. D’Hondt, son Secrétaire Générale et Mr. Dupuis, doyen des participants aux congrés, nous sommes heureux de vous voir aujourd’hui parmi nous. In all the years that followed, the Congresses were held at regular intervals till 1963 under the direction of a “Comité Permanent”. For many years, French was the official language of the congresses. Great names were in the chair of these meetings and keynote lectures were presented by such names like von Virchow, Agassiz, Metschnikov, Grassé, Romer, Julian Huxley and others, It has been a long and meritorious history of which I shall mention only the beginnings and the final moments. Right from the beginning, in Paris, on the initiative of Raphael Blanchard, the Congresses dealt with problems of Zoological Nomenclature. In due time the International Committee of Zoological Nomenclature started to work in the framework of the congresses, routinely submitting its reports to the Congress Assemblies. The attendance increased from congress to congress. From a few tens in Paris and in Moscow – where there were almost only Russian zoologists from all parts of the then Russian Empire - to 700 in Budapest in 1927. The London Congress set a target of 1000, but there were 1400 members and 400 associates. Finally in Washington the number reached 2500. Clearly, there was a problem with the big numbers. In parallel, the number of sessions and sections increased, to keep abreast with the growing specialization. In London in 1958 there were 8-9 daily parallel sections of communications. In Washington an attempt was made to organize symposia instead.

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There were no less than 29 such symposia. The problem of unifying subjects became even more important than the numbers of participants. In London, the Darwin-Wallace Centenary of that year provided for a unifying framework. The congress in Washington in 1963 chose as its symbol the Phoenix bird and the goal was “a phoenix-like rebirth of breadth of vision in the study of animal life”, in the words of its president Alfred Romer. Or according to the General Secretary Gairdner Moment, the Phoenix was a symbol of the ”organism reborn from its homogenized macromolecules”. The Phoenix did not rise. What happened? The Washington Congress decided that the Board of the Division of Zoology of the newly founded IUBS, would assume in the future the role of the Comité Permanent and would be responsible for the maintaining and the continuity of Zoological Congresses. This did not work. There was an invitation from New Delhi, which was withdrawn for lack of funds. The new International Congresses of Systematic and Evolutionary Biology ICSEB, took over what in the view of many has been the role of the Zoological Congresses. The International Committee of Zoological Nomenclature became an independent organism in IUBS. A gallant effort was made in 1972 by Vaissière and the French colleagues to convene a XVIIth Congress in Monte Carlo. Attendance was poor and the proceedings never left the xerox stage. A long hiatus started. The care for the “vanishing species” was central in Washington. Instead, what followed, was the vanishing of Zoology from the international academic scene. Names were even changed in order to avoid the word “Zoology”! This unbearable situation appeared in its full light after the 1992 Rio de Janeiro Conference and the ensuing Convention on Biodiversity. Now, after nearly three decades since Monte Carlo, the computer revolution entirely changed the situation. With rapid communication and interchange, a reunification of the splinter specialties of Zoology became easily possible. The concept of an integrative zoology, synthesizing data and results ranging from molecular biology to behavior, gained wide acceptance. Cybernetics became the means which could raise again the Phoenix of unified zoology on “wwwings”! This Congress sprung into life entirely through the world-wide web. What started from my letter exchange with Rosa Polymeni found within a few months a world wide positive response. On our web page and with the care of Stuart Poss, the congress turned rapidly into a vibrant virtual, electronic reality. My thanks to both of you, dear friends. The Congress in Moscow in 1893 obtained a financial support of 7,000 gold roubles personally from Emperor Alexander II and the Czarevitsch Nikolas. Seventy years later the Congress in Washington had a budget of 256,000 $ (old dollars!) of which 200,000 came from the US Government through the NSF and of which 100,000 alone were spent in travel grants! Athens started from nothing. I wish to give my full appreciation and thanks to the Hellenic Zoological Society, our host, which had the courage to sign the blank check of the XVIIIth Congress. We received back the sponsorship of IUBS and this has been a major moral buster. For this, our thanks are due especially to Marwalee Wake and Talal Younes. We have received a grant from UNESCO/MAB, thanks to Peter Bridgewater,

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for the publications of the Congress. National Zoological Societies have also contributed from their meager means. But the present Congress owes its existence almost exclusively to the voluntary enthusiasm of its participants and their registration fees. We are especially thankful to the 20 organizers of the symposia and discussions and to the over 100 invited speakers who did not receive resources from us. Special thanks are due to Sandro Minelli and to Philip Tubbs who brought the meeting of the International Committee on Zoological Nomenclature back to the venue of the Congress of Zoology. The symposium program of this Congress strove to present a cross-zoological picture of the many levels of zoological inquiry, both horizontal and vertical. We shall have a very good selection of such symposia. I am only sorry that ethology is not sufficiently represented. The four General Discussions on stringent issues of zoological science and education policy are an innovation which has to prove its justification for the future. The world community of zoologists has to regain its say in international science politics. Due to the special panoramic structure of the Congress, specific aspects of faunal conservation, important as they are, could not be sufficiently represented. Hopefully this will be done in the future. Attendance here in Athens is far from the incommunicable thousands in the last congresses. Perhaps too far. But if we will succeed to create a precedent and a framework which will conveniently re-unite on line Phoenix-like, all the zoologists and make them interact, both virtually and in future reunions, this Athens Congress will really be a new start. During this week we shall have plenty of opportunities in the lecture halls and outside, to discuss the ways in which Zoology should be put back again on the academic world map. The world of culture has changed much since the Washington congress. It needs an active zoological thinking in order to redefine our relation to the living world in face of the dangers of destruction and in order to respond to the onslaught of creationism and to the ethical mysticism of the “deep green” philosophy. All this we shall try to synthesise in the General Assembly which will take place on Friday. Unlike the sleepy Business Meetings of regularly functioning organisms, this meeting should represent the quintessence of our efforts here. Please attend! Let us work together for the successful proceedings of this Congress and reach a fruitful closing session. Long live Zoology, the rich and integrated science of animal evolution and of the human roots! Thank you all for coming.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Opening Remarks The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. xv-xvi, 2003

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Opening Remarks Rosa Polymeni, Secretary, Organizing Committee

Distinguished Minister of Agriculture, Distinguished Rector of the University of Athens, Dear colleagues, In 1963, Prof. Romer, the president of the last official ICZ in Washington stressed the disintegration of Zoology stating that “today each biologist has a much better knowledge of some fraction of the picture. But few of us can or do make any attempt to fit the pieces together. The animal has dissolved into fragments; and so has the Science of Zoology”. Four and a half years ago we were wondering if this opening ceremony would ever happen. It is exactly then, that I received a letter from Prof. Por –a very serious letter as he characterized it - proposing to help him for the realization of the 18th (New) International Congress of Zoology. The purposes of this idea are to resist against the extreme specialization which very often develops into a blind alley, to bring forward again the rich unifying aspects of Zoology, and to reverse the crisis in the professional Zoological education. These facts which the last 20 years I had the sad opportunity to feel and realize in my own professional domain, pushed me to answer the fatal “Yes”. At the very beginning we tried to bring back in action the International Zoological Society. And it worked out! The very hopeful fact is that the new generations of Zoologists responded too. But, believe me, it has not been easy. Very soon the Hellenic Zoological Society entered the game officially. My colleagues in Athens, especially Dr. Spyros Sfenthourakis, who worked on a 24 hour basis, and also Prof. Tasos Legakis and Prof. Maria Legaki, worked very hard to keep the Congress going. The financial matters have been the real nightmare of the Congress. We started from the absolute zero. Thanks to Professor Por’s continuous injections of optimism we were able to overcome our normal pessimism and reach our goals. We have received support from many Zoological Societies and Organizations, especially from the French Zoological Society, who founded the institution of the International Congresses of Zoology. This fact invigorated us to keep on and conclude our efforts. Chers collègues, de la Société Zoologique de France, nous sommes très heureux de vous avoir parmis nous. Nous souhaitons de pouvoir reétablir ensemble et continuer l’institution des Congrès Internationaux de Zoologie.

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I would like to express my thanks to all the conveners and invited speakers who came to support the Congress by their own means, as well as the to the representatives of UNESCO, the International Committee of the Zoological Nomenclature and the International Union of Biological Sciences. Special thanks to our Section of Zoology Marine Biology and to the University of Athens, under the aegis of which we accomplished the realization of the Congress. The list would very long in order to mention all the moral and, more infrequent, financial support that we have received. Despite our good intentions, some mistakes, omissions or delays may have occurred. We take full responsibility for these and we ask for your understanding. I wish this congress will prove fruitful and will support effectively the discipline of Zoology. Thank you

A General Review of Zoological Trends During the 20th Century

Invited Lectures

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

A General Review of Zoological Trends The During the 20th of Century 3 New Panorama Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 3-13, 2003

A General Review of Zoological Trends During the 20th Century E. R. Pianka Integrative Biology C0930, University of Texas at Austin, Patterson Labs, 24th at Speedway Austin, TX 78712-1064 U.S.A. E-mail: [email protected]

Abstract Enormous progress has been made in zoology during the 20th century, largely due to a multitude of clever new technological advances: electron microscopes, oscilloscopes, radioisotopes, radiotelemetry, digital and satellite imagery, PCR and DNA sequencing, global positioning systems (GPS), rapid and affordable travel, unimaginable computing prowess, faxes and email. All this new technology has allowed zoologists to study things previously impossible. The century began with the rediscovery of Mendelian genetics, followed by the discovery of DNA structure, the genetic code itself, instinct and animal behavior, speciation, hybrids, parthenoforms, a new previously unknown Kingdom of chemosynthetic organisms, restriction enzymes, cloning, genetic engineering, genetic control of development, and understanding of metabolic pathways. One of the strongest recurrent themes in biology this century has been to consider all sorts of phenomena within the context of natural selection. Phylogenetic systematics has revitalized many areas of biology, forcing and facilitating an evolutionary approach. Evolution provides the conceptual backbone of zoology. Zoologists study phenomena that range across vastly different spatial and temporal scales, from molecules to cells to organisms to populations to communities and entire ecosystems. Like other scientists, most zoologists have rushed to embrace the reductionistic approach. Too often, workers at different levels look somewhat askance at the next higher level of approach. The reason for this hesitancy to accept the next higher level may be that one must slur over interesting detail at one’s own level in order to practice biology at the next level up. Each level of approach offers distinct advantages but suffers from its own problems. Molecular biologists cannot “see” the objects of their studies, but they can do experiments in Eppendorf tubes in small spaces in a matter of hours. An experiment is planned before lunch, executed that afternoon and results are analyzed that evening or the next day. Rapid progress can be made with such a compressed timetable. Other sorts of biology require more space and greater patience. Funding for zoological research is strongly skewed towards molecular biology. We should all attempt to couple our approach to higher levels and we should be more tolerant of others working at higher levels of approach.

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Funding should be spread more equitably across disciplines. We have made impressive strides in understanding phenomena at most levels of approach in biology, but the approach at the community-ecosystem level lags far behind others. Much more thought needs to be devoted towards attempts to connect community properties with those of individuals in populations. Examples of how community-level properties could emerge from attributes of individuals are given.

Introduction First, I’d like to thank the organizing committee for inviting me to present this opening address to the 18th International Congress of Zoology. I suspect that my Greek friends Petros Lymberakis, Moysis Mylonas, and Efstratios Valakos, were instrumental in my being chosen and I’d like to thank them as well. I did not choose this title — it was “assigned” to me by the organizers. How could anyone review a subject as broad as zoology over an entire century? It’s the kind of challenging thing one would expect somebody like Ernst Mayr to do. I guess if you live long enough, you get a chance at something like this. Obviously, I won’t be able to mention many things zoologists have discovered during the past one hundred years. Please, do forgive me if I fail to mention your own favorite area of research! I’ve been a zoologist for only 35 to 40 years and I’ve seen rather massive changes and new developments during just these four decades. For example, when I entered graduate school in 1960, numerical taxonomy was just being invented. It quickly replaced “old fashioned classical systematics,” and then, just as quickly, phenetics was swept away by phylogenetics, which has endured and become entrenched during the last several decades. Zoology has become Obsolete I fear that I must begin with some bad news for all zoologists. Zoology is rapidly becoming obsolete! Let me illustrate this with the example of my own ill-fated department at the University of Texas. It seems as if people get restless towards the end of centuries and they like to reorganize things to become more “modern.” From 1892 to 1899, the University of Texas had a “School of Biology” which included botanists, geologists, and zoologists. As the end of the century drew near, in 1899, the University reorganized and created three new “departments” of botany, geology, and zoology. These departments thrived and became recognized as among the best in the world. But last year, in 1999, the powers that be at my University got restless and decided to reorganize biology once again. [After all, it was the end of another century!] The once proud Department of Zoology where I spent the last 32 productive years was abolished on the eve of its 100th birthday! Our Departments of Botany and Microbiology were also eliminated. We went back a century to the old 1899 plan and created a new “School of Biological Sciences.” Departments were out now, replaced with four “Sections.” Their major motivation must have been to emphasize molecular biology since two of the four new sections are:

A General Review of Zoological Trends During the 20th Century

and

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Molecular Cell and Developmental Biology Molecular Genetics and Microbiology

Another strangely narrow small section is Neurobiology. All three of these sections adopt a reductionistic approach. In the fourth section, mine, we embrace an explicit anti-reductionistic approach. We call it Integrative Biology Our group includes botanists and zoologists who work on ecology, evolution and organismal biology. We understand the need to integrate across levels of organization and taxonomic groups. We engage in interdisciplinary research (for example, I’m an ecologist and I have a small DNA laboratory). We hope that zoological research will flourish in our section. These four sections are expected to serve as focal points for research, but all activities including faculty hiring and promotion, undergraduate and graduate programs, teaching and advising, are now coordinated through the overarching School of Biological Sciences, which has its own Director and staff. The School is a sort of “super” department for the biological sciences. What do you suppose they’ll do when 2099 rolls around?? If, indeed, humans haven’t gone extinct by then! Techniques and Technological Breakthroughs When you think about the past century, the first things that pop into mind are new techniques and technological breakthroughs. Electron micrographs allow us to see and study phenomena at a microscopic level. Satellite imagery has given us “macrographs” that allow us to see and study very large phenomena like El Niño. Satellite imagery has been available long enough now (since 1972) that chronosequences can be used to follow cyclical phenomena such as fire succession in grasslands. Oscilloscopes are a relatively old invention that greatly enhanced the ability of physiologists to study neural phenomena. Isotopes allow us to follow the movements of molecules through an organism or an ecosystem and to follow a given cell or cell lineage through development as well as many other things like carbon 14 dating. Modern molecular biotechnological tools, such as restriction enzymes and gene splicing, now enable geneticists to transfer particular genes from one organism to another using vectors such as plasmids and various viruses. Human insulin and growth hormone are now routinely produced in chemostats of E. coli bacteria that have had human genes spliced into their genomes. Transgenic cows produce milk containing medically useful proteins such as human blood clotting factors (useful for hemophiliacs!) Genetically altered transgenic bacteria have been used as living vaccines that confer resistance to particular diseases such as typhoid. Such recombinant DNA technology has also enabled us to produce useful new life forms such as pollutant-eating bacteria that can help us to clean up what’s left of our environment. Pest resistance and nitrogen-fixing genes are

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being spliced into crop plants with the hope of vastly increasing yields. Any day now, some enterprising genetic engineer will transplant elephant growth genes into cattle to make bigger and better cows! You want a bigger chicken: I’ll transplant some ostrich genes into chickens! We are audaciously bypassing natural selection and creating whatever phenotypes we think best. A large percentage of US crops (corn, soybeans, cotton, tomatoes, etc) are genetically engineered and are now being grown commercially. More transgenic organisms will eventually be designed for release into nature. Genetically engineered organisms could have adverse effects on other species in natural ecosystems. We already have enough natural pests and certainly don’t want to make any new ones! Unfortunately, we still know far too little to engineer ecological systems intelligently. Obviously genetic engineers should work hand in hand with ecological engineers (a nearly non-existent breed)! Radiotelemetry has advanced to the point that very small transmitters can be attached to small animals and used to follow their movements. Large animals like sea turtles and whales now carry devices that transmit their locations to satellites which download the data at prescribed times and positions. You can now be anywhere and follow a whale’s migration from the arctic to the Antarctic. In many ways, this is the best time ever to be a zoologist. We have easy access to anywhere in the world via rapid and affordable travel. We can go almost anywhere anytime and there are still bits and pieces of wilderness left scattered around the globe. Global positioning systems (GPS), invented for military purposes, now allow us to get relatively exact co-ordinates for any spot on earth quickly and with ease. Modern biological technological tools such as the polymerase chain reaction (PCR) allow us to amplify tiny amounts of DNA, which can now be sequenced relatively easily and inexpensively. DNA sequences can be used to establish degrees of relatedness among animals and to recover robust phylogenies, which can be used to infer probable evolutionary pathways. I have been using computers and the Internet ever since their inception. I learned FORTRAN in grad school during the early 1960’s and I wrote my own programs to do statistical analyses on one of the first vacuum tube IBMs (it took up a entire large room and could not even match one of today’s low-end desktop personal computers). The speed with which personal computers have been improved is awesome. Just a few years ago, I treasured floppy disks. Now I almost never use them, only 100 meg zip disks. When my hard drive failed, I upgraded from 4 gigs to 38 gigs for only $289. Recently we got a 400 megahertz G4 which processes 128 byte bits rather than 32 byte bits. Being twice as fast and processing 4 times as much information at each step makes this computer eight times faster than last year’s. Such computing prowess was unimaginable a mere decade ago. People reading this 10 years from now will undoubtedly laugh at how modest my fancy new computer was! Today you can collaborate with people around the world with ease using faxes and email. While your colleague on the other side of earth is sleeping, you work, emailing it to him at the end of your day. Then, he or she plugs away at it while you sleep. Together, you can work around the clock!

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Major Developments Now let’s consider some other major developments in zoology during the last century. Of course, the century began with the rediscovery of Mendelian genetics and genetics has been a focal point all though the 1900’s. Herman Mueller discovered that x-rays cause mutations. Mid century came the discovery of the structure of DNA, then the genetic code itself, the machinery of genetics, transcription, translation, etc. Instinct and animal behavior came to the fore, as did speciation, hybridization and parthenogenesis. The late W. D. Hamilton (and others) developed the idea of kin selection and inclusive fitness. Studies of sexual selection abounded (Andersson 1994). A new kingdom of chemosynthetic organisms was discovered. Metabolic processes such as the Kreb’s cycle were understood for the first time. These still need to be placed in an evolutionary perspective ... how do metabolic pathways evolve? Why are they sometimes dismantled? The vast majority of mammals can synthesize ascorbic acid but humans cannot and must supplement their diets with Vitamin C. Many aspects of the genetic control of development, including developmental plasticity and canalization, have been elucidated, but much more remains to be learned. Phylogenetic systematics has revitalized many areas of biology and has both forced and facilitated an evolutionary approach. Evolution is Our Conceptual Backbone One of the strongest recurrent themes in biology this century has been to consider all sorts of phenomena within the context of Darwinian natural selection. Dobzhansky (1971) said that “nothing in biology makes sense except in the light of evolution.” Evolution provides the conceptual backbone of all zoology. Natural Selection: the ultimate inventor Natural selection is surely the ultimate inventor: a short list of some of its many patents includes flight, fusiform shapes, celestial navigation, echolocation, insulation, infrared sensors, hypodermic needles, plus a wide variety of pharmeceuticals including analgesics, antibiotics, diuretics, emetics, laxatives, and tranquilizers. As another example of natural selection, consider gecko feet. These lizards can run up a pane of glass and even run upside down across a ceiling. Scanning electron micrographs show literally millions of elaborate very fine hairlike setae, each bearing tiny hooks and hundreds of spatulae which allow these lizards to gain purchase on almost any surface including very smooth ones (Hiller 1976). A single individual gecko can have as many as a billion spatulae! Several mechanisms of adhesion have been proposed, including suction, glue, electrostatic attraction, and friction. Gecko feet still stick in a vacuum, eliminating suction. Gecko feet have no glands, making glue most unlikely. Experiments using x-rays to ionize air have eliminated the possibility of electrostatic attraction. A smooth pane of glass offers very little in the way of friction, although friction would certainly be quite important when climbing on any rough surface.

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In a very interesting recent study published in Nature (Autumn et al. 2000) of gecko setae, scientists removed a single seta from a large Tokay gecko and under a microscope glued it with epoxy to an extremely fine wire. Each seta ends in hundreds of spatulae, which press up and conform to the substrate. Direct forces of setal attachment were measured with an extremely tiny (about 100 x 100 micrometers) high-tech microelectromechanical sensor (a two dimensional “dual-axis piezoresistive cantilever fabricated on a single-crystalline silicon wafer”). Earlier work that had rejected two previously proposed mechanisms of adhesion, suction and friction (Hiller 1968), had demonstrated that intermolecular forces, or van der Waal’s forces, provided the adhesion. Van der Waals forces are basically like gravitational forces, but acting between molecules. Autumn et al’s amazing high-tech study provided indirect support for such intermolecular forces. Van der Waal’s forces require exceedingly intimate contact between a gecko’s spatulae and the surface and they are extremely weak at distances greater than atomic distance gaps. These authors estimate that if a gecko’s entire billion spatulae were simultaneously engaged with substrate molecules, the force holding a gecko to the substrate would be over 500 pounds per square inch! With such powerful forces, one might expect geckos to be plastered against their substrates unable to move. During a powerful cyclone on Mauritius (Vinson & Vinson 1969), Phelsuma day geckos were actually beaten to death by the furious flapping of leaves they were on — but these dead geckos nevertheless remained firmly attached to the leaves! How do geckos manage to break such strong bonds? How do they control their powerful feet and toes? Autumn et al. (2000) liken the complex behavior of toe uncurling during attachment to blowing up an inflating party favor, whereas toe peeling during detachment is analogous to removing a piece of tape from a surface. During running, geckos peel the tips of their toes away from a smooth surface. Toe peeling may have two effects. First, it could put an individual seta in an orientation or at a critical angle that aids in its release. Second, toe peeling concentrates the detachment force on only a small subset of all attached setae at any instant (Autumn et al. 2000). Indeed, one of the great remaining mysteries is why don’t such clinging toe pads pick up all sorts of debris? These authors comment that manufacture of such small, closely packed arrays mimicking gecko setae is currently beyond the limits of human technology. Nevertheless, the natural technology of gecko foot-hairs could provide biological inspiration for future design of remarkably effective re-usable dry adhesives. Perhaps one day, people wearing gecko skin gloves will climb cliffs and buildings! If so, natural selection will hold the patent! Biological Hierarchies: Time and Space Scales Zoology has a complex hierarchical organization. Zoologists study phenomena that range across vastly different spatial and temporal scales, from molecules to cells to organisms to populations to communities and entire ecosystems (Fig. 1). Across this broad range of scale, factors vary by many orders of magnitude. Emergent properties arise at each level. For example, glycolysis is a property shared by some metabolic pathways but it is not a property of a molecule. Dominance or recessiveness are properties

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Fig. 1. Diagrammatic representation of the time-space scaling of various biological phenomena. Community and ecosystem phenomena occur over longer time spans and more vast areas than suborganismal- and organismal-level processes and entities. Most subdisciplines of biology take a narrow reductionistic approach. A broader integrative approach across all these levels of organization must be adopted.

shared by some genes but not of nucleotides. Sexual behavior is a property shared by some organisms but not of a gene. Sex ratio and population density are properties of groups of organisms but not of single animals. Food web connectance is a property of a community but not a property of a population. Dan Brooks (1988) gave a nice example: (1) individuals move and disperse during their lifetimes, (2) over the lifetimes of multiple individuals, immigration and emigration take place between and among populations, giving rise to metapopulation structure, (3) over still longer time and space scales, geographical ranges shift in response to changing climates and geotectonic movements, ultimately leading to geographical patterns of diversity. Molecular biology is small and fast. You can do multiple experiments in a few rooms using tiny Eppendorf tubes. In some cases, a researcher can plan an experiment while driving to work, execute the experiment early in the AM, go to lunch, and analyze the results later that afternoon. The next day a paper can be written and submitted to Nature. Simple causality reigns in molecules. In contrast, community ecology requires lots of space and time. It may take decades to acquire results. Community ecology is not for the impatient or feint of heart. Multiple causality is the rule and constitutes an effective roadblock. People are impatient — they want results, recognition and fame NOW, not later. Many scientists adopt a reductionistic approach: take something apart into its component pieces and then try to put it back together again. The molecules are in motion,

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but knowing their positions and paths, can you extrapolate to explain phenomena at higher levels? An alternative integrative approach attempts to understand an entire complex entity across levels of organization, although some scientists express disdain for such a perspective. Both the reductionistic and integrative approaches can offer insights, often of very different sorts, into how a biological entity operates. As we race to embrace molecular biology, many have been neglecting higher levels of approach. Indeed, it is worse than simple benign neglect. People working at each level actually express disdain for those struggling to work at higher levels. Molecular biologists think cell biology is sloppy because it necessarily slurs over interesting detail. Cell biologists find physiology crude. Organismal biologists wonder how population biologists can gloss over so much important biology of organisms. Population biologists scoff at community ecologists. Such narrow-minded snobbery towards higher levels of approach is inadvisable and unacceptable. It has resulted in funding being diverted more and more towards molecular biology and away from other disciplines like ecology. Worse, traditional areas of zoology like comparative anatomy and physiology are no longer deemed important and therefore are not attracting new graduate students. “Ology” courses, such as protozoology, entomology, ichthyology, herpetology, etc. have disappeared from curricula everywhere. When everyone has become a molecular biologist, who is going to be able to tell molecular biologists what they are studying. Who will describe new species? Understanding molecular interactions seldom provides great insights into evolutionary forces molding adaptations. This is perilous because all levels of approach are necessary to truly understand any biological phenomenon. We need to integrate from molecules to communities. Proximate versus Ultimate Factors Consider the question “Why do migratory birds fly south in the autumn?” A physiologist tells us that a bird compares photoperiod against its internal biological clock. Decreasing day length stimulates hormonal changes, which in turn alter bird behavior with an increase in restlessness. Eventually this “Wanderlust” gets the upper hand and the birds head south. In contrast, an evolutionist would most likely explain that, by virtue of reduced winter mortality, those birds that flew south lived longer and therefore left more offspring than their non-migratory ancestors. Over a long period of time, natural selection resulted in intricate patterns of migratory behavior, including the evolution of celestial navigation, by means of differential reproductive success. The physiologist’s answer concerns the mechanism by which avian migratory behavior is influenced by immediate environmental factors, whereas the evolutionist’s response is couched in terms of what might be called the strategy by which individual birds have left the most offspring in response to long-term consistent patterns of environmental change (i.e., spring bloom, high winter mortality). The difference between them is in outlook, between thinking in an “ecological” time scale (now time) or in an “evolutionary” time scale (geological time). At the physiologist’s level of approach to science the first answer is complete, as is the evolutionist’s answer at her or his own level. Ernst Mayr (1961) termed these the “how?” and “why?” approaches to biology.

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They have also been called the “functional” and “evolutionary” explanations and the “proximate” and “ultimate” factors influencing an event (Baker 1938). The first involves short-term cues whereas the second is a long-term strategy for passing on genes. Neither is more correct; a really thorough answer to any question must include both, although often only the first can be examined by direct experiment. Nor are those two ways of looking at biological phenomena mutually exclusive; behavioral, physiological and ecological events can always be profitably considered from within an evolutionary framework and vice versa. To understand avian migration, one needs to know about both immediate mechanisms and evolutionary forces. We should all attempt to couple our approach to higher levels and we should be more tolerant of others working at higher levels of approach. Funding needs to be spread more evenly across all levels of approach rather than most of it being devoted to molecular biology. Community Ecology Community structure concerns all the various ways in which members of communities relate to and interact with one another, as well as any community-level properties that emerge from these interactions. Just as populations have properties that transcend those of the individuals comprising them, communities have both structure and properties that are not possessed by their component populations. You can think of a community as a complex network of interacting populations. Ecologists are not very interested in captive animals. Their subjects are wild organisms in natural settings, with a normal environment in which that particular creature has evolved and to which it has become adapted. Rolston (1985) made a useful analogy: he likened life on earth to a book written in a language that humans can barely read. Each page in this book of life represents a species, describing not only its phylogenetic relationships, but also its interactions with its physical and biotic environments, as well as its relationships with its competitors, parasites, predators, and prey. Each chapter represents a biome with pages describing all of its component species. Zoologists are just now acquiring the skills necessary to read and decipher this book, but the poor book is tattered and torn, pages are missing (extinct species such as passenger pigeons), and entire chapters have been ripped out (e.g., the tall grass prairies of midwestern North America). There is considerable urgency to study wild organisms in pristine natural habitats now. We must save as much of this vanishing book of life as possible. We must also read it before it is gone forever. Community ecology has to attract population ecologists who are well versed in natural selection. It has become the province of systems ecologists and ecosystem engineers: more born-again population ecologists should become community ecologists. Community ecology is doubtlessly one of the most difficult kinds of biology, but it has obvious utility as we approach oversaturation of this planet. Moreover, data must be gathered now because so many systems are vanishing. Community ecology is also very promising. Major new discoveries, potentially things as important as DNA and natural selection, remain undiscovered because biologists have shied away from this discipline. Community ecologists are still in the process of developing their vocabulary. Identification of appropriate aggregate variables or macrodescriptors (Orians 1980) is

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essential, but constitutes a double-edged sword; macrodescriptors allow progress but simultaneously constrain the directions that can be pursued. To be most useful, macrodescriptors must simplify population-level processes while retaining their essence without fatal oversimplification. Examples include trophic structure, connectance, rates of energy fixation and flow, ecological efficiency, diversity, stability, distributions of relative importance among species, guild structure, successional stages, and so on. At this early stage in community ecology, we should not become overly locked in by words and concepts until we are confident that we are going in the most fruitful directions. Moreover, a diversity of approaches seems desirable. Even the trophic level concept should not be inviolate. A major pitfall for community ecologists is that communities are not designed directly by natural selection (as are individual organisms). We must keep clearly in mind that natural selection operates by differential reproductive success of individual organisms. It is tempting, but dangerously misleading, to view ecosystems as “superorganisms” that have been “designed” for efficient and orderly function. Antagonistic and asymmetric interactions at the level of individuals and populations (such as competition, predation, parasitism and even mutualisms) must frequently impair certain aspects of ecosystem performance while enhancing other properties. Much more thought needs to be devoted towards attempts to connect community properties with those of individuals in populations (Pianka 1992). Terrestrial succession offers a possible example of how community-level properties could emerge from those of individuals. For an individual plant, a fast rate of photosynthesis and hence a rapid growth rate and high rate of reproduction are presumably incompatible with shade tolerance, and hence competitive ability in a light-limited situation. In contrast, shade tolerance and an ability to compete require slower rates of photosynthesis, growth and reproduction as well as relatively larger offspring. Such physiological trade-offs at the level of individuals could very well dictate many of the sequential patterns of species replacement (i.e., colonizing species to climax species) that characterize terrestrial succession. Another example concerns ecological energetics. Only about 10% to 15% of the energy at any given trophic level is available to the next higher trophic level (an “ecological efficiency” of 0.10 to 0.15). This low efficiency has become a sort of rule for how natural communities behave. Genetic engineers tinkering with plant genes hope to increase ecological efficiency by making transgenic crop plants. Why are natural communities so inefficient? Natural selection operating on individual predators favors more efficient predators — this in turn increases efficiency of flow of energy up through the trophic levels but reduces a system’s stability. In homogeneous simple predator-prey systems, efficient predators harvest their prey to overexploitation, driving it extinct and then starving to death themselves. Selection operating on individual prey always favors escape ability, which reduces energy flow and enhances stability, exactly the reverse effects as those operating on predators. Heterogeneous complex habitats offer hiding places where prey can take refuge from predators, thus reducing energy flow and enhancing stability. In the coevolutionary arms’ race between a predator and its prey, the prey must remain a step ahead of their predators, or they are overharvested to extinction. As a corollary, community-level properties of ecological

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efficiency and community stability may in fact be inversely related precisely because natural selection operates at the level of individual predators and prey. Thus, the apparent constancy and low level of ecological efficiency observed in natural ecosystems could be a result of the “compromise” that must be reached between coevolving prey and their predators. Humans seem to feel that we do not have to obey ecological rules — we think that we are somehow above nature. Soon we may find out that we are not! Acknowledgments I am grateful to the organizers for inviting me to give this address. I thank Michael Pianka, Monica Swartz, David Hillis, and Antone Jacobson for reading earlier drafts and for suggesting improvements. Aaron Bauer gave me the Vinson and Vinson reference. References ANDERSSON M. 1994. Sexual Selection. Princeton University Press. AUTUMN K., LIANG Y.A., HSIEH S.T., ZESCH W., CHAN W.P., KENNY T.W., FEARING R. & R. J. FULL 2000. Adhesive force of a single gecko foot-hair. Nature 405: 681-685. BAKER J. R. 1938. The Evolution of Breeding Systems. In, Evolution, essays presented to E. S. Goodrich. Oxford University Press, London. BROOKS D.R. 1988. Scaling effects in historical biogeography: A new view of space, time, and form. Syst. Zool. 38: 237-244. DOBZHANSKY T. 1973. Nothing in biology makes sense except in the light of evolution. Amer. Biol. Teacher 35: 125-129. HAMILTON W.D. 1964. The genetical evolution of social behavior (two parts). J. Theoret. Biol. 7: 1-52. HILLER U. 1968. Untersuchungen zum Feinbau zur Funktion der Haftborsten von Reptilien. Z. Morph. Tiere 62: 307-362. HILLER U. 1976. Comparative studies on the functional morphology of two gekkonid lizards. J. Bombay Nat. Hist. Soc. 73: 278-282. MAYR E. 1961. Cause and effect in biology. Science 134: 1501-1506. ORIANS G.H. 1980. Micro and macro in ecological theory. BioScience 30: 79. PIANKA E.R. 1992. The State of the Art in Community Ecology. In Adler K. (ed.), Herpetology. Current Research on the Biology of Amphibians and Reptiles. Proceedings of the First World Congress of Herpetology at Canterbury. Contributions to Herpetology, Number 9, pp. 141-162. ROLSTON H. 1985. Duties to endangered species. BioScience 35: 718-726. VINSON J. & J.-M. VINSON. 1969. The saurian fauna of the Mascarene Islands. Mauritius Inst. Bull. 6: 203-320.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) On Species and Speciation The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 15-18, 2003

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On Species and Speciation E. O. Wiley Department of Ecology & Evolutionary Biology, Natural History Museum and Biodiversity Research Center The University of Kansas, Lawrence, KS 66045 USA

Abstract Discovering the mode and tempo of speciation requires detailed knowledge of the relationships and distributions patterns among species in clades where extinction and dispersal have not affected the original speciation pattern. It also requires adoption of a species concept that provides an ontology appropriate to the processes thought to be at work. Modern phylogenetic and biogeographic methods, coupled with a lineage concept of species, have allowed systematists to corroborate Wallace’s hypothesis that the predominant biogeographic pattern between sister species is a pattern of allopatry, suggesting that vicariance is the predominant mode of speciation.

Wallace (1855) concluded that the most likely place one would find the closest relative of a particular species was in an adjacent region and not in the sympatry. This observation was used by later workers to develop what we know recognize as Model I allopatric speciation (e.g., Jordan 1905, literature summaries by Bush 1975 and Wiley 1981). However, determining the frequency of this mode of speciation compared to other possible modes was not possible until the development of modern phylogenetic techniques for reconstructing the evolutionary relationships among species and the identification of monophyletic groups of species (Hennig 1966). The combination of a robust phylogenetic tree and a detailed knowledge of the distributions of species within a monophyletic group permit both qualitative and analytical studies of the relative frequency of modes of speciation (Wiley 1981, Lynch 1989). Wiley (1981) outlined the patterns of phylogeny and biogeography one might expect if a particular clade of group of clades speciated. For example, patterns of phylogeny and biogeography repeated between groups of unrelated clades suggests vicariant, Mode I, speciation, while unresolved polytomies between closest relatives suggest peripheral isolation and persistence of the central ancestral species. Wiley and Mayden (1985) investigated a number of patterns of North American freshwater and coastal marine fishes and found an over whelming pattern of allopatry between sister species. Similar patterns are found in many groups of North American freshwater fishes (e.g. Mayden 1988, Wood & Mayden 1993, Grady & LeGrande 1992). Similar patterns also have been

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demonstrated in several vertebrate groups, as summarized by Lynch (1989: frogs, fishes, and birds) and Chesser and Zink (1994: birds). The predominance of allopatry between sister species even extends to the deep ocean where Myia and Nashida (1996) have shown that the majority of sister species of the mesopelagic fishes of the genus Cyclothone are largely to entirely allopatric in their distributional patterns. Determining the tempo and mode of speciation is not easy. The first requirement is a robust phylogenetic hypothesis at the species level. The second requirement is a detailed knowledge of the distribution of populations in each species. The third requirement is that extinction or a change in habitat is not giving a false picture of the original pattern of speciation. That is, if extinction is common and the extinct relative is not included in the analysis, one might very well conclude that sympatry was common among recent sister relatives when, in fact, actual sister species were allopartic when both were extant. Conversely, movement of sister species into non-contiguous refugia might give the impression of allopatry, even if the species had speciated via some sympatric speciation mechanism. So, such analyses are by no means straightforward. There is another complicating factor, the manner in which systematists conceive of species as entities (species taxa). Among those who conceive of species as existing in nature (realists as opposed to nominalists), the very perception of the pattern of speciation can differ because two investigators have adopted different species concepts. Consider, for example, the patterns of variation and distribution of a small clade of topminnows, the Fundulus nottii group (Wiley & Hall 1975, Wiley 1977). Agassiz (1854) originally described five species. Mayr (1963) considered the clade as a single polytypic species and he considered the recognition of five species as an example of typological thinking. Wiley (1977) recognized five species. (I do not know if these are the same five species originally described by Agassiz as the types were lost of misplaced.) Obviously, the difference between Mayr’s and Wiley’s interpretations lies in the adoption of different species concepts. Adding to the possible confusion is the ontological status of the species themselves. Hennig (1966), Ghiselin (1966, 1974), Hull (1976) and others have asserted that species taxa are individual entities and neither sets nor natural kinds. Although the claim that species taxa are individuals rather than kinds or sets may be “radical,” the properties of species taxa seem to fit this concept. Species taxa, unlike natural kinds, have particular origins and extinctions. They are not timeless, but time bound. Species taxa, unlike sets, can change their parts over time. Under this concept, an individual organism is not a member of a species taxon, but rather, it is a part of a species taxon. And, groups of species taxa make up the parts of a monophyletic groups rather than being members of a monophyletic group. Further, species and other taxa do not function within scientific theories in a manner similar to natural kinds (Wiley 1989). If species are, logically, individuals, then what of species concepts? There are a plethora of such concepts (see review by Mayden & Wood 1995). Some, often described as “operational”, are designed to help investigators discover species but fail to capture the relationship between macroevolutionary theory and the relationship of individual species to that theory. They usually describe properties thought to be characteristic of species, such as sufficient morphological distinctiveness or possession of an apomorphy. Examples include the morphological species concept and various version of the

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phylogenetic species concept. Obviously, if particular species are individuals and not kinds, then such species concepts, which treat species as kinds or sets, cannot serve as general concepts regardless of their utility to particular systematists. In other words, while it is true that many species are morphologically distinct, or have one or more apomorphies, it is also true that such characterizations do no actually define a natural kind “species” that exists in some logical relationships with the underlying theories of descent with modification and speciation. Species are not apomorphies nor are they smallest or largest clusters (sets) of organisms or populations. However, I do not wish to imply that such concepts are useless. They may very well allow us to discover new species that will be shown by later investigation to have more interesting biological characteristics. Others attempt to derive the kind species directly from a process theory. This is the usual way natural kinds are defined in science. One cites biological characteristics such as isolated breeding system (e.g., Biological Species Concept; Dobzhansky 1937, Mayr 1942) or continuity of lineages through time (e.g., Evolutionary Species Concept: Simpson 1961, Wiley 1978) that are judged to delimit members who play a significant role in natural processes (anagenesis, speciation, etc,). Fundulus nottii is a member of the kind “evolutionary species” because it is hypothesized to have properties that define the kind “evolutionary species.” That is, it forms a lineage though time that has descended from an ancestral lineage via a speciation event. Such concepts can be judged based on whether they capture a more or less complete picture of species as they are found in nature. For example, the Biological Species Concept is not wrong just because we recognize many species that have entirely allopartic distributions. It is simply incomplete. A lineage concept seems to be much more general. Macroevolutionary processes produce descent trees composed of ancestral and descendant lineages. Species are those lineages (Hennig 1966). Acknowledgements Thanks to my Greek hosts for a wonderful meeting and the organizers for inviting me. Thanks also to Keith Coleman, David Hull, and Rick Mayden for many hours of conversation on the “species question.” References BUSH G.L. 1975. Modes of animal speciation. Annual Review of Ecology and Systematics 6: 339364. CHESSER R.T. & R.M. ZINK 1994. Modes of speciation in birds: a test of Lynch’s (1989) method. Evolution 48: 490-497. CRACRAFT J. 1983. Species concepts and speciation analysis. Current Ornithology I:159-187. DOBZHANSKY T. 1937. Genetics and the Origin of Species. Columbia University Press, New York, NY. GHISELIN M.T. 1966. On psychologism on the logic of taxonomic controversies. Systematic Zoology 15: 207-215. GHISELIN M.T. 1974. A radical solution to the species problem. Systematic Zoology 23: 536-544.

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GRADY J.M. & W.H. LeGRANDE 1992. Phylogenetic relationships, modes of speciation, and historical biogeography of the madtom catfishes, genus Noturus Rafinesque (Siluriformes: Ictaluridae). In Mayden R.L. (ed.), Systematics, Historical Ecology, and North American Freshwater Fishes. Stanford University Press, Stanford, CA, pp. 747-777. HENNIG W. 1966. Phylogenetic Systematics, Urbana, Illinois, University of Illinois Press, Urbana, IL. HULL D.L. 1976. Are species really individuals? Systematic Zoology 25: 174-191. JORDAN D.S. 1905. On the origin of species through isolation. Science 22: 545-562. LYNCH J.D. 1989. The gauge of speciation: on the frequencies of modes of speciation. In Otte D. & J.A. Endler (eds.), Speciation and its Consequences. Sinauer Associates, Sunderland, MA, pp. 527-553. MAYDEN R.L. 1988. Vicariant biogeography, parsimony, and evolution in North American freshwater fishes. Systematic Zoology 37: 331-357. MAYDEN R.L. & R.M. WOOD 1995. Systematics, species concepts, and the ESU in biodiversity and conservation biology. In Nielson J. (ed.), Evolution and the Aquatic Ecosystem: Defining Unique Units in Population Conservation. American Fisheries Society, Bethesda, MD, pp. 58113. MAYR E. 1942. Systematics and the Origin of Species. Columbia University Press, New York, NY. MAYR E. 1963. Animal Species and Evolution. Harvard University Press, Cambridge, MA. MYIA M. & M. NASHIDA 1996. Molecular phylogenetic perspective on the evolution of the deep-sea genus Cyclothone (Stomiiformes: Gonostomatidae). Ichthyological Research 43(4): 375398. WALLACE A.R. 1855. On the law which has regulated the introduction of new species. Annals and Magazine of Natural History 16(2): 184-196. WILEY E.O. 1977. The phylogeny and systematics of the Fundulus nottii species group (Teleostei: Cyprinodontidae). Occasional Papers Museum of Natural History, University of Kansas. 67: 1-31. WILEY E.O. 1978. The evolutionary species concept reconsidered. Systematic Zoology 27: 17-26. WILEY E.O. 1981. Phylogenetics. The Theory and Practice of Phylogenetic Systematics. Wiley-Interscience, New York, NY. WILEY E.O. 1989. Kinds, individuals, and theories. In Ruse M. (ed.), What the Philosophy of Biology? Kluwer Academic Publ., Dordrecht, The Netherlands, pp. 289-300. WILEY E.O. & D.D. HALL. 1975. Fundulus blariae, a new species of the Fundulus nottii complex. American Museum Novitates 2577: 1-14. WOOD R.M., & R.L. MAYDEN. 1993. Systematics of the Etheostoma jordani species group (Teleostei: Percidae), with descriptions of three new species. Bulletin of the Alabama Museum of Natural History 16: 29-44.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Aristotle: DescriptorThe Animalium Princeps! New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 19-25, 2003

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Aristotle: Descriptor Animalium Princeps! J. St. J. S. Buckeridge Earth & Oceanic Sciences Research Centre, Auckland University of Technology, P.O. Box 92006, Auckland, New Zealand. E-mail: [email protected]

Abstract Aristotle’s “formative world” was dominated by Platonic dogma, in which reality was perceived as an entirely transcendent and immaterial realm of ideal entities. Aristotle’s challenge of this was total and enduring: He replaced Plato’s ideals with concrete reality, empirical veracity and a world of independent substances defined by qualities. A lifelong fascination and wonderment of nature, combined with his “New World view” paradigm, led to Aristotle’s establishment of the foundations of modern zoology. Throughout his life, he was consumed with the “need to know”. This lead to his developing the elements of a zoological classification, which, although paying some regard to physical differences between organisms, is more a consideration of animal behaviour. Aristotle’s prime aim was not to develop a tidy systematic taxonomy; rather, he constructed a system through which he could ascertain the causes of observed phenomena. A pre-occupation with causes lead Aristotle to base his classification of animals upon reproduction, with the highest level of organisms, the internally viviparous, closest to perfection. Although this aspect of his work is less palatable to modern zoologists, we should view Aristotle’s extraordinary advances in heredity and animal reproduction in light of the intellectual and technical resources available to him. It is appropriate then, to conclude that Aristotle was “Descriptor Animalium Princeps” - the founder of modern zoology, and arguably the greatest biologist of all time. Today’s re-evaluation of phylogeny, on the basis of DNA, necessitates an even greater paradigm shift than that from Aristotle’s causal classification to one of systematic taxonomy. However, we must not forget the extraordinary variety of nature. And this, in twentyfirst century terms, is perhaps Aristotle’s great legacy: an imperative to adopt a sustainable stewardship of nature, so that future generations can, with wonderment, enjoy the beauty and richness of the earth’s biodiversity.

Introduction Aristotle was born in 384 B.C. at Stageira, a small Greek settlement in Chalkidiki. The state of Chalkidiki lay at the north-east end of the Aegean Sea, adjacent to Macedonia,

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into which it was absorbed in 348 B.C. (Hammond 1986). The Greek States, each of which previously had jealously guarded its individual autonomy, were soon about to become part of an empire that would extend from the western Mediterranean to India. It was a time of expansion – military, politically and intellectually. Aristotle was to play a significant part in this, for in 343 B.C., Philip of Macedon appointed him to tutor his young son, Alexander, (the future Alexander the Great). The personal relationship between Aristotle and Alexander is uncertain, especially in later years, although Hammond (loc. cit.), contends that Aristotle was responsible for the instilling of a love for Greek culture, daring speculation and amazing versatility in Alexander during his formative years. According to Novikov (1998), the friendly relationship between Alexander and Aristotle was eventually destroyed by “court conspiracies”. None-theless, Alexander and Philip acknowledged their great debt to Aristotle by richly rewarding him, and rebuilding the ruined settlement of Stageira. Of particular significance for the advancement of early science was Alexander’s endowment of a great library for Aristotle. Further, during his eastern campaigns, he permitted the collection of a large amount of materials and data for Aristotle and his students. Philip’s choice of tutor to his son clearly confirms Aristotle’s pre-eminence as one of the great thinkers and teachers of his time. But Aristotle was not an intellectual island; rather, his education was part of a pedagogic continuum that can at least be extended back to Socrates. Although Aristotle’s father was a learned man and could have been expected to influence his son’s education (he was court physician to Amyntas II, father of Philip), he died when Aristotle was still a child. The most significant teacher then, was Plato, a student of Socrates. In 368 B.C., a seventeen-year-old Aristotle entered Plato’s Academy in Athens. He was to remain a member of the Academy for 20 years, until the death of Plato in 348 BC, at which time he went to live in a small Greek state in Asia Minor near the island of Lesvos. According to Singer (1959), the following five years were pivotal in the formulation of Aristotle’s ideas of zoology, as he had both sufficient time for study and an ideal natural environment in which to undertake it. In 336 BC, following the assassination of Philip and the accession of his pupil Alexander to the throne of Macedon, Aristotle returned to Athens, and established his own school, the Lyceum. Here, in a garden setting, near a temple to Apollo (Lykeios = light-bearing, an epithet of Apollo), a peripatetic Aristotle was to teach and research philosophy for a further fourteen years. He left his beloved Lyceum, and Athens in 322 BC, shortly after the death of Alexander, and died in the following year, aged 63 years. There are two distinct foci within Aristotle’s scientific works: one toward an understanding of the physical universe, the other toward biology. Interestingly it was the former that, until the sixteenth century, formed the basis of man’s understanding of the cosmos; whilst the latter, for two millennia, was largely ignored. Today, Aristotle’s geo-centric model of the cosmos is rarely discussed, except as an historical curiosity, whilst there is increasing interest in his biological works, which still demonstrate a keen appreciation of natural systems. Although Aristotle’s legacy to science is extraordinarily broad, it is to biology, especially zoology, that he contributed most. His curiosity and wonderment of the natural world endures, and is emulated by those of us who have inherited his passion and perspective of the natural sciences:

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“Εν πασι τοιζ ϕυσιχοιζ ενεστι τι θαυµαστον” In all the things of Nature, there is something marvellous. [Parts of Animals, 645a: 16.]

After Aristotle’s death, his library and works were collected and concealed in a cellar in Asia Minor. They remained there until the first century BC, when they were reassembled and edited by Andronicus of Rhodes. Our current knowledge of Aristotle is based upon Andronicus’ edition. Aristotle’s Intellectual Inheritance The Classical Greek Intellectual Environment: Although Aristotle lacked the physical accoutrements that one could anticipate in a modern zoological laboratory, his intellectual environment was none-the-less impressive, as contemporary Greek thought was very much focussed by a desire to understand the forms and functions of nature. There were distractions, such as the Pluralists (e.g. Empedocles, c. 493-433 BC), who saw matter as comprising four “elements”: fire, air, water and earth, and the Eleatics (e.g. Parmenides c. 475 BC), who perceived reality as “single and changeless”. Interestingly, it does not appear that philosophies such as these particularly hindered Aristotle’s understanding of zoological science. A very compelling intellectual environment existed however, for Aristotle during his “formative years” – a time when he was tutored by Plato. For Plato, and his teacher, Socrates, were very much opposed to research into nature. Rather, the focus of their study was the behaviour of man, particularly the determination of the nature of an individual’s actions, which upon death, would ensure that the soul returned to heaven. The methodology employed by both Socrates and Plato was the dialectic, a process in which analytical discussion through criticism is used to pursue knowledge. This, rather than any pre-occupation with nature, is the legacy of Socrates and Plato to Aristotle, as the dialectic established a platform for the development of science. Rationalism and Empiricism: Plato’s basic philosophical perception was that the ultimate reality lay in the world of intelligence, i.e. in “ideas”. He perceived great inspiration in mathematics, especially in the field of geometry, where the certainty of geometry was likened to timeless perfection. Plato’s pre-occupation with mathematics was anathema to Aristotle, who contended that perfect forms were of no use as models for living organisms, or the processes operating within them. Aristotle asks the following, which succinctly states the limitations of mathematics in understanding the natural world: … indeed one might ask this question, ... why a boy may become a mathematician, but not a wise man or a natural scientist. It is because the objects of mathematics exist by abstraction, while the first principles of these other subjects come from experience. [Nichomachean Ethics 9. 1142a: 16-19]

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Experience, based upon observations of natural phenomena, followed by deliberation of any interpretations, was the way that Aristotle saw as best leading to an understanding of nature. Aristotle’s perspective was that ideas could not exist separate from their physical embodiment. Rather, all things possess a striving to attain their telos, or true nature. The mechanism through which the Greeks believed teleology was achieved was through a mystical vitalism. Interestingly, although modern science dismisses the vitalism concept, there is still no broadly accepted theory as to the nature or essence of “living”. Plato was a rationalist, in the sense that reason, derived from the dialectic, is the foundation of certainty in knowledge. Although Aristotle inherited aspects of this, he was able to lay the foundations of empirical science: where understanding is based upon observation, in which reality is defined within the world of perceptible concrete objects, and where the real world is one of independent substances, each characterised by qualities. Thus Aristotle’s emphasis was on form and function, through which he attempted to define the fundamental nature of an object. Our modern concept of empiricism is firmly founded in both observation and experimentation. It is perhaps a little surprising then, that Aristotle himself did not develop the art of experimentation. None-the-less, he did provide the framework for it, as Strato, one of his successors, and head of the Lyceum in Athens, became the first to establish an experimental technique (Checkland 1993). Tutor and student: There are indeed great differences in the philosophies of Plato and his student, Aristotle. It is a testament to the compassion and vision of Plato that this was achievable, and a testament to the tenacity and originality of Aristotle, that an intellectual transformation that was to last two millennia, was achieved in one lifetime. One of the most informative images of the relationship between them can be gained from the painting, Stanza della Segnatura : La Scuola d’Atene, by Raphael, painted between 1509-1511. In the fresco, Plato stands pointing heavenward (i.e. to the One), while Aristotle gesticulates downward, with his fingers splayed, he is thus focussed upon the earth – the diversity of life and the demands that the material world makes upon us. Aristotle and Nature Aristotle’s primary objective was to understand the “nature of nature”. His perception of nature was an almost imperceptible, but continuous growth and development towards perfection (in form). In order to achieve this understanding he recognised two fundamental postulates: that nature is changing, and that nature can be classified. His approach was thus systematic, and it is this that places him as first zoologist in the modern genre. Pangenesis: Aristotle was responsible for a singular leap in the understanding of how organisms replicate. Philosophers such as Hippocrates, who had formulated the theory of pangenenis, were of the opinion that the means by which heredity functioned was located within all parts of the body (hence pangenis). In this sense, Hippocrates and his adherents believed that form was derived from the existing organism. Aristotle demolishes pangenesis elegantly, with the example of a two-legged child born of a onelegged warrior. He was adamant that the process of inheritance involved a “potentiality

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to develop”, and that every substance possesses a form, and is possessed by a form. In this, he is remarkably close to how we currently view heredity. Systematics: Aristotle considered that it was not possible to develop a systematic classification on the basis of physical differences, as many characters do not appear to be exclusive to one group. e.g. the possession of a placenta, a characteristic of mammals, is not restricted to the mammals. Aristotle describes a selachian (now known as the “placental dogfish”, Mustelus laevis), that was both vivaparous and had what he perceived to be a placenta. Importantly, he did not place this fish with the mammals, as other characteristics, especially behaviour of the fish, clearly placed it amongst the sharks. This example is significant in that it illustrates the importance Aristotle placed on observation. Singer (1959) points out that most naturalists dismissed the possibility of a “placental dogfish”, and that it was not until the nineteenth century that the German biologist Johannes Müller proved that Aristotle had been correct. None-the-less, Aristotle did group animals, although not in a formal systematic way. By grouping animals that possessed characters that were commonly found in combination, he believed that he would be able to ascertain the causes of observed phenomena. This pre-occupation with causes led him to group animals on the basis of their methods of reproduction, with the highest level of organisms, the vivapara, closest to perfection. Evolution: Aristotle was not in the strict sense an “evolutionist”, although some (e.g. Singer, 1959), believed he may have become one if he had lived another decade. What Aristotle did achieve in determining the relationships between organisms is remarkable, and serves as a first step in defining an evolutionary framework. He concluded that living things could be arranged in a Scala Naturae (i.e. a “ladder of nature”), of ascending worth and complexity, in which grades within the hierarchy are not rigorously separated (Fig. 1). It is important to note however, that the Scala Naturae is not “taxonomic”, nor Man Mammals Cetaceans Reptiles & Fish Cephalopods Crustaceans Insects Molluscs Medusoids Zoophytes Ascidians Sponges

Inanimate Matter Fig. 1. The “Scala Naturae”, demonstrating how nature proceeds gradually and imperceptibly from things lifeless to animal life. Based on the books of Aristotle. (After Singer 1941, Barnes 1982).

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was it intended to be. Rather, the groups were divided on the basis of “movements”. As one drops down the scala, there is a progressive weakening of movements, and a commensurate reduction in the nature of the soul. Man was perceived as the closest to “perfection”, as he possessed the highest form of soul (i.e. a rational soul). Inanimate matter by contrast, could not move of its own accord, and of course it possessed no soul. Zoology as a “working science”: The greatest impediment to understanding nature would be a lack of a formal nomenclature to describe the features, behaviour and organisms under study. Aristotle’s nomenclature was well developed, sufficiently so to permit reconstruction of many of his diagrams (the originals no longer exist), e.g. the mammalian generative and urinary systems described in History of Animals, have been reproduced in Singer (1959). Aristotle’s descriptions confirm that he had a very good appreciation of form and function, and this is demonstrated over a wide range of taxa, including cephalopods, teleosts, sharks and cetaceans. Indeed, in Parts of Animals, he uses the term “genus” and “species” (genos and eidos), in a similar manner to that of the present. It is appropriate then, to give credit to Aristotle for development of two key areas of science: scientific illustration and a sophisticated scientific nomenclature. Without these, zoology could not be a “working science”. Epilogue The current pre-occupation and “narrow” focus of many modern zoologists is clearly at the molecular level. This necessitates an emphasis on the geometric disposition of certain protein and carbohydrate groups, rather than a quest to understand relationships through form and function. Perhaps we are in danger, through reductio ad absurdum, of failing to understand the real picture. We should be mindful of Aristotle who concluded [Nichomachean Ethics 9. 1142a], that mastery of natural science is best achieved though wisdom (and experience), rather than by abstraction, (through which he perceived the path to mathematical competence to be achievable). Acknowledgements I thank Emeritus Professor John Morton, Castor Bay, Auckland, for encouragement and thoughtful advice during the preparation of the manuscript. Financial support to attend the XVIII (New) Congress of Zoology was provided by a grant from the Deputy Vice Chancellor’s Discretionary Fund, Auckland University of Technology, New Zealand. References Note: Apart from quotes from Nichomachean Ethics, which are cited as such, references to Aristotle’s works have been taken from selected translations provided in Ackrill, and McKeon. These two texts are listed below, but the works are cited in the text using the well-established numbering system for Aristotle’s work.

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ARISTOTLE 1985. Nichomachean Ethics. Transl. T. Irwin. Hackett Publishing, Indianapolis, 441 pp. ARISTOTLE 1987. A New Aristotle Reader. Ed. by J.L. Ackrill. Princeton University Press, New Jersey, 580 pp. BARNES J. 1982. Aristotle. Oxford University Press, Oxford, 101 pp. CHECKLAND P.B. 1993. Systems Thinking, Systems Practice. John Wiley and Sons, Chichester, 330 pp. HAMMOND N.G.L. 1986. A History of Greece to 332 B.C. 3rd Edition. Oxford University Press, Oxford, 691 pp. McKEON R. 1992 (ed.). Introduction to Aristotle. Modern Library Edition, Random House, New York, 712 pp. NOVIKOV I.D. 1998. The River of Time. Transl. from Russian by V. Kisin. Cambridge University Press, Cambridge, 275 pp. SINGER C. 1941. A Short History of Science to the Nineteenth Century. Dover Publications, Mineola, New York, 399 pp. SINGER C. 1959. A History of Biology to About the Year 1900. A General Introduction to the Study of Living Things. 3rd Revised Edition. Abelard-Schuman, London, 580 pp.

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The Persistent Progression: a New View on Animal Evolution F.D. Por Hebrew University of Jerusalem, 91904 Jerusalem, Israel. E-mail: [email protected]

Abstract Animal evolution is seen today through the dilemma of two reigning views. The first sees animal evolution as a shallow sequence of contingent accidents and catastrophic extinctions. The second, accepting a progressive trend in this evolution, sees a hidden vitalistic or deistic force at work. I propose a third way, which accepts progressivism, but considers it to be a historical consequence of directional dissipative thermodynamic processes which are acting on the globe. The animals have a crucial role in stimulating the gradual expansion of the biosphere and the increasingly efficient recycling within it. The different animal phyla, irreversibly marked by their morpho-physiological signatures are the selective and selected players in this process. The terrestrial environment, once colonised, provided for maximum biomass and highest animal efficiency and complexity. The thermoregulating vertebrates and among them the human species selected out as the recent culmination of this evolution.

Introduction Zoologists looking at the animal world parented the idea of Evolution seen as a structural unfolding of life in time. Aristotle, the father of zoology was also the first evolutionist, as he wrote ‘Nature proceeds from the inanimate to the animal in small steps... a continuity (συνεχεια)”. At the “Darwin Centenary” XVth International Congress of Zoology in London (1958), Julian Huxley said in his keynote lecture that evolution is a progressive ‘natural process of irreversible change, which generates novelty, variety and increase of organisation’ (Huxley 1959). By a strange historical twist, half a century later, the scions of Neo-Darwinism of which Huxley was one of the founders, consistently oppose the idea of progressive evolution in the animal kingdom. All animal species and even all beings are ‘equally evolved’ (Margulis 1998) since the only objective value criterion is selective success. As a consequence of the reigning reductionism, all beings are ‘organisms’ on an equal footing, from bacteria to elephants, and they represent only different strategies to succeed in the struggle for existence. Modern biology seems to ignore the need for a theory of Zoology

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that tries to explain the evolutionary sequence from unicellular to multi-cellular animal organisms, followed by the appearance of animals with complex structure and complicated behaviour and lately by the appearance of the human species. Deprived of any qualitative dimension, evolution as seen by many today, is nothing more than shallow transformism. Evolution is presented by the reigning view as a sequence of sudden (‘punctuated’) aleatory modifications, the products of which are reshuffled again and again by equally unforeseeable and sudden mass-extinction events, primarily of extra-terrestrial causation. This doctrine of saltations and of contingent catastrophes is opposed to Darwinian uniformitarianism and actualism. It is a neo-catastrophism masterly popularised in the many writings of S.J. Gould (1995 and passim). The zoological community has been strangely silent in face of these ideological positions that have invaded all the media of popularisation. Today, as the relationship between humans and the rest of the biosphere is of wide interest and extreme acuity, it has to be openly debated whether humanity is an accidental, ephemeral, and even deleterious side-product of evolution, soon to disappear, or on the contrary, a natural, logical and irreversible result of it. Unfortunately also, a concept of transformism caused by contingency alone, makes a singularly poor argument against the recent aggressive rise of creationism. Fitful and stray transformism is philosophically primitive, perhaps on equal footing with creationism. An alternative theory of animal evolution, which has been published in extenso (Por 1994), is succinctly presented here. Animal evolution is seen as a predictable, persistent process that is progressively channelled. In this view the humans represent a natural consequence of organic evolution. The conflict between the two views of extremely relevant philosophical and operative significance ought to be solved in the field of zoology alone. The result should be a unifying theory of zoology, consistent with a general theory of cosmic and global evolution, without having to take recourse to idealistic and vitalistic concepts. Evolutionary irreversibility and channelling The biosphere is an open, dissipative thermodynamic system in the sense of Prigogine. It operates with the external source of solar energy, and with the thermal sink is the surrounding space. Like other such systems and within the given constraints, the biosphere evolved away from an original high entropy state of structural simplicity. Progressive animal evolution is a consequent stage in this evolution, which previously led already to the organisation of living matter, and in sequence to the raise of the eukaryotic cell. With each step, more energy expending structures evolved and the information content in each new system increased. The emergence of the complex animal organisms led to an increase of the energy greed per unit by several orders of magnitude. Being part of a global progressive thermodynamic dissipative process, progressive animal evolution can be called, with some reserve, a ‘telematic’ process in the sense of Mayr’s (1974) terminology. The whole process has been channelled by natural selection already from the biochemical level. Left-handed amino acids were selected and the four-nucleotide bases

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of the genetic code were chosen among the many of them. Some time in the late Proterozoic, the photosynthesising RuBISCo enzyme of the green plants became globally predominant. Although fairly inefficient, it has been never surpassed. Likewise the basic energy storage and exchange phospho-nucleotide molecule ATP was selected. Its energycarrying capacity is the official tender of the living world. This singled-out twosome of the energy fixing and of the energy-storing molecules, constitutes the constraining mould which delimited all further biological evolution. At each organic level, evolution proceeds in a roughly similar way. New mutations undergo testing in the selective natural environments. The selected solution, often the best available at that time, becomes irreversibly fixed, limits the contingent liberty of future mutations and hence canalises the consequent evolutionary process. This is a somewhat expanded rendering of the well-known Dollo’s Law. Like in a game of chess, each evolutionary step is a move which cannot be taken back and which inevitably influences the whole sequence of the game. The Biosphere and Global Evolution During its existence, the biosphere was exposed to an increase of more than 30% in solar irradiation, as our central star advanced in its own predetermined stellar evolution. Had the biosphere not been able to buffer this increase, the result would have been a thermal death in an overheated atmosphere, perhaps similar to our sister planet Venus. By the end of the Proterozoic the active tectonism of continental accretion was nearly completed and on the mature globe a new phase of plate-tectonic shifting of the existing continents started instead. Around and on the stable established continents, biological evolution could gain continuity and momentum. This has been probably impossible in the times of the ‘peristable’ Archean micro-continents. Biological evolution has been a follow-up of a necessary tectonic evolution. At the end of the Varangerian, after a last phase of global volcanic paroxysm, the atmospheric CO2 concentration stood probably at some 350 times the present value. In the following the dominant process started to be the gradual extraction of CO2 through fixation of reduced carbon by the expanding volume of global organic biomass. This ‘ice house’ process has been probably essential in balancing the impact of the further increase in radiation and maintained global temperatures for the last 600 million years within limits compatible with multicellular life. The atmosphere maintained a ‘complex equilibrium (between) the production of Oxygen from CO2 by plants and regeneration of CO2 by respiration of animals’ (Brown & Mussett 1981). This is the basic ‘Gaian’ feedback process of Lovelock. Since the limits of biochemical efficiency are irreversibly set, the progressive accumulation of live biomass and of other forms of biologically reduced carbon could proceed only along two avenues of liberty. Firstly, the vegetation expanded to the whole global surface. Secondly, the processes of recycling and renewal of biological fixation by the plants was accelerated, refined and globalised. In both these processes the activity of animal organisms has been essential. As a result the global energy capturing green cover grew in extension and the total volume of the energy flow increased.

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The Age of the Animals Among the kingdoms of the living organisms, Kingdom Animalia contains and contained by far the largest number of species. This seems to be paradoxical, since the biosphere existed for most of its history without the presence of animal organisms and functioned only through the simple linear cycle of prokaryotic producers and prokaryotic decomposers. The animal organisms, which appeared in the last billion years, added an apparent complication to the cycle, by interposing different levels of consumers and various links in the food chain. To some, the exuberant flourishing of the animal world is an unnecessary ‘little blimp’ on the body of the laborious producers and decomposers and the biosphere could have gone along quiet well without them (Gould 1996). However, in fact, reprocessing of the organic product through a complicated food web of hungry consumers is much more efficient than the old linear cycle. Unlike bacteria and fungi, which mainly feed on dead organic matter, the animals are killing, engulfing and digesting their food organisms alive and without delay. Moreover, they are generally highly motile organisms, which detect and approach their prey, actively spanning distances unheard of by plants, fungi and bacteria. Some aquatic animals developed advanced techniques to trap suspended food particles. The complicated multi-level food webs ensure that little of the organic production is being lost unrecycled. The big diversity of the animal organisms corresponds to as many channels of recycling specific food items, everywhere in space and during all the time. Feeding on live prey includes all the food objects, from bacteria to plants and of course to other animals. I called animal activity in all its varied facets ‘harpactic activity’ (from the Greek ‘Harpagein’). Darwinian fight for survival and natural selection gained a dramatic and rich content with the rise of the animals. A colony of unicells cannot allow itself to become senescent; it has to maintain a logarithmic growth in order to replace predatory loss. Rapid growth in order to replace the losses and increased body mass of the prey organisms became widespread means of defence. Suddenly, the biological world became replete also with physical and chemical defence devices, rapidly improving in response to the improving performance of the predatory animals. A seemingly endless chain of positive feedback effects resulted in what Vermeij (1987) aptly called ‘escalation’. Over time, action and reaction became more and more rapid and complex and reached the present breathtaking speeds. Gradually also, the behavioural means became more important in this race. The importance of the animals as promoters of bacterial decomposition cannot be estimated enough. On the large extents of the oceanic bottoms they facilitated decomposition, by digging after the dead organic mater in the marine sediments and liberating carbon dioxide. In the continental soils animal reworking results also in massive CO2 restitution to the atmosphere. A runaway depletion of carbon in the atmosphere is being probably balanced by harpactic activity. Due to harpactic activity metabolic rates increased in the organisms over time (Maiorana & Van Vallen 1990) and there has been an increase in per capita energy use, a stepwise economic expansion of the multicellular animals (Vermeij 1987).

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Expansion as an escape Harpactic activity has been most probably the main stimulus for the expansion of the biosphere. One of the most frequent ways to avoid predatory pressure has always been the escape to novel and more extreme environments, out of the reach of the predators. Temporary escape from predation compensated for the extra metabolic cost required by the unfamiliar environment. But the animal consumers always follow after some delay and the same cycle of predation and defence and of escape starts again. One can rightly suspect that without the incentive of escaping predation, there would have been no expansions of the biosphere from the marginal belts of the coastal shallows to a recent almost complete global covering. With the appearance of the animals in the Phanerozoic, diversity, mode and tempo of biotic evolution entered in a qualitatively new phase. The brackish waters, the first to be colonised by the expanding marine biota have probably always been the most productive environments. The continents themselves rapidly turned to be extremely productive. Today they produce 3 times more organic matter than the seas, although they represent proportionally much less than half of the oceanic cover of the globe. As plant production flourished, animals found ample food resources in the estuaries and on land for their complicate and costly functions and structures. In their turn, they facilitate renewed production. Progressive Animality Lotka (1922) wrote: ‘Evolution proceeds in such direction as to make the total energy flux through the system a maximum compatible with the constraints’. It is the animals that turned the modern biosphere into an interwoven global system of energy fluxes. The higher an animal consumer is situated in the food chain or food pyramid, the more mobile, the more sensorial alert it is, the more space it covers in search of its prey Some oceanic fish migrate from shore to shore. Migrating birds visit seasonally, ecosystems situated at the antipodes. The essence of animality in the biospheric context is aggressive consumption of live organisms, sensory capacity to detect the food resources, mechanical means to approach the prey and liberty to move among different environments in search for food. Improvement in these capacities is the yardstick of progressive animal evolution. The trend to progressively improve ‘animality’ is however far from universal in the animal kingdom. It is not a broad front in which all the animal types participate. The critics of progressivism often imply universality of progress, a zoological ‘orthogenesis’, for the sole purpose of knocking it down. Neither is progress in the animal kingdom, as also often assumed, a relay race in which each phylum hands the torch to the following one, a modern replay of the classical Aristotelian scale of life. Many branches of the animal world diverged into specialised side alleys, for instance progressively improving adaptation to sedentary or to parasitic life styles. To use another athletic comparison, progress in the animal world resembles more a cross-country marathon race in which a whole crowd starts out and gradually, as most of the participants remain behind or leave the race, the leaders run in a single thin and distanced file.

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Phyletic Selection The runners in the race are the phyla and the classes of the animal kingdom. Every phylum or class is defined by a set of irreversibly fixed physiological and morphological properties. These represent the ‘Bauplan’ of each of these major taxonomic units. The 35 animal phyla exist since at least the early Cambrian. Classes within them have been defined later. Each and every animal species bears the limitations and freedoms of the phylum or class to which it belongs. Therefore the individual success or failure of the totality of the component species is summing-up as the success or the failure of the phylum. As a result, natural selections acts also at the level of the phyla, provided of course, that the phylum as we define it, is a monophyletic natural unit. Every echinoderm is invariably penta-radial. No flatworm ever developed a skeleton and all are limited to gliding movement. All the nematodes are structurally obliged to move on a wet substrate and therefore cannot live in the plankton. Five phyla and several classes, with all their multitude of species are irreversibly condemned to sedentary life. So are the countless species of the parasitic phyla and classes. It results that natural selection does not play its hand with jolly jokers or wildcat cards, but only with a limited set of cards, which belong to a certain suite and colour. In the Cambrian shallow sea environments, all the 35 phyla were represented by a relatively low number of species each. As life branched-out into the different environments and evolutionary history proceeded, many of the phyla remained confined to the oceanic waters and several of them saw their diversity reduced to a handful of species. On the contrary, three phyla, namely the Mollusca, Arthropoda and Chordata, with their classes, expanded over all the environments of metazoan life and showed a disproportionate increase in species numbers. On their account, also parasites, Platyhelminthes, Nematoda and several classes of ‘Protozoa’ (see below) have achieved hyper-diversity. The top-heavy emergence of these few privileged hyper-diverse phyla is one of the most significant results of the Phanerozoic animal evolution. These phyla were selected out of the many because of the elements of adaptive freedom of their Bauplans. Natural selection acts day-by day at the level of the species. Phyletic selection acts cumulatively over long periods of time and especially in the critical time-periods, called ‘global extinctions’ by the neocatastrophists, when legions of species of a phylum are being felled. The Osmotic Hurdle Expansion into brackish-estuarine and fresh inland waters required adaptation to low, fluctuating and unpredictable salinities. To achieve this, the animals had to secure the osmotic hoemostasis of their internal liquid milieu. Many phyla and classes of marine animals, such as the corals, the echinoderms, the brachiopods, the cephalopods and the sea squirts are stenohaline, fundamentally unable to osmoregulate. Most of the animal phyla remained in fact confined to the seas. Only the good osmoregulators, different phyla of worms, the crustaceans and arachnids, the shells and the snails and most of the fish classes could produce species able colonise the estuaries and in the following also the fresh waters. The high biological productivity of the land-locked waters amply

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compensates for the considerable metabolic costs of osmoregulation. The stenohaline marine phyla confined to the oceans live on the contrary, in an oligotrophic, nutrient poor environment, albeit much less energy-demanding. The capacity to osmoregulate has been a long-term asset also in the marine environment, since seawater has a stable high salinity and a buffered temperature, at least on the secular scale. But during critical periods of the tectonic history of the globe, oceanic salinity and temperatures fluctuated sharply and even dissolved oxygen could be at times deficient. Though extra-terrestrial factors, such as massive meteorite falls possibly joined in, the crisis periods were nothing but rare instances of congruence of extreme fluctuations in several earthly, tellurian environmental factors at once. With the exception of the below-mentioned demise of the dinosaurs, the catastrophes of the neo-catastrophists have been all reported as impacting the marine biota. Phyla unable to osmoregulate were severely castigated and many of their classes extinguished. The frequent near-extinctions suffered for example by the echinoderms and by the cephalopods, were due to the fact that all of their species are strictly stenohaline. The coral reef taxa both stenohaline and stenothermic were also gravely touched and it sometimes took millions of years before they could recover. Euryhaline phyla could always survive fluctuations of marine salinity and temperature in refugia of semi-secluded marginal environments. As a matter of fact the dominant euryhaline phyla emerged hardened from each global crisis. Unlike Russian roulettes in which the rule of natural selection was suddenly suspended and replaced by the rule of sheer chance (see for instance Eldredge 1999), the crisis times have been the major events of phyletic selection in the oceans. Into the Open Air Only mobile and osmoregulating animals could emerge onto the dry land. Moreover, only animals that could not keep their body fluids protected against evaporative loss, animals that developed homeohydric capacity, could leave the protection of the wet soils. A watertight body cover and a water-saving excretion were needed for this. Out in the open, also a skeleton was essential to oppose gravity, to withstand the blowing winds and to serve as a lever for efficient movement. Only the class of the gastropods, the arthropods and the vertebrates could colonise open land. However, the snails use their external conch only as a protection against exsiccation, and not as a lever for their musculature. When moving, they have to extend and creep flatworm-like, loosing big amounts of water. It is understandable that the gastropods remained by and large marginal players in the terrestrial ecosystems. Two phyla, the arthropods and the vertebrates remained in the race, capable of using all the rich vegetal biomass that developed on land since the Carboniferous. They could take also unlimited use of the free plentiful oxygen for the full respiratory use of the ingested food. Unlike their aquatic ancestors who had often to face oligotrophic conditions and lack of dissolved oxygen for their respiratory metabolism, the land animals had all the food resources needed in order to develop extremes of energy dissipative complexity. The most dramatic and extreme chapters in animal evolution, both in terms of complexity as well as functional and behavioural achievements could have happened only on land.

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A Constrained Host It seems paradoxical to assert that despite their hyper-diversity of many millions of species presently living; the arthropods have had their own dramatic limitations in the progressive evolutionary race and in phyletic selection. Their armour-like exoskeleton has to be shed periodically and in their inter-moult periods the soft-skinned arthropods are exposed to predation and to water loss. Small body size was required for better hiding in these critical periods. Among the three great arthropod classes, the crustaceans never fully adapted to terrestrial life, because of functional shortcomings of their own. The arachnids and the insects developed good aerial respiration, a watertight epicuticle and water-saving purine-based excretion. However the arachnids remained morphologically restrained to predatory life. The tracheal system for air breathing, which the insects developed to maximum efficiency, is not a centralised respiratory system. Above a certain critical body mass, oxygen supply is impossible for the muscles, which exclusively need full aerobic conditions. The diffuse tracheal system ‘replaced’ also a proper circulatory system and functional decentralisation characterises the insect body functions. A further premium for small size was set with the appearance of the agile raptorial reptiles in the Permian. As a consequence, the advanced insects specialise in miniaturisation. Owing to some not fully elucidated constraints, insects colonised the high seas only in a few exceptional cases. Vertebrate Primacy The vertebrates were thus left in the cross-country marathon of progressive evolution towards the highest complexity and animal efficiency. They probably displayed right from the beginning a morpho-physiological type with the minimal set of constraints, with maximal liberty to adapt to the requirements of the biotic and abiotic media. The success of the vertebrates could have been foreseen right from the beginning, among others because of the possession of the multiple HOX gene sets. Gould (1989) speculated that the presence in the Cambrian Burgess shale of only one isolated chordate, Pikaia, is a proof that the future success of the vertebrates was pure contingency. Lately, chordate remnants were found much more in abundance in the older Chenjian site and the widespread Cambrian fossil conodont animals have been also identified recently as chordates. In the Silurian the jawed fishes, some of them gigantic, already dominated the seas. In a continuous sequence, uninterrupted by the extinction events, their descendants, the bony fishes and other vertebrates are today the masters of the aquatic world. Subservience It is often and rightly being asked, why the vertebrates are ‘chauvinistically’ singledout, when there are so many more species in other phyla, notably among the arthropods and the nematodes. The large hosts of these animals are mostly subservient to the higher terrestrial vegetation and to the vertebrates. With increasing success, higher complexity

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and more refined homeostatic functions, the vertebrates turned into hosts and were exploited by many species of organic beings, starting with the bacteria. The fact that there are today many more bacteria and other simple organisms than at the start of the Phanerozoic is not a proof that evolution is not progressive (Gould 1996). Quite on the contrary, the recent huge diversity of these organisms depends on the existence of the advanced plants and animals. Without the opportunities created by the newfangled complex host organisms, the biosphere would still be dominated by a few conservative species of prokaryotes, as it has been during pre-Phanerozoic times (Schopf 1995). Among the Platyhelminthes, the Nematoda, and many classes of ‘Protozoa’, there are many more subservient parasitic species than free-living species. All of these depend on the more complex animal and vegetal organisms. Very much of the insect hyperdiversity is due to the relationship with the flowering plants. Several insect orders and many families are parasites and commensals of terrestrial vertebrates: blood-suckers, feather and hair eaters, nest parasites, dung eaters and even tear-lickers. Tens of thousands of mite and tick species are also subservients of vertebrates and higher plants. The geometric multiplication of the subservient phyla and classes on the expense of the relatively few highly organised top-organisms is another important evolutionary trend of the Phanerozoic times. It is worth mentioning that, baring a few picturesque exceptions, there are no parasites among the vertebrates. Hot Blood The sub-aerial environments, unlike the aquatic ones, are always prone to extreme temperature changes and fluctuations. The efficiency of the biochemical processes increases with body heat and low temperatures induce lethargy. Therefore homeothermy, at the highest possible temperature has been an important terrestrial adaptation. The homeothermic animals evolved to live a thermal brinkmanship at the highest tolerable temperatures, avoiding heat death, which starts soon above 400C. Many insects bask in the sun before taking to the wings. Bumblebees even conserve their muscle-generated body heat below an insulating fur-like body cover. But small size prevents insects from maintaining high temperatures for a prolonged time. Ectothermy, the technique of heating-up by basking in the sun was extensively used by the big reptiles of the Jurassic-Cretaceous times. This has been an exceptional and long period of about 70 million years with pole-to-pole high temperatures and very little seasonal fluctuations. Impressive body sizes were reached by the dinosaurs during this period, enabling them to maintain inertially the accumulated solar heat in their massive bodies over night and during the year-long summers. Fairly high and constant temperatures in the guts of the Mesozoic reptiles and later of the mammals promoted the establishment there of large colonies of cellulose-splitting bacteria and protists. This symbiotic relationship enabled the vertebrates to consume large quantities of vegetal cellulose, which they could not digest alone before. The stage was set for large-scale terrestrial herbivory. The joint processing of the terrestrial vegetal biomass by the vertebrate + bacteria couple opened an unlimited energy supply for the more and more energy-avid vertebrate organisms.

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Whereas the Mesozoic dinosaurs were diurnal sun-basking giants, in the nocturnal and shaded arboreal environments the forefathers of the birds and of the mammals developed active, endothermic thermoregulation. Towards the end of the Cretaceous, as world temperatures fell and the dry savannah forests were replaced by wet rainforests, spanning to the poles, the ‘dinosaur-strategy’ became anachronistic. In the cool and wet forests of the Paleocene, huge reptiles could neither move nor keep warm. They were on their way out even without the helping hand of a catastrophic meteorite impact. During the downward trend of world climate, which continues from the Eocene till now, only small ‘cold-blooded’ reptiles could survive on land, leading a life of alternative activity and of torpor. The turn of the endothermic vertebrates came. Birds and mammals rely on the heat produced by a variety of body functions, its conservation by an insulating plumage or pelt and, if needed, active means of avoiding overheating. Internal heat production needs massive feed since about 90% of their food intake dissipates as heat production. Endotherms are the most energy wasting and complex organisms ever to be produced. In compensation they are continuously foraging, often day and night and generally irrespective of climate changes. Jointly with their gut micro-organisms, they are the most efficient recyclers of vegetal biomass, and massive CO2 producers. The Behavioural Attribute To the dismay of the reductionists, only animals behave. Behaviour is a fundamental animal property and an advance in behavioural performance and complexity is perhaps the main indicator of animal progress. In most of the animal world, patterns of behaviour are innate, genetically transmitted functions of the central nervous system. Learning, memorising and horizontal experience transmissions, superposed on the innate behaviour, are the indices of the most advanced animals. The smallness of the insect brain and the consequent small number of neurons in their central nervous system limited the insects on the level of behavioural automatons. Each species represents one behavioural pattern. Perhaps herein lies another reason for their hyper-diversity. The insects escaped this limitation only in their complicated ‘multibrain’ societies. That learning and memorising is not an accidental attribute of the higher vertebrates, but exists in the potentiality of behavioural evolution itself, is shown by the fact that such capacities appeared independently also among the Cephalopoda, at the other end of the animal panorama. They are rightly considered to be the apex of invertebrate evolution. The cephalopods have very voluminous brains, extremely developed eyes, ‘manipulating’ tentacles, and their learning, discriminating and memorising capacities are competing with those of a mammal. They carry, however, the irreversibly limiting deficiency of their respiratory pigment, the haemocyanin. They could not avoid this basic shortcoming even though their circulatory system is anatomically more perfected than that of any vertebrate. Furthermore, the squids and octopuses are all stenohaline and limited to the relatively poor oceanic food supplies. Understandably all are also aggressive and nimble predators, perhaps the only real competitors the fish ever had.

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But their wound-up metabolism extracts its price: without any internal food reserves the cephalopods live one year or maximum two (except Nautilus, the living fossil). The wisest octopus cannot accumulate much experience and does not pass it on to its progeny. The Fettered Birds Back again to the vertebrates by the fiat of phyletic selection, we have to see in endothermy the major factor that promoted the functions of the brain in the birds and in the mammals. An internal medium of stable and optimal temperature enables the brain to accumulate memorised individual experience. The brains expand progressively in relation to the body size and this expansion, even taken with a grain of salt, is a good general measure for progressive vertebrate evolution. Memory build-up and exchange of experience within the group, weaning of the progeny became a new type of information transmission, superposed on the genetic one. Nothing likely can happen among the small cold-blooded reptiles, which as suggestively expressed, forget during the stupor of the cool nights what they experienced over day, not to speak of the long seasons of hibernation. Perhaps the dinosaurs, able to preserve their resilient sunlit body heat, were wiser. The birds and the mammals, representing two separate lines of reptile descendants, have achieved independently endothermy, by somewhat different but equifinal ways. This speaks again against the alleged orthogenesis of progressive evolution. On the contrary it proves that the same circumstances encourage parallel adaptations, wherever and whenever possible. Once again phyletic selection acted, and this time fated to turn the birds into an evolutionary dead end, even if a glorious one. Extreme adaptation to flight severely limited the birds. Flight limits their size to around 15 kg and all their anatomical structure is surrendered to the aerodynamic needs. Small heads, lack of dentition, reduced pneumatic skeleton, lack of manipulating fore limbs, all this are flightinduced. Although the metabolic energy production in the birds is higher than in the mammals, the lack of homeothermy of the nestlings is also a limiting aspect. The need of highenergy food limits birds to insectivorous, frugi- and granivorous or to outright nectar feeding. Besides perhaps the geese, no bird lives on heavy loads of slow-digesting leafy food. The average positioning of the birds in the terrestrial food chains is between that of the small insects and that of the large mammals. Both birds and mammals achieved high capacity of learning, again each through a different and morphological evolution of their brains. The bird forebrain developed a ‘neostriatum’ as site of the advanced co-ordinated behaviour. Unlike the mammalian neocortex, it is growing inwards and is space-limited. According to Allman (1999), the birds have only one stereotypic visual map and limited possibilities to change their stereotypic behaviour. Still they are champions in sound imitation. Human Primacy It is evident that from among the mammals it was the Primates, a very old Paleocene order that had the best preconditions to produce the first rational animal. Gould tries to

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make us believe that in the unlikely event of a reshuffling of the cards of animal evolution, the results would be completely unforeseen products. To the contrary, I believe that under similar gross environmental circumstances, the result can be again only a terrestrial endothermic vertebrate, perhaps even primate looking. The use of fire has been most important in human evolution. Of the most overwhelming importance has been the human cooking pot, which hydrolyses cellulose and prepares for human consumption the most refractory or poisonous vegetal materials. A unique and perfectly omnivorous animal machine was born. Humans became the only natural cosmopolite metazoan species ever to appear, when a baby was recently born even on the Antarctic continent. All the available sources of energy of the globe, biological and physical, are being gradually captured in the service of the human consumption. The soon 10 billion strong human population, represents the most complex and energy dissipating monospecific biomass ever produced. For better and for worse all the species of the globe fell under the subservient human bondage. The humans have selected out several tens of animal and several hundreds of plant species and turned them into new biological entities, cultigens, a kind of pseudospecies able to survive and flourish only under human care. Many of these are of a rare beauty, which competes with that of the ‘wild’ species. Continuing an old evolutionary story, unaccounted numbers of species from bacteria to mammals became subservient profiteers of the successful human population and of its anthropic environments. Out of them tens of thousands of species accompanied willy-nilly the humans and reached a cosmopolite distribution. With the emergence of the humans, the biosphere reached a new stage in its evolution, comparable to the other big steps, like the emergence of the eukaryotes or of the metazoans. True to their animal ascendancy, the humans have now almost completed the integration of the biosphere into a unique global supermarket. On a globe wide open to human agency, the whole evolutionary process has come to a near standstill. What is being called wildlife survives today only due to the varied degrees of human goodwill. Some natural speciation will still continue among the undesired camp followers, but the large sweep of evolution is over. This is a novel evolutionary stage, as irreversible as any other has been (Por 1996). Humanity is bound to produce, like each previous new stage, a breakthrough in the energy capture and transformation on the globe, both by transgenic increase in the primary production and by the liberation of new energy sources like hydrogen burning and ‘clean’ atomic fusion. To Gould (1996) the humans might be a small twig, a mere Christmas bauble on the tree of life. But in real time this twig came to overshadow the whole tree. The humans, the first and probably only rational species produced by evolution, will survive and dominate the globe until it will start to be engulfed by the red giant-turned sun. By than we shall have had already colonised space. References ALLMAN J.M. 1999. Evolving Brains. Scientific American Library, New York BROWN G.C.& A.E. MUSSETT 1981. The Inaccessible Earth. Unwin Hyman.

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ELDREDGE N. 1999. The Pattern of Evolution. Freeman and Co. New York GOULD S.J. 1989. Wonderful Life. The Burgess Shale and the Nature of History. Norton. GOULD S.J. 1995. Tempo and Mode in the Macroevolutionary Reconstruction of Darwinism. In Fitch W.M. & Ayala F.J. (eds), Tempo and Mode in Evolution. Genetics and Paleontology 50 Years after Simpson. National Academy Press, pp. 125-144. GOULD S.J. 1996. Full House. The Spread of Excellency from Plato to Darwin. Harmony Books. HUXLEY J. 1959. The Emergence of Darwinism. Inaugural Lecture. In Hewer H.R. & Riley N.D. (eds.), Proceedings of the XVth International Congress of Zoology. William Cowles, London. LOTKA A.J. 1922. Contribution to the energetics of evolution. Proceedings National Academy of Sciences 8:147-152. MAIORANA V.C. & L. VAN VALEN 1990. Energy and Community Evolution. In Dudley E.C. (ed.), The Unity of Evolutionary Biology. Dioscorides Press, pp. 655-665. MARGULIS L. 1998. The Symbiotic Planet. A New Look at Evolution. Phoenix Books. MAYR E. 1974. Teleological and Teleonomic: a new analysis. Boston Studies in the Philosopy of Sciences 14: 91-117. POR F.D. 1994. Animal Achievement. A Unifying Theory of Zoology. Balaban Publishers, Rehovot. POR F.D. 1996. Diversity, subservience and the future of evolution. Israel Journal of Zoology 24(2): 455-463. SCHOPF J.W. 1995. Disparate rates, differing fates. Tempo and mode of evolution changed from the Precambrian to the Phanerozoic. In Fitch W.M. & Ayala F.J. (eds.), Tempo and Mode of Evolution. Genetics and Paleontology 50 Years after Simpson. National Academy Press, pp. 41-61. VERMEIJ G.J. 1987. Evolution and Escalation. An Ecological History of Life. Princeton University Press.

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© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Introduction: The new paleontological panorama 43 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 43-44, 2003

Introduction: The new paleontological panorama J. Bergström Swedish Museum of Natural History, Stockholm, Sweden

Although not very clear from our daily newspapers, there is more to palaentology than finding even bigger or more horrible dinosaurs. The last decades have provided us with an unprecedented number of new lagerstätten, that is, occurrences of well-preserved fossils that can give exceptional glimpses of the morphology and construction of old forms of life. Fossil animals usually are represented only by biomineralized shells and skeletons, but in the variety of different kinds of lagerstätten also soft tissues can be found. The understanding of the fossilization processes is a science in its own. Quite a large proportion of the lagerstätten is from the Cambrian. This has the surprising result that some marine groups known from today but are virtually unknown from fossils through much of the last 500 million years can be retrieved from rocks some 500-540 million years old. Not only can we recognize them, but some of them yield surprising detail on the µm scale. We have now to realize that we can get information that would hardly be possible to get from the field of comparative morphology in zoology, or from genetics. An example is the new understanding of the arthropod coxa. It was thought to be the most proximal segment of the leg in all arthropods – but “orsten” material demonstrates how the coxa came into being first only in the crustacean lineage through the growth of a ring-shaped sclerite from a new endite where there was before only soft skin. A somewhat similar revolution occurred in vertebrates during the transition from fish to amphibian. Although the evolution from fin to leg is a classical textbook example of gradual evolution, even the most amphibian-like osteolepiform fish had fin rays where the first amphibian had fully developed digits. Evidence from developmental genetics shows that HoxD genes cause a 2-phase growth pattern in tetrapods, but only a single phase in fishes. Thus a genetic “invention” seems to have created the digits by “doubling” the bone formation procedure, and the digits are a completely new structure without any forerunner. Data squeeze this event into a 5 million year interval, which would be very short if only gradual evolution were involved, but time in excess for a genetic “invention”. It is not long ago that we learned that birds most probably evolved directly from dinosaurs. The oldest birds used to be a handful of specimens from Solnhofen in Germany. Recently, nicely preserved bird specimens in the oldest Cretaceous of China have been found in the order of a thousand. The Chinese deposits are also yielding small carnivorous

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dinosaurs with the skin covered by feathers. Thus, birds apparently inherited feathers from their ancestors and made a new use of them. In dinosaurs, feathers should have been for insulation and display. Ultimately, one paper provides an example of the more broad-scaled faunal analyses that can be done as data are accumulating. This gives us new possibilities to understand how communities were composed and how they evolved. The example is taken from mammal faunas in Eurasia during pre-glacial times, thus in times with great changes in climate and vegetation. These are glimpses from the papers in the palaeontology session. They demonstrate that a great evolution has taken part not only in animals, but also in our capability to extract and deal with their remains.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Fossils, Developmental Patterning And The Of Tetrapods 45 TheOrigin New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 45-54, 2003

Fossils, developmental patterning and the origin of tetrapods P. E. Ahlberg Department of Palaeontology, The Natural History Museum, Cromwell Road, London SW7 5BD, U.K. E-mail: [email protected]

Abstract Our understanding of the origin of tetrapods has improved greatly in recent years, due to new fossil discoveries and improved phylogenetic analyses. The move from water to land occurred gradually within the upper part of the tetrapod stem group, during a 15-20 million year time interval from the late Middle Devonian to the Early Carboniferous. The first stage, represented by Panderichthys, involved transformation of the body and head to a “crocodile-like” shape; this may reflect a move into very shallow water. This was followed by the rapid evolution of defining tetrapod characters such as limbs and sacrum. However, the earliest limbed vertebrates, represented by Acanthostega and Ichthyostega, still retained many aquatic adaptations including a tail fin and fish-like lateral line canals. Real terrestriality probably only evolved at the base of the tetrapod crown group in the Early Carboniferous. Developmental genetics shows that the production of digits is dependent on a late distal phase of Hox expression in the limbs, which seems to be unique to tetrapods. This expression phase, which probably originated in the stem group between the Panderichthys and Acanthostega + Ichthyostega nodes, may have been prompted by the duplication of a promoter for Bone Morphogenesis Protein receptor IB.

Introduction The origin of tetrapods or land vertebrates, which occurred during the Late Devonian period between about 370 and 355 million years ago, was one of the most important events in vertebrate history (Ahlberg & Milner 1994). It generated a new “kind” of animal, with a morphology and lifestyle radically different from those of its ancestors, and sparked an evolutionary radiation that has continued to the present day and now comprises more than 24000 living species of amphibians, reptiles, birds and mammals. It is also what can be termed a “major morphological transition”, that is an evolutionary transformation of such magnitude that it is difficult to establish detailed homologies between the ancestral and derived conditions, explain how the derived condition was generated, understand the selection pressures involved, or infer the morphology and

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mode of life of unknown intermediate forms. As such it represents an important class of evolutionary problem, which also includes the origin of the different animal phyla - as demonstrated for example by the contributions in this volume on the origin of the Arthropoda. Until recently the origin of tetrapods was not well understood, but new fossil discoveries (Coates & Clack 1990, 1991, Coates 1996, Clack 1994, 1998, Ahlberg 1991, 1995, 1998, Ahlberg et al. 1994, 2000) and revisions of existing material (Ahlberg & Clack 1998) coupled with detailed cladistic analyses (Coates 1996, Ahlberg & Clack 1998, Ahlberg & Johanson 1998) have created a much clearer picture of the transformation. It is now possible to map the sequence of character change from fish to tetrapod, to describe certain intermediate morphological states, and to define approximate time frames for these changes. At the same time, the science of developmental genetics has advanced to a point where it allows tentative hypotheses to be framed about the genetic basis for some of the morphological innovations. A sketch synthesis of these approaches, applied to the origin of limbs, is presented below as an illustration of the potential of “evolutionary developmental phylogenetics”. The phylogenetic context The “origin of tetrapods”, in the morphological and ecological sense, was an event within the tetrapod stem group. As such it conflicts with much of current taxonomic usage (De Queiroz & Gauthier 1990, Patterson 1994), which would pin the term “Tetrapoda” either to the tetrapod crown group or total group. A brief explanation of these terms may therefore be helpful (Fig. 1). The tetrapod crown group comprises the living tetrapod groups - Lissamphibia (containing frogs, salamanders and caecilians) and Amniota (containing reptiles,

Fig. 1: Schematic cladogram of a Recent group and its Recent sister group, illustrating the concepts of crown group, stem group and total group. Note that all these concepts are defined in relation to Recent taxa and that the stem group consists, by definition, entirely of extinct forms.

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mammals and birds) - and the fossil members of these lineages. Fossil evidence indicates that the crown group originated during the Early Carboniferous (Ahlberg & Milner 1994). The closest living relatives of the tetrapods are probably the lungfishes, Dipnoi (Cloutier & Ahlberg 1996), so the tetrapod total group comprises all animals that are more closely related to living tetrapods than to lungfishes. The lungfish and tetrapod lineages appear to have separated by the Early Devonian, as the earliest known lungfishes are of this age (Denison 1968, Campbell & Barwick 1984). Numerous members of the tetrapod stem group (the total group minus the crown group; see Fig. 1) are known from the Devonian and Carboniferous. Basal stem group members have paired fins, were apparently wholly aquatic, and would subjectively be described as “fishes”, whereas derived forms have limbs, were probably at least partly terrestrial, and would be described as “tetrapods”. This is a situation comparable with that of birds (Currie, this volume), where the upper part of the bird stem group contains Archaeopteryx and other obvious “primitive birds”, whereas the base of the stem group contains some very un-birdlike dinosaurs such as Diplodocus. When I discuss the “origin of tetrapods” in this paper I refer to the morphological and ecological transition within the stem group. As the stem group by definition consists exclusively of fossil forms, the detailed phylogeny is based on morphological data and the chronology on stratigraphic evidence rather than molecular divergence dates. The morphological transformation The morphological transformation from the base of the tetrapod stem group to the base of the crown group can be summarised as follows (Fig. 2): Losses: Median fins and tail fin, dermal fin rays (lepidotrichia) of paired fins, bony gill cover, internal gills in adult, bones connecting top of shoulder girdle to back of head. Gains: Digits on paired appendages, sacrum (connection between pelvis and vertebral column), zygapophyses (articulations between neural arches in vertebral column). Modifications of existing structures: Braincase reconstructed so that intracranial joint between orbitotemporal and otic regions disappears, hyomandibula modified into stapes, skull modified into “crocodile-like” morphology with dorsal eyes and long snout, lateral line canals less deeply incised into dermal bones, rib cage greatly expanded, scapulocoracoid (endoskeletal part of shoulder girdle) and pelvis greatly enlarged, hind limb enlarged, tail lengthened. These transformations did not of course occur all at once. Looking at the stem group in more detail (Fig. 3), we can discern the following stages: Rhizodonts and osteolepiforms: These fishes form the basal part of the stem group (Johanson & Ahlberg 1998, Ahlberg & Johanson 1998). They have certain tetrapod characteristics, such as an internal nostril or choana (Jarvik 1980), which are not present in other fish groups, but show no obvious adaptations toward life on land. Eusthenopteron (Fig. 2A) is a representative osteolepiform. Panderichthys: This genus, from the late Middle Devonian of Latvia (Fig. 4A), has a crocodile-like skull shape and has lost the median fins but not the tail fin (Vorobyeva & Schultze 1991). The intracranial joint has been immobilised by fusion of the skull roof,

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Fig. 2: Two members of the tetrapod stem group, Eusthenopteron (an “osteolepiform fish”) and Ichthyostega (an “early tetrapod”), illustrating the main morphological changes of the fish-tetrapod transition. Structures labelled in A disappear during the transition; those labelled in B are morphological novelties of tetrapods. Further changes from B to the tetrapod crown group include the loss of the lepidotrichial tail fin and the reduction of the foot from 7 to 5 digits. (The hand of Ichthyostega is unknown.) A modified from Jarvik (1980); B from Coates & Clack 1995.

Fig. 3: Schematic representation of the tetrapod stem group. Note that the “osteolepiforms” are a paraphyletic group. Eusthenopteron is one of the most derived osteolepiforms.

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Fig. 4: A, Panderichthys, the most derived “fish” in the tetrapod stem group, showing tetrapod-like body form coupled with paired fins of osteolepiform type (pectoral fin shown in detail). Lateral view. B, Acanthostega, a Devonian “tetrapod” from the stem group, showing a similar body form but with 8-digit limbs (forelimb shown in detail). Dorsolateral view. Note that Acanthostega has a larger tail fin and smaller rib cage than Ichthyostega. A from Ahlberg & Milner (1994) and Vorobyeva (1992); B from Coates (1996).

but persists internally; the braincase and hyomandibula are still unmodified (Ahlberg et al. 1996). The ribs are moderately enlarged (Vorobyeva & Schultze 1991). Panderichthys retains paired fins, a bony gill cover, and a connection between the shoulder girdle and the back of the skull. It has a short tail, a pelvis that is apparently not connected to the backbone (pers. obs.), and no zygapophyses (Vorobyeva & Schultze 1991). Acanthostega and Ichthyostega: These forms, from the latest Devonian of Greenland, are the earliest limbed vertebrates known from complete skeletons (Fig. 2B, 4B). However, fragmentary earlier genera such as Elginerpeton from the middle Late Devonian of Scotland (Ahlberg 1995, 1998) seem to be essentially similar. These animals have all the tetrapod characters of Panderichthys, and in addition have a tetrapod braincase without an intracranial joint, a stapes rather than a hyomandibula, no bony gill cover, limbs with seven or eight digits, greatly enlarged scapulocoracoid and pelvis, contact between pelvis and vertebral column, no bone contact between shoulder girdle and skull, moderately (Acanthostega) or greatly (Ichthyostega) expanded ribcage, a long tail, and poorly developed zygapophyses (Coates & Clack 1990, 1995, Coates 1996, Clack 1994, 1998). However, they retain some fish-like characters such as a lepidotrichial tail fin and deeply incised lateral line canals which open to the surface through series of discrete pores. Acanthostega retained internal gills as an adult (Coates & Clack 1991).

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Early Carboniferous tetrapods: A number of Early Carboniferous tetrapods such as Greererpeton (Smithson 1982, Godfrey 1989) and Whatcheeria (Lombard & Bolt 1995) fall into the top of the stem group or the very base of the crown group. They have all the tetrapod characteristics of Acanthostega and Ichthyostega, and in addition have shallow lateral line grooves, limbs with four or five digits, a further enlarged scapulocoracoid, well developed zygapophyses, and no tail fin. They probably lacked internal gills as adults. As can be seen from this summary, the lower part of the tetrapod stem group shows no obvious trend towards terrestrial life and only modest morphological change. The first step in the transition is the evolution (in the common ancestor of Panderichthys and tetrapods) of a tetrapod-like head and body form. This is followed by an episode of major character change between the Panderichthys node and the Acanthostega + Ichthyostega node, where the paired fins turn into limbs (initially with seven or eight digits) and much of the internal skeleton is redesigned; this corresponds to the conventionally recognised “origin of tetrapods”. Finally, in the uppermost part of the stem group, the tail fin is lost, the number of digits is reduced, and the lateral line canals become more superficial - probably because the skin is growing thicker. It can be added that all tetrapod stem group members were predators, and that those from the Panderichthys node to the top of the stem group are all relatively large, about 0.8 to 1.5 metres in length. It is interesting to note that all the most dramatic changes are compressed onto one internode, between Panderichthys and Acanthostega + Ichthyostega (but in fact probably between Panderichthys and Elginerpeton; see Fig. 3). The time interval between Panderichthys and Elginerpeton is only about 5 million years (Ahlberg & Milner 1994, Ahlberg et al. 1996), suggesting that these changes took place remarkably quickly. This phenomenon needs to be considered from both ecological and developmental genetic perspectives. Environment and mode of life Almost all Devonian members of the tetrapod stem group come from “Old Red Sandstone” sediments. These represent a range of non-marine and marginal marine environments from lakes and rivers to lagoons, estuaries and deltas (see for example Kuršs 1992, Prichonnet et al. 1996, Chidiac 1996, and references therein), and frequently contain fossil land plants. It is not yet possible to determine whether the fish-tetrapod transition took place in a freshwater or brackish environment, but it seems unlikely to have occurred in a fully marine setting. The rhizodonts and osteolepiforms were clearly wholly aquatic, occupying a range of predatory fish niches, though outgroup comparison with lungfishes, coelacanths and primitive living actinopterygians indicate that they had functional lungs. Panderichthys gives the first hint of a changing mode of life: its dorsoventrally flattened body, loss of median fins, and eyes positioned on top the head under raised “eyebrows”, suggest that it may have operated in very shallow water, possibly using its raised eyes to look out above the surface. The earliest tetrapods, such as Acanthostega and Ichthyostega, differ significantly from Panderichthys in internal anatomy, particularly as regards the braincase, hyoid arch and

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limb girdles (Vorobyeva & Schultze 1991, Ahlberg et al. 1996), but their gross morphology is not very different. This is particularly true for Acanthostega, which has a large symmetrical tail fin, weak limbs, and persistent internal gills. These tetrapods were clearly still largely aquatic, and may have had a similar lifestyle to Panderichthys. Limbs may thus have evolved primarily for locomotion in shallow water rather than on land (Coates & Clack 1995). However, many of the tetrapod features which first appear at this point in the stem group (zygapophyses, sacrum, expanded ribcage, detachment of shoulder girdle from head) are functionally related to weight support. This suggests that the internode between Panderichthys and the earliest tetrapods was characterised by a rapid shift towards more activity in mechanically non-supportive conditions (water too shallow to support the body, or dry land). This trend continued up into the tetrapod crown group, with the loss of primitive aquatic features such as the tail fin, but even at this early stage of tetrapod evolution some taxa such as Crassigyrinus (Panchen & Smithson 1990) reverted to a more wholly aquatic life. Evolution and developmental patterning At present, the only part of tetrapod anatomy that is well enough understood in developmental genetic terms to permit an attempt at an evolutionary-developmental synthesis, is the limb skeleton. However, the principles underlying this synthesis will also be applicable to other parts of the anatomy once enough developmental data have been assembled. Morphological evidence: The osteolepiform part of the tetrapod stem group is characterised by paired fin endoskeletons in which humerus/femur, radius/tibia, ulna/ fibula and some wrist/ankle bones can be identified with confidence, but which lack digits. The appendage is terminated distally by a fin web supported by dermal lepidotrichia. Panderichthys shows essentially the same condition, though the distal part of the endoskeleton is somewhat simplified (Fig. 4C). The proximal elements, by contrast are more tetrapod-like in shape than those of osteolepiforms. In Acanthostega and Ichthyostega, the lepidotrichia have disappeared and an array of seven or eight endoskeletal digits has sprouted from the distal end of the appendage (Fig. 4D). The proximal elements on the other hand have not been greatly modified. Finally, in postDevonian tetrapods the number of digits is reduced to four or five. Overall, it appears that the osteolepiform / Panderichthys fin endoskeleton is equivalent to the arm/leg but not the hand/foot, and that the digits appeared rather abruptly as an addition to the distal end of the endoskeleton at the same time as the lepidotrichia disappeared. Developmental genetic evidence: Early appendage development is essentially similar in teleost fishes and tetrapods; in both groups it is, for example, associated with the expression of Hoxd-11 and Hoxd-13 in the posterior part of the appendage bud, and anteroposterior asymmetry is regulated by Sonic hedgehog (Sordino et al. 1995, Shubin et al. 1997). However, tetrapods also show a late phase of expression of the Hoxd genes in the distal part of the limb bud, in the area where the digits develop (Sordino et al. 1995, Shubin et al. 1997). There is no corresponding late phase in teleosts. Recently, it has been shown that distal mesenchyme proliferation in the tetrapod limb bud (which creates the mesenchyme in which the distal Hox expression occurs) is dependent on a distal

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promoter of Bone Morphogenesis Protein (BMP) receptor type IB (Baur et al. 2000). The corresponding, possibly paralogous proximal promoter is involved in the regulation of brain development (Baur et al. 2000). Synthesis: The conservation of early appendage development between recent teleost fishes and tetrapods implies that the same developmental pathways were present in the tetrapod stem group. This inference is supported by the morphological stability of the proximal endoskeletal elements throughout the tetrapod stem group, which suggests that the patterning of these elements did not change significantly across the fish-tetrapod transition. We can infer that distal mesenchyme proliferation and late distal Hox expression originated at the internode between Panderichthys and Ichthyostega + Acanthostega, possibly in connection with duplication of a promoter of BMP receptor IB. Questions for the future: This tentative synthesis still leaves many questions unanswered. Some of the more important ones are: a) Is the distal promoter of BMP receptor IB really unique to tetrapods? b) The proximal appendage endoskeleton of sarcopterygian fishes (not only members of the tetrapod stem group, but also other groups like coelacanths and lungfishes) contains elements (humerus/femur, radius/tibia, ulna/fibula) which cannot be identified in the teleost fin. Thus, in addition to the gene expressions that are conserved between teleosts and tetrapods (and therefore general to the Osteichthyes as a whole) there must exist a more restricted set of sarcopterygian expression patterns regulating the morphology of these elements. What are these expression patterns? c) How does the distal mesenchyme proliferation and emergence of digits relate to the loss of the lepidotrichia? Are the two directly linked, or simply coincident? d) What controls digit number? e) Given that the proximal promoter of BMP receptor IB in tetrapods is involved in brain patterning, could there be a genetic link between the evolution of digits and the simultaneous rebuilding of the tetrapod braincase? Conclusion Recent work has done much to illuminate the details of the origin of tetrapods. We can now see that it was a fairly gradual affair, with the transition from aquatic to fully terrestrial life stretching across the upper part of the stem group from the Panderichthys node to the base of the crown group and occupying a time interval of about 15-20 million years from the late Middle Devonian to the Early Carboniferous. However, within this extended period of change lies a brief pulse of much more rapid and dramatic morphological evolution, the traditionally recognised “origin of tetrapods”, which occupied about 5 million years and corresponds to the internode between Panderichthys and Elginerpeton. This phase of rapid change was probably driven by selection pressure for terrestrial or extreme shallow-water competence, but it is possible that pleiotropic effects due to linked gene expression patterns were also involved in some of the changes. Phylogenetic and developmental genetic data can at present only be synthesised (in a preliminary manner) in relation to the evolution of limbs. However, in the long run it will be possible to extend this synthesis to other parts of the anatomy such as the braincase and middle ear; this will be crucial to understanding

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how our distant ancestors generated the new morphologies that allowed them to make the transition from water to land. References AHLBERG P.E. 1991. Tetrapod or near-tetrapod fossils from the Upper Devonian of Scotland. Nature 354: 298-301. AHLBERG P.E. 1995. Elginerpeton pancheni and the earliest tetrapod clade. Nature 373: 420-425. AHLBERG P.E. 1998. Postcranial stem tetrapod remains from the Devonian of Scat Craig, Morayshire, Scotland. Zoological Journal of the Linnean Society 122: 99-141. AHLBERG P.E. & J.A. CLACK 1998. Lower jaws, lower tetrapods - a review based on the Devonian genus Acanthostega. Transactions of the Royal Society of Edinburgh: Earth Sciences 89: 11-46. AHLBERG P.E. & Z. JOHANSON 1998. Osteolepiforms and the ancestry of tetrapods. Nature 395: 792-794 AHLBERG P.E. & A.R. MILNER 1994. The origin and early diversification of tetrapods. Nature 368: 507-514. ˇ E. & O. LEBEDEV 1994. The first tetrapod finds from the DevoAHLBERG, P.E., LUKŠEVI CS nian (Upper Famennian) of Latvia. Philosophical Transactions of the Royal Society of London B 343: 303-328. ˇ E. & J.A. CLACK 1996. Rapid braincase evolution between PanderAHLBERG P.E., LUKŠEVICS ichthys and the earliest tetrapods. Nature 381: 61-64 ˇ E. & E. MARK-KURIK 2000. A near-tetrapod from the Baltic MidAHLBERG P.E., LUKŠEVICS dle Devonian. Palaeontology 43: 533-548. BAUR S.T., MAI J.J. & S.M. DYMECKI 2000. Combinatorial signalling through BMP receptor IB and GDF5: shaping of the distal mouse limb and the genetics of distal limb diversity. Development 127: 605-619 CAMPBELL K.S.W. & R.E. BARWICK 1984. Speonesydrion, an early Devonian dipnoan with primitive tooth plates. Palaeoichthyologica 2: 1-48. CHIDIAC Y. 1996. Paleoenvironmental interpretation of the Escuminac Formation based on geochemical evidence. In Schultze H.-P. & R. Cloutier (eds), Devonian Fishes and Plants of Miguasha, Quebec, Canada. Verlag Dr. Friedrich Pfeil, München, pp. 47-53. CLACK J.A. 1994. Earliest known tetrapod braincase and the evolution of the stapes and fenestra ovalis. Nature 369: 392-94. CLACK J.A. 1998. The neurocranium of Acanthostega gunnari Jarvik and the evolution of the otic region in tetrapods. Zoological Journal of the Linnean Society 122: 61-97. CLOUTIER R. & P.E. AHLBERG 1996. Morphology, characters and the interrelationships of basal sarcopterygians. In Stiassny M.L.J., Parenti L.R. & G.D. Johnson (eds), Interrelationships of Fishes. Academic Press, San Diego, pp. 445-479. COATES M.I. 1996. The Devonian tetrapod Acanthostega gunnari Jarvik: postcranial anatomy, basal tetrapod interrelationships and patterns of skeletal evolution. Transactions of the Royal Society of Edinburgh: Earth Sciences 87: 363-421. COATES M.I. & J.A. CLACK 1990. Polydactyly in the earliest known tetrapod limbs. Nature 347: 66-69. COATES M.I. & J.A. CLACK 1991. Fish-like gills and breathing in the earliest known tetrapod. Nature 352: 234-36.

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COATES M.I. & J.A. CLACK 1995. Romer’s gap: tetrapod origins and terrestriality. Bulletin du Muséum National d’Histoire Naturelle, Paris, 4e série 17(C) (1-4): 373-388. DENISON R.H. 1968. Early Devonian lungfishes from Wyoming, Utah and Idaho. Fieldiana, Geology 17: 353-413. DE QUEIROZ K. & J. GAUTHIER 1990. Phylogeny as a central principle in taxonomy: phylogenetic definitions of taxon names. Systematic Zoology 23: 449-480. GODFREY S.J. 1989. The postcranial skeletal anatomy of the Carboniferous tetrapod Greererpeton burkemorani Romer 1969. Philosophical Transactions of the Royal Society of London B 323: 75-133. LOMBARD R.E. & J.R. BOLT 1995. A new primitive tetrapod Whatcheeria deltae from the Lower Carboniferous of Iowa. Palaeontology 38: 471-94. JARVIK E. 1980. Basic Structure and Evolution of Vertebrates, volume 1. Academic Press, London, 575 p. JOHANSON Z. & P.E. AHLBERG 1998. A complete primitive rhizodont from Australia. Nature 394: 569-572 KURŠS V. 1992. Depositional environment and burial conditions of fish remains in Baltic Middle Devonian. In Mark-Kurik E. (ed.), Fossil Fishes as Living Animals. Academia 1, Tallinn, pp. 251-264. PANCHEN A.L. & T.R. SMITHSON 1990. The pelvic girdle and hind limb of Crassigyrinus scoticus (Lydekker) from the Scottish Carboniferous and the origin of the tetrapod pelvic skeleton. Transactions of the Royal Society of Edinburgh: Earth Sciences 81: 31-44. PATTERSON C. 1994. Bony fishes. In Spencer R.S. (ed.), Major Features of Vertebrate Evolution (short courses in paleontology 7). Paleontological Society, pp. 57-84. PRICHONNET G., DI VERGILIO M. & Y. CHIDIAC 1996. Stratigraphical, sedimentological and paleontological context of the Escuminac Formation: Paleoenvironmental hypotheses. In Schultze H.-P. & R. Cloutier (eds), Devonian Fishes and Plants of Miguasha, Quebec, Canada. Verlag Dr. Friedrich Pfeil, München, pp. 23-36. SHUBIN N., TABIN C. & S. CARROLL 1997. Fossils, genes and the evolution of animal limbs. Nature 388: 639-648. SMITHSON T.R. 1982. The cranial morphology of Greererpeton burkemorani Romer (Amphibia: Temnospondyli). Zoological Journal of the Linnean Society 76: 29-90. SORDINO P., VAN DER HOEVEN F. & D. DUBOULE 1995. Hox gene expression in teleost fins and the origin of vertebrate digits. Nature 375: 678-681. VOROBYEVA E.I. 1992. The role of development and function in formation of “tetrapod-like” pectoral fins. Zhurnal Obshei Biologii 53: 149-158. VOROBYEVA E.I. & H.P. SCHULTZE 1991. Description and systematics of panderichthyid fishes with comments on their relationship to tetrapods. In Schultze H.-P. & L. Trueb (eds), Origins of the Higher Groups of Tetrapods: Controversy and Consensus. Cornell Publishing Associates, Ithaca, pp. 68-109.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Feathered dinosaurs and thePanorama origin ofof birds The New Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 55-60, 2003

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Feathered dinosaurs and the origin of birds P.J. Currie Royal Tyrrell Museum of Palaeontology, Box 7500, Drumheller, Alberta T0J 0Y0, Canada

Abstract Since 1996, specimens from at least six families of non-avian theropod dinosaurs have been found with preserved feathers and feather-like structures. Feathers seem to have originated from simple branching structures in primitive coelurosaurian dinosaurs like Sinosauropteryx, where they presumably served as insulation. From these developed longer, stiffer structures on the arms of velociraptorine dromaeosaurids like Sinornithosaurus and Beipiaosaurus. These may have been display structures, but may also have functioned to cover eggs in the nests of brooding females. True feathers are found on the arms and tails of the non-avian theropods Caudipteryx and Protarchaeopteryx, and may represent more elaborate display structures. These were relatively short and had symmetrical vanes, and were still clearly not adapted for flight. Archaeopteryx, the earliest bird, is the first true flier known in the theropod-bird lineage. Theropods less derived than Archaeopteryx should be considered as non-avians, whereas those more derived are true birds.

With the development and spread in the 1970s of the idea that dinosaurs might be warm-blooded animals that were the direct ancestors of birds (Bakker 1975, Ostrom 1974, Paul 1988), paleontologists started to consider the possibility that some dinosaurs had feathers. Logic suggested that if dinosaurs were warm-blooded, then smaller ones would have needed insulation to help stabilize their body temperatures. Furthermore, if theropods were the ancestors of birds, it would make sense that the insulation used by dinosaurs would have been some form of feather. Most paleontologists believe that feathers had to have developed before birds were able to incorporate them into their flight mechanism. Although we normally think of birds as being the only animals covered with feathers, in truth it is their form of powered flight that sets them apart from all other life forms. Warm-blooded dinosaurs and the dinosaurian origin of birds were two of the biggest controversies in palaeontology at the end of the twentieth century. When first proposed, there were far more people opposed to these hypotheses than there were in support of them. This trend has reversed now, largely because of some remarkable specimens from northeastern China that appeared after the discovery of an extremely rich source of early Cretaceous bird fossils (Chiappe et al. 1999). These specimens include fossilized feathers, which only preserve under exceptional circumstances (Davis & Briggs 1995).

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In 1996, Ji & Ji announced the discovery of the first “feathered” dinosaur, Sinosauropteryx prima, in a paper published in Chinese. Additional specimens of this small, chicken-sized animal were discovered and described shortly after (Chen et al. 1998, Ji & Ji 1997b). A downy coat of simple branching structures covered the head, neck, thorax, limbs and tail of Sinosauropteryx (Currie & Chen 2001), and presumably functioned to insulate the animal. Although Chen et al (1998) took a conservative view and referred to these as “integumentary structures”, everyone was aware that they represent potential protofeathers. This triggered escalation in the controversies on the ancestry of birds and warm-bloodedness in dinosaurs (Brush et al. 1997, Currie 1997, 2000, Ruben et al. 1997). More “feathered” dinosaurs have been subsequently described, and represent theropod families distinct from that of Sinosauropteryx. Beipiaosaurus inexpectus (Xu et al. 1999a) is a larger, approximately man-sized dinosaur that had a relatively small head with leaf-like teeth, a long neck, long arms and a relatively short tail. This animal belongs to an unusual group of theropod dinosaurs that are called therizinosaurs. Its body was covered with structures similar to those that covered Sinosauropteryx, but it also has long, stiff, feather-like structures on the backs of its arms. Another “feathered” dinosaur is Sinornithosaurus millenii (Xu et al. 1999b, Ji et al. 2001), which is a dog-sized animal with sharp serrated teeth and raptorial claws. The pattern of body covering is similar to that of Beipiaosaurus. It is a dromaeosaurid that is closely related to Velociraptor. Microraptor zhaoianus (Xu et al. 2000) is another “feathered” dromaeosaurid from Liaoning. This animal was smaller than Archaeopteryx, has a shorter tail than other dromaeosaurids, and has several adaptations in the feet that suggest it may have been arboreal. As in Sinosauropteryx (Currie & Chen 2001) and Sinornithosaurus (Xu et al. 2001), the “feathers” of Microraptor seem to have been simple branching structures. Protarchaeopteryx robusta is a feathered theropod described by Ji & Ji (1997a). Whereas Sinosauropteryx is a small dinosaur with short arms and an extremely long tail, Protarchaeopteryx has long arms and a relatively short tail. In addition to having downy, feather-like structures covering its body, Protarchaeopteryx also has long quill-like feathers at the end of the tail. With this animal, there is no doubt concerning the identification of true feathers, each of which has a central rachis, barbs and barbules. Another feathered dinosaur was also discovered in 1997, and was originally misidentified as Protarchaeopteryx. In addition to having long feathers at the end of the tail, it has true feathers behind the arms. The six specimens recovered represent another species of feathered dinosaur (Ji et al. 1998), now known as Caudipteryx zoui. Distantly related to Oviraptor (Barsbold et al. 2000), Caudipteryx was a turkey-sized dinosaur with relatively long legs that suggest it was strongly cursorial. The feathers make its arms look like rudimentary wings, although the feathers, which have symmetrical vanes, and arms are both too short to have allowed it to fly. It is more likely that the elongate feathers on the arms and at the end of the tail were used for display (Currie 1998). Dinosaurs were highly visual animals that evolved a fantastic array of ornamentation (crests, frills, horns, spikes, etc.) to attract mates, warn potential rivals, and otherwise enhance their interactive behavior. Once dinosaurs had acquired feathers for insulation, it would have been relatively easy to adapt them into display structures that are lightweight, strong, colorful, and can be shed and replaced. Display may not have been

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the only function for these longer, stiffened feathers. Specimens of the related Oviraptor have been found on nests of eggs (Dong & Currie 1996), and taphonomic analysis suggests that long feathers on the backs of the arms might have helped protect the eggs from the elements (Hopp & Orsen 1998). The presence of a fan of feathers on the end of the tail presumably led to the reduction in number of tail vertebrae in these oviraptorosaurs, and even to the development of a pygostyle in Nomingia (Barsbold et al. 2000). There are now six species of “feathered” dinosaurs from northeastern China. Evidence also shows that alvarezsaurids (which are classified by different workers as either nonavian theropods or as birds) had feathers (Schweitzer et al. 1997), as well as the oviraptorosaur Nomingia (Barsbold et al. 2000). More “feathered” dinosaurs are in the process of being described from northeastern China, where the sedimentary rocks are extremely fossiliferous and are being excavated on an unprecedented scale. The described “feathered” species represent six different families of non-avian theropods. Sinosauropteryx is a compsognathid theropod, closely related to the European Compsognathus (Ostrom 1978). Protarchaeopteryx is only known from a single specimen, but might be the non-avian theropod most closely related to the earliest bird, Archaeopteryx. Both Caudipteryx and Nomingia are oviraptorosaurs, but represent two distinct families. Beipiaosaurus is a therizinosauroid, whereas Sinornithosaurus and Microraptor are dromaeosaurids. As already pointed out, alvarezsaurids are sometimes classified as non-avian theropods and sometimes as birds. All of these families belong to the Coelurosauria (Hutchinson & Padian 1997). The fact that the known “feathered” dinosaurs represent such a diverse assemblage of coelurosaurians strongly suggests that many, if not most, of the meat-eating dinosaurs were probably feathered. Because Tyrannosaurus is on the coelurosaurian branch of the Theropoda (Holtz 1994, 2000), it is possible that even it had feathers somewhere on its body at some stage in its life. Such a large animal would not have needed feathers for insulation as an adult, and pebbly skin impressions with no indication of feathers are preserved for the related tyrannosaurs Gorgosaurus and Daspletosaurus. However, it is not impossible that newborn tyrannosaurs might have had some sort of insulating down, or that the adults used feathers somewhere on their bodies for display. The presence of feathers on dinosaurs does not by itself prove that birds came from dinosaurs. There is much stronger evidence in the skeleton to suggest that birds and dinosaurs are more closely related to each other than either is to any other type of animal (Gauthier 1986, Chiappe 1995, Holtz 2000, Sumida & Brochu 2000). However, feathers are such complex structures that the discovery of feathered dinosaurs has done far more to convince people that birds are living representatives of the Dinosauria than all of the details of skeletal anatomy have. Still, not everyone is convinced (see, for example, Feduccia 1996, Ruben et al. 1997), and some of these people are more vocal than the majority of paleontologists who accept that birds descended from dinosaurs. However, lack of a convincing alternative for bird ancestry, and use of circular reasoning undermine their arguments. For example, they have long argued that the structure of the ankle is different in theropod dinosaurs and birds. Although they claim that Caudipteryx is a secondarily flightless bird (Jones et al. 2000) because it has feathers, this animal has the same ankle structure as theropod dinosaurs like Velociraptor and Tyrannosaurus.

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Fig. 1. Phylogenetic relationships of some feathered theropods.

Most scientists agree Archaeopteryx is the earliest known bird, and therefore represents the dividing line between dinosaurs and birds. Related animals more derived or advanced than Archaeopteryx are birds. But species that are more primitive are not. Using this concept, birds are animals that fly using wings that incorporate specialized feathers, or are derived from such animals. Feathers separate birds from all other living animals, but they cannot be used to define birds because they would also have been present in avian ancestors that could not fly. Birds could be diagnosed as all feathered animals, but we would then have to reclassify all feathered dinosaurs as birds, as well as all of their direct descendants. Because preservation of feathers is rare, we do not know, and may never know, how widespread feathers were amongst other types of dinosaurs. Although an adult Tyrannosaurus rex probably did not have feathers, its ancestors and closest relatives did (Fig. 1). Rather than classify this animal as a bird, it would be more logical to emphasize flight, rather than feathers, in the diagnosis of Aves. The fact that we are having trouble classifying many of the new “feathered” fossils emphasizes how closely related dinosaurs and birds are to each other. As we draw towards consensus on the ancestry of birds, attention is shifting to equally interesting problems – the evolution of feathers (Prum 1999), and the origin of flight.

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References BAKKER R.T. 1975. Dinosaur renaissance. Scientific American 232(4): 58-78. BARSBOLD R., OSMÓLSKA H., WATABE M., CURRIE P.J. & K. TSOGTBAATAR 2000. A new oviraptorosaur (Dinosauria, Theropoda) from Mongolia: the first dinosaur with a pygostyle. Acta Palaeontologica Polonica 45: 97-106. BRUSH A., MARTIN L. D., OSTROM J. H., & P. WELLNHOFER 1997. Bird or Dinosaur? — statement of a team of specialists. Episodes 20: 46. CHEN P.J. DONG Z.M. & S.N. ZHENG 1998. An exceptionally well-preserved theropod dinosaur from the Yixian Formation of China. Nature 391: 147-152. CHIAPPE L.M. 1995. The first 85 million years of avian evolution. Nature 378: 353. CHIAPPE L.M., JI S.-A., JI Q. & M.A. NORELL 1999. Anatomy and systematics of the Confuciusornithidae (Theropoda: Aves) from the Late Mesozoic of northeastern China. American Museum of Natural History, Bulletin 242: 1-89. CURRIE P.J. 1997. Feathered dinosaurs. In Currie P.J. & K. Padian (eds), The Encyclopedia of Dinosaurs. Academic Press, San Diego, p. 241. CURRIE P.J. 1998. Caudipteryx revealed. National Geographic Magazine 194 (1): pp. 86-89. CURRIE P.J. 2000. Feathered dinosaurs. In Gregory S. (ed.), The Scientific American Book of Dinosaurs. Paul. St. Martin’s Press, New York, pp. 183-189. CURRIE P.J. & P.-J. CHEN (2001). Anatomy of Sinosauropteryx prima from Liaoning, northeastern China. Canadian Journal of Earth Sciences 38: 1705-1727. DAVIS P.G. & D.E.G. BRIGGS 1995. Fossilization of feathers. Geology 23: 783-786. DONG Z.M., & P.J. CURRIE 1996. On the discovery of an oviraptorid skeleton on a nest of eggs at Bayan Mandahu, Inner Mongolia, People’s Republic of China. Canadian Journal of Earth Sciences 33: 631-636. FEDUCCIA A. 1996. The Origin and Evolution of Birds. Yale University Press. 420p. GAUTHIER J. 1986. Saurischian monophyly and the origin of birds. In Padian K. (ed.), The Origin of Birds and the Evolution of Flight. California Academy of Sciences, San Francisco, pp. 1-55. HOLTZ T.R. 1994. The phylogenetic position of the Tyrannosauridae: implications for theropod systematics. Journal of Paleontology 68: 1100-1117. HOLTZ T.R., Jr. 2000. A new phylogeny of the carnivorous dinosaurs. Gaia 15: 5-61. HOPP T. & M. ORSEN 1998. Dinosaur brooding behavior and the origin of flight feathers. In Wolberg D.L., Gittis K., Miller S., Carey L. & A.Raynor (eds), Dinofest International Symposium, Program and Abstracts, Academy of Natural Sciences, Philadelphia, p. 27. HUTCHINSON J.R. & K. PADIAN 1997. Coelurosauria. In Currie P.J. & K. Padian (eds), Encyclopedia of Dinosaurs. Academic Press, San Diego, pp. 129-133. JI Q., CURRIE P.J., NORELL M.A. & S.-A. JI 1998. Two feathered dinosaurs from northeastern China. Nature 393: 753-761. Ji Q. & Ji S. A. 1996. On discovery of the earliest bird fossil in China and the origin of birds. Chinese Geology 233: 30-33 (in Chinese). JI Q. & S.-A. JI 1997a. Protarchaeopterygid bird (Protarchaeopteryx gen. nov.) - fossil remains of archaeopterygids from China. Chinese Geology 238: 38-41 (in Chinese). JI Q. & S.-A. JI 1997b. Advances in the study of the avian Sinosauropteryx prima. Chinese Geology 242: 30-32 (in Chinese).

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JI Q., NORELL M.A., GAO K.-Q., JI S.-A. & D. REN 2001. The distribution of integumentary structures in a feathered dinosaur. Nature 410: 1084-1088. JONES T. D., FARLOW J.O., RUBEN J.A., HENDERSON D.M. & W.J. HILLENIUS 2000. Cursoriality in bipedal archosaurs. Nature 406: 716-718. OSTROM J.H. 1974. Reply to “Dinosaurs as reptiles”. Evolution 28: 491-493. OSTROM J.H. 1978. The osteology of Compsognathus longipes Wagner. Zitteliana 4: 73-118. PAUL G.S. 1988. Predatory Dinosaurs of the World. Simon and Schuster, New York, 464p. PRUM R.O. 1999. Development and evolutionary origin of feathers. Journal of Experimental Zoology 285: 291-306. RUBEN J.A., JONES T.D. & N.R. GEIST 1997. Lung structure and ventilation in theropod dinosaurs and early birds. Science 278: 1267-1270. SCHWEITZER M.H., WATT J., FORSTER C., NORELL M. & L. CHIAPPE 1997. Keratinous structures preserved with two Late Cretaceous avian theropods from Madagascar and Mongolia. Journal of Vertebrate Paleontology 17: 74A. SUMIDA S.S. & C.A. BROCHU 2000. Phylogenetic context for the origin of feathers. American Zoologist 40: 486-503. XU X., TANG Z.-L. & X.-L. WANG 1999a. A therizinosaur dinosaur with integumentary structures from China. Nature 399: 350-354. XU X., WANG X.-L. & X.-C. WU 1999b. A dromaeosaurid dinosaur with a filamentous integument from the Yixian Formation of China. Nature 401: 262-266. XU X., ZHOU Z.-H. & R.O. PRUM 2001. Branched integumental structures in Sinornithosaurus and the origin of feathers. Nature 410: 200-204.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Evolution of Dental Capability in Eurasian ... Evolution 61 TheWestern New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 61-67, 2003

Evolution of Dental Capability in Western Eurasian Large Mammal Plant-Eaters 22-2 Million Years Ago: A Case for Environmental Forcing Mediated by Biotic Processes M. Fortelius Department of Geology, University of Helsinki, P.O. Box 11, FIN-00014 Helsinki, Finland

Abstract The development of computerised databases of fossil organisms that include ecomorphological information on species as well as the conventional data on locality occurrence and taxonomy has enabled “taxon-free” study of community structure and evolution. The Neogene large mammal plant-eater communities of western Eurasia can be shown to exhibit strong geographic and temporal patterns of dental capability (molar crown height times number of cutting edges), with the proportion of dentally capable forms increasing over time as well as in geographic gradients from east to west and south to north. These patterns correspond to an expected general increase in environmental harshness beginning in the interior of the continent and progressively advancing over time towards the climatically moderated oceanic rim. Consistent differences between climatically continental and maritime areas are seen during 20 million years in both body size and dental morphology, with parallel trends advancing at the same rate but at different levels. Closer scrutiny of the dental functional evolution of individual groups shows that the gradual increase seen at the community level is underlain by a considerable diversity of morphologically distinct solutions and rates of change. The combination of long-term trends of change and sustained regional contrasts is strong evidence for direct and precise environmental control over the community structure and functional evolution of the herbivorous large mammals of the Neogene. The fact that the patterns are re-established after brief turnover-driven anomalies seen at about 10 and 5 Ma reinforces the impression of environmental forcing mediated by biotic processes being the main factor responsible for them.

Introduction From the point of view of understanding the relationship between long-term evolution and community structure any example of long-term evolutionary change in a persisting biotic community is of interest, especially if the changes observed can be related to function and environmental conditions in some relatively straightforward fashion.

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Suitable fossil data compilations are rare, but the NOW database (Fortelius et al. 1996) of Neogene Old World mammals offers data that do lend themselves to such an investigation. The NOW data are especially useful because they are “taxon free” (Damuth 1992) in the sense that all species carry morphological and inferred functional attributes, so that analyses can be carried out regardless of which individual taxa happen to be represented in any given interval or geographic location. The NOW data also offer a relatively dense sampling of Western Eurasia, with multiple occurrences of individual taxa in temporally and geographically defined subsets. This in combination with the taxon-free characterization of species allows the use of a computationally trivial but useful methodological ploy, which we have previously referred to as “SPLOC-analysis” (Fortelius & Hokkanen 2001). In contrast to traditional analyses based on faunal lists, SPLOC-analysis makes use of all the available data and weights the impact of individual species in proportion to their frequency of locality occurrence, a non-specific proxy for geographic range and/or local abundance. The most suitable functional complex offered by fossil mammals for this purpose is the chewing apparatus, and in particular the molar dentition. In this study two aspects of the molar dentition were selected: hypsodonty and the number and orientation of cutting edges on the occlusal surface, the latter based on the “crown type” scheme of Jernvall (1995). Neither aspect offers a direct one-to-one relationship with either food or environment, but both have well understood functional roles, and both do show strong general correlation patterns with both diet and habitat (Van Valen 1960, Fortelius 1985, Janis 1988, Janis & Fortelius 1988, Solounias et al. 1994, Popowics & Fortelius 1997, Jernvall et al. 2000, Damuth et al. 2000, Fortelius & Solounias 2000). Using these variables singly and in different combinations offers a means of functional dissection of the temporal, geographic and taxonomic changes observed, and suggests possible causal interpretations of the patterns. Materials and Methods The data used were downloaded from the NOW database on March 9, 2000. A subset limited to Eurasia west of 60 degrees longitude and the time interval 22-2 Ma was selected (Fig. 1). Based on previous work (Fortelius et al. 1996, Fortelius & Hokkanen 2001) the geographic area was divided into blocks, East separated from West at 20 degrees eastern longitude and North from South at 45 degrees northern latitude. All Insectivora, Chiroptera, Rodentia, Lagomorpha, Pinnipedia and Cetacea were deleted from the dataset, as were all indeterminate and “cf.” species attributions. All analyses reported here were limited to species characterized as plant-eaters or plant-dominated omnivores. Hypsodonty was ranked according to the following scheme: brachydont=1, mesodont=2, hypsodont or hypselodont=3. Molar crowns were scored according to the crown type scheme of Jernvall (1995), and the number of longitudinal and transverse lophs was extracted from the crown type formulae. Molar capability was defined as ranked hypsodonty times total loph count. The dataset is available from the author, and the latest public NOW dataset can be downloaded from http://www.helsinki.fi/science/ now/, where further details regarding the data may also be obtained. Mean values for lophedness, hypsodonty and molar capability were calculated for subsets of the data

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Fig. 1. Map of Western Eurasia showing localities included in the study. The vertical line at 20 degrees eastern longitude separates West from East, the horizontal line at 45 degrees northern latitude separates North from South (see under Materials and Methods). Width of field ca 5 500 km.

defined by time units, designed to correspond to the biochronologic MN-units according to the correlation tables of Steininger et al. (1996). Results Selected results for overall trends are shown in Figures 2-3. Longitudinal and transverse lophedness are negatively correlated in the interval studied (Fig. 2), as would be expected from the strong known association between transverse lophs and leaf-eating, a relationship violated only by advanced elephantids at the end of the study interval. Transverse lophedness accordingly peaks in the “forested” Middle Miocene, and both graphs show rapid shifts (in opposite directions) at 10 Ma, the major mammal turnover event known in western and central Europe as the “Vallesian Crisis” (Agustí & MoyàSolà 1990). Total loph count (Fig. 3) shows virtually no change except for a rise at the end, while hypsodonty shows a sustained rise, again including a rapid shift at 10 Ma. Fig. 4 shows the evolution of mean molar capability separately for the East and West blocks. Molar capability is persistently higher in the more continental (seasonal) East

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Fig. 2. Bivariate scatter plot of longitudinal and transverse loph counts against time units. Fitted lines are LOWESS-smoothed with tension 0.5.

Fig. 3. Bivariate scatter plot of hypsodonty and total loph count against time units. Fitted lines are LOWESS-smoothed with tension 0.5.

except for two turnover events, the Vallesian Crisis at 10 Ma and the Miocene-Pliocene boundary at 5 Ma. Fig. 5 shows the same for the North and South blocks. Molar capability is consistently higher in South than in North except for an interval around 10-7 Ma, beginning with the Vallesian Crisis and continuing into a time that is very poorly sampled for Europe north of the Alps. For both comparisons the difference between blocks is statistically highly significant (Kruskal-Wallis test, P<0.001) for the whole dataset but not for most individual age units. Fig. 6 shows a breakdown by order of the long-term trend in molar capability. This is one example of many showing that the regular summary patterns are composed of a subsets that each behave differently. Discussion and Conclusions Space restrictions preclude any deeper discussion of these patterns or their causation, but it may be briefly noted that they do carry several interesting implications. First, the general trend of all changes seen is what would be expected from the climatic changes that occurred during the Neogene: from a plant-eater’s perspective, the world became progressively harsher and the dentition progressively more capable. What changed most was hypsodonty, suggesting that the main change was a non-specific one, demanding greater dental durability in general, and that increased cutting ability per chewing cycle, although present, was only a secondary demand. Secondly, the fact that persistent geographic contrasts persisted during the entire interval strongly suggests that the dental composition of the plant-eater community was under direct and detailed environmental control, reflecting at each point in time climatic and vegetational gradients of both continentality (East-West) and temperature (North-South). Thirdly, the fact that the summary curves appear smooth despite the asynchronous changes seen for individual groups indicates that the effect of chance and constraints operating at lower levels are largely cancelled out at the level of the community. These analyses cannot alone resolve the relative effect on community-level changes of environmental forcing and biotic

Evolution of Dental Capability in Western Eurasian ...

Fig. 4. Evolution of mean molar capability in East (E) and West (W). Upper diagram is a scatter plot with LOWESS-smoothed lines fitted (). Lower diagram shows lines through the means for time units, with standard error of the mean indicated by vertical bars (0.95).

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Fig. 5. Evolution of mean molar capability in North (N) and South (S). Upper diagram is a scatter plot with LOWESS-smoothed lines fitted (as in Figs 2-3). Lower diagram shows lines through the means for time units, with standard error of the mean indicated by vertical bars (0.95).

Fig. 6. Bivariate scatter plot of mean molar capability against time unit, divided by taxonomic order Fitted lines are LOWESS-smoothed with tension 0.5.

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processes (such as competition or escalation), but the near-linear change seen at the community level would be easier to reconcile with models that include a significant component of internal biotic processes than with models based only on direct environmental forcing. The fact that the few and geologically brief anomalies in these geographic patterns correspond to known episodes of faunal turnover reinforces the impression that the normal state is maintained by biotic processes acting with sufficient delay for its effect to be visible at the coarse resolution available. Acknowledgements I thank the Advisory Board members of the NOW database for past and ongoing collaboration. I’m grateful to John Damuth, Jukka Jernvall and Ray Bernor for discussion of the results reported here. This paper is a contribution from the Valio Armas Korvenkontio Unit of Dental Anatomy in Relation to Evolutionary Theory and the Academy of Finland project nr 34080. References AGUSTÍ J. & S. MOYÀ-SOLÀ 1990. Mammal extinctions in the Vallesian (Upper Miocene). In Kauffman E.G. & O.H. Walliser (eds), Extinctions, Events in Earth History. Lecture Notes in Earth Sciences, 30. Springer-Verlag, Berlin - New York, pp. 425-432. DAMUTH J. 1992 Taxon-free characterization of animal communities. In Behrensmeyer A.K., Damuth J., DiMichele W.A., Potts R., Sues H. & S.L. Wing (eds), Terrestrial Ecosystems Through Time. University of Chicago Press, Chicago, pp. 183-203. DAMUTH J., JANIS C.M. & J.M. THEODOR 2000. Miocene ungulates and terrestrial primary productivity: Where have all the browsers gone? Proc. Natl. Acad. Sci. 97: 7899-7904. FORTELIUS M. 1985. Ungulate cheek teeth: developmental, functional, and evolutionary interrelations. Acta Zool. Fennica 180: 1-76. FORTELIUS M. & A. HOKKANEN 2001. The trophic context of hominoid occurrence in the later Miocene of western Eurasia: a primate-free view. In de Bonis L., Koufos G. & P. Andrews (eds), Hominoid Evolution and Climatic Change in Europe. Cambridge University Press, Cambridge. pp. 19-47. FORTELIUS M. & N. SOLOUNIAS 2000. Functional characterization of ungulate molars using the abrasion-attrition wear gradient: a new method for reconstructing paleodiets. Amer. Mus. Novitates 3301: 1-36. FORTELIUS M., WERDELIN L. ANDREWS P., BERNOR R.L., GENTRY A., HUMPHREY L., MITTMANN H.-W. & S. VIRANTA 1996. Provinciality, diversity, turnover and paleoecology in land mammal faunas of the later Miocene of Western Eurasia. In Bernor R.L., Fahlbusch V. & H.-V.Mittmann (eds), The Evolution of Western Eurasian Neogene Mammal Faunas. Columbia University Press, New York, pp. 414-448 JANIS C.M. 1988. An estimation of tooth volume and hypsodonty indices in ungulate mammals and the correlation of these factors with dietary preferences. Mus. Natl. Hist. Nat. Mem. Sér. C 53. In Russel D.E. Santorio J.P. & D. Signogneu-Russel (eds), Teeth Revisited: Proceedings of the VII International Symposium on Dental Morphology. Paris: Museum National d’Histoire Naturelle Press, pp. 367-387.

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JANIS C. & M. FORTELIUS 1988. On the means whereby mammals achieve increased functional durability of their dentitions, with special reference to limiting factors. Biol. Rev. 63: 197-230. JERNVALL J. 1995. Mammalian molar cusp patterns: Developmental mechanisms of diversity Acta Zool. Fennica 198: 1-61. JERNVALL J., HUNTER J.P. & M. FORTELIUS 2000. Trends in the evolution of molar crown types in ungulate mammals: evidence from the northern hemisphere. In Teaford M.F., Smith M.M. & M.W.J. Ferguson (eds), Development, Function and Evolution of Teeth. Cambridge University Press, Cambridge, pp. 269-281. POPOWICS T. & M. FORTELIUS 1997. On the cutting edge: Tooth blade sharpness in herbivorous and faunivorous mammals. Acta Zool. Fennica 34: 73-88. SOLOUNIAS N., FORTELIUS M. & P. FREEMAN 1994. Molar wear rates in ruminants: a new approach. Ann. Zool. Fennici 31: 219-227. STEININGER F.F., BERGGREN W.A., KENT D.V., BERNOR R.L., SEN S. & J. AGUSTI 1996. Circum-Mediterranean Neogene (Miocene-Pliocene) marine-continental chronologic correlations of European mammal units. In Bernor R.L., Fahlbusch V. & H.-W. Mittmann (eds), The Evolution of Western Eurasian Neogene Mammal Faunas. Columbia University Press, New York, pp. 7-46. VAN VALEN L. 1960. A functional index of hypsodonty. Evolution 14: 531-532.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Cambrian ‘Orsten’-type Arthropods and the Phylogeny of Crustacea 69 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 69-87, 2003

Cambrian ‘Orsten’-type preserved Arthropods and the Phylogeny of Crustacea Dieter Waloszek Section for Biosystematic Documentation, University of Ulm, Helmholtzstraße 20, D-89081 Ulm, Germany. E-mail: [email protected] http://biosys-serv.biologie.uni-ulm.de/

Abstract ‘Orsten’-type preservation refers to the finds of three-dimensional, phosphatized Cambrian microscopic arthropod fossils with soft parts, most of which specimens are smaller than one millimeter. Such unique material permits the reconstruction of the early phylogeny of the Crustacea and thereby the recognition of progressive modification of the cephalic locomotory and feeding system as one of the major evolutionary strategies of this group. ‘Orsten’ fossils also help to re-evaluate traditional hypotheses of arthropod phylogeny. Since the record of ‘Orsten’-type fossils assignable to the late stem lineage of the Eucrustacea ranges down to the Early Cambrian, it is proposed that the stem species of Crustacea existed in the late Pre-Cambrian and therefore that all prior branchings down the metazoan lineage had occurred even earlier. The record of exceptionally preserved Cambrian fossils is still scattered and limited to a few lagerstätten, more or less, exclusively of the Chengjiang, Burgess-shale or ‘Orsten’ type. We predict that the most promising fossils to hunt for in the future to improve our still fragmentary knowledge of the early phylogeny of Arthropoda and, particularly, of the evolutionary paths to the major arthropod groups with living derivatives, Chelicerata, Atelocerata and Crustacea, are those of the ‘Orsten’-type of preservation.

‘Orsten’ and ‘Orsten’-type fossils Stinkstones, meaning “smelling stones” because of their characteristic smell of rotten eggs due to their high content of organic matter when cracked, are a special type of bituminous concretionary limestones found in southern Sweden, in the upper part of the black-shale sequence (alum shales) deposited from the Early to Late Cambrian (ca. 520-490 Mya). They are locally called ‘orsten’, possibly a combination of orne = male pig and sten = stone (Bergström, personal communication). We use the term ‘Orsten’ to denote a special type of lagerstätten yielding three-dimensionally preserved secondarily phosphatized cuticles of mainly arthropods that are etched from such limestones using

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mild acetic acid. The first ‘Orsten’ lagerstätten from the Upper Cambrian were discovered in southern Sweden (Öland, Västergötland) by Klaus Müller, University of Bonn, in the 1970s (see e.g., Müller 1979, 1985, 1990) and have, since then, yielded more than 50.000 of this type of bodily preserved minute fossils. Similarly preserved material has also been found in other regions of the World, such as North and South America, Siberia, and Australia, and in rocks of Early Cambrian to the Early Cretaceous age (listed in Walossek & Müller 1997). ‘Orsten’-type fossilization offers potential for the recovery of, principally, arthropod or arthropod-like cuticles, possibly only the outermost layer, the epicuticle. Details preserved, which may be smaller than one µm, range from eyes, limbs, hairs, gland openings, to even minute sensilla and bristles such as the secondary bristles on filtratory setae of limbs, which are much narrower than half a Micron. The interior of ‘Orsten’type preserved fossils is, in most cases, empty or filled by amorphous crystalline matter. Rarely, specimens are much larger than a millimeter (Weitschat 1986 for ammonites) or massive (Müller et al. 1995 for a Middle Cambrian tardigrade). In one case even musculature has been beautifully preserved, such as in larval Pentastomida (tongue worms) from rocks of Lower Ordovician age (Andres 1989). None of the Swedish ‘Orsten’ specimens is larger than three mm. The best-preserved animals are between 100 µm and 700 µm long. Thus, not unexpectedly, the ‘Orsten’ fossils represent mainly postembryonic stages, in some cases found in sets of successive stages (e.g., Müller & Walossek 1988 for Bredocaris admirabilis Müller, 1983, and Walossek 1993 for Rehbachiella kinnekullensis Müller, 1983, which had 30 instars to reach a juvenile stage). More rarely, small-sized adults could be identified such as of Bredocaris admirabilis (Müller & Walossek 1988) or the two species of Skaracarida (Müller & Walossek 1985). ‘Orsten’-type preservation is generally much superior to most other types of fossil preservation because it preserves the whole external morphology and topology of structures and structural systems of the animals including ontogenetic information of the morphogenesis of structures. This enables full reconstruction of the morphology of these animals with a high degree of confidence and completeness, and therefore to speculate with some assurance about their life habits. The majority of the ‘Orsten’ animals possibly represent temporary or permanent components of a soft-bottom living meiofauna (Müller & Walossek 1991). Clearly fossils of this kind can also serve as a toolbox for phylogenetic analyses (e.g. Walossek 1993, 1999, and herein; Walossek & Müller 1990, 1997, 1998; Maas et al. 2003). Toward the reconstruction of the phylogeny of the Crustacea In 1983 Klaus Müller and I started a still ongoing joint project on the arthropods of the Swedish ‘Orsten’ fauna, representing a specific part of the fauna from the Upper Cambrian Alum Shale Sea (representative forms in Figure 1). We have been able to identify, on the basis of shared apomorphies in the sense of synapomorphies, several ‘Orsten’ forms as early representatives of the so-called crown group of Crustacea, the Eucrustacea sensu Walossek (1999). The 0.85 mm long Bredocaris admirabilis Müller, 1983 (Fig. 1A for the supposed adult and B for the first recognized larva, a metanauplius with initial maxillulae) is related to the Thecostraca, a group that includes the barnacles

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Fig. 1. Examples of ‘Orsten’ crustaceans. A, B, Bredocaris admirabilis Müller, 1983; A, presumed adult (approx. 850 µm long; from Müller & Walossek 1988); B, earliest stage, a metanauplius with buds of maxillulae; approx. 170 µm long; same reference). C, D Rehbachiella kinnekullensis Müller, 1983; C, advanced larval stage (TS12; body length approx. 1 mm; from Walossek 1993); D, dorsal organ of first larval stage, the orthonauplius, arrows point to sensilla (specimen ca. 170 µm long; same paper). E, F, Martinssonia elongata Müller & Walossek, 1986; E, largest stage, arrow points to incomplete fusion of maxillary segment to head (body length approx. 1.5 mm; from Müller & Walossek 1986a); F, first larval stage (non-feeding) of the same species, arrow points to functional 4th limb (body length approx. 250 µm; from same article). G, H, Hesslandonid phosphatocopine Hesslandona unisulcata Müller, 1982; G, advanced instar (UB 658, shield length approx. 700 µm; from Müller 1982); H, presumed earliest larval stage of Vestrogothia spinata Müller, 1964, Wa 249 (approx. 300 µm long; from Maas et al. 2003). Abbreviations: a1, antennula; a2, antenna; an, anus; bas, basis, cox, coxa; en, endopod; end, endite; eye, lateral or facetted eye; ex, exopod; fu, furca; cs, head shield; ini, initial; la; labrum; md, mandible; mx1, maxillula; mx2, maxilla; pe, proximal endite; stn, sternum; thp, thoracopods.

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and represents an in-group of the Maxillopoda (Müller & Walossek 1988; Walossek 1993, Walossek & Müller 1998). The two species of the Skaracarida are 1,2 and 0,7 mm long, indicative of a life at different vertical levels in the flocculent bottom zone (Müller & Walossek 1985). Skaracarida form a possibly monophyletic group with Mystacocarida and Copepoda (cf. Walossek & Müller 1998). Rehbachiella kinnekullensis is a representative of the Branchiopoda (Fig. 1C for a TS12 instar and D showing the dorsal organ of the first instar, a true orthonauplius). Its largest instar was approximately 2 mm long (cf. Walossek 1993). Work on other ‘Orsten’ eucrustaceans, such as Dala peilertae Müller, 1983 and Walossekia quinquespinosa Müller, 1983, is in progress. We have also described forms that do not easily fit into the current scheme of the Crustacea, at least as that group was traditionally understood. Such forms are recognized as representatives of the early evolutionary lineage of the Eucrustacea. They comprise the 1,5 mm long Cambropachycope clarksoni Walossek & Müller, 1990, Goticaris longispinosa Walossek & Müller, 1990, possibly also 1.5 mm long when adult, Martinssonia elongata Müller & Walossek, 1986, also 1.5 mm long but in its largest known, most likely still immature stage (Fig. 1E shows the latest known and 1F the first instar, a non-feeding head larva type; see Walossek & Müller 1990 for re-evaluation of its phylogenetic position), the 1 mm long Henningsmoenicaris scutula (Walossek & Müller, 1990), and Cambrocaris baltica Walossek & Szaniawski, 1991, a form discovered in a bore hole core from Poland and known only from the anterior body portion. All of these species share and also lack some key characters known from Recent and fossil eucrustaceans. These shared features, not known from any other arthropods, formed the basis of a new characterization of the monophylum Crustacea (see Walossek & Müller 1990) and the discussion of the evolutionary path towards the crown-group, the Eucrustacea sensu Walossek (1999). According to this new scheme, the ground pattern (GP) of Crustacea embraces at least the following autapomorphies (Figure 2, node 1): • Antennula (first antenna, a1) composed of a few segments and special distal setae acting for locomotion and feeding (plesiomorphic state (ples.): multi-annulated, sensorial, filamentous, as in GP, of Euarthropoda); • Basis of post-antennular limbs bearing a movable, setae-bearing endite (= proximal endite, PE) medioproximally (ples.: only basis, as in GP of Euarthropoda; examples: trilobites, all Chelicerata); • Exopod (ex) of post-antennular limbs may be multi-annulated, ca. each annulus with 1 seta pointing towards the endopod (ples.: leaf-shaped with circum-marginal setation, as in GP of Euarthropoda); • Number of endopod articles of post-antennular limbs maximally 5 (ples.: 7, as in GP of Euarthropoda). These new features, particularly the design of the antennula, the appearance of a proximal endite, and the development of exopods with many annuli having their setae against the endopod, indicate significant changes in the locomotory and feeding system (see below). Crustacea also retain euarthropod features as plesiomorphies in their ground pattern such as: • Head including antennular and 3 limb-bearing metameres (limbs as described above);

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• Mouth exposed, located at rear of a prominent hypostome (reduced secondarily in Cambropachycope clarksoni and Goticaris longispinosa; • Sternites behind mouth separate; • Tail ends in a spinose or plated non-metameric piece with ventral pre-terminal anus; • Larva is of the head-larva type, having antennulae and 3 pairs of functional limbs. The presence of a ‘head larva’ is a plesiomorphy of all derivatives of the stem lineage of the Eucrustacea, named above (Walossek in preparation) that dates back to the ground pattern of Euarthropoda (Walossek & Müller 1997; example in Fig. 1F). A similar ‘head larva’ is present still today in the ‘protonymph’, the hatching stage of Pantopoda (see Müller & Walossek 1986b for an Upper Cambrian pycnogonid larva, and Walossek and Dunlop 2001 for discussion of this larva in the context of chelicerate phylogeny). Increased knowledge of the largest group of putative crustaceans and most abundant components in the ‘Orsten’ material, the phosphatocopines has yielded a more detailed picture of the later crustacean lineage than was possible in 1990. Phosphatocopina (examples in Fig. 1G, H) and Eucrustacea share a number of synapomorphies characterizing a yet unnamed monophylum (Figure 2, node 2 and N.N. 1, will be named in Siveter et al. 2003), such as: • Labrum (la) occurs, as a fleshy outgrowth, at the rear of the hypostome and above the mouth (ples.: only hypostome, as in GP of Crustacea, retained from GP of Euarthropoda); • Atrium oris (ao) present as a funnel-shaped depression of the mouth area (ples.: mouth occurs at the rear of the hypostome, as in GP of Crustacea and retained from GP of Euarthropoda); • Sternum, as a sclerotic plate, consists of the sternites of the antennal to maxillulary segments (ples: isolated sternites, as in GP of Crustacea, as retained from GP of Euarthropoda); • Fine hairs on sternum and sides of labrum (ples.: fine hairs absent; sternum and labrum absent in GP of Crustacea); • Paragnaths (pgn) as outgrowths of the mandibular sternite (ples.: lacking in GP of Crustacea); • Proximal endite of antenna (second antenna, a2) and mandible (md) enlarged and more strongly sclerotized to form a separate stem portion, coxa, proximal to the basis (ples.: only basis with proximal endite in GP of Crustacea; only basis in GP of Euarthropoda); • 5 limb-bearing head segments, including the segment of the trunk-limb shaped maxilla (mx2) (ples.: 4 limb-bearing head segments in GP of Crustacea, as retained from GP of Euarthropoda). The status of the head shield, which at this stage in the evolution of the Crustacea, is fully fused with the segment of the so-called maxillae (second maxillae, mx2) dorsally (so-called because these limbs specialize as mouthparts much later and well within the evolutionary lines of the Eucrustacea, see below) remains uncertain. At least some of the derivatives of the early stem lineage of Crustacea also seem to have obtained this feature, such as Henningsmoenicaris scutula and, partly also Martinssonia elongata (Fig. 1E, arrow; see also Fig. 5, middle). The plesiomorphic state has only the maxillula incorporated within the head (and head shield), as in the ground pattern of the

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Crustacea and Euarthropoda (see also Scholtz 1997), as can be seen in Cambropachycope clarksoni and Goticaris longispinosa. It is also uncertain when the internal 3-cupped naupliar eye appeared (ples.: absent). Plesiomorphic retention of euarthropod features is also apparent in the design of the tail end and in the non-specialization of all of the postmandibular limbs. The Phosphatocopina were long interpreted as Ostracoda (and still by some workers, e.g. Hinz-Schallreuter 1998) because they possess a bivalved head shield. The ventral details, known so far only from larval stages up to 1 mm, while adults may grow to 5 mm in shield length, demonstrate that Phosphatocopina can be characterized as a monophylum, based on a large set of autapomorphies (Figure 2, node 3; Walossek 1999; Maas et al. 2003). Examples are: • Head shield bivalved, enclosing the body (ples.: simple, roof-shaped shield in GP of N.N. 1); • Head shield primarily phosphatic (ples: consisting of non-mineralized shield cuticle in GP of N.N. 1); • Antennula much reduced in size, segmentation and setation (ples.: approx. as long as the subsequent schizoramous limbs in GP of Crustacea, even longer in GP of Euarthropoda); • Number of trunk segments reduced to <6 (ples: >10 segments in GP of Crustacea). Phosphatocopina have retained several characters from the ground pattern of N.N. 1 that clearly demarcate them off from the Eucrustacea, such as the undifferentiated maxillula (first maxilla, mx1) (of trunk-limb shape) and the retention of a ‘head larva’ (see below; cf. Maas et al. 2003). The Eucrustacea includes all groups with extant descendants. This group can be characterized as a monophylum by autapomorphies in their ground pattern (Figure 2, node 4) such as: • Maxillula modified into a ‘mouthpart’ (ples.: trunk-limb-shaped first post-mandibular limb, with basis and proximal endite, as in GP of N.N. 1); • Tail ending in a conical telson with a terminal anus and carrying articulated leafshaped furcal rami (but see below) with marginal setation (ples.: spinose end piece with the anus opening ventrally and pre-terminally, as in GP of N.N. 1, furcal rami lacking in GP of N.N. 1); most likely this telson carries a pair of latero-caudal spines and a pair of ventro-caudal spines (since present in various eucrustacean groups), and at least, two larval features: • Hatching stage is a nauplius larva (orthonauplius), termed ‘short-head’ larva by Walossek & Müller (1990) and having three pairs of limbs, antennulae, antennae and mandibles, (ples.: ‘head larva’ with 4 pairs of functional limbs, as in GP of N.N. 1, Crustacea and Euarthropoda); • Nauplius with a supra-anal flap carrying a dorso-caudal spine (ples.: not present). This characteristic eucrustacean nauplius larva acts as a kind of ”locomotive” for the developing trunk, which takes over locomotory and feeding functions much later during postembryonic growth (in Artemia salina this change occurs very late in its

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post-larval differentiation phase; see Walossek 1993). Nauplius and larval development as a whole play an important role in the understanding of the evolution and phylogeny of crustaceans (see Dahms 2000 for a recent review). The earliest phosphatocopine larva also has a labrum, sternum and paragnath humps (N.N. 1 features), but, in addition, one more pair of limbs (Fig. 1H), a feature further confirming the placement of this group as the sister group to the Eucrustacea. It follows that the eucrustacean nauplius is not an ancient larval type that was present in the ground pattern of the Euarthropoda or at least Mandibulata (Lauterbach in several papers between the early 1970iest and mid 1980ies). It is implicit that it could develop not before the two postantennular limbs, the antenna and mandible, and not just the mandible, had received coxae and not before the other features, such as the labrum, atrium oris and setation pattern, had also been developed in the stem species of N.N. 1 (cf. Walossek & Müller 1997, Walossek 1999). The furca remains a problem because latest finds of structures that may be furcae have been found in phosphatocopines (Maas et al. 2003) and might, therefore, push this character down to the GP of N.N. 1. Though incorporated into the head, the second postmandibular limb, the maxilla was still a trunk limb in the ground pattern of Eucrustacea, and not modified into a ‘mouth part’. Still today the Cephalocarida, as a living example, have unspecialized limbs on the maxillary segment (Sanders 1963; see Walossek 1993 for an extensive discussion of this matter). Accordingly, two pairs of “antennae” = feelers and two pairs of “maxillae” = mouthparts neither characterize the Crustacea nor the Eucrustacea. In the light of the similarity of the first two pairs of post-antennular limbs N.N. 1 (Fig. 2, node 2) could be regarded as ‘Di-Mandibulata’. This brings us also to the problem of the affinities of atelocerates or, depending on authors, insects, with the crustaceans or any in-group and the discussion of a taxon ‘Mandibulata’, as was recently brought into focus again particularly by developmental biologists (examples: Abzhanov et al. 1999; Abzanov & Kaufman 2000). This cannot be the place of an extensive discussion, but at least the feature ‘structural identity of antennae and mandibles’ is a critical one because no other limbs have, at the evolutionary stage of N.N. 1, coxal portions on their limbs. As listed below, such limb-stem portions occur only in certain eucrustacean groups in different ways and cannot be regarded as a groundpattern feature of Crustacea. There is also no such distinctive “pre-gnathal” region in the ground pattern of Crustacea and even still not in that of the Eucrustacea, as some Atelocerata- or Malacostraca-centered workers still favor (e.g., Scholtz 1997). Moreover, in many of these works on the problem of mandibles and the relationships of Crustacea (not clarifying which group or branching they mean) to Atelocerata (frequently using insects as taxon for sister-group hypothesis and not specifically discussing the myriapods) the mandible - a limb in the series – either seems mismatched with its coxa – a portion of the stem – or it is neglected or not known (Popadic et al. 1996; Scholtz et al. 1998) that “gnathobasic” can just be anything from: • a spine-bearing or dentate flattened, obliquely oriented median edge of a coxal portion below a basis (N.N. 1: antenna + mandible), • a straight spinose median edge of the basis (e.g., trilobites, naraoids, anomalocarids, chelicerates),

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• a fusion product of coxa and basis (in later larval stages of phosphatocopines), or • a dentate median edge that expands along the whole limb and involves all still separate portions, coxa, basis and endopod segments (Middle Cambrian phosphatocopines, see Walossek et al. 1996). A far more differentiated view of this matter is highly recommended if we want to advance in this discussion. As a matter of fact, the limb stems of Atelocerata have never been specifically investigated under this perspective, and thus it has remained uncertain for any of the postmandibular limbs as well if the basal limb portion refers/can be homologized to/with the coxal or basis portion. Again, features like the ‘proximal endite’, the ‘labrum’ - in the above sense and not mismatched (as in, e.g., Scholtz 1997) with the euarthropod hypostome (see Walossek 1993 and Dewel et al. 1999 for a recent discussion), the setation pattern, the antennula as a feeding device, the second head limb, exopods, and a nauplius larva are characters simply missing in the Atelocerata. Lack gives not any positive indication of shared structures in the sense of synapomorphies, as demanded in a serious evaluation, and, therefore, does not add to the problem of systematic relationships with Crustacea. And there are more problems to be solved. The Eucrustacea branched into two major lineages, the Entomostraca and the Malacostraca. Both groups can be characterized as monophyla by sets of autapomorphies in their ground patterns. For one of the features, the shape of the maxillula, it is noteworthy that it is different in the two taxa and it is also different from that in the Phosphatocopina. The phosphatocopine maxillula comprises a basis with one spinose

Fig. 2. Proposed scheme of relationships within the Crustacea. Nodes specified in the text.

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median protrusion, a proximal endite and two rami, regarded to represent the plesiomorphic state. That of malacostracans has a coxa and basis, both with a narrow vertical median edge with spines, and the two rami, while entomostracans have a basis with three setiferous lobate endites plus the proximal endite and rami. Thus, here are two apomorphic states, one for each eucrustacean group. Malacostraca have in their ground pattern a large number of autapomorphies (Figure 2, node 5) and therefore have since long been accepted as a monophylum. Examples of autapomorphies are: • Adult antennula comprising a divided stem + 2 flagella (ples.: antennula uniramous and comprising a few segment, as in GP of Eucrustacea); • Antenna modifies during larval development from a locomotory device to a sensorial organ (ples.: locomotory and feeding function retained, limb stem and rami as in GP of Eucrustacea, Entomostraca and N.N. 1); • Mandibular palp present (ples.), but comprising only the basis + the 2-segmented endopod maximally (ples.: coxa + basis + 2 rami, as in GP of Eucrustacea and N.N. 1); • Maxillula with a 2-divided limb stem (ples.: only proximal endite proximal to basis, as in GP of N.N. 1 - status in GP of Eucrustacea uncertain); • Maxilla with a 2-divided limb stem, each portion medially split into 2 endites (ples.: only proximal endite proximal to basis in GP of N.N. 1 – status of Eucrustacea uncertain); • Tagmatisation of the thorax in 2 portions (ples.: undivided set of trunk segments with limbs similar in design, as in GP of Entomostraca and Eucrustacea); • Anterior 8 thoracopods with limb stem comprising a coxa + basis (ples.: basis with a proximal endite, as in GP of Eucrustacea, retained from GP of N.N. 1); • Gonopores fixed to thoracic segment 6 in females and 8 in males (status uncertain, since all others different). Entomostraca have been, by most arthropod workers, regarded as a “non-phylogenetic assemblage” of crustacean groups on the way to the so-called “higher” Crustacea, the Malacostraca. This view may be mainly conditioned by apparent plesiomorphies in the ground pattern of Entomostraca retained from earlier levels, such the large anamorphic larval sequence or the long, regularly segmented trunk. The various subsequent structural changes within the major entomostracan lineages (Cephalocarida and N.N. 2 = Maxillopoda and Branchiopoda, see Fig. 2; see also Walossek & Müller 1998) that led to quite distinctive morphologies, may have made it difficult for many students of Crustacea and Arthropoda to accept common ancestry because by the overestimating of structural differences or loss of features in certain in-groups (these are nothing more than autapomorphies of particular groups). Yet there are several autapomorphic features that characterize Entomostraca as a monophylum and demonstrate their sister-group status with regard to the Malacostraca (Figure 2, node 6), such as: • Adult mandible lacking a palp (ples.: with coxa + palp comprising the basis + 2 rami, as in GP of Eucrustacea and N.N. 1, palp retained in Malacostraca, but without exopod);

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• Maxillula with a proximal endite (ples.) and a basis with 3 additional lobate setiferous endites medially (ples.: only proximal endite proximal to endites-less basis in GP of N.N. 1, but GP of Eucrustacea unclear); • Maxilla and all thoracopods with a proximal endite (ples.) and an elongate, littlesclerotized basis carrying several lobate endites medially in long axis of the limb (ples.: only proximal endite and an endites-less basis, as in GP of Eucrustacea, retained from GP of N.N. 1); • Each basipodal endite with a set of setae in 3 sets, possibly all mobile due to internal musculature running into the endites (ples.: no such endites). Entomostraca have in their groundpattern a limb-less region, the so-called abdomen, comprising several ring-shaped segments, which clearly set them off of Malacostraca (Walossek & Müller 1997, fig. 12.13). The status of this feature remains, however, uncertain, because there is at least one segment externally in Malacostraca, possibly more originally (e.g., Olesen & Walossek 2000) and it is similarly uncertain for Eucrustacea and N.N. 1 because also several fossil arthropods have such limb-less regions (e.g., Fuxianhuia protensa, first described in Hou 1987). As with the early evolution of Crustacea, also here did the major changes occur in the locomotory and feeding system. Even more, also the C-shaped curvature of the limb stems of all post-maxillulary limbs and their arrangement in a sucking-chamber system may be a further autapomorphy in the ground pattern of Entomostraca, as known from Cephalocarida (Sanders 1963) and Branchiopoda (Walossek 1993), while filtratory mechanisms require still more features, and they are assembled, e.g., in the ground pattern of Branchiopoda or in cirripedes convergently (Walossek 1993). This elaborate locomotory and feeding trunk apparatus of the ‘Orsten’ Rehbachiella kinnekullensis Müller, 1983, with combs of setulae on the filter setae at the Micron scale (details in Fig. 3) and the inwards folding of the trunk sternites to form a deep, V-shaped filter groove, was well adapted for filtration and identifies the fossil as a representative of the Branchiopoda (Walossek 1993; Olesen 1999), in contrast to claims by Wägele (2000). This author also

Fig. 3. Details of the filter apparatus of Rehbachiella kinnekullensis Müller, 1983. A, Regular setulae on the filter setae. B, Comb seta for particle grooming. C, Whip setae of the proximal endites pointing into the filter groove (from Walossek 1993).

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wrongly depicts an antenna of this fossil as having a proximal endite to demonstrate its non-crustacean position (and as a stem-lineage “mandibulate”), although this form as others clearly have a coxa (see Walossek 1993 and numerous SEM images and drawings there). Another feature of R. kinnekullensis is its exclusively larval organ, the so-called dorsal or neck organ (Fig. 1H). This feature of all living Branchiopoda serves in their larvae as an osmoregulatory organ and may eventually be either retained, reduced or modified. Limb evolution and the development of locomotory and feeding structures Limb design in arthropod phylogeny has been controversially discussed for more than a century, often involving speculative hypotheses and erroneous observations. In an important but often overlooked contribution to this debate, Cisne (1975) demonstrated that the trilobite limb – representative for the euarthropod type of limb – consists of only an undivided limb stem (named basis herein, see below) and two rami, that is, a seven-segmented endopod (as the distal elongation of the stem) and a leaf-shaped exopod arising from the slanting outer rim of the stem. Endopods and exopods are completely different from one another and most likely also had a different genesis (see Walossek & Müller 1997 for discussion and references). Starting from such a design, and considering the ‘Orsten’ evidence as a toolbox of features, broadly similar limbs occur in representatives of the stem lineage of the Eucrustacea, but these possess one more element, a lobate setiferous protrusion = endite at the inner proximal edge of the limb stem, the ‘proximal endite’ sensu Walossek & Müller (1990). Because the large proximal limb portion or stem carries the two rami, we regard it as the basis – and no longer as the coxa, as it was traditionally named (also by Cisne). The special, movable and setae-bearing proximal endite may have been used for proximal food transportation, thus initiating a new feeding method in the early crustacean lineage. Notably, the basis and proximal endite in stem-lineage derivatives such as Martinssonia elongata have even the same basic setation pattern as in the antenna of a living barnacle nauplius (Walossek 1999, fig. 7). The antennula and the exopods of at least the next subsequent limbs (being multi-annulated) were also modified in order to achieve a new locomotory and feeding system, one that was possibly used for sweepnet feeding rather than for shoveling in food from the posterior, as in euarthropods. The proximal endite also permitted the de-coupling of the feeding activities from the locomotive activities of the distal parts of the limbs. Subsequently, in the stem lineage of Crustacea another crucial change included the recessment of the mouth into an atrium oris. The mouth was originally opening at the rear of the bulge-like hypostome (a sclerotic plate, see Müller & Walossek 1987 for Agnostus pisiformis and discussion of hypostomes there; see also Walossek & Müller 1990 and the hypostome-origin hypothesis by Dewel et al. 1999). This organizational level was retained in the ‘Orsten’ representatives of the eucrustacean stem lineage and in their larvae. Another new feature was the covering of the atrium oris by a bulging structure containing slime glands and chemoreceptors (e.g., Cannon 1922; Zaffagnini & Zeni 1987), the labrum sensu Walossek & Müller (1990), which has been interpreted by these authors to be the extruded mouth membrane, as was

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present e.g. in Agnostus pisiformis (Müller & Walossek 1987). Accordingly the labrum should not be mismatched with the hypostome, a character of the ground pattern of Euarthropoda. Yet the hypostome is retained in the Crustacea and sited in front of the labrum (and is not exchanged by it) as the anchoring structure for the two anterior appendages, the antennae; see Walossek & Müller 1990; Schulz 1976, for the head capsule of Ostracoda). The dual recognition of the presence of the ‘proximal endite’ and the stem as a basis for the rami rather than the coxa, it was then possible to re-interpret the traditional models of limb design and limb evolution in crustaceans and arthropods as a whole (e.g. Walossek 1993, 1999; Walossek & Müller 1990, 1997). The proximal endite, present in the two post-antennular limbs of feeding eucrustacean nauplii undergoes progressive transformation into a large, sclerotic portion proximal to the basis, the so-called ‘coxa’ (see, e.g., for the mandible of Rehbachiella kinnekullensis in Walossek 1993, fig. 17 ). Yet this sclerotization of the proximal endite occurs only in certain limbs and not necessarily includes the formation of a median “grinding plate”. In Crustacea such coxal structures occur only: • in the second antenna and mandible at N.N. 1 level (Figure 2) and are retained in the Eucrustacea; • in the maxillula and in the first eight thoracopods of Malacostraca (no more feeding armature in any Eumalacostraca); but: • not in the first and second maxillae in the ground pattern of Entomostraca; • not in the thoracopods in the ground pattern of Entomostraca (see for the trunk limb of Rehbachiella kinnekullensis in Walossek 1993, fig. 27 ); • most likely not in the second maxilla in the ground pattern of Malacostraca. Notably, we were unable to detect any coxal or endite portion in any of the malacostracan pleopods (März unpublished diploma work). Nearly a century ago Calman (1909: p. 51) had already hinted to the presence of this separate proximal endite, but regarded it as a ”primitive” trait of Branchiopoda. Transformed to current terminology, it implies that branchiopodan thoracopods – and this can be expanded to basically all entomostracans – did not modify the proximal endite into a coxa but that they retained its plesiomorphic shape; that is, such a limb consists only of a basis and its proximal endite plus the two rami. In fact, this limb type became modified in another direction by considerable elongation of the basis and by the subdivision of its inner rim into a set of small setiferous, and basically even movable endites (see Walossek 1999, fig. 9), as can be seen in Cephalocarida, Branchiopoda, and the ‘Orsten’ maxillopods Bredocaris admirabilis and Dala peilertae Müller, 1983 (for a reconstruction see Walossek & Müller 1998). Both the phosphatocopines and the eucrustaceans have the antenna and mandible a coxa and basis drawn out medially into a spine-bearing protrusion (sometimes called gnathite). Even today the antenna, and not the mandible, plays the major role in the food intake of a feeding nauplius, such as in barnacles or copepods. Within the Eucrustacea the antenna has become modified into a large variety of different forms and to achieve many different functions, and, furthermore, the mandible also underwent

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transformation by enhancing its coxal portion as the major organ of food destruction, while reducing the ”palp” (= basis + rami). In the entomostracans, this process became completed at the adult state, with the exception of the most possibly paedomorphic Maxillopoda. In contrast in the Malacostraca the ”palp” is retained as a basically tripartite setiferous ramus during later larval development (possibly for cleaning). Olesen & Walossek (2000) demonstrate that in the investigated Nebalia species (Phyllocarida, Malacostraca) the palp comprises the basis and a two-segmented endopod, therefore this condition is considered to be plesiomorphic with respect to that in Entomostraca. This single feature can serve well to characterize the sister-group relationship of Malacostraca and Entomostraca (Fig. 2). Our hypothesis of the evolution of arthropod appendage types (Fig. 4), is based on the limb morphology from Cambrian fossils and all Recent groups. The terminology used and the standardized color scheme applied facilitates the easy recognition and homology of the different limb portions and their modifications in the various crustacean and other arthropod groups. It must be emphasized that, when applying this scheme it must be tested against each individual limb, and that there is no “crustacean limb”. This scheme abandons the idea of a ”pre-coxa” in any arthropod limb since if there is no

Fig. 4. Arthropod limb evolution and modifications towards the Crustacea. Abbreviations: bas, basis, cox, coxa; en, endopod; ex, exopod; stem, limb stem; number refer to podomeres (modified from several sources, such as Hou & Bergström 1998; Walossek 1993, 1999).

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coxa originally, there cannot be a pre-coxa either (for discussion of misidentifications of the pre-coxa see Walossek & Müller 1997). The terminology of Boxshall (1997), reintroducing pre-coxa refers to a specific model of subdivision of the copepodan maxillula and was not intended to address to the ground pattern of Crustacea or even Arthropoda (Boxshall pers. communication 1998). With the change of features around the mouth (labrum, setulae, sternum, paragnaths, etc.) the limbs also became modified to take on new functions in the locomotory and feeding system along the evolving crustacean lineage. Lastly, food brought in by means of the antennae and mandibles, and assisted by the antennulae, and/or by the trunk limbs, was transported into the vicinity of the mouth by the maxillulae and finally by the coxal grinding plates of the mandibles. The functionality of the mandibular coxae becomes supported by the sloping paragnath humps, the carpet of fine hairs on the sternum and at the flanks of the labrum and the atrium oris. Further aids developed in this context are slime glands and proprioreceptors at the rear of the labrum (Fig. 5; all are apomorphies of N.N. 1, see Fig. 2, see also Maas et al. 2003). All this demonstrates the high priority placed on efficient food gathering, leading to these significant changes particularly in the head region of crustaceans. On the other hand, the limbs around the head-trunk border, the maxillulae and maxillae, are of importance for evaluating the divergence of the major lineages of the Crustacea. Only the maxillula becomes modified to a “mouth part” at the level of Eucrustacea, while the maxilla retains its trunk-limb design for much longer, any further modifications occurring well within the different lineages. Apart from the living Cephalocarida, also ‘Orsten’ fossils such as Bredocaris admirabilis, Rehbachiella kinnekullensis and Dala peilertae depict this plesiomorphic organizational level (cf. Walossek 1993), while the Skaracarida have specialized maxillulae, maxillae and maxillipeds forming a complex cephalo-thoracic

Fig. 5. Evolutionary changes in the locomotory and feeding system. Left: Euarthropod level – post-antennular limbs similar, basis shovels food toward the mouth, locomotion and feeding coupled. Middle: Stemlineage level of Eucrustacea - antennula involved in locomotion and feeding, post-antennular limbs with a proximal endite for proximal food manipulation. Right: eucrustacean level – antenna and mandible with coxa and basis and annulated exopods, labrum at rear of hypostome, with slime glands and chemoreceptors, mouth recessed in atrium oris, post-oral sternites fused to a single sternum, maxillula modified to a further mouthpart (still not specialized at N.N. 1 level, otherwise similar). Abbreviations: a1, antennula; hyp, hypostome; la; labrum; md cox, coxa of mandible; mo mouth; pe, proximal endite; numbers refer to limb-bearing head segments; stippled line refers to progressive change in head-segment equipment in the crustacean lineage (from Walossek 1999).

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feeding and locomotory apparatus. This is one of the characters shared with Mystacocarida and Copepoda, considered as synapomorphic of these groups (Walossek & Müller 1998). Concluding remarks This short review, as the written form of my lecture at the Athens conference is my personal and ‘Orsten’-based view of arthropod and crustacean phylogeny and likely one that touches only a fraction of the evidence. Nevertheless, the ‘Orsten’ arthropods, with their large and detailed data set, demonstrates clearly the potential of exceptionally well preserved fossils and, thus, the contribution of palaeontology to phylogenetic analysis. Students in the field should use these data obtained from existing animals as a toolbox for further analyses of structural details, since it permits a clearer recognition of structures in other fossil and facilitates comparative morphology and phylogenetic analysis. The reconstruction of ground patterns is an essential part of any phylogenetic reconstruction (see also Scholtz 1997). Such a ground pattern, however, cannot be a kind of paper model, but should include the whole set of features of organisms at a certain evolutionary level, that is, it combines plesiomorphies as well as apomorphies. Such methodology view is demonstrated in the reconstruction of the evolutionary path within the Crustacea, in which structures are successively added and modified in response to an enhanced feeding and locomotory system mainly of the head. A key feature in this scenario is the specific change on every limb along the sequence. This differentiated view, possible only after detailed investigations of these fossils, renders claims by Lauterbach (e.g., 1988) and his followers that the ‘Orsten’ fossils as a whole are nothing but a bunch of stem-line mandibulates useless. As claimed earlier, there is neither a general coxa-basis subdivision in any postantennular limb at the stem-lineage level of the Crustacea, as traditionally postulated, and this hold only for the antenna and mandible at the level of N.N. 1 (Fig. 2). Even in the living forms there is simply no group that has coxae on all post-antennular limbs, though it is clear that subdivisions of the limb stems can occur due to special functional needs (e.g., malacostracans throughout, on maxillula and thoracopods 1-8; copepods on maxillula and swimming limbs cf. Boxshall 1997; notostracan Branchiopoda on the anterior 11 trunk limbs; see, e.g., Walossek 1993). With regard to the timing of the appearance of Crustacea in the history of life, it is noteworthy that the differentiated investigations of the Cambrian ‘Orsten’ forms demonstrated that there are ones that share characters with particular eucrustacean groups, such as Maxillopoda and Branchiopoda. This implies that, although they are hitherto not recorded (according to Hou & Bergström 1997 no crustaceans have been found in the Chengjiang faunas so far), by the Upper Cambrian the other eucrustacean groups, e.g. Malacostraca and Cephalocarida, must also have cooccurred. Moreover, the findings of phosphatocopine specimens from the Early Cambrian (Hinz 1987, Siveter et al. 2001, 2003), almost as early as the earliest record of arthropods, demonstrates that the evolutionary lineage of the Crustacea must have started considerably before the Atdabanian - to my view even before in the

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Pre-Cambrian. From this we can estimate that all further, earlier branchings of gastroneuralian metazoans occurred even before that of Crustacea. In the light of the paucity of records of such well preserved fossils, however, our view on early crustacean evolution remains still limited, particularly with regard to the knowledge of the early lineages of Recent taxa. Nevertheless I predict that our knowledge of Cambrian crustacean diversity will grow and that representatives of other taxa will be uncovered once people start to investigate more suitable environments and rock types, particularly of the ‘Orsten’ type. Acknowledgements My gratitude goes to the organizers of this conference and, particularly, to Jan Bergström, Stockholm, for inviting me to Athens and for reviewing an early draft of this paper. Geoffrey A. Boxshall, London and Andreas Maas, Ulm, gave valuable comments, Greg Edgecombe, Sidney, and David Siveter, Leicester, kindly reviewed the manuscript and improved the language. The ‘Orsten´ project, under the leadership of K.J. Müller, Bonn, has been continuously supported by the Deutsche Forschungsgemeinschaft until 1995 and now is continued in Ulm, again supported by a grant from the D.F.G. for the Phosphatocopina project. All ‘Orsten’ material belongs to the Institute of Palaeontology, Bonn, but is currently kept at the University of Ulm for research. References Abzhanov A., Popadic A. & Kaufman T.C. 1999. Chelicerate Hox genes and the homology of arthropod segments. Evolution & Development 1(2): 77-89. Abzhanov A. & Kaufman T.C. 2000. Embryonic expression of the Hox genes of the crayfish Procambarus clarkii (Crustacea, Decapoda). Evolution & Development 2(5): 271-283. Andres D. 1989. Phosphatisierte Fossilien aus dem unteren Ordoviz von Südschweden. Berliner geowissenschaftliche Abhandlungen (A) 10: 9-19. Boxshall, G.A. 1998. 13. Comparative limb morphology in major crustacean groups: the coxabasis joint in postmandibular limbs. In: Fortey R.A. & Thomas R.H. (eds), Arthropod Relationships. Systematics Association Special Volume 55. Chapman & Hall, London,pp. 156-167. Calman C. 1909. Part VII. Appendiculata. 3rd. vol. Crustacea. In: Lankester R. (ed), A Treatise on Zoology. Adam & Charles Black, London. 346 pp. Cannon H.G. 1922. On the labral glands of a cladoceran (Simocephalus vetulus), with a description of its mode of feeding. Quarterly Journal of Microscopical Science 66: 213-234. Cisne J.L. 1975. Anatomy of Triarthrus and the relationships of the Trilobita. Fossils and Strata 4: 45-63. Dahms H. 2000. Phylogenetic implications of the Crustacean nauplius. Hydrobiologia 417: 91-99. Dewel R., Budd G., Castano D.F. & Dewel W.C. 1999. The Organization of the Subesophageal Nervous System in Tardigrades: Insides into the Evolution of the Arthropod Hypostome and Tritocerebrum. Zoologischer Anzeiger 238: 191-203. Hinz I. 1987. The Lower Cambrian microfauna of Comley and Rushton, Shropshire/England. Palaeontographica A 198(1-3): 41-100.

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Hinz-Schallreuter I. 1998. Population structure, life strategies and systematics of phosphatocope ostracods from the Middle Cambrian of Bornholm. Mitteilungen aus dem Museum für Naturkunde Berlin, Geowissenschaftliche Reihe 1: 103-134. Hou Xianguang 1987. Three new large arthropods from the Lower Cambrian Chengjiang, Eastern Yunnan. Acta Palaeontologica Sinica 26(3), 272-285. Hou Xianguang & Bergström J. 1997. Arthropods of the Lower Cambrian Chengjiang fauna, southwest China. Fossils and Strata 45: 1-116. Hou Xianguang & Bergström J. 1998. Chapter 4: Chengjiang arthropods and their bearing on early arthropod evolution. In: Edgecombe G.D. (ed), Arthropod Fossils and Phylogeny. Columbia University Press, New York, pp. 151-184. Lauterbach K.-E. 1988. Zur Position angeblicher Crustacea aus dem Ober-Kambrium im Phylogentischen System der Mandibulata (Arthropoda). Verhandlungen des naturwissenschaftlichen Verlags in Hamburg (NF) 30: 409-467. Maas A., Waloszek, D. & Müller, K.J. 2003. Morphology, Ontogeny and Phylogeny of the Phosphatocopina (Crustacea) from the Upper Cambrian ‘Orsten’ of Sweden. Fossils and Strata 50: 1-200. Müller K.J. 1964. Ostracoda (Bradorina) mit phosphatischen Gehäusen aus dem Oberkambrium von Schweden. Neues Jahrbuch der Geologie und Paläontologie, Abhandlungen 121(1): 1-46. Müller K.J. 1979. Phosphatocopine ostracodes with preserved appendages from the Upper Cambrian of Sweden. Lethaia 12: 1-27. Müller K.J. 1982. Hesslandona unisulcata sp.nov. with phosphatised appendages from Upper Cambrian ‘Orsten’ of Sweden. In: Bate R.H., Robinson E. & Sheppard L.M. (eds), Fossil and Recent Ostracods. Ellis Horwood, Chichester, pp. 276-304. Müller K.J. 1983. Crustacea with preserved soft parts from the Upper Cambrian of Sweden. Lethaia 16: 93-109. Müller K.J. 1985. Exceptional preservation in calcareous nodules. Philosophical Transactions of the Royal Society of London B 311: 67-73. Müller K.J. 1990. 3.11.3. Upper Cambrian ‘Orsten’. In: Briggs D.E.G. & Crowther, P.R. (eds), Palaeobiology, a synthesis. Blackwell Scientific Publications, Oxford, London, Edinburgh, Boston, Melbourne, pp. 274-277. Müller K.J., & Walossek D. 1985. Skaracarida, a new order of Crustacea from the Upper Cambrian of Västergötland, Sweden. Fossils and Strata 17: 1-65. Müller K.J. & Walossek D. 1986a. Martinssonia elongata gen. et sp. n., a crustacean-like euarthropod from the Upper Cambrian ‘Orsten’ of Sweden. Zoologica Scripta 15(1): 73-92. Müller K.J. & Walossek D. 1986b. Arthropod larvae from the Upper Cambrian of Sweden. Transactions of the Royal Society of Edinburgh: Earth Sciences 77: 157-179. Müller K.J. & Walossek D. 1987. Morphology, ontogeny and life habit of Agnostus pisiformis from the Upper Cambrian of Sweden. Fossils and Strata 19: 1-124; pls. 1-33. Müller K.J. & Walossek D. 1988. External morphology and larval development of the Upper Cambrian maxillopod Bredocaris admirabilis. Fossils & Strata 23: 1-70. Müller K.J. & Walossek D. 1991. Ein Blick durch das -Fenster in die Arthropodenwelt vor 500 Millionen Jahren. Verhandlungen der Deutschen Zoologischen Gesellschaft 84: 281-294. Müller K.J., Walossek D. & Zakharov A. 1995. “Orsten” type phosphatized soft-integument preservation and a new record from the Middle Cambrian Kuonamka Formation in Siberia. Neues Jahrbuch für Geologie und Paläontologie Abhandlungen 197(1): 101-118.

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Olesen J. 1999. Larval and post-larval development of the branchiopod clam shrimp Cyclestheria hislopi (Barird, 1859) (Crustacea, Branchiopoda, Conchostraca, Spinicaudata). Acta Zoologica (Stockholm) 80: 163-184. Olesen J. & Walossek D.: Limb ontogeny and trunk segmentation in Nebalia species (Crustacea, Malacostraca, Leptostraca). Zoomorphology 120: 47-64. Popadic A. Rusch D., Peterson M., Rogers B.T. & Kaufman T.C. 1996. Origin of the mandible. Nature 380: 395. Sanders H.L. 1963. The Cephalocarida. Functional Morphology, Larval Development, Comparative External Anatomy. Memoirs of the Connecticut Academy of Arts & Sciences 15: 1-80. Scholtz G. 1997. 24. Cleavage, germ band formation and head segmentation: the ground pattern of the Euarthropoda. In: Fortey, R. A. & Thomas, R. H. (eds), Arthropod Relationships, Systematics Association Special Series Volume 55. Chapman & Hall, London, pp. 317-332. Scholtz G., Mittmann B. & Gerberding M. 1998. The pattern of distal-less expression in the mouthparts of crustaceans, myriapods and insects: new evidence for a gnathobasic mandible and the common origin of Mandibulata. Journal of Developmental Biology 42(6): 801-810. [Schulz, K. 1976. Das Chitinskelett der Podocopida (Ostracoda, Crustacea) und die Frage der Metamerie dieser Gruppe. Unpublished thesis, University of Hamburg: 1-167.] Siveter D.J., Williams M. & Waloszek D. 2001. A Phosphatocopid Crustacean with Appendages from the Lower Cambrian. Science 293: 479-481. Siveter D.J., Waloszek D. & Williams M. 2003. An early Cambrian phosphatocopid crustacean with three-dimensionally preserved soft-parts from Shropshire, England. In Lane P.D., Fortey R.A. & Siveter D.J. (eds), Proceedings of the Third International Symposium on Trilobites and their relatives. Special papers in Palaeontology. Published by the Palaeontological Association. Wägele W. 2000. Grundlagen der phylogenetischen Systematik. Pfeil, München: 1-315. Walossek D. 1993. The Upper Cambrian Rehbachiella kinnekullensis and the phylogeny of Branchiopoda and Crustacea. Fossils and Strata 32: 1-202. Walossek D. 1999: On the Cambrian diversity of Crustacea. In: Schram F.R. & von Vaupel Klein J.C. (eds), Crustaceans and the Biodiversity Crisis, Proceedings of the 4th International Crustacean Congress, Amsterdam, The Netherlands, July 20-24, 1998 vol. 1. Brill Academic Publishers, Leiden, pp. 3-27. Waloszek, D. and Dunlop, J. 2002: A larval sea spider (Arthropoda: Pycnogonida) from the Upper Cambrian ‘Orsten’ of Sweden, and the phylogenetic position of pycnogonids. - Palaeontology 45(3): 421-446. Walossek D. & Müller K.J. 1990. Stem-lineage crustaceans from the Upper Cambrian of Sweden and their bearing upon the position of Agnostus. Lethaia 23(4): 409-427. Walossek D. & Müller K.J. 1997: Cambrian ‘Orsten´-type arthropods and the phylogeny of Crustacea. In: Fortey R.R. & Thomas R. (eds), Arthropod relationships, Systematics Association Special Volume 55. Chapman & Hall, London, pp. 139-153. Walossek D. & Müller K.J. 1998: Early Arthropod Phylogeny in the Light of the Cambrian ‘Orsten´ Fossils. In: Edgecombe G. (ed-in-chief): Arthropod Fossils and Phylogeny. Columbia University Press, New York, pp. 185-231.

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Weitschat W. 1986: Phosphatisierte Ammonideen aus der Mittleren Trias von Central-Spitzbergen. Mitteilungen aus dem Geologisch-Paläontologischen Institut der Universität Hamburg 61: 249-279. Zaffagnini F. & Zeni C. 1987. Ultrastructural Investigations on the Labral Glands of Daphnia obtusa (Crustacea, Cladocera). Journal of Morphology 193: 23-33.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Cambrian arthropods: a lesson inThe convergent evolution 89 New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 89-96, 2003

Cambrian arthropods: a lesson in convergent evolution J. Bergström1 & Hou Xianguang2 1. Swedish Museum of Natural History, Stockholm, Sweden 2. Research Center for Chengjiang Biota, Yunnan University, Kunming, China

Abstract Convergent or parallel evolution is very common, perhaps much more so than generally understood. The primary radiation of a new group of organisms commonly involves the origination of numerous lineages which from the start are fairly similar to one another, but soon develop their specific features. In Cambrian arthropods parallel trends include dorsal migration of the eyes, formation of a head by the addition of post-antennal segments, formation of a bivalved carapace, and abandoning of mud-engulfing habits with the specialisation of head appendages. It is notable that evolution has often chosen slightly different solutions to achieve the same result.

The Cambrian radiation of arthropods has been intensely studied since the find of the Burgess Shale fauna more than a century ago. Much more data has accumulated after the find of the Chengjiang fauna in 1984 (see Hou & Bergström 1997). The Burgess Shale is Middle Cambrian in age, the Chengjiang fauna Early Cambrian. The latter is thus older and brings us deeper into the center of radiation. It is also preserved with more contrast in colour and relief. A combination of evidence from the two therefore means that, for instance, morphological interpretations gain in accuracy. Below follows an overview of a few cases of parallel evolution of particular features in the early arthropods. For details the reader is referred to the description of the Chengjiang arthropod fauna by Hou & Bergström (1997, with references). The case of the dorsal migration of compound eyes One character that is prone to a directed evolutionary trend is the position of the paired eyes. They were very commonly anterior in position in the oldest arthropods, placed beneath the edge of the head shield. With time other solutions become more common, but an anterior position is still found in many crustaceans. In Fig. 1 there is an emphasis on the derived states, which means that there may be a false impression of a rarity of the supposed plesiomorphic state.

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In the lineage leading to the crustaceans, we meet the (supposed) plesiomorphic state in ‘stem-group crustaceans’ such as the Cambrian Henningsmoenicaris (Fig. 1; Walossek & Müller 1990). We meet it also in the anostracans, which belong to the basic crustacean group of branchiopods. In another branchiopod group, the Notostraca, the eyes in principle are ventral, but in fact are situated in a deep pocket penetrating the head so that they look upwards through the dorsal carapace. The maxillopod groups commonly have lost their paired eyes, but they appear to be present in their plesiomorphic position in the Cambrian Bredocaris (Müller & Walossek 1988). The maxillopodan ostracodes have often lost their compound eyes, but when preserved they look through the bivalved carapace (e.g. Cypridina). Most subgroups of the advanced crustacean subgroup of Malacostraca retain the plesiomorphic state. The eyes have moved up onto the dorsal shield in isopods (e.g. Serolis) and amphipods (e.g. Gammarus). These two groups are generally considered not to have a shared origin, and the eye condition thus must have been reached independently. Thus at least four groups of crustaceans have moved from the plesiomorphic state of eye position and now have their eyes looking upwards, and have done so in three quite different ways. In trilobite-like arthropods it is usually possible to state the presence of compound eyes only when they are placed on the carapace, since the ventral side is rarely preserved. However, new well-preserved material from the Lower Cambrian Chengjiang fauna in China has given us new glimpses into the ventral side. Thus, for instance, Retifacies lacked dorsal eyes, but had stalked ventral eyes emerging from the ventral side (Hou & Bergström 1997, Figs 48A, 50, 51). Another genus, Xandarella, had its compound eyes on the dorsal side, but an open cleft extending from the lateral margin of the shield to the eye indicates the possibility that the eye had migrated in from the side and ultimately from the underside (Hou & Bergström 1997, Figs 68D-E, 69-73). Another genus, Cindarella, provides strong evidence that this interpretation is correct (Ramsköld et al. 1997). Cindarella is quite close to Xandarella, sharing general morphologic features as well as a unique arrangement with posterior tergites covering more than one segment. It differs mainly in having stalked eyes which extend from the ventral side to look out beyond the margin of the carapace. Cindarella – Xandarella thus provide us with an example of how the eyes could move from the ventral to the dorsal surface. In Sidneyia the eye has a position somewhat intermediate to that of Cindarella and Xandarella, but still somewhat different in that it is between the head shield and the succeeding tergite. The head in this genus is exceptionally short, including the antennal segment but no leg segments. These examples demonstrate that migration of the eyes towards a dorsal position took place in trilobite-like arthropods as well as in the crustacean group. Since the former have usually a wide carapace extending laterally, it is easy to understand the need to move the eyes from a ventral surface with very limited view possibilities. We thus find dorsal eyes in a number of additional trilobite-like groups (Fig. 1). One example is Skioldia. In this genus the eyes are fairly close to the anterior margin. It is therefore tempting to see the position as the result of migration from a position similar to that in Retifacies, rather than from a more lateral position such as in the Cindarella – Xandarella sequence. In virtually all trilobite-like groups and in the chelicerates the end result of the eye

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Fig. 1. Evolutionary migration of the compound eyes from under the margin of the head shield to a dorsal position (to the right of the dotted line). This happened along different paths in different groups. The eyes appear commonly to have migrated over the shield margin, as can be seen in the closely related Cindarella and Xandarella. In the crustacean notostracans the eyes are in a ‘pocket’ invaginated from the ventral side, and in the ostracodes (Cypridina) the eyes look through the carapace fold. The eyes generally look perpendicularly through the shield, but in trilobites (Triarthrus) a fissure in the shield opened up for a horisontal view. A problem is that many Cambrian arthropods lacking dorsal eyes are considered as blind because the underside is unknown. The left side is therefore under-represented by examples.

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migration is an eye looking perpendicularly through the carapace surface. The only exception is the trilobites (e.g., Triarthrus), in which the eye looked horisontally through a slit in the carapace. This was modified in many advanced trilobites, but is certainly characteristic of early forms. We can therefore state that there was a very strong trend among early arthropods to shift the position of the compound eye from a position beneath the margin of the head shield to one on the shield, or in cases to one where the eye was looking through the shield. This trend was strongest in the more flattened arthropods. The case of head formation There are reasons to believe that the head in the shared ancestor included only the antennal segment in addition to the presegmental acron because in many Cambrian arthropods the antenna is the only specialised appendage; such a head is indeed preserved in Sidneyia. From this state there was an accretion of segments to the head in a large number of small groups. It is notable that, for instance, the lamellipedians do not share a single number of head segments, but there is 1, 2, 3, 4, 5 and 6 additional pairs in different groups, in addition to groups where there is a shield covering the entire animal and no specific secondary head developed. Since there are much more than 5 subgroups of lamellipedians, and many other arthropod groups, it is evident that the different head sizes have been reached independently in different groups. The ‘mean’ number of about 3-4 postantennal segments shared by any two groups thus cannot in itself be used as evidence of a shared origin. It is also clear that there is no phylogenetic sequence with addition of segments one by one, since in each group the addition appears to have occurred all at once, or at least more or less so. Thus, for instance, Sidneyia may well be close to chelicerates, although the former lacks post-antennal segments in the head, and chelicerates have five to six (and have lost the antenna). At the same time there is no evidence for a close relationship between chelicerates and Xandarella, although the latter has six (or seven, depending on how a dwarfed segment is counted) post-antennal segments in the head. The case of the bivalved carapace Another case of parallel evolution in arthropods is the formation of a bivalved carapace. In talking about a carapace, we have to leave the formal definition behind, since it is uncertain if there is any carapace that fulfils the formal requirements (i.e., that it is a fold from the maxillary segment). Here it is thus considered to be a fold in the anterior part of the animal, whatever segment(s) it comes from. Crustaceans have either an undivided shield or carapace or a bivalved carapace. The latter was developed in some ‘stem-group crustaceans (e.g. Hesslandona, Waptia (Chuandianella), see Fig. 2) and developed twice in the branchiopods, namely in conchostracans (e.g. Laevicaudata in Fig. 2) and the extinct protocaridids (Protocaris, Branchiocaris and other Cambrian genera). Among maxillopod groups, a bivalved carapace evolved in the ostracodes (e.g. Cypridina). Most malacostracan groups (not isopods and amphipods) have a carapace. It is bivalved only in phyllocarids (e.g. Paranebalia), where there is a complication in the

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Fig. 2. Arthropods may have a head shield without a fold, or a folded carapace. In the latter case the carapace may be bivalved, that is, separated into left and right halves (periphery outside the dotted line). This has happened repeatedly in a number of different groups and in somewhat different ways. Arthropods known to lack a bivalved carapace are greatly under-represented in the figure. The lack of illustrations in the central part is therefore not because of lack of information.

shape of a median rostrum and, in some fossil forms, an additional median plate. It appears clear that the bivalved condition has evolved at least four times in the crustaceans and presumably at least twice in ‘stem-line crustaceans’. A bilobed carapace is not seen in any trilobite-like arthropod, but it does occur in a number of Cambrian forms of more or less unknown affinities. Some of these groups,

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represented in Fig. 2 by Isoxys, Kunmingella and Tuzoia, are ostracode-like forms with the entire body shielded by the carapace. Others, such as Vetulicola, Occacaris and Canadaspis, have an abdomen protruding posteriorly. Of the latter, Vetulicola has lateral fissures in the carapace, and the two valves appear to be united ventrally. Its abdomen is hinged to the carapace. This appears also to be the case in Occacaris. The construction of the biramous limb, where known, shows that these forms are mostly not closely related. In addition, the hinge between carapace and abdomen in Vetulicola, and the lateral fissures and probable ventral fusion is also alien to at least most other arthropods. Again there is reason to believe that a bivalved carapace came into being several times also in this ”group” of arthropods. The case of the loss of mud-engulfing habits The find of the Chengjiang fauna in the Lower Cambrian of China has directed our attention to the food and feeding manners of the oldest arthropods. Perhaps the greatest surprise was that a high proportion of them have the gut clearly filled with mud (Hou & Bergström 1997, pp. 101, 111). Previously, based on evidence only from the Burgess Shale, where the situation is less obvious, Briggs & Whittington (1985, p. 152) had considered the possibility but hesitated to believe in it. As a rule, these species lack specialisation of the appendages of the head (with the exception of the antenna). On the other hand, species with specialised grasping appendages of the head as a rule do not have mud in the gut. In several cases we can instead recognise a dark staining and/or formation of apatite (e.g. Briggs & Whittington 1985). In some cases there are even recogniseable fragments of animal shells or skeletons in the gut (Sidneyia, Utahcaris). It therefore appears evident that many Cambrian arthropods were mud-engulfers, whereas many others selected organic food. The lamellipedians (or trilobitomorphs) include many members with apparently mudengulfing habits (Naraoia, Retifacies, Squamacula, Xandarella, Almenia, Sinoburius, and trinucleids among trilobites), but also many others with a selected organic food (Marrella, Kuamaia, Emeraldella, Sidneyia, Emeraldella, most(?) trilobites). Some forms which may be stem-group crustaceans (Plenocaris, Clypecaris) were mud-engulfers, whereas another (Chuandianella) was not. Supposed crustaceans (Odaraia, Pectocaris) show no mud filling. Several members of a wide array of early off-shoots are found with mud-filled guts (Vetulicola, Canadaspis (at least one species), Perspicaris, Molaria, Burgessia, Chuandianella), whereas others show evidence of selected feeding (Utahcaris, Fortiforceps, Sanctacaris, Leanchoilia, Yohoia). Fuxianhuia, with grasping appendages, is seen with both dark grains and mud and appears to have had a mixed diet. Mud-engulfing appears to be the plesiomorphic state. Evidence to this end is the combination of mud-engulfing with a lack of leg specialisation, and also the distinct evolutionary trend from the common occurrence of mud-engulfing in the Cambrian to the virtual absence of such habits in younger arthropods. The presence of both mudengulfing and selected feeding in the different main groups is strong indication that evolution resulted in more advanced habits on a broad front, with numerous cases of parallelism.

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The case of leg segment reduction Legs of some early Cambrian arthropods have a comparably large number of podomeres or segments, all of which are very similar to one another. Thus Fuxianhuia has some 20 segments, Fortiforceps around 15. Canadaspis, sometimes regarded as a malacostracan crustacean but with some decidedly primitive features, had around 13 segments. Modern land arthropods have a much lower number, and the podomeres are individually unique in shape. Again there is considerable reason to believe in parallel evolution on a broad front. Convergent arthropodization? The Cambrian anomalocaridids share some features with arthropods, and there is a tendency to regard them as related, and even the latter as derived from the former (e.g. Budd 1998). However, as we have seen above, some “typical” arthropod features such as differentiation of mouthparts, loss of mud-eating habits, and dorsal position of eyes were not yet present in the first arthropods, but were there in the supposed anomalocaridid ancestors. The latter also show a stable number of body segments, quite unlike the situation in early arthropods. On the other hand, it is also difficult to derive anomalocaridids from arthropods, since the latter are more advanced in leg segmentation and in the development of dorsal skeletal plates. It is much more parsimonious to derive both from a shared ancestor with initial segmentation. The situation with multi-leg body “segments” in fuxianhiids and xandarellids indicates that segmentation in the earliest arthropods was perhaps not yet fully developed in the earliest arthropods (see Hou & Bergström 1979, Figs 7, 9A, F,11C-D, 12-14, 68-73, and Ramsköld et al. 1997). A derivation even from unrelated sources therefore appears equally possible. Conclusion The general conclusion to be drawn from these and many other examples is that convergence, or parallel evolution, is not exceptional but the general rule. Organisms have an amazing capacity to find similar or the same solution to the same problem over and over again. For those who believe that a similarity, or identical character state, is a synapomorphy until the opposite is proven, this should be a mementum. The uncertainty of arthropod origins means that there is the problem to identify morphological and other features of the ancestral arthropod. However, we can make certain judgements from evolutionary trends. Some trilobites had an impressive serial similarity with only two appendage morphologies, antennae and legs. A crayfish has 18 differently shaped appendage pairs. There can also be a great simplification. In both cases evolution obviously resulted in a more complicated function of the homeobox system. Identical serial similarity is obviously primitive, even when the individual segment may appear complicated. This holds true for body segments as well as for limb segments (podomeres). This is why body limbs in Fuxianhuia must be considered primitive, even if there is specialisation in the head. This is also why a trilobite, or Naraoia, is more primitive in its leg formation (and homeobox system) than any anomalocaridid.

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It also appears that such a primitive morphology is associated with mud-engulfing, which in itself is judged to be primitive since it is common only in the Cambrian. References BRIGGS D.E.G. & H.B. WHITTINGTON 1985. Modes of life of arthropods from the Burgess Shale, British Columbia. Trans. R. Soc. Edinburgh: Earth Sciences 76: 149-160. BUDD G. 1998. Stem group arthropods from the Lower Cambrian Sirius Passet fauna of North Greenland. In Fortey R.A. & R.H. Thomas (eds), Arthropod Relationships. Systematic Association Special Volume Series 55. Chapman & Hall, London, pp. 125-138. HOU X.-G. & J. BERGSTRÖM 1997. Arthropods of the Lower Cambrian Chengjiang fauna, southwest China. Fossils and Strata 45: 1-116. MÜLLER K.J. & D. WALOSSEK 1988. External morphology and larval development of the Upper Cambrian maxillopod Bredocaris admirabilis. Fossils and Strata 23:1-70. RAMSKÖLD L., CHEN JUNYUAN, EDGECOMBE G.D. & ZHOU GUIQING 1997. Cindarella and the arachnate clade Xandarellida (Arthropoda, Early Cambrian) from China. Transactions of the Royal Society of Edinburgh: Earth Sciences 88: 19-38. SHU D.-G., CONWAY MORRIS S., HAN J., CHEN L., ZHANG X.-L., ZHANG Z.-F., LIU H.-Q., LI Y. & LIU J.-N. 2001: Primitive deuterostomes from the Chengjiang Lagerstätte (Lower Cambrian, China). Nature 414, 22 November 2001, pp. 419-424. WALOSSEK D. & K.J. MÜLLER 1990. Upper Cambrian stem-lineage crustaceans and their bearing upon the monophyletic origin of Crustacea and the position of Agnostus. Lethaia 23(4): 409-427.

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Molecular Macroevolution

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers The urmetazoa: Molecular biological studies with living fossils - Porifera 99 The New Panorama of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 99-104, 2003

The urmetazoa: Molecular biological studies with living fossils - Porifera W.E.G. Müller & I.M. Müller Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany

Abstract The origin of Metazoa remained – until recently – the most enigmatic of all phylogenetic problems. Sponges [Porifera] as “living fossils” are positioned at the base of the multicellular animals. During the last few years cDNAs/genes coding for informative proteins have been isolated and characterized from sponges, especially from the marine demosponges Suberites domuncula and Geodia cydonium. The analyses of their deduced amino acid sequences allowed a molecular biological resolution of the monophyly of Metazoa. Molecules of the extracellular matrix, cell-surface receptors, elements of nerve system/sensory cells, homologs/ modules of an immune system as well as morphogens (myotrophin) classify the Porifera as true Metazoa. The hypothetical ancestral animal, the URMETAZOA, from which the metazoan lineages diverged (more than 600 MYA) may have had the following characteristics: cell adhesion molecules with intracellular signal transduction pathways - morphogens/ growth factors forming gradients - a functional immune system - a primordial nerve cell/ receptor system. The structure and function of the molecules identified are evolutionary novelties and must have been present in the hypothetical ancestral Metazoa, the URMETAZOA; hence they are restricted to the metazoan phyla only.

The Sponges Multicellularity of Viridiplantae, Fungi and Metazoa arose in the Proterozoic ≈1,000 MYA. Focusing on the earliest Metazoa, the sponges, it is documented that the major poriferan taxa existed already since the Early Cambrian (Atdabatian) (reviewed in Mehl et al. 1998). Even though it might be argued that Porifera are not the first metazoan phylum which evolved, they are witnesses of an evolutionary step that occurred during the maturation of the Metazoa near the Proterozoic-Phanerozoic boundary, close to 1,000 MYA. In this respect they can be considered as living fossils (Müller 1998). In the last years our group has analyzed genes of sponges in order to obtain an insight into the genomic organization as well as the function of genes coding for functional proteins. In detail, genes from Demospongiae, Suberites domuncula and Geodia cydonium, from

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Calcarea, Sycon raphanus, as well as from Hexactinellida, Aphrocallistes vastus, have been analyzed also with the aim to describe the hypothetical ancestral Metazoa, the URMETAZOA. In earlier studies it was postulated that the level of evolution is correlated with DNA complexity, implying that increase in DNA complexity parallels with the number of different genes in a given organism. However, later it was realized that the correlation between genome size [C value] and “advancement” is poor. More reliable than DNA complexity as a reflection of complexity of a given organism is the number of genes. The genome size had been determined in the marine sponges Geodia cydonium and Suberites domuncula (Imsiecke et al. 1995). In both species the DNA content has been found to be 3.5 pg/cell, corresponding to a C value of 1,670,000 kb. The number of chromosomes is 32 in the diploid state [S. domuncula] (Imsiecke et al. 1995). The G. cydonium DNA was further characterized by density gradient centrifugation (BartmannLindholm et al. 1997). An extreme heterogeneity of the DNA composition was observed which mainly contains single copy DNA. The amount of fast re-associating repetitive DNA represents less than 10%. In an approach to understand these findings, the gene density in the sponge genome was estimated. The 7.2 kb long gene cluster in G. cydonium formed of three tyrosine kinase genes comprises 4.4 kb exon stretches and 2.8 kb of introns (Müller et al. 1999b). Considering other parts of the genome sequenced in G. cydonium a rough estimation reveals a value of 4,000 b/gene. This value would imply that G. cydonium contains ≈400,000 genes (1,700 Mb: 4,000 b/gene) and hence more than 4-times more than human (3,300 Mb: 50,000 b/gene) and 20-fold more than Caenorhabditis elegans (97 Mb: 5,000 b/gene). The question arises why sponges are apparently so rich in genes. One explanation might be that these animals “shuttle” domains within the genome, mediated by recombination processes perhaps driven by the process of crossing over during mitosis. Two examples for an unusual domain arrangement in sponges should be mentioned. In G. cydonium a cDNA was cloned, encoding a putative “multiadhesive protein” which comprises three interesting modules; (i) a fibronectin-, (ii) a scavenger receptor cysteinerich- and (iii) a short consensus repeat-module (Pahler et al. 1998). Recently, another cDNA was isolated from S. domuncula whose open reading frame comprises only two domains, the epidermal growth factor-like domain and the C2 domain. The evolutionary novelties in sponge protein Since the introduction of molecular biological methods, such as cloning of cDNAs coding for informative proteins, the monophyly of Metazoa – including the Porifera – could be established (Müller et al. 1994, Müller 1995). A vast number of evolutionary novelties has been identified in Metazoa. Metazoan cells are provided with the possibilities to interact with each other via highly developed and diverse receptor and signaling systems, mainly guided by kinases. In addition, they are provided with novel extracellular organic skeletal elements, with collagen as the most prominent, as well as with innovative immune molecules and morphogens. Examples for characteristic metazoan cell adhesion molecules identified already in sponges are: C-type lectin (to be published) or collagen (Diehl-Seifert et al. 1985, Schröder et al. 2000); receptors: integrins

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(Pancer et al. 1997, Wimmer et al. 1999a, 1999b), receptor tyrosine kinases, or sevenspanning G protein-linked receptors (Perovic et al. 1999). Potential sponge immune molecules have been identified which share high similarity/identity with molecules involved in the immune system in mammals (reviewed in Müller et al. 1999a). Experiments with the marine sponges G. cydonium and S. domuncula have been performed on tissue (auto- and allografting) as well as on a cellular level. Thereby elements of the innate immune system have been identified: molecules which have sequence similarity to macrophage-derived cytokine-like molecules (allograft inflammatory factor 1) or the cytokine-like molecule (glutathione peroxidase) (Kruse et al. 1999). The (2-5)A synthetase system has been studied in detail (Kuusksalu et al. 1995, 1998); the first invertebrate (25)A synthetase from the marine sponge G. cydonium was cloned (Wiens et al. 1999). Precursors of an adaptive immune system in sponges are also present, e.g. cytokinerelated molecules, or molecules that contain polymorphic immunoglobulin-like domains (Blumbach et al. 1999, Müller et al. 1999c). Until recently no conclusive evidence has been presented for the existence of neuronal-like elements in sponges. We succeeded to demonstrate that isolated cells from the sponge G. cydonium react to the excitatory amino acid glutamate with an increase in the concentration of intracellular calcium (Perovic et al. 1999). Furthermore, the cDNA for the sponge metabotropic glutamate receptor-like protein was cloned (Perovic et al. 1999). In addition, a morphogen was identified in S. domuncula, the myotrophin-like molecule, (Schröder et al. 2000). Besides these characteristic metazoan molecules, also proteins have been isolated from sponges which are apparently unique among the Metazoa: the potential ethylene-responsive protein (Krasko et al. 1999) and silicatein (Krasko et al. 2000). Conclusion - Sponges are reference animals for the urmetazoa With the establishment of the monophyly of all Metazoa it appears to be timely to formulate the common features of the metazoan ancestor. The major novelties that characterize the hypothetical ancestral animal of Metazoa, the Urmetazoa, are the following: extracellular matrix molecules, transmembrane adhesion molecules, G-protein linked transmembrane receptors, receptor tyrosine kinases, morphogens, neuronal receptor(s) and immune molecules. Taken together, the overwhelming molecular evidence indicates that during the transition to Metazoa a qualitative set of novel properties has emerged which were used as a repertoire for the evolution into the different metazoan phyla including sponges; these novelties are the characteristics of the metazoan animal ancestor, the Urmetazoa (Fig.1). A further outcome of the molecular biological studies with sponges is the indication that several molecules, found to be required only for multicellular organisms, are present both in Metazoa and also in Viridiplantae/Fungi, suggesting that the major kingdoms of multicellular organisms had a common ancestor, the Urmulticellularia. Acknowledgements Supported by the Deutsche Forschungsgemeinschaft [Mü 348/12-4 and Mü/14-1] and the International Human Frontier Science Program [RG-333/96-M].

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Fig. 1. Postulated evolution of URMETAZOA from a common ancestor of multicellular organisms, the URMULTICELLULARIA. It is proposed that from unicellular organisms (Schütze et al. 1999) the multicellular Viridiplantae/Fungi evolved which are provided with sophisticated signal transduction- and adhesion molecules. The invention of new proteins, novelties, including cell-cell/matrix-, signal transduction-, immune-, neuronal- and morphogenetic molecules, which are proposed to have been present in the hypothetical metazoan ancestor, the URMETAZOA, allowed the branching into the Porifera and later to the Radiata and the Urbilateria.

References Bartmann-Lindholm C., Geisert M., Güngerich U., Müller W.E.G. & D. Weinblum 1997. Nuclear DNA fractions with grossly different base ratios in the genome of the marine sponge Geodia cydonium. Progr. Colloid Polym. Sci. 107: 122-126. Blumbach B., Diehl-Seifert B., Seack J., Steffen R., Müller I.M. & W.E.G. Müller 1999. Cloning and expression of new receptors belonging to the immunoglobulin superfamily from the marine sponge Geodia cydonium. Immunogenetics 49: 751-763.

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DIEHL-SEIFERT B., KURELEC B., ZAHN R.K., DORN A., JERECEVIC B., UHLENBRUCK G. & W.E.G. MÜLLER 1985. Attachment of sponge cells to collagen substrata: effect of a collagen assembly factor. J. Cell Science 79: 271-285. IMSIECKE G., CUSTODIO M., BOROJEVIC R., STEFFEN R., MOUSTAFA M.A. & W.E.G. MÜLLER 1995. Genome size and chromosomes in marine sponges [Suberites domuncula, Geodia cydonium]. Cell Biol. Intern. 19: 995-1000. KRASKO A., SCHRÖDER H.C., PEROVIC S., STEFFEN R., KRUSE M., REICHERT W., MÜLLER I.M. & W.E.G. MÜLLER 1999. Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis. J. Biol. Chem. 274: 31524-31530. KRASKO A., LORENZ B., BATEL R., SCHRÖDER H.C., MÜLLER I.M. & W.E.G. MÜLLER 2000. Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin. Europ. J. Biochem. 267: 4878-4887. KRUSE M., STEFFEN R., BATEL R., MÜLLER I.M. & W.E.G. MÜLLER 1999. Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto- and allograft response in marine sponges. J. Cell Sci. 112: 4305-4313. KUUSKSALU A., PIHLAK A., MÜLLER W.E.G. & M. KELVE 1995. The (2-5)oligoadenylate synthetase is present in the lowest multicellular organisms, the marine sponges. Demonstration of the existence and identification of its reaction products. Eur. J. Biochem. 232: 351-357. KUUSKSALU A., SUBBI J., PEHK T., REINTAMM T., MÜLLER W.E.G. & M. KELVE 1998. (2'5')Oligoadenylate synthetase in marine sponges: Identification of its reaction products. Eur. J. Biochem. 257: 420-426. MEHL D., MÜLLER I. & W.E.G. MÜLLER 1998. Molecular biological and palaeontological evidence that Eumetazoa, including Porifera (sponges), are of monophyletic origin. In:Watanabe Y. & N. Fusetani (eds), Sponge Science - Multidisciplinary Perspectives. Springer-Verlag, Tokyo, pp. 133-156. MÜLLER W.E.G. 1995. Molecular phylogeny of Metazoa (animals): monophyletic origin. Naturwiss. 82: 321-329. MÜLLER W.E.G. 1998. Origin of Metazoa: sponges as living fossils. Naturwiss. 85: 11-25. MÜLLER W.E.G., BLUMBACH B. & I.M. MÜLLER 1999a. Evolution of the innate and adaptive immune systems: relationships between potential immune molecules in the lowest metazoan phylum [Porifera] and those in vertebrates. Transplantation 68: 1215-1227. MÜLLER W.E.G., KRUSE M., BLUMBACH B., SKOROKHOD A. & I.M. MÜLLER 1999b. Gene structure and function of the tyrosine kinase(s) in the marine sponge Geodia cydonium: An autapomorphic character of Metazoa. Gene 238: 179-193 MÜLLER W.E.G., MÜLLER I.M. & V. GAMULIN 1994. On the monophyletic evolution of the Metazoa. Brazil. J. Med. Biol. Res. 27: 2083-2096. MÜLLER W.E.G., PEROVIC S., WILKESMAN J., KRUSE M., MÜLLER I.M. & R. BATEL 1999c. Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s). DNA & Cell Biol. 18: 885-893. PAHLER S., BLUMBACH B., MÜLLER I. & W.E.G. MÜLLER 1998. A putative multiadhesive basal lamina protein from the marine sponge Geodia cydonium: cloning of the cDNA encoding a fibronectin-, an SRCR- as well as a complement control protein module. J. Exp. Zool. 282: 32-343. PANCER Z., KRUSE M., MÜLLER I. & W.E.G. MÜLLER 1997. On the origin of adhesion receptors of Metazoa: cloning of the integrin subunit cDNA from the sponge Geodia cydonium. Molec. Biol. Evol. 14: 391-398.

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PEROVIC S., PROKIC I., KRASKO A., MÜLLER I.M. W.E.G. & MÜLLER 1999. Origin of neuronal-like receptors in Metazoa: cloning of a metabotropic glutamate/GABA-like receptor from the marine sponge Geodia cydonium. Cell & Tissue Res. 296: 395-404. SCHRÖDER H.C., KRASKO A., BATEL R., SKOROKHOD A., PAHLER S., KRUSE M., MÜLLER I.M. & W.E.G. MÜLLER 2000. Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula. FASEB J. 14(13): 2022-2031. SCHÜTZE J., REIS CUSTODIO M., EFREMOVA S.M., MÜLLER I.M. & W.E.G. MÜLLER 1999. Evolutionary relationship of Metazoa within the eukaryotes based on molecular data from Porifera. Proc. Royal Society Lond. B 266: 63-73. WIENS M., KUUSKSALU A., KELVE M. & W.E.G. MÜLLER 1999. Origin of the interferon-inducible (2’-5’)oligoadenylate synthetases: cloning of the (2’-5’)oligoadenylate synthetase from the marine sponge Geodia cydonium. FEBS Letters 462: 12-18. WIMMER W., BLUMBACH B., DIEHL-SEIFERT B., KOZIOL C., BATEL R., STEFFEN R., MÜLLER I.M. & W.E.G. MÜLLER 1999a. Increased expression of integrin and receptor tyrosine kinase genes during autograft fusion in the sponge Geodia cydonium. Cell Adhesion. Commun. 7: 111-124. WIMMER W., PEROVIC S., KRUSE M., KRASKO A., BATEL R. & W.E.G. MÜLLER 1999b. Origin of the integrin-mediated signal transduction: functional studies with cell cultures from the sponge Suberites domuncula. Europ. J. Biochem. 260: 156-165.

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The Integrative Approach in Zoological Evolution

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Evolutionary dynamics of host plant range in the butterflyThe tribe ... Evolution 107 NewNymphalini Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 107-114, 2003

Evolutionary dynamics of host plant range in the butterfly tribe Nymphalini (Insecta, Lepidoptera, Nymphalidae) S. Nylin Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail: [email protected].

Abstract I outline results of research on host plant range performed with a focus on the unusually polyphagous comma butterfly Polygonia c-album and its relatives in the tribe Nymphalini, combining phylogenetic and experimental approaches. I present a hypothesis of phylogenetic relationships within the Nymphalini, and show that all taxa near the root of this tribe are specialists on the plant family Urticaceae and relatives. The host plant range in the lineage leading to P. c-album was evidently later broadened to include other plant families. Experimental attempts to establish newly hatched larvae on non-hosts used by related species suggest that ancestral host plants sometimes are retained as part of a “potential” host plant range of larvae, a fact which may explain both the conservatism and evolutionary dynamics in the host plant range of ovipositing females. Comparisons between species and populations in the tribe Nymphalini suggest that constraints on the gathering and processing of information are likely to be factors of general importance driving the evolution of specialization.

Introduction Most phytophagous insects are specialists, sometimes on a single plant species, sometimes on a set of related plants (Thompson 1994). In butterflies the typical pattern is that larvae of a given species feed on members of one plant family or a couple of closely related and/or chemically similar plant families (Ehrlich & Raven 1964). In addition, closely related species of insects often feed on closely related plant taxa (but are specialists on a subset of them), as is the case in the butterflies (Ehrlich & Raven 1964, Janz & Nylin 1998). Ehrlich & Raven (1964) used this pattern as evidence for their co-evolutionary scenario, where plants escape from herbivory when they evolve a new chemical defence and radiate into new species, whereupon an insect eventually breaks through this defence and radiates onto the new set of plant species. The general questions raised by these patterns have challenged researchers in the decades since the publication of this seminal paper: Why the high degree of specialization, when a broader range of

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host plants can increase realized fecundity and ensures that adequate resources are more often available for offspring? Why the evident bonds with a particular plant group, which seem to persist over long time scales (Ehrlich & Raven 1964, Janz & Nylin 1998, Nylin & Janz 1999, Janz et al. 2001)? Why the pattern of specialization on related, but not identical, plant taxa - suggesting a pattern of frequent host shifts followed by rapid specialization? Is co-evolution between insects and plants really involved in creating these patterns? Here I give an overview of some results from butterflies, addressing these questions through a combined comparative and experimental approach. The focal species The comma butterfly, Polygonia c-album (L.), is extremely polyphagous when compared to the great majority of butterflies. Females of the same population, such as the one in the Stockholm area of Sweden, may oviposit on plants in at least seven families – some of them only distantly related – and larvae can survive to adulthood on all of them. This exceptional host plant range provides a focus for our butterfly-plant studies (Nylin & Janz 1999, Janz et al. 2001). We can study the reasons for the exception to the rule of specificity, as well as causes of within-species variation in specificity. This may give insights into the evolution of specialization in phytophagous insects. The most preferred hosts belong to the order Urticales: stinging nettle (Urtica dioica), elm (Ulmus glabra) and hop (Humulus lupulus) in the families Urticaceae, Ulmaceae and Cannabidaceae, respectively. Slightly less preferred hosts are Salix caprea (Salicaceae) and a few closely related species of Salix, and Ribes spp. in the Grossulariaceae (currants and gooseberries). The least preferred hosts are in the Betulales: Corylus avellana (Corylaceae) and Betula pubescens (Betulaceae), but oviposition will take place also on these plants in the Swedish population (Nylin 1988, Janz et al. 1994, Nylin et al. 2000). The other common birch in Sweden, B. pendula, is lethal to larvae and not oviposited on (Nylin & Janz 1993, Nylin et al. 2000). Such patterns demonstrate that P. c-album is not an indiscriminate generalist but will oviposit on only a few select species, despite the broad host plant range. There is also a good overall correlation between the preferences of females for a particular plant (when given a simultaneous choice) and the performance of offspring on them, measured as larval survival and growth rate (Nylin 1988, Janz et al. 1994). Studied populations vary in specificity but not in preference ranking. Thus, populations from Sweden, Norway, Estonia and England all prefer the hosts in Urticales on average (individuals differ), but this preference is much more evident in the English population than in the others, perhaps because in this population there is a partial second generation. This puts a selective premium on fast growth, which is only possible on hosts in Urticales (Nylin 1988, Janz & Nylin 1997, Janz 1998, Nylin et al. 2000 and Nylin S., Janz N. & G.H. Nygren, unpublished observations). The tribe Nymphalini - major evolutionary patterns Host plant patterns in the butterfly tribe Nymphalini, of which Polygonia is a member, makes it clear that current host plant range in P. c-album must be seen in a historical

Evolutionary dynamics of host plant range in the butterfly tribe Nymphalini ...

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Ny mp ha lis Po Ka lygo Ro nisk nia dd a ia

Ag lai s

Va ne ss a Ba ss a An ris ta na rti a Ina ch is

Hy pa M nar yn tia es Sy m br Ar ent h as ch ia ni a Cy nt hia

context (Fig.1). According to Harvey (1991) the tribe consists of the genera Hypanartia, Araschnia, Mynes, Symbrenthia, Antanartia, Bassaris, Vanessa, Cynthia, Inachis, Aglais, Nymphalis, Kaniska and Polygonia. Out of these 13 genera (Roddia was more recently proposed as a monotypic genus, see Nylin et al. 2001), no less than nine feed exclusively on plants in Urticales, most importantly on the Urticaceae (Nylin & Janz 1999, Janz et al. 2001). Three additional genera (including Polygonia) have strong ties with these plant families, besides other hosts. Only the monotypic Kaniska has switched hosts completely, to the monocotyledon family Smilacaceae. Furthermore, all of the remaining host plant families used by P. c-album are shared by other species of Polygonia, and most of them by the species of Nymphalis as well. Hence, for a complete understanding of current host plant range in P. c-album a phylogenetic analysis of host plant patterns in the Nymphalini is necessary (Nylin & Janz 1999, Janz et al. 2001). An analysis of Nymphalini phylogeny (Fig.1, Nylin et al. 2001) based on total evidence from morphology, ecology and behaviour, as well as the mitochondrial gene “nd1” and the nuclear gene “wingless”, suggests the following: Polygonia is in a derived position in the tribe, close to Nymphalis itself. The most basal genera in the tribe seem to be Hypanartia, Araschnia, Mynes and Symbrenthia. All of these genera feed on trees and herbs in Urticaceae and, in the case of Hypanartia, also on trees in Ulmaceae. Approaching Polygonia, the next clade apparently consists of Antanartia, Bassaris, Vanessa and Cynthia, all of which are specialists on Urticaceae except for the polyphagous Cynthia. Species in

Urticales only Also new hosts used Fig. 1. A hypothesis of phylogenetic relationships in the butterfly tribe Nymphalini at the level of genera based on morphological, ecological and molecular evidence (see Nylin et al. 2001) showing that the ties with hosts in Urticales probably are ancestral in the tribe. Host expansions are relatively recent and seen only in the genus Cynthia and in the “N-P clade”, i.e. Nymphalis+Polygonia+Kaniska+Roddia.

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this genus utilize herbs in the Urticaceae but in addition a wide range of herbs in e.g. Asteraceae and Malvaceae. Even closer to Polygonia, species in the clade Inachis + Aglais feed exclusively on herbaceous Urticaceae and Cannabidaceae. Hence, there is little doubt that the ties with Urticaceae and other Urticales are ancient in the tribe. In all probability the ancestor of the tribe was a specialist on hosts in the Urticales (most likely Urticaceae itself) and all genera close to the root retain this ancestral host plant range. Colonizations of other plant families are more recent, and have occurred in Cynthia and in the clade Nymphalis + Roddia (= “Nymphalis vau-album” or “Polygonia lalbum”) + Kaniska + Polygonia (henceforth referred to as the “N-P clade”) (Nylin & Janz 1999, Janz et al. 2001, Nylin et al. 2001). Evolution of host plant range in Nymphalini If current host plant families are traced on the species-level phylogeny, using character optimization techniques (Brooks & McLennan 1991, Maddison & Maddison 1992) the result is a very dynamic picture of the evolution of host plant relationships (Janz et al. 2001). Even if gains of plant families (colonizations) are weighed twice as high as losses the optimizations suggest numerous colonizations and losses of the plant families used in the tribe. The plant families can be divided into three groups: 1) The families in Urticales. 2) The other typical “Nymphalis-Polygonia hosts”, i.e. Salicaceae, Betulaceae, Grossulariaceae, Ericaceae and Rosaceae (all of which are shared by several species in the N-P clade). 3) Other families, namely Asteraceae, Malvaceae, Rhamnaceae and Smilacaceae. No matter the weighting scheme, the untypical families in the third group apparently have been colonized only once each and relatively recently. Urticaceae may have been colonized only once (in an ancestor of the tribe) or up to five times if gains and losses are given equal weight. The other families in Urticales are suggested by the optimizations to have been colonized on several occasions, and this is true also for the other “N-P hosts”. In total there seems to have been 20-30 colonizations within these eight families (depending on the weighting) and only four outside of them! Hence, on one hand we have an apparently simple picture of host plant evolution in the tribe, with an ancestral use of Urticales followed by colonizations of Salicaceae and Betulaceae in the ancestor of the N-P clade, and later Grossulariaceae and Ericaceae in an ancestor or early member of Polygonia. On the other hand we have optimizations instead suggesting numerous colonizations. However, the fact that this is true only for the families in groups 1 and 2 above, the “typical” hosts in the tribe, suggests that these colonizations may not be truly independent. For instance, Grossulariaceae is not used by any butterflies outside of Polygonia. It does not seem likely that there have been two wholly independent colonizations within this genus, even though this is suggested by all optimizations. Instead, there are three possibilities. Firstly, some other shared characteristic of members of this genus may “pre-adapt” them to relatively easily colonize the Grossulariaceae, i.e. a case of parallel evolution. Secondly, there may have been a single early colonization followed by several losses, leaving two distant sections of Polygonia with this host plant association today. Thirdly, Grossulariaceae may have been lost as a host by one of the sections and later re-colonized. It seems probable that a host used in the past would be more easily to colonize than an entirely novel host. This third possibility would also neatly explain the

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general dynamics in the tribe, with much apparent colonization of shared host plant families. The possibilities are not mutually exclusive. In order to illuminate these possibilities further, we performed establishment tests with hatchlings of 20 butterfly taxa (Janz et al. 2001). If it could be shown that larvae are able to feed on plants which are not currently used for oviposition by females, this could be taken as evidence for any of the three scenarios just described. The details which species can feed on which plants - could help us to choose between them. We could test only a few (5-10) larvae with each plant family (nine families, groups 1-2 above and Asteraceae), so even when results were negative other genotypes might have been able to feed on the plant. Despite this limitation of the methodology, there were some positive results. Thus, four species that are not known to utilize Urticaceae as host plants showed some ability to feed on U. dioica. Also, P. c-album was found to be able to reach adulthood on Vaccinium myrtillus of the Ericaceae, a family used by other Polygonia and one which character optimization suggests was used earlier in the lineage leading to P. c-album. Such results lend support to the second and third scenarios above, the shared host plants may have been colonized only once at an early stage and are kept in the potential host plant range of larvae and could potentially be re-colonized. This would explain the persisting bonds with a particular plant family despite a dynamic picture of gains and losses. However, other positive results fit the scenario of “parallel evolution”. V. indica showed an unexpected ability to feed on Salicaceae (an “N-P host”) and N. polychloros on Ericaceae (a Polygonia host). Thus, members of the tribe may be “preadapted” to feed on plant families that have evidently not yet been colonized by ovipositing females. We constructed a new host data set that included (besides plants known to be used by females) also plants found to be edible in these trials with larvae - the “potential range” vs. the earlier “actual range”. It could have been predicted from the second and particularly the third scenario that this would result in fewer colonization events when the data is optimized onto the phylogeny (because plants are not actually lost and recolonized, but kept in the potential range). This was however not the case. There were fewer colonizations of e.g. Urticaceae, but instead more of e.g. Salicaceae and Ericaceae. Another way to study the role of the potential range is to see what happens when a new plant family is colonized. Is there an “immediate” shift to the new plant, or is the host plant range only expanded? By “immediate” I mean over phylogenetic time scales, i.e. the shift is complete in the species before the next speciation event, rather than the ancestral host being kept at least in some populations. Again, it could be predicted from the third scenario that the ratio of “expansions” vs. such “shifts” should be higher when the potential range is optimized rather than the actual range, because it is the essence of the hypothesis that potential ranges are kept. This was not the case, as the ratio was only slightly higher. For both data sets, expansions greatly (and statistically significantly) outnumbered shifts, illustrating the general trend towards increased polyphagy in the tribe. This also suggests that rapid host plant shifts (as would be expected from some evolutionary scenarios, like specialization driven by physiological trade-offs between plants), may be rare. It can be concluded from this that there was some, but limited, evidence that ancestral host plants are kept in the potential range of larvae. On the other hand the methodology

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used (see Janz et al. 2001) could only pick up fixed species characteristics and is thus very conservative with respect to this hypothesis. A completely different picture may have been found with large samples from several populations. Why specialization? As can be seen from the above, the trend in this tribe is towards increased polyphagy. Still, many species are relatively specialized, even within the “N-P clade”. Moreover, they are specialists on different families, suggesting multiple recent processes of specialization after a polyphagous phase. Most species have specialized on one of the families in the Urticales, suggesting that this ancestral host clade may remain the most suitable one over long periods of time. Others have specialized on novel families: K. canace on Smilacaceae and N. californica on Rhamnaceae (it is interesting to note that such innovations only have occurred in the most polyphagous sections of the tribe: in the N-P clade and in Cynthia). Why this trends towards re-specialization, after the apparent widening of the range early in the evolution of the N-P clade? Some possibilities are: Firstly, some hosts are bound to be better than others (resulting in higher fitness) and often the advantages of using only the best host should outweigh the possible costs in e.g. decreased oviposition rate and less spreading of risks. It seems reasonable that this “best” host should often be the one that the lineage once specialized on. Secondly, optimal performance may not be possible on more than one plant simultaneously. Attention has focused on physiological efficiency, and there has been relatively little success in finding evidence for the hypothesis that “the jack of all trades is a master of none” (but see Joshi & Thompson 1995). However, there are many other aspects of performance than growth efficiency, e.g. optimal timing of the developmental period and “choice” of developmental pathway (Wedell et al. 1997, Tikkanen et al. 2000). It may not be possible to evolve reaction norms that ensure optimal development on each of a wide range of host plants, on top of variation in e.g. temperature (see also Via & Lande 1985). Thirdly, fast and accurate choices of plant species and individuals may not be possible if the range is too wide. This “information-processing hypothesis” suggests that there are limits to how much information can be handled accurately by neural machinery, a fact that could favour specialization. The idea is not new (Levins & MacArthur 1969) but is has recently gained new ground (Bernays & Wcislo 1994, Bernays 1996, Bernays 1998). We have found support for it in experiments comparing species in the Nymphalini and populations of P. c-album differing in host plant specificity. Specialists made fewer mistakes when given the choice between individuals of U. dioica differing in quality (Janz & Nylin 1997) or between host plants and non-hosts that are very similar in appearance (Nylin et al. 2000). Of course, this does not imply that information-processing constraints is the only explanation of host plant utilization patterns in the Nymphalini. For instance, polyphagy may be favoured by the migratory habits of a species like Cynthia cardui, and in other cases host plant phenology, structure and morphology, as well as the life history characteristics of butterfly species (e.g. clutch size) are likely to interact with the evolution of host plant range. However, constraints on host plant search behaviour is potentially a more general explanation, and for this reason of great interest.

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Conclusions Host plant range in the butterfly tribe Nymphalini is evolutionarily dynamic; ranges expand and contract again. Ancestral host plants are often kept in the diet after colonizations of new plants, leading to an expansion of the range. Later, sometimes after considerable time, re-specialization occurs. Such specialization is often on the ancestral host or a close relative, creating a pattern of conservatism that may even obliterate all traces of the polyphagous phase. However, shifts to more distantly related plants also seems to occur more easily when associated with episodes of polyphagy. There is no evidence that co-evolution plays a major role in butterfly-plant interactions (Janz & Nylin 1998) but the species diversity of the “N-P-clade” could suggest that such episodes of polyphagy also promote speciation, an interpretation that follows the spirit of Ehrlich & Raven’s classic paper on co-evolution (1964). This is because one explanation of such patterns could be that host range expansions under some circumstances may permit also geographical expansions, followed by vicariance phenomena and genetically isolated populations that later retain only part of the ancestral host plant range. This possibility is currently under study. Acknowledgements I thank my team-members; Niklas Janz, Klas Nyblom, Anders Bergström and Georg H. Nygren, for letting me give an overview of our joint results under my own name. This research was supported by grants from the Swedish Natural Sciences Research Council. References BERNAYS E.A. 1996. Selective attention and host-plant specialization. Entomol. exp. appl. 80: 125-131. BERNAYS E.A. 1998. The value of being a resource specialist: behavioral support for a neural hypothesis. Am. Nat. 151: 451-464. BERNAYS E.A. & W.T. WCISLO 1994. Sensory capabilities, information processing, and resource specialization. Q. Rev. Biol. 69: 187-204. BROOKS D.R. & D.H. MCLENNAN 1991. Phylogeny, Ecology, and Behavior. A Research Program in Comparative Biology. Chicago University Press, Chicago. EHRLICH, P. R. & P.H. RAVEN 1964. Butterflies and plants: a study in coevolution. Evolution 18: 586-608. HARVEY D.J. 1991. Higher classification of the Nymphalidae. In Nijhout H.F. (ed), The Development and Evolution of Butterfly Wing Patterns. Smithsonian University Press, Washington, pp. 255-276. JANZ N. 1998. Sex-linked inheritance of host-plant specialization in a polyphagous butterfly. Proc. R. Soc. B 265: 1-4. JANZ N., NYBLOM K. & S. NYLIN 2001. Evolutionary dynamics of host-plant specialization: a case study of the tribe Nymphalini. Evolution 55: 783-796.

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JANZ N. & NYLIN, S. 1997 The role of female search behaviour in determining host plant range in plant feeding insects: a test of the information processing hypothesis. Proc. R. Soc. B. 264: 701-707. JANZ N. & NYLIN, S. 1998. Butterflies and plants: a phylogenetic study. Evolution 52: 486-502. JANZ N., NYLIN S. & N. WEDELL 1994. Host plant utilization in the comma butterfly: sources of variation and evolutionary implications. Oecologia 99: 132-140. JOSHI A. & J.N. THOMPSON 1995. Trade-offs and the evolution of host specialization. Evol. Ecol. 9: 82-92. LEVINS R. & R.H. MACARTHUR 1969. A hypotheses to explain the incidence of monophagy. Ecology 50: 910-911. MADDISON W.P. & D.R. MADDISON 1992. MacClade: Analysis of Phylogeny and Character Evolution. Version 3. Sinauer, Sunderland. NYLIN S. 1988. Host plant specialization and seasonality in a polyphagous butterfly, Polygonia c-album (Nymphalidae). Oikos 53: 381-386. NYLIN S., BERGSTRÖM A. & N. JANZ 2000. Butterfly host plant choice in the face of possible confusion. J. Insect Behav. 13: 469-482. NYLIN S. & N. JANZ 1993. Oviposition preference and larval performance in Polygonia c-album (Lepidoptera: Nymphalidae) - the choice between bad and worse. Ecol. Entomol. 18: 394-398. NYLIN S. & N. JANZ 1999. The ecology and evolution of host plant range: butterflies as a model group. In Drent R., Brown V.K & H. Olff (eds), Herbivores, Plants and Predators. Blackwell, Oxford, pp. 31-54. NYLIN S., NYBLOM K., RONQUIST F., JANZ N., BELICEK J. & K. KÄLLERSJÖ 2001. Phylogeny of Polygonia, Nymphalis and related butterflies (Lepidoptera: Nymphalidae): a total-evidence analysis. Zool. J. Linn. Soc. 132: 441-468. THOMPSON J.N. 1994. The Coevolutionary Process. Chicago University Press, Chicago. TIKKANEN O.-P., NIEMELÄ P. & J. KERÄNEN 2000. Growth and development of a generalist insect herbivore, Operophtera brumata, on original and alternative host plants. Oecologia 122: 529-536. VIA S. & R. LANDE 1985. Genotype-environment interaction and the evolution of phenotypic plasticity. Evolution 39: 505-522. WEDELL N., NYLIN S. & N. JANZ 1997. Effects of larval host plant and sex on the propensity to enter diapause in the comma butterfly. Oikos 78: 569-575.

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© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Comparative Immunology of the Kingdom 117 The Animal New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 117-125, 2003

Comparative Immunology of the Animal Kingdom E.L. Cooper Laboratory of Comparative Immunology, Department of Neurobiology, School of Medicine, University of California, Los Angeles, USA. E-mail: [email protected]

Abstract Comparative Immunology has gained wide acceptance in biology, as an offspring of immunology and an amalgam of immunology and zoology. The prescient experiments of Metchnikoff on phagocytosis in invertebrates during the 19th century served to splinter immunology into its two main components: CELLULAR and HUMORAL. There is much interest in the immune system of invertebrates as representing early models or precursors of the innate system of vertebrates in contrast to the more highly evolved adaptive system. The symposium on Comparative Immunology of the Animal Kingdom provided an overview of current and crucial topics essential for understanding basic components of immune systems.

Comparative immunology is central to zoology When immunology began to flower in the 1960s, there was an attempt to amalgamate zoology (Cooper 1975, Azzolina et al. 1981, Honeycutt 1997) with concepts and techniques of immunologists. Thus comparative immunology emerged at a time when there was minimal coordinated research aimed at understanding the evolution of immune mechanisms (Cooper 1976, 1990, Cooper & Nisbet-Brown 1993). Some of this work began by discoveries of graft rejection in earthworms (Cooper 1968, Hildemann & Cooper 1970, Acton et al. 1972, Bibel 1988, Vetvicka et al. 1993) and of lymphoid organs in frog larvae. We advocated that immune responses be examined in animal models other than mammals (Gershwin & Cooper 1978, Cooper & van Muiswinkel 1982, Cooper & Brazier 1982, Cooper & Wright 1984, Cooper et al. 1987, Greenberg 1987). Confirming the concept of self/non-self, there has emerged the view of two major animal immune systems: invertebrates possess natural, non-adaptive, innate, non-clonal, nonanticipatory responses whereas those of vertebrates are induced, adaptive, acquired, clonal, anticipatory. All animals from protozoans to humans have solved the threat of extinction by having evolved an immune-defense strategy that ensures the capacity to react against foreign, non-self microorganisms and cancer that disturb the homeostatic self. Invertebrate-type innate immune responses evolved first and they characterize the

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metazoans, (Cooper 1974, 1996a,b, Beck et al. 1994, 2000, Flajnik 1994, Humphreys & Reinherz 1994, Beck & Habicht 1996, Rinkevich & Müller 1996). These rapid natural responses act immediately and are often essential for slower more specific adaptive vertebrate-type immune responses to occur. As components of the innate immune system, there is an emphasis on several major steps in the evolutionary process: 1) recognition, 2) the phagocytic cell, 3) the natural killer (NK) cell that can rapidly destroy cancer. When vertebrates evolved, beginning with fish, thymus-controlled T cells first appeared and so did bone-marrow-derived B cells that are precursors of plasma cells that synthesize and secrete antibodies (first found in amphibians with long bones) (Zapata & Cooper 1990). The IMMUNE system of non-coelomate animals Stotz et al. (this volume) afirm that animals and plants share certain protective mechanisms against invasive microorganisms. Whereas innate immunity is common to both phyla, only gnathostome vertebrates recently invented an adaptive immune system. Molecular genetic studies revealed that similar signaling pathways regulate the production of antimicrobial molecules in plants, insects, and mammals. [As will be reviewed by Roch in this symposium, molluscs produce and are protected by antimicrobial molecules. This is apparently the situation in all invertebrates]. Moreover, there is evidence for a link between self-defense and developmental processes. For instance homologs of toll and dorsal, originally discovered as determinants of embryonic patterning, regulate antimicrobial responses in insects and mammals. Thus, axis formation and immunity have apparently coevolved since the beginning of multicellular life. The freshwater polyp Hydra offers the possibility to test this evolutionary hypothesis, (Stoltz et al., this volume). This group has already identified genes that are involved in axis formation of cnidarians. One of them, a bmp1 / tolloid homologue, determines the anterior-posterior axis of bilaterians. With relevance to this symposium and to isolate defense-related molecules, it is essential to establish specific interactions between Hydra and bacterial pathogens. And to do this, immune responses in Hydra must be characterized via differential gene expression and peptide profiling. To link the immune system, genetic dissection of pathogenicity is possible in the bacteria that are pathogenic for Hydra. It is feasible to study the evolution of innate immunity because Hydra can discriminate between self and nonself, as xenograft rejection shows. In addition, the cellular machinery mediating defense responses, such as phagocytosis and apoptosis, is present in Hydra. Recent advances in plant biology suggest that resistance to insects as well as disease utilize similar signaling pathways. However, there may be conflicting effects of different enemies on host defense. Immune responses in coelomates Contrasts or functional similarities of innate immune mechanisms Earthworms and vertebrates The ability to maintain individual integrity especially by means of self/non-self discrimination and immune mechanisms is an elementary prerequisite for the existence of all multicellular organisms (Kauschke, this volume). Concerning successful survival

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of numerous, recent invertebrates, we must recognize their success due to efficient immune mechanisms despite the absence of immunoglobulins. The advantage of acquired immunity in vertebrates is long term action based on immunoglobulins and memory cells. Although innate immunity is rapid and short-lived, it is of tremendous importance even for vertebrates, including mammals. Phagocytosis, brown body formation (granuloma-like), graft rejection and NK-like activity are the most prominent cellular immune reactions in earthworms effected by coelomocytes in collaboration with humoral factors. Lectins, antimicrobial peptides and pore forming proteins as well as proteases are important humoral components. Both, cellular and humoral immune reactions in earthworms are increased in response to an inflammation or intracoelomic injection of foreign material. Certain earthworm immune mechanisms, like NK-like activity and pore formation resemble functionally perforin and/or complement of vertebrates. To find similar immunological phenomena in vertebrates and invertebrates is not surprising, since immune reactivity results from a long biological evolution that acts efficiently in preserving effective structures and functions. To substantiate their phylogenetic relationship by using molecular methods is currently an exciting task in comparative immunology. Defining the immune system of leeches Moving to another annelid, this time the Hirudinea, the leech immune system, like that in all animals, recognizes foreign antigens and responds with a wide repertoire of reactions (de Eguileor, this volume). Leeches can utilize a different system against different injected micro (bacterial lipopolysaccharide) and macro (protozoa, yeasts, and latex beads) antigens. Each injection of antigens first provokes a migration of macrophage-like, NK-like cells and granulocytes towards the non-self material and later different responses (degranulation, phagocytosis and encapsulation) take place depending upon the type of antigen. The migrating cells, involved in these series of processes, have similar behavior and CD markers of macrophage, NK and granulocytes of many invertebrates and vertebrates. Auto, allo and xenografts in Hirudo medicinalis are studied. While autogenic grafts may become “absorbed” in the self-host, allo and xenografts are destroyed by synergistic work of migrating cells. Concurrently, after a short time from the graft, there is a beginning of vasculo/angiogenic activity and new blood vessels move from the botryoidal tissue (localized between gut and body wall among muscle fibers) to reach the graft. The immune system precursors appear inside the neovessels and mAb anti-human CD 33, 34, 117 can characterize them. Angiostatin administration in grafted leeches inhibits angiogenesis and, in its presence, graft tissues are destroyed in a longer period of time. Interactions between the immune and nervous system The situation in annelids According to Salzet et al. (this volume), there is today growing evidence that the nervous and the immune systems can exchange information, mainly through small

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molecules, either cytokines or neuropeptides. Furthermore, it appears that some socalled neurotransmitters like neuropeptides can function as endogenous messengers of the immune system, and that they most likely participate in an important part in the regulation of the various components of the immune response. In this context, it is widely accepted that all organisms have processes that maintain their state of health. Failure of these processes, such as those involving naturally occurring antibacterial peptides, may lead to pathological events. The presence of antibacterial peptides on both proenkephalin invertebrate (Leeches) and vertebrate (Human) neuropeptide precursors such like enkelytin, peptide B, further supports the hypothesis that some of neuropeptide precursors are implicated in immune response. Indeed, their peptides, with their high antibacterial activities further associate opioid peptides with immune related activities. We surmise that immune signaling molecule may lead to enhanced proenkephalin proteolytic processing by prohormone convertase freeing both opioid peptides and antibacterial peptides during innate immune response. However, because it is necessary to modulate inflammation, invertebrates like leeches are also able to synthesize a panoply of messengers that modulate inflammation e.g. endocannabinoids, opiates and proopiomelanocortin derived peptides such like adrenocorticotrophin and melanostimulating hormone. This demonstrates that the equilibrium between the stimulation and the inhibition of the immune response has evolved sooner than previously thought. Tunicates: an evolutionary perspective Some of the same conclusions have been reached by Pestarino (this volume) with respect to the other major group of invertebrates. Several observations support the bidirectional communication between the immune and neuroendocrine systems both in vertebrates and invertebrates. The finding of mutual hormonal control mechanisms supports the mechanistic basis for a neuroimmune axis. Moreover, it is now well known that such bi-directional communication occurs as a result of the immune and neuroendocrine systems sharing a common set not only of hormones but also of receptors. The molecular characterization of the shared ligands, receptors and second messengers, provides further evidences on the structural and functional basis of neuro-endocrinimmune interactions. More recently, genes predominantly expressed in neuroendocrine and immune systems have been cloned and characterized. Efforts to understand how the immune system can influence nervous system function are hampered by the complexity of mammalian nervous and immune systems. Therefore, useful model systems to investigate the cellular mechanisms involved in neural-immune interactions have been found in invertebrates such as mollusks and protochordates. Immune- and neuro-active molecules and relative receptors have been also described in unicellular eukaryotes. Therefore, it seems that common chemical messengers responsible for neuroimmune interactions could have evolved early during evolution.

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Experimental analyses of immune responses to potential internal threats and external pathogens Cytotoxicity as measured by invertebrate effectors against various targets including cancer cells According to Parrinello et al. (this volume), cell killing is a basic aspect of immune responses. During evolution, cell-killing activity emerged in primitive phagocytes and developed as immune mechanisms dissociated from phagocytosis. Protostome and deuterostome invertebrate models are presented for comparing cytotoxic reactions and effector cells as assayed against allogeneic and xenogeneic (including erythrocytes and tumor cells) targets. Although, functional and structural analogies between invertebrate humoral and cellular lysins and vertebrate perforin model have been revealed, diversity of some cytotoxic proteins and polypeptides are discussed. On the other hand, recent data on amino acid sequences and gene expression indicate that the invertebrate lyric mechanism may involve complement-related proteins. Homologues of complement components in sea urchin and a mannan-binding proteins-associated serine protease in ascidians suggest an “archeo” C system in deuterostome ancestors. Also the melanogenetic pathway phenoloxidase-dependent may be source of cytolytic molecules. Following the recognition of microbial polysaccharides, in insects and ascidians, hemocytes generate reactive forms of oxygen or quinoid intermediates by a prophenoloxidase (proPO) system which needs an activating cascade modulated by serine proteases and their inhibitors. Recent sequence analysis suggests that arthropod proPOs form one separate group and the deuterostome zymogen belongs to the vertebrate type of tyrosinase. The differences between cell death and necrosis of the target cells will be examined. Unique antibacterial peptides in mollusks are structurally related to insects but functionally to mammals Up to now, the considerations have dealt mostly with experimental situations that describe how invertebrates may handle antigens. Now we turn to a well-analyzed model to describe the existing mechanisms for handling microbial antigens, some of which are pathogenic. According to Roch et al. (this volume), marine mollusks are capable of several immune reactions belonging to innate immunity. Three groups of cationic, cysteinerich, 4 kDa peptides were recently identified in mussels: defensins, mytilins and myticins, including several isoforms that possess complementary antimicrobial properties. The 3D structure of both native and synthesized defensins revealed their close relationship with arthropod defensins. Even if sharing the same name, vertebrate defensins are of totally different molecular structure. Mussel peptides are synthesized as precursors in circulating cells before being stored as mature forms in hemocyte granules. The entire precursors share the same organization related to the horseshoe crab tachiplesin. The peptides are involved in several levels of the anti-infectious response, (I) intra-cellular on phagocytosed bacteria, (ii) extra cellular on a later systemic response. Meanwhile, mytilins are also present in enterocytes from which they can be released to act on gut microflora. Confocal microscopy revealed the presence of hemocytes containing only

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mytilins, only defensins, both peptides and no peptide. Even if defensins are structurally related to those of arthropods, the involvement of mussel peptides in anti-infectious responses strongly differs from other invertebrate models. According to their behavior, mussel hemocytes appeared more closely related to mammalian monocyte/ macrophages than to insect hemocytes. Perspectives on the Evolution of the immune system The title of our symposium may be somewhat misleading for persons interested in the full spectrum of immune systems. Our purpose is to view current works as representing a mere miniscule component of the larger field. We have considered primarily invertebrate immune systems and how they are integrated in the whole organism with due consideration afforded to interactions where they are known to occur. Linkages between the nervous and immune systems are beginning to be unraveled as integrated in certain complex invertebrates. It is our belief that if we analyze the immune systems of invertebrates we will come closer to realizing the panorama of immune reactivities, especially how all of them probably evolved. We now have available techniques and assays that are current and easily accessible (Stolen et al. 1992-1997, Wiesner et al. 1998). We will grow to understand the possible evolutionary pressures that caused the immune system to parallel in its development the complexity and diversity of animals in which it is housed (Wright & Cooper 1976, Lauder 1982, Desowitz 1987, Kelsoe & Schultz 1987 Langman 1989, Sima & Vetvicka 1990, Warr & Cohen 1991). A safe first guess is one of survival, perhaps equal in impact to fecundity. Thus the immune system cannot be any more simple or complex than the animal in which it resides. This concerns the animal’s structure, environmental niche and position in the phylogenetic scale. Invertebrates are exceedingly diverse and numerous estimates reveal nearly 2 million species, classified in more than 20 phyla from unicellular organisms up to the complex, multicellular protostomes and deuterostomes. In this context, clearly Darwin contributed to the development of comparative immunology at a time when this kind of emphasis was not considered (Cooper 1982, Lewin 1982, Ayala 1997). It is therefore not surprising to find less diverse immunodefense responses whose effector mechanisms remain to be completely elucidated. Our symposium did not pretend to offer the very last word on all invertebrates and certainly there will be only fleeting acknowledgements of or comparisons with analogous or homologous cells and molecular responses in vertebrates. It was our aim to dispel the myth that immune responses are solely vertebrate attributes, leaving invertebrates defenseless in similar worlds, with shared environments (Zelikoff et al. 1997). We should have also revealed that the essential innate immune system of vertebrates is an updated vestige of the ancient system of phagocytosis, so commonly attributed to invertebrates. Invertebrates are more complex and it is safe to assume that there may be even glimmers of adaptive immune reactivity if we look with finer less biased lenses. In the historical context of all immunology, we should recall that roughly 100 years ago, the simple act of phagocytosis discovered by Metchnikoff in Messina heralded the first major split in the monolithic immunology of the 19th century. It seems that in the 21st century there is an effort to coalesce innate and adaptive immunity with

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invertebrates again providing the necessary glue, (Hoffmann et al. 1994, Söderhall et al. 1996). Finally, we may come to realize that a new system of classification of invertebrates based on immune capacities rather than embryological evidence may be more suitable (Wake 1994). References ACTON R.T., EVANS E.E., WEINHEIMER P.F., COOPER E.L., CAMPBELL R.D., PROWSE R.H., BIZOT M., STEWART J.E. & FULLER G.M. et al. 1972. Invertebrate Immune Defense Mechanisms. MSS Information Corp., New York, 204p. AYALA F.J. 1997. Ascent by natural selection. Science 275:495-496. (Review of: Ruse M. 1996 Monad to Man: the Concept of Progress in Evolutionary Biology. Harvard University Press, Cambridge, MA. 628p.) AZZOLINA L.S., TRIDENTE G. & E.L. COOPER (eds) 1981. Proceedings of the Verona Workshop, 16-17, July, 1980. Dev. Comp. Immunol., 5 Suppl. 1: 1-180. BECK G., COOPER E.L., HABICHT G.S. & J.J. MARCHALONIS (eds) 1994. Primordial immunity: Foundations for the vertebrate immune system. Ann. N. Y. Acad. Sci. 712: 1-376. Beck G., Cooper E.L. & M. Sugumaran (EDS.) 2000. PHYLOGENETIC PERSPECTIVES ON THE VERTEBRATE IMMUNE SYSTEM. PLENUM PUBLISHERS NEW YORK, NY, 383P. BECK G. & G.S. HABICHT 1996. Immunity and the invertebrates. Scientific American 275: 60-66. BIBEL D.J. 1988. Milestones in Immunology: A Historical Exploration, Foreword by Arthur M. Silverstein, Sci. Tech. Madison, WI. Comparative Pathology of Inflammation (self-nonself) Discovery of graft rejection in earthworms. 170-173. COOPER E.L. 1968. Transplantation immunity in annelids. I. Rejection of xenografts exchanged between Lumbricus terrestris and Eisenia foetida. Transplantation 6: 322-337. COOPER E.L. (ed.) 1974. Invertebrate Immunology. Contemporary Topics in Immunobiology vol. 4. Plenum Press, New York, 299p. COOPER E.L. (ed.) 1975. Developmental Immunology. Am. Zool. 15: 35. COOPER E.L. 1976. Comparative Immunology. Prentice- Hall, Englewood Cliffs, N.J. 338p. COOPER E.L. 1982. General Immunology. Pergamon Press, New York, 343p. COOPER E.L. 1982. Did Darwinism help comparative immunology? Am. Zool. 22: 890. COOPER E.L. (ed.) 1990. Comparative Immunology. Bioscience 40: 720-768. COOPER E.L. 1990. General Immunology. (Japanese translation) Nishimura Co. Ltd., Japan, 324p. COOPER E.L. 1996a (ed.) Invertebrate immune responses: Cells and Molecular Products. Adv. Comp. Environ. Physiol. 23: 1-216. COOPER E.L. 1996b (ed.) Invertebrate immune responses: Cell Activities and the Environment. Adv. Comp. Environ. Physiol. 24: 1-249. COOPER E.L. & M.A.B. BRAZIER (eds) 1982. Developmental Immunology: Clinical Problems and Aging. UCLA Forum in Medical Sciences, Vol. 25, Academic Press, New York, 321p. COOPER E.L., LANGLET C. & J. BIERNE (eds) 1987. Developmental and Comparative Immunology, Alan R. Liss, N.Y., 180p. COOPER E.L. & E. NISBET-BROWN (eds) 1993. Developmental Immunology. Oxford University Press New York. 480p. COOPER E.L. (editor-in-chief) & VAN MUISWINKEL W.B. (guest editor) 1982. Immunology and Immunization of Fish. Dev. Comp. Immunol., Suppl. 2: 255p.

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COOPER E.L. & R.K. WRIGHT (eds) 1984. Aspects of Developmental and Comparative Immunology II. Proc. 2nd Int. Congr. Int. Soc. of Dev. Comp. Immunol., Suppl. 3, Pergamon Press, New York, 280 pp. DESOWITZ R.S. 1987. The Thorn in the Starfish: The Immune System and How it Works. W.W. Norton, New York. 270p. FLAJNIK M. 1994. Lines of Defense. Science 265: 1254-1255. GERSHWIN M.E. & E.L. COOPER (eds) 1978. Animal Models of Comparative and Developmental Aspects of Immunity and Disease. Pergamon Press, New York, 396p. GREENBERG A.H. (ed.) 1987. Invertebrate Models: Cell Receptors & Cell Communication. Karger, Basel. 270p. HILDEMANN W.H. & E.L. COOPER (eds) 1970. Phylogeny of Transplantation Reactions. Transplant. Proc. 2: 179-341. HOFFMANN J.A., JANEWAY C.A. & S. NATORI (eds) 1994. Phylogenetic Perspectives in Immunity: The Insect Host Defense. R.G. Landes Co. Austin TX, 197p. HONEYCUTT R.L. 1997. Evolutionary issues. Science 275: 36-37. (Review of Ferraris J.D. & S.R. Palumbi 1996. Molecular Zoology: Advances, Strategies and Protocols. Wiley-Liss, New York, 580p.) HUMPHREYS T. & E.L. REINHERZ 1994. Invertebrate immune recognition, natural immunity and the evolution of positive selection. Immunology Today. 15: 316-320. KELSOE G. & D.H. SCHULZE 1987. Evolution and Vertebrate Immunity. University of Texas Press, Austin, 469p. LANGMAN R.E. 1989. The Immune System: Evolutionary Principles Guide our Understanding of this Complex Biological Defense System. Academic Press, Inc. San Diego, 209p. LAUDER G.V. 1982. Historical biology. Science 218: 781-782. (Review of Joysey K.A. & A.E. Friday (eds) 1980. Problems of Phylogenetic Reconstruction. Academic Press, New York, 442p. LEWIN R. 1982. Darwin died at a most propitious time. Science 217: 717-718. REINISCH C.L. & G.W. LITMAN 1989. Evolutionary Immunobiology. Immunol. Today. 10: 278281. RINKEVICH B. & W.E.G. MULLER (eds) 1996. Progress in Molecular and Sub-cellular Biology 15: Invertebrate Immunology. Springer-Verlag, Heidelberg, 250p. SIMA P. & V. VETVICKA 1990. Evolution of Immune Reactions. CRC Press, Boca Raton, FL, 247p. SODERHALL K., IWANAGA S. & G.R. VASTA (eds) 1996. New Directions in Invertebrate Immunology. SOS Publications, Fair Haven, NJ, 494p. STOLEN J.S. & T.C. FLETCHER (eds) 1994. Modulators of Fish Immune Responses. Breckenridge Series I SOS Publications. Fair Haven, NJ, 252p. STOLEN J.S., FLETCHER T.C., ANDERSON D.P., KAATTARI S.L. & A.F. ROWLEY (eds) 1992. Techniques in Fish Immunology (FITC II). SOS Publications. Fair Haven, NJ, 196p. + Appendix 8p. STOLEN J.S., FLETCHER T.C., ANDERSON D.P., KAATTARI S.L., ZELLIKOFF J.T. & S.A. SMITH (eds) 1997. Techniques in Fish Immunology (FITC III). SOS Publications. Fair Haven, NJ, 208p. + Appendix 18p. STOLEN J.S., FLETCHER T.C., ANDERSON D.P., ROBERSON B.S. & W.B. VAN MUISWINKEL (eds) 1993. Techniques in Fish Immunology (FITC I). SOS Publications. Fair Haven, NJ, 197p. STOLEN J.S., FLETCHER T.C., BAYNE C.J., SECOMBES C.J., ZELIKOFF J.T., TWERDOK L.E. & D.P. ANDERSON (eds) 1996. Modulators of Immune Responses: the Evolutionary Trail. Breckenridge Series 2: SOS Publications. Fair Haven, NJ, 600p.

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STOLEN J.S., FLETCHER T.C., SMITH S.A., ZELIKOFF J.T., ANDERSON D.P., SODERHALL K., KAATTARI S.L. & B.A. PERKINS-WEEKS (eds) 1995. Techniques in Fish Immunology: Fish Immunology Technical Communications (FITC IV). SOS Publications. Fair Haven, NJ, 258p. + Appendix 42p. VETVICKA V., SIMA P., COOPER E.L., BILEJ M. & P. ROCH 1993. Immunology of Annelids. CRC Press, Boca Raton Florida, 300p. WAKE D.B. 1994. Comparative terminology. Science 265: 268-269. (Review of Hall B.K. (ed) 1994. The Hierarchical Basis of Comparative Biology. Academic Press, San Diego, CA, 483p.) WARR G.W. & N. COHEN (eds) 1991. Phylogenesis of Immune Functions. CRC Press, Boca Raton, FL, 326p. WIESNER A, DUNPHY G.B., MARMARAS V.J., MORISHIMA I, SUGUMARAN M & M. YAMAKAWA (eds) 1998. Techniques in Insect Immunology (FITC V). SOS Publications, Fair Haven, NJ, 304p. WRIGHT R.K. & E.L. COOPER (eds) 1976. Phylogeny of Thymus and Bone Marrow-Bursa Cells. Elsevier/North Holland, Amsterdam, 325p. ZAPATA A.G. & E.L. COOPER 1990. The Immune System: Comparative Histophysiology. John Wiley and Sons, Chichester, 335p. ZELIKOFF J.T., LYNCH J.M. & J. SHEPERS 1997. Ecotoxicology: Responses, Biomarkers & Risk Assessment. SOS Publications. Fair Haven, NJ, 534p. (Roundtable Discussion, 18p.).

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Novel approaches for the analysis of immune reactions Tunicate ... Evolution 127 The New in Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 127-132, 2003

Novel approaches for the analysis of immune reactions in Tunicate and Cnidarian model organisms H.U. Stotz1, R. Augustin1, K. Khalturin1, S. Kuznetsov1, B. Rinkevich2, J. Schröder3, & T.C.G. Bosch1 1. Zoologisches Institut, Christian-Albrechts-Universität (Biozentrum), Am Botanischen Garten 9, 24098 Kiel, Germany. E-mail: [email protected] 2. National Institute of Oceanography, Tel Shikmona, P.O. Box 8030, Haifa 31080, Israel 3. Klinik für Dermatologie, Venerologie und Allergologie Christian-Albrechts-Universität, Schittenhelmstr. 7, D-24105 Kiel

Abstract All multicellular organisms share innate immunity against microorganisms. Adaptive immunity probably evolved first in an ancestral gnathostome. Molecular studies of innate immunity reveal similar pathways regulating synthesis of antimicrobial molecules in plants, insects, and mammals. Evidence for direct links between immune-defense and developmental regulatory processes was discovered in Drosophila, suggesting a common evolutionary origin of axis formation and immunity. To understand immune system evolution Hydra offers analytic approaches to ancestral innate immunity and Botryllus for molecular responses during allorecognition. Hydra can discriminate self from non-self and may reveal regulatory mechanisms common to development that evolved from those controlling immuno-defense reactions.

Two unusual organisms to study immune reactions: Hydra, the most basal eumetazoan, and Botryllus, the ancestral chordate Hydra belongs to the Cnidaria, the most basal eumetazoan phylum, which arose approximately 600 million years ago (Fig. 1A). Cnidaria are the first animals in metazoan evolution that have a defined body plan including a tissue layer construction, a nervous system, and a single axis with radial symmetry. This axis is composed of a head, a body column and a foot. Hydra is diploblastic consisting of two epithelia, the ectoderm and the endoderm surrounding a gastric cavity. There are about 20 cell types distributed among 3 cell lineages (Bosch 1998). Each of the epithelial layers is made up of a cell lineage, while the remaining cells are part of the interstitial cell lineage, which reside among the epithelial cells of both layers. Multipotent interstitial stem cells give rise to neurons, nematocytes, secretory cells

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Fig. 1. Evolution of the immune system in metazoans with special emphasis on tunicates and the cnidarian genus Hydra. (A) Phylogenetic relationships among the animal kingdom. Developments of key components of the immune system are indicated. Cnidarians undergo apoptosis and engage in phagocytosis. Whereas complement systems are found exclusively in deuterostomes, coagulation cascades exist in other invertebrates as well. Adaptive immunity is a novel feature of jawed vertebrates. Our research emphasizes comparative immunology in tunicates and cnidarians, shown in bold. (B) Hydra, shown on the left, is an experimentally accessible member of the cnidarian phylum. We have demonstrated cellular mechanisms of immuno self-defense, such as phagocytosis in epitheliomuscular cells shown on the right. Arrows indicate phagosomes.

and gametes (Bosch & David 1986). Hydra is amenable to a variety of tissue and cell manipulations that are useful for analyzing developmental processes. Breakthroughs of the past decade include (i) a detailed description of processes governing Hydra development at the cell and tissue level (summarized in Bosch, 1998); (ii) characterization of the molecular basis of these processes (e.g., Martinez et al. 1997,

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Technau & Bode 1997, Endl et al. 1999); and (iii) technical advances such as the generation of loss-of-function phenotypes (Lohmann et al. 1999) and identification of large numbers of genes by the Hydra EST project. Botryllus schlosseri (Pallas) is a cosmopolitan, encrusting, colonial protochordate (Tunicata). The protochordates are dimorphic organisms that alternate between nonfeeding, short-lived, pelagic, chordate-like larvae and sessile, filter-feeding, adult ascidians. However, even adult ascidians share two important characters with chordates, the endostyle (the ciliated groove of the filter feeding apparatus) and gill openings of the branchial basket, both of which are homologues of the vertebrate gill slits. These and other characters are in support of a common evolutionary origin of vertebrates and protochordates (Berrill 1955) and provide clues to the diversification of developmental and immunological traits in vertebrates, including mammals. Whereas complement systems are found exclusively in deuterostomes, coagulation cascades exist in invertebrates as well (Iwanaga et al. 1998). Allorecognition features of Botryllus schlosseri Recent interest in colonial ascidians has centered on consequences of allogeneic recognition. Pairs of colonies meet and either fuse along their contacting, peripheral ampullae to form a vascular parabiont (cytomyctical chimera), or develop cytotoxic lesions in the contact zone (reviewed in Taneda 1985, Weissman et al. 1990, Rinkevich 1992). This allorecognition is a codominant trait and genetically controlled by a single fusibility/histocompatibility (Fu/HC) locus. The degree of polymorphism at this locus resembles genes of the vertebrate major histocompatibility complex (Rinkevich et al. 1995). Wild Botryllus colonies are usually heterozygotic for the Fu/HC locus. Thus, such colonies can be designated as AB at this locus. Unlike vertebrate histocompatibility, a Botryllus AB colony has the capacity to fuse with any other colony carrying at least one of the two alleles at its fusibility locus (namely: AB, AA, BB, AX, XB). Rejecting colonies share no Fu/HC allele, suggesting that Fu/HC alleles determine self/nonself recognition in Botryllus species. Immune reactions in Hydra As any other multicellular organism, Hydra is confronted with a variety of microbial and protozoan parasites. For instance, Hydramoeba hydroxena (Entz) is a serious pathogen of the genus Hydra both in the laboratory and in the wild. Different Hydra species vary in resistance to this protozoan, suggesting underlying genetic diversity in defense mechanisms (Rice 1960, Stiven 1973). In addition, bacteria and fungi are significant Hydra pathogens both in culture and in the wild. While nothing is presently known about the molecular basis of disease resistance in Hydra, cellular aspects of immune reactions are well characterized. Immune responses in Hydra are apparently mediated by epitheliomuscular cells, which function as phagocytes (Bosch & David 1986) recognizing and reacting to nonself cells (Fig. 1B). Epitheliomuscular cells are also responsible for morphogenesis (Weinziger et al. 1994) and perform important homeostatic functions by eliminating apoptotic cells (Cikala et al. 1999).

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Novel approaches to study immune reactions in Hydra and Botryllus Recent molecular analyses in the emerging field of comparative and evolutionary immunology reveal common mechanisms underlying innate immunity in protostome and deuterostome invertebrates (Cooper & Bosch 2000). Studies in basal metazoan groups outside Bilateria are required to gain insights into the evolution of immunity.

Fig. 2. Potential effects of BS-cadherin during histocompatibility. (A) The domain structure cadherin from Botryllus schlosseri is indicated. The presence of a signal sequence suggests processing in the endoplasmic reticulum. Activation of BS-cadherin presumably occurs via removal of a pro peptide. Like other cadherins, the extracellular domain of BS-cadherin consists of five repeats. A transmembrane domain separates the extracellular region from the cytoplasmic tail. (B) Cadherins mediate calcium-dependent cell-cell adhesions and establish cell polarity. Interactions with actin filaments through catenin are required for cell adhesion. In addition, cadherin is influenced by developmental signaling pathways, such as wingless/Wnt, through the action of â-catenin. Conversely, cadherin may indirectly influence cell fate and motility through interaction with â-catenin as well. The role of BS-cadherin during allogeneic rejection in Botryllus is not resolved.

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Hydra, a member of the most basal eumetazoan phylum Cnidaria, represents an attractive model to trace the evolutionary conservation of immune-related pathways. We have started to examine immune reactions in Hydra (i) by identifying target genes with altered expression in response to microbial challenges using differential display (Bosch & Lohmann 1998) and (ii) by identifying antimicrobial peptides by MALDI-TOF that are induced in response to microbial attack. The mechanism controlling activation of Hydra immune-related genes will be compared to those that have already been identified to participate in regulation of development. The approach will not only reveal the nature of the ancient defense mechanism but also allow insight into the relationship between innate immunity and development. The protochordate allorecognition system has long been compared with the vertebrate major histocompatibility complex (Fagan & Weissman 1998). In order to elucidate molecular mechanisms involved in the Botryllus rejection process and to obtain information regarding immune-like responses in phylogenetically older chordates, we applied the colony allorecognition assay (Rinkevich et al. 1995) and differential display between two incompatible Botryllus colonies. By comparing mRNA expression between the histoincompatible, activated colony and its naive parts, we identified a cadherin encoding gene (Fig. 2A) which is expressed at high levels during allogeneic rejection (Levi et al. 1997). Cadherins are cell surface membrane glycoproteins that mediate homophilic calcium dependent cell-cell adhesions and thus qualify to play a major role in the Botryllus histocompatibility process (Fig. 2B). Currently we are engaged in (i) characterizing genes from Botryllus that respond to allogeneic contact; (ii) studying the functional role of BS-cadherin during allogeneic responses; and (iii) identifying signal(s) that specify nonself recognition using marker genes whose expression is altered in response to allogeneic contact. Acknowledgements The work is supported by grants from the Deutsche Forschungsgemeinschaft (to T.C.G. B.), the Daimler-Benz Foundation (to S.K.) and the MINERVA Center for Marine Invertebrate Immunology and Developmental Biology. References BERRILL N.J. 1955. The Origin of Vertebrates. Clarendon Press, Oxford. BOSCH T.C.G. 1998. Hydra. In Ferretti P. & J. Géraudie (eds), Cellular and Molecular Basis of Regeneration: From Invertebrates to Humans. Wiley & Sons Ltd., Sussex, pp. 111-134. BOSCH T.C.G. & C.N. DAVID 1986. Immunocompetence in Hydra: Epithelial cells recognize self-nonself and react against it. J. Exp. Zool. 238: 225-234. BOSCH T.C.G. & J.U. LOHMANN 1998. Identification of differentially expressed genes by nonradioactive differential display of messenger RNA. Methods Mol. Biol. 86: 153-60. CIKALA M., WILM B., HOBMAYER E., BÖTTGER A. & C.N. DAVID 1999. Identification of caspases and apoptosis in the simple metazoan Hydra. Current Biol. 9: 959-962. COOPER E.L. & T.C.G. BOSCH 2000. Ontogeny recapitulates phylogeny: comparative immunology in Germany. Exp Clin Immunogenet. 27: 77-82.

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ENDL I., LOHMANN J. & T.C.G. BOSCH 1999. Head specific gene expression in Hydra: Complexity of DNA/protein interactions at the promoter of ks1 is inversely correlated to the head activation potential. Proc. Natl. Acad. Sci. USA 96: 1445-1450. FAGAN M.B. & I.L. WEISSMAN 1998. Linkage analysis of HSP70 genes and historecognition locus in Botryllus schlosseri. Immunogenetics 47: 468-76. IWANAGA S., KAWABATA S. & T. MUTA 1998. New types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions. J. Biochem (Tokyo) 123: 1-15. LEVI L., DOUEK J., OSMAN M., BOSCH T.C.G. & B. RINKEVICH 1997. Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri. Gene 200: 117-123. LOHMANN J.U., ENDL I. & T.C.G. BOSCH 1999. Silencing of developmental genes in Hydra. Dev. Biol. 214: 211-214. MARTINEZ, D.E., DIRKSEN M.L., BODE P.M., JAMRICH M., STEELE R.E. & H.R. BODE 1997. Budhead, a fork head/HNF-3 homolog, is expressed during axis formation and head specification in hydra. Dev. Biol. 192(2): 523- 536. RICE N.E. 1960. Hydramoeba hydroxena (Entz) a parasite on the fresh water medusa Craspedacusta sowerbii Lankester, and its pathogenicity for Hydra cauliculata Hyman. J. Protozool. 7: 151-156. RINKEVICH B. 1992. Aspects of the incompatibility nature in botryllid ascidians. Anim. Biol. 1: 17-28. RINKEVICH B., PORAT R. & M. GOREN 1995. Allorecognition elements on a urochordate histocompatibility locus indicate unprecedented extensive polymorphism. Proc. Roy. Soc. Lond. B 259: 319-324. STIVEN A.E. 1973. Hydra-Hydramoeba: A model system for the study of epizoic processes. Curr. Top. Comp. Pathobiol. 2: 145-212. TECHNAU U. & H.R. BODE 1999. HyBra1, a Brachyury homologue, acts during head formation in Hydra. Development 126: 999-1010. TANEDA Y. 1985. Simultaneous occurrence of fusion and nonfusion reaction in two colonies in contact of the compound ascidian, Botryllus primigenus. Dev. Comp. Immunol. 9: 371-375. WEISSMAN, I.L., SAITO Y. & B. RINKEVICH 1990. Allorecognition histocompatibility in a protochordate species: Is the relationship to MHC semantic or structural? Immun. Rev. 113: 227-241.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Does Functional Similarity of Certain Innate Immune Mechanisms ... Evolution 133 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 133-138, 2003

Does Functional Similarity of Certain Innate Immune Mechanisms of Invertebrates and Vertebrates Point to their Phylogenetic Relation? E. Kauschke1 & W. Mohrig2 1. Zoological Institute and Museum, Ernst-Moritz-Arndt-University Greifswald, J. -S. -Bach-Straße 11/12, D-17489 Greifswald, Germany. E-mail: [email protected] 2. Bahnhofstraße 53, D-17489 Greifswald, Germany

Abstract The ability to maintain individual integrity by means of self/non-self discrimination and immune mechanisms is an elementary prerequisite for the existence of all multicellular organisms. The enormous number of recent invertebrate species reflects success and efficiency of innate immune mechanisms in invertebrates although they lack immunoglobulins and specificity. The advantage of adaptive immunity is long term action based on immunoglobulins and memory cells, but innate immunity is of tremendous importance even for mammals. To find similar immunological phenomena in vertebrates and invertebrates is not surprising, since all result from biological evolution that acts efficiently in preserving essential structures and functions.

Introduction: Evolution Generally evolution means development, and development is a process of optimizing established structures as well as their adaptation to new conditions due to selection. Often surprising new functions are developed in that process. Nature is using a fundamental principle in evolution; it is economical and efficient. A very impressive example with strong consequences in this sense is the evolution of the notochord. The principle of economy and efficiency not only applies to evolution of morphological structures, it is also true at the biochemical level and is refereed to as molecular evolution. Recognition and immunodefense responses as exemplified by phagocytosis Without a doubt, molecules that effect immune mechanisms are interesting for phylogenetic analysis since they are important for the fitness of organisms as well as having a high selection significance. Generally, we differentiate immune mechanisms as cellular and humoral responses. This applies to innate immune mechanisms, which

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are present in all organisms, invertebrates and vertebrates, as well as to the adaptive immune mechanisms. Adaptive immunity is based on immunoglobulins and characterized by specificity and immunological memory, which have been demonstrated clearly in vertebrates and with questionable results in certain invertebrates. Circulating cells (hemocytes or coelomocytes or leukocytes) in the body fluid cause cellular reactions in invertebrates. They are comparable to macrophages and granulocytes of mammals morphologically and functionally. Phagocytosis is a primary function of these cells. Receptors as well as other molecules on phagocytes mediate recognition of potential pathogenic material and induce its intracellular incorporation and elimination. In contrast, particles too large for intracellular ingestion become immobilized and sequestered by encapsulation, a response that resembles granuloma formation in vertebrates. As an example, in earthworms, the resulting capsules consist of numerous layers of phagocytes and are known as brown bodies (Quaglino et al. 1996). Cytotoxic responses that are more elaborate In contrast to the more simple, non-specific phagocytic encapsulation response, other cells eliminate foreign materials including cells and tissues by a response referred to as cytotoxicity. Cytotoxic cells establish membranous contacts with target cells, which results in pore formation within target membranes. Enzymes are released through these pores ultimately causing target cell death. One group of effector cells, of long-term evolutionary significance, is called natural killer (NK) cells. The pore-forming molecule is perforin, a protein related to C9, another pore-forming protein of the complement cascade in mammals. Earthworm coelomocytes are also able to establish intimate membrane contact with target cells (K562) resulting in death as revealed by electron microscopy (Quaglino et al. 1996). Our investigations demonstrated that a monoclonal perforin-antibody binds to 17% of earthworm coelomocytes (Kauschke et al., 2001). The cross-reacting material occurs in a homogeneous pattern distributed throughout the cytoplasm of small coelomocytes (Fig.1). Small coelomocytes were shown to be positive also for certain cell differentiation markers (CD11a+, CD45RA+, CD45RO+, CDw49b+, CD54+) as well as β2-m+ and Thy-1+ (Cossarizza et al. 1996) whereas the more phagocytic large coelomocytes were negative for these markers. Moreover, cytokines (IL-1α, TNF-α) and POMC-derived peptide like molecules (ACTH, β-endorphin, α-MSH) have been found in large cells (Cooper et al. 1995). Western blot analysis of cell lysates using perforin antibody resulted in two protein bands with molecular weights of about 75 kDa, which resembles that of mammalian perforin. Although the molecular relationship of these molecules to mammalian perforin remains to be shown, it becomes obvious that NK-cells or NK-like cells as well as the effective mechanism of pore formation may be a phylogenetically old immune mechanism. Pore formation Pore formation is an efficient defense strategy as revealed by perforin and components of the complement cascade. Complement activation is a multi factorial mechanism,

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induced by antibodies as well as by other recognition molecules. The most important step for destruction of a target membrane after binding is the oligomerization of homologous protein molecules, their insertion into the target membrane and the formation of a pore. Even bacteria utilize pore formation to effect tissue destruction, as demonstrated for Staphylococcus α-toxin (Arbuthnott et al. 1973). In our investigations, we showed the mechanism of pore formation for cytolytic proteins discovered in earthworm coelomic fluid (Fig.2). Electron microscopy of sheep erythrocytes and phospholipid vesicles reveal pore like structures. Pores are formed by oligomerization of at least six monomers of the lytic protein, Eiseniapore, as concluded from the molecular

Fig. 1. Perforin-like activity in earthworm coelomocytes. Immunostaining of E. fetida coelomocytes and antiperforin-(P1-8)-mab. A: POD labeling (x400), B: POD labeling (x1000), C/D: TEM immunogold labeling.

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weight of about 228 kDa of a macromolecular complex, obtained from lipid vesicles, incubated with Eiseniapore followed by detergent treatment (Lange et al. 1997, Lange et al. 1999). Humoral immunodefense responses effected by antibacterial peptides Humoral immune molecules in invertebrates are antibacterial proteins and peptides, cytolytic molecules, lectins, proteases and other enzymes. Especially antimicrobial

Fig. 2. Pore formation by oligomerization of a cytolytic protein called Eiseniapore. A and B: Electron micrograph of negatively stained membranes incubated with EP; A - pore formation on erythrocyte ghosts; B - pore formation on phospholipid vesicles (egg-PC /Chol / SM); arrows indicate pore formation. C: SDS-PAGE of (1) E. fetida coelomic fluid, (2) Eisenia pore (EP) and (3) macro molecular complex of polymerized EP.

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defense by means of antimicrobial proteins and peptides reflects similarity in vertebrates and invertebrates. Lysozyme, which at first was described for secretions and serum samples of mammals is a well known antibacterial molecules. In 1968, Meßner and Mohrig described lysozyme for the first time as an antibacterial factor in the hemolymph of insects (Mohrig & Meßner 1968). Today we know that lysozyme occurs in vertebrates, invertebrates, and even in plants. Moreover, the phylogenetic tree of lysozyme has been published, which indicates the relationship of lysozymes in insects and vertebrates (Hughes 1998). The role of lectins Different from vertebrates, invertebrates fight infections and defend their integrity without immunoglobulins. It seems reasonable that lectins may act as substitute defense molecules in invertebrates, which effect self/non-self recognition by carbohydrate discrimination. Their occurrence in the body fluid as well as in cell membranes and their promoting effect upon pathogen incorporation or encapsulation, has been shown for many invertebrates. Lectins are defined as sugar binding proteins of non-immune origin, which agglutinate cells and/or precipitate glycoconjugates (Goldstein et al. 1980). The carbohydrate binding activity of lectins is due to the carbohydrate recognition domain (CDR), which shows significant similarity for all animal lectins (Dickamer & Taylor 1993), including the mannose binding lectin (MBL), a key player in pathogen recognition in vertebrates. Perspectives on invertebrate immune mechanisms Analysis of invertebrate immune mechanisms offers uniquely, original or primitive features, but their efficiency in immunodefense is perhaps reflected in their positive selection. Therefore comparative immunologist should always be open for breakthroughs, as we know from Drosophila. Very similar activation mechanisms have been described for Drosophila hemocytes and mammalian macrophages that leads to the expression of proteins involved in immunity (Hoffmann et al. 1999). The efficiency of innate immune mechanisms in invertebrates has been often underestimated due to the impressive effectiveness and specificity of acquired immunity based on immunoglobulins, complicated regulatory mechanisms as well as immunological memory. The advantage of specific immunity is its long lasting effect due to memory cells that facilitate the elimination of second infections rapidly. Nevertheless the immediate action of phagocytes and / or humoral compounds causes recognition and a spontaneous elimination of invading microorganisms. The advantage centers around the relatively long period required for the delayed production of immunoglobulins; their presence ensures a chance to win the competition against reproduction and toxin production by pathogens. Because of the existence of more than two million recent animal species that consist of more than 95% invertebrates, neither the existence nor efficiency of immune mechanisms can be denied for invertebrates. To show their phylogenetic relationships remains an exciting subject for further research.

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References ARBUTHNOTT J.P., FREER J.H. & B. BILLCLIFFE 1973. Lipid-induced polymerization of staphylococcal α–toxin. J. General Microbiol. 75: 309-319. COOPER E.L., FRANCHINI A. & E. OTTAVIANI 1995. Earthworm coelomocytes possess immunoreactive cytokines and POMC-derived peptides. Anim. Biol. 4: 25-29. COSSARIZZA A., COOPER E.L., SUZUKI M.M., SALVIOLI S., CAPRI M., GRI G., QUAGLINO D. & C. FRANCESCHI 1996. Earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines. Exp. Cell. Res. 224: 174-182. DICKAMER K. & M.E. TAYLOR 1993. Biology of animal lectins. Ann. Rev. Cell. Biol. 9: 237-264. GOLDSTEIN J.J., HUGHES R.C., MONSIGNY M., OSAWA T. & N. SHARON 1980. What should be called a lectin? Nature 285: 66. HOFFMANN J.A., KAFATOS C.F., JANEWAY C.A. & R.A.B. EZEKOWITZ 1999. Phylogenetic perspectives in innate immunity. Science 284: 1313-1318. HUGHES A.L. 1998. Protein phylogenies provide evidence of a radical discontinuity between arthropod and vertebrate immune system. Immunogenetics 47: 283-296. KAUSCHKE E., KOMIYAMA K., MORO I., EUE I., KÖNIG S. & E.L. COOPER 2001. Evidence for Perforin-like activity associated with earthworm leukocytes. Zoology. 104: 13-24. LANGE S., KAUSCHKE E., MOHRIG W. & E.L. COOPER 1999. Biochemical characteristics of Eiseniapore, a pore-forming protein in the coelomic fluid of earthworms. Eur. J. Biochem. 262: 547-556. LANGE S., NUESSLER F., KAUSCHKE E., LUTSCH G., COOPER E.L. & A. HERRMANN 1997. Interaction of earthworm hemolysin with lipid membranes requires sphingolipids. J. Biol. Chem. 272: 20884-20892. MOHRIG W. & B. MEßNER 1968. Immunreaktionen bei Insekten I. Lysozym als grundlegender antibakterieller Faktor im humoralen Abwehrmechanismus der Insekten. Biol. Zentralbl. 87: 439-470. QUAGLINO D., COOPER E.L., SALVIOLI S., CAPRI M, SUZUKI M.M., RONCHETTI I.P., FRANCESCHI C. & A. COSSARIZZA 1996. Earthworm coelomocytes in vitro: cellular features and granuloma formation during cytotoxicity against the tumor target K562. Eur. J. Cell Biol. 70: 278-288.

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State of the art for the immune system in leeches 139 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 139-145, 2003

State of the art for the immune system in leeches M. de Eguileor1, A. Grimaldi1, G. Tettamanti1, R. Valvassori1 & E.L. Cooper2 1. Department of Structural and Functional Biology, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy. E-mail: [email protected] 2. Laboratory of Comparative Immunology, Department of Neurobiology, School of Medicine, University of California, Los Angeles, Los Angeles California 90095-1763, USA

Abstract The leech immune system recognizes and responds to foreign antigens. We have identified and classified migrating cells as macrophage-like, NK-like and granulocytes. These cells are positive for CD 25, while activated macrophage-like cells are positive for CD 61, 68, 14, 11b and 11c. Activated NK-like cells are positive for CD 56, 57, 16 and granulocytes are positive for CD 11b, 11c. Western blots revealed these similarities of CD-like leech molecules to their mammalian counterparts. Leech responses to several challenges, (e.g. living and non-living antigens), suggests that these antigenic stimuli may evoke immune reactions functionally similar to those of invertebrates and vertebrates. Leech immune system and anatomical features of the body wall Why study the immune system of leeches since there is substantial data concerning other annelids? Unlike oligochaetes and polychaetes that have large coelomic cavities from which immune cells can flow, leeches are parenchymatous and can only respond by immune cells migrating into the extracellular matrix similar to that of vertebrates (Stein et al. 1977, Porchet-Henneré 1990, Porchet-Henneré et al., 1992, Quaglino et al. 1996). Leeches are well known aquatic iteroparus animals. Their muscular cutaneous sac contains several structures embedded in a loose connective tissue. In addition, the body wall is composed of substantial numbers of muscle fibers grouped in close adjacent fields and in the thickness of this muscular sac blood vessels are almost absent (Mann 1962, Sawyer 1986). Leeches are characterized by a long life span up to several years. Since they survive for extended periods, they have evolved a mechanism that responds against possible damages. These include not only small or large wounds but also the attack of microorganisms including bacteria, parasites, viruses and fungi (de Eguileor et al. 1999).

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Responses against living or non-living foreign antigens Leeches have an immune system that can recognize foreign antigens and can respond with a wide repertoire of reactions in relation to non-self. We can identify different responses in relation to inflammation, to living or non-living foreign antigens, large explants and grafts (allo and xenografts). Thus the massive migration and significant increase of cells involved in immuno-defense processes, are the “leit-motif” for every kind of evoked responses. All migrating cell types are generally visible in the few blood vessels, in lacunae and in connective tissue. However, when cells migrate they increase numerically in the connective tissue of lesioned areas. Thus it is relatively easy to follow any modification, i.e. migration and/or heightened cell activity linked to wound responses in the leech’s body due to their relative anatomical simplicity (Fig. 1). Shortly after mild injury, inflammation begins with a migration of numerous cells, from the center of the body corresponding to the gut region, towards the lesioned surface (Fig. 2). These cells reach the wound area shifting among the adjacent longitudinal muscle fields and forming a plug. In relation to time elapsed after a lesion, all cells increase in number as supported by BrdU incorporation (Fig. 3). Criteria for identification of migrating cells We have used several criteria to identify all migrating cells and to classify them as macrophage-like, NK-like and granulocytes. First, we utilized morphological and histochemical evidence: migrating macrophage-like show typical ruffled surface and projections as well as typical phagolysosomes (Fig. 4). NK-like cells have a large nucleus and granules in the cytoplasm (Fig.5). Two different granule types occur in the cytoplasm, large irregular (Fig. 6) or small round electron dense (Fig. 7). Macrophage and NK-like cells are strongly positive for NADH-tetrazolium reductase, Periodic acid Schiff, Oil Red O (O.R.O), acid and alkaline phosphatase (de Eguileor et al., 1998). We have also confirmed the morphological and histochemical identification of three main cell types using antibodies directed against human cell and CD-markers. We selected these antibodies mainly because some of them have been tested in invertebrates and vertebrates by other invertebrate immunologists, (Roch et al. 1983, Mansour & Cooper 1984, Mansour et al. 1985, Saad & Cooper 1990, Franceschi et al. 1991, Negm et al. 1991a,b, 1992, Cossarizza et al. 1996, Blanco et al. 1997, de Eguileor et al. 2000). Furthermore the selected markers although not entirely specific, could be used within a panel of immune system cells. All migrating cells are positive for CD 25 (Fig. 8), while activated macrophage-like cells are positive for CD 61, 68, 14, 11b and 11c. Activated NK-like cells are positive for CD 56, 57, 16 and granulocytes are positive for CD 11b and 11c (de Eguileor et al. 2000). Western blot analysis stressed such similarities and the molecular weights of CD-like leech molecules are strikingly similar to their mammalian counterparts (de Eguileor et al. 2000). Inflammatory responses Macrophage-like cells are the first that appear in lesioned areas, in sufficient numbers to reduce a small aperture by linking wound edges with pseudopodia that act as bridges,

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Fig. 1. Cross-section of unlesioned Glossiphonia complanata. Epithelium (E) and thick layer of muscles (M) with fibers make the muscular cutaneous sac circularly (C), obliquely (O) and longitudinally (L) disposed. Fig. 2. Semithin cross-section of lesioned G. complanata. Migrating cells move from gut region towards superficial lesion areas shifting among the muscle fields (arrowheads). M: muscles. Fig.3. Semithin section of lesioned G. complanata: migrating cells increase numerically. As BrdU technique demonstrates, the nuclei of S-phase cells are positively stained (arrowheads). M: muscles. Fig. 4. Activated macrophage-like cell shows a ruffled surface, projections (arrowheads) and phagolysosomes in the cytoplasm. Fig. 5. NK-like cells show a large nucleus and granules in the cytoplasm. Figs 6–7. Granulocytes have cytoplasm filled with large electron-dense granules or with small roundish granules. Fig. 8. Cryosection, superimposed confocal image. The CD25 is detected with indirect immunofluorescence. The signal is related to migrating cells located in the area between adjacent muscle fields.

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directly with epithelial cells or indirectly with other macrophage-like cells (Fig. 9). Later, macrophage-like cells engaged in phagocytosis, NK-like cells having cytolytic activity and large granulocytes with different behavior as judged by their dimension and shape of granules, crowd under the lesioned surface filling it and forming a plug. LPS, injected into unlesioned leech or topically administered on the lesioned area causes a fivefold increase of migrating cells in comparison to non-treated leeches (de Eguileor et al. 2000). Different responses against non-self in leeches Defining which types of responses to living or non-living foreign antigens that can occur in leeches is a problem. Host cells involved in immuno-defense reactions are the same as those activated in the non-specific inflammatory response to wounds. Phagocytosis of foreign living and non-living objects (experimentally induced responses by injecting yeast or sulfate spheres 2µm) is a simple mechanism to engulf and eliminate non-self and it is also evidence of a non-self recognition system (Fig. 10). Encapsulation and melanization are two other systems utilized when foreign bodies are too large to be phagocytosed (groups of spheres or parasites can be initially encapsulated by migrating cells and afterwards melanized). A thick melanin layer adheres to parasites’ surface (Fig. 11). The black capsule surrounding the parasite can be decolored with hydrogen peroxide, suggesting the presence of melanin (de Eguileor et al. 2000). After injection of protozoa, continuous sheets form around unicellular parasites. This coat is due to degranulation of granulocytes that contain large irregular granules. In addition to the different types of responses, closely correlated with different types of antigens, possible antibacterial substances (Salzet et al., this volume) can be produced by granulocytes with small round granules. They become functional and degranulate when a large dose of Escherichia coli is injected subcutaneously. Responses to large explants or grafts A wide range of connected and/or sequential events that begin with a classic inflammatory response reacts against large explants and grafts. At 24h after surgical lesion, there is impressive angiogenic activity and new vessels move from the gut region, among muscle fields, towards the lesioned area (Fig. 12). Vessels travel over the same route as that of migrating cells and serve as a piping system that can rapidly move large numbers of cells involved in immune defense. Massive angiogenesis is due to botryoidal tissue, a characteristic component of Arynchobdellids (localized in loose connective tissue between gut and body wall sac) (de Eguileor et al. 2000). Organization and functioning of botryoidal tissue, composed of large, granular botryoidal cells and flattened endothelial cells, is closely related to different demands during a leech’s life, i.e. to its undamaged or wounded condition. This tissue is normally involved in maintenance but it becomes particularly active after large explants or deep wounding. In normal conditions, most botryoidal cells are organized to form clustered cells ropes and only a few define a cavity. Instead, in wounded leeches most botryoidal tissue cells change shape and finally form new capillary vessels (Fig. 13). Botryoidal cells concurrently secrete iron (contained in granules) into the new cavity. Once this is complete, the function of botryoidal cells is still unfinished. The

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Fig. 9. Scanning microscope photograph. Macrophage-like cells, acting as bridges, link wound edges with their pseudopodia. Fig. 10. Superimposed confocal image of cells engaged in phagocytosis of fluorescent spheres. Fig. 11. A thick melanin coat (arrowheads) surrounds the parasite. Fig. 12. Semithin section of Hirudo medicinalis., easily showing angiogenic activity. Numerous new vessels (arrowheads) occupy the whole body wall thickness. M: muscles. Figs. 13-13a. Semithin section of lesioned or grafted leeches. Botryoidal tissue changes its rope-like appearance forming new capillary vessels.

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contact area of botryoidal cells and the basal lamina decreases until there is complete detachment. Botryoidal cells containing only melanin granules can freely move in the circulating fluid and can be mobilized towards a lesioned area where melanin will be utilized in immuno-defense processes such as encapsulation. The immuno-defense responses of leeches to various treatments, i.e. injection of living and non-living antigens, explants and grafts, suggests that these stimuli evoke pathways analogous to those observed in many other invertebrates and vertebrates. References BLANCO G.A., ESCALADA A.M., ALVAREZ E. & S. HAJOS 1997 LPS-induced stimulation of phagocytosis in the sipunculan worm Themiste petricola: possible involvement of human CD14, CD11b and CD11c cross-reactive molecules. Dev. Comp. Immunol 21: 349-362. COSSARIZZA A., COOPER E.L., SUZUKI M.M., SALVIOLI S., CAPRI M., GRI G., QUAGLINO D. & C. FRANCESCHI 1996. Earthworm leukocytes that are not phagocytic and cross-react with several human epitopes can kill human tumor cell lines. Exptl. Cell Res. 224: 174-182. DE EGUILEOR M., GRIMALDI A., TETTAMANTI G., BOSELLI A., SCAR G., VALVASSORI R., COOPER E.L. & G. LANZAVECCHIA 2000. Lipopolysaccharide-dependent induction of leech leukocytes that cross react with vertebrate cellular differentiation markers. Tissue & Cell 32: 437-445. DE EGUILEOR M., GRIMALDI A., TETTAMANTI G., VALVASSORI R., COOPER E.L. & G. LANZAVECCHIA 2000. Different types of response to foreign antigens by leech leukocytes. Tissue & Cell 32: 40-48. DE EGUILEOR M., TETTAMANTI G., GRIMALDI A., LURATI S., BOSELLI A., SCARÌ G., VALVASSORI R., COOPER E.L., & G. LANZAVECCHIA 1999. Histopatological changes after induced injury in leeches. J. Invert. Pathol. 74: 14-28. FRANCESCHI C., COSSARIZZA A., MONTI D. & E. OTTAVIANI 1991 Cytotoxicity and immunocyte markers in cells from the freshwater snail Planorbarius corneus (L) (Gastropoda, Pulmonata): implications for the evolution of the natural killer cells. Eur. J. Immunol 21: 489-493. MANN K.H. 1962. Leeches (Hirudinea): Their Structure, Physiology, Ecology and Embryology. Pergamon Press. Oxford. MANSOUR M.H. & E.L. COOPER 1984. Serological and partial characterization of a Thy-l homolog in tunicates. Eur. J. Immunol. 14: 1031-1039. MANSOUR M.H., DELANGE R. & E.L. COOPER 1985. Isolation, purification and amino acid composition of the tunicate hemocyte Thy-1 homolog. J. Biol. Chem. 260: 2681- 2686. NEGM H.I., MANSOUR M.H. & E.L. COOPER 1991a. Identification and structural characterization of Lyt-1 glycoproteins from tunicate hemocytes and mouse thymocytes. Comp. Biochem. Physiol. 99B: 741-749. NEGM H.I., MANSOUR M.H. & E.L. COOPER 1991b. Serological characterization and partial purification of a Lyt-1 homolog in tunicate hemocytes. Biol. Cell. 72: 249-257. NEGM H.I., MANSOUR M.H & E.L. COOPER 1992. Identification and structural characterization of a Lyt-2/3 homolog in tunicates. Comp. Biochem. Physiol. 101B: 55-67. PORCHET-HENNERÉ E. 1990. Cooperation between different coelomocyte populations during the encapsulation response of Nereis diversicolor demonstrated by using monoclonal antibodies. J. Invert. Pathol. 56: 353-361.

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PORCHET-HENNERÉ H., DUGIMONT T. & A. FISHER 1992. Natural killer cells in a lower invertebrate, Nereis diversicolor. Eur. J. Cell Biol. 58: 99-107. QUAGLINO D., COOPER E.L., SALVIOLI S. CAPRI M., SUZUKI M.M., RONCHETTI I.P., FRANCESCHI C. & A. COSSARIZZA 1996. Earthworm coelomocytes in vitro: cellular features and “granuloma” formation during cytotoxic activity against the mammalian tumor cell target K562. Eur. J. Cell Biol. 70: 278-288. ROCH P., COOPER E.L. & D.P. ESKINAZI 1983. Serological evidences for a membrane structure related to human 2-microglobulin expressed by certain earthworm leukocytes. Eur. J. Immunol. 13: 1037-1042. SAAD A.H. & E. COOPER 1990. Evidence for a Thy-1 like molecule expressed on earthworm leukocytes. Zool. Sci. 7: 217-222 SAWYER R.T. 1986. Leech Biology and Behaviour 1: Anatomy, Physiology and Behaviour. Oxford Scientific Publications, Oxford. STEIN E., AVTALION R.R. & E.L. COOPER 1977. The coelomocytes of the earthworm Lumbricus terrestris: morphology and phagocytic properties. J. Morph. 153: 467-478.

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Comparison of Molecular Neuroimmune Processes Between ... Evolution 147 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 147-157, 2003

Comparison of Molecular Neuroimmune Processes Between Leeches and Human M. Salzet1*, A. Tasiemski1, Ch. Lefebvre1 & E.L. Cooper2 1. Laboratoire d’Endocrinologie des Annélides, UPRES-A CNRS 8017, SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cédex, France. E-mail: [email protected] 2. Laboratory of Comparative Immunology, Department of Neurobiology School of Medicine, University of California, Los Angeles, California 90095-1763, USA

Abstract During evolution, invertebrates and vertebrates have maintained common signaling molecules such as neuropeptides. Complete hormonal-enzymatic systems for the biosynthesis of opioid peptides have been found in both the central nervous system (CNS) and immune systems (IS) of these animals. These signaling molecules act as immunomodulators in circulating blood. In vertebrates, release occurs during stress (cognitive or pathogens), which triggers the hypothalamo-hypophysial-adrenal (HPA) axis. These neuropeptides are also employed as conserved messengers that initiate and stimulate innate immune responses in invertebrates and in humans. Thus, cross talk between nervous and immune systems has an ancient evolutionary origin essential to homeostasis

Introduction: The neuroendocrinimmune system Neuroimmunological interactions can be considered essentially as a bi-directional exchange of information effected by classes of molecules, which were originally thought to be restricted to neural, endocrine or immune systems (Salzet et al. 2000a, Salzet 2000), (Fig. 1). These include neuropeptides such as corticotrophin releasing hormone (CRH), adrenocorticotrophic hormone (ACTH), monoamines (epinephrine, norepinephrine and dopamine), glucocorticoids, free radicals, cytokines such as interleukins IL-1, IL-6 and tumor necrosis factor (TNF) á, opioid peptides, opiates and endocannabinoids (Stefano et al. 1996, Weigent & Blalock 1997, Salzet et al. 2000a,b, c). Cytokines were originally described as small molecules allowing the precise cooperation between immunocompetent cells during the immune response involving vascular elements. Neuropeptides, originally described in the CNS, were also found to be expressed by immune cells and exhibit a number of immunomodulatory properties (Merril et al. 1996, Elmquist et al. 1997, Salzet et al. 2000a). A comparison of various immunocytes with

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Fig. 1. Interactions between the nervous and the immune systems in mammals. In case of information exchanges, the nervous system (which is stimulated by cognitive stresses) can control the immune one through the hypothalamo-hypophysio-adrenal axis. At the contrary, the immune response (for example, the bacteria challenge is induced by lipopolysaccharides) can alert the brain in order to trigger some process like fever. These interactions implicate molecules like neuropeptides and cytokines. It is interesting to note that some members of these two families have been found in both nervous and immune systems. This co-localization reinforces the idea that neuropeptides and cytokines are the principal messengers of this bi-directional communication. (With permission from Medicine and Sciences)

neuroendocrine cells from evolutionarily diverse organisms has been the consequence of these observations Immunocytes were shown to bear “receptors” for several neurohormones and hypothalamic releasing factors (Stefano et al. 1996, Weigent & Blalock 1997). They are also able to produce neurohormones after processing by pro-protein

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convertases, in response to neuroendocrine stimulation (Salzet et al., 2000a). Such overlapping elements between classic neuroendocrine and immune systems strongly suggest a level of cross talk that is mediated by a common currency of signaling molecules. The questions that arose concern the presence of such cross talk in invertebrates and a comparison with the vertebrate system. Annelid Immune System Modulation The effects on the immune system registered after injections of either lipopolysaccharide (LPS), a potent immune stimulator agent or morphine, a potent endocrine immunosuppressor, can be easily followed by the differential–display HPLC coupled to Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDITOF-MS). We performed these experiments in our annelid model, the leech Theromyzon tessulatum. Compared to controls (Fig. 2a), profiles obtained with LPS (Fig. 2b) or morphine (Fig. 2c) showed high differential display patterns. In leeches injected with LPS, several peaks emerged and some corresponded to previously isolated antimicrobial peptides e.g. peptide B (Tasiemski et al. 2000a), theromacin (Tasiemski et al. unpublished data) from leech coelomic fluid. By contrast, after morphine injections we found that most peaks emerging after LPS injections decreased and new sets of peaks appeared. Responses of annelid immune system to antimicrobial antigen stimulation Innate immune responses The most interesting results were those related to an antibacterial peptide (peptide B) present on a neuropeptide precursor i.e., the proenkephalin. This peptide is present at the C-terminal side of the leech proenkephalin (Salzet & Stefano 1997, Tasiemski et al. 2000a). This peptide is present in both neural tissue (neurons), immunocytes, and in nephridia (Tasiemski et al. 2000a). Levels of both peptide B and Methionine-enkephalin (Met-enkephalin) are significantly increased in response to LPS, surgical trauma, bacterial exposures or electric chocks at the brain level (Tasiemski et al. 2000a). Furthermore, the leech and vertebrate peptide were found to be very similar, exhibiting high sequence homology (95-98%) (Goumon et al. 1996, 1998, Tasiemski et al. 2000a). It is strongly antibacterial towards Gram + bacteria as indicated by in vivo and in vitro experiments (Goumon et al. 1996, 1998, Tasiemski et al. 2000a). It is important to note that proenkephalin-derived peptides are released independently of the external stimulus e.g., LPS (derived from Gram – bacteria), surgical trauma and electric shocks directed to neural tissues. All these treatments cause an increase in circulating levels of Gram + antibacterial peptide B and Met-enkephalin (Tasiemski et al. 2000a). Thus, we can surmise that the co-processing and liberation of peptide B and Met-enkephalin represents a unified neuroimmune protective response to an immediate threat to the organism, regardless of the form the stimulation takes (Stefano et al. 1998a, Tasiemski et al. 2000a, Salzet & Tasiemski 2000). Clearly, bacteria may accompany the above stimuli. Thus, to avoid the complication of a bacterial infection, peptide B is released for either specific bacterial assaults or as a precautionary action, whereas Metenkephalin stimulates or activates immunocytes during the initial stages of the response

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Fig. 2. Global analysis of peptides in coelomic fluid of the leech by MALDI-TOF MS Analysis of (the) peptide populations contained in coelomic fluid of the leech T. tessulatum was performed after acidic extraction. Samples were then directly mixed in the matrix then applied onto the target and exposed to an electron impact with a laser. A differential display analysis can be then performed by comparison of the mass spectra obtained for controls injected with LPS (100 µM/animal) or morphine (100 µM/animal). Arrows indicate significant changes observed by mass spectra comparison.

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(Tasiemski et al. 2000a, Salzet & Tasiemski 2000). Thus, this unified neuroimmune response would provide a highly beneficial survival strategy at the time it is most needed, at the beginning of a pro-inflammatory process (Fig. 3). A simultaneous presence of Met-enkephalin is equally important for this response. Met-enkephalin can be envisioned to activate immunocytes and to provide a chemotactic signal for further immunocyte recruitment (Stefano et al. 1996, Stefano et al. 1997, Tasiemski et al. 2000a, Salzet & Tasiemski 2000). However, this process may take many minutes to accomplish, hence the presence of bactericidal peptide B can during this latent period. Moreover, in this scenario, peptide B breaks down with time, it liberates the heptapeptide Met-enkephalinArg-Phe or the antibacterial peptide, enkelytin. Met-enkephalin-Arg-Phe is able to interact with the ä2 opioid receptor, as found in our earlier studies and in the displacement data, ensuring a continuation of the immunocyte-activated state, including chemotaxis (Stefano et al. 1989, 1991, 1995, Stefano & Salzet 1999, Tasiemski et al. 2000a). Comparative aspects of proenkephalin derived peptides in immunity In lower vertebrates e.g. amphibians, they contain in their dermal glands like in human skin (Harder et al. 1997), a variety of antibacterial peptides and neuropeptides related to opioids e.g. dermorphin and dermenkephalin, which are also released during stress (Vouille et al. 1997; Amiche et al. 1999). In humans, we demonstrated that in nonpathogenic models in which inflammatory processes occur in the absence of bacterial

Fig. 3. Simplified representation of methionine enkephalin’s (Met-Enk) immunoregulatory role. Based on the literature noted in the text we surmise that the proenkephalin precursor present in immunocytes, following immunocyte stimulation, i.e., lipopolysaccharide, is released in the hemolymph. Here, depending on the concentration and the identity of specific enzymes found in the hemolymph including immunocytes, the precursor is processed into its active but smaller peptides. In Met-Enk case, this molecule can stimulate a cytokine presence as well as stimulate more immunocytes to enter an area (chemotaxis) or wander in an area randomly once its concentration is very high (chemokinesis).

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infection, such as during coronary cardiology by-pass, time-course experiments have shown the presence of enkelytin, peptide B and opioids (methionine-enkephalin and methionine-enkephalin-Arg-Phe) in plasma before surgery (Tasiemski et al. 2000b). Their amounts are greatly increased just after skin incision. However, the origin of these peptides is not yet clear. We know that monocytes contain proenkephalin like T cells and B cells (Padros et al. 1989, Kamphuis et al. 1998). These cells are able to release proenkephalin derived peptides during inflammation by action of prohormone convertase (Vinendrola et al. 1990, Salzet et al. 2000a). However, we exclude that these peptides can be derived from skin (Nissen et al. 1997) or from the adrenals (Metz-Boutigue et al. 1998). Whatever their origin, proenkephalin-derived peptides are released immediately and are implicated in human innate immune response like in invertebrates. Taken together, these results demonstrates that a neuropeptide precursor like proenkephalin is implicated in innate immune responses. It is able to release antibacterial peptides that act to cover the latent period and in turn to stimulate the immune response. Moreover, it is implicated in the initiation of the immune response by releasing a neuropeptide, the Met-enkephalin that can now be considered as a cytokine. Responses of annelid immune system to endocrine stimulation: immunosuppression We previously demonstrated in leech brain, the presence of a morphine-like substance (Laurent et al. 2000) and a complete endogenous cannabinoid system (Salzet et al. 2000b, Matias et al. 2000). According to results, we confirmed our previous evidence supporting the involvement of morphine and cannabinoids as immune response inhibitors (Salzet et al. 1997) (Fig. 2c). Morphine and anandamide after binding to their own receptors (µ3 (Stefano et al., 1993) and CB1-like receptors (Stefano et al. 1997a), quickly stimulate transient calcium release (Nieto-Fernadez et al. 1999), then NO production by the constitutive NO synthase (cNOs) (Salzet et al. 1997, 1998b, 1998c, Stefano et al. 1997b, Salzet 2000). This NO production stimulates at both translational and transcriptional levels POMC, ACE and prohormone convertase (PC) genes related to POMC processing in ACTH and á-MSH peptide production (Salzet et al. 1997). By either an autocrine or paracrine action and binding to its own receptor (MCR-like), á-MSH down regulates invertebrate immunocytes as in humans (Lipton & Catania 1997, Salzet 2000). Furthermore, in this context, the hydrophilic form of leech ACE allows the ACTH conversion in á-MSH (Fig. 4, Laurent et al. 1998). In parallel, NO inhibits the stimulatory pathway, which is triggered by cytokines (interleukin 1 (IL-1)) through inhibition of the iNOs by action at the cyclase adenylate level (Laurent et al. 2000). Moreover, we demonstrate that in leech ganglia, injection of LPS provoked after a prolonged latency period of 24 hours, a significant increase of ganglionic morphine-like levels (from 2.4 + 1.1 pmol/gm to 78 + 12.3 pmol/gm (P<0.005, LPS injected 1 µg/ml). This phenomenon occurs at a concentration and time-dependent manner, demonstrating that bi-directional communication also occurs in leeches (Laurent et al. 2000). This confirms that cross talk between immune and CNS systems also exists in invertebrates. This communication acts to inhibit the immune response. CNS messengers therefore base molecular neuroimmune processes in leeches on equilibrium between stimulation and modulation of the immune response.

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Panel 1

Panel 2

A

B

Fig. 4. Panel 1: Effect of morphine (10-6 M) and anandamide (10-6 M) on the levels of ACTH- and á-MSH-like peptides in presence of phosphoramidon (Neutral endopeptidase inhibitor) or captopril (ACE inhibitor). Each experiment was run in triplicate and mean values were combined to obtain the mean of the means (SEM: P<0.01). Panel 2: (A) Time course experiments using Western blot analysis techniques of apparition in immunocyte membrane proteins solubilization in detergent rich fraction of leech ACE forms after morphine injection. a: control; b: 5 min post-injection, c: 15 min; d: 30 min; e: 45 min, f: 60 min. (B) Electrophoresis analysis of immunocytes membrane proteins solubilization in detergent poor fraction after morphine injection. a: control; b: 5 min postinjection, c: 15 min; d: 30 min; e: 45 min, f: 60 min.

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Comparative aspect of immunosuppressive responses in invertebrates and humans Knowing the above results found in invertebrates, it is interesting to determine if some of these molecular events can also occur in vertebrates during infections by pathogens or during cancer. Clearly we can use invertebrate models to understand human diseases. Morphine and cancer We recently demonstrated that human monocytes like invertebrates immunocytes contain a new opiate receptor subtype designated m3 (Stefano et al. 1993, Chuang et al. 1995, Fimiani et al. 2000). This subtype was found in lung, non-small lung cell carcinoma and granulocytes (Fimiani et al. 2000). In all positive human tissues analyzed, the identified transcripts appear to be the same but their size differs significantly from known µ opiate receptor subtypes, strongly suggesting that it represents a splice variant. Our binding studies indicate that this µ receptor is opiate alkaloid selective and opioid peptide insensitive. It is important to note that in those tissues that have the µ3 receptor, this receptor is coupled to NO release and that in tumors, morphine-stimulated NO release appears not to be under any feedback regulation. Finally, we observed multiple messages by Northern analysis. Studies now underway to sequence the larger PCR product will help us to determine if these cells have multiple types of µ receptors. We can now deduce that µ receptors are a central target for immunoregulation and inflammatory responses, with morphine being responsible for the suppression of these functions. The specific role of morphine in the modulation of cellular responsiveness to immunostimulating molecules is just now emerging. In fact, glucocorticoid/ mineralcorticoid and cytokine response elements (i.e., NF-interleukin IL-6 and NF-GMb) have been found in the mouse µ receptor gene, consistent with the fact that µ opioid receptors appear to be upregulated both by gonadal steroid hormones and IL-1â (Zhou & Hamme 1995). These data provide novel experimental findings regarding the expression of the µ opioid receptor gene in humans, thus supporting the view of more intriguing functions for these receptors in general homeostasis and immune surveillance in both invertebrates and humans. Evolution of neuropeptides Neuropeptides are widespread in the animal kingdom, including protozoans. The similar amino acid sequences of certain neuropeptides in vertebrates and invertebrates and conservation of their biological function demonstrate that they are stable and ancient in evolutionary terms. The enzymes implicated in their biosynthesis and catabolism are also well conserved during evolution. We demonstrated that except for receptors, which are of course important to establish the presence of a complete hormono-enzymatic system like RAS in invertebrates, most of its components are already present in invertebrates. Thus, given the wealth of information now emerging on these mammalianlike neuroendocrine processes found in invertebrates (Salzet et al. 2000a, Salzet 2000), it would appear that this system, in all probability, originated in “simple” animals (Salzet

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et al. 2000a,b,c, Salzet 2000). Therefore, in addition to its historical origin, it may be correct to designate the mammalian neuroendocrine system as invertebrate-like. Acknowledgements This work was supported in part by the MNERT, the CNRS, the ANVAR Nord Pas de Calais, the FEDER, the Conseil Régional de la Région Nord-Pas De Calais, and the NIH Fogarty INT 00045 grant. Edwin L. Cooper is partially supported by a NATO grant 971128 and is the recipient of the Alexander Von Humboldt Prize from Germany. References AMICHE M., SEON A.A., PIERRE T.N. & P. NICOLAS 1999. The dermaseptin precursors: a protein family with a common preproregion and a variable C-terminal antimicrobial domain. FEBS Lett. 45: 352-356. ANDERSON R., RABSON A., SHER R. & H. KOORNHOF 1976. Defective neutrophil motility in children with measles. J. Pediatrics 89(1): 27-32 CHUANG T.K., KILLAM K.F., CHUANG L.F., KUNG H.F., SHENG W.S., CHAO C.C., YU L. & R.Y CHUANG 1995. Mu opioid receptor gene expression in immune cells. Biochem. Biophys. Res. Commun. 216: 922-928. ELMQUIST J.K., SCAMMEL T.E., SAPER C.B. 1997. Mechanisms of CNS response to systemic immune challenge: the febrile response. Trends in Neurosci. 20: 565-570. FIMIANI C., ARCURI E., SANTONI A., RIALAS C.M., BILFINGER T.V., PETER D., SALZET B. & G.B. STEFANO 1999. The opiate µ3 receptor expression in non-neural human tissues: binding and coupling to nitric oxide release. Cancer Lett. 146: 45-51. FIMIANI C., MATTOKS D., CAVANI F., SALZET M., DEUTSCH D.G., PRYOR S., BILFINGER T.V. & G.B. STEFANO 1999. Morphine and anandamide stimulate intracellular calcium transiens in human arterial endothelial cells: coupling to nitric oxide release. Cell Signal. 11: 189-193. Goumon Y., Lugardon K., Kieffer B., Lefevre J.F., Van Dorsselaer A., Aunis D. & M.H. MetzBoutigue 1998. Characterization of antibacterial COOH-terminal proenkephalin-A-derived peptides (PEAP) in infectious fluids. Importance of enkelytin, the antibacterial PEAP209-237 secreted by stimulated chromaffin cells. J. Biol. Chem. 273(45): 29847-29856 GOUMON Y., STRUB J. M., MONIATTE M., NULLANS G., POTEUR, L., HUBERT P., VAN DORSSELAER A., AUNIS D. & M.H. METZ-BOUTIGUE. 1996. The C-terminal proenkephalin-A diphosphorylated peptide (209-237) from adrenal medullary chromaffin granules possesses antibacterial activity. Eur J Biochem 235(3): 516-525. HARDER J., BARTELS J., CHRISTOPHERS E. & J.M. SCHRÖDER 1997. A peptide antibiotic from human skin. Nature 387: 861. KAMPHUIS S., ERIKSSON F., KAVELAARS A., ZIJLSTRA J., VAN DE POL M., KUIS W. & C.J. HEIJNEN 1998. Role of endogenous pro-enkephalin A derived peptides in human T cell proliferation and monocyte IL-6 production. J. Neuroimmunol 84: 53-60. LAURENT V., SALZET B., VERGER-BOCQUET M., BERNET F. & M. SALZET 2000. Morphinelike substance in leech ganglia:neuroimmune implications. Eur. J. Biochem. 267: 2354-2362. LAURENT V., STEFANO G.B. & M. SALZET 1998. Leech angiotensin converting enzyme. Ann. N. Y. Acad. Sci. 839: 500-502.

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LIPTON J.M. & A. CATANIA 1997. Anti-inflammatory actions of the neuroimmunomodulator alpha-MSH. Immunol. Today 18: 140-145. MAGAZINE H.I., LIU Y., BILFINGER T.V., FRICCHIONE G.L. & G.B. STEFANO 1996. Morphine-induced conformational changes in human monocytes, granulocytes, and endothelial cells and in invertebrate immunocytes and microglia are mediated by nitric oxide. J. Immunol. 156: 4845-4853. MAGLIULO E. & R. BENZI-CIPELLI 1975. Impaired leukotaxis in viral hepatitis B. New England J. Med. 293: 303-304 MATIAS I., BISOGNO T., MELCK D., VANDENBULCKE F., VERGER-BOCQUET M., DE PETROCELLIS L., SERGHERAERT C., BRETON C., DI MARZO V. & M. SALZET (2000). Evidence for a cannabinoid system in the central nervous system of the leech Hirudo medicinalis. Mol. Brain Res. MATTOCKS D., SALZET M., SALZET B. & G.B. STEFANO. 1997. Anandamide-induced conformational changes in leech and mussel immunocytes are mediated by nitric oxide. Animal Biol. 6: 73-77. MERRILL J.E. & E.N. BENVENISTE 1996. Cytokines in inflammatory brain lesions: helpful and harmful. Trends in Neurosci. 19: 331-338. METZ-BOUTIGUE M.H., GOUMON Y., LUGARDON K., STRUB J.M. & D. AUNIS 1998. Antibacterial peptides are present in chromaffin cell secretory granules. Cell Mol Neurobiol 18: 249266. NIETO-FERNANDEZ F.E., MATTOKS D., CAVANI F., SALZET M. & G.B. STEFANO 1999. Morphine coupling to invertebrate immunocyte Nitric oxide release is dependant on intracellular calcium transients. Comp Biochem Physiol B Biochem Mol Biol.123: 295-299. NISSAN J.B. 1997. Enkephalin-like immunoreactivity in human skin is found selectively in a fraction of CD68-positive dermal cells: increase in enkephalin-positive cells in lesional psoriasis. Arch Dermatol Res 289: 265-271. PADROS M.R., VINDROLA O., ZUNSZAIN P., FAINBOIN L., FINKIELMAN S. & V.E. NAHMOD 1989. Mitogenic activation of the human lymphocytes induces the release of proenkephalin derived peptides. Life Sci 45: 1805-1811. SALZET M. 2000. Molecular Neuroimmune Processes. Brain Res. Rev. 34: 69-79. SALZET M., BRETON C., BISOGNO T. & V. DI MARZO 2000c. Comparative biology of endogenous Endocannabinoids system: possible role in immune response. Eur. J. Biochem. 267: 49174927. SALZET M., CAPRON A. & G.B. STEFANO 2000b. Molecular cross-talk in host parasite relationships: schistosomes or leeches -host interactions. Parasitol. Today 12: 536-540. SALZET M., SALZET B., COCQUERELLE C., VERGER-BOCQUET M., PRYOR S., LAURENT V. & G.B. STEFANO 1997. Biochemical and molecular characterization of ACTH, its precursor and receptor in the leech Theromyzon tessulatum : morphine increases ACTH levels. J. Immunol. 159: 5400-5411. SALZET M. & G.B. STEFANO 1997. Invertebrate proenkephalin: Delta opioid binding sites in leech ganglia and immunocytes. Brain Res 768: 232-240. SALZET M., VIEAU D. & R. DAY. 2000a. Cross-talk between nervous and immune systems through the animal kingdom: focus on opioids. Trends in Neurosc. 23: 500-505. SALZET M., VIEAU D. & G.B. STEFANO 1999. Serpins: an evolutionarily conserved survival strategy. Immunol. Today 20: 541-544.

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STEFANO G.B., CADET P. & B. SCHARRER 1989. Stimulatory effects of opioid neuropeptides on locomotory activity and conformational changes in invertebrate and human immunocytes: Evidence for a subtype of delta receptor. Proc. Natl. Acad. Sci. USA 86: 6307-6311. STEFANO G.B., CASARES F. & Y. LIU 1995. Naltrindole sensitive d2 opioid receptor mediates invertebrate immunocyte activation. Acta Hungaria 46: 321-327. STEFANO G.B. & M. SALZET 1999. Invertebrate opioid precursors: Evolutionary conservation and the significance of enzymatic processing. Intern. Rev. Cytol. 187: 261-286. STEFANO G.B., SALZET B. & G. FRICCHIONE 1998a. Enkelytin and opioid peptide association in invertebrate and vertebrate: Immune activation and pain. Immunol. Today 19: 243-48. STEFANO G.B., SALZET B., RIALAS C.M., POPE M., KUSTKA A., NEENAN K., PRYOR S. & M. SALZET 1997b. Mophine and anandamide-stimulates nitric oxide production inhibits presynaptic dopamine release. Brain Res. 763: 63-68. STEFANO G.B., SALZET-RAVEILLON B. & M. SALZET 1997a. Leech CNS cannabinoid receptor is coupled to nitric oxide release: high sequence homology with mammals. Brain Res. 753: 219-224. STEFANO G.B., SCHARRER B., BILFINGER T.V., SALZET M. & G.L. FRICCHIONE 1996b. A novel view of opiate tolerance. Adv. Neuroimmunol. 6: 265-277. STEFANO G.B., SCHARRER B., SMITH E.M., HUGHES T.K., MAGAZINE H.I., BILFINGER T.V., HARTMAN A., FRICCHIONE G.L., LIU Y. & M.H. MAKMAN 1996a. Opioid and opiate immunoregulatory processes. Crit. Rev. in Immuno. 16: 109-144. STEFANO G.B., SHIPP M.A. & B. SCHARRER 1991. A possible immunoregulatory function for Met-enkephalin-Arg6-Phe7 involving human and invertebrate granulocytes. J Neuroimmunol. 31: 97-103. TASIEMKI A., SALZET M., BENSON H., FRICCHIONE G.L., BILFINGER T.V., GOUMON Y., METZ-BOUTIGUE M.H., AUNIS D. & G.B. STEFANO 2000b. The presence of antibacterial peptides in human plasma during coronary artery bypass surgery. J Neuroimmunol. 109: 228235. TASIEMSKI, A., VERGER-BOCQUET, M., CADET M., GOUMON Y., METZ-BOUTIGUE M.H., AUNIS D., STEFANO G.B. & M. SALZET 2000a. Proenkephalin and innate immunity in invertebrates: the antibacterial peptide, peptide B. Mol. Brain Res. 76: 237-252. VINDROLA O., PADROS M.R., STERIN-PRYNC A., ASE A., FINKIELMAN S. & V. NAHMOD 1990. Proenkephalin system in human polymorphonuclear cells. Production and release of a novel 1.0-kD peptide derived from synenkephalin. J. Clin. Invest. 86: 531-537. VOUILLE V., AMICHE M., NICOLAS P. 1997. Structure of genes for dermaseptins B, antimicrobial peptides from frog skin. Exon-1 encoded prepropeptide is conserved in genes for peptides of highly different structures and activities. FEBS Lett. 91: 27-32. ZHOU L. & HAMMER R.P. Jr. 1995. Gonadal steroid hormones upregulate medial preoptic mureceptors in the rat. Eur. J. Pharmacol. 278: 271-280.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Bidirectional communication between the immune and ... Evolution 159 Theneuroendocrine New Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 159-166, 2003

Bidirectional communication between the immune and neuroendocrine systems: an evolutionary perspective M. Pestarino Dipartimento di Biologia Sperimentale, Ambientale e Applicata, Sezione di Neuroendocrinologia e Biologia dello Sviluppo, Università di Genova, viale Benedetto XV 5, 16132 Genova, Italy. E-mail: [email protected]

Abstract Bi-directional communication exists between animal’s immune and neuroendocrine systems; they share common hormones and receptors. Molecular characterization of their ligands, receptors and second messengers, provides further evidence of the structural and functional basis of neuroendocrinimmune interactions. Genes expressed in neuroendocrine and immune systems have been cloned and characterized. Understanding how the immune system influences the nervous system’s function has been hampered by mammalian. Alternative models for investigating cellular mechanisms of neural-immune interactions exist in mollusks and protochordates. Because immuno- and neuro-active molecules and receptors have also been described in unicellular eukaryotes, common messengers responsible for interactions probably evolved early during evolution.

Early history of the neuroendocrinimmune system The first experimental studies demonstrating an influence of the immune system on endocrine function were carried out by Bliss et al. (1954) and by Wexler et al. (1957). They demonstrated that administration of bacterial endotoxin or pyrogen increased plasma concentrations of adrenal corticosteroids in humans and rats. Later on, Chowers et al. (1966) postulated that pyrogen stimulated the secretion of an endogenous leukocytic substance responsible for increasing corticosteroid release. But only 25 years ago, Besedovsky et al. (1975) suggested that the immune system acts in concert with the nervous and endocrine systems to constitute an interactive network. In particular, Besedovsky proved that the immune system (IS) ‘talks back’ to the central nervous system (CNS), by activation of the hypothalamic–pituitary–adrenal (HPA) axis during immune responses. Moreover defects in this circuitry predispose pathogenic immune reactions in animal models with spontaneous or induced autoimmune diseases. Recent evidence indicates that neuroimmune interactions are modulated by a variety of signal transduction events involving cytokines, neurotransmitters, neurosteroids,

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neuropeptides, cyclic nucleotides, calcium and protein kinases. These molecules mediate cross talk between the three physiological systems to maintain homeostasis (Fig. 1). When pathological challenge or stress interferes, these systems become deregulated, often resulting in aberrant immune, neuronal or endocrine responses. Cytokines in the vertebrate brain Numerous immunoregulatory molecules, including interleukin-1α (IL1α), interleukin1β (IL1β), interleukin-3 (IL3), interleukin-6 (IL6), interferon (IFN) and tumor necrosis factor (TNF) are able to affect several hypothalamic functions, such as neuroendocrine secretions, sleep, thermoregulation and feeding in mammals (Koenig 1991). By contrast, these immune cell-derived factors are not capable of crossing the blood-brain barrier because of their large size. Therefore, local production of cytokines within the brain must be considered. In fact, their local production is responsible for fever and sleep induction and for hormone secretion. Peripheral stimuli can induce the local production of cytokines that act as neuromodulators and/or neurotransmitters in the brain. On the other hand, it is generally accepted that LPS stimulates IL1β production, in fact IL1β has been demonstrated in the cerebro-spinal fluid (CSF) of cats after the administration of bacterial lipopolysaccharide (LPS) (Coceani et al. 1988). From these data it is evident that neuroimmune responses are bi-directional with feedback loops, and are both facilitatory and inhibitory. Moreover, alterations of the neuroimmune axis are generally

Fig. 1. Molecular interactions in neuroimmune modulation, both during homeostasis and when defects in the interaction result in disease (redrawn from Chambers & Schauenstein 2000).

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small in magnitude and duration, and cross talk occurs between neurally derived substances and lymphokines. Considerable interest has been focused on IL1 because it is a highly pleiotropic molecule able to elicit many of the responses associated with acute and chronic inflammatory diseases. In most species studied, there are two different forms of IL1, α and β, which differ substantially in amino acid sequence and immunoreactivity, but not, in biological activity. Recently, a third form of IL1 has been identified and characterized by cDNA cloning. This form known as IL1Ra appears to be an IL1 antagonist able to induce biological responses (Zahedi et al. 1991). Immunoreactive IL1β has been found in nerve cell bodies and fibers within the hypothalamus, hippocampus and other regions of the mammalian brain. Moreover, IL1β mRNA has also been reported in these regions suggesting that this cytokine is synthesized in neuronal elements. Additionally, both astrocytes and microglia contain IL1β and its specific mRNA. IL1α appears to be produced exclusively by activated glial cells in the brain. Therefore IL1β is the predominant brain cytokine, possibly functioning as a neurotransmitter in some brain regions, such as the hypothalamus, and as a glial-derived growth factor exerting less specific effects that aid compensatory mechanisms following brain trauma. Brain cells such as astrocytes, oligodendrocytes, microglia, motor and sensory neurons, and pituitary cells respond to cytokines. Typically neuron-rich brain areas (the granule cell layer and the pyramidal cell layer of the dentate gyrus, the pyramidal cell layer of the hippocampus and the granule cell layer of the cerebellum) have been found to have high-density binding. Moderately high binding occurs in the anterior dorsal thalamus and ventromedial hypothalamus. Only a low density of IL1 binding has been found in the median eminence, the nucleus accumbens, the anterior hypothalamus, the mammillary and pontine nuclei, and the deep layers of the olfactory tubercle. The best known effect of IL1 on the CNS is its ability to induce fever during an inflammatory response, to affect other neuronal cell functions and to modify animal behavior such as modulation of food intake, induction of analgesia, increasing of CRF release and to regulate cell growth and differentiation. IL1 also exerts a stimulatory effect on ACTH secretion as well as on β-endorphin release. In particular actions of IL1 on the brain form part of an immunoregulatory loop in which IL1 secreted by cells of the immune system activates the HPA axis in some manner (Dunn 1990). The resulting glucocorticoid production limits the production of IL1 and immune activation. Therefore IL1 acts as a messenger signaling the brain that an immune activation is in progress and thus triggering a stress response. Cytokines in invertebrates As demonstrated by numerous studies, cytokines are present not only in vertebrates but also in invertebrates (Raftos et al. 1991a,b, 1992, 1998, Kelly et. al. 1992, 1993a,b, Beck et al. 1993). In Mollusks, interesting and effective experimental investigations have demonstrated that cytokine-like factors control many aspects of molluscan host defense responses (Ottaviani et al. 1993). Moreover, several studies have shown the expression of IL-1, TNF and IL-6 in hemocytes of deuterostome and protostome invertebrate species (Table 1). Invertebrate cytokine-like factors share many biological activities with their

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vertebrate counterparts. For example, as in mammalian species, invertebrate cytokinelike factors are expressed in response to antigenic stimuli as lipopolysaccharide (LPS) and zymosan. Furthermore, similarities in the structure and function of invertebrate and mammalian cytokines have been demonstrated. For example, human IL-1 has a molecular weight of 17,500 and two major charged forms. IL-1 isolated from a variety of deuterostome and protostome invertebrates has a molecular weight of between 18,000 and 22,000 and at least two major charged forms. At the same time, some invertebrate cytokine-like factors (e.g. IL-1) show biological activity when assayed in vertebrate systems. More recently, human IL-1 and IL-6 have been shown to induce changes in protein phosphorylation in the leech CNS. The ability of invertebrate cytokine-like molecules to be active in vertebrate systems and vice versa suggests that there is conservation of the three-dimensional structure required for interaction with vertebrate cytokine receptors. However, the extent of homology between vertebrate and invertebrate cytokine-like factors awaits more definitive analysis such as isolation and sequencing of the cDNA clones that encode them. Table 1. Examples of cytokine-like factors found in a variety of protostome and deuterostome invertebrates.

PHYLA

CYTOKINE-LIKE

MOLECULES

Mollusca Aplysia californica Planorbarius corneus Viviparus ater Biomphalaria glabrata Mytilus edulis

IL-1β, TNF-α IL-1·, IL-1β, IL-2, IL-6, TNF-α IL-1·, IL-1β, IL-2, IL-6, TNF-α IL-1β, TNF-α IL-1·, IL-1β, IL-2, IL-6, TNF-α

Samia cynthia Antheraea polyphemus Hyalophora cecropia Calliphora vomitoria

haemokinin haemokinin haemokinin TNF-α

Insecta

Annelida Eisenia foetida Echinodermata Asterias forbesi Pisaster ochraceus Tunicata Botryllus schlosseri Styela plicata Molgula occidentalis Ciona intestinalis Amaroucium pellucidum Styela clava Modified after Ottaviani & Franceschi (1997)

IL-1·, TNF-α IL-1, IL-6 IL-1 IL-1 IL-1β IL-1 IL-1 IL-1 IL-1β

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Tunicates are invertebrate deuterostomes characterized by some vertebrate features and they have been for a long time a useful model system to study the evolutionary pathways of chordates and in particular the evolution of neural-immune interactions at cellular and molecular levels. As demonstrated by numerous studies (for review see Cooper et al. 1992) ascidians do not express immunoglobulin antibodies but they have lectin-mediated complement pathway evolved before antibody-based complement activation mechanisms. It has also shown that ascidians express serine proteases that are closely related to vertebrate mannose binding lectin-associated serine protease (MASPs). More recently, calcium-independent agglutinin probably belonging to the galectin family has been found in the colonial ascidian, Botryllus schlosseri (Ballarin et al. 2000), and a collectin-like protein has been identified in Styela plicata (Nair et al. 2000). At the same time, cytokines have been found in tunicates. In particular, IL1β and its mRNA have been found in hemocytes and in neurons of the cerebral ganglion of the ascidian S. plicata (Pestarino et al. 1997, DeAnna et al. 1998). Neurotransmitters and the immune response In mammals, substantial evidence indicates that nervous, endocrine and immune systems communicate. Experimental results allow us to understand some of the main aspects of the neuro-endocrine-immune pathways such as the ability of neurally derived factors to modulate lymphocyte function. Lymphocytes are able to perceive neural modulation by interacting with neurotransmitters, neurohormones or neuropeptides, because lymphocytes have receptors for such signal molecules. Moreover lymphocytes express neurotransmitter receptors such as the serotonin receptors 5HT1A (Ferriere et al. 1996) and 5HT3 (Meyniel et al. 1997) and can synthesize catecholamines and acetylcholine. By contrast, receptors for immune-derived substances are also present in lymphocytes, and therefore the modulation of lymphocyte responses is based on crosstalk between second messengers (Roszman & Carlson 1991). There is substantial evidence confirming that neuropeptides and neurotransmitters are produced also by blood cells of nonmammalian vertebrates and invertebrates (see for review Ottaviani & Franceschi 1997). In particular, also protochordate hemocytes are also known to produce different neuropeptides (see for review Pestarino 1991). Messengers involved in communication If a bioregulator acts as inter- and intracellular messengers, receptor transformation must be involved. For instance, the action of cAMP as second messenger suggests that an internalization process takes place, because in E.coli, cAMP is produced in the same cell in which it exerts its effect by autocrine action and forms a complex with an extracellular specific protein. This protein is homologous to protein kinases, which are intracellular cAMP receptors in unicellular eukaryotes. With the development of multicellularity, cAMP cannot act as a cell-to-cell messenger because of its intrinsic chemical instability and can only carry information as an intracellular second messenger by means of cytosolic cAMPdependent protein kinases. The presence of membrane and cytosolic cAMP receptors suggests the development of three transitional stages during evolution (Table 2).

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Table 2. Evolutionary transition of cAMP-receptors: from membrane to cytosolic environment.

O RGAN ISM

TYPE OF CAMP

CAMP

RECEPTOR

FUN CTION S

Mem brane form Mem brane and cytosolic form s Cytosolic form

First m essenger First and second m essenger Second m essenger

IN TERACTION C AMP/ C AMP RECEPTOR

PROKARYOTE EUKARYOTE UN ICELLULAR

EUKARYOTE MULTICELLULAR

Escherichia coli Dyctiostelium discoideum Vertebrates and invertebrates

Au tocrine Paracrine End ocrine

Modified after Stoka (1999)

Perspectives on evolution and communication A phylogenetic analysis of distribution, biological functions and mechanisms of action of informational molecules is a logical way to gain insight into evolutionary trends and history (Cooper 1992). To explain the evolution of intercellular signalling, Geenen et al. (1981) proposed a model consisting of a primitive stage of cell-cell adhesion followed by a most complex neural networks. Interactions among signal molecules produced by nervous, endocrine and immune systems are based mainly on four physiological mechanisms: such as autocrine, paracrine, endocrine and neuroendocrine. But also an intracrine model seems to be present. Because identical molecular messages (TRH, LHRH, opioids, insulin and somatostain) are present in unicellular and multicellular organisms and in plants, intercellular communication may not be unique to organisms that possess well-developed neuroendocrinimmune systems. Thus nervous, endocrine and immune systems have had a common and early evolutionary origin. This “unifying theory of intercellular communication”, suggests unity of many, if not all, forms of intercellular communication (LeRoith et al. 1992). References BALLARIN L., TONELLO C., & A. SABBADIN 2000. Humoral opsonin from the colonial ascidian Botryllus schlosseri as a member of the galectin family. Mar. Biol. 136: 823-827. BECK G., O’BRIEN R.F., HABICHT G.S., STILLMAN D.L., COOPER E.L. & D.A. RAFTOS 1993. Invertebrate cytokines III: Invertebrate interleukin-1-like molecules stimulate phagocytosis by tunicate and echinoderm cells. Cell. Immunol. 146: 284-299. BESEDOVSKY H.O., SORKIN E., KELLER M. & J. MULLER 1975. Changes in blood hormone levels during the immune response. J. Proc. Soc. Exp. Biol. Med. 150: 466-70. BLISS E.L., MIGEON C.J., EIK-NES K., SANBERG A.A. & L.T. SAMUELS 1954. The effect of insulin, histamine, bacterial pyrogen, and the an abuse-alcohol reaction upon the levels of 17hydroxycorticosteroids in the peripheral blood of man. Metabolism 3: 493-497. CHAMBERS D.A. & K. SCHAUNENSTEIN 2000. Mindful immunology: neuroimmunomodulation. Immunol. Today 21: 168-170. CHOWERS I., HAMMEL H.T., EISENMAN J., ABRAMS R.M. & S.M. McCANN 1966. Comparison of effect of environmental and preoptic heating and pyrogen on plasma cortisol. Am. J. Physiol. 210: 606-610.

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CLATWORTHY A.L. 1998. Neural-Immune interactions–An evolutionary perspective. Neuroimmunomodulation 5: 136-142. COOPER E.L. 1992. Overview of immunoevolution. Boll. Zool. 59: 119-128. COOPER E. L., RINKEVICH B., UHLENBRUCK G. & P. VALEMBOIS 1992. Invertebrate Immunity: another viewpoint. Scand. J. Immunol. 35: 247-266. DEANNA E., MASINI M.A., STURLA M., CANDIANI S. & M. PESTARINO 1998. Expression of interleukin1β in blood cells of the ascidian Styela plicata. Animal Biol. 7: 91. DUNN A.J. 1990. Interleukin-1 as a stimulator of hormone secretion. Prog. NeuroendocrinImmunol. 3: 26-34. GEENEN V., MARTENS H., ROBERT F., LEGROS J.J., DEFRESNE P., BONIVER J., MARTIAL J., LEFEVRE P.J. & P. FRANCHIMONT 1991. Thymic cryptocrine signaling and the immune recognition of self neuroendocrine functions. Prog. NeuroendocrinImmunol. 4: 135-142. KELLY K.L., COOPER E.L. & D.A. RAFTOS 1992. Purification and characterization of a humoral opsonin from the solitary urochordate Styela clava. Comp. Biochem. Physiol. 103B: 749-753. KELLY K.L., COOPER E.L. & D.A. RAFTOS 1993a. Cytokine-like activities of a humoral opsonin from the solitary urochordate Styela clava. Zool. Sci. 10: 57-64. KELLY K.L., COOPER E.L. & D.A. RAFTOS 1993b. A humoral opsonin from the solitary urochordate Styela clava. Dev. Comp. Immunol. 17: 29-39. KOENIG J.I. 1991. Presence of cytokines in the hypothalamic-pituitary axis. Prog. NeuroendocrinImmunol. 4: 143-153. LEROITH D., SHEMER J., & C.T. ROBERTS 1992. Evolutionary origins of intercellular communication systems: implications for mammalian biology. Horm. Res. 38: 1-6. NAIR S.V., PEARCE S., GREEN P.L., MAHAJAN D., NEWTON R. & D.A. RAFTOS 2000. A collectin-like protein from tunicates. Comp. Biochem. Physiol. 125B: 279-289. OTTAVIANI E. & C. FRANCESCHI 1997. The invertebrate phagocytic immunocyte: clues to a common evolution of immune and neuroendocrine systems. Immunol. Today 18: 169-174. OTTAVIANI E., FRANCHINI A. & C. FRANCESCHI 1993. Presence of several cytokine-like molecules in molluscan hemocytes. Biochem. Byophys. Res. Commun. 195: 984-988. PESTARINO M. 1991. The neuroendocrine and immune system in protochordates. Adv. Neuroimmunol. 1: 114-123. PESTARINO M., DE ANNA E., MASINI M.A. & M. STURLA 1997. Localization of interleukin-1β mRNA in the cerebral ganglion of the protochordate, Styela plicata. Neurosci. Lett. 222: 151-154. RAFTOS DA, STILLMAN D.L. & E.L. COOPER 1998 Chemotactic responses of tunicate (Urochordata, Ascidiacea) hemocytes in vitro. J Invertebr Pathol 72: 44-49. RAFTOS D.A., COOPER E.L., HABICHT G.S. & G. BECK 1991a. Invertebrate cytokines: Tunicate cell proliferation stimulated by an interleukin 1-like molecule. Proc. Natl. Acad. Sci. USA 88: 9518-9522. RAFTOS D.A., COOPER E.L., STILLMAN D.L., HABICHT G.S. & G. BECK 1992. Invertebrate cytokines II: Release of interleukin-1-like molecules from tunicate hemocytes stimulated with zymosan. Lymphokine and Cytokine Research. 11: 235-240. RAFTOS D.A., STILLMAN D.L. & E.L. COOPER 1991b. Interleukin-2 and phytohemagglutinin stimulate the proliferation of tunicate cells. Immunol. Cell Biol. 69: 225-234. ROSZMAN T.L. & S.L. CARLSON 1991. Neural-immune interactions: circuits and networks. Prog. Neuroendocrinimmunol. 4: 69-78. ROTH J. & D. LEROITH 1997. Chemical cross talk. The Sciences 27: 50-55.

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STEFANO G.B., SALZET B. & G.L. FRICCHIONE 1998. Enkeletin and opioid peptide association in invertebrates and vertebrates: immune activation and pain. Immunol. Today 19: 265-268. STOKA A.M. 1999. Phylogeny and evolution of chemical communication: an endocrine approach. J. Mol. Endocrinol. 22: 207-225. WEXLER B.C., DOLGIN A.E., & E.W. TRYCZYNSKI 1957. Effects of a bacterial polysaccharide (pyrogen) on the pituitary-adrenal axis: further aspects of hypophyseal-mediated control of response. Endocrinology 61: 300-306. ZAHEDI M., SELDIN M.F., RITS M., Ezekowitz R.A.B. & A.S. WHITEHEAD 1991. Mouse IL-1 receptor antagonist: molecular characterization, gene mapping and expression of mRNA in vivo and in vitro. J. Immunol. 146: 4278-4281.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

How do cells of the invertebrate immune systems kill other cells? 167 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 167-175, 2003

How do cells of the invertebrate immune systems kill other cells? N. Parrinello1, M. Cammarata1, V. Arizza1, M. Vazzana1 & E.L. Cooper 2 1. Department of Animal Biology, University of Palermo, Palermo, Italy. E-mail: [email protected] 2. Laboratory of Comparative Immunology, Department of Neurobiology, University of California, Los Angeles, USA

Abstract Various cytotoxic cells and molecules succeeded in invertebrates and vertebrates. Both pore-forming system and prophenoloxidase activating cascade represent ancient killing mechanisms. Lytic peptides and proteins kill a variety of foreign cells acting according to the barrel-stave model. In phylogenetically distant groups (insect, ascidians, mammals), products (radical oxygen metabolites or quinones) from the melanogenetic pathway may be responsible for cytotoxic activity. In spite of functional similarity to mammal tyrosinase, phenoloxidases are more similar in their sequence to hemocyanin. In tunicates prophenoloxidase-containing cells may be activated by restricted and unrestricted recognition systems in producing cytotoxicity.

Introduction The invertebrates to survive in almost any kind of habitats developed a variety of successful defences. Structural and functional analogies as well as diversities between cytotoxic mechanisms and molecules witness the experiences of each species that live in a given environment. Cytotoxic activity arose very early in the evolution and appeared at all the phylogenetic levels. The first killing by an eukaryotic cell was intracellular, during phagocytosis against bacteria, while released toxins enhanced defence potentialities becoming active towards eukaryotic cells. Then, several cytotoxic mechanisms emerged and various toxic factors were experienced against a panel of cell targets eg. oxygen radical metabolites or nitric oxide, pore-forming peptides and proteins, proteinases. In this paper we show some aspects of pore forming systems, and the phenoloxidase linked cytotoxicity. Both are ancient killing mechanisms emerged independently in various animal groups.

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Pore-forming peptides: amoebopores and defensins In cytoplasmic granules of Entoamoeba histolytica, a family of peptides termed amoebopores are contained to fight intracellular microbial growth within the digestive vacuoles (reviewed by Leippe 1999). They act by forming transmembrane pores (Fig. 1). This molecular armament kills both engulfed bacteria in phagosomes and destroys host cells when cell-cell contact stimulates granular exocytosis. Sequence studies witness the need of functionally important structural units such as amphipatic alpha helices and intramolecular disulphide bonds that stabilize the structure. These structures form a prototype of an optimized folding plan for the killing of a variety of foreign cells. Similar to amoebopores they appear to be defensins of mammals, molluscs and insects (Hubert et al. 1996, Leippe 1999). A sequence analysis, using the six cysteine residues as landmarks, reveals marked differences between defensins from two molluscs, between insect and mollusc defensins as well as differences in the three dimensional structure between insect and mammal defensins have been found. The above mentioned data are in favour of an independent origin. Pore-forming proteins In mammals, cytotoxic T lymphocytes and NK cells kill targets including virus infected cells, tumour cell lines, parasites, and microorganisms. They use granule exocytosis and multiple mechanisms. Both utilize a pore-forming protein, the perforin, secreted following the interaction between effector-target cells (Podack & Tschopp 1982, Selsted & Ouellette 1995). The lysis results from the perforation of target cell membranes according to the “barrel-stave” model in which channels are formed by progressive oligomerization of a variable number of monomers around a central pore (Fig. 1). Plasma membrane permeabilization in itself can cause sufficient ionic imbalance to lead to the death of some targets. In addition, pores provide access for other granule-derived mediators. The mechanism is analogous to that produced by membrane attack complex of complement. In invertebrates, proteins usually named hemolysins, have been revealed by in vitro assay against not nucleated cells like erythrocytes (reviewed by Canicattì 1990). Lysin

A

B

Fig.1. Schematic illustrations of helical elements of amoebapores in phospholipids bilayers (A) and perforin-mediated target cell lysis (B)

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monomers polymerize on the target membrane bilayer opening channels leading to cell destruction. In cnidaria, molluscs, annelids and echinoderms, cytolytic factors produce erythrocyte membrane lesions. Irregular pores from 5 to 25 nm have been observed in the erythrocyte membrane by hemolysins from mussel, the echinoderm Holothuria polii, the annelids Eisenia fetida and Nereis diversicolor (Roch et al. 1989, Canicattì 1990, Hubert et al. 1997). In H. polii and Paracentrotus lividus hemolysins are contained in amoebocyte/ phagocyte granules (Pagliara et al. 1993). The hemolytic activity presents common characteristics: rapid time course, sigmoid dose-response curve, sensitivity to temperature and a wide range of pH stability. Subunit molecular weight and composition give diversity. Relationships between cytolytic complexes have not been shown. Lectins can also be pore-forming molecules. One among four C-type lectins from the holothurian Cucumaria echinata lyses rabbit and human erythrocytes, tumor cells and may be toxic to microbes (Hatakeyama et al. 1995). This lectin is a D-galactoside specific monomeric protein that has more than one sugar-binding site. Limulin is a sialic acid-specific lectin isolated from Limulus polyphemus that lyses sheep red blood cells (Asokan &Armstrong 1999). This lectin binds phosphorylethanolamine and probably forms aggregates of 300 kDa on erythrocyte membrane. The cytolysis is modulated by fast-form a2-macroglobulin (Armstrong & Quigley 1999). Following the binding to sugars on the target membrane, cytolytic lectins aggregate to form ion-permeable transmembrane pores. The target cells were ruptured by colloid osmotic shock. The phenoloxidase-linked cytotoxic activity derived from the melanogenetic pathway following an activating cascade. Melanogenesis is a protective ubiquitous process. In invertebrates, melanization may have additional roles including bacteria immobilization, wound repair and encapsulation (Soderhall & Cerenius 1998). It is a common response to parasites. Although melanin has not been shown in all the examined species the main enzyme, phenoloxidase (oxidoreductase) and intermediates from the melanogenetic pathway may be released from hemocytes in several body districts. In spite of functional similarity to mammal tyrosinase, phenoloxidase are more similar in their sequence to hemocyanin. In Table 1 we show the comparative matrix of arthropod phenoloxidase zymogens, Halocynthia tyrosinase, mammal tyrosinase and arthropod hemocyanins. The tyrosinase from Halocynthia roretzi larvae is an exception. It shows 48-49 per cent homology with mammalian tyrosinase. The presence of two functional copper-binding sites suggests that they diverged from a copper-containing protein. In mammals, tyrosinase initiates melanin synthesis by hydroxylating tyrosine to dihyLdihydroxyphenylalanine (dopa) and further oxydizing dopa to dopaquinone that is converted to dihydroxyindole-2-carboxilic acid or dihydroxyindole to indole-5,6quinone. Mammals can use both the indoles to produce melanin. In invertebrates, the phenoloxidase oxidizes phenols to dihydroxyindole to quinones that polymerize to form melanins. In immune responses, this enzyme is the end product of a prophenoloxidase

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Table 1. Comparative matrix of arthropod hemocyanins, prophenoloxidases, Halocynthia roretzi tyrosinase, mammalian tyrosinases; homology level (%) was calculated by DNASIS software.

Hemocyanins

Pro-PO

Tyrosinase (H.roretzi)

Hemocyanins

59-69

Pro-PO

-

46-59

46-47

3 -3

-------

45-46

33-3

-

Tyrosinase (H.roretzi) Tyrosinases (mammals)

Tyrosinases (mammals)

68-88

References: Linzen et al. 1985, Kwon et al. 1987, Yamamoto et al. 1989, Lang et al. 1991, Inagaki et al. 1994, Aspan et al. 1995, Fujimoto et al. 1995, Kawabata et al. 1995, Sato et al. 1997.

(proPO)-activating system. In crustaceans, the proenzyme may be activated inside or outside the hemocytes by β1,3-glucan, peptidoglycans, lipopolysaccharides, from fungi and microorganisms (Johansson & Soderhall 1996). Biological functions includes melanization, antimicrobial activity, degranulation factors, cell adhesion factors, opsonins, clotting. Recognition occurs through humoral or cellular factors that activate a serine protease that cleaves proPO at a specific peptide bond (Fig. 2). Phenoloxidasecontaining hemocytes can act as cytotoxic cells in insect immune reactions; radical oxygen intermediates generated from the melanogenetic pathway are cytotoxic factors (Nappi & Vass 1993). In mammals, some studies on the potential therapy of malignant melanoma showed that intermediates of melanin metabolism within melanocytes were potentially cytotoxic (Smit et al. 1992). During melanogenesis, phenolic compounds may be converted into toxic products (eg. reactive ortho-quinone). A phenoloxidase-linked cytotoxic mechanism is present in tunicates. Tunicates have a crucial phylogenetic position between invertebrates and vertebrates. They possess immune responses that depend on polymorphic loci that control inflammatory reactions, contact reaction between allogeneic and xenogeneic hemocytes, and allograft reactions. These occur in solitary ascidians in which codominant multiple alleles (at least six) of a single gene locus encode cellular histocompatibility antigens, non fusion reaction (NFR) as is the case in colonial ascidians. When two colonies are placed in mutual contact, histocompatibility appears to be controlled by a single locus (Fu/HC) with approximately 100 codominatly expressed alleles (reviewed by Parrinello 1996). The ascidian hemocytes are classified into several groups. They are usually known as hemoblasts and lymphocyte-like cells that are stem cells, hyaline amoebocytes, granular amoebocytes with small or large granules, signet ring cells with a single large vacuole, compartment cells that contain a variable number of large round and angular vacuoles distributed at the cell’s periphery (Wright 1981). Morula cells (8-16 µm, MC) may assume a berry-like or morula appearance because they contain variable numbers

How do cells of the invertebrate immune systems kill other cells?

β-1,3-glucans

Peptidoglycan

Lipopolysaccharide

Recognition molecules

Mediators of immuno defence

171

β-glucan binding protein LPS-binding protein Söderhäll and Cerenius 1997

Serine proteases Protease inhibitors

Phenoloxidase

proPO Phenols

O2

Transitional-metal ions Quinones

Cytotoxic quinonoid compounds

Cytotoxic oxygen radicals Melanin Fig. 2. A model for the activation of the proPO system in ascidians.

of tightly packed vacuoles (2-3 µm) or globules that contain dense inclusions. In several ascidian species, phenoloxidase is contained in morula cells in which polyphenols that act as substrates are found. The PO activity has been assayed in hemocyte lysate supernatants by reacting with dopa or 3-methyl-2-benzothiazolonehydrazone (MBTH) following proenzyme activation by β-1,3 glucan, LPS and proteolysis by trypsin (Arizza et al. 1995). Cytochemical reactions have been performed by means of dopa and MBTH oxidation (Cammarata et al. 1997, Ballarin et al. 1998). Phenoloxidase-containing hemocytes are involved in immune reactions. In colonial ascidians (Botryllus primigenius, B. schlosseri, Botrylloides simodensis, B. fuscus, B. violaceus) NFR, morula cells can recognize allogeneic humoral factors diffusing from incompatible colonies, move chemotactically, infiltrate into the fused tunic where they disintegrate outside and inside of the ampullae and discharge their vacuolar contents (Shirae 1999). In the solitary ascidian (Styela plicata) morula cells and lymphocyte-like

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cells (LLCs), are mainly involved in tunic transplantation rejection (see Parrinello 1996). LLCs accumulate around allogeneic blood vessels and are capable of a specific adaptive responses. Morula cells are the most frequent in autografts, first-set and second-set allografts. In allografts their numbers are significantly higher than those of LLCs. Based upon certain morphological and cytochemical data on hemocytes derived from hemolymph, we suggest that morula cells develop from LLCs (Parrinello et al., 2001). Their massive presence could be related to disruption of allogeneic tissue. Another ascidian Ciona intestinalis possesses a specific cell type called “Univacuolar Refringent Granule Cell” (URG) that may be added to the cell line that includes morula cells. URG is characterized by a unique granule that is refringent to light microscopy. Electron microscopy reveal homogeneous and compact finely granular material within the vacuole, the cytoplasm is reduced and the nucleus is flattened and eccentric (De Leo 1992). Both hemocytes show phenoloxidase activity. When phenoloxidase activity of hemocyte lysates was examined, LPS laminarin and, proteinases enhance enzyme activity, while enzyme inhibitors abolish the activity (Arizza et al. 1995). To examine the immunological properties of PO-containing hemocytes, enriched cell populations separated by a discontinuous Percoll gradient have been assayed. Both S. plicata morula cells and C. intestinalis URGs possess PO-linked spontaneous cytotoxic activity when activated by erythrocytes or tumor cells (Cammarata et al. 1997). When assayed against rabbit erythrocytes, both cell types prepared in a suitable medium release toxic factors that form lytic plaques in erythrocyte monolayers. One effector hemocyte can rapidly (few minutes) act on several erythrocytes (E: T ratio of 1:8 and 1:10). Cytotoxicity is related to phenoloxidase activity (Fig. 2). As analyzed by SDS-PAGE, this enzyme appears to be localized at 121kDa and 175 kDa. Phenoloxidase inhibitors, phenylthiourea, tropolone and sodium benzoate inhibit cytotoxic activity. Differences between cytotoxic cells of the two ascidians concern effectiveness of phenolthiourea, sphingomyelin inhibition and target cell types. These differences may be related to the variety of target membrane components. Both Ciona and Styela species have been crucial models in understanding various aspects of cytotoxicity in relation to various kinds of targets, (Kelly et al. 1992, Peddie & Smith 1993, Cammarata et al. 1995, Raftos & Hutchinson 1995). Different factors from phenoloxidase pathway are cytotoxic in solitary and colonial ascidians As already shown in vertebrates, cytotoxic activity may be related to quinones and semiquinones. They can cause lipid peroxidation, react with -SH groups, reduce oxygen to superoxide anions, giving rise to peroxide, hydroxylradicals, and singlet oxygen. The formation of quinone metilure that is highly cytotoxic cannot be excluded. In Ciona and Styela, treatments with the antioxidant enzymes, superoxide dismutase and catalase, with sodium nitroprusside that is an inhibitor of endogenous superoxide dismutase, were ineffective. These treatments rule out oxy-radicals as being responsible for spontaneous cytotoxicity, while MBTH inhibition, a quinone scavenger, supports the view that quinone compounds may be the effector cytotoxic molecules. Since inhibition of nitric oxide synthase by nitro-l-arginine did not affect the activity, even nitric oxide

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activity was ruled out. On the contrary, a PO-linked mechanism mainly due to oxidative stress by oxy radical metabolites has been found in Botryllus schlosseri NFR (Ballarin et al. 1998). Perspectives on cytotoxic mechanisms Various cytotoxic mechanisms and molecules are highly effective in invertebrates and vertebrates. Pore-forming system is an ancient killing mechanism. Lytic peptides and proteins with functionally important structural units, such as amphipathic α-helices and stabilizing intramolecular disulphide bonds, represent an optimized folding plan for the killing a variety of foreign cells acting according to the barrel-stave model. Melanogenesis is also an ancient defence pathway that assumed various immune roles following a prophenoloxidase activating cascade. The main enzymes, prophenoloxidase and tyrosinase, diverged early in the evolution from a common copper-containing protein. Products from the melanogenetic pathway may be responsible for cytotoxic activity. In phylogenetically distant groups (e.g. insects, ascidians, mammals) radical oxygen metabolites or quinone products may be responsible of cytotoxic activity. In tunicates prophenoloxidase-containing cells may be activated by restricted and unrestricted recognition systems in producing cytotoxicity in non-fusion reactions and tunic allograft responses. In vitro experiments have revealed that radical oxygen metabolites in colonial ascidians or quinone products in solitary ascidians are toxins. Acknowledgement This study was supported by a grant to MURST 60% to Nicolò Parrinello. Edwin L. Cooper is partially supported by a NATO grant 971128 and is the recipient of the Alexander Von Humboldt Prize from Germany References ARIZZA V., CAMMARATA M., TOMASINO M.C., & N. PARRINELLO 1995. Phenoloxidase characterization in vacuolar hemocytes from the solitary ascidians Styela plicata. J. Invert. Pathol. 66: 297-302. ARMSTRONG P.B. & J.P. QUIGLEY 1999. a2-macroglobulin: an evolutionary conserved arm of the innate immune system. Dev. Comp. Immunol. 23: 375-390. ASOKAN R. & P.B. ARMSTRONG 1999. Cellular mechanisms of hemolysis by the protein Limulin, a sialic-acid-specific lectin from the plasma of American horseshoe crab, Limulus polyphemus. Biol. Bull. 197: 275-276. ASPAN A., HUANG T.S., CERENIUS L., & K. SÖDERHÄLL 1995. cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc. Natl. Acad. Sci. USA 92: 939-943. BALLARIN L., CIMA F., & A. A. SABBADIN 1998. Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri. Dev. Comp. Immunol. 22: 479-492.

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PARRINELLO N. 1996. Cytotoxic Activity of Tunicate Hemocytes. In Rinkevich B. and W.E.G. Muller (eds.), Invertebrate immunology. Springer, Berlin, pp. 190-216. PARRINELLO N., CAMMARATA M., VAZZANA M., ARIZZA V., VIZZINI V., & E.L. COOPER 2001. Immunological activity of ascidian hemocytes. In H. Sawada, H. Yokosawa and C.C. Lambert (eds.), The Biology of Ascidians Springer-Verlag Tokio pp. 395-401. PARRINELLO N., GRIMALDI M., VAZZANA M. & M. CAMMARATA in press. Properties of PO-dependent cytotoxic activity of ascidian hemocytes against mammalian erythrocytes and K562 tumor cells. J. Inv. Pathol. PEDDIE C.M. & V.J. SMITH 1993. In vitro spontaneous cytotoxic activity against mammalian target cells by the hemocytes of the solitary ascidian, Ciona intestinalis. J. Exp. Zool, 267: 616-623. PODACK E.R. & J. TSCHOPP 1982. Circular polymerization of the ninth component of complement. J. Biol. Chem. 257: 15204-15212. RAFTOS D.A. & A. HUTCNINSON 1995 Cytotoxicity reactions in the solitary tunicate Styela plicata. Dev. Comp. Immunol. 19: 463-471. ROCH P., CANICATTÌ C., & VALEMBOIS P. 1989. Interactions between hemolysins and sheep red blood cell membrane. Bioch. Biophys. Acta 983: 193-198. SATO S., MASUYA H., NUMAKUNAI T., SATO N., IKEO K., GOJOBORO T., TAMURA K., IDE H., TAKEUCHI T. & H. YAMAMOTO 1997. Ascidian tyrosinase gene: its unique structure and expression in the developing brain. Dev. Dyn. 208: 363-374. SELSTED M.E. & A.J. OUELETTE 1995. Defensins in granules of phagocytic and non-phagocytic cells. Trends. Cell Biol. 50: 114-119. SHIRAE M., HIROSE E. & Y. SAITO 1999. Behavior of hemocytes in the allorejection reaction in two compound ascidians, Botryllus scalaris and Symplegma reptans. Biol. Bull. 197: 188-197. SMIT N.P.M., PETERS K., MENKO W., WESTERORHOF W., PAVEL S. & P.A. RILEY 1992. Cytotoxicity of a selected series of substituted phenols toward cultured melanoma cells. Melanoma Res. 2: 295-300. SODERHALL K. & L. CERENIUS 1998. Role of the prophenoloxidase-activating system in invertebrate immunity. Cur. Opinion Immunol. 10: 23-28. WRIGHT R.K. 1981. Urochordates In Ratcliffe NA & AF Rowley (eds.), Invertebrate Blood Cells. Academic Press, New York, vol.2, pp. 565-626. YAMAMOTO H., TAKEUCHI S., KUDO T., SATO C. & T. TAKEUCHI 1989. Melanin production in cultured albino melanocytes transfected with mouse tyrosinase cDNA. Jpn. Genet. 66: 121-135.

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Originality of the Mytilus (Bivalve Mollusc) antibacterial peptides ... Evolution 177 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 177-184, 2003

Originality of the Mytilus (Bivalve Mollusc) antibacterial peptides: structurally related to Insects but involved as in Mammals Ph. Roch1, G. Mitta1*, F. Vandenbulcke2, M. Salzet2, A. Aumelas3, Y.-S. Yang3, A. Chavanieu3 & B. Calas3 1. Défense et Résistance chez les Invertébrés Marins (DRIM), Université de Montpellier 2, cc 80, 34095 Montpellier cedex 5, France. E-mail: [email protected] * Present address: Biologie des Populations d’Helminthes Parasites, Université de Perpignan, France 2. Laboratoire d’Endocrinologie des Annélides, Université de Lille 1, France 3. Centre de Biochimie Structurale, Université de Montpellier 1, France

Abstract Marine molluscs (mussels) have innate immune capacities: three groups of cationic, cysteine-rich, 4kDa peptides: defensins, mytilins and myticins, including isoforms with complementary antimicrobial properties. Three-dimensional structure of native and synthetic defensins revealed relationship with arthropod defensins. In contrast to arthropods, mussel peptides are continuously produced as precursors in hemocytes, before storage in granules. Confocal microscopy revealed hemocytes that contain defensins, or mytilins, both peptides and none. In response to infection, mussel peptides are involved both, (i) intra cellular on phagocytosed bacteria, and (ii) extra cellular on later systemic response. According to their behaviour towards infection, mussel hemocytes are apparently more closely related to mammal monocyte/macrophages than to insect-arthropod hemocytes. In addition, enterocytes contain mytilins that are released against gut microflora.

Introduction: Diseases in Molluscs From time to time, populations of cultivated molluscs, mainly the oysters, Crassostrea gigas, C. virginica, Ostrea edulis, and the mussels, Mytilus edulis, M. galloprovincialis, suffer from severe mortalities. Major phenomena responsible for the deleterious impact can be arranged into two groups. The first includes all modifications of the environment due to human activities and the second involves the occurrence of several diseases that result from an extended variety of pathogens. Among the high prevalence of diseases, one can observe two causes: (i) increasing bed density that enhances risks to encounter sick animals and favor contamination, and (ii) weakened animals caused by various stresses such as density, insufficient nutrients, pollution, temperature, salinity.

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Antimicrobial peptides are components of the innate immune effector repertoire. Since their discovery in the moth, Hyalophora cecropia, by Hans Boman’s group, in 1981 (see Boman 1995 for review), more than 400 peptides have been reported so far and are still being continuously discovered. Antimicrobial peptides are therefore most probably present in all living creatures, from plants to mammals. Even if the molecular structures are diverse, some common features allow their classification into a restricted number of families. In addition, peptides with different molecular structures can be similarly involved in the anti-infectious response. Diversity of the Mytilus antibacterial peptides Based on the pioneering work done in insects, several small proteins of 4 kDa were purified from mussel plasma and hemocyte granules according to specific characteristics (Charlet et al. 1996, Hubert et al. 1996). Eluting acid extract prepared from hemocyte granules in a reverse phase HPLC resulted in about 10 active fractions. Routinely, growth inhibition has been observed against Gram + Micrococcus luteus and the fungus Fusarium oxysporum. Elevated numbers of active fractions revealed the presence of several distinct molecules. Most peaks shared both antibacterial and anti fungal activities, whereas other peaks were only antibacterial. Complete sequence of the molecules revealed that they are of similar size and composed of 36-40 amino acids (Table 1). The presence of 6-8 cysteines is one remarkable, common feature (Roch 1999). Based on primary amino acid sequence homologies, mussel peptides have been arranged into 3 families: defensins related to arthropod defensins, Table 1. Diversity of mussel antimicrobial peptides (completed from Hubert et al. 1996, Charlet et al. 1996, Mitta et al. 1999a, 1999b, 2000a, Roch 1999).

Peptides Defensins Pep tid e A Pep tid e B MGD1 (Defensin A) MGD2 (Defensin B) Mytilins A B C D G1 (E)

My t ilus

N umber of amino acids

kD a

N umber of cysteines

edulis edulis

37 (p artial) 35 (p artial)

4.3 4.4

6 6

galloprovincialis galloprovincialis

39 39

4.4 as m RN A

8 8

edulis edulis-galloprovincialis galloprovincialis galloprovincialis galloprovincialis

34 34 31 (p artial) 34 36

3.7 3.9 4.2 3.8 4.1

8 8 7 (partial) 8 8

galloprovincialis galloprovincialis edulis galloprovincialis

40 40 32 (p artial) not sequenced

4.4 4.5 6.2 15

8 8 p robably 12 u nknow n

Myticins A B Mytim icin Antibacterial

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Fig. 1. Complete amino acid sequences of defensins, mytilins and myticins isolated from the Mediterranean mussel, Mytilus galloprovincialis, and structure of defensin MGD-1 as determined by 1H-NMR (PDB code: 1FJN) of both native and synthetic MGD-1. Note the presence of an α helix followed by two antiparallel β strands linked by four disulfide bonds indicated in white and labeled (partial from Mitta et al. 1999a, 1999b, 2000a & Yang et al. 2000).

mytilins and myticins that are originals. In M. galloprovincialis, all the peptides, regardless of the family, possess 8 cysteines arranged in specific conserved arrays (Mitta et al. 1999a, 1999b, 2000a) as reported for M. edulis mytilins. Meanwhile, M. edulis arthropod defensin related peptides A and B contain only 6 cysteines (Charlet et al. 1996). Slight differences in other amino acids determine several isoforms (Fig. 1). Genes encoding defensin B and mytilin B have been cloned and sequenced, revealing that both genes share the same organization including four exons and three introns (Mitta et al. 2000b). Three-dimensional structure of M. galloprovincialis defensins (MGD) Due to its homology with arthropod defensins, but with the distinction of two extra cysteines, we decided to determine the structure of mussel defensins. Chemical synthesis of MGD-1 was done in solid phase using the Fmoc (9-Fluorenylmethoxycarbonyl) strategy. The correct formation of the four disulphide bonds was deduced both from the similarity of the 1H-NMR spectra of the native and synthetic MGD-1 and their similar anti Gram + activity, (Yang et al. 2000). The solution structure of MGD-1 was established by 1H-NMR (Fig. 1) and appeared closely related to that of arthropod Phormia terranovae defensin A (Cornet et al. 1995) with an a helix followed by two anti-parallel b strands linked by three disulfide bonds constituting a typical cystine-stabilised-ab motif. Differences with arthropod defensin A consists in a shorter N-terminal loop and in a fourth disulfide bond (21-38) that attaches the N-terminal and the C-terminal components of the two β strands.

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Even if sharing protective activities against invading microorganisms, primary sequences and structures can be very different from one peptide family to another. Confusion exists with respect to the term defensin. For instance, whereas mammalian α and β human defensins consist of 3 b sheets linked by disulfide bonds (Hill et al. 1991), both arthropod, mussel and plant defensins (originally called thionins, Broekaert et al. 1995) possess an a helix and 2 b strands with a similar CS-ab motif (Hoffmann et al. 1999). Complementary antimicrobial activities Tested in vitro against a variety of microorganisms, all peptides inhibited the growth of Gram + bacteria. None were active against protozoa nor against Gram – bacteria, except against several Vibrios and a slight activity towards E. coli D31, an LPS mutant strain not representative of Gram – cell wall composition (Mitta et al. 2000a). According to isoforms, defensins, mytilins and myticins may or may not be active against fungi. For instance, mytilins B and D inhibited in vitro growth of Fusarium oxysporum; mytilins C and E exerted no effect. Similarly, myticin B was active, whereas myticin A was not. Consequently, activity spectra of mussel peptides appear to be extensive due to isoforms that possess broad target specificity. All mussel peptides possess bactericidal effects but with strong differences between isoforms. In vitro tested against Gram + M. luteus, defensin A and mytilin C killed all bacteria in less than 3 minutes after contact (Mitta et al. 2000a). By contrast, myticin A required 2 hours of contact and mytilin E more than 6 hours, revealing different kinetics of activity (Mitta et al. 1999a). In fact, apparently different families and within a family, the different isoforms possess complementary activities, both in terms of target specificity and kinetics. This might explain why mussels possess such a diversity of antimicrobial peptides. Expression in hemocytes Expression of defensin, mytilin and myticin genes is strictly restricted to hemocytes as demonstrated by Northern blots using mRNA prepared from circulating hemocytes, mantle, foot, labial palp, gill, hepatopancreas and adductor muscle. Faint bands of similar mobility might be observed especially in the mantle, due to the presence of infiltrating hemocytes that cannot be totally eliminated when extracting mRNA. To detect exceptions we used polyclonal anti-defensin and polyclonal anti-mytilin antibodies and found crossreacting material inside enterocytes that surround digestive lumens (Mitta et al. 1999b 2000c), as reported in mammals for Paneth cells (Ouellette & Selsted 1996) and in insects (Lehane et al. 1997). The labeled molecule was not identified, but one can hypothesize that antimicrobial peptides are secreted into the gut to kill potential pathogens and/or to participate in the degradation of proteins for nutrition. Immunochemistry at both optical and ultrastructural levels showed that defensins are located primarily in vesicles of a hemocytic granulocyte subtype that contains small granules and in large clear granules of another granulocytic subtype (Mitta et al. 1999b). Immunofluorescence performed with polyclonal anti-mytilin antibodies indicated that labeling is restricted to one granulocyte subtype. Inside hemocytes, two structures were labeled: large dense granules of 0.5-1.5 µm and multivesicular

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structures of 3-8 µm (Mitta et al. 2000a). Confocal microscopic observations performed with both anti-defensin and anti-mytilin antibodies on separated and merged images revealed that at least these two peptide families can be detected: (i) in different hemocyte subtypes, (ii) in the same granulocyte but in different intra cellular compartments, and (iii) even in the same compartment (Mitta et al. 2000c). Defensinlabeling was detected as intensely fluorescent small spots distributed throughout the cytoplasm and surrounding the nucleus. Mytilin-immune reactivity was also detected throughout the cytoplasm, the labeling being more intense at the cell periphery. Different hemocytes can be positive for defensins (16 %) or mytilins (37 %), but both immune reactivities often appeared within the same hemocyte, packed in different compartments (11 %) or in the same compartment (21 %). It is important that 15 % of circulating hemocytes did not stain for defensins or mytilins. Dynamics of the reaction Injecting heat killed Vibrio alginolyticus into the adductor muscle resulted in a rapid decrease in the number of circulating hemocytes as soon as one-hour post-injection. These numbers will not return to baseline even 24 h after challenge. In fact, circulating hemocytes accumulated at the injection site as demonstrated by histological localization of mytilin positive cells suggesting recruitment of peptide bearing hemocytes at the site of injury (Mitta et al. 2000a). Mytilin plasma content did not change during the first hours following bacteria injection but dramatically increased between 24 and 72 h post injection. In fact, we observed hemocyte peripheral migration of peptide and exocytosis pictures 24 h post injection, suggesting an active process of secretion that results in higher content of peptides in the circulation (Mitta et al. 1999b). None of these modifications neither in circulating hemocyte numbers nor in peptide plasma content were observed following saline injection. Consequently, the response is somewhat specific depending upon the injected material, whatever it is living or dead. Co-localization of peptides and bacteria Bacteria and peptides were simultaneously observed using double immune labeling in confocal microscopy of hemocytes incubated in vitro with heat-killed V. alginolyticus. On the first minute of contact, some bacteria were found simply adsorbed onto the hemocyte membrane. Few were internalized suggesting phagocytosis. Furthermore, in some cases, mytilin-reactivity and bacteria were found closely associated (Mitta et al. 2000c). The number of internalized bacteria increased with time, as well as the number of mytilin and bacteria co-localization events. In contrast, the number of absorbed bacteria, which increased in the first 10 min of contact, decreased in 20 min, at the time when numerous bacteria were internalized. Sub-cellular location of bacteria and peptides were investigated by electron microscopy. In some cases, phagocytosed bacteria were sequestered in phagosomes. In numerous cases, mytilin-labeling was observed inside cellular organelles that also contained bacteria, resembling phagolysosomes. Open contacts between mytilincontaining granules and phagosome-containing bacteria argue in favor of their

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fusion. Within phagolysosomes, bacteria may encounter high antimicrobial peptide concentrations suitable to kill them. The model In contrast to arthropods, mussel antimicrobial peptides are continuously synthesized and stored as mature forms in different hemocytes. Consequently, large amounts of active peptides are rapidly available, before any neo-synthesis occurs. In addition, hemocytes were not infiltrating equally into various tissues. For instance, mytilin and myticin expressing hemocytes are well represented in gills, whereas no defensin positive hemocytes have been found. The fact that various peptides are transported to different organs might be according to the pathogen, driven by an unknown danger signal. Since defensin immune reactivity has never been detected in phagocytosing cells, the granulocyte subtype that contains only mytilins seems to be the only one involved in phagocytosis of bacteria. Both defensin and mytilin concentrations increased in the plasma 24 h after challenge, being released by hemocytes through exocytosis. Colocalization in the same granules supports the hypothesis that both peptide families may be simultaneously released. Consequently, the granulocytes containing only mytilins and those that contain both mytilins and defensins are probably involved at different stages of the anti-infectious response: a. Cells containing mytilins (37 % of total circulating hemocytes): involved in an early phase response by migrating towards infectious sites, phagocytosing microorganisms and destroying them in phagolysosomes; b. Cells containing both mytilins and defensins (32 %): involved 24 h after contact by releasing both peptides into the plasma; c. Cells containing defensins (16 %): not involved in phagocytosis but releasing the peptide 24 h later; d. Cells not containing defensins or mytilins (15 %): unknown activity. In contrast to the fruit-fly, Drosophila melanogaster, where a variety of peptides are rapidly synthesized by the fat body and immediately secreted (Hoffmann et al. 1996), and to the chelicerate, Tachypleus tridentatus, where hemocytes immediately degranulate releasing the stored peptides (Shigenaga et al. 1990), the mussel model exhibits similarities with those described for human neutrophil α-defensin (Ganz et al. 1985) and the mussel hemocytes with cells of the human monocyte/macrophage lineage. Acknowledgements This work was partially supported by a grant from the Commission of the European Communities (FAIR-CT97-3691). DRIM is a joint research unit funded by the Institut Français pour l’Exploitation de la Mer (IFREMER), the Centre National de la Recherche Scientifique (CNRS) and the University of Montpellier 2. GM was supported by a doctoral grant from IFREMER.

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References BOMAN H. 1995. Peptide antibiotics and their role in innate immunity. Ann. Rev. Immunol. 13: 61-92. BROEKAERT W.F., TERRAS F.R.G., CAMMUE B.P.A. & R.W. OSBORN 1995. Plant defensins: novel antimicrobial peptides as components of the host defence system. Plant Physiol. 108: 1353-1358. CHARLET M., CHERNYSH S., PHILIPPE H., HÉTRU C., HOFFMANN J.A. & P. BULET 1996. Innate immunity: Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc Mytilus edulis. J. Biol. Chem. 271: 21808-21813. CORNET B., BONMATIN J.M., HÉTRU C., HOFFMANN J.A., PTAK M. & F. VOVELLE 1995. Refined tree-dimensional solution structure of insect defensin A. Structure 3: 435-448. GANZ T., SELSTED M.E., SZKLAREK D., HARWIG S.S., DAHER K., BAINTON D.F. & R.I. LEHRER 1985. Defensins. Natural peptide antibiotics of human neutrophils. J. Clin. Invest. 76: 1427-1435. HILL C.P., YEE J., SELSTED M.E. & D. EISENBERG 1991. Crystal structure of defensin HNP-3, an amphiphilic dimer: mechanisms of membrane permeabilization. Science 251: 1481-1485. HOFFMANN J.A., KAFATOS F.C., JANEWAY JR C.A. & R.A.B. EZEKOWITZ 1999. Phylogenetic perspectives in innate immunity. Science 284: 1313-1318. HOFFMANN J.A., REICHART J.M. & C. HÉTRU 1996. Innate immunity in higher insects. Cur. Opin. Immunol. 8: 8-13. HUBERT F., NOËL T. & Ph. ROCH 1996. A member of the arthropod defensin family from edible Mediterranean mussels, Mytilus galloprovincialis. Eur. J. Biochem. 240: 302-306. LEHANE M.J., WU D. & S.M. LEHANE 1997. Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans. Proc. Nat. Acad. Sci. USA 94: 11502-11507. MITTA G., HUBERT F., DYRYNDA E.A., BOUDRY P. & Ph. ROCH 2000b. Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis. Develop. Comp. Immunol. 24: 381-393. MITTA G., HUBERT F., NOËL T. & Ph. ROCH 1999a. Myticin, a novel cysteine-rich antimicrobial peptide isolated from hemocytes and plasma of the mussel, Mytilus galloprovincialis. Eur. J. Biochem. 265: 71-78. MITTA G., VANDENBULCKE F., HUBERT F. & Ph. ROCH 1999b. Mussel defensins are synthesised and processed in granulocytes then released into plasma after bacterial challenge. J. Cell Sci. 112: 4233-4242. MITTA G., VANDENBULCKE F., HUBERT F., SALZET M. & Ph. ROCH 2000a. Involvement of mytilins in mussel antimicrobial defense. J. Biol. Chem. 275: 12954-12962. MITTA G., VANDENBULCKE F., NOËL T., ROMESTAND B., BEAUVILLAIN J.C., SALZET M. & Ph. ROCH 2000c. Differential distribution and defence involvement of antimicrobial peptides in mussel. J. Cell Sci. 113: 2759-2769. OUELLETTE A.J. & M.E. SELSTED 1996. Paneth cell defensins: Endogenous peptide components of intestinal host defense. FASEB J. 10: 1280-1289. ROCH Ph. 1999. Defense mechanisms and disease prevention in farmed marine invertebrates. Aquaculture 172: 125-145.

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SHIGENAGA T., MUTA T., TOH Y., TOKUNAGA F. & S. IWANAGA 1990. Antimicrobial tachyplesin peptide precursor cDNA cloning and cellular localisation in the horseshoe crab, Tachypleus tridentatus. J. Biol. Chem. 265: 21350-21354. YANG Y.S., MITTA G., CHAVANIEU A., CALAS B., SANCHEZ J.F., ROCH Ph. & A. AUMELAS 2000. Solution structure and activity of the synthetic four disulfide bond Mediterranean mussel defensin, MGD-1. Biochemistry 39: 14436-14447.

Evolution of body axis segmentation in the bilaterian radiation

Evolution as Reflected in Embryonic Development

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Evolution of body axis segmentation in theThe bilaterian radiation 187 New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 187-195, 2003

Evolution of body axis segmentation in the bilaterian radiation M. Shankland Section of Molecular Cell and Developmental Biology, Institute of Cellular and Molecular Biology, 1 University Station CO930, University of Texas at Austin, Austin, Texas 78712, USA. E-mail: [email protected]

Abstract The recent discovery that some of the same genes are expressed during the segmentation of arthropod, chordate and annelid embryos has led to the idea that segmentation is homologous between these groups and its origin deeply rooted in the bilaterian radiation. However, these arguments are based entirely on the presence of interphyletic similarities, and do not consider the relative likelihood of the alternative explanation, i.e. homoplasy. Only a small percentage of Drosophila segmentation genes appear to function as segmentation genes in other phyla, consistent with what would be expected as a result of homoplastic cooptation. In addition, a detailed comparison of engrailed gene products in fly and leech embryos reveals only a superficial similarity in the patterns of gene expression, and no apparent similarity in morphogenetic function or downstream genes. Thus, at the present time there is not a strong case for a conservation of segmentation mechanisms between any of these three phyla, and additional studies will be required to distinguish whether their segmentation is homologous or homoplastic.

There are many widely conserved features in the body plan of bilaterian animals (McGinnis & Krumlauf 1992), but there is considerable debate as to how many times body axis segmentation has arisen in this group. Some authors (De Robertis 1997, Kimmel 1996) view segmentation as a primitive feature of “urbilateria” (i.e. the last common ancestor of extant Bilateria), but others envision segmentation of the anteroposterior (AP) axis evolving multiple times within different parts of the bilaterian radiation (Patel et al. 1989, Willmer 1990). One of the most widespread and influential views of the twentieth century was that of Hyman (1940-1967), who proposed that annelids and arthropods are descended from a common segmented protostome ancestor, and that the segmentation found in chordates evolved independently. In this model the segmentation of annelids and arthropods is homologous - and should display an underlying commonality of developmental mechanism - whereas the segmentation of chordates is homoplastic to that of other phyla.

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Several aspects of this model have come into question since the advent of modern molecular biology. First, molecular phylogenies indicate that the annelids and arthropods are distantly related within the protostomes. The current wisdom is that Bilateria can be subdivided into three great clades (Aguinaldo et al. 1997, de Rosa et al. 1999), with the annelids, arthropods and chordates dispersed among them. All three of these clades include both segmented and unsegmented taxa, and the most parsimonious explanation for that character state distribution is that segmentation evolved independently on three separate occasions. On the other hand, molecular developmental analysis has revealed certain similarities in the genetic pathways used by arthropods, chordates, and annelids to generate segmentation. These mechanistic similarities have led other authors to the opposite conclusion, namely that segmentation evolved only once and is a deeply rooted homology that extends back to urbilateria (Kimmel 1996, De Robertis 1997). The aim of this article is to re-examine the latter idea. Arguments based on homology are inherently seductive because they offer a clear-cut explanation for similarity - i.e. common descent. But it is clear that homoplasy can shape organs or body plans towards a common end, and it is not always obvious what selective pressures or developmental constraints account for independent evolutionary trajectories converging on the same morphological endpoint. As discussed below, the arguments used to support an interphyletic conservation of segmentation simply discount homoplasy out of hand, and as such make no attempt to meet our most basic expectations of significance. Hox genes and segmentation A problem with many arguments in support of homology is that they include similarities that are not pertinent to the comparison at hand. The concept of homology hinges upon the identification of synapomorphies (shared derived characters), which should not be conflated with symplesiomorphies (shared ancestral characters). Consider the significance - or lack thereof - of the Hox gene cluster with regards to the interphyletic homology of segmentation. Annelids, arthropods and chordates all possess Hox gene clusters containing a number of orthologous genes (Graham et al. 1989, Kourakis et al. 1997). The genes within these clusters are expressed in nested domains along the AP axis, and the AP ordering of those expression domains is nearly identical between phyla. And in those segmented species in which genetics is feasible it has been shown that differential Hox gene expression brings about the morphological diversification of otherwise homologous segments, a phenomenon known as ‘segment identity’ (McGinnis & Krumlauf 1992, Le Mouellic et al. 1992, Bell et al. 1999). It is not uncommon to hear these interphyletic similarities in Hox gene utilization construed as evidence that segmentation is homologous between phyla. But this idea overlooks the fact that the role of the Hox gene cluster in the patterning of the AP axis is not a synapomorphy of the overtly segmented bilaterians, but rather of the bilaterian animals as a whole (Slack et al. 1993). Hox clusters are also found in unsegmented bilaterians, in which they show the same conserved chromosomal ordering, the same colinearity of gene expression along the AP axis, and a similar role in specification of positional identity along the AP axis (Salser & Kenyon 1994). In fact the distinction between Hox gene function in segmented and unsegmented animals is largely semantic

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- i.e. the “segment identity” of segmented animals is simply another name for regional identity. Thus, conservation of Hox gene function among segmented animals is but a reflection of a wider conservation, and is only relevant to the evolutionary origin of segmentation if we assume a priori that urbilateria was segmented. Interphyletic conservation of segmentation genes To accurately infer the evolutionary origin of segmentation, we must compare the developmental mechanisms that actually generate segmental repeats. An enormous amount has been learned about the genetic basis of segmentation in the arthropod Drosophila (reviewed by Martinez Arias 1993), and if we had a similarly detailed knowledge of the segmentation process in representative annelid and chordate species it would probably be obvious (or at least we can hope so) whether the underlying developmental mechanisms are homologous or homoplastic. But there is no annelid or chordate in which segmentation is understood at anything approaching the same level of mechanistic detail, and arguments in favor of interphyletic homology generally focus on similarities in the utilization of a few genes (Wedeen & Weisblat 1991, Müller et al. 1996, Holland et al. 1997). For some authors (Palmeirim et al. 1997), the fact that any gene serves as a ‘segmentation gene’ in two different phyla constitutes a compelling argument for homology of the segmentation process as a whole. But what we need to ask in this sort of situation is whether the alternative explanation - parallel cooptation of orthologous genes into independently evolved segmentation mechanisms - is sufficiently unlikely to be rejected. The fruit-fly has 1.36 X 104 protein-coding genes (Rubin et al. 2000), and there are about 40 of these genes which are directly involved in the generation of segmental iteration - i.e. their mutation yields a pair-rule or segment polarity phenotype (Martinez Arias 1993, Perrimon et al. 1996). What if we imagine a second animal species with a similar genomic complexity, assume that there is a 1:1 orthology of genes between the two species, and suppose that the ancestors of this Species X evolved segmentation independently of the arthropods? If each of the two lineages co-opted 40 segmentation genes at random, there would be a probability P < 0.01 that two or more of their segmentation genes would be orthologous. But not all genes are equally likely to be coopted to generate segmentation. Some types of protein (transcription factors; cell signaling molecules) are much more likely than others to be utilized as the developmental regulators of tissue patterning. In addition, it is clear that some molecular pathways are frequently co-opted by nonhomologous tissues to serve the same developmental function - e.g. the role of Notchdelta signaling in lateral inhibition (Greenwald 1998). The same could be true of genetic pathways that were preadapted to the generation of iterated morphologies in urbilateria, and hence preferentially likely to be co-opted whenever segmentation evolved among its descendants. There is no straightforward way to account for these genetic biases, but we can ask what would happen if only 10% of protein-coding genes are available to be co-opted as segmentation genes? Given this one restriction, random cooptation of genes into two independently evolved segmentation mechanisms would be expected to select three or more orthologous genes (out of a total of 40 segmentation genes) more than

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one-tenth of the time, i.e. P > 0.1. Viewed from this vantage point, it would seem rash to discount homoplastic segmentation as a plausible explanation solely because two phyletically distant species happen to use one or even a few of the same genes to generate segmentation. Vertebrate orthologues have been identified and characterized for nearly all of the known Drosophila pair-rule and segment polarity genes, and only one, hairy, has vertebrate orthologues that appear to be closely tied to the process of segmentation (Müller et al. 1996, Palmeirim et al. 1997). Evidence of another potentially conserved segmentation gene comes from analysis of the cephalochordate Amphioxus (Holland et al. 1997), which expresses its engrailed (en) orthologue in a segmentally repeated pattern in its anteriormost somites. But this latter observation is not as compelling given that the Amphioxus en gene is only expressed in a few segments, and en is clearly not required for normal segmentation in vertebrates. In either case, the available data indicate that only a small fraction of the Drosophila segmentation genes may be serving as segmentation genes in chordates as well, and - as discussed above - such a finding is not a strong argument against the independent evolution of segmentation in these two groups. However, one could make a compelling argument for homology of the segmentation process even if only a small percentage of genes are conserved so long as those genes are utilized in some similar way that is unlikely to have arisen by homoplasy. Indeed, this very argument was put forward following early reports that the hairy orthologue her1 displays “pair-rule” expression in zebrafish embryos (Müller et al. 1996), similar to the pair-rule expression of the fruit-fly gene. However, subsequent work has indicated that expression of her1 (and other veretebrate orthologues of hairy) does not consist of pair-rule stripes, but rather of travelling waves of changing width that occur at a temporal periodicity of once per segment (Holley et al. 2000). Thus the expression pattern of fruitfly and vertebrate hairy genes is not remarkably similar, and inferences based on such similarity have little foundation. I will focus the remainder of this presentation on the segmentation of annelids and arthropods, where an apparent conservation of en gene expression during embryonic segmentation has also been portrayed as evidence of a deeply rooted homology. Role of the en gene in annelid and arthropod segmentation The annelids and arthropods were frequently portrayed as sister groups (together forming the “Articulata”) during much of the twentieth century precisely because they are both segmented. Recent molecular phylogenies suggest that these two phyla are in fact much more distantly related (Aguinaldo et al. 1997, de Rosa et al. 1999), still it is possible that their segmentation is homologous if it were a primitive character that has been lost or obscured on intervening branches of the phylogenetic tree. It should also be noted that only a few potential segmentation genes have been characterized in annelids to date, so that the observation of any genetic similarity in the segmentation process is of potentially great significance. At the time of this writing our molecular understanding of annelid segmentation is almost entirely restricted to leeches of the genus Helobdella. Orthologues of several Drosophila segmentation genes have been identified and characterized, and while some

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of these genes do not play any obvious role in leech segmentation (Savage & Shankland 1996, Pilon & Weisblat 1997, also see below), this is not true of all. In Drosophila, the en gene encodes a transcription factor that plays a pivotal role in the process of segment polarity specification, being expressed in transverse stripes that demarcate the posterior compartment of each segmental primordium (DiNardo et al. 1988). In the leech Helobdella triserialis, the en gene is also expressed in segmentally organized stripes that span the width of the germinal band, leading a number of authors to propose an evolutionarily homologous role for this gene in the segmentation process (Wedeen & Weisblat 1991, Lans et al. 1993, Shankland 1994, Ramirez et al. 1995). However, there are a number of interphyletic differences in the relationship between en expression and morphological segmentation in leech and fly, and recent studies suggest that there are also profound interphyletic differences in the morphogenetic function of this gene: (1) In the fly, en expression precedes morphological segmentation. But in leech en expression first appears after the onset of segmentally iterated patterns of cell division (Lans et al. 1993). (2) In the fly, en-expressing cells form a sharply bounded tissue compartment lying immediately anterior to the segment border. In contrast, the en-expressing sublineages of the leech germinal band contribute descendants that spread over both sides of the segment border (compare Lans et al. 1993 with Weisblat & Shankland 1985 and Shankland 1987). (3) In the fly, en-expressing cells in the posterior compartment do not intermingle with other, non-expressing cells. In contrast, en-expressing cells in the leech germinal band give rise to descendants that intermix with more anterior and posterior sublineages that never express en (compare Lans et al. 1993 with Weisblat & Shankland 1985). (4) In the fly, the expression and function of en persists through both embryonic and larval development. In contrast, en expression lasts only 2-3 cell cycles in leech (Lans et al. 1993). (5) In the fly embryo, en-expressing cells initiate both direct (Heemskerk & DiNardo 1994) and indirect (Lawrence et al. 1996) cell interactions that specify the positional identities of non-expressing cells located in the next anterior and next posterior segmental repeats. In contrast, a laser cell ablation analysis of segment polarity specification in the ectoderm of the Helobdella embryo reveals no evidence of segment polarity signals being conveyed along the AP axis, nor do the en-expressing cells appear to be required for the specification of normal positional values in their anterior or posterior neighbors (Seaver & Shankland 2000, 2001). In addition, the expression of en does not appear to play any role in the morphological segmentation of the leech nervous system (Shain et al. 2000). (6) In the fly, en-expressing cells influence the fate of neighbouring cells by the expression and secretion of the intercellular signalling protein ‘hedgehog’ (hh) (DiNardo et al. 1988, Heemskerk & DiNardo 1994). An orthologue of the Drosophila hh gene has been identified in the leech Helobdella robusta, and in situ hybridization reveals high levels of expression in the developing gut, reproductive tract, and certain other organs (D. Kang, D. Li, D.A. Weisblat & M. Shankland, unpublished results). But to date, no expression of leech hh RNA has been detected in the cells known to express en during the early stages of segmentation, consistent with the absence of detectable cell interactions.

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Taken together these observations reveal many differences in the utilization and function of the en gene. In fact the only evidence that en is playing a conserved role in the segmentation of leeches and flies is that both species express the gene in segmentally organized stripes. Is this similarity significantly unlikely to have arisen by homoplasy? A random sampling of Drosophila cDNAs reveals that 87% of the gene products expressed at the onset of segmentation show segmentally repeating patterns of expression, including ‘housekeeping’ gene products that do not regulate segmental morphogenesis per se (Liang & Biggin 1998). It goes without saying that patterns of gene expression do not necessarily indicate gene function, and this problem is almost certainly exacerbated here because the defining characteristic of segmentation - structural periodicity - is a pervasive feature of the AP axis that is likely to leave its imprint on the expression patterns of many downstream genes. In short, the mere fact that both arthropods and leeches express this one gene in transverse stripes is not the sort of remarkable similarity that should lead us to exclude homoplasy out of hand. Conclusions The arguments that have been offered in support of a deeply rooted homology of segmentation focus almost entirely on interphyletic similarities. But in general, these arguments have made little or no attempt to exclude the alternative explanation, i.e. that some of the same genes were independently co-opted into mechanisms of segmentation that are homoplastic. In fact, it is not at all obvious that homoplasy can be excluded as a viable explanation for the genetic similarities reported to date in the segmentation of vertebrate chordates and arthropods, or of arthropods and annelids. It is possible that the observed similarities are remnants of a shared ancestral segmentation mechanism that has undergone extensive derivation, but at the present those similarities are few in number and superficial in detail. While it is tempting to take a bold stance on controversial issues, there are many reasons why we should remain open-minded in this case. First, the accuracy of evolutionary inferences is very much dependent on the completeness of the data sets being compared, and we only have a detailed mechanistic understanding of segmentation in one organism, the fruit-fly Drosophila, although our knowledge of vertebrate segmentation is growing rapidly. But with respect to the annelids, there can be no question that we must keep an open mind as new genetic data come to light. Second, it is difficult to compare similarities and differences between phyla without a solid understanding of variation within phyla, and we should be hesitant to make wide-ranging conclusions based on the comparison of a few experimentally amenable species. For instance, some aspects of the Drosophila segmentation pathway are not even conserved in other insects (Patel et al. 1992) - much less arthropods as a whole - and at the time of this writing exceedingly little is known about segmentation genes in annelids other than the leech. Significant advances in the data set available for comparison will take time, and there will be no moratorium on evolutionary speculation in the interim. Still, it is useful to remember that one of the goals of the scientific approach is to avoid the overinterpretation of fragmentary data, and to strive instead to define and acquire data sets that will ultimately lead to meaningful answers.

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MARTINEZ ARIAS A. 1993. Development and patterning of the larval epidermis of Drosophila. In Bate M. & A. Martinez Arias (eds), The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Plainview, NY, pp.517-608. MCGINNIS W. & R. KRUMLAUF 1992. Homeobox genes and axial patterning. Cell 68: 283-302. MÜLLER M., WEIZSÄCKER E.V. & J.A. CAMPOS-ORTEGA 1996. Expression domains of a zebrafish homologue of the Drosophila pair-rule gene hairy correspond to primordia of alternating somites. Development 122: 2071-2078. PALMEIRIM I., HENRIQUE D., ISH-HOROWICZ D. & O. POURQUIE 1997. Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91: 639-648. PATEL N.H., BALL E.E. & C.S. GOODMAN 1992. Changing role of even-skipped during the evolution of insect pattern formation. Nature 357: 339-342. PATEL N.H., MARTIN-BLANCO E., COLEMAN K.G., POOLE S.J., ELLIS M.C., KORNBERG T.B. & C.S. GOODMAN 1989. Expression of engrailed proteins in arthropods, annelids and chordates. Cell 58: 955-968. PERRIMON N., LANJUIN A., ARNOLD C. & E. NOLL 1996. Zygotic lethal mutations with maternal effect phenotypes in Drosophila melanogaster. II. Loci on the second and third chromosomes identified by P-element-induced mutations. Genetics 144: 1681-1692. PILON M. & D.A. WEISBLAT 1997. A nanos homolog in leech. Development 124: 1771-1780. RAMIREZ F.-A., WEDEEN C.J., STUART D.K., LANS D. & D.A. WEISBLAT 1995. Identification of a neurogenic sublineage required for CNS segmentation in an Annelid. Development 121: 2091-2097. RUBIN G.M., YANDELL M.D., WORTMAN J.R., MIKLOS G.L.G., NELSON C.R., HARIHARAN I.K., FORTINI M.E., LI P.W., APWEILER R., FLEISCHMANN W., CHERRY J.M., HENIKOFF S., SKUPSKI M.P., MISRA S., ASHBURNER M., BIRNEY E., BOGUSKI M.S., BRODY T., BROKSTEIN P., CELNIKER S.E., CHERVITZ S.A., COATES D., CRAVCHIK A., GABRIELIAN A., GALLE R.F., GELBART W.M., GEORGE R.A., GOLDSTEIN L.S., GONG F., GUAN P., HARRIS N.L., HAY B.A., HOSKINS R.A., LI J., LI Z., HYNES R.O., JONES S.J., KUEHL P.M., LEMAITRE B., LITTLETON J.T., MORRISON D.K., MUNGALL C., O’FARRELL P.H., PICKERAL O.K., SHUE C., VOSSHALL L.B., ZHANG J., ZHAO Q., ZHENG X.H. & S. LEWIS 2000. Comparative genomics of the eurkaryotes. Science 287: 2204-2215. SALSER S.J. & C. KENYON 1994. Patterning C. elegans: homeotic cluster genes, cell fates and cell migrations. Trends Genetics 10: 159-164. SAVAGE R.M. & M. SHANKLAND 1996. Identification and characterization of a hunchback orthologue, Lzf2, and its expression during leech embryogenesis. Dev.Biol. 175: 205-217. SEAVER E.C. & M. SHANKLAND 2000. Leech segmental repeats develop normally in the absence of signals from either anterior or posterior segments. Dev. Biol. 224: 339-353. SEAVER E.C. & M. SHANKLAND 2001. Establishment of segment polarity in the ectoderm of the leech Helobdella. Development 128: 1629-1641. SHAIN D.H., STUART D.K., HUANG F.Z. & D.A. WEISBLAT 2000. Segmentation of the central nervous system in leech. Development 127: 735-744. SHANKLAND M. 1987. Differentiation of the O and P cell lines in the embryo of the leech. II. Genealogical relationship of descendant pattern elements in alternative developmental pathways. Dev. Biol. 123: 97-107. SHANKLAND M. 1994. Leech segmentation: a molecular perspective. BioEssays 16: 801-808.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Behavioural, genetic and evolutionary interactionsThe between cuckoos ... Evolution 199 New Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 199-204, 2003

Behavioural, genetic and evolutionary interactions between cuckoos and their hosts A.P. Møller Laboratoire de Parasitologie Evolutive, CNRS UMR 7103, Université Pierre et Marie Curie, 7 quai St. Bernard, Case 237, F-75252 Paris Cedex 05, France. E-mail: [email protected]

Abstract Brood parasitic cuckoos exploit the parental care of their hosts to rear offspring, and parasitised hosts have little or no reproductive success. Hence, there is intense natural selection against parasitism, resulting in the evolution of host ejection behaviour and subsequent cuckoo egg mimicry. The coevolutionary interactions between cuckoos and their hosts provide a model system for the study of antagonistic coevolution, as shown by three different recent studies. (1) Facultative virulence. Cuckoos may respond aggressively to the egg ejection behaviour of hosts by destruction of the clutch of the host. This mafia-like behaviour can force the host to accept rather than eject cuckoo offspring, and such acceptance may be an evolutionary stable strategy for the host because the benefits of acceptance exceed the costs. (2) Antagonistic anti-parasite defences. Hosts often use many different defences against parasites such as nest defence and egg recognition and ejection, and these defences are hierarchical in the sense that avoidance of parasitism takes priority over eviction of parasites. Magpie Pica pica hosts of the great spotted cuckoo Clamator glandarius show a range of defences that are antagonistic because individuals that are specialising at defending themselves in one particular way are less suited for other kinds of defence. (3) Gene flow and population structure. Cuckoo hosts may eject cuckoo eggs even in allopatry with the cuckoo, suggesting that ejection behaviour may arise because of gene flow from sympatric host populations. In a study of European populations of magpies the response of magpie hosts to cuckoo eggs depended on conditions in the local population, but also on conditions in neighbouring populations. Hence, we can only understand coevolutionary interactions between parasites and their hosts by considering the interaction in a geographical perspective.

Introduction Brood parasitic cuckoos exploit the parental care of their hosts to rear offspring, and parasitised hosts have little or no reproductive success due to the exploitative nature of this interspecific interaction (Rothstein 1990, Johnsgard 1997). Hence, there is intense natural selection against parasitism, resulting in the evolution of host ejection behaviour

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and subsequent cuckoo egg mimicry (Rothstein 1990, Johnsgard 1997). The coevolutionary interactions between cuckoos and their hosts provide a model system for the study of antagonistic coevolution (Rothstein 1990). Studies of coevolution are best performed by adopting a number of different research methods in a number of different sites. The main reason for the need of this approach is that current interactions between host and parasite will not only depend upon interactions in the focal site, but also upon interactions elsewhere due to gene flow among populations. Here I present three different recent results on coevolution from studies of cuckoos that are of general interest to evolutionary biologists working on host-parasite interactions: (1) The evolution of facultative virulence; (2) antagonistic anti-parasite defences; and (3) gene flow and geographic structure. The evolution of facultative virulence Hosts of brood parasitic birds often respond to parasitism by ejection of the egg of the parasite (Rothstein 1990, Johnsgard 1997). Cuckoos may respond aggressively to the egg ejection behaviour of hosts by predation on the clutch of the host (Zahavi 1979). However, such destruction is not necessarily predation if the function of the behaviour is to change the response of the host. Great spotted cuckoos Clamator glandarius lay eggs in the nests of a range of hosts, in Europe mainly the magpie Pica pica and to some extent the carrion crow Corvus corone (Soler et al. 1998c). The offspring of the cuckoo is reared together with those of the host. However, damage of host eggs during laying by the cuckoo and severe competition between cuckoo and magpie nestlings result in parasitised nests suffering a reduction in reproductive success by more than 50% (Soler et al. 1998c). Magpies will relay in a new nest in the same territory or lay a new clutch the subsequent year. Great spotted cuckoos have large home ranges covering the territories of many different magpies (Soler et al. 1998c), and parasite and host may thus interact repeatedly during several breeding seasons. In a first experiment to test for facultative virulence, Soler et al. (1995) ejected the egg of the great spotted cuckoo from half of the parasitised nests of the magpie host, but left it in the control nests. Nest contents were destroyed in 69% of experimental nests, but in 11% of control nests. Nest contents were often left in the nest as cracked eggs or pecked nestlings without these being eaten by the perpetrator, suggesting that the culprit was not an ordinary nest predator like a corvid. In several instances, Soler et al. (1995) observed that a great spotted cuckoo entered a magpie nest with undamaged eggs or nestlings, while the contents were destroyed after the cuckoo left, showing that it was the cuckoo that damaged the nest contents. In several cases, Soler et al. observed that it was the female cuckoo, which actually laid the egg, that subsequently destroyed the clutch of the host after removal of the cuckoo egg. Finally, the experiment showed that magpies had as large reproductive success during a season when accepting a cuckoo egg as when ejecting it. Thus, this study demonstrated that the parasite changed its virulence in response to host behaviour, and that hosts benefited from accepting the cuckoo egg. In a second experiment, Soler et al. (1999b) tested for repeatability of magpie responses to cuckoo eggs. In one study area without cuckoos present magpies responded similarly to the presence of a cuckoo egg in the first and the replacement clutch after the first

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clutch of eggs had been destroyed after the experimental ejection test. However, in a second study area with many cuckoos present magpies significantly changed their behaviour from rejection to acceptance between the first and the replacement clutch. Thus, cuckoos could change the behaviour of the host by increasing the level of virulence when the host was resistant, and the host responded to this behaviour depending on the presence of parasites. Mafia-like behaviour can force the host to accept rather than eject cuckoo offspring, and such acceptance may be an evolutionary stable strategy if the host benefits from this behaviour (Soler et al. 1998a). In the literature on intraspecific and interspecific interactions there are many examples of such flexible parasite behaviour, and cases of facultative virulence may be more common than previously thought (review in Soler et al. 1998a). Antagonistic anti-parasite defences Hosts often use many different defences against brood parasites such as nest defence and egg recognition and ejection, and these defences are ordered hierarchically in the sense that avoidance of parasitism takes priority over eviction of parasites once infected (Hochberg 1997). Brood parasitic cuckoos considerably reduce the reproductive success of their hosts, which therefore have developed defences against the brood parasite. The first line of defence consists of protecting the nest against parasitism. The second line of defence is the ability of the host to recognise and reject the egg of the brood parasite from their nest, once this has been parasitised. These two defensive tactics are costly and will be counteracted by brood parasites. While a failure of effective nest defence implies successful parasitism and, therefore, great fitness loss to the host, a host that recognises parasitic eggs has the opportunity to reduce the effect of parasitism by removing the parasitic egg. We hypothesised that hosts, which recognise cuckoo eggs should defend their nest at a lower level than non-recognisers because the latter also recognise adult cuckoos and thereby avoid parasitism (Soler et al. 1998b). Magpie hosts that rejected model eggs of the great spotted cuckoo showed lower levels of nest defence when exposed to a live great spotted cuckoo than magpies that did not recognise the parasite. However, the levels of defence of recognisers and non-recognisers against a carrion crow were similar (Soler et al. 1998b). These results suggest that hosts specialise in anti-parasite defence, and that different kinds of defence are antagonistically expressed. While nest-defence mechanisms may be ancestral, egg recognition and rejection may have evolved at a subsequent stage in the coevolutionary process (Soler et al. 1998b). However, recognition ability of the host should no longer occur when brood parasites manage to bypass egg recognition and rejection. The antagonistic defences described here appear to result from behavioural trade-offs rather than the lost of an ability to adopt a particular defence. Gene flow and geographic population structure Cuckoo hosts sometimes eject model cuckoo eggs even in allopatry with the cuckoo (e. g., Soler & Møller 1990, Briskie et al. 1992), suggesting that ejection behaviour may

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arise because of gene flow from neighbouring populations. In a long-term study of 15 European populations of magpies, we recorded the response of hosts to parasitism with mimetic and non-mimetic model cuckoo eggs. This experimental method provides reliable results in terms of egg ejection or nest desertion that resemble the responses to real cuckoo eggs (Soler et al. 1999a). We subsequently analysed the response of magpie hosts in sympatry and allopatry with the great spotted cuckoo. Ejection rate of mimetic model eggs was indeed significantly greater in sympatry than in allopatry (mean ejection rate in sympatry: 37.8%; mean ejection rate in allopatry: 19.8%), particularly for nonmimetic model eggs (Soler et al. 1999a). There is significant gene flow among magpie populations which are structured according to a model of isolation by distance (Martínez et al. 1999). We used Mantel tests to investigate the response of magpie hosts to cuckoo model eggs in relation to conditions in the local population and in neighbouring populations. This statistical technique allows partitioning of the variance in ejection rate into effects due to local interactions and effects due to gene flow from neighbouring populations. We have found that the level of resistance by magpie hosts to parasitism by the great spotted cuckoo depends on interactions in the local population, but also on interactions in neighbouring populations (J.J. Soler, J.G. Martínez, M. Soler & A.P. Møller unpublished data). This finding supports recent theoretical developments emphasising the importance of spatial patterns of coevolutionary interactions (Nuismer et al. 1999, Thompson 1998, 1999). A study of coevolution between populations of a snail and its trematode parasite suggests that gene flow of host and parasite may affect levels of interaction (Dybdahl & Lively 1996), as shown for the magpie and the great spotted cuckoo (Martínez et al. 1999). Magpies have significantly greater gene flow in sympatry than in allopatry (Martínez et al. 1999). Since the level of local adaptation in the interaction between hosts and parasites depends on the relative levels of gene flow of the two interacting parties (Gandon et al. 1996), we can only predict the level of local adaptation when having estimates of gene flow. Hence, we can only understand coevolutionary interactions between parasites and their hosts by considering the interaction in a geographic perspective. General conclusion All three case studies presented here emphasise the complexity of interactions between hosts and parasites and the importance of continuing studies beyond what appears to be the current level of interaction. Therefore, they emphasise the need of detailed investigations. In the first study on facultative virulence, we could have concluded that parasites show a given level of virulence based on the first line of interactions between host and parasite. However, by extending the study to include repeated interactions it was possible to demonstrate the interdependent flexibility of host and parasite behaviour. In the second case study on antagonistic defences by hosts of brood parasites we could have concluded that hosts use a range of different defences. However, it was only by quantifying these defences in the same host individuals that we were able to show that specialisation on one kind of defence reduces the effectiveness of other kinds of defence. In the third case study on gene flow and anti-parasite defences we could have quantified

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the level of defence in a local population. However, it was only by including information from neighbouring populations that we were able to demonstrate that local levels of defence depend not only on local interactions, but also on interactions between hosts and parasites in more distant populations. Acknowledgements I would like to thank S. Morand for inviting me to present my research at the XVIII International Congress of Zoology in Athens, Greece. References BRISKIE J.V., SEALY S.G. & K.A. HOBSON 1992. Behavioral defenses against avian brood parasitism in sympatric and allopatric host populations. Evolution 46: 334-340. DYBDAHL M.F. & C.M. LIVELY 1996. The geography of coevolution: comparative population structures for a snail and its trematode parasite. Evolution 50: 2264-2275. GANDON S., CAPOWIEZ Y., DUBOIS Y., MICHALAKIS Y. & I. OLIVIERI 1996. Local adaptation and gene-for-gene coevolution in a metapopulation model. Proc. R. Soc. Lond. B 263: 1003-1009. HOCHBERG M.E. 1997. Hide or fight? The competitive evolution of concealment and encapsulation in parasitoid-host associations. Oikos 80: 342-352. JOHNSGARD P.A. 1997. The Avian Brood Parasites. Oxford University Press, New York. MARTÍNEZ J.G., SOLER J.J., SOLER M., MØLLER A.P. & T. BURKE 1999. Comparative population structure and gene flow of a brood parasite the great spotted cuckoo (Clamator glandarius), and its primary host, the magpie (Pica pica). Evolution 53: 269-278. NUISMER S.L., THOMPSON J.N. & R. GOMULKIEWICZ 1999. Gene flow and geographically structured coevolution. Proc. R. Soc. Lond. B 266: 605-609. ROTHSTEIN S.I. 1990. A model system for studying coevolution: avian brood parasitism. Annu. Rev. Ecol. Syst. 21: 481-508. SOLER J.J., MARTÍNEZ J.G., SOLER M. & A.P. MØLLER 1999a. Rejection behaviour of European magpie populations in relation to genetic and geographic variation: an experimental test of rejecter-gene flow. Evolution 53: 947-956. SOLER J.J., MØLLER A.P. & M. SOLER 1998a. Mafia behaviour and the evolution of facultative virulence. J. theor. Biol. 191: 267-277. SOLER J.J., SOLER M., PÉREZ-CONTRERAS T., ARAGON S. & A.P. MØLLER 1998b. Antagonistic anti-parasite defenses: Nest defense and egg rejection in the magpie host of the great spotted cuckoo. Behav. Ecol. 10: 707-713. SOLER J.J., SORCI G., SOLER M. & A.P. MØLLER 1999b. Change in host rejection behavior mediated by the predatory behavior of its brood parasite. Behav. Ecol. 10: 275-280. SOLER M. & A.P. MØLLER 1990. Duration of sympatry and coevolution between great spotted cuckoo and its magpie host. Nature 343: 748-750. SOLER M., SOLER J.J. & J.G. MARTÍNEZ 1998c. Duration of sympatry and coevolution between the great spotted cuckoo Clamator glandarius and its primary host, the magpie Pica pica. In Robinson S.K. & S.I. Rothstein (eds), The Ecology and Evolution of Brood Parasitism. Oxford University Press, New York, pp. 113-128.

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SOLER M., SOLER J.J., MARTÍNEZ J.G. & A.P. MØLLER 1995. Magpie host manipulation by great spotted cuckoos: evidence for an avian mafia? Evolution 49: 770-775. THOMPSON J.N. 1998. The population biology of coevolution. Res. Popul. Ecol. 40: 159-166. THOMPSON J.N. 1999. Specific hypotheses on the geographic mosaic of coevolution. Am. Nat. 153: S1-S14. ZAHAVI A. 1979. Parasitism and nest predation in parasitic cuckoos. Am. Nat. 113: 157-159.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Phenotypic Manipulation and Parasite-Mediated Host Evolution 205 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 205-212, 2003

Phenotypic Manipulation and Parasite-Mediated Host Evolution R. Poulin Department of Zoology, University of Otago, P. O. Box 56, Dunedin, New Zealand. E-mail: [email protected]

Abstract The ability of many parasites to manipulate the phenotype of their host in order to facilitate their own transmission has become a paradigm in the area of host-parasite evolutionary ecology. After three decades of investigation focusing mainly on the adaptive nature of this phenomenon for the parasites, two recent lines of research are bringing new aspects of it in the limelight. First, the magnitude and ubiquity of parasite-induced changes in host phenotype may be less spectacular than originally believed. The magnitude of published estimates of parasite-induced alterations in host behaviour has been decreasing steadily and significantly over the years, an observation that may be explained in part by publication biases. The existence of the phenomenon is not in doubt, but its quantitative importance needs a review. Second, the altered phenotype of parasitised hosts can have consequences for host evolution. The altered phenotypes of hosts can mask the existing genetic variation in the host population and possibly slow down natural selection by weakening the link between host genotype and host phenotype.

Introduction Natural selection proceeds by the differential survival and reproductive success of organisms with different phenotypic characteristics. Thus its eventual evolutionary effects on genotypes are mediated by ecological processes acting on phenotypes, the immediate targets of selection. Many parasitic organisms are notorious for their ability to alter the phenotype of their hosts. In particular, many parasitic helminths with complex life cycles are known to modify the colouration, morphology, behaviour or spatial distribution of their intermediate hosts in ways that facilitate their transmission to their next host. Past research in this area has focused mainly on the adaptiveness of the phenomenon for parasites (Dobson 1988, Moore & Gotelli 1990, Poulin 1994a, 1995, Kuris 1997, Brown 1999) and on the physiological mechanisms involved (Hurd 1990, Thompson & Kavaliers 1994). Very little consideration has been given to the possibility that parasites, by altering host phenotype and therefore the raw material of host

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evolution, could directly or indirectly influence the evolution of host traits not involved in defence against infection (Poulin & Thomas 1999). At the same time, parasites are usually ignored in studies of environmental forces causing phenotypic variation and modulating evolution, i.e. the so-called Baldwin effect (see Robinson & Dukas 1999). If phenotype manipulation by parasites is widespread, its evolutionary consequences for host evolution may be very important. In this paper, I will briefly explore the potential of phenotype-altering parasites to influence the evolution of their hosts. First, I will comment on the available quantitative estimates of parasite-induced changes in host phenotypic traits. Second, I will discuss the many ways in which changes in host phenotype caused by parasitism can influence rates of host evolution. Parasites and host phenotypes over time Parasites from a wide range of taxa are known to modify one or more phenotypic traits of their hosts (Moore & Gotelli 1990). An analysis of available quantitative estimates of the effect of parasites on host traits suggests that most parasites have small to moderate, but statistically significant, effects on host phenotype (Poulin 1994b). The validity of this conclusion rests on the assumption that published estimates of the effects of parasites are an unbiased sample of the true effects of parasites in nature. This may not be the case, however. Elsewhere I showed that the magnitude of published estimates of the effects of parasitism on host behaviour has decreased over the years (Poulin 2000). First, using an updated version of the data set of Poulin (1994b) that was gathered for different purposes, I found a negative correlation between quantitative effect sizes of parasites and the year in which they were published, among 137 comparisons of the behaviour of infected and uninfected hosts (Poulin 2000). This result holds only for the subset of 107 comparisons involving parasites that can apparently benefit from altering host phenotype. Second, using 14 published estimates of the increase in transmission via predation achieved by parasites that alter host phenotype (compiled for other purposes by Thomas et al. 1998), I again found a significant decrease in these estimates over time (Poulin 2000). These are not measures of changes in host phenotype per se, but they indicate how important parasite-induced changes in host traits can be for selection (i.e. predation) on the host. Both these analyses suggest that while on average the published estimates of parasite-induced changes in host phenotype are not trivial, they are getting smaller over time. Jennions & Møller (2002) recently re-examined earlier claims that effect sizes vary with year of publication in studies of some aspects of sexual selection. Using a multiple regression approach, they showed that the year effects reported by Alatalo et al. (1997) and Simmons et al. (1999) disappeared once the influence of other potentially confounding variables was considered in the statistical models. Jennions & Møller (2002) correctly pointed out that it is impossible to infer causation from a simple correlation between two variables, especially when the relationship disappears in more complex regression models. Here, following Jennions & Møller’s (2002) recommendations, I re-analyse the data used in Poulin (2000). In the first analysis, effect sizes of parasites on host behaviour

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were log-transformed and served as the dependent variable in a multiple regression. The independent variables were log-transformed sample size, year of publication, host taxon (a dummy variable coded as 0 for invertebrates and 1 for vertebrates), and parasite taxon (another dummy variable coded as 0 for non-acanthocephalans and 1 for acanthocephalans). Distinguishing between acanthocephalans and other helminth parasites is important because acanthocephalans are considered masters of host modification (Moore 1984). When data on all 137 comparisons were included, the negative effect of year of publication remained significant, even with the effects of other variables being controlled statistically (Table 1). Interestingly, when including only the 107 comparisons involving systems in which the parasite infects an intermediate host and gets transmitted to its next host when the latter preys on the intermediate host, the effect of year of publication remains strong. But when including only the 30 comparisons from systems with other transmission modes and in which changes in host behaviour have no obvious adaptive value, year of publication no longer has a significant effect (Table 1). These results fully support the simpler correlation analyses of Poulin (2000). The multiple regressions also indicate that the effect size of parasitism on host behaviour tends to be smaller in vertebrate than invertebrate hosts, and tends to decrease with increasing sample size (Table 1).

Table 1. Multiple regression analyses of the effects of four independent variables on the effect size of parasitism on host behaviour.

Independent variable

partial regression coefficient

t

P

All comparisons (F4, 132 = 6.046, r2 = 0.155, P =0.0002) Year of publication Log sample size Host taxon Parasite taxon

-0.346 -0.222 -0.219 -0.167

4.220 2.537 2.330 1.841

0.0001 0.0124 0.0213 0.0680

Only systems with transmission by predation (F4, 102 = 10.996, r2 = 0.301, P =0.0001) Year of publication Log sample size Host taxon Parasite taxon

-0.496 -0.257 0.097 0.025

5.911 2.849 0.979 0.246

0.0001 0.0053 0.3299 0.8059

Only systems with other types of transmission (F4, 25 = 1.684, r2 = 0.212, P =0.1851) Year of publication Log sample size Host taxon Parasite taxon

-0.189 -0.153 -0.580 0.008

0.937 0.706 2.427 0.040

0.3575 0.4864 0.0227 0.9683

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In a second analysis, the multiple regression approach was applied to the estimates of increases in transmission via predation achieved by parasites that alter host phenotype. To the 14 estimates included by Poulin (2000) and obtained from the list in Thomas et al. (1998), I added the results of two more recent studies (Thomas & Poulin 1998, Webster et al. 2000) to consider all estimates available to date. Adding these two estimates only reinforced the negative correlation between the estimates and the year of publication (rs = -0.699, N = 16, P = 0.0068). For the multiple regression, the estimates of increases in the probability of the host being eaten by a predator as a result of infection (i.e. increases in the probability of the parasite being transmitted) were used as the dependent variable. As above, log-transformed sample size (total number of hosts eaten during the experimental tests), year of publication, and host and parasite taxa (coded as before) were used as independent variables. The effect of year of publication did not come out as significant in the multiple regression (Table 2). It must be noted, however, that its partial regression coefficient was negative and was the closest of the 4 independent variables to approach statistical significance. Detecting the influence of one variable in a multivariate analysis with so few observations is not easy, and the possibility that this effect exists cannot be ruled out. What do these trends mean? Their explanation may lie in the way that scientists react to attractive new ideas. Following the article of Holmes & Bethel (1972) in which the ability of parasites to modify the phenotype of their hosts to their own ends was beautifully illustrated, the enthusiasm of parasitologists for the idea grew rapidly. It is only in the last decade that the phenomenon received more critical scrutiny (Moore & Gotelli 1990, Poulin 1995). Perhaps the early enthusiasm resulted in a bias in the sort of results that were submitted to journals and eventually published, with the more subtle effects only finding their way into the literature in recent years. This sort of cyclic life of scientific paradigms is not uncommon in the history of science (Kuhn 1996). Whatever the real explanation for the year effects reported here and in Poulin (2000), the existence of the phenomenon of host phenotype modification by parasites is not in doubt. The average magnitude of these modifications, however, may have been inflated by early reports; it will gradually be adjusted by the many subtle effects now being published. Even if a case by case approach is necessary to evaluate the effects of parasites, we will find that there are still many systems in which parasites have marked effects on host phenotype, and perhaps important effects on host evolution as well.

Table 2. Multiple regression analysis of the effects of four independent variables on estimates of parasiteinduced increases in rates of transmission by predation.

Independent variable (F4, 11 = 2.388, r2 = 0.465, P =0.1143) Year of publication Log sample size Host taxon Parasite taxon

partial regression coefficient

t

P

-0.413 0.017 0.302 0.333

1.420 0.075 1.296 1.102

0.1834 0.9419 0.2215 0.2940

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Parasites and evolving host phenotypes Beyond its fitness benefits for the manipulating parasite, the alteration in host phenotype can have serious implications for the evolution of the host itself. Clearly, host manipulation by parasites can change the shape of the frequency distributions of various continuous phenotypic variables within the host population (Poulin & Thomas 1999). Infection by a manipulating parasite can increase or decrease the mean value of a phenotypic trait and increase its variance in the overall host population. This will of course depend on the prevalence or abundance of the manipulating parasite. If it is very common, the frequency distribution of host phenotypic traits may remain normal but shift toward higher or lower values; if the parasite is only moderately common, the distribution of host traits is likely to become skewed (Poulin & Thomas 1999). The importance of this effect is that because of the parasite, the observed distribution of host traits in the population no longer reflects the distribution expected from host genotypes. This parasite-mediated uncoupling between host genotype and phenotype can disrupt selection acting on hosts. The strong background noise generated by parasites renders selection myopic, i.e. blind to the genotype and only capable of seeing and acting on the presently expressed phenotypes. One possible consequence would be a slowing down of evolutionary (genetic) changes in specific host traits from generation to generation (Poulin & Thomas 1999). It is also possible that parasitism, as other external influences on host phenotypes, can have different effects on the evolution of specific phenotypic traits (Robinson & Dukas 1999). For instance, in a host population experiencing directional selection on a given trait, if the effect of parasitic infections is to shift trait values toward the optimal values, the effect of the parasites could be to increase the mean fitness of the population with respect to that trait (Fig. 1). Of course, other pathological effects of the parasites could cancel out this effect. The important point is that the phenotypic shift induced by parasites can correspond to the direction in which selection is pushing the host population, just as it can act in the opposite direction. In more extreme cases, manipulating parasites cause such a large shift in host phenotype that they split the population into two morphotypes, i.e. the frequency distribution of certain host phenotypic traits becomes truly bimodal. This is true of certain helminths that alter the colouration or morphology of their intermediate hosts to the point that a human observer can easily separate parasitized individuals from healthy ones simply by looking at them (e.g., Hindsbo 1972, Plateaux 1972, LoBue & Bell 1993). In other systems, manipulating parasites cause a shift in the spatial distribution of their hosts, such that parasitized individuals are segregated in space from healthy conspecifics. For example, a trematode causes its amphipod intermediate hosts to move toward the water surface whereas unparasitized amphipods stay at the bottom of their lagoon habitats (Helluy 1984). This marked spatial segregation causes parasitized amphipods to mate assortatively with one another, whereas uninfected amphipods tend to mate only with other uninfected conspecifics (Thomas et al. 1995). The evolutionary effects of spatial segregation and reduced gene flow between parasitized and healthy individuals in a host population as a consequence of manipulation by a parasite have received very little attention to date (Poulin & Thomas 1999).

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Fig. 1. Frequency distributions of host phenotypic trait values in two hypothetical host populations, one without parasites, and one affected by parasites that shift the mean value of the trait toward the right. Under directional selection for larger trait values, the expected fitness that an individual host can achieve will increase at higher trait values, all else being equal. In this case, the parasitized population would have higher mean fitness than the non-parasitized population (inspired from Robinson & Dukas 1999).

Conclusion Many parasites cause marked changes in the phenotypes of their hosts. These effects have been well documented over the past three decades. Despite a tendency for recent estimates of the magnitude of parasite effects on host phenotypes to be smaller than earlier ones, the existence of this phenomenon is well established. Whether or not these phenotypic changes speed up or slow down host evolution remains unknown, however. There has been no attempt to date at quantifying these sorts of secondary evolutionary effects of parasite manipulation of host phenotypes. A first step could be to use population genetics models, to quantify the effect of phenotype alterations on evolutionary changes in host genotypes. Several well-studied host-parasite systems involve invertebrate intermediate hosts that have relatively short generation times and that can easily be studied in nature or in the laboratory. A second step could thus be to study the evolution of certain phenotypic traits in parasitized and unparasitized populations to see if and how they diverge. This promises to be an interesting research direction, as well as an important extension of previous research on parasite-induced changes in host phenotypes. References ALATALO R.V., J. MAPPES & M.A. ELGAR 1997. Heritabilities and paradigm shifts. Nature 385: 402-403.

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BROWN S.P. 1999. Cooperation and conflict in host-manipulating parasites. Proc. R. Soc. London B 266: 1899-1904. DOBSON A.P. 1988. The population biology of parasite-induced changes in host behavior. Q. Rev. Biol. 63: 139-165. HELLUY S. 1984. Relations hôtes-parasites du trématode Microphallus papillorobustus (Rankin, 1940). III. Facteurs impliqués dans les modifications du comportement des Gammarus hôtes intermédiaires et tests de prédation. Ann. Parasitol. Hum. Comp. 59: 41-56. HINDSBO O. 1972. Effects of Polymorphus (Acanthocephala) on colour and behaviour of Gammarus lacustris. Nature 238: 333. HOLMES J.C. & W.M. BETHEL 1972. Modification of intermediate host behaviour by parasites. In Canning E.U. & C.A. Wright (eds), Behavioural Aspects of Parasite Transmission. Academic Press, London, pp. 123-149. HURD H. 1990. Physiological and behavioural interactions between parasites and invertebrate hosts. Adv. Parasitol. 29: 271-318. JENNIONS M.D. & A.P. MØLLER (2002). Relationships fade with time: a meta-analysis of temporal trends in publication in ecology and evolution. Proc. R. Soc. London B. 269: 43-48. KUHN T.S. 1996. The Structure of Scientific Revolutions, third edition. University of Chicago Press, Chicago. KURIS A.M. 1997. Host behavior modification: an evolutionary perspective. In Beckage N.E. (ed.), Parasites and Pathogens: Effects on Host Hormones and Behavior. Chapman & Hall, New York, pp. 293-315. LOBUE C.P. & M.A. BELL 1993. Phenotypic manipulation by the cestode parasite Schistocephalus solidus of its intermediate host, Gasterosteus aculeatus, the threespine stickleback. Am. Nat. 142: 725-735. MOORE J. 1984. Altered behavioral responses in intermediate hosts: an acanthocephalan parasite strategy. Am. Nat. 123: 572-577. MOORE J. & N.J. GOTELLI 1990. A phylogenetic perspective on the evolution of altered host behaviours: a critical look at the manipulation hypothesis. In Barnard C.J. & J.M. Behnke (eds), Parasitism and Host Behaviour. Taylor & Francis, London, pp. 193-233. PLATEAUX L. 1972. Sur les modifications produites chez une fourmi par la présence d’un parasite cestode. Ann. Sci. Nat. Zool. Biol. Anim. 14: 203-220. POULIN R. 1994a. The evolution of parasite manipulation of host behaviour: a theoretical analysis. Parasitology 109: S109-S118. POULIN R. 1994b. Meta-analysis of parasite-induced behavioural changes. Anim. Behav. 48: 137146. POULIN R. 1995. ‘Adaptive’ changes in the behaviour of parasitized animals: a critical review. Int. J. Parasitol. 25: 1371-1383. POULIN R. 2000. Manipulation of host behaviour by parasites: a weakening paradigm? Proc. R. Soc. London B 267: 787-792. POULIN R. & F. THOMAS 1999. Phenotypic variability induced by parasites: extent and evolutionary implications. Parasitol. Today 15: 28-32. ROBINSON B.W. & R. DUKAS 1999. The influence of phenotypic modifications on evolution: the Baldwin effect and modern perspectives. Oikos 85: 582-589. SIMMONS L.W., TOMKINS J.L., KOTIAHO J.S. & J. HUNT 1999. Fluctuating paradigm. Proc. R. Soc. London B 266: 593-595.

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THOMAS F. & R. POULIN 1998. Manipulation of a mollusc by a trophically transmitted parasite: convergent evolution or phylogenetic inheritance? Parasitology 116: 431-436. THOMAS F., RENAUD F., DEROTHE J.M., LAMBERT A., DE MEEÜS T. & F. CÉZILLY 1995. Assortative pairing in Gammarus insensibilis (Amphipoda) infected by a trematode parasite. Oecologia 104: 259-264. THOMAS F., RENAUD F. & R. POULIN 1998. Exploitation of manipulators: ‘hitch-hiking’ as a parasite transmission strategy. Anim. Behav. 56: 199-206. THOMPSON S.N. & M. KAVALIERS 1994. Physiological bases for parasite-induced alterations of host behaviour. Parasitology 109: S119-S138. WEBSTER J.P., GOWTAGE-SEQUEIRA S., BERDOY M. & H. HURD 2000. Predation of beetles (Tenebrio molitor) infected with tapeworms (Hymenolepis diminuta): a note of caution for the manipulation hypothesis. Parasitology 120: 313-318.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Parasites and the evolution of host life Panorama history of traits 213 The New Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 213-218, 2003

Parasites and the evolution of host life history traits S. Morand Centre de Biologie et d’Ecologie Tropicale et Méditerranéenne (UMR 5555 CNRS), Université de Perpignan, 66860 Perpignan Cedex France. E-mail: [email protected]

Abstract Numerous studies have emphasised the potential roles of parasites to regulate the population dynamics of their hosts. Parasites decrease the reproduction and the survival of their hosts, which should invest in defences in order to resist and/or to diminish the debilitating impacts of pathogens. Parasites are ubiquitous and no free-living organisms seem to escape parasite infection, although there is a great variance in parasite infection both in abundance and species richness among host species. Hence, parasites should be seen as major driven evolutionary forces. Parasites seem to have driven the evolution of the adaptive immune system. The investment in defences should be linked to the pressures of parasites. Then, if defences are costly, there should be a major influence of parasites on the evolution of host life-history traits. I give several examples, which seem to support that parasites may drive the evolution of host life traits.

Introduction Parasites and pathogens have the ability to regulate the population dynamics of their hosts (Grenfell & Dobson 1995). Parasites may have an influence on many aspects of host’s fitness, such as survival and reproduction (Poulin 1999). Parasites and pathogens are also thought to play a great role in the evolution of sexuality and behaviour of their hosts (see Combes 1995 for various examples). Parasites are ubiquitous and no freeliving organisms seem to escape parasite infection, although there is a great variance in parasite infection both in abundance and species richness among host species (Poulin 1995, 1998, Morand 2000). Hence, parasites may be seen as major driven evolutionary forces. However, the first question we should answer is how to measure parasite pressures? Parasite infections vary through time (between seasons and years) and space (between populations). Moreover, the impact of a given parasite infection may also vary according to its virulence that may differ among strains within species. It is then often difficult in natural conditions to rely a given parasite’s infection to an impact on host’ fitness (Pampoulie et al. 1999).

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An alternative view is to hypothesise that the influences of parasites should increase with parasite diversity, such as parasite species richness, a given host must face. Then parasite species richness should be a good predictor of the influences of parasites and pathogens on the evolution of host life history traits (Morand 2000). Tests of the influence of parasites should be conducted at the level of species (or among populations) and must be conducted using comparative methods (Brooks & McLennan 1991, Harvey & Pagel 1991). In the present paper, I illustrate how parasites may influence several aspects of host life traits, namely age at maturity, energy, longevity and investment in immune organs. Parasites and host’s age at maturity The age at maturity is an important schedule in the life cycle of organisms. The maximisation of fitness implies that this life cycle should be optimal for age at reproduction (Roff 1992). Increased adversity with age

A

Fitness

α*

B

Residuals of contrasts in fish age at maturity (year in ln, controlled for host size)

Age at maturity

.8 n=22 r=0.420 p=0.046

.6 .4 .2 0 -.2 -.4 -.6 -.5 -.4 -.3 -.2 -.1

0

.1

.2

.3

.4

Residuals of contrasts in species richness (controlled for sample size)

.5

Fig. 1. A. The optimal age at maturity is dependent of the lifetime reproductive success, and any increase of adversity with age should select for earlier maturity. B. Parasites that accumulate through age (such as larval stages of parasites) should lead hosts to mature earlier, which is found using fish larval parasites as a test.

Parasites and the evolution of host life history traits

215

The optimal age at reproduction depends of the lifetime reproductive success. Hence, any increase of adversity with age should select for earlier maturity. If parasites live longer than their hosts, one can hypothesise than hosts should mature earlier, as parasites may accumulate with age (Fig. 1A). For example, Lafferty (1993) showed that a marine snail matures at smaller sizes when level of parasitism is high. I test this hypothesis using larval parasites in fish. For this, I compiled data on larval parasite species richness of 33 fish species, for which I obtained information on age at maturity and maximum length (Winemiller & Rose 1992). Using the independent contrasts method, I found that fish harbouring a high larval parasite species richness mature earlier (Fig. 1B). Parasites and the evolution of the evolution of immune defences Vertebrates have developed an original defence system for differentiating self and non-self, i.e. parasites and pathogens. This defence is based on the recognition of a peptide or its three-dimensional motif, and requires three molecules, the major histocompatibility complex (MHC), the T-cell receptor and the immunoglobulin molecules (Ig or antibodies). The function of the MHC molecules is to present peptides to T lymphocytes of the immune system and Klein (1991) emphasised that MHC genes are not neutral and that their persistence and the level of their polymorphism are explained by balancing selection caused by parasites and pathogens (Takahata 1990). Then, both host complexity and parasite pressures should be invoked to explain the diversity of antibodies and MHC molecules. Indeed, lower vertebrates (sharks, fish and herptiles) posses a small number of specific antibodies than birds and mammals, respectively fewer than 500,000 as compared between 107 and 109 (Du Pasquier 1982, see Frost 1999). In the same manner, the emergence of the MHC in the lower vertebrates was followed by dramatic expansion and duplication of MHC genes in birds and mammals (Klein 1991). The MHC evolution concerns both the genes organisation and the nucleotide sequence and by consequences the diversity of MHC molecules. In parallel, the pressures of parasites have increased as revealed by using parasite species richness as an indicator. Helminth species richness are low in reptiles and amphibians, from 3.7 to 6.7 parasite species per host species and high in birds (14.0) and mammals (12.0) (Morand 2000, Poulin & Morand 2000) (Fig. 2). The correlations between the increase of the antibodies repertoire, the expansion of MHC loci and the parasite pressures (Fig. 2) highly support the hypothesis that the parasites are the agents that have driven the immune system of vertebrates (Klein 1991). Although the helminths are not the only pathogens that may have an effect, we may expect a co-variation in parasite diversity. Host species harbouring a high species richness of helminths should also harbour a high diversity of other parasites such as protozoans, bacteria or viruses. Klein (1991) argued that, in order to have an impact on MHC evolution, a parasite must coevolve over a long period of time, also emphasising on which parasite may play this role. We may argue that it is not a parasite species or a parasite group per se (say trypanosomes as exemplified by Klein 1991) that is responsible for MHC evolution, but

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25 20

Mean Parasite species richness

15

95 79

10 152

31

5

36

122

5

Chondrichthyes

Osteichthyes

Caudata

Salientia

Mammalia

Squamata

Expansion of Class I & Class II MHC Loci

Aves

0

(Klein, 1991)

Increase in the antibody repertoire (DuPasquier, 1982)

Fig. 2. Expansion of the MHC genes and increase of the antibodies repertoire in relation with parasite pressure (from Combes & Morand 1999).

rather a community of parasite species, i.e. parasite species richness (but see Paterson et al. 1998). Parasites and investment in immune function The development of organs involved in immunity should be proportional to the parasite pressures. Comparative studies showed that bird species that are likely exposed to parasites such as dichromatic species, migratory species, or species that live in the tropics display large immune organs (such as spleen or bursa of Fabricius) (Møller 1998, Møller & Errizøe 1998, Møller et al. 1998). John (1995) found a positive relationship between the prevalence of nematode parasites and the relative weight of spleen in birds. Morand & Poulin (2000) also found a positive relationship between the relative mass of spleen and the nematode species richness (controlled for host sampling size and host bid mass), supporting the view that the investment in immune organs is dependent on parasite pressures. Moreover, Morand & Poulin (2000) confirmed the existence of a trade-

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off between the investment in reproduction and the investment in defences as they found a negative relationship between the relative testes mass and the relative spleen mass. Parasites and metabolism Hosts should invest in immune functions in order to control and to reduce the damages the parasites may induce. However, the immune function is energetically costly to the host (Lochmiller & Deerenberg 2000). This implies that hosts should allocate their energy between reproduction, immunity and survival. Morand & Harvey (2000) investigated the link between the basal metabolic rate of mammals and the parasite pressures. They hypothesised that mammal species submitted to great infection, estimated by the number of potential parasite species, should invest in high BMR in order to mount a costly immune response. Because BMR and immune functions decline intraspecifically with age, they put forward the second hypothesis that parasites have a negative influence on host longevity. Morand & Harvey (2000) found that hosts suffering from high parasite species richness have a lower life expectancy and have a higher BMR. Conclusion The examples illustrated here seem to support the hypothesis that parasites influence the evolution of host life-history traits through the maintenance of a competent immune system. The immune system, and particularly the adaptive immune system seems to have evolved in response to the parasite pressures. The maintenance of a competent immune system is costly, and the result showing that there is a link between the increase of BMR and parasite diversity seems to confirm this. Moreover, this competence decreases with ageing and as suggested by Hamilton (1966) mortality and senescence are likely to be under the influence of evolutionary force. References BROOKS D.R & D.A. MCLENNAN 1991. Phylogeny, Ecology, and Behaviour: A Research Program in Comparative Biology. The University of Chicago Press, Chicago. COMBES C. 1995. Interactions durables. Masson, Paris. COMBES C. & S. MORAND 1999. Do parasites live in extreme environments ? Constructing hostile niches and living in them. Parasitology 119: S107-S110. DU PASQUIER L. 1982. Antibody diversity in lower vertebrates-why is it so restricted? Nature 296: 311-313. FROST S.D.W. 1999. The Immune system as an inducible defense. In Tollrian R. & C.D Harvell (eds), The Ecology and Evolution of Inducible Defenses. Princeton University Press, Princeton, pp. 104-126. GRENFELL B.T. & A.P. DOBSON 1995. Ecology of Infectious Diseases in Natural Populations. Cambridge University Press, Cambridge HAMILTON W.D. 1966. The moulding of senescence by natural selection. Journal of Theoretical Biology 12: 12-45.

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HARVEY P.H. & M. PAGEL 1991. The Comparative Method in Evolutionary Biology. Oxford University Press, Oxford. HOCHBERG M.E., MICHALAKIS Y. & T. DE MEEUS 1992. Parasitism as a constraint on the rate of life-history evolution. Journal of Evolutionary Biology 5: 491-504. KLEIN J. 1991. Of HLA, Tryps, and selection: an essay on coevolution of MHC and parasites. Human Immunology 30: 247-258. LAFFERTY K.D. 1993. The marine snail, Cerithidea californica, matures at smaller sizes where parsitism is high. Oikos 82: 265-270. LOCHMILLER R.L. & C. DEERENBERG 2000.Trade-offs in evolutionary immunology: just what is the cost of immunity? Oikos 88: 87-98. MORAND S. 2000. Wormy word: comparative tests of theoretical hypotheses on parasite species richness. In Poulin R., Morand S. & A. Skorping (eds), Evolutionary Biology of Host-ParasiteRelationships: Reality Meets Models. Elsevier, Amsterdam, pp 63-79. MORAND S & P.H. HARVEY 2000 Mammalian metabolism, longevity and parasite species richness. Proceedings Royal Society London, B267: 1999-2003. MORAND S. & R. POULIN 2000. Nematode parasite species richness and the evolution of spleen size in birds. Canadian Journal of Zoology 78(8): 1356-1360. PATERSON S., WILSON K. & J.M. PEMBERTON 1998. Major histocompatibility complex variation associated with juvenile survival and parasite resistance in a large unmanaged ungulate population (Ovis aries L.). Proceedings of the National Academy of Sciences, USA 95: 3714-3719. PAMPOULIE C., MORAND S., LAMBERT A., ROSECCHI E., BOUCHEREAU J.-L. & A.J. CRIVELLI 1999. Influence of the Trematode Aphalloïdes cœlomicola (Dollfus, Chabaud & Golvan, 1957) on the fecundity and survival of Pomatoschistus microps (Krøyer, 1838) (Teleostei, Gobiidæ). Parasitology 119: 61-69. PATERSON S., WILSON K. & J.M. PEMBERTON 1998. Major histocompatibility complex variation associated with juvenile survival and parasite resistance in a large unmanaged ungulate population (Ovis aries L.). Proceedings of the National Academy of Sciences, USA 95: 3714-3719. POULIN R. 1999. The functional importance of parasites in animal communities: many roles at many levels? International Journal for Parasitology 29: 903-914. POULIN R & S MORAND 2000. The diversity of parasites. Quartely Review of Biology 75(3): 277293. ROFF 1992. The Evolution of Life Histories, Theory and Analysis. Chapman and Hall, New York. TAKAHATA N. 1990. A simple genealogical structure of strongly allelic lines and trans-species evolution of polymorphism. Proceedings of the National Academy of Sciences, USA 87: 24192423. WINEMILLER K.O. & K.A. ROSE 1992. Patterns of life history diversification in North Americam Fishes: implications for population regulation. Canadian Journal of Fisheries and Aquatic Sciences 49: 2196-2218.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Parasites and the evolution of cleaning symbioses amongoffish 219 The New Panorama Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 219-226, 2003

Parasites and the evolution of cleaning symbioses among fish I. M. Côté School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ UK. E-mail: [email protected]

Abstract Cleaning symbioses among fish involve the removal by small fish of ectoparasites and other materials from the body of larger, cooperating individuals. In this paper, I review the effects of ectoparasites on cleaner and client behaviour and assess their role in the evolution of interspecific cleaning interactions. Specifically, I ask whether client ectoparasite load can explain both cleaner preferences for specific clients and variability in client inclination to visit and adopt solicitation poses at cleaning stations. Overall, there is some evidence for an impact of ectoparasites on most cleaner and client behaviours. In largescale regional comparisons, areas with naturally high client ectoparasite loads were found to harbour more species of obligate cleaners and exhibit higher cleaning rates, suggesting a global effect of ectoparasites on cleaning interactions. However, as long as the benefits of cleaning for clients have not been established widely, generalisations about the role of ectoparasites remain premature. Research should now focus on determining the conditions under which cleaning is not mutualistic and understanding how cleaning interactions are maintained despite apparent cheating by cleaners and clients. These new lines of enquiry may yield general insight into the evolution of interspecific symbioses.

Introduction Cleaning symbioses are widespread among animals and are perhaps best and most abundantly represented in marine fishes (Côté 2000). Cleaning usually involves smallbodied cleaners which remove ectoparasites, diseased and injured tissue, and other particles from the body surface or buccal cavity of larger-sized, co-operating clients (Feder 1966). Cleaning interactions usually occur at traditional sites called cleaning stations (Youngbluth 1968), and they often begin with the cleaner fish performing a ‘dance’, a vertical zig-zag swimming pattern which appears to attract clients (Potts 1973). Most clients, in turn, pose to solicit cleaning. These poses are often species-specific and typically entail an immobile head-stand or tail-stand position with all fins spread and the opercula flared (Losey 1971, Côté et al. 1998). These poses may be accompanied by

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dramatic colour changes by the clients (Feder 1966). The cleaner may then swim onto and ‘inspect’ the client, and may start to ingest material gleaned from the client’s body, mouth or gills, or return to the station without feeding. Cleaning interactions may also be terminated by clients, twitching to signal their intention to depart (Feder 1966). This sequence of behaviours can vary. In particular, client poses may be assumed after cleaner inspection has begun (Losey 1971). Since the 1950’s, when Randall (1955, 1958) first found ectoparasites in the gut of cleaners, cleaning symbioses have been widely held as textbook examples of mutualism (e.g. Trivers 1971, McFarland 1985, Begon et al. 1990, Thompson 1994). The benefits to each party seem clear: cleaners gain food from cleaning, while clients are rid of ectoparasites. While the former is easy to verify, and indeed some cleaners glean their entire daily food requirements from cleaning activity (e.g. Grutter 1996a), showing the latter has proved more problematic. Only recently has a significant reduction in ectoparasites been shown in one client species (the half-and-half wrasse, Hemigymnus melapterus) interacting with one cleaner species (the blue-streak cleaner wrasse, Labroides dimidiatus) (Grutter 1999). Earlier experimental studies found that parasite loads of clients is not affected by cleaner activity (Gorlick et al. 1987, Grutter 1996b) and that removal of cleaners does not result in perceptible changes in fish communities (Losey 1972, Grutter 1997a). These studies, especially those by Grutter, were generally well replicated and were statistically powerful enough to detect even small effects. Finally, it is becoming clear that cleaners remove not only parasites but mucus and scales from their clients (e.g. Gorlick 1980, Arnal & Côté 2000). This may be a general phenomenon among organisms which were once thought to be exclusively mutualistic (e.g. red-billed oxpeckers, Weeks 1999, 2000). How important, then, are parasites for the evolution of cleaning symbioses among fishes? In this paper, I first review evidence for the role of ectoparasites in determining the behaviour of clients and cleaners. I then search for large-scale patterns of ectoparasite abundance and cleaning activity. Finally, I consider the implications of these results for our understanding of the role of parasites in cleaning symbioses. Parasites and client behaviour Client behaviour during cleaning interactions can be divided into two main components, namely visits to cleaning stations and incitation poses. Do clients with more ectoparasites visit cleaners more often? This has been examined surprisingly seldom in a way that allows one to consider only client behaviour, i.e. by recording all visits to cleaning stations regardless of whether they result in inspection or not. In laboratory experiments, Losey (1979) found that parasitised surgeonfish Zebrasoma flavescens visited a cleaner model more often in captivity than chemically deparasitised conspecifics; however, clean and parasitised butterflyfish Chaetodon auriga spent similar amounts of time with the cleaner model. Using field data and body size as a substitute for ectoparasite load, since these two variables are usually intercorrelated (Poulin 2000), Arnal et al. (2000) found no correlation between client length and the rate of visits to cleaning stations operated by Caribbean cleaning gobies (Elacatinus spp.), either in cross-species or in phylogenetically controlled analyses. However, when client

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ectoparasite load was measured directly, more heavily parasitised species did seek cleaning gobies more often than expected on the basis of their abundance on the reef (Arnal et al. 2001). Do clients with more ectoparasites pose more? This relationship may be expected since posing increases the likelihood of being cleaned (Côté et al. 1998). However, client posing and attractiveness to cleaners need not necessarily covary positively. Thus, one could predict that clients which are very attractive to cleaners (e.g. because of their high ectoparasite loads) might need to pose less to be cleaned than those with fewer parasites. A similar situation has been suggested in models of animal courtship where attractive males may be selected to court less than unattractive males (Reynolds 1993). There is at present little evidence for a relationship between posing and ectoparasites. In the laboratory, Losey (1979) found that parasitised surgeonfish Z. flavescens exhibited less posing in the form of body tilting but more in the form of erecting fins than chemically cleaned surgeonfish when exposed to a cleaner model. By contrast, parasitised butterflyfish C. auriga showed similar amounts of body tilting but less fin erections than deparasitised conspecifics. Similarly ambiguous results were obtained with another butterflyfish C. lunula (Losey 1971). In the field, Arnal et al. (2000, 2001) found no relationship between interspecific variation in posing for Elacatinus gobies and either client body size or direct assessments of client ectoparasite loads. However, Caribbean yellowtail damselfish (Microspathodon chrysurus) spend more time posing for cleaners when they have higher ectoparasite loads (Sikkel et al. 2000). Parasites and cleaner behaviour It is clear that all clients are not equal for cleaners. Some clients may pose for several minutes without being inspected, while others are inspected by cleaners without solicitation (Losey 1971, 1974, Arnal et al. 2000). Brown chromis (Chromis multilineata) and yellow goatfish (Mulloidichthys martinicus), for example, are inspected on nearly 100% of their visits to cleaning stations in the Caribbean, whereas grunts are only inspected on 10-20% of visits (Arnal & Côté 1998). Could these differences in apparent preferences by cleaners be related to client ectoparasite load? In the field, Arnal et al. (2000, 2001) found no relationship between the frequency of cleaning (controlled for number of visits) by gobies and client body size, a usual correlate of ectoparasite load (Poulin 2000). But Gorlick (1984) provided experimental evidence for an effect of ectoparasite load on client choice by cleaners. When presented with infected and chemically cleaned clients, cleaner wrasses L. phthirophagus spent more time with parasite-ridden clients. Moreover, client preference could be reversed by changing the ectoparasite load of the client species. Inspection duration is another measure by which cleaner preference for specific clients can be measured. In Australia, the total amount of time cleaner wrasses L. dimidiatus spent inspecting 11 species of clients each day increased with client ectoparasite load and client body surface area (Grutter 1995). This also held intraspecifically, with larger individuals being cleaned for longer than smaller conspecifics (Grutter 1995). Similarly, Caribbean cleaning gobies spent more time inspecting large-bodied clients (Arnal et al. 2000). However, the Caribbean relationship becomes non-significant when ectoparasite

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load is considered instead of its correlate, body size (Arnal et al. 2001). Moreover, total inspection time per day may be related to client abundance on the reef. When inspection time of Australian wrasses is expressed per inspection bout (as in Arnal et al. 2000), inspection time is no longer significantly related to ectoparasite load (r2 = 0.22, F1,9 = 2.58, P = 0.14; reanalysed from Grutter 1995). The importance of ectoparasites in determining foraging time is therefore not clear. Large-scale relationships between parasites and cleaning If ectoparasites are generally important for cleaning symbioses, one may make a series of predictions relating to the interrelationship between parasites and behaviour on a large scale. Thus, in areas of naturally high levels of ectoparasitism: Prediction 1 – There should be relatively more obligate cleaners (i.e. species that rely virtually completely on cleaning for their subsistence), since an abundance of ectoparasites may permit foraging specialisation. Prediction 2 – Cleaner densities should be higher, since the ‘carrying capacity’ for individuals with this foraging mode may be higher. Prediction 3 – Cleaning rates should be higher, since it has been shown earlier that heavily parasitised fish sought cleaners more frequency. Data on ectoparasite loads of client fish are patchy, but a selective survey of the literature yielded estimates of mean gnathiid abundance for Puerto Rico (4.4 gnathiids client-1; Losey 1974), Hawaii (1.72; Losey 1972), Australia (5.5; Grutter & Poulin 1998), Barbados (0.9; Sikkel et al. 2000), California (3; Hobson 1971) and Noumea (7.6; Grutter 1999b). Because a priori predictions were made, directed tests were carried out, with the critical value α = γ +δ = 0.05, where γ, the probability of rejecting H0 in the anticipated direction, equals 0.4, and δ, the probability of rejecting H0 in the unanticipated direction, equals 0.1 (Rice & Gaines 1994). Prediction 1 – There should be relatively more obligate cleaners. The proportion of obligate cleaners varied from 5% in Barbados to 42% in Australia, and the total number of cleaning species ranged from 10 to 19 (Fig. 1). There was no significant relationship between the proportion of all cleaning species which are obligate cleaners and gnathiid load on clients (r2 = 0.21, F1,4 = 1.07, Pdir = 0.23), but the absolute number of obligate cleaning species tended to increase with increasing gnathiid load (r2 = 0.56, F1,4 = 5.1, Pdir = 0.06; Fig. 1). Prediction 2 – Cleaner densities should be higher. Cleaner densities varied widely among locations, ranging from 0.6 to 13 individuals m-2. There was no significant relationship between cleaner densities and client gnathiid load (r2 = 0.14, F1,3 = 0.52, Pdir = 0.33). Prediction 3 – Cleaning rates should be higher. The number of clients cleaned h-1 increased significantly with client gnathiid load (r2 = 0.72, F1,3 = 10.4, Pdir = 0.03; Fig. 2).

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Fig. 1. Relationship between the number of species of obligate cleaners and mean gnathiid number on clients. Information on cleaner diversity was derived from Côté, 2000.

Fig. 2. Relationship between cleaning rate, expressed as the number of clients cleaned h-1, and mean gnathiid number on clients.

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These analyses are preliminary, and the potential effect of confounding variables, such as the general covariance of parasite intensity, species diversity and latitude, remains to be elucidated. In addition, the impact of spatial autocorrelation, which can lead to non-independence of data points, needs to be assessed carefully. Sites within geographic region may be expected to be more similar to each other than sites among regions. It is therefore not surprisingly to find such similarity between Noumea and Australia (Figs 1 and 2). Additional data will allow analyses to be carried out within region, which should alleviate the problem. At any rate, the preliminary analyses above suggest that large-scale patterns of client behaviour and cleaner species diversity may be related to client ectoparasite load. Adaptive significance of interspecific cleaning symbioses: where do we go from here? The previous sections show that there is at least some evidence of an influence of ectoparasites on most behaviours of cleaners and clients, and large-scale relationships between ectoparasites and cleaning symbioses are emerging. The recent studies by Grutter (1995, 1997, 1999a) of Labroides dimidiatus on the Great Barrier Reef have demonstrated unequivocally that some cleaning symbioses are mutualistic, at least in an area with naturally high rates of parasitism. Can these results be generalised to encompass all cleaning symbioses? The answer is probably not. The variable nature of outcomes in symbiotic interactions is now being recognised, not only for cleaning interactions but for a wide range of interspecific symbioses (e.g. Bronstein 1994). Whether a relationship is mutualistic depends on environmental conditions, as well as on the state of each participant, and a given mutualistic interaction may at times become more akin to parasitism given a shift in these conditions. This may lead to a dynamic, geographic or temporal mosaic of symbiotic outcomes (Thompson 1994), which may be difficult to interpret with smallscale, snapshot studies. I suggest that the way forward in understanding cleaning symbioses, particularly with respect to ectoparasites, lies in a three-pronged approach. First, we need to establish the generality of the benefit of ectoparasite removal for clients. Second, the conditions under which this benefit is not realised must be understood. In this context, the role of ectoparasites may be crucial. Finally, we should ask how cleaning relationships are maintained under these conditions. Evidence is now mounting to suggest that cleaners are indeed opportunistic and respond to low ectoparasite availability by foraging on alternative client-generated material, such as scales and mucus (Grutter 1997b, Arnal & Côté 2000). Whether this tips the cleaning relationship within the realm of parasitism by cleaners will depend on the cost to clients of having such items removed from their body. This cost is currently unmeasured. Acknowledgements I thank Serge Morand for organising the symposium and kindly inviting me. I gratefully acknowledge the support of a Royal Society travel grant to attend the Congress.

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References ARNAL C. & I.M. CÔTÉ 1998. Interactions between cleaning gobies and territorial damselfish on coral reefs. Animal Behaviour 55: 1429-1442. ARNAL C. & I.M. CÔTÉ (2000). Diet of broadstripe cleaning gobies on a Barbadian reef. Journal of Fish Biology 57: 1075-1082. ARNAL C., CÔTÉ I.M., SASAL P. & S. MORAND 2000. Cleaner-client interactions on a Caribbean reef: influence of correlates of parasitism. Behavioral Ecology and Sociobiology 47: 353-358 ARNAL C., CÔTÉ I.M. & S. MORAND 2001. Why clean and be cleaned? The importance of client ectoparasites and mucus in a marine cleaning symbiosis. Behavioral Ecology and Sociobiology 51: 1-7. BEGON M., HARPER J.L. & C.R. TOWNSEND 1990. Ecology: Individuals, Populations and Communities. Blackwell, London. BRONSTEIN J.L. 1994. Conditional outcomes in mutualistic interactions. Trends in Ecology and Evolution 9: 214-217. CÔTÉ I.M. 2000. Evolution and ecology of cleaning symbioses in the sea. Oceanography and Marine Biology Annual Review 38: 311-355. CÔTÉ I.M., ARNAL C. & J.D. REYNOLDS 1998. Variation in posing behaviour among fish species visiting cleaning stations. Journal of Fish Biology 53 (Supplement A): 256-266. FEDER H.M. 1966. Cleaning symbiosis in the marine environment. In Henry S.M. (ed.), Symbiosis. Academic Press, New York, pp.327-380. GORLICK D.L. 1980. Ingestion of host fish surface mucus by the Hawaiian cleaning wrasse, Labroides phthirophagus (Labridae), and its effect on host species preference. Copeia 1980: 863868. GORLICK D.L. 1984. Preference for ectoparasite-infected host fishes by the Hawaiian cleaning wrasse, Labroides phthirophagus (Labridae). Copeia 1983: 758-762. GORLICK D.L, ATKINS P.D. & G.S. LOSEY 1978. Cleaning stations as water holes, garbage dumps, and sites for the evolution of reciprocal altruism? American Naturalist 112: 341-353. GORLICK D.L, ATKINS P.D. & G.S. LOSEY 1987. Effect of cleaning by Labroides dimidiatus (Labridae) on an ectoparasite population infecting Pomacentrus vaiuli (Pomacentridae) at Enewetak Atoll. Copeia 1987: 41-45. GRUTTER A.S. 1995. Relationship between cleaning rates and ectoparasite loads in coral reef fishes. Marine Ecology Progress Series 118: 51-58. GRUTTER A.S. 1996a. Parasite removal rates by the cleaner wrasse Labroides dimidiatus. Marine Ecology Progress Series 130: 61-70. GRUTTER A.S. 1996b. Experimental demonstration of no effect by the cleaner wrasse Labroides dimidiatus (Cuvier and Valenciennes) on the host fish Pomacentrus moluccensis (Bleeker). Journal of Experimental Marine Biology and Ecology 196: 285-298. GRUTTER A.S. 1997a. Effect of the removal of cleaner fish on the abundance and species composition of reef fish. Oecologia 111: 137-143. GRUTTER A.S. 1997b. Spatiotemporal variation and feeding selectivity in the diet of the cleaner fish Labroides dimidiatus. Copeia 1997: 346-355. GRUTTER A.S. 1999a. Cleaner fish really do clean. Nature 398: 672-673. GRUTTER A.S. 1999b. Fish cleaning behaviour in Noumea, New Caledonia. Marine and Freshwater Research 50: 209-212.

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GRUTTER A.S & R. POULIN 1998. Intraspecific and interspecific relationships between host size and the abundance of parasitic larval gnathiid isopods on coral reef fishes. Marine Ecology Progress Series 164: 263-271. HOBSON E.S. 1971. Cleaning symbiosis among California inshore fishes. Fishery Bulletin 69: 491-523. LOSEY G.S. 1971. Communication between fishes in cleaning symbiosis. In Cheng T.C. (ed.), Aspects of the Biology of Symbiosis. University Park Press, Baltimore, pp. 45-76. LOSEY G.S. 1972. The ecological importance of cleaning symbiosis. Copeia 1972: 820-833. LOSEY G.S. 1974. Cleaning symbiosis in Puerto Rico with comparison to the tropical Pacific. Copeia 1974: 960-970. LOSEY G.S. 1979. Fish cleaning symbiosis: proximate causes of host behaviour. Animal Behaviour 27: 669-685. McFARLAND D. 1985. Animal Behavior. Benjamin Cummings, Menlo Park. POTTS G.W. 1973. The ethology of Labroides dimidiatus (Cuv. and Val.) (Labridae; Pisces) on Aldabra. Animal Behaviour 21: 250-291. POULIN R. 2000. Variation in the intraspecific relationship between fish length and intensity of parasitic infection: biological and statistical causes. Journal of Fish Biology 56: 123-137. RANDALL J.E. 1955. Fishes of the Gilbert Islands. Atoll Research Bulletin 47: 143-144. RANDALL J.E. 1958. A review of the labrid fish genus Labroides, with description of two new species and notes on ecology. Pacific Science 12: 327-347. REYNOLDS J.D. 1993. Should attractive individuals court more? Theory and a test. American Naturalist 141: 914-927. RICE W.R. & S.D. GAINES 1994. ‘Heads I win, tails you lose’: testing directional alternative hypotheses in ecological and evolutionary research. Trends in Ecology and Evolution 9: 235-237. SIKKEL P.C., FULLER C.A. & W. HUNTE 2000. Habitat/sex differences in time at cleaning stations and ectoparasite loads in a Caribbean reef fish. Marine Ecology Progress Series 193: 191199. THOMPSON J.N. 1994. The Coevolutionary Process. University of Chicago Press, Chicago. TRIVERS R.L. 1971. The evolution of reciprocal altruism. Quarterly Review of Biology 46: 35-57. WEEKS P. 1999. Interactions between red-billed oxpeckers, Buphagus erythrorhynchus, and domestic cattle, Bos taurus, in Zimbabwe. Animal Behaviour 58: 1253-1259. WEEKS P. 2000. Red-billed oxpeckers: vampires or tickbirds? Behavioral Ecology 11: 154-160. YOUNGBLUTH M.J. 1968. Aspects of the ecology and ethology of the cleaning fish, Labroides phthirophagus Randall. Zeitschrift für Tierpsychologie 25: 915-932.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Host behaviour: the first of defense The Newline Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 227-234, 2003

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Host behaviour: the first line of defense C. Combes Université de Perpignan, Avenue de Villeneuve, 66860 Perpignan, France. E-mail: [email protected]

Abstract From the patterns of exchange of information between parasites and hosts, the paper is a reflection on the relative importance of the first line of defense of hosts (behavioural, but not only) and the second line of defense (immune, but not only). As a rule, natural selection is rarely efficient at the behavioural level (encounter filter) because infective stages transmit only weak signals, or no signal at all, which could be analysed by the target hosts. Defense is thus principally devoted to immunity (compatibility filter). Exceptions are listed, related to the fact that: a) some parasites are big enough or numerous enough to be detectable, b) some hosts change habitat under the parasite pressure, c) other potential hosts take advantage of a visible change provoked in infected hosts by the parasite, to avoid contagion, d) other hosts modify life-history traits to minimize encounters with the parasite. A particular case is that of human culture which “rehabilitates” the first line of defense with the aim of controlling human parasitic diseases.

Introduction: Parasites differ from predators In predator-prey systems, predators are continuously trying to find prey, whereas prey are trying to escape predators. As any kind of interspecific relationships, finding and escaping depend on information that is exchanged between the two partners. In both the predator and the prey, natural selection is expected to retain behavioural adaptations, which serve: 1) to emit as few signals (information) as possible to “the other”; 2) to pick up as many signals as possible from “the other”. Information is conveyed by visual, chemical and physical stimuli. As a rule, the game of life consists in emitting the least, and detecting the most (in some cases, however, the release of information by a predator is used to capture prey: in cheaters for instance, the game consists in emitting deceiving signals; cheating is an adaptation like any other behavioural trait….)

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In parasite-host systems, the game could be theoretically the same, since the parasite takes the place of the predator and looks for a host, whereas the potential host takes the place of the prey and tries to avoid infective stages. In reality, things are different (Fig. 1).

Fig. 1. The unique line of defense of prey (top) and the two lines of defense of hosts (bottom). In parasitehost systems, selection can favour the first or the second line, depending on systems and circumstances.

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Prey have only one line of defense: if a rodent is caught by a fox, no second chance is offered to the rodent. The selection can act only at the level of the “encounter filter” as defined by Combes (1995, 1997, 2001). On the contrary, hosts have two lines of defense. The first is exactly parallel to the unique line of defense of prey: avoiding being “caught” by the parasite. The second line plays its role after infection (see Combes 2000). By preening, grooming, and the sophisticated mechanisms of immunity, a host has at its disposal a battery of weapons to get rid of the parasite. The selection acts at the level of two successive filters: one of encounter and one of compatibility (Fig. 1). The question is then: since a second line of defense is possible, does the first line still play a role in host defense? Logically, behaviour could be expected to play a comparable defensive role in providing both a host with means to escape parasites and in providing a prey with means to escape predators. Over the course of evolution, it seems that many hosts have invested more in the second than in the first line, i.e. have acquired a sophisticated immune system. Conversely, there has rarely been selection of an adaptive behaviour. Three explanations can be proposed to account for this phenomenon: One is that many loci are usually involved in a genetically determined behaviour, which implies that selection of behavioural changes can only be slow. Another is that behavioural changes are often very costly; for instance modifying the type of prey that are commonly captured may necessitate many ethological, physiological and even morphological changes in the predator. The selection of the first vs the second line of defense is a matter of cost of behaviour vs cost of immunity (regarding cost of immunity, see Rigby & Moret 2000). But the most likely explanation is that infective stages of parasites are usually difficult to see or otherwise detect; in other words, the potential hosts are well equiped to pick up information, but signals emitted by infective stages of parasites are often virtually non-existant: a human receives no detectable signal from an Entamoeba cyst, from a Schistosoma cercaria, or from a Trichinella larva (for a review of life-history strategies of parasites themselves, see Poulin 1998). Against such parasites, hosts have only one line of defense, the one that we call “the second”. There are four main exceptions to the above statement. The first is because some parasites are actually detectable in such a way that individual responses of potential hosts may occur. The second is related to a change of habitat under the pressure of parasites, either as an individual response or as a fixed behaviour (individuals show the trait even in the absence of the parasite). The third is related to a change of lifehistory traits in populations submitted to a high pressure from parasites. The last one is the intervention of culture in humans, which rehabilitates defensive behaviour. In all these cases, the first line of defense plays a significant role in arms races between parasites and their hosts. Direct detection of the presence of parasites by individual hosts By exception, certain parasites in search of hosts are big and/or noisy.

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In parasitoid-insect systems, where it is the adult which looks for a suitable host, an exchange of detailed information is possible between the agressor and the potential victim. The information is conveyed by chemical volatiles or by air vibrations. For instance, accurate acoustical methods allowed Meyhöfer et al. (1997) and Meyhöfer and Casas (1999) to demonstrate that vibratory signals are used not only by the parasitoid to find its target, but also by the target-host itself, a leafminer caterpillar, to escape the wasp: the caterpillar detects the noise made by the wasp and immediately tries to escape in the galeries of the leaf. The authors indicate that the complicated design of the galeries of the leafminers is an additional adaptation, which enhances the chances of escaping the parasitoid. Cuckoos can be detected, directly or indirectly, by some passerine birds. Cuckoos need a tree to stake-out the nests of their prospective hosts (Fig. 2). When the host nest is on the ground the parents may chose a site far from trees in order to escape surveillance and avoid being parasitized (Alvarez 1993). Rufous warblers (Cercotrichas galactotes) for instance nest in littoral plants and show preferences for sites far from trees, even to the point of competing with others for these low risk habitats (Oien et al. 1996) Birds can also detect small parasites if they are sufficiently aggregated. Richner et al. (1993) have demonstrated by a series of experiments that great tits (Parus major) are capable of detecting whether a nest box is “parasitized” by fleas or not and that consequently they use significantly more often clean boxes. Cattle behaviour relative to ticks shows some similarities with great tit behaviour. Sutherst et al. (1986) studied defense mechanisms against the tick, Boophilus microplus, experimentally. Different numbers of tick larvae were set in circles on a pasture. Groups

Fig. 2. An imaginary landscape, inspired by the data of Alvarez (1993): nests of rufous warblers, symbolized by small ovals on the ground, are built far from trees in order to escape surveillance by cuckoos.

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of bulls were used and their behaviour was observed. It was shown that bulls avoided grazing in circles with a high density of tick larvae. Bulls could detect larvae because, as the hosts approached, the ticks, which are dark brown, moved onto the upper surface of grass blades. Cattle are also capable of limiting infection by larvae of the nematode, Dictyocaulus viviparus, but in this case there is no real detection of the parasites. Instead, what has been selected for has been the avoidance of ingesting grass growing in the immediate proximity of bovine feces (Michel 1955). Indirect detection of the presence of parasites by individual hosts Finally, deficiencies in the elaboration of secondary sexual traits (colours, feathers, parades) are used as signals by potential mates to choose a partner. Whereas in certain cases this behaviour is adaptive to provide the offspring with “good genes” (hypothesis of Hamilton & Zuk 1982), in other cases, it is simply a strategy to avoid personal contamination. Clayton (1990) has shown that the choice of a sexual partner in Columba livia is influenced by the duration of the courtship display of males and that this reduces the risk of contamination by Phthiroptera, although these and their damage are strictly invisible from the outside. Here the signal points out the parasite but is involuntarily emitted by a contagious host. Since these kinds of signals (long displays, etc.) are costly, Zahavi (1977), Zahavi and Zahavi (1997) consider them as “honest”, i.e. non-susceptible to cheating. Refuge in space Changes in habitat have certainly played a role in host defense through evolution, especially in the world of parasitoids. Hochberg and Hawkins (1992) showed that the less accessible plant of an insect larva, the fewer the number of parasitoid wasps that will attack it. Some species, however, do not have the parasite richness which seems in agreement with their easy accessibility; it is probable that these species have developed the second line of defense… Hochberg (1997) has discussed the conditions, especially density of parasitoids, which make advantageous to “hide” (first line of defense) or “fight” (second line) in parasitoid-hosts associations. The change of habitat is at selective advantage where parasite pressure is high. For instance, reindeer, Rangifer tarandus, form herds with some groups having their calving grounds and summer pastures overlapping, and others having their calving grounds and summer pastures separated by hundreds of kilometers. Folstad et al. (1991) found that there was a negative correlation between the amplitude of the migration and the abundance of the parasitic dipteran Hypoderma tarandi, in Norway. Migratory herds drop fewer larvae within their summer pastures than non-migratory ones and thus harbour fewer parasites. Reindeer with migratory behaviour have a selective advantage over sedentary ones, so that parasite pressure possibly selects “genes of migration”. Interactions between habitat choice and parasitism have been also reported in other vertebrates like birds and lizards (see Boulinier & Lemel 1997, Danchin et al. 1998, Sorci et al. 1994, Van Vuren 1996).

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Refuge in time In recent years it has been increasingly perceived that the encounter filter can also be closed, at least partially, by adaptive changes in life-history traits of hosts (see Koella 2000). For instance, the selection of rapid maturation under the pressure of parasites has been demonstrated by Lafferty (1993) in the mollusk Cerithidea californica. These gastropods are intermediate hosts of trematodes, many of which are castrators. It was shown that the size at maturity was small in localities where prevalence of larval trematodes was high. In order to verify that this was due to selection and not to environmental factors, the author exchanged mollusks between populations and was able to show that descendants of the moved mollusks reached maturity as in their original population. The selection of rapid maturation is a component of the first line of defense because, even if the parasites finish up infecting the snails, they do it at an age when immune defense is no longer vital, since the reproduction of the snail has already occurred. Adaptive changes in life-history traits are different from behavioral adaptations previously considered, but have a comparable effect regarding fitness: they decrease the probability of contact with the parasite during the time when genes can be transmitted to the next generation. A new weapon: culture There is a particular case in which the first line of defense can play a paramount role in parasite avoidance. This case is that of humans. Humans are not better at detecting tiny infective stages of parasites than other species, but they accumulate knowledge on them, thanks to the lamarckian process of transmission of acquired cognitive characters. Cultural traits capable of limiting infection, for instance being aware of where and when infective stages, although invisible, are present in the environment, are vertically heritable (from parents to children) and horizontally transmittable (between unrelated people). By elucidating life-cycles and transmission features, humans can intervene directly on parasite transmission rate and decrease it. They can filter or boil water, wash hands and vegetables, control vectors, educate people, drain swamps, etc. Some of these actions belong to what Laland et al. (2000) call “niche construction”: in the case of humans, modifying the environment in an adequate way can be a very efficient first line of defense against parasitic tropical diseases, especially those which are water-dependent. Even if the second line of defense also benefits from scientific discoveries (therapeutics), it appears that culture has re-established (in the sense of “rehabilitated”) the importance of the first line of defense, which had been otherwise somewhat neglected by evolution. Conclusion As a rule, non-human hosts can close the encounter and the compatibility filters only if they pick up a signal from the presence of the parasite (an infective stage for the

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encounter filter, a resident parasite for the compatibility filter). This explains why selection gave parasites the ability to be as discrete as possible all along their life-cycle. With the exceptions I summarized above, infective stages of parasites are minute and extremely avaricious of emitting any detectable information (being tiny provides in fact two advantages: being invisible and being numerous). This makes the first line of defense a difficult one in most cases. Later, when parasites are on or in their hosts, their adaptative strategy will consist again to make them invisible to cells and molecules involved in immunity (they will for instance disguise them by coating their tegument with host molecules, etc.). Giving the least possible information to their hosts is thus a leit-motiv of parasite adaptive strategies. This strategy is totally reversed when parasites have interest in being conspicuous in order to insure their transmission; they will for instance manipulate the phenotype of their vectors to make them more attractive or palatable to predators in which they must complete their development (see Dawkins 1982, Combes 1991, Moore 1995). In such cases, using the first line of defense is an even more difficult challenge for hosts. Acknowledgments: I thank Robert Poulin for his comments on an earlier draft of this manuscript. References ALVAREZ F. 1993. Proximity of trees facilitates parasitism by cuckoos Cuculus canorus on rufous warblers Cercotrichas galactotes. Ibis 135: 331. BOULINIER T. & J-Y. LEMEL 1997. Spatial and temporal variations of factors affecting breeding habitat quality in colonial birds: some consequences for dispersal and habitat selection. Acta Oecol. 17: 531-552. CLAYTON D.H. 1990. Mate choice in experimentally parasitized rock doves: lousy males lose. Am. Zool. 30: 251-262. COMBES C. 1991. Ethological aspects of parasite transmission. Am. Nat. 138: 866-880. COMBES C. 1995. Interactions Durables. Ecologie et Evolution du Parasitisme. Masson, Paris. COMBES C. 1997. Fitness of Parasites. Pathology and Selection. Int. J. Parasitol. 27: 1-10. COMBES C. 2000. Parasites, hosts, questions. In Poulin R., Morand S. & A. Skorping (eds), Evolutionary Biology of Host-Parasite Relationships: Theory meets Reality. Elsevier, Amsterdam, pp. 1-8. COMBES C. 2001. Paratism. The Ecology and Evolution of Intimade Interactions. The University of Chicago Press, Chicago. DANCHIN E. T., BOULINIER T. & M. MASSOT 1998. Conspecific reproductive success and breeding habitat selection: implications for the study of coloniality. Ecology 79: 2415-2428. DAWKINS R. 1982. The Extended Phenotype. Oxford University Press. FOLSTAD I., NILSSEN A.C., HALVORSEN O. & J. ANDERSEN 1991. Parasite-avoidance: the cause of post-calving migrations in Rangifer? Can. J. Zool. 69: 2423-2429. HAMILTON W.D. & M. ZUK 1982. Heritable true fitness and bright birds: a role for parasites? Science 218: 384-386.

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HOCHBERG M.E. 1997. Hide or fight? The competitive evolution of concealment and encapsulation in parasitoid-host associations. Oikos 80: 342-352. HOCHBERG M.E. & B.A. HAWKINS 1992. Refuges as a predictor of parasitoid density. Science 255: 973-976. KOELLA J.C. 2000. Coevolution of parasite life cycles and host life-histories. In Poulin R., Morand S. & A. Skorping (eds), Evolutionary Biology of Host-Parasite Relationships: Theory Meets Reality. Elsevier, Amsterdam, pp. 185-200. LAFFERTY K.D. 1993. The marine snail, Cerithidea californica, matures at smaller sizes where parasitism is high. Oikos 68: 3-11. LALAND K.N., ODLING-SMEE J. & M.W. FELDMAN 2000. Niche construction, biological evolution and cultural change. Behav. Brain Sci. 23: 131-175. MEYHÖFER R. & J. CASAS 1999. Vibratory stimuli in host location by parasitic wasps. J. Insect Physiol. 45: 967-971. MEYHÖFER R., CASAS J., & S. DORN 1997. Vibration mediated interactions in a host-parasitoid system. Proc. R. Soc. London B 264: 261-266. MICHEL M. 1955. Parasitological significance of bovine grazing behaviour. Nature 175: 10881089. MOORE J. 1995. The behavior of parasitized animals. When an ant… is not an ant. Bioscience 45: 89-96. OIEN I. J., HONZA M., MOKSNES A. & E. ROSKAFT 1996. The risk of parasitism in relation to the distance from reed warbler nests to cuckoo perches. J. Anim. Ecol. 65: 147-153. POULIN R. 1998. Evolutionary Ecology of Parasites. From Individuals to Communities. Chapman & Hall, London. RICHNER H., OPPLIGER A. & P. CHRISTE 1993. Effect of an ectoparasite on the reproduction in great tits. J. Anim. Ecol. 62: 703-710. RIGBY M.C. & Y. MORET 2000. Life-history trade-offs with immune defenses. In Poulin R., Morand S. & A. Skorping (eds), Evolutionary Biology of Host-Parasite Relationships: Theory Meets Reality. Elsevier, Amsterdam, pp. 129-142. SORCI G., MASSOT M. & J. CLOBERT 1994. Maternal parasite load increases sprint speed and philopatry in female offspring of the common lizard. Am. Nat. 144: 153-164. SUTHERST R.W., FLOYD R.B., BOURNE A.S. & J.J. DALLWITZ 1986. Cattle grazing behaviour regulates tick populations. Experientia 42: 12-15. VAN VUREN D. 1996. Ectoparasites, fitness and social behaviour of yellow-bellied marmots. Ethology 102: 686-694. ZAHAVI A. 1977. The cost of honesty. J. Theor. Biol. 67: 603-605. ZAHAVI A. & A. ZAHAVI 1997. The Handicap Principle. Oxford University Press.

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers TreeMap: an algorithm to maximize the number of codivergences when ... Evolution 235 The New Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 235-240, 2003

TreeMap: an algorithm to maximize the number of codivergences when reconstructing the history of an associate and its host J.P. Hugot Muséum National d’Histoire Naturelle, Institut de Systématique, FR 1541 du CNRS, Biosystématique et Coévolution chez les Nématodes Parasites, 75231 Paris cedex 05, France. E-mail: [email protected]

Abstract Because closely related organisms show extensive similarities in the parasites associated with them, it should be possible to base genealogical conclusions on parasite data. In the first attempt to accomplish this, the parasite data were converted into binary characters using additive binary coding. The concept of maximizing the amount of co-divergence when mapping the respective trees of two associates on each other was introduced later, in order to recover the history of a particular association. The basic principles of TREEMAP, an algorithm to find all reconstructions that maximize the number of co-divergences, in the particular case of a host-parasite assemblage, are presented first. Then, an example is given of how this program can be used to investigate the common history of several associates.

Because closely related organisms show extensive similarities in the parasite associated with them, it should be possible to base genealogical conclusions on parasite data. In the first attempt to accomplish this the parasite data were converted into binary characters using additive binary coding: this method was proposed as Brooks Parsimony Analysis (BPA) (Brooks 1981, Wiley 1988). After Ronquist and Nylin (1990), Page (1994) introduced the concept of maximizing the amount of codivergence (shared history), when mapping each other the respective trees of two associates for recovering the history of a particular association. Later, Page (1995) developed TreeMap: an algorithm to find all reconstructions that maximize the number of codivergences in the particular case of a host-parasite assemblage. In the following, the basic principles of TreeMap algorithm are first presented. Then, an exemple is given of how this program can be used to investigate (i) the common history of the primates and their oxyurids, (ii) how to explain the presence of three species parasitic of squirrel in a family that contains primarily primate parasites.

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TreeMap: how does that work? TreeMap Page (1994) is a free sofware available at: http://taxonomy.zoology.gla.ac.uk/rod/rod.html. TreeMap maximizes the amount of codivergence (shared history), when superimposing each other the respective trees of two associates in order to recover the history of a particular association. TreeMap exact search algorithm proposes all these scenarios, which admit the maximum amount of cospeciations. Following Page (1996), speciation events are divided into three categories (Fig. 1): cospeciation, duplication and host switch. To built evolutionary scenarios a fourth category is needed: sorting events cover any case in which a parasite species can be expected on one host species and has not been observed. A supplementary category of speciation event can be added: cophylogeny without cospeciation which covers any case in which a parasite species can be expected to have been inherited from the common ancestor of its primary host and of one (or more) of its secondary hosts. If the absence of other arguments, which could support or refute a particular scenario, the sole criterion to choose the most reliable one is the parsimony of hypotheses. As each scenario exhibits the same number of cospeciations, they have to be compared optimization of codivergence events = cophylogeny with cospeciation host switch or host transfer of a parasite to a non-closely related host, followed by a speciation of the parasite duplication or speciation of a parasite without corresponding speciation of the host sorting events : any case in which a parasite species is expected and has not been recorded host switch or inheritance of the same parasite species from the common ancestor of the hosts = cophylogeny without cospeciation

Fig. 1. TreeMap: how does that work?

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using the other three kinds of evolutionary events, scenarios with the lowest number of extra hypotheses (evolutionary events other than cospeciations), can be considered the most parsimonious. Compared phylogeny of the pinworm parasite of primates and their hosts A morphologically based cladistic analysis of the Enterobiinae, which includes most of the Oxyuridae parasitic in Primates, allows a reevaluation of the Cameron’s hypothesis of close coevolution with cospeciation between hosts and parasites. The three genera separated in the Enterobiinae fit with one of the suborders defined in Primates: Lemuricola with the Strepsirhini, Trypanoxyuris with the Platyrrhini, and Enterobius with the Catarrhini (Fig. 2). Inside each of the three main groups, the subdivisions observed in the parasite tree also fit with many of the subdivisions generally accepted within the Primate order. These results confirm the subgroups previously described in the subfamily and support Cameron’s hypothesis in its aspect of association by descent. Although the classification of the Enterobiinae generally closely underlines the classification of Primates, several discordances also are observed. They have been discussed case by case, using computed reconstruction scenarios (Hugot 1999).

Fig. 2. Tree reconciliation parasite species versus host species. Parasite tree on the right (after Hugot 1999), host tree after Purvis (1995).

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What can Explain the Presence of Squirrel Parasites in the Enterobiinae? Squirrel parasites are classified into two genera (Fig. 2). Xeroxyuris has a single species whose host, Xerus inauris, is a ground squirrel living in the dry steppes of southern Africa. Rodentoxyuris has two species: R. sciuri has been recorded from S. vulgaris, from Spain to Kamchatka. R. bicristata has been recorded from the North American squirrels: Glaucomys volans, G. sabrinus, Sciurus niger, S. carolinensis and S. aberti. In America the respective ranges of S. niger, S. carolinensis, and G. volans are widely overlapping, from the East coast west to the Rocky Mountains and north to the Canadian border. In addition, G. volans has montane populations scattered from Mexico to Honduras. G. sabrinus has a very different range overlapping with the respective ranges of the other three American squirrels around the Great Lakes and in the Appalachian Mountains. The phylogenetic relationships between Glaucomys spp. and the other squirrels still are debated. Glaucomys has been proposed to be classified together with all the other flying squirrels in a different subfamily within the Sciuridae; this supposes that the flying squirrels are a monophyletic group (Johnson-Murray 1977, Thorington 1984). Conversely, some authors have suggested that Glaucomys could be more closely related to other holarctic squirrels, particularly to the Sciurini (Gorgas 1967). These different hypothesis can be represented using two different topologies: (1) Glaucomys spp. as the sister group of Sciurus spp. + Xerus, (2) Xerus as the sister group of Sciurus spp. + Glaucomys spp. Several scenarios were obtained using Treemap. On Figure 3 are represented the most parsimonious scenario obtained using either topology 1 (Fig. 3A) or 2 (Fig. 3B). Thus, the reconstruction of the parasite tree proposed by scenarios A and B is independent of which phyletic relationships can be hypothesized between Sciurus and Glaucomys. To be valid, this reconstruction requires two assumptions: (i) a first host switching from the Platyrrhini to the ancestor of Sciurus spp., and (ii) a later host switching from Sciurus to Glaucomys. Because Sciurus probably migrated into the Neotropics during the late Tertiary, the first assumption seems valid. Considering the present distribution of Glaucomys spp. and Sciurus spp. in the Nearctic, the second assumption also is acceptable. Consequently, the scenario represented on Figures 3 looks acceptable. Finally, the cladistic analysis does not support close relationships between the squirrel parasites and suggests an early separation from the Enterobiinae for the first one (Xeroxyuris), and a host switching from the Callitrichidae to the Sciurini at the end of the Tertiary, when terrestrial connections were re-established between North and South America, for the second one (Rodentoxyuris). The simplest explanation for the distribution of this genus is a parallel evolution and dispersal of Rodentoxyuris with Sciurus spp., in the Holarctic. In the Asiatic part of its range, S. vulgaris is not parasitized by Rodentoxyuris but by a different genus of pinworm, Syphabulea, which is closely related to another subfamily: the Syphaciinae. The range of this last group extends from southeast Asia to North America and to Spain and parazites squirrels exclusively (Hugot & Feliu 1990). In the Holarctic part of its range Syphabulea, which is a parasite for Sciurus, Sciurotamias, Tamiasciurus and Glaucomys spp. (Hugot 1988), challenges Rodentoxyuris in the Sciuridae and separates the range of Rodentoxyuris into two disconnected area. This could explain the appearance of a different species of Rodentoxyuris in the Western Palearctic.

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Fig. 3. The most parsimonious scenario obtained using either topology 1 (A) or 2 (B), explaining the presence of squirrel parasites in the Enterobiinae. S. = Sciurus; G. = Glaucomys; X. = Xeroxyuris; R. = Rodentoxyuris.

Conclusion TreeMap allows: - to visualize all these scenarios which are maximizing the amount of codivergence and also to produce manually less parsimonious ones, - to clearly assess which scenarios are really supported by the sources studies and to discuss their validity by comparison with paleogeographical data. TreeMap also can be used for simulation studies or to test several phylogenetic hypotheses. Thus TreeMap appears to be a powerful tool to elucidate the history of two associates on the basis of their hypothesized individual history. References BROOKS D.R. 1981. Hennig’s parasitological method: a proposed solution. Systematic Zoology 30: 229-249. GORGAS M. 1967. Vergleichend-anatomische Untersuchungen am Magen-Darm-Kanal der Sciuromorpha, Hystricomorpha and Caviomorpha (Rodentia). Z. Wiss. Zool. 175: 237-404.

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HUGOT J.P. 1988. Les nématodes Syphaciinae parasites de rongeurs et de lagomorphes. Taxonomie. Zoogéographie. Evolution. Mém. Mus. Natl. Hist. Nat. Paris sér. A zool. 141:1-153. HUGOT J.P. 1999. Primates and their pinworm parasites: The Cameron hypothesis revisited. Systematic Zoology 48: 523-546. HUGOT J.P. & C. FELIU 1990. Description de Syphabulea mascomai n. sp. Analyse du genre Syphabulea. Systematic Parasitology 17: 219-230. JOHNSON-MURRAY J.L. 1977. Myology of the gliding membranes of some petauristine rodents (Genera: Glaucomys, Petaurista, Petinomys, and Pteromys). Journal of Mammalogy 58: 374-384. PAGE R.D.M. 1994. Maps between trees and cladistic analysis of historical associations among genes, organisms, and areas. Systematic Biology 43: 58-77. PAGE R.D.M. 1995. Parallel phylogenies: Reconstructing the history of host-parasite assemblages. Cladistics 10: 155-173. PAGE R.D.M. 1996. Temporal congruence revisited: comparison of mitochondrial DNA sequence divergence in cospeciating pocket gophers and their chewing lice. Systematic Biology 45: 151-167. RONQUIST F. & S. NYLIN 1990. Process and pattern in the evolution of species associations. Systematic Zoology 39: 323-344. THORINGTON R.W. Jr. 1984. Flying squirrels are monophyletic. Science 225: 1048-1050. WILEY E.O. 1988. Parsimony analysis and vicariance biogeography. Systematic Zoology 37: 271-290.

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Diverse perspectives on the Protozoan – Metazoan transition G. Shields1 & W. Foissner2 1. School of Earth Sciences, James Cook University, Townsville, Queensland 4811, Australia. E-mail: [email protected] 2. Institut für Zoologie, Universität Salzburg, Salzburg, Austria. E-mail: [email protected]

Introduction In order to represent adequately the increasingly interdisciplinary nature of evolutionary research, five scientists with strikingly different backgrounds were invited to record their individual perspectives on the Protozoan-Metazoan transition. This was no easy request considering the broadness of the subject and it is pertinent to note how freely the authors cross disciplines in their narratives, frequently covering common ground, but from quite different viewpoints. Comparative anatomy, molecular biology, geochemistry, paleontology and geology can all be found within these contributions. Here we outline four major issues that have emerged recently as “challenges and stimulants” to our understanding of the Protozoan-Metazoan transition. 1. The brush phenomenon problem Recent exponential increases in information, whether taxonomic, morphological, molecular or paleontological, might be expected to shed light on the pathway(s) from protozoa to metazoa. However, each new package of information or research direction, while leading to greater knowledge overall, tends to blur rather than clarify the evolutionary pathways of protozoan and metazoan taxa. Increasingly, the data do not produce an evolutionary “tree” but more of a “bush” or “brush”. Impressive examples of this problem can be found in papers by Cavalier-Smith (1998) on protists and by Abouheif et al. (1998) on multicellular organisms. The brush phenomenon is scaleindependent, that is, it can be observed between kingdoms and phyla as well as families and genera. Accordingly, common mechanisms may be at work, possibly involving parallel and convergent evolution. As more taxa are analyzed, more hidden convergences can accumulate that disturb the tree, especially in molecular systematics, where plesiomorphies and apomorphies cannot yet be distinguished. In addition, molecularbased tree construction has encountered other obstacles, such as long-branch attraction (Morin, 2000) and the unsuitability of most molecules for kingdom-scale phylogenetic

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analyses (Abouheif et al. 1998), which necessitate the successful marriage of morphological (ultrastructural) and molecular techniques in any modern study. Despite the recent advances outlined below, it is not clear at present how this brush phenomenon can be circumvented. 2. Hunting the ancestral metazoan The hunt for the ancestral metazoan has been the focus of much speculation in the published literature. Rieger (this volume) takes the reader through a comprehensive survey of hypotheses that explain the sequence of changes, which led from unicellular to multicellular organization. In agreement with the emerging consensus from molecular phylogeny (Hackstein, this volume), Rieger concludes on the basis of ultrastructure that unicellular organisms similar to choanoflagellates are the most likely candidates for the ancestral metazoan. Recently, this hypothesis has been invigorated following the publication of morphological and molecular evidence that a flagellate, Ancryomonas, could be the closest extant relative of the common ancestor to the metazoans, fungi and choanoflagellates (Atkins et al. 2000). Other major events in metazoan diversification are also discussed below. For example, the introduction of a biphasic lifestyle, common in primitive animals such as porifera and bryozoa, with a microscopic, ciliated, dispersive larval stage and a macroscopic, colonial adult stage, is proposed to have been one such significant event (Rieger, this volume). This idea is further embellished by Dewel et al. (this volume) who maintain that the “morphology of living metazoans indicates that the adult bilaterian ancestor was both macroscopic and complex” (see also Budd & Jensen 2000). Indeed, it is argued that the fossil record, in the form of the large, latest Neoproterozoic Ediacaran fauna (580 – 540 Ma), may lend support to this idea. However, it is still far from clear whether these enigmatic frond- and disc-like fossils were in any way ancestral to later lines of extant phyla (Knoll & Carroll 1999). The recent discovery of new taphonomic windows for the preservation of microscopic metazoans, such as phosphatized embryos (Bengtson, this volume), will certainly help in establishing the relevance or irrelevance of Ediacaran fossils to later metazoan diversification during the Cambrian explosion. 3. Molecular phylogeny vs. Fossil records: how real is the Cambrian explosion? Paleontologists worldwide have recently been able to reach agreement on a number of important issues regarding the fossil record (Bengtson, this volume). Most importantly, all reports of pre-580 Ma metazoan fossils are now widely considered to be of dubious nature. This is especially important as it comes in the light of rapidly improving knowledge of our global fossil archive and serves to firmly close the door on the possibility of a long, but hidden history of macroscopic metazoan evolution. This consensus has led many to consider the “Cambrian explosion” of greatly increasing metazoan diversity between 545 Ma and 510 Ma to represent a real evolutionary event, and one that needs to be incorporated into future hypotheses of metazoan origins. However, molecular studies generally do not support such a late branching of metazoan phyla (Bengtson and Hackstein, both this volume), while studies that have, on the basis

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of irresolvable nodes, lent weight to the notion of a period of very rapid evolutionary expansion, are themselves open to ambiguous interpretation (Abouheif et al. 1998). The joint ancestor of the protostomes and deuterostomes is consistently estimated by “molecular clocks” to have existed more than 670 million years ago. This highlights a possible gap in the current fossil record, especially if one considers Rieger’s arguments (this volume) that the ancestral bilaterian possessed a macroscopic adult phase, which ought certainly to have left a recognizable fossil record during the long period between 580 and >670 Ma. This apparent contradiction can be partially resolved by assuming a long, hidden history of microscopic metazoan evolution (Fortey et al. 1997, Peterson & Davidson 2000) but a hypothesis based on a lack of evidence is unsatisfactory. Bengtson (this volume) makes the important point that distinguishing features are acquired successively throughout the evolution of a lineage, causing the first members of that lineage to go unrecognized, if indeed any representatives of crown-group phyla were present during the earliest phases of metazoan diversity expansion (Budd & Jensen 2000). However, at the present time there are no fossil candidates for an ancestral metazoan within this crucial pre-Ediacaran period. Although the lack of macroscopic metazoans in the Precambrian awaits definitive interpretation, more recent research has significantly narrowed the gaps between fossil and molecular-based estimates of metazoan branching (Ayala et al. 2000, Smith & Peterson, 2002), leaving us optimistic that this will continue. 4. Unraveling the geological context behind early metazoan evolution By analogy to Phanerozoic evolutionary events, such as the Permian-Triassic boundary, the geological context is likely to have been an exceedingly important factor in early metazoan evolution (Knoll & Carroll 1999). Bengtson, Brasier, Dewel et al., and Hackstein (all this volume) all emphasize the role of massive climate upheaval in causing potential population bottlenecks. Global glaciations, such as occurred during the late Neoproterozoic, are likely to have led to small, isolated gene pools, thus helping to maximize mutation rates. Intense glaciations repeatedly disrupted Earth’s surface environment between 720 and 580 Ma, causing ocean anoxia, and massive sea-level change (Brasier, this volume). Although increases in atmospheric oxygen concentrations may have acted as a trigger for higher metazoan evolution (Runnegar 1991), repeated environmental stress over millions of years caused by fluctuating oxygen and nutrient levels (Brasier, this volume) may have had a greater effect on the pace and nature of early metazoan evolution. Bearing such environmental instability in mind, it is certainly worth considering that the early adoption of a biphasic lifestyle (Rieger, this volume) would have proved advantageous to the first true metazoans. References ABOUHEIF E., ZARDOYA R. & A. MEYER 1998. Limitations of metazoan 18S rRNA sequence data: Implications for reconstructing a phylogeny of the animal kingdom and inferring the reality of the Cambrian explosion. J. Mol. Evol. 47: 394-405.

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ATKINS M.S., McARTHUR A.G. & A.P. TESKE 2000. Ancryomonadidina: a new phylogenetic linkage among the protozoa closely related to the common ancestor of metazoans, fungi and choanoflagellates (Opisthokonta). J. Mol. Evol. 51: 278-285. AYALA F.J., RZHETSKY A. & F.J. AYALA 2000. Origin of the metazoan phyla: molecular clocks confirm paleontological estimates. Proc. Natl. Acad. Sci. USA 95: 606-611. BUDD G.E. & S. JENSEN 2000. A critical reappraisal of the fossil record of the bilaterian phyla. Biol. Rev. 75: 253–295. CAVALIER-SMITH T. 1998. A revised six-kingdom system of life. Biol. Rev. 73: 203-266. FORTEY R.A., BRIGGS D.E.G. & M.A. WILLS 1997. The Cambrian evolutionary ‘explosion’ recalibrated. BioEssays 19: 429–432. KNOLL A.H. & S.B. CARROLL 1999. Early animal evolution: emerging views from comparative biology and geology. Science 284: 2129-2137. MORIN L. 2000. Long branch attraction effects and the status of “basal Eukaryotes”: phylogeny and structural analysis of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis. J. Eukaryot. Microbiol. 47: 167-177. PETERSON K.J. & E.H. DAVIDSON 2000. Regulatory evolution and the origin of the bilaterians. Proc. Natl. Acad. Sci. USA 95: 4430-4433. RUNNEGAR B. 1991. Precambrian oxygen levels estimated from the biochemistry and physiology of early eukaryotes. Palaeogeography, Palaeoclimatology, Palaeoecology 71: 97-111. SMITH, A.B. & K.J. PETERSON 2002. Dating the time of origin of major clades: molecular clocks and the fossil record. Ann. Rev. Earth Planet. Sci. 30: 65-88.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

The phenotypic transition from uni- to multicellular animals 247 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 247-258, 2003

The phenotypic transition from uni- to multicellular animals R.M. Rieger Department of Zoology and Limnology, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria. E-mail: [email protected]

Abstract This contribution reviews three processes leading from unicellular to multicellular organization as well as models proposed for the early differentiation of the metazoan phenotype. Phenotypic features of choanoflagellates and ciliated cells in basal metazoans are discussed. The significance of the extracellular matrix (ECM) and especially of the monociliated cell as an ancestral feature of the metazoan body plan is emphasized. New evidence suggests that certain avenues to multiciliary cells go back to the onset of metazoan evolution. It is concluded that unicellular organisms similar to choanoflagellates are the most likely candidates among unicellular eukaryotes for having given rise to multicellular animals.

Introduction The topic reviewed here deals with an ancient event in the history of animal life and has stimulated phylogenentic discussions for many years (Willmer 1990, Denis 1995, Dewel 2000, Baldauf et al. 2000, Atkins et al. 2001, Cavalier-Smith 2001, Nielsen 2001 and references therein). Here I want to point out the three processes that could have led from the phenotype of unicellular eukaryotes to that of premetazoan cell colonies and to multicellular animals. The current developments in unravelling molecular aspects and reproductive strategies at the onset of multicellular organization are not considered (see eg., Rieger 1994a, Szathmáry 1994, Maynard Smith & Szathmáry 1999, Kerzberg & Wolpert 1998, Müller 2001). Rather, the hypotheses are discussed for the differentiation of the phenotype from premetazoan cell colonies to multicellular animals as well as the significance of cytological features of the first somatic metazoan cell for understanding the steps to the early metazoan radiation. Three scenarios for deriving the metazoan phenotype from unicellular eukaryotes It is generally accepted that three processes could have produced the phenotype of the early metazoans: (1) cell clones produced by cell divisions, (2) cellularization of

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multinucleated cells, (3) cell aggregations (Fig. 1). The formation of cell clones on/within an extracellular matrix (ECM) is widely accepted as the basis for the appearance of multicellular animals (Rieger & Weyrer 1998 and references therein). Formation of cell clones also played a role at the origin of multicellular plants and fungi. For multicellularity of metazoans and metaphytes it is becoming evident that cells in the clones were connected initially by cytoplasmic bridges. A well-known example of this is given by the cytoplasmic bridges connecting all cells in the colony in basal multicellular Chlorophyta such as Volvox. More elaborate variations of interconnections are seen in the primary (remnants of cell division) and secondary (fusions of cells) plasmodesmata in higher plants (Sitte et al. 1998). The Hexactinellida, likely the sister group of all other extant metazoans (Müller 2001 and references therein) feature interconnections in the form of plasmic bridges apparently resulting both from incomplete cell divisions and from secondary fusions of cells (Mackie & Singla 1983, Mackie 1990, Reiswig & Mehl 1991). Similar intercellular connections with plugstructures are known from choanoflagellates (Hibberd 1975) and also between the fiber cells of Trichoplax (in Grell & Ruthmann 1991). In this context it should be promising to

Fig. 1. Processes that could have led from unicellular eukaryotes to multicellular organization. A) Complete or incomplete cell divisions of unicellular ancestors within a common extracellular matrix (ECM). B) Cellularization of a multinucleated cell. C) Aggregations of cells attracted by chemical signals. Modified after Westheide & Rieger (1996).

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test the hypothesis that cytoplasmic bridges in multicellular animals were replaced soon thereafter by gap junctions. This would represent a significant event for the evolution of independent, yet physiologically linked, cells. In addition to this direct cell-cell communication spot- or band-shaped indirect junctions were formed for controlling paracellular pathways and providing structural support. The unique, complex macromolecular structure of the ECM was a key factor in the origin of division cell colonies of the Metazoa (Rieger & Weyrer 1998). The ECM facilitated movements of individual cells within the colony and provided a substrate for chemical communication during development and in the adult in the form of intercellular gradients (Wolpert 1998, Gilbert 2000). All basic molecular components of the ECM, collagens, proteoglycans and specific glycoproteins (eg. fibronectins) are already present in the Porifera (eg., Morris 1993, Rieger 1994a, Garonne 1998, Müller 2001). Among animals with the simplest organization, only Trichoplax adhaerens lacks any evidence of ECM (Rieger & Weyrer 1998). It is not resolved entirely whether this lack is primitive or derived, but some molecular and phenotypic data suggest the latter (eg. Ax 1996, Zrzavy et al. 1998) Models for the phenotype of early multicellular animals (Fig. 2). Molecular biology has substantially supported the monophyly of the Metazoa, including the Parazoa (Müller 2001 and references therein). The presence of at least some ciliated cells, either for locomotion or for producing water currents through an internal cavity- or canal-system of the body characterizes all anatomical models at the protozoan/metazoan boundary. This characteristic – as well as the lack of a cell wall distinguishes the early metazoans from early fungi. Many ideas about the early differentiation of metazoans were formulated already during the second half of the nineteenth century, concurrent with the advent of new methods for light histological and embryological studies. Several of these ideas are still valid today in the light of new taxa and ultrastructural and molecular data (see Rieger & Weyrer 1998, Rieger & Ladurner 2001). All these models propose a small ciliated vermiform organism with an internal cavity resulting from one invagination (or single cell ingressions) from the outermost layer of cells as the ancestral metazoan. The Gallertoid-hypothesis was one of the first to emphasize the importance of the development of the ECM and thereby of the novel mechanical properties for the metazoan body plan (Gutmann 1966, Gutmann 1981). Based on comparative ultrastructural studies, I have proposed the early establishment in metazoans of a biphasic life cycle in which a ciliated, dispersive larval stage alternated with a macroscopic, modular or colonial adult (Rieger 1994a,b). As the most basal extant metazoans suggest, tissue diversification arose first in organisms subjected to two selection regimes, in the adult and larva, and not in a small ciliated organism alone. The internal cavity system could have been developed by fusion of multiple invaginations (“multi-gastrulae”), which arose from asexually produced daughter colonies by processes as seen in Volvox. Modular organization is an important characteristic also during the further evolution of the Metazoa. The concept of the importance of colonial or modular

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Fig. 2. Proposed models for the early evolution of the metazoan phenotype. A) Haeckel´s Gastrea hypothesis. B) Blütschli´s Placula hypothesis. C) Metschnikoff´s Phagocytella hypothesis. D) Lankester´s and v. Graff´s Planula hypothesis. E) Gutmann´s and Grasshoff´s Gallertoid hypothesis. F) Rieger´s hypothesis of plesiomorphy of biphasic life cycle. Modified after Rieger & Weyrer (1998).

organization during the transition from diploblastic to triploblastic Eumetazoa was further elaborated recently by Dewel (2000). The monociliated cell as ancestral cell type of all somatic cells and of germ cells of the Metazoa (Fig. 3) Ultrastructural characteristics of monociliated cells were first studied comparatively in larval echinoderms (Nørrevang & Wingstrand 1970), in the epidermis of various gastrotrichs and in the polychaete Owenia fusiformis (for lit. see Rieger 1976, Gardiner 1992). In a series of excellent papers, Holley (1982, 1983, 1984, 1985, 1986) has compared monociliated epithelia in lower and higher Metazoa and discussed their functional aspects. Nielsen (1987, 2001) has given an overview of ciliated epithelia and the distribution of monociliated versus multiciliated cells in the Metazoa. Yet the picture of variability remains incomplete, and detailed investigations, such as that by Woollacott and Pinto (1995) will be needed for understanding the evolution of cell and tissue differentiation in animals. Generally, monociliated cells in metazoan epithelia are bilaterally symmetrical. The ciliary apparatus, the direction of the ciliary stroke, nucleus and Golgi complex mark the plane of symmetry. The basal foot and the accessory centriole indicate the posterior part of the cell. The vertical rootlet is connected to the basal body of the cilium at its anterior pole. Monociliated cells in epithelial configuration (i. e. in unspecialized epithelia used in the production of water currents) occur in approximately 95% of the poriferan, 100% of the placozoan, 75% of the coelenterate, 10% of the deuterostome, and in 0.5% of the

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protostome species (data taken from lit. in Rieger 1976, Westheide & Rieger 1996, Nielsen 1987, 2001). The dominance of monociliated cells in Porifera, Placozoa and radiates strongly supports the proposal that this was the original cell type of the Metazoa. In the search for the sister group of the Metazoa among the unicellular eukaryotes the monociliated cell type limits the possible choices. A comparison of monociliated cells in the taxa of the lower Metazoa shows the following: In the Parazoa (see lit. and summary in Harrison & De Vos 1991, Woollacott & Pinto 1995, Boury-Esnault 1999) the choanocyte has a well-developed microvillar collar (30 -

Fig. 3. Important features of monociliated cells in Metazoa. A1, A2: ciliary base of eumetazoan monociliated cells, showing bilateral organization. In A2 the ciliary effective stroke is towards the right. B1 - B5: monociliated cells of Parazoa (B1,B2) and Eumetazoa (B3-B5), explanations in text. A1, A2 modified after Holley (1984). B1-B5 modified after Westheide & Rieger (1996) and Rieger & Weyrer (1998).

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40 microvilli), with the character being absent in larvae. An accessory centriole may or may not be present. A basal foot is usually present. Three different designs are known from sponge larvae, where well developed microtubular, laminated, or striated fibers are present (see Woollacott & Pinto 1995, p. 257-258 for details). Striated rootlets are generally missing in choanocytes of adults, but are present in certain larvae of the Calcarea. A bilateral ciliary vane occurs in many choanocytes, but appears to be absent in the monociliated cells of the larvae. In sponge choanocytes the flagellum beat in a planar undulating wave, in the larvae Nielsen (2001, p.42) point to the fact that “…their cilia show the effective-stroke beating pattern characteristic of all planktonic larvae...” (see Woollacott & Pinto for a different terminology). If present, the accessory centriole is situated “caudally” to the basal body, that is, on the downstream side of the effective planar beat of the flagellum or of the ciliary effective stroke. According to Woollacott and Pinto (1995) - The accessory centrioles in the larvae are directed either perpendicular to, or in the plane of, the effective stroke of the cilium. However, the larvae of Tedania ignis and Dysidea etherina show that the accessory centrioles are most commonly placed into the direction of the effective stroke (Rieger 1994a, Rieger & Weyrer 1998, unpublished observations) The monociliated cells of sponges are unique, and differ from those in the other metazoans (eg. presence or absence of accessory centriole, variation of position of accessory centriol). In addition, the ancestral structure of the somatic cell in the Parazoa became specialized very early into two directions: 1). The larval “monociliated” cell developed a deeply penetrating fibrous rootlet that then became elaborated. Among larvae, the loss of a microvillar collar and the occurrence of a striated rootlet fiber among certain species suggest that the primary function of these cells was enhancement of the locomotory function in the larva. 2) In the adult, a loss of cilia occurred in most species in the pinacoderm, but choanocytes remained similar to the ancestral stage, adapted for filter feeding of particles of the size of bacteria. The decrease in number of microvilli in the collar of monociliated cells is evident above the level of Parazoa. This may be a consequence of the change in the size of food consumed by coelenterates and most bilaterians (Rieger & Weyrer 1998). In order to understand the history of the terms “choanocyte-like or monociliated cells” in higher metazoans it may be of interest to mention here the retention of microvillar collars by some protonephridial terminal cells, which probably also filter small particles and macromolecules. Multiciliated cells have been found so far only in the Trichimella-larva of the Hexactinellida (Boury-Esnault 1999). Because of the syncytial tissue organization of this taxon multiciliarity here might be a consequence of cytoplasmic bridges between monociliated cells. These cells, however, lack accessory centrioles, as in most multiciliary cells in eumetazoans (Rieger 1976, Nielsen 1987). Placozoa, according to Grell & Ruthmann (1991), exhibit a monociliated design of the upper and lower epithelioid layers with interdispersed gand cells. The flagellar base is surrounded by a depression, microvillar collars are lacking and the striated rootlet is well developed and penetrates deeply into the cell. The accessory centriole is oriented apparently as in higher animals (see below). In the Cnidaria the monociliated cell is a basic somatic cell type (see e.g., Rieger 1976, Holley 1982, Westheide & Rieger 1996, Nielsen 2001). In this taxon, epithelio-muscle cells,

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gland cells, sensory cells, even certain nerve cells may show a monociliated organization. Accessory centrioles in Cnidaria and in all higher Eumetazoa are generally situated perpendicular to the direction of the effective stroke of the cilium. Although monociliated cells dominate in cnidarians, gastrodermal cells in Anthozoa may be multiciliated, with accessory centrioles (Rieger 1976, Nielsen 2001, own unpublished results on various octocorallians). Multiciliated cells occur also in the tentacles of a hydromedusa (see lit. in Nielsen 2001). Another exception is the occurrence of biciliated epidermal cells in the gastrodermal epitheliomuscle cells in Hydra (Tardent 1984). It seems that published data on ciliation of the planula larvae have revealed only monociliated cells (eg. Hydrozoa, see Thomas & Edwards 1991, Scyphozoa, see Leish-Laurie & Suchy 1991, for Anthozoa see Fautin & Mariscal 1991, Vicki Martin, person. comm.). In the Ctenophora, multiciliated cells, such as macrocilia and combs are the epidermal ciliated cells used for producing water currents plates (see Hernadez-Nicaise 1991). Except for sensory cells, epidermal monociliated epithelia appear to be absent in this taxon. In Bilateria the monociliated cell can still be found, but multiciliated cells have increased significantly (Rieger 1976, Nielsen 1987, 2001). Groups that possess exclusively monociliated cells are: all Gnathostomulida (Lammert 1991), certain gastrotrich taxa (Rieger 1976, Ruppert 1991) the annelid genera Owenia and Magelona (Gardiner 1992, own unpublished results), almost all echinoderms, the Brachiopoda, Phoronida, Chaetognatha, Pterobranchia, and the Cephalochordata (see Holley 1984, Westheide & Rieger 1996). A multiple origin for multiciliated cells in bilaterians is generally accepted. Nevertheless, a phylogenetic analysis based on non-ciliary characters (Meyer & Bartolomaeus 1996) suggests a reversal of multi- into monociliated epithelia. However, the validity of some of the conclusions made by Meyer and Bartolomaeus have subsequently been questioned (Merz & Woodin 2000, Salvini-Plawen 2000). Nielsen (2001) assumes that the multiciliated stage is an apomorphy for all protostome taxa and suggests a reversal to the primitive monociliated stage for the Gastrotricha, the Gnathostomulida and Owenia among the Annelida. Interesting as this assumption is, in the case of the Gastrotricha and Gnathostomulida Rieger (1976) already provided ample evidence that the monociliated condition must be the plesiomorphic condition in those groups. It may be worth to stress here that multiciliated cells in Bilateria in general seem to have not much less than 10 cilia per cells and monociliated epithelia may have some biciliated cells (see e.g., Gardiner 1978). In summary, the original type of somatic cells and female and male germ cells had the organization of the monociliated cell. Although this interpretation is widespread in the literature for the somatic cell and the male gamete type (Barnes 1985, Ax 1996, Westheide & Rieger 1996, Nielsen 2001), it is much less well known that recent data suggest the same interpretation for female gametes (Frick & Ruppert 1996, 1997, Frick et al. 1996). Conclusions While a comprehensive model of the premetazoan is premature, some phenotypic aspects can be identified that might stimulate research on structures and regulatory factors at the transition between protozoans and metazoans.

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Choanoflagellates – sister group of the Metazoa? From the data presented here, I deduce that the unicellular eukaryote ancestor was either a dimastigote flagellate that lost one ciliary shaft and developed a bilateral symmetry, or was a uniflagellate, bilaterally symmetrical flagellate. In either case, one basal body was an accessory centriole, and the other bore the cilium. The ciliary basal body bore a basal foot on the downstream side of the effective beat of the flagellum. A basal foot is rare in protists, but present in choanoflagellates where it may be more complex (Woollacott & Pinto 1995). Accessory centrioles are known also in several choanoflagellates (see Woollacott & Pinto 1995 for lit.). The ciliary basal apparatus has been reconstructed recently in the choanoflagellates Monosiga and Desmarella (Karpov & Leadbeater 1998). This organization of the ciliary base may well represent the ancestral structure of all monociliated cells of higher Metazoa: the accessory centriole is situated below the basal body, the striated fibrillar rootlet extends from the Golgi body to the accessory centriole and a fibrillar connection between accessory centriole and basal body exists. In detail, differences remain when comparing these choanoflagellates with monociliated cells in larval and adult sponges (see e.g. Woollacott & Pinto 1995) and more comparative ultrastructural work is needed to clarify this issue. If Metazoa (and Fungi) are derived from choanoflagellates (see Atkins et al. 2000, Baldauf et al. 2000, Cavalier-Smith 2001), the changes to the somatic cell of metazoans would have occurred primarily in the larva (reduction of collar, origin of fibrous main rootlet), the choanocyte in the adult may have remained similar to the ancestral choanoflagellates. The presence of cytoplasmic bridges with plugs in the choanoflagellate Codosigna (Hibberd 1975), resembling the bridges in the most primitive extant metazoans (Hexactinellida, Mackie & Singla 1983), certainly corroborates such a proposal. However, some arguments questioning a sister-group relationship of choanoflagellates and metazoans remain (e.g. distinct differences in the structure of the “flagellar vanes”, see Ax 1996). No “true” metazoan organism without ECM. In premetazoan cell colonies some cells must have invaded the ECM to form more complex supportive tissues and a new space for storing reproductive cells; therefore, the original premetazoan cell colonies had to be a three-dimensional organism held together by a collagenous ECM (Morris 1993, Rieger 1994a, Müller 2001). Colonial organization in general occurs often among flagellate protists, but a well-developed ECM is not always present. Examples of colonial protists with a well-developed ECM (but with different chemical composition) are the volvocaceans, certain choanoflagellates and colonial heterotrophic flagellates such as Uroglena, or Spongomonas. (see Patterson & Larsen 1991). Obviously there is a need for more comparative studies on the molecular composition of the metazoan ECM and that of colonial protists. Collagen has been reported from fungal fimbria (Celerin et al. 1996), which may support other molecular evidence that animals are more closely related to Choanozoa and the Fungi than to plants (see e.g., Cavalier Smith 2001). Monociliated cells and the origin of centrioles: The three-dimensional complex structure of the flagellar basal apparatus was important for the functioning of the unicellular ancestor and remained so in early metazoan cell colonies. Gradually this flagellar base became also an important center for specifying the positional relationships

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of cells in early multicellular animals. The spatial relationship between the basal body, the accessory centriole and other organelles may have played a role in the original patterning of the cells. The centrioles of animal cells and their role in cell division can be seen as a further step in the evolution of the basal ciliary apparatus. Multiciliated cells occurred very early in metazoan evolution. The finding of multiciliated cells (Boury-Esnault 1999) in what is very likely the most primitive metazoan taxon (the Hexactinellida see lit. in Müller 2001) adds a new perspective to the question of the origin of multiciliated cells during the early steps in metazoan evolution. Incomplete cell divisions and secondary fusion of cell-cell processes as seen in the Hexactinellida seem to be the first cytological metazoan design. Multiciliarity in the hexactinellid larva could be a direct product of fusion or incomplete separation of monociliated cells. However, plasmodial and syncytial tissue design was soon displaced in animal evolution by true cellularity (see above). Plasmatic bridges may be present in colonial Choanoflagellates (see above), but apparently only between uniflagellated cells. Thus, the monociliated cell must still be seen as the ancestral cell type from which soma and germ cells in the Metazoa arose. Multiciliarity - even if now known to have occurred during the very early phase of metazoan evolution - developed convergently many times from the monociliated condition. Acknowledgements Thanks are due to W. Foissner and G. Shields for the invitation to this symposium. I also thank in particular Ed Ruppert (Clemson University) and Gunde Rieger but also S. Tyler (Univ. of Maine at Orono) and P. Ladurner (Univ. of Innsbruck) for valuable comments. The final preparation of figures was carried out by R. Gschwentner (Univ. of Innsbruck). Supported by FWF, grant P13060-BIO. References ATKINS M.S., McARTHUR A.G. & A.P. TESKE 2000. Ancyromonadida: A new phylogenetic lineage among the Protozoa closely related to the common ancestor of metazoans, fungi, and choanophlagellates (Opisthokonta). J. Mol. Evol. 51: 278-285. AX P. 1996. Multicellular Animals. A New Approach to the Phylogenetic Order in Nature. Springer, Berlin, Heidelberg, New York, 340 p. BALDAUF S.L., ROGER A.J., WENK-SIEFERT I. & W.F. DOOLITTLE 2000. A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 290: 972-977. BARNES R.D. 1985. Current perspectives on the origins and relationships of lower invertebrates. In Conway Morris S., George J.D., Gibson R. & H.M. Platt (eds), The Origins and Relationships of Lower Invertebrates. Clarendon Press, Oxford, pp. 360-367. BOURY-ESNAULT N., EFREMOVA S., BÉZAC C. & J. VACELET 1999. Reproduction of a hexactinellid sponge: first description of gastrulation by cellular delamination in the Porifera. Invert. Reprod. Develop. 35(3): 187-201. CAVALIER-SMITH T. 2001. What are Fungi? In McLaughlin D.J., McLaughlin E.G. & P.A. Lemke (eds), The Mycota VII, Part A, Systematics and Evolution. Springer-Verlag Berlin Heidelberg, pp. 3-37.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers From Famine to Feast: a context for the protozoan-metazoan transition 259 The New Panorama of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 259-268, 2003

From Famine to Feast: a context for the protozoan-metazoan transition M. Brasier Department of Earth Sciences, Oxford University, UK

Abstract Very long periods of environmental stability are suggested by the carbon isotopic and palaeoclimatic record prior to c. 1000 Ma. It was during this time that eukaryotes, including protozoans, are likely to have emerged. This steady state was repeatedly disrupted in the prelude to the ‘Cambrian Explosion’ up to 540 Ma, as shown by stable isotopes and evidence for extreme, possibly ‘snowball earth’ glaciations. Filter feeding by multicellular sponges appears to have evolved during this time of inferred eutrophication. Multiple mass extinctions may then have accelerated evolutionary rates, leading towards metazoan phyla.

Introduction The aim of this paper is to draw attention to what the geological record appears to be saying about the timing and environmental conditions relating to the emergence of protistan heterotrophy and animal multicellularity. It explores the possibility that both innovations were driven forward by extrinsic factors that shifted environmental parameters from stable and oligotrophic towards more oscillatory, seasonal and eutrophic conditions, especially between c. 1000 and 500 Ma. The article begins with evidence for the timing of emergence of foraminifera, dinoflagellates, algae and sponges, as obtained from both the rock record and studies of molecular phylogeny. This is followed by a discussion of the geological context. The fossils vs. molecules debate The fossil record arguably suggests that multicellularity in animals appeared rather late in the history of life, within a short interval between c. 600 and 500 Ma ago, known as the Cambrian explosion. Indeed, some would put the earliest undisputed evidence for animals as late as 565 Ma or even close to 543 Ma, at the Precambrian-Cambrian boundary (Knoll & Caroll 1999). Molecular data can be marshaled in support of this ‘big bang’ model. Some have inferred that the polytomies (i.e. the unresolved nodes) and weakly

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supported nodes among protostome and deuterostome clades on the metazoan tree may be the result of a big bang in the evolution of animal phyla, culminating in the Cambrian Explosion (Erwin 1991, Phillipe et al. 1994; Cavalier-Smith 2000). It can, however, be argued that the Cambrian explosion conflicts with evidence based on the calibrated rates of sequence divergence and inferred deep-time divergence between protozoans and metazoans (e.g. Wray et al. 1996). Some palaeontologists have speculated that early metazoans left no fossil record because they were microscopic meiofauna, or lived as microplankton (Fortey et al. 1997). Against this, however, is the view that the 18s RNA molecule alone is an unsuitable candidate for reconstructing the phylogeny of the metazoa and that the polytomies observed within the 18S RNA phylogenies are not reliable evidence for inferring evolutionary rates (Abouheif et al. 1998). Foraminiferid origins This possible mismatch between the fossil record and the molecular evidence is amply illustrated by evidence from the foraminifera. These are one of the most diverse groups of unicellular eukaryotes with probably the best known fossil record of any group (Tappan & Loeblich 1988, Pawlowski et al. 1996). They are characterized by granuloreticulose pseudopodia and, generally, by the formation of a protective external test. Phylogenetic analysis of both the small 18S rDNA and the large 29-rDNA ribosomal RNA genes place the foraminifera as a monophyletic clade deep within the eukaryotic tree, later than the amitochondrial Archezoa and earlier than the Euglenozoa and other mitochrondria-bearing phyla (Pawlowski et al. 1996, 1997). The earliest ubdoubted Foraminiferal microfossils are agglutinated tubes of Platysolenites sp., which appeared in the Nemakit-Daldynian stage, just above the base of the Cambrian across the north Atlantic region (McIlroy et al. 2001). These had a delicate proloculus followed by single, flexible tube and could be regarded as an ancestral stock of the Astrorhizida, while simple, spheroidal agglutinated forms occur in rocks of younger Lower Cambrian age (McIlroy et al. 2001). A greater diversity of agglutinated tests, including Ammodiscus and Turitellella, has also been recorded from questionably earliest Cambrian rocks in Senegal (Culver 1994). Molecular evidence indicates that foraminifera could have existed much earlier than the Cambrian explosion, as naked amoeboid cells, or with poorly preserved organic or agglutinated tests (Pawlowski et al. 1997, Langer 1999). An alternative and provocative view is that the appearance of fossil foraminiferids in the Cambrian did not long postdate the evolutionary radiation of most eukaryote groups (Cavalier-Smith 1999, 2000). Unfortunately, it may not be possible to reconstruct exactly when the Foraminifera diverged from the eukaryote root stock using ‘molecular clocks’, because of their highly variable rates of rDNA evolution, which can be up to ten times those known from plants (Pawlowski et al. 1997, Langer 1999). Several lines of evidence, however, support the concept of late stage skeletalization in the foraminifera, especially during the Cambrian explosion. The discovery of fossilized ciliates in Triassic amber (Foissner et al. 1999), for example, reminds us of the incompleteness of the fossil record of naked protists. Some foraminiferid stocks (the Miliolina) also appear to have evolved from unpreserved organic-walled ancestors as late as the Carboniferous (e.g., Tappan & Loeblich 1988,

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Pawlowski et al. 1997). More importantly, it can now be argued that the loricae of testate amoebae occur in rocks as old as c. 800-740 Ma (Porter and Knoll 2000). This is significant because molecular evidence suggests that both filose and lobose testate amoebae are likely to have branched off later than foraminifera, within the ‘crown’ of the eukaryotic tree (e.g., Pawlowski et al. 1997, Porter & Knoll 2000). The radiation of ‘crown’ eukaryotes Branching patterns in the molecular phylogenies of eukaryotes suggest that red algae, green algae, chromophyte algae, ciliates, dinoflagellates, plasmodia, fungi and diploblastic animals all diverged rapidly and relatively late within the history of the domain, at some point between the foraminifer and the sponges (Sogin 1994, Pawlowski et al. 1997). Perhaps the earliest convincing date for this radiation is provided by dinosteroid biomarkers of a kind consistent with dinoflagellate eukaryote phytoplankton, in rocks as old as the Mesoproterozoic McMinn Formation of Australia (Moldowan et al. 2000), estimated at some 1340 Ma old (Schopf & Klein 1992). Multicellular fossils of algal grade, with some of the features of rhodophyte algae, first appear in the fossil record c. 1200 Ma ago (Butterfield et al. 1990, Butterfield 2000a), while xanthophyte-like algae occur close to 1000 Ma (Woods et al. 1998). Between c. 1000 and 720 Ma, an increase is then seen in the size, complexity and diversity of organic-walled microfossils called ‘acritarchs’, taken to be the cysts or growth stages of prasinophytes, chrysophytes or extinct groups of algae (Schopf & Klein 1992, Knoll 1994). Some bear a marked resemblance to dinoflagellate cysts (Butterfield & Rainbird 1998), while others from the Proterozoic Doushantuo Formation (c. 565 Ma) compare closely with much younger ‘mazuelloid’ microfossils of Ordovician-Devonian age (Zhou et al. 2001). At least some of these could have been the remains of giant protistan phytoplankton. An intriguing suggestion for a gradual increase in microfossil diversity near to 1000 Ma is that it marks the development of a sex-based life cycle (Knoll 1994) and/or the effects of an increase in atmospheric oxygen and/or nitrate levels (Porter & Knoll 2000). An opposing view is that all these comparisons with extant eukaryotic groups are misleading; none were true eukaryotes and the evolution of plastids may have taken place as late as 600 Ma ago (Cavalier-Smith 1999, 2000). Sponges and metazoan origins The first important step, from unicellular protists to sponge-grade multicellular animals, arguably took place via colonial aggregation of genetically identical unicellular clones (Rieger & Weyrer 1998). Molecular evidence confirms that sponges must lie close to the ancestral group of the monophyletic metazoan clade to which they belong (Abouheir et al. 1998, Muller et al. 1998). Furthermore, the 18S rRNA data suggest that sponges share a common ancestor with their sister group, the choanoflagellates (Cavalier-Smith et al. 1996); and that, among sponges, hexactinellids are the most primitive (Muller et al. 1998). The latter differ from other sponges in having a syncytial structure, though whether this is a primitive or secondary feature is not known (Muller et al. 1998).

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Late Precambrian-Cambrian hydrocarbons (c. 600 and 500 Ma) commonly contain high concentrations of biomarkers found in modern demosponges, suggesting that such sponges may have flourished during this time interval (e.g., McCaffrey et al. 1994), though this evidence may not be wholly diagnostic. Isolated spicule-like structures in cherts from the lower part of the Doushantuo Formation of southern China (c. 560 Ma) are poorly illustrated (Ding et al. 1988) and remain questionable, while spicule-like structures from the younger, Dengying Formation of southern China (Steiner et al. 1993) are poorly preserved and illustrations appear to include casts or moulds of filamentous cyanobacteria or algae. Also uncertain as yet are those structures compared with sponge gemmules bearing monaxon spicules found in c. 560 Ma phosphorites from south China (Li et al. 1998). It is still necessary to be cautious about these because small spherical, spicule-bearing bodies could also be protistan (cf. choanoflagellates; e.g., Leadbeater 1986); they lack evidence for growth into adult sponges; and an origin of the spicules from inorganic mineral growth needs to be discounted. Recent claims have also been made for possible metazoan embryos in the same beds (Xao et al. 1998, 1999), though they had previously been compared with Volvox-like chlorophyte colonies (Xue et al. 1998). These comprise phosphatically-preserved spheroidal microfossils with cleavagelike sutures, whose embryonic nature is suggested by cellular subdivisions which are independent of size. The cleavage patterns are rather unusual when compared with most metazoans, however, and there is a puzzling absence of embryos beyond the 32 cell stage (cf. Conway Morris 1998). Given the limited number of ways in which embryonic cells can cleave, leading to the very real possibilities of convergence, the interpretation of these structures as metazoan (and even as an extant type of metazoan) is suggestive rather than compelling. The affinity of these microfossils lies beyond resolution until transitions to adult forms are found (cf. Zhao & Bengtson 1998). Discoidal fossils with possible hexactinellid impressions have been reported from c. 550 Ma old rocks in South Australia (Gehling & Rigby 1996), though the preservational morphology of these fossils is more like that of other, problematical, Late Proterozoic, discoidal Ediacaran fossils. Well-preserved clusters of six-rayed hexactinellid sponge spicules have been found in c. 545 Ma cherts in Mongolia, shortly before the Precambrian-Cambrian boundary and approximately coeval with the latest Ediacaran biota elsewhere. (Brasier et al. 1997). The earliest isolated demosponge spicules (as yet undescribed) occur in the basal Cambrian Nemakit-Daldynian stage of Iran (Brasier 1992). Calcareous sponges are not yet known until the Tommotian and later in the early Cambrian (e.g., Brasier 1992). From famine to feast The geological and molecular records, when taken together, suggest that unpreserved foraminiferid- and dinoflagellate-like protozoans could have evolved at some time prior to 1340 Ma (Fig.1). The transformation to animal multicellularity is not well supported in the fossil and biomarker record, however, until as late as c. 600-580 Ma. These evolutionary steps can now be put into a geological context. Between c. 1950 and 1400 Ma, glacial deposits appear to be lacking from Earth’s climatic record, and carbon isotopic values from carbonates appear extremely stable. This suggests that the nutrient (P, N, Fe) cycles, the carbon cycle and climate were all

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Fig. 1. From famine to feast: how environmental changes could have driven evolutionary changes from Protozoa to Metazoa through the early Proterozoic to Cambrian. Adapted from sources cited in the text.

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held in a remarkably steady and extremely ‘oligotrophic’ state. Rates of primary production are likely to have been comparitively low at this time. I have conjectured elsewhere that climatically driven mass extinctions during this interval were rare in comparison with later times, and that this, coupled with the fiercely oligotrophic regime, favoured symbiogenesis of plastids within the cells of eukaryote autrotrophs (Brasier & Lindsay 1998, Brasier 2000). Under such conditions, a heterotrophic life habit may not have been sustainable for long periods within the water column (Porter & Knoll 2000). Heterotrophic feeding could, however, have been favoured by a facultatively autotrophic life habit (as in modern, planktonic dinoflagellates) or perhaps by a benthic grazing habit (as in modern foraminifera). Both groups are inferred to have originated in this interval. Strontium isotopes, palaeomagnetism and basin analysis show that the interval between c. 1000 and 500 Ma was a time of major plate tectonic activity, with breakup of the supercontinent Rodinia at some time after c. 900 Ma, and amalgamation of the supercontinent Gondwana between c. 560 and 510 Ma (Brasier & Lindsay 2000). The late Neoproterozoic was also a time of extreme climatic oscillation. Multiple global glaciations of an apparently widespread to global nature (Hoffman et al. 1998; Kennedy et al. 1998) were separated by prolonged non-glacial intervals that reached down to equatorial latitudes, making them among the most extreme in earth history. This has led to the concept of the ‘snowball earth’, in which the whole surface of the ocean was frozen over (Hoffman et al. 1998), and to alternative arguments for extreme obliquity of the earth’s axis of rotation, so that equatorial latitudes received less solar insolation than did the pole (Schmidt & Williams 1995). Carbon isotopes in carbonate rocks track these climatic oscillations with considerable fidelity. Extremely positive δ13C values tend to precede the glacials, falling to negative values just beneath and immediately above them (Kennedy et al. 1998). This evidence suggests a situation in which maximal rates of productivity and carbon burial occurred in the lead up to the glaciations, bringing about the removal of large amounts of atmospheric CO2, which then led to drastic cooling of the earth’s surface. Rates of primary productivity could have fallen to very low levels during peak glaciation (Hoffman et al. 1998). Deglaciation may then have been triggered by the build-up of volcanically-derived CO2, leading perhaps to ‘supergreenhouse’ conditions. Both atmospheric warming, and the rapid release of CO2, HCO3' and CH4 to the surface during deglaciation, could have brought about the global deposition of cap carbonates. Direct evidence for glaciations is unproven in the rock record after c. 560 Ma. But carbon isotopic oscillations of decreasing amplitude continued throughout the latest Precambrian and well into the Cambrian (Brasier et al. 1994, Brasier & Sukhov 1998). These later negative carbon isotopic excursions tend to be associated with phosphorites or other evidence for oceanic upwelling, suggesting that climatic oscillations continued through the Cambrian, though they were much less extreme than before. What parameter could have caused the climate to shift from steady state towards chaos after c. 1000 Ma? One possibility of relevance here is the inferred increase in maximum cell size and the trend towards multicellularity (Knoll 1994). Both could, perhaps, have helped to amplify rates of carbon burial (Brasier 2000). These increasingly dramatic oscillations in climate and the carbon cycle (and by implication, the nutrient cycles) can be argued to have impinged upon biosphere

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evolution in three main ways (Brasier 2000). Firstly, increased carbon burial could have led to a rise in levels of atmospheric oxygen, favouring the development of multicellularity (though the evidence for this is still largely circumstantial). Secondly, increased rates of oceanic circulation and increasing eutrophication could have brought to an end the ‘boring billion’ years of oligotrophic regime, allowing purely heterotrophic planktonic life habits, and longer food chains. This ‘nutrient-stimulus’ model is supported by evidence for an increase in the size and diversity of putatively phytoplanktonic microfossil cysts after c. 1000 Ma; by high levels of dinosterane in Neoproterozoic and Cambrian sediments; by nutrient markers such as widespread chert-phosphorite-black shale facies and hydrocarbons near the Precambrian-Cambrian boundary; and by an inferred mesotrophic to eutrophic, often filter-feeding ecology for many Cambrian metazoans (see Brasier & Lindsay 1998, Butterfield 2000b, Moldowan et al. 2000, Porter & Knoll 2000, Zhuravlev 2000, McIlroy et al. 2001, Zhou et al. 2001). These observations can be reconciled by a model involving an increase in the abundance, variety and particle size of the (mainly protistan) planktonic food supply. If this food supply was eutrophic in nature (i.e. episodic, perhaps seasonal, with large swings between high and low availability), this could have encouraged a trend towards encystment and larger maximum cell size in protists. Trophic famine and feast could also have been a factor in the encouragement of increased body mass in benthic heterotrophs, leading to coloniality in protists and to ultimately multicellularity in metazoans. In this respect, it may be significant that the earliest strategy adopted by multicellular animals (i.e., the sponges) was that of passive filter feeders. Such a trophic strategy appears consistent with an episodically abundant supply of pico- and microplankton (Zhuravlev 2000). Seen from this viewpoint, the primary role of the spicular skeleton was to elevate the sponge colony higher within the water column (rather than to protect the colony from predation). Macroevolutionary forces A connection is likely to have existed between increasing eutrophication, as suggested above, and the extreme oscillations in climate which were also taking place. Together with associated sea level fluctuations, sharp changes in climate can be predicted to have accelerated the rates of evolutionary turnover. Major population bottlenecks can be argued to have affected the biosphere on at least four occasions between c. 720 and 543 Ma (Brasier 2000, Knoll 2000, Zhou et al. 2001): 1, in relation to the Sturtian glaciations (c. 720 Ma), with implicit acritarch extinctions. 2, in relation to the Marinoan glaciations (c. 600-580 Ma), with documented acritarch extinctions. 3, in relation to the Moelv glaciation (c. 570 Ma), involving global extinction of the diverse, giant Doushantuo microflora. 4, Across the Precambrian-Cambrian boundary (c. 543 Ma), when there was a sharp δ13C negative excursion and major elements of the Ediacara fauna and the Cloudina ‘reefal’ biota pobably became extinct. Eukaryote populations could have been reduced to such small gene pools at these times that macromutations took root, rapidly accelerating the rates of evolution. Very rapid evolutionary rebounds after these extinctions are also likely to have incorporated founder effects. The common posession of hox genes in deuterostomes, ecdysozoans and lophotrochozoans, for example, is

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Origin and Diversification of the Metazoa: Superorganisms among ... Evolution 269 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 269-276, 2003

Origin and Diversification of the Metazoa: Superorganisms among the Ediacarans R.A. Dewel1, W.C. Dewel1 & F.K. McKinney2 1 2

Department of Biology, Appalachian State University, Boone, NC 28608, USA. Department of Geology, Appalachian State University, Boone, NC 28608, USA. E-mail: [email protected]

Abstract The origin and diversification of the Metazoa is persistently enigmatic, principally because the body plans of, for example, poriferans, cnidarians and bilaterians are so disparate that it is difficult to discern how they could be related. The enormous morphological gaps separating these taxa have led by default to the view that the ancestors of the Metazoa, Eumetazoa, and Bilateria were minute larvae or flatworm-like organisms. Such ancestors, however, have been formulated with the assumption that complex bilaterian body plans arose gradually and convergently in bilaterian lineages. An alternative hypothesis is presented here based on the 1) shared genetic and morphologic complexity of protostomes and deuterostomes, 2) propensity for basal metazoans to form colonies and for colonies to become integrated into “superorganisms”, and 3) construction of the Ediacarans, many of which are modular and display the same patterns of growth and degrees of integration found in colonial metazoans. The hypothesis proposes that the stem species of the Eumetazoa possessed a colonial organization and that the ancestor of the Bilateria acquired a complex, segmented and coelomate body plan through individuation of a colonial Ediacaran. The fossil record has not been silent on the early transitions of metazoans. The Ediacarans can be mapped onto the stem groups of basal metazoans to reveal an astonishingly clear picture of how complexity was assembled early in macroscopic ancestors through colonial budding and individuation.

Introduction Since the time of Haeckel’s Gastraea Theory (1874) biologists have been satisfied with a narrow phylogenetic concept for the early evolution of the Metazoa. This concept stipulates that the ancestors of basal metazoan taxa were simple larvae or flatworm-like animals, such as the microscopic acoelomate that commonly is considered to be the bilaterian ancestor (see Rieger et al. 1991, for review). Such hypothetical ancestors, however, have been created with the assumption that metazoan complexity arose gradually and

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that the simplest taxa are the most primitive. These presumptions now have been challenged by the discovery that protostomes and deuterostomes share complicated developmental programs such as the Hox cluster controlling axial patterning. They are also contradicted by the morphology of living metazoans indicating that the bilaterian ancestor was macroscopic and complex (Dewel 2000) and by a Cambrian fossil record suggesting that a majority of body plans were macroscopic and adapted for the benthic environment (Dzik 1993, Gehling 1991, Valentine et al. 1999, Budd & Jensen 2000). Second, simple ancestors have been postulated by default because the morphological discontinuities between poriferans, cnidarians, and bilaterians seem to be insurmountable, and the transformations proposed for uniting them inevitably rely on simple ancestors. Neither the fossil record nor functional morphology is perceived as providing the necessary intermediates (Conway Morris 1998a, 2000). Finally, hypothetical ancestors reveal little about how or when animals became complex. The possibility that morphological complexity arose early in the radiation of Metazoa can not be dismissed since both functional and morphological considerations argue that the characters typifying bilaterians evolved in large rather than small animals (Budd & Jensen 2000, Dewel 2000). It is unlikely that genetics or any other discipline alone can paint a clear picture of the critical transitions in metazoan evolution. Molecular data must not be overvalued while other lines of evidence are ignored. The remarkable similarity in the morphology of complex bilaterians is sufficient reason to consider that they emerged from ancestors that were structurally as well as genetically complex. To ignore this evidence is to assume that larval or flatworm-like ancestors possessed a special capacity to assemble preadaptively the genetic framework of bilaterians in the absence of selection on macroscopic body plans (Conway Morris 1998b). Nevertheless, what the ancestors of Cnidaria, Porifera, and Bilateria were like is unclear. If they were of centimetric size, they probably would have left a fossil record. Although this record may be difficult to interpret because these organisms were undoubtedly different from their living descendents, it potentially can reveal early events in the metazoan radiation. The phenotypes of fossil organisms, especially the Ediacarans from the late Neoproterozoic, cannot be ignored. Potentially they can close the conceptual gaps left by molecular studies (Conway Morris 2000). Observations and Discussion The Ediacaran biota encompasses an extraordinary assemblage of Late Neoproterozoic soft-bodied Problematica. The earliest forms dating from below the Marinoan glaciation at about 600 Ma are simple centimeter sized discs (Hofmann et al. 1990). These forms preceded larger, radially symmetrical discs (Shields 1999, Martin et al. 2000) that are taxonomically unstable (see Ivantsov & Grazhdankin 1997, Gehling 2000, for discussions on their interpretation). Although the latter frequently are described as medusoid, they lack the distinguishing attributes of cnidarian medusae. Later appearing Ediacarans dating from about 565 Ma are diverse and include complex modular forms. Some of these are reminiscent of recent taxa such as the colonial frond-like pennatulids while others are cephalized and “segmented” and have been described as annelids, arthropods, or molluscs. The appearance of the mollusc-like Kimberella and other bilaterian-like fossils

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such as Spriggina is correlated with the diversification of trace fossils including grazing traces at about 555 Ma and suggests that mobile bilaterians were present by the end of the Neoproterozoic (Valentine 1994, Fedonkin & Waggoner 1997, Jensen 1997, Gehling 1999, Martin et al. 2000). One of the most puzzling attributes of the Ediacarans is that many of them are highly compartmented. Although their peculiar structure is usually attributed to their being constructed of unique materials, such striking modularity is characteristic of colonial metazoans and is a compelling reason to consider that the modular Ediacarans evolved as the bud colonies of a solitary ancestor. Possible candidates for this ancestor are the stratigraphically older discoid forms. The construction of the discs is consistent with grades of organization ranging from large choanoflagellate colonies to simple sponges or cnidarians. It is reasonable to suppose that organisms having these grades of organization could grow by asexual budding and could have given rise to the compartmented Ediacarans.

Fig. 1. A, B. A poorly integrated encrusting cheilostome bryozoan, Onychocella angulosa (Reuss), with two types of zooids, autozooids and avicularia (teardrop-shaped polymorphs). C, D. Selenaria punctata Tenison Woods, a close relative of O. angulosa that is highly integrated, with avicularia, scattered among autozooids over most of the surface of the dome-shaped colony, and successive peripheral bands of female and male zooids, kenozooids mixed with avicularia at the mature colony margin.

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If modular Ediacarans have a colonial organization, it is essential to examine carefully the attributes of colonial organisms (Fig. 1). A colony of individuals represents a unit of selection, and as such, undergoes adaptive evolution as a single entity. Over time selection for the individuality of the colony is counterbalanced by selection for the autonomy of the individual. In some clades the former can lead to a well-documented trend toward greater colony integration. In metazoan bud colonies this trend may include the 1) formation of physical and chemical links between modules, 2) specialization of modules (loss of modular autonomy through polymorphism), 3) development of extramodular structures, 4) development of coordinated activity, and 5) evolution of mobile bilaterally symmetrical morphotypes [see McKinney & Jackson (1989) for a discussion of intergration in bryozoans]. An example of individuation of colonies can be seen in a pair of cheilostome bryozoans, one which is primitive and the other highly derived (Fig. 1). The genus Onychocella includes species that have indeterminate form and size and that consist of one or two types of zooids (Figs. 1A,B). In contrast the closely related genus Selenaria has tightly specified colony shapes and sizes and precise distribution of several specialized polymorphs (Figs. 1C,D). There are multiple other instances of transition from poorly integrated colonies of essentially monomorphic modules to highly integrated complex and commonly mobile colonies among living cnidarians, bryozoans, and ascidians.

Fig. 2. Summary of trends exhibited by colonial organisms and a comparison of the level of “individuation” achieved by living “superorganisms” and the postulated Ediacaran ancestor of the Bilateria. The emergent individual would arise through increasing levels of integration involving 1) evolution of extramodular structures, 2) specialization of modules, 3) development of intermodular chemical and structural communication. All three processes would contribute to the formation of organs and organ systems in an organism having a higher level of nested hierarchical complexity than individuals in a nonintegrated colony.

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Fig. 3. Relationship between module autonomy and colony integration. The relationship is inverse but probably not linear as drawn. Placement of the bryozoan taxa and range and position of the colonial superorganisms are intended to be only relative. For example, the levels of integration and autonomy would be different for modern superorganisms and Ediacarans.

Integration of the colony creates a new “superorganism” exhibiting a higher level of nested hierarchical complexity (Figs. 2, 3). The former individuals cooperate in resource sharing, defensive behavior and locomotion (Mackie 1986). This cooperation may involve formation of common blood vascular, digestive, and neuromuscular systems. Unit “zooids” also trend toward greater specialization and dependency, as they become subordinate to the emergent colonial organism. Frequently the former individuals in the colony lose their autonomy and eventually function as organs within a new entity. While there are modern colonial superorganisms, none (with the possible exception of the chondrophorines) has achieved a level of integration that hinders its identity as a colony. This lack of complete integration in recent metazoans may result from the failure of selection for the new individual to proceed to completion (Fig. 3). Perhaps predation and competition in the more tightly packed ecosystems of the Phanerozoic have favored retention of zooid individuality or autonomy. If modular Ediacarans were bud colonies they would exhibit the same morphological trends evident in modern colonial organisms (Fig. 1). This prediction appears to be supported by the fossil record (Fig. 4). The oldest Ediacarans are small simple discs, some of which are arranged in clumps or uniserial chains (Hofmann et al. 1990, Gehling et al. 2000) suggesting a budding pattern of growth. Other Ediacarans such as Windermeria, Pteridinium, Ernietta, and Phyllozoon are clearly composed of replicated individuals.

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Fig. 4. A time scale of the late Neoproterozoic/Cambrian boundary showing some Ediacaran and Cambrian fossil assemblages. Simple disc-like Ediacarans are separated chronologically from more diverse assemblages containing modular forms by a period of approximately 40 million years that included the Marinoan glaciation. The shorter period of less than 10 Ma separating more recent Ediacarans from the complex bilaterians of the Cambrian may be more apparent than real since trace fossils, which were presumably formed by bilaterians, diversify in the late Neoproterozoic, and Ediacaran fronds have been found in Cambrian deposits (Jensen et al. 1998). Representative taxa are (from bottom) Nimibia and Vendella sp. (Hofmann et al. 1990), Charniodiscus, Spriggina, Kimberella, Yunnanozoon, Thaumaptilon, and Eoredlichia. Adapted from Shields (1999) and Martin et al. (2000).

Although the modules are arranged in a variety of patterns, they seem to be poorly integrated, lacking polymorphisms, extramodular structures, and “cephalization”. Others such as Ventogyrus with a common system of ducts (Ivantsov & Grazhdankin 1997) and the frond-like Charnia, Glaessnerina, Charniodiscus, Swartpuntia, Rangea, and perhaps an Ediacaran holdover from the Cambrian, Thaumaptilon (Conway Morris 1993), have extramodular or polymorphic structures including a holdfast, stalk, and rachis. In addition, Spriggina, Vendomia, Mialsemia, Bomakellia, and Kimberella are cephalized and have bilaterally symmetrical slug-like morphologies that may have been mobile. If the modular Ediacarans are interpreted as colonial organisms they provide an entirely new perspective on the evolution of metazoans. The earliest disc-like forms, which could have provided a framework for the development of cell specializations including epithelia and complex connective tissue (see Müller 1998) for apomorphies of the Metazoa), would correspond to a single module. The later colonial Ediacarans would have furnished through duplication and integration of modules a mechanism for increasing nested or hierarchical complexity and the body organization of triploblasts.

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If this scenario is correct, then the Ediacarans would constitute a paraphyletic grade of organization encompassing the stem groups (possibly some crown groups) of the Porifera, Ctenophora, Cnidaria and Bilateria (Fig.4). Because the levels of integration that produce frond and slug morphotypes or mobility in colonial lineages have evolved repeatedly, it is reasonable to consider that in the ecologically open environment of the late Neoproterozoic these levels were sufficient to create the individuated ancestor of the Bilateria. Summary The Ediacaran biota is profoundly mysterious. Although there is a growing realization that they should not be grouped into a single taxon (Petalonamae, Vendobiota etc.), the debate continues on what the group as a whole represents. It is argued here that many Ediacarans were in essence colonial organisms. The early disc-like forms would be comparable to solitary individuals, perhaps with a choanoflagellate or simple sponge grade of organization, while the modular forms would represent the bud colonies of those entities. The modular Ediacarans exhibit a trend toward higher levels of integration that is closely similar to one evident in colonial metazoans. Because this trend can lead to the development of colonial superorganisms, it is reasonable to postulate that it could have been responsible for the individuation of the last common ancestor of the Bilateria. Thus the Ediacarans could represent a remarkable experiment in multicellularity and coloniality that profoundly impacted the evolution of metazoan body plans and was a harbinger of the radiation of complex triploblasts in the Cambrian. Acknowledgments We thank Graham Budd for stimulating discussions during early stages of the development of the “colony theory”, Robert Creed for help with Figure 3, and are especially grateful to Paul Taylor for providing scanning electron micrographs of Onychocella angulosa and Selenaria punctata. We also thank the College of Arts & Sciences, Graduate Studies and Research, and International Programs at Appalachian State University for financial assistance. References BUDD G.E. & S. JENSEN 2000. A critical reappraisal of the fossil record of the bilaterian phyla. Biol. Rev. 75: 253-295. CONWAY MORRIS S. 1993. Ediacaran-like fossils in Cambrian Burgess Shale-type faunas of North America. Palaeontol. 36: 593-635. CONWAY MORRIS S. 1998a. The Crucible of Creation. The Burgess Shale and the Rise of Animals. Oxford University Press, Oxford. 242p. CONWAY MORRIS S. 1998b. Palaeontology: grasping the opportunities in the science of the twenty-first century. Geobios 30: 895-904. CONWAY MORRIS S. 2000. Evolution: bringing molecules into the fold. Cell 100: 1-11.

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DEWEL R.A. 2000. Colonial origin for Eumetazoa: major morphological transitions and the origin of bilaterian complexity. J. Morphol. 243: 35-74. DZIK J. 1993. Early metazoan evolution and the meaning of its fossil record. In Hecht M.K. (ed), Evol. Biol. 27: 339-386. FEDONKIN M.A. & B.M. WAGGONER 1997. The Late Precambrian fossil Kimberella is a mollusc-like bilaterian organism. Nature 388: 869-871. GEHLING J.G. 1991. The case for Ediacaran fossil roots to the metazoan tree. Mem.Geol. Soc. India 20: 181-224. GEHLING J.G., NARBONNE G.M. & M.M. ANDERSON 2000. The first named Ediacaran body fossil, Aspidella Terranovica. Palaeontol. 43: 427-456. GEHLING J.G, NARBONNE G.M., JENSEN S. & M.L. DROSER 1999. Simultaneous appearance of Ediacaran Trace fossils and bilateral body fossils: implications for animal evolution. GSA Abst. Prog. 1999: A-362. HAECKEL E. 1874. Die Gastraea-Theorie, die phylogenetische Classification des Thierreichs und die Homologie der Keimblätter. Jena Z. Naturwiss. 8: 1-55. HOFMANN H.J., NARBONNE G.M. & J.D. AIKEN 1990. Ediacaran remains from intertillite beds in northwestern Canada. Geology 18: 1199-1202. IVANTSOV A-YU & D.V. GRAZHDANKIN 1997. A representative of the Petalonamae from the Upper Vendian of the Arkhangelsk region. Paleontol. J. 31: 1-10. JENSEN S. 1997. Trace fossils from the Lower Cambrian Mickwitzia sandstone, southern central Sweden. Fossils & Strata 42: 1-111. MACKIE G.O. 1986. From aggregates to integrates: physiological aspects of modularity in colonial animals. Phil. Trans. R. Soc. Lond. B 313: 175-196. MARTIN M.W., GRAZHDANKIN D.V., BOWRING S.A., EVANS D.A.D., FEDONKIN M.A. & J.L. KIRSCHVINK 2000. Age of Neoproterozoic bilatarian body and trace fossils, White Sea, Russia: implications for metazoan evolution. Science 288: 841-845. MCKINNEY F.K. & J.B.C. JACKSON 1989. Bryozoan Evolution. Unwin Hyman, Boston. 238p. MÜLLER M. 1998. Origin of Metazoa: sponges as living fossils. Naturwiss. 85: 11-25. RIEGER R.M., HASZPRUNAR G. & P. SCHUCHERT 1991. On the origin of the Bilateria: traditional views and recent alternative concepts. In Simonetta A.M. & S. Conway Morris (eds), The Early Evolution of Metazoa and the Significance of Problematic Taxa. Clarendon Press, Oxford, pp. 107-113. SHIELDS G. 1999. Working towards a new staratigraphic calibration scheme for the Neoproterozoic-Cambrian. Eclogae geol. Helv. 92: 221-233. VALENTINE J.W. 1994. Late Precambrian bilaterians: grades and clades. Proc. Natl. Acad. Sci. 91: 6751-6757. VALENTINE J.W., JABLONSKI D. & D.H. ERWIN 1999. Fossils, molecules and embryos: new perspectives on the Cambrian explosion. Development 126: 851-859.

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The protozoan-metazoan boundary: a molecular biologist’s view 277 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 277-288, 2003

The protozoan-metazoan boundary: a molecular biologist’s view J.H.P. Hackstein Dept. Evolutionary Microbiology, Fac. Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands

Abstract Due to the poor fossil record, the analysis of the protozoan-metazoan boundary has to rely predominantly on the phylogenetic analysis of DNA sequences of extent protozoa, fungi and animals. The deep history of this event, however, might seriously flaw such an analysis. A phylogenetic analysis of the small subunit of the ribosomal genes, for example, can only provide a limited view because of the rather constrained information content of this gene. Protein phylogenies, on the other hand, can have rather contrasting outcomes, since gene duplications, lateral gene transfer, and a changing compartmentalisation of metabolic pathways can blur our view of evolutionary history. Whole genome studies are much more informative, but rather expensive and, consequently, they will be restricted to a few model organisms and those of commercial interest. Thus, the currently available information about complete genomes is very limited, and will remain fragmentary for many years. Notwithstanding, the available data support a close evolutionary relationship between animals and fungi, and suggest that the metazoans originated deep in the Precambrian.

There is compelling evidence that life on earth had a unique origin. Molecular data, especially, leave us in no doubt that bacteria, archaea, and eukaryotes are sister groups that share a common ancestry (Woese et al. 1990). However, due to substantial exchange of genes between bacteria, archaea, and eukaryotes, a remarkable chimaerism of their genomes arose in the course of evolution that has prohibited any convincing “rooting” of the “tree-of-life” until now (Doolittle 1999, Eisen 2000, Woese 2000). Similarly, a monophyletic origin of eukaryotes is likely, but the events that created the first eukaryote are highly controversial (Martin & Müller 1998, Doolittle 1999, Lopes-Garcia & Moreira 1999). Also, there is growing evidence for a monophyletic origin of the “metazoans”, the “true”, multicellular animals (Aravind & Subramanian 1999, Adoutte et al. 1999, 2000, Baldauf 1999, Baldauf et al. 2000). Here, I will summarise the currently available knowledge about the evolutionary origin of metazoans, and discuss the potential flaws and caveats of molecular phylogenies.

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Obviously, a molecular biologist’s approach to the unravelling of the elusive origins of metazoans has to rely on DNA sequence analysis of present-day organisms. This fact creates a fundamental problem since an overwhelming biodiversity is still characteristic for life on earth. Just the metazoans - together with their sister groups of protozoa, sponges and fungi are believed to account for considerably more than 1 million different species (Patterson 1999, see also Hackstein 1997 for discussion). It is obvious that even the most intensive and well-funded molecular approaches cannot cope adequately with this diversity. In other words, molecular studies on the evolution of metazoans are notoriously fragmentary, and will remain so for many years. The textbook definition of a “metazoan” requires multicellularity in connection with cellular differentiation, i.e. the presence of cells with different functions and fates in one and the same individual. Such multicellularity requires the evolution of a large repertoire of sensing and signalling pathways, receptors and ligands (Meyerowitz 1999). Notably, there is persuasive evidence that multicellularity evolved repeatedly and independently in animals, several taxa of algae, plants and fungi (Baldauf 1999, Meyerowitz 1999, Baldauf et al. 2000). Consequently, careful comparisons between the different modes of becoming “multicellular” can help to identify common ancestries, but also recognizing the existence of alternative solutions for the same problems. Lastly, and this is a trivial statement that nevertheless has to be recalled constantly - when dealing with “evolution”, we are looking at history that happened (Pagel 1999). Evolutionary biology cannot provide a rationale for a mechanistic process that, in a reproducible and testable way, gave rise to the present-day biodiversity. Consequently, we have to look for “historical” information in any available form to unravel the elusive protozoan-metazoan boundary. The fossil record allows only a faint glimpse at the early evolution of eukaryotes (Knoll & Carroll 1999, Schopf 2000). However, novel techniques have been developed that enable the study of “molecular fossils”. They indicate eukaryotic metabolic activities in deep Precambrian shales (e.g. 2700 Ma ago; Brocks et al 1999, House et al. 2000). The situation with fossilized metazoans is more satisfying. However, the fossil record prior to the “Cambrian explosion” is scarce and controversial (cf. Conway-Morris 2000, Schopf 2000). Especially, the discovery of Proterozoic embryos –without the concomitant discovery of identifiable adults – is problematic (Bengtson & Yue, 1997, Xiao et al. 1998, Chen et al. 2000, see Bengtson 1998 and Conway-Morris 1998 for discussion). The available palaeontological data seem to support the conclusion that around the time of “Cambrian explosion” something dramatically happened that led to the “sudden” appearance of all present-day animal taxa in the geological record. The evolving concept of the “Snowball Earth”, i.e. of worldwide low latitude glaciations, can provide a scenario for an understanding of the biological implications and consequences (Jenner 2000, Kerr 2000, Peterson et al. 2000, Runnegar 2000). It is conceivable that global low latitude glaciations were able to cause unprecedented massextinctions that were followed by massive radiations of the survivors when more moderate environmental conditions were restored. The dating of the Cambrian explosion and the Snowball Earth is becoming more and more precise (Kirschvink et al. 2000). However, the information deduced from DNA sequence analysis still does not permit conclusions that are (1) in complete agreement with the palaeontological data, and (2) broadly accepted in the scientific world. A number of molecular data can be brought

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into agreement with the geological time scale – no doubt. However, the analysis of different datasets and, even more baffling, simply the use of different mathematical models or algorithms for the analysis of the same datasets can have widely diverging outcomes (c.f. Wray et al. 1996, Ayala et al. 1998). Before discussing the molecular aspects of the protozoan- metazoan boundary in more detail, I will briefly summarize the contribution of molecular biology to the current knowledge about the early evolution of the major animal phyla. Notably, molecular phylogenies have triggered quite a number of revolutions that changed our perception of life on earth substantially. For example, it was not until the publication of Woese’s pioneering paper on the phylogenetic analysis of the ribosomal RNA genes that “Archaea” were recognized as a distinct kingdom that united organisms as different as thermophiles, halophiles, and methanogens (Woese et al. 1990). Similarly, molecular phylogenies were pivotal for the current view of metazoan phylogeny. In particular, the perception of the poriferans (sponges) as true metazoans has to be mentioned here (Borchiellini et al. 1998, Ono et al. 1999, Schutze et al. 1999, Suga et al. 1999). Also, the concept of a clade of “ecdysozoa” that unite animals as different as nematodes and arthropods into one monophyletic group arose from molecular studies (Aguinaldo et al. 1997). It is not surprising that, initially, molecular phylogenies that came up with nematodes and arthropods as close relatives were regarded as flawed. Later, more and more arguments, also from classical zoological disciplines supported the “heretical” concept of uniting all those animals into a single clade that “moult”, i.e. that undergo “ecdysis” at one or other point of their life-cycle (e.g. de Rosa et al. 1999). Substantial differences in the composition of the cuticle (e.g. chitin in arthropods, and collagen in nematodes) appeared not to be enough for rejecting this hypothesis. Lastly, molecular phylogenies provided the key-information for the identification of microsporidia as highly modified fungi (Baldauf 1999, Baldauf et al. 2000, van de Peer et al. 2000). Molecular phylogenies also functioned as an eye-opener for the establishment of another important taxonomic group, i.e. the “lophotrophozoa” (Halanynch et al. 1995). This clade unites annelids, molluscs, and a number of “minor” taxa into the “Lophotrophozoa”. Members of this taxon are characterized by a trochophora larva and, in some subgroups, by a lophotrochophor, a filter-feeding apparatus. Not completely unexpected, the “Deuterostomes”, i.e. chordates, hemichordates, and echinodermata, comprise the third major phylum that is supported by molecular phylogenies (Adoutte et al. 1999, 2000, Cameron et al. 2000; Peterson et al. 2000). The position of the diploblastic animals, i.e. the cnidarians, ctenophores, and sponges, with respect to the “triploblastic” phyla of protostomes (i.e. ecdysozoa, lophotrophozoa), cannot be resolved with the currently available data. Also, a few “minor” taxa remain to be placed. It is obvious that a number of these polytomies will persist for some time. Although the discussion about the validity of the concept and the placement of one or the other taxon continues, it is reasonable to assume that not only the two diploblastic taxa, but also the three major phyla of triploblastic animals underwent a substantial radiation immediately after the evolutionary “big-bang” (Balavoine & Adoutte 1998, Adoutte et al. 1999). In other words, these five major taxa must have existed before this event. However, despite the unquestionable contributions of molecular phylogenies to our understanding of the evolution of eukaryotes, the debates on the protozoan – metazoan boundary are still

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controversial. Therefore, I will summarise the problems that can arise when one is studying these early events of metazoan evolution with molecular means. 1. Bottlenecks. Evidence is evolving that the “Cambrian Explosion” might have been triggered by a preceding dramatic extinction caused by a “Snowball Earth”, i.e. global low latitude glaciations of an unprecedented scale. There is evidence for at least two global Precambrian catastrophic events that potentially caused enormous extinctions threatening the existence of life on earth (Hoffmann et al. 1998, Kerr 2000, Kirschvink et al. 2000, Runnegar 2000). It is known that in more recent times, the Permian and the K/T mass extinctions had dramatical consequences for evolution, since fossil evidence revealed that plethora of species became eradicated while the few surviving taxa underwent rapid radiation after each mass-extinction. Notably, all molecular phylogenies have to rely on the few lines that survived all these mass-extinctions that happened to the biosphere from the dawn of biological evolution on. Because of these evolutionary bottlenecks with the subsequent radiations, the available information is extremely biased (Fig. 1). 2. Long Branch Attraction. Substitution rates can differ substantially among the different taxa, species, and genes. High rates of substitution cause “long branches” that disturb the topology of the phylogenetic tree. It is possible that the Long Branch Attraction leads to highly supported but nevertheless completely wrong placements of species (Embley & Hirt 1998, Philippe et al. 2000, Stiller & Hall 1999). Long Branch Attraction, for example, was the major cause for the wrong placement of the Microsporidia at the base of the phylogenetic tree (see below). Also, the unresolved phylogenetic position of the acoela (platyhelminthes) is predominantly caused by the high nucleotide exchange rates that are characteristic for most of the species (Carranza et al. 1997, Ruiz-Trillo et al. 1999, Berney et al. 2000). However, these effects can be compensated by correcting for the enhanced exchange rates or by using exclusively species with low rates of nucleotide exchange (Philippe & Germot 2000). In any case, all applicable algorithms and rigorous statistical tests must be applied to come to reliable trees – if there are sufficient reliable data available (Pagel 1999, Bromham et al. 2000, Steel & Penny 2000). 3. Sequence heterogeneity. The base composition of the DNA of the various organisms can vary broadly. Especially substantial differences in the G+C content can lead to tree construction artefacts (Galtier & Gouy 1995). However, the discussions go on to what extent G+C biases can flaw the phylogenetic reconstructions (Philippe et al. 2000). 4. Mutation saturation. Intuitively, one can imagine that the accumulation of mutations over extended periods can cause a loss of phylogenetic information. It is possible to measure the extent of mutational saturation and to identify the limits of the reliability. It is obvious that the resolving power depends on the length of the sequence and the number of informative positions (Abouheif et al. 1998, Philippe & Laurent 1998, Adoutte et al. 2000). Therefore, phylogenetic analysis of concatenated (protein) sequences will help to analyse the early events in the evolution of the eukaryotes (Baldauf et al. 2000).

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5. Paralogues and lateral gene transfer: The phylogenetic analysis of multigene families can be hampered by difficulties in the discrimination between orthologues, paralogues and “unrelated” genes (Gogarten & Olendzenski 1999, Pagel 1999). 6. Variable molecular clocks: Mutational rates can differ substantially not only between organisms and genes, but also in time. Since molecular clock determinations are extrapolations of a few points that allow palaeontological calibration, the conclusions might be seriously flawed – because of molecular and palaeontological misinterpretations (Dollittle et al. 1996, Wray et al. 1996; Feng et al 1997; Wright &

Fig. 1. Cartoon illustrating some of the consequences of mass-extinctions on the phylogeny of life on earth. The horizontal black bars indicate subsequent mass-extinctions. The few survivors that escape extinction pass through the “holes” and undergo subsequent radiations. Since every mass-extinction represents a new “filter”, our view on the evolutionary history is extremly biased. It has to be realized that present-day biodiversity can only provide very limited information about the early events that resulted in the evolution of multicellular animals. ?: an example for a whole taxon that became extinct in early evolution.

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Lynn 1997, Ayala et al. 1998; Bromham et al. 1998, Gu & Li 1998, Lee 1999, Wang et al. 1999, Kollman & Doolittle 2000). This compilation of the inherent problems of molecular phylogenies might suggest that molecular phylogenies can only marginally, if at all, contribute to an understanding of the early evolution of eukaryotes. The most pessimistic conclusion might be that the currently available data do not permit any analysis of a Cambrian or Precambrian “big bang” of eukaryotes. However, it has been shown that a careful analysis of the available data can nevertheless contribute to a deeper understanding of evolution – albeit that a certain number of polytomies cannot be resolved without the results of the forthcoming genome projects (Pagel 1999, Adoutte et al. 2000). These genome projects, proteome analyses, and extended DNA sequencing efforts will contribute substantially to an exponentially growing data set (Aravind & Subramanian 1999, Meyerowitz 1999). Mitochondrial and chloroplast genomes will provide additional information (Lang et al. 1999, http://megasun.bch.umontreal.ca/ogmp/projects/other/mtcomp.html; http:// megasun.bch.umontreal.ca/People/lang/FMGP/FMGP.html). The analysis of DNA signature sequences and insertions of, for example, introns, retroviruses, and other mobile elements, will provide information that is unbiased by Long Branch Attraction, differences in base composition and evolutionary rates (c.f. Baldauf 1999, Adoutte et al. 2000). Lastly, the analysis of transcription factors, homeotic (and other developmental) genes including an analysis of their temporal and spatial expression patterns will have an enormous impact for the understanding of the protozoan-metazoan boundary and the evolution of the various animal phyla (de Rosa et al. 1999, Knoll & Carroll 1999, Gauchat et al. 2000, Hobmayer et al. 2000, Peterson & Davidson 2000, Peterson et al. 2000). What can molecular biology tell us about the protozoan-metazoan boundary? A number of data concerning the protozoan-metazoan boundary are still controversial – however, the compilation of the published evidence shown below, to my opinion, can provide the paraphernalia for creating a panorama of the origin of the metazoans. 1. Molecular phylogenies support deep branching of the major metazoan phyla: at least three taxa of triploblastic animals (i.e. ecdysozoa, lophotrophorates, and deuterostomes), two taxa of diploblastic animals (i.e. Ctenophors and Cnidarians) and the poriferans (sponges) are likely to have a Precambrian origin (ConwayMorris 1998, 2000, Li et al. 1998, Schopf 2000). It is likely that besides these metazoan phyla, fungi, several algal and protist taxa existed in the Precambrian (Roger 1999, Baldauf et al. 2000). All these lines radiated independently in the early Cambrian, suggesting strongly that these lines existed already in the Precambrian (Fig. 1; cf. Adoutte et al. 2000, Conway-Morris 2000, Dewel 2000, Peterson et al. 2000). 2. The broad repertoire of homeotic genes and transcription factors that is shared by proto- and deuterostomes, strongly suggests that the last common ancestor of both phyla was of a remarkable complexity (cf. de Rosa et al. 1999, Hobmayer et al. 2000, Miller et al. 2000). There is no convincing evidence for “intermediate taxa” since it is likely that most, if not all of the “primitive taxa”, might be the result of secondary

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reduction (Adoutte et al. 1999, 2000). Maximal indirect development is widespread among the various metazoan taxa. The concept of “set-aside-cells” for maximal indirect development has a large heuristic potential for the understanding of metazoan evolution (Jenner 2000, Peterson et al. 2000). It might also explain the absence of macroscopic fossils in the Proterozoic. 3. Fungi are the sister group of the metazoans. This evidence is supported not only by many phylogenies, but also by an analysis of DNA insertions (e.g. Baldauf 1999; Baldauf et al. 2000), and mitochondrial genomes (http://megasun.bch.umontreal.ca./ People/lang/FMGP/phylogeny.html). Both, animals and fungi evolved a broad repertoire of G-proteins that are involved in cellular signalling (Ono et al. 1999, Schutze et al. 1999, Suga et al. 1999). In protists and plants such proteins are rare, if not absent. Genome projects will reveal whether protists and plants lack G-proteins completely. Fungi, i.e. Ascomycota, Basidiomycota, Zygomycota and Chytridiomycota are monophyletic and include unicellular flagellated forms. This observation is based on molecular and ultrastructural criteria (see Lang et al. 1999 for discussion). It could not yet be established by phylogenetic analysis of several genes whether the myxozoa (slime moulds) are “fungi”. However, there is little doubt, that microsporidia are curious “fungi” (Baldauf et al. 2000, van de Peer et al. 2000). The presence of duplicated annexin genes in animals, fungi and slime molds (and their potential absence in plants and protozoa) argues for a close relationship between fungi and slime molds and, in addition, strongly supports a common origin of animals and fungi (Braun et al. 1998). Consequently, the last common ancestor of metazoans and fungi might pinpoint the protozoan-metazoan boundary. Choanoflagellates might represent such a potential common ancestor of both metazoans and fungi. The taxon Ancyromonadina, a new lineage among protozoa, might provide a link between metazoans, fungi and choanoflagellates (Atkins et al. 2000), and support the concept of the “Opisthokonta” (Cavalier-Smith 1998). 4. A number of molecular clock approaches dates the metazoan origins before 1000 Ma, and the origin of the eukaryotic cell before 1500 Ma (Bromham et al. 1998, Wang et al. 1999). These data are highly controversial, since other calculations come to much younger estimates – near to the Precambrian-Cambrian border (Feng et al. 1997, Ayala et al. 1998, Gu & Li 1998). Since fossils of multicellular red algae have been dated to an age of 1200 Ma (Butterfield 2000), and molecular fossils (biological lipids in Archaean shales) might indicate the existence of eukaryotes as early as 2700 Ma before present (Brocks et al. 1999), estimations of more than 1000 Ma for the protozoan-metazoan boundary seem not unrealistic. What did the first metazoan look like, and what triggered the evolution of metazoans? It has been shown that the Permian and K/T mass extinctions triggered massive radiations of animals and plants. It is likely that global low latitude glaciations, i.e. the “Snowball Earth”, triggered the Cambrian explosion - after a mass extinction of Precambrian forms of life. However, fossils, molecular phylogenies and evolutionarydevelopmental genetic data, consistently argue that complex, multicellular forms of life existed before the Cambrian explosion. The available evidence suggests that diploblastic

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cnidarians and sponges, as well as triploblastic ecdysozoa, lophophorates and deuterostomes existed before the Cambrian explosion. Moreover, the extant representatives of these taxa possess an elaborate set of developmental genes. For example, a comparative molecular genetic analysis reveals the existence of many shared families of “developmental” genes. Notably, also a diversification of G-proteins, PTK’s (protein tyrosine kinases), and PTPs (protein tyrosine phosphatases) did occur before the (Precambrian) separation of cnidarians and sponges (Schutz et al. 1998, Ono et al. 1999, Suga et al. 1999, Dewel 2000). The discovery of well-preserved Precambrian (Ediacarian) fossilized embryos and cleavage stages supports the concept that the maximal indirect development of many animal species was an “early” invention (Li et al. 1998). Multicellularity had been invented much earlier (Li et al. 1998, Meyerowitz 1999) as recently described fossils of red algae of an approximate age of 1200 Ma years revealed (Butterfield 2000). Comparative genomics and phylogenetic analysis argue that multicellularity has been invented several times and independently in plants and animals, but potentially also in fungi and three different taxa of algae (Meyerowitz 1999, Baldauf et al. 2000). It is unlikely, that the rise of atmospheric oxygen concentrations was a major trigger for these revolutionary inventions since already the most ‘”primitive” unicellular eukaryotes must have possessed mitochondria. They evolved when atmospheric oxygen concentrations were much lower than present day (Schopf & Klein 1992). One might speculate as to whether Proterozoic, global catastrophes were the major cause for the subsequent phases of evolutionary progress. On the other hand, more and more evidence arises that “evolutionary progress” can be accompanied by secondary reductions and losses of plesiomorphic (ancient-shared) characters. The loss of mitochondria (or their transformation into “mitosomes” (or “cryptons”) in Entamoeba, and their evolution into hydrogenosomes in anaerobic ciliates and chytridiomycete fungi (Akhmanova et al. 1998, Hackstein et al. 1999, Müller 2000, Rotte et al 2000, van Hoek et al. 2000) are examples of a reductional evolution. The evolution of parasites such as microsporidia and myxozoa provides additional examples for the assumption that evolution does not necessarily result in organisms of higher complexity. This new view on evolution became only possible by the rise of molecular genetics, genomics, proteomics and molecular phylogenetics. If used in a careful manner and on a broad basis, these methods will be able to answer many questions about evolution of life on earth, including the protozoan-metazoan boundary. References ABOUHEIF E., ZARDOYA R. & A. MEYER 1998. Limitations of metazoan 18S rRNA sequence data: Implications for reconstructing a phylogeny of the animal kingdom and inferring the reality of the Cambrian explosion. J. Mol. Evol. 47: 394-405. ADOUTTE A., BALAVOINE G., LARTILLOT N. & R. DE ROSA 1999. Animal evolution - the end of the intermediate taxa? Trends Genetics 15: 104-108. ADOUTTE A., BALAVOINE G., LARTILLOT N., LESPINET O., PRUD’HOMME B. & R. DE ROSA 2000. The new animal phylogeny: Reliability and implications. Proc. Nat. Acad. Sci. USA 97: 4453-4456.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Tracing metazoan roots thePanorama fossil record Thein New of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 289-300, 2003

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Tracing metazoan roots in the fossil record S. Bengtson Department of Palaeozoology, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden. E-mail: [email protected]

Abstract Taken literally, the fossil record tells us that animals first appeared on Earth less than 600 million years ago, in the prelude to the Cambrian explosion. Estimates based on gene sequence comparisons suggest that the protostome and deuterostome lineages diverged at least a hundred million and perhaps almost a thousand million years earlier. The apparent incongruence between molecular and palaeontological data is to some extent to be expected: The former relate to genetic distances, whereas the latter trace the appearances of phenotypic characters. Characters are acquired sequentially during the evolution of a lineage, and the first members of a lineage are likely to lack the features characteristic of the crown group. However, this difference between the dating methods is unlikely to explain all of the discrepancy. The recent discoveries of fossilized metazoan embryos in early Cambrian and late Neoproterozoic rocks provide a tool to trace metazoan roots in the fossil record, and to bring together evolutionary and developmental biology in the study of extinct early animals. Evolution is constrained by geological events, and a proposed runaway global glaciation, ‘Snowball Earth’, ending about 600 million years ago may represent a bottleneck after which metazoan evolution could finally gather momentum.

Introduction Digging up roots can be a frustrating experience: always deeper ones to be found. The fossil record never really yields the oldest members of any particular taxon. Sometimes we may assume that we are quite close, however. At least this is true for the Phanerozoic, where there is some consistency in fossilization potential through time. At the start of that golden age of ubiquitous fossils, however, things are not so clear. There is in fact an extraordinary uncertainty regarding the timing of the events that are at the focus of the present section, the transition from Protozoa to Metazoa. Recent published estimates of the age of initial divergence of extant metazoan lineages, and thus the minimum age of the protist–metazoan transition, vary from less than 600 million to some 1500 million years, a period of uncertainty almost twice as long as the known fossil record of animals (Fig. 1).

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Fig. 1. Comparisons of recently published estimates of the ranges of stem-group metazoans and four major metazoan clades. The upper four sets of ranges are based on palaeontological, the remainder mainly on molecular data. The vertical dotted line marks the Precambrian–Cambrian boundary and the broad vertical band the Cryogenian period of more-or-less global glaciations (including ‘Snowball-Earth’-like events). The change in conclusions by one group of palaeontologists (Fortey et al. 1996, 1997) represents a ‘recalibration’ on the basis of molecular data. The phylogeny of the taxa outlined in the legend approximates the general consensus (though see Collins 1998 regarding possible paraphyly of the Porifera) and is used as a framework for the diagram (even though all of the taxa are not included in all analyses). The horizonal bands represent the cited authors’ estimates of the ranges as given in text, tables or diagrams. ‘Explicit uncertainty’ is that stated by the authors; ‘implicit uncertainty’ marks an uncertainty not stated by the authors but necessitated by the phylogenetic context. The upper-range limit for the (by definition extinct) stem-group metazoans has been arbitrarily set to the Precambrian–Cambrian boundary (in agreement with the commonly held notion that the Ediacaran biota includes stem-group metazoans).

An integrated interpretation of evolutionary and environmental events based on fossils as well as on geological and chemical data from the rock record is presented by Brasier (this volume). The present chapter deals with some of the major uncertainties, highlighting promises and pitfalls in the interpretation of the fossil record.

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Fossils, molecules, and the age of animals For a long time following Darwin, palaeontologists tended to regard the sudden appearance of animal fossils at the Precambrian–Cambrian boundary (currently dated at 543 Ma) as a preservational artifact: animals had a long Precambrian history that was not revealed by the fossil record. This view was effectively challenged by Preston Cloud (1948, 1968), who argued that the fossil record indeed tells us essentially what happened, that the metazoans did not have a long Precambrian history, and basically that there is no need to postulate something which is not necessitated by theory and for which there is no evidence. Cloud’s once heretic views attracted followers, particularly when the rejuvenated interest in tempo and mode in evolution in the 1970s made rapid evolutionary transitions palatable. Parallel evidence from skeletal fossils, trace fossils, organic microfossils, and soft-tissue preservation confirmed a dramatic biotic turnover near the beginning of the Cambrian (Stanley 1976), and the growth of a respectable but doggedly microbial fossil record of Precambrian life seemed to round up the case (e.g., Schopf & Klein 1992). The reality of the ‘Cambrian explosion’ is thus not in dispute among palaeontologists. A major reorganization of the biosphere obviously took place. Numerous questions still surround the event, however, not least the question of how closely it is connected with the origins of the major metazoan lineages. By the 1980s an opinion seemed widely established that at least the bilaterians did not by much predate the ‘Cambrian explosion’. Durham’s (1971, 1978) extrapolation of Phanerozoic evolutionary rates to suggest a long Precambrian history of the metazoans was perhaps the last serious attempt by a palaeontologist to apply a uniform-rate gradualistic model directly to the fossil record to demonstrate that the perceived suddenness of the event was an artifact of preservation. Yet an analogous approach from molecular biology, pioneered already two decades ago by the palaeontologist Runnegar (1982) and brought to full force during the last few years (Wray et al. 1996, Nikoh et al. 1997, Ayala et al. 1998, Bromham et al. 1998, Gu 1998, Lynch 1999, Wang et al. 1999), using molecular-clock-based calculations, has persistently put the dates of emergence of major animal lineages far beyond the ages documented from the fossil record. The molecular ages vary widely among themselves (Fig. 1), but even the youngest of them (Ayala et al. 1998) leaves a gap of about a hundred million years to the oldest dates indicated by fossils. At present, then, there is a substantial spread between the ‘old-animals’ and the ‘young-animals’ interpretations. The former tend to be represented by molecular biologists, the latter by palaeontologists (though exceptions occur, e.g., the palaeontologists Fortey et al. 1997). The watershed between the two views coincides with the late Proterozoic low-latitude glaciations, commonly interpreted as run-away global glaciations, ‘Snowball Earth’ (Kirschvink 1992, Hoffman et al. 1998), ending at about 600 Ma. A number of recent palaeontological studies (Fortey et al. 1996, Knoll & Carroll 1999, Valentine et al. 1999, Budd & Jensen 2000, Conway Morris 2000) would accommodate most or all of metazoan diversification after this event (cf. Fig. 1). A prerequisite for such interpretations is that the divergence times suggested by molecular biologists are unreliable or worse (as conceded also by some biologists; cf. Ayala 1999, Lee 1999). The widely divergent results of the molecular studies may seem to support

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this view. Nevertheless, unless the consistently ‘too old’ dates yielded by comparisons of gene sequences can be clearly shown to be due to fundamental and systematic methodological errors, they cannot easily be dismissed from the discussion. Conversely, the ‘old-animals’ interpretations have to assume that the fossil record is unreliable, hundreds of millions of years of animal evolution having gone by without at trace. Dismissing the absence of fossils as evidence for absence of animals may be justified by the fact that a number of small and soft metazoan phyla indeed have a poor or nonexistent fossil record even though phylogenetic relationships suggest that they have existed at least throughout the Phanerozoic (e.g., Valentine et al. 1999, fig. 3). However, to explain why not a single one of the early metazoan lineages brought fourth animals large or hard enough to be fossilized calls for specific evolutionary (Davidson et al. 1995, Peterson et al. 2000) or environmental (Runnegar 2000) models. Such models typically require that the early animals were all very small and not prone to fossilization. However, as argued forcefully by, e.g., Valentine et al. (1999) and particularly Budd and Jensen (2000), many of the characters shared by modern metazoan phyla would not likely have evolved in animals of millimetre-size or below. The question is thus currently unresolved. The fossil record and the molecular data give different answers, and we need to understand both better in order to collate them. At least part of the answer concerns methodology, as suggested by the fact that analogous conflicts exist with regard to the origin of mammals and of flowering plants in the Phanerozoic (e.g., Foote et al. 1999), but the question of metazoan origins also has unique aspects that do not apply to the later Phanerozoic events. Lineages and characters First an obvious caveat: In phylogenetic analysis, separation of an evolutionary lineage is recognized when it acquires its first distinguishing character, not its last. The first members of a clade may look nothing like later ones, which may have acquired a score of other characters that now help us to recognize them. The first member of the metazoan clade would thus in actual appearance be closest to a protist (or perhaps to the fungal sister group; cf. Baldauf 1999, Baldauf et al. 2000), and if found fossilized it would hardly be recognized as a metazoan ancestor. The recognition of a lineage’s presence in the fossil record typically comes when it has evolved most or all of the (fossilizable) characters used to recognize later members of the lineage. This distinction is generally clear to scientists involved in reconstructions of the evolutionary history of organismic groups (e.g., Runnegar 1996, Valentine et al. 1999, and many others). Nonetheless, confusion will persist as long as taxa are understood as groups of organisms with similar genomes and morphotypes shared through common inheritance, but defined as holophyletic (in the sense of Ashlock 1971; ‘monophyletic’ in cladistic terminology) clades of a phylogenetic tree of life, the structure of which is determined exclusively by inferred branching order, not by genetic or morphological distances. Regardless of potential confusion, however, there will always be a natural gap between the time of divergence of a clade from its sister group and the age of the oldest fossils recognized as belonging to that clade. The crucial point in the discussion is whether this gap can (Peterson K. et al. 2000) or cannot (Valentine et al. 1999, Budd & Jensen 2000) be very long.

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Dealing with extinct forms Another fact almost universally recognized but commonly sacrificed for methodological convenience, is that most lineages become extinct, most of them early in their history. This is mainly because a lineage evolves out of a single founding species, and during the time of low initial diversity chances are great that extinctions of the few species will terminate the lineage. Thus the longevity distribution of taxa is highly skewed in favour of short-lived forms (Raup 1991). Molecular phylogenetic trees, from the universal ‘Tree of Life’ to any small subset thereof, are based on lineages that survive today. Sepkoski’s (1997) database of fossil genera of marine metazoans in the Phanerozoic counts about 2,500 genera in the Quaternary and about 250 (excluding the anomalous archaeocyathan peak) during the interval from the late Early Cambrian until the Ordovician radiation. If the Cambrian/ Quaternary 1:10 ratio of genera is representative also for species, the 105–107 extant marine metazoan species (Sepkoski 1997 ) should correspond to 104–106 marine species in the Cambrian. If we accept Budd and Jensen’s (2000) conclusion that few if any crowngroup phyla are present in the Early Cambrian, then as few as about 30 (corresponding to the approximate number of living phyla) Cambrian species, or 3x10-2–3‰ of the total number of Cambrian marine metazoan species, would have living descendants and thus be represented in molecular phylogenies. The overwhelming dominance of extinct vs. surviving lineages during the early phases of metazoan diversification has been suggested as a partial explanation of the large number of problematic taxa around the Precambrian–Cambrian boundary (Stanley 1976, Bengtson 1977). Bengtson (1986a) argued that metazoan phyla are essentially historical concepts solely based on living taxa and fundamentally bounded by ignorance (‘major units of uncertain systematic position’), that therefore they are not a suitable tool to analyze the history of metazoan diversification, and that the method of pigeonholing problematic taxa into the least dissimilar Recent phylum leads to a pruning of the phylogenetic bush (containing a large number of short-lived early taxa) into a tree (exclusively containing living phyla). In 1989, the theme of early high diversities and the dangers of ‘shoehorning’ was expounded by Gould in his famous book on the Burgess Shale. At about the same time that Gould’s book was published, however, the pendulum was forcefully swung back toward the side of phylogenetic ‘pruning’ (‘pigeonholing’, ‘shoehorning’) by three influential developments: (1) Discoveries of new early Cambrian conservation lagerstätten, in particular the Chengjiang fauna of southern China, gave evidence that some of the more celebrated Cambrian problematica in fact shared probable synapomorphies with living phyla (e.g., Ramsköld & Hou 1991). (2) Molecular systematists started to provide reproducible phylogenies of the animal kingdom (Field et al. 1988 and a deluge of subsequent papers; see, e.g., Adoutte et al. 2000, Baldauf et al. 2000 for recent examples and references), thereby focusing attention to the phylogeny of surviving lineages. (3) The popularity of parsimony-based methods in phylogenetic analysis downplayed the possibility of character similarities being due to convergence (Budd & Jensen’s (2000, p.281) statement that ‘to assume that two structures are not homologous despite their similarity appears to be methodologically suspect’ reflects this sentiment well).

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The idea of animal taxa outside the established hierarchy of Recent phyla is thus currently unpopular. Budd and Jensen (2000) stressed that all animals either have a crown-group or a stem-group relationship to living phyla (and further that this relationship is obscured by assigning fossils to extinct phyla). This is logically uncontestable, but it basically mirrors the methodological principles applied and thus circumvents the question of whether the few lineages that happened to have survived until the present provide valid patterns for the hundreds of thousands times more numerous ones that did not. In evolution, fundamental innovations tend to be introduced early in the history of a clade; what follows is pruning of lineages and tinkering with characters (cf. Miklos & Campbell 1994). Unless early metazoan evolution was constrained and channeled towards extant anatomical and morphological types in a way that could only be described as teleological, the early lineages would represent a considerably more disparate anatomical space than the few that came to be the founders of living phyla. Misapplication of the parsimony principle may lead us fundamentally astray, particularly when we are dealing with poorly preserved fossils having few, simple, and convergence-prone characters. Fossilization potential of early animals Animals lack cell walls, the outer envelopes that cover the cells of most other organisms. A small animal without skeleton or cuticle has a miniscule chance of being preserved as fossil. Yet there is now quite a body of knowledge about where and how to look for fossilized non-skeletal remains. Recent discoveries of fossilized cellular tissues from early animals suggest that we may be able to obtain data from the fossil record in order to resolve the controversial issue of a long (or short) Proterozoic history of small, larva-sized metazoans. Phosphatization of cellular tissue One of the most exquisite kinds of fossil preservation results from phosphatization. The series of studies by Müller and Walossek (see references in Walossek 1993) on Upper Cambrian limestone rich in organic matter and calcium phosphate demonstrated how small animals, particularly arthropod larvae, can be preserved in stunning detail, down to features of sub-micrometer size. In most of these cases only cuticles seem to be preserved, but other instances of phosphatization demonstrate that also less resilient tissues may be fossilized. Recent discoveries of metazoan embryos in Cambrian and Neoproterozoic phosphatic rocks (Zhang & Pratt 1994, Bengtson & Yue 1997, Li et al. 1998, Xiao et al. 1998, Yue & Bengtson 1999, Xiao & Knoll 2000a, b) point towards an avenue of investigation into the early metazoans that is independent of whether the animals have attained a certain structural complexity. The new examples include fossilized early embryonic stages with recognizable cleavage patterns. Such developmental stages must have been present even in the earliest multicellular animals, and there seems to be no reason why they could not also be preserved from the very beginnings of animal multicellularity. Sedimentary phosphorites are rare in sequences older than the massive phosphoritization event at the Precambrian–Cambrian transition (Cook & Shergold 1986), but the most exquisite fossil preservation through phosphatization in fact seems to occur in rocks that are not

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particularly strongly phosphatized. Early diagenetic phosphatization of organisms may be an extremely localized phenomenon (Briggs & Wilby 1996), and it appears feasible to search for such occurrences throughout the Proterozoic sequences. The oldest embryonic tissues reported so far are from the ca. 570 Ma old Doushantuo phosphorite deposits in southern China (Li et al. 1998, Xiao et al. 1998, Chen et al. 2000, Xiao & Knoll 2000a, b). This is too young to bear direct evidence on whether or not metazoans diversified before or after the great Neoproterozoic glaciations (cf. Fig. 1). However, the mere presence there of metazoan embryos or minute juveniles/adults is of great potential significance for the idea that small early metazoans have hitherto gone undetected and that they may be found in deposits older than those containing larger animal-like fossils (most or all Ediacara-type deposits are younger than the Doushantuo Formation; cf. Martin et al. 2000). Chen et al. (2000) reported a diversity of embryo-like fossils from the Doushantuo, including cnidarian and bilaterian gastrulae, and suggested this as evidence for a long pre-Ediacaran history of bilaterian diversification, as required by the model of metazoan evolution proposed by coauthors of that article (Davidson et al. 1995, Peterson et al. 1997, 2000). As argued by Xiao et al. (2000), however, the ‘epithelial layers of cells’ are better interpreted as apatite coatings of the kind that is common in phosphatized fossils (see, e.g., Yue & Bengtson 1999, fig. 9), and the alleged diversity of embryo fossils may be explained by post-mortem alteration and phosphate encrustation on globular microfossils. Cellular tissue may indeed be preserved by such coatings. This can be seen in Fig. 2, which shows a fractured specimen of an early Cambrian embryo in which the cell

Fig. 2. Section through phosphatized embryo from the Lower Cambrian Dengying Formation of Shizhonggou, southern Shaanxi, China (locality details in Yue & Bengtson 1999). Scanning electron microscope images. National Geological Museum of China No. NGMC IV9801. An outer layer of cells has been preserved by an internal coating of centripetally growing apatite (calcium phosphate). Note polygonal surface pattern reflecting the boundaries between the cells. cb – cell boundary; fa – fibronormal apatite; sph – spherulite formed by the growth of apatite.

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boundaries have been encrusted by centripetally growing apatite. The orientation of the apatite crystallites is caused by fibronormal growth on the encrusted surface, however, rather than reflecting the original polarity of cells, as suggested by Chen et al. (2000) for the Doushantuo fossils. Although post-mortem changes may produce strong alterations of original morphology and cleavage pattern, careful studies of numerous and wellpreserved embryos have the potential to open a major window to the evolution and development of the earliest metazoans. Ancient sponges? The monophyly of the Metazoa including the Porifera seems well supported by molecular sequences (Müller 1995, Müller et al. 1998, Müller et al. 1999). The sponges are often treated as a holophyletic clade, although a recent analysis of complete 18S rRNA sequences suggested that the Demospongea and Hexactinellida form a sister group to the remaining metazoans including the Calcarea (Collins 1998). Known sponges typically have a resilient supporting skeleton consisting of spongin (a kind of collagen) and/or mineralized calcareous or opaline spicules, sometimes also of a calcareous basal skeleton, all preservable in fossils. The basal position of sponges in the metazoan tree and their propensity for fossilization make them obvious targets when searching for early crown-group metazoans in the fossil record. Well-preserved sponge spicules with a structure and morphology hardly distinguishable from those of extant sponges are known from most parts of the Cambrian (Bengtson 1986b, Bengtson et al. 1990, Mehl 1998). However, although a large number of reports have been published on alleged Proterozoic sponges, few of them stand up to scrutiny. Among the more probable examples are structures resembling hexactinellid spicules in optical thin sections from the late Neoproterozoic of Mongolia (Brasier et al. 1997). Zhou et al. (1998), however, demonstrated that similar structures in other rocks of that age consist of arsenopyrite crystals, and scanning electron microscopy of these does not suggest any biological mediation in their formation. The most convincing published examples of Proterozoic sponges are hexactinellid-like fossils in the Ediacara biota (Gehling & Rigby 1996) and millimetre-sized bodies containing minute monaxon spicules in the Doushantuo Formation (Li et al. 1998). Even these are controversial, however (e.g., Xiao et al. 2000), and a conservative assessment of the fossil record would suggest that sponges, in spite of their alleged basal position, did not diversify much before the other metazoans. Biomarkers (molecular fossils) specific to metazoan taxa are poorly known, but the relative abundance of certain steroids (24-isopropylcholestanes vs. 24-npropylcholestanes) has been used to indicate a prevalence of sponges in the late Neoproterozoic and early Cambrian biotas (McCaffrey et al. 1994). Although currently fraught with considerable uncertainty with regard to metazoans, the use of taxon-specific biomarkers offers a way of tracking taxa through geological time that is partly independent of morphological fossils (Moldowan & Jacobson 2000). Also, this approach may bring molecular phylogenetics closer to the rock record and thus help resolve some of the discrepancies that currently plague the study of the origins of major taxonomic groups.

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Conclusion We do not know how deep metazoan roots are, i.e. when the transition from protist to metazoan and the radiation(s) of metazoans took place. Data are in severe conflict, and answers are selected mostly on the basis of the researcher’s background (are fossils ‘better’ than molecular sequences, or vice versa?) and on whether there is a specific model of animal evolution to defend. Given that there is a long Precambrian record of microbial fossils but that the indubitable fossil record of animals begins only after the late Neoproteorozoic glaciations (‘Snowball Earth’), the burden of proof currently would seem to lie on those who advocate a long Proterozoic history of the metazoans. Extrapolations of substitution rates in lineages are not likely to be conclusive in themselves, but the accelerating availability of gene sequences should lead to better precision in the estimates of divergence times or, alternatively, to the realization that no reliable results can be attained by these methods. However, the ‘young animals’ hypotheses are in theory falsifiable by just a single well-documented find of a definitive metazoan in rocks predating the Neoproterozoic glaciations. At present, the molecular data provide a healthy challenge to the ‘palaeontological viewpoint’ that the fossil record is reasonably reliable in these questions, but to turn the tide conclusively hard evidence would be needed from actual fossils or from reliable chemical biomarkers that metazoans existed a significant time before the Neoproterozoic glaciations. The increasing understanding of fossilization of non-skeletal tissues, and the improving methods of chemical analysis of rocks and fossils may provide some of the tools. Proving the non-existence of ancient animals will be more difficult, but if well-designed searches of such evidence consistently fail, the ‘young animals’ hypotheses will at least be strengthened. The ever-present danger is in the over-application of the models derived from the study of present life and present environments. The present may be a good key to the not-too-distant past, but very old locks may need some more creative tinkering than just a brand-new key to be opened properly. References ADOUTTE A., BALAVOINE G., LARTILLOT N., LESPINET O., PRUD’HOMME B. & R. de ROSA 2000. The new animal phylogeny: Reliability and implications. Proceedings of the National Academy of Sciences 97: 4453–4456. ASHLOCK P.D. 1971. Monophyly and associated terms. Systematic Zoology 20: 63–69. AYALA F.J. 1999. Molecular clock mirages. BioEssays 21: 71–75. AYALA F.J., RZHETSKY A. & F.J. AYALA 1998. Origin of the metazoan phyla: Molecular clocks confirm paleontological estimates. Proceedings of the National Academy of Sciences, USA 95: 606–611. BALDAUF S.L. 1999. A search for the origins of animals and fungi: Comparing and combining molecular data. American Naturalist 154: 178–188. BALDAUF S.L., ROGER A.J., WENK-SIEFERT I. & W.F. DOOLITTLE 2000. A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 290: 972–977. BENGTSON S. 1977. Aspects of problematic fossils in the early Palaeozoic. Acta Universitatis Upsaliensis. Abstracts of Uppsala Dissertations from the Faculty of Science 415: 1–71.

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Wild and domestic mammals in Sardinia 303 Theholocenic New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 303-308, 2003

Wild and domestic mammals in holocenic Sardinia B. Wilkens & F. Delussu Department of History, University of Sassari, Italy

Abstract Archaeozoological research is still at an initial stage in Sardinia. Consequently in a project for the study of Sardinian fauna during the Holocene, in recent years, four Neolithic and Chalcolithic, three Bronze age, three Iron age, four Roman and nine medieval sites have been studied. Of the mammal species living on the island in the Upper Palaeolithic, one lagomorph (Prolagus sardus) and some rodents are still present during the Neolithic. Already in Neolithic, alongside the domestic fauna, other wild species imported by man appear. Prolagus and the Tyrrhenicola survive at least up to the Iron age. The importation of new species, both domestic and wild, is not limited to prehistory, but continues in the following centuries. Some species such as the fallow deer, are known only from the Middle ages.

Archaeozoological research is in an initial phase in Sardinia. Of the faunal remains coming from excavations conducted in the past only a small part has been examined and often in an inappropriate way by non-specialists. In recent years, numerous projects regarding the population of the island during the Holocene by wild and domestic animals have been conducted. The projects have concentrated above all on the central-northern part and regard sites from the Neolithic to the Medieval period. In all, the bone remains from 23 sites, concentrated in the central-northern part, have been studied and where possible the data from publications of other authors has been checked. Despite the new information from these works, the situation is still uncertain with regards to the earliest phases, the Neolithic and Chalcolithic. The data obtained from recent studies, while of fundamental importance, are of a provisional nature as the fauna examined is not of a great number and the sites examined insufficient for the formulation of general rules. Neolithic and Chalcolithic The substitution of the Pleistocene fauna with that of today was a process, which extended over a notable length of time and should not be considered as having been

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concluded in the first phases of the Neolithic. At around the end of the Pleistocene the extinction of almost all the terrestrial mammals of the endemic fauna can be observed, of which only a few species belonging to the orders of the insectivores, the rodents and the lagomorphs survive. From the data presently available it can be stated that the principal domestic species of which no possible Pleistocene ancestors exist in situ arrive in Sardinia during the Neolithic, between the 6th and 5th millennia BC, imported by man: the dog (Canis familiaris), the pig (Sus scrofa domesticus), the ox (Bos taurus), the sheep (Ovis aries) and the goat (Capra hircus). Perhaps even during the Middle Neolithic (Vigne 1988), a part of the pigs and a part of the sheep return to the wild, giving origin to the wild boar and mouflon respectively. From the old pleistocenic fauna (cfr. Hofmeijer et al. 1987) the Episoriculus similis (Insectivores), Rhagamys orthodon (Muridae), Tyrrhenicola henseli (Microtinae) and Prolagus sardus (Lagomorphs) still survive while the pleistocenic deer (Megaloceros cazioti) is substituted, probably in an uncertain period in the Neolithic, perhaps towards the end, by Cervus elaphus introduced by man. Other animals already present in the Neolithic are the fox and the hedgehog. In the Neolithic levels of the Grotta del Guano (Sanges & Alcover 1980) and Grotta Corbeddu (Sanges 1987) all the fauna characteristic of this period have been found, to which a large part of the micromammals that currently populate the island are to be added. Other sites with Neolithic/Chalcholithic levels are Grotta Filiestru (Levine 1983), Grotta di Punta del Quadro, Alghero, the small cave in via Besta in Sassari etc. As regards the metrical and morphological characteristics of the mammals of these earliest phases, given the scarcity and the fragmentary nature of the material which we have studied, it has been possible only to recover some metrical data regarding the domestic animals. These are subjects of rather important dimensions and, given the fact that the Sardinian mouflon derives with a degree of probability from the Neolithic sheep, it is possible that the Sardinian domestic sheep were similar to the Neolithic animals in central and southern Italy, which have characteristics close to those of the mouflon. The Bronze Age For the Bronze Age information comes from four Nuragic settlements: Madonna del Rimedio - Oristano (Santoni & Wilkens 1996), Serra Niedda - Sorso (Wilkens 2000), Nuraghe Miuddu - Birori (Delussu 1997), Brunku Madugui - Gesturi (Fonzo 1986). The species which are present in highest numbers are: the caprovines, bovines, pigs, amongst the domestic animals; and the red deer, boar and mouflon amongst the wild animals. Of the micromammals, the Prolagus sardus and Tyrrhenicola henseli still survive. In the case of Sierra Niedda the presence of a tibia of hare is important in that it attests the presence of the animal on the island for the first time. Unfortunately, this piece comes from a level which is disturbed by the presence of Black- figure pottery and hence its dating to the Bronze age is not certain, even though the appearance of the hare on the island is of a period which is quite early. The domestic species are generally fairly small in dimensions, a characteristic they have in common with the domestic stock on the italian peninsula during the Bronze Age. The bovines may be identified as being of the brachyceros type, often of very small size.

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The goats had horns which were without or nearly without torsion, possessed by both sexes, although larger in the males. Fairly robust horns of sub-triangular section belonging to male sheep have been found. Iron Age The fauna types of this period are still those typical of the nuragic period (pig, ox, caprovine, / boar, red deer, mouflon) which are flanked by the horse, present among the remains dating to the Early Iron Age of the nuragic santuary of S. Antonio di Siligo (Delussu, 2000) and which has not yet been attested before the Iron Age. In this period the edible dormouse also appears (at S. Antonio and S. Imbenia) (Manconi, 2000), which may also have been important in the diet. Amongst the micromammals Prolagus sardus (at S. Antonio, S. Imbenia, Nuraghe Aeddos, Genna Maria and Is Paras) and Tyrrhenicola henseli (at Is Paras) are still present, though perhaps close to extinction. A small amount of remains has recently been recovered from Nuraghe Aeddos in Orotelli (Delussu, in study). Another fragment of hare has come from the Final Bronze/ Iron I levels of this nuraghe, which confirms the fact that the species was already present in Sardinia in this phase. The domestic animals are still of slender build, while the boar is bigger than both the contemporary pigs and the modern Sardinian boar: a scapula has come from the Nuraghe Is Paras (Zedda et al. 1997) the attribution of which to the boar is without doubt, as it regards a subject injured by a lance that produced a 15mm circular lesion on the scapula in question. The dimensions of this scapula are notably superior to those attested for the pigs. Roman Period For this phase the best information (Manconi 1990, 1996, Wilkens 1996) come from Olbia (4th c.BC to 3rd c. AD), from Porto Torres (Columeau 1984, Manconi, in study, Delussu, in press b) and from the Nuraghe Mannu (Delussu, in press) both of the 3rd-7th c. AD; the faunal remains relating to ritual offering dating to the 2nd c. AD come from Genoni-S.Antine (Wilkens, in preparation). The donkey and the cock appear for the first time in the Roman period. The donkey, of a small size, already displays metrical and morphological characteristics which are similar to those of the modern Sardinian donkey. Important information regarding dogs, that in this period are already notably differentiated in terms of morphology, has come from Olbia (Manconi 1996). The remains of 14 goats, 5 bovines, 8 pigs and 7 dogs have come from the well of the Roman period of S.Antine (Wilkens, in preparation). The material is in a good state of conservation so that it has been possible to study the morphological characteristics of these domestic animals. The bovines are larger than those attested in the preceding period. The pigs are very different from those considered, in the Roman period, a valued breed, such as those portrayed in the scenes of the suovitaurilia and described by the Latin authors: in the case of S. Antine the size is so small that comparison with other contemporaries cannot be carried out. Even the crania of the males are small and of

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straight profile, with slightly developed canines and reduced diastema. It is probable that this regards a local breed which had not been widely selected and which certainly was not widespread in the island, as the pigs in Olbia are of a good size. The dogs are long-snouted, with slender limbs. Amongst the goats the males have marked frontal protuberances, robust horn cores of elliptical section with rounded edges. The wild cat (Felix lybica) has been identified amongst the wild animals, therefore we must assume that the arrival of the domestic cat took place in a previous period as the wild animal originated from the latter (Vigne 1988). Medieval For the high medieval we have information from S. Filitica (Delussu, 1999) on the North coast, from the Nuraghe Urpes (Webster 1987) and the church of S. Eulalia in Cagliari (Zedda, in study). The definitive appearance of all the species that currently populate the island took place during the medieval and post-medieval centuries, amongst which the rabbit, which has been identified for the first time at Geridu, a 14th century village close to Sassari. The fallow deer (Dama dama L.), that appears in the 14th century at Geridu (Delussu 1996) and has been found in abundance at Saccargia and S. Maria di Cea, deserves special attention. The abundance of this species in the medieval leads us to suppose its introduction took place at least some centuries previously but for now we have no earlier indication of the animal, perhaps as a result of the difficulty of distinguishing this animal from the red deer at the level of bone analysis. With regards to this we are conducting a project on current material and excavation material from Sardinia and the Italian peninsula, to underline the distinctive characteristics of the single bone. The Sardinian fallow deer became extinct in the 1960’s, as a result of excessive hunting. Besides the abandoned village of Geridu, further information on the period in question comes from the study of the faunal remains from the bastions of Alghero, and from its historical centre, from the castle of Bosa (Delussu, 2000), from the excavation in the Duomo and Palazzo Ducale (Delussu, 2000) and the market of Sassari (Wilkens, in preparation), from the excavations in the well of the monastery of S. Maria di Cea close to Banari (Wilkens & Delussu, 2000) and, finally from the excavations of the monastery of Saccargia situated some kilometres from Sassari (Baldino & Conti, in press). The recurring faunal association is given by the presence of pigs, oxen, caprovines and equines (horse and donkey), together with red deer, fallow deer and mouflon. To this the presence of leporines, birds, amongst which the chicken, and marine molluscs must be added. The marine turtle has also been found at Saccargia. The domestic species are generally small in size: in particular the pigs still display rustic characteristics, with straight but shortened snout profiles, small canines and slender build. The bovines are of medium-small size, with small horns, very similar to some indigenous bovines currently living in some parts of Sardinia. The sheep are also quite close, in terms of size, to the breed currently to be found in Sardinia, from which they may be differentiated by the robust male horns. The goats are of medium size, with crooked horns. The remains of a large dog of robust build and large head have come from Palazzo Ducale in Sassari. Other dog remains from Saccargia demonstrate the

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existence of notable metrical and morphological variety that probably indicates the existence of different breeds. Deer and fallow deer are of reasonable dimensions although smaller than the continental animals. The donkeys display all the characteristics of the modern Sardinian breed of small size, while a subject of larger size has come from Alghero. Remains of horse, of which several breeds probably existed, are not common. References Baldino B., Conti C. in press. Resti faunistici dal Convento di Saccargia (Sassari), Proceedings of the 3° Convegno Nazionale di Archeozoologia, Siracusa, 3-5 novembre 2000. Columeau P. 1984. La faune archéologique. In Villedieu F. (ed.), Turris Libisonis. Fouille d’un site romain tardif à Porto Torres, Sardaigne. BAR International Series, 224: 345-351. Delussu F. 1996. Il villaggio medievale di Geridu (Sorso, SS). Campagne di scavo 1995/1996: relazione preliminare - I resti faunistici. A cura di M. Milanese. Archeologia Medievale, XXIII: 530-533. Delussu F. 1997. Le faune dell’Età del Bronzo del Nuraghe Miuddu. Rassegna di Archeologia, 14: 189-204. Delussu F., 1999, I reperti faunistici, In Rovina D. (ed.) L’insediamento altomedievale di Santa Filitica (Sorso-SS)- interventi 1980-1989 e campagna di scavo 1997. Relazione preliminare. Archeologia Medievale 26: 202-207. Delussu F. 2000. Lo stato attuale degli studi sulle faune oloceniche della Sardegna centrosettentrionale, Atti del 2° Convegno Nazionale di Archeozoologia, Asti 14-16 novembre 1997: 183-192 Delussu F. in press. I resti faunistici del Nuraghe Mannu. Bollettino di Archeologia 1996/97. Delussu F., in press b. Produzione e consumo dei prodotti animali nell’ambito dell’economia di Turris Libisonis (Porto Torres - SS) in età imperiale. Proceedings of the 3° Convegno Nazionale di Archeozoologia, Siracusa, 3-5 novembre 2000. Fonzo O. 1986, Reperti faunistici in Marmilla e Campidano nell’età del Bronzo e nella prima età del Ferro. Atti del 2° Convegno di Studi “Un millennio di relazioni fra la Sardegna e i Paesi del Mediterraneo”, Selargius-Cagliari. Hofmeijer G. K., Martini F., Sanges M., Sondaar P.Y. & A. Ulzega 1987. La fine del Pleistocene nella Grotta Corbeddu in Sardegna. Fossili umani, aspetti Paleontologici e cultura materiale. Rivista di Scienze Preistoriche, XLI, 1-2: 1-36. Levine M. 1983. La fauna di Filiestru (trincea D). In Trump D.H. (ed.), La grotta di Filiestru a Bonuighinu, Mara (SS). Quaderni-13 - Soprintendenza ai Beni archeologici per le province di Sassari e Nuoro. Sassari, pp. 112-131. Manconi F. 1990. Olbia. Un’area sacra sotto Corso Umberto n. 138: i resti faunistici. In Mastino A. (ed.), L’Africa Romana VII. Sassari, pp. 505-510. Manconi F. 1996. Olbia. Su Cuguttu 1992: i reperti faunistici. In Mastino A. & P. Ruggeri (eds), Da Olbìa ad Olbia, 2500 anni di storia di una città mediterranea. Sassari, pp. 447-460. Manconi F. 2000. La fauna dell’Età del Ferro degli scavi 1988 e 1990 del nuraghe S.Imbenia di Alghero (Sassari). Atti del 2° Convegno Nazionale di Archeozoologia, Asti 14-16 novembre 1997: 267-277. Sanges M. 1987. Gli strati del Neolitico Antico e Medio nella Grotta Corbeddu di Oliena (Nuoro). Nota Preliminare. In Il Neolitico in Italia. Firenze, pp 825-830.

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Sanges M. & J.A. Alcover 1980. Noticia sobre la microfauna vertebrada holocenica de la Grotta Su Guanu o Gonagosula (Oliena, Sardenya). ENDINS 7: 57-62. Santoni V. & B. Wilkens 1996. Il complesso nuragico “La Madonna del Rimedio” di Oristano, Quaderni della Soprintendenza Archeologica di Cagliari-Oristano 13: 29-43. Vigne J.D. 1988. Les mammifères post-glaciares de Corse-étude archéozoologique. CNRS, Paris. Webster G.S. 1987. Vertebrate faunal remains. In Studies in Nuragic Archaeology: Village Excavations at Nuraghe Urpes and Nuraghe Toscono in West-Central Sardinia. BAR International Series 373: 69-161. Wilkens B. 1996 a. Un saggio di scavo sull’Acropoli di Olbia: la fauna. In Mastino A. & P. Ruggeri (eds), Da Olbìa ad Olbia, 2500 anni di storia di una città mediterranea. Sassari, pp. 353-355. Wilkens B. 1996 b. Conserves de poisson à partir de quatre amphores romaines. Archaeofauna 5: 165-169. Wilkens B. 2000. Resti rituali dal pozzo sacro di Serra Niedda (SS). Atti del 2° Convegno Nazionale di Archeozoologia, Asti 1997: 263-266. Wilkens B., Delussu F. 2000. Resti ossei dal convento di S.Maria di Seve (Banari - SS). Archeologia Medievale, 27: 311-313. Wilkens B. in preparation. Archeozoologia. Manuale per lo studio dei resti faunistici dell’area mediterranea. Zedda M., Farina V., Sanna Passino E., Careddu G., Delussu F. & B. Wilkens 1997. Morphological and pathological characteristics of a scapula suggesting a Bronze Age wild boar hunt. Proceedings of the 15° Meeting of the European Society of Veterinary Pathology, Sassari-Alghero, p. 203.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

People and animals in the early Neolithic in Central Europe. New ... Evolution 309 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 309-317, 2003

People and animals in the early Neolithic in Central Europe. New approach to animal bones assemblages from farming settlements Arkadiusz Marciniak ´ sw. ´ ´ Poland Institute of Prehistory, University of Pozna n, Marcin 78, 61-809 Pozna n,

Abstract The paper presents results of studies of animal bone assemblages from the early Neolithic in Central Europe focused upon social relations between humans and animals, food and its preparation and consumption as well as refuse disposal patterns. It is assumed that animals were maintained and consumed in ways that accented social relationships, such as creating identity and highlighting ancestry, inequalities, social rules and status. The empirical examples from Kujavia region in Central Poland are provided. Faunal assemblages were analyzed on the level of particular sites and features. The following methods were used: correlation between body part representation and structural bone density, the Modified General Utility Index, and the Marrow Index as well as anatomical body part distribution, and species composition. The impact of taphonomic factors was evaluated prior to its further interpretation.

Introduction This paper is explicitly aimed at overcoming the ‘economic’ bias of European archaeozoology. I want to focus on the social context of animal use by recognizing that animals were maintained and consumed in ways that accented social relationships, such as those creating identity, highlighting ancestry, inequalities, importance of gender, negotiating social roles, links, and evaluating and/or maintaining social status. Social factors influence particularly the following kind of animal patterns: the use of domestic vs. wild animals, carcass distribution, food preparation, and discard. When one looks at archaeological literature over the last few decades addressing the role of animals in prehistoric society, one is to be surprised at how easily animal bone assemblages are incorporated in models relating to herd management, subsistence, nutrition, and adaptation. The faunal remains, being debris of certain activities, were usually regarded as the patterned residues of these activities, but were treated as representation of the economic system that structured these activities (see Barrett 2000: 63). Consequently, the studies of prehistoric fauna were focused on the larger scale that

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is more distanced from the archaeological record. Small-scale events were given little interest and all of them were explained by long-term changes. Archaeological and faunal materials I would like to discuss here a couple of examples from early and late Danubian tradition in Central Europe, more specifically the Band Pottery Culture from Kujavia region in the North European Plain. The early phase of the LBK complex is characterized by remarkable uniformity over vast geographic distances. This is most notable in case of distinctive timber rectangular longhouses, pottery, polish stone adzes and chipped stone assemblages. The dominant scenario of the LBK phenomenon stresses that these groups were the first agricultural communities in the area of Central Europe settled on loesses or other fertile soils. These groups present homogeneous socio-cultural system, formed and undergoing transformations in the zone of spread of an agricultural economy from the „Near East centre” (e.g. Bogucki 1988). The end of early Neolithic, namely the later stage of the LBK, was characterized by expansion of farming communities into new areas and both large trapezoidal houses and big settlements accompanied this process. The process of constructing small communities was of local character and carried out relatively independently as part of a wider tendency characterized by the convergent development. This does not necessarily mean that these groups lived in isolation. On the contrary, they drew from the richness of their cultural tradition, mainly Danubian Neolithic, in order to construct their own identities, ancestry in a unique and specific way. Longhouses and its attendant material culture as well as cattle were the means through which the first lowland Neolithic communities of the central European woodlands were created (see more in Marciniak 2000). The analysis is based upon 6 settlements from the early Band Pottery Culture · (Bozejewice, site 22; Łojewo, site 35; Miechowice, site 7; Radojewice, site 29; Siniarzewo, · site 1; and Zegotki, site 2) and 4 settlements from the late Band Pottery Culture · (Kuczkowo, site 5; Siniarzewo, site 1; Węgierce, site 12; and Zegotki, site 3). All of them come from Kujavia region in central Poland. In total, 16,436 animal bones were collected and 9,413 (57,3%) of them were identified. 11,530 (70,2%) bones come from the earlier period while 4906 (29,8%) from the later period. I have chosen for the analysis only those settlement sites, which contained a considerable number of animal bones and were properly excavated and the material was correctly recorded. It is required that all archaeological and faunal data to be attributed to given strata and/or feature which make statistical and contextual analyses possible. Methods The most appropriate strategy, in which the social dimension of human-animal relationships in prehistoric farming communities can effectively be addressed, is analysis and interpretation of horizontal distribution of skeletal parts, both within a given settlement and microregion. It provides an information about functional utilization of animals and their parts in different places within the settlement and allows us to get into the social dimension of animal exploitation. Furthermore, the context in which bone

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materials are deposited is directly responsible for their preservation. It also enables us to recognize a group predilection toward a particular method of refuse disposal. This should be prerequisite of all analyses before we pursue this any further. This kind of analysis has to be supplemented by the study of the context of other features such as dwelling structures, burial ground and numerous archaeological data. The observed distribution of anatomical parts in archaeological context is a result of potentially complex set of cultural and natural processes. Thus, it is necessary to discern the impact of these factors upon observed frequency of anatomical parts. They provide ‘frames of reference’ in searching of explanations for the frequencies of skeletal parts composition. Some of them are based upon results of actualistic studies, but they do not draw out all potential causes. The analysis of collected data comprised: (1) a correlation between density and body part representation (Lyman 1984), (2) a correlation between the Modified General Utility Index (Binford 1978) and body part representation, (3) a correlation between the Marrow Index (Binford 1978) and body part representation. The results made then possible to look at (4) anatomical body part distribution, and then (5) species composition. An impact of depositional and postdepositional factors upon faunal materials has been widely studied and discussed in the literature (e.g. as summarized recently by Lyman 1994). The scope of these transformations is often considerable and it has been shown that a given assemblage might have been formed by other factors, e.g. natural than those which supposedly should have been reflected in studied deposits. The differential preservation might depend upon bone density. Thus, the study of density driven attrition can be a valuable factor in assessing whether the observed variability is caused by differential density of particular anatomical parts. In order to discern this pattern a correlation between the frequency of each skeletal part and the structural density values was calculated. I have used for this calculation a structural bone density of deer (Odocoileus spp.) and sheep (Ovis aries) bones as measured by Lyman (1984), and North American bison (Bison bison) measured by Kreutzer (1992). The analysis of frequencies of skeletal parts is often aimed at recognition of various strategies of human use of food. The tools for such studies were provided by Binford (1978) who measured amount of meat (weight of fat and muscle tissue) and marrow (marrow cavity volume multiplied by the percentage of fatty acids in the marrow) of particular skeletal parts of two domestic sheep (Ovis aries) and one caribou (Rangifer tarandus). This led to the calculation of food utility indeces of the anatomical parts (see detailed calculation of indices in Binford 1978:74). In order to discern these relations in analyzed assemblages a correlation between the Modified General Utility Index and the Marrow Index for anatomical parts and frequency of these fragments was calculated. Values for sheep and caribou, as proposed by Binford (1978), were used. Analysis of anatomical part representation was proceeded by ordering of animal body into 7 categories based upon proposal by Stiner (1991; see there for more details) with futher modifications: (1) horn/antler, head, neck, (2) axial column below the neck, (3) upper front limbs, (4) lower front limbs, (5) upper hind limbs, (6) lower hind limbs, and (7) feet.

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Results Analysis of studied materials reveals a lack of correlation, calculated by Spearman’s correlation coefficient, between bone density of anatomical parts and frequency of these fragments, both in the early and late Band Pottery assemblages. In some cases, statistically significant negative correlation between these two variables was observed. This means that the most numerous bones in this assemblage are those that are the most fragile. Taphonomic processes mediated by the structural density of the skeletal parts had not influenced the frequencies of skeletal parts. Thus, one could conclude that the observed frequency of bones was not caused by factors acting upon an assemblage after bones were deposited. It was rather a result of cultural factors prior to its deposition, namely an intentional deposition of particular segments of animal bodies. A correlation between the Modifed General Utility Index and the Marrow Index, and body part representation was calculated using Spearman’s correlation coefficient for all settlements sites as well as all features where number of animal bones made such an analysis statistically valid. Analysis was conduced for cattle, sheep/goat, and pigs only as other species were represented by such a small number of bones that analysis of this type proved to be impossible. In the early LBK assemblages, there was no statistically positive correlation between the Modified General Utility Index and body part representation for analyzed species, both for particular settlements and features/strata at these sites. There was only one exception, namely a significant positive correlation between these two variables for cattle · at feature 153 at Bo zejewice, site 22. Situation is rather similar in the late phase of the LBK. Generally, there is no significant positive correlation between these two variables for whole sites, while there are some discrepancies in particular features. In only three cases out of 43 this correlation was positive and significant. In two of them it referred to pig (feature · 32 and the whole site at Zegotki) and one to cattle (feature 1, layer 1 at Węgierce). These results show that domesticated animals in the LBK, both in its early and late phases, were not in the first place used according to contemporary nutritional standards. Similarly to the Modified General Utility Index, in majority of the early LBK assemblages there was no significant correlation between the Marrow Index and body part representation irrespective of the context from which the bones come from. However, in 15 cases (out of 54) this correlation was positive and significant and it referred both to cattle (8 cases) and sheep/goat (7 cases). Analysis of data from the late LBK reveals also a general lack of correlation between these two variables. However, a number of cases, in which this relationship is positive, are smaller than in the early LBK. This was only observed in 5 cases out of 28 and characterized both cattle and sheep/goat. Analysis of the values of the correlation coefficients of particular species in the same features reveals considerable differences between different features and their content existing at the same sites of the late LBK. To sum up, a general lack of correlation confirmed previous observation that domesticated animals in the LBK, both in its early and late phases, were not used according to contemporary nutritional standards but consumption of marrow was considerable. Having analyzed theses three ‘frames of reference’, offered by actualistic studies, in searching of explanation of the frequencies of skeletal parts it is necessary now to look

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at the body part representation itself. Closer look at data from the early LBK for cattle reveals very uniform pattern that can be called leglessness. It is characterized by a firm predominance of the first two anatomical categories (see previous chapter), namely: horn/antler, head, and neck, and axial column below the neck (Fig.1). Limb bones, both front and hind, as well as feet bones are represented in small numbers. The pattern is almost identical both for particular settlements and features/strata at these sites and · existing deviations are small. They can be seen e.g. at Bo zejewice site 22, feature 153. The most different pattern is observed at Radojewice, site 29 and Siniarzewo, site 1. The pattern for sheep/goat differs considerably from cattle what is shown in more numerous and differentiated frequency of leg bones (Fig.2). Additionally, there are differences among settlements and particular features. The pattern for pig is closer to cattle than to sheep/goat, however it lacks the regularity observed for cattle. Interestingly, deposition of body parts of particular species was different in the same feature e.g. body parts for cattle were considerably different than those of sheep/goat. The body part distribution pattern for the late LBK is more complex. With regard to cattle, one can distinguish three general patterns: (1) a firm dominance of head/neck bones and axial column below the neck resembling the pattern from the early phase, (2) predominance of head/neck bones with small and very similar representation of other anatomical categories, (3) equal representation of all categories except for feet. Particular body fragments appear also in other configurations (Fig.3). All these patterns can be traced at the same settlement in its different features. Thus, one can conclude that importance of cattle and deposition patterns of these bones were considerably varied. The distribution of sheep/goat body parts was also varied, however it was different than that of cattle except for one case. The following patterns can be discerned: (1) a firm predominance of head/neck bones and axial column below the neck resembling the cattle pattern from the early LBK (these proportions, however, are not that regular

Fig. 1. Miechowice, site 7. Cattle - body part representation.

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Fig. 2. Bo|ejewice, site 22. Sheep/goat – body part representation.

Fig. 3. Siniarzewo, site 1, feature 30. Cattle – body part representation.

like in cattle), (2) predominance of head/neck bones with small representation of other categories – this is the most dominant, (3) dominance of one of two other anatomical segments. Body part representation of pigs from the late LBK is characterized by predominance of head/neck bones and axial column below the neck (Fig.4). This is relatively regular and is similar to part distribution of cattle from the early LBK. Other proportions were also observed namely a dominance of head/neck bones and lower front limbs or

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Fig. 4. Siniarzewo, site 1. Pig – body part representation.

predominance of lower hind limbs. One has to remember, however, that these bones are not numerous, and thus the results might not be representative for the whole period. The last analytical step comprises species composition. The most frequently represented species at the LBK settlements is cattle. Cattle bones comprise about 90% or more of all bones. Cattle were followed by sheep/goat. Other domesticated species such as pig are rarely represented, and their number is not higher than bones of wild animals. Wild animals, such as deer, roe deer, aurochs, wild boar are also rarely represented. These proportions are almost identical at all analyzed settlements as well as particular features and strata in these features. The species composition at the late LBK settlements is not that uniform. While cattle were the dominant species at all settlements, composition of species in particular features was considerably differentiated. In some of them, we observed dominance of cattle followed by sheep/goat and then pig, which is identical to the early phases of the LBK; in others sheep/goat dominated followed by cattle and pig or alternatively dominance of pig was observed followed by sheep/goat and then cattle. In other features, number of cattle bones is equal to number of sheep/goat bones. Generally, the number of pigs increased and they became much more popular than in the early phases. Discussion Animals in the early Neolithic, especially cattle, were the very basis for maintaining and creating group’s identity as well as security in a new ‘frontier’ situation and unknown environment. A commonly shared opinion that the early cattle exploitation was meat focused is completely unjustified and has to be rejected. Cattle became an important social resource and when killed, provided food for ceremonial practices (Thomas 1999: 74). These animals were probably slaughtered around longhouses, but ‘communal

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feasting facilities’ might have existed outside longhouses, and it is where food was consumed. Wild animals were slaughtered outside of longhouses and possibly consumed at the ridge of the settlement or outside. Analysis of anatomical distribution shows considerable similarity of cattle and pig that clearly differs from sheep/goat. At the same time, anthropological accounts show that mutton is universally eaten and is not a subject of any taboo as pork or beef (Ryder 1984: 194). This may indicate that in the early Neolithic sheep/goat was consumed in an ordinary fashion and this consumption was not regulated by ideology. At the same time, the use of pig as a meat animal has to be questioned. I would argue that peculiar way of treating of pigs and cattle (developed later in the form of sophisticated cultural and religious regulations) have begun as early as the beginning of the Neolithic. Taphonomy analysis reveals lack of weathering of early Neolithic bones, which implies that bones were directly deposited in pits and that the early LBK sites were thoroughly cleaned. On the other hand, the late LBK faunal remains reveal large number of weathered bones indicating their long presence on the surface before the final deposition. This supports some indications that interiors of long houses in the early Neolithic in Central Europe were cleaned out (Milisauskas 1986: 117), and refuse deposited in pits around houses. However, later phases of the Danubian tradition are characterized by removing a dirt far from the houses. The rubbish was clearly separated from the house and deposited towards the edges of the settlement (e.g. in Inden-Lamersdorf) (Hodder 1990: 128). More generally, it seems that communal identity was of crucial importance for the early LBK communities. The communities were of egalitarian character with consensual decision-making. A village/settlement was the basic social unit creating definable groups. In the late LBK household became a basic social entity and it is discernible archaeologically in the form of household clusters that comprise house, human graves, storage facilities and rubbish pits. The period of construction of identity and descent involved mobilization of external cultural resources like the cattle, the idea of house, and exotic resources such as flint and copper. Cattle as well as houses had the potential to bring a world into being and continually reproduced human relations in and with that world. The historical trajectory of the Kujavia region caused cattle and longhouse to became a „cultural object” incorporating an „extended” form of signification (Giddens 1987: 100) and a means for creation of identity of early farmers. This early phase was later replaced by the period of stabilization when the common identity was set up and configuration of previously mobilized and further recontextualised resources provided conceptual means for these groups. References BARRETT J.C. 2000. A thesis on agency. In Dobres M.-A. & J. Robb (eds), Agency in Archaeology. Routledge, London and New York, pp. 61-68. BOGUCKI P. 1988. Forest Farmers and Stockherders. Early Agriculture and its Consequences in NorthCentral Europe. Cambridge University Press, Cambridge. BINFORD L.R. 1978. Nunamiut Ethnoarchaeology. Academic Press, New York. GIDDENS, A. 1987. Social Theory and Modern Sociology. Cambridge University Press, Cambridge.

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GRYGIEL R. & P. BOGUCKI 1997. Early farmers in north-central Europe: 1989-1994 excavations at Osłonki, Poland. Journal of Field Archaeology 24: 161-178. HASTORF C.A. 1991. Gender, space and food in prehistory. In Gero J.M. & W.M. Conkey (eds), Engendering Archaeology. Women and Prehistory. Basil Blackwell, Oxford, pp. 132-158. HODDER I. 1990. The Domestication of Europe: Structure and Contingency in Neolithic Societies. Blackwell, Oxford. HODDER I. 1997. Architecture and meaning: the example of Neolithic houses and tombs. In Parker Pearson M. & C. Richard (eds), Architecture and Order. Approaches to Social Space. Routledge, London and New York. KREUTZER L.A. 1992. Bison and deer mineral densities: comparisons and implications for the interpretation of archaeological faunas. Journal of Archaeological Science 19: 271-294. LAST J. 1996. Neolithic houses - A Central European perspective. In Darvill T. & J. Thomas (eds), Neolithic Houses in Northwest Europe and Beyond. Oxbow Monographs, Oxford, pp. 27-40. LYMAN L.R. 1984. Bone density and differential survivorship of fossil classes. Journal of Anthropological Archaeology 3: 259-299. LYMAN L.R. 1994. Vertebrate Taphonomy. Cambridge University Press, Cambridge. MARCINIAK A. 2000. Living space. Construction of social complexity in Central European communities. In Richie A. (ed.), Neolithic Orkney and its European Context. McDonald Monographs in Archaeology, Cambridge, pp. 333-346. MILISAUSKAS S. 1986. Early Neolithic Settlement and Society at Olszanica. University of Michigan Press, Ann Arbor. RYDER M.J. 1984. Livestock products: Skins and fleeces. In Mercer R. (ed.), Farming Practice in British Prehistory. Edinburgh University Press, Edinburgh, pp. 182-209. THOMAS J.S. 1996. Time, Culture and Identity. Routledge, London and New York. THOMAS J.S. 1999. An economy of substances in earlier Neolithic Britain. In Robb J. (ed.), Material Symbols: Culture and Economy in Prehistory. Southern Illinois University Press, Carbondale, pp.79-80.

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© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Libbie Hyman and Invertebrate Zoology thePanorama 20th Century 321 TheinNew of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 321-328, 2003

Libbie Hyman and Invertebrate Zoology in the 20th Century J.E. Winston Virginia Museum of Natural History, 1001 Douglas Ave., Martinsville, VA 24112, USA. E-mail: [email protected]

Abstract American zoologist Dr. Libbie Henrietta Hyman, 1888-1969, daughter of immigrant Jewish parents, grew up in Fort Dodge, Iowa, where she acquired an early interest in flowers and wildlife. A high school teacher encouraged her to apply for a scholarship at the University of Chicago. The lack of good manuals for the comparative anatomy and zoology labs she taught as a graduate student led her to develop her own laboratory manuals, published by the University of Chicago Press. After receiving her Ph.D. from Chicago in 1915, she continued to work in the Zoology Department as an assistant to Professor Charles Manning Child. In 1931, with Child nearing retirement, she was finally able to pursue the invertebrate work that interested her most, a treatise on invertebrate zoology. She settled in New York where she became a Research Associate in G. K. Noble’s Department of Experimental Biology at the American Museum of Natural History. This unpaid honorary position supplied her with office space and use of the magnificent AMNH library, and she was able to live frugally on the royalties from her lab manuals and part-time writing and editing jobs. During the summers she traveled to marine labs where she observed and drew living organisms and gathered information about various invertebrate groups from her colleagues; the rest of the year she devoted to literature research and writing. Her 6 volume treatise, The Invertebrates, was published between 1940 and 1967 by McGraw-Hill, and for many years set the standard for invertebrate zoology courses and other books on the subject. Her work was influential for its thorough literature review, uniformity of approach, comprehensive illustration, and thoughtful synthesis of phylogenetic relationships for each group covered.

Family and Childhood Libbie Henrietta Hyman (Fig.1) was born on December 6, 1888, in Des Moines, Iowa, the youngest child, and only daughter of Sabina and Joseph Hyman, Jewish immigrants to the U. S. Her father, Joseph Hyman, came from the town of Konin (now in Poland). After a few years spent learning tailoring in London, he moved to the United States where he and a friend started a successful clothing store in Des Moines. Her mother,

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Fig. 1. Invertebrate zoologist Libbie Henrietta Hyman shown at various stages of her life: baby, schoolgirl, young woman, and elderly woman (Photographs from archives of the Department of Invertebrates, American Museum of Natural History).

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Sabina Neumann, was born in the town of Stettin (Germany). She met and married Joseph, 20 years her senior, in Des Moines. Libbie’s father later went into business on his own, moving the family first to Sioux Falls, South Dakota, then to Fort Dodge, Iowa, where Libbie and her brothers Arthur and Samuel spent their childhood years (Winston 1999b). In her short autobiography (Hyman and Hutchinson 1991) Libbie describes her father as a better scholar than businessman, providing a home full of books, but constantly worried about family finances. Her mother was unaffectionate and traditional, her insistence on housekeeping training for Libbie instilled in the girl a lifelong dislike of housework. What young Libbie preferred was to roam the fields and woods around Fort Dodge, learning about local plant and animal life. Her scientific awakening came with the discovery of the meaning of plant families, when, trying to identify wild flowers with the help of a copy of Gray’s botany belonging to one of her brothers, she realized that the blossoms of two different flowers, unalike in size and color, had the same structure. But, after graduating as valedictorian of the class of 1905 at Fort Dodge High School, her options seemed limited. She was working in a local factory, pasting labels on oatmeal boxes, when a chance encounter with her high school German teacher, Mary Crawford, made her aware of the tuition scholarships the new University of Chicago had established for deserving mid-western students. With her teacher’s help she obtained one of these scholarships, and, in the autumn of 1906, moved to Chicago, where she worked her way through four undergraduate years at the University. Her early desire to major in botany was discouraged by antisemitism in that department, but she was welcome in the Zoology Department (Winston 1999b). After graduating in 1910, she went on to do her graduate work in zoology under Professor Charles Manning Child. Dr. Child was a protégé of the University of Chicago Zoology Department’s first chairman, Charles Otis Whitman, who had himself been especially recruited by U. C.’s brilliant president, William Rainey Harper, as part of his plan to build a great research university. Child and Whitman shared a similar academic heritage, both had received their doctorates from the University of Leipzig, Whitman in 1884 and Child in 1894. As department chairman Whitman stressed the importance of morphology and physiology through whole organism research, as well as cooperative research by department members (Maienschein 1999). Early Career – Chicago Years Libbie Hyman received her Ph. D. in 1915, with a dissertation entitled, “An analysis of the process of regeneration in certain microdrilous oligochaetes.” She then stayed with the department for another 16 years as Child’s assistant. This position, although lacking in prestige, gave her the opportunity to carry out and publish a large amount of original experimental research, and to participate fully in what Jane Maienschein has termed the “Chicago Style” of biology which emphasized the study of whole organisms and populations, physiology and its relation to organism structure, and cooperative and comparative study. Child had begun his career studying the regulation of development, but about the time Libbie Hyman started her graduate work he was developing his “gradient concept”, the idea that an anteroposterior or axial gradient

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characterizes an organism’s basic functioning. Libbie Hyman’s own work complemented that of Child, taking his basic concept and applying it to different organisms and different physiological processes. Her work focused on physiological studies, especially of planaria, including experimental studies of regeneration, feeding, starvation, and oxygen consumption. According to Jane Maienschein, however, Hyman carefully presented her research in ways that made the experiments and their results more important than their role in the gradient theory, whereas Child’s papers always emphasized his theory. Libbie’s papers clearly placed her work in context, explaining how her evidence supported or questioned existing ideas, carefully described her experiments, basically treating them as extended observations, and remained ever attentive to the facts of morphology, systematics, natural history and physiology (Maienschein 1991). Her care in writing up her experimental work may have stemmed from her experience as a writer of laboratory manuals. As a graduate teaching assistant she had been asked to develop laboratory manuals for the Zoology Department’s laboratories in zoology and vertebrate anatomy. At that time the department was switching the emphasis of its students’ laboratory work from a typological approach to the comparative method, but no good comparative manuals were available. The mimeographed notes Libbie produced were so well done and useful to Chicago students that the University Press decided to publish them for general sale. A Laboratory Manual for Elementary Zoology appeared in June1919, and Comparative Vertebrate Anatomy in 1922 (Wake 1999, Winston1999b). Later Career – The Invertebrates Child and Hyman were a productive team, producing 3-4 papers per year. However, by 1931, Child was department chairman and almost retirement age. Libbie was also ready for a change. After her father’s death in 1907, Libbie’s family had moved to Chicago, and for many years she had supported and kept house for mother and brothers. Her mother’s death in 1930, plus Libbie’s increasing financial independence thanks to the royalties from her laboratory manuals, made it possible for her to quit her job in order to take on the project she wanted most to complete – a treatise on invertebrate zoology. She left Chicago in 1931 and after touring Europe for several months settled in New York City where she contacted Gladwyn Kingsley Noble, founder and head of the new Department of Experimental Biology at the American Museum of Natural History. Noble, who had met Libbie in Chicago, responded with an offer of space in his department. Eventually he arranged her appointment as an AMNH Research Associate, an honorary unpaid position that provided her with office space and library privileges. She was able to get on with her treatise, working at the AMNH during the winter months, and, in spring and summer, traveling to various marine laboratories, as well as doing field work in streams and parks in the New York area. For example, the summer of 1935 found her at the Bermuda Biological Station, where Libbie, an excellent swimmer, was even able to dive using a surface air supplied helmet apparatus. In1936, she visited Hopkins Marine Station, and her California friends Ralph and Mildred Buchsbaum, Ed Ricketts, and A. E. Galigher. In 1937 she traveled to Mount Desert Island, Maine and in 1938 to the University of Washington’s Friday Harbor Laboratories. Letters to her friend

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Martin Burkenroad show that she greatly enjoyed her marine excursions, but found them hard on her health, a bungled sinus operation in early adulthood had left her with severe sinus problems, and she also suffered from a digestive ailment (Schram 1993, Winston 1999b). The first volume of The Invertebrates, Protozoa to Ctenophora, was published by McGrawHill in 1940. Although it was not labeled as volume 1 of a series, it is clear from its preface that Libbie had realized that it would take several volumes to accomplish her goal of covering all the invertebrate groups. She ended her preface modestly, “Whether I shall proceed with this treatise will depend upon the reception accorded the present volume. I ask charity for its imperfections in view of the labor involved.”(Hyman 1940, p. vi). She need not have worried; the books had an immediate positive reception from invertebrate zoologists, and in 1941, the University of Chicago awarded her the first of her honorary degrees. At this time she was also able to achieve something she had long wanted for herself, a house with a garden. She bought a house in Millwood, New York, where she could spend her free time gardening and landscaping its grounds. The two hour commute required her to change her work schedule, taking the train into New York City to the Museum for only four extended work days each week, but her Millwood years were nevertheless extremely productive. After volume 1 was published she did reluctantly take time off from treatise work to revise her comparative anatomy manual (Wake 1999). Once this was accomplished she returned to production of The Invertebrates. Volumes 2, Platyhelminthes and Rhynchocoela, and 3, Acanthocephala, Aschelminthes, and Entoprocta were both published in 1951. In 1952, at 64, feeling increasing pressure to get on with her work more rapidly, she gave up her house, and returned to the city, to an apartment hotel not far from the AMNH. Volume 4 of The Invertebrates, The Echinodermata, appeared in 1954, and Volume 5, Smaller Coelomate Groups in 1959. Her method of manuscript production was simple. Using the resources of the AMNH library she thoroughly surveyed the literature for each group, noting the important points on index cards. She then typed the manuscript directly on her Oliver typewriter, making no preliminary written draft. She produced most of the illustrations herself, working either from the literature or live specimens (Winston 1999b). Once back in the city, Libbie lived an active life, going to concerts with friends from the AMNH, library, developing a personal art collection, and attending scientific meetings. Her happiest times were still probably those she spent at marine laboratories. She taught in the invertebrate courses at the Marine Biological Laboratory at Woods Hole, Mass. from the 1940s to the early 1950s. Those who attempted to visit her in New York often found her abrupt, or even rude, impatient with interruptions to her work. Those who met her in the marine laboratory saw a different person, one who delighted in living invertebrates. She did take time to help many students, even some of secondary school age, and to offer both scientific advice and financial support to graduate students she considered serious scientists. Honors had begun to come her way, including membership in the National Academy of Sciences in 1951. The Eliot Medal, 1951, and the Gold Medal of the Linnaean Society, 1961. Finally, academic job offers came as well, but she claimed that she had no interest in accepting them (Winston 1999b).

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In 1956, on a visit to Ernst and Eveline Marcus in São Paulo, to gather information for volume 5, she started showing the symptoms of Parkinson’s disease. The disease’s progression put an end to her travels, and she spent the last years of her life in a wheelchair, with nursing attendance. Volume 6, Mollusca I, published in 1967 marked the end of The Invertebrates, “I now retire from the field, satisfied that I have accomplished my original purpose, to stimulate the study of invertebrates.” (Hyman 1967, p. v). Libbie Hyman died at age 81, on August 3,1969. Contribution to 20th Century Invertebrate Zoology Libbie Hyman’s contribution to 20th century invertebrate zoology was six-fold: 1) Her first contribution came in graduate school with her development of a manual for the elementary zoology laboratories at the University of Chicago (Hyman 1919). This manual went through a second edition (1926), and was reprinted by the University of Chicago Press up through the 1940s, instructing and influencing generations of zoology students. 2) Her second contribution was the body of experimental work she produced during her 16 years as Child’s assistant in the Zoology Department at Chicago. 3) Her third contribution was her work on land planarian taxonomy. According to Robert Ogren (1999) her work “placed land planarian taxonomy on a solid basis” by including the necessary histology of body and copulatory apparatus in her descriptions. Libbie Hyman described 27 new species and two new genera of land planarians. Perhaps even more importantly, she served as an authority and promoter of work on land planarians, encouraging young workers to study and publish in the field. 4) She also produced a large body of work on other marine and freshwater Turbellaria, over 60 papers in all. Seth Tyler (1999) analyzed recent developments in flatworm systematics in the light of the phylogenetic system she presented in the treatise. His conclusion: “by no means has her system been supplanted by newer cladistic or any other systems, rather it has been incrementally improved with discovery of new characters of ultrastructure and molecular biology and with application of cladistic paradigms for defining evolutionary relationships as precisely as possible.” 5) Her most important contribution was undoubtedly The Invertebrates. The books’ abundant illustrations, and uniform format, incorporating historical background, characteristics, classification, morphology, physiology, development, ecology, and phylogenetic considerations for each group, ensured their immediate and enduring value to zoologists (Morse 1999). Linguistically challenged North American zoologists in particular appreciated her work for its summary and interpretation of the European zoological literature. But zoologists world-wide, even those who could read French or German, also relied on her interpretation of that literature, which she had available in the fine AMNH library, but which, in the days before modern photocopying and interlibrary loan service, was inaccessible to many zoologists. 6) Finally, she contributed both directly and indirectly to the training of a new generation in ways beyond her published works. As Trish Morse (1999) has pointed out, her visibility, because of her treatise, her position (even though unpaid) at a major museum, and her teaching in the Marine Biological Laboratory summer courses, made

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her a role model to young women interested in invertebrate zoology at a time when there were few women professors around. As early as 1943 she was biographically portrayed in a book intended to interest girls in careers in science, American Women of Science, by Edna Yost (1943). Libbie Hyman faithfully responded to queries from students — the scribbled postcards with her responses to questions are still prized by their recipients. In addition, according to those who knew her well, she even supported some deserving students financially. We will never know exactly why Libbie Hyman chose to devote her career to The Invertebrates. The golden age of comparative zoology, the transformation of morphology by evolutionism, began in the 1860s in Germany, and was over by the end of the 19th century (Bowler 1996, Nyhart 1995). But when a discipline is no longer cutting edge is when it becomes incorporated into textbooks. Libbie Hyman’s zoological education at Chicago under the direction of Child and Whitman, both educated at Leipzig, places her firmly in that great comparative tradition. From that background, plus her graduate student success in transforming Chicago’s laboratory manuals to reflect the comparative method, might have come the germ of her great treatise. Her opus bridges the gap between the 1870s and the 1970s, carrying the tradition of functional and evolutionary morphology into the renascent evolutionary developmental and systematic studies of the second half of the twentieth century. The Invertebrates is now out-dated; its references half a century or more old. New technologies of scanning and transmission electron microscopy, as well as molecular genetics, have created a huge new body of information to absorbed. Probably no one person could now attempt to accomplish a complete review of all the invertebrates (note the 15 multi-authored volumes just for Microscopic Anatomy of Invertebrates). But, as we create 21st century zoology, let us hope we can do so with the enthusiasm, intelligence, and tenacity personified by Libbie Henrietta Hyman. Acknowledgments Much presented here results from a symposium: Libbie Henrietta Hyman: Life and Contributions, organized by Rachel Fink and I for the1991 meeting of the American Society of Zoologists, and later published as an AMNH Novitate (Winston 1999a). Thanks to all who shared their reminiscences of and correspondence with Libbie Hyman both before and after that event. References Bowler P.J. 1996. Life’s Splendid Drama. University of Chicago Press, Chicago, 525p. Hyman L.H. 1919. A Laboratory Manual for Elementary Zoology. University of Chicago Press, Chicago, 149p. Hyman L.H. 1922. A Laboratory Manual for Comparative Vertebrate Anatomy. University of Chicago Press, Chicago, 380p. Hyman L.H. 1940. The Invertebrates. Vol. 1. Protozoa through Ctenophora. McGraw-Hill, New York, 726p. Hyman L.H.1951a. The Invertebrates. Vol. 2. Platyhelminthes and Rhynchocoela. McGraw-Hill, New York, 550p.

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Hyman L.H.1951b. The Invertebrates. Vol. 3. Acanthocephala, Aschelminthes, and Entoprocta. McGrawHill, New York, 572p. Hyman L.H.1955. The Invertebrates. Vol. 4. The Echinodermata. McGraw-Hill, New York, 763p. Hyman L.H. 1959. The Invertebrates. Vol. 5. Smaller Coelomate Grups. McGraw-Hill, New York, 783p. Hyman L.H. 1967. The Invertebrates. Vol. 6. Mollusca I. McGraw-Hill, New York, 792p. Hyman L.H. & G.E. Hutchinson 1991. Libbie Henrietta Hyman, December 6, 1888—August 3, 1969. Biogr. Mem. Nat. Acad. 60: 103-114. Maienschein J. 1999. Libbie Hyman at the University of Chicago. Amer. Mus. Novit. No. 3277: 25-32. Morse M.P. 1999. Libbie Henrietta Hyman: her influence on teaching and research in invertebrate zoology. Amer. Mus. Novit. No. 3277: 48-52. Nyhart L.H. 1995. Biology Takes Form: Animal Morphology and the German Universities 1800-1900. University of Chicago Press, Chicago, 414p. Ogren R.E. 1999. Contributions of Libbie H. Hyman to knowledge of land planarians: relating personal experiences (Tricladida: Terricola). Amer. Mus. Novit. No. 3277: 39-47. Schram F.R. 1993. A correspondence between Martin Burkenroad and Libbie Hyman: or, whatever did happen to Libbie Hyman’s lingerie. In Truesdale F. (ed.), History of Carcinology. A. A. Balkema, Rotterdam, pp.119—142 Tyler S. 1999. Systematics of the flatworms—Libbie Hyman’s influence on current views. Amer. Mus. Novit. No. 3277: 52-66. Wake M.H. 1999. Libbie Hyman and comparative vertebrate anatomy. Amer. Mus. Novit. No. 3277: 39. Winston J.E. (ed) 1999a. Libbie Henrietta Hyman: Life and Contributions. Amer. Mus. Novit. No. 3277: 1-66. Winston J.E. 1999b. Libbie Hyman and the American Museum of Natural History. Amer. Mus. Novit. No. 3277: 12-32. Yost E. 1943. American Women of Science. Frederick A. Stokes Company, New York, 231p.

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Konrad Lorenz, Niko Tinbergen, and the founding of ethology as aThe scientific discipline 329 New Panorama of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 329-336, 2003

Konrad Lorenz, Niko Tinbergen, and the founding of ethology as a scientific discipline R.W. Burkhardt Department of History, University of Illinois at Urbana-Champaign, USA

Abstract The establishment of ethology as a scientific discipline in the 20th century was in significant measure the work of the Austrian biologist Konrad Lorenz and the Dutch biologist Niko Tinbergen. Lorenz is properly credited with having been the bold, intuitive theorist who laid the field’s initial conceptual foundations. Tinbergen contributed experimental and analytical talents that were an invaluable complement to Lorenz’s early theory-building, and he was also after the Second World War the more important of the two when it came to guiding ethology’s continued development. The different methodologies of the animal-raiser and the field naturalist shaped Lorenz’s and Tinbergen’s respective perceptions of their field. Since the 1960s, Tinbergen’s definition of ethology as the biological study of behavior and his enumeration of the “four questions of ethology” have continued to provide an organizing vision for students of animal behavior.

Among the more conspicuous achievements within the broad domain of zoology in the twentieth century was the establishment of ethology, the biological study of behavior, as a legitimate scientific enterprise. When the century began, few zoologists concerned themselves seriously with behavior. By the mid-1960s, specialists in animal behavior were regarded as essential components of any major zoology department. Prominent among the factors that led to this change were the discipline-building efforts of two scientists in particular – the Austrian zoologist Konrad Lorenz (1903-1989) and the Dutchturned British zoologist Niko Tinbergen (1907-1988). They reached the height of their fame in 1973 when they shared with Karl von Frisch the Nobel Prize for Medicine or Physiology for their respective contributions to the study of animal behavior. The complementary scientific talents and practices of Lorenz and Tinbergen There is a common understanding of the respective roles that Lorenz and Tinbergen played in the construction of ethology as a new science. Briefly stated, Lorenz was the bold, intuitive theorist who laid the broad conceptual foundations of ethology in the 1930s. Tinbergen, for his part, was the critical analyst who helped Lorenz clarify his

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ideas, who figured out how to subject ethological theories to experimental verification, and who thereby made ethology into a respectable science. This formulation is useful as far as it goes. It needs to supplemented, however, by attention to the continued development of ethology in the period after 1940, when Tinbergen himself made important theoretical contributions to the field (e.g, in his concepts of displacement activities and the hierarchical organization of different major instinct systems). Emphasis also needs to be placed on an important difference in the scientific practices that informed the work of the two men. As Lorenz liked to put it, he in his own research practices was essentially a farmer, while Tinbergen was essentially a hunter. To be more specific, Lorenz liked raising and breeding animals, nurturing them when they were ill, and having them as companions. Tinbergen preferred stalking animals in the field, matching wits with them, and discovering how the details of their behavior contributed to their survival. If one asks where Lorenz was most “at home” as a researcher, the answer, literally, was at his own home, that is, at the research station he built at his father’s home in Altenberg, Austria. There, with jackdaws nesting in the attic and geese and other birds moving relatively freely in and about the house and surrounding aviaries, he made the vast majority of the observations on which his theories were based. Tinbergen, in contrast, was most at home as a scientific researcher when he was out in the field. Although he also conducted important laboratory studies, it was as a field naturalist that he felt most complete as a researcher and as a person. The differences in their respective practices ultimately led the two men to make significantly different contributions to the biology of behavior (Burkhardt 1999). Lorenz’s early work Lorenz’s practices as a biologist were continuous with those he developed in his youth as an animal lover. He maintained indeed that no one who was not an animal lover could ever have the patience to watch animals long enough to make useful observations on their behavior. To this love of animals he added an appreciation for the comparative method, which he learned as a medical student at the University of Vienna from the comparative anatomist Ferdinand Hochstetter (Nisbet 1976, Lorenz 1985, Wuketits 1990). With Hochstetter’s encouragement, Lorenz concluded that the comparative method could be applied to animal behavior patterns just as effectively as it could be applied to animal structures. In other words, behavior patterns could be used just like organs to reconstruct phylogenies. Lorenz soon discovered that he was not the first to have come to this idea. Charles Otis Whitman in the United States and Oskar Heinroth in Germany had already based researches of their own upon it. Lorenz would later call this idea “the Archimedean point” from which modern animal behavior studies developed (Lorenz 1978). But Lorenz’s early studies did much more than give him clues about the evolutionary history of the animals he studied. They also gave him the opportunity to think about the physiological causation of instincts. In a series of seminal papers on instincts in birds (1931, 1932, 1935), he began setting forth a conceptual framework for analyzing the causes of instinctive behavior. The major elements of this theoretical system included inborn motor patterns, innate releasing mechanisms, and external releasing stimuli. He also called special attention to the phenomenon of imprinting. This work quickly established Lorenz’s reputation within

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German ornithological circles. Soon he had opportunities to address wider audiences. In 1936 he was invited to lecture on instinct at Harnackhaus, the cultural center of the Kaiser Wilhelm Gesellschaft, in Berlin. Discussions with the physiologist Erich von Holst immediately after his Harnackhaus lecture led Lorenz to develop the idea of endogenously generated action specific energies. This idea of energies or fluids that build up within the organism until they ultimately require release was later represented in Lorenz’s famous psycho-hydraulic model of instinctive action (Lorenz 1950). Tinbergen’s early work Lorenz and Tinbergen first met in November 1936 at a symposium on instinct held at the University of Leiden. Lorenz at this time was a thirty-three year old Austrian zoologist whose star was rapidly rising in the German-speaking scientific world but who still lacked a paid academic position. Tinbergen was a twenty-nine year old Dutch zoologist who occupied the modest position of Assistant in the Zoology Department of the University of Leiden. The common interest of the two young scientists was the study of animal behavior, in particular the study of animal instincts. They imagined themselves establishing a physiologically-oriented, objectivistic science of behavior distinct from the more psychological and subjectivistic approaches pursued by the older speakers at the Leiden conference (Roëll 1996, Burkhardt 1997). Tinbergen, like Lorenz, had been an ardent naturalist as a youth. His formation as a naturalist, however, was appreciably different from Lorenz’s. A strong and special tradition of outdoor, nature studies flourished in Holland in the early twentieth century, and Tinbergen became part of it. In doing so, he came to love fieldwork. In contrast, academic and museum-based zoology, such as studies of comparative anatomy, left him cold. Fortunately, he was allowed to write his doctoral thesis on a field study: the orientation behavior of the bee wolf, Philanthus triangulum. He modeled this research in part on the work of Karl von Frisch. Appointed as an assistant in the zoology department at Leiden, he established a program of teaching and research there that featured both field and laboratory studies of behavior. Beginning in 1935 this included a special sixweek “practical” in the spring for third-year undergraduates. It was in this course that he and his students conducted research on the reproductive behavior of the three-spined stickleback. In the summers Tinbergen took students to a field camp and directed their researches on insects and birds (Tinbergen 1985, Roëll 1996). Tinbergen thus did not need Lorenz to introduce him to the study of animal behavior. Nevertheless, Lorenz’s influence upon Tinbergen was profound. From Tinbergen’s perspective, Lorenz was bringing order out of chaos, providing animal behavior studies with a powerful theoretical framework and focus, and reforming animal psychology by making biological questions central to the enterprise. Tinbergen found all this highly inspiring. Lorenz in turn was excited by the results Tinbergen reported to him on the stickleback work that Tinbergen had just completed with his student, Joost ter Pelkwijk. They had used “dummies” to determine the different sign stimuli to which the sticklebacks responded in fighting, courting, spawning, and so forth. Lorenz was greatly impressed by what he heard. Tinbergen arranged to take a research leave the following spring (1937) to go work with Lorenz in Altenberg. They spent an invigorating three

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and a half months together, during which time they established a life-long friendship. It was there that they conducted their classic experiments on the egg-rolling behavior of the grey-lag goose (Lorenz & Tinbergen 1938). They also experimented on how young birds react to simulated predators. War-time experiences and post-war recovery Tinbergen and Lorenz stayed in scientific contact with one another as the political situation in Europe worsened, and they remained in touch even after Germany invaded Holland. That contact was broken, however, in 1942, when Tinbergen was incarcerated in a German prisoner of war camp for having resisted the Nazification of Leiden University. He remained a prisoner for two years. After the war, his experiences during the occupation made him reluctant to resume relations with German scientists immediately. He felt particularly ambivalent about Lorenz because he was aware that his former friend and colleague had expressed pro-Nazi sentiments in several wartime papers. Then came the news that Lorenz was missing on the eastern front and presumed dead. Tinbergen lamented the loss of the man whom he regarded as the leader of his field. As it eventually turned out, however, Lorenz was not dead. He had been captured by the Russians and was in a Russian prisoner of war camp. But he was not able to return to Austria until 1948. By that time Tinbergen had put wartime feelings aside for the sake of rebuilding ethology as an international science, and he and Lorenz successfully resumed their scientific relations and their friendship. Tinbergen was arguably the more important of the two men when it came to furthering ethology’s development in the postwar period. He founded the journal Behaviour, which began publication in 1947. In 1949 he left Leiden (and a professorship of experimental biology that had been specially created for him) to take up a lectureship at Oxford. There he developed a major center for animal behavior studies and promoted ethology to the English-speaking world. 1951 saw the publication of his book, The Study of Instinct, the first systematic, booklength treatment of ethology as a whole. On through the 1950s and 1960s, Tinbergen was the individual who worked hardest for the field’s coordinated growth. Promoting ethology as “the biological study of behavior,” he insisted that for the discipline to thrive it needed to address simultaneously the questions of physiological causation, individual development, evolutionary history, and function. His classic statement of the “four questions” of ethology appeared in his paper, “On aims and methods of ethology” (Tinbergen 1963). Before the war Tinbergen’s own work had focussed above all on issues of behavioral causation (Tinbergen 1942). After he arrived in Oxford he began to pay more attention to questions of behavioral evolution and function (Burkhardt 1983). His comparative and experimental studies of the survival value of behavior patterns played a significant role in the development of modern behavioral ecology. Like Lorenz, Tinbergen used not only popular writings but also films to communicate his work more broadly. Styles of practice and specific authority claims The lasting differences between Tinbergen’s and Lorenz’s contributions to ethology reflected their different styles of science practice. Lorenz based his claims about being

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able to speak authoritatively about behavior on his particular methods of animal rearing and observation. As Lorenz saw it, the scientific raiser of animals had special advantages over other practitioners in terms of what his daily activities, maintained over long periods of time, allowed him to witness (Lorenz 1941). The comparative psychologist, Lorenz explained, tended to use animals that were either so domesticated or so frightened by the conditions in which they were placed that they never displayed the full range of natural instinctive behavior patterns of their species. The museum taxonomist was at a different sort of disadvantage. Because the specimens he worked with were not living, he did not have behavior patterns to aid him, along with morphological patterns when it came to reconstructing phylogenies. As for the field biologist, Lorenz said, his work was not well suited to comparative behavior analysis because it did not lend itself to watching closely related species of animal side by side on a day to day basis over extended periods of time. The place to study animal behavior, Lorenz confidently concluded, was in a zoo or in a special facility like his own at Altenberg – or later in the research facilities that the Max Planck Gesellschaft established for him in the 1950s, first at Buldern in Westphalia and then more permanently at Seewiesen near Starnberg in Bavaria. Tinbergen, in contrast, always remained a field naturalist at heart, even when he was having considerable success studying sticklebacks in aquaria. This field orientation allowed him to bring to ethology an ecological dimension that Lorenz’s work basically lacked. Shortly after moving to Oxford, and partly in response to prodding from Lorenz, Tinbergen decided he should take up comparative studies similar to Lorenz’s studies of ducks and geese. But Tinbergen was not just interested in identifying homologies. He wanted to see how behavior had evolved in particular ecological settings. Working with his graduate and post-doctoral students, he began doing comparative studies of both gulls and sticklebacks. The fieldwork on gulls proved especially illuminating. Already a specialist himself on the behavior of the herring gull, he put his students to work on related birds, most notably the black-headed gull and the kittiwake. Esther Cullen, conducting an intensive study of the kittiwake, identified in this species a whole host of behavioral peculiarities that appeared to be “corollaries” to the way the species had gained protection from predators by breeding on the narrow ledges of steep cliffs. Among these distinctive behavioral features were specific releasers (such as special head-turning movements that served to “appease” conspecifics), specialized fighting movements, distinctive nest-building behavior, the non-removal of egg shells from the nest site, and more (Cullen 1957). Tinbergen promoted these findings as an excellent illustration of the way that selection for change in one character could have repercussions on other characters as well. Tinbergen and field studies Through the 1950s Tinbergen’s work expanded beyond its earlier emphasis on physiological causation. He studied the evolution and ritualization of signals and then the ways that the behavioral repertoires of different species reflect their adaptation to different selective pressures. He eventually concluded that experimental field studies of behavioral function were what appealed to him most. He was especially pleased by the results generated by a study of egg-shell removal in the black-headed gull. In his

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words (1962: 114): “. . . the picture that emerges is one of great complexity and beautiful adaptedness. It has further become clear that at least some of the different means of defense are not fully compatible with each other, and that the total system has the character of a compromise between various, in part directly conflicting, demands.” What was safest for the parents, he explained, was not necessarily safest for the brood. What helped protect against one kind of predator did not necessarily work against other kinds of predators. A few years later, criticizing V. C. Wynne-Edwards’ idea of group selection, Tinbergen concluded: “If our field studies have convinced me of one thing, it is of the fact that the imagination of even the best field biologist falls far short of the reality; what has been found so far about the nature of selection pressures ought to make us realize how little we know of their true nature and of their variety.” He went on to say: “’field craft’, atrophied alarmingly even among biologists, is in urgent need of redevelopment” (1967: 56-57). Through his field studies of adaptations and selection pressures, Tinbergen helped stimulate the development of modern behavioral ecology. In the meantime, he continued to maintain that for ethology to thrive it needed to develop simultaneously on all four of biology’s main fronts, that is, it needed to attend to questions of physiological causation, individual development, evolutionary history, and function. While recognizing the importance of laboratory studies, and continuing to direct lab studies as part of the program of researches at Oxford, he urged that it was only in field studies that the four questions of biology could be pursued together. Lorenz and Tinbergen and the continuing development of ethology Lorenz made his greatest conceptual contributions to ethology in the 1930s. Aside from his later idea of the “innate schoolmarm” or “innate disposition to learning”, most of the views he promulgated in the postwar period were essentially restatements of his earlier ideas. To many scientists of the postwar generation, his notions of “innate drives”, “action-specific energies”, and the like seemed to have outlived their usefulness. Lorenz remained, nonetheless, a charismatic leader who continued to attract talented new recruits to the field. He was always a major presence at the international ethological congresses. Through lectures and popular writings, he also exerted a considerable influence on thinkers in neighboring fields. Tinbergen continued to be one of Lorenz’s greatest defenders, even if he recognized flaws in aspects of Lorenz’s theorizing and various polemical writings. Indeed the two men remained warm friends to the end of their lives. Paired together, the farmer and the hunter, the broad, intuitive theory-builder and the more painstaking analyst and experimenter, served as the chief architects of ethology’s establishment as a scientific discipline in the twentieth century. Back in the 1930s, it had been Lorenz who lectured to the German society of animal psychologists on the importance of bringing biological questions to bear on the study of animal behavior (Lorenz 1937). In the 1950s and 1960s it was above all Tinbergen who carried this campaign further. He promoted the simultaneous treatment of the four questions of causation, development, function, and evolution as the surest way to ensure ethology’s long-term vitality. In the face of increasing specialization in all areas of biology, this continues to provide a major, organizing vision for students of animal behavior. It constitutes one of the major guiding visions of twentieth-century zoology.

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References BURKHARDT R.W. 1983. The development of an evolutionary ethology. In Bendall D.S. (ed.), Evolution from Molecules to Men. Cambridge University Press, Cambridge, pp. 431-444. BURKHARDT R.W. 1997. The founders of ethology and the problem of animal subjective experience. In Dol M. et al. (eds), Animal Consciousness and Animal Ethics: Perspectives from the Netherlands. Van Gorcum, Assen, pp. 1-13. BURKHARDT R.W. 1999. Ethology, natural history, the life sciences, and the problem of place. Journal of the History of Biology 32: 489-508. CULLEN E. 1957. Adaptations in the kittiwake to cliff-nesting. The Ibis 99: 275-302. LORENZ K.Z. 1931. Beiträge zur Ethologie sozialer Corviden. Journal für Ornithologie 79: 67-127. LORENZ K.Z. 1932. Betrachtungen über das Erkennen der arteigenen Triebhandlungen der Vögel. Journal für Ornithologie 80: 50-98. LORENZ K.Z. 1935. Der Kumpan in der Umwelt des Vogels: der Artegenosse als auslösendes Moment sozialer Verhaltungsweisen. Journal für Ornithologie 83: 137-213, 289-413. LORENZ K.Z. 1937. Biologische Fragestellung in der Tierpsychologie. Zeitschrift für Tierpsychologie 1: 24-32. LORENZ K.Z. 1941. Vergleichende Bewegungsstudien an Anatiden. Journal für Ornithologie 89. Ergänzungsband 3: 194-293. LORENZ K.Z. 1950. The comparative method in studying innate behaviour patterns. Symposia of the Society for Experimental Biology 4: 221-268. LORENZ K.Z. 1978. Vergleichende Verhaltensforschung. Grundlagen der Ethologie. Springer-Verlag, New York and Vienna. 307 pp. LORENZ K.Z. 1985. My family and other animals. In Dewsbury D. A. (ed.), Leaders in the Study of Animal Behavior. Autobiographical Perspectives. Bucknell University Press, Lewisburg, and Associated University Presses, London and Toronton, pp. 259-297. LORENZ K.Z. & N. TINBERGEN 1938. Taxis und Instinkthandlung in der Eirollbewegung der Graugans. Zeitschrift für Tierpsychologie 2: 1-29. NISBETT A. 1976. Konrad Lorenz. Harcourt Brace Jovanovich, New York and London, 240p. ROËLL R. 1996. De Wereld van Instinct, Niko Tinbergen en het Ontstaan van de Ethologie in Nederland (1920-1950). Erasmus Publishing, Rotterdam. English translation, 2000: The World of Instinct. Niko Tinbergen and the Rise of Ethology in the Netherlands (1920-1950). Van Gorcum, Assen, The Netherlands. TINBERGEN N. 1942. An objectivistic study of the innate behaviour of animals. Bibliotheca Biotheoretica 1: 39-98. TINBERGEN N. 1951. The Study of Instinct. Clarendon Press, Oxford, 228 pp. TINBERGEN N. 1963. On aims and methods of ethology. Zeitschrift für Tierpsychologie 20: 410-433. TINBERGEN N. 1967. Adaptive features of the black-headed gull, Larus ridibundus L. Proc. XIV Int. Orn. Congr. Blackwell Science Publications, Oxford and Edinburgh, pp. 43-59. TINBERGEN N. 1985. Watching and wondering. In Dewsbury D.A. (ed.), Leaders in the Study of Animal Behavior. Autobiographical Perspectives. Bucknell University Press, Lewisburg, and Associated University Presses, London and Toronto, pp. 431-463.

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TINBERGEN N., BROEKHUYSEN G.J., FEEKES F., HOUGHTON J.C.W., KRUUK H. & E. SZULC 1962. Egg shell removal by the black-headed gull, Larus ridibundus L.: a behaviour component of camouflage. Behaviour 19: 74-117. WUKETITS F. M. 1990. Konrad Lorenz. Leben und Werk eines großen Naturforschers. Piper, München and Zurich, 286p.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

From scientific natural history to ecosystem The research:changing ... Evolution 337 New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 337-344, 2003

From scientific natural history to ecosystem research: changing roles of the animal in the history of animal ecology Kurt Jax Department of Landscape Ecology, Technische Universität München-Weihenstephan, D-85350 Freising, Germany and Interdepartmental Center for Ethics in the Sciences and Humanities, Universität Tübingen, Germany. E-mail: [email protected]*

Abstract During the history of animal ecology, the perspective on the animal has changed gradually from a very concrete perception of specific species or even individuals to an increasing abstraction of these entities as merely functional elements of ecosystems. This thesis is demonstrated by focusing on three key figures of animal ecology which represent both different approaches to the perception of the animal and at the same time initiated or influenced decisive new theoretical and methodological directions in animal ecology: Karl Semper, Charles S. Elton and George E. Hutchinson. For Semper, the focus was on the specific animals as a means to apply and test Darwinian theory. The influential early work of Elton marks an important transition point for animal ecology, with the community concept as an organizing scheme for the whole discipline. At the middle of the century, Hutchinson, by his own work and through that of his students (in particular R. Lindeman and H.T. Odum), introduced the biogeochemical approach into ecology, emphasizing the roles of animals as components of the flow of energy and matter. The consequences of these different approaches for the perception of the individual animal are analyzed with respect to theoretical/methodological consequences, not the least in the light of conservation issues.

Introduction The 20th century covers not just part of the history of animal ecology but almost the whole of it, as ecology as a self-conscious science originated not before the 1880s and 1890s. In this paper I follow the fate and the role of the individual animal during this * Current address: UFE Center for Environmental Research Leipzig-Halle, Interdisciplinary Department of Conservation Biology and Natural Resources, Permoserstr. 15, D-04318 Leipzig, Germany

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history and illustrate this with three key figures of this specialty, namely Karl Semper, Charles Elton and George Evelyn Hutchinson. They represent both different approaches to the perception of the animal and at the same time initiated or influenced decisive new theoretical and methodological directions in animal ecology. The thesis which is developed here is that there has been an gradual change of emphasis, from a very concrete perception of specific species or even individuals to an increasing abstraction of these entities as merely functional elements of ecosystems. I will then discuss these developments with respect to the gains and losses which they have brought to ecological methodology and to issues of biological conservation. Karl Semper: Animal ecology and evolutionary theory In 1866 Ernst Haeckel introduced the word “ecology” (German: Oekologie) in the course of his voluminous work about the General Morphology of Organisms. In attempting to structure the whole realm of biology, he defined ecology as a branch of physiology, namely that part of physiology, which does not deal with the inner physiology of the organism but with its “outer physiology”. That is, ecology deals with the relations of the organism to its outside world, or, as he also expressed it, to all its “conditions of existence” (Haeckel 1866: vol.2: 286). Haeckel himself, however, made no further contributions to this subject area. The first extensive treatment on animal ecology was instead published by another German zoologist, namely by Karl Semper (1832-1893) in Würzburg. Semper was a morphologist whose research program was centered around Darwin’s theory of evolution to which he adhered. He was convinced that it was necessary to collect more empirical evidence for Darwin’s theory saying that “it had already enough been philosophized by the Darwinists and the task would now come into its right to verify the hypotheses thus gained by means of exact investigations” (Semper 1880: Vol.1: v; translation K.J.). This comment was also a sideswipe at the speculative thinking of Haeckel. In consequence Semper thus tried to combine the historical and comparative approach of evolutionary theory and morphology with the causal and even experimental research of the contemporary physiological biology, both in embryology, and – to a lesser degree – with respect to ecology (Nyhart 1995: 177ff.). As early as 1868 he postulated that it was necessary to investigate “the influence of temperature, light, heat, humidity, nutrition, etc. on the living animal” and to find “ecological laws” (Semper 1868: 228f., quoted after Nyhart 1995: 179 & 309). The culmination of this branch of his studies was a lecture series which he held in 1877 at the Lowell-Institute near Boston and which subsequently was published in 1880 (German edition) and 1881 (English edition) respectively. This book, entitled “Animal life as affected by the natural conditions of existence” (German title: “Die natürlichen Existenzbedingungen der Thiere”) was the first book on animal ecology. With Semper’s emphasis on explaining the morphological adaptations of animals to their environment – in itself a means to give evidence to the causal mechanisms of evolution – it comes as no wonder that Semper’s book is essentially autecological, dealing with the individual organism (or the species which it represents). Although his treatment is – rightly – credited with being the first to point to food chains and trophic pyramids, concepts which were later formalized by Shelford and Elton, this was only done in passing, namely in dealing

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with food as one of the influences of the “inanimate” (sic!) environment1. We can thus say, that for Semper ecology served as a tool to explain morphology and evolution. If we look the other way, with the focus on ecology itself, we might justifiably say that here Darwinian evolutionary theory served as the structuring idea for ecological research. Charles Elton: The community as an organizing principle for animal ecology The approach to explain the distribution of animals as a consequence of their physiology was also at the core of Victor Shelford’s research. Shelford, however, although he explicitly referred to Semper in his own definition of ecology (Shelford 1913: 1), was skeptical of any morphological as well as evolutionary considerations and subscribed to an actualistic fit between the physiology of animals and their distribution. But it soon turned out that the use of this approach to explain the occurrence of different animal species in particular locations fell short of the expectations placed in it. It thus was not Victor Shelford but the Oxford zoologist Charles Elton (1901-1991) who developed a new structuring notion for the study of animal ecology2. Unsatisfied with the results of the physiological approach of Shelford, which Elton tried to apply in his early studies of animal communities at Spitsbergen, he centered his studies around the (animal) community as the central integrative concept, which he – in contrast to the early Shelford – saw basically as an interactive unit characterized and formed through the biotic interactions of the organisms. In his famous textbook “Animal Ecology” published in 1927 and written in only 85 days (Cox 1979: 78f.), he developed a set of four “organizing principles” which helped to structure theory and research. These principles were the food chain/food web, food size relations, the pyramid of numbers, and the ecological niche. None of these principles was really an original idea of Elton; all of them had been alluded to by former authors like Semper, Shelford or – as to the niche – Joseph Grinnell. However, it was Elton’s merit to sharpen them and to organize them into a framework that could be used to structure theory and empirical research in a way that allowed to connect diverse data sets. While these concepts were rather marginal to the earlier authors and were more used to illustrate particular circumstances, they became core ideas for Elton. In looking back from today they in fact successfully served the aims he had designed them for: as guiding principles for research. “Animal Ecology” was a tremendous success that influenced many ecologists and was – reprinted almost unchanged – in stock up to the 1980s. Elton perceived of ecology as “scientific natural history” (Elton 1927: 1), a notion to which Semper would surely have agreed. However, the book introduced in fact a new approach to animal ecology and a new perspective on the individual animal. On the one hand, the community served to understand the individual animal (and for Elton also the animal population3) and what it

1 Semper justified this with the argument that food was developing its impact on the animal only when it was finally dead matter (Semper 1880: vol.1: 46f.). 2 For more details on the biography and work of Elton see Cox (1979), Crowcroft (1991), Jax (2001). 3 The population was also a unit to which Elton devoted much of his work. It did, however, never gain the status of a unit around which ecology as a whole could be organized.

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was doing in nature, giving strong emphasis to interactions with organisms of other species. On the other hand, the community itself became a focus and an object of the study. Elton’s concept of the niche, for example, was one of the role of an animal in a community, not just the requirements or the performance of an animal as in the later niche definition of Hutchinson (1957). Elton compared the niche to the occupation of a species. This was a significant change in perspective, at least as to animal ecology and its theory. As a step aside, it must be mentioned that the idea of the community had been popular in plant ecology long since and that also aquatic ecologists, as Karl August Möbius (1877), Stephen Forbes (1887), or August Thienemann (1918) had already developed and applied such ideas. The latter, however, understood the community (or the biocoenosis) much more ontologically, sometimes even as “organism of higher order”. Eltons approach was more pragmatic and he always was conscious that the concept was a tool4. During the 1920’s, the emphasis of animal ecology thus in general both broadened as well as it shifted towards ecological units beyond the individual animal. Within these units, the individuals were increasingly viewed with respect to their functional roles for the whole of the community, especially as the first classifications beyond that of the biological species were – although tentatively – developed, for which Elton’s niches or Thienemann’s distinction between producers, consumers, and decomposers (Thienemann 1926) are important examples. George Evelyn Hutchinson: from the community to the ecosystem While the individual animal (species) was in most cases still a visible actor in the larger unit of the animal community, a further and radical abstraction occurred with the rise of modern ecosystem theory. The term “ecosystem” had been first coined by the British botanist Arthur Tansley in 1935, although ideas of a unit composed of both organisms and their abiotic environment had been proposed by other authors before him (see Jax 1998 for more details about this and the following). One of the important innovations in Tansley’s concept was that he considered the ecosystem to be a system “in the sense of physics”, (Tansley 1935: 299) in which abiotic and biotic elements were given equal rank. This was a radical departure from the community concept, although Tansley himself never took the necessary steps to operationalize this notion in his research. Nor did others at first. Tansley’s idea lay dormant for almost 7 years, until it was put into a research program. The key figure for this development was again a zoologist, namely George Evelyn Hutchinson5 (1903-1991). It is fair to say that it was not

4 Möbius‘ concept of the biocoenosis (German: Biozönose), which was used at first mostly in zoology, remained largely restricted to the German-speaking countries, where it was, however, a very influential concept either. It seems however, that the more specific meaning of the concept, which included properties like self-regulation and a „biocoenotic equilibrium”, led the idea to be less productive than the broad and – sometimes even vague – community concept which Elton put forward and which often represented more a perspective than a concrete unit (Jax 2002). 5 For details on Hutchinson’s life and work see e.g. Edmondson (1971, 1993), Hutchinson (1979), Schwarz & Schwoerbel (2001).

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Hutchinson himself who implemented the first ecosystem research in the sense in which it would become popular later. But looking from today, Hutchinson was almost like a strong lens, which collected different lines of knowledge and research that had been lying separate until then, transformed them into something new and sent them out as new rays of research through the work of his many students. These students and the generations they educated again (see Kohn 1971) where as influential as they were diverse in their research, among them another extremely influential (community-) animal ecologist, namely Robert H. MacArthur. As to the ecosystem concept, Hutchinson first of all was mentor of Raymond Laurel Lindeman, who was the first to apply Tansley’s ecosystem concept as a research approach (Lindeman 1942). The idea to supplement the classical food-web approach of earlier authors with a perspective of the flow of energy and the energy content of different compartments, can be traced back to the works of Chancey Juday and to the direct influence of Hutchinson. The most radical formulation of an ecosystem approach was developed by Hutchinson’s PhD-student Howard T. Odum (see Taylor 1988). Using the ideas of biogeochemistry, as developed by the Russian scientist Vladimir Vernadsky, which Hutchinson had adopted for western ecology (Hutchinson 1948), Odum perceived of the ecosystem as a mere system of flows of matter and especially energy. In this kind of (eco)systems organisms are aggregated into functional compartments, that is, abstracted with respect to their role in the transformation of energy and matter (see e.g. Odum 1983), so that Odum sometimes even referred to them as “ecocatalysts” (Taylor 1988: 226). Hutchinson thus stands at the crossroads towards a development of ecology in which the abstraction of the organism reaches its culmination, in which it becomes almost completely invisible and seems to be substitutable by numbers of biomass and its energetic contents. Conclusions and questions We can draw several conclusions from this brief tour through the history of animal ecology. Firstly, the realm of animal ecology was strongly broadened trough the 20th century. Starting almost exclusively as autecology during the period of Semper, the domain of objects became supplemented by the study of communities and populations in the work of Elton and his contemporaries. By the mid-century, under the influence not the least of Hutchinson, the different threads of ecology became more and more merged into a more general ecology, transcending also the classical boundaries of animal and plant ecology, aquatic and terrestrial ecology. At the same time, the ecosystem became a new focus of research. These periods in animal ecology were also dominated by different major research goals and organizing ideas, for which the three scientists stand as important key figures (Table 1). This was evolutionary theory and in particular adaptation for Semper, the interaction patterns of animals for Elton (who also was still interested in evolutionary aspects), and finally the flow of energy and matter for Hutchinson and his successors. The importance of the individual species decreased in this order, and from being the focus for Semper almost turned into invisibility in the work of some systems ecologists. Of course, this a rather rough sketch of animal ecology and its history. Today all of the different levels of research are carried out: investigations on the physiological and

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Table 1. Unifying ideas and focus units in animal ecology.

Organizing principle Semper Elton Hutchinson

Focus unit

Ad ap tation

Ind ivid u al

Biotic interactions

Com m u nity

Flow of energy and m atter

Ecosystem

behavioral ecology of animals as well as research on animal populations, animal communities, and ecosystems. But the shift in emphasis is there and brings up some important questions, namely: what are the gains and losses of abstracting from individual species? Is the new emphasis on “biodiversity” a revival of organismic ecology, i.e. ecology focused on the individual and the species? And what, if anything, might integrate the different specialties of animal ecology and of ecology as a whole? What we have gained by shifting our views towards an abstraction from the specific species is a reduction of the great complexity of ecological phenomena and the ability to answer questions about particular of these phenomena. The description of a lifecontaining segment of space as an ecosystem under the perspective of the flows of energy and matter occurring there can register as gains that it allows, for example, to make statements about how changes in the human use of an area will affect the productive potential in this place. On the side of the losses, however, we find that the fate of particular species during such changes becomes inaccessible, but it includes also an abandonment of the potentials to derive basic methodological criteria for an understanding of the whole system via the properties of the organisms (see also Jax 1996). Today, there is an increasing demand expressed by ecologists to find schemes that integrate the different approaches, as recent publications show (see especially Allen et al. 1992, Jones & Lawton 1995). In the past there have been several attempts to find a unifying “currency” for ecology, the most prominent of which were “energy” or “information”. H.T. Odum, for example, tried to explain almost everything, up to human societies and religions, in terms of the “hard” currency of energy (see e.g. Odum 1971). The problem is, however, that in fact this perspective is not sufficient to explain and predict many phenomena. For many phenomena, the specific characteristics of the species involved, like generation times or particular behaviors, matter very much. The same is valid outside the realm of pure science, in conservation; people not only value energy flow or systems, regardless of the specific species which compose them. Species and individuals matter to them more than just “functions” – which does not deny that the sustainable conservation of resource and an environment suited for human health and wellbeing is also important. To value “ecosystems” as ends in itself is also problematic both from an scientific and an ethical perspective (Cahen 1988, Shrader-Frechette & McCoy 1994). The new emphasis on research and conservation of a biodiversity in it very breadth is surely also a reaction to the feeling that species matter. The new biodiversity debate was from its onset a political one and not primarily a scientific (Takacs 1996). But a new look at individuals and species can also aid a search for the question of the integrating element of ecology. If we look for a unifying currency it should not be energy or some other physical variable, but the organism and its properties. This is not a new

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idea, but still one which has not been developed to its end. Ecology depends on the individual organism. All phenomena with which ecology deals are either brought about by organisms or at least are viewed in their consequences to organisms. Ecology is thus by necessity a biological science. Moreover MacMahon et al. (1978) have shown already more than two decades ago that the organism is the only unit which connects several possible hierarchies: a hierarchy based on the exchange of matter and energy, a phylogenetic hierarchy, a coevolutionary hierarchy and a physiological-anatomical one. To view and compare also communities and even ecosystems from the perspectives of the organism involved can bring new insights both for the theory and application of ecology (Jax 1996). To develop such theoretical approaches, which transcend beyond the boundaries of the now classical subdivisions of ecological research, as they have slowly developed during the 20th century, is a challenging task for the new century. The diversification and extension of the domain of ecological studies was important; it is now the time to integrate them again. This task might bring us full circle again to the roots of animal ecology, at least with respect to the importance of the animal in animal ecology. References ALLEN T.F.H. & T.W. HOEKSTRA 1992. Toward a Unified Ecology. Columbia University Press, New York. 384 p. CAHEN H. 1988. Against the moral considerability of ecosystems. Environ. Ethics 10: 195-216. COX D.L. 1979. Charles Elton and the Emergence of Modern Ecology. PhD. Thesis, Washington University, Washington. 232p. CROWCROFT P. 1991. Elton’s ecologists. A history of the Bureau of Animal Population. Chicago University Press, Chicago. 177p. EDMONDSON W.T. 1993. Eulogy for G. Evelyn Hutchinson. Verh. Int. Ver. Limnol. 25: 49-55. EDMONDSON Y. 1971. Some components of the Hutchinson legend. Limnol. Oceanogr. 16: 157-172. ELTON C. 1927. Animal Ecology. Sidgwick & Jackson, London. 207p. FORBES S.A. 1887. The lake as a microcosm. Bull. Peoria Sci. Assoc. 1887: 77-87. reprinted 1925 in: Illinois Nat. Hist Surv. Bull. 15: 537-550. HAECKEL E. 1866. Generelle Morphologie der Organismen. Georg Reimer, Berlin. Vol. 2. 604p. HUTCHINSON G.E. 1948. Circular causal systems in ecology. Ann. New York Acad. Sci. 50: 221-246. HUTCHINSON G.E. 1957. Concluding remarks. Cold Spring Harbor Symp. Quant. Biol. 22: 415-427. HUTCHINSON G.E. 1979. The Kindly Fruits of the Earth. Recollections of an Embryo Ecologist. Yale University Press, New Haven & London. 264p. JAX K. 1996. Über die Leblosigkeit ökologischer Systeme. Gedanken zur Rolle des Individuellen Organismus in der Ökologie. In Ingensiep H.-W. & R. Hoppe-Sailer (eds), NaturStücke. Edition Tertium, Ostfildern, pp 209-230. JAX K. 1998. Holocoen and ecosystem. On the origin and historical consequences of two concepts. J. Hist. Biol. 31: 113-142. JAX K. 2002. Die Einheiten der Ökologie. Analyse, Methodenentwicklung und Anwendung in Ökologie und Naturschutz. Peter Lang Verlag, Frankfurt/Main. 249p.

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JAX K. (2001). Charles Sutherland Elton. In Jahn I. & M. Schmitt (eds), Darwin & Co. Eine Geschichte der Biologie in Portraits. Vol. 2. Beck, München. pp. 233-250, 533-534. JONES C.G. & J.H. LAWTON 1995. Linking species and ecosystems. Chapman & Hall, New York, 387p. KOHN A.J. 1971. Phylogeny and biogeography of HUTCHINSONIA: G.E. Hutchinson’s influence through his doctoral students. Limnol. Oceanogr. 16: 173-176. LINDEMAN R.L. 1942. The trophic-dynamic aspect of ecology. Ecology 23: 399-417. MACMAHON J.A., PHILLIPS D.L., ROBINSON J.V. & D.J. SCHIMPF 1978. Levels of biological organization: an organism-centered approach. BioScience 28: 700-704. MÖBIUS K.A. 1877. Die Auster und die Austernwirtschaft. Wiegandt, Hempel & Parey, Berlin. 126p. NYHART L.K. 1995. Biology Takes Form. Animal Morphology and the German Universities, 18001900. University of Chicago Press, Chicago. 414p. ODUM H.T. 1971. Environment, Power and Society. Wiley Interscience, New York. 331p. ODUM H.T. 1983. Systems Ecology. An introduction. Wiley, New York. 644p. SCHWARZ A. & J. SCHWOERBEL (2001). George Evelyn Hutchinson. In Jahn I. & M. Schmitt (eds), Darwin & Co. Eine Geschichte der Biologie in Portraits. Beck, München. SHRADER-FRECHETTE K.S. & E.D. MCCOY 1994. How the tail wags the dog: how value judgements determine ecological science. Environmental Values 3: 107-120. SEMPER K. 1868. Reisen im Archipel der Phillipinen, Zweiter Theil. Wissenschaftliche Resultate. Vol. 1, Holothurien. W. Engelmann, Leipzig. SEMPER K. 1880. Die natürlichen Existenzbedingungen der Thiere. Brockhaus, Leipzig. 2vols. SHELFORD V.E. 1913. Animal Communities in Temperate America. University of Chicago Press, Chicago. TAKACS D. 1996. The Idea of Biodiversity. Philosophies of Paradise. John Hopkins University Press, Baltimore, London. TANSLEY A.G. 1935. The use and abuse of vegetational concepts and terms. Ecology 16: 284-307. TAYLOR P.J. 1988. Technocratic optimism, H.T. Odum, and the partial transformation of ecological metaphor after World War II. J. Hist. Biol. 21: 213-244. THIENEMANN A. 1918. Lebensgemeinschaft und Lebensraum. Naturwiss. Wochenschrift, NF 17: 281-303. THIENEMANN A. 1926. Der Nahrungskreislauf im Wasser. Verh. Dtsch. Zool. Ges. 31. Jahresversammlung Kiel 25.-27.5.1926: 29-79.

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) HommageThe à Pierre-Paul Grassé New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 345-350, 2003

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Hommage à Pierre-Paul Grassé M. Delsol1, Ch. Noirot2, J. Génermont3 & J.-L. d’Hondt4 1. Laboratoire de Biologie animale, Faculté catholique de Lyon, 24, rue du Plat, 69288 Lyon Cedex 2, France 2. UMR 5548 : Développement - communication chimique, Université de Bourgogne, 6, boulevard Gabriel, 21000 Dijon, France 3. UPRES-A 8079 : Écologie, systématique et évolution, Institut de biologie animale intégrative et cellulaire, bâtiment 446, Université de Paris-Sud, 91405 Orsay Cedex, France 4. Laboratoire de Biologie des invertébrés marins et Malacologie, Muséum national d’histoire naturelle, 57, rue Cuvier, 75231 Paris Cedex 05, France

Pierre-Paul Grassé, ce maître de la zoologie au vingtième siècle, méritait absolument qu’un hommage lui soit rendu lors du présent congrès qui rassemble pour la première fois depuis 1963 des zoologistes du monde entier. Nous tenons, au nom des zoologistes français, à remercier les organisateurs d’avoir pensé à inclure un tel hommage dans le programme. Le rôle majeur joué par P.-P. Grassé dans les progrès des sciences naturelles tient aussi bien à l’importance de ses propres découvertes qu’à l’impact de ses idées, fruit de son immense érudition et de son exceptionnel niveau d’appréhension de l’univers zoologique dans toute sa complexité. Il paraît impossible qu’un naturaliste puisse avoir de la zoologie une connaissance plus vaste et plus profonde que la sienne. Les étapes de la carrière de P.-P. Grassé Pierre-Paul Grassé naquit à Périgueux le 27 novembre 1895 et c’est dans son Périgord natal qu’il mourut le 9 juillet 1985. Des conversations que ses élèves ont eues avec lui, il ressort qu’il fut dans sa jeunesse fortement influencé par la vaste culture et la soif de comprendre la nature de ce monde d’un grand-père sculpteur qui vivait de son art. Il entreprit simultanément, à Bordeaux, des études de médecine et de sciences naturelles. La médecine reposait alors principalement sur un éventail de connaissances assemblées depuis des millénaires par des hommes souvent remarquables, mais elle n’était qu’à peine pénétrée par la biologie. C’est pourquoi il la délaissa au profit des sciences naturelles pour lesquelles il nourrissait une véritable passion. Ainsi devint-il, comme il aimait à le dire, un « naturaliste ». Il suivit à cette époque les cours et fréquenta le laboratoire de Jean de Feytaud dont les travaux étaient en partie consacrés aux termites. Après une interruption due à la première guerre mondiale, il fréquente à Paris le laboratoire d’Étienne Rabaud. Venu à Montpellier, il y acquiert une solide formation en

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cytologie grâce à un séjour de trois ans dans le laboratoire d’Octave Duboscq. À la retraite de celui-ci, il vient s’initier auprès d’Eugène Bataillon à la biologie du développement, s’intéressant notamment à la parthénogenèse expérimentale et aux phénomènes de néoténie. Il se familiarise en outre avec l’éthologie grâce à François Picard. Il est chargé de cours en 1923 et soutient sa thèse en 1926. Il crée en 1928 le laboratoire d’entomologie de l’école d’agriculture de Montpellier. De cette époque date son intérêt pour l’entomologie agricole et pour l’éthologie des insectes sociaux, ainsi que pour l’évolution animale. Il publie quelques notes sur les orthoptères du Périgord, sa région d’origine à laquelle il est très attaché. Il y fondera du reste quelques années plus tard une station de terrain dans une localité déjà reconnue comme un haut-lieu de la préhistoire, Les Eyzies. Dès 1929, grâce à la qualité de sa thèse et à son dynamisme, il accède à la chaire de zoologie de la faculté des sciences de Clermont-Ferrand, succédant à un spécialiste du développement des bryozoaires, Louis Calvet. En 1937, il est nommé maître de conférences à Paris où il devient en 1940 titulaire de la prestigieuse chaire, aujoud’hui malheureusement disparue, d’évolution des êtres organisés, succédant à Maurice Caullery. Entre autres distinctions, il reçoit en 1935 le prix Gadeau de Kerville de la société zoologique de France et en 1940 le prix Cuvier de l’académie des sciences. Il est président de la société zoologique de France en 1939, président de la société entomologique de France en 1941. Il entre à l’académie des sciences, dont il deviendra plus tard président, en 1948. Sur son épée d’académicien, il fait graver sa devise « connaître », résumant ainsi, selon la biographe Rachelle Laurand, sa passion et l’objectif de toute sa vie. Il n’est pas question d’énumérer ici toutes les responsabilités qu’il eut l’occasion d’exercer, notamment au centre national de la recherche scientifique et dans les instances universitaires, ni tous les titres qui l’honorèrent. Mentionnons seulement qu’il dirigea cinq périodiques scientifiques, dont « Annales des sciences naturelles » et « Insectes sociaux », qu’il fut docteur honoris causa de sept universités étrangères, membre de nombreuses associations scientifiques, membre honoraire de plusieurs académies. La liste de ses publications ne comporte pas moins de 414 titres. Ses travaux ont d’abord porté sur la protistologie, la parasitologie, la cytologie. Il comprit cependant dès les années 1930 que la cytologie serait prochainement renouvelée par l’emploi en biologie du microscope électronique récemment découvert. C’est une des raisons pour lesquelles il orienta dès lors ses recherches sur les insectes sociaux. Il ne revint à la cytologie qu’après 1950, exploitant les possibilités offertes par la microscopie électronique. Il devait du reste créer le service commun de microscopie électronique de l’université de Paris. Le Zoologiste Nous présentons ici un résumé très succinct des apports de P.-P. Grassé à différents domaines de la zoologie, tant par des recherches originales que par des travaux de synthèse et de diffusion des connaissances.

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Protistologie Dès le début de sa carrière, il s’intéressa aux êtres unicellulaires ou protistes, leur consacrant notamment sa thèse de doctorat ès sciences, « Contribution à l’étude des flagellés parasites », qui porte sur la cytologie et les cycles biologiques de diverses espèces appartenant à des genres aussi différents que Proteromonas, Monocercomonas, Trichomonas, Chilomastix, etc. Ce travail s’est poursuivi par des recherches sur les flagellés symbiotes des termites, principalement des espèces françaises, et par une importante monographie réalisée en collaboration avec Octave Duboscq sur l’appareil parabasal des flagellés de nos jours regroupés au sein de l’ensemble des Parabasalia. Seul ou en collaboration avec des élèves, notamment André Hollande, il a apporté une contribution considérable à la connaissance des flagellés symbiotes des termites, d’abord au moyen de la microscopie photonique, puis en utilisant les ressources de la microscopie électronique, ce qui bouleversa bon nombre d’idées antérieures. Son intérêt pour les parasites le conduisit à se tourner vers les sporozoaires. Il a du reste rédigé personnellement une large part des chapitres consacrés à ce groupe dans son traité de zoologie. Ses travaux de protistologie se sont poursuivis jusqu’en 1978, date à laquelle, à 83 ans, il discuta des caractères originaux des myxosporidies, montrant qu’il s’agissait de pluricellulaires, et non de protistes, ce qu’a depuis lors confirmé la biologie moléculaire, et créant pour ces êtres l’embranchement des myxozoaires. Les insectes sociaux Dans le cadre de ses recherches sur les flagellés, P.-P. Grassé entreprit en 1934 un voyage en Afrique occidentale française pour étudier la microfaune de nouvelles espèces de termites. D’emblée passionné par ces insectes, il en fit un de ses sujets de recherches favoris. À l’issue d’une de ses missions en Afrique, il créa en 1957 un laboratoire à Makokou, sous le nom de mission biologique au Gabon. Son intérêt s’est très vite étendu à l’ensemble des insectes sociaux et au phénomène social en général. Il engagea de nombreux élèves dans des recherches sur ces thèmes. Il fonda en 1951, avec K. Gösswald, l’union internationale pour l’étude des insectes sociaux, puis créa en 1954 la revue « Insectes sociaux ». Ses recherches originales ont essentiellement porté sur les termites. Celles de la période 1945-1962 ont été pour la plupart réalisées en collaboration avec Charles Noirot. La systématique ne constitue qu’un aspect relativement mineur de l’œuvre de P.P. Grassé. Il faut néanmoins souligner la création de la sous-famille Apicotermitinae, car elle repose sur la prise en compte, outre de certains traits du comportement constructeur, de caractères anatomiques du tube digestif qui sont actuellement largement utilisés dans la systématique des termites. Plus importants sont les travaux sur la symbiose. Les flagellés des termites dits inférieurs sont ordinairement éliminés à chaque mue et réacquis par trophallaxie avec l’« aliment proctodéal », bien distinct des excréments. Chez certaines espèces, la mue imaginale fait exception, de telle sorte que les essaimants sont automatiquement pourvus en symbiotes. Les macrotermitinés, termites champignonnistes, édifient des meules à champignons, faites d’excréments d’un type particulier, émis après un transit intestinal

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très bref, qui servent de substrat nutritif à une agaricacée du genre Termitomyces. Des ouvriers consomment la partie inférieure de la meule, tandis que d’autres ajoutent des matérieux frais à la partie supérieure. Le polymorphisme a été étudié par P.-P. Grassé dans le genre Kalotermes qui ne possède pas d’ouvriers vrais. Leur rôle est tenu par des larves âgées dont beaucoup prolongent leur vie larvaire par des mues stationnaires, voire régressives, devenant ainsi des « pseudergates » qui restent aptes à se différencier ultérieurement en imagos. Dans le même genre, des sexués de remplacement, néoténiques, apparaissent grâce à une mue particulière de certaines larves. L’étude plus générale du polymorphisme des termites a été poursuivie par des élèves de P.-P. Grassé. Les recherches sur les nids ont montré qu’un nid est un espace clos, ne s’ouvrant à l’extérieur que de façon très temporaire. Les « cheminées » que comportent certains nids sont en relation avec un réseau de canaux topologiquement extérieur au nid luimême. Les échanges gazeux se font par diffusion au travers de parois poreuses. Dès ses premières recherches, P.-P. Grassé s’est attaché à l’observation précise de la vie des insectes sur le terrain. Il a par la suite insisté sur l’importance des études menées dans des conditions aussi proches que possible des conditions naturelles. C’est dans cet état d’esprit qu’il a lui-même analysé le comportement d’essaimage et surtout le comportement constructeur des termites. De ses propres résultats et de la synthèse d’autres résultats sur ce dernier point est née la théorie de la stigmergie, selon laquelle c’est le travail déjà accompli qui oriente le travail futur. La coordination entre les activités des individus participant à la construction ou à la reconstruction d’un nid est ainsi indirecte, l’activité d’un ouvrier étant focalisée sur les ébauches déjà construites. Ce concept est maintenant utilisé dans les travaux de modélisation mathématique des comportements de groupe. Une autre contribution de P.-P. Grassé à la compréhension générale du phénomène social chez les animaux est le développement de la notion d’effet de groupe, relative aux modifications du comportement, de la physiologie et même du développement qui résultent des échanges de stimulations sensorielles entre individus groupés. L’ouvrage en trois volumes Termitologia (1982-1986) constitue, bien qu’écrit en français, un ouvrage de référence pour les termitologues du monde entier. Sous l’influence d’un tel maître à penser s’est développée une école française sur les insectes sociaux qui reste aujourd’hui très vivante. Le laboratoire d’évolution des êtres organisés que P.-P. Grassé dirigea pendant près de trente ans a été le siège de travaux non seulement sur les termites, mais aussi sur les blattes, les criquets, les fourmis, les guêpes, les halictes, devenant ainsi un puissant moteur pour le développement des recherches sur les sociétés animales. Signalons enfin que P.-P. Grassé fut à l’origine de la création dans l’université française d’un enseignement de psychophysiologie grâce auquel les futurs psychologues et les futurs biologistes s’initient conjointement à l’étude du comportement animal. L’évolution P.-P. Grassé a été durant toute sa carrière un ardent défenseur de l’idée d’évolution, face au scepticisme de divers milieux, y compris parmi les scientifiques. Il est

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incontestable que, par ses conférences, par ses écrits, en particulier par ceux qui étaient destinés à un large public, il a contribué à asseoir cette idée dans les cercles cultivés de notre pays. Cependant sa formation et ses goûts de naturaliste l’ayant tenu relativement éloigné de l’approche génétique des mécanismes de l’évolution, il est resté attaché à une conception néolamarckienne de moins en moins crédible et c’est là le seul bémol qui doit figurer dans le présent hommage. Le traité de zoologie P.-P. Grassé fut auteur ou co-auteur de nombreux ouvrages, le premier étant le « Précis de biologie animale », rédigé en collaboration avec Max Aron, qui fut grâce à plusieurs rééditions le livre de chevet de nombreux étudiants de premier cycle universitaire pendant plus d’un quart de siècle. Son œuvre la plus marquante est néanmoins le « Traité de zoologie » auquel ont collaboré sous sa direction plus de 300 auteurs. C’est son esprit d’universalité qui a conduit P.-P. Grassé à concevoir cet ouvrage encyclopédique. Comme l’a écrit Maurice Durchon, il a souhaité réaliser « la synthèse des connaissances zoologiques de notre temps, œuvre collective à laquelle collaborerait l’élite de nos zoologistes et biologistes, qui montrerait au monde la vitalité de la science française ». Selon Rachelle Laurand, il aurait pensé à un tel travail dès 1938. En 1942, il se rendit à Bruxelles pour s’assurer que ses amis P. Brien, A. Dalcq et P. Girard pourraient l’aider dans son entreprise. Il savait en effet, il l’a écrit dans la préface du premier volume, qu’aucun des précédents traités de zoologie n’était arrivé à son terme. Le projet initial prévoyait dix-sept volumes ou tomes, mais la zoologie a pris depuis 1942, comme toutes les disciplines scientifiques, une ampleur que personne ne soupçonnait. Aussi le traité s’accrut-il d’année en année en conservant son plan d’origine, un tome étant amené à se subdiviser en deux ou plusieurs « fascicules ». Se voyant vieillir, P.-P. Grassé eut la sagesse de déléguer, de nommer des sous-directeurs afin d’achever son œuvre. Au total, 47 fascicules ont été publiés, avec une seule lacune par rapport au projet initial : le volume consacré aux myriapodes et à de petits groupes d’arachnides n’a pas vu le jour. Autres lacunes, mais celles-ci n’apparaissent qu’à la lumière de progrès récents, des groupes de métazoaires tels que les placozoaires, les cycliophores, les myzostomides, les vestimentifères, etc., ne figurent pas dans le traité ou y sont trop sommairement traités, et il en est de même chez les unicellulaires. Malgré cela, ce qu’a réussi P.-P. Grassé est sans commune mesure avec ce que ses prédécesseurs avaient pu réaliser. Au-delà de la Zoologie Il est indispensable d’ajouter que P.-P. Grassé était bien plus qu’un zoologiste. Il a publié plusieurs essais de philosophie et a laissé une somme considérable de manuscrits, de notes de toutes sortes sur ses voyages, ses recherches, ses réflexions. Il s’exprimait dans une langue française d’une exceptionnelle qualité littéraire, employant un vocabulaire d’une inhabituelle richesse, dont la conférence prononcée à l’occasion du centième anniversaire de la société zoologique de France constitue une remarquable illustration.

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Et il y avait encore dans ce personnage illustre, qui avait amassé une immense bibliothèque dans son château dominant les rives de la Dordogne, un troisième homme. Il aimait rire, il aimait les bons côtés de la vie. Dans sa jeunesse, il organisait des farces d’étudiants comme on n’en fait plus. Il appréciait le théâtre, la peinture, la poésie, les courses de taureaux. Il aimait la bonne chère, dont le Périgord, son pays, est une des capitales. Il a trouvé le temps d’écrire un livre de cuisine et de gastronomie périgourdines qui prend place au milieu de l’œuvre immense du zoologiste. Il s’était lié d’amitié avec plusieurs des personnalités qui ont marqué notre époque, scientifiques comme Étienne Wolff, Jean Rostand, Germaine Cousin, Jean Piveteau ou André Thomas, ou écrivains tels Jean Fourastié, Jean Dutourd ou André Maurois. Enfin, il aimait ses amis et ses élèves. Ce polémiste éloquent, indépendant et libre, ayant le goût du spectacle et de la formule percutante, doté d’un remarquable sens de l’humour et d’une vivacité d’esprit exceptionnelle, était profondément bienveillant. Il accueillait avec chaleur et simplicité ses jeunes collègues, savait les aider et leur rendre service. Il avait eu la douleur de perdre deux enfants. Sa fille Isabelle garde de lui le souvenir d’un père merveilleux. C’est un tel souvenir que conservent de lui tant ses élèves directs que tous ceux qui ont eu le privilège de bénéficier de ses conseils et de ses qualités humaines, ses fils spirituels. References Laurand (R.) – 1991 – P.P. Grassé: « Un zoologiste se penche sur l’évovution » Mémoire de maîtrise. Université Lyon III, 164 pages.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

The Invisible Subject: Zoology and the Evolutionary Synthesis 351 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 351-358, 2003

The Invisible Subject: Zoology and the Evolutionary Synthesis V.B. Smocovitis Dept. History, University of Florida, Gainesville, FL 32611, USA. E-mail: [email protected]

Abstract Although the subject of botany and the evolutionary synthesis has been explored by historians, the counterpart subject of zoology and the evolutionary synthesis has received little or no significant discussion. Following a historical assessment of the evolutionary synthesis, the paper engages in a comparative study between the contributions of botany and zoology to the synthesis. It then highlights some of the major contributions made by zoologists and directs readers to further areas for historical inquiry. The argument is made that zoology and the synthesis are considered to be so closely synonymous that the two are frequently equated, so much so, that the subject has been rendered invisible.

Introduction The invitation to contribute a paper on the subject of “zoology and the evolutionary synthesis” took me by surprise for at least two reasons. For one thing, although I have studied the wider evolutionary synthesis as a historian, I am presently concentrating more narrowly on the role that botanists have played in twentieth century evolutionary science. I am presently writing a biography of the late George Ledyard Stebbins, one of the pioneers of plant evolutionary biology and the botanical architect of the evolutionary synthesis. For another thing, the subject is so enormous, as I hope I will convince you in this presentation, that it would be impossible to address it properly in a hefty scholarly volume, let alone compress it in any substantive manner for a 20-25 minute presentation. As I reflected on the proposed topic, however, I realized that the subject was so rich that it really deserved an opening discussion in a forum exactly like the International Congress of Zoology. The topic becomes more intriguing still when we realize that only very recently has it even been acknowledged as a topic at all. It has received only the most brief consideration in the form of a personal memoir/recollection in the form of an interview with the German biologist Wolf Herre (Hossfield 1999). Though the brief article is titled “Zoologie und Synthetische Theorie,” it concentrates primarily on the personal reflections of Herre

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concerning the German context of the evolutionary synthesis and does not address zoology and the synthesis directly. Far more revealing of “zoology and the evolutionary synthesis” is the coverage given to it in the most impressive scholarly work on the evolutionary synthesis to date. This is a collection of papers that appeared in 1980 that is edited by Ernst Mayr and William B. Provine and titled The Evolutionary Synthesis. Perspectives on the Unification of Biology. A new printing was recently made available in 1998 with a brief new preface by Mayr. Despite the fact that the volume has a significant essay written by Stebbins on “botany and the evolutionary synthesis,” there is no formal comparable essay on zoology and synthesis; and nowhere in the volume is the subject directly addressed. Although key zoologists who contributed to the synthesis are noted, cited heavily, and their contributions are acknowledged, their common field remains unrecognized. Included here are individuals like Theodosius Dobzhansky, Julian Huxley, Ernst Mayr, Bernhard Rensch and G. G. Simpson among others. Even more revealing is the fact that “Part One” of the book titled, “Different Biological Disciplines and the Synthesis” lists the following disciplines as playing a vital role in the synthesis: genetics, cytology, embryology, systematics, botany, paleontology, and morphology, but once again there is no mention of zoology. More revealing still, is the index to volume. Although there is an entry under “botany,” which sends interested readers to the essay by Stebbins, there is no comparable entry under zoology. It may seem ironic indeed that the topic has never been addressed—almost rendered invisible— given that the history of evolution has been dominated by the presence of Ernst Mayr, one of the great zoologists of the twentieth century and the foremost historians of the evolutionary synthesis. My sense, however, is that it is not an accident that the subject has never really been addressed formally. What I will suggest instead is that the absence of zoology—as a category for historical analysis— in both the Mayr and Provine volume and in our historical consciousness is the result of its close identification with the achievements of the synthesis and with the fact that Ernst Mayr has written the historical account. In his mind, “zoology,” which represents his area of expertise was virtually synonymous with the synthesis. Given the space restrictions and the fact that the topic is enormous, what I hope to do is to first engage in a comparative history between the contributions of botany (which is better known) and zoology to the evolutionary synthesis and then to highlight some of the major features of the relationship of zoology—and zoologists— to the synthesis. I also hope to ask pertinent questions especially about the absence of substantive historical work on the subject that may lead to more detailed scholarly consideration in the future. What was the Evolutionary Synthesis? It might be appropriate to begin with the obvious first question. What exactly was the evolutionary synthesis? The answer to this question has eluded a veritable army of scientists, philosophers and historians of science (Mayr & Provine 1980, Mayr 1982, Mayr 1993, Burian 1988, Gayon 1990, Gayon 1992). Although some of the recent scholarship may have helped us understand some of the social or cultural aspects of the synthesis, it remains a controversial subject (Smocovitis 1992, 1994, 1996, Cain 1993,

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Ruse 1996, Junker & Engels 1999). All scholars agree that it was what William B. Provine (1980) has described as “an intellectual event of first-order magnitude.” First a brief historical background to the understanding of the synthesis. The story of the synthesis begins in the mid-nineteenth century. The fact that the diversity of life is the product of evolution—in Darwin’s own terms descent with modification—was almost universally accepted by biologists soon after the publication of Darwin’s On the Origin of Species in 1859. Although Darwin’s specific explanation—descent with modification by the novel mechanism of natural selection was immediately adopted by colleagues like Joseph Hooker, Asa Gray, E.B. Poulton, August Weismann and of course his co-discoverer Alfred Russel Wallace, it was rejected by most biologists for a number of reasons including the fact that little direct evidence existed. Darwin knew this and spent the remainder of his life completing monograph-studies, many of which were botanical in nature—in support of phenomena like adaptation and natural selection. After 1870, after this work was published, natural selection appeared to gain in popularity only to suffer a demise again by the turn of the century when a number of alternative mechanisms were proposed. Only a handful of individuals were ardent selectionists, and these included Darwin’s codiscoverer, Alfred Russel Wallace. Varied schools of thought arose in support of some of these alternative mechanisms and included belief in saltationism, orthogenesis (directed evolution including nomogenesis), mutation theory, and neo-Lamarckism. So popular were these alternatives to natural selection, that they led to a serious challenge to traditional Darwinian theory. Surveying this period, Julian Huxley in 1942 used the phrase the “Eclipse of Darwinism” to explain the demise of natural selection (Huxley 1942). These divisions were only deepened and rendered more complex at the turn of the century by what Ernst Mayr and historian Garland Allen have repeatedly described as “a widening chasm” between naturalist-systematists or field workers, and the new breed of laboratory -oriented practitioner of experimental biology represented most by the new generation of geneticists (Mayr, 1982; Allen, 1979). These were such vastly different communities—cultures in fact—that, in addition, to using different scientific methods, they also spoke different scientific languages, had different scientific training, and held even to different standards of evidence or what counted as “good” science. Many of the younger geneticists saw the older systematics or natural history as unrigorous, poor or even bad sciences because they did not rely on laboratory experimentation or quantification. There were more than a few celebrated disagreements between biologists as a result of these divisions. How these disagreements shaped the course of evolutionary biology at the turn at the turn of the century was directly addressed by Provine in his 1971 book, The Origins of Theoretical Population Genetics. Various attempts to reach a consensus about the actual or even the dominant mechanism of evolution failed in the 1920s. In the interval of time between approximately 1936-1950, however, the disagreements appeared to diminish, the number of alternative mechanisms appeared to diminish as well, and a seemingly new theory was synthesized that incorporated Darwinian selection theory with Mendelian genetics in a way that accounted for the diversity of life on earth. In the process natural selection emerged as the dominant mechanism of evolution, and the theoretical commitments to the continuity between microevolution and macroevolution (two terms that gained currency during the synthesis) were made.

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The synthesis officially began with the establishment of the mathematical models of the theorists R. A. Fisher, J. B. S Haldane and Sewall Wright who both explored and mathematically demonstrated the efficacy of natural selection in a range of theoretical conditions (Provine 1971, Provine 1986). Their work was followed by the publication of Theodosius Dobzhansky’s Genetics and the Origin of Species in 1937, which was the foundational volume that led to the birth of evolutionary genetics. Dobzhansky had also drawn on his Russian predecessors (like Sergei Chetverikov), who had pioneered populational approaches to evolution in the nineteen-teens and twenties, but who had been either exiled, dispersed or killed during the Stalinist period (Adams 1994). Dobzhansky’s synthetic book was followed by a string of books that successively built on Dobzhansky’s evolutionary framework or amended the framework in some way. In 1942 avian systematist Ernst Mayr wrote Systematics and the Origin of Species. The Viewpoint of a Zoologist; the counterpart to this volume, the viewpoint of a botanist was offered by Edgar Anderson, who did not complete a book-length manuscript. In 1942 Julian Huxley, wrote The Modern Synthesis, which officially announced the arrival of the new synthetic theory; in 1944, vertebrate paleontologist George Gaylord Simpson wrote Tempo and Mode in Evolution, and in 1950 George Ledyard Stebbins completed his enormous Variation and Evolution in Plants. Others were involved including Bernhard Rensch, who wrote Neure Problem der Abstammunglehre in 1947, C. D. Darlington and others. It should be noted here that with the exception of Stebbins and Darlington, the above scientists were zoologists in training. Dobzhansky’s book had great international effect as well, and was instrumental in launching a European synthesis (Junker & Engels 1999). Simultaneous to the publication of these synthetic volumes, there also took place an organizational synthesis that saw the first international society for the study of evolution established in the United States, the Society for the Study of Evolution. Britain was unable to win the bid since it was recovering from heavy war-related damages like the absence of paper (Smocovitis 1994). The society was launched beginning in March 1946 and led to the establishment of the journal Evolution. Many—in fact most— of the organizational leaders were zoologists, including individuals like Alfred Emerson and especially Ernst Mayr, who were both orchestrators and who served as organizational pivots. Of the first seven elected presidents of the SSE, only two were botanists—Stebbins and E. B. Babcock. The others included Simpson, J. T. Patterson, N.D. Newell, and Theodosius Dobzhansky. It therefore seems clear that zoology, and zoologists, led the way in efforts to organize evolutionary study in the United States. The clear dominance of zoology—and zoologists—over botanists during the period of the evolutionary synthesis is easy to document by examination of simple numbers of participants and authors. But it is much harder to understand conceptual advances made by zoologists who contributed to the evolutionary synthesis. Here I will begin to explore this topic by first concentrating on the “rival” science of botany and its contributions, and then offering a comparative approach that highlights zoology. Conceptual Contributions Although much of the early work in genetics was the result of work on botanical study—Mendel himself was a botanist—the tide turned in favor of animal genetics with

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the discovery of that most perfect experimental organism—the dew-loving black stomached fly—Drosophila melanogaster. The success of Drosophila melanogaster as an experimental system in the hands of geneticist Thomas Hunt Morgan—and his school of fly-room geneticists in the first two decades of the twentieth century largely eclipsed comparable efforts to understand Mendelian genetics and later the genetic basis of evolutionary change in plant model organisms (Allen 1975, 1978, 1979, Kohler 1994). We all know Morgan and his organism, but few recall the pioneering work on the genus Crepis—a lowly weed in the Compositae family—through the efforts of Berkeley geneticist E. B. Babcock (Smocovitis 1997). One problem with plants as experimental model organisms was that their genetic systems were far more complex and difficult to understand than in animal systems. One recalls the widespread confusion precipitated by De Vries when he watched what he thought was the sudden appearance of new elementary species in Oenothera lamarckiana, the evening primrose. It took plant geneticists over twenty-some years to determine that Oenothera did not just “throw off such elementary species” but was in fact a permanent translocation heterozygote. Thus overall in genetics—we may say that although plants made notable contributions, animal systems like Drosophila sp. seemed to dominate conceptual advances in genetics that later fed into the synthesis. For similar reasons, that is, the overwhelming complexity of plant genetic systems, evolution in plants tended to support a range of oftentimes contradictory theories. Unlike many zoologists who in fact did reject Neo-Lamarckism like Ernst Mayr, botanists continued to uphold neo-Lamarckian theories well into the 1930s and 1940s. One reason for this is the result of the fact that plants have open or indeterminate developmental systems—such a system made it difficult to separate phenotypic from genotypic variation rendering plant evolution susceptible to neo-Lamarckism. Additional phenomena demonstrated widely in the plant world like frequent hybridization, polyploidy and apomixis—a method of asexual reproduction—and most importantly the interplay between all three made plants less tractable model study organisms. Added on to this was the phenomenon of cytoplasmic inheritance, and the greater plasticity of phenotypic responses in plants. Finding a coherent evolutionary theory for the plant world was thus far more difficult than that in the animal world. All these biological complexities also led increasingly to serious differences of opinion between botanists about proper taxonomic methods. Many taxonomists preferred a strictly Linnaean taxonomic methodology while others entertained a dynamic, ecological view of species. By the 1930’s many botanists were divided into the herbarium worker who preferred the Linnaean scheme and the more ecologically minded field experimentalist or naturalist. One of the pioneers of plant evolution the Swede Göte Turesson pointed these divisions out clearly in the late 1920’s Turesson’s own area of expertise—the study of plant adaptation to varied environments in the science called genecology—was in fact considered a discipline separate from botany! When Berkeley taxonomist Harvey Monroe Hall teamed up with the ecologist Frederic Clements to reform the practice of plant taxonomy into a more ecologically attuned study in their 1923 book, The Phylogenetic Method in Taxonomy, they ended up writing what amounted to a taxonomic manifesto. Both lost considerable status in the eyes of the staunch herbarium workers who continued to uphold the static Linnaean scheme. No such

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divisions existed in zoology since the middle of the nineteenth century when fieldoriented collecting practices were introduced into zoology. By the twentieth century, a populational field-oriented mode of collecting and identifying specimens was routine practice. No doubt, the fact that animals could locomote—move from area to area rather than maintain a stationary existence— may have additionally contributed to a more dynamic view of species and in turn a more dynamic view of speciation. Even simple collecting practices in systematics differed widely between zoologists and botanists. As Ernst Mayr has repeatedly pointed out, it was zoologists who collected specimens and arranged them in terms of series in order to appreciate the full range of variation in species. In zoology, it had been standard practice since the middle of the nineteenth-century to collect population samples or “series” as the museum workers named them. This tradition did not exist in botany. Duplicates in plants were very often samples from the same plant. The botanist who introduced the method of “mass collecting” to botany and who introduced a wholesale reappreciation of patterns of variation and devising methods to measure variation was Edgar Anderson. He had in turn appropriated the method from individuals like his close friends the zoologists Alfred Kinsey and Norman Fassett. It was in fact, the work of zoological systematists who had paved the way for one of the key aspects of the evolutionary synthesis—the switch from typological to populational thinking. The transition from typological or static essentialistic thinking about the natural world to a dynamic variational and populational understanding of the natural world was surely one of the vital components of the synthesis. Though botanists made notable contributions to areas like “biosystematics,” in the 1940s, it was a zoologist, Julian Huxley, who first announced this dynamic, new evolutionary approach to taxonomy in his pivotal and influential book in 1940 titled The New Systematics. The differences were only widened after the mid-1930s when zoologists like Dobzhansky and Mayr laid the groundwork for the biological species concept (BSC), thus recognizing the definition of species strictly in terms of sterility barriers and the existence of other isolating mechanisms. Though it was generally acknowledged by all zoologists, it should be noted that protozoologists like T. M. Sonneborn challenged its general applicability to all animals (Sonneborn, 1957) and maintained that it had limited use mostly to birds and mammals. Even though there was some notable disagreement between zoologists like Mayr and Sonnenborn, zoologists as a whole accepted the BSC. Botanists, however, argued vociferously between themselves and with zoologists about the applicability of the BSC to the frequently hybridizing species of plants. The Invisible Subject We can then highlight a few of the major features of the contributions that zoology made to the evolutionary synthesis: 1. Understanding the mechanism of Mendelian heredity. 2. Laying the foundation for the Biological Species Concept (BSC). 3. Transforming evolution and biology from a static or typological study to a dynamic populational area of study. Related to this was a move towards a view of collecting

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practices and systematic study that stressed variational evolution and that led ultimately to the “new systematics.” There are of course numerous other contributions that we could discuss—such as Simpson’s insights into the tempo and mode of evolution from his observations of vertebrate fossils, or perhaps Mayr’s famous recognition of “founder events,” and their importance in understanding speciation, but I think the above three suit the comparative purposes of this paper since they were not the result of botanical study. Given that zoology—and zoologists are such vital subjects in the evolutionary synthesis, then why has zoology been left out of this history? This is a good question: I suggest it has much to do with the fact that Mayr—both as the historian and zoologist who has been closest to the subject could not imagine that the topic of zoology and synthesis could be separated for historical analysis; they are one and the same. Following a similar logic, Mayr draws a line of demarcation around botany as a category for analysis; this is a conversation that only one outside the category of botany, but firmly inside the category of zoology might understand. Only from the perspective of a zoologist did the category botany exist in the 1940s; hence Mayr’s repeated assertions that botany was very different from all the other biological disciplines and was somehow “delayed” in entering the synthesis (Mayr 1980). In this sense, I might suggest that the subject of zoology and the evolutionary synthesis has been rendered invisible by virtue of its own success; zoologists dominated the synthesis as both a scientific event and a historical event. Additionally as well, Mayr’s sense of the word “zoology” should be given close critical examination and its assumptions lain bare. There are at least two that deserve consideration. First, by zoology, Mayr generally means the zoology of birds and mammals, and not other animal systems like protozoa. Second, by zoology, Mayr generally means the zoological tradition that fed ultimately to the synthetic theory. This was the tradition in systematics that was dominated by his German predecessors like E. Streseman and later B. Rensch. The rival tradition that dominated German zoology after E. Haeckel that concentrated on morphology and phylogeny, what Mayr has characterized as the continental “idealist” morphological tradition, is not included in historical considerations. My overall sense is that it would be a most useful undertaking to explore the general subject of “zoology and the synthesis” in a full historical project. Any such attempt to do so must keep in mind definitional aspects of these two terms and the fact that much of the history of biology has been dominated by the leading historical player, Ernst Mayr. References ADAMS M. (ed.) 1994. The Evolution of Theodosius Dobzhansky. Princeton, Princeton University Press. ALLEN G. 1975. Life Science in the Twentieth Century. Wiley, New York. Rep. ed. Cambridge University Press, Cambridge, 1978. ALLEN G. 1978. Thomas Hunt Morgan: The Man and His Science. Princeton University Press, Princeton.

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ALLEN G. 1979. Naturalists and experimentalists: the genotype and the phenotype. Studies in the History of Biology 3: 179-209. BURIAN D. 1988. Challenges to the Evolutionary Synthesis. Evolutionary Biology 23: 247-288. CAIN J.A. 1993. Common Problems and Cooperative Solutions. Isis 84: 1-25. GAYON J. 1990. Critics and criticisms of the Modern Synthesis: The Viewpoint of a Philosopher. Evolutionary Biology 24: 1-49. GAYON J. 1992. Darwin et l’Après Darwin. Éditions Kimé, Paris. HALL F. & F. CLEMENTS 1923. The Phylogenetic Method in Taxonomy. Carnegie Institution of Washington no. 326. HOSSFIELD U. 1999. Zoologie und Synthetische Theorie: Interview mit Wolf Herre. In Th. Junker & E.-M. Engels (eds), Die Entstehung der Synthetischen Theorie. Beiträge zur Geschichte der Evolutionsbiologie in Deutschland 1930-1950. Verlag für Wissenschaft und Bildung, Berlin, 1999, pp. 241-257. JUNKER TH. & E.-M. ENGELS (eds) 1999. Die Entstehung der Synthetischen Theorie. Beiträge zur Geschichte der Evolutionsbiologie in Deutschland 1930-1950. Verlag für Wissenschaft und Bildung, Berlin. HUXLEY J. (ed.) 1940. The New Systematics. Clarendon Press, Oxford. HUXLEY J. 1942. Evolution: The Modern Synthesis. London, Allen and Unwin. KOHLER R.E. 1994. Lords of the Fly: Drosophila Genetics and the Experimental Life. Chicago University Press, Chicago. MAYR E. 1982. The Growth of Biological Thought. Belknap Press, Cambridge, Mass. MAYR E. 1993. What was the evolutionary synthesis? Trends in Ecology and Evolution 8:31-34. MAYR E. & W.B. PROVINE (eds) 1980. The Evolutionary Synthesis. Perspectives on the Unification of Biology. Harvard University Press, Cambridge, Mass. Reprinted with a new Preface by Ernst Mayr, 1998. PROVINE W.B. 1971. The Origins of Theoretical Population Genetics. University of Chicago Press, Chicago. PROVINE W.B. 1986. Sewall Wright and Evolutionary Biology. University of Chicago Press, Chicago. RUSE M. 1996. Monad to Man. Harvard University Press, Cambridge, Mass. SMOCOVITIS V.B. 1992. Unifying Biology: The Evolutionary Synthesis and Evolutionary Biology. Journal of the History of Biology 25: 1-65. SMOCOVITIS V.B. 1994. Organizing Evolution: Founding the Society for the Study of Evolution (1939-1950). Journal of the History of Biology 27: 241-309. . SMOCOVITIS V.B. 1996. Unifying Biology: The Evolutionary Synthesis and Evolutionary Biology. Princeton, Princeton University Press. SMOCOVITIS V.B. 1997. G. Ledyard Stebbins, Jr. and the Evolutionary Synthesis (1924-1950). American Journal of Botany 84: 1625-1637. SONNEBORN T.M. 1957. Breeding systems, Reproductive Methods, and Species Problems in Protozoa. In E. Mayr (ed.), The Species Problem. American Association for the Advancement of Science, Washington, pp. 155-324.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Our evolving understanding of biodiversity through history and ofitsAnimal ... Evolution 359 The New Panorama Proc. 18th Int. Congr. Zoology, pp. 359-368, 2003

Our evolving understanding of biodiversity through history and its impact on the recognition of higher taxa of Metazoa F.R. Schram Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Mauritskade 57, NL-1092 AD Amsterdam, Netherlands

Abstract Although animal taxonomy began officially with the work of Linnaeus in 1758, the recognition and naming of the animal phyla was largely a phenomenon of the middle and late 19th century. By 1900 we had essentially delineated most of the major phyla. For the next 40 years, there was only one phylum level name added to the roster of animal higher taxa. However, since World War II we have added roughly one new phylum every decade. Similar patterns can be seen at class and subclass levels. This renaissance can be attributed to three things: 1) advances in microscopy and electronic imaging, 2) the interest in exploration of previously ignored habitats such as the deep sea, meio- and groundwater fauna, and 3) the publication of Hyman’s The Invertebrates, instrumental in rekindling an interest in invertebrate studies. Currently, a conceptual breakthrough is occurring. From striving to find “new” higher taxa, an increasing importance is now placed on identifying monophyletic groups. Well-known phyla are being subsumed into other taxa, long established groups are emerging as paraphyletic assemblages, and more comprehensive analyses are hinting at polyphyly of familiar taxa. Biodiversity in the 21st century will have an entirely different meaning than in the past.

Introduction A dictum of epistemology is that we don’t really begin to understand a thing until we put a name to it. So it is with our understanding of animal diversity and in particular how we conceptualize what we refer to as the “higher taxa” of the Metazoa. Although animal taxonomy began “officially” with the work of Linnaeus in 1758, the recognition and naming of the animal phyla was largely a phenomenon that historically unfolded over a long period of time extending even to the end of the 20th century. We’ve all been amazed at the announcement of “new phyla” like the Cycliophora (Funch & Kristensen 1995) or the new class Micrognathozoa (Kristensen & Funch 2000), but this is a process that actually has its roots in Cuvier’s (1812) recognition of his four “embranchments.”

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Fig. 1. Cumulative growth of “phyla” level higher taxa of Metazoa as self-standing concepts by decades from 1758 to the present. Four phases of this growth are recognized: Early Consolidation (1758-1850), Darwinian Era (1850-1900), Total Disinterest (1900-1945), Hyman Era (1945-1995).

This chronicle of recognizing higher taxa of metazoans has not been one of steady pace. In Fig. 1, I chart the historical accumulation of our currently recognized animal “phyla.” Charting this is not as easy as it appears. On the one hand, defining a “Phylum” is a very subjective process; no clear criteria prevail. On the other hand, sometimes the early recognition of a group occurred under a name different from that which we now use, e.g., the Lophophorata (Phoronida, Ectoprocta, Brachiopoda) was known throughout most of the late 19th and early 20th centuries as the Molluscoidea. Even so, the traditionally recognized three groups of lophophorates date as clear concepts from the 1880s. Finally, for varying periods, groups perceived as distinct were still subsumed within larger units. For example, Echiura, long the subject of debate as to its higher taxonomic and phylogenetic affinities, only emerged as a self-standing phylum in 1940, while the group in which it long had been placed, Gephyrea (also including Priapulida and Sipuncula), has totally fallen from use. [Currently, there appear some suggestions that perhaps the Echiura is merely a sub-group of the Annelida.] Consequently, what Fig. 1 charts is the graphic history of “phyla-level” taxa as they came to be recognized as separate distinct units, though not necessarily as “phyla” per se. I believe four phases in this history can be recognized. Early Consolidation (1758-1850) The first phase is a period of early consolidation extending from the Systema Naturae of Linnaeus (1758) to around 1850. This is a period in which biologists were getting acquainted with Nature as an object of scientific study. The time was dominated by simple systems of classification. Linnaeus recognized only 6 “classis”: Mammalia, Aves,

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Amphibia, Pisces, Insecta, and Vermes. Linnaeus clearly had a modern understanding of the Vertebrata, but he mingled all the non-insectan invertebrates under either his Insecta Aptera (including spiders and most crustaceans), or his five types of Vermes. These latter consist of the Intestina (many of the worms), Mollusca (but only some molluscs along with most echinoderms and more worms), Testacea (most of the mollusks, plus serpulids and barnacles), Lithophyta (corals), and Zoophyta (everything else). By the early 1800s, Cuvier (1812) erected four embranchments: Zoophytes or Radiata, Articulés, Mollusques, and Vertébrés, and each of these had four classes. While we today find Cuvier’s quaternary symmetry rather quaint, it was nevertheless a system of classification that found wide acceptance, and even at the end of the century we still see aspects of it reflected in the phylum taxonomy of Zittel (1900). This epoch was an exciting time for zoology. The big question zoologists were attempting to address during the early period of consolidation was, “Just what is the structural plan of … [substitute any name]?” There certainly was an on-going subdivision of the Linnaean classes. However, it was a time when researchers were thinking about the larger scale issues involved in comparing various animal forms. These are more difficult questions than one would think. While “Just what is the plan of a mollusk?” was quickly resolved between the time of Linnaeus and Cuvier, “Just what is the unifying plan of a worm?” was not really clarified until late in the century. Some might argue that the question of “what is a worm” is still not resolved, others still that it is a facetious question altogether. It was, however, a period in which scientists were focusing on trying to describe basic structural plans, and, by engaging in rather deep and sophisticated comparisons of anatomy, arriving at conceptions of structural plans by which major groups could be defined. We might also characterize this first epoch as the “French Era” since the most prominent figures active at the time were people like Buffon, Lamarck, Cuvier, and Geoffroy Saint-Hilaire, figures who moved zoology from an activity of cataloging the “divine plan” to a nascent “evolutionary,” natural historical science directed towards delineating form. Darwinian Era (1850-1900) The second phase I term the Darwinian Era, extending from 1850 to 1900. This was a period of phylogenetic exploration, triggered in large part by the publications of Charles Darwin. It was a time in which zoologists were actively charting the diversity of life within an evolutionary context. Rather than focusing on abstract anatomical plan, scientists through this period increasingly delved into genealogy. Things were named, not so much because they conformed to type, but because they were related in some way to something else. The era found the “cutting edges” of zoology in the fields of Comparative Anatomy and Comparative Embryology. The big question addressed in this period is, “What are the Phyla?” The last half of the 19th century was a time dominated in this regard by German scientists. This is not to denigrate the contributions of other communities of scientists at the time, but names like Haeckel, Gegenbauer, Claus, Grobben, and Hatschek dominated the field. It was a time important for us, since by the close of the 19th century almost all of our modern phyla were formally recognized either as overtly separate taxa, or at least their

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distinct nature implied. Not only did zoology have by the end of the 19th century a “modern” array of phyla, but also the classification of metazoans was then employed to help organize other fields of knowledge. As one example, the classic monograph of Korschelt and Heider (1893), Lehrbuch der vergleichenden Entwicklungsgeschichte der wirbellosen Thiere, employed the phylogeny of Haeckel and the taxonomy of Hatschek to interpret and organize knowledge of the early embryonic patterns of development of animal groups. Total Disinterest (1900-1945) By the early 1900s, this 19th century’s intense interest in cataloging patterns of animal diversity and relating the components to each other began to fade. A phase followed, which I characterize as a period of total disinterest, extending from 1900 through to about 1945. “Total disinterest” may be too strong a characterization; obviously, systematics continued apace. While the pattern of an early 20th century flat plateau in the cumulative numbers of classes and subclasses mirrors that already noted for phyla (Fig. 2a), the cumulating numbers of lower taxonomic categories continued to grow unabated (Fig. 2b). Nevertheless, the first half of the 20th century is a period highlighted not by studies of animal diversity or large-scale genealogical relationships, but by the flowering of genetics and other experimental sciences. It was a period dominated by “the synthesis,” and the building of the Synthetic Theory of Evolution was perceived as a more exciting scientific venture to be involved in rather than charting biodiversity. Having seen the broad patterns of animal evolution laid out in the 19th century, zoologists in the early 20th century became more interested in answering the big question, “What are the processes of evolution?” Hyman Era (1945-1995) With the end of World War II a change can be noted. I term this, the Hyman Era, extending from roughly 1945 to 1995. The publication of Hyman’s series, The Invertebrates, helped rekindle an interest in studying animal diversity, especially among the invertebrates. Hyman, of course, was not the lone cause of this rekindling, for the period also was dominated by an increasing use of technology. New instruments, such as SEM and TEM, as well as more advanced forms of light microscopy, produced an ever more sophisticated and detailed understanding of animal form and diversity. Furthermore, new devices such as deep-sea submersibles and scuba allowed exploration of habitats little studied in previous phases. The accumulation of collections from deep-sea, meiofauna, and cave and ground-water habitats allowed a resumption of growth in the accumulation of new higher taxa. From the 1940s on, we find roughly one new phylum per decade entering the literature, and similar figures for class and subclass taxa. Conceptual Shift in Biodiversity Studies While the details of my graphic analysis might be debated, I think we could agree that one can characterize the 19th and 20th centuries in regards to accumulating knowledge of biodiversity as reflected in numbers of recognized higher taxa as a time of alternating boom and bust cycles. There were periods of slow or no growth, followed by periods of

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Fig. 2. A) Cumulative growth of Class and Subclass of Crustacea by decades from 1758 to the present; phases labeled are the same as those in Fig. 1. B) Cumulative growth of Orders of Crustacea by decades from 1758 to the present.

knowledge explosion. However, all throughout these roughly two and a half centuries there was a focus on describing biodiversity in terms of body plans that characterize higher level animal taxa. Effectively, I perceive in those centuries an obsession with recognizing “the new” — new phyla, new classes, new orders. In other words, zoologists were preoccupied with finding new “boxes” into which they could sort the diversity of life. Irregardless of the boom and bust cycles, irregardless of the new techniques of study, irregardless of little explored habitats made accessible, the entire 237 year period between 1758 to 1995 has been one constrained by what the “first scientists of biodiversity” did: name (Linnaeus), and categorize (Cuvier). However, I think something new and fundamentally different is now, at the opening of the 21st century, afoot. Throughout the 1990s, two new aspects of technology have

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been making inroads to provide us with a new paradigm for biodiversity studies: 1) computer based cladistic analyses, and 2) new sources of data derived from molecule sequencing and developmental genetics. Together, I believe these have brought about a conceptual shift in our science. In the 1990s, we have once again become tree builders. These, however, are not the gnarled oaks of Haeckel, but the precise cladograms of Hennig. Now when we construct trees within the framework of this newest technology, we are not interested in phyla per se. Rather, we are interested in establishing monophyletic groups. Constructing trees is not easy. As the first to publish a comprehensive tree of all phyla (Meglitsch & Schram 1991, Schram, 1991) I can attest to this. Indeed, my last attempt at it (Schram 1997) I no longer believe in. The whole field of metazoan phylogenetics is “up for grabs” with conflicting data and interpretations appearing in the literature constantly. For example, new sources of molecular data and more careful analyses of morphological data are suggesting that our carefully constructed series of coelomate worm phyla may be all wrong, i.e., the phyla Echiura (McHugh 1997) and Pogonophora (Rouse & Fauchald 1995, 1997, Rouse 2001) should probably be subsumed back into the Annelida, and the class Polychaeta is likely a paraphyletic grade rather than a monophyletic clade. Even the status of long established phyla and classes are being called into question (Table 1). Several authors, on the basis of 18S rDNA, have uncovered possible paraphyly of Porifera (Collins 1998). In addition, 18S rDNA as well as ultrastructural anatomy appears to indicate that either Cnidaria are paraphyletic with regard to the enigmatic Myxozoa (Zrzavy et al. 1998), or they are highly derived cnidarians and should assume a position within that phylum. I would suggest that this shift away from naming new higher taxa and towards defining monophyletic groups is a major revolution with profound implications for Table 1. A partial listing of suggested instances of paraphyly among higher taxa of Metazoa. These refer to cases where paraphyly is positively supported by either chiefly molecular or good morphological phylogenetic data.

Taxon Porifera

D ata 18S rRN A, aa seq. PKC

Cnid aria Platyhelm inthes Turbellaria Rotifera Acanthocep hala Cru stacea

18S rDN A 18S rDN A u ltrastructure 18S rDN A 18S rRN A 18S rDN A, RN A pol. II, ef-1 alp ha, m itochond rial genes m orphology (fossil & Recent) 18S rDN A 18S rDN A 18S rDN A 18S rDN A ef-1 alpha

Maxillop od a Brachiopod a Ascid iacea Enterop neusta Annelid a, Polychaeta

References e.g., Cavalier-Sm ith et al. 1996, Kru se et al.. 1998, Collins 1998 Zrzavy et al. 1998 Littlew ood et al. 1999 Ehlers 1985 Garey et al. 1996, 1998 Garcia-Varela et al. 2000 García-Machad o et al. 1999, Shultz & Regier 2000 Schram & H of 1998, Spears & Abele 1997 Cohen 2000 Sw alla et al. 2000 Cam eron et al. 2000 McH u gh 1997, Kojim a 1998

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future studies of biodiversity. We are watching many “primitive” higher taxa, groups that are characterized more by plesiomorphies rather than with apomorphies, being reinterpreted as actually paraphyletic series. We have two options in dealing with this process. First, we can re-define our paraphyla and their crown groups into “bigger boxes” — superphyla. Thus, for example, we could re-define Cnidaria to be able to include the Myxozoa, as noted above. The result in this case would be a monophyletic clade with a sort of superphylum one might term the “Myxocnidaria” This alternative becomes less feasible when the paraphyla lie on the main stem of metazoans. For example, how do we re-define taxa to remove a paraphyletic series of sponges from the base of the entire Metazoa? A second option would be to simply recognize smaller, but more easily definable, monophyletic groups. Thus, Calcarea and “Demohexactinellispongia” might enter the list of metazoan monophyletic groups as distinct entities. The “classes of Cnidaria” (Anthozoa, Scyphozoa, Cubozoa, Hydrozoa, and Polypodium) along side the Myxozoa could be treated as co-equal monophyletic groups among the diploblasts. Furthermore, as we come to abandon partial analyses of animal relationships for more comprehensive analyses (even total evidence approaches - see Jenner & Schram 1999 and Jenner 2000) we may find that some of our “primitive paraphyla” may actually emerge as “polyphyla.” For example, some analyses based on 18S rDNA suggest that the platyhelminth group Acoelomorpha might not be sister taxa to rhabditophoran Platyhelminthes, but rather stands in opposition to various groupings of bilaterians (Ruiz-Trillo I. et al. 1999), although Berney et al. (2000) would argue against this. Certainly, as these polyphyla achieve official recognition, we will need to establish the separate monophyletic status of the constituent groups. In the end, we must come to accept that the higher categories that we have used so faithfully for more than 240 years are really quite arbitrary. We can continue to use the old names of course, for they still have value in terms of their usefulness to catalogue information. However, systems of classification are not equivalent to genealogies, taxonomic catalogues are not family trees. While some day we may use our new knowledge of phylogeny to establish a more natural system of higher classification for the metazoans, we should not use an as yet unsatisfactory and incomplete level of understanding to formalize genealogical relationships.

Conclusion Finally, we might consider this point. Will there be a Phase V on the chart in Fig. 1? What does the 21st century hold for us in terms of our evolving sense of animal biodiversity? I want to suggest that as we come to shift away from classifying biodiversity into neat little “boxes,” that is, as we give up our strict adherence to the old phyla, classes, and subclasses, we will focus on recognizing monophyletic groups. As a result, we will indeed enter into a new “boom cycle” (Fig. 3). If I will be permitted to augur history, I would venture to predict that as we approach mid-century we will have identified in excess of 100 monophyletic, “phylum-like” groups among the Metazoa. Furthermore, these may or may not correspond to the currently accepted phyla. Indeed,

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Fig. 3. Projected growth of monophyletic groups of Metazoa (Phase V) superimposed on the cumulative growth pattern of phyla from Fig. 1. We might expect that by mid-century, something in excess of 100 monophyletic groups of metazoans will be definitively recognized, which may or may not resemble in whole or in part the currently recognized phyla.

the implications of this adjustment in our overview of animal biodiversity will be quite profound for the way in which we will teach zoology, the manner we shall plan research programs, and how we will make decisions concerning biodiversity conservation exciting times for us indeed! Acknowledgements I wish to thank the organizers of the congress for suggesting the broad subject of this symposium and inviting my participation as an organizer. I am also grateful to my co-

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organizer, Michael Schmidt of the Alexander König Museum, Bonn, for shouldering the greater burden in putting together the list of speakers. I also wish to acknowledge the help of my colleague Ronald Jenner, Zoological Museum, University of Amsterdam, for his help and input towards the development of this paper, and Dr. Claus Nielsen and one other reviewer for their helpful suggestions in revising the manuscript. References BERNEY C., PAWLOWSKI J. & L. ZANINETTI 2000. Elongation Factor 1-alpha sequences do not support an early divergence of the Acoela. Molecular Biology and Evolution 17: 1032-1039. CAMERON C.B., GAREY J.R. & B.J. SWALLA 2000. Evolution of the chordate body plan: new insights from phylogenetic analyses of deuterostome phyla. Proceedings of the National Academy of Science, USA 97: 4469-4474. CAVALIER-SMITH T., CHAO E.E., BUORYESNAULT, N. & J. VACELET 1996. Sponge phylogeny, animal monophyly, and the origin of the nervous system: 18S rRNA evidence. Canadian Journal of Zoology 74: 2031-2045. COHEN B.L. 2000. Monophyly of brachiopods and phoronids: reconciliation of molecular evidence with Linnaean classification (the subphylum Phoroniformea nov.). Proceedings of the Royal Society, London (B)267: 225-231. COLLINS L.G. 1998. Evaluating multiple alternative hypotheses for the origin of Bilateria: an analysis of 18S rRNA molecular evidence. Proceedings of the National Academy of Science, USA 95: 15458-15463. CUVIER G. 1812. Sur un nouveau rapprochment à établir entre les classes qui composent le Régne animal. Annales du Muséum d’Histoire Naturelle 19: 73-84. EHLERS U. 1985. Phylogenetic relationships within the Platyhelminthes. In Conway Morris S. et al. (eds), The Origins and Relationships of the Lower Invertebrates. Clarendon Press, Oxford, pp. 143-158. FUNCH P. & R.M. KRISTENSEN 1995. Cycliophora is a new phylum with affinities to Entoprocta and Ectoprocta. Nature 378: 711-714. GARCIA-MACHADO E., PEMPERA M., DENNEBOUY N., OLIVA-SUAREZ M., MOUNOLOU J.C. & M. MONNEROT 1999. Mitochondrial genes collectively suggest the paraphyly of Crustacea with respect to Insecta. Journal of Molecular Evolution 49: 142-149. GARCÍA-VARELA M., PÉREZ-PONCE DE LEÓN G., DE LA TORRE P., CUMMINGS M.P., SARMA S.S.S. & J.P. LACLETTE 2000. Phylogenetic relationships of Acanthocephala based on analysis of 18S ribosomal RNA gene sequences. Journal of Molecular Evolution 50: 532-540. GAREY J.R., NEA T.J., NONNEMACHER M.R. & S.A. NADLER 1996. Molecular evidence for Acanthocephala as a subtaxon of Rotifera. Journal of Molecular Evolution 43: 287-292. GAREY J.R., SCHMIDT-RHAESA A., NEAR T.J. & N.A. NADLER 1998. The evolutionary relationships of rotifers and acanthocephalans. Hydrobiologia 387: 83-91. KOJIMA S. 1998. Paraphyletic status of Polychaeta suggested by phylogenetic analysis based on the amino acid sequences of elongation factor-1 alpha. Molecular Phylogeny and Evolution 9: 255-261. HYMAN L.H. 1940-1967. The Invertebrates. McGraw-Hill, New York. 6 volumes. JENNER R.A. 2000. Evolution of animal body plans: the role of metazoan phylogeny at the interface between pattern and process. Evolution & Development 2: 208-221.

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JENNER R.A. & F.R. SCHRAM 1999. The grand game of metazoan phylogeny: rules and strategies. Biological Reviews 74: 121-142. KORSCHELT E. & K. HEIDER 1893. Lehrbuch der vergleichenden Entwicklungsgeschichte der wirbellosen Thiere. Gustav Fischer Verlag, Jena. 1509 p. KRISTENSEN R.M. & P. FUNCH 1995. Micrognathozoa: a new class with complicated jaws like those of Rotifera and Gnathostomulida. Journal of Morphology 246: 1-49. KRUSE M., LEYS S.P., MUELLER I.M. & W.E.G. MUELLER 1998. Phylogenetic position of the Hexactinellida within the phylum Porifera based on the amino acid sequence of the protein kinase C from Rhabdocalyptus dawsoni. Journal of Molecular Evolution 46: 721-728. LINNAEUS C. 1798. Systema Naturae, editio decima. Laurentus Salvius, Holmiae. LITTLEWOOD D.T.J., ROHDE K. & K.A. CLOUGH 1999. The interrelationships of all major groups of Platyhelminthes: phylogenetic evidence from morphology and molecules. Biological Journal of the Linnean Society 66: 75-114. McHUGH D. 1997. Molecular evidence that echiurans and pogonophorans are derived annelids. Proceedings of the National Academy of Science, USA 94: 8006-8009. MEGLITSCH P. & F.R. SCHRAM 1991. Invertebrate Zoology. Oxford Univ. Press, New York, 623 p. ROUSE G.W. & K. FAUCHALD 1995. The articulation of annelids. Zoologica Scripta 24: 269-301. ROUSE G.W. & K. FAUCHALD 1997. Cladistics and polychaetes. Zoologica Scripta 26: 139-204 ROUSE G.W. 2001. A cladistic analysis of Siboglinidae Caullery, 1914 (Polychaeta, Annelida): formerly the phyla Pogonophora and Vestimentifera. Zoological Journal of the Linnean Society 132: 55-80. RUIZ-TRILLO I., RIUTORT M., LITTLEWOOD D.T.J., HERNIOU E.A. & J. BAGUNÀ 1999. Acoel flatworms: earliest extant bilaterian metazoans, not members of Platyhelminthes. Science 283: 1919-1923. SCHRAM F.R. 1991. Cladistic analysis of metazoan phyla and the placement of fossil problematica. In Simonetta A. & S. Conway Morris (eds), The Early Evolution of Metazoa and the Significance of Problematic Taxa. Cambridge Univ. Press, Cambridge, pp. 35-46. SCHRAM F.R. 1997. Of cavities - and kings. Contributions to Zoology 67: 143-150. SCHRAM F.R. & C.H.J. HOF 1998. Fossil taxa and the relationships of major crustacean groups. In Edgecombe G. (ed.), Arthropod Fossils and Phylogeny. Columbia Univ. Press, New York, pp. 273-302. SHULTZ J.W. & J.C. REGIER 2000. Phylogenetic analysis of arthropods using two nuclear protein-encoding genes support a crustacean + hexapod clade. Proceedings of the Royal Society, London (B)267: 1011-1019. SPEARS T. & L.G. ABELE 1997. Crustacean phylogeny inferred from 18S rDNA. In Fortey R. & R.H. Thomas (eds), Arthropod Relationships. Chapman & Hall, London, pp. 169-187. SWALLA B.J., CAMERON C.B., CORLEY L.S. & J.R. GAREY 2000. Urochordates are monophyletic within the deuterostomes. Systematic Biology 49: 52-64. ZITTEL K.A. von. 1900. Text-book of Palaeontology, vol. I. MacMillan, London. 706 p. ZRZAVY J., MIHULKA S., KEPKA P., BEZDEK A. & D. TIETZ 1998. Phylogeny of the Metazoa based on morphological and 18S ribosomal DNA evidence. Cladistics 14: 249-285.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Willi Hennig and The theNew RisePanorama of Cladistics of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 369-379, 2003

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Willi Hennig and the Rise of Cladistics M. Schmitt Zoologisches Forschungsinstitut und Museum Alexander Koenig, Adenauerallee 160, D-53113 Bonn, Germany. E-mail: [email protected]

Abstract Willi Hennig was born and grew up in humble circumstances. He had the benefits of a good education largely due to his mother’s ambition. His interest in systematics dates back to the secondary level. He had the luck to be supported by a schoolteacher who recognized that Hennig should be trained by a more experienced person. Thus, Willi Hennig got in contact with Wilhelm Meise and Fritz van Emden at Dresden Museum before entering university. These two systematists, and after 1933 Klaus Günther (successor of Fritz van Emden), were Willi Hennig’s primary mentors, and there were the major influences on the development of Hennig’s scientific reasoning. Although the development of Willi Hennig’s method of phylogenetic systematics was largely stimulated by Wilhelm Meise and Klaus Günther, certain elements of this method were already discussed and published prior to Willi Hennig’s “Grundzüge einer Theorie der Phylogenetischen Systematik” in 1950. Since Willi Hennig seemingly cited the sources of his developing method, e.g. papers of Adolf Naef and Walter Zimmermann, I believe that he would certainly have mentioned Daniele Rosa’s book of 1918, entitled “Ologenesi - Nuova Teoria dell’ Evoluzione e della Distribuzione dei Viventi”, if he had had knowledge of this book AND at the same time had found it to contain ideas decisive to the new method. Finally, the argument between Ernst Mayr and Willi Hennig in 1974 and some major post-Hennigian modifications of phylogenetic systematics are outlined. Here, I attempt to evaluate Willi Hennig’s contribution to present-day cladistics. On one hand, Willi Hennig’s papers are definitely basic and crucial to the history of modern systematics. On the other hand, the flaws in his original method should not be neglected.

Introduction The present-day paradigm in biological systematics, the so-called cladistic method, is based on the works of Willi Hennig. Ever since Linnaeus, there was no single person who changed the face and the role of biological systematics more dramaticly than he did. In order to reach a comprehensive understanding of this school of systematics, is it

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reasonable to examine the family background, basic education and scientific training of Willi Hennig. In the following, I focus on the early development of phylogenetic reasoning and the origins of the method of phylogenetic systematics designed by him. One question asked is: From which premises and from which background Willi Hennig started when outlining the fundamentals of his method? Another question examined: To which extend other scientists - teachers, friends, colleagues, authors of papers Hennig read - contributed to the method specifically credited to him? And finally: How influential Willi Hennig really was, how much of his original ideas is still active in the modern, computer-based systematics? Life and professional career of Willi Hennig Willi Hennig was born 20.4.1913 in the village of Dürrhennersdorf in the province of Saxonia. His parents lived under humble circumstances: his father, Karl Ernst Emil Hennig (28.8.1873-28.12.1947) was a railroad worker and advanced to a civil service position in the regional railway administration. His mother, Marie Emma Hennig, was the illegitimate child of a servant, suffered from this social stigma, and tried to compensate for it by devoting an extraordinarily high amount of energy to the education to her three sons Emil Hans Willi, Fritz Rudolf (5.3.1915-24.11.1990) and Karl Herbert (24.4.1917-ca. January, 1943, listed as a missing person). Willi Hennig was especially subject to his mother’s ambitions. In addition, he was diligent and intelligent, as was already recognized during his first class at elementary school (all information on Willi Hennig’s family background, childhood and youth from Vogel & Xylander 1999). When Willi Hennig entered the university of Leipzig in 1932, he already had considerable experience with entomological systematics. Through several years, from about 1927 to 1932, he lived in the home of one of his schoolteachers, M. Rost, as a boarder. Mr. Rost was an acquaintance of Wilhelm Meise (12.09.1901-24.08.2002), at that time the curator of all taxa except insects at the Dresden State Museum of Natural History. This teacher introduced Willi Hennig to Wilhelm Meise because Hennig wanted to learn more zoology, and especially systematics, than his schoolteacher was able to teach him. Meise first entrusted Hennig with the task of compiling an alphabetical index of the fishes in the British Museum (Natural History). When he realized that Hennig was talented and motivated, he prompted him to undertake serious empirical investigations. Since Meise was interested in flying non-bird tetrapods, he stimulated Willi Hennig to study “flying” snakes and lizards. These studies yielded two publications on snakes, co-authored by Meise and Hennig (1932, 1935), and a revision of the lizard genus Draco (Hennig 1936a,b). The snakes investigated belong to the genera Dendrelaphis (at that time called Dendrophis) and Chrysopelea. Both genera include snakes that are able to jump, or better to fling or hurl themselves into the air from tree limbs, thereby stretching their body. The Chrysopelea-species spread out ribs to both sides of the body, thus gliding over a certain distance. The agamids of the genus Draco also produce sort of wings by expanding their ribs. Willi Hennig pounced avidly on these challenges, and the papers produced demonstrate that he succeeded surprisingly well for his age, and these papers on reptiles are still useful up today. Here, Willi Hennig learned to cope with nomenclatorial

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problems, with taxonomic descriptions, and with zoogeographic data. The revision of the genus Draco, 67 pages long, is an example of careful observing and measuring and show that Willi Hennig mastered quite a lot of statistics (in later papers, he rarely returned to statistical analyses). In these early papers, Willi Hennig and Wilhelm Meise followed an entirely conventional concept of “systematics”. They started with Alpha-taxonomy and geographical distribution, they used the term “monophyletic” in the pre-Hennigian sense of “single origin in evolution”; there is no indication of distinguishing between primitive and derived traits. However, here lay the beginnings of the first major contributions Willi Hennig made to systematics: to define “relationship” in a strictly genealogic way, i.e. distinguishing relationship from similarity. However, he did not invent this distinction, but rather took it from the writings of Adolf Naef (1.5.1883-15.5.1949), especially from his papers published in 1917 and 1919. Wilhelm Meise reports that Willi Hennig was already interested in insects when he first contacted with him. Willi Hennig wrote in a curriculum vitae accompanying an application for a grant around 1930 that he was already accustomed to museum entomology since the 5th year at the Gymnasium. Thus, Meise referred the young pupil to Fritz Isidor van Emden (03.10.1898-02.09.1958) who was curator of insects at that time. Willi Hennig had heard of van Emden through his schoolteacher (M. Rost). Willi Hennig quickly joined Fritz van Emden, who had his lab on the first floor, and Wilhelm Meise wrote me that Hennig regularly disappeared upstairs to meet van Emden. Perhaps as early as 1932, but at the latest in 1934 (according to Wilhelm Meise’s letter), Fritz van Emden encouraged Willi Hennig to begin a revision of the dipteran family Tylidae, which Hennig completed in 1934 and published it in two parts on more than 300 pages until 1936. Before finishing his doctoral thesis (1936: on the copulation apparatus and the system of the Tanypezidae - acalyptrate Diptera -, supervised by Paul Buchner, 12.04.1886-19.10.1978), Willi Hennig published not only the papers on reptiles but also the revision of the Tylidae and four other - shorter - papers on dipterans. Fritz van Emden had to leave the museum due to the racial laws of the Nazi government (and luckily emigrated first to the Netherlands and later to Great Britain) on 30.09.1933 (Hennig 1960). In 1934, Klaus Günther (07.10.1907-01.08.1975) from Berlin became his successor as the entomology curator. Willi Hennig kept his connection to the Dresden museum and met there with Klaus Günther. There are few letters from that time, and there are no witnesses to these encounters. However, from later letters and from statements of contemporaries it is completely clear that the two men became close friends before 1939 (i.e. before World War II; see also Hennig 1976). It is not clear who stimulated Willi Hennig’s development of phylogenetic systematics. Either Wilhelm Meise (a very modest person who did not claim to be the one), or Fritz van Emden, or Klaus Günther must be considered. It is possible that Hennig spoke to none of them, nor to anybody else, about his scientific ideas. However, this is not very probable, and if we look at the extensive and detailed correspondence between Hennig and Günther after 1963 (Schmitt 1996), it was usually Willi Hennig asking for advice, not vice versa. Thus, it is likely that Klaus Günther played a certain part in the considerable step Hennig made in 1936(c), when he wrote on the relations between geographic distribution and systematic classification of some dipteran families as a

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contribution to the problem of classifying taxa of higher order. Here, Hennig clearly distinguishes primitive characters as “independently retained inheritance from the ancestor of all tylids” from the “progressive characters shared by Tylinae and Neriinae”. The latter type of characters “especially suggests” closer phylogenetic relationships of the two subfamilies mentioned. Also in this paper, we find an explicit statement that a systematic classification must be done according to phylogenetic relationship rather than to morphological differentiation. Willi Hennig received his military training from winter of 1938 to spring of 1939 and was called up at the beginning of the war. After he was wounded in 1942, he was appointed a military entomologist in Italy. His job was to control malaria and other epidemics. While he published little on taxonomy and only few papers on pest arthropods (not just insects but also ticks), he must have thought a lot about methodology. He was taken prisoner of war (POW) in May 1945 in Lignano, Upper Italy, by British troops, and stayed in British captivity until October. However, he was not confined to but was taken out of the POW camp by a British officer and put into the British anti-malariaservice only after a few weeks of captivity (see the notes of Willi Hennig’s second son, Bernd Hennig, on http://www.cladistics.org/about/hennig.html). During the time as a British POW, he wrote the manuscript of his Grundzüge einer Theorie der Phylogenetischen Systematik. He wrote in pencil or coloured crayon in an Italian exercise book on squared paper. According to Schlee (1978, who had interviewed Willi Hennig’s widow), Hennig completed the manuscript during the Berlin blockade in 1948, gluing some additional pages into the exercise book. Short parts of the manuscript were published in 1947 and 1949. Only the 1949 paper is of methodological interest. Here, he introduced the terms “apomorph” and “plesiomorph” and others (“apoök” - “plesioök”, “apochor” “plesiochor”), as terms regarding taxa. But he mentioned that in cases of “specialization crossings” these terms might apply to characters only. Due to paper shortage, the book did not appear until 1950, which makes it somewhat difficult to trace the first publication date of several terms coined by Hennig. In his 1949 paper, he wrote that he already had defined them in different publications, but full citations are missing. After WWII, Willi Hennig received a temporary appointment at Leipzig University as the replacement of Prof. Hempelmann and later Prof. Paul Buchner (his doctoral supervisor). But he always wanted to return to Berlin and finally succeeded in getting a position at Deutsches Entomologisches Institut (East Berlin) (01.04.1947) where he became head of the department of systematic entomology and vice-director on 01.11.1949. He received his habilitation in zoology from Brandenburgische Landeshochschule Potsdam and was entitled “professor” on 10.10.1951. He lived in West Berlin and worked at the Deutsches Entomologisches Institut until the erection of the Berlin Wall (13.08.1961). For personal and political reasons he did not want to move to East Berlin. Thus, he accepted a temporary extraordinary professorship at the Technische Universität Berlin (West). However, before a permanent solution could be established in Berlin, he was offered an attractive position as head of a department of phylogenetic investigation at Staatliches Museum für Naturkunde, Stuttgart, opened ad personam. This department was housed provisionally in the city of Ludwigsburg, 15 km north of Stuttgart. Here, he worked and lived until the night of 05.11.1976, when he died from a sudden heart attack.

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‘Phylogenetic Systematics’ and ‘Hologenesis’ Since Willi Hennig wrote the manuscript of his fundamental work when he was a prisoner of war, he had little access to scientific literature. He had to rely on his memory and on the letters his wife, Irma Hennig (29.08.1910-26.04.2000), was allowed to send him. He asked her from time to time to copy - by hand - some passages of papers he needed. Unfortunately, neither he nor his wife kept all of these letters after he returned to Germany. Thus, there is no record of whether or not Willi Hennig took notice of Daniele Rosa’s (29.10.1857-28.04.1944) so-called theory of Hologenesis. According to A. Kluge (pers. comm. 2001), “it has been suggested (....) that key elements of phylogenetic systematics are the same as those in Rosa’s (1918) little known publication on the theory of hologenesis, and some have even speculated that Hennig learned of those details during the time he spent in Italy (...). Indeed, it seems certain that Hennig visited Rosa’s former student Giuseppe Colosi in Florence, and that he read Rosa’s papers at the Institute Library (Baroni-Urbani 1990: 2). According to B. Lanza (pers. comm.), Hennig’s visit to Florence occurred a few years after World War II, but before the 1950 publication of his “Grundzüge”. This description is supported by G. J. Nelson (pers. comm. 1999), with the exception of the dating of Hennig’s encounter with G. Colosi. Rosa had published a book on “La riduzione progressiva della variabilità” (the progressive reduction of variability) in 1899. A German translation had appeared in 1903. Willi Hennig cited this book in 1950 as well as in 1966. In this publication, Rosa treated rules or laws of evolution. Willi Hennig mentioned Rosa when he discussed possible tools of determining the direction of character transformation (character polarisation). In 1918, Daniele Rosa published the book “Ologenesi - Nuova Teoria dell’ Evoluzione e della Distribuzione dei Viventi”. A preliminary note had already appeared in 1912, and a summary was published in French in 1923 (see also 1988). A French translation of the book was published in 1931. In this “theory”, Rosa states that species always split into two descendants, and that of these two always one will change evolutionarily at a higher rate than the other. He called the faster changing line “linea precoce” (precocious), the slower changing one “linea tardiva” (tardy). The precocious line should on one hand evolve at a higher rate, but on the other hand keep inferior organisation relative to the tardy line. I could not find a theoretically convincing substantiation for this statement. My impression is that Rosa stated these relations axiomaticly. It is certainly important that Rosa did not provide any empirical criterion as to how to distinguish between the precocious and the tardy line, nor did he even intimate that his “theory” would offer a practical tool for systematics (at least not in the 1923 paper). According to the 1923 summary, there are two points in Rosa’s ideas superficially resembling aspects of Hennig’s method: (1) the ‘law of ramification’ that causes species obligatorily to split into two daughter species, and the normally dichotomous design of Hennigian cladograms. However, Hennig discussed in detail the mode of speciation without starting from dichotomous splitting. On the contrary, in his 1966 book he states explicitly that “if phylogenetic systematics starts out from a dichotomous differentiation of the phylogenetic tree, this is primarily no more than a methodological principle” (p. 210). (2) Rosa’s distinction of a “linea precoce” and a “linea tardiva”, and Hennig’s

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conceptions of apomorph and plesiomorph. However, Hennig referred not at all to a ‘law of different evolutionary speed’. From p. 88 in his 1966 book it becomes clear that he regarded character transformation as a prerequisite for systematists in order to distinguish different species: if we can recognize two (or more) species where before was only one, then at least one character must have changed in each additional lineage. There is no idea of an obligatorily faster evolving lineage whatsoever. All of his pre-1950 publications demonstrate that Willi Hennig was primarily a taxonomist who wanted to reach a theoretically better substantiated order than the previous ones. His interests in evolutionary processes were clearly motivated by his desire to give good reasons for his hypotheses on character polarisation. And he wanted to polarise characters in order to establish monophyletic (= holophyletic) taxa. Therefore, it is unlikely that he came across Rosa’s book in the course of his literature search. Since Willi Hennig, from his 1950 book on, conscientiously cited other sources that contributed to his method (e.g. Adolf Naef’s papers and the seminal paper of Zimmermann 1937), it is pure speculation to insinuate that he had knowledge of Rosa’s “theory” but dismissed it. Thus, even if he had known Rosa’s “theory”, he would possibly - and understandably - not have seen the need to cite it. However, it remains highly improbable that he ever saw Rosa’s book: When I tried to borrow it from a public academic library, it turned out that it is not kept in any German university library. Moreover, in a discussion thread on the list TAXACOM ([email protected]) in 1999, it was mentioned that it was not even present in the library of the Natural History Museum, London, before Chris Humphries brought a private xerocopy from Italy and transferred it to the museum’s library some years before 1999. Under these auspices, it is the more stunning that Bernhard Rensch cited Rosa’s book in 1947, and Walter Zimmermann cited it in 1953. The latter listed Rosa in the references and mentioned him when discussing internal causes of evolution, but not when he introduced his own “Hologenie”. I assume that none of them actually read Rosa’s book but relied on the summaries of it. By the way, George Gaylord Simpson cited the French translation in his 1944 book on “Tempo and Mode of Evolution”. After all, Willi Hennig wrote the core parts of his Grundzüge in 1945, during his time as a POW. As shown above, he had clear conceptions of the basics of his method much earlier than that. The manuscript was completed in 1948. Thus, a visit of Florence ‘after 1945 but before 1950’ is irrelevant to the question of what he possibly learned from reading Rosa’s book. The Mayr-Hennig debate Willi Hennig provided several examples for his method in papers on the phylogenic system of certain taxa of insects, mostly dipterans. This might be one of the reasons that he received little attention from outside the entomological community. Only when his review paper of 1965 and his book of 1966 were published, did he reach a wider audience. Though there are numerous letters of and to Klaus Günther, there cannot be any doubt that Hennig developed his method alone. He sent a German version of his 1965 paper to Klaus Günther who admired it very much and suggested only minor changes. He would clearly not have written what he did if he had seen the manuscript beforehand. It is also clear that Lars Zakarias Brundin (30.05.1907-17.11.1993), a Swedish dipterist

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who likewise spoke English and German, contributed considerably to the spreading of Hennig’s ideas into the English speaking community (Hull 1989; Dupuis 1979, 1984). The manuscript of Hennig’s reply to Ernst Mayr’s demand (1974) to analyse but not to classify cladistically was sent several times from Ludwigsburg to Berlin and back. Unfortunately, the final version is lost. However, from the letters exchanged during this phase, it can be inferred that Klaus Günther contributed a considerable part to the basic line and wording of this paper. Initially, he had thought to write the paper himself in order to stand by his friend. Thus, he planned to include paragraphs from a letter of Willi Hennig. But finally, Willi Hennig wrote the first draft, and Klaus Günther commented on it. Several phrases were doubtlessly contributed by him (e.g. “the most useful generalizations and explanations” - in German “die weitesttragenden Generalisationen und Exhaustionen”, and the old-fashioned term “epistemiologisch” for epistemological). Günther’s highly sophisticated language allows distinguishing between the two authors - Hennig used a rather conventional but laborious vocabulary (Schmitt 1996). Be it as it is, even if Klaus Günther put in some of his wit and knowledge, there are no indications that Willi Hennig had taken crucial portions of his concepts from other authors, let alone without citing them. Post-hennigian Cladistics What today is widely accepted as “cladistics” is definitely not identical with Willi Hennig’s original concept of “phylogenetic sytematics”. The term ‘cladistics’ was coined by Ernst Mayr in 1974 and refers to ‘cladistic’ (Cain & Harrison 1960) but stems from Julian Huxley’s term “clade”. Hennig’s ideas are probably most accurately and congenially transferred into English by Wiley (1981). According to Dupuis (1984), they form the foundation of present-day cladistics. Since Hennig’s untimely death in 1976, the phylogenetic method developed by Hennig has undergone at least two major modifications: (1) The elaboration of the out-group comparison method (Wiley 1981: 139, Watrous & Wheeler 1981). Hennig - and many others - had used this method implicitly (his “criterion of the distribution of chartacters among taxa”, Hennig & Schlee 1978) but did certainly not consider this “criterion” to be the most useful one. The outgroup comparison method reached high impact through its implementation in computer programmes based on parsimony algorithms (e.g. Farris 1988, see Forey et al. 1992). The most important consequence of applying these computer programmes is, in my opinion, that polarisation is not longer done for each character separately (which means in the ideal case: independently) but by labelling one of the rows of the data matrix ‘out-group’. Consequently, there is no longer need for a priori reasoning over the possibilities of evolutionary transformation, i.e. before the tree calculating programme is executed. (2) The so-called ‘transformation of cladistics’ (Nelson 1979, Platnick 1979, Patterson 1980) the result of which (“pattern cladism”) implies a strict separation of the analysis of the pattern formed by the distribution of the characters analysed through the taxa under consideration from the explanation of this pattern by reference to evolutionary processes. Although there are certainly good arguments for such a strict separation, it was and is not accepted throughout, especially not in Willi Hennig’s country (e.g. Ax

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1984). As Hull (1989) pointed out, pattern cladists have departed from Hennig in several respects, up to the total reversion of one of Hennig’s original views [concerning the logical and historical precedence of a non-phylogenetic system over the phylogenetic one, a position held by Platnick (1985) but deemed “absolutely wrong” by Hennig (1966: 11)]. To a great extend, the overwhelming success of Hennig’s method is due to the fact that it can be formalized and, therefore, executed by a computer. This is also true for the parsimony based “Wagner groundplan divergence method”. This method had “received ... no attention from zoologists until Kluge and Farris (1969) used Wagner’s basic method to produce a phylogenetic computer algorithm...” (Wiley 1981: 176f.). The “grand alliance between the Wagner tradition and the Hennig tradition” (E.O. Wiley, pers. comm. 2001) formed the basis for the revolution of systematics during the last three decades of the 20th century. Some major flaws and weaknesses of Willi Hennig’s phylogenetic systematics cannot be neglected. First, he declined to provide a general empirical tool for character polarisation. The “criteria” he gave in 1966 (paleontological order, ontogenetic sequence, geographic distribution, and correlation of transformation series) are either theoretically obsolete (the last) or they can only be applied in special cases. This disadvantage was only eliminated by the method of out-group comparison (e.g., Wiley 1981, Watrous & Wheeler 1981). Second, he was rather vague concerning the relation between his concept of synapomorphy and the traditional homology concept. This led to a considerable degree of confusion and to views far apart from Hennig’s own ideas, which were so close to traditional continental European usage that he saw no need for clarifying his position explicitly. Third, Willi Hennig never gave up the idea that a - well substantiated phylogenetic hypothesis should be converted into a taxonomic classification. Here, necessarily a conflict arose between the limited number of taxonomic ranks and the discovered number of splitting events; each of them would require a separate rank for the resulting taxon. Hennig tried to cope with this problem in his Stammesgeschichte der Insekten (1969) but his solution (superlong sequences of numbers) was not convincing. Another irritating aspect is the high frequency of nomenclatorial changes, according to the publication of new phylogenetic hypotheses. Up to the present, these unsolved problems stimulate unconventional suggestions, e.g. consequent renunciation of Linnean ranks and invariant use of current names (De Queiroz & Gauthier 1992). Even if little is left of Willi Hennig’s original principles in modern cladistics, there is no doubt that he turned systematics from a craft or an art into a scientific enterprise that fits into the Popperian hypothetico-deductivistic model. He worked out a clear terminology and argumentation scheme (Wiley 1981, Richter & Meier 1994) and by this helped to eliminate or at least reduce arbitrariness in this field of science. Acknowledgements Gareth J. Nelson (Melbourne, Australia) directed me to several biographic sources I had overlooked and provided a description of Willi Hennig’s visit of G. Colosi. Gabriele Uhl (Bonn, Germany), Arnold G. Kluge (Ann Arbor, Michigan, USA), and E.O. Wiley (Lawrence, Kansas, USA) carefully read the manuscript and contributed considerably

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to its improvement. E.O. Wiley refined the English of an earlier version of the manuscript. I thank all of them for their valuable help. References [Necrologies have been published by Ax (1977a, b), Byers (1977), Kiriakoff (1977), Kühne (1978), Schlee (1977, 1978), and Schuh & Wygodzinsky (1977), a list of publications compiled by Wolfgang Hennig appeared anonymously in Beiträge zur Entomologie (1978). Additional biographical notes are given by Peters (1995), Pont (1981), Schlee (1981), Dupuis (1990), Schmitt (2001) and Vogel & Xylander (1999)] ANONYMOUS [Wolfgang Hennig] 1978. In memoriam: Willi Hennig (*20.4.1913 †5.11.1976). Beitr. Entomol. 28: 169-177. AX P. 1977a. Professor Dr.Dr.h.c. Willi Hennig † Zoomorphol. 87: 1-2. AX P. 1977b. Willi Hennig 20.4.1913 bis 5.11.1976. Verh. dt. zool. Ges. 70: 346-347. AX P. 1984. Das Phylogenetische System. Gustav Fischer, Stuttgart - New York, 349p. BARONI-URBANI C. 1990. Search for the evolutionary roots of cladistics (a simplified conspectus of hologenetic theory). Pp. 1-7. Osaka Group for the Study of Dynamic Structures (newsletter), November. BYERS G.W. 1977. In memoriam Willi Hennig (1913-1976). J. Kansas entomol. Soc. 50: 272-274. CAIN A.J. & G.A. HARRISON 1960. Phyletic weighting. Proc. zool. Soc. London 135: 1-31. DE QUEIROZ K. & J. GAUTHIER 1992. Phylogenetic taxonomy. Ann. Rev. Ecol. Syst. 23: 449-480. DUPUIS C. 1979. La “systématique phylogénétique” de W. Hennig (Historique, discussion, choix de références). Cahiers des Naturalistes. Bull. des Naturalistes Parisiens N.S. 34: 1-69 (1978). DUPUIS C. 1984. Willi Hennig’s impact on taxonomic thought. Ann. Rev. Ecol. Syst. 15 : 1-24. DUPUIS C. 1990. Hennig, Emil Hans Willi. In Holmes F.L. (ed.), Dictionary of Scientific Biography Vol. 17, Suppl. 2. Charles Scribner’s Sons, New York, pp. 407-410. FARRIS J.S. 1988. Hennig86 version 1.5, Computer Programme and Manual, publ. by the author. FOREY P.L., HUMPHRIES C.J., KITCHING I.J., SCOTLAND R.W., SIEBERT D.J. & D.M. WILLIAMS 1992. Cladistics. A Practical Course in Systematics (Systematics Association Publications 10). Clarendon Press, Oxford, XI+191p. HENNIG W. (1934/35): Revision der Tyliden (Dipt., Acalypt.) I. Teil: Die Taeniapterinae Amerikas. Stettiner ent. Ztg. 95: 6-108, 294-330 (1934), 96: 27-67 (1935). HENNIG W. (1935/36): Revision der Tyliden (Dipt., Acalypt.) II. Teil: Die außeramerikanischen Taeniapterinae, die Trepidariinae und Tylinae. Konowia 14: 68-92, 192-216, 289-310 (1935), 15: 129-144, 201-239 (1936). HENNIG W. 1936a. Revision der Gattung Draco (Agamidae). Temminckia 1: 153-220. HENNIG W. 1936b. Über einige Gesetzmäßigkeiten der geographischen Variation in der Reptiliengattung Draco L.: „parallele“ und „konvergente“ Rassenbildung. Biol. Zbl. 56: 549-559. HENNIG W. 1936c. Beziehungen zwischen geographischer Verbreitung und systematischer Gliederung bei einigen Dipterenfamilien: ein Beitrag zum Problem der Gliederung systematischer Kategorien höherer Ordnung. Zool. Anz. 116: 161-175. HENNIG W. 1947. Probleme der biologischen Systematik. Forschungen und Fortschritte 21/23: 276-279.

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HENNIG W. 1949. Zur Klärung einiger Begriffe der phylogenetischen Systematik. Forschungen und Fortschritte 25: 136-138. HENNIG W. 1950. Grundzüge einer Theorie der phylogenetischen Systematik. Deutscher Zentralverlag, Berlin, [VI]+370p. HENNIG W. 1960. F.I. van Emden †. Zool. Anz. Suppl. 23(Verh. dt. zool. Ges. 1959): 528-529. HENNIG W. 1965. Phylogenetic Systematics. Ann. Rev. Ent. 10: 97-116. HENNIG W. 1966. Phylogenetic Systematics. Univ.Illinois Press, Urbana, IV+263p. HENNIG W. 1969. Die Stammesgeschichte der Insekten. Waldemar Kramer, Frankfurt am Main, 436p. HENNIG W. 1974. Kritische Bemerkungen zur Frage “Cladistic analysis or cladistic classification?”. Z. zool. Syst. Evolutionsforsch. 12: 279-294. (Engl.: “Cladistic analysis or cladistic classification ?” A reply to Ernst Mayr. Syst. Zool. 24: 244-256). HENNIG W. 1976. Klaus Günther 7.10.1907 bis 1.8.1975. Verh. dt. zool. Ges. 1976: 297-298. HENNIG W. 1984. Aufgaben und Probleme stammesgeschichtlicher Forschung (ed. by Wolfgang Hennig). Paul Parey, Berlin – Hamburg, 65p. HENNIG W. & D. SCHLEE 1978. Abriss der phylogenetischen Systematik. Stuttgarter Beitr.Naturk., Ser. A (Biol.), 319: 1-11. HULL D.L. 1989. The evolution of phylogenetic systematics. In Fernholm B., Bremer K. & H. Jörnvall (eds) The Hierarchy of Life. Elsevier (Excerpta Medica), Amsterdam etc., pp. 3-15. KIRIAKOFF S.G. 1977. Necrologie: Willi Hennig (1913-1976). Bull. Ann. Soc. r. Ent. Belgique 113: 240-243. KLUGE A.G. & J.S. FARRIS 1969. Quantitative phyletics and the evolution of anurans. Syst. Zool. 18: 1-32. KÜHNE W.G. 1978. Willi Hennig 1913-1976: Die Schaffung einer Wissenschaftstheorie. Entomologica Germanica 4: 374-376. MAYR E. 1974. Cladistic analysis or cladistic classification. Z. zool. Syst. Evolutionsforsch. 12: 94-128. MAYR E. 1984. Die Entwicklung der biologischen Gedankenwelt. Springer, Berlin etc. (Amer. Orig. 1982), XXI+766p. MEISE W. & W. HENNIG 1932. Die Schlangengattung Dendrophis. Zool. Anz. 99: 273-297. MEISE W. & W. HENNIG 1935. Zur Kenntnis von Dendrophis und Chrysopelea. Zool. Anz. 109: 138-150. NAEF A. 1917. Die individuelle Entwicklung organischer Formen als Urkunde ihrer Stammesgeschichte. Gustav Fischer, Jena, 77p. NAEF A. 1919. Idealistische Morphologie und Phylogenetik (Zur Methodik der systematischen Morphologie). Gustav Fischer, Jena, VI+77p. NELSON. J. 1979. Cladistic analysis and synthesis: Principles and definitions, with a historical note on Adanson’s Familles des Plantes (1763-1764). Syst. Zool. 28: 1-21. PATTERSON C. 1980. Cladistics. Biologist 27: 234-240. PETERS G. 1995. Über Willi Hennig als Forscherpersönlichkeit. Sber. Ges. naturf. Freunde Berlin N.F. 34: 3-10. PLATNICK N.I. 1979. Philosophy and the transformation of cladistics. Syst. Zool. 28: 537-546. PLATNICK N.I. 1985. Philosophy and the transformation of cladistics revisited. Cladistics 1: 87-94. PONT A.C. 1981. Foreword. In Hennig W. Insect Phylogeny. John Wiley & Sons, Chichester etc., pp. IX-XI. RENSCH B. 1947. Neuere Probleme der Abstammungslehre. Ferdinand Enke, Stuttgart. VII+407p.

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RICHTER S. & R. MEIER 1994. The development of phylogenetic concepts in Hennig’s early theoretical publications (1947-1966). Syst. Biol. 43: 212-221. ROSA D. 1903. Die Progressive Reduktion der Variabilität und ihre Beziehungen zum Aussterben und zur Entstehung der Arten. Gustav Fischer, Jena (Ital. Orig.: La riduzione progressiva della variabilità. C. Clausen, Torino 1899),III+106p. ROSA D. 1923. Qu’est-ce que l’hologénèse? Scientia - Rivista di Scienza 33: 113-124. ROSA D. 1988. The theory of hologenesis (1909-1918). Rivista di Biologia - Biology Forum 81: 613-615. SCHLEE D. 1977. Willi Hennig. Jahresh. Ges. Naturk. Württemberg 132: 196-197. SCHLEE D. 1978. In Memoriam Willi Hennig 1913-1976. Eine biographische Skizze. Entomologica Germanica 4: 377-391. SCHLEE D. 1981. Introduction to the English Edition. In Hennig W. Insect Phylogeny. John Wiley & Sons, Chichester etc., pp. XIII-XV. SCHMITT M. 1996. Klaus Günthers Bedeutung für die Phylogenetische Systematik. Sber. Ges. naturf. Freunde Berlin N.F. 35: 13-25. SCHMITT M. 2001. Willi Hennig. In Jahn I. & M. Schmitt (eds), Darwin & Co. vol. 2. C.H. Beck, München, pp. 316-343, 541-546. SCHUH R.T. & P. WYGODZINSKY 1977. Willi Hennig. Syst. Zool. 26: 104-105. SIMPSON G.G. 1944. Tempo and Mode in Evolution. Columbia University Press, New York. VOGEL J. & W.R. XYLANDER 1999. Willi Hennig – Ein Oberlausitzer Naturforscher mit Weltgeltung. Recherchen zu seiner Familiengeschichte sowie Kinder- und Jugendzeit. Ber. naturf. Ges. Oberlausitz 7/8: 131-141. WATROUS L.E. & Q.D. WHEELER 1981. The out-group comparison method of character analysis. Syst. Zool. 30: 1-11. WILEY E.O. 1981. Phylogenetics. The Theory and Practice of Phylogenetic Systematics. Wiley, New York etc. XV+439p. ZIMMERMANN W. 1937. Arbeitsweise der botanischen Phylogenetik und anderer Gruppierungswissenschaften. In Abderhalden E. (ed.), Handbuch der biologischen Arbeitsmethoden Abt. 9, Teil 3, 2. Hälfte. Urban & Schwarzenberg, Berlin - Wien, pp. 941-1053. ZIMMERMANN W. 1953. Evolution - die Geschichte ihrer Probleme und Erkenntnisse. Carl Alber, Freiburg - München. IX+623p.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Willi Hennig and The theNew RisePanorama of Cladistics of Animal Evolution Proc. 18th Int. Congr. Zoology, p. 383, 2003

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Diversity, Endemism and Conservation Priorities in Madagascar W.R. Lourenço Dépt. de Systématique et Evolution, section Arthropodes (Arachnologie), Museum National d’Histoire Naturelle, 61 rue Buffon, 75231 Paris cedex 05, France. E-mail: [email protected]

Madagascar can be considered as the first priority in the world in matter of conservation of the biodiversity. The country contains more genetic information in a given unit of surface than any other region in the world. The lost of one hectare of forest in Madagascar has a much more negative effect to the global biodiversity than it has in any other region in the world. The forests of Madagascar are severely threatened by several human activities e.g. agriculture, cattle raising and forest extraction, and most negative estimation give up to 80% of the original forest cover already destroyed, and this since men arrived to Madagascar between 1500 and 2000 years ago. The planification for conservation has motivated a very important reaction to these needs in conservation. Since 1985 several conferences and workshop took place to allowed discussion among experts in different disciplines, and can be cited in particular: Antananarivo 1995; Chicago 1995; Paris 1995, and Paris 1999. The aim of the symposium « Diversity, Endemism and Conservation Priorities in Madagascar » which was proposed during the International Congress of Zoology was to bring, at least, a sample of this information to a broader zoological public.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers The remarkable levels of diversity and endemicity in the scorpion fauna ... Evolution 385 The New Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 385-391, 2003

The remarkable levels of diversity and endemicity in the scorpion fauna of Madagascar W.R. Lourenço Dépt. de Systématique et Evolution, section Arthropodes (Arachnologie), Muséum National d’Histoire Naturelle, 61 rue de Buffon 75005 Paris, France. E-mail: [email protected]

Abstract Madagascar is one of the most biologically diverse regions on Earth. To understand such diversity, the biogeographic patterns of several groups of plants and animals have been and are constantly being analyzed. Certain areas of the island exhibit especially high diversity and endemism. In the case of scorpions, the northern region is considered to be a possible epicenter of diversity and endemicity in Madagascar. Biologists always seek a rational basis on which to decide which centers of both diversity and endemism should receive the highest priority for conservation. This kind of choice requires some basic knowledge of the faunistic elements present in the region in which the study is conducted. For invertebrates, the lack of comprehensive systematic surveys across the island remains an important impediment for understanding certain biogeographical aspects. For this reason, most research on tropical applied biogeography in Madagascar is based mainly on a limited number of taxa, primarily vertebrates. Since 1994 it has been possible to put together relevant data on patterns of geographical distribution, taxonomy, diversity and endemicity of Malagasy scorpions. In this conference I will propose a clear view of the diversity and endemic level of Malagasy scorpions, and suggest their possible implication in conservation programs.

Historical Introduction Contributions to the knowledge of the scorpion fauna of Madagascar began with the contributions by Gervais (1844), Pocock (1889, 1890, 1893, 1896), Kraepelin (1894, 1896, 1901), and Birula (1903). Other publications followed such as Fage (1929, 1946), Roewer (1943), Millot (1949), Vachon (1969, 1979) and Legendre (1972). However, it was only by the middle of the 1990s that a precise inventory project started to be developed by Lourenço (e.g., 1995, 1996a,b,c,d), with the description of several new taxa. Among others, one new family, seven new genera and 26 new species have been described in the space of only five years (see also Lourenço 1997, 1998a,b, 1999a,b, 2000a,b, Lourenço & Goodman 1999a,b).

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With the use of more sophisticated methods of collecting (e.g. pit-fall traps, extraction by Winkler or detection with ultra-violet light), the number of taxa within the Malagasy scorpion fauna will be multiplied by two or even three. Madagascar is one of the most biologically diverse regions on Earth (see Vachon 1953, Paulian 1961, Lourenço 1996a, Goodman & Patterson 1997). To understand such diversity, the biogeographic patterns of several groups of plants and animals have been and are constantly being analyzed (Lourenço 1996c,d,e, Ganzhorn et al. 1997; Goodman & Patterson 1997). Although these patterns vary somewhat, certain regions exhibit especially high levels of endemism (i.e. a large number of species native to a localized region). One region with a high level of local endemism is the northern portion of the island, while other endemic centers occur in the southeast and southwest or in the central highlands. The northern region has, however, been considered by several authors, and particularly in the case of scorpions, to be the epicenter of diversity in Madagascar (Lourenço 1996b). However, current information may be biased by the fact that the northern region is one of the most extensively investigated on the island. Biologists now seek a rational basis on which to decide which centers, both of diversity and of endemism, should receive the highest priority for conservation (Ganzhorn et al. 1997). Such a choice needs, a priori, a basic knowledge of most of the elements of the taxon on which it is based, at least for the region in which the study has been conducted. Biosystematic biology as well as historical and ecological factors need be considered. For this reason, most research on tropical evolutionary biogeography, both in Madagascar and in other areas of the world, is largely based on a limited number of taxa, primarily of vertebrates and woody-plants. Only recently, has it been possible to assemble much relevant data on the patterns of geographical distribution, differentiation, and life history of Malagasy scorpions (Lourenço 1996c, Lourenço & Cloudsley-Thompson 1998). In this respect, Malagasy scorpions are still relatively poorly known compared with those of other parts of the world such as the Neotropics (Lourenço 1991a,b, 1994). In this paper my aim is to present a clear view of the diversity, importance and endemic levels of Malagasy scorpions, and to indicate their possible utilization in conservation programs. Furthermore, I will attempt to illustrate how studies of scorpion populations can contribute to an emerging consensus as to which of the Malagasy biomes should be preserved. Diversity and endemism of the scorpion fauna of Madagascar A total of four families, 11 genera and 39 species have been listed from Madagascar (Lourenço 2000a,b). From this total number the level of endemism is extremely important with: * two endemic families (Heteroscorpionidae and Microcharmidae) = 50% * 10 endemic genera (Opisthacanthus is the only genus with an extralimital distribution) = 91% * 39 endemic species = 100% From this proposed table, a comparative analysis can be presented of the diversity and endemicity of the scorpion fauna of Madagascar in relation with other well-studied

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Fig. 1. Graph showing the progression of the knowledge of the Madagascan scorpion fauna in different periods.

regions of the world with similar surface areas. These values indicate that the scorpion fauna of Madagascar has a moderate level of diversity. The total number of native species (39) is low when compared to Baja California (61), or with countries having a much smaller surface area, such as Ecuador (36). The number of families (4) is also low; but other countries such as Colombia also has only 4 families, and the same holds for Brazil which has a surface area 16 times larger than Madagascar. The total number of genera in Madagascar, however, is significantly greater (11) when compared with that of Ecuador (8), or even of Baja California (11). Even Brazil has a number of genera only slightly greater (16) than Madagascar. Moreover, the species inventory of Baja California and Ecuador is more complete than in Madagascar. As far as endemicity is concerned, the picture is quite different. Madagascar is a region of the world, which possesses a remarkable level of endemic scorpion species. Of the 39 native species of scorpions all are endemic to Madagascar. Ten genera out of 11 are also endemic to Madagascar. The percentages of endemics are 91% for genera and 100% for species are the highest known in the world. Can scorpions be a useful tool in conservation programs? Although investigation on a worldwide basis of scorpion biogeography began only recently in comparison with studies of other groups (Lourenço 1996f), the data obtained have been remarkably consistent with the general pattern observed in other taxa.

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Malagasy scorpions exhibit a very high level of endemism within quite localized areas (endemic centers). For instance, with the exception of Grosphus madagascariensis (Gervais) (the most common scorpion in the island), no species of scorpion occurs in both the northern and southern portions of the island. Moreover, no species is known from both the eastern and western coastal regions. Furthermore, certain areas are of particular importance for their levels of endemics: These are: in the north — Sambirano, Nosy Be, Marojejy, Anjanaharibe-Sud and the Montagne d’Ambre. In the south-west: Toliara region. In the south- east: Tolagnaro region, Andohahela. In the Central Highlands: Ankaratra.

Fig. 2. Major areas of scorpion diversity and endemicity in Madagascar.

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Conclusions Although current knowledge on Malagasy scorpions can be considered preliminary, the following characteristics of this fauna can be suggested: 1. The majority of taxa found in Madagascar correspond with primitive or archaic lineages that no longer exist in most other regions of the world. 2. Most genera are not very speciose. However, it is reasonable to expect the future discovery of several new species in some genera of microscorpions. 3. The number of recorded species shows a moderate level of diversity for the island; however, many areas of the island have yet to be inventoried. 4. The total number of genera is significant, even when compared with other well-studied regions of the world. 5. The most remarkable characteristic of the scorpion fauna of Madagascar is the impressive level of endemicity, both in species and in genera. This supports the hypothesis of the very early isolation of the island from other landmasses. The natural habitats on Madagascar include a large number of biomes. The biological diversity within these habitats is among the highest in the world, both for plants and animals. In recent years, studies of species diversity have evolved in parallel with the application of conservation programs. A basic understanding of the biodiversity in the different biomes of Madagascar is now available. Since it is highly likely that only a small portion of the remaining biomes will be effectively protected, future studies should be directed towards the identification of those areas that present the highest diversity so that they can be selected for conservation. These studies should provide the data necessary for determining the location, size and number of conservation units (parks and reserves) to be designated, so that the biodiversity of Madagascar can be, at least, partially preserved. In recent years, the distributions of several species of scorpions have been defined and centers of endemism identified. Scorpions appear to provide an important and useful model (in the sense of Noss 1990) in multidisciplinary programs directed toward the understanding of patterns of biodiversity and evolutionary biogeography. Acknowledgements In this chapter, I benefited from the comments of Dr. Steven M. Goodman, Field Museum of Natural History, to whom I express all my thanks. References Birula A. 1903. Sur une nouvelle espèce de scorpions provenant de l’isle Madagascar. Annals Museum St. Petersburg 7(1): 10-11. Fage L. 1929. Les Scorpions de Madagascar. Faune des Colonies françaises 3. Société d’Editions Géographiques Maritimes Coloniales, Paris, pp. 637-694. Fage L. 1946. Complément à la faune des Arachnides de Madagascar. Bulletin du Muséum National d’Histoire Naturelle, Paris, 2è sér. 18(3): 256-267.

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Ganzhorn J.U., Rakotosamimanana B., Hannah L., Hough J., Iyer L., Olivieri S., Rajaobelina S., Rodstrom C. & G. Tilkin 1997. Priorities for biodiversity conservation in Madagascar. Primate Report 48-1: 1-81. Gervais P. 1844. Remarques sur la famille des Scorpions. Archives du Muséum d’Histoire Naturelle, Paris 4: 201-240. Goodman S.M. & B.D. Patterson (eds) 1997. Natural Change and Human Impact in Madagascar. Smithsonian Institution Press, Washington, D.C. 432p. Kraepelin K. 1894. Revision der Scorpione. II. Scorpionidae und Bothriuridae. Jahrb. Hamburg. Wissensch. Anst. 11: 1-248. Kraepelin K. 1896. Neue und Weniger bekannte Scorpione. Jahrb. Hamburg Wissensch. Anst. 13: 121-146. Kraepelin K. 1901. Ueber einige neue Gliederspinnen. Abh. Geb. Naturwiss. 16: 3-17. Legendre R. 1972. Les Arachnides de Madagascar. In Richard-Vindard G. & R. Battistini (eds), Biogeography and Ecology in Madagascar. Monographiae biologicae, Dr. W. Junk B.V., publ., The Hague, pp. 427-457. Lourenço W.R. 1991a. La « Province » biogéographique guyanaise; étude de la biodiversité et des centres d’endémisme en vue de la conservation des patrimoines génétiques. C. R. Soc. Biogéogr. 67: 113-131. Lourenço W.R. 1991b. Biogéographie évolutive, écologie et les stratégies biodémographiques des Scorpions néotropicaux C.R. Soc. Biogéogr. 67: 171-190. Lourenço W.R. 1994. Biogeographic patterns of tropical South American scorpions. Stud. Neotr. Fauna Envir. 29(4): 219-231. Lourenço W.R. 1995. Description de trois nouveaux genres et quatre nouvelles espèces de Scorpions Buthidae de Madagascar. Bulletin du Muséum National d’Histoire Naturelle, Paris, 4e sér. 17(1-2): 95-106. Lourenço W.R. 1996a. Microcharmus hauseri, nouvelle espèce de Scorpion de Madagascar (Scorpiones, Buthidae). Rev. suisse Zool. 103(2): 319-322. Lourenço W. R. 1996b. A new species of Tityobuthus from Madagascar (Scorpiones, Buthidae). Bolletino del Museo Regionale di Scienze Naturali, Torino 14(1): 267-273. Lourenço W.R. 1996c. Scorpions (Chelicerata, Scorpiones). Faune de Madagascar. M.N.H.N., Paris. 102p. Lourenço W.R. 1996d. Origins and affinities of the scorpion fauna of Madagascar. In Lourenço W.R. (ed.), Biogéographie de Madagascar. Editions de l’ORSTOM, Paris, pp. 441-455. Lourenço W.R. (ed.) 1996e. Biogéographie de Madagascar. Editions de l’ORSTOM, Paris. 588p. Lourenço W.R. 1996f. The biogeography of scorpions. Rev. suisse Zool. Vol. hors sér. 437-448. Lourenço W.R. 1997. Another new species of Tityobuthus from Madagascar (Scorpiones, Buthidae). Entomologische Mitteilungen aus dem Zoologischen Museum Hamburg 12(155): 147-151. Lourenço W.R. 1998a. Description of a new species of scorpion from the Réserve Spéciale d’Anjanaharibe-Sud, Madagascar. Fieldiana: Zoology, new series 90: 69-72. Lourenço W.R. 1998b. Une nouvelle famille est nécessaire pour des microscorpions humicoles de Madagascar et d’Afrique. Comptes Rendus de l’Académie des Sciences, Paris, Sciences de la Vie 321: 845-848. Lourenço W. R. 1999a. Un modèle de distribution géographique présenté par les scorpions du genre Microcharmus Lourenço, avec la description d’une nouvelle espèce. Comptes Rendus de l’Académie des Sciences, Paris, Sciences de la Vie 322: 843-846.

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Lourenço W.R. 1999b. A new species of Grosphus Simon (Scorpiones, Buthidae), the first record of an intertidal scorpion from Madagascar. Entomologische Mitteilungen aus dem Zoologischen Museum Hamburg 13(161): 133-138. Lourenço W.R. 2000a. More about the Buthoidea of Madagascar, with special references to the genus Tityobuthus Pocock (Scorpiones, Buthidae). Rev. suisse Zool. 107(4) : 721-736. Lourenço W.R. 2000b. Un nouveau genre de scorpion malgache, maillon possible entre les Microcharmidae et les Buthidae. Comptes Rendus de l’Académie des Sciences, Paris, Sciences de la Vie 323(10): 877-881. Lourenço W.R. & J.L. Cloudsley-Thompson 1998. Notes on the ecology and postembryonic developmment of Grosphus flavopiceus Kraepelin, 1901 (Scorpiones, Buthidae) from the Ankarana Mountain range in the north of Madagascar. Biogeographica 74(4): 183-187. Lourenço W.R. & S.M. Goodman 1999a. Taxonomic and ecological observations on the scorpions collected in the Réserve Naturelle Intégrale d’Andohahela, Madagascar. Fieldiana: Zoology, new series 94: 149-153. Lourenço W.R. & S.M. Goodman 1999b. Taxonomic and ecological observations on the scorpions collected in the Forest of Ankazomivady-Ambositra and on the « RS d’Ivohibe », Madagascar. Revista de Biologia Tropical 47(3): 475-482. Millot J. 1949. Revue générale des Arachnides de Madagascar. Mémoires de l’Institute Scientifique de Madagascar, Sér. A 1(2): 137-155. Noss R.F. 1990. Indicators for monitoring biodiversity: a hierarchical approach. Conservation Biology 4: 355-364. Paulian R. 1961. La zoogéographie de Madagascar et des îles voisines. Faune de Madagascar 13: 1-484. Pocock R.I. 1889. Notes on some Buthidae, new and old. Annals and Magazine of Natural History, ser. 6, 3: 334-351. Pocock R.I. 1890. A revision of the genera of scorpions of the family Buthidae, with descriptions of some South-African species. Proceedings of the Zoological Society, London 11: 114-141. Pocock R.I. 1893. Notes on the classification of Scorpions followed by some observations upon synonymy, with descriptions of genera and species. Annals and Magazine of Natural History ser. 6, 12: 303-330. Pocock R.I. 1896. Notes on some Ethiopian species of Ischnurinae contained in the collection of the British Museum. Annals and Magazine of Natural History ser. 6, 17: 312-319. Roewer F.C. 1943. Über eine neuerworbene Sammlung von Skorpionen des Natur-Museums Senckenberg. Senckenbergiana 26(4): 205-244. Vachon M. (ed.) 1953. Contribution à l’étude du peuplement de Madagascar. Mém. Soc. Biogéographie, nouv. sér. 1: 358p. Vachon M. 1969. Grosphus griveaudi, nouvelle espèce de Scorpion Buthidae Malgache. Bulletin du Muséum National d’Histoire Naturelle, Paris 2è sér. 4(2): 476-483. Vachon M. 1979. Remarques biogéographiques sur la faune des Scorpions de Madagascar à propos de l’utilisation de caractères trichobothriotaxiques permettant la distinction des genres Odonturus Karsch, 1879 et Tityobuthus Pocock, 1893. Comptes-Rendus du Vème Colloque d’Arachnologie d’Expression Française, Barcelona: 217-224.

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Biogeographic relations and life history characteristics of vertebrate communities in littoral forests of Madagascar J.U. Ganzhorn1, S.M. Goodman2, J.-B. Ramanamanjato3, D. Rakotondravony4 & B. Rakotosamimanana5 1. Zoologisches Institut, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany. E-mail: [email protected] 2. Field Museum of Natural History, Chicago IL 60605, USA and WWF Madagascar, B.P. 738, Antananarivo (101), Madagascar. E-mail: [email protected] 3. QIT Madagascar Minerals, B.P. 225, Tolagnaro (614), Madagascar 4. Département de Biologie Animale, B.P. 906, Université d’Antananarivo, Antananarivo (101), Madagascar 5. Département d’Anthropologie, B.P. 906, Université d’Antananarivo, Antananarivo (101), Madagascar

Abstract The littoral forests of Madagascar resting on sandy soils have been classified as an ecosystem that is distinct from the evergreen eastern rain forests generally growing on lateritic soils. There is no endemic land vertebrate species to the eastern littoral forest. Nevertheless, the vertebrate communities of the littoral forests are distinct from the communities of the eastern rain forest belt. For lemurs, the littoral forest communities represent subsets of the communities in the nearby eastern rain forest. For lipotyphlans, rodents, birds, and amphibians, littoral forest communities are more similar between sites, located several hundred kilometers from one another than to the nearest eastern rain forest site just a few kilometers away. On a functional level, lemur communities of littoral forests contain relatively fewer frugivores but more omnivorous species than eastern rain forests. Among birds, there is a higher percentage of seed-eating and omnivorous species but a lower percentage of insectivorous or vertebrate-eating species in littoral forests than in the eastern rain forest. Littoral forests have a lower percentage of terrestrial rodent species but a higher percentage of terrestrial bird and amphibian species than eastern rain forest sites. For the time being it is unclear whether these differences are due to different resource availability in littoral versus eastern rain forest sites or consequences of unspecified fragmentation effects.

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Introduction The eastern littoral forests of Madagascar correspond biogeographically to sites near sea level on sand within Madagascar’s humid bioclimatic region as defined by Cornet (1974 in Schatz 2000). Assuming that littoral forest once occupied an area averaging 3 km in width along the ca. 1,500 kilometers of coastline between Vohémar and Fort Dauphin this vegetation formation once covered an area of ca. 4,500 km2. Today, no more than 500 km2 remain, distributed in patches of a few hundred ha at best (Du Puy & Moat 1996). The original surface area of littoral forest was sufficiently large to allow evolution of species endemic to this forest type among higher plants (Rabevohitra et al. 1998, Dumetz 1999, Schatz 2000, Schatz et al. 2000). Among invertebrates, there are endemic species restricted to littoral forests, e.g. for ants (Fisher et al. 1998, Fisher & Girman 2000). Relatively few vertebrate species occur in this ecosystem and not one of them is endemic to this forest type. Nevertheless, the vertebrate species composition in littoral forests appears distinct from the communities found in the adjacent eastern rain forests, which rest on lateritic soils. In general, littoral forest vertebrate communities are composed of a reduced set of species locally specific to the nearby eastern rain forests and a small set of widespread species. The low number of vertebrate species could be an inherent characteristic of littoral forests related to resource availability, or it could be the result of recent fragmentation of the littoral forest that did not allow the more vulnerable species to persist in the remaining forest patches (Ganzhorn et al. 2000a,b). In the present paper we summarize taxonomic affinities and compare the structure of vertebrate communities of littoral and eastern rain forests on a gross functional level. We assume that these functional characteristics reflect the availability of resources and thus provide insight into a better understanding of why the littoral forest communities are distinct from the eastern rain forest. Methods Database Analyses were restricted to vertebrate taxa: amphibians, birds, rodents (only native species), lipotyphlans (tenrecs and shrews; only native species), and lemurs from littoral forests along the east coast of Madagascar. Surveys were not available for all taxa at all sites. The information available was condensed into presence-absence data. Species composition was used for the littoral forests of Mandena, Manafiafy, Tampolo, and Sahaka. Eastern rain forest sites were Andohahela (Parcel I), Andringitra, Ranomafana, Analamazaotra, Anjozorobe/Andranomay, Zahamena, Ambatovaky, Anjanaharibe-Sud, Marojejy, Masoala, and Montagne d’Ambre. Data were taken from Langrand (1990), Goodman (1996, 1998, 1999), Goodman et al. (1997), Rakotondravony and Goodman (1998), Ratsirarson and Goodman (1998), Ganzhorn et al. (2000), Goodman and Rakotondravony (2000), Ramanamanjato (2000), and Ratsirarson and Goodman (2000). These publications give details on survey sites and associated coordinates. Since not all taxa considered here were equally inventoried at all sites, different numbers of eastern rain forest sites were used depending on the taxon.

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Taxonomic affinities Species presence absence data were used to calculate Jaccard’s indices of community similarities between sites. These indices were then used in single linkage cluster analyses to illustrate similarities between littoral and eastern rain forest sites. More details are given by Ganzhorn et al. (2000b). Functional classification Species were classified based on their “life style” and diet. The “life style” categories are based on the most common way of foraging for a given species. For this we discriminated between feeding in trees (arboreal), feeding on the ground (terrestrial), or feeding in trees as well as on the ground. Dietary categories were: seed-eaters and fruit-eaters, folivores, insectivores, omnivores, and predators on vertebrates. Analyses of dietary categories were only performed for birds and lemurs. Categorizations follow Glaw and Vences (1994), Wilmé (1996), Ganzhorn (1997), Ramanamajato (2000), and Goodman et al. (unpubl.). The percentage of occurrence of these functional types was compared between littoral and eastern rain forest sites. We applied cluster analyses to illustrate taxonomic affinities, F- and t-tests or the non-parametric Mann Whitney U test for statistical analyses. All tests are two-tailed. Rerults Details of the analyses on taxonomic affinities have been published previously (Ganzhorn et al. 2000b). The results are summarized here to provide a complete picture of the taxonomic and functional affinities of the littoral forest vertebrates. Taxonomic affinities Fig.1 illustrates the taxonomic affinities of littoral forests. Amphibian, bird, and lipotyphlan communities of littoral forests are more similar among each other than to eastern rain forest sites close by. The affinities of rodent communities were not included in the figure. The littoral forests along Madagascar’s east coast surveyed so far contain only a single native species, Eliurus webbi. Thus, rodent “communities” of littoral forests are also more similar to each other than to any other site in the eastern rain forest. In contrast, lemur communities of a given littoral forest site group with the geographically closest eastern rain forest site, suggesting different colonization history for these different taxonomic groups. Life Style The percentage of terrestrial amphibian species is 15% higher in littoral forests than in the eastern rain forest (Fig.2). This difference is not quite significant (t-test: t = 2.17, df = 5, p = 0.08). When species are pooled that are found on the ground as well as in trees, the variance in the datasets is reduced, and though the mean values do not change

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Fig. 1. Taxonomic affinities between littoral forests and the closest eastern rain forest sites. Littoral forests are: Mandena, Manafiafy, Tampolo, and Sahaka. The closest eastern rain forest sites are Andohahela (for Mandena and Manafiafy), Ambatovaky (for Tampolo), and Daraina (for Sahaka); from Ganzhorn et al. (2000b).

Fig. 2. Means and standard deviations of percentages of terrestrial species in different taxa in littoral forests (open squares) and eastern rain forests (black squares).

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markedly, the difference in the percentage of terrestrial and terrestrial/arboreal species between the two types of forest becomes significant (t = 2.68, df = 5, p = 0.04). The percentage of terrestrial bird species is also higher in littoral forests than in the eastern rain forest. Again, the difference is insignificant, when based on exclusively terrestrial species (t = 1.46), but becomes significant when species are included that forage on the ground as well as in trees (t = 3.31, df = 11, p = 0.007). There is no difference in the proportion of terrestrial lipotyphlans between littoral and eastern rain forests. In contrast to the other groups considered, the percentage of terrestrial rodents is lower in littoral forests than in the eastern rain forest. This difference is highly significant (t = 3.46, df = 9, p = 0.007). The analysis of rodents should not be given too much weight because, as mentioned above, all rodent “communities” consist only of Eliurus webbi, the only endemic rodent species found in littoral forests of the east coast so far. Since modern lemur species show too little variation in this trait, similar analyses were not performed for these animals. Diet Dietary differences were analyzed only for birds and lemurs. Bird communities of the littoral forests have a higher percentage of seed/fruit-eating and omnivorous species, but lower percentages of insectivorous species and species predating on vertebrates. All these differences are significant with p < 0.01 ( t > 3.15 for all comparisons, df = 11; Fig.3).

Fig. 3. Means and standard deviations of percentages of bird species in different dietary guilds in littoral forests (open squares) and eastern rain forests (black squares).

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Fig. 4. Means and standard deviations of percentages of lemur species in different dietary guilds in littoral forests (open squares) and eastern rain forests (black squares).

In littoral forests lemur communities have significantly lower percentages of frugivores than in the eastern rain forest (t = 6.65, df = 11, p < 0.001). The percentage of omnivores is higher than in eastern rain forests (t = 2.35, df = 11, p = 0.04; Fig.4). Discussion Previous analyses have shown that the littoral forest growing along the sandy coastal plain of eastern Madagascar is taxonomically distinct from eastern rain forests on lateritic soils with respect to plants (Schatz 2000, Schatz et al. 2000), ants (Fisher et al. 1998, Fisher & Girman 2000), and vertebrates (Ganzhorn et al. 2000b). This taxonomic distinction is paralleled by functional differences in the composition of vertebrate communities. In littoral forests relatively few amphibians and bird species are arboreal and a high percentage of these communities consists of species that make some use of the ground. The small mammal communities are very impoverished in the littoral forests surveyed so far. Eliurus webbi is the only native rodent species found in these forests along the east coast. The lipotyphlan community is composed of the widespread and very generalised Tenrec ecaudatus, Setifer setosus, and Suncus madagascariensis. In Mandena, Microgale pusilla has been caught recently after several thousand trapnights (Ramanamanjato & Goodman, unpubl.). On the basis of these features it can be hypothesized that when taken separately, the arboreal as well as the terrestrial habitat offer fewer possibilities for niche diversification or resource availability in the littoral forests than in the eastern rain forest. If this were true, species that can make use of both habitats have an advantage. In support of this

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interpretation, omnivorous species are better represented in littoral forests than specialized predators or fruit-eating species, indicating also that the littoral forest does not offer possibilities for dietary specializations in the same way as the eastern rain forest. However, without detailed comparative studies on resource distributions in the different vegetation types, all interpretations must remain speculative. From a conservation point of view, the littoral forests of Madagascar are a matter of grave concern. Human activities and natural catastrophes (cyclones and fire) have reduced this forest type, so that today, no more than 500 km2 remain in total (Du Puy & Moat 1996). Their figure is based on vegetation maps of Faramalala (1995) which in turn, used satellite images obtained during the 1970s. Thus, today, the actual surface covered by this forest type is likely to be much smaller. Due to its remnant character and substantial floristic endemism, the littoral forests of Madagascar’s east coast were ranked among the habitat types of highest conservation priority in a workshop setting research and conservation priorities for Madagascar’s second environmental action plan (Rakotosamimanana & Ganzhorn 1995, Ganzhorn et al. 1997). This classification as a critical ecosystem for conservation action receives now further support from the present and previous analyses of vertebrate communities, which show that the littoral forests are also distinct with respect to taxonomic and functional characteristics (Ganzhorn et al. 2000b). Yet, despite their distinctiveness, the littoral forests are among the most highly threatened and impacted vegetation types in Madagascar, and are almost surely condemned to extinction unless immediate steps are taken to preserve them. Acknowledgements We thank the Commission Tripartite, the Direction des Eaux et Forêts, and the Association Nationale pour la Gestion des Aires Protégées for their continued support and authorizations to work at various sites in Madagascar. We would like to thank Wilson Lourenço for his invitation to join this symposium, Urs Thalmann for presenting the study, and the organizers of the congress to make all this possible. The work of the authors has been supported by the Deutsche Forschungsgemeinschaft, QIT Madagascar Minerals, Margot Marsh Biodiversity Foundation, Volkswagen Stiftung, John D. and Catherine T. MacArthur Foundation, and the National Geographic Society. References DUMETZ N. 1999. High plant diversity of lowland rainforest vestiges in eastern Madagascar. Biodiv. Cons. 8: 273-315. DU PUY D.J. & J. MOAT 1996. A refined classification of the primary vegetation of Madagascar based on the underlying geology: using GIS to map its distribution and to assess its conservation status. In Lourenço W.R. (ed.), Biogeography of Madagascar. ORSTOM, Paris, pp. 205-218. FARAMALALA M.H. 1995. Formations végétales et domaine forestier national de Madagascar. CI Washington, DEF, CNRE and FTM, Antananarivo. Scale 1: 1 000 000. FISHER B.L. & G.J. GIRMAN 2000. Biogeography of ants in eastern Madagascar. In Lourenço W.R. & S.M. Goodman (eds.), Biogeography of Madagascar. Mémoires de la Société de Biogéographie, Paris, pp. 331-344.

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FISHER B.L., RATSIRARSON H.J. & S. RAZAFIMANDIMBY 1998. Les fourmis (Hymenoptères: Formicidae). In Ratsirarson J. & S.M. Goodman (eds), Inventaire Biologique de la Forêt Littorale de Tampolo, Recherches pour le Développement. Séries Sciences biologiques ed., vol. 14. Centre d’Information et de Documentation Scientifique et Technique, Antananarivo, pp. 107-131. GANZHORN J.U. 1997. Test of Fox’s assembly rule for functional groups in lemur communities in Madagascar. J. Zool., Lond. 241: 533-542. GANZHORN J.U., GOODMAN S.M., RAMANAMANJATO J.-M., RALISON J., RAKOTONDRAVONY D. & B. RAKOTOSAMIMANANA 2000a. Effects of fragmentation and assessing minimum viable populations of lemurs in Madagascar. In Rheinwald G. (ed), Isolated Vertebrate Communities in the Tropics, Bonn. zool. Monogr. vol. 45. Museum Alexander Koenig, Bonn, 265-272. GANZHORN J.U., GOODMAN S.M., RAMANAMANJATO J.-M., RALISON J., RAKOTONDRAVONY D., RAKOTOSAMIMANANA B. & D. VALLAN 2000b. Vertebrate species in fragmented littoral forests of Madagascar. In Lourenço, W.R. & S.M. Goodman (eds.), Biogeography of Madagascar. Mémoires de la Société de Biogéographie, Paris, pp. 155-164. GANZHORN J.U., RAKOTOSAMIMANANA B., HANNAH L., HOUGH J., IYER L., OLIVIERI S., RAJAOBELINA S., RODSTROM C. & G. TILKIN 1997. Priorities for Biodiversity Conservation in Madagascar, Primate Report 48-1, Göttingen. GLAW F. & M. VENCES 1994. Amphibians and Reptiles of Madagascar, 2nd ed. Eigenverlag, Köln. GOODMAN S.M. 1996. A floral and faunal inventory of the eastern slopes of the Réserve Naturelle Intégrale d’Andringitra, Madagascar: with reference to elevational variation, Fieldiana: Zoology, New Series, vol. 85. Field Museum of Natural History, Chicago. GOODMAN S.M. 1998. A Floral and Faunal Inventory of the Réserve Spéciale d’Anjanaharibe-Sud, Madagascar: with Reference to Elevational Variation, Zoology, New Series, vol. 90. Field Museum of Natural History, Chicago. GOODMAN S.M. 1999. A Floral and Faunal Inventory of the Réserve Naturelle Intégrale d’Andohahela, Madagascar: with reference to elevational variation, Fieldiana: Zoology, new series, vol. 94. Field Museum Natural History, Chicago. GOODMAN S.M., GANZHORN J.U., OLSON L.E., PIDGEON M. & V. SOARIMALALA 1997. Annual variation in species diversity and relative density of rodents and insectivores in the Parc National de la Montagne d’Ambre, Madagascar. Ecotropica 3: 109-118. GOODMAN S.M. & D. RAKOTONDRAVONY 2000. The effects of forest fragmentation and isolation on insectivorous small mammals (Lipotyphla) on the Central High Plateau of Madagascar. J. Zool., Lond. 250: 193-200. LANGRAND O. 1990. Guide to the Birds of Madagascar. Yale University Press, New Haven. RABEVOHITRA R., WILMÉ L., LOWRY P.P.II & G.E. SCHATZ 1998. La diversité floristique et l’importance de la conservation des forêts littorales de la côte est. In Ratsirarson J. & S.M. Goodman (eds), Inventaire Biologique de la Forêt Littorale de Tampolo, Recherches pour le Développement. Séries Sciences biologiques, vol. 14. Centre d’Information et de Documentation Scientifique et Technique, Antananarivo, pp 65-99. RAKOTONDRAVONY D. & S.M. GOODMAN 1998. Inventaire Biologique, Forêt Andranomay, Anjozorobé, Recherches pour le Développement, Série Sciences biologiques, vol. 13. Centre d’Information et de Documentation Scientifique et Technique, Antananarivo. RAKOTOSAMIMANANA B. & J.U. GANZHORN 1995. Rapport final de l’Atelier Scientifique sur la Définition des Priorités de Conservation de la Diversité Biologique à Madagascar, 10-

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14 Avril 1995, Hotel Panorama, Antananarivo. Projet PRIF-FEM/GEF, ONE, Direction des Eaux et Forêts, ANGAP, PNUD, Conservation International, Antananarivo. RAMANAMANJATO J.-B. 2000. Fragmentation effects on reptile and amphibian diversity in the littoral forest of southeastern Madagascar. In Rheinwald G. (ed.), Isolated Vertebrate Communities in the Tropics, Bonn. zool. Monogr., vol. 45. Museum Alexander Koenig, Bonn, pp. 299-310. RATSIRARSON J. & S.M. GOODMAN 1998. Inventaire Biologique de la Forêt Littorale de Tampolo (Fenoarivo Atsinanana), Recherches pour le Développement. Série Sciences biologiques, vol. 14. Centre d’Information et de Documentation Scientifique et Technique, Antananarivo. RATSIRARSON J. & S.M. GOODMAN 2000. Monographie de la Forêt d’Ambohitantely, Série Sciences Biologiques, vol. 16. Centre d’Information et de Documentation scientifique et technique, Antananarivo. SCHATZ G.E. 2000. Endemism in the Malagasy tree flora. In Lourenço W.R. & S. M. Goodman (eds), Biogeography of Madagascar. Mémoires de la Société de Biogéographie, Paris, pp. 1-9. SCHATZ G.E., BIRKINSHAW C., LOWRY P.P.I.I., RANDRIANTFIKA F. & F. RATOVOSON 2000. The endemic plant families of Madagascar project: integrating taxonomy and conservation. In Lourenço W.R. & S. M. Goodman (eds), Biogeography of Madagascar. Mémoires de la Société de Biogéographie, Paris, pp. 11-24. WILME L. 1996. Composition and characteristics of bird communities in Madagascar. In Lourenço W.R. (ed.), Biogeography of Madagascar. ORSTOM, Paris, pp. 349-362.

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© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

The amphibians and reptiles of Madagascar: diversity, ... Evolution 403 The New threats Panoramaand of Animal Proc. 18th Int. Congr. Zoology, pp. 403-408, 2003

The amphibians and reptiles of Madagascar: diversity, threats and conservation perspectives F. Andreone Museo Regionale di Scienze Naturali, Sezione di Zoologia, Via G. Giolitti, 36, I-10123 Torino, Italy. E-mail: [email protected]

Abstract The diverse and speciose herpetofauna of Madagascar is presented here, taking into account its peculiar traits, endemicity and conservation priorities. Some aspects concerning safeguard are discussed in detail, especially taking into account the habitat alteration and fragmentation, the capture for the international pet-trade, and the local use as food. Actions suggested to support conservation are: (i) study of endangered species; (ii) prosecution of field surveys, (iii) assessment of the herpetological interest in protected areas, (iv) education (in collaboration with local institutions).

Introduction Amphibians and reptiles are among the most peculiar vertebrates of Madagascar, since they have a high species number and a great sensitivity to habitat alteration. Despite the almost inexorable deforestation and habitat alteration which affect Madagascar, the number of Malagasy amphibians and reptiles is continuously increasing, mainly due to the accelerated discovery and description of new taxa, in conjunction with a renewed interest in field surveys and inventories. The conservation of these organisms, therefore, is urgently needed. In this paper we shortly overview the herpetological diversity of Madagascar, with some recommendations on their conservation. The amphibian diversity Neither bufonids (toads), nor caecilians (Gymnophiona) live on Madagascar. Frogs (“sahona” or “bakaka” in Malagasy), are well represented, with more than 182 species, 179 of which are endemic to Madagascar (Glaw & Vences 2000). Although there is evidence that amphibians are declining all around the world (e.g., Houlahan et al. 2000), there is anyway the prevision of a further increase in species numbers (Hanken 1999), mainly due to new discoveries, as it just happens in Madagascar. The above-mentioned numbers are therefore to be intended as preliminary. Madagascan anurans belong to

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few families, of which they represent a particular off-shot. The most recently accepted classification assigns them to four families: Mantellidae, Ranidae, Microhylidae, and Hyperoliidae (Glaw & Vences, 1994; Vences, 2001). Although in the past the mantellids were considered as subfamily either of Ranidae or Rhacophorinae, they are now treated as a distinct family endemic of Madagascar, respectively with three subfamilies: Mantellinae, Boophinae, and Laliostominae. Mantellinae include two genera, Mantidactylus and Mantella. Mantidactylus is represented by frogs with terrestrial-scansorial (e.g., M. opiparis, M. malagasius), aquatic or semiaquatic (e.g., M. lugubris, M. majori, M. grandidieri), and arboreal habits (e.g., M. aglavei, or the species allied to M. pulcher). The peculiar genus Mantella shows several derived characters, among which a contrasting aposematic coloration associated with toxic skin, mirmecophagy and some aspects of parental care (Vences et al., 1999). The Boophinae include the single genus Boophis, which is composed by approximately forty species, breeding in forest streams or in lentic waters (e.g., Boophis tephraeomystax, B. xerophilus, B. lichenoides). The Laliostominae include Laliostoma labrosum (formerly Tomopterna labrosa), typical of dry regions of the country, and three Aglyptodactylus species, inhabiting rain and dry-forests. The “true” Ranidae from Madagascar are represented by two non-endemic species. Ptychadena mascareniensis is present also on mainland Africa and in some Mascarene islands. It is a species which colonises open habitats and ricefields. On the other hand, the huge Hoplobatrachus tigerinus was introduced to Madagascar by Arab traders, and is currently quite abundant in the NW area, with some findings referring also for the central plateau. Among the three subfamilies of Malagasy microhylids, Cophylinae and Scaphiophryninae are endemic, while the Dyscophinae are said to be related to the Oriental genus Calluella. The subfamily Scaphyophrininae includes the genera Scaphiophryne and the aquatic Paradoxophyla. Among the Cophylinae the genera Anodonthyla, Platypelis and Cophyla and Plethodontohyla are independent of running water and ponds for spawning, using water-filled tree-holes or bamboo internodes or having a water-independent direct development. The taxonomic status of Malagasy microhylids is at a very preliminary point, and for many taxa little is known about their distribution and variability. Hyperoliidae are represented by the endemic genus Heterixalus (8 species), which is closely related to the mainland African Hyperolius and to Tachynemis from Seychelles. Hyperoliids only rarely penetrate in pristine rainforests, preferring marshes, swampy areas, ricefields, and in general are savannah colonisers. The reptile diversity The current number of Malagasy species is 333 (including marine genera), 305 of which are endemic to Madagascar (Glaw & Vences 2000). Lizards are represented by Gekkonidae, Scincidae, Gerrhosauridae, Chameleonidae, and Opluridae. Chameleons find in Madagascar their greatest diversity with more than 70 known species (up to 50% of the known world species), all endemic, and including three genera: Calumma, Furcifer, and Brookesia. The Opluridae (genera Chalarodon and Oplurus) show phylogenetic affinities to “iguanids” from Central and South-America (to which they are sometimes ascribed). Gekkonidae include some of the most outstanding Malagasy lizards, namely

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the day-geckos of the genus Phelsuma, and the highly cryptic Uroplatus. The terrestrial ophidiofauna in Madagascar is composed by Typhlopidae, Colubridae, and Boidae. Malagasy Boidae (three species) were included by Kluge (1991) in a single genus (Boa), together with a fourth species (B. constrictor) from Central and South-America, but recent works of M. Vences and collaborators indicate that the Malagasy boids should retain the former classification (one Sanzinia and two Acrantophis species). Colubrids of Madagascar are adapted to a wide habitat range, with taxa from open and semi-desertic areas (e.g., Mimophis mahfalensis), and species from forest areas (e.g., Pseudoxyrhopus, Langaha, Liopholidophis). In general, anyway, Malagasy snakes occupy many ecological niches, and show a surprising similarity in terms of guild composition to snake communities of other rainforests around the world (Andreone & Luiselli 2000). Living terrestrial turtles are included in the genera Astrochelys (formerly Geochelone), Pyxis, and Kinyxis, while two extinct giant species belong to the genus Dipsochelys (Pedrono et al., 2000). Freshwater turtles include Erymnochelys madagascariensis, Pelomedusa subrufa, Pelusios subniger and P. castanoides. Finally sea turtles belong to the families Cheloniidae (genera Chelonia, Caretta, Eretmochelys, Lepidochelys) and Dermochelydae (Dermochelys coriacea), while the single crocodile species present in Madagascar is Crocodylus niloticus. Threats affecting Malagasy herpetofauna The conservation of habitats and species is evidently one of the top priorities in Madagascar. Although it is difficult to state here clear and defined strategies to follow, I limit myself to a few considerations of the most important and evident threats, which affect the herpetofauna. 1. Habitat alteration and fragmentation - This is the major conservation threat shared by large part of the Malagasy wildlife. The forest destruction is quickly followed by the disappearance of several species, due to the transformation of pristine ecosystems to a rather sterile and monotonous environment. Between these two ecological “extremes” there is a wide variety of alterations closely related to habitat destruction, among which the fragmentation of originally larger forest habitats. The surface reduction is followed by a reduced genetic exchange and, in general, by a lowering in habitat diversity. A study carried out in the RS d’Ambohitantely (Central Madagascar), a site where the original plateau-forest has been fragmented, and which now consists of hundreds of forest islands (Vallan 2000) showed that amphibian taxa with a breeding strategy independent of free water (such as certain microhylids), are present in forest fragments of 30 ha or more. The species which are rather tied to forest streams, are able to survive in even smaller fragments as a consequence of generally “stable” ecological conditions around a water body, while terrestrial habitats are more subject to climatic fluctuations (and dryness) typical of small fragments (Andreone 1999). 2. Pet trade - Reptiles (e.g., chameleons and turtles) are especially subject to legal and illegal trade. As an example we may quote the several thousands of Furcifer pardalis exported in the last years, as well as many other species of skinks, lizards, geckos (especially Phelsuma), and snakes. Concerning amphibians the rate of commercial exploitation is much lower than reptiles, and limited to a handful of attractive and coloured species. This is the case of the “famous” Mantella aurantiaca, which indeed is

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one of the most requested and attractive species. Other species are interesting for the international trade, such as those belonging to the genus Scaphiophryne, some Boophis and Mantidactylus. Of a greatest interest is the golden toad Dyscophus antongili, which is the only Malagasy amphibian included in CITES I. This species, anyway, is limited to an area around Maroantsetra, in NE Madagascar, but the limits of this extension are currently unknown. A detailed analysis of the status of the botanical and zoological species subject to international trade is currently being realised by Rahagalala & Randrianasolo (in press). 3. Bush-meat market - Differently from mainland Africa (e.g., Akani et al. 1998), in Madagascar there is no specialised and widely extended “bush-meat market”, and wild species, although subject to capture as supplementary food source, do not usually become subjects of a regular exchange or selling. Hunting associated to man-induced environmental alterations have been the most important causes for extinctions of several lemur, large mammal and bird species. Less known is the situation concerning reptiles and amphibians, since we are aware of only a few Malagasy species extinct by man (the giant tortoises Dipsochelys grandidieri and D. abrupta) (Bour 1994). This extinction was a direct result of over-hunting rather than recent climatic change (Pedrono et al. 2000). Among the existing species there is in general an utilisation of boid snakes for food, but usually only by Chinese communities. On the other hand, there is a capture at a local scale of large Mantidactylus species (M. grandidieri, M. guttulatus, M. microtympanum). Although these captures are not dangerous per se, they may accelerate local extinctions when the rainforest habitat is already disturbed. The capture of sea turtles (mainly Chelonia mydas) and freshwater turtles (e.g., Erymnochelys madagascariensis) for food is frequent at several areas of Madagascar, although the effect on the abundance of these species is not well known. According to Garcia (in preparation) E. madagascariensis appears to be sold on the markets of Ankarafantsika Reserve (NW Madagascar). On the other hand the utilization of Pelusios castanoides and Pelomedusa subrufa as food is occasional. One of the main potential threats for E. madagascariensis is the possibility of export to the Asiatic market of food. Traditional fishing techniques on lakes and rivers affect the wild populations of this species. The intensive pressure on the wetlands leaves no choice to the breeders and young turtles to escape nets and traps. On some areas (e.g. Besalampy) the egg collection is added to the other pressures. Handicraft items derived from turtles and crocodiles and traditionally sold at “zoma” (market) are frequently seen at Antananarivo. At Ankarafantsika, according to Garcia (in preparation), crocodile meat and skin are economically less interesting than the oil from the fat. This is very appreciated locally for its big range of medical use. The meat is directly consumed in the village and the skin is sometimes sold to an intermediary. Conservation recommendations Indeed amphibians and reptiles are among the most diverse vertebrates of Madagascar and their study needs much more attention, since in many cases they are key organisms in establishing conservation priorities and categorizing protected areas. It is therefore suggested that some urgent actions should be undertaken. They are listed here consequently.

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Study of endangered species - Little is known about the biology and ecological requirements of many of the species considered as endangered or included in CITES appendices. Field studies should be urgently carried out on the following taxa: (i) Mantella (distribution of little known species and in particular of M. cowani and M. bernhardi), (ii) Dyscophus antongili and D. guineti (distribution of antongili, ecology of both the species, genetic differentiation between them), (iii) Sanzinia and Acrantophis (population dynamics, ecology, genetic differentiation between the populations), (iv) Calumma and Furcifer species (in particular F. pardalis, F. verrucosus, F. oustaleti: population dynamics, structure, effect of harvesting), (v) terrestrial turtles, in particular Astrochelys yniphora, G. radiata, Pyxis arachnoides, P. laticauda (captive breeding, ecology, distribution). Field surveys - Biological inventories (especially in remote and neglected areas) are badly needed, and must be continued and supported by Malagasy and foreign organisations. Concerning amphibians it is very important that the international coordinators of the DAPTF (Declining Amphibian Task Force) concretely undertake a conservation program on these vertebrates, especially supporting (by means of international funding) long-term studies on the amphibians, up to now neglected in Malagasy conservation programs. An important step in this project is to monitor the possible population decrease in a few selected areas, with the support of Malagasy and foreign institutions and researchers. Protected areas reinforcement - “Important areas for the herpetological conservation” similarly to what has been done for birds (Projet ZICOMA 1999) might be established, taking into account on one side the existence of “peculiar” species and on the other a high species diversity. The system of protected areas in Madagascar should be consequently reinforced taking also into account the diversity richness in terms of amphibians and reptiles. Education & training - The importance of amphibians and reptiles in terms of biodiversity is little known at local scale. Educational programs (including leaflets, exhibitions, university courses) should be made, most likely with the aid of Malagasy institutions (e.g., PBZT, University, MEF, ANGAP), and NGOs (e.g., WWF, WCS, CI). An important tool for conservation through education concerns the realisation of informative material at some selected nature reserves, showing in detail the species of amphibians and reptiles present and visible there, perhaps together with a local natural history museum. Acknowledgements Special thanks to W. R. Lourenço and the organizers of the XVIIIth ICZ at Athens for having been so kind to invite me to speak about Madagascar. Then I thank all the friends who accompanied me in the field and gave me useful advice. Among others G. Aprea, F. Glaw, F. Mattioli, H. Randriamahazo, J.E. Randrianirina, D. Vallan, M. Vences. Much help was obtained from G. Garcia, who gave unpublished information. A. Carpenter provided bibliographic material as well. Thanks also to the organizations who supported my research (WCS, WWF, PBZT) and to MEF and ANGAP for permission to visit the protected areas, and for collecting and exportation permits. Last but not least I would like to acknowledge my family, whose assistance and encouragements were always welcome and necessary.

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References AKANI G.C., LUISELLI L., ANGELICI F.M. & E. POLITANO 1998. Bushmen and herpetofauna: notes on amphibians and reptiles traded in bush-meat markets of local people in the Niger Delta (Port Harcourt, Rivers State, Nigeria). Anthropozoologica 27: 21-26. ANDREONE F. 1999. Madagascan amphibians. In Yamagishi S. (ed.), Madagascar Animals - Their Wonderful Adaptive Radiations. Tokyo: Shoukabou co. ltd, pp. 213-261. ANDREONE F. & L. LUISELLI 2000. Are there shared patterns of specific diversity, abundance, and guild structure in snake communities of tropical forests of Madagascar and continental Africa? Terre et Vie (Revue d’Ecologie). BLOMMERS-SCHLOSSER R.M.A. & C.P. BLANC 1991. Amphibiens - (première partie). Faune de Madagascar 75(1): 1-379. BOUR R. 1994. Récherches sur des animaux doublement disparus: les tortues géantes subfossiles de Madagascar. Mémoire EPHE 19: 1-253. GLAW F. & M. VENCES 1994. A Fieldguide to the Amphibians and Reptiles of Madagascar. Vences und Glaw Verlags, Cologne. 480 p. GLAW F. & M. VENCES 2000. Current counts of species diversity and endemism of Malagasy amphibians and reptiles. In Lourenço W.R. & S.M. Goodman (eds.), Diversity and Endemism in Madagascar. Mémoires de la Société de Biogéographie, pp. 243-248. GLAW F., VENCES M. & W. BÖHME 1998. Systematic revision of the genus Aglyptodactylus Boulenger, 1919 (Amphibia: Ranidae), and analysis of its phylogenetic relationships to other Madagascan ranid genera (Tomopterna, Boophis, Mantidactylus, and Mantella). Journal of Zoological Systematic and Evolutionary Research 36: 17-37. HANKEN J. 1999. Why are there so many new amphibian species when amphibians are declining? Trends in Ecology and Evolution 14 (1): 7-8. HOULAHAN J.E., FINDLAY C.S., SCHIMDT B.R., MEYERS A. & S.L. KUZMIN 2000. Quantitative evidence for global amphibian population declines. Nature 404: 752-755. KLUGE A.G. 1991. Boine snake phylogeny and research cycles. Misc. Publs Mus. Zool. Univ. Michigan 178: 1-58. PEDRONO M., SAROVY A., SMITH L.L. & R. BOUR (2000). The status and conservation of endemic Malagasy chelonians: an historical perspective. In Lourenço W.R. & S.M. Goodman (eds), Diversity and Endemism in Madagascar. Mémoires de la Société de Biogéographie, pp. 249-260. PROJET ZICOMA (1999). Les Zones d’Importance pour la Conservation des Oiseaux à Madagascar. Projet ZICOMA, Antananarivo, 266 p. RAHAGALALA T. & H. RANDRIANASOLO (in press). Le statut des espèces de flore et de faune malagasy sujets au commerce international et la mise en place d’une base de données permanente. WCS Madagascar. VALLAN D. 2000. Influence of forest fragmentation on amphibian diversity in the nature reserve of Ambohitantely, highland Madagascar. Biological Conservation 96: 31-43. VENCES M. & F. GLAW 2001. When molecules claim for taxonomic change: New proposals on the classification of Old World treefrogs. Spixiana 24: 85-92. VENCES M., GLAW F. & W. BÖHME 1999. A review of the genus Mantella (Anura, Ranidae, Mantellinae): taxonomy, distribution and conservation of Malagasy poison frogs. Alytes 17(12): 3-72.

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT An Publishers integrative approach to the study of diversity and regional endemism in lemurs ... Evolution 409 The New Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 409-418, 2003

An integrative approach to the study of diversity and regional endemism in lemurs (Primates, Mammalia) and their conservation U. Thalmann Anthropological Institute, Zürich University, Winterthurerstr. 190, CH–8057 Zürich, Switzerland. E-mail: [email protected]

Abstract Madagascar, the fourth largest island on earth, is a ‘Megadiversity’ and ‘hottest Hotspot’ region for biodiversity conservation due to the combination of high diversity, endemism, and degree of threat. Lemurs, a group of primates endemic to the island, may serve as flagship and umbrella species to support conservation incentives but their diversity and distribution is insufficiently known. I first summarize new discoveries made in recent years before focusing on the zoogeographic region of central western Madagascar. I present a cladistic method that allows for a better assessment of taxon diversity and distribution, and facilitates definition of priority regions for conservation and research. A more participatory approach to promote education and research in partnership between industrialized and developing countries is proposed to enhance and anchor the cause of conservation at the provincial level in Madagascar.

Introduction The island of Madagascar has recently been identified as one of three ‘hottest Hotspot’ regions for biodiversity conservation world-wide due to the combination of high diversity, endemism, and degree of threat (Myers et al. 2000). Lemurs, a group of primates endemic to Madagascar, are among the most famous and conspicuous animals on this island. With 33 (Garbutt 1999) to 35 (Rumpler & Ravoarimanana 2000) already recognized extant species, six new additional species and 5 resurrected synonyms (Groves 2000, Rasoloarison et al. 2000, Thalmann & Geissmann 2000), lemurs are the most diverse group of mammals on Madagascar. At least 17 additional lemur species have gone extinct in late Pleistocene/Holocene time (Godfrey et al. 1999), and with these already 2 to 3 entire families or subfamilies, respectively (Tattersall 1982, Garbutt 1999). All extinct forms were large. The largest may have reached a body mass of 160 - 200 kg (e.g., Mittermeier et al. 1994).

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Available evidence of climatic conditions since the end of the Pleistocene does not indicate that Madagascar’s fauna was under unprecedented environmental stress in this period. It is thus impossible to avoid the conclusion that human activity following the first incursion onto the island some two thousand years ago played a critical role in the elimination of several dozens of mammal and other animal species of larger body size than their surviving relatives (Tattersall 1999). Despite the disastrous rate of ongoing environmental destruction that menaces Madagascar’s unique fauna and flora, new discoveries of animals and plants are being reported at an increasing rate, including those of new mammals. Here, I first summarize some recent discoveries relating to lemurs, and evaluate the reasons for this development and the problems that arise when assessing the diversity of lemurs. I then focus on the lemur diversity of central western Madagascar, a region of high lemur species richness (Fig. 1). In a third part I highlight and emphasize the pressing need for enhanced research and education in situ - in Madagascar. I present a conceptual approach for a Research Partnership Program focusing on lemurs with the provincial University of Mahajanga in northwestern Madagascar (Fig. 1).

Fig. 1. Central western Madagascar.

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Recent Discoveries and Developments In 1987, the Golden bamboo lemur (Hapalemur aureus Meier et al., 1987) was described, followed one year later by the Golden crowned or Tattersall’s sifaka (Propithecus tattersalli Simons, 1988). Both species are of medium size, and at least Tattersall’s sifaka is fully diurnal. Unsurprisingly, new discoveries and distribution refinements have also been achieved for nocturnal lemurs. The enigmatic nocturnal Aye-aye (Daubentonia madagascariensis E. Geoffroy, 1795), once thought to be one of the rarest primates world-wide has now been reported from many localities including protected areas. Other populations of nocturnal lemurs, the small and rare Hairyeared dwarf lemur [Allocebus trichotis (Günther 1875). Meier & Albignac 1991, Rakotoarison 1998, Schütz & Goodman 1998] and the smallest known primate, a pygmy mouse lemur, (Schmid & Kappeler 1994), were also re-discovered. An isolated population of Woolly lemurs (Avahi Jourdan, 1834) still awaits description (Thalmann & Geissmann 2000). A detailed examination of museum specimens has revealed several sub-species of Phaner furcifer (Blainville, 1870) (Groves & Tattersall 1991), and karyotype analyses have demonstrated the existence of several subspecies of Lepilemur septentrionalis Rumpler & Albignac, 1975 (Rumpler & Ravoarimanana 2000). Most recently, Microcebus ravelobensis has been described (Zimmermann et al. 1998), and in Volume 21, Issue 6 of the International Journal of Primatology a new woolly lemur (Avahi unicolor Thalmann & Geissmann, 2000), two new Dwarf lemurs (Cheirogaleus minusculus and C. ravus Groves, 2000), three Mouse lemurs (Microcebus berthae, M. tavaratra, M. sambiranensis Rasoloarison et al., 2000). Five synonyms have been resurrected as valid taxa (Groves 2000, Rasoloarison et al. 2000). Many new findings concern nocturnal lemurs. This is not surprising since they are on average smaller than diurnal species (Martin 1990), and exhibit less conspicuous differences in their external morphology (e.g. Groves & Tattersall 1991) making taxon identification based on field sightings particularly difficult, or impossible. Despite powerful genetic methods, important problems of assessing lemur diversity still persist. This is demonstrated by the selected cases given in Table 1. Distinction of Table 1. Distinction between different lemur taxa.

Distinction between Propithecus verreauxi coquereli Propithecus tattersalli Cheirogaleus sp. Microcebus sp. Lepilemur edwardsi Lepilemur ruficaudatus 1 2 3 4 5

Supported based on...? Morphology Karyotype mtDNA yes yes1 no2 yes

no3

yes2

'no'4

yes5

yes2

Poorman 1983, Simons 1988. Pastorini 2000. Petter et al. 1977. Morphological differences suggest subspecies distinction (Tattersall 1982). Ishak et al. 1992, Bachmann et al., in press.

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Propithecus verreauxi coquereli Milne-Edwards, 1867 and P. tattersalli is highly suggestive based on external morphology and karyotype (Poorman 1983, Simons 1988), but is not supported by mitochondrial DNA (mtDNA) analysis (Pastorini 2000). For the nocturnal genera Cheirogaleus and Microcebus distinction is evident based on morphology and mtDNA (Petter et al. 1977, Pastorini 2000) but karyotypes are indistinguishable (Petter et al. 1977). The two species Lepilemur edwardsi Forsyth Major, 1894 and L. ruficaudatus A. Grandidier, 1867 can hardly be distinguished morphologically (Tattersall 1982), but are distinguishable based on karyotype and mtDNA (Ishak et al. 1992, Bachmann et al. 2000). An assessment of the diversity of lemurs requires the combination of ‘classic’ and modern methods for the adequate identification of localized lemur populations. Both a combination of field research and sampling of material for karyotype and genetic laboratory analysis, as well as the re-evaluation of museum collections are necessary. New discoveries and developments can be attributed to the combination of four main factors: 1) Intensified field research, including surveys in remote regions of difficult access. This was favored by a political opening of Madagascar in the early eighties. 2) Increased application of comprehensive methods to characterize populations encompassing morphology, behavior, and different genetic methods. 3) Re-evaluation of museum collections. 4) Increased application of operational concepts for taxon recognition by field biologists (e.g. Phylogenetic Species Concept, Groves 2001). Lemurs in Central Western Madagascar Central western Madagascar (Fig. 1), bounded by the west coast, the Tsiribihina River to the south, the Betsiboka River to the north, and the central highlands, is a distinct zoogeographic region within Madagascar (Martin 1972, 1995). Ten years ago it was still one of the least researched areas of Madagascar, even though several reserves of different protection status are located within this region. While only 7 lemur species were reported from the Strict Nature Reserve and National Park of Bemaraha in 1989 (Nicoll & Langrand 1989), at least 12 species are known to occur in this reserve today (Table 2). However, the systematic status of several of the reported populations remains problematic. Improved survey data and the advent of cladistic analysis software (e.g. PAUP 4.0, Swofford 1998) provide a fruitful basis for improved biogeographic analyses and an extended application to conservation biology. In essence, cladistic biogeography uses the same approach as cladistic phylogeny but instead of maximizing character congruence (minimizing homoplasy) between taxa (clades), species congruence between localities (clades) is maximized (minimizing dispersal events). In biogeography, the method is also known as Parsimony Analysis of Endemism (Rosen 1988, Forey et al. 1992, Humphries & Parenti 1999). I have applied cladistic biogeography in an extended form allowing for predictions of the probable systematic status of lemur populations. From the literature and my own surveys I derived a present/absent data matrix for lemurs for 28 predominantly western localities. I then varied the systematic status of problematic taxa in the matrix to find a more congruent biogeographic pattern. For example: In the literature it is indicated that Lepilemur edwardsi is present in central western Madagascar north of the Tsiribihina

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Table 2. Lemurs reported from the National Park and Strict Nature Reserve Bemaraha (Fig. 1) in the years 1989 and 2000.

19891 Microcebus murinus

Mirza coquereli Phaner furcifer Hapalemur griseus occidentalis Lepilemur edwardsi Eulemur fulvus rufus Propithecus verreauxi deckeni

1 2

20002 Microcebus murinus Microcebus myoxinus Cheirogaleus medius Cheirogaleus cf. major Mirza coquereli Phaner furcifer cf. pallescens Hapalemur griseus cf. occidentalis Lepilemur ? ruficaudatus Avahi n. sp. Eulemur fulvus rufus Daubentonia madagascariensis Propithecus verreauxi deckeni ? 3rd Microcebus species ? Eulemur mongoz

Nicoll & Langrand 1989. for references see Thalmann 2000.

River (Mittermeier et al. 1994), while L. ruficaudatus occurs south of the Tsiribihina. It is not known though which species actually occurs between the rivers Manambolo and Tsiribihina (Tattersall 1982, Garbutt 1999). Replacing Lepilemur edwardsi at all localities in central western Madagascar (i.e., north of the Tsiribihina River) with L. ruficaudatus results in a more parsimonious cladogram. This suggests that Lepilemur ruficaudatus occurs also across the Tsiribihina River at least as far north as the Manambolo River. Incidentally, this has been confirmed independently through karyotype and DNA analysis by Bachmann et al. (2000). It is not predictable though where the northern distribution limit is to be expected. Recent mtDNA data indicate, that an undescribed species of Lepilemur may be present south of the Mahavavy River (Pastorini 2000). Empirical evidence, thus confirms the suitability of the applied method which is explained in more detail elsewhere (Thalmann 2000). In addition, cladistic biogeography analysis allows for inferences to be made regarding the causes for the absence of expected species in certain localities: Absence is either due to local extinction caused by various factors (e.g. hunting, habitat destruction) or unsuitable ecological conditions, and in some cases it is due to incomplete surveys. The latter case may especially apply to nocturnal and/or behaviorally cryptic species, which may easily be overlooked. The advantage of this method is that we can derive founded hypotheses regarding the diversity and distribution of lemurs which allow for predictions to be made that can then be tested. Thus, we may approach the ‘real’ diversity, distribution and biogeographic pattern for lemurs through an iterative process. Although this method further facilitates focus of research endeavors on certain problematic lemur populations and regions, we should remain alert to entirely new discoveries. The method is based on what we believe to know. We cannot know what unpredictable surprises may await us in the field.

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Based on empirical evidence, cladistic biogeography is a powerful tool for conservation biology. It allows us to recognize regions or localities of high endemism, to predict the systematic status of problematic populations, and to evaluate the accuracy of surveys. However, the theoretical background to the method needs further investigation before we can fully understand why it actually works, and where its limitations are. Most forests in western Madagascar are of the deciduous seasonally dry type (Du Puy & Moat 1996). Locally, especially rich subhumid and riverine forests exist (Rakotoarison et al. 1993, Müller et al. 2000). Many of these forest types have already disappeared or are degraded. With its species richness and mixture of a typical western lemur community combined with the conserved influence of the central highlands (Thalmann 2000), central western Madagascar merits special attention as a region of high lemur diversity, and very likely biodiversity in general. Conservation: Research and Education in Partnership In the preceding sections I have outlined some new discoveries on lemurs that have been made despite the disastrous rate at which forests in Madagascar are disappearing. To contribute to the exploration, discoveries and research relating to the natural history of Madagascar, a diversity of skills and approaches are needed. These include the development of research projects to seek funding for actual realization, data analysis, the communication and publication of results, and to make eventual inferences for conservation. Apart from the necessary means and research facilities a considerable know-how is required. This know-how is not as widely distributed in Madagascar as it should and could be. A pilot teaching project ‘Introduction to Physical Anthropology/Primatology’ conducted at a provincial university in Madagascar (Thalmann & Zaramody 1999) revealed that 4th-year students of Natural Sciences had a surprisingly feeble knowledge of the precious wildlife on Madagascar. Many students indicated lemurs for any continent, one also for Antarctica. Students were aware though, that forests are ecologically important, that they benefit from forests, and that forests in Madagascar are threatened. Students participating in the lecture came from almost all over Madagascar. Hence, improved knowledge and skills would potentially be spread across the island (Thalmann & Zaramody 1999). Of course, the problems of immediate concern in Madagascar are much more of a daily-life and socio-economical rather than academic nature. However, skills acquired in scientific methodology are to some degree transferable and applicable to a wide variety of topics. An ecology-training program of the World Wide Fund for Nature (WWF) in the capital Antananarivo produced encouraging results. 26 out of 28 students who finished the program have obtained jobs in the conservation sector (S.M. Goodman personal communication). I have developed a concept for a Research Partnership (sensu KFPE 1998) addressing research, research capacity and institution building, and especially emphasizing transversal aspects that are of general importance (Fig. 2): Planning, developing and realizing projects, introduction and application of Information Technology to open-up

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Fig. 2. Conceptual approach for a Research Partnership with a Developing Country.

possibilities for international communication and co-operation, and use of information available on the Internet, to account for modern developments. Lemurs are perfectly suited for research and education in a Research Partnership. They are fancy research subjects in the north and favorite zoo animals that attract much attention and may serve as flagship species for conservation incentives in Madagascar (Durbin 1999) and internationally (WWF 1999). They can facilitate fund-raising for and by Malagasy students and scientists. As forest dwelling animals they may in addition serve as umbrella

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species. Simply said: Lemur conservation equals forest conservation, and thus conservation of the less conspicuous wildlife. The crucial role of forests in preventing erosion, regulating the water household, and providing other goods is well known. The first milestone on the way to such a partnership will be the joint and fully participatory development of the program in Madagascar (Fig. 2: Project 1). If this preparatory phase produces promising results (commitment, engagement, positive administrative environment for the Research Partnership) and funding becomes available, the actual program can be realized. If successful, the approach could then serve as a model for replication. However, many obstacles, which are not directly linked to research on lemurs, have to be overcome first before a costly Research Partnership can be successfully implemented. The loss of Madagascar’s wildlife would amount to the loss of about 3% of worldwide biodiversity but would be one of the highest possible losses per surface unit (Myers et al. 2000). Undoubtedly, the planet earth would be poorer without lemurs but would continue to exist. For Madagascar though, the loss of its adapted and highly endemic special biodiversity would almost be total. This should always be kept in mind by the policy and decision makers concerned. Acknowledgment Special thanks go to Wilson Lourenço for the invitation to contribute to the symposium on Madagascar, and the organizing committee of the congress. Without the most appreciated support of Bob Martin ground laying fieldwork in Madagascar would have been impossible. I thank Alexandra Müller, Christophe Soligo, Wilson Lourenço and an anonymous reviewer for helpful comments on the manuscript. I am grateful for financial support by the Société Suisse d’Anthropologie and the A.H. Schultz Foundation. References BACHMANN L., RUMPLER Y., GANZHORN J.U. & J. TOMIUK 2000. Genetic differentiation among natural populations of Lepilemur ruficaudatus. Int. J. Primatol. 21: 853-864. DU PUY D.J. & J. MOAT 1996. A refined classification of the primary vegetation of Madagascar based on the underlying geology: using GIS to map its distribution and to assess its conservation status. In Lourenço W.R. (ed), Biogéographie de Madagascar. ORSTOM, Paris, pp. 205218, 3 maps. DURBIN J.C. 1999. Lemurs as flagships for conservation in Madagascar. In Rakotosamimanana B., Rasamimanana H., Ganzhorn J.U. & S.M. Goodman (eds), New Directions in Lemur Studies. Kluwer Academic/Plenum Publishers, New York, pp. 269-281. FOREY P.L., HUMPHRIES C.J., KITCHING I.J., SCOTLAND R.W., SIEBERT, D.J. & D.M. WILLIAMS (eds) 1992. Cladistics: A Practical Course in Systematics. Clarendon Press, Oxford. 191p. GARBUTT N. 1999. Mammals of Madagascar. Pica Press, Sussex. 320p. GODFREY L.R., JUNGERS W.L., SIMONS P.S. & B. RAKOTOSAMIMANANA 1999. Past and present distribution of lemurs in Madagascar. In Rakotosamimanana B., Rasamimanana H., Ganzhorn J.U. & S.M. Goodman (eds), New Directions in Lemur Studies. Kluwer Academic/ Plenum Publishers, New York, pp. 19-53.

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GROVES C.P. 2000. The genus Cheirogaleus: unrecognised biodiversity in dwarf lemurs. Int. J. Primatol. 21: 943-962. GROVES C.P. 2001. Primate Taxonomy. Smithsonian Institution Press, Washington. 368p. GROVES C.P. & I. TATTERSALL 1991. Geographical variation in the fork-marked lemur, Phaner furcifer (Primates, Cheirogaleidae). Folia Primatol. 56: 39-49. HUMPHRIES C.J. & L.R. PARENTI 1999. Cladistic Biogeography (2nd ed.). Oxford University Press, Oxford. 187p. ISHAK B., WARTER S., DUTRILLAUX B. & Y. RUMPLER 1992. Chromosomal rearrangements and speciation of sportive lemurs (Lepilemur species). Folia Primatol. 58: 121-130. KFPE (Swiss Commission for Research Partnerships with Developing Countries) 1998. Guidelines for Research in Partnership with Developing Countries. KFPE, Berne. 56p. (Available in E/ F/G via www.kfpe.unibe.ch) MARTIN R.D. 1972. Adaptive radiation and behaviour of the Malagasy lemurs. Phil. Trans. Royal Soc. London 264(B): 295-352. MARTIN R.D. 1990. Primate Origins and Evolution: A Phylogenetic Reconstruction. Chapman and Hall, London. 804p. MARTIN R.D. 1995. Prosimians: From obscurity to extinction? In Alterman L., Doyle G.A. & M.K. Izard (eds), Creatures of the Dark: The Nocturnal Prosimians. Plenum Press, New York, pp. 535-563. MEIER B. & R. ALBIGNAC 1991. Rediscovery of Allocebus trichotis Günther 1875 (Primates) in northeast Madagascar. Folia Primatol. 56: 57-63. MITTERMEIER R.A., TATTERSALL I., KONSTANT W.R., MEYERS D.M. & R.B. MAST 1994. Lemurs of Madagascar. Conservation International, Washington DC. 356p. MÜLLER P., VELO A., RAHELARISOA E.-O., ZARAMODY A. & D.J. CURTIS 2000. Surveys of five sympatric lemurs at Anjamena, north-west Madagascar. African J. Ecol. 38: 248-257. MYERS N., MITTERMEIER R.A., MITTERMEIER C.G., FONSECA G.A.B. & J. KENT 2000. Biodiversity hotspots for conservation priorities. Nature London 403: 853-858. NICOLL M.E. & O. LANGRAND 1989. Madagascar: Revue de la Conservation et des Aires Protégées. WWF, Gland (Switzerland). 374p. PASTORINI J. 2000. Molecular Systematics of Lemurs. PhD thesis, University of Zürich, Zürich (Switzerland). 182p. PETTER J.-J., ALBIGNAC R. & Y. RUMPLER 1977. Mammifères Lémuriens (Primates Prosimiens). ORSTOM/CNRS, Paris. 513p. POORMAN P.A. 1983. The banded chromosomes of coquerel’s sifaka, Propithecus verreauxi coquereli (Primates, Indriidae). Int. J. Primatol. 4: 419-425. RAKOTOARISON N. 1998. Recent discoveries of the Hairy-eared dwarf lemur (Allocebus trichotis). Lemur News 3: 21. RAKOTOARISON N., MUTSCHLER T. & U. THALMANN 1993. Lemurs in Bemaraha (World Heritage Landscape, western Madagascar. Oryx 27: 35-40. RASOLOARISON R., GOODMAN S.M. & J.U. GANZHORN 2000. A taxonomic revision of mouse lemurs (Microcebus) occurring in the western portion of Madagascar. Int. J. Primatol. 21: 963-1019. ROSEN B.R. 1988. From fossils to earth history: Applied historical biogeography. In Myers A. & P. Giller (eds), Analytical Biogeography: an Integrated Approach to the Study of Animal and Plant Distribution. Chapman Hall, London, pp. 437-481.

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RUMPLER Y. & B. RAVAOARIMANANA 2000. Cytogenetic and molecular studies are necessary preliminaries for lemur conservation. In Lourenço W.R. & S.M. Goodman (eds), Diversity and Endemism in Madagascar. Mémoires de la Société de Biogéographie, Paris, pp. 181-189. SCHMID J. & P.M. KAPPELER 1994. Sympatric mouse lemurs (Microcebus spp.) in Western Madagascar. Folia Primatol. 63: 162-170. SCHÜTZ H. & S. GOODMAN 1998. Photographic evidence of Allocebus trichotis in the Réserve Spéciale d’Anjanaharibe-Sud. Lemur News 3: 21-22. SIMONS E.L. 1988. A new species of Propithecus (Primates) from northeast Madagascar. Folia Primatol. 50: 143-151. SWOFFORD D.L. 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland (Massachusetts), Software. TATTERSALL I. 1982. The Primates of Madagascar. Columbia University Press, New York. 382p. TATTERSALL I. 1999. Patterns of origin and extinction in the mammal fauna of Madagascar. In Reumer J.W.F. & J. De Vos (eds), Elephants Have a Snorkel! Papers in Honour of Paul Y. Sondaar. DENINSEA 7: 303-311. THALMANN U. 2000. Lemur diversity and distribution in western Madagascar - Inferences and predictions using a cladistic approach. In Lourenço W.R. & S.M. Goodman (eds), Diversity and Endemism in Madagascar. Mémoires de la Société de Biogéographie, Paris, pp. 191-202. THALMANN U. & T. GEISSMANN 2000. Distribution and geographic variation in the western woolly lemur (Avahi occidentalis) with description of a new species (Avahi unicolor). Int. J. Primatol. 21: 915-941. THALMANN U. & A. ZARAMODY 1999. Teaching primatology at the Université de Mahajanga - experiences, results, and evaluation of a pilot project. In Rakotosamimanana B., Rasamimanana H., Ganzhorn J.U. & S.M. Goodman (eds), New Directions in Lemur Studies. Kluwer Academic/Plenum Publishers, New York, pp. 249-268. WWF (Worldwide Fund for Nature) 1999. Dossier Madagaskar. WWF Magazin Schweiz 5/ 1999: 1-23. ZIMMERMANN E., CEPOK S., RAKOTOARISON N., ZIETEMANN V. & U. RADESPIEL 1998. Sympatric mouse lemurs in north-west Madagascar: A new rufous mouse lemur species (Microcebus ravelobensis). Folia Primatol. 69: 106-111.

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Comparative Biology of Sperm Storage in Vertebrates

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© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Sperm Storage in the Class Chondrichthyes & Class Osteichthyes 421 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 421-429, 2003

Sperm Storage in the Class Chondrichthyes & Class Osteichthyes W.C. Hamlett1, H. Greven2 & J. Schindler3 1. South Bend Center for Medical Education, Indiana University School of Medicine, B-10 Haggar Hall, Notre Dame, in 45556, U.S.A. E-mail: [email protected] 2. Institut für Zoomorphologie und Zellbiologie der Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany 3. Institut für Anatomie der Universität Regensburg, 93040 Regensburg, Germany

Abstract This brief review will address sperm storage in male and female Chondrichthyes and Osteichthyes. In Chondrichthyes depending on the species , either spermatozeugmata or spermatophores are formed in the male genital ducts. Sperm sequentially pass through the epididymis, ductus deferens and seminal vesicles. The bulk of the seminal matrix is contributed by secretions of the accessory Leydig gland. Sperm masses are stored in the seminal vesicles. Internal fertilization is effected by the insertion of a clasper and transport of the sperm mass to the female reproductive tract assisted by smooth muscle contraction of the seminal vesicles. In the placental smooth hound, Mustelus canis, sperm embed in the uterine epithelium adjacent to the former placental attachment sites. This is viewed as a transient event and does not represent sperm storage but more likely sperm activation. Sperm are found throughout gestation in the terminal zone of the oviducal gland. In male Osteichthyes, in particularly the Poeciliidae and Hemiramphidae, sperm are stored in both the efferent and main testis ducts. Internal fertilization is effected by the male gonopodium. Spermatozeugmata matrix dissolves rapidly and sperm are initially found in all parts of the female reproductive tract. In Poeciliidae, sperm are stored for short periods of time in “delle”, depressions in the ovarian epithelium above the ovary. Longer term sperm storage occurs within the cranial and caudal parts of the ovary.

Sperm storage in male Chondrichthyes In Chondrichthyans the internal reproductive organs of the male include the testes, genital ducts (including the efferent ductules, epididymis, ductus deferens, seminal vesicle) and Leydig’s gland, a modified portion of the kidney. A siphon sac occurs in sharks and an alkaline gland occurs in skates and rays. In the elasmobranch testis, supporting Sertoli cells mature in concert with a synchronously developing isogenetic

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clones of germ cells. The term spermatoblast refers to a single Sertoli cell and its associated germ cells. Many spermatoblasts together form the spermatocyst. As individual spermatozoa pass through the male genital ducts they align laterally and become associated with matrix produced largely by secretory activity of the Leydig gland, as well as minor contributions from epithelial cells of the epididymis and ductus deferens. In Heterodontus (shark) (Jones & Lin 1993) and Callorhynchus (chimaera) (Hamlett et al. 2002a), only the seminal vesicle has a muscular tunic, all other segments of the duct system convey sperm via ciliary activity. The result of association of laterally bundled sperm with matrix produces spermatozeugmata or spermatophores, depending on the type of reproduction of the particular species, which are stored in the seminal vesicles until copulation. In skates and stingrays, the paired alkaline glands are almost always distended and filled with a clear fluid. The epithelium maintains a 100-fold concentration gradient of hydroxyl ions and a 50-fold gradient of carbon dioxide from plasma to gland lumen and maintains a pH of 9.2. It appears to secrete hydroxyl ions buffered by carbon dioxide (Maren et al. 1963, Smith 1985) and has been suggested that the gland secretions neutralizes the acid urine, pH 5.8, and may be involved in sperm protection (Smith 1929) or the formation of copulatory plugs of skates (Callard 1988). In sharks, at copulation, secretions of the siphon sac gland are transported down the clasper groove to blend with spermatozeugmata. Each siphon sac opens through the apopyle to the clasper groove (Gilbert & Heath 1972). In mature animals, the sacs contain a sticky fluid secreted by goblet cells of the surface epithelium. The secretion has a pH of 5.8. Mann (1960) demonstrated that siphon sac secretion of mature S. acanthias contains a high concentration of serotonin (5-hydroxytryptamine). In sexually immature males, serotonin was absent or present only in trace. The siphons in these immature specimens contained some 200 times less serotonin than in sexually mature males. In other vertebrates, serotonin is a powerful stimulator of smooth muscle contraction. Serotonin stimulates isolated rat uterus and when administered to dogs intravenously (Mann 1960). Serotonin may play a role in copulation and ejaculation in male elasmobranchs. Mann & Prosser (1963) demonstrated that siphon sac secretion of 5-hydroxytryptamine in Squalus uterus in vitro caused uterine contractions amounts. They suggested that during copulation 5hydroxytryptamine caused uterine contractions that aid sperm transport in the female. Batoids lack a siphon sac gland but possess a clasper gland. Secretions of the clasper gland blend with alkaline gland secretions and spermatozeugmata. Contraction of the clasper gland sac forces secretion down the clasper groove where it is mixed with spermatozoa from the cloaca. Fresh clasper gland secretion is rich in muco- and glycoprotein and phospholipid and has a slightly acid pH. It is concluded that the gland secretion seals the clasper groove into a closed tube. This is supported by the fact that clasper gland secretions coagulate upon contact with seawater. This protects the semen from dilution in seawater and prevents escape before reaching the hypopyle. Other functions of the clasper gland secretions include provision of a transport medium for the sperm and lubrication to facilitate clasper insertion (Hamlett 1999) Recently, Hamlett et al. (2002a) have presented the first ultrastructural description of sperm storage and spermatophore formation in the male elephant fish, Callorhynchus milii. Jones & Hamlett (2001) have used a panel of 20 lectins to investigate glycan expression in the male genital

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ducts and spermatozeugmata in the clear nose skate, Raja eglanteria. There data shows marked changes in glycan expression along the length of the male tract, generally shown by a progressive decrease in expression. This no doubt is associated with spermatozeugmata formation and sperm nourishment. Sperm storage in female Chondrichthyes Chondrichthyes possess a unique oviducal gland that produces egg jelly, the tertiary egg envelope and harbors sperm. Analysis of the microscopic structure of oviducal glands from a variety of chondrichthyes with differing modes of reproduction has established a consistent zonation within the gland (Hamlett et al. 1998, 1999a, Hamlett & Koob 1999). From anterior to posterior there are club and papillary zones that produce egg jellies that surround the fertilized egg. These are followed by the baffle zone characterized by a series of tubular glands that produce the tertiary egg envelope as a liquid crystal polymer. This polymer is extruded between paired baffle plates as it enters transverse grooves of the gland that lead to the gland lumen. Babel (1967) remarked that the shell secreting function of the oviducal gland in Urolophus halleri has been lost with the acquisition of viviparity and Hamlett et al. (1998) determined that no baffle plates occur in Urolophus jamaicensis and that this animal does not produce an egg envelope. The last zone of the oviducal gland is the terminal zone which is the actual site of sperm storage. Metten (1939) described sperm storage in Scyliorhinus canicula as occurring in the shell secreting tubules. This is contrary to observations of Hamlett et al. (1999a, 1998). Metten examined the oviducal gland of the oviparous shark, Scyliorhinus canicula from animals in various stages of secretion of the tertiary egg envelope. He cited Hobson’s (1930) work in the skate and stated that ova were found in the upper oviducts, between the ostium and oviducal gland, whilst egg capsules were three-quarters completed. In his own observations in S. canicula, Metten reported that in fish with ova in the coelom or upper oviduct, the egg capsule was half secreted or less. He believed that fertilization and egg capsule secretion occur simultaneously and that the shell secreting tubules provided some nutrient material in the capsule substance. He noted that some sperm were incorporated into the egg capsule substance and that it hardened immediately upon leaving the glands. He also claimed that sperm in the bottom of shell secreting tubules actively secreting egg capsule material were in the process of “turning around” to exit the glands along with the egg capsule material. In studies of the same animal, Knight et al. (1996) examined the structure of the shell secreting tubules of the oviducal gland and concluded that fertilization must occur in the upper oviduct or abdominal cavity. He noted sperm in the baffle zone tubules in animals secreting egg capsule but did not notice sperm in the caudal segment of the oviducal gland corresponding to the terminal zone. He indicated that the tubules in this segment of the gland secreted sulphated and neutral mucopolysaccharides. Metten (1939) did not recognize the terminal zone but pictured a broad caudal region of the oviducal gland that he indicated had short mucous glands. This corresponds to the terminal zone. The histological organization of the oviducal gland of two other oviparous chondrichthyes have been studied. In the skate, Raja eglanteria (Hamlett et al. 1999b) and the holocephalan elephant fish, Callorhynchus milii (Smith 2001) terminal

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zone tubules are shorter than in Mustelus canis and do not sweep to the periphery of baffle zone tubules. Their surface area is large due to the width of the terminal zone, not the depth of each gland, as in viviparous species. In both Raja and Callorhynchus sperm have been observed in the terminal zone and no sperm were seen in the baffle zone. Metten’s results show incidental sperm occurrence in baffle zone tubules and sperm being purged from the tubules with secretion of egg capsule. The refilling of gland tubules may be the result of repeated inseminations. Recently, Conrath (2000) has studied various aspects of the reproductive biology in M. canis and reported observations on sperm storage. She made transverse histological sections of the caudal one-third of the oviducal gland from samples collected throughout the year and consistently found sperm in the oviducal gland, specifically the terminal zone. The fate of spermatozoa deposited within the female reproductive tract reproductive tract has been described in the smooth hound, M. canis (Hamlett et al. 2002b). Evidence of sperm-uterine association is presented as well as documentation of sperm storage specifically in the terminal zone of the oviducal gland. Immediately postpartum the placental-uterine attachment sites, now termed uterine or placental scars, begin to remodel to a mucous epithelium for the next gestational cycle. Sperm become embedded in the uterine epithelium adjacent to placental scars. Bundled sperm then occur throughout gestation in the terminal zone of the oviducal gland. Fertilization is presumed to occur in the anterior oviduct above the oviducal gland. The physiological mechanisms that mediate sperm-uterus attachment, release and storage in the terminal zone of the oviducal gland are currently under investigation. Storrie et al. (2001) reported on sperm in the oviducal gland in Mustelus antarcticus during different periods of gestation. Their evidence demonstrated sperm storage exclusively in the terminal zone, although transient occurrence of sperm was noted in other gland tubules in animals not actively secreting jelly or egg envelope. Noteworthy is the fact that terminal zone sperm were found in both mature (pregnant, non-pregnant and postpartum) and immature (prior to first ovulation) animals throughout the year. Efforts are being directed at elucidating whether terminal zone sperm are being stored for prolonged periods from a single mating event with one or more males or result from multiple matings throughout the year. Feldheim et al. (2001) have recently applied genetic analysis using DNA microsatellite loci developed for lemon sharks, Negaprion brevirostris, to investigate the possibility of multiple paternity. Their results demonstrated that at least three males sired a single litter. Various workers have commented on uterine sperm, generally immediately after insemination. Metten (1944) reported uterine digestion of sperm in the dogfish, Scyliorhinus canicula. Our observations in M. canis do not confirm Metten’s (1944) conclusions. Leesa et al. (1986) observed sperm in the uterus and oviducal gland of Rhinobatos horkelii from Brazil after birth and subsequent copulation. The published micrograph of sperm in the oviducal gland does not allow determination of what precise zone of the oviducal gland was involved but it appears that the sperm are in the gland lumen. Fishelson & Baranes (1998) described the folded endometrium of gravid placental Iago omanensis as forming simple tubular glands at the bases of the folds. They saw aggregations of sperm in the tubules but did not report sperm being embedded in the uterus.

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Sperm storage in male Osteichthyes Viviparous fishes generally have elongate sperm while externally fertilizing fishes have sperm with a round head. In male viviparous Osteichthyes spermatozeugmata are formed. In the guppy, Poecilia reticulata, spermatozeugmata are ovoid structures measuring 135-235 ¼m in diameter. Sperm nuclei are arranged at the periphery with the tails projecting toward the center of the mass (in goodeids it is the reverse). The number of sperm in each spermatozeugmata seems to be species specific. Matrix is secreted by the former cyst epithelium. Periodic acid-Schiff positive testicular secretions help maintain the integrity of spermatozeugmata while efferent ducts secrete acid Alcian blue positive and periodic acid-Schiff positive mucopolysaccharides including N-acethyl galactosamine, N-acetyl glucosamine and N-acetyl neuramic acid (Greven, unpublished). Spermatozeugmata are stored in the main testis duct and up to 400 can be removed from a single male by artificial stimulation. Ageing of sperm probably also occurs. Gardiner (1978a) described spermatophore formation in the shiner surfperch, Cymatogaster aggregata. He describes the matrix supporting the sperm as being proteinaceous and are produced by efferent sperm duct epithelial cells. Many Sertoli cells degenerate causing the cyst wall to collapse. However, many Sertoli cells do not break down but assume the configuration of columnar duct cells. Spermatophores are intact within the testicular ducts but rapidly dissolve within the female ducts in response to increased pH. Gardiner (1978a) referred to the sperm aggregates in Cymatogaster as spermatophores, based on the occurrence of an extracellular capsule visible only with transmission electron microscopy. The integrity of the cortex is not apparent from his micrographs and most contemporary authors would term them spermatozeugmata. Sperm storage in female Osteichthyes In 4 teleost families, Poeciliidae, Anablepidae, Goodeidae and Hemiramphidae the brood develops in the ovary as no Müllerian ducts are formed. Oocytes develop in ovarian follicles surrounded by a reduced zona radiata. After fertilization, embryos develop either in the follicle (intrafollicular gestation in Poeciliidae, Anableps and some Hemoramphidae) or eggs, which in most cases are already fertilized, are discharged from the follicle and develop in the ovary (intraovarian gestation in Goodeidae, Jenynsia and some Hemiramphidae). Hogarth & Sursham, (1972) suggested the ovary may be a favorable site for sperm survival because estradiol impeded allograft rejection. In some viviparous Osteichthyes, in particular the Poeciliids and Halfbeaks, sperm are stored within folds of the inner ovarian epithelium. In P. reticulata once transferred to the female, spermatozeugmata dissolve within 1 minute in the genital sinus and/ or lower portion of the oviduct (Greven, unpublished). Spermatozoa migrate to the ovarian cavity within 10-15 minutes. They are stored in the caudal and cranial parts of the ovarian cavity, as well as in small depressions called “delle” in the ovarian epithelium immediately above the oocytes. Large “true” storage occurs in the cranial and caudal part of the ovary as sperm are deeply embedded in epithelial invaginations. Sperm are not deeply embedded in the delle. Sperm may remain viable for at least one year in the guppy.

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Paris et al. (1998) described sperm storage in the swordtail, Xiphophorus helleri. They described long term sperm storage occurring in epithelial folds of the cranial and caudal parts of the ovary and short term sperm storage occurring in delle. Stored sperm are deeply embedded in ovarian epithelial cells. Sperm release starts when sperm move to the apical part of the cell. This movement is caused by coalescence of small basal vacuoles into a single large vacuole that pushes the sperm to the lumen for release. In female Halfbeaks sperm are also embedded in invaginations of the ovarian epithelium but the major site of storage has not yet been determined. Sperm also occur in delle above oocytes. Ultrastructural details are not available and duration of storage is unknown. In Nomorhamphus celebensis 8 litters were produced after a single successful insemination and 7 litters were produced in Dermogenys pusillus (Greven, unpublished). In female Cymatogaster aggregata insemination and ovulation are separated by about 6 months Females are inseminated in the summer and sperm are stored in the ovarian lumen. Following fertilization, embryos develop in the ovarian lumen for about 6 months (Gardiner 1978b). Ovarian epithelium has dilated intercellular spaces containing extracellular material during much of the year. The dilations increase in volume in the months prior to ovulation and fertilization and decrease during the months of embryogenesis and gestation. Gardiner concludes that the extracellular material is released into the ovarian lumen to serve a nutrient function for the developing embryos. Sperm storage occurs when sperm are associated with sperm pockets of the ovarian epithelium. The cells lining the sperm pocket do not develop intercellular dilations characteristic of most of the ovarian epithelium and sperm are associated only with sperm pocket cells. Recently Potter & Kramer (2000) reported ultrastructural observations of sperm storage in the platyfish, Xiphophorus maculatus. They describe sperm as being associated with epithelial cells of the oviduct and some epithelial cells taking up sperm into their cytoplasm. Their conclusions of sperm digestion are based on unconvincing electron micrographs. What is purported to be sperm incorporation may simply be a sectioning artifact of embedded sperm. The fact that no lysosomes were detected supports sperm embedding and not sperm incorporation. Koya et al. (1995, 1997, 2002) described ultrastructural details of sperm storage in the marine sculpin, Alcichthys alcicornis, a teleost with a reproductive mode termed internal gametic association. In this mode sperm are introduced into the female tract at copulation. Sperm enter the micropyle of eggs that have been ovulated into the ovarian cavity. Penetration of sperm into egg cytoplasm does not occur until after the eggs have been spawned into seawater. The ovarian fluid plays an important role in activation of sperm motility. Ovarian fluid originates from cells of the ovarian cavity including ovigerous lamella epithelium and ovarian wall epithelium. Both epithelia secrete proteinaceous material during the spawning season. Sperm are maintained in the ovarian cavity, floating in the ovarian fluid during the spawning season. Sperm in the ovarian cavity are isolated from the maternal immune system by tight junctions between adjacent ovarian epithelial cells during spawning season. After spawning the junctional complexes break down and remaining sperm are phagocytosed by macrophages invading the ovarian cavity after the spawning season.

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Conclusion Sperm storage and association either into spermatophores or spermatozeugmata occurs in viviparous chondrichthyes and osteichthyes. Sperm masses helps to decrease sperm loss during copulation in an aqueous environment. Substances in the matrix no doubt provided nourishment to the sperm. In chondrichthyes the majority of sperm matrix is provided by secretions of the Leydig gland and sperm masses are stored in the seminal vesicles of the male. Insemination is effected by insertion of a clasper into the female. Interesting structural parallels between sperm-uterine association in the smoothhound, M. canis, and the mammalian oviducal sperm reservoir suggest similar functions that might include sperm activation. Sperm storage per se in effected by the terminal zone tubules in the oviducal gland. Fertilization then occurs in the oviduct above the oviducal gland before the egg is coated with egg jellies and the tertiary egg envelope. In osteichthyes insemination is generally effected by insertion of a gonopodium. Storage of sperm, often for prolonged periods of time, occurs in the ovary of some viviparous osteichthyes. In some species sperm float freely in the ovarian lumen while in others sperm become embedded in ovarian epithelium. Some species have long term sperm storage in cranial and caudal parts of the ovary and short term sperm storage in delle. Ovarian epithelial cells provide nourishment for the sperm. Due to the large suite of reproductive adaptations and the large number of species, more detailed studies in a comparative context are necessary. References Babel J.S. 1967. Reproduction, life history, and ecology of the round stingray, Urolophus halleri Cooper. Calif. Fish Game Bull. 137: 1-104. Callard G.V. 1988. Reproductive physiology: B The male. In Shuttleworth T. (ed.), Physiology of Elasmobranch Fishes. New York: Springer, pp.292-317 Conrath C.L. 2000. Population Dynamics of the Smooth Dogfish, Mustelus canis, in the Northwest Atlantic. M.Sc. Thesis, Virginia Institute of Marine Science. 93p. Feldheim K.A., Gruber S.H. & M.V. Ashley 2001. Multiple paternity of a lemon shark litter (Chondrichthyes: Carcharhinidae). Copeia 2001: 781-786. Fishelson L. & A. Baranes 1998.Observations on the Oman shark, Iago omanensis (Triakidae), with emphasis on the morphological and cytological changes of the oviduct and yolk sac during gestation. J. Morph. 236: 151-165. Gardiner D.M. 1978a. The origin and fate of spermatophores in the viviparous teleost Cymatogaster aggregata (Perciformes: Embiotocidae). J. Morph. 155:157-172. Gardiner D.M. 1978b. Cyclic changes in fine structure of the epithelium lining the ovary of the viviparous teleost, Cymatogaster aggregata (Perciformes: Embiotocidae). J Morph. 156: 367-380. Gilbert, P.W. & G.W. Heath 1972. The clasper-siphon sac mechanism in Squalus acanthias and Mustelus canis. Comp. Biochem. Physiol. 42A: 97-119. Hamlett W.C. 1999. Male reproductive system, In Hamlett W.C. (ed), Sharks, skates, and rays: the biology of elasmobranch fishes. Baltimore, MD: The Johns Hopkins Univ Press, pp. 444-470.

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HAMLETT W.C., KNIGHT D.P., KOOB T.J., JEZIOR M., LUONG T., ROZYCKI T., BRUNETTE N. & M.K. HYSELL 1998. Survey of oviducal gland structure and function in elasmobranchs. J. Exp. Zool. 282: 399-420. HAMLETT W.C., HYSELL M.K., JEZIOR M., ROZYCKI T., BRUNETTE N. & K. TUMILTY 1999a. Fundamental zonation in elasmobranch oviducal glands. In Seret B. & J.-Y. Sire (eds), 5th Indo-Pacific Fish Conference Noumea 1997. ed. Paris: French Ichthyological Society and ORSTOM, pp. 271-280. HAMLETT W.C., HYSELL M.K., ROZYCKI T., BRUNETTE N., TUMILTY K., HENDERSON A. & J. DUNNE 1999b. Sperm aggregation and spermatozeugmata formation in the male genital ducts in the clearnose skate, Raja eglanteria. In Seret B. & J.-Y. Sire (eds), 5th Indo-Pacific Fish Conference Noumea 1997. ed. Paris: French Ichthyological Society and ORSTOM, pp. 281-291. HAMLETT W.C. & T. KOOB 1999. Female reproductive system. In Hamlett W.C. (ed.), Sharks, Skates and Rays: Biology of Elasmobranch Fishes. Baltimore, MD: The Johns Hopkins University Press, pp. 398-443. HAMLETT W.C., MUSICK J.A., HYSELL C.K. & D.M. SEVER 2002b. Uterine epithelial-sperm interaction, endometrial cycle and sperm storage in the terminal zone of the oviducal gland in the placental smoothhound, Mustelus canis. J. Exp. Zool. 292: 129-144. HAMLETT W.C., REARDON M., CLARK J. & T.I. WALKER 2002a. Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii. J. Exp. Zool. 292:111-128. HOBSON A.D. 1930. A note on the formation of the egg case of the skate. J. Mar. Biol. Ass. U.K. 16: 577-581. HOGARTH P.J. & C.M. SURSHAM 1972. Antigenicity of Poecilia sperm. Experentia 28: 463. JONES C.J.P. & W.C. HAMLETT 2001. Glycosylation of the male genital ducts and spermatozeugmata formation in the clearnose skate, Raja eglanteria. Proc XVI Internat Symp Morph Sci, Sun City, South Africa, p. 11. JONES R.C. & M. LIN 1993. Structure and functions of the genital ducts of the male Port Jackson shark, Heterodontus portusjacksoni. Environ. Biol.Fish. 38: 127-138. KNIGHT D.P., FENG D. & M. STEWART 1996. Structure and function of the selachian egg case. Biol. Rev. 71: 81-111. KOYA Y., MUNEHARA H. & K. TAKANO 1997. Sperm storage and degradation in the ovary of a marine copulating scilpin, Alcichthys alcicornis (Teleostei: Scorpaeniformes): role of intercellular junctions between inner ovarian epithelial cells. J. Morph. 233: 153-163. KOYA Y., MUNEHARA H. & K. TAKANO 2002. Sperm storage and motility in the ovary of the marine sculpin Alcichthys alcicornis (Teleostei: Scorpaeniformes), with internal gametic association. J. Exp. Zool. 292: 145-155. KOYA Y., TAKANO K. & H. TAKAHASHI 1995. Annual changes in fine structure of inner epithelial lining of the ovary of a marine sculpin, Alcicthys alcicornis (Teloestei: Scorpaeniformes), with internal gametic association. J. Morph. 223: 85-97. LEESA R.P., VOOREN C.M. & J. LA HAYE 1986. Desenvolvimento e ciclo sexual das femeas, migraçoes e fecundidae da viola Rhinobatos horkelii (Müller & Henle, 1841) do sul do Brasil. Atlantico, Rio Grande 8: 5-34. MANN T. 1960. Serotonin 5-hydroxytryptamine in the male reproductive tract of the spiny dogfish. Nature 188: 941-942.

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MANN T. & C.L. PROSSER 1963. Uterine response to 5-hydroxytryptamine in the clasper siphon secretion of the spiny dogfish Squalus acanthias. Biol. Bull. 125: 384-385 MAREN T.H., RAWLS J.A., BURGER J.W. & A.C. MYERS 1963. C The alkaline Marshall’s gland of the skate. Comp. Biochem. Physiol. 10: 1-16. METTEN H. 1939. Studies on the reproduction of the dogfish. Phil. Trans. Roy. Soc. London 230: 217-238. METTEN H. 1944. The fate of spermatozoa in the female dogfish (Scyliorhinus canicula). Quart. J. Microsc. Sci. 84: 283-295. PARIS F., PAAßEN U. & V. BLÜM 1998. Spermienspeicherung bei weiblichen Schwertträgern (Xiphophorus helleri). Sperm storage in the female swordtail (Xiphophorus helleri). Verhandlungen der Gesellschaft für Ichthyologie 1: 157-165. POTTER H. & C.R. KRAMER 2000. Ultrastructural observations on sperm storage in the ovary of the platyfish, Xiphophorus maculatus (Teleostei: Poeciliidae): the role of the duct epithelium. J. Morph. 245: 110-129. SMITH H.W. 1929. The composition of the body fluids of elasmobranchs. J. Biol. Chem. 81: 407-419. SMITH P.L. 1985. Electrolyte transport by alkaline gland of little skate Raja erinacea. Am. J. Physiol. 248: R346-352. SMITH R. 2001. The Reproductive Biology of the Female Elephant Fish, Callorhynchus milii, with Particular Reference to the Oviducal Gland. Honors thesis, Dept Zool, The Univ Melbourne, Parkville, Victoria, Australia, pp. 1-60. STORRIE M.T., WALKER T.I., LAURENSON L. & W.C. HAMLETT 2001. Observations of sperm in the oviducal gland of the gummy shark, Mustelus antarcticus. 6th Indo-Pacific Fish Conf. Sci. Prog., Durban, South Africa, abst. 51.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Sperm Storage inThe theNew Class Amphibia Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 431-438, 2003

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Sperm Storage in the Class Amphibia D.M. Sever1, L.C. Rania1 & R. Brizzi2 1. Department of Biology, Saint Mary’s College, Notre Dame, Indiana 46556 USA 2. Department of Animal Biology and Genetics, University of Florence, Florence, Italy

Abstract The three orders of extant amphibians are Caudata, Anura, and Apoda. In salamanders and newts (Caudata), absence of sperm storage in females is the ancestral condition (three families). In the derived condition, sperm storage occurs in cloacal glands called spermathecae, and their possession is a synapomorphy for females in the suborder Salamandroidea (seven families). The anatomy and phylogeny of caudate spermathecae have been studied extensively (reviewed by Sever & Brizzi 1998). Internal fertilization has convergently evolved in a few frogs and toads (Anura), but females of just one species, Ascaphus truei are known to possess oviducal sperm storage tubules (Ssts). Ssts of A. truei are similar anatomically to such glands in squamate reptiles. This similarity is convergence perhaps due to design constraints imposed by the basic structure of the vertebrate oviduct. Although all caecilians (Apoda) apparently have internal fertilization and many are viviparous, female sperm storage is unknown.

Introduction The Lissamphibia (extant amphibians) consists of three orders, Caudata or Urodela (salamanders and newts, 500 species), Anura (frogs and toads, 4800 species), and Apoda or Gymnophiona (caecilians, 165 species). Sperm storage by females occurs in most species of salamanders, one species of frog, and is unknown in the caecilians. The anatomy of sperm storage glands has been extensively studied in salamanders and was recently reviewed by Sever & Brizzi (1998). Ascaphus truei (Stejneger, 1899) is the only frog in which female sperm storage is known, and this phenomenon has been studied by Noble (1925), Van Dijk (1955, 1959), Metter (1964), and Sever et al. (2001). Caudata Sperm storage occurs in all females found in the seven families of salamanders that comprise the suborder Salamandroidea (Sever 1991a, 1994). Instead of oviductal sperm storage, however, sperm are stored in cloacal glands called spermathecae. Possession of

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a cloaca is considered the ancestral condition for vertebrates (Wake 1979, 1987), but salamanders are the only vertebrates in which cloacal sperm storage glands have evolved (Sever 1994). The ancestral condition for salamanders is lack of sperm storage glands, a condition found in three families with external fertilization, Sirenidae, Hynobiidae, and Cryptobranchidae (Sever 1991b, 1994, Sever et al. 1996b). Sever & Brizzi (1998) mapped 14 characters involved with sperm storage in spermathecae on a phylogeny of salamander families taken from Larson & Dimmick (1993). The only character with definite phyletic value is whether sperm storage occurs in a “complex spermatheca” composed of a single compound tubulo-alveolar gland (Plethodontidae) or in “simple spermathecae” consisting of numerous simple tubular glands (other families). The variation in complex spermathecae of plethodontids was described by Sever (2000). The annual cycle of sperm storage has been studied at the ultrastructural level in two Plethodontidae (Sever 1997, Sever & Brunette 1993), three Salamandridae (Brizzi et al. 1995, Sever et al. 1996a, 1999, 2001), two Ambystomatidae (Sever 1995, Sever & Kloepfer 1993, Sever et al. 1995), one Amphiumidae (Sever et al. 1996c), and one Proteidae (Sever & Bart 1996). Studies still need to be done on representatives of the Rhyacotritonidae and Dicamptodontidae. Thus, female sperm storage has been studied in only 2% of the known species of salamanders, and much diversity exists among the few species that have been studied in reproductive habits and sperm storage characters (Sever & Brizzi 1998). More comparative work is needed before we resolve any of the questions concerning the significance of variability in sperm storage mechanisms among salamanders. Sever & Brizzi (1998) concluded that: (1) sperm storage is an ancient trait in salamanders, evolving in the common ancestor of all the current families in the Salamandroidea; (2) some of the differences observed among taxa in spermathecal characters may not be phyletically informative but related to other species-specific reproductive adaptations; (3) sperm storage is apparently obligatory prior to fertilization in salamandroids so that the duration of effective sperm storage must be considered in any study on the reproduction of these taxa; and (4) storage of sperm facilitates multiple matings and provides the conditions for sperm competition within the spermathecae of salamanders. Anura The presence of sperm in the lumen of the oviducts and in oviducal glands of females of the frog Ascaphus truei was first reported by Noble (1925). Ascaphus truei is the sole member of the family Ascaphidae and is generally considered the sister taxon of all other anurans (Ford & Cannatella 1993). Ascaphus truei is associated with cold, clear mountain streams in disjunct populations in the Cascade Mountains west to the coast from southern British Columbia to northwest California; in the Blue Mountains of southwestern Washington and northeastern Oregon; and in the Rocky Mountains of northern Idaho and western Montana (Metter 1968)1. Of the 4000+ species of anurans, A. truei is the only 1

The Rocky Mountain populations of Ascaphus have recently been recognized as a new species, A. montanus (Nielson et al. 2001).

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species known to engage in copulation. The male possesses a “tail” that, when engorged, forms a sulcus for passage of sperm and is inserted in the cloaca of the female (Noble 1925, Noble & Putnam 1931, Slater 1931). Copulation has been assumed to be an adaptation that ensures fertilization in fast-moving water (Stebbins & Cohen 1995). Posterior to a short, aglandular infundibular region, the oviduct of A. truei possesses: (1) a proximal, convoluted ampullary region where intrinsic tubular glands secrete gelatinous envelopes around eggs; (2) a middle ovisac region where fertilization occurs; and (3) a distal oviductal sinus formed by medial junction of the ovisacs. An oviductal sinus has previously been described in Frogs only for the viviparous African bufonid Nimbaphrynoides occidentalis (Xavier 1973). Sperm storage tubules (Ssts) occur in the anterior portions of the ovisacs and consist of simple tubular glands. Ssts and the rest of the oviductal lining stain positively with the periodic acid-Schiff’s procedure for neutral carbohydrates, and this reaction is especially intense in reproductively active females. Sperm were found in the Ssts of gravid females as well as some non-vitellogenic females. The sperm are in orderly bundles in the Ssts, and although occasionally sperm nuclei were embedded in the epithelium, no evidence for spermiophagy was found. Apoda Apparently all caecilians have internal fertilization. The male possesses a cloacal structure called the phallodeum that is everted from the cloaca and serves for intromission of sperm into the female cloaca during copulation (Wake 1979). Many caecilians are viviparous, a derived condition within the Apoda (Wilkinson & Nussbaum 1998). We hypothesize that sperm storage occurs in the oviducts of female caecilians, but no observations on sperm in the oviduct of a female caecilian have yet been made. Comparative biology To date, Ascaphus truei is the only amphibian in which oviductal sperm storage has been reported. Indeed, the only other anamniotes in which oviductal sperm storage is known are elasmobranchs (Pratt 1993, Hamlett et al. 1998, Hamlett & Koob 1999, Hamlett et al. 1999) in the class Chondrichthyes, which is not considered the sister taxon of Amphibia. Females of some teleosts in the Osteichthyes store sperm (Howarth 1974), but they lack homologues to the oviduct (Kardong 1995). Instead sperm are stored in the ovary or a gonaduct (ovarian duct) formed from ovarian tissue (Howarth 1974, Constanz 1989). The extant representatives of Actinistia and Dipnoi, descendant taxa of sarcopterygiian sister groups of amphibians (Schultze 1994), possess oviducts (Millot & Anthony 1960, Wake 1987), and Latimeria is viviparous (Smith et al. 1975) indicating that fertilization is internal (Fig. 1). Sperm storage, however, has not been reported in Latimeria or any of the extant lungfish. Thus, no neontological evidence exists for sperm storage as the ancestral state for amphibians. The Lissamphibia is generally considered monophyletic. Most evidence supports a frog + salamander clade (Pough et al. 1998, Fig. 1). As noted previously, sperm storage is unknown in female caecilians, even though internal fertilization apparently occurs in all taxa (Fig. 1), and many caecilians are viviparous (Wilkinson & Nussbaum 1998). In

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Fig. 1. “Scenariogram” showing distribution of internal fertilization and sperm storage in the Lissamphibia with extant sacropterygiians (descendent taxa of piscine ancestors to amphibians) as outgroups. Within the amphibians, sperm storage glands evolved independently in the cloaca in one suborder of salamanders (Salamandroidea) and in the oviduct of one species of frog (Ascaphus truei). From Sever et al. (2001).

salamanders, cloacal sperm storage in spermathecae is a synapomorphy for the Salamandroidea (Fig. 1), and is unique within vertebrates. Aside from Ascaphus, only a few anurans have internal fertilization, with sperm transfer accomplished by cloacal apposition. These species include Mertensophryne micranotis (Grandison & Ashe 1983) and four species of “Nectophrynoides” (sensu Wake 1980) within the Bufonidae from Africa, and Eleurodactylus jasperi (Wake 1978) and E. coqui (Townsend et al. 1981) within the Leptodactylidae from Puerto Rico. Obviously, research needs to be done to determine whether oviductal sperm storage occurs in caecilians and internal fertilizing bufonids and leptodactylids. Ascaphus truei, however, is not the sister taxon of any caecilian or of

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the other internal fertilizing frogs (Fig. 1), so oviducal sperm storage must be considered independently derived in A. truei. Thus, oviductal sperm storage in Ascaphus truei is a classic example of homoplasy through convergence (Sanderson & Hufford 1996). Structural and functional similarities in sperm storage between A. truei and other vertebrates with oviductal sperm storage therefore are not based upon direct descent but related either to similar functional adaptations and/or to internal design restraints (Wake 1991). In the latter case, structural and physiological constraints on the basic vertebrate oviduct and sperm morphologies may limit the options for expression of oviductal sperm storage. The group of anamniotes phyletically closest to frogs and with which frogs share the most developmental similarities (the closest generative system, Wake 1996) is the Caudata. Numerous differences, however, occur between the spermathecae of salamanders and the Ssts of Ascaphus truei. The distal portions of the spermathecae of salamanders are typically alveolar, lack cilia, and possess basal myoepithelium (Sever & Brizzi 1998). Secretory activity in salamander spermathecae is sometimes regionalized and seasonal, depending upon the taxa (Sever 1994). A great deal of variation also occurs in reaction to carbohydrate stains with, however, most species showing AB+ reactions for carboxylated glycosaminoglycans (Sever 1994). In some forms the sperm are in orderly arrays in the spermathecae (Sever and Hamlett 1998) whereas in others, sperm are in tangled masses (Sever et al. 1999). Alignment of sperm may depend to some degree upon the anatomy of the spermatheca (more orderly in compound glands than simple tubular). Spermiophagy by the spermathecal epithelium has been described in various taxa of salamanders (Sever & Brizzi 1998). The Ssts of Ascaphus truei more closely resemble those of squamate reptiles (Fox 1956, Girling et al. 1997, Sever & Ryan 1999). Oviductal sperm storage glands are known from all groups in the reptile-bird clade except Amphisbaenia (in which they likely occur) and Rhynchocephalia (Gist & Jones 1987). Like reptiles, the Ssts of A. truei are simply continuations of the oviducal lining, and contain ciliated non-secretory cells and nonciliated secretory cells. Myoepithelium is absent, but the oviduct possesses layers of smooth muscle (tunica muscularis) superficial to the mucosa. The linings and glands of reptilian oviducts are generally described as PAS+, like those of A. truei, with little reaction to acidic mucosubstances. Sperm in the Ssts of A. truei are generally in close alignment, although in squamates, this condition varies (Fox 1956, Sever & Ryan 1999). Although sperm nuclei are sometimes found embedded in Ssts of reptiles (Sever & Ryan 1999) and of A. truei, no evidence exists for spermiophagy in these taxa. In conclusion, oviducal Ssts in distantly related vertebrate taxa show more similarities than exist between Ssts in Ascaphus truei and spermathecae in salamanders, members of sister taxa. The basic structure of the vertebrate oviduct, therefore, may limit the range of features associated with oviducal sperm storage. For a more extensive discussion, see Sever (2001). References BRIZZI R., DELFINO G., SELMI M.G. & D.M. SEVER 1995. Spermathecae of Salamandrina terdigitata (Amphibia: Salamandridae): Patterns of sperm storage and degradation. J. Morphol. 232: 21-33.

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CONSTANZ G.D. 1989. Reproductive biology of poeciliid fishes. In Meffe G.K. & F.F. Snelson, Jr. (eds), Ecology and Evolution of Livebearing Fishes, (Poeciliidae). Prentice Hall, New Jersey, pp. 33-50. FORD L.S. & D.C. CANNATELLA 1993. The major clades of frogs. Herpetol. Monogr. 7: 94-117. FOX W. 1956. Seminal receptacles of snakes. Anat. Rec. 124: 519-539. GIRLING J.E., CREE A. & L.J. GUILLETTE, Jr. 1997. Oviductal structure in a viviparous New Zealand Gecko, Hoplodactylus maculatus. J. Morphol. 234: 51-68. GIST D.H. & J.M. JONES 1987. Storage of sperm in the reptilian oviduct. Scann. Microscop. 1: 1839-1849. GRANDISON A.G.C. & S. ASHE 1983. The distribution, behavioural ecology and breeding strategy of the pygmy toad, Mertenosphryne micranotis (Lov.). Bull. Brit. Mus. Nat. Hist. (Zool.) 45: 85-93. HAMLETT W.C., KNIGHT D.P., KOOB T.J., JEZIOR M., LUOUG T., ROZYCKI T., BRUNETTE N. & M.K. HYSELL 1999. Survey of oviducal gland structure and function in elasmobranchs. J. Exper. Zool. 282: 399-420. HAMLETT W.C. & T.J. KOOB 1999. Chapter 15. Female reproductive cycle. In Hamlett W.D. (ed), Sharks, Skates and Rays: the Biology of Elasmobranch Fishes. John Hopkins Univ. Press, Maryland, pp. 315-345. HAMLETT W.C., SEVER D. & C. HYSELL 1999. Gestational plasticity of the uterus in placental sharks. Placenta 20: A28. HOWARTH B., Jr. 1974. Sperm storage: as a function of the female reproductive tract. In Johnson A.D. & C.W. Foley (eds), The Oviduct and Its Functions. Academic Press, New York, pp. 237-270. KARDONG K.V. 1995. Vertebrates Comparative Anatomy Function Evolution. W. C. Brown, Dubuque, Iowa. LARSON A. & W.W. DIMMICK 1993. Phylogenetic relationships of the salamander families: An analysis of congruence among morphological and molecular characters. Herpetol. Monogr. 7: 77-94. METTER D.E. 1964. On breeding and sperm retention in Ascaphus. Copeia 1964: 710-711. METTER D.E. 1968. Ascaphus and A. truei. Cat. Amer. Amphib. Rept. 69: 1-2. MILLOT J. & J. ANTHONY 1960. Appareil genital et reproduction des coelacanthes. C. R. Hebd. Seanc. Acad. Sci. Paris D 251: 442-443. NELSON M., LOHMAN K. & J. SULLIVAN 2001. Phylogeography of the tailed Frog (Ascaphus truei): implications for the biogeography of the Pacific northewest. Evolution 55: 147-160. NOBLE G.K. 1925. An outline of the relation of ontogeny to phylogeny within the Amphibia I. Amer. Mus. Novitates 165: 1-17. NOBLE G. K. & P.G. PUTNAM 1931. Observations on the life history of Ascaphus truei Stejneger. Copeia 1931: 97-101 POUGH F.H., ANDREWS R.M., CADLE J.E., CRUMP M.L., SAVITZKY A.H. & K.D. WELLS 1998. Herpetology. Prentice Hall, Upper Saddle River, New Jersey. PRATT H.L. 1993. The storage of spermatozoa in the OGs of western North Atlantic Sharks. Enivron. Biol. Fishes 38:139-149. SANDERSON M.J. & L. HUFFORD (eds) 1996. Homoplasy: the Recurrence of Similarity in Evolution. Academic Press, San Diego. SCHULTZE H-P. 1994. Comparison of hypotheses on the relationships of sarcopterygians. Syst. Biol. 43: 155-173.

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SEVER D.M. 1991a. Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata). I. Evolution at the family level. Herpetologica 47: 165-193. SEVER D.M. 1991b. Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata). II. Cryptobranchidae, Hynobiidae, and Sirenidae. J. Morphol. 207: 283-301. SEVER D.M. 1994. Observations on regionalization of secretory activity in the spermathecae of salamanders and comments on phylogeny of sperm storage in female salamanders. Herpetologica 50: 383-397. SEVER D.M. 1995. Spermathecae of Ambystoma tigrinum (Amphibia: Caudata): Development and a role for the secretions. J. Herpetol. 29: 243-255. SEVER D.M. 1997. Sperm storage in the spermatheca of the red-back salamander, Plethodon cinereus (Amphibia: Plethodontidae). J. Morphol. 234: 131-146. SEVER D.M. 2000. Sperm storage in female plethodontids with especial reference to the Desmognathinae. In Bruce R.C., Jaeger R. & L.C. Houck (eds), The Biology of Plethodontid Salamanders. Kluwer Academic/Plenum, New York, pp. 345-369. SEVER D.M. 2002. Femail sperm storage in amphibians. J. Exp. Zool. 292: 165-179. SEVER D.M. & H.L. BART, Jr. 1996. Ultrastructure of the spermathecae of Necturus beyeri (Amphibia: Proteidae) in relation to its breeding season. Copeia 1996: 927-937. SEVER D.M. & R. BRIZZI 1998. Comparative biology of sperm storage in female Salamanders. J. Exp. Zool. 282: 460-476. SEVER D.M. & N.S. BRUNETTE 1993. Regionalization of eccrine and spermiophagic activity in spermathecae of the salamander Eurycea cirrigera (Amphibia: Plethodontidae). J. Morphol. 217: 161-170. SEVER D.M., DOODY J.S., REDDISH C.A., WENNER M.M. & D.R. CHURCH 1996c. Sperm storage in spermathecae of the great lamper eel, Amphiuma tridactylum (Caudata: Amphiumidae). J. Morphol. 230: 79-97. SEVER D.M., HALLIDAY T., MORIARTY E.C. & B. ARANO 2001. Sperm storage in females of the smooth newt (Triturus v. vulgaris L.): II. Ultrastructure of the spermathecae after the breeding season. Acta Zoologica 82(1): 49-56. SEVER D.M., HALLIDAY T., WAIGHTS V., BROWN J., DAVIES H.A. & E.C. MORIARTY 1999. Sperm storage in females of the smooth newt (Triturus v. vulgaris L.): I. Ultrastructure of the spermathecae during the breeding season. J. Exp. Zool. 283: 51-70. SEVER D.M & W.C. HAMLETT 1998. Sperm aggregations in the spermatheca of female desmognathine salamanders (Amphibia: Urodela: Plethodontidae). J. Morphol. 238: 143-155. SEVER D.M. & N.M. KLOEPFER 1993. Spermathecal cytology of Ambystoma opacum (Amphibia: Ambystomatidae) and the phylogeny of sperm storage organs in female salamanders. J. Morphol. 217: 115-127. SEVER D.M., KRENZ J.D., JOHNSON K.M. & L.C. RANIA 1995. Morphology and evolutionary implications of secretion and sperm storage in spermathecae of the salamander Ambystoma opacum (Amphibia: Ambystomatidae). J. Morphol. 223: 35-46. SEVER D.M., MORIARTY E.C., RANIA L.C. & W.C. HAMLETT 2001. Sperm storage in the oviduct of the internal fertilizing frog Ascaphus truei. J. Morphol. 248: 1-21. SEVER D.M., RANIA L.C. & J.D. KRENZ 1996a. Annual cycle of sperm storage in spermathecae of the red-spotted newt, Notophthalmus viridescens (Amphibia: Salamandridae). J. Morphol. 227: 155-170.

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SEVER D.M., RANIA L.C. & J.D. KRENZ 1996b. Reproduction of the salamander Siren intermedia Le Conte with especial reference to oviducal anatomy and mode of fertilization. J. Morphol. 227: 335-348. SEVER D.M. & T.J. RYAN 1999. Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea): Part I. Evidence for oviducal sperm storage. J. Morphol. 241: 1-18. SLATER J.R. 1931. The mating of Ascaphus truei Stejneger. Copeia 1931: 62-63. SMITH C.L., RAND C.S., SCHAEFFER B. & J. ATZ 1975. Latimeria, the living coelacanth, is ovoviviparous. Science 190: 1105-1106. STEBBINS R.C. & N.W. COHEN 1995. A Natural History of Amphibians. Princeton Univ Press, Princeton, Mew Jersey. STEJNEGER L. 1899. Description of a new genus and species of discoglossid toad from North America. Proc. U. S. Nat. Mus. 21: 899. TOWNSEND D.S., STEWART M.M., POUGH F.H. & P.F. BRUSSARD 1981. Internal Fertilization in an oviparous frog. Science 212: 469-471. VAN DIJK D.E. 1955. The “tail” of Ascaphus: A historical resume and new histological-anatomical details. Ann. Univ. Stellenbosch 31: 1-71. VAN DIJK D.E. 1959. On the cloacal region of Anuran in particular of larval Ascaphus. Ann. Univ. Stellenbosch 35: 169-249. WAKE D.B. 1991. Homoplasy: The result of natural selection, or evidence of design limitations? Am. Nat. 138: 543-567. WAKE D.B. 1996. Introduction. In Sanderson M.J. & L. Hufford (eds), Homoplasy: The Recurrence of Similarity in Evolution. Academic Press, San Diego, pp. xvii-xxv. WAKE M.H. 1978. The reproductive biology of Eleutherodactylus jasperi (Amphibia, Anura, Leptodactylidae), with coments on the evolution of live-bearing Systems. J. Herpetol. 12: 121-133. WAKE M.H. 1979. The comparative anatomy of the urogenital system. In Wake M.H. (ed.), Hyman’s Comparative Vertebrate Anatomy, 3rd ed. Univ. Chicago Press, Chicago, pp. 555-614. WAKE M.H. 1980. The reproductive biology of Nectophrynoides malcolmi (Amphibia: Bufonidae), with comments on the evolution of reproductive modes in the genus Nectophrynoides. Copeia 1980: 193-209. WAKE M.H. 1987. Urogenital morphology of dipnoans, with comparisons to other fishes and to amphibians. In Bemis W.E., Burggren W.W. & N.E. Kemp (eds), The Biology and Evolution of Lungfishes. A. R. Liss, New York, pp. 199-216. WILKINSON M. & R.A. NUSSBAUM 1998. Caecilian viviparity and amniote origins. J. Nat. Hist. 32: 1403-1409. XAVIER F. 1973. Le cycle des voies genitales Femelles de Nectophrynoides occidentalis Angel, amphibien anoure vivipare. Z. Zellf. 140: 509-534.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Sperm StorageThe in New the Class Reptilia Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 439-446, 2003

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Sperm Storage in the Class Reptilia D.M. Sever1 & W.C. Hamlett2 1. Department of Biology, Saint Mary’s College, Notre Dame, Indiana 46556 USA 2. Indiana University School of Medicine, South Bend Center for Medical Education, Notre Dame, Indiana 46556 USA

Abstract Reptilia contains some 7200 species of amniotes whose monophyly is supported by both morphological and molecular characters although sister-group relationships among the four major groups containing extant taxa remain controversial. These groups are Chelonia (turtles), Crocodilia, Rhynchocephalia, and Squamata. The Squamata is the largest group (6900 species) and contains Amphisbaenia, Sauria (lizards), and Serpentes (snakes). Internal fertilization and oviparity most likely are symplesiomorphies for modern reptiles, and viviparity has evolved independently numerous times in Sauria and Serpentes. Oviducal sperm storage is known in females of all the above taxa except Amphisbaenia. In rhynchocephalians and crocodilians, however, sperm storage is poorly studied, and specialized sperm storage tubules (Ssts) are unknown. Ssts arose independently in Chelonia and Squamata. Turtles possess albumen-secreting glands in the anterior half of the oviduct (the tuba or isthmus), and the most distal of these glands also serve as Ssts; in addition, some turtles possess Ssts in the adjacent segment of the oviduct, the uterus. Squamates lack albumen-secreting glands, and the ancestral state is possession of Ssts in the posterior infundibulum (uterine tube). Secondarily, iguanids have evolved vaginal Ssts.

Introduction The Class Reptilia contains some 7200 species and includes Crocodilia (22 species), Chelonia (turtles, 260 species) Rhynchocephalia (2 species), Amphisbaenia (135 species), Sauria (lizards, 3200 species), and Serpentes (snakes, 1800 species; Pough et al. 1998). Amphisbaenids, lizards, and snakes comprise the Squamata, and rhynchocephalians are grouped with squamates to form the Lepidosauria. Crocodilians are grouped with birds in the Archosauria, traditionally considered the sister taxon to Lepidosauria, and Chelonia usually is deemed the sister taxon to Archosauria + Lepidosauria (Fig. 1; Pough et al. 1998). Recent molecular work, however, indicates that Chelonia and Archosauria are closest sister-taxa, and thus Chelonia + Crocodilia is the sister group of Squamata (Fig. 1; Hedges & Poling 1999, Kumazawa & Nishida 1999). Rhynchocephalians may have their closest

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Fig. 1. Recent molecular studies have changed the way we traditionally view the phyletic relationships among amniotes. We adopt the view that turtles form sister group relationships with crocodilians and birds. Since we have no data on the presence of Ssts in crocodilians, we omit them form the cladogram that we use to trace reproductive characters. From Sever and Hamlett (2002).

sister-group relationships with Chelonia + Archosauria rather than Squamata (Hedges & Poling 1999), thus casting doubt on the Lepidosauria as a natural group. In this paper we use the molecular phylogenetic hypothesis ((Chelonia + Archosauria) (Squamata)) to trace evolution of sperm storage characters in reptiles using McClade 3.0 (Maddison & Maddison 1992). This paper is limited to female sperm storage in the oviduct. While little is known on this topic, even fewer observations exist on male sperm storage. Results and discussion Crocodilia.—Davenport (1995) reported that a female caiman (Paleosuchus palpebrosus) laid 16 eggs, in at least one of which an embryo developed, 488 days after isolation from a male. Ferguson (1985) failed to find sperm storage structures in the alligator (Alligator mississippiensis) or the crocodile (Crocodylus niloticus). Palmer & Guillette (1992) reported that the oviduct of A. mississippiensis has separate uterine regions for formation of the eggshell membranes and calcareous layer similar to those of birds and unlike other reptiles. Sperm storage tubules (Ssts) in birds are uterovaginal, and this area should be searched for similar structures in crocodilians. Rhynchocephalia.—Dawbin (1962) stated that 10 months may elapse between copulation and oviposition in the tuatara (Sphenodon punctatus), implying long-term

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sperm storage may occur (St. Girons 1973). No sperm storage structures, however, have been found in the oviduct (Gabe & Saint-Girons 1964). Amphisbaenia.—To our knowledge, no histological descriptions exist of the oviduct of any amphisbaenid, and we know of no observations on the potential for sperm storage. Chelonia.—The first description of Ssts in turtles was in the box turtle (Terrapene carolina) by Hattan & Gist (1975). Subsequently, Ssts were reported from 12 species representing six families (Gist & Jones 1989), and such structures probably exist in the remaining seven families. Turtles have an extensive area in the anterior half of the oviduct, called the tuba by Gist & Jones (1987), in which albumen-secreting glands occur. In the most caudal end of this region some albumen glands also serve as Ssts. In two species, Sternotherus odoratus and Gopherus polyphemus, Ssts also have been reported from the uterine region of the oviduct near the uterovaginal junction (Palmer & Guillette 1988, Gist & Congdon 1998). The only ultrastructural observations on the Ssts of turtles are TEM studies on T. carolina by Gist & Fischer (1993). In turtles: (1) sperm are found in glands located at the periphery and not the center of major glandular regions; (2) TEM of the albumen glands of T. carolina show that tubules containing sperm are identical in ultrastructure to those that do not; (3) Ssts are therefore sites of sperm residence rather than tubules specialized for the maintenance of stored sperm; and (4) despite the lack of glands “specialized” for sperm storage, turtles have perhaps the longest periods of effective sperm storage (up to four years) known among vertebrates. Serpentes.—The first observations on sperm storage in the oviduct of a female snake was made by Rahn (1940) but specialized Ssts were not described until Fox (1956). No Ssts were found in the brown tree snake (Boiga irregularis) by Bull et al. (1997), but these glands are no doubt widespread in snakes, and numerous accounts, often anecdotal, exist on long-term sperm storage in various species (reviewed by Sever & Ryan, 1999). The only ultrastructural studies on the Ssts of snakes concern TEM of the garter snake, Thamnophis sirtalis (Hoffman & Wimsatt 1972), SEM of the ringneck snake, Diadophis punctatus (Perkins & Palmer 1996), and both TEM and SEM of the black swamp snake, Seminatrix pygaea (Sever & Ryan 1999). Ssts occur in a short region between the infundibulum and the uterus called the “uterine tube” (Perkins & Palmer 1996) or simply “the posterior infundibulum” to distinguish this region from the more anterior, aglandular regions of the infundibulum (Fox 1956, Blackburn 1998, Sever & Ryan 1999). In many snakes in temperate regions, however, mating occurs in fall and the sperm are held over-winter in vaginal or uterine sperm receptacles, and migrate to the tubal Ssts for a brief period of storage prior to ovulation in the spring (Halpert et al. 1982, Aldridge 1992, Perkins & Palmer 1996). Snake Ssts are usually characterized as compound alveolar but Sever & Ryan (1999) described them as simple or compound tubular in S. pygaea. As reported for turtles, the glands used for sperm storage do not appear specialized for this function. The epithelium of the glands and surrounding oviduct are similar in histology and consist of alternating ciliated and non-ciliated secretory cells (Sever & Ryan 1999). Sauria.—In lizards, Ssts were first discovered and histologically described in the oviducts of the chameleons, Chamaeleo basiliscus, C. chamaeleon, and C. lateralis (Saint Girons 1962) and the green anole, Anolis carolinensis (Fox 1963). Other literature concerning histological studies on lizard Ssts is given in Table 1. The only ultrastructural

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studies are SEM of the SST area in A. carolinensis (Conner & Crews 1980) and several species of geckos (Girling et al. 1997, 1998), and TEM studies on Ssts of the gecko Acanthodactylus scutellatus (Bou-Resli et al. 1981) and the brown anole, Anolis sagrei (Sever and Hamlett 2002). Both vaginal and infundibular (tubal) Ssts are known (Table 1), and some species in the Iguanidae (Cuellar 1966) and Agamidae (Saint Girons 1973) apparently have Ssts in both areas. Comparative biology.—Character states were determined for location of Ssts and various other reproductive characters for reptiles, birds, and mammals (Table 2). Birds are well known to possess Ssts at the uterovaginal junction (Bakst 1987), and mammals either lack Ssts, or store sperm in the uterus or the uterotubal (isthmus) region (e.g., Racey 1979, Bedford & Breed 1994, Suarez 1998). Mapping of these characters on the phylogeny chosen for this study reveals that an ancestral state for Ssts in amniotes cannot be determined and that Ssts evolved independently in birds, turtles, squamates, and mammals (Fig. 2). The ancestral state for squamates is possession of infundibular Ssts. The lizard data from Table 1 can be mapped on a phylogeny of lizards taken from Pough et al. (1998). The result (Fig. 3) indicates that vaginal Ssts are a specialization found in the Iguania, and that some iguanids also have either retained infundibular Ssts or re-evolved them. Conclusions Sperm storage evolved independently in Chelonia and Squamata. The ancestral state for turtles is storage in posterior albumen-producing glands. The ancestral state for squamates is receptacles in the posterior infundibulum (uterine tube). Vaginal Ssts are Table 1. Literature on location of sperm storage tubules (Ssts) in lizards. From Sever and Hamlett (2002).

D1 Family

D4

Reference Infundibulum

Vagina

Agamidae

X

X

Anguidae

X

Chameleonidae

Saint Girons 1973, Kumari et al. 1990 Saint Girons 1973

X

Saint Girons 1962

Eublepharidae

X

Cuellar 1966

Gekkonidae

X

Cuellar 1966; Bou-Resli et al. l981; Murphy-Walker & Haley 1996, Girling et al. 1997

Iguanidae

X

Polychrotidae Scincidae

X

X

Cuellar 1966, Adams & Cooper 1988

X

Fox 1962, Conner & Crews 1980 Saint Girons 1962, Schaefer & Roeding 1973

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Table 2. Character states and their polarities for sperm storage tubules (Ssts) and some other reproductive characters in reptiles. From Sever and Hamlett (2002).

A – Fertilization 0 – external 1 – internal B – Ovary 0 – solid 1 – hollow C – Albumen 0 – present 1 – absent D – Ssts 0 – absent 1 – posterior infundibulum 2 – tubal albumen glands 3 – uterovaginal 4 – vaginal 5 – uterus or uterotubal *

Birds Turtles Snakes Lizards – 1 Lizards – 2 Mammals – 1 Mammals – 2

A

B

C

D

1 1 1 1 1 1 1

0 0 1 1 1 0 0

0 0 1 1 1 0 0

3 2* 1 1 4 0 5

uterine glands also reported as Ssts in two species of turtles

Fig. 2. Ssts evolved independently in birds, turtles, squamates, and mammals. Among squamates, the ancestral state is infundibular Ssts, and vaginal Ssts have subsequently evolved in some lizards. From Sever and Hamlett (2002).

secondarily derived in iguanids. Oviducal Ssts in birds (+ crocodilians?) and mammals are also independently derived. Any similarities among these taxa in anatomy of the Ssts arise from convergence perhaps due to design constraints of the vertebrate oviduct. Sperm production, mating, and ovulation are often out of phase with one another in reptiles (Schuett 1992). Thus female (as well as male) sperm storage is an obligatory part of the reproductive cycle in many species. We know very little about sperm storage in reptiles. Ultrastructural studies are essential to study sperm/epithelial interactions

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Fig. 3. Vaginal Ssts are a specialization found only in the Iguania. Some iguanids also have either retained infundibular Ssts or re-evolved them. From Sever and Hamlett (2002).

in Ssts; yet out of 7200+ species of reptiles, we have TEM observations on just one turtle, two lizards, and two snakes. We know virtually nothing about the physiology of sperm storage in reptiles, i.e., how viability is maintained and capacitation achieved. Since reptiles are the stem amniote group, they are useful models to include in comparative analyses of oviductal sperm storage mechanisms in birds and mammals. For a more extensive discussion, see Sever and Hamlett (2002). References ADAMS C.S. & W.E. COOPER, Jr. 1988. Oviductal morphology and sperm storage in the keeled earless lizard, Holbrookia propinqua. Herpetologica 44: 190-197. ALDRIDGE R.D. 1992. Oviductal anatomy and seasonal sperm storage in the southeastern crowned snake (Tantilla coronata). Copeia 1992: 1103-1106. BAKST M.R. 1987. Anatomical basis of sperm-storage in the avian oviduct. Scann. Microscop. 1: 1257-1266. BEDFORD J.M. & BREED W.G. 1994. Regulated storage and subsequent transformation of spermatozoa in the Fallopian tubes of an Australian marsupial, Sminthopsis crassicaudata. Biol. Reprod. 50: 845-854. BLACKBURN D.G. 1998. Structure, function, and evolution of the oviducts of squamate reptiles, with special reference to viviparity and placentation. J. Exper. Zool. 282: 560-617. BOU RESLI M.N., BISHAY L.F. & N.S. AL-ZAID 1981. Observations on the fine structure of the sperm storage crypts in the lizard Acanthodactylus scutellatus hardyi. Arch. Biol. (Bruxelles) 92: 287-298.

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BULL K.H., MASON R.T. & J. WHITTIER 1997. Seasonal testicular development and sperm storage in tropical and subtropical populations of the brown tree snake (Boiga irregularis). Aust. J. Zool. 45: 479-488. CONNER J. & D. CREWS 1980. Sperm transfer and storage in the lizard, Anolis carolinensis. J. Morphol. 163: 331-348. CUELLAR O. 1966. Oviductal anatomy and sperm storage structures in lizards. J. Morphol. 130: 129-136. DAVENPORT M. 1995. Evidence of possible sperm storage in the caiman, Paleosuchus palpebrosus. Herp. Rev. 26: 14-15. DAWBIN W.H. 1962. The tuatara in its natural habitat. Endeavour 21: 16-24. FERGUSON M.W.J. 1985. Reproductive biology and embryology of the crocodilians. In Gans C., Billett F. & P.F.A. Maderson (eds), Biology of the Reptilia, Vo. 14, Development A. John Wiley & Sons, New York, pp. 329-491. FOX W. 1956. Seminal receptacles of snakes. Anat. Rec. 124: 519-539. FOX W. 1963. Special tubules for sperm storage in female lizards. Nature 198: 500-501. GABE M. & H. SAINT GIRONS 1964. Histologie de Sphenodon punctatus. Centre National de la Recherche Scientifique, Paris. GIRLING J.E., CREE A. & L.J. GUILLETTE, Jr. 1997. Oviductal structure in a viviparous New Zealand gecko, Hoplodactylus maculatus. J. Morphol. 234: 51-68. GIRLING J.E., CREE A. & L.J. GUILLETTE, Jr. 1998. Oviductal structure in four species of gekkonid lizard differing in parity mode and eggshell structure. Reprod. Fertil. Dev. 10: 139-154. GIST D.H. & J.D. CONGDON 1998. Oviductal sperm storage as a reproductive tactic of turtles. J. Exper. Zool. 282: 526-534. GIST D.H. & E.N. FISCHER 1993. Fine structure of the sperm storage tubules in the box turtle oviduct. J. Reprod. Fert. 97: 463-468. GIST D.H. & J.M. JONES 1987. Storage of sperm in the reptilian oviduct. Scann. Microscop. 1: 1839-1849. GIST D.H. & J.M. JONES 1989. Sperm storage within the oviduct of turtles. J. Morphol. 199: 379-384. HALPERT A.P., GARSTKA W.R. & D. CREWS 1982. Sperm transport and storage and its relationship to the annual cycle of the female red-sided garter snake, Thamnophis sirtalis parietalis. J. Morphol. 174: 149-159. HATTAN L.R. & D.H. GIST 1975. Seminal receptacles in the eastern box turtle, Terrapene carolina. Copeia 1975: 505-510. HEDGES S.B. & L.L. POLING 1999. A molecular phylogeny of reptiles. Science 283: 998-1001. HOFFMAN L.H. & W.A. WIMSATT 1972. Histochemical and electron microscopic observations on the sperm receptacles in the garter snake oviduct. Amer. J. Anat. 134: 71-96. KUMARI T.R.S., SARKAR H.B.D. & T. SHIVANANDAPPA 1990. Histology and histochemistry of the oviductal sperm storage pockets of the agamid lizard Calotes versicolor. J. Morphol. 203: 97-106. KUMAZAWA Y. & M. NISHIDA 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink: statistical evidence for archosaurian affinity of turtles. Mol. Biol. Evol. 16: 784-792. MADDISON W.P. & D.R. MADDISON 1992. MacClade Analysis of Phylogeny and Character Evolution Version 3. Sinauer Associates, Inc., Sunderland, Massachusetts. 398p.

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MURPHY-WALKER S. & S.R. HALEY 1996. Functional sperm storage duration in female Hemidactylus frenatus (family Gekkonidae). Herpetologica 52: 365-373. PALMER B.D. & L.J. GUILLETTE, Jr. 1988. Histology and functional morphology of the female reproductive tract of the tortoise Gopherus polyphemus. Amer. J. Anat. 183: 200-211. PALMER B.D. & L.J. GUILLETTE, Jr. 1992. Alligators provide evidence for the evolution of an archosaurian mode of oviparity. Biol. Reprod. 46: 39-47. PERKINS M.J. & B.D. PALMER 1996. Histology and functional morphology of the oviduct of an oviparous snake, Diadophis punctatus. J. Morphol. 227: 67-79. POUGH F.H., ANDREWS R.M., CADLE J.E., CRUMP M.L., SAVITZKY A.H. & K.D. WELLS 1998. Herpetology. Prentice Hall, Upper Saddle River, New Jersey. 577p. RACEY P.A. 1979. The prolonged storage and survival of spermatozoa in Chiroptera. J. Reprod. Fert. 56: 391-402. RAHN H. 1940. Sperm viability in the uterus of the garter snake Thamnophis. Copeia 1940: 109-115. SAINT GIRONS H. 1962. Presence de receptacles seminaux chez les cameleons. Beaufortia 9: 165-172. SAINT GIRONS H. 1973. Sperm survival and transport in the female genital tract of reptiles. In Hafez E.S.E. & C.G. Thibault (eds), The Biology of Spermatozoa. Karger, Basel, Switzerland, pp. 105—113. SCHAFER G.C. & C. E. ROEDING 1973. Evidence for vaginal sperm storage in the mole skink, Eumeces egregius. Copeia 1973: 346-347. SCHUETT G.W. 1992. Is long-term sperm storage an important component of the reproductive biology of temperate pitvipers? In Campbell J.A. & E.D. Brodie, Jr. (eds), Biology of the Pitvipers. Selva, Austin, Texas, pp. 169—184. SEVER D.M. & W.C. HAMLETT 2002. Female Sperm storage in reptiles. J. Exp. Zool. 292: 187-199. SEVER D.M. & T.J. RYAN 1999. Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea): Part I. Evidence for oviducal sperm storage. J. Morphol. 241: 1-18. SUAREZ S.S. 1998. The modulation of sperm function by the oviductal epithelium. Biol. Reprod. 58: 1102-1104.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Oviducal Sperm Storage in Turkeys (Meleagris Gallopavo) ... Evolution 447 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 447-450, 2003

Oviducal Sperm Storage in Turkeys (Meleagris Gallopavo): The Infundibulum as a secondary Sperm Storage Site, or is it? M.R. Bakst Germplasm and Gamete Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705-2350 USA. E-mail: [email protected]

Abstract Oviducal sperm storage is the basis for sustained fertility in the turkey following a single artificial insemination. While sperm storage tubules (SST) localized in the uterovaginal junction (UVJ) are the primary sperm storage sites, it has been hypothesized that the infundibulum at the anterior end of the oviduct also serves as a site of sperm storage. To determine if the infundibulum is a sperm storage site, hens were inseminated with sperm stained with a nuclear fluorescent dye. Forty-eight hours later the infundibulum was examined using fluorescence light microscopy. No more than 1 to 3 sperm were observed in the infundibulum. The absence of sperm in significant numbers at the infundibulum indicates that while this region is the site of fertilization, it does not function as an oviducal sperm storage site.

Introduction The capacity for oviducal sperm storage in poultry eliminates the need for daily inseminations or copulations in order to obtain a succession of fertile eggs (for reviews see 1-4). A lingering question regarding oviducal sperm during the course of the daily ovulatory cycle is the timing and fate of sperm release from the sperm storage tubules (SST), particularly with respect to ovulation. Once released from the SST, where is the final destination of the oviducal sperm? Current dogma, at least as suggested by Bakst (1981), supports the hypothesis that the infundibulum and the initial 2 cm of the magnum are secondary sperm storage sites. For these to be “secondary sperm storage sites”, it must be accepted that the SST at the uterovaginal junction (UVJ) are the “primary sperm sites”. The latter has been unequivocal demonstrated (for review, see Bakst et al. 1994). Conversely, the existence and possible role of the “secondary sperm storage sites” at the infundibulum needs to be re-examined in the context of published observations.

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The new panorama of animal evolution

Van Drimmelen (1946) was the first to suggest that sustained fertility in the bird was due to oviducal sperm storage in the infundibulum. Following intraperitoneal inseminations he found sperm in the anterior end of the oviduct, and concluded that the infundibulum was the site of oviducal sperm storage. It took nearly two more decades before the uterovaginal junction (UVJ) was examined and found to contain sperm storage tubules (SST) (see Bakst et al. 1994, for review). In 1966, Ogasawara et al and Van Krey et al., showed that intramagnal inseminations, like the intraperitoneal inseminations, resulted in large numbers of sperm at the infundibulum. Interestingly, while large numbers of sperm in the infundibulum increased the duration of fertility, it also increased the incidence of early embryonic mortality. In the following paper, I present data that shows that while sperm may be present in the infundibulum, it is questionable whether the infundibulum is truly a sperm storage region. Data is also presented which shows that the distribution of sperm in the infundibular mucosa is random. Materials and Methods Large White breeder turkeys (British United Turkeys of America), 40-45 wk old were used in this study. Turkeys were maintained in environmentally controlled houses on a 14:10 light:dark photoperiod and housed individually either in cages (fowls and turkey hens) or in groups of 8 to 10 in pens (turkey males). Feed and water were provided ad libitum. Semen was collected and stained prior to inseminations with Hoechst 33342 (90 nM) according to the procedure of Bakst (1994). Within one hour after laying, hens were inseminated with a single deep (4-6 cm) intravaginal insemination of about 250 million sperm. Nine hens were euthanized by cervical dislocation 48 hr after insemination. Small segments (about 2-3 mm) of mucosal folds were excised from the proximal, middle and distal region of the infundibulum. Within each of these regions, 3 samples were removed equidistant from each other. To determine the presence or absence of sperm, each of the 9 mucosal samples per hen was prepared as a squash preparation. For each specimen slide, the presence or absence of sperm in randomly chosen microscopic fields was determined by fluorescence microscopy (40X objective). These data were subjected to a logistic regression and significant differences were identified by general ANOVA and pairwise contrast likelihood ratio tests on generalized logits using Proc Logistic in SAS Version 8 (Stokes et al. 1995). Results and Discussion It has been proposed that the infundibulum functions as a secondary sperm storage site in addition to being the site of fertilization in the avian oviduct (Bakst 1981, Bakst et al. 1994). As a secondary sperm storage site, sperm released from the SST would accumulate at the infundibulum during the daily ovulatory cycle. However, in the present study sperm were rarely observed in the infundibulum. No more than 1 to 3 sperm were observed in an infundibulum in any hen and of the 164 fields examined, only 30 contained sperm. Furthermore, no significant differences (p=0.88) were observed in the

Oviducal Sperm Storage in Turkeys (Meleagris Sperm Gallopavo): StorageThe in the Infundibulum Class Mammalia as ...

449

distribution of sperm between the proximal, middle and distal regions of the infundibulum. Should one consider the infundibulum a specialized region for oviducal sperm storage or is this region a site where sperm reside temporarily within duration of the daily ovulatory cycle? According to Bakst et al (1994) once a hen is in egg production, sperm residing in the SST are released continuously over the course of the daily ovulatory cycle. These sperm ascend to the infundibulum, reside either between apposed mucosal folds or in the subepithelial tubular glands and accumulate in number between successive ovulations. However, while sperm are found in the lumen of each of the oviducal segment throughout the daily ovulatory cycle (Bakst 1981), the population of sperm at the infundibulum at any one period remains sparse. The only evidence that the infundibulum possesses a significant sperm storage capacity is either following an intramagnal or intrauterine insemination or if the hen is inseminated immediately after oviposition. Under such circumstances, it is clear that the transitional tubular glands in the distal infundibulum are engorged with sperm. However, this state has been linked to poor fertility and early embryonic mortality either as a result of poor quality sperm or pathological polyspermy. Whether or not the infundibulum is a true sperm storage site needs to be addressed in the context of what oviducal sperm storage affords to the hen in terms of reproductive success. The ramifications of oviducal sperm storage within the UVJ SST include the following: eliminates the need for synchronization of copulation with ovulation; sperm transfer to hen is not necessary for production of fertile eggs over one or more clutches; provides reservoir for selected sperm; and, affords protection to sperm during daily ovulatory cycle. Given the paucity of sperm observed within the infundibulum, and the likelihood that the majority of sperm in the infundibulum at the time of ovulation interact with and become incorporated with the ovum, it is unlikely that any of these functions can succeed. Therefore, it is suggested that the infundibulum not be considered a sperm storage site analogous to the UVJ SST. References BAKST M.R. 1981. Sperm recovery from oviducts of turkeys at known intervals after insemination and oviposition. J. Reprod. Fert. 62: 159-164. BAKST M.R. 1983. Fate of turkey spermatozoa after intrainfundibular and intramagnal inseminations. J. Reprod. Fert. 67: 315-317. BAKST M.R. 1994. Fate of fluorescent stained sperm following insemination: New light on oviducal sperm transport and storage in the turkey. Biol. Reprod. 50: 987-992. BAKST M.R., WISHART G., & J.P. BRILLARD 1994. Oviducal sperm selection, transport, and storage in poultry. Poult. Sci. Rev. 5: 117-143. OGASAWARA F.X., LORENZ F.W. & L.W. BOBR 1966. Distribution of spermatozoa in the oviduct and fertility in the domestic birds. III. Intra-uterine insemination of semen from lowfecundity cocks. J. Reprod. Fertil. 11: 33-41. STOKES M.E., DAVIS C.S. & G.G. KOCH 1995. Categorical Data Analysis Using the SAS System. Cary, NC: SAS Institute Inc., 499p.

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VAN DRIMMELEN G.C. 1946. Sperm nests in the oviduct of the domestic hen. J. S. African Vet. Med. Assoc. 17: 42-52. VAN KREY H.P., OGASAWARA F.X. & F.W. LORENZ 1966. Distribution of spermatozoa in the oviduct and fertility in the domestic birds. IV. Fertility of spermatozoa from infundibular and uterovagainal glands. J. Reprod. Fert. 11: 257-262.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Sperm Storage in The theNew Class Mammalia Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 451-458, 2003

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Sperm Storage in the Class Mammalia S.S. Suarez Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14583, U.S.A. E-mail: [email protected]

Absrtact In most mammalian species, mating is confined to a specific period in the estrous cycle when the female is receptive to males. Once deposited in the female reproductive tract, sperm must survive until ovulation. For some species, this can be a matter of minutes (such as when mice mate during the ovulatory period), while in others (such as bats) it can be a matter of months. However, the principal site of sperm storage in all nonprimates that have been investigated is the oviduct. Storage takes place in special crypts or within mucosal folds. In many eutherian mammals, sperm are held within mucosal folds near the uterotubal junction by binding to the epithelial surface. This binding has been demonstrated to involve carbohydrate recognition in the hamster (Mesocricetus auratus), horse (Equus caballus), and cow (Bos taurus). In these species, a lectin-like molecule on the surface of sperm binds to a specific carbohydrate moiety on the surface of the epithelium. This interaction somehow serves to maintain the fertility of sperm while they are being held. As the time of ovulation approaches, sperm lose binding affinity for the carbohydrate and are released. The loss is associated with a process known as capacitation; that is, attaining the capacity to undergo the acrosome reaction and fertilize the oocyte. In some marsupials and insectivores, sperm are stored in distinct oviductal crypts, but they do not bind to the epithelium. A storage site has not been identified in the human fallopian tube, but incubation of human sperm with tubal epithelium in vitro prolongs sperm survival.

Introduction In most eutherian mammals, oocytes are fertilized within a few hours of ovulation. In contrast, sperm may have to survive for days, or even months, within the female. In mice, fertilization takes place within hours of mating, but in some species of bats, mating takes place during winter and fertilization occurs in the spring (Hoskin et al. 1996, Bernard & Cumming 1997, Bernard et al. 1997). Sperm are terminally differentiated cells deprived of an active nucleus and a synthetic apparatus; therefore, they must survive those long periods without benefit of the renewal mechanisms available to other cells.

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The new panorama of animal evolution

Overview of Sperm Transport in the Female Mammal The semen of humans and other primates (Harper 1994), and cattle and other ruminants (Hawk 1987, Harper 1994), is deposited in the cranial vagina at the external os of the cervix. In contrast, boars, stallions, and dogs deposit semen directly into the uterus (Harper 1994). Rodent semen is deposited in the vagina, but is rapidly moved en masse into the uterus (Bedford & Yanagimachi 1992). From the uterus, sperm must pass through the uterotubal junction into the isthmus of the oviduct. The junction presents a selective barrier to sperm in many mammals. Its lumen is tortuous and narrow and there are many folds in the mucosa lining the lumen, some of which form dead-end passages (Hook & Hafez 1968, Hafez & Black 1969, Wrobel et al. 1993, Suarez et al. 1997). In some species, smooth muscle and/or a vascular plexus in the wall of the junction that resembles erectile tissue may serve to reduce the lumen at certain times (Wrobel et al. 1993). An additional barrier may be present in the form of viscous mucus, which has been described in rabbits (Jansen & Bajpai 1982), pigs (Suarez et al. 1991), cattle (Suarez et al. 1997), and humans (Jansen 1980). Thus, the junction may act as a valve that closes more tightly at specific times. As such, it can serve to filter out morphologically abnormal sperm or sperm with poor motility. In pigs (Baker & Degen 1972), rats (Gaddum-Rosse 1981), and hamsters (Smith et al. 1988), highly motile sperm pass through the uterotubal junction much more successfully than immotile sperm or those lacking progressive motility. Oviductal sperm reservoir Upon entering the oviduct, sperm become trapped to form a reservoir. The sperm reservoir was first discovered in hamsters by Yanagimachi & Chang (1963) and has since been reported to exist in a variety of mammals (rabbits: Harper 1973, Overstreet et al. 1978, pigs: Hunter 1981, sheep: Hunter & Nichol 1983, cattle: Hunter & Wilmut 1984). The oviductal reservoir of sperm may serve a number of functions. First, it may prevent polyspermic fertilization by allowing only a few sperm at a time to reach the oocyte in the ampulla. Sperm numbers were increased at the site of fertilization in the pig by surgical insemination directly into the oviduct (Polge et al. 1970, Hunter 1973); by resecting the oviduct to bypass the reservoir (Hunter & Leglise 1971); or by administering progesterone into the muscularis to inhibit contractions (Hunter 1972). This increased the incidence of polyspermy. Second, the oviductal reservoir maintains the fertility of sperm between the onset of estrus and ovulation. Sperm fertility and motility were maintained longer in vitro when sperm were incubated with oviductal epithelium (cattle: Pollard et al. 1991, Chian & Sirard 1994, pigs: Suarez et al. 1990, horses: Ellington et al. 1993, humans: Kervancioglu et al. 1994). Third, the processes of capacitation may be regulated within the reservoir. Capacitation is defined as a set of changes in the sperm plasma membrane that enables a sperm to undergo the acrosome reaction (Yanagimachi 1994). Capacitation of bull sperm was enhanced by incubation in medium conditioned by oviductal epithelium (Chian et al. 1995) or in oviduct fluid (Mahmoud & Parrish 1996). There is evidence for several species of eutherian mammals that the oviductal reservoir is created by binding of sperm to oviductal epithelium. Motile sperm have been observed

Sperm Storage in the Class Mammalia

453

Fig. 1. Scanning electron micrograph of bovine sperm in the oviductal isthmus. The sperm are located in grooves created by mucosal folds. They appear to be stuck to cilia, as observed with living tissue in vitro (micrograph by R. Lefebvre, H.-C. Ho & S.S. Suarez)

to bind to oviductal epithelium in cattle (Suarez et al. 1990, Lefebvre et al. 1995a), mice (Suarez 1987), hamsters (Smith & Yanagimachi 1991), pigs (Suarez et al. 1991), and horses (Thomas et al. 1994). The trapping action of the sperm-binding moieties may be enhanced by the narrow, sometimes mucus-filled lumen of the uterotubal junction and initial segment of isthmus, which slow the progress of sperm and increase their contact with the mucosal epithelium. Sperm binding to oviductal epithelium is a specific interaction, in which sperm attach to certain carbohydrates expressed on the mucosal surface. Fetuin and its terminal sugar, sialic acid, were found to inhibit binding of hamster sperm to epithelium (DeMott et al. 1995). Fetuin-binding sites were localized to the head of hamster sperm and fetuinbinding proteins were detected in extracts of sperm proteins (DeMott et al. 1995). These data indicate that there is a protein on the head of hamster sperm that binds sialic acid and is responsible for attachment of sperm to the epithelium. Binding of stallion sperm to explants of oviductal epithelium was inhibited by asialofetuin and its terminally expressed sugar, galactose (Lefebvre et al. 1995b, Dobrinski et al. 1996). Bull sperm binding to oviductal epithelium was blocked by fucoidan and its component fucose (Lefebvre et al. 1997). Fluorescent fucosylated molecules were seen to bind to sperm heads (Suarez et al. 1998, Revah et al. 2000). Pretreatment of bovine epithelium with fucosidase, but not galactosidase, reduced sperm binding (Lefebvre et al. 1997). Thus, carbohydrate involvement in sperm binding to epithelium appears to be a widespread phenomenon, although the particular carbohydrate within the binding site varies according to species. The species differences may not seem so unusual when one considers that a single amino

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acid residue can determine the carbohydrate specificity of a lectin (Revelle et al. 1996), and that closely-related animal lectins have different carbohydrate specificities (Weiss 1994). How are sperm released from the reservoir? Theoretically, sperm could be released if the carbohydrate ligand is lost from the mucosal surface or if sperm lose the capacity to bind the ligand. It was found that changes in the hormonal state of oviductal epithelium related to impending ovulation did not reduce sperm binding (Lefebvre et al. 1995a); therefore, it appears that the epithelium does not release sperm by losing binding sites. Instead, current evidence indicates that a change in the sperm brings about its release. Capacitation involves changes in the plasma membrane over the sperm head and, therefore, could lead to sperm release by eliminating or modifying binding molecules on the head. Hyperactivation (a change in sperm flagellar movement pattern that occurs in the oviduct) may provide the force necessary for overcoming the attraction between sperm and oviductal epithelium. Smith & Yanagimachi (1991) reported that capacitated/ hyperactivated hamster sperm were not trapped when infused into oviducts. Videomicroscopy of transilluminated mouse oviducts revealed that only hyperactivated sperm detached from epithelium (DeMott & Suarez 1992). When bull sperm were capacitated, binding to explants of oviductal epithelium was significantly reduced (Lefebvre et al. 1996). Thus, it is evident that changes in the sperm head surface are responsible for loss of binding affinity, while the pull produced by hyperactivation may enhance the ability of sperm to release. Epithelial secretions initiated by signals of impending ovulation could enhance sperm capacitation and hyperactivation, thereby bringing about sperm release. Soluble oviductal factors were found to enhance capacitation of bull sperm (Chian et al. 1995, Mahmoud & Parrish 1996). The molecule on sperm that is responsible for binding to the epithelium is lost or loses carbohydrate binding affinity in capacitated sperm. Capacitated hamster sperm and protein extracts show reduced binding of fetuin (DeMott et al. 1995). When bull sperm were capacitated in vitro, they no longer labelled with fucosylated probes (Revah et al. 2000). A fucose-binding protein obtained by affinity purification of bull sperm extracts, was reduced in extracts of capacitated sperm (Ignotz et al. 2001). Marsupials and Insectivores In marsupial mammals (Bedford 1991, Taggart 1994) and some insectivores (Bedford et al. 1997a,b), sperm are stored in mucosal crypts in the oviduct; however, the sperm heads do not attach to the epithelium in the crypts. Many of the sperm in the tubules of the marsupial Sminthopsis crassicuadata were observed to be immotile (Bedford & Breed 1994), and so motility suppression may serve to hold sperm within crypts until ovulation. In the primitive eutherian mammals, the insectivores, some species possess distinctive bubble-like outpocketings of the oviduct wall in the caudal ampulla. Sperm enter these structures and do not adhere to the epithelium (Bedford et al. 1997a,b). If one assumes that the insectivores possess primitive reproductive characteristics, distinctive storage

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structures seem to have been lost from the oviduct in the course of evolution of eutherian mammals and replaced by trapping sperm via binding them to the epithelium. Humans So far, there has been no conclusive evidence for a distinct oviductal sperm reservoir in humans (Williams et al. 1993). Data of sperm distribution in the tubes of women have not provided a clear picture of the events of sperm transport and sperm have not been found to be concentrated in a specific region (e.g., Williams et al. 1993). In vitro, human sperm do not bind as readily to oviductal epithelium as other species (Yeung et al. 1994, Pacey et al. 1995, Murray & Smith 1997). Nevertheless, human sperm viability is maintained by incubation with oviductal epithelium (Murray & Smith 1997), as it is in species in which there is strong binding of sperm to epithelium. One possibility is that the human cervix serves as the site of a sperm reservoir. The lumen of the human cervix is 3 cm in length and lined by extensive mucosal folds. Human sperm must travel only a few cm through the uterus to reach the uterotubal junction, which is shaped rather like a funnel and is not guarded by mucosal folds as in other species (Hafez & Black 1969). The evidence against a cervical reservoir is that very few sperm have been recovered from human uteri 24 hours after coitus (Rubenstein et al. 1951, Moyer et al. 1970) and leukocytic infiltration of the cervix and uterus becomes significant several hours after coitus (Thompson et al. 1992), thereby presenting a barrier to passage of sperm stored in the cervix. Unless sperm are protected from phagocytosis (and they might be!), it is unlikely that they could travel from the cervical reservoir to the oviduct 24 hours post coitus. Alternatively, human sperm could be retained in the oviduct, but not in a distinct reservoir and not by binding tightly to the mucosa. The mucosal folds of the human oviductal lumen are small in the caudal isthmus and increase in size and complexity towards the ovary, thus offering increasingly greater barriers to the advancement of sperm. Sperm progress could be also be slowed by the mucus in the isthmus (Jansen 1980) and by sticking lightly to the mucosa (Pacey et al. 1995). So, rather than having a distinct reservoir, human sperm advancement to the site of fertilization could be slowed in such a manner so as to increase the chances that a few will be present at the site of fertilization when ovulation occurs. References BAKER R.D. & A.A. DEGEN 1972. Transport of live and dead boar spermatozoa within the reproductive tract of gilts. J. Reprod. Fert. 28: 369-377. BEDFORD J.M. 1991. The coevolution of mammalian gametes. In Dunbar B.S. & M.G. O’Rand (eds), A Comparative Overview of Mammalian Fertilization. Plenum Press, New York, pp. 3-35. BEDFORD J.M. & W.G. BREED 1994. Regulated storage and subsequent transformation of spermatozoa in the fallopian tubes of an Australian marsupial. Sminthopsis crassicaudata. Biol. Reprod. 50: 845-854. BEDFORD J.M. & R. YANAGIMACHI 1992. Initiation of sperm motility after mating in the rat. J. Androl. 13: 444-449.

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BEDFORD J.M., MOCK O.B. & D.M. PHILLIPS 1997a. Unusual ampullary sperm crypts, and behavior and role of the cumulus oophorus, in the oviduct of the least shrew, Cryptotis parva. Biol. Reprod. 56: 1255-1267. BEDFORD J.M., PHILLIPS D.M. & H. MOVER-LEV 1997b. Novel sperm crypts and behavior of gametes in the fallopian tube of the white-toothed shrew, Crocidura russula Monacha. J.Exptl. Zool. 277: 262-273. BERNARD R.T.F. & G.S. CUMMING 1997. African bats: Evolution of reproductive patterns and delays. Quarterly Review. Biol. 72: 253-274. BERNARD R.T.F., HAPPOLD D.C.D. & M. HAPPOLD 1997. Sperm storage in a seasonally reproducing African vespertilionid, the banana bat (Pipistrellus nanus) from Malawi. J. Zool. Lond. 241: 161-174. CHIAN R.I.-C., LAPOINTE S. & M.-A. SIRARD 1995. Capacitation in vitro of bovine spermatozoa by oviduct cell monolayer conditioned medium. Mol. Reprod. Dev. 42: 318-324. CHIAN R.-C. & M.-A. SIRARD 1994. Fertilizing ability of bovine spermatozoa cocultured with oviduct epithelial cells. Biol. Reprod. 52: 156-162. DEMOTT R.P., LEFEBVRE R. & S.S. SUAREZ 1995. Carbohydrates mediate the adherence of hamster sperm to oviductal epithelium. Biol Reprod 52: 1395-1403. DEMOTT R.P. & S.S. SUAREZ 1992. Hyperactivated sperm progress in the mouse oviduct. Biol. Reprod. 46: 779-785. DOBRINSKI I., IGNOTZ G.G., THOMAS P.G.A. & B.A. BALL 1996. Role of carbohydrates in the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells in vitro. Amer. J. Vet. Res. 57: 1635-1639. GADDUM-ROSSE P. 1981. Some observations on sperm transport through the uterotubal junction of the rat. Amer. J. Anat. 160: 333-341. HAFEZ E.S.E. & D.L. BLACK 1969. The mammalian uterotubal junction. In Hafez E.S.E. & R.J. Blandau (eds), The Mammalian Oviduct: Comparative Biology and Methodology. The University of Chicago Press, Chicago, pp. 85-128. HARPER M.J.K. 1994. Gamete and zygote transport. In Knobil E. & J.D. Neill (eds), The Physiology of Reproduction, 2nd ed., Raven Press, Ltd, New York, pp. 123-187. HAWK H.W. 1987. Sperm survival and transport in the female reproductive tract. J. Dairy Sci. 66: 2645-2660. HOOK S.J. & E.S.E. HAFEZ 1968. A comparative study of the mammalian uterotubal junction. J. Morphol. 125: 159-184. HOSKEN D.J., O’SHEA J.E. & M.A. BLACKBERRY 1996. Blood plasma concentrations of progesterone, sperm storage and sperm viability and fertility in Gould’s wattled bat (Chalinolobus gouldii). J. Reprod. Fert. 108: 171-177. HUNTER R.H.F. 1972. Local action of progesterone leading to polyspermic fertilization in pigs. J. Reprod. Fert. 31: 433-444. HUNTER R.H.F. 1973. Polyspermic fertilization in pigs after tubal deposition of excessive numbers of spermatozoa. J. Exp. Zool. 183: 57-64. HUNTER R.H.F. 1981. Sperm transport and reservoirs in the pig oviduct in relation to the time of ovulation. J. Reprod. Fert. 63: 109-117. HUNTER R.H.F. & P.C. LEGLISE 1971. Polyspermic fertilization following tubal surgery in pigs, with particular reference to the role of the isthmus. J. Reprod. Fert. 24: 233-246.

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HUNTER R.H.F. & R. NICHOL 1983. Transport of spermatozoa in the sheep oviduct: preovulatory sequestering of cells in the caudal isthmus. J. Exp. Zool. 228: 121-128. HUNTER R.H.F. & I. WILMUT 1984. Sperm transport in the cow: periovulatory redistribution of viable cells within the oviduct. Reprod. Nutr. Devel. 24: 597-608. IGNOTZ G.G., LO M.C., PEREZ C.L., GWATHMEY T.M. & S.S. SUAREZ 2001. Characterization of a fucose-binding protein from bull sperm and seminal plasma that may be responsible for formation of the oviductal sperm reservoir. Biol. Reprod. 64: 1806-1811. JANSEN R.P.S. 1980. Cyclic changes on the human fallopian tubes isthmus and their functional importance. Am. J. Obstet. Gynecol. 136: 292-308. JANSEN R.P.S. & V.K. BAJPAI 1982 Oviduct acid mucus glycoproteins in the estrous rabbit: ultrastructure and histochemistry. Biol. Reprod. 26: 155-168. KERVANCIOGLU M.E., DJAHANBAKHCH O. & R.J. AITKEN 1994. Epithelial cell coculture and the induction of sperm capacitation. Fert. Steril. 61: 1103-1108. LEFEBVRE R., CHENOWETH P.J., DROST M., LECLEAR C.T., MACCUBBIN M., DUTTON J.T. & S.S. SUAREZ 1995a. Characterization of the oviductal sperm reservoir in cattle. Biol. Reprod. 53: 1066-1074. LEFEBVRE R., DEMOTT R.P., SUAREZ, S.S. & J.C. SAMPER 1995b. Specific inhibition of equine sperm binding to oviductal epithelium. Equine Reproduction VI, Biol. Reprod. Mono. 1: 689-696. LEFEBVRE R., LO M.C. & S.S. SUAREZ 1997. Bovine sperm binding to oviductal epithelium involves fucose recognition. Biol. Reprod. 56: 1198-1204. LEFEBVRE R. & S.S. SUAREZ 1996. Effect of capacitation on bull sperm binding to homologous oviductal epithelium. Biol. Reprod. 54: 575-582. MAHMOUD A.I. & J.J. PARRISH 1996. Oviduct fluid and heparin induce similar surface changes in bovine sperm during capacitation. Mol. Reprod. Devel. 43: 554-560. MOYER D.L., RIMDUSIT S. D.R. MISHELL, Jr. 1970. Sperm distribution and degradation in the human female reproductive tract. Obstet. Gynecol. 35: 831-840. MURRAY S.C. & T.T. SMITH 1997. Sperm interaction with Fallopian tube apical plasma membrane enhances sperm motility and delays capacitation. Fertil. Steril. 68: 352-357. OVERSTREET J.W., COOPER G.W. & D.F. KATZ 1978. Sperm transport in the reproductive tract of the female rabbit: II. The sustained phase of transport. Biol. Reprod. 19: 115-132. PACEY A.A., HILL C.J., SCUDAMORE I.W., WARREN M.A., BARRATT C.L.R. & I.D. COOKE 1995. The interaction in vitro of human spermatozoa with epithelial cells from the human uterine (Fallopian) tube. Human Reprod. 10: 360-366. POLGE C., SALAMON S. & I. WILMUT 1970. Fertilizing capacity of frozen boar semen following surgical insemination.Veterinary Rec. 87: 424-428. POLLARD J.W., PLANTE C., KING W.A., HANSEN P.J., BETTERIDGE K.J. & S.S. SUAREZ 1991. Fertilizing capacity of bovine sperm may be maintained by binding to oviductal epithelial cells. Biol. Reprod. 44: 102-107. REVAH I., SUAREZ S.S., FLESCH F.M., COLENBRANDER B. & B.M. GADELLA 2000. The physiological state of bull sperm affects fucose- and mannose-binding properties. Biol. Reprod. 62: 1010-1015. REVELLE B.M., SCOTT D. & P.J. BECK 1996. Single amino acid residues in the E- and P-selectin epidermal growth factor domains can determine carbohydrate binding specificity. J. Biol. Chem. 271: 16160-16170.

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RUBENSTEIN B.B., STRAUSS H., LAZARUS M.L. & H. HANKIN 1951. Sperm survival in women. Fert. Steril. 2: 15-19. SMITH T.T., KOYANAGI F. & R. YANAGIMACHI 1988. Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination. Biol. Reprod. 37: 225-234. SMITH T.T. & R. YANAGIMACHI 1991. Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct. J. Reprod. Fertil. 91: 567-573. SUAREZ S.S. 1987. Sperm transport and motility in the mouse oviduct: observations in situ. Biol. Reprod. 36: 203-210. SUAREZ S.S., BROCKMAN, K. & R. LEFEBVRE 1997. Distribution of mucus and sperm in bovine oviducts after artificial insemination. Biol. Reprod. 56: 447-453. SUAREZ S.S., DROST M., REDFERN K. & W. GOTTLIEB 1990 Sperm motility in the oviduct. In Bavister B.D., Cummins J. & E.R.S. Roldan (eds), Fertilization in Mammals. Serono Symposia, Norwell, pp. 111-124. SUAREZ S.S., REDFERN K., RAYNOR P., MARTIN F. & D.M. PHILLIPS 1991. Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir. Biol. Reprod. 44: 998-1004. SUAREZ S.S., REVAH I., LO M., KØLLE S. 1998. Bull sperm binding to oviductal epithelium is mediated by a Ca2+-dependent lectin on sperm which recognizes Lewis-A trisaccharide. Biol. Reprod. 59: 39-44. TAGGART D.A. 1994. A comparison of sperm and embryo transport in the female reproductive tract of marsupial and eutherian mammals. Reprod. Fertil. Dev. 6: 451-472. THOMPSON L.A., BARRATT C.L.R., BOLTON A.E. & I.D. COOK 1992. The leukocytic reaction of the human uterine cervix. Amer. J. Reprod. Immunol. 28: 85-89. WEISS W.I. 1994. Recognition of cell surface carbohydrates by C-type animal lectins. In Metcalf B.W., Dalton B.J. & G. Poste (eds), Cellular Adhesion. Plenum Press, New York. WILLIAMS M., HILL C.J., SCUDAMORE I., DUNPHY B., COOKE I.D. & C.L.R. BARRATT 1993. Sperm numbers and distribution within the human Fallopian tube around ovulation. Human Reprod. 8: 2019-2026. WROBEL K.-H., KUJAT R. & G. FEHLE 1993. The bovine tubouterine junction: general organization and surface morphology. Cell. Tissue Res. 271: 227-239. YANAGIMACHI R. 1994. Mammalian fertilization. In Knobil, E. & J.D. Neill (eds), The Physiology of Reproduction. Raven Press, Ltd., New York, pp. 189-317. YANAGIMACHI R. & M.C. CHANG 1963. Sperm ascent through the oviduct of the hamster and rabbit in relation to the time of ovulation. J. Reprod. Fertil. 6: 413-420. YEUNG W.S.B., NG V.K.H., LAU E.Y.L. & P.C. HO 1994. Human oviductal cells and their conditioned medium maintain the motility and hyperactivation of human spermatozoa in vitro. Human. Reprod. 9: 656-660.

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© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Integrative approaches to phylogenetic relationships of arthropods ... Evolution 461 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 461-466, 2003

Integrative approaches to phylogenetic relationships of arthropods: Introduction to the Symposium A. Schmidt-Rhaesa Zoomorphology and Systematics, University of Bielefeld, P.O. Box 100131, 33501 Bielefeld, Germany. E-mail: [email protected]

Abstract The traditional hypothesis of a closer relationship between panarthropods (Euarthropoda, Tardigrada, Onychophora) and annelids in a taxon Articulata has been challenged by molecular systematical data. These favour a monophyletic taxon Ecdysozoa that contains all moulting animals and indicate a closer relationship of panarthropods and e.g. nematodes and priapulids. These competing hypotheses stimulate a large-scale discussion of all available data. The morphological characters that have been named for Articulata have to be critically discussed and compared to probable autapomorphies of Ecdysozoa. Molecular systematical data have to be complemented by more data, especially from different genes (to date, most information is available for the 18S rDNA gene). Very important for the discussion are developmental biological data, especially by comparing segmentation patterns in different metazoan taxa, because segmentation is the most convincing character complex supporting Articulata. Furthermore, palaeontological data can be consulted to elucidate the problem. The diversity of stem group arthropods known today might make it possible to draw conclusions about the last common ancestor of Panarthropoda and to compare it in the light of the Articulata/Ecdysozoa problem. This diversity of approaches illustrates the character of modern systematics: complex systematical problems call for an integration of comparative analyses from different disciplines. The symposium reviews the contribution of Comparative Morphology, Molecular Systematics, Developmental Biology and Palaeontology to the Articulata/Ecdysozoa problem.

In 1817, in his famous “La Régne Animal”, Georges Cuvier named his third division of the animal kingdom “the articulated animals” and united under this annelids and arthropods. Although every now and then different associations were proposed, the hypothesis of a close relationship of annelids and arthropods remained one of the most stable through history. Therefore it was surprising to find that, after molecular genetical methods became widespread in phylogenetic research, there was hardly any signal for a monophyletic group Articulata. After the genes and the methodology became better known, an alternative hypothesis arose: a close relationship of arthropods with other

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moulting animals, e.g. nematodes and priapulids (Aguinaldo et al. 1997). This taxon was named Ecdysozoa. There are two reasons for the late emergence of the Ecdysozoa-hypothesis: the long absence of hypotheses of phylogenetic relationships of the aschelminth (nemathelminth, pseudocoelomate) taxa and long-branch effects in molecular systematical analyses. For a long time the names Aschelminthes, Nemathelminthes or “pseudocoelomates” were only loose bonds for vague associations of groups that could not be assigned to other taxa. Gastrotricha, Nematoda, Nematomorpha, Priapulida, Kinorhyncha, Loricifera, Rotifera and Acanthocephala were regularly assigned to this grouping and some authors also added Tardigrada (Ruppert & Barnes 1994), entoprocts and gnathostomulids (Brusca & Brusca 1990) or chaetognaths (Wallace et al. 1995, 1996). The first attempt to systematize this grouping using the methodology of phylogenetic systematics was made by Lorenzen (1985). In the mid-90s, three groups arrived in parallel to partly similar conclusions about phylogenetic relationships: Nielsen (1995), Ehlers et al. (1996) and Wallace et al. (1995, 1996). All authors agreed in a monophyletic taxon containing Gastrotricha, Nematoda, Nematomorpha, Priapulida, Kinorhyncha and Loricifera, which was named Cycloneuralia by Nielsen (1995) but Nemathelminthes by Ahlrichs (1995) and Ehlers et al. (1996) (or Nemathelminthes s.str. by Neuhaus 1994). According to Wallace et al. (1996), the sister group of Nemathelminthes/Cycloneuralia is Gnathostomulida, followed by (Acanthocephala + Rotifera), according to Nielsen (1995) the sister groups are Chaetognatha and (Acanthocephala + Rotifera) in an unresolved trichotomy. After Ahlrichs (1995) (see also Ehlers et al. 1995, Ahlrichs 1997), Rotifera and Acanthocephala belong to another clade with a close relationship to Gnathostomulida, which belong, due to spiral cleavage of gnathostomulids (Riedl 1969), into the Spiralia. The new discovery of Limnognathia (Micrognathozoa; Kristensen & Funch 2000) and further investigations on gnathostomulids (Herlyn & Ehlers 1997, Sørensen 2000) support this hypothesis. This paraphyly of “Aschelminthes” has also been shown by 18S rDNA sequence comparisons (Winnepenninckx et al. 1995). As has been mentioned, different names have been proposed in parallel by Nielsen (1995) and Ahlrichs (1995). The monophylum of (Nematoda, Nematomorpha, Priapulida, Kinorhyncha and Loricifera) was named Cycloneuralia by Ahlrichs (1995) and Introverta by Nielsen (1995), this taxon plus Gastrotricha is named Nemathelminthes by Ahlrichs (1995) and Cycloneuralia by Nielsen (1995). I regard the name Introverta as not fortunate, because it is not at all sure if an introvert (an in- and evaginable anterior body part) was present in the common ancestor of this taxon (Schmidt-Rhaesa 1998) and therefore encourage to apply the names sensu Ahlrichs: Cycloneuralia and Nemathelminthes. Long-branch effects are caused in molecular systematical analyses if taxa with widely differing rates of substitution are analyzed. High substitution rates are named as such when a sequence accumulated a high number of substitutions compared with other taxa in the analysis. If sequence diversity is translated into branch length in graphical illustrations, long branches connect these “fast evolving” taxa with other nodes in the tree. Long-branch taxa very often cause serious problems. First they are difficult to align with other sequences and therefore reduce the quality of the statements of positional homologies. In the analysis, long-branch taxa will often cluster unreliably, a problem known as the “Felsenstein zone” (Felsenstein 1978, Swofford et al. 1996, Page & Holmes

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1998). Probably due to homoplasies and symplesiomorphies, sequences of long-branch taxa cluster either with other fast evolving sequences or move down the tree into basal positions. Examples are rhabditid nematodes (e.g. Garey et al. 1996), chaetognaths (Halanych 1996), myxozoans (Smothers et al. 1994) and gnathostomulids (Littlewood et al. 1998) which all have extremaly long branches and may cluster together unreliably (Littlewood et al. 1998). The current state in the discussion about Articulata or Ecdysozoa remains exciting. From the perspective of molecular systematics, most evidence for Ecdysozoa comes from the 18S rDNA gene (e.g. Aguinaldo et al. 1997, Eernisse 1997, Zrzavy et al. 1998), but the strength of this evidence has also been doubted (Wägele et al. 1999). Further analyses using elongation factor-1α (McHugh 1997), Hox-genes (De Rosa et al. 1999) or β-Thymosin (Manuel et al. 2000) also support Ecdysozoa. Analyses from other genes may not support Ecdysozoa: Goodman et al. (1988) find evidence for Articulata analysing globin sequences, an analysis of a concatenated sequence from 10 nuclear genes (Hausdorf 2000) and another of 50 genes (Wang et al. 1999) do not support monophyly of Ecdysozoa. However, none of these analyses included representatives of all three taxa in question (Annelida, Panarthropoda, Cycloneuralia) and the standard representative of Cycloneuralia, the nematode Caenorhabditis elegans, is known to have many fast-evolving genes (Garey, this volume). Mushegian et al. (1998) found in an analysis of sequences of 42 genes that faster evolving genes often do not support Ecdysozoa while slower evolving genes do. The evidence from comparative morphology seems to be extremely difficult to evaluate. Articulata is supported by characters that more or less belong to the context of segmentation, while characters supporting Ecdysozoa are connected to the complex of cuticle and moulting (Schmidt-Rhaesa et al. 1998). For all characters in discussion, examples can be found where this character likely evolved convergently within Metazoa. Examples are the embryological segmentation patterns in chordates (Patel et al. 1989) which resembles the one in annelids and arthropods or moulting with ecdysteroid hormones in leeches (Sauber et al. 1983) which resembles moulting in panarthropods and cycloneuralians (for a partial moulting in the oligochaete Tubificoides benedii see Giere et al. 1988). One difficulty is that the ground patterns (ancestral states) for annelids and panarthropods are not clearly worked out, which renders a comparison of characters more difficult. One promising approach may arise from the analysis of basal panarthropods, i.e. tardigrades and onychophorans. The basal panarthropods are also interesting from the fossil perspective, because numerous fossils that had been evaluated as “weird wonders” (such as Anomalocaris, Hallucigenia and Opabinia, see Gould 1989) have now been included into Panarthropoda as stem group fossils (e.g. Budd 1996, 1997, 1998, Dewel & Dewel 1997). This may shed light on the reconstruction of the panarthropod ancestor and make it possible to compare it to either annelids or cycloneuralians. Very promising are the current advances in developmental biology. We are beginning to understand the interactions of the genetical and phenotypical level during embryology. Interesting for the Articulata-Ecdysozoa-problem is especially the understanding of segmentation, because segmentation is often regarded as a character of high complexity that is unlikely to evolve convergently. Molecular developmental biology might find

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details for the comparison of annelids, panathropods and cycloneuralians that are able to support or contradict hypotheses of homology. The preceding reflections illustrate that different disciplines are currently contributing to the solution of the Articulata/Ecdysozoa-problem. Apart from Molecular Genetics, Comparative Morphology, Palaeontology and Developmental Biology, any other discipline with a comparative approach is likely to make valuable contributions to this (and other) problem(s). For example, a broad physiological approach comparing the hormonal basis of moulting is desirable. This Special Symposium „ Integrative approaches to phylogenetic relationships of arthropods” brings together specialists of Molecular Genetics (J.R. Garey), Comparative Morphology (R.M. Kristensen), Palaeontology (G. Budd) and Developmental Biology (G. Scholtz) to review the current state in the discussion of the Articulata/Ecdysozoaproblem. Acknowledgements Many thanks to the Deutsche Zoologische Gesellschaft (DZG), which supported this symposium with financial support for the two german contributors. References AGUINALDO A.M.A., TURBEVILLE J.M., LINFORD L.S., RIVERA M.C., GAREY J.R., RAFF R.A. & J.A. LAKE 1997. Evidence for a clade of nematodes, arthropods, and other molting animals. Nature 387: 489-493. AHLRICHS W. 1995. Ultrastruktur und Phylogenie von Seison nebaliae (Grube 1859) und Seison annulatus (Claus 1876). Hypothesen zu phylogenetischen Verwandtschaftsverhältnissen innerhalb der Bilateria. Cuvillier Verlag, Göttingen. 310p. AHLRICHS W. 1997. Epidermal ultrastructure of Seison nebaliae and Seison annulatus, and a comparison of epidermal structures within the Gnathifera. Zoomorphology 117: 41-48. BRUSCA R.C. & G.J. BRUSCA 1990. Invertebrates. Sinauer, Sunderland. 922p. BUDD G.E. 1996. The morphology of Opabinia regalis and the reconstruction of the arthropod stem-group. Lethaia 29: 1-14. BUDD G.E. 1997. Stem group arthropods from the Lower Cambrian Sirius Passet fauna of North Greenland. In Fortey R.A. & R.H. Thomas (eds), Arthropod Relationships. Syst. Assoc. Spec. Vol. Ser. 55. Chapman and Hall, London, pp. 125-138. BUDD G.E. 1998. Arthropod body-plan evolution in the Cambrian with an example from anomalocaridid muscle. Lethaia 31: 197-210. CUVIER G. 1817. Le régne animal. Vol. II. Deterville, Paris. 532p. DE ROSA R., GRENIER J.K., ANDREEVA T., COOK C.E., ADOUTTE A., AKAM A. CARROLL S.B. & G. BALAVOINE 1999. Hox genes in brachiopods and priapulids and protostome evolution. Nature 399: 772-776. DEWEL R.A. & W.C. DEWEL 1997. The place of tardigrades in arthropod evolution. In Fortey R.A. & R.H. Thomas (eds), Arthropod Relationships. Syst. Assoc. Spec. Vol. Ser. 55. Chapman and Hall, London, pp. 109-123.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Comparative Morphology: Do the ultrastructuralThe investigations ofAnimal ... Evolution 467 New Panorama of Proc. 18th Int. Congr. Zoology, pp. 467-477, 2003

Comparative Morphology: Do the ultrastructural investigations of Loricifera and Tardigrada support the clade Ecdysozoa? R.M. Kristensen Department of Invertebrate Zoology, Zoological Museum, University of Copenhagen, Copenhagen, Denmark. E-mail: [email protected].

Abstract The loriciferans are one of the most recently discovered groups of microscopic marine animals. They have complex alimentary, reproductive, excretory, nervous and muscular systems and possess a molting cuticle with chitin. The head of the adults may have up to 235 multicellular appendages (scalids) covered by cuticle, and the larvae develop leg-like locomotory spines and terminal toes. The loriciferans make up their own phylum, but are usually considered related to the phyla Kinorhyncha and Priapulida in the taxon Scalidophora. Telescoping of the mouth cone, annulation of the flexible buccal tube, and three rows of placoids in the triradiate myo-epithelial pharynx bulb are found only in Loricifera and Tardigrada. The unique long muscle attachment fibers found in Loricifera may be an adaptation to small size although similar long fibers have been described in the legs of marine Arthrotardigrada. The similarities between Panarthropoda (including Tardigrada) and Cycloneuralia (including Loricifera) could be true homologies rather than surprising analogies. Panarthropoda may have developed from Cycloneuralia, which possess a molting cuticle, and not from annelid-like animals which lack a “true” molting cuticle. Tardigrada may have plesiomorphic characters in common with Cycloneuralia that may have been lost in other panarthropods. The new morphological data on Loricifera and Tardigrada may therefore support the clade Ecdysozoa, a monophylum including all molting invertebrates, originally based only on molecular data.

Introduction In many recent textbooks, Panarthropoda (Arthropoda, Onychophora, Tardigrada) is regarded as a sister-group to Annelida in the taxon Articulata (Nielsen 1995), but this view is not supported by molecular data (Aguinaldo et al. 1997). Recently the older theory (Rauther 1909) hypothesizing a relationship between arthropods and some groups of aschelminths (Cycloneuralia) has gained support, primarily based on analysis of 18S rRNA gene sequences. Comparisons of these molecular data suggest that all taxa with

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ecdysis (Nematoda, Nematomorpha, Priapulida, Kinorhyncha, Loricifera and Panarthropoda) should be included in a monophyletic taxon Ecdysozoa (for review, see Schmidt-Rhaesa et al. 1998). There is also new fossil evidence from the so-called Burgess Shale Fauna (e.g. Anomalocaris and Opabinia) which suggest surprising affinities between Cycloneuralia (especially Kinorhyncha, Priapulida and Loricifera) and Panarthropoda (Kristensen 1991a, Budd 1996). Therefore, this paper will briefly consider the morphology of Tardigrada (Panarthropoda) and Loricifera (Cycloneuralia) based mainly on new ultrastructural research, which may support the clade Ecdysozoa. There are two major schools of thought concerning the phylogenetic position of Tardigrada. One school (“the American school”) places the tardigrades within the polyphyletic group Aschelminthes (Crowe et al. 1970, Dewel & Clark 1973, Ruppert & Barnes 1994), the second school (“the European school”) places them within Panarthropoda (Kristensen 1976, 1981, Møbjerg & Dahl 1996, Nielsen 1997), while molecular analyses suggest that tardigrades are related to both arthropods and some aschelminth groups (Garey et al. 1996, Garey, this volume). This discussion will focus on a new general tardigrade bauplan based on TEM-data which is central to a phylogenetic discussion of Ecdysozoa (for review, see Dewel & Dewel 1997, Schmidt-Rhaesa et al. 1998). Traditionally, the following tardigrade characteristics indicate arthropod relationships: segmented legs, a procuticle containing chitin (Kristensen & Neuhaus 1999), a very complex molting cycle (Kristensen 1976), sensillae of arthropod type (Kristensen 1981), cross-striated muscles, Malpighian tubules (Møbjerg & Dahl 1996), rectal pads, and a peritrophic membrane (Greven 1982). Furthermore, tardigrades are strictly segmented animals, consisting of three to four fused head segments and always four trunk segments (for review, see Dewel et al. 1993, 1999). However, some Cycloneuralia also show many of these so-called arthropod characters, e.g. Kinorhyncha, Priapulida and Loricifera have chitin (shown by WGA-gold labelling technique) in the procuticle (Neuhaus et al. 1996, 1997 ) and Kinorhyncha display metamery in the nervous system, in the muscular arrangement, and in the distribution of cuticular plates (Kristensen & Higgins 1991). Furthermore, all kinorhynchs have 13 segments. They hatch with 11 segments and add two segments during the first two molts (Neuhaus 1995), similar but perhaps not homologous to teloblastic growth in both Annelida and Arthropoda. Finally chitin is also present in the larval cuticle of Nematomorpha but not of the adult, and in the pharyngeal cuticle of nematodes (Neuhaus et al. 1996). The morphology of Loricifera (Fig. 1) Adult loriciferans are bilaterally symmetrical marine metazoans and between 108485 µm long. The body is divided into five regions: mouth cone, head (introvert), neck, thorax and abdomen. The first four regions may be retractable into the loricate abdominal region. The internal armature and sense organs of the mouth cone are always arranged in a strict tri-or hexagonal pattern, the external armature varies from 7-9 outer oral styles to 6-16 thin oral ridges. An extrusible cuticularized buccal canal is centered within the mouth cone. The mouth cone can be withdrawn but not everted. The head or introvert is eversible and may be armed with up to nine variable distinct rings of sensory or

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Fig. 1. Nanaloricus mysticus (Loricifera, Cycloneuralia) from France. Dorsal view of female with fully extended mouth cone and telescoping mouth tube. Abbreviations: an, anus; bu, buccal tube; cv, clavoscalid; du, glandular duct; dv, dorsalventral muscle; gu, midgut; fl, flosculum; fu, oral furca; gl, epidermal gland; lg, lorical spike gland; mo, mouth cone; mt, mouth tube; nu, nucleus; oo, ovary; os2, seconcondary oral ridge; pr1- pr2, protonephridia in the gonads; sr2-sr9, scalids rings, ts1-ts2, trichoscalids (Modified from Kristensen 1991b).

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locomotory appendages (scalids). Each scalid is multicellular with several ciliary sensory structures and muscles, which attach to the introvert. The first row of scalids consists of multiciliary club, blunt-shaped or blunt-tipped chemoreceptors, the clavoscalids. The last ring of scalids on the neck consists of 15 uniciliary, flat trichoscalids. Seven of the trichoscalids may be double as in the genus Nanaloricus. The thorax is accordion-like without any appendages and joins with the abdomen. The abdomen is covered with a more or less cuticularized lorica (girdle). The lorica consists of six plates with hollow spines or at least 22 folds or plica. Sensory structures called flosculi are often located posteriorly on the lorica. Saccated dorsal gonads open terminally. The protonephridia may be located inside the gonads. Terminal cells are always uniciliary. The straight digestive system consists of a foregut and hindgut lined with cuticle and a midgut covered with microvilli. Also present is a triradiate myoepithelial pharynx bulb, which may have five placoids in three rows (cuticularized rods with chitin as found in the tardigrades). Postlarvae, when they are present (apparently lacking in Pliciloricus), are without toes, setae and locomotory spines on the abdomen. The postlarva often appears similar to the adults, but always with fewer scalids, a thinner lorical cuticle, and absent of gonads. The immature stages in the life history of Loricifera were known first and named “Higgins-larvae”. The Higgins-larvae are 50-402 µm long and with the same body regions as the adults. The mouth cone is without scalids and may wear 6-12 oral stylets, internal armature always in a strict tri- or hexagonal pattern. The eversible head always has 8 clavoscalids in the first ring and up to 7 rings of spinoscalids. The neck lacks trichoscalids and may be a closing apparatus. The accordion-shaped thorax has plates formed from transverse and longitudinal folds. The lorica is longitudinally folded with two or three locomotory setae present on the anterior/ventral part. Two or three setae are present on the posterior margin of the lorica. Two lateral and one middorsal flosculi may be present close to the dorsal anus. A single pair of protonephridia is located laterally. The seven monociliary terminal cells in each nephridium are connected to a duct cell, which opens near or into the dorsal rectum. Toes located caudally with a pair of large adhesive glands are located basally in the abdomen. Since the description of the type species Nanaloricus mysticus in 1983 from shallow water, 11 species representing three genera and two families have been described, but about 90 other undescribed species are currently known. The phylum Loricifera still consists of a single order, Nanaloricida (Kristensen 1983) with two described families, but several new families and one new order are under description. The Nanaloricidae consists of two genera each with three species (only one genus and two species are currently described). The adults of these species have sexually dimorphic differences in the first ring of appendages (the clavoscalids); all are similarly uniramus in the female, but in males, the middorsal, dorsolateral and ventrolateral pairs of clavoscalids are divided into three branches. The lorica consists of six plates forming a strongly coronate anterior margin. The family Pliciloricidae has two described genera, Pliciloricus and Rugiloricus (see Higgins & Kristensen 1986). Family characteristics of the adults include the presence of two kinds of unbranched clavoscalids, a “double organ” consisting of two variously fused and modified midventral second-ring head appendages (Kristensen 1991b), and a lorica with 22-60 longitudinal folds or plicae.

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Most of the biology of Loricifera is still unknown. At least 2-5 larval instars exist before metamorphosis, but the life-cycle can be very complicated, involving a postlarva. Females and males are of the same size (108-485 µm), but sexual dimorphism is present in the scalids. Seminal receptacles have been found in one undescribed species of Nanaloricus. Free eggs of the genus Pliciloricus are found in the sediment. The larval stage always has a set of two “toes” and may be seen free-swimming (Nanaloricus) or found adhering strongly to sand grains with glue from the toe glands (Pliciloricus). In one species of the genus Rugiloricus, the larva produces a mature ovary (neoteny), and develops 4-8 new larvae inside the old larval exuvium. The neotenous larvae look exactly like the larvae that arise from the free-laying egg. The Rugiloricus-larvae appear to be common most of the year and outnumber the adults 1:100. In an undescribed order from Faroe Bank, the female keeps two layers of larval exuviae, when she develops the mature ovary with 10-45 eggs. The eggs and later the larvae develop successively inside the female. The tissue of the female completely disintegrates after the last egg with chorion is formed and only the layers of the cuticle exist as a shelter for developing eggs and larvae. This phenomenon, in which a larval female develops larvae, which consume her from the inside, is called paedogenesis. Phylogeny of Loricifera The new ultrastructural data has shown that the loriciferans indeed have their own “Bauplan” in the animal kingdom. Unfortunately these data have not resolved the position of Loricifera in the phylogenetic system, but many “Cycloneuralia” characters are present in the sensory structures, cuticle and glands. The fine structure of the mouth cone, the introvert and the circumenteric brain indicate that the phylum is a monophyletic group of meiofaunal animals, related to Kinorhyncha and Priapulida in the monophyletic group Scalidophora. Annulation of the flexible buccal tube, telescoping of the mouth cone and three rows of placoids in the triradiated myo-epithelial pharyngeal bulb are found only in Tardigrada and Loricifera. These similarities between tardigrades and loriciferans may be considered as plesiomorphic characters. However, the circumenteric brain of the Scalidophora (Loricifera, Kinorhyncha and Priapulida) is very different from the trilobed brain of Tardigrada, which is a true arthropod character. The body of a loriciferan is only about one quarter of a millimeter in length, approximately the size of many protozoans. Despite this, the loriciferan body is composed of thousands of cells, and the head bears 200-400 specialized complex sensory and locomotory appendages (Kristensen 1995) operated by tiny muscles and complex cuticular structures. The body also houses complex gut, reproductive, excretory, nervous and muscle systems. Such complexity in such a small animal is a paradox of design in the animal kingdom, and many cell biologists suspect that loriciferans must have developed from a much larger animal. The morphology of Tardigrada (Fig. 2) Tardigrades are a phylum of microscopic, multicellular coelomates with 4 pairs of segmented or telescoping legs. With an adult size of 80-1200 µm (most marine species

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Fig. 2. Parastygarctus nov. sp (Tardigrada, Panarthropoda) from Australia. Dorsal view of male with fully extended telescoping mouth cone. Note also the telescoping legs. Abbreviations: at, cuticular muscle attachment; an, anus; bu, buccal tube; cE, cirrus E; cf, cirrophorus; co, coxa; ec, external cirrus; fe, femur; fl, flagellum; go, gonopore; hp, head plate; ic, internal cirrus; lc, lateral cirrus; m1-m2, median plates; mc, median cirrus; mo, mouth cone; np, neck plate; op, cuticular opening of sensory structure; pII-pIV, segmental plates; pc, primary clava; pe, pedestal of clava; sA, spine of head plate; sB1-sD1, secondary spines of segmental plates, sB2-sE2, primary spines of segmental plates, sc, secondary clava; se4, fourth leg senseorgan; sp, scapus of cE; ti, tibia.

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are only 100-300 µm) they are among the smallest Metazoa. The body is bilaterally symmetrical and can be dorsolaterally flattened to cylindrical in shape. There are five distinct body segments including a cephalic segment and four trunk segments each bearing a pair of segmented legs. The terrestrial and limnic forms have reduced segmentation in their stumpy legs with two to four claws, while marine forms may have telescoping segmental legs with up to 13 claws or 4-6 toes with complex claws. Other marine tardigrades have rod-shaped adhesive discs or round suction discs also inserted on the foot via toes. Both dorsal and ventral body-cuticle may have segmental plates with spines or the outer segmentation can be reduced to a smooth cuticle without appendages. The very complex cuticle contains mucopolysaccharides with tannin protein in the epicuticle and chitin in the procuticle. Wax is reported from the layer (intracuticle) between the epicuticle and the procuticle in some terrestrial species. The epicuticle may have hourglass-shaped to tubular pillars. The cuticle is shed periodically (simplex stage) and a molting cycle exists in the typically arthropod fashion (Kristensen 1976). When the new body cuticle is formed, the cuticle of the digestive system is also re-synthesized. Molting of the cuticle continues after the animals are mature. The toes, claws and the cuticular part of the legs (tarsus) are formed in the so-called claw glands. The claw glands may be homologous with the stylet glands along the pharyngeal apparatus, which are also known as salivary glands. The digestive system consists of three major parts: the foregut (ectodermal origin), the midgut (mesodermal origin) and the hindgut (ectodermal origin). The foregut is a complex feeding structure and consists of a mouth cavity, buccal tube, pharyngeal apparatus with placoids and esophagus, which are all lined with cuticle. The telescoping mouth cone can be located terminally or ventrally close to the first pair of legs. A stylet mechanism is associated with the buccal tube, which in the plesiomorphic condition consists of CaCO3-encrusted stylets and stylet supports. It is possible that the stylets and the stylet supports could be greatly reduced mouth appendages that originally functioned as mandibles. The somatic musculature consists of separate muscle bands, each composed of a single mesodermic cell. All arthrotardigrades that have been investigated have cross-striated muscles, while terrestrial heterotardigrades and eutardigrades have obliquely striated muscles. Myoepitheal cells are found in the ectodermal pharyngeal bulb and in the female rosette cells surrounding the gonopore. The nervous system is distinctly metameric, consisting of the three-lobed brain, the subpharyngeal ganglion and the four ventral trunk ganglia. Characteristic to all tardigrades is the paired commensure that connects the protocerebrum to the first ventral trunk ganglion. Both hetero- and eutardigrades may have eye spots, usually located inside the protocerebrum. Antennae are totally lacking in all tardigrades. The sensory structures found on the head are not antennae, but appear to be homologous to single arthropod sensilla (Kristensen 1981). Tardigrades lack respiratory organs or gas exchange structures. Gaseous exchange takes place trough the epidermal cells and the complex cuticle. The general shape of the gonad is rather constant in tardigrades although the wall structure of mature gonads is different in eutardigrades and heterotardigrades. Both the single ovary and the single

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testis are only surrounded by a thin basal lamina in heterotardigrades, while the eutardigrades may have a germinal epithelium as well as muscles related to the dorsal gonad. The phylum comprises three classes: Heterotardigrada, Mesotardigrada and Eutardigrada. Heterotardigrada, which consists of the two orders Arthrotardigrada and Echiniscoidea, have cephalic appendages, called cirri (mechanoreceptors) and clavae (chemoreceptors). The arthrotardigrades are marine forms, which usually have a median cirrus and telescopic legs with or without toes, while the echiniscoids are terrestrial armored and marine unarmored forms. The echiniscids have no median cirrus and the legs are without toes. All heterotardigrades have separated gonopore (anterior) and anus (posterior). The females of many marine heterotardigrades have two cuticuclelined seminal receptacles. All heterotardigrades lack Malpighian tubules, but in marine arthrotardigrades the segmental trunk glands or segmental glands may have the same excretory function as the coxal glands in Crustacea. In Eutardigrada, which consists of the two orders Parachela and Apochela, the external part of the cephalic sensory structure is absent. The cuticle is not armored and the stumpy legs are without toes. The so-called double claws of eutardigrades differentiate into a primary and secondary branch. Some soil eutardigrades are snake-like with reduced legs and claws. In the clawless eutardigrade from Western Australia, Apodibius serventyi all legs are reduced to stumpy buds without claws (Morgan & Nicholls 1986). The eutardigrades have a common opening, the cloaca, for gametes, excretory products and feces. Some female eutardigrades may have a single internal seminal receptacle opening close to the rectum. All eutardigrades have three Malpighian tubules at the junction between the mid- and hindgut. The Malpighian tubules may have both an excretory and osmoregulary function. The class Mesotardigrada was established on the basis of a single species found in a hot sulfur spring in Japan (Rahm 1937). The mesotardigrades were reported to have Malpighian tubules and a cloaca as in eutardigrades, but claws and cephalic cirri as heterotardigrades. The type material does not exist and the species description has been strongly criticized. Hermaphrodism has been reported in representatives of most families of eutardigrades (for review, see Bertolani 1992). Only one species of marine heterotardigrades is hermaphroditic and all others are dioecious. Males are unknown in many species of the heterotardigrade genus Echiniscus. Sexes are not always easily distinguished externally but males can be smaller, and in heterotardigrades, the chemoreceptors (clavae) can be longer. Internal fertilization by copulation is present in most marine species. Eggs are round or oval and possess a smooth or ornamented shell. Ornamented eggs are generally laid free and are found in many eutardigrade genera and in the heterotardigrade genus Oreella. All other known heterotardigrades lay smooth eggs either free or into the exuvium. Cleavage is holoblastic and does not follow patterns seen in other Protostomia. Cleavage is said to be indeterminate, and mesoderm formation is by enterocoely (Marcus 1929). It has recently been suggested that tardigrades have a modified spiral cleavage (Eibye-Jacobsen 1996/97). Her observations are based on ultrastructural research. Furthermore, the enterocoelous formation of the four mesodermal sacs shown by Marcus

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(1929) may be a misinterpretation. Reinvestigations of the original 8 µm thick sections made by Marcus indicate that the sacs are the precursors of the claw glands. The phylogeny of Tardigrada The phylogenetic position of tardigrades is still uncertain. They have similarities to Cycloneuralia, especially nematodes, loriciferans and kinorhynchs. Recent ultrastructural investigations show that these similarities may be convergences, because of similarity in life style. Tardigrades may be truely scizocoelomate and have a metameric nervous system consisting of a three-segmented brain, subpharyngeal ganglion and four trunk ganglia (one for each trunk segment). Furthermore, the ultrastructure of the sense organs (Kristensen 1981) indicates that the group should be placed close to arthropod-line. The arthropodization, i.e the evolution of articulated limbs may have taken place only once. If so, the peculiar telescoping legs of Tardigrada are homologous with the true segmented legs of Euarthropoda. The other possibility that evolution of articulated limbs may have taken place several times cannot be ruled out totally (Fryer 1992). Conclusion The position of Loricifera remains still controversial, but the close relation of loriciferans to kinorhynchs and priapulids can hardly be doubted. However, the telescoping of the mouth cone, annulation of the flexible buccal tube, and the triradiated myo-epithelial pharyngeal bulb with placoids are found only in Tardigrada and Loricifera (Kristensen 1991a). The similarities between panarthropods and Cycloneuralia could be true homologies rather than surprising analogies. Proarthropods (tardigrades) may have developed from cycloneuralians and not from annelid-like animals without a true moulting cuticle. Thus, the similarities between Tardigrada and Cycloneuralia could be true homologies, autapomorphic for the entire group (including arthropods) but plesiomorphic in each subgroup. In this case, the new morphological observations of Loricifera and Tardigrada presented here suggests that Cycloneuralia could be the sister group to Panarthropoda. These results support the existence of Ecdysozoa as a monophyletic group, suggesting that the clade is not an “artifact” as some authors (including me) have stated (Wägele et al. 1999). References AGUINALDO A.M.A., TURBEVILLE J.M., LINFORD L.S., RIVERA M.C., GAREY J.R., RAFF R.A & J.A. LAKE 1997. Evidence for a clade of nematodes, arthropods, and other molting animals. Nature 387: 489-493. BERTOLANI R. 1992. Tardigrada. In Adiyodi K.G. & R.G. Adiyodi (eds), Reproductive Biology of Invertebrates, vol.5: Sexual Differentiation and Behavior. Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi, pp 255-266. BUDD G.E. 1996. The morphology of Opabinia regalis and the reconstruction of the arthropod stem-group. Lethaia 29: 1-14.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Arthropods as ecdysozoans: fossil evidence 479 Thethe New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 479-487, 2003

Arthropods as ecdysozoans: the fossil evidence G.E. Budd Dept of Earth Sciences (Historical Geology and Palaeontology), Norbyvägen 22, Uppsala, SE-752 36, Sweden. E-mail: [email protected]

Abstract The conventional view of the fossil record revealing little or nothing about the early evolution of the phyla is gradually being modified by ongoing findings of exceptionally preserved Cambrian fossils, together with a more precise understanding of their systematics. The ‘stem-’ and ‘crown-’ group concepts have proved fruitful in distinguishing basal, extinct members of clades from members that share the body plan of extant taxa. Many Cambrian fossils may indeed be placed into the stem- and crown-groups of the phyla. The morphological concept of the extant phyla can thus be extended basally towards the branching points of pairs of related phyla. The arthropods offer an excellent demonstration of these possibilities, and a suite of fossils is now available that demonstrate the early stages of arthropod evolution, which almost certainly took place early in the Cambrian. Phylogenetic reconstructions of the basal arthropod suggest it was an annulated and not fully-segmented worm-like organism with a terminal mouth and very limited cephalization, lending considerable support to the ‘Ecdysozoa’ concept. The Cambrian fossil record also reveals a rich array of scalidophoran taxa, which although as yet poorly understood, offer the potential for a much clearer understanding of basal morphology in this clade too. It may on this basis be possible to recognise stemgroup Ecdysozoa and thus to ground the entire Ecdysozoa concept on a concrete historical foundation.

Introduction The evolutionary origins of very high level groups such as phyla have long been problematic, and the classical solution of simply looking in the fossil record in the hope of finding ‘ancestral’ forms or ‘missing links’ has long been discredited (e.g. Patterson 1981). However, as Patterson himself recognised, the entire field of highlevel metazoan relationships has been transformed by the advent of molecular systematics (Patterson 1990). No one - especially now - would claim that this technological breakthrough has solved all problems, not least because of problems of

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resolution. Rather, it seems better to regard its importance as lying in its ability to generate new hypotheses of relationship, or to place new emphasis on old ones. In turn, this can and has greatly invigorated other fields of study to look afresh at problems that had previously stagnated. Molecular systematics has not, however, been the only advance of the last ten years or so. There has been a reflorescence of interest in evolutionary aspects of morphology including development in the field that has become known as ‘Evolutionary developmental biology’ (e.g. Hall 1998), which has placed emphasis on comparative aspects of these studies. From the perspective of this present contribution though, the most significant advance has been the remarkable numbers of new fossils that have been described from the Cambrian. Combined with a slow recognition of the potential contribution they can make to the debate, this wealth of data is lying waiting, and largely untapped, as a distinctive strand to the complex endeavour of understanding metazoan relationships. Fossils and the origin of phyla Fossils of relevance to the origins of the phyla are almost entirely from the Cambrian period of some 543-500 million years ago. As discussed by Budd & Jensen (2000), ‘relevance’ in this context implies that the relevant fossils lie within the stem-groups of the phyla whose origins are being investigated (see Budd 2001a for practical considerations of how this is achieved). It has been the belated recognition of this fact that has hampered attempts to understand the Cambrian fossil record. Given a search image provided by the extant phyla, it was inevitable that some fossils would be assignable to them, but many others would simply be problematic, possessing strange mixtures of characters that apparently precluded them from systematization. Such fossils were usually either placed in limbo as ‘Problematica’, or asserted to be ‘new phyla’ - the apotheosis of this unhelpful process being Gould’s Wonderful Life of 1989. This is somewhat surprising in that Hennig’s views of the systematisation of fossils had already entered the English-speaking world, especially when Jefferies (1979) introduced the stem- and crown-group concepts. With this recognition, that all fossils lie in the stem group of an extant crown group at one level or another, the systematic potential of taxa could be transformed (Smith 1994). As far as the neontologist systematist is concerned though, the placement of fossils within stem-lineages is the recognition that they will share many features - but not all - with the crown groups. These fossils, far from being mere morphological curiosities, offer unique information about the origins of body plans - and in such a way may offer insights into the problems posed by the systematics of extant organisms. The fossil record of the arthropods offers considerable scope for insights of this sort. Herein, “Euarthropoda” = least inclusive group containing Chelicerata, Myriapoda, Crustacea, Insecta; Pycnogonida; “Arthropoda” = Onychophora + Euarthropoda. The Tardigrada are also likely to lie within the Arthropoda (e.g. Budd 2001a) as the sister group to the Euarthropoda, with Tardigrada + Euarthropoda forming the Tactopoda (Budd 2001a).

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The arthropod fossil record A reasonable amount of progress has been made in reconstructing the ground-plan of the Euarthropoda (Scholtz 1997, Walossek 1999). However, several Cambrian taxa cannot on this basis be assigned to the crown-group of the Euarthropoda, including many very ’arthropod’ looking taxa, and they must presently be considered to lie near the top of the stem-group. Apart from this surprising possibility, there are a considerable number of rather less arthropod-like taxa that can also be reasonably considered to be stem-group euarthropods. These include the famous Burgess Shale problematica Anomalocaris Whiteaves, 1892 and Opabinia Walcott, 1912 and taxa from other Cambrian exceptionally-preserved faunas such as various Chengjiang taxa (e.g. Parapeytoia Hou et al., 1995) and two important taxa from the Sirius Passet fauna, Pambdelurion Budd, 1997 and Kerygmachela Budd, 1993 (see Budd 1999 for discussion and review). Although these taxa have been controversial (Budd 1999 reviews the various theories), two views are most relevant to the issue at hand here: first, that these taxa are not closely related to the arthropods, but lie within an aschelminth clade consisting of priapulids, nematodes, kinorhynchs (and other putative cycloneuralians) and tardigrades (Hou et al. 1995); secondly, that these taxa lie within the stem-group of the arthropods and show both lobopod and arthropod characters (see analysis in Budd 1999). The rest of this paper is devoted to showing how these two viewpoints are reconcilable within an ecdysozoan framework, and thus to demonstrate that the fossil record of early arthropod evolution supports the ecdysozoan concept. ‘Ecdysozoan’ characters of early arthropod-like fossils. i) The mouth parts Hou et al. (1995) discuss the difficulties involved in assigning the anomalocaridids to the arthropods, pointing to the very unusual mouth structures present in Anomalocaris and their similarities to those seen in, for example, kinorhynchs; similar observations were made by Dewel & Dewel (1996) when they compared tardigrade and anomalocaridid mouthparts. As discussed by Budd (1999), broadly similar circum-oral mouthparts are indeed known from Pambdelurion, Kerygmachela and perhaps Opabinia. The extant onychophorans also possess circum-oral lips that are extended during feeding. In at least a very broad sense then, such a structure is thus present in all arthropods apart from the crown-group of the Euarthropoda. While the presence of a circum-oral structure of this type, and a potentially suctorial mouth part (Budd 1999) thus seems important to point to, it does not necessarily mean that these taxa should be moved wholesale to the aschelminths. Rather, in the ecdysozoan concept, this feature is an inherited plesiomorphy, with a single loss or transformation sometime after the anomalocaridids branched off in the euarthropod stem group. An even greater problem to the ‘aschelminth’ solution to anomalocaridid relationships (and indeed, to ’euarthropod’ solutions) is posed by Kerygmachela, because although clearly anomalocaridid-like, it also shares undoubted properties with lobopods and, in particular, the rather onychophoran-like Aysheaia Walcott 1911 (Budd 1999). If the

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Anomalocaris-like taxa are to be shifted from a stem-group arthropod position, then they must drag Kerygmachela with them, and thus almost certainly the Cambrian lobopods and the onychophorans. This would be tantamout in the first case to supporting the ecdysozoan concept (i.e. moving all the arthropods into the ‘aschelminths’) or in the second case, to inverting arthropod evolution and deriving onychophorans from within the euarthropod clade (see Ballard et al. 1992 for the single support for such a view; and Wägele & Stanjek 1995 for a refutation). Whilst it is thus possible to devise alternative models for one or other of the Anomalocaris-like taxa from a stem-group position, this task is made considerably harder by considering all of the relevant taxa - especially the lobopodan and presumably rather basal Kerygmachela (Budd 1999). If these points are accepted, then it becomes inevitable that the arthropods are seen to have evolved from a lobopodous grade of organisation, represented today by the onychophorans and probably the tardigrades, but with retained ecdysozoan-like characters. I have already mentioned two of these, the circum-oral mouth parts and the suctorial pharynx; but there are at least two critical others: mouth position and segmentation. ii) Mouth position in basal arthropods. Whilst the Articulata concept suggests that arthropods and annelids share a primitively pre-oral, unsegmented region that is referred to as the ‘acron’ in arthropods, this structure has proved to be remarkably hard to pin down (Scholtz 1997). There appears to be no trace of it in the Onychophora (Anderson 1973), and recent investigations have also failed to reveal it (e.g. Eriksson & Budd 2001). The fossil record reveals why this is the case: the structure in arthropods in all likelihood does not exist. All Cambrian lobopods possess a terminal mouth with no apparent preoral structure, including Aysheaia, and – critically - Kerygmachela. Together with the tardigrades (if their affinities lie broadly here), this evidence strongly suggests that the mouth of the euarthropods has become secondarily rotated, and thus that all the apparently pre-oral structures in the arthropod head are originally derived - within the clade - from post-oral, segmental structures (see Eriksson & Budd 2001, for discussion of the unusual situation in onychophorans, which may be intepreted as a modified circum-oesophageal ring). iii) Segmentation in basal panarthropods Here, I use ’segmentation’ in the sense of serial repetition, and no more. This leaves the possibility open, then, of enquiring what is serially repeated, and how this repetition is controlled. In such a way it is possible to get away from sterile arguments about whether or not a particular organism is ‘truly’ segmented or not (Budd 2001b). After this character analysis, it is then possible to consider homology. This subtlety of use is increasingly demanded as it becomes clearer that whether or not an organism has part of it segmented is a matter of degree, and therefore evolving segmentation could be a creeping process as well as an on-off switch. As discussed by Budd (1999), the point of interest in the arthropods is provided by the onychophorans, which although clearly segmented in the sense of growth by serially repeated coelomic compartments that give rise to (some) segmented adult structures, the epidermal ectodermal structures are in

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general not segmented in the way that those of arthropods and annelids are. In the absence of consideration of the fossil record, it has proved possible to dismiss this lack as a derived character. Cambrian fossils, however, make this much less likely. First, at least Aysheaia of the Cambrian fossils shows an apparently identical state to the onychophorans, and secondly (Budd 1996), none of the other lobopods show proper segmental division of the epidermis either. The best they manage is a heteronomous annulation (Ramsköld 1992), where the epidermis is divided into micro-annulated and non-annulated regions lying above the limbs. This state persists in Kerygmachela too; and epidermal segmentation can in general not be demonstrated in the Anomalocarislike taxa either. As none of these taxa are assignable to the stem-group of the Onychophora (Budd 1999), it is clear that lack of epidermal segmentation was much more widely distributed in Cambrian arthropods than now. The implication of these observations is clear. Although segmentation genes are widespread, the structural expression of their effect as classically seen in eaurthropods and annelids (although in both it can be highly modified) is not fully homologous between the two. At least some of the similarities between arthropods and annelids as far as segmentation is concerned may be demonstrable convergences. Fossil ecdysozoans? The fossil record could concretely support the Ecdysozoa if it were to provide examples of taxa lying within its stem-group. The obvious difficulty with this aim is that the groundplan of the Ecdysozoa is not well-established, so recognising fossils that would only possess some aspects of it might be very problematic. Nevertheless, several taxa from the Cambrian are suggestive of at least cycloneuralian affinity. Fossil worms from the Burgess Shale were described by Conway Morris (1977) as priapulids. More fossils have been assigned to the problematic group Palaeoscolecidae (Whittard 1953), which are recognised now to bear a introvert similar to that present in the Introverta of Nielsen (1995), from exceptionally well-preserved taxa from the Chengjiang fauna (Hou & Bergström 1994). Recently Conway Morris (1997), following on previous suggestions (Dzik 1993), agreed that the supposed Burgess Shale priapulid Louisella Walcott 1911 was also best referred to the Palaeoscolecida. The affinities of the palaeoscolecids (and potentially the more priapulid-like Burgess Shale forms) remain in my view open to question. Hou & Bergström (1994), with some hesitation, suggested that although intermediate in their characteristics, the palaeoscolecids were most similar to at least larval nematomorphs (but see SchmidtRhaesa 1998 for doubts about their nematomorph affinitites); whilst Conway Morris (1997) inclined towards a priapulid affinity, partly on the evidence from Louisella. However, a stem- and crown- group formulation suggests that there are many more possibilities for their systematic position. The resemblance of many of these forms to the priapulids might simply be plesiomorphic, and the relevant characters may only appear synapomorphic because the priapulids (and the presumably rather derived Nematomorpha) are the only large living members of the Cycloneuralia. In fact, I know of no characters that unite the Palaeoscolecids as a monophyletic clade, nor that unite them to any crown-group of cycloneuralians. This lack suggests that they variously lie

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in the stem-groups of various cycloneuralian crown-groups, or even in the stem-group of the Cycloneuralia itself. The groundplan of the Ecdysozoa The very presence of several large, rather disparate cycloneuralian-like worms in the Cambrian-Silurian suggests that cycloneuralians may not necessarily have a small groundplan body size (as in Schmidt-Rhaesa et al. 1998). This, then, brings up the final issue of the ecdysozoan groundplan. Although it has been considered that in the Ecdysozoan concept, tardigrades are likely to be basal within the arthropods, the fossil evidence has suggested otherwise to some palaeontologists (e.g. Dzik & Krumbiegel 1989, Dzik 1993, Waggoner 1996, Budd 1999). In the discussion above, the groundplan of the

Fig. 1. A composite phylogeny of the Ecdysozoa based on data and trees published by Budd (1996, 1999); Nielsen (1995); Schmidt-Rhaesa et al. (1998), Lemburg (1999) and Eriksson & Budd (2001). Shaded areas indicate regions of the tree probably represented by Cambrian fossils. Some taxa placement (e.g. within the ’Scalidophora’) are controverted, but not essential to the general evolution of important characters). Selected important apomorphy acquisitions (see also the above references for other characters): 1. (inherited plesiomorphies plus apomorphies) large body size; terminal mouth; micro-annulated outer surface; lack of epidermal segmentation; ?separated lateral nerve cords; triradial muscular pharynx; ecdysis with ecdysteroids; loss of locomotory cilia; epicuticle secretion by epidermal microvilli; ?external sclerites; circular body-wall musculature; 2. Introvert; ’cycloneuralian’ brain consisting of circum-oral nerve ring; 3. lobopodous appendages; 4. Small size; non-inversible mouth cone. 5. Ventral mouth; other onychophoran autapomorphies (e.g. slime glands); 6. Loss of circum-oral structures; ectodermal segmentation. It should be noted that on this analysis, the monophyly of the Cycloneuralia is not particularly well-supported, as the circum-oral nerve ring may be present in the stem-group of the Arthropoda (Eriksson & Budd 2001), as, in broad terms, may some sort of introvert (Budd 1999, fig. 24 and p. 271). These two features may thus be acquired at node 1.

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Arthropoda would be a broadly onychophoran-like worm with external micro-annulations but no epidermal segmentation; an anterior mouth with a muscular pharynx and some sort of circum-oral structures (perhaps with a pair of differentiated frontal appendages which were not necessarily antenna) and a series of lobopodous limbs. Essentially, such an organism would not be too dissimilar to something like a priapulid or palaeoscolecid with limbs (Dzik 1993). If so, then there is no particular reason why some of the Cambrian worms do not in fact lie within the Ecdysozoa stem group (see Fig.1 for summary of possible relationships and evolutionary transitions). One further point of interest is that, in this analysis, morphological monophyly of the Cycloneuralia is difficult to support. The arthropod groundplan brain structure may be similar to that of the Cycloneuralia (Eriksson & Budd 2001); and the eversible ’introvert’ may also find an equivalent in basal Panarthropods (see e.g. Budd 1999, Fig.24 and discussion, p. 271). Summary Cambrian fossils provide critical and as yet only partially-tapped information concerning the origin of phyla and interphyletic relationships. These allow the reconstruction of ever-larger scions embracing more and more taxa in the stem groups of phyla, and allowing a better estimate of the groundplan of the entire clade. Such an endeavour is possible with the arthropods, and a rather complete sequence of fossils is now available. They demonstrate that several important ‘Articulata’ characters such as a non-terminal mouth and full segmentation are derived within the Panarthropoda, and thus cannot be seen as Articulata synapomorphies. Conversely, the inferred basal condition of an annulated, non-septate worm-like organism with anterior mouth, suctorial pharynx and lacking ectodermal segmentation, is more consistent with the Ecdysozoa concept, a judgement that is supported by the retention of cycloneuralianlike characters within the Panarthropod clade. The presence of several types of large, rather cycloneuralian-like worms in the Lower Palaeozoic fossil record hints that large body size may be more widely distributed in the Cycloneuralia than presently considered, and that even the groundplan might be large. Although the Ecdysozoa has largely been a concept rooted in molecular phylogeny (Aguinaldo et al. 1997) the fossil record thus provides potentially critically important morphological data for assessing its viability. Acknowledgements I thank Andreas Schmidt-Rhaesa for the opportunity to participate in the symposium and for helpful discussions, together with Jan Bergström, Dieter Walossek, James Garey, Reinhardt Kristensen, Gerhardt Scholtz and Claus Nielsen. This work was funded by the Swedish Natural Sciences Research Council (NFR, now VR). References AGUINALDO A.M.A., TURBEVILLE J.M., LINFORD L.S., RIVERA M.C., GAREY J.R., RAFF R.A. & J.A. LAKE 1997. Evidence for a clade of nematodes, arthropods and other moulting animals. Nature 387: 489-493.

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SCHMIDT-RHAESA A. 1998. Phylogenetic relationships of the Nematomorpha - a discussion of current hypotheses. Zool. Anz. 236: 203-216. SCHMIDT-RHAESA A., BARTOLOMAEUS T., LEMBURG C., EHLERS U. & J. R. GAREY 1998. The position of the Arthropoda in the phylogenetic system. J. Morph. 238: 263-285. SCHOLTZ G. 1997. Cleavage, germ band formation and head segmentation: the ground pattern of the Euarthropoda. Syst. Assoc. Spec. Vol. 55: 317-332. SMITH A.B. 1994. Systematics and the Fossil Record: Documenting Evolutionary Pathways. Blackwell Scientific, Oxford. 223p. WÄGELE J.W. & G. STANJEK 1995. Arthropod phylogeny inferred from partial 12S rRNA revisited - monophyly of the Tracheata depends on sequence alignment. J. Zool. Syst. Evol. Res. 33: 75-80. WAGGONER B.M. 1996. Phylogenetic hypotheses of the relationships of arthropods to Precambrian and Cambrian problematic fossil taxa. Syst. Biol. 45: 190-222. WALOSSEK D. 1999. On the Cambrian diversity of Crustacea. In Schram F.R. & J. C. von Vaupel Klein (eds), Crustaceans and the Biodiversity Crisis. Brill, Leiden, pp. 3-27. WHITTARD W.F. 1953. Palaescolex piscatorum gen et sp. nov., a worm from the Tremadocian of Shropshire. Q. J. Geol. Soc. London 109: 125-135.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Is the taxon Articulata obsolete? Arguments in favour of a close ... Evolution 489 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 489-501, 2003

Is the taxon Articulata obsolete? Arguments in favour of a close relationship between annelids and arthropods G. Scholtz Humboldt-Universität zu Berlin, Institut für Biologie/Vergleichende Zoologie, Philippstr. 13, 10115 Berlin, Germany. E-mail: [email protected]

Abstract The traditional view that annelids and arthropods (Articulata) are closely related has been challenged by phylogenetic analyses using molecular data sets. The outcome of these studies is a clade of moulting animals (Ecdysozoa) comprising arthropods and some taxa of the nemathelminth worms. The implication of the Ecdysozoa hypothesis is that the type of segmentation found in annelids and arthropods must be either convergent or an ancestral feature of protostomes or even bilaterians. This review discusses the morphological and developmental evidences for both hypotheses. Morphologically the taxon Ecdysozoa is not well supported. In contrast to this, there are numerous characters indicating that segmentation of annelids and arthropods is homologous and apomorphic for a taxon Articulata.

Introduction Ever since Cuvier (1817) annelids and arthropods have been unified in the taxon Articulata. Cuvier erected this taxon on the basis of the evident similarities in the body organisation of the two groups, which is characterised by repeated morphological units along the antero-posterior body axis, the so-called segments. This traditional view was supported by more recent phylogenetic-systematic studies in which segmentation has been considered as being a synapomorphy unifying annelids and arthropods (e.g. Weygoldt 1986, Rouse & Fauchauld 1997, Scholtz 1997, Ax 1999). According to these analyses none of the other protostome groups shows comparable segmentation patterns and segmentation of the deuterostome chordates is convergent based on parsimony considerations and differences in the ontogenetic and morphological patterns. Recently, the Articulata-hypothesis has been challenged by cladistic analyses using 18S rDNA data (Aguinaldo et al. 1997, Giribet & Wheeler 1999). In these studies the arthropods appeared as close relatives of several Nemathelminthes taxa. Because the members of the resulting clade share the character of moulting a cuticle it has been named Ecdysozoa

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(Aguinaldo et al. 1997). This idea is not new, as long ago as 1909 Rauther propagated the hypothesis of a close relationship between arthropods and nematodes based on morphological studies. Furthermore, in a cladistic analysis of morphological data Eernisse et al. (1992) came to similar results. However, since the publication of the paper of Aguinaldo et al. (1997) there is a sudden acceptance of the Ecdysozoa in the scientific community and many papers have been published discussing the idea and its implications for the view of bilaterian evolution from the perspectives of morphology, genetic data, Hox genes, and palaeontology (e.g. Schmidt-Rhaesa et al. 1998, Adoutte et al. 1999, De Rosa et al. 1999, Budd & Jensen 2000, Valentine & Collins 2000). One major problem of the Ecdysozoa-hypothesis is that if it is correct, the segmentation we find in annelids and arthropods must be either a convergent or a very ancient character which occurred already either in the stem species of the protostomes or even in that of the Bilateria. Both alternatives have been discussed by various authors (e.g. Holland et al. 1997, Schmidt-Rhaesa et al. 1998, Davis & Patel 1999). However, morphologically the Ecdysozoa find only weak support by characters such as a chitinous cuticle, which is shed under the influence of ecdysone and by the absence of locomotory cilia (SchmidtRhaesa et al. 1998). Moreover, the molecular support is not unambiguous (Wägele et al. 1999) and the use of 18S r DNA has been shown to be problematical at the levels of the Metazoa (Abouheif et al. 1998) and of the Arthropoda (Giribet & Ribera 2000). Here, I want to discuss the questions of whether segmentation of annelids and arthropods is homologous and apomorphic for a taxon Articulata. Furthermore, characters not related to the complex of segmentation are evaluated which might add support for a close relationship between annelids and arthropods. Arthropod monophyly, annelid monophyly Prerequisite for the following discussion is the acceptance of a monophyletic taxon Arthropoda comprising at least Onychophora and Euarthropoda. There is increasing evidence for arthropod monophyly based on morphological, developmental and molecular data (e,g. Nielsen 1995, Ax 1999, Edgecombe et al. 2000, Giribet & Ribera 2000, Scholtz 2001). The position of the Tardigrada is still unclear but there is some evidence that they are the sister group of the euarthropods (Nielsen 1995, Ax 1999). The monophyly of annelids is still a problematic issue (see Westheide 1997). However, most authors discuss at least some characters as support for a monophyletic Annelida (Westheide 1997, Rouse & Fauchauld 1997, Dohle 1999). To make homology plausible: the complexity test The question of what is a segment is correlated with the problem of homology. Some remarks on homologisation have to be made first. The test for the homology of structures is complexity (Riedl 1975, Dohle 1989). Homologisation of characters can be done best when the character under question can be subdivided into substructures, which together show a distinct pattern such as a set of morphological features, a cell pattern, a bristle pattern, or a DNA sequence to give a few examples. The complexity of this pattern can be shown by proving the independence

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of individual substructures. This is done by comparing the patterns of substructures in different taxa. If individual substructures of the pattern under comparison have been altered or lost between the taxa (i.e. during evolution) without an effect on the general pattern, the independence of these particular substructures is shown. The fact that the general pattern is maintained shows the complexity of this general pattern, which again makes it unlikely that the information producing this pattern, arose convergently. Thus, complexity of similarity makes homology likely or plausible. The substructures can also be the subject of a homology analysis applying the same type of complexity test. This hierarchical approach of evaluating the patterns and sub-patterns under comparison makes the assumption of an independent evolution of these patterns very unlikely (see Riedl 1975, Dohle 1976, 1989, Scholtz 1984). Another important aspect of homologisation concerns the asymmetry between similarity and difference. The question must be: how many substructures of a pattern must be similar to claim homology of this pattern? The question is not: how many differences must occur to reject the possibility of homology? Against this background the problem of homology of arthropod and annelid segmentation has to be discussed. What is a segment? The crucial question to start with is: what is a segment? What do we mean by segmentation? Is a segment the region of embryonic gene expression? Is it characterised by genetic regulatory networks? Does it represent a physiological unit? Is it defined by clonal restrictions? Is it a morphological unit? How are all these levels related to each other? According to different scientific backgrounds the answer will be a different one. A segment for a molecular geneticist is something different from the segment of a morphologist. Lawrence (1992: 91) defines a segment of the ectoderm “as a pair of compartments, one anterior and one posterior”. Kroiher et al. (2000: 485) define segmentation as “the formation of a periodic pattern of paralogous blocks of cells”. In particular, the latter definition is certainly too general in order to address questions of homology between segmentation in different taxa because it comprises virtually all cases of repeated structures along the body axis. Therefore, the most meaningful definition of a segment in this context is the classical morphological definition of a segment. A segment is a repeated body unit, which can be defined by a set of sub-structures or characters occurring together, these are in the case of annelids and arthropods the following features (see Goodrich 1897): - an outer annulus - one pair of coelomic sacs - one pair of ventral ganglia with commissures, lateral nerves, and connectives - one pair of metanephridia - a set of muscles - one pair of appendages All these structures together characterise segments in arthropods and annelids (but not in chordates). One could now argue, if you want to make a metameric body organisation, all these characters together are necessarily linked and the information

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basal to segment formation is just complicated but not complex. However all the listed parts of a segment can be altered individually up to entire loss, and there are numerous examples of segments where one or more of these characters are absent - we find segments without ganglia, without nephridia, without outer annulus etc. This proves that the suite of characters that makes up a segment is highly complex (because in many cases most of the characters appear together although they do not have to) and an independent evolution of the segment is therefore unlikely. But we can go further and apply this test to the level of the sub-characters that make up a segment. One example is the structure of the ganglia. They are composed of paired neuropils with ventrolaterally lying perikarya. This is not so evident in some groups such as Onychophora or oligochaetes. But even in these cases a neuromere structure is still recognisable (Schürmann 1995). Between the neuropils are the transverse commissures and the ganglia of adjacent segment are connected by a pair of longitudinal connectives. The number of commissures varies but in many cases we find two large commissures per segment in annelids and arthropods (Hanström 1928, Whitington 1996, Harzsch et al. 1997, Müller 1999, Müller & Westheide 2000). The ganglia are equipped with three main (large) lateral nerves in many arthropods, clitellates and polychaetes (Hanström 1928, Hessling & Westheide 1999, Müller 1999). Hanström (1928: 303) considered this as being a good character to unify annelids and arthropods. However, Müller (1999) expresses some doubt because in polychaetes the number of segmental nerves varies to a high degree. A situation, which is also found in arthropods (Heckmann & Kutsch 1995). In this case the problem is the homologisation of the smaller segmental nerves between different taxa. A median nerve running through all ganglia is a character shared by most annelids and arthropods (Hanström 1928, Harzsch et al. 1997, Müller 1999). Unfortunately we do not have data on individual homologous neurons between annelids and arthropods, which are comparable with respect to their position, to their axon morphology and to the expression of transmitters or genes. At least for arthropods there is growing evidence for conservation of characters like this (Whitington 1996, Gerberding & Scholtz 1999, DumanScheel & Patel 1999) – a promising field for further studies to find possible homologues between annelids and arthropods. It is evident that some comparisons of substructures are problematic because the complexity of the similarity is not very high. For instance, the homology between annelid parapodia and arthropod lobopodia and arthropodia is very doubtful (Lauterbach 1978, Westheide 1997). However, the general pattern of segmentation in annelids and arthropods makes a convergent evolution unlikely. To make an even stronger point one can include developmental data. Evidence from development Cell proliferation and segmentation One can discriminate two developmental processes crucial for segment formation in annelids and arthropods. One is the generation of competent cellular material along the antero-posterior body axis. The other is the subdivision of the body into metamerically

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repeated units (Dohle 1972, Scholtz 1992). Often these processes are described as teloblastic formation of segments (Anderson 1973, Nielsen 1995). However, true teloblasts (large stem cells at the posterior end of the germ band giving rise to smaller descendants in anterior direction by unequal divisions) occur only in clitellate annelids (Dohle 1999) and convergently in malacostracan crustaceans (Dohle & Scholtz 1988) and are certainly derived within arthropods and annelids (Scholtz 1997). The material for segmentation is formed by proliferation in a preanal growth zone, which comprises the ectoderm and the mesoderm. This growth consists of basically two steps (at least in the ectoderm), a posterior cell proliferation and an intercalary cell division or rearrangement spread all over the length of the embryo. This has been clearly demonstrated in clitellates and malacostracan crustaceans where the stereotyped cell division patterns make an analysis of this kind possible (Dohle & Scholtz 1988, Shankland 1999). The segmentation process in annelids and arthropods follows mainly an anteroposterior gradient with the more anterior segments being the most differentiated whereas the posterior segments develop last. In both processes the ventral side of the embryo shows a higher degree of differentiation than the dorsal side. In yolky eggs of annelids and arthropods this leads to the convergent evolution of ventral germ bands with a dorsal side consisting of extraembryonic ectoderm (Scholtz 1997). Neurogenesis The segmental ganglia of annelids and arthropods originate from paired longitudinal cell strands on each side of the embryonic midline (Bate 1976, Dohle & Scholtz 1988, Scholtz & Dohle 1996, Shain et al. 1998). Together with the other aspects of segmentation, the cells of these strands show iterated specifications and form the segmental ganglion anlagen by internalisation. This means that already before, or at the latest with, the internalisation the ganglia and their primordia are already individualised. Although arthropod ganglia are homologous the process of internalisation differs between various arthropod taxa. We find metameric paired invaginations in onychophorans, chelicerates, and myriapods (Anderson 1973, Whitington et al. 1991). Crustaceans and insects differentiate fields of neuroblasts, which produce neuronal precursors by asymmetric divisions (Bate 1976, Dohle & Scholtz 1988). Little is known for annelids in this respect. It seems as if irregular cell divisions lead to a thickening of the ganglion anlagen but this has to be confirmed by further investigations. Coelom formation The mesoderm is also first proliferated and then segmentally subdivided. It forms paired lateral strands left and right of the midgut anlage. In these strands hollow spaces develop in antero-posterior direction (Anderson 1966, 1973). This so-called schizocoely shows a very similar pattern in annelids and arthropods. Recent studies in Onychophorans revealed that even at the ultrastructural level the epithelia of these coelomic spaces are very similar between arthropods and annelids with the exception

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that the onychophoran coelothel seems to remain in a more undifferentiated state (Bartolomaeus & Ruhberg 1999). It must be stressed, however, that during development the coelomic spaces of arthropods are highly transformed, subdivided, and reduced (“mixocoel”) (Dohle 1979). Only in onychophorans are parts of them connected with ciliated funnel bearing metamerically repeated metanephridia (Storch & Ruhberg 1993). Cell level Cell lineage studies have shown for leeches as representatives of annelids and for malacostracan crustaceans among the arthropods that the morphological segments do not match the genealogical units at the cellular levels. The descendants of the primary blast cells of the leech and of the ectodermal transverse cell rows in malacostracans contribute to parts of two adjacent segments (Shankland 1999, Dohle & Scholtz 1988, Scholtz & Dohle 1996). This resembles the parasegment of Drosophila which is the primary metameric unit based on lineage restrictions and gene expression. This parasegment also does not match the segment but contributes to parts of two adjacent segments (Lawrence 1992). So it seems a general principle for segmentation in annelids and insects that the segments are composed of cells from different origins. For arthropods and annelids a longitudinal chain of unpaired midline cells has been described which participates in the formation of ganglia (McGlade-McCulloh et al. 1990, Klämbt et al. 1992, Gerberding & Scholtz 1999). The midline described for vertebrates lies on the dorsal side (Arendt & Nübler-Jung 1999) and it is not clear whether it consists of a one-cell wide chain. Segmentation genes It has been shown for numerous representatives of all major euarthropod groups (except myriapods) that the segment polarity gene engrailed is expressed in transverse stripes in the posterior portion of forming segments (Patel et al. 1989a,b, Scholtz & Dohle 1996, Damen et al. 1998, Peterson et al. 1998, Telford & Thomas 1998, Queinnec et al. 1999). In addition, there is a secondary neuronal expression in the ganglion anlagen. Interestingly, at least in the midline the neuronal engrailed expression is not found in clones deriving from cells of the early segmental expression (Gerberding & Scholtz 1999). A corresponding pattern of dual engrailed expression has been described for leech embryos. As in euarthropods, engrailed is first expressed in transverse stripes in the posterior of the segment anlagen followed by distinct neuronal expression in the ganglia (Wedeen & Weisblat 1991, Lans et al. 1993). Again, there is no general clonal continuity between the early ectodermal and the neuronal expression (Lans et al. 1993). Despite the highly similar expression pattern there seem to be differences in the role of engrailed expressing cells on the regulation of cell fate of neighbouring cells between leeches and Drosophila (Seaver & Shankland 2000). However, this does not necessarily contradict homology of the engrailed pattern but might be due to the highly derived stereotyped cell division pattern found in leeches and similar processes might be true for malacostracan crustaceans as well. Differences in underlying developmental processes do not refute homology of resulting patterns (Dohle & Scholtz 1988, Scholtz & Dohle

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1996). The engrailed expression described for an onychophoran is different since it seems to be restricted to the mesoderm and perhaps the neurogenetic region (Wedeen et al. 1997). This is difficult to interpret but since the patterns found in leeches and euarthropods are so similar it seems likely that onychophorans are derived in this respect. In chordates a segmental pattern of engrailed expression has been found in the lancelet Branchiostoma (Holland et al. 1997). However, AmphiEn expression is restricted to the first eight mesodermal somites and does not show a stripe pattern. The expression in the nervous system of Branchiostoma is very different to that observed in annelids and arthropods showing no segmental repeats (Holland et al. 1997). Hox genes The anterior boundary of the expression of the Hox genes labial, proboscipedia, Deformed, sex combs reduced, Antennapedia, and the combined domains of Ultrabithorax and abdominalA is by and large conserved throughout the euarthropods (Abzhanov & Kaufman 1999, 2000, review by Scholtz 2001). The assumption of a conserved anterior boundary led some authors to re-interpret the homology between chelicerate and mandibulate segments (Damen et al. 1998, Telford & Thomas 1998, Damen & Tautz 1999). If we compare this general Hox pattern with that of annelids, the leech Helobdella and the polychaete Chaetopterus, we find again a striking similarity of anterior boundaries (Kourakis et al. 1997, Shankland 1999, Irvine & Martindale 2000). On the basis that the prostomial ganglion of annelids is homologous to the protocerebrum of arthropods and that the antennal segment of arthropods is the anteriormost true segment (Scholtz 1997), the annelid pattern is in good register with the general euarthropod pattern. For instance, in all cases the anterior boundary of labial and proboscipedia expression is found in the region of the 1st or 2nd segments. The only exception is seen in Drosophila where the anterior border of proboscipedia expression is in the 4th segment. Deformed expression is found from the middle of the 2nd segment in the leech, at the border between 2nd and 3rd segments in Crustacea, Insecta, and Chelicerata and in the posterior of the 3rd segment in Chaetopterus. The anterior border of sex combs reduced expression spans the region from the middle of the 3rd segment in the leech representative, the anterior border of the 4th segment in crustaceans and chelicerates, and the anterior border of the 5th segment in Drosophila. The anterior border of Antennapedia expression is restricted to the 4th or 5th segments and the combined expression of Ultrabithorax and abdominal-A is seen in the 6th and 7th segments in the leech and the arthropods studied. Again, the onychophoran studied concerning the expression of Ultrabithorax/abdominal-A does not fit into this pattern showing expression only in posterior segments (Grenier et al. 1997). As in the case of the engrailed expression pattern this seems one of the numerous apomorphies of Onychophora. Even if the slight differences in segmental register are considered, the resemblance between annelids and arthropods is astonishing. It is not trivial that the relative size class of segments in relation to Hox gene expression is the same in annelids and arthropods. A comparison with Hox gene expression in chordates reveals distinct differences with respect to the segmental register (Holland & GarciaFernandez 1996, Prince et al. 1998a,b).

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Additional, non-segmental characters For many groups of arthropods and annelids the presence of mushroom bodies (corpora pedunculata) in the anterior brain region has been described (Hanström 1928, Åkesson 1963, Strausfeld et al. 1995, Yoshida-Noro et al. 2000). These neuropil regions are characterised by their shape, their bundles of fibers, and specific arrangements of so-called globuli cells. The position and shape of the stomatogastric nervous system is similar between many annelids and arthropods (Hanström 1928). Annelids and arthropods possess a long, tube-like dorsal heart with a postero-anterior blood flow. The heart is formed embryologically between the dorsal parts of the paired coelomic spaces (Siewing 1969, Dohle 1979). The longitudinal muscles of the body wall form distinct bands (Rouse & Fauchauld 1997, Ax 1999). The complex of segmentation in annelids and arthropods: an apomorphy for the Articulata All the listed characters strongly suggest homology of the specific segmentation of annelids and arthropods. Most of these characters find no correspondence in other animal taxa. In some cases this might be simply due to a lack of knowledge about developmental processes in other animal groups. But given that characters such as the longitudinal growth of the body is achieved by a preanal proliferation zone and intercalary growth might also occur in other taxa there is still enough evidence that the combination of all genetic, cellular, developmental, and morphological character related to segmentation as well as the other characters listed are synapomorphies for annelids and arthropods. A scenario for the evolution of segmentation There are all sorts of serially repeated structures along the body axis of several bilaterian groups. These characters comprise elements of the nervous system (e.g. Platyhelminthes, Solenogastres, Kinorhyncha, Nematoda), muscle patterns (e.g. Monoplacophora, Kinorhyncha), shell structures (e.g. Polyplacophora), gonads (e.g. Nemertini) or nephridia (e.g. Monoplacophora). This repeated pattern is certainly also reflected at the level of embryonic gene expression. But it means stretching the term too far if all this is called segmentation (see above). The bilaterian stem species must have been some kind of worm-like creature and one can expect that it already showed repeated elements along the body axis, perhaps in the nervous system, and that these repeated elements were formed on the basis of serially expressed genes. However, there is no evidence at all that this bilaterian stem species was segmented in the way defined above (see also Jenner 2000). Otherwise, one has to assume numerous reductions and losses of segmentation in most animal lineages. There are many hypotheses about the origin of segmentation as we find it in annelids and arthropods (Remane 1950, Clark 1977, Newman 1993, Westheide 1997, Davis & Patel 1999). The fact that single features of segmentation can be lost independent of others offers the possibility to conclude that segmentation evolved via a stepwise inclusion of structures. The starting point might

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have been the nervous system with repeated neurons or other elements. This assumption is based on the fact that many segmentation genes are also expressed in the developing nervous system (Patel 1994). In addition, there is good evidence that regional specification in the body of stem lineage bilaterians arose first in the nervous system (Deutsch & Le Guyader 1998). In the course of evolution more and more structures became involved in the repeated pattern until the complete set of segmental characters we find in annelids and arthropods was achieved. This scenario is supported on phylogenetic grounds. Given that the Articulata is monophyletic, its sister group is the Mollusca (Ax 1999). Among the molluscs, the representatives of the early branches, the Caudofoveata, Polyplacophora, Solenogastres, and Monoplacophora which together do not form a monophyletic group show a central nervous system comprising repeated elements, in particular, in the pedal region (Götting 1974). This makes it likely that the type of segmentation we find in annelids and arthropods evolved from a situation as is found in these molluscan taxa and which was still present in the lineage leading to the Articulata. Acknowledgements I thank the organisers of the 18th International Zoological Congress in Athens and, in particular Andreas Schmidt-Rhaesa, for inviting me to give a talk on the Articulata/ Ecdysozoa problem. Graham Budd, Wolfgang Dohle, Günter Purschke, and Andreas Schmidt-Rhaesa helped to improve the manuscript with their valuable comments. I am very grateful to Graham Budd for many fruitful discussions and for permanently challenging my views on arthropod evolution. References ABOUHEIF E., ZARDOYA R. & A. MEYER 1998. Limitations of metazoan 18S rRNA sequence data: implications for reconstructing a phylogeny of the animal kingdom and inferring the reality of the Cambrian explosion. J. Mol. Evol. 47: 394-405. ABZHANOV A. & T.C. KAUFMAN 1999. Homeotic genes and the arthropod head: expression patterns of the labial, proboscipedia, and Deformed genes in crustaceans and insects. Proc. Natl. Acad. Sci. USA 96: 10224-10229. ABZHANOV A. & T.C. KAUFMAN 2000. Crustacean (malacostracan) Hox genes and the evolution of the arthropod trunk. Development 127: 2239-2249. ADOUTTE A., BALAVOINE G., LARTILLOT N. & R. DE ROSA 1999. Animal evolution, the end of the intermediate taxa? TIG 15: 104-108. AGUINALDO A.M.A., TURBEVILLE J.M., LINFORD L.S., RIVERA M.C., GAREY J.R., RAFF R.A. & J.A. LAKE 1997. Evidence for a clade of nematodes, arthropods and other moulting animals. Nature 387: 489-493. ÅKESSON B. 1963. The comparative morphology and embryology of the head in scale worms (Aphroditidae, Polychaeta). Ark. Zool. 16: 125-163. ANDERSON D.T. 1966. The comparative embryology of the Polychaeta. Acta Zool. 47: 1-42. ANDERSON D.T. 1973. Embryology and Phylogeny in Annelids and Arthropods. Pergamon Press, Oxford. 495p.

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MÜLLER M.C. 1999. Das Nervensystem der Polychaeten: Immunhistochemische Untersuchungen an ausgewählten Taxa. PhD thesis, Universität Osnabrück. 402p. MÜLLER M.C. & W. WESTHEIDE 2000. Structure of the nervous system of Myzostoma cirriferum (Annelida) as revealed by immunohistochemistry and cLSM analyses. J. Morph. 245: 87-98. NEWMAN S.A. 1993. Is segmentation generic? BioEssays 15: 277-283. NIELSEN C. 1995. Animal Evolution. Oxford University Press, Oxford. 467p. PATEL N.H. 1994. Developmental evolution: insights from studies of insect segmentation. Science 266: 581-590. PATEL N.H., KORNBERG T.B. & C.S. GOODMAN 1989a. Expression of engrailed during segmentation in grasshopper and crayfish. Development 107: 201-212. PATEL N.H., MARTIN-BLANCO E., COLEMAN K.G., POOLE S.J., ELLIS M.C., KORNBERG T.B. & C.S. GOODMAN 1989b. Expression of engrailed proteins in arthropods, annelids, and chordates. Cell 58: 955-968. PETERSON M.D., POPADIC A. & T.C. KAUFMAN 1998. The expression of two engrailed-related genes in an apterygote insect and a phylogenetic analysis of insect engrailed-related genes. Dev. Genes Evol. 208: 547-557. PRINCE V.E., JOLY L., EKKER M. & R.K. HO 1998a. Zebrafish hox genes: Genomic organisation and modified colinear expression patterns in the trunk. Development 125: 407-420. PRINCE V.E., MOENS C.B., KIMMEL C.B. & R.K. HO 1998b. Zebrafish hox genes: Expression in the hindbrain region of wild-type and mutants for the segmentation gene, valentino. Development 125: 393-406. QUEINNEC E., MOUCHEL VIELH E., GUIMONNEAU M., GIBERT J.-M., TURQUIER Y. & J.S. DEUTSCH 1999. Cloning and expression of the engrailed.a gene of the barnacle Sacculina carcini. Dev. Genes Evol. 209: 180-185. RAUTHER M. 1909. Morphologie und Verwandtschaftsbeziehungen der Nematoden. Ergeb. Fortschr. Zool. 1: 491-596. REMANE A. 1950. Die Entstehung der Metamerie der Wirbellosen. Verh. Dtsch. Zool. Ges. 1949 Mainz, 16-23. RIEDL R. 1975. Die Ordnung des Lebendigen. Parey, Hamburg. 372p. ROUSE G.W. & K. FAUCHAULD 1997. Cladistics and polychaetes. Zool. Scripta 26: 139-204. SCHMIDT-RHAESA A., BARTOLOMAEUS T., LEMBURG C., EHLERS U. & J.R. GAREY 1998. The position of the Arthropoda in the phylogenetic system. J. Morph. 238: 263-285. SCHOLTZ G. 1984. Untersuchungen zur Bildung und Differenzierung des postnauplialen Keimstreifs von Neomysis integer Leach (Crustacea, Malacostraca, Peracarida). Zool. Jb. Anat. 112: 295-349. SCHOLTZ G. 1992. Cell lineage studies in the crayfish Cherax destructor (Crustacea, Decapoda): germ band formation, segmentation, and early neurogenesis. Roux‘s Arch. Dev. Biol. 202: 36-48. SCHOLTZ G. 1997. Cleavage, germ band formation and head segmentation: the ground pattern of the Euarthropoda. In Fortey R.A. & R.H. Thomas (eds), Arthropod Relationships. Chapman and Hall, London, pp. 317-332. SCHOLTZ G. 2001. Evolution of developmental patterns in arthropods – the analysis of gene expression and its bearing on morphology and phylogenetics. Zoology 103: 99-111. SCHOLTZ G. & W. DOHLE 1996. Cell lineage and cell fate in crustacean embryos - a comparative approach. Int. J. Dev. Biol. 40: 211-220.

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S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Ecdysozoa: the evidence forA.aLegakis, close relationship between ... Evolution 503 The New arthropods Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 503-509, 2003

Ecdysozoa: the evidence for a close relationship between arthropods and nematodes J.R. Garey Department of Biology, University of South Florida, Tampa, FL 33613, USA. E-mail: [email protected]

Abstract The hypothesis that molting animals form a monophyletic group known as Ecdysozoa is directly opposed to Articulata, in which segmented protostomes form a monophyletic taxon. According to morphology, nematodes were once considered to be basal bilaterians, but more recent ultrastructural and cladistic studies have led to the widely accepted hypothesis that nematodes belong among the protostomes. Early molecular studies also suggested that nematodes were basal bilaterians but more recent evidence suggests that this was an artifact of “long branch attraction” and 18S rRNA gene, total evidence, and hox gene studies all support Ecdysozoa. The branching pattern within Ecdysozoa has been difficult to elucidate, but it now appears that priapulids and kinorhynchs form the earliest branching clade, followed by nematodes + nematomorphs, and finally the panarthropods. This suggests that Cycloneuralia is paraphyletic and that arthropods are the most derived of the ecdysozoans.

Introduction The idea that molting animals form a monophyletic group known as Ecdysozoa is directly opposed to the traditional view that molting has evolved multiple times and that segmented protostomes form a monophyletic group known as Articulata. Why has it taken so long for Ecdysozoa to reveal itself? Cuvier (1817) originated Articulata as one of only four taxa into which he fit all metazoans. The idea of a “progression” from organisms with simple annelid-like segments to more complex and specialized arthropod-like segments has proven irresistible. Since Cuvier, the validity of Articulata has been assumed by virtually all invertebrate zoologists and has only recently been questioned. Many metazoan phyla are represented by species that are reduced in size and secondarily simple in body organization. The result was that these “simple” taxa (including nematodes) were often lumped together as “aschelminths” because of difficulty in finding characters useful for determining their relationship to other

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metazoans. The advent of ultrastructural analyses, molecular phylogenetic methods, and evolutionary developmental biology have all provided clues to the existence of Ecdysozoa. The discovery of Ecdysozoa Early molecular analyses of metazoan phylogeny (e.g. Field et al. 1988) utilized the 18S rRNA gene (rDNA) and suffered from poor taxon sampling and a general lack of understanding of how unequal rates of nucleotide substitution in different taxa could confound the tree-making process. It was clear in nearly all molecular studies that arthropods and annelids were not sister taxa, but these early studies incorrectly placed nematodes and flatworms as basal bilaterians due to the phenomenon of long branch attraction, in which more rapidly evolving sequences were attracted to the long branch between the diploblastic animals and the triploblasts (e.g. Winnepenninckx et al. 1995). In 1997, Aguinaldo et al. published a molecular analysis in which rDNA sequences from more slowly evolving species of flatworms and nematodes were used along with more sophisticated tree making techniques. These analyses provided the startling finding that all molting animals formed a monophyletic clade within the protostomes named Ecdysozoa. The phylogenetic position of nematodes within Metazoa There are many confounding reports in the literature that claim to prove or disprove the Ecdysozoa concept (e.g. Giribet et al. 2000, Hausdorf 2000, Wang et al. 1999, Zrzavy et al. 1998). Most of the arguments “disproving” Ecdysozoa center on the use of gene sequences from Caenorhabditis elegans and other rhabditid nematodes. The following discussion is intended to assist in the interpretation of the confusing literature concerning molecular phylogenetic studies of metazoans. Nematodes have traditionally been considered pseudocoelomates, often placed as basal bilaterians within the aschelminths, prior to the protostome/deuterostome split. The idea that nematodes represent a basal “pre-eucoelomate” bilaterian has been discarded and widely accepted morphology based cladistic studies have placed nematodes within the protostomes or in a sister group to the protostomes (Brusca & Brusca 1991, Nielsen 1995). The Articulata hypothesis does not require that nematodes be basal bilaterians, and the placement of nematodes within Protostomia is consistent with both the Articulata and the Ecdysozoa hypotheses. Unfortunately, the first molecular studies of metazoan phylogeny utilized 18S ribosomal RNA gene sequences (rDNA) from rhabditid nematodes. Rhabditid nematodes appear to evolve at a much faster rate than most other metazoans, and “long branch attraction” incorrectly places rhabiditid nematodes as basal bilaterians. This fast rate of nucleotide substitution observed in rhabditid rDNA appears to extend to the majority of protein coding genes as well (Mushegian et al. 1998), so molecular studies that use C. elegans as the nematode representative will consistently but incorrectly show nematodes as basal bilaterians (e.g. Wang et al. 1999, Hausdorf 2000). Nematoda is an extremely diverse taxon, and rDNA sequences are now available from over 100 nematode species.

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There is a clear relationship between the rate of evolution and the position of nematodes within the metazoan tree, in which the fast evolving sequences show nematodes as basal bilaterians, while the more slowly evolving sequences place nematodes as protostomes, near the arthropods (Fig. 1). The problem of long branch attraction is difficult because it affects both the alignment and the tree making method. Many tree making methods have been “proven” to be immune to long branch attraction in computer simulation studies, but generally these studies simulate nucleotide substitution within a set of pre-aligned sequences so that the alignment is perfect. In real studies of metazoan phylogeny, aligning rDNA sequences from taxa representing all of metazoan diversity is problematic, especially when there are widely varying rates of evolution, resulting in less than ideal alignments. Alignments can be improved using knowledge of rRNA secondary structure, but this is rarely done.

Fig. 1. Analysis showing the effect of evolutionary rate of rDNA within a nematode species to its position among Metazoa. Each point represents the 18S rDNA branch length from an internal node to a nematode species relative to three reference taxa (Saccharomyces cerevisiae, Antedon serrata and Glycera americana). This branch length (horizontal axis) is plotted against the length of the internal branch (vertical axis) of the tree that places nematodes as protostomes. The slow evolving nematode sequences (upper left) consistently support nematodes as protostomes (filled squares) while the fast evolving nematode sequences (lower right) support nematodes as basal to protostomes (hollow squares). The analysis was carried out using four cluster analysis (Kumar 1995) for each of 54 nematode rDNA sequences from Blaxter et al. 1998).

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Fig. 2. The relative rate test. A: If the outgroup is close to the two ingroups, then differences in evolutionary distance from the outgroup to ingroup 1 compared to the distance from the outgroup to ingroup 2 can easily be detected. B: If the outgroup is too distant, then the difference in distance from the outgroup to ingroup 1 compared to the distance from the outgroup to ingroup 2 will be more difficult to detect and likely be statistically insignificant, incorrectly suggesting that unequal evolutionary rates are not a factor.

A recent trend has been to test sequence data sets for unequal evolutionary rates with the relative rate test. Ingroup sequences are compared in all pairwise combinations to an outgroup, usually yeast. The evolutionary distance from the outgroup to each of two ingroup taxa is measured (Fig. 2A). If the two distances are statistically indistinguishable, then it is assumed that unequal rates are not present. The problem is in the choice of the outgroup. If the outgroup is too distant, then statistical tests will fail to find a significant difference in the distance to each ingroup and it will be incorrectly determined that long branch attraction will not be a factor (Fig. 2B). What is the relationship of taxa within Ecdysozoa? Studies that use rDNA to analyze metazoan phylogeny find good support for Ecdysozoa if great care is taken with sequence alignment and by avoiding representative species with particularly high substitution rates (e.g. rhabditid nematodes). Molecular studies have failed to resolve the topology within Ecdysozoa, presumably because rDNA cannot resolve branching patterns at that level. The “slowly” evolving nematode used by Aguinaldo et al. (1997) was Trichinella spiralis. While slower evolving than most rhabditid nematodes, its rDNA sequence produces the longest branch in the tree, and rDNA

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sequences from more slowly evolving nematodes have been published (Blaxter et al. 1998). By carefully eliminating the fastest evolving sequences, using secondary structure assisted alignments, correcting for site to site variation in substitution rate and using appropriate outgroups, the topology within Edysozoa can be revealed (Fig. 3). Ultrastructural and molecular analyses have demonstrated that aschelminths are polyphyletic and composed of several groups. One group (Cycloneuralia) includes only molting animals: priapulids, nematodes, nematomorphs, kinorhynchs, and loriciferans. Panarthropoda is another widely accepted group of molting animals and includes arthropods, onycophorans and tardigrades. Conceptually, the key to Ecdysozoa is to place Cycloneuralia and Panarthropoda together as sister taxa. An important question has been to determine if Cycloneuralia and Panarthropoda are sister taxa or if somehow they merge in a more complicated fashion. Fig. 3 reveals that Cycloneuralia is paraphyletic and that Arthropoda is highly derived. Interestingly, Panarthropoda is the

Fig. 3. Neighbor Joining analysis of the topology within Ecdysozoa. Branches are drawn according to scale and bootstrap values are shown at each node. The arrows show that the position of the onychophoran sequence was uncertain (see text for details). 18S rRNA gene sequences were selected following preliminary analysis to exclude fast evolving taxa. The slow evolving taxa were aligned according to a secondary structure model, and analyzed using the MEGA program using Kimura 2-parameter distances using a gamma correction (α=0.72, see Winnepenninckyx et al. 1995 for details). Genbank accession numbers are shown to the right of each taxon name.

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sister group to Nematoda + Nematomorpha. There is still some question concerning the branching order of tardigrades and onychophorans. There are currently only two onychophoran rDNA sequences available and the even the slowest evolving onychophoran sequence (Euperipatoides leuckarti) forms the longest branch in Fig. 3. This sequence makes the tree somewhat unstable and results in extremely low bootstrap support for its position in the tree (see arrows in Fig. 3). When the onychophoran sequence is eliminated from the analysis, all the nodes among the ecdysozoan phyla are highly supported (77-97%, not shown). Other evidence for Ecdysozoa Ecdysozoa is also supported by several combined molecular and morphological analyses (e.g. see Giribet et al. 2000, and Zrzavy et al. 1998). The molecular evidence supporting Ecdysozoa extends to hox gene clusters as well. It has been shown that there is a particular type of hox gene found in ecdysozoans but absent in non-molting protostomes, while another kind of hox gene is found in non-molting protostomes but not in ecdysozoans (de Rosa et al. 1999). Developmental biologists have also discovered similarities in homeobox expression in arthropods and chordates which suggest that either segmentation predates the protostome/deuterostome split, or that segmentation has appeared convergently among different metazoans (Holland et al. 1997). The implication of this finding is that annelids and arthropods need not be sister taxa. In morphological studies, the characters thus far that support Ecdysozoa are related to molting, while characters that support Articulata are related to segmentation (SchmidtRhaesa et al. 1998). For the near future it will be important to (1) analyze multiple protein coding genes from slow evolving nematode taxa, (2) find morphological characters that are independent of molting and segmentation and (3) investigate the patterns of gene expression responsible for molting and segmentation. References AGUINALDO A.M., TURBEVILLE J.M., LINFORD L.S., RIVERA M.C., GAREY J.R., RAFF R.A. & J.A. LAKE 1997. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387: 489-493. BLAXTER M.L., DE LEY P., GAREY J.R., LIU L.X., SCHELDEMAN P., VIERSTRAETE A., VANFLETEREN J.R., MACKEY L.Y., DORRIS M., FRISSE L.M., VIDA J.T. & W.K. THOMAS 1998. A molecular evolutionary framework for the phylum Nematoda. Nature 392: 71-74. BRUSCA R.C. & G.J. BRUSCA 1990. Invertebrates. Sinauer Associates, Sunderland MA, 922p. CUVIER G.B. 1817. La Régne Animal. Vol. II. Deterville, Paris. 532p. DE ROSA R., GRENIER J.K., ANDREEVA T., COOK C.E., ADOUTTE A., AKAM M., CARROLL S.B. & G. BALAVOINE 1999. Hox genes in brachiopods and priapulids and protostome evolution. Nature 399: 772-776. FIELD K.G., OLSEN G.J., LANE D.J., GIOVANNONI S.J., GHISELIN M.T., RAFF E.C., PACE N.R. & R.A. RAFF 1988. Molecular phylogeny of the animal kingdom. Science 239: 748-753. GIRIBET G., DISTEL D.L., POLZ M., STERRER W. & W.C. WHEELER 2000. Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cyclio-

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phora, Plathelminthes, and Chaetognatha: A combined approach of 18S rDNA sequences and morphology. Syst. Biol. 49: 539-562. HAUSDORF B. 2000. Early evolution of the Bilateria. Syst. Biol. 49:130-142. HOLLAND L.Z., KENE M. & N.A. WILLIAMS 1997. Sequence and embryonic expression of the amphioxus engrailed gene (AmphiEn): The metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development 124: 1723-1732. KUMAR S. 1995. PHYLTEST: Phylogeny Hypothesis Testing Using the Minimum Evolution Method. The Pennsylvania State University, University Park, PA. 16802. NIELSEN C. 1995. Animal Evolution. Oxford University Press, Oxford, 597p. SCHMIDT-RHAESA A., EHLERS U., BARTOLOMAEUS T., LEMBURG C. & J.R. GAREY 1998. The phylogenetic position of the Arthropoda. J. Morphol. 238: 263-285. MUSHEGIAN A.R., GAREY J.R., MARTIN J. & L. LIU 1998. Large-scale taxonomic profiling of eukaryotic model organisms: a comparison of orthologous proteins encoded by the human, fly, nematode, and yeast genomes. Genome Research 8: 590-598. WANG D.Y.C., KUMAR S., & S.B. HEDGES 1999. Divergence time estimates for the early history of animal phyla and the origin of plants, animals and fungi. Proc. Royal Soc. Lond. Series B. 266: 163-171. WINNEPENNINCKX B., BACKELJAU T., MACKEY L.Y., BROOKS J.M., DE WACHTER R., KUMAR S. & J.R. GAREY 1995. 18S rRNA data indicate that the aschelminthes are polyphyletic and consist of at least three distinct clades. Molec. Biol. Evol. 12: 1132-1137. ZRZAVY J., MIHULKA S., KEPKA P., BEZDEK A. & D. TIETZ 1998. Phylogeny of the Metazoa based on morphological and 18S ribosomal DNA evidence. Cladistics 14: 249-285.

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Zoological Implications of the Discovery of Geothermally-driven Communities

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Adaptations of hydrothermal vent organisms to environment 513 Thetheir New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 513-517, 2003

Adaptations of hydrothermal vent organisms to their environment F. Gaill, M. Zbinden & F. Pradillon UMR 7622, CNRS, Université Pierre et Marie Curie, 7 Quai Saint Bernard Paris 75005 France

Abstract Deep-sea hydrothermal vent organisms are often cited as examples of adaptation to extreme environmental conditions. After a brief review of what is known about the biology of the vent communities, we will focus our purpose on some adaptations of vent organisms submitted to high temperatures. Several characteristics of the Alvinella pompejana Desbruyères & Laubier (1980) collagen suggest an adaptation to the physical and chemical characteristics of their environment. The molecular basis of such adaptation will be analyzed on the collagen from protective surfaces of the so-called Pompeii worms (Alvinella pompejana) and additional data about the colonisation process involving the worm exoskeleton will be described.

Introduction The discovery of deep-sea vent fauna has given new insights into several important zoological aspects (Tunnicliffe 1991). These hydrothermal vents are one of the most unusual habitats found on earth. Vents are surrounded by a dense community, which is supported by primary production through chemoautotrophic bacteria. Most of this fauna is composed of sessile animals that harbor bacteria as intracellular symbionts. Such geothermally-driven communities are dependent on the reduced sulfur compounds, found in the emerging hot hydrothermal fluid (up to 400°C), which are the main energy source for free-living and symbiotic bacteria (Humphris et al. 1995). The vent communities Since the primary food source of the vent community is locally produced, it has been suggested that these communities are largely independent of environmental changes at the earth surface and not subject to the same evolutionary pressures than other organisms. Supporting this hypothesis is the fact that most of the organisms found at the vents are endemic (Tunnicliffe 1991). This observation raises also the question of how old these ecosystems are and when animals first started to be associated with vents. These

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communities may have escaped the mass extinctions affecting surface dwelling organisms. Even though our knowledge about the zoology of this fauna is increasing, we still do not know what the life cycles of these animals are, what their larvae look like, and where they are found (Humphris et al. 1995). Understanding the dispersal, colonisation and succession of species in vent and cold seep habitats is a great challenge for the future and will shed light about major questions such as the origin of life, evolution of symbiotrophy, diversity of physiological adaptations and molecular phylogeny. The vent surroundings: an extreme environment The hydrothermal environment is harsh, considering the pressure (260 atmospheres), temperature (350°C) and toxicity of the hydrothermal fluid, which is acid, anoxic, and rich in metallic sulfides (Tunnicliffe 1991). Deep-sea hydrothermal vent organisms are often cited as examples of adaptation to extreme environmental conditions (Gaill et al. 1991, Dalhoff et al. 1991; Gaill 1993). Alvinella pompejana (Desbruyères & Laubier 1980) is a polychaetous annelid from the East Pacific Rise inhabiting one of the most extreme environment of the earth for animals (Desbruyères et al. 1998). These organisms live in tubes they secrete at the surface of the smoker and are considered as the most eurythermal metazoans presently known (Cary et al. 1998). This review will focus on the main molecular (collagen) and exoskeletal characteristics (tube) of the protective surfaces, which preserve these worms from the environmental stresses. Molecular adaptation to the temperature Collagens are among the most ubiquitous proteins found in the animal kingdom. Several features of the A. pompejana collagen suggest an adaptation to the hydrothermal vent environment: thermostability (Gaill et al. 1995), but also barostability and the associated enzymatic processes which appear to be optimized under anoxic conditions (Kaule et al. 1998).Whereas the interstitial collagen of coastal polychaete worms (Arenicola marina) is denatured at 28°C, the collagen of A. pompejana remains stable at 45°C and is thus the most thermostable fibrillar collagen known up to now (Gaill et al. 1995). In contrast with what was previously expected, Sicot et al. (2000) have shown that the worm collagen stability process would be the same as what is known in vertebrate and human fibrillar collagen. Moreover, all the stabilizing factors known to day are amplified in the A. pompejana collagen, including the percentage of stabilizing triplets, the proline content and the frequency of hydroxyproline in the Y position of the GlyX-Y triplets. Phylogenetic analysis realized on vent and coastal collagen worms have shown that two parts of the same molecule, the central helical domain and the C terminal propeptide, have evolve at different rates. Since the helical chain, present in the extracellular matrix, is likely the only part submitted to thermal selective pressure, Sicot et al. (2000) have taken this differential evolutionary rate as evidence for an adaptive process at the molecular level.

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Diversity of thermostability process Results obtained on the second type of worm fibrillar collagen, the cuticular one (Gaill et al. 1991), indicate that the same type of protein family, the fibrillar collagen, would exhibit two different strategies of thermal stability. The first is that which would have had a great success in the course of evolution - and that is the one found in the alvinellid interstitial collagen, relying on the proline hydroxylation of the residues in the Y position of the GXY triplets. The second and one which seems to be original up to now, is that which relies on the glycosylation of the threonine in the same position of the triplet (Mann et al. 1996). Both examples underline the importance of post-translational processes in the molecular stability. No one knows to day if these differences are phylogenetically related or collagen type specific and additional data are needed on the cuticular collagen sequence to answer this question. Temperature and colonisation process Biochemical studies obtained at the molecular and cellular level indicate that the Pompeii worms would support a 30°C thermal range (20-50°C), which is twice less that which would exist in the tube habitat (60°C, Cary et al. 1998) outside the worm tissues (Fig.1). In order to study the colonisation process with non-invasive methods, a specific device was designed by one of us. This device, the so-called TRAC for Titanium Ring for Alvinellid Colonization, consists in a hollowed titanium cylindrical structure (25 cm in diameter and 15 cm in height). Several TRAC were deployed on white smokers and recovered during successive cruises. The results indicate that the Pompeii worms are the first colonizers when the temperature range of the smoker wall is about 30-40°C (Taylor et al. 1999) and various analyses of the biogeoassemblages obtained with the alvinellid colonies are still in progress. Temperature time recorders associated with the TRAC indicate a thermal gradient of about 10°C/cm smaller as estimated from the literature (Fig.1 in the alvinellid colony thickness. Short-term experiments have shown that a biogeoassemblage, including alvinellids, tubes and mineral sulfides, was colonizing the TRAC in a temperature range of about 40°C (48°-93°C), for the most basal part of the TRAC. Such range was about twice less at the upper one facing the seawater. The tube micro-ecosystem Because of their position on active sulphide chimney walls, these organisms are facing mineral precipitations resulting from the fluctuating thermal and chemical hydrothermal fluid/sea water interactions (Fig.1). The tubes they secrete are characterized by a tremendous chemical and thermal stability, their structure being still preserved at 80°C (Gaill & Hunt 1991). These extracellular matrices protect the worm tissues from the mechanical stress generated by the rain of mineral particles. Furthermore, Zbinden et al. (2000) have shown that the more a tube is mineralized, the more iron-rich is its outer face. In contrast, the only particles observed within the tube thickness are zinc sulphides

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Fig. 1. Alvinella pompejana thermal environment according to literature data reviewed in Desbruyères et al. 1998 and Cary et al. 1998. Top: schematic drawing of Alvinella pompejana in its tube on the wall of a chimney (A: animal; T: tube; M: mineral; I: interface; SW: seawater). Bottom graph compiles punctual temperature values from literature data. The overall mean gradient (10°C/cm) is indicated below the temperature graph.

of remarkably constant composition whatever the tube considered. These results indicate that, by structuring the fluid-seawater interface, these exoskeletons allow the animals to regulate their own physical microsurroundings. Complementary approaches have been recently used in order to evaluate the in vivo temperature tolerance of the hot ‘pole’ hydrothermal vent fauna Shillito et al. 2001 or the thermal range of the alvinellid developmental process (Pradillon et al. 2001). It is obvious that such studies will bring in the future new insights into adaptational strategies used by life to colonize deep-sea hydrothermal vents. Acknowledgements We thank D. Desbruyères, L. Mullineaux and C. Fisher for samples collected from the East Pacific Rise during various oceanographic cruises.

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References CARY S., SHANK T. & J: STEIN 1998. Worms bask in extreme temperatures. Nature 391: 545-546. DAHLHOFF E., O’BRIEN J., SOMERO G. & R. VETTER 1991. Temperature effects on mitochondria from hydrothermal vent invertebrates: evidence for adaptation to elevated and variable habitat temperatures. Physiol. Zool. 64(6): 1490-1508. DESBRUYÈRES D., CHEVALDONNÉ P., ALAYSE A.M., JOLLIVET D., LALLIER F., JOUINTOULMOND C., ZAL F., SARRADIN P.M., COSSON R., CAPRAIS J.C., ARNDT C., O’BRIEN J., GUEZENNEC J., HOURDEZ S., RISO R., GAILL F., LAUBIER L. & A. TOULMOND 1998. Biology and ecology of the “Pompeii worm” (Alvinella pompejana Desbruyères & Laubier), a normal dweller of an extreme deep-sea environment: A synthesis of current knowledge and recent developments. Deep-Sea Res. 45: 383-422. DESBRUYÈRES D. & L. LAUBIER 1980. Alvinella pompejana gen. sp. nov., Ampharetidae abberrant des sources hydrothermales de la ride Est-Pacifique. Oceanol. Acta 3: 267-274. GAILL F. 1993. Aspects of life development at deep sea hydrothermal vents. FASEB J. 7: 558-565. GAILL F. & S. HUNT 1991. The biology of Annelid worms from high temperature hydrothermal vent regions. Rev. Aquat. Sci. 4: 107-137. GAILL F., MANN K., WIEDEMANN H., ENGEL J. & R. TIMPL 1995. Structural comparison of cuticle and interstitial collagens from annelids living in shallow seawater and at deep-sea hydrothermal vents. J. Mol. Biol. 246: 284-294. GAILL F., WIEDEMANN H., MANN K., KÖHN K., TIMPL R. & J. ENGEL 1991. Molecular characterization of cuticule and interstitial collagens from worms collected at deep-sea hydrothermal vents. J. Mol. Biol. 221: 157-163. HUMPHRIS S., ZIERENBERG R., MULLINEAUX L. & R. THOMSON 1995. Seafloor hydrothermal system: Physical, chemical, biological, and geological interactions. Geophysical Monograph 91, American Geophysical Union, Washington. 466p. KAULE G., TIMPL R., GAILL F. & V. GUNZLER 1998. Prolyl activity in tissue homogenates of annelids from deep-sea hydrothermal vents. Matrix Biol. 17: 205-212. MANN K., MECHLING D., BACHINGER H., ECKERSON C., GAILL F. & R. TIMPL 1996. Glycosylated threonine but not 4-hydroxyproline dominates the triple helix stabilizing positions in the sequence of a hydrothermal vent worm cuticle collagen. J. Mol. Biol. 261: 255-266. PRADILLON F., SHILLITO B., YOUNG C.M. & F. GAILL 2001. Developmental arrest in vent worm embryos. Nature 413: 698-699. SHILLITO B., JOLLIVET D., SARRADIN P.M., RODIER P., LALLIER F.H., DESBRUYERES D. & F. GAILL 2001. Temperature resistance of Hesiolyra bergi, a polychaetous annelid living on deep-sea vent smoker walls. Mar. Ecol. Prog. Ser. 216: 141-149. SICOT F., MESNAGE M., MASSELOT M., EXPOSITO J., GARRONE R., DEUTSCH J. & F. GAILL 2000. Molecular adaptation to an extreme environment: Origin of the thermal stability of the Pompeii worm collagen. J. Mol. Biol. 302: 811-820. TAYLOR C.D., WIRSEN C.O. & F. GAILL 1999. Rapid microbial production of filamentous sulfur mats at hydrothermal vents. Appl. Environment. Microbiol. 65: 2253-2255. TUNNICLIFFE V. 1991. The biology of hydrothermal vents: Ecology and evolution. Oceanog. Mar. Biol. Ann. Rev. 29: 319-407. ZBINDEN M., MARTINEZ I., GUYOT F., COMPÉRE P., CAMBON-BONAVITA M.A. & F. GAILL 2000. Characteristics of mineral particles associated with the Pompeii worm tubes. Biol. Cell 92(5): 382.

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The Role of Symbiosis in Physiology and Evolution

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© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

The Role of Symbiosis in Physiology Evolution 521 The Newand Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 521-522, 2003

The Role of Symbiosis in Physiology and Evolution P. Nardon & A. Heddi UMR INRA/INSA de Lyon - Biologie Fonctionnelle, Insectes et Interactions (BF2I) - Bâtiment Louis Pasteur - 20, avenue Albert Einstein - 69621 Villeurbanne Cedex - France

The symposium on symbiosis is devoted to the presentation of different models of symbiotic associations, and their role in the physiology and especially evolution of organisms. The association host/symbiote creates a new biological unit, the symbiocosm, itself submitted to natural selection. Different types of association exist. The symbiotes can be outside (ectosymbiosis) or inside the host, in the intestine or in some invaginations in tegument (endosymbiosis), or inside the cells (endocytobiosis). In integrated symbioses the symbiote (a bacterium) is perfectly controlled by the host (location and number), and behaves as a new cell organelle, only transmitted to the progeny by the mother. In other cases the symbiote is not perfectly controlled and invade most cells (Wolbachia), but not all the host population. Six models have been presented: in the Squid-Vibrio model (extracellular symbiosis), the bacterium is transmitted horizontally, or cyclically, and can be grown in vitro. The colonization of the luminous organ modifies this one. There are processes of recognition and specificity. Another endosymbiosis is the termite symbiosis. The gut lumen harbours protozoa and/or bacteria. That help the digestion. Transmission is horizontal. A coevolution host/symbiote is highly probable. Among endocytobioses the first model will be a protozoan, the amoeba, living symbiotically with a bacterium. The formation of the symbiosis has been observed in the laboratory: the bacterium, which was firstly a parasite, has coevolved with the host cell so that to become obligate. We have the story of an integration. The role of symbiote in co-evolution is also spectacular with the Wolbachia model, a bacterium associated with of insects and other invertebrates. An integrated and obligate symbiosis in insect will be presented with the aphid model. The changes of the symbiotic bacterium during symbiosis are spectacular (notably the reduction of the genome). Furthermore, for the 1st time, this prokaryotic genome has been sequenced. The first conclusions, at the molecular level, have been communicated. A last model is the weevil (Coleoptera), where symbiosis is perfectly integrated, but surprinsingly not always obligate. The comparison of symbiotic and aposymbiotic (= without symbiotes) strains allows to appreciate the exact role of

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symbiosis. The symbiocosm is controlled by interactions of four different genomes: nuclear, mitochondrial and symbiotic (principal endocytobiote and Wolbachia). Symbioses appear as important factors of evolution. Symbiology must be recognized as an important field of zoology, botany and genetics. What we call “an animal” is in reality a community of numerous organisms.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Wolbachia: Symbionts as Reproductive Parasites 523 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 523-526, 2003

Wolbachia: Symbionts as Reproductive Parasites K. Bourtzis Department of Environmental and Natural Resources Management, University of Ioannina, 2 Seferi St., 30100 Agrinio, Greece & Insect Molecular Genetics Group, Institute of Molecular Biology and Biotechnology, Vassilika Vouton, PO Box 1527, 71100 Heraklion, Crete, Greece. E-mail: [email protected]

Abstract Wolbachia is a group of intracellular maternally inherited bacteria that are able to invade and maintain themselves in an enormous range of invertebrate species. These bacteria are associated with a number of reproductive alterations including induction of parthenogenesis, feminization of genetic males, male-killing, and most commonly induction of cytoplasmic incompatibility (CI), a form of embryonic lethality in crosses between infected males and females of different Wolbachia infection status. The biology of these bacteria is discussed, with an emphasis on their potential role in speciation and the use of Wolbachia-induced reproductive abnormalities for the development of novel, environmentally friendly, biotechnological approaches for the control of arthropod pests and modification of beneficial arthropod species.

Introduction Wolbachia is a group of obligatory intracellular maternally transmitted bacteria that infect a wide range of invertebrate species, including insects, mites, crustaceans and nematodes (for recent reviews: Werren 1997a, Bourtzis & O’Neill 1998, Bourtzis & Braig 1999, Stouthamer et al. 1999). Recent PCR surveys suggest that perhaps over 20% of the arthropod species may be Wolbachia-infected, rendering this bacterium the most ubiquitous intracellular symbiont as yet described. The ability of these bacteria to infect and persist into such a variety of invertebrate species may be related with their potential to “escape” the innate immune system of their hosts (Bourtzis et al. 2000). By using the 16S rDNA gene as phylogenetic marker, it was shown that Wolbachia belongs to the αProteobacteria, the bacterial group from where the modern mitochondria are descended and are very closely related with a number of mammalian pathogens such as Anaplasma, Ehrlichia and Rickettsiae (O’Neill et al. 1992). By using faster evolving protein coding genes, such as ftsZ and wsp, it was possible to divide the Wolbachia strains into four major groups, A, B, C, and D. Wolbachia strains infecting arthropods belong to A and B

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groups while the nematode Wolbachia strains belong to C and D groups (Werren et al. 1995, Bandi et al. 1998, Zhou et al. 1998). Interestingly, the phylogenies between Wolbachia and its arthropod hosts are not congruent suggesting horizontal transmission events. On the other hand, the phylogenies between Wolbachia and its nematode hosts are congruent indicating a mutualistic association. Wolbachia in Nematodes In filarial nematodes, Wolbachia is present in hypodermis and reproductive tissues. Tetracycline treatments inhibit development in early stages and reduce worm fertility, confirming the mutualistic association between the bacteria and their nematode hosts. Recent studies showed that an endotoxin or lipopolysaccharide (LPS) from Wolbachia is a major cause of inflammatory responses induced directly by the filarial nematode. These data indicate that Wolbachia may be considered as potential target for control of filarial nematodes (Taylor & Hoerauf 1999, Taylor et al. 2000). Wolbachia in Arthropods Wolbachia infections in arthropods are known to induce a number of reproductive alterations in their hosts, such as feminization of genetic males to reproductively competent females, killing of males, parthenogenesis, and cytoplasmic incompatibility (for recent reviews: Werren 1997a, Bourtzis & O’Neill 1998, Bourtzis & Braig 1999, Stouthamer et al. 1999). Elimination of Wolbachia via antibiotic treatment of the infected hosts results in the restoration of normal reproductive phenotypes. The most common of these phenotypes is cytoplasmic incompatibility (CI) which can be either unidirectional or bidirectional. Unidirectional CI is expressed when an infected male is crossed with a female of different Wolbachia infection status and is lethal to the developing embryo, but in insects with haplodiploid sex determination (Hymenoptera), the end result of CI is a sex ratio shift to the haploid sex, which is usually male. The reciprocal cross is fully compatible, as are crosses between infected individuals. Although the mechanism by which Wolbachia causes CI has not yet been fully elucidated, the available genetic evidence suggest that CI apparently involves at least two bacterial genes; a “modification” and a “rescue” gene. According to this model, bacteria present in the testes modify the developing sperm. The same bacterial strain must then be present in the fertilized egg to rescue this modification. If rescue does not occur, then incompatibility between the egg and sperm occurs resulting in early embryonic death. Bidirectional CI usually occurs in crosses between infected individuals harboring different strains of Wolbachia. In this case, the different bacterial strains have different modification-rescue systems. Wolbachia and Speciation Werren (1997b) has recently reviewed the ways by which Wolbachia-induced reproductive alterations could contribute to speciation. First, bidirectional CI may lead to reproductive isolation between two populations; their subsequent genetic divergence could

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result to speciation. Second, unidirectional CI combined with other reproductive isolating mechanisms in the reciprocal direction may also result in bidirectional reproductive isolation. Moreover, if a parthenogenesis-inducing Wolbachia strain remains for a long period of time in a population, it may contribute, combined with deleterious mutations in the genes involved in sexual reproduction, to the production of an asexual species. Applied Biology of Wolbachia It has been proposed that Wolbachia infections can be used in an applied context (Beard et al. 1993; Bourtzis & Braig 1999). For example, Wolbachia-induced CI can be used in several ways: a) to directly suppress natural arthropod populations of economic and health importance, b) as a tool to spread genetically modified strains into wild arthropod populations (e.g. a whitefly strain which can not transmit a virus in plants) and c) as an expression vector, once a genetic transformation system for this bacterium is developed. There is an ongoing intensive collaborative effort of eight laboratories from six European countries through the European Wolbachia project, funded by the European Union, to identify Wolbachia and host genes that may be used for the development of novel, environmentally friendly, biotechnological approaches for the manipulation of arthropod species. Towards this goal, the project aims at the identification of Wolbachia and host genes that are involved in host-Wolbachia interaction and in the induction of cytoplasmic incompatibility, parthenogenesis and feminization by using an integrated genomics, proteomics and post-genomics approach. The characterization of these genes will be a major breakthrough towards the understanding of Wolbachia-host symbiotic associations and the evolution of intracellular symbiosis. References BANDI C., ANDERSON T.J.C., GENCHI C. & M.L. BLAXTER 1998. Phylogeny of Wolbachia in filiarial nematodes. Proc. R. Soc. Lond. B Biol. Sci. 265: 2407-2413. BEARD C.B., O’NEILL S.L., TESH R.B., RICHARDS F.F. & S. AKSOY 1993. Modification of arthropod vector competence via symbiotic bacteria. Parasitol. Today 9: 179-183. BOURTZIS K. & H.R. BRAIG 1999. The many faces of Wolbachia. In Raoult D. & T. Hackstadt (eds), Rickettsiae and Rickettsial Diseases at the Turn of the Third Millennium. Elsevier, Amsterdam, pp. 199-219. BOURTZIS K. & S.L. O’NEILL 1998. Wolbachia infections and arthropod reproduction. Bioscience 48: 287-293. BOURTZIS K., PETTIGREW M.M. & S.L. O’NEILL 2000. Wolbachia neither induces nor suppresses antibacterial peptides. Insect Molec. Biol. 9(6): 635-640. O’NEILL S.L., GIORDANO R., COLBERT A.M., KARR T.L. & H.M. ROBERTSON 1992. 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc. Natl. Acad. Sci. U.S.A 89: 2699-702. STOUTHAMER R., BREEUWER J.A.J. & G.D.D. HURST 1999. Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annu. Rev. Microbiol. 53: 71-102. TAYLOR M.J., BANDI C., HOERAUF A.M. & J. LAZDINS 2000. Wolbachia bacteria of filarial nematodes: a target for control? Parasitol. Today 16: 179-180.

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TAYLOR M.J. &. A.M. HOERAUF 1999. Wolbachia bacteria of filarial nematodes. Parasitol. Today 15: 437-442. WERREN J.H. 1997a. Biology of Wolbachia. Annu. Rev. Entomol. 42: 587-609. WERREN J.H. 1997b. Wolbachia and speciation. In Howard D. & S. Berlocher (eds), Endless forms: Species and Speciation. Oxford University Press, Oxford, pp. 245-260. WERREN J.H., ZHANG W. & L.R. GUO 1995. Evolution and phylogeny of Wolbachia – reproductive parasites of arthropods. Proc. R. Soc. Lond. B Biol. Sci. 261: 55-63. ZHOU W., ROUSSET F. & S.L. O’NEILL 1998. Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc. R. Soc. Lond. B Biol. Sci. 265: 509-515.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

The weevil’s symbiocosm and its four intracellular genomes 527 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 527-534, 2003

The weevil’s symbiocosm and its four intracellular genomes A. Heddi UMR INRA/INSA de Lyon 0203 – Biologie Fonctionnelle, Insectes et Interactions (BF2I). INSA de Lyon - Bâtiment L. Pasteur - 20, Avenue Albert Einstein, France. E-mail: [email protected]

Abstract The weevil symbiocosm is composed of as many as four different genomes: nuclear, mitochondrial, principal endosymbiont and Wolbachia. The principal endocytobiont, which belongs to the proteobacteria γ-subgroup, is present constantly in the bacteriocytes of all weevils studied. It improves significantly the physiology and the behavior of the insect by interacting with the mitochondrial energetic pathways. Wolbachia, the α-proteobacterium that is widespread in arthropods, does not infect all weevil strains. However, it is disseminated throughout the body cells of infected strains, particularly in high density in the germ cells where it causes nucleo-cytoplasmic incompatibility, changing therefore weevil reproduction. The coexistence of two distinct types of intracellular bacteria at different levels of symbiont integration illustrates the complexity of animal tissue and indicates the predominant roles of prokaryote on eukaryote cell evolution.

Introduction Symbiosis is a widespread phenomenon in arthropods and particularly in insects (Buchner 1965). It involves most often species feeding on nutritionally unbalanced diets such as phloem sap, wood, blood and cereals. It is believed that symbionts help to compensate these nutritional deficiencies by interacting with several metabolic pathways of the insect, allowing thereby the insect to adapt to such poor environment. Symbionts could be prokaryotic (bacteria) or eukaryotic (yeast, protozoan), external (ant/fungi association) or internal (termite, cockroach). Intracellular symbiosis, or endocytobiosis, belongs to the last group and exhibits a sophisticated system where prokaryotes and eukaryotes are intimately interacting to fashion the phenotype of a so-called symbiocosm (Nardon & Grenier 1993). Generally, neither the host nor the symbiont is able to develop independently. The host has developed, during the life history of the association, specific cells, called bacteriocytes, which form the bacteriome organ. The bacteriome harbors the bacteria and protects them from the insect immunity. Phylogenetically, bacteriome-

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associated symbionts of many insects like aphids, ants and tsetse flies, have been shown to present parallel evolutionary histories with their respective host species (Schroder et al. 1996; Baumann et al. 1997; Chen et al. 1999). The oldest association described so far is that of the aphids with an Enterobacteriaceae called Buchnera. It has been estimated that it was established 200 to 250 million years ago (Moran et al. 1994). In the cereal weevils Sitophilus, symbiosis was discovered by Pierantoni (1927) and has been studied by several authors (Nardon & Grenier 1988). First studies performed between the 1930’s and the 1950’s period by Mansour (1930), Tiegs & Murray (1938), were focused mainly on the histological characterization of the intracellular bacteria within the Sitophilus genus, named Calandra at that time. A complete description of insect embryonic and post-embryonic development, in relation to the symbiont transmission and localization, was achieved. Until the 1970’s, no significant progress was noticeable with regard to the role of bacteria on the host’s biology, except for the studies on the fine structure and the anatomy of the weevil bacteriomes that were carried on by Muscgrave and collaborators (Musgrave & Miller 1953). Baker (1974) attempted for the first time to study the bacterial role in the beginning of the 70’s but the real progress started since Nardon (1973) succeeded in obtaining a viable aposymbiotic strain from a symbiotic one by heat treatment. Comparative works between these two strains have permitted to understand how intracellular bacteria interact with the weevils at the physiological level. Following, I will develop the molecular characterization of the weevil’s endocytobionts and summarize their respective role on the insect’s biology. Molecular and in situ characterization of the weevil intracellular bacteria Intracellular bacteria of the weevil were identified by PCR using 16S rDNA universal eubacterial primers on total DNA extracted from the weevil larva (Heddi et al. 1999). Two types of sequences were obtained, one of which falls into the γ-subdivision of the proteobacteria and the other type belongs to the α-proteobacteria of the Wolbachia group. Fluorescence in situ hybridization revealed that the former is the bacteriome-associated symbiont and was arbitrarily named Sitophilus oryzae principal endocytobiont (or SOPE) (Heddi et al. 1998). Fig. 1 positions SOPE and Wolbachia relative to several representatives of the α and γ subgroups of proteobacteria. The weevil’s Wolbachia forms a monophyletic group with those of other insects, which are in turn closely related to the Rickettsia group. SOPE is found within the Enterobacteriaceae and shares a mean of 95.0 % sequence homology with Escherichia coli, and 87 % sequence homology with Buchnera aphidicola, the endocytobionts of aphids. SOPE is also monophyletic within the Enterobacteriaceae family suggesting that the establishment of symbiosis may have occurred before the host species differentiation. Nevertheless, more sequence information (concerning the symbiosis status of the ancestral species of Sitophilus) is needed to precisely estimate the age of the symbiosis association in Sitophilus evolution. This work is currently investigated on the Rhynchophiridae super family that includes the Sitophilini tribe. Fluorescence in situ hybridization (or FISH), using specific oligonucleotide probes for Wolbachia and SOPE, was carried out on sections performed at different localization of the insect body and at different embryonic stages. This work has revealed that SOPE

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Fig. 1: Phylogenetic tree of the SOPE group and Wolbachia based on 16S rDNA genes. Alignments were realized with Clustal W software. Phylogenetic trees were created using the neighbour-joining program of Clustal W software. Genetic distances (percent divergence) were calculated excluding all positions with gaps. Confidence limits on grouping were determined with Clustal W bootstrapping technique. Bacillus subtilis, a Gram positive bacterium, was used as an outgroup. Bacteria are followed by GeneBank accession number; W.: Wolbachia; E.: Endocytobiont.

and Wolbachia exhibit different features concerning their distribution within the weevils. As already described by Nardon (1971), SOPE is concentrated at the posterior pole of the oocyte and the new laid embryo. From 72 hours of post-embryonic development, SOPE is limited to the bacteriocytes that start forming the bacteriome (unpublished data). After the fourth day, the first instar larva emerges with a completely developed bacteriome that attaches to the junction of the fore- and midgut. The bacteriomes increase their shape but occupy the same position all over the following three larval stages. During the nymph metamorphosis, the larval bacteriome degenerates and some bacteriocytes succeed to reach the apex of mesenteric caeca where they develop many bacteriomes. One-month-old adults are devoid of SOPE except in the female germ lines, where they are constantly present and transfer to the offspring through the oocyte. Wolbachia, in contrast, does not show any specific tissue localization at any time of insect development (Heddi et al. 1999). It is disseminated throughout the whole body of the insect including the muscle, adipocyte and intestine, and even in the bacteriocytes coexisting with SOPE. However, the Wolbachia is found in particularly high density in the male and female germ cells, located around the periplasmic membrane of the oocyte and surrounding many spermatid nuclei in the testis.

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Influence of Wolbachia and SOPE on the weevil’s biology This study was made possible by a selective elimination of both SOPE and Wolbachia. Heat treatment was shown to disrupt the former only and tetracycline treatment for two generations removes both bacteria. - SOPE improves the physiological traits of the insect by a nutritional way Nardon and coworkers have demonstrated that SOPE elimination from symbiotic insects results in diminished fertility, increased development time and flying inaptitude of the aposymbiotic weevils (Nardon 1973, Nardon & Grenier 1988, Grenier et al. 1994). Furthermore, they have shown that symbionts provide their host with amino acids (Wicker & Nardon 1982) and vitamins such as pantothenic acid (part of Coenzyme A), riboflavin (precursor of NAD+-FAD) and biotin (Wicker 1983). As these vitamins are involved in mitochondrial enzyme activities and particularly in those from the oxidative phosphorylation (OXPHOS) pathway, we have tested the effect of SOPE on mitochondrial energetic metabolism. In this experimental context, we have shown that the respiratory control ratio (RCR) was increased in mitochondria isolated from the symbiotic strain as compared to the aposymbiotic one of Sitophilus oryzae (Heddi et al. 1991). Moreover, six mitochondrial enzymes were investigated and their specific activities were higher in the symbiotic strain (Heddi et al. 1993a). To find out how symbiotic bacteria interact with mitochondrial enzymatic activities we have tested two hypotheses: i. nutritional one, symbiotic bacteria could be involved in the biosynthesis of enzymes and coenzymes of mitochondria by supplementing their host with vitamins; ii. molecular one, symbiotic bacteria could interact with mitochondria at the gene expression level. To test the first hypothesis, weevils were reared during one generation on wheat flour artificial pellets complemented (or not) with pantothenic acid and riboflavin, and four enzymatic activities were measured on mitochondria isolated from symbiotic and aposymbiotic strains (Fig. 2). The results were consistent with the nutritional hypothesis as the differences between symbiotic and aposymbiotic insects (at specific activity level) were attenuated, whatever the enzyme being measured, when riboflavin and pantothenic acid were added to the diet. The second hypothesis was investigated with Southerm and Northern blot analyses using a PCR probe encompassing COI and COII genes of mitochondrial DNA. This experiment revealed no difference between symbiotic and aposymbiotic weevil at both mitochondrial gene replication and expression levels (Fig. 3). Hence, symbiotic bacteria seem to have no molecular interaction with the mitochondrial genome. We therefore concluded that SOPE is probably involved by a nutritional way to increase mitochondrial energetic metabolism and thereby to improve the insect physiological traits. - Wolbachia alters the weevil’s reproduction Wolbachia is an α-proteobacterium closely related to the parasitic group of Rickettsia. It is widespread in arthropods and may infect 15-20 % of insect species (Werren 1997). Its role was studied in several species belonging to different groups of arthropods. It

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Fig. 2: Specific enzymatic activities of mitochondrial suspensions. Insects were reared during one generation on wheat flour artificial pellets complemented or not with vitamins. A: control pellets, B: pellets supplemented with pantothenic acid (2.38 mg/kg of wheat flour) and C with riboflavin (1.88 mg/kg of wheat flour). SCR: succinate cytochrome c reductase (y unit = µmol mg-1s-1), GCR: glycerol-3-phosphate cytochrome c reductase (y = 0.5µmol mg-1s-1), PDH: pyruvate dehydrogenase (y = 0.1µmol mg-1s-1), KGDH: α-ketoglutarate dehydrogenase (y = 0.2µmol mg-1s-1). No significant difference between symbiotic and aposymbiotic insects (t test, α= 0.05)

was shown that Wolbachia alters the host’s reproduction in three ways: cytoplasmic incompatibility (Breeuwers & Werren 1990, O’Neill & Karr 1990, Stouthamer et al. 1993), parthenogenesis (Stouthamer et al. 1993) and feminization of genetic males (Rigaud et al. 1991). In the weevil’s populations, more that 40% are totally or partially infected with Wolbachia. The study of its role was realized on a Chinese strain, treated with heat in order to remove SOPE and to retain Wolbachia. Genetic crossings between individuals either treated (to remove Wolbachia) or not treated with tetracycline have shown that Wolbachia behaves differently than SOPE in the weevil’s symbiocosm (Heddi et al. 1999). First, because Wolbachia does not seem to have much involvement in weevil physiology (development time and fertility). Second, unidirectional cytoplasmic incompatibility is seen when females, but not males, are treated with tetracycline; tetracycline treatment of males re-establishes high levels of fertility, which are almost restored to normal. We

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Fig. 3: Southern (A) and Northern (B) blot analyses of SOPE-symbiotic and SOPE-aposymbiotic S. oryzae strains. A: total DNA was extracted as described by Heddi et al. (1998). Three µg of each sample were digested with Sac I and the blot was hybridized with a PCR probe encompassing COI and COII mitochondrial genes. The blot was washed and rehybridized with 18S rDNA probe. B: total RNA were extracted and blotted as described by Heddi et al. (1993b). The blot was hybridized with the COI/COII probe, washed and rehybridized with the 18S rDNA probe for normalization. Lanes 1 and 3 are symbiotic strains originating from China and France, respectively. Lanes 2 and 4 are the SOPE-aposymbiotic strains isolated by the heat treatment from 1 and 3 respectively.

have noticed that Wolbachia induces more that 75 % of cytoplasmic incompatibility in the Chinese population of S. oryzae. From a basic perspective, this is an intriguing phenomenon in that the bacterium, by inducing cytoplasmic incompatibility, could provide a reproductive barrier between symbiotic and aposymbiotic weevils, favoring sympatric speciation. Conclusion The symbiocosm of the weevil Sitophilus exhibits an adapted model for discussing the concept of the “biological individual”. Four intracellular genomes coexist within the same “organism” and interact at different levels to direct its physiology and its reproduction. SOPE induces specific tissue (the bacteriome) and is deeply involved in the physiology of the insect. Wolbachia does not need any specific tissue (like bacteriocytes) to develop and to transmit in the weevil. In return, it interacts specifically with the host germ lines and causes reproduction alteration. The rising question is how should we consider such an entity, where different permanent genomes share the same space and are transmitted vertically to the progeny? For instance, most pluricellular organisms harbor generally many more prokaryotic cells than eukaryotic ones. In Sitophilus we have estimated, for SOPE, 3 x 106 bacteria per weevil larva, which represents tenfold the number of eukaryotic cells. Moreover, all Sitophilus strains, from the three most representative species S. oryzae, S. zeamais and S. granarius, are naturally symbiotic except one S. granarius strain that was discovered by Mansour (1935) in Egypt. From a symbiogenesis point of view, pluricellular organisms

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could be considered as a mosaic of cells, belonging to different species, and having integrated the entity serially throughout evolution. Such integration has occurred many times in species evolution and continues “today” in eukaryotic cells. The most important consequence of these perpetual genome acquisitions is the production of more viable offspring in fluctuating environmental conditions. References BUCHNER P. 1965. Endosymbiosis of Animals with Plant Microorganisms. New York, London, Sidney: Wiley, J. and Sons, Intersciences Publishers, 909p. BAKER J.E. 1974. Differential sterol utilization by larvae of Sitophilus oryzae and Sitophilus granarius. Ann. Entomol. Soc. Am. 67: 591-594. BAUMANN P., MORAN N.A. & L. BAUMANN 1997. The evolution and genetics of aphid endosymbionts. BioScience 47: 12-19. BREEUWERS J.A.J. & J.H. WERREN 1990. Microorganisms associated with chromosome destruction and reproductive isolation between two insect species. Nature 346: 558-560. CHEN X.A., LI S. & S. AKSOY 1999. Concordant evolution of a symbiont with its host insect species: Molecular phylogeny of genus Glossina and its bacteriome-associated endosymbiont, Wigglesworthia glossinidia. J. Mol. Evol. 48(1): 49-58 GRENIER A.M., NARDON C. et al. 1994. The role of symbiotes in flight activity of Sitophilus weevils. Entomol. exp. appl. 70: 201-208. HEDDI A., CHARLES H., KHATCHADOURIAN C., BONNOT G. & P. NARDON 1998. Molecular characterization of the principal symbiotic bacteria of the weevil Sitophilus oryzae: A peculiar G - C content of an endocytobiotic DNA. J. Mol. Evol. 47(1): 52-61. HEDDI A., GRENIER A.M., KHATCHADOURIAN C., CHARLES H. & P. NARDON 1999. Four intracellular genomes direct weevil biology: Nuclear, mitochondrial, principal endosymbionts, and Wolbachia. Proc. Natl. Acad. Sci., USA 96: 6814-6819. HEDDI A., LEFEBVRE F. et al. 1991. The influence of symbiosis on the respiratory control ratio (RCR) and the ADP/O ratio in the adult weevil Sitophilus oryzae (Coleoptera, Curculionidae). Endocytobiosis & Cell. Res. 8: 61-73. HEDDI A., LEFEBVRE F. & P. NARDON 1993a. Effect of endocytobiotic bacteria on mitochondrial enzymatic activities in the weevil Sitophilus oryzae. Insect Biochem. Molec. Biol. 23(3): 403-411. HEDDI A., LESTIENNE, P., WALLACE D.C. & G. STEPIEN 1993b. Mitochondrial DNA expression in mitochondrial myopathies and coordinated expression of nuclear genes involved in ATP production. J. Biol. Chem. 268: 12156-12163. MANSOUR K. 1930. Preliminary studies on the bacterial cell mass (accessory cell mass) of Calandra oryzae: the rice wevil. Q. J. Microsc. Sci. 73: 421-436. MANSOUR K. 1935. On the microorganisms free and the infected Calandra granaria. Bull. Soc. Roy. Entomol. Egypt 19: 290-306. MORAN N.A., BAUMANN P. & C. VON DOHLEN 1994. Use of DNA sequences to reconstruct the history of the association between members of the Sternorrhyncha (Homoptera) and their bacterial endosymbionts. Eur. J. Entomol. 91: 79-83. MUSGRAVE A.J. & J.J. MILLER 1953. Some microoganisms associated with the weevils, Sitophilus granarius and Sitophilus oryzae. I - Distribution and description of the organisms. Can. Ent. 85: 387-390.

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NARDON P. 1971. Contribution a l’étude des symbiotes ovariens de Sitophilus saskii: localisation, histochimie et ultrastructure chez la femelle adulte. C. R. Acad. Sci. Paris 272D: 29752978. NARDON P. 1973. Obtention d’une souche asymbiotique chez le charançon Sitophilus sasakii Tak: différentes méthodes d’obtention et comparaison avec la souche symbiotique d’origine. C. R. Acad. Sci. Paris 277D: 981-984. NARDON P. & A.M. GRENIER 1988. Genetical and biochemical interactions between the host and its endosymbiotes in the weevil Sitophilus (Coleoptera Curculionidae) and other related species. In Scannerini S., Smith D., Bonfante-Fasolo P. & Gianinazzi-Pearson V. (eds), Cell to Cell Signals in Plant, Animal and Microbial Symbiosis. Springer-Verlag Berlin, pp. 255-270. O’NEILL S. L. & T.L. KARR 1990. Bidirectional incompatibility between conspecific populations of Drosophila simulans. Nature 348: 178-180. PIERANTONI U. 1927. L’organo simbiotico nello sviluppo di Calandra oryzae. Rend. Reale Acad. Sci. Fis. mat. Napoli 35(3a): 244-250. RIGAUD T., SOUTY-GROSSET C., RAIMOND R., MOCQUARD J.P. & P. JUCHAULT 1991. Feminizing endocytobiosis in the terrestrial crustacean Armadilidium vulgare Latr.: recent acquisistion. Endocytobiosis and Cell Res. 7: 259-273. SCHRODER D., DEPPISCH H., OBERMAYER M., KROHNE G., STACKEBRANDT E., HOLLDOBLER B., GOEBEL W. & R. GROSS 1996. Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural characterization. Mol. Microbiol. 21(3): 479-489. STOUTHAMER R., BREEUWERS J.A.J., LUCK R.F. & J.H. WERREN 1993. Molecular identification of microorganisms associated with parthenogenesis. Nature 361: 247-252. TIEGS O.N. & F.U. MURRAY 1938. Embryonic development of Calandra oryzae. Q. J. Microsc. Sci. 80: 159-284. WERREN J.H. 1997. Biology of Wolbachia. Ann. Rev. Entomol. 42: 587-609. WICKER C. 1983. Differential vitamin and choline requirements of symbiotic and aposymbiotic S. oryzae (Coleoptera : Curculionidae). Comp. Biochem. physiol. 76A: 177-182. WICKER C. & P. NARDON 1982. Development responses of symbiotic and aposymbiotic weevil Sitophilus oryzae L. (Coleoptera, Curculionidae) to a diet supplemented with aromatic amino acids. J. Insect Physiol. 28(12): 1021-1024.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Characteristic features of the genome of an aphid endosymbiotic ... Evolution 535 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 535-540, 2003

Characteristic features of the genome of an aphid endosymbiotic bacterium, Buchnera H. Ishikawa Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan 113-0033

Abstract Buchnera, an intracellular symbiotic bacterium of aphids, is a close relative of Escherichia coli, but it contains more than 100 genomic copies per cell, and its genome size is only a seventh of that of E. coli. The complete genome sequence of Buchnera revealed that its gene repertoire is quite different from those of parasitic bacteria, such as Mycoplasma, Rickettsia, and Chlamydia, though their genome sizes have been reduced to similar extents. While the parasitic bacteria have lost most genes for the biosynthesis of amino acids, Buchnera retains many of those genes. In particular, its gene repertoire is characterized by the richness in the genes for the biosynthesis of essential amino acids that the host insect is not able to synthesize, reflecting a nutritional role played by the symbiont. In contrast, curiously enough, Buchnera lacks almost all genes for the citric acid cycle, though many lines of evidence suggest that Buchnera is an aerobic bacterium. To account for the apparent lack of the citric acid cycle, there are four alternatives: 1, Acquisition of sufficient NADH through glycolysis; 2, importation of electron donors from the outside; 3, importation of relevant enzymes; 4; utilization of multifunctional proteins.

Introduction Most aphid species (Homoptera, Insecta) harbor prokaryotic intra-cellular symbionts, Buchnera (Munson et al. 1991), in their bacteriocytes (Ishikawa 1989). The intracellular symbiosis between Buchnera and aphids probably dates back as far as 250 million years ago (Moran et al. 1993), and represents one of the most mutualistic associations between a bacteirum and the eukaryotic cell (Ishikawa 1996, Baumann et al. 1997). Indeed, the association is so intimate and obligate that neither partner can any longer reproduce independently (Ishikawa 1989). Since the initial infection, endosymbionts have been vertically transmitted through generations of the host insects, and the bacterial and insect partners appear to have diversified in parallel (Moran & Baumann 1994). Phylogenetic analyses based on numerous genes, including 16S rDNA, of Buchnera from a number of aphid species have revealed that these bacteria belong to the ³ E. coli (Munson

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et al. 1991). Here I briefly describe how cospeciation of Buchnera with the host insect has modified the symbiont’s genome in the course of intracellular symbiosis over 250 million years. Materials and methods A long-established parthenogenetic clone of pea aphids, Acyrthosiphon pisum (Harris), was maintained on young broad bean seedlings at 15oC with a 16 hr photoperiod. Young apterous aphids were dissected in buffer A (35 mM Tris-HCl, pH 7.5, 25 mM KCl, 10 mM MgCl2, 250 mM sucrose) under a microscope. Bacteriocytes were freed from the insect body and collected by manual suction with a thin glass capillary. Buchnera cells were freed by pipetting bacteriocytes in buffer A and passing the suspension through an isopore membrane with a pore size of 3 ¼m to remove the nuclei of the bacteriocytes (Sasaki & Ishikawa 1995). Buchnera cells were washed in buffer A and collected by centrifugation at 1500 x g. After these procedures, virtually no contaminant particles were observed. Buchnera cells were treated with lysozyme and Proteinase K, and their DNA was extracted according to the standard procedures of the phenol/chloroform/isoamyl alcohol method. Subsequently, they were treated with RNase A, followed by phenol/ chloroform/isoamyl alcohol (Murray & Thompson 1980). Genome size and G+C content In an effort to characterize the Buchnera genome, we estimated its size by pulse-field gel electrophoresis of restriction fragments from the Buchnera DNA (Charles & Ishikawa 1999). As a result, it was shown that the Buchnera genome was a circular double-stranded DNA whose size was around 650 kb, about one-seventh of that of E. coli. A plausible explanation for the genome shrinkage of intracellular bacteria is an old rule of evolution and natural selection, “use it or lose it” (Maniloff 1996). This seems exactly true for parasitic bacteria, such as Rickettsia, Borrelia, and Chlamydia. Since these organisms enjoy an abundant supply of nutrients from their hosts, predictably the machinery responsible for their syntheses would decay. As a consequence, their genomes would tend to lose the relevant genes, leading to reduction of genome sizes. However, this is not applicable to Buchnera, which not only retains the ability to synthesize amino acids, but also provides them to the host (Sasaki & Ishikawa 1995, Nakabachi & Ishikawa 1997). It is interesting to know what caused the genome shrinkage in Buchnera and which kinds of genes are missing from its genome. One characteristic feature of the Buchnera genome is an overall A+T enrichment (Ishikawa 1987, Ohtaka & Ishikawa 1993). A negative correlation between genome size and AT content has been reported for many bacteria (Heddi et al. 1998). In eukaryotes, pseudogenes, in general, are more AT rich and smaller in size than their functional homologs (Gu & Li 1995). It is likely that the loss of DNA stability due to AT accumulation tends to favor the deletion process rather than insertion, and thus, the reduction in size of the Buchnera genome can be a neutral consequence of the accumulation of A and T.

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Genomic copy number The genome size of Buchnera is only a seventh that of E. coli, a close relative. Notwithstanding this, Buchnera cells are much larger in volume than E. coli cells, and do not divide as frequently as free-living bacteria. These observations led us to suspect that, unlike E. coli and other bacteria, Buchnera may be a bacterium with many copies of the genomic DNA in a single genome. This possibility was tested by three different methods, namely dot-blot hybridization, fluorimetry using a video-intensified microscope photon-counting system, and real-time quantitative PCR (Komaki & Ishikawa 1999, 2000). As a result, we obtained convincing evidence that each cell of Buchnera contains an average of 120 genomic copies. A dramatic reduction in the genome size (Charles & Ishikawa 1999) accompanied by an exceptional increase in the genomic copy number (Komaki & Ishikawa 1999) in Buchnera is reminiscent of eukaryotic cell organelles, such as mitochondria and chloroplasts (Gray et al. 1999). Loss of the ability to divide outside the eukaryotic cell is also a common attribute of Buchnera and these organelles. It is possible that these changes are an inevitable consequence for the prokaryotes that have been housed in eukaryotic cytoplasm for an evolutionary length of time. From an evolutionary point of view, the high copy number of the Buchnera genome may explain how this symbiotic bacterium has slowed down Muller’s ratchet (Muller 1964), which otherwise would be serious to small and asexual populations such as that of Buchnera (Ohta 1987; Moran 1996). It is possible that these genomic copies are homologous, rather than identical, to each other. This, in turn, raises the possibility that different copies carry distinct alleles like homologous chromosomes of eukaryotes. Provided that recombination between these homologous copies takes place, it will effectively remove mildly deleterious mutations that otherwise accumulate in the population of Buchnera. Thus, multiple genomes may enable Buchnera to utilize mechanisms somewhat similar to sexuality to avoid the deleterious effect caused by the bottleneck that symbionts undergo every time when transmitted to a next generation of the host. Gene repertoire The entire sequence of the Buchnera genome was determined using the whole genome random shotgun sequencing method. It turned out that the genome is composed of one 640,681 kb chromosome and two small plasmids (Shigenobu et al. 2000). The genome was shown to contain 583 ORFs, which were compared against a non-redundant protein database and their biological roles were assigned. Here I focus on genes for the biosynthesis of amino acids and those involved in energy metabolism. The Buchnera genome contains as many as 54 genes for the biosynthesis of amino acids. This forms a striking contrast to gene repertoires of parasitic bacteria, which contain only a few genes involved in amino acid synthesis in their similar-sized or larger genomes (Fraser et al. 1995, Andersson et al. 1998, Stephens et al. 1998). Another characteristic feature of Buchnera is that the genes for biosyntheses of amino acids essential for the aphid host are present, but those for nonessential amino acids are almost completely

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missing. This suggests that Buchnera provides the host with what the host cannot synthesize, and conversely, the host provides the symbiont with what Buchnera cannot synthesize. In addition, as precursors of some essential amino acids are nonessential amino acids, such as glutamate and aspartate, the biosynthetic pathways of both the host and symbiont are not only complementary, but also mutually dependent. This gene repertoire of the Buchnera genome is consistent with experimental evidence that aphids recycle the amino group of nitrogenous waste products as glutamine, which Buchnera uses as a substrate for the synthesis of essential amino acids (Sasaki & Ishikawa 1995; Douglas 1998). The genome data clearly indicate that Buchnera is an aerobic bacterium. This seems reasonable as this bacterium inhabits the bacteriocyte, which receives an ample supply of oxygen through the trachea and contains many mitochondria in the cytoplasm. Buchnera has complete gene sets for the glycolytic pathway, the pentose phosphate cycle, and aerobic respiration. In the Buchnera genome, the NADH dehydrogenase operon and the cytochrome o operon are conserved with the same gene arrangement as in the E. coli genome. Buchnera has an F0F1 type ATP synthase operon, suggesting that this bacterium is able to produce ATP using the proton electrochemical gradient generated by the electron transfer system (ETS). In contrast, Buchnera lacks genes responsible for fermentation and anaerobic respiration. Although all these results indicate that Buchnera respires aerobically, to our surprise, Buchnera does not have a gene set for the citric acid cycle except genes for the 2-oxoglutarate dehydrogenase complex. It is interesting to know whether or not Buchnera respires aerobically without operating the citric acid cycle. It is possible that Buchnera obtains an amount of NADH enough to operate ETS through the glycolytic pathway as the symbiont is accessible to plenty of sugar that the host ingests. It is also possible that Buchnera imports electron donors indirectly from the host cytoplasm or mitochondria in the vicinity. Alternatively, Buchnera may operate the citric acid cycle without having its relevant gene set. In this case, one possibility is importation of relevant enzymes from the host cytoplasm as do mitochondria. The fourth possibility is the utilization of multifunctional proteins, which may operate the cycle in place of the original enzymes encoded by genes lost through evolution. References ANDERSSON S.G.E., ZOMORODIPOUR A., ANDERSSON J.O., SICHERITZ-PONTEN T., ALSMARK C.M.U., PODOWSKI R.M., NØSLUND A.K., ERIKSSON A.-C., WINKLER H.H. & C.G. KURLAND 1998. The genome sequence of Rickettsia prowazekki and the origin of mitochondria. Nature 396: 133-140. BAUMANN P., MORAN N.A. & L. BAUMANN 1997. The evolution and genetics of aphid endosymbionts. BioScience 47: 12-20. CHARLES H. & H. ISHIKAWA 1999. Physical and genetic map of the genome of Buchnera, the primary endosymbiont of the pea aphid Acyrthosiphon pisum. J. Mol. Evol. 48: 142-150. DOUGLAS A.E. 1998. Nutritional interactions in insect-microbial symbiosis: Aphids and their symbiotic bacteria. Ann. Rev. Entomol. 43: 17-37.

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FRASER C.M., GOCAYNE J.D., WHITE O., ADAMS M.D., CLAYTON R.A., FLEISCHMANN R.D., BULT C.J., KERLAVAGE A.R., SUTTON G., KELLEY J.M., FRITCHMAN J.L., WEIDMAN J.F., SMALL K.V., SANDUSKY M., FUHRMANN J.L., NGUYEN D.T., UTTERBACK T., SAUDEK D.M., PHILLIPS C.A., MERRICK J.M., TOMB J., DOUGHERTY B.A., BOTT K.F., HU P.C., LUCIER T.S., PETERSON S.N., SMITH H.O. & J.C. VENTER 1995. The minimal gene complement of Mycoplasma genitalium. Science 270: 397-403. GRAY M.W., BURGER G. & B.F. LANG 1999. Mitochondrial evolution. Nature 283: 1476-1481. GU X. & W.H. LI 1995. The size distribution of insertions and deletions in human and rodent pseudogenes suggest the logarithmic gap penalty for sequence alignment. J. Mol. Evol. 40: 464-473. HEDDI A., CHARLES H., KHATCHADOURIAN C., BONNOT G. & P. NARDON 1998. Molecular characterization of the principal symbiotic bacteria of the weevil Sitophilus oryzae: A peculiar G - C content of an endocytobiotic DNA. J. Mol. Evol. 47(1): 52-61. ISHIKAWA H. 1987. Nucleotide composition and kinetic complexity of the genomic DNA of an intracellular symbiont in the pea aphid Acyrthosiphon pisum. J. Mol. Evol. 24: 205-211. ISHIKAWA H. 1989. Biochemical and molecular aspects of endosymbiosis in insects. Int. Rev. Cytol. 116: 1-45. ISHIKAWA H. 1996. Intracellular symbiosis in insects. In Colwell R.R., Simidu U. & K. Ohwada (eds), Microbial Diversity in Time and Space. New York, Plenum Press, pp. 93-100. KOMAKI K. & H. ISHIKAWA 1999. Intracellular bacterial symbionts of aphids possess many genomic copies per bacterium. J. Mol. Evol. 48: 717-722. KOMAKI K. & H. ISHIKAWA 2000. Genomic copy number of intracellular bacterial symbionts of aphids varies in response to developmental stage and morph of their host. Insect Biochem. Mol. Biol. 30: 253-258. MANILOFF J. 1996. The minimal cell genome: “On being the right size.” Proc. Natl. Acad. Sci. USA 93: 10004-10006. MORAN N.A. 1996. Accelerated evolution and Muller’s ratchet in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 93: 2873-2878. MORAN N.A. & P. BAUMANN 1994. Phylogenetics of cytoplasmically inherited microorganisms of arthropods. Trends Ecol. Evol. 9: 15-20. MORAN N.A., MUNSON M.A., BAUMANN P. & H. ISHIKAWA 1993. A molecular clock in endosymbiotic bacteria is calibrated using insect host. Proc. Roy. Soc. Lond. B 253: 167-171. MULLER H.J. 1964. The relation of recombination to mutational advance. Mutat. Res. 1: 2-9. MUNSON M. A., BAUMANN P. & M.G. KINSEY 1991. Buchnera gen. nov. and Buchnera aphidicola sp. nov., a taxon consisting of the mycetocyte-associated, primary endosymbionts of aphids. Int. J. Syst. Bacteriol. 41: 566-568. MURRAY M. G. & W.F. THOMPSON 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321-4325. NAKABACHI A. & H. ISHIKAWA 1998. Differential display of mRNAs related to amino acid metabolism in the endosymbiotic system of aphids. Insect Biochem. Mol. Biol. 27: 1057-1062. OHTA T. 1987. Very slightly deleterious mutations and the molecular clock. J. Mol. Evol. 26: 1-6. OHTAKA C. & H. ISHIKAWA 1993. Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont. J. Mol. Evol. 36: 121-126. SASAKI T. & H. ISHIKAWA 1995. Production of essential amino acids from glutamate by mycetocyte symbionts of the pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 41: 41-46.

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SHIGENOBU S., WATANABE H., HATTORI M., SAKAKI Y. & H. ISHIKAWA 2000. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407: 81-86. STEPHENS R.S., KALMAN S., LAMMEL C., FAN J., MARATHE R., ARAVIND L., MITCHELL W., OLINGER L., TATUSOV R.L., ZHAO Q., KOONIN E.V. & R.W. DAVIS 1998. Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282: 754-759.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Integration of bacterial endosymbionts in amoebae 541 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 541-547, 2003

Integration of bacterial endosymbionts in amoebae K.W. Jeon Department of Biochemistry, University of Tennessee, Knoxville, TN 37996-0840, USA. E-mail: [email protected]

Abstract An amoeba-bacteria symbiosis system is described in which the host and symbionts became associated spontaneously and developed a stable symbiosis within a few years under continuous observation. The symbionts survive inside amoebae, bring about hosts’ cellular character changes, and cause the host’s dependence for survival within a short period of time. Such changes caused by symbiosis can be reproduced under laboratory conditions. These changes arise as a result of the production of new macromolecules by both the host and symbionts or by an apparent suppression of some gene expression. One notable example is the transcription inhibition of the amoeba’s S-adenosylmethionine synthetase gene by symbionts. Integration of bacteria in amoebae is a good model with which to study various steps involved in the initial establishment and maintenance of endosymbiosis, that lead to changes in cellular phenotypic characters and the acquisition of new cell components.

I. Background A strain of Amoeba proteus became infected with an unidentified strain of Gramnegative X-bacteria in 1966 (Jeon & Lorch 1967). When the bacteria first infected amoebae, they were virulent and killed whole cultures of newly infected amoebae. At first, the infecting bacteria numbered more than 100,000 per amoeba, occupying up to 20% of the cell volume. While most of the D amoebae infected by X-bacteria died, a few of them survived and X-bacteria became less virulent. The number of symbionts in each xD amoeba was stabilized at about 42,000, occupying about 10% of the host cell volume. Thus, both the host and infective organisms changed to establish a stable symbiosis. Within a few years, host xD amoebae became dependent on their newly acquired endosymbionts for survival (Jeon & Jeon 1976). The presence of X-bacteria caused changes in several physiological and phenotypic characters in host amoebae, and such changes including the host’s dependence could be experimentally reproduced at any time under laboratory conditions. When X-bacteria isolated from xD amoebae were

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introduced into symbiont-free amoebae of the same or different strain, they quickly established a stable symbiosis and influenced the physiology and phenotypic characters of their new hosts. X-Bacteria were no longer virulent and the host xD amoebae grew normally under standard culture conditions. Physiological and molecular bases for the symbiotic interactions between X-bacteria and amoebae have been studied extensively and much information has been obtained. However, there are still many unanswered questions, including the reasons for the mutual dependence between the symbiotic partners. II. What is known The following is a summary of what is known about amoeba/X-bacteria symbiosis: 1. Symbionts: X-Bacteria are Gram-negative rods (~0.5 x 2 µm), enclosed in symbiosomes of varying sizes. Their ultrastructure is similar to that of other free-living or symbiotic Gram-negative bacteria. Within symbiosomes, X-bacteria are embedded in a matrix of fibrous matter that contains an 85-kDa protein (Kim & Jeon 1995). Phylogenetically, X-bacteria are very close to L. pneumophila and C. burnetii as determined on the basis of amino acid sequences of GroEL analogs. 2. Symbionts’ Survival inside Hosts: X-Bacteria appear to use two strategies in insuring their survival and multiplication within the host: a) resistance to lysosomal enzymes and b) modification of symbiosome membranes to prevent lysosome-symbiosome fusion. Isolated X-bacteria are also resistant to lysing agents in vitro. It is suspected that one or both of the plasmids present in X-bacteria (Han & Jeon 1980) plays a role in protecting X-bacteria from digestion by the host. X-bacteria’s infectivity may also be correlated with the presence or absence of plasmid DNAs in X-bacteria since plasmid-free X-bacteria do not infect other amoebae. At least two symbiosome membrane components produced by X-bacteria, a 96-kDa protein (Ahn et al. 1990) and lipopolysaccharides (LPS) (Choi & Jeon 1992) are involved in the prevention of lysosomal fusion with symbiosomes. Double-labeling experiments using anti-LPS mAb and a mAb against the lysosomal-membrane protein show that masking symbiosome-membrane components with an anti-LPS mAb permits lysosomes to fuse with symbiosomes (Kim et al. 1994). 3. Effect of Symbiosis on the Host: The presence of X-bacteria causes various physiological changes in host amoebae including: a) accelerated cell growth during the initial phase of experimental infection, b) increased sensitivity to starvation, over-feeding, and crowding, c) newly acquired temperature sensitivity above 26° C, one degree higher than their optimum growth temperature, d) altered nucleocytoplasmic compatibility between nuclei of xD amoebae and the cytoplasm of D amoebae, e) newly acquired nuclear lethal effect exerted by xD-amoeba nuclei on D amoebae, and f) new symbiontsynthesized proteins in the host cytoplasm. 4. Induced Infection: Isolated X-bacteria may be transferred into other amoebae either by microinjection or by induced phagocytosis. Most of the X-Bacteria thus introduced are digested, but a few survive and establish symbiosis. Newly infected host amoebae become dependent on their new symbionts within 200 cell generations (18 months) (Jeon & Ahn 1978).

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5. Mutual Dependence: X-Bacteria and established xD amoebae are mutually dependent for survival. X-Bacteria cannot live outside amoebae although they may stay viable for several days at 4° C. If X-bacteria are removed from xD amoebae, the latter lose viability within two weeks but X-bacteria-depleted amoebae may be rescued by reintroducing X-bacteria. X-Bacteria selectively accumulate host-produced actin, spectrin and myosin on their surfaces, but their roles are not known. 6. Genes of Symbionts: There are several genes that seem to be involved in the symbiosis between X-bacteria and amoebae. a. The s29x gene: The cytosol of xD amoebae contains a large amount of X-bacteriaproduced 29-kDa protein (S29x). The s29x gene has promoter sequences that are different from known bacterial consensus promoter sequences (Pak & Jeon 1996, Pak & Jeon 1997). The s29x gene has an ORF of 774 nucleotides, coding for 258 amino acids, equivalent to Mr of 29,968, and the gene shares no sequence identity with any other known genes. Inside amoebae, S29x is synthesized by X-bacteria and is transported to the amoeba cytoplasm, and the protein also enters the amoeba’s nucleus. While S29x moves easily across X-bacterial membranes and amoeba’s nuclear envelopes, the protein does not have any potential signal sequences at the N-terminus or trans-membrane domains. The function of S29x is not yet known but the protein may indirectly regulate a symbiont’s gene(s) involved in bacterial infectivity or their intracellular survival. b. The groELSx genes: X-Bacteria contain much GroELx and the groELx gene is controlled by a novel and potent second promoter (P2) in addition to the heat-shock consensus promoter (P1) (Ahn et al. 1994, Lee et al. 1996). The P2 promoter present in the groESx gene enhances the expression of groELx and other fusion genes in transformed E. coli. Transformed E. coli produce a large amount of GroELx and they can grow at their normally lethal temperature, 45° C. It is speculated that X-Bacteria survive inside amoebae by having potent heat-shock promoters in their groEx operon that are used to synthesize a large amount of GroELx, using it as a chaperonin. Thus, possession of such potent promoters would be a useful adaptation for X-bacteria in living inside the potentially hostile environment of amoeba’s cytoplasm. In endosymbiosis, host cells usually provide a suitable “shelter ” and supply important material needs for endosymbionts, but they present many difficulties for symbionts to survive. Thus, any would-be intracellular symbionts must have fitness traits to overcome the adverse conditions in order to colonize a host cell. It is becoming apparent that heat-shock proteins are widely involved in host-symbiont interactions (Hoffman & Garduno 1999, Radwanska et al. 2000). It is assumed that the roles of GroELx of X-bacteria are similar to those found in other cells or cell organelles and that the protein provides some protective benefits to symbionts as well as stabilizing macromolecules imported from the host cell cytoplasm. Unlike the aphid endosymbionts that synthesize only one protein, symbionin (Ishikawa 1984), X-bacteria synthesize many other proteins as well. However, they must import some proteins from the host cytoplasm since they are dependent on the host for survival. Thus, the GroEx protein might be involved in the transport and stabilization of such imported proteins. c. The mipx gene: X-Bacteria contain a macrophage infectivity potentiator (Mip)-like protein (Mipx ) (Oh & Jeon 1999). The Mipx protein contains amino acids corresponding to the peptidyl-prolyl cis/trans isomerase (PPIase) activity region at its C-terminus as in

544

The new panorama of animal evolution

some other pathogenic microorganisms (Rahfeld et al. 1996). Recombinant Mipx protein produced by E. coli transformed with mipx exhibit the PPIase enzyme activity and the activity is inhibited by the immunosuppressive drug, FK506. When X-bacteria are pretreated with FK506, X-bacteria’s infectivity in amoebae is reduced to one-third that of the control group. These results suggest that Mipx is involved in the initial infection of X-bacteria in amoebae (Oh & Jeon 1999) as in other organisms (Masuzawa et al. 1997, Seshu et al. 1997, Harb & Abu Kwaik 2000). The mipx gene has a sequence identity of 79% and 74% with genes of Legionella micdadei and L. pneumophila, respectively. III. A Case for Symbionts’ Control of Host’s Gene Expression When D amoebae are infected with X-bacteria, amoebae lose several proteins, among which a 45-kDa S-adenosyl-L-methionine synthetase (SAMS) is most prominent (Choi et al. 1997). Once infected with X-bacteria, xD amoebae stop transcribing the sams gene and producing their own SAMS within a few weeks after infection. However, xD amoebae possess about half the level of SAMS enzyme activity found in D amoebae, and it appears that there is an alternative source for the enzyme in infected amoebae. The mechanism for the suppression of amoeba’s sams gene expression caused by bacterial infection is not known, but it is apparent that X-bacteria inhibit the transcription of sams and the synthesis of SAMS. Meanwhile, it has been found that the sams gene is present in xD amoebae and its complete nucleotide sequence has been determined, together with sams genes of D amoebae and X-bacteria (T.J. Jeon & K.W. Jeon, unpubl. data). The nucleotide sequence of xD amoeba’s sams gene is identical to that of D-amoeba’s. The enzyme SAMS mediates the formation of S-adenosylmethionine (AdoMet) from methionine and ATP, and AdoMet is a key molecule in methylation reactions and polyamine biosynthesis in various organisms. Thus, for example, it is required for continuous growth of Pneumocystis carinii (Merali et al. 2000), and lack of AdoMet results in a cell division defect in E. coli (Newman et al. 2000). Mutations affecting the biosynthesis of AdoMet reduce DNA methylation in Neurospora crassa (Roberts & Selker 1995). Also, deficiency in DNA methylation causes partial differentiation in mammalian cells (Razin & Riggs 1980) and Chlamydomonas (Sager et al. 1981). In yeast, the level of AdoMet affects meiosis and sporulation (Varma et al. 1985). Since AdoMet is the precursor for many essential cellular constituents, a mutation causing an extreme deficiency of AdoMet would be lethal. It could be assumed that an amoeba’s inability to transcribe its sams gene would be equally fatal. This would explain why host amoebae become dependent on symbionts since the symbionts supplement what xD amoebae themselves no longer produce following infection by X-bacteria. As a result, symbiotic X-bacteria would become essential cellular components of xD amoebae by supplementing a genetic defect that is brought about by an action of X-bacteria themselves. This is the first reported example in which symbionts alter the host’s gene expression and block the production of an essential protein. It is not clear how the transcription of sams of infected amoebae is suppressed by Xbacteria. One could postulate the following scheme for the symbiont-host relationship in xD amoebae, involving SAMS (Fig. 1): Infection of amoebae by X-bacteria would initiate a change in the amoeba’s sams gene by a DNA-binding protein(s), blocking its

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Fig. 1. A schematic diagram to show possible interactions between an amoeba and X-bacteria, in which the symbionts block the transcription of amoeba’s sams gene and supply their own SAMS for the amoeba’s use (Adapted from Choi et al. 1997).

transcription. Then in the absence of amoeba’s SAMS, X-bacteria would supplement the genetic defect they caused by providing their hosts with an alternative source of SAMS. Thus, when X-bacteria are removed from xD amoebae, amoebae would be deficient in SAMS, and hence methylating reactions using AdoMet as the methyl group donor would stop. If such interference occurred in the processing of pre-rRNA methylation, the result would be an abnormal morphology of amoeba’s nucleoli as has been actually observed (Lorch & Jeon 1980). When X-bacteria are removed, xD amoebae exhibit irreversible nucleolar abnormalities and the findings suggest that X-bacteria may supply the needed SAMS enzyme. Thus, xD amoebae would not recover unless live X-bacteria are reintroduced. Otherwise, the damaged nucleus would have to be transplanted into normal xD cytoplasm for recovery. Work is in progress to elucidate the mechanism by examining the inhibition of gene expression in xD amoebae compared to that in D amoebae, by genomic footprinting and other approaches. By comparing genomic footprints of D and xD amoebae, it should be possible to detect a protected site(s) on or near the gene. The X-bacteria-produced S29x protein is a potential candidate to be such a DNA-binding regulatory protein. IV. Concluding Remarks The amoeba/X-bacteria symbiosis system is unique and novel, in which the host and symbionts became associated and developed a stable symbiosis within a few years under

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The new panorama of animal evolution

continuous observation. This symbiosis system is unique in that the history of the establishment of symbiosis is known, symbionts bring about the host’s dependence for survival within a short period of time, and cellular character changes caused by symbiosis can be reproduced ad libitum under laboratory conditions. The symbionts, X-bacteria, are different from other known bacterial inclusions in their large number per host cell, their morphology including the membranes surrounding them, and their relationship to the host nuclear genome. The system is well suited for studying both the process of symbiont integration into host cells and the consequences, that include the acquisition of new cell components and changes in cellular phenotypic characters arising from the production of new macromolecules by both the host and symbionts. Acknowledgments I thank Dr. Bruce D. McKee for his critical reading of the manuscript. The research work in my own laboratory was supported by grants from the US National Science Foundation. References AHN G.S., CHOI E.Y. & K.W. JEON 1990. A symbiosome-membrane-specific protein in symbiontbearing Amoeba proteus as studied with a monoclonal antibody. Endocyt. Cell Res. 7: 45-50. AHN T.I., LIM S.T., LEEU H.K. & K.W. JEON 1994. A novel strong promoter of the groEx operon of symbiotic bacteria in Amoeba proteus. Gene 128: 43-49. CHOI E.Y. & K.W. JEON 1992. Bacterial-endosymbiont-derived lipopolysaccharides on amoeba symbiosome membranes. J. Protozool. 39: 205-210. CHOI J.Y., LEE T.W., JEON K.W. & T.I. AHN 1997. Evidence for symbiont-induced alteration of a host’s gene expression: Irreversible loss of SAM synthetase from Amoeba proteus. J. Eukaryot. Microbiol. 44: 412-419. HAN J.H. & K.W. JEON 1980. Isolation and partial characterization of two plasmid DNAs from endosymbiotic bacteria in Amoeba proteus. J. Bacteriol. 141: 1466-1469. HARB O.S. & Y. ABU KWAIK 2000. Characterization of a macrophage-specific infectivity locus (milA) of Legionella pneumophila. Infect. Immun. 68: 368-376. HOFFMAN P.S. & R.A. GARDUNO 1999. Surface-assocated heat shock proteins of Legionella pneumophila and Helicobacter pylori: roles in pathogenesis and immunity. Infect. Dis. Obster. Gynecol. 7: 58-63. ISHIKAWA H. 1984. Characterization of the protein species synthesized in vivo and in vitro by an aphid endosymbiont. Insect Biochem. 14: 417-425. JEON K.W. & T.I. AHN 1978. Temperature sensitivity: A cell character determined by obligate endosymbionts in amoebas. Science 202: 635-637. JEON K.W. & M.S. JEON 1976. Endosymbiosis in amoebae: Recently established endosymbionts have become required cytoplasmic components. J. Cellular Physiol. 89: 337-347. JEON K.W. & I.J. LORCH 1967. Unusual intra-cellular bacterial infection in large, free-living amoebae. Exp. Cell Res. 48: 236-240. KIM K.J. & K.W. JEON 1995. A novel protein involved in the amoeba-bacteria symbiosis. Molec. Biol. Cell Suppl. 6: 108a.

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KIM K.J., NA Y.E. & K.W. JEON 1994. Bacterial endosymbiont-derived lipopolysaccharides and a protein on symbiosome membranes in newly infected amoebae and their roles in lysosomesymbiosome fusion. Infect. Immun. 62: 65-71. LEE J.E., LIM S.T. & T.I. AHN 1996. Characterization of promoter sequences for strong expression of groEx in Escherichia coli. J. Microbiol. 34: 15-22. LORCH I.J. & K.W. JEON 1980. Resuscitation of amoebae deprived of essential symbiotes: Micrurgical studies. J. Protozool. 27: 423-426. MASUZAWA T., SAWAKI K., NAGAOKA H., AKIYAMA M., HIRAI K. & Y. YANAGIHARA 1997. Relationship between pathogenicity of Coxiella burnetii isolates and gene sequences of the macrophage infectivity potentiator (Cbmip) and secsor-like protein (qrsA). FEBS Micriol. Lett. 154: 201-205. MERALI S., VARGAS D., FRANKLIN M. & A.B. CLARKSON Jr. 2000. S-Adenosylmethionine and Pneumocystis carinii. J. Biol. Chem. 275: 14958-14963. NEWMAN E.B., BUDMAN L.I., CHAN E.C., GREENE R.C., LIN R.T., WORDRINGH C.L., & R. D’ARI 2000. Lack of S-adenosylmethionine results in a cell division defect in Escherichia coli. J. Bacteriol. 180: 3614-3619. OH S.W. & K.W. JEON 1999. The Mip protein and its gene of endosymbionts in Amoeba proteus. Endocyt. Cell Res. 13: 87-104. PAK J.W. & K.W. JEON 1996. The s29x gene of symbiotic bacteria in Amoeba proteus with a novel promoter. Gene 171: 89-93. PAK J.W. & K.W. JEON 1997. A symbiont-produced protein and bacterial symbiosis in Amoeba proteus. J. Eukaryot. Microbiol. 44: 614-619. RADWANSKA M., MAGEZ A., STIJLEMANS B., GEUSKENS M. & E. PAYS 2000. Comparative analysis of antibody responses against HSP60, invariant surface glycoprotein 70, and variant surface glycoprotein reveals a complex antigen-specific pattern of immunoglobulin insotype switching during infection by Trypanosoma brucei. Infect. Immun. 68: 848-860. RAHFELD J.U., RUCKNAGEL K.P., STOLLER G., HORNE S.M., SHIERHORN A., YOUNG K.D. & G. FISHER 1996. Isolation and amino acid sequence of a new 22-kDa FKBP-like peptidylprolyl cis/trans isomerase of Escherichia coli. J. Biol. Chem. 271: 22130-22138. RAZIN A. & A.D. RIGGS 1980. DNA methylation and gene function. Science 210: 604-610. ROBERTS C.J. & E.U. SELKER 1995. Mutations affecting the biosynthesis of S-adenosylmetionine cause reduction of DNA methylation in Neurospora crassa. Nuc. Acids Res. 23: 4818-4826. SAGER R.C., GRABOWY C. & H. SAND 1981. The MAT-1 gene in Chlamydomonas regulates DNA methylation during gametogenesis. Cell 24: 41-47. SESHU J., MCIVOR K.L. & L.P. MALLAVIA 1997. Antibodies are generated during infection to Coxiella burnetii macrophage infectivity potentiator protein (Cb-Mip). Microbiol. Immunol. 41: 371-376. VARMA A., FREESE E.B. & E. FREESE 1985. Partial deprivation of GTP initiates meiosis and sporulation in Saccharomyces cerevisiae. Mol. Gen. Genet. 201: 1-6.

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Diversification and Evolutionary Ecology

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Disclosure of songbird diversity in the Palearctic/Oriental transition 551 The New Panorama zone of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 551-558, 2003

Disclosure of songbird diversity in the Palearctic/Oriental transition zone J. Martens & M. Päckert Institut für Zoologie, Johannes Gutenberg-Universität Mainz, Saarstr. 21, D-55099 Mainz, Germany. E-mail: [email protected]

Abstract Songbird diversity of temperate and tropical SE Asia seems to be considerably richer than previously believed. Apart from species concepts used, underestimation of song diversity results from discovery of hitherto unknown species and more precise judgement on morphological, acoustical and molecular genetic characters of i) populations of highly disjunct area parts of large transcontinental distributions; ii) small peripheral disjunct populations; iii) sympatric, even syntopic hitherto misplaced populations. Examples of the three categories seem to be quite common. Under both species concepts now widely used in ornithology, extant species numbers and local diversity are underestimated. Examples are presented for the genera Regulus (crests), Certhia (treecreepers) and Seicercus (golden-spectacled warblers).

Introduction Songbird taxonomy and evolutionary history at the species and genus levels have mainly been based on traditional characters, in particular external morphology. More recently, characters specified by bioacoustics, molecular genetics and behaviour in contact zones have become increasingly important in revealing the complexity of taxa, but these modern techniques have rarely been applied to bird faunas of temperate and tropical/ subtropical Asia (Stattersfield et al. 1998: 76). Now a number of astonishing findings have been made, several of which will concern us here. In this context, the question of species concepts is crucial. The number of species in a classification depends on the concept applied, especially in the field of ornithology, where problems on “species” have always been playing a major role (Mayr 1993, Eck 1996, Mayden 1997, Zink 1997, Zink & McKitrick 1997). Presently, the diversity of viewpoints and practices are already hampering everyday taxonomy. Here, we shall adhere to Mayr’s concept of biospecies (BSC), otherwise known as the isolation concept of species. However, we aim to demonstrate the systematic position of certain populations

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The new panorama of animal evolution

within the framework of other interpretations - primarily the phylogenetic species concept (PSC). We shall also demonstrate how arbitrary taxonomic decisions under both concepts may be. General remarks Acoustics On a small scale, vocalizations of songbirds tend to vary within local populations. They form dialects. Dialects have little or no significance with respect to speciation processes and several dialects may occur in the same population. Regiolects, however, encompass large continental populations. Regiolect differences may be considerable, beyond the level of dialects, and communication between regiolect carriers is reduced or even impossible in the extreme case. Regiolects developed over long periods of time, and their carrier populations are old. To alter regiolects special mechanisms and population structures are required (Martens 1996). Regiolects have a strong evolutionary impact. Molecular genetics Molecular genetics has provided powerful tools to demonstrate long-lasting independent development of allopatric populations. In songbirds, a sequence divergence of 2-3% of the cytochrome b gene is considered efficient to distinguish species (Helbig et al. 1995). This value is corroborated by closely related, often sympatric species which do not interbreed, hence the BSC applies. Consequently, this percentage value is often used as the main, often the only proof of species identity under the PSC, and, incorrectly, even under the BSC. When applied to East Asian bird faunas, in an area where Palearctic and Oriental regions meet, the concept of regiolects and the results of molecular biology, though rarely employed so far, have yielded surprising results. The Goldcrests (Regulus regulus) This is a genus of holarctic distribution which comprises only five species. Two occur sympatrically in W Europe and two others in N America, and there is a relict population of R. goodfellowi in Taiwan. To focus on the widespread Goldcrest (Regulus regulus) alone, its distribution is transcontinental from the Azores and Canary islands to Japan, with large gaps in between (Fig. 1). Morphological characters of the various goldcrest subspecies are not conspicuous, and they give little evidence of evolutionary pathways (Vaurie 1959, Cramp & Brooks 1992). Most remarkably, territorial songs of R. regulus vary considerably between the large allopatric areas to form distinct regiolects. In field tests, alien regiolect songs are not well “understood” because response-eliciting parameters differ between them (Martens & al. 1998). This may cause difficulties when birds - in potential sympatry areas - start to form breeding pairs.

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Fig. 1. Distributional area of the Goldcrest (Regulus regulus) (from Martens et al. 1998). Percentage values indicate cytochrome b gene haplotype divergence (pairwise differences, uncorrected, 500 bp) between various R. regulus subspecies as indicated by arrows. E.g., differences between Azorean ssp. azoricus, sanctaemariae and inermis (areas extralimital) and mainland ssp. regulus is between 0.6 and 0.8 %, difference between ssp. teneriffae and the latter 2.2%.

A syntactical development of goldcrest regiolect songtypes across the Palearctic is evident. Apparently basic are songs, which are represented by subspecies in central Asia and China. Originating from these, two developmental pathways can be traced. One led to E Siberian and Japanese populations and is traceable even in N America in the song of a close ally, the Golden-crowned kinglet (R. satrapa). The other cline runs to the W Palearctic. Thus, acoustics can draw a pretty good picture of the possible evolutionary history of Palearctic goldcrests and shed some light on Nearctic populations, too (Martens et al. 1998). Is this view supported by molecular genetics? Genetic distance values for the cytochrome b gene between nine subspecies of the goldcrest are high, ranging from 0.6 to 6.2% (Päckert & al. in press). They are highest between E Asian populations and the widespread western nominate R. regulus. They are lowest between the Atlantic island populations and the European mainland (Fig. 1). According to the BSC, all the allopatric subspecies are to be regarded as one species only, being morphologically highly similar and representing each other geographically. Their potential inbreeding, however, cannot be proven due to lack of contact zones. Partisans of the PSC will certainly raise several of these populations to species rank because of their marked genetical differences. But to adopt a certain level of differentiation as defining so called “species” causes difficulties. Differences of about 2.2% distance as found between Canarian and European mainland populations of the Goldcrest are believed to be on the borderline between the status of being specifically distinct or not. Azorean populations, also on Atlantic Islands, differ even less, but are diagnosable, even according to song. Differences between West and East Palearctic subspecies are greater, exceeding 5 % and clearly fit the criterion of good diagnosability. So, where to draw correct species limits along this continuous line of increasing distance values?

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The new panorama of animal evolution

This case clearly indicates that the BSC is advantageous in putting together populations that are evidently closely related. No new insight is gained by taking out single populations and declaring them “species”. A transcontinental distributional pattern like that of the goldcrest is atomized into several independent species, but the uniting perspective for this evolutionary unit is lost. However, we cannot deny strong interpopulation differences of Goldcrests (Table 1), which may indicate species limits even under the BSC. The Brown-throated treecreeper (Certhia discolor) The treecreepers are a family with only six presently recognized species. Their center of diversity is in the Himalayan region, where four species occur, mostly separated by altitude and, correspondingly, by habitats (Martens 1981). Due to their bark-living cryptic habits, they are difficult to tell apart. A population of the SE Asian C. discolor was only recently discovered in Sichuan, W China, far from the population in the tropical parts of the Himalayas, Burma and Thailand. This very localized subspecies, C. d. tianquanensis, represents a high-altitude population which is remarkably distinct with respect to contour feather proportions, coloration, vocalizations and genetics. The genetic distance from nominate discolor of the Himalayas is as high as 8.8%. C. tianquanensis song differs by its extremely high frequency and the unique turn from ascending to descending notes within a single strophe (Martens et al., 2002). Moreover, the distributional area of tianquanensis is situated at high altitudes in W/C China, where other Chinese bird endemics also occur. These have close relatives in the northern Palearctic such as the Chinese hazelgrouse (Bonasa [bonasia] sewerzovi), David’s Woodowl (Strix uralensis davidi) and the Sichuan Jay (Perisoreus [infaustus] internigrans). Unlike these examples, populations closely related to tianquanensis clearly live under tropical/subtropical conditions. Thus, tianquanensis may well have reached species level - according to both species concepts under consideration. Allopatry obscures the correct view according to the BSC. The Golden-spectacled warblers (Seicercus burkii complex) A common, widespread species along the entire Himalayan chain, China, Burma and Vietnam, the Golden-spectacled warbler turned out to be a swarm of sibling species. Most exciting is the fact that several siblings live sympatrically, but normally are separated vertically (Alström & Olsson 1999, Martens et al. 1999). Rarely, up to three species have been found living together in the same forest patch during the breeding period (Martens et al., in press), but normally only in contact zones. Why do we have to consider the Golden-spectacled Warbler a swarm of species rather than subspecies? a) There are morphological differences, but these are slight and detectable only by thorough comparison of captured individuals. To compare, e.g., the shape of the yellow eye-ring one even needs to look at living birds from close up (Martens & Eck 2000). As slight as the differences may be, they are constant over large areas and show only little variation. Morphology alone, however, hardly allows species limits to be identified.

acoustics x x x x x x x x x x x x x x x

morphology x x x (x) x x x x x x x x x x

M irafra assamica

A nthus novaeseelandiae

Phylloscopus proregulus

P. tenellipes

P. inornatus

P. collybita

Seicercus burkii

Bradypterus thoracicus

Regulus regulus

Parus caeruleus

P. cyanus/P. flavipectus

P. ater/P. melanolophus

P. rubidiventris

P. montanus

Sitta frontalis

Certhia discolor

Remiz pendulinus

Corvus macrorhynchos

species taxa

x

-

-

x

-

x

x

x

-

-

x

x

x

x

-

x

x

x

distribution

-

-

x

-

-

-

x

x

x

-

x

x

-

-

-

-

-

genetics

Martens et al. 2000

H arrap & Qu inn 1996

Martens et al. (2002)

H arrap & Qu inn 1996

Ku zjm ina 1972

Martens 1971

Martens & Eck 1995

Salzbu rger et al. (2002)

Salzbu rger et al. (2002)

Päckert et al. (in p ress)

Rou nd & Loskot 1995

Martens et al.1999

H elbig et al. 1996

Svensson 1992

Martens 1988

Alström & Olsson 1989

Glutz v. Blotzheim & Bau er 1995

Alström 1998

authors

3 - 4 (BSC, PSC)

4 (PSC)

2 (BSC, PSC)

3 (PSC)

2 (PSC)

2 (BSC, PSC)

1 (BSC)

1 (BSC)

2 (BSC, PSC)

3 - 5 (PSC)

2 (PSC)

6 - 8 (BSC, PSC)

4 - 5 (BSC, PSC)

2 (PSC)

2 (BSC, PSC)

3 (BSC, PSC)

5 (BSC, PSC)

3 - 4 (PSC)

numbers of species likely to be accepted

Table 1. Selected bird species with area parts in or close to the Oriental (Indomalayan) region which were tentatively split into several species or lumped into one species on grounds of morphology, vocalizations, distribution [parapatry, sympatry] and molecular genetics. “BSC” (Biological species concept) and “PSC” (Phylogenetic species concept) indicate the validity of the newly erected species under both species concept.

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b) Vocalisations, calls and song, differ considerably between the species, and fall into several categories. Because these normally cannot be heard at the same spot, the differences were not noticed until recently. c) The mitochondrial genome, mainly the cyt-b gene, shows a high degree of differentiation, with distances ranging between 5 and 8 %. They clearly indicate species status of the carrier populations under the PSC and the BSC (sympatry proven and absence of gene flow!). The large genetic distances indicate that the individual species are not young, but are up to about 4 million years old. As is generally understood, the molecular tree also contains phylogenetic information. When compared with the acoustic findings, we can clearly see congruence: On the same branch are situated populations that display the same syntax form of song, i.e. elementgroup song versus the trill type of song (details in Martens et al. 1999, Martens & Eck 2000). Thus, the song structure and genetic data point in the same direction with respect to relationship and age of the forms concerned. It has also become clear that at least the syntax characters of song have remained constant over long periods of time. Indeed, the evolutionary history of this species swarm must be long and complex. We have to assume that there were multiple refuge events even during early glacial periods, which separated many populations and gave rise to independent developments. There must also have been multiple postglacial immigration events, which resulted in the present complex pattern of partly allopatric, partly sympatric distribution. The ecological requirements of the present species are partly still very similar and do not allow large-scale syntopy. But syntopy occurs along sharp lines of secondary contact, and local occurrence of two and even three species on the breeding grounds without interbreeding has been confirmed. Discussion The implications of the above examples are twofold: - Subtropical/temperate Asian avifaunas are considerably more complex than formerly believed. The state of knowledge is still rather basic and, as a consequence, in many cases species limits are not yet correctly documented. This holds especially true for species with large areas and allegedly wide-band ecological requirements including broad altitudinal belts. To a certain extent, especially where allopatric populations are concerned, the way species limits are to be drawn depends on the species concept applied. But even under the broader BSC the inventory of songbirds in the temperate/tropical SE Asian transitional zone is far from being complete. Table 1 lists cases of species which recently had to be split into several species. - The more thorough and detailed investigations on populations are carried out, the more apparent clearly diagnosable and biologically relevant characters (contour feather ratios, voice, ecological needs, genetic markers) become evident. This causes problems of definition for both species concepts: i) In the BSC, it becomes evident how distinct peripheral populations already are with respect to vocalizations and genetics. Morphology does not necessarily reflect such highly advanced states. Consequently, even under the BSC peripheral populations will

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be raised to species level - despite allopatry and lack of zones of secondary contact like in Certhia discolor/C. tianquanensis. The treatment of assemblages of highly diverse populations of geographic representatives like in R. regulus remains arbitrary. ii) In the PSC, because high-resolution analysis of populations is now possible with a resulting enhanced diagnosability of single populations, the number of “species” is steadily increasing (Eck 2000). They are difficult to survey in terms of relationships and evolutionary history, and the consequence is a troublesome instability of everyday taxonomy and nomenclature. Acknowledgments Extended field trips to Asia were made possible by grants of German Academic Exchange Service (DAAD), German Research Society (DFG), Feldbausch Foundation at Fachbereich Biologie of Mainz University, Forschungskommission of Deutsche Ornithologen-Gesellschaft and Gesellschaft für Tropenornithologie. Logistic support was provided by former Soviet Academy of Sciences, Moscow and several outlying branches thereof and Chinese Academy of Sciences, Beijing. To all institutions and related individuals we express our sincere thanks. This paper is part of the series Results of the Himalaya Expeditions of J. Martens, No. 240. - For No. 239 see: Bull. Amer. Mus. Nat. Hist. 2002. References ALSTRÖM P. 1998. Taxonomy of the Mirafra assamica complex. Forktail 13: 97-107 ALSTRÖM P. & U. OLSSON 1990. Taxonomy of the Phylloscopus proregulus Complex. Bull. B. O. C. 110: 38-43. ALSTRÖM P. & U. OLSSON 1999. The Golden-spectacled Warbler: a complex of sibling species, including a previously undescribed species. Ibis 141: 545-568. CRAMP C. & D.J. BROOKS (eds) 1992. Handbook of the Birds of Europe, the Middle East and North Africa. Vol. 6. Oxford University Press, Oxford. ECK S. 1996. Die paläarktischen Vögel - Geospezies und Biospezies. Zool. Abh. Staatl. Mus. Tierkde. Dresden 49 (Suppl.), 103p. ECK S. 2000. Die neuen Vogelarten der Paläarktis. Zool. Abh. Staatl. Mus. Tierkde. Dresden 51: 105-118. GLUTZ v. BLOTZHEIM U.N. & K.M. BAUER 1995. Handbuch der Vögel Mitteleuropas. Bd 10/II, Passeriformes (1.Teil). Aula, Wiesbaden. HELBIG A. J., MARTENS J., HENNING F., SCHOTTLER B., SEIBOLD I. & M. WINK 1996. Phylogeny and species limits in the Palaearctic Chiffchaff Phylloscopus collybita complex: mitochondrial genetic differentiation and bioacoustic evidence. Ibis 138: 650-666. HELBIG A.J., SEIBOLD I., MARTENS J. & M. WINK 1995. Genetic differentiation and phylogenetic relationships of Bonelli’s Warbler Phyloscopus bonelli and Green Warbler P. nitidus. J. Avian Biol. 26: 139-153. HARRAP S. & D. QUINN 1996. Tits, nuthatches & treecreepers. Christopher Helm, London. KUZJMINA M.A. 1972. Paridae. In Korelew M.N. & A.F. Kowshar (eds), The Birds of Kazakhstan, vol. IV. Alma-Ata, pp. 264-311 (in Russian). MARTENS J. 1971. Artstatus von Parus rufonuchalis Blyth. J. Ornith. 112: 451-458.

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MARTENS J. 1981. Lautäußerungen der Baumläufer des Himalaya und zur akustischen Evolution in der Gattung Certhia. Behaviour 77: 287-318. MARTENS J. 1988. Phyllocopus borealoides Portenko - ein verkannter Laubsänger der Ost-Paläarktis. J. Ornith. 129: 343-351. MARTENS J. 1996: Vocalizations and speciation in Palearctic birds. In Kroodsma D.E. & E.H. Miller (eds), Ecology and Evolution of Acoustic Communication in Birds. Cornell, Ithaca, London, pp. 221-240. MARTENS J., BÖHNER J. & K. HAMMERSCHMIDT 2000. Calls of the Jungle Crow (Corvus macrorhynchos s. l.) as a taxonomic character. J. Ornith. 141: 275-284. MARTENS J. & S. ECK 1995. Towards an ornithology of the Himalayas. Systematics, ecology and vocalizations of Nepal birds. Bonner Zool. Monogr. 38, 445p. MARTENS J. & S. ECK 2000. Der Seicercus burkii-Komplex im Himalaya und China oder: Schätzen wir die Diversität der Singvögel falsch ein? Ornithol. Anz. 39: 1-14. MARTENS J., ECK S., PÄCKERT M. & Y.-H. SUN 1999. The Golden-spectacled Warbler Seicercus burkii - a species swarm (Aves: Passeriformes: Sylviidae) Part 1. Zool. Abh. Staatl. Mus. Tierkde Dresden 50: 281-327. MARTENS J., ECK S., PÄCKERT M. & Y.-H. SUN 2002. Certhia tianquanensis Li, a treecreeper with relict distribution in Sichuan, China. J. Orinith. 143: 440-456. MARTENS J., ECK S., PÄCKERT M. & Y.-H. SUN (in press): Methods of systematic and taxonomic research on passerine birds: the timely example of the Seicercus burtii complex (Sylviidae). Part 2. Bonner Zool. Beitr. MARTENS J., PÄCKERT M., NAZARENKO A.A., VALCHUK O. & N. KAWAJI 1998. Comparative bioacoustics of territorial song in the Goldcrest (Regulus regulus) and its implications for the intrageneric phylogeny of the genus Regulus (Aves: Passeriformes: Regulidae). Zool. Abh. Staatl. Mus. Tierkde. Dresden 50: 99-128. MAYDEN R.L. 1997. A hierarchy of species concepts: the denouement in the saga of the species problem. In: Claridge M.F., Dawah H.A. & M.R. Wolson (eds), Species. The Units of Biodivertsity. Chapman & Hall, London, pp. 381-424. MAYR E. 1993. Fifty years of progress of research on species and speciation. Proc. Calif. Acad. Sci. 48: 131-140. PÄCKERT M., MARTENS J., KOSUCH J., NAZARENKO A.A. & M. VEITH (in press). Phylogenetic signal in the songs of Crests and Kinglets (Aves: Regulus). Evolution. ROUND P. D. & V. LOSKOT 1995. A reappraisal of the taxonomy of the Spotted Bush-Warbler Bradypterus thoracicus. Forktail 10: 159-172. SALZBURGER W., MARTENS J. & C. STURMBAUER 2002. Molecular phylogeny of the Blue Tit (Parus caeruleus) and Azure Tit (Parus cyanus) assemblage inferred from cytochrome b sequences. Mol. Phyl. Evol. STATTERFIELD A. J., CROSBY M.J., LONG, A.J. & D.C. WEGE 1998. Endemic bird areas of the world. BirdLife Conservation series No. 7. Birdlife International, Cambridge. SVENSSON L. 1992. Identification Guide for European Passerines. 4th ed. Stockholm. VAURIE C. 1959. The Birds of the Palearctic Fauna. A systematic reference. Passeriformes. Witherby, London. ZINK R.M. 1997. Species concepts. Bull. B.O.C. 117: 97-109. ZINK R.M. & M.C. McKITRICK 1997. The debate over species concepts and its implications for ornithology. Auk 112: 701-719.

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Zoological Education in New Zealand: a 21st The Century perspective 561 New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 561-567, 2003

Zoological Education in New Zealand: a 21st Century perspective J. St J. S. Buckeridge Earth & Oceanic Sciences Research Centre, Auckland University of Technology, P.O. Box 92006, Auckland, New Zealand. E-mail: [email protected]

Abstract Reductionist thinking has, until recently, pervaded much of Western scientific culture. Although this necessary cognitive phase led to an agreeably sophisticated understanding of the nature of our world, it is also perceived to have resulted in a degradation of the biosphere. In response, a need to adopt a systems (or holistic) approach to natural resources management arose in the mid 20th century, and this thinking was particularly prevalent in zoology. As a consequence, many zoology curricula abandoned traditional zoological foundations, particularly systematic taxonomy, anatomy and functional morphology. A new brand of biology, focussing on molecular biology, biochemistry and ecology, increasingly subsumed the traditional subjects. This paper examines the effects of these changes in a New Zealand context, where the vocational opportunities that currently exist for zoologists, and the qualities of recent graduates is briefly reviewed. In order to determine the likely profile of new “zoology” graduates, a pilot questionnaire was designed to test understanding of some basic zoological principals. The questionnaire had a deliberate bias toward systematic taxonomy - this in view of the local need for scientists with an understanding of systematics in areas such as biosecurity and assessment of environmental effects. Two groups, senior secondary school pupils, and third year university students were asked to complete the questionnaire. The results of the survey are disappointing as they show little improvement in the knowledge of systematic taxonomy in the two - three years of university education.

Introduction During the seventeenth century, the philosopher Descartes concluded that the only way in which phenomena (e.g. the environment) could be understood was through a reductionist approach, i.e. that only by breaking a problem down to its simplest elements could it be comprehended. This reductionism subsequently pervaded much of Western culture, in particular the education system, for in order to understand anything, it was necessary to disassemble it, either literally or figuratively (Buckeridge 1994). In the

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middle of the twentieth century, a paradigm shift began, partially fuelled by a perceived knowledge that technology (and business) was ravaging the biosphere. In biology, the response to this was to move away from detail, and to adopt a systems approach to science. System approaches are indeed appropriate in many spheres of scientific endeavor – in some, such as the assessments of environmental effects, they are essential. However, it appears that the educational curriculum pendulum is swinging wildly, for in the wild rush to embrace “systems thinking” we may have forgotten to lay good, sound foundations in science. Ample anecdotal evidence exists to suggest that current zoological education is not producing sufficient graduates of the “right kind”. In New Zealand during the middlelate twentieth century, there was a perceived need for marine ecologists, and the universities responded with enthusiasm, the catch phrase was for zoology to be “holistic” and environmentally sensitive. A consequence of this was to remove much of the comparative anatomy, functional morphology and systematics. The lack of new graduates with these skills is testament to the effectiveness of the new curricula. None-the-less, much of the understanding of the “systematic crisis” is anecdotal, and as such any deductions based upon this sit uncomfortably in the world of science. Real data were needed, and this paper summarises a pilot study to evaluate the depth of systematic taxonomy knowledge of both senior secondary school pupils and senior undergraduate students. In both cases, the students surveyed were “majors” in biological or palaeontological sciences. Current issues A somewhat simplistic measure of whether there is a shortage of biosystematists in a country may be determined through a “job market analysis”. Although employment opportunities can certainly be skewed by government initiatives, especially funding priorities, they none-the-less provide a crude indication of the state of a scientific discipline. As a consequence, consideration was given to the number of employment opportunities in systematic biology and/or palaeontology that had arisen over the last 12 months. In light of the results of this, it was hoped to determine whether there is a crisis, and if there is a crisis, if it really matters. Crisis or calm? A time of crisis in this sense is here defined as a period when there are significant vocational opportunities for systematic biologists/palaeontologists, and no, or few, replacements being produced in the university sector. In New Zealand, there are three areas where graduates in systematic biology could expect to find employment. (Note that private consultancies may employ biosystematists, but are more likely to seek ecologists):

Government research institutes, of which the National Institute of Water and Atmospheric Research (NIWA), Landcare, and the Institute of Geological and Nuclear Sciences (IGNS) probably provide the greatest opportunities. In the last

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six months, one position in marine taxonomy was advertised by NIWA. This attracted about 20 applicants, five of whom were considered to be “first class”, with well-established credentials in systematic zoology. Of the remainder, two were still completing doctorates. A further position, managing a marine taxonomy database, attracted almost 40 applicants, a significant number of whom were considered “weak”. Over the same period, an appointment in palaeontology was made by IGNS. However, the new scientist had previously served two post-doctoral posts, and was advised that the appointment was conditional upon the securing of external funding. Fortunately the applicant was successful.

Museums. Wellington’s Museum of New Zealand (Te Papa), is the largest in the country, but has not made any recent appointments in systematic biology. In the last two years, the Auckland Museum, one of the larger regional museums, has downsized its marine section, with the loss of two biosystematists. The Canterbury Museum has been proactive in encouraging university emeriti to join as adjuncts, and at this time is well endowed with taxonomic expertise at little or no cost.

Universities. In the last 12 months, there have been more than 100 redundancies in science faculties, particularly outside Auckland. This reflects reduced funding and lower enrolments in science generally, especially in biological and agricultural disciplines. Of the country’s larger universities, two, Auckland University of Technology and the University of Auckland have had no recent vacancies in biosystematics, and there is no likelihood of new positions in the short to medium term. Further, the University of Canterbury has appointed no biosystematists in either the Departments of Zoology or Plant and Microbial Sciences in the last 12 months.

Graduates in the pipeline There are now very few opportunities to study zoological taxonomy in New Zealand universities. (There are currently no post-graduate students in this field at the Auckland University of Technology, and only two, one in fish taxonomy, the other in invertebrate palaeontology, at the University of Auckland). Reasons for the lack of interest reflect concerns about employment, and poor levels of remuneration. However, the average age of the current cohort of systematic biologists and palaeontologists in New Zealand is now over 50. In the next decade, a significant number of these scientists will retire, and there are currently very few replacements in the university pipeline (Gordon 2000). A place for biosystematists The New Zealand Government has recently advised of a priority, through its Foundation for Research, Science and Technology, to maintain capabilities in biosystematics. In part this reflects a commitment to (international) obligations as part of the management of the current Economic Exclusion Zone. There are however, even more compelling reasons for this, and these include the potential to make informed

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decisions about conservation of taxa, to manage natural resources effectively, to maintain appropriate biosecurity at our borders, and to understand the origins of the region’s biota. In summary, a broad capability in biosystematics is crucial to the social and economic future of the nation. The Pilot Study: Evaluating the knowledge base The questionnaire (see appendix) was designed to provide a quick assessment of the level of current students’ knowledge in biosystematics. Two groups of students were surveyed. The first group, enrolled in secondary school biology classes, comprised 56 students, each with an average age of about 17.5. A second group of 51, with an average age of about 20, was made up of students who were enrolled in either a biology or palaeontology class at university level. The two secondary schools surveyed are considered to be “academic”. The universities were the two inner city Auckland campuses. The questionnaire was in “multiple-choice” format, with most questions designed to test concepts within systematic biology. Two questions (6 and 8) were included as a means of assessing basic biology and biological reasoning. The Results of the Questionnaire Overall (and perhaps fortunately for those of us in the university sector), the university students scored better than secondary school students (Fig.1). Of particular interest, were the areas where both groups scored poorly, e.g. question 2, which demonstrates a fundamental misunderstanding of how binomial nomenclature is applied. Of those in error, 32% of secondary school students, and 29% of university students believed that Hibolithes minor and Hipparionyx minor were the same species. The answers to Question 4 were also of concern, as 43% of secondary students, and 35% of university students believed that the Mammalia was an order. Perhaps students view the “authors of concepts” as less important than the concepts themselves, and this, with the high profile of Darwin, may account for the fact that 26% overall thought

100 80 60 40 20 0 1

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Fig. 1. Survey results. The X-axis gives the question number (see Appendix), the Y-axis the percentage of students who answered each question correctly. The white bar represents Secondary School students, the black bar, university students.

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that he had devised the system of binomial nomenclature. The only part in which university students scored both poorly, and comparatively badly, was Question 8, which asked about the characteristics of a single species that inhabited a range of environments. Of those that gave an answer, 13% believed that individuals in warmer climates would be larger. The result is a little surprising, as the correct answer involved analytical thinking. Finally, Question 10 illustrated that there is considerable confusion about “types”, as 36% of secondary students and 35% of university students believed that a holotype was a group of specimens. Analysis Neither secondary school nor university students scored particularly well in the questionnaire, confirming earlier perceptions about a lack of good biosystematic knowledge in young people electing to study in the biosciences. Graduates in the natural sciences clearly need good environmental stewardship skills, and these form the basis of many biological curricula. Although much of general zoology has become holistic, that is, “broader”, it has also become “shallower”. Graduates know a great deal about the environment, but without good systematic taxonomy, lack the skills to address 21st century crises such as the biodiversity drop and biosecurity enforcement. Current graduates are undoubtedly good fish physiologists, ecologists, biotechnologists and aquaculturalists. But they are certainly not being given exposure to comparative anatomy. Further, an incomplete knowledge of comparative anatomy and embryology will surely lead to more mistakes in studies such as cladistics where electronic manipulation of data is paramount. Conclusions The modern zoology student must learn taxonomic skills in the laboratory and field so that proficiency in systematics can be attained, whilst at the same time maintaining an appreciation of biological systems. Although the electronic resources that now complement the learning environment may mitigate this somewhat daunting task, the evidence is not being demonstrated at undergraduate level, where appreciation of systematic principles appears to be poor. Failure to address a devalued zoology curriculum can only have very negative effects in sub-disciplines such as Conservation and Biosecurity, the flow on effects being both social and economic. The need for change however, is not necessarily felt with the same passion by all of those who teach in the natural sciences. Further, it is clear that without the support of a wide spectrum of practitioners within the natural sciences, we will not succeed in changing the curriculum, i.e. we must convince the wider profession that a biosystematics crisis exists before we address the wider issues of inequity in funding. Finally, we must be ever vigilant of the pedagogic pendulum: the need for greater emphasis on biosystematics does not reduce a need for good molecular biologists.

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References BUCKERIDGE J.S. 1994. From Reductionist to Systems Thinking: The Engineering Imperative. AEESEAP Journal of Engineering Education 24(1-2): 49-51. GORDON D. P. 2000. 1000, 200, or 50 more years? Can we shorten the time to inventory biodiversity? Pacific World 57: 39-43.

Appendix Earth & Oceanic Science Research Centre Auckland University of Technology Multiple Choice Questions 1. Circle the genus name in the following: Conus geographicus Linnaeus, 1758. 2. Circle the letter before the name pair(s) below that represent the same species: a Felis concolor Felis catus b Hibolithes minor Hipparionyx minor c Belemnopsis aucklandica trechmanni Belemnopsis aucklandica aucklandica d Canis familiaris Canis lupus e Rattus rattus Balanus balanus 3. Lumbricus terrestris is: a an earthworm b a hardwood c a softwood d an isopod e a mollusc 4. Homo sapiens belongs to which order: a Hominidae b Primates c Vertebrata d Mammalia e Chordata 5. Which of the following is a prokaryote? a Tobacco Mosaic Virus

b c d e

Streptococcus a poriferan an annelid a reptile

6. Egg laying is not a characteristic of: a a platypus b a sparrow c a cockroach d a placental e a frog 7. The system of binomial nomenclature was devised by: a Aristotle b Darwin c Huxley d Wallace e Linnaeus 8. Within a single species that inhabits a range of environments: a the average size of individuals tends to be larger in warm climates. b protruding parts of animals (ears, tail) will be shorter in cold climates. c colours of individuals tend to be lighter in moist warm climates. d the internal body temperature of individuals will be lower in cold climates.

Zoological Education in New Zealand: a 21st Century perspective

e body fat will be less in individuals that inhabit cold climates 9. A species is a group of organisms that: a are anatomically very alike b have similar metabolisms c are actually or potentially interbreeding natural populations d are reproductively isolated e show no morphological similarity 10. A holotype is: a a group of specimens upon which a species-group taxon is based.

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b a single specimen from the location where the first individual of a particular species was found. c a single specimen upon which a species-group taxon is based. d a group of specimens chosen to represent a species when the original(s), upon which the species was founded, is lost. e the location at which the first individual of a species was discovered.

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A New Engine For a Holistic Zoology Education inNew thePanorama 21st Century 569 The of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 569-574, 2003

A New Engine For a Holistic Zoology Education in the 21st Century J. Azariah Department of Zoology, University of Madras, Chennai 600 020, India. E-mail: [email protected] Founder President, All India Bioethics Association, New No.4, 8th Lane, Indiranagar, Chennai 600 020. India.

Abstract A need to reshape zoology education in India has been felt for many years. Currently, few students opt to study the discipline of zoology in their undergraduate program. There is considerable diversity in Indian zoology curricula. Administrative regulation is seen as the only way in which to develop a meaningful zoology curriculum. Since the new century is likely to be the “age of biology” there is an immediate need to reshape biology education to meet the challenges that are raised by science and technology. In view of the rapid revolution in the field of information technology, it is necessary to integrate computer literacy with biological science literacy. Any such attempt must also take job opportunities into consideration.

Introduction It is commonly said that India is a land of diversity in unity. But in the area of zoology education there is nothing but diversity, with no proper directive principles to provide a meaningful direction for students to progress. It is customary for Indian universities to update their curricula “with ill defined halfhearted attempts to incorporate modern branches of zoology” (UGC 1990). A national committee formulated by the University Grants Commission while screening the undergraduate (UG) zoology syllabi found “an unenvious scenario of tremendous diversity in course content pattern and scope. The spectrum of diversity (together with the generalized syndrome of persisting with classic zoology with emphasis on descriptive aspects and an overall apathy to incorporate modern areas of biological sciences) has resulted in creating an archaic aura. Apparently, students who go through such a course find the subject less dynamic and pedantic and the course as a whole, proves pitifully inadequate in providing conceptual information.” (UGC 1990). Therefore a generalized syllabus and a uniform pattern of examination were evolved for implementation by

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the universities in India but it was never implemented successfully. There were three main reasons, (i) Management/Universities, (ii) Teachers, (iii) students. A hierarchical model did not deliver the expected results because it did not take into account the need and aspirations of the upcoming student generation. In a model consisting of concentric rings, students occupy the central ring space followed by the teachers and the management/universities. The problem of diversity Diversity still persists. Some universities offer traditional subjects like botany and zoology while others offer a unified course of biological sciences. The latter may have subdivisions like animal science and plant science. Currently the theme of life sciences is being advocated. Such a diverse picture has become complicated by the introduction of new systems of curricular development in the autonomous colleges and the selffinancing colleges. There is both the British pattern of education (with annual examination) and the American pattern of education (with semester based credit system). Other variations such as semester with terminal examination without any credits may also coexist, in addition to variations in course duration and title. It could be a threeyear UG course or a five-year integrated course in Life Sciences leading to a masters degree (MSc). The problem is compounded with the number of core (main/optional) papers a student can take at the UG level, in some universities it is one major (zoology) and in others it is three (zoology, botany, chemistry). There is an inability to change the “continued emphasis on descriptive and classical zoology and a general apathy and reluctance to incorporate newer areas of biological sciences”. The current trend is to have a system of credit based semester system (CBSS). Only some universities have adopted this system of education. The British system of education is followed where teaching colleges are affiliated to the main universities; the latter may adopt the CBSS whereas the former may still continue with the annual or semester with terminal examinations, e.g. University of Madras. Is biology a subject like physics and chemistry? Physics and chemistry were the main players of the early 20th century. However, this new 21st century is called the “age of biology”. The title “biology” was used for the first time during the year 1802 by the German physician Gottfried Reihold Treviranus (Zeiss 1995). He titled his book as ‘Biologie oder Philosophie der lebenden Natur für Naturforscher und Ärzte’ (Biology or philosophy of the living Nature for Scientists and Physicians). It is significant to note that biology was equated with philosophy and nature was characterized as a living entity. The current thinking and teaching of zoology has deviated from these ideals. Cambridge University created the very first chair in zoology in 1866. The first Asian Zoological Research Laboratory was instituted at the University of Madras in 1927, which later became the Department of Zoology as it incorporated teaching with research. With its continued efforts in teaching and research, the Department of Zoology, University of Madras, was the only institution in India to adopt the semester pattern with credit system (UGC 1990). Such was the case till 1997. In this system a student requires 80 credits to qualify for the Masters degree. This pattern, which was distinguished as the Special Zoology, provided room to introduce almost all the modern subjects. The quality

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of students was par excellence. Students with a Masters degree in zoology were able to find suitable jobs and new affiliations were given by the University of Madras to start a new Masters program in zoology in aided colleges. In many colleges there were two batches of students in the undergraduate course. Current state However, the above trend started to decline with the advent of self-financed colleges (colleges who collect heavy fees and capitation fees - a specified amount of money to be given to run the college), which started only job-oriented courses like biochemistry, microbiology and environmental sciences. The critical point was reached during mid 1990s when no new affiliation to start any course in zoology was sought by any college. Therefore, in 1996-97 a need to revitalize the curriculum of zoology was determined during the University Leadership in College Science Improvement Program (COSIPULP). In this programme college professors were invited to the Zoology Department and were exposed to modem branches of zoology. The group felt that there is a lack of “qualitative levels of explanation that now can be reached through inter-disciplinary contact” in the Indian curriculum in zoology. The contributions by the Biological Science Curriculum Study (BSCS) in the USA, revolutionized the teaching of biology by developing a three-level biology textbook: the Yellow, Green and Blue versions. Using these books many Summer Institutes were conducted for college lecturers at the University of Madras from 1960 to 1969. These courses were held for a period of 45 days during the summer vacation with emphasis on experimental approach in biology. Further educational materials prepared by the Nuffield Science Foundation, UK were also used. These attempts in teacher education created sustaining interest in zoology teachers and contributed to capacity building for improvement of biology teaching in colleges. However, with the cessation of these vital activities, the commitment of zoology teachers to teach zoology with conviction stated to erode. Quality teachers in zoology are currently a “rare and endangered species”. This is one of the contributory factors for the decline of zoology and for the lack of interest among students to choose zoology for their career. It may be pointed out that the inflow of students in to B.Sc. zoology course is reducing, due either to the availability of other job-oriented courses or because the current zoology courses are not attracting the students. Students’ view - a career During earlier centuries knowledge was pursued for its own sake and there was some degree of job satisfaction for many students. In today’s India, it is said that about 60- 70% of jobs require a working knowledge of computers. Moreover, specialized subjects can provide only limited job opportunities. It is also highly doubtful whether most students can anticipate job satisfaction. Currently, students pay more attention to the job opportunities a science course can offer. After a creditable matriculation pass, a student is faced with two options: to study a professional course like medicine or take a bachelor’s degree program in science. In the latter, the first choice of a student is to seek admission into job oriented courses like

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microbiology and biochemistry, failing which a student may opt for a zoology course. In some colleges, zoology courses are tagged with vocational subjects like Industrial Fish and Fisheries, Industrial Microbiology, Sericulture, Preparation of Museum Specimens, Vermi-culture and composting, Biological Specimens and Slide Preparation, Laboratory Instrumentation and Biotechnology. These courses may offer opportunities for self-employment. Another variation is that of the medium of instruction. Generally a student prefers an English medium course, while others opt for the course in the vernacular (Tamil) and for this stream of students there is no postgraduate (PG) course in zoology in Tamil. English is the medium of instruction in all PG courses. What are the most promising directions zoology major can take after graduation? After securing a BSc degree in zoology a candidate is faced with some hard choices. It is difficult to get a job with just a bachelor’s degree in zoology. Additional qualifications in obtaining a diploma in Medical Laboratory Technology or a masters degree in business management or a Corporate Secretarianship may increase the job opportunities. After a masters degree in zoology, the prospects of getting a job are still diminished. With an additional degree in education one may get a job as a teacher in matriculation schools where zoology/botany is taught. The recent financial downturn has led to unfilled teaching positions; further, governmental regulations for a lecturer’s jobs in colleges hamper job prospects of a zoology graduate. These regulations are that an aspirant, even a PhD degree holder, must pass special tests like State Level Educational Testing (SLET) and the national level National Eligibility Test (NET) conducted by the UGC. Clearing these tests also makes a student eligible for getting financial assistance in doing research leading to the doctoral degree. Curriculum development It is not easy, therefore, to provide a new image of zoology in an Indian context. This is because job opportunities are poor and the current curriculum is out of date. A student’s major reason for not choosing zoology is that of the frustration of not finding a job after graduation. Is this drive common to students in both the Western and Eastern countries? If so, should we develop a universal (global) zoology curriculum or should we develop a country specific curriculum? It is suggested that we create a global initiative for zoology teaching to facilitate the development of a suitable curriculum and to promote “mental and coping skills” in students to face the challenges of the 21st century. Part of the role of an academic is to modify existing courses or to create such courses that will generate job opportunities for the students. Advantage was taken of the (COSIPULP) run by the Department of Zoology, University of Madras, under the leadership of the author of this paper. A group of college professors was exposed to various modern frontiers of zoology, leading to a new awareness that the present undergraduate curriculum in zoology was lacking much of the new image of modern zoology. A dedicated and select group of college professors worked alongside the author of this paper and produced a new curriculum with a new face. The new interdisciplinary curriculum, while retaining the identity of the discipline, changed the engine of zoology to computer based and computer mediated zoology. While the team of curriculum changers exhibited a positive attitude, there were other managerial difficulties due to strict regulations that govern the bachelor’s course in general.

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The university has prescribed a certain upper limit for the total marks (1,800) for a three-year undergraduate program, so more papers could not be introduced. It was proposed to reduce the total marks of each question paper in any given course by 50% increasing the number of subjects without violating the required total. It was pointed out that this would increase teachers’ workload. As a compromise, two related subjects were offered under one course paper, sacrificing the depth of a subject against breadth. In order to accommodate new subjects in frontier areas in zoology and modernize the discipline, some classical subjects have to be dropped. The working group then faced the most difficult situation of “What subjects are to be dropped?” The guideline given by Schumacher (1975) in the chapter entitled “The Greatest Resource - Education” was followed whenever possible. The working group felt that the new curricular approach must be that of “Life Oriented Value Education (LOVE)” as suggested by Azariah (1991). The curriculum must ignite a love for nature and its biotic resources with special reference to peaceful coexistence with our fellow humans. In this context, subjects like bioethics are of enormous relevance. The new curriculum includes core subjects like “Mathematics, Statistics and Computation in Life Sciences” and other elective papers like “Computers for Biologists” and “Bioethics”. The finished curriculum has to be approved by the Academic Council and by the Senate of the University. There was a strong debate for introducing mathematics! Should a zoology student study mathematics which he/she originally “wanted to avoid” by taking a science subject? Finally, the new curriculum per se was approved and introduced. However, continued resistance led to removal of mathematics. Those zoology professors who taught classical zoology did not relish the time required to prepare fresh material for class teaching. The COSIP-ULP curriculum development group also prepared new masters programs in zoology, entitled “Masters in Computer Applications in Natural Science”. The future The 21st century is going to embrace a life style of “webcentrism”. There is a great future for a zoology (biology) student with computer education as there is an explosion of job opportunities in the field of Information Technology (IT) in both the national and international markets. The new image of zoology must include an integrative approach in biology education. There should be more on line courses in zoology. While changing to this new engine, any curriculum must include aspects like (1) inquiry (2) technology (3) problem based learning and (4) skill development to cope up with the new challenges of the 21st century. Although there is no easy solution to the current problem of change of interest in career development, it is always “ better late than never “. This is a critical period in the life of zoology as a curricular subject. It is proposed here that a new set of text books with computer based approaches be developed and to organize both Summer and Winter schools in modem zoology so as to enthuse both the teacher and the taught.

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References AZARIAH J. 1991. Philosophy of ecological degradation. In Ecology and Development. Chetti D.D. (ed.), Evangelical Lutheran Church of India. Gurukul Theological College and Research Institute. Chennai, pp. 65- 74. KERKUT G.A. 1960. Implications of Evolution. Pergamon Press. SCHUMACHER E.F. 1975. Small is Beautiful. Perennial Library. Part 2: 79-102. UGC 1990. Report of the Curriculum Development Center in Zoology. University Grants Commission, New Delhi, pp. 655. ZEISS F. 1995. Natuurlijke Historieen Geschiedenis von de Biologie van Aristoteles tot Darwin, Boom, Meppel.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

The Crisis In Teaching Of Zoology: The Experience 575 The Israeli New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 575-579, 2003

The Crisis In Teaching Of Zoology: The Israeli Experience F.D. Por Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. E-mail: [email protected]

Abstract The history, the shortcomings and the present crisis of zoological education in Israel, both at the academic and secondary level, are discussed in light of the local circumstances and the present international requirements.

Introduction The faunal inventory of Israel, the central core of the biogeographic landbridge of the Old World, does not contain many endemic species. However it represents a mixture of species of Palaearctic, Ethiopian, and even Oriental taxa living side-by-side. Along the coasts Atlantic Boreal and Indo Pacific tropical marine species are mixing (Por 1975, Tchernov & Yom-Tov 1988). To be a systematist in Israel requires a very comprehensive knowledge of the entire Old World fauna. Moreover, the dynamics of this faunal interpenetration are very active, especially under the present enhanced anthropic changes. Israeli Zoological Education: A History 1. The “Turtox” Period Israel is an “exotic” country with a newly immigrant population. Zoological teaching in Israel started tentatively by the end of the 1920’s with European teachers, (mainly from German). Knowledge and methodology were transplanted from abroad, with no time available to structure curricula based on local fauna. Identifications were made, very often wrongly, on the basis of existing European faunal monographs, laboratory praxis was based on imported specimens and preparations supplied by “Turtox” Inc. and even wall posters of anatomy of European animals were used. The fact that academic life started in full only in the late 1930’s is reflected in the curriculum, where experimental “modern” zoology is promoted at the expense of “classical” descriptive” zoology. Zoology in Israel skipped the venerable 19th century – the phase of basic descriptive regional stocktaking. Nonetheless, collecting activity was intensive and collections were sent abroad for identification. Understandably, the

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response was slow and very patchy, depending on the availability and the time of specialists abroad. Bodenheimer’s (1937) bold generalizations were based on shaky taxonomic ground. During this period, teaching of zoology at the Hebrew University, Israel’s first university, was structured like that in Germany: two full years of undergraduate courses in invertebrate and vertebrate zoology and a full year course of cytology-histology and of embryology. Comparative anatomy was taught as an advanced course. 2. The “Faunistics” Period During the 1960’s, a new trend became evident. New, Israel-born generations, started to explore the local fauna and developed a sentimental need to protect it. At this stage, the new Society for the Protection of Nature (a NGO in modern terms) and the Nature Reserves Authority started to function and to attract the enthusiasm and involvement of tens of thousands of young Israelis and naturalists. The Society opened a whole network of field-schools (see below). The “field” institutes Haifa, Tiberias (on the Sea of Galilee),the Desert Studies Institute in Sede Boqer and especially Eilat, on the Red Sea, were inaugurated. The developing academic curricula in Jerusalem and afterwards in Tel Aviv responded by launching a series of courses based on much fieldwork and on the study of the faunal diversity. These, with a slight derogatory touch, were christened “faunistic” courses. They included courses on bird, fish, insect, aquatic organisms, etc. These courses were developed partly at the expense of the cytology, embryology and even introductory freshman courses on invertebrates and vertebrates. The elementary and secondary grade studies of the state schools (there were and are few private schools) were shaped in this period around a unique compulsory curriculum, co-ordinated with that of the universities and using the facilities of the field schools. During this period, the zoological collections started to organize under the umbrella of the Academy of Sciences, although they remained within the frame of the Universities of Jerusalem and Tel Aviv. The “Fauna et Flora Palaestina” monographs started to be published by the Academy. It was also during this period that a number of zoologists at the Universities of Israel specialized in different animal taxa: the various vertebrate taxa, but also coelenterates, molluscs, worms, crustaceans, various orders of insects, arachnids and echinoderms. 3. The “Supermarket” Period During the late 80’s, this positive trend was reversed. No less than 6 universities with biology curricula functioned in a country of 5 million. Moreover, courses of experimental biology were duplicated at several faculties. For example Biochemistry, Genetics and Physiology were taught simultaneously at the Natural Sciences, Medical and Agronomy faculties of the Hebrew University. Since the budget and new positions did not increase proportionally, new faculty members could be added by cutting down on the positions of “ descriptive” zoology and botany. The same happened, even more drastically, in the curricula. Anatomy, cytology and even embryology disappeared from the teaching

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programmes and the basic zoological education was left within strange mongrel-courses called “animal biology” or “from the cell to the organism and the community”. In an easy and cheap way, these courses incorporated disjointed portions of lectures by available teachers. The result was, not surprisingly, a smorgasbord “supermarket” with fragments of information and a variety of approaches competing for the personal preference of each student, rather than a didactically constructed course. With the exception of Tel Aviv, departments of Zoology were subsequently dismantled or amalgamated. However, even in this new “interdisciplinary” form, many departments decreased in size; for example, the Department of Evolution, Systematics and Ecology at the Hebrew University went down from a staff of 12 zoologists and botanists in 1992, to an active staff of 5 in 2000. At the same time, the positions of the curators for the collections were cutback to dangerous levels. Concomitantly, less and less research money went the way of zoology. This is indeed a scenario, which is known with small variations from all over the world! Public pre-academic education, with basically unchanged curricula, received fewer zoology-trained teachers with field experience. The overwhelming majority of new biology teachers are now graduates in experimental fields, who failed to go into research and who are unable and/or unwilling to take their pupils into the field. While there is still the practice that each higher class makes a yearly week-long excursion to one of the field schools (where they are being guided and taught by local rangers) the quality of these rangers is also declining because of the decline of the university training they receive. There still persists the so-called “biotope project” for the biology classes, in which each student has to make a term project presenting the biodiversity in a certain environment. But without the teachers who are able to supervise these projects, the biotope projects are often “sub-contracted” to paid outside instructors. In complete contrast, the young generation is increasingly involved in hiking, diving, bird watching and nature protection in general. First graders at universities are disappointed by the fact that the universities do not satisfy their expectations for receiving field-related courses. The few remaining staff in field zoology and botany is often overburdened with thesis supervision. Further, the Israel Journal of Zoology is becoming more of a regional publication, with Greek and Turkish research papers often outnumbering the Israeli ones. The Israel Society of Zoology still holds annual meetings, but the contributions presented there are chiefly student projects and field observations. Virtual versus Hands-on Zoology There is a problematic contradiction between the need to protect animal species and the moral need to avoid suffering of animals and hands-on zoology. In Israel, nature protection rules are very severely enforced. This is all too natural in this small country, which furthermore is characterized by the intricate co-existence of biomes of different biogeographic realms. This is exemplified by the need to protect the 2 km long fringing coral reef of the Gulf of Aqaba in Eilat, the care for the palearctic freshwater fauna of the Jordan headwaters and the Ethiopian fauna of the Dead Sea oases. Permits to collect for research purposes are very difficult to obtain. To allow collecting or field experimenting

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for didactic purposes is nearly impossible. Bureaucracy and faulty education of the nature rangers is often an insurmountable obstacle. Animal rights considerations are limiting the praxis of animal dissections and this moral qualm often includes insect “impaling” and formalin provoked “genocide” of a zooplankton sample. Nature films and virtual video-dissections are only a limited solace to the lack of zoological praxis. But efforts should be made in order to explain that the practical knowledge of the animal species, by collecting, rearing, dissecting and experimenting on them, is a prerequisite to their efficient protection in nature. In a similar way, dissection of human cadavers is a necessary, if unpleasant, part of the education of every physician. Resisting Evolution Denial The anti-evolutionist crusade of Judeo-Christian fundamentalism has understandably its own local brand in Israel, perhaps supported by the stories of the Old Testament, which contain a whole bestiary of the local fauna. One of the important ways to fight off creationism is the study of the waxing and waning of this fauna in prehistoric and historic times. Teaching of embryology, comparative anatomy, ethology and palaeontology is important in order to supply the young generation with sufficient background to support the evolutionist arguments. Since these disciplines have by and large disappeared from the curricula in Israel, one cannot expect that only the dinosaur stories on television will convince. Local creationists, perhaps different from their peers abroad are especially active in the denial of zoological evolution and are more ready to accept as non-heretical the transformism in the bacterial and vegetal world. Zoological education therefore gains an added importance. While Darwinism is still uniquely reigning in the curricula of the public school system, “Creation Science” has freely penetrated the religious school systems, whether public or private. The Biodiversity Crisis in Israel Israel is today one of the leading countries in biotechnology but has less than a dozen creative zoological systematists. The worldwide awareness of the crisis of biodiversity has caught us completely unawares. The causes can be seen in a lack of tradition in natural sciences on one hand and in an ebullient echoing of the scientific trends in the West on the other. The basic duty of knowing and presenting the treasures of the diversity of its own national territory has not been fulfilled. Like a metallic giant on feet of clay, we are competing for scientific primacy in many biological fields with the industrialized world, but our biodiversity is less known than in many Third World countries. Under the impulse of the Convention on Biodiversity (CBD), of which it is a signatory, Israel has to rethink and restructure the zoological education in the country. Since universities are mainly budgeted from public money, a co-ordinated effort should be possible. Unlike the unfettered proliferation of branches of biological teaching at the universities, a national blueprint plan should be embraced. This should support and stimulate already existing inter-university projects like the GIS project (Kadmon 2000),

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the National Collections framework (Ferber 1985) and the inter-university laboratory in Eilat. It must also designate national priorities in the teaching and research at the different universities. Instead of having moribund zoology dispersed in several universities, positions should be earmarked and created for a single, central national powerhouse of zoological teaching and research. The same should be done with botany, palaeontology and descriptive geology. With a comprehensive taxonomical training, the quality and the prognosis value of many environmental impact statements will improve. This is necessary, since Israel is both a central faunal corridor of the Old World and a country with a very high population density and industrialization. The major task for Israeli zoology is to maintain this central corridor practicable and open for future faunal exchanges (Por 1987). References BODENHEIMER F.S. 1937. Prodromus Faunae Palaestinae. Imprimerie de l’Institut d’Archeologie Orientale Cairo. FERBER I. (ed.). 1985. Israel Collections of Natural History. Israel Academy of Sciences and Humanities, Jerusalem. KADMON R. 2000. BioGIS Program. Hebrew University, Jerusalem. POR F.D. 1975. An Outline of the Zoogeography of the Levant. Zoologica Scripta 4: 5-20. POR F.D. 1987. The Levantine Landbridge: Historical and Present Patterns. In Krupp F., Schneider W. & R. Kinzelbach (eds), Proceedings of the Symposium on the Fauna and Zoogeography of the Middle East. Ludwig Reichert, Wiesbaden, pp. 23-28. TCHERNOV E. & Y. YOM TOV 1988 (eds.). The Zoogeography of Israel. The Distribution and Abundance at a Zoogeographical Crossroad. Dr.W.Junk Publishers, Dordrecht. 600pp.

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Libbie Hyman and Invertebrate Zoology thePanorama 20th Century 581 TheinNew of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 581-582, 2003

Ways for improving modern zoological education: overview of the session J.St.J.S. Buckeridge Earth & Oceanic Sciences Research Centre, Auckland University of Technology, P.O. Box 92006, Auckland, New Zealand. E-mail: [email protected]

Overview: Three papers were presented with lively discussion. The participants clearly identified that there were problems in zoological education, and that on the whole these were not confined to one or a few countries. The problems: Four key areas were recognised as being under threat - the quality of texts, the quality of teachers, the nature of zoology teaching and the availability of traditional zoology courses. In particular: • there is inconsistency in zoology texts – both from the content and from the immediacy of the information therein. • most teachers at secondary school level lack confidence in “matters zoological”. • there is a decrease in the amount of “hands on” zoology teaching – both in secondary schools and in the universities. • there are fewer zoology courses now available in universities. • overall, there is a perception that we are facing a degradation in the zoological knowledge base. Reasons for the problems: It was agreed that a lack of funding was high amongst the factors leading to a decline in the “zoological learning environment”. However, some parties believe this is in part alleviated by electronic media, i.e. virtual zoology. Nonethe-less, no monitor can equate to the sensation of holding a garden snail, or perhaps a spider in one’s hand. It was stressed that the very nature of zoology should ensure that it be a practical subject. There is also a growing international concern for the environment, for instead of encouraging young people to study zoology, this appears to work in the reverse - there is a growing tendency to believe that conservation issues are best solved by “fencing off” wilderness areas. Of some concern too was the current ascendancy of

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the “animal rights movement”, which opposes any activities that could lead to animal discomfort. This movement is strong in its opposition to the dissection of animals and to some degree, of breeding animals for laboratory study. There is also a strong public perception that there are few jobs in zoology. Finally, there is the competition from today’s glamour subjects in biology: biotechnology, cell biology and molecular biology. Outcomes: The meeting agreed that it was possible to frame a number of recommendations. These are: Fieldwork: 1. The importance of fieldwork is paramount in zoology education. Field trips should be organised early in a course to capture the interest of students. Importantly, senior professors must be encouraged to participate. 2. It was also considered important for universities to address field related issues such as health and safety, including the provision of guidelines regarding liability following any mishaps. Laboratories: 3. Animal dissections were confirmed as integral to future zoological education. However, where possible dissections should use “cultivated” organisms, thus reducing conservation concerns. Promotion of Zoology: 4. Professional zoologists are to be encouraged to highlight the importance of zoology. This should be from the perspective of what zoologists have done in the past (e.g. understanding the nature and diversity of life), to what we can do in the future (e.g. helping to sustain life on the planet). We should encourage secondary school teachers onto the campus to take part in interactive “refresher courses”. We must make use of the international network (and the human and other resources therein), to support development of new curricula. Finally, we must more effectively utilise the media (radio, television and the press) to further our aims.

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Coordinated Development and Use of Collections Databases 585 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 585-589, 2003

Coordinated Development and Use of Collections Databases S.G. Poss Gulf Coast Research Laboratory Museum, Institute of Marine Sciences, University of Southern Mississippi, P.O. Box 7000, Ocean Springs, MS,39566-7000, USA. E-mail: [email protected]

Abstract Advances in networking technologies present zoologists with unprecedented access to voluminous information about organisms. It also presents zoologists with the challenge of creating, managing, and organizing a rapidly expanding base of knowledge in dynamic and complex ways, while balancing the contrasting needs for increased specialization and synthesis. Natural history collections and their associated databases are of particular significance to meeting this challenge, because they have served historically as zoological archives and because they provide resources essential to the development of “taxon-centric” electronic documents and of indices to zoological information. Direct links within such documents to specific collections records assure the accuracy and repeatability of associations made among data sets taken from organisms, inferences drawn from such data, and the names used to refer to these organisms. Integration of historical collections data with other kinds of zoological knowledge in a distributed network environment requires reassessment of traditional methods of creating and sharing information. It also requires close cooperation among workers from many zoological disciplines, as well as with computer scientists, librarians, and educators.

Computer technology has redefined how zoologists practice and view their profession. Its power presents zoologists with unprecedented opportunities to access and manipulate information at the frontiers of our science. However, this same power also presents zoologists with one their greatest challenges. With the advent of widespread networking, zoologists are confronted with how to best manage and organize a rapidly radiating diversity of new zoological knowledge in dynamic and complex ways, while simultaneously balancing the need for increasing specialization and refinement on one hand with the need for integration and synthesis on the other. Solutions to this problem are particularly acute for those who seek to build or use information management systems for research and education. Although methods for organizing and managing widely dispersed data of varying complexity and structure

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are familiar to computer scientists (Abiteboul et al. 1999, McLaughlin 2000), tools associated with XML and related technologies are only beginning to be employed within the zoological community. Implementations making use of these methods are impeded by the complexity involved with associating inherently different kinds of biological information. Difficulties posed by such complexity are compounded by the staggering variety of organisms, whose biology is often as unique or as highly specialized as the organisms themselves. Also, differing traditions and needs among the various subdisciplines of zoology have led to a mixture of approaches involving a variety of largely independent data structures and models, many not explicitly specified. Sub-disciplines with a reductionist focus, such as molecular biology, have produced a number of highly collaborative yet specialized databases. In contrast, sub-disciplines, such as systematics and ecology that require synthesis of widely disparate kinds of information, have developed more fragmentary or partially overlapping efforts to amass and integrate knowledge. Among the latter sub-disciplines, information associated with natural history collections and libraries are of particular importance, because they have served historically as zoological archives. However, unlike libraries and other institutions that more recently serve as managers of various specialized databases, natural history collections are permanent repositories of specimens that can be used to confirm whether or not the appropriate, valid name has been associated with information provided for a given species. Unfortunately, like libraries, chronic under-funding has made it difficult for collections staff to keep up with the rapid pace of new discoveries and information, while also meeting their primary obligation to archive increasingly extensive holdings. Unless rectified, this could seriously retard efforts to develop effective means to network zoological knowledge. Although still poorly inventoried on a global scale, collections also provide an irreplaceable wealth of comparative information about historically complex faunal assemblages (e.g. Wallace 1863: 234, Poss & Collette 1995, Pietsch & Anderson 1997). Collections are essential to assessing the consequences of the accelerating pace of anthropogenically induced biotic change (eg. Carelton 1989, Kareiva et al. 1993, Poss 1999). Collections, particularly collections of type-specimens, serve as the basis of Zoological Nomenclature (ICZN 1999: 75-86, Articles 71-76), without which there is no scientific means to assure the consistency of names applied in traditional zoological literature from one study to the next. Likewise, collections increasingly play an essential role in assuring the verifiability of “atomic” elements of distributed and dynamically-created “taxon-centric” electronic documents and nomenclatural indices. With wholesale loss of biodiversity and fundamental shifts of biotic composition within ecosystems (e.g. Elliot & Norse 1993, Huston 1994, Pauly et al. 2000), there is greater need to rapidly access information about the biology of species. Consequently, interest in making such data more accessible has intensified. Since such collections also provide the only documented and verifiable record of historical distributions, they also serve as a reference foundation for the study of spatial associations of organisms evaluated through the use of geographic information system (GIS) technology. Thus, collections data are being increasingly used to study and manage organisms throughout their geographic ranges. It can be expected that the merger of both electronic publishing and GIS technology will further accentuate the fundamental importance of natural history collections as primary sources of scientific information about

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species. Recent technological progress in both computer imaging and computer assisted measurement (e.g. Cromwell & Poss 2001) also underscore the critical need to incorporate support for the physical maintenance and utilization of specimens within collections. It is the specimens themselves that ultimately serve as fundamental sources of information about organisms and not their redefinition as “data” or other abstract representations within systems designed to accumulate and access zoological knowledge. Like names in the traditional scientific literature, usage of the latter can only be interpreted and verified through examination of specimens. Hence, specimen vouchering for zoological studies of all kinds assumes an even greater importance. Because the entire process of retrieving and applying zoological information rests on the assumption of consistently accurate identification of taxa on which observations are made, support will also be required to expand the pool of trained taxonomists to both confirm the identify voucher materials and to adequately curate these collections. Hopefully this trend is analogous to the recent need in functional genomics and bioinformatics, where increasing employment opportunities exist for “database curators” whose services are required to annotate and maintain the integrity of a broad range of molecular databases. Advances in procedures for automatic identification are also needed, because there is an acute shortage of taxonomists and traditional methods of identification can be extremely time-consuming even for experts. Likewise, rigorous standard laboratory practices for species identification must be developed and used, particularly for studies in which it is impractical or not cost effective to retain voucher specimens. Capabilities and limitations of existing systems suggest that future designs will refine mechanisms for more highly structured machine-assisted interaction among zoologists in all sub-disciplines. Modern computing typically no longer involves only a single user interacting with a stand-alone computer. Likewise, the client-server paradigm of a user (client) interacting with a shared mainframe (server), perhaps via middle-ware that may form a “3-tiered” architecture, has also now been superceded. “N-tiered” server architectures have emerged, where “clients” may be other server or application processes interacting with one another, rather than a human user. Such architectures often operate in a distributed environment where users, algorithms, and data may reside anywhere on a network. Because the logic inherent in programming such systems is complicated, efforts to simplifying electronic document production attempt, where possible, to fully separate content, presentation style, and the logic used in document construction. Such reorganization further accentuates the need to reassess traditional methods of creating and sharing information. Zoologists among all sub-disciplines are now confronted with the challenge of reassessing how they gather and share information. To meet this challenge, the zoological community must coordinate effort toward formulation of a new “network architecture” for zoology. Like the infrastructure of traditional publication, this architecture must remain both open to attract and train new scientists using a variety of hardware platforms and flexible to efficiently celebrate and make use of new discovery. By establishing a scientific basis for the association of zoological names and morphological features with other kinds of special collections and zoological data, historical collections data and the specimens upon which they are based provide a means to consistently integrate taxonspecific information.

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However, for such a reference system to work as intended, the quality and physical integrity of these collections must be of paramount concern. The emergence of a more pervasive and fully functional network architecture for zoology, particularly as new options for electronic publication are pursued, will undoubtedly lead to reconsideration of how traditional zoological institutions, professional societies, and scientists interact, with each other and with the those outside the scientific community. Hopefully, with such changes, increased appreciation will emerge as to why the use of natural history collections are essential to assuring that zoology remains a science. Natural history collections permit verification in zoology and without verification there can be no science. Given their greater inherent complexity, design considerations for distributed computing demand increased attention to both practical as well as theoretical issues. Hence, it is unlikely that systems of truly general utility to zoologists will be built by computer scientists, librarians, or educators, without significant input from a broad spectrum of practicing zoologists. Coordinated involvement of specialists from many disciplines will be needed to adapt complex data structures for use within sophisticated networked programming environments that are capable of tracking and distributing enormous quantities of information to a variety of users, who have differentially fluency with terminology used in zoology. Many organisms have limited distributions or exhibit local variation. Because such organisms and phenomena are often best known in detail by workers in a given region, it is also unlikely that universally useful systems will emerge solely through the activities that take place only at large research centers specializing in bioinformatics. Investigators at institutions of all sizes and locations, including laboratory experimentalists, field biologists, and theoreticians will be needed, if a sufficiently comprehensive network is to be built that is capable of meeting zoology’s greatest challenges, such as the evolutionary origins and functions of myriad genes, predicting the consequences of global change, or preventing catastrophic loss of biodiversity. To tackle such problems coordinated effort by workers studying zoological phenomena at all zoological scales from molecular to that of ecosystems is needed. It behooves zoologists of all persuasions and geographic origins to become involved in addressing aspects of how such a zoological network can be best constructed, so that their own work and sub-disciplines will be reflected in the emerging reality of a truly new Zoology, and so that during their own lifetimes, they may usefully apply knowledge made instantaneously available through the use of networking technologies. Even at this early stage of development of such a network, older zoologists can be continuously amazed that knowledge, now available on the Internet at the click of a button, was undreamed of just a few years before. Likewise, the number of web-sites dealing with such issues, through which younger zoologists can achieve professional distinction, now number into the thousands, with new opportunities emerging daily. References Abiteboul S. Buneman P. & D. Suciu 1999. Data on the Web: From Relations to Semistructured Data and XML. Morgan Kauffman, 258p.

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Carelton 1989. Man’s role in changing the face of the ocean: Biological invasions and implications for conservation of near-shore environments. Conservation Biology 3(3):265-373. Cromwell R.L. & S.G. Poss 2001. Accurate 3D-Morphological Measurement Using a Structured Light Range Sensor. Gulf and Caribbean Science 13: 19-35. Huston M.A. 1994. Biological Diversity. The Coexistence of Species on Changing Landscapes. Cambridge University Press, 681p. International Commission on Zoological Nomenclature 1999. International Code of Zoological Nomenclature, Fourth Edition, 306p. Kareiva P.M., Kingsolver J.G. & R.B. Huey (eds) 1993. Biotic Interaction and Global Change. Sinauer Associates, Inc., 559p. McLaughlin B. 2000. Java and XML. O’Rielly, 479p. Norse E.A. 1993. Global Marine Biological Diversity. A Strategy for Building Conservation into Decision Making. Island Press, Washington, D. C., 383p. Pauly D., Christensen V., Froese R. & M.L. Palomares 2000. Fishing Down Aquatic Food Webs. American Scientist 88: 46-51. Pietsch T.W. & W.D. Anderson Jr. 1997. Collection Building in Ichthyology and Herpetology. American Society of Ichthyologists and Herpetologists, Special Publication Number 3, 593p. Poss S.G. 1999. Coastal Fish Distribution and Diversity in the Southeastern United States: How is it changing? Assoc. Southeastern Biologists Coastal Ecology Symposium. ASB Bulletin 46(4): 320-330. Poss S.G. & B.B. Collette 1995. Second Survey of Fish Collections in the United States and Canada. Copeia 1995(1): 48-70. Wallace A.R. 1863. On the physical geography of the Malay Archipelago. J. Roy. Geogr. Soc. 33(8): 217-234.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Evolutionary Paleontology and Informatics: The Neogene Marine Biota ... Evolution 591 The New Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 591-601, 2003

Evolutionary Paleontology and Informatics: The Neogene Marine Biota of Tropical America (NMITA) Database H. Fortunato Smithsonian Tropical Research Institute, P.O.Box 2072, Balboa, Panama. E-mail: [email protected]

Abstract Basing biodiversity and evolutionary studies of multiple, higher-level taxa on well dated samples and consistently identified material is essential for the reliability of any work. The complex and sophisticated analyzes of biotic changes through geologic time that are currently being done will profit from the availability of integrated database systems, allowing the access to all kinds of data in a faster and more efficient way. The Neogene Marine Biota of Tropical America (NMITA) database has image and synoptic information on taxa collected as part of several multi-taxa survey programs designed to assess marine biodiversity in Tropical America over the past 20 million years. Taxonomic, geochronologic and geographic occurrence data for more than 1,800 taxa from several taxonomic groups (gastropods, bivalves, Cheilostome and Cyclostome bryozoans, ostracodes, benthic foraminifera, solitary and reef building corals, and fish) is currently available in an enterprise relational database system. High quality images, synonyms, morphologic matrices, maps, and stratigraphic columns are some of the information that can be requested. Illustrated glossaries of morphologic terms and identification tools are being developed. The information can be used both in research and in education, as well as by amateurs. The NMITA database can be accessed at http://nmita.geology.uiowa.edu.

Introduction The question of how changes in environments and geographic isolation affect organisms, populations and communities cannot be fully understood without a good knowledge of past environments and biodiversity. To achieve this goal, evolutionary paleoecological studies have become more quantitative, relying on very sophisticated methods for analyzing and interpreting rates and patterns of change in biodiversity through time. As analyzes and methods have become more complex, the paleontological community is faced with the necessity of managing an ever growing paleontological database and making sure that such data is available to all practitioners for the purpose of research and education.

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The new panorama of animal evolution

Being such an incredibly information-rich and integrative field, the management of data in paleontology has always been problematic. These problems have grown during the last several decades mainly due to lack of attention paid earlier to both data management and collections computerization, as well as to the decline in trained systematists. Although such problems appear in other scientific areas (biologists, for example, had to redo their ways of sharing data with the increase of molecular studies), paleontological data is so much more complex and diverse that a considerably more complex and flexible system in terms of data-handling capabilities is required. To this end, the field of paleosystematics is becoming one of the more relevant in our community (details in Paleo21 Report, 1997, http://www.nhm.ac.uk/hosted_site/paleonet/ paleo21/). More and more museums, universities, government geological and paleontological survey agencies, have become actively engaged in the computerization of collections, many of which start to be available through the World Wide Web. The challenge of our times it is not just to computerize but to integrate all these fast appearing databases in a way that will allow fast storage, updating and retrieval of paleontological data to be used in both research and education. This paper presents the approach used to design and implement one of these database projects, the Neogene Marine Biota of Tropical America (NMITA), and how it can be used in evolutionary and systematic studies, as well as in education. Scope Of The Project Collections The main objective of the NMITA project it is to create an on-line biotic database that could provide a modern electronic alternative to the usual systematic monographic publication. It is based on specimens rather than literature citations and can be used as a model for distribution of taxonomic information not just in paleontology, but in any other biological field as well. Currently, NMITA contains high quality images as well as taxonomic and distribution (geochronologic and geographic) information on specimens collected as part of several large ongoing multi-taxa collecting projects. This information is fundamental for the consistent taxonomic identification of the marine taxa inhabiting in the shallow waters of Tropical America during the last 20 million years. The present contents of the NMITA database is based on collections made mainly by the Panama Paleontology Project (PPP) based at the Smithsonian Tropical Research Institute (STRI), Panama, and the Neogene Paleontology of the northern Dominican Republic Project based at the Naturhistorisches Museum, Basel, Switzerland. These projects are unique in their rigorous sampling and age determination protocols designed to estimate relative abundances of taxa within each stratigraphic horizon. The PPP is a multidisciplinary project aimed to study the nature, timing, and magnitude of the evolutionary events that shaped the history of the shallow water marine biota of Tropical America during the last 20 million years. It was started in 1986 by A.G. Coates and J.B.C. Jackson, and since then, more than 5,000 samples containing over a million of specimens have been collected and processed. After processing, specimens

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are sent for identification and further systematic and evolutionary studies to a team of more than 30 scientists in residence in eight countries. After the completion of the studies, all specimens are deposited in the Naturhistorisches Museum, Basel, Switzerland (mollusks and fish mainly) and the U.S. National Museum of Natural History (all other taxa) where these collections are permanently curated and can be accessed. The PPP collections have already been used as the basis for two volumes synthesizing ongoing taxonomic and evolutionary studies in the region (Jackson et al. 1996, Collins & Coates 1999) as well as many other individual and multi-authored works listed at the PPP site (http://www.fiu.edu/~collinsl/). Besides of the use of standard sampling and processing protocols, information on paleobatimetry, stratigraphic columns and age determination are also standardized and integrate data on planktonic foraminifera, nannofossils, paleomagnetics and strontium isotopes. Detailed information on the PPP collections and data can be obtained at the project web site (see address above). The Dominican Republic project (sometimes just called DR project) was funded in 1978 by P. Jung and J. Saunders of the Naturhistorisches Museum, Basel, Switzerland. It covered a 10 million year Neogene Caribbean sequence in the northern part of the Dominican Republic where several hundred samples were collected along nine river sections in the Cibao valley. Seventeen systematic monographs have been published on this material, along with stratigraphic and age information based mainly in planktonic foraminifera and nannofossils. Classic studies, like the ones on punctuated evolution (Cheetham 1986) among others, used these collections extensively. Material from several other smaller projects [Budd’s collections from Jamaica, Curaçao and Bahamas (Budd & McNeil 1998, Budd et al. 1998), Jackson’s collections from Venezuela and Trinidad, Fortunato’s collections from Venezuela] that follow the same protocols have also been incorporated into the database. A third major project that will be added to NMITA in the near future is the Recent Marine Survey of Central America, also based at STRI, Panama. The aim of this survey it is to provide a baseline (or ‘time zero’) for use in the evolutionary and biodiversity studies that are being conducted using the fossil collections. The need for such baseline arose when the group of people working with the PPP realized that a comparable detailed taxonomic and occurrence data set was mostly unavailable for the living taxa with the exception, perhaps, of corals. Understanding the taxonomic and ecological distribution of recent taxa is fundamental to evaluate the effects that the Neogene rise of the Isthmus of Panama had in the marine shallow water biota of the region. Taxa More than 5,500 images and partial systematic and occurrence information are currently available in NMITA (Table 1, Fig. 1). Data is available for over 1,800 taxa belonging to nine taxonomic groups: bivalves (~300 genera and subgenera); gastropods (~600 genera and subgenera of muricids, marginellids, and columbellids); Cheilostome and Cyclostome bryozoans (~300 and ~50 species); azooxanthelate and zooxanthelate corals (~50 and 189 species); benthonic foraminifera (~100 species); ostracodes (~300 species); and fish (~230 species).

Taxonomic group

gastropod m olluscs (m arginellid s) gastropod m olluscs (m u ricids)

50 species

R. N ehm , N MB

D. Miller, N MB

N MB

N MB

J. Tod d, BM(N H )

200 (100 light, 100 SEM) 405 (369 light, 36 SEM)

150 (all SEM) 600 (400 light, 200 SEM)

900 (all SEM)

~120 sp ecies

~120 sp ecies

400 (300 light, 100 SEM) 400 (300 light, 100 SEM)

299 900 (600 genera/ su bge light, 300 nera SEM)

O.Agu ilera, 230 sp ecies UN EFM

S.D.Cairns, N MN H

N MN H A.H . 300 sp ecies (Paleobiology); Cheetham , N MB J. Sanner, N MN H N MN H Pau l Taylor, 50 species (Paleobiology) BM(N H ) N MN H A.F.Bu d d , 175 sp ecies (Inv.Zoology); Univ. Iow a N MB

Primary contributors

Total # Estimated images to total # N eogene and be input into Quaternary database Tropical American taxa 2 3 4 5 N MN H L.S. Collins, 100 sp ecies 250 (all (Paleobiology) FIU SEM)

Institution housing the collections

corals N MN H (azooxanthellate) (Inv.Zoology); N MB fish N MB (elasm obranch teeth; teleostean otoliths & teeth) bivalve m ollu sks N MB

cyclostom e bryozoans corals (zooxanthellate)

cheilostom e bryozoans

1 benthic foram iniferans

A

6

50 sp ecies

7 100 sp ecies, >10000 sp ecim ens 235 sp ecies, 340000 sp ecim ens

PPP collections to be included

~120 species

299 genera / su bgenera; >50,000 sp ecim ens ----

----

~120 species

----

20 sp ecies, 20 sp ecies, 1600 specim ens 1800 sp ecim ens 84 sp ecies 66 sp ecies, 500 sp ecim ens

85 sp ecies, 85 sp ecies, 2500 specim ens 2500 sp ecim ens

----

132 sp ecies, >20,000 sp ecim ens

----

D R project collections to be included

8

----

----

Jam aica, Carriacou: 20 sp ecies, >500 sp ecim ens Venezuela: 90 sp ecies, 900 sp ecim ens -Trinid ad : 92 sp ecies, 2500 sp ecim ens ----

Jam aica, Cu racao, Baham as: 50-90 sp ecies, >4500 sp ecim ens

----

Venezuela and Trinid ad

----

Other collections

static pages com p lete; d ynam ic p ages for 38 sp ecies com p lete and are in p rogress for the rem aining sp ecies static pages com p lete

<5% com plete

static pages com p lete

10 ~30% com p lete

status

in progress

m u ricid_genera

biv_id form

~30% com p lete

~30% com p lete

~60% com p lete

OTOLITH _FAMILY; ~60% com p lete OTOLITH _SPECIES

static p ages only

typ e_genu s; coral_sp ecies

in progress

static p ages only

9 FORAM_SPECIES

D ynamic search *

Table 1. A. List of primary contributors, repository institutions for NMITA collections. B. Estimated number of taxa and images currently available in the database.

594 The new panorama of animal evolution

2

3 4 H .Fortu nato, ~120 sp ecies STRI; P.Jung, N MB

N MN H T.M.Cronin, 300 sp ecies (Paleobiology); USGS; N MB P.Borne, LSU

N MB

600 (all SEM)

5 440 (330 light, 110 SEM)

Taxonomic group

Estimated total # N eogene and Quaternary Tropical American taxa foram s 100 cheilostom e bryozoans 300 cyclostom e bryozoans 50 corals (zooxanthellate) 175 corals (azooxanthellate) 50 Fish 230 bivalve m ollu scs 299 gastrop od m ollu scs 120 (m arginellid s) gastrop od m ollu scs 120 (m u ricid s) gastrop od m ollu scs 110 (colu m bellids) ostracod es 300 1854

B

30% 100% 1% 100% 100% 60% 60% 30% 30% 10% 10%

400 440 600 5245

status (total)

150 sp ecies, >5000 sp ecim ens

0%

0%

30%

30% 0% 0% 22% 0% 60% 60% 0%

dynamic

30 985,9

11

36

30 300 0,5 175 50 138 179,4 36

total taxa

60 2903,5

44

120

75 900 1,5 600 200 243 540 120

total images

0 421,9

0

36

30 0 0 38,5 0 138 179,4 0

0 1110

0

120

75 0 0 132 0 243 540 0

dynamic images

<5% com plete

10 ~10% com p lete

dynamic taxa

8 9 Venezuela: 2 sp ecies, in progress >1600 sp ecim ens; Dom inican R. and Barbad os: 2 species, 15 sp ecim ens; Gulf of California throu gh Ecu ad or and Galap agos: 30 sp ecies, > 10000 sp ecim ens >200 species, ---in progress >5000 sp ecim ens

6 7 8 species, 23 sp ecies; 3000 specim ens >5000 sp ecim ens

Total # images to be input into database 250 900 150 600 200 405 900 400

* [http://nmita.geology.uiowa.edu:8001/ows-bin/owa/…]

ostracodes

1 gastropod m olluscs (colu m bellids)

A

Table 1. Continued.

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The new panorama of animal evolution

Fig. 1. NMITA Homepage (http://nmita.geology.uiowa.edu).

Besides of an illustrated inventory of the major taxonomic groups present on the collections, the database also documents taxonomic concepts used in the identification, annotated lists of all names, common synonyms, images of representative specimens, and a taxonomic bibliography. A new and unique part of the project is the morphologic descriptions. These will be highly structured and formatted as character matrices. Characters and their states will be linked to a pick-list, which contains all possible states for each character. Descriptions of the included taxa will use only these predefined terms. NMITA will also provide illustrated glossaries of morphologic characters used for each group. Stratigraphic and geographic distribution data used in NMITA is mostly of an interpretative nature. The raw data used for the interpretations can be accessed by links to the PPP database and to bibliographic sources. Paleoenvironmental data will be available for selected groups that are useful as indicators. Structure Of The Project Data model and equipment The NMITA database model consists of 71 entities linking taxon, morphologic, specimen, and occurrence data. This model follows the Association of Systematics Collections Information Model for the Biological Collections (ASC 1993). The model has six main subject areas (Fig. 2): taxon, specimen, locality, literature, morphology, and illustration. The main innovation are the last two areas which include fields for morphologic characters, measurements, different kinds of illustrations (ranging from

Evolutionary Paleontology and Informatics: The Neogene Marine Biota ...

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Fig. 2. Summary of the main subject areas in the NMITA database model.

photographs in standard orientations for a given group, to drawings, SEMs, cladograms, graphics, field photographs, etc.). Reduced, denormalized versions of the full model are being used for prototype development. Currently, each taxonomic group has its own model and database system, depending of the type of information that is contributed. These models have a similar basic structure and are integrated into the main database management system.

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The new panorama of animal evolution

Information for each database system is entered into a spreadsheet and loaded into Oracle. Presently, NMITA provides links to the PPP database, museum catalog data, and the Atlas of Scleractinian Morphology (ASM 1999-2000), which is being compiled by the Scleractinian Treatise Steering group as part of the Treatise on Invertebrate Paleontology. An integrated WWW and enterprise relational database management system server based at the University of Iowa hosts NMITA. This system runs in an IBM RS6000 43P140 machine with AIX4. The software used is Oracle Web Application server 2.1 and Oracle 7.3. Maps and images are processed using Arc-Info and Adobe Photoshop. Regular backups are made on tape and CD-Rom. Monthly summaries on web visits are done with Analog 3.0. The data Data for NMITA are submitted by the different groups working on the collections. Although most of them are members of the PPP, this is not a requirement, as collections are available to anyone who wants to use them. Contributors submit TIFF and spreadsheet data files. These are processed and made available in the WWW by a team of people at the University of Iowa. Images are the most important component of the project. They are submitted either as 8-bit grayscale (black and white photos) or as 24-bit true color mode (color photos). Resolution is usually 2,000 x 2,000 pixels, although it may depend of the features illustrated. Original images are processed and converted into GIF format. Three different sizes are available to the user: regular size (450 x 450 pixels), large size (900 x 900 pixels), and thumbnail size (150 x 150 pixels). The minimum information provided for each taxonomic group is: a hierarchical list of taxa including at least class, order, family, and genus; a bibliography (all systematics citations will follow the Journal of Paleontology format); information about the illustrated specimen, type specimen and authorship of each lowest level taxon, as well as its geographic and geochronologic occurrence. The main subject area of NMITA is taxon pages generated by the system and linking all the other areas. These pages should reflect the lowest taxonomic rank used in biodiversity analyzes for the taxonomic group in question. For most groups, these pages are at generic and species level. In mollusks the lowest rank being analyzed are genera and subgenera, and the highest families. Pages listing and comparing other rank-order taxa can also be requested by the user. The information available for each lowest-rank taxon usually consists of: name/ author/date; images of representative specimens in standard orientations and following various preparatorial techniques; information about the type specimen (i.e. synonyms, type locality, museum catalog numbers, etc.); a list of morphologic characters linked to an illustrated glossary; stratigraphic and general geographic occurrence as well as age information. Pages for a given higher-rank taxonomic unit (i.e. genera, family, etc.) contain name/author/date, thumbnail images of all lowest-rank taxon belonging to that higher-rank unit, and a matrix with a summary of morphologic differences among these lowest-rank taxa.

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Accessing the information NMITA uses both dynamic and static, pre-defined queries to generate taxon pages. Procedures use PL/SQL (Procedural Language/Standard Query Language) routines designed and compiled with the Oracle Schema manager. Depending on the group queried, such procedures integrate almost instantaneously from a few to over 30 fields distributed in several tables in the database. The result are web pages formatted with a title, taxon name, author and publication date, as well as several tables with images, specimen information, synonyms, character matrices linked to the glossary, stratigraphic and geographic distribution. An example of a PL/SQL application is given in Figure 3. It queries the Oracle database for all bivalve genera and subgenera available in the reference collection of the Neogene of Tropical America assembled by the PPP and housed at the Naturhistorisches Museum in Basel, Switzerland. The result are tables with the available information on the queried taxon, including images of selected specimens, museum catalog and locality numbers, geographic and geochronologic distribution. NMITA has two types of identification tools: traditional polychotomous keys, using hypertext links, and dynamic searches, which work with the morphologic matrices available for each taxon. In both cases, the morphologic data is linked to illustrated glossaries of terms and to definitions for characters and states used. The dynamic searches work through a pick list of a priori defined character states. Users can choose a set and will obtain a list of generic names that correspond to the selected options. These names can then be searched to obtain species pages with images, etc. These tools should help users (researchers, students, etc.) with the identification of their collections directly online. Lists of taxa can also be obtained by selecting a geographic region or stratigraphic horizon and clicking on maps and/or on stratigraphic columns. From these lists, users can go to the images, character matrices, or any other part of the database. The geographic information used in NMITA was downloaded from Pen State University Libraries Digital Chart of the World, and complemented with scanned 1:50,000 locality maps. Both data sets were processed with ARC/INFO, converted to the same coordinate system, and used to produce clickable maps. These maps are linked to the Oracle database and can be queried for information on geologic ages and occurrence of taxa at a given locality.

Fig. 3. Model currently in use to generate web pages for the bivalve genera and subgenera included in the Neogene Caribbean reference collection.

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The new panorama of animal evolution

Development of NMITA The plans for the future development of this project include several main areas. Being images one of the most important elements, the improvement of their quality is a priority. Images should be of such quality as to actually replace the specimens, thus eliminating or greatly reducing the need for costly museum loans. Another important area is the development of tools enabling researchers to analyze data on-line. We plan to integrate the Oracle database with a set of procedures specifically designed for paleoecological analyzes [Statistical Analyzes of Paleontological Occurrence Data (STATPOD), Johnson & McCormick 1995]. This package allows the user to calculate stratigraphic range intervals and evolutionary rates through time using the occurrence data stored in NMITA. It also includes randomization tests and confidence intervals to evaluate the evolutionary patterns obtained. The development of the search and identification tools is another important area to develop. Keys that use data in the DELTA format will be integrated. This will allow the use of a wider and more flexible coding system for the descriptions, as well as direct conversion of the character set available in NMITA into cladistic programs like PAUP and MacClade (Dallwitz 1980, Dalwitz et al. 1993-1999). The use of the DELTA format will also allow the implementation of identification keys using Pollyclave, a CGI program written in ‘C’ (http://prod.library.itoronto.ca/pollyclave/). These keys will be integrated with the NMITA database and linked to the morphologic glossaries and taxon pages. Features like character weighting, handling of missing data and numeric variables, listing of similarities and differences among taxa will also be available to users. Two procedures using Pollyclave are currently being developed one for the reef building corals of the family Faviidae, and another for the solitary coral genera Anomocora and Asterosmilia. NMITA will continue and improve its data sharing capabilities with available on-line museum catalogs. We plan on continuously updating the database with data on new groups, as well as on already existing ones. Information on locality and geochronology will be expanded and its presentation and quality improved. Currently, taxonomic data, maps, and stratigraphic columns from Limon, Costa Rica, and Bocas del Toro, Panama, are being added to the database. Another important area for NMITA developers is the educational. Graduate students can use the material available in the database to check for groups that need work. They can also use it as a learning tool in systematics and a starting point to construct phylogenies for selected clades. In the future, NMITA will offer new laboratory exercises for use in undergraduate courses in systematic and evolutionary paleontology. A major step will be the addition in the near future of the recent Caribbean bivalve reference collection, which is being assembled now at STRI, Panama, and at Scripps Oceanographic Institute, La Jolla, California. This will provide users with a baseline for analyzes of biodiversity and abundance of the most common bivalve taxa during the last 25 million years in the Tropical American region.

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Acknowledgments NMITA is the product of a team of people who contribute their data, write programs, enter and process data, process, prepare and curate collections and specimens. We are grateful to all researchers listed in Table 1 who have made their data available in NMITA. S. P. Jones and J. S. Klaus wrote the queries; T. Adrain and V. Baeder-Helmke processed images and other data. The support personnel of the Naturhistorisches Museum, Basel (R. Panchaud, A. Heitz, S. Dahint), and STRI (Y. Ventocilla, S. Velotti, M. Alvarez, F. Rodriguez, R. Chong, J. Ceballos) were responsible for sample processing, and specimen preparing and photographing. I specially thank T. Adrain who helped with the figures for this paper and N. Budd for compiling table 1. This project is funded by a grant from the Biotic Surveys and Inventories Program at the National Science Foundation and by the University of Iowa. References ASC COMMITTEE ON COMPUTERIZATION AND NETWORKING. 1993. An Information Model for Biological Collections, Report of the Biological Collections Data Standards Workshop, August 18-24, 1992 (http://biodiversity.uno.edu). ASM. 1999-2000. Atlas of Scleractinian Microstructure (ASM) assembled by the Scleractinian Treatise Steering Group (compilers: B.R. Rosen & K.G. Johnson) (http://cayoagua.ucsd.edu/ASM/). CHEETHAM A.H. 1986. Tempo of evolution in a Neogene bryozoan: rates of morphometric change within and across species boundaries. Paleobiology 12: 190-202. COLLINS L.S. & A.G. COATES (eds) 1999. A Paleobiotic Survey of the Caribbean Faunas from the Neogene of the Isthmus of Panama. Bull. Am. Paleo. 357: 351p. DALLWITZ M.J., PAINE T.A. & E.J. ZURCHER 1993-1999. User’s guide to the DELTA system: a general system for processing taxonomic descriptions. 4th edition (http://biodiversity.uno.edu/ delta/). DALLWITZ M.J., PAINE T.A. & E.J. ZURCHER 1998. Interactive keys. In Bridge P. et al. (eds), Information Technology, Plant Pathology and Biodiversity. CAB International, Wallingford, p.201-212. JACKSON J.B.C., BUDD A.F. & A.G. COATES (eds) 1996. Evolution and Environments in Tropical America. University of Chicago Press, Chicago, 425p. JOHNSON K.G. & T. MCCORMICK. 1999. The quantitative description of biotic change using paleontological databases. In Harper D.A.T. (ed.), Numerical Paleobiology. John Wiley & Sons, Chichester, UK, p. 227-248. PALEONTOLOGY IN THE 21ST CENTURY. 1997. Reports and Recommendations of the International Senckenberg Conference and Workshop. (http://www.nhm.ac.uk/hosted_sites/paleonet/paleo21/).

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S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers The Register Of CollectionsA.OfLegakis, European Marine Species: Overview 603 The New An Panorama of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 603-609, 2003

The Register Of Collections Of European Marine Species: An Overview A. Legakis1 & C.S. Emblow2 1. Zoological Museum, Dept. of Biology, Univ. of Athens, Panepistimioupolis, GR-157 84 Athens, Greece 2. Ecological Consultancy Services Ltd. (EcoServe), 17 Rathfarnham Road, Terenure, Dublin 6W, Ireland

Introduction There are a large number of collections throughout Europe that include a variety of marine organisms from bacteria to algae to mammals. These collections may be old or new, large or small, private or public. They may contain type specimens, single significant collections of expeditions or lifetime research, reference collections of national importance or large time-series of collections from particular locations. A large number of people are involved both as scientists and as technicians, and a lot of money has been invested in the collection, conservation and maintenance of specimens. Some of these collections are open to the public and contribute to education and public awareness in environmental matters. Others may form the basis of research efforts that increase our knowledge of the natural world, and help us conserve marine biodiversity and use marine resources in a sustainable way. In order to manage collections more efficiently, we need today to assess the status of these collections, to find their strengths and deficiencies, to look at their problems and help them propose solutions. Methodology In order to compile the register of collections of European marine organisms within the framework of the EC funded project “European Rigister of Marine Species (ERMS)” (Costello et al. 2001), a questionnaire was distributed to the scientists that had been identified in the compilation of the Register of Taxonomic and Identification Experts and to the institutions known to host marine collections. The list of institutes identified from the BioCise project was used as a starting point. The results will be used to start a voluntary register of marine species collections in Europe. In addition, a data entry form on the project website is to be made available.

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The new panorama of animal evolution

Results Of the 500 people contacted, replies were received from people in 80 institutes. The majority was from universities and the government (museums and institutes) sector (Table 1, Fig. 1). Of the 32 government institutions, 15 are museums or herbaria (47%) while the rest (53%) are scientific institutes. Of the 41 university collections, 17 are university museums or herbaria (41%). In total, 40% of all the collections belong to museums or herbaria. Table 1. Type of institute holding marine collections.

Private Government (museums, institutes) Societies, NGO’s Universities

4 32 3 41

Private Government Society, NGO University

Fig. 1. Type of institution holding marine collections.

Number of people employed The collections can be divided into three separate classes according to the number of people employed. A small number of collections (13%) are large employing 20-150 scientists and 20-150 technicians. A larger number (27%) are medium sized with 5-20 scientists and 5-20 technicians. The largest percentage (60%) has a low number of employees, 0-4 scientists and 0-4 technicians. The ratio between scientists and technicians is 2:1 in the highest and in the lowest class while the medium class has a 1:1 ratio.

The Register Of Collections Of European Marine Species: An Overview

605

Geographic coverage The majority of the institutes had collections with a global coverage of marine species whilst few had collections covering just Europe or the Atlantic (Table 2, Fig. 2). Table 2. Geographic coverage.

World Europe Atlantic Local seas (e.g. Mediterranean, Black, Baltic etc.) National and local

40 4 1 15 20

World Europe Atlantic Local Seas National & local

Fig. 2. Geographic coverage of collections.

Content of collections Of the institutes that responded 50 (63%) of them had major marine collections and 47 (59%) of them held type specimens. Over half of them, 44 (55%) had old collections, which required some form of updating. Presence and extent of coverage of collection catalogues Only 29 of the collections where fully catalogued on paper whilst most where partly catalogued. A number where not catalogued on paper (Table 3, Fig. 3). Electronic catalogues were less complete only 8 collections being fully covered and the majority part covered or not catalogued at all.

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Table 3. Presence and extent of coverage of collection catalogues.

Paper catalogues Full coverage Part coverage No coverage Electronic catalogues Full coverage Part coverage No coverage

29 35 16 8 38 31

40 35 30 25 Paper

20

Electronic

15 10 5 0 Full

Part

None

Fig. 3. Extent of coverage of collection catalogues.

Availability of loans The majority of collections where available to researchers and students but not to the general public, whilst a few were only available to institute staff members. Nine collections were not available to anyone (Table 4, Fig. 4). Table 4. Availability of loans.

No availability Staff only Researchers Students Public

9 4 63 29 5

The Register Of Collections Of European Marine Species: An Overview

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None Staff only Researchers Students Public

Fig. 4. No of collections available to various groups.

Species covered in collections The majority of the collections had between 0 and 1000 species in their collections. Only eight institutes had collections containing more than 10000 species (Table 5, Fig. 5). These large collections were at Leiden, Copenhagen, London, Paris, Hamburg, Brussels, Stockholm, Rolan coll., Spain There is confusion in the number of species and specimens reported by the collections. Some mention only European species while others include all the species in their collections. However, the general trend of a large number of small collections and a small number of large collections, arising from the analysis of the data is still valid whether we talk of European or world species. Looking at the ratio between specimens and species we notice that for the most common groups such as Mollusca, Crustacea and Annelida this ratio usually lays between 50:1 Table 5. Number of species.

0-1000 1001-2000 2001-3000 3001-4000 4001-5000 5001-6000 6001-7000 7001-8000 8001-9000 9001-10000 >10000

33 coll. 8 2 1 0 1 2 1 0 2 8

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35 No of colections

30 25 20 15 10 5

0,

00

0

00 00 -1

01 90

>1

0 00

0 -9 01

01 70

80

-7

-8

00

00

0

0 01 60

50

01

-5

-6

00

00

0

0 00 40 01

-4 30

01

-3

00

0 01 20

-2

00

00 01

10

010

0

0

Fig. 5. Number of species in the collections.

and 800:1. The less common groups have a ratio between 1:1 and 30:1. Some rare groups may attain high ratios because the particular collections have a strong interest in them. In order to give an idea for a particular group, the algal collections were investigated in detail. Most of them have 100-500 species. Small (0-100 species) and medium (101500 species) collections have a ratio of specimens:species of 7:1 on average. Large collections (>500 species) have a ratio of higher than 10:1, reaching 30:1. Table 6. Number of species, for Algal collections.

0-100 101-500 > 500

1 12 3

Problems with collections A range of problems was identified with the current state of collections. The greatest problems were the lack of scientific and technical assistance and lack of funding. Other problems were perhaps results from these two (Table 7). Discussion From the analysis of the questionnaires it is evident that the European collections of marine organisms can be divided into three categories according to the size of their collections and the number of people employed by the institute holding them. A few institutions can be classified as large. These are in most cases, large government or university museums. They have large collections, they employ a high number of scientists and technicians and their

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Table 7. Problems with collections.

Lack of scientific & technical assistance Lack of funding Slow cataloguing Lack of maintenance of collections Lack of space and storage facilities Poorly labelled collections Lack of laboratory cultures No management after retirement of senior scientist Lack of literature resources Doubtful determinations Poor presentation to the public

28 8 6 6 5 3 2 2 1 1 1

geographic coverage is worldwide. All of them have important major collections and many type specimens. All of them also have old collections that have not been updated. The majority of the collections are small. Some of them are private (although there are some very large private collections); some are collections in research institutes, university laboratories or local museums. Most of them are understaffed having sometimes no technicians and no scientific staff apart from the person responsible. However, they may have important collections or type specimens and they may be the only or one of the few collections existing nation-wide. The problems facing marine collections are not related to their size. Both large and small institutions complain about the lack of staff, the lack of funding and the backlogging of catalogues. Although not mentioned specifically, most of the problems are related to lack of funding, whether it is lack of staff, maintenance of collections or storage space. To mention specifically the problem of inventories, most of the collections have either partly or fully catalogued their specimens in paper but are lagging behind in the creation of the electronic version of these catalogues. Also, most of the collections loan their material to researchers and to some extent, to students. It is quite possible that with more staff and a better organisation, more collections would be able to loan their material to a larger number of interested persons. The high number of institutions having worldwide collections, almost 50%, indicates the global importance of the European marine collections. Most of these collections have arisen from expeditions carried out by European institutions in all marine areas of the world and contain many data on the biodiversity of non-European countries. On the other hand, an equally significant number of institutions (45%) are concentrated on local seas or at the national or even very local level. This highlights the importance of national and regional collections and the responsibility of countries to support their collections and to collaborate at the regional level with neighbouring countries. References COSTELLO M.J., EMBLOW & R. WHITE (eds.) 2001. European Register of Marine Species. A checklist of the marine species in Europe and a bibliography of guides to their identification. Museum National d’Histoire Naturelle, Paris, 463 pp.

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© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Use Them Or Lose Them: The Need to Make Collection Databases ... Evolution 611 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 611-617, 2003

Use Them Or Lose Them: The Need to Make Collection Databases Publicly Available R. Froese1 & R. Reyes Jr.2 1

Institut of Marine Research, Düsternbrooker Weg 20, 24105 Kiel, Germany. E-mail: [email protected] 2 WorldFish Center, MC P.O. Box 2631, 0718 Makati City, Philippines. E-mai: [email protected]

Abstract Twelve fish collection databases have been made available online through a common search interface at www.fishbase.org/SearchFishCollections.cfm. Copies of the collection databases have been integrated completely or in part into a standard format contained in FishBase, a large relational database covering all recent fishes (www.fishbase.org). For those collections that were already online, record-to-record links were established to enable users to look up additional information in the original database. Scientific names used in the collection databases were matched against FishBase names and attached to valid species. The 0.6 million accessions from 12 independent collections covered over 18,000 species. Of the records which could be matched with FishBase names, 18% (3-29%) were listed under synonyms or misspelling. For 16% (1-32%) of the records no match was found because they were not identified to species level or were new misspellings not contained within the 70,000 synonyms compiled in FishBase. As a result of the integration a search for a valid species or higher taxa found all records that matched any of the recorded synonyms or misspellings. Similarly, country names and geographic regions were standardized and can now be used for searches. About 35% of the records had coordinates with them and can now be used in point maps. About 10% of the records had no coordinates and insufficient information to assign a country without further research. Records with assigned country or region were compared with the known range of the species, and this was used to assign a reliability indicator that marks questionable records, which may either be misidentifications or range extensions. The compiled collection databases are currently being used by several developing countries in their efforts to establish inventories of their species, as required by the Convention on Biological Diversity, and thus provide an efficient tool to repatriate biodiversity data to the countries of origin in a cost-effective way.

Introduction At the beginning of the 21st Century there is increasing recognition of the crucial role that specimen collections can play for the understanding of biotic diversity and our

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quest to turn the current destructive use of the living planet into a harmonized one. In order to live up to such expectations collections have to be cleaned-up and made publicly available. Public use as manifested, e.g., in ‘hits’ in the Internet and participation in international programs, can be a much more powerful advocate for an adequate share of public funds than theoretical argumentation about the importance to science. Collections that decide to remain ‘restricted’ will miss out on current opportunities and may find it difficult to obtain an adequate role in the future as others will have taken the lead and the recognition. The main problems that collections are facing are the often daunting tasks of (1) computerizing the collection, (2) correcting and standardizing scientific names and localities, (3) verifying identifications, and (4) making the information publicly available in a user-friendly interface. Computerization (item 1) is usually less difficult and time-consuming than anticipated. For example, it took just one person-year to digitize the hand-written catalog with 125,000 lots of the fish collection of the Natural History Museum (BMNH). Items (2) to (4) are usually more demanding, and in this contribution we describe our preliminary experiences with a project to make fish collections publicly available. Material and Methods The data presented in this study stem from twelve fish collection databases listed in Table 1. The procedures for matching names are described in Froese (1997) and the ones for assigning reliability indicators in Froese et al. (1999). These procedures draw mainly on reference data in FishBase (www.fishbase.org), version of August 2000, a large biological database containing practically all known fish species (Froese & Pauly 2000), and on the database that forms the basis of Catalog of Fishes (Eschmeyer 1998, www.calacademy.org/ research/ichthyology/catalog/ main.htm), version of November 1999. Results and Discussion As of August 2000 FishBase contained 12 collection databases from 9 countries with about 600,000 collection records (see Table 1). Despite the variety of formats and software used, it took normally one week to transfer a collection database into the OCCURRENCE table of FishBase and to clean-up most of the obvious errors such as misspellings. One precondition for the integration of different collection databases is the standardization of scientific names and localities. For the checking and matching of scientific names we used the approach described by Froese (1997), which is implemented as the ‘Check names’ routine in the CD-ROM version of FishBase. This routine compares the scientific names used in the collections with known synonyms and misspellings as contained in the SYNONYMS table of FishBase and in the Catalog of Fishes. It establishes a link between unambiguous synonyms in the collections and matching synonyms in FishBase, which are linked to current names (fig. 1). These links remain operational even if the valid name in FishBase is changed. For non-matching collection names such as new misspellings or new combinations, the ‘Check names’ routine suggests a list of possible correct names for manual correction in the collection database. Applying this procedure resulted in 84% of the records being linked to about 18,000 valid species in FishBase.

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Table 1. Fish collections contained in FishBase as of August 2000. Online refers to the Internet availability of the contributing database. Record numbers marked with * are incomplete.

Museum BMN H BPBM CAS CICIMAR-IPN GCRL ISH MN H N MRAC N MK N RM UBC ZMH 12 Collections

City Lond on, UK H onolu lu, H aw aii, USA San Francisco, California, USA La Paz, Mexico Ocean Sp rings, Mississip pi, USA H am burg, Germ any Paris, France Bru ssels, Belgium N airobi, Kenya Stockholm , Sw ed en Vancou ver, Canad a H am burg, Germ any 9 Countries

FishBase Species Platichthys flesus

Online No No No No No No Yes No No Yes No Yes 3 online

FishBase Synonyms

Accessions 125212 *9027 143319 4515 28471 *3922 98,331 *46259 1115 35512 24602 20233 0.6 million

Species 11932 2332 11503 486 2801 307 9,623 1537 349 4069 2072 4130 18,000

D atabase MS Access MS Access MUSE MS Excel Dbase MS Access Oracle MS Access MS Excel MUSE MS Access File Maker 6 databases

Mu seum Collection

Pleuronectes lu scus Pleuronectes luscu s

Fig. 1. Simplified database schema for linking scientific names in collection databases to currently valid names.

About 18% of these names were synonyms or misspellings. The 16% unmatched names were either not identified to the species level in the contributing databases, or were new combinations or misspellings not easily placed into synonymies. We tried to implement three standards for localities: large geographic areas as used by FAO for continents and oceans; English ISO names of countries and islands; and regular geographical coordinates. The collections followed a variety of standards for larger areas, and we had to assign the FAO areas based on stated countries, localities and environment (marine or freshwater). Standardization of country names started with printing a unique list of names used in a given collection. Most of the necessary corrections such as typographic errors or translation from other languages were obvious. Assignment of historical names to current countries or islands was more demanding but achievable for the majority of cases. Names of smaller islands were often difficult to trace, especially when no coordinates or other geographic indicators were given. Electronic gazetteers such as the Encarta World Atlas with 1.2 million place names were helpful for this task.

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Typographical errors in coordinates were common and not easily detected. We applied the methods discussed in Froese et al. (1999) to detect points that were outside the range of a given FAO area or country, or that placed marine species on land, freshwater species in the ocean, or, e.g., tropical species in other climate zones. We also used a routine in FishBase to detect records that were outside the established range of a species, and thus either erroneous, range extensions, or misidentifications. About 35% of the records had coordinates with them and can now be used in point maps (Fig. 2). About 10% of the records had no coordinates and insufficient information to assign a country without further research. The most important question about collection records is whether the identification of the specimen is correct. We applied the method of Froese et al. (1999) which compared every collection record with reference information in FishBase and assigned a reliability NIACC indicator, detailing the confidence in the given scientific Name, Identifier, Area, Country and Coordinates in a five digit number, where every digit stands for one of these categories and numbers indicate probability of correctness, with some variation between categories, as: 1 = high, 2 = medium, 3 = low (probably wrong), 4 = no data available, and 5 = not yet evaluated. The most common combination was NIACC 14114 (18 % of records with matching names in FishBase), suggesting reliable records within the known range, but without evaluation of the identifier and without coordinates. Other telling combinations were

Fig. 2. Map with about 175,000 fish collection sites as contained in FishBase. Black dots mark probably erroneous records.

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NIACC 11111 (735 records), referring to records with unambiguous scientific name, a family expert as identifier, and reported from within the known range of the species. In contrast, NIACC 23331 (28 records) indicated records where the species had been misidentified before, the identifier was unknown, and the locality (with consistent coordinates) was clearly outside the established range, i.e., most likely these were misidentifications. The NIACC 11131 (46 records), on the other hand, suggested a wellidentified species occurring in a new country within its broader distributional range. A remaining problem with our approach to incorporating collection databases in FishBase is the difficulty of establishing a functioning updating loop where the errors found by the FishBase team and communicated to the contributing collections are corrected in the originating databases, and improved versions of those databases are sent to FishBase, e.g., every year, to completely replace the previous versions. In our experience curators were for various reasons often unable to verify and apply the corrections we sent them. An alternative approach where the collection databases remain at their respective locations and are polled when needed through the Internet is unsatisfactory in comparison as the standardization step is skipped and records with non-matching scientific or locality names will not be detected nor corrected, thus leaving a large number of records unused. Also, such distributed approach depends on all databases being available on-line and all Internet connections being faster than various time-out limits in order to create reproducible results. Maybe the best solution is a hybrid approach where only part of the data (name, date, locality, coordinates, identifier) are standardized and held and updated centrally for fast access with reproducible results, and where another part with additional information is linked and maintained in the contributing database only. This may evolve into a distributed system as more databases become available online, data standards are adopted and implemented, and speed and reliability of the Internet connections improves. Table 2. Percentages of valid names, synonyms or misspellings, and unmatched names in 12 collection databases, sorted by valid names. The last row gives overall values and the range within the different databases.

Valid % 39 54 55 60 67 74 75 76 77 82 83 96 66 (39-96)

Synonyms / misspellings % 29 16 27 11 19 14 14 20 14 10 9 3 18 (3-29)

N o match % 32 30 18 29 14 12 11 4 9 8 8 1 16 (1-32)

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The new panorama of animal evolution

In summary, tools are available to largely clean-up fish collection databases and to make them available for research and public use. However, curators will need support to make the necessary corrections and standardizations in their respective databases. Eschmeyer (1998) listed 457 fish collections of which 64 had home pages in the Internet and 24 could be searched through their own interface in August 2000. The NEODAT Project (www.neodat.org) provided access to eight of these online databases plus an additional 16 collections restricted to neotropical fishes. The total number of fish accessions (= lots) in all existing collections was estimated at about 10 million by Froese et al. (1999). The twelve databases and 0.6 million records investigated in this study thus present only about 6% of the total and therefore the various trends we describe may change as more collections are added. Currently the apparent gaps of fish collection records shown in Fig. 2 reflect more the intermediate status of our work than biodiversity patterns. We expect the patterns to become more meaningful once a threshold of, e.g., 2 million accessions is surpassed. Several additional museums have already declared their willingness to make their collections available through FishBase (e.g. AMNH, RUSI, UF). Negotiations are underway to join forces with other initiatives that aim to bring collection databases together, namely the Neodat project (www.neodat.org), the Fishnet project (habanero.nhm.ukans.edu/fishnet), and the European Natural History Specimen Information Network (www.nhm.ac.uk/science/rco/enhsin). The compiled collection databases are being used by several developing countries in their efforts to establish inventories of their species (see Ramjohn 1999 as example), as required by the Convention on Biological Diversity. Access through one interface thus provides an efficient tool in an effort to repatriate biodiversity data to the countries of origin in a cost-effective and useful manner. The public interest in scientific information on fishes is often underestimated. The FishBase web site entertained over half a million hits and 40,000 user sessions per month during 2000. In the two years since August 1998 the site had altogether 206,000 unique visitors of whom 34,000 visited more than once and used the database for an average of 14 minutes per session. If the voluntary entries in the FishBase guest book are taken as a measure then one third of the users were individuals (non-scientists), one third came from Universities, and the rest were from the private sector, governments, and nongovernmental organizations. Museum data are featured at several locations in the FishBase web site, including various biodiversity maps. Links to the home pages of the museums and to online collections are provided whenever museum data are shown, thus giving visible credit to the contributing institutions. This is even true for maps, where a click on a point will show the underlying data. Please contact the first author if you want to contribute to the approach described in this study. Acknowledgements We thank the contributing databases for their trust in and support of the FishBase vision. We thank the FishBase team for encoding the reference data that allowed us to do this study. We thank Nicolas Bailly and Sven Kullander for comments on the manuscript. This research was conducted partly under the joint Fisheries Research Initiative of African, Caribbean and Pacific (ACP) countries with the European Union

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and was sponsored in the framework of the capacity building project ‘Strengthening of fisheries and biodiversity management in ACP countries’ (7.ACP.RPR.545). It was also partly conducted under the ‘Census of Marine Fishes’ project funded by the Sloan Foundation. WorldFish Center Contribution No. 1572. References ESCHMEYER W.N. (ed.) 1998. Catalog of Fishes. Special Publication, California Academy of Sciences, San Francisco, 3 vols., 2905p. FROESE R. 1997. An algorithm for identifying misspellings and synonyms in lists of scientific names of fishes. Cybium 21(3): 265-280. FROESE R., BAILLY N., CORONADO G., PRUVOST P., REYES R.Jr. & J.-C. HUREAU 1999. A new procedure to evaluate fish collection databases. In Séret B. & J.-Y. Sire (eds), Proc. 5th IndoPac. Fish Conf., Soc. Fr. Ichthyol., Paris, pp. 697-705. FROESE R. & D. PAULY (eds) 2000. FishBase 2000: Concepts, Design and Data Sources. ICLARM, Los Bãnos, Philippines. 333p. RAMJOHN D.D. 1999. Checklist of Coastal and Marine Fishes of Trinidad and Tobago. Marine Fishery Analysis Unit, Fisheries Division, Ministry of Agriculture, Land and Marine Resources, Trinidad and Tobago. Fisheries Information Series No. 8, 151p.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Distributed Information Systems and Predictive Biogeography: Putting ... Evolution 619 The New Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 619-624, 2003

Distributed Information Systems and Predictive Biogeography: Putting Natural History Collections to Work in the 21st Century E.O. Wiley & A.Townsend Peterson Department of Ecology and Evolutionary Biology and Biodiversity Research Center, The University of Kansas, Lawrence, KS 66045 USA

Abstract Natural history collections contain the most comprehensive description of biodiversity on earth. Until recently, extracting this information and using it has been an extremely time-consuming operation, even when the collections are computerized. New technologies are permitting zoologists and others to gain access to museum data on a world-wide scale and other new technologies are allowing us to synthesize and analyze these data in novel ways. In this talk, we review these new technologies and show how they can be used to study different aspects of both biogeography and ecology. Finally, we introduce a distributed network specifically suited for the study of marine and freshwater fishes and suggest some uses that can be made with the data provided by this network.

Scientific collections housed at world natural history museums constitute the most complete and authoritative record of global biodiversity (PCAST 1998, Krishtalka & Humphrey 1998, 2001) available to both scientists and decision-makers. The value of these collections lies in the fact that records are tied directly to actual voucher specimens. Changes in taxonomic names and discovery of new species can always be related to actual specimens available to all qualified investigators. The problem has been that access to these data has been hampered by our inability to integrate the data from different museums and other authoritative databases seamlessly into a single interoperable information system. Efficient access to such data can furnish investigators with baseline data regarding where and when collections have been made and thus where species are found. Clearly, this information provides an important tool in planning future research and making policy decisions concerning biodiversity issues. Segments of the museum community have worked over the past years to computerize their data records and make these records available to the scientific community. One segment in particular, the ichthyological community, has led the effort to make data-sharing a reality through initiatives such as FISHGOPHER, MUSE and NEODAT projects (all accessible from http://www.neodat.org/). These older

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technologies depend on each museum adopting the same database program and data structure. However, most museums active in computerizing their records have adopted different database programs and database structures, making interoperability difficult or impossible to achieve on the large scale necessary to achieve the goal of a virtual world-wide resource. A different approach emerged with the adoption of the concept of distributed database computing and the evolution of protocols allowing servers to communicate to each other in a fashion that allows seamless integration of data from databases of different structures but similar standards. A detailed understanding of distributed database technology is not necessary, but a superficial description may help clarify how the network operates. The Species Analyst is a client program developed by Dr. David Vieglais at the University of Kansas Biodiversity Research Center that is based on the same technology as that commonly used for bibliographic searches; ANSI/NISO Z39.50 (NISO 1995, Lynch 1997). Z39.50 protocols define a standard way for computers to communicate for the purpose of information transfer and retrieval. A computer operating as a “client” submits a request to another computer acting as a “server.” Software on the server performs a search on the database, generates a set of records that meets the criteria of the search request, and returns the results to the client for processing. Distributed data networks that use ANSI/NISO Z39.50 technology are able to share information because they share a common core of information fields that defines the kinds of data that are being searched. The Species Analyst, as well as component projects such as FISHNET, uses the Darwin Core as the criterion for searching databases. The Darwin Core is a list of fields common to all museum specimen records: genus name, species epithet, date of collection, collector, locality, latitude, longitude, and higher taxonomic names such as family, order, etc. The Darwin Core is dynamic, so a number of fields specific to, for example, marine biodiversity will soon be added, such as ocean basin and depth. Not all museum records carry all information, nor is it necessary that they do so in order to be queried. A search is initiated from a client computer using a software program called a “ZClient application.” The Species Analyst receives a request, such as a request to search all databases for records of a particular species. It processes the request and broadcasts the request to all linked databases specified by the user. When a linked server (termed a “Zserver”) receives the request, a program installed on the server searches its associated database(s), compiles and sorts the information, and returns it to The Species Analyst application. The Species Analyst can then process the information into a format requested by the user or a format specified by the client software application. The Z-server software provides a layer of abstraction based on the definitions in the Darwin Core that provides a common interface to databases being served, and a common result set structure for information being returned. Imagine a single Z-server serving two databases, one for fishes and another for corals. Now, imagine that although these databases contain similar kinds of information, their data structure is different (i.e., they are idiosyncratic relative to each other), or perhaps they are entered into different database management programs. In this case, we would have two pieces of Z39.50 software, each configured to read its associated database, and each configured to put the data into a common format as defined by the Darwin Core. Both packages of

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information could then be returned to the client computer in a common format, perhaps responding to a query regarding marine life of a particular sector of the ocean. The Species Analyst provides for return of data in a variety of formats. In the WWW implementation (http://habanero.nhm.ukans.edu/zportal/tsasimple.asp), TSA presents an html page that summarizes the results of the search. The summary includes: 1) a list the databases queried, 2) options for downloading the data (html summary table, Excel table, xml, shape file for use in geographical information system programs), and 3) a summary spot map of all georeferenced data. It also includes links specific for that species to Genbank, ITIS (the Integrates Taxonomic Information System) and the Zoological Record. Although most queries using The Species Analyst are in terms of species, it can also use used to query records or genera and families, and to query across all taxa for specific regions. Any software program compliant with Z39.50 standards can be used in conjunction with The Species Analyst by installing a Species Analyst client program that works with it. For example, Species Analyst clients have been developed for Excel and ESRI ArcView. By using a client directly attached to such an application as Excel, the user can search for data directly, without the need to access the TSA WWW site. In such a case, the data are returned in the format appropriate to the application (e.g., an Excel file or a shape file). An Example: The FISHNET Partnership Direct access to museum data requires their entry in electronic databases. The ichthyological community has been active for over twenty years in electronic capture of specimen data through such initiatives as the MUSE and NEODAT projects. These early steps positioned the community to play a leadership role in making museum data accessible. However, their full use has been hindered by lack of efficient mechanisms for search and retrieval of information from geographically scattered databases with idiosyncratic database structures. The FISHNET community is a partnership of 23 large and small museums who have agreed to form a partnership to share data in a distributed information environment using The Species Analyst. FISHNET is not an entity: At one level, it is simply a series of databases available through The Species Analyst. At another level, it is an active consortium of ichthyologists and information technologists driving The Species Analyst and its associated Darwin Core to provide new functionalities for use by the ichthyological community. At yet another level, it provides resources to partner institutions to enable them to serve much more biological information than simply fish collection records. As a distributed database system, FISHNET has several advantages. It does not require partners to have data in the same database structure or to use the same database management system. The distributed structure of the network allows partner institutions to maintain control of their data locally, and make available only those data they wish to be queried. Not only are the data distributed, but also the work, with most of the computing burden borne by local data servers, allowing for almost infinite expandability without slowing down the system. Further, data are always current, since they are available as soon as entered or as soon as the database replica is updated. Finally, as a component of the broader Species Analyst partnership composed of databases for other

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taxa (mammals, birds, insects, other invertebrates, plants, etc.), cross-taxon queries and analyses become possible. For example, we can query for fish species and for other taxa simultaneously, making feasible detection of correlations among organisms, such as predator and prey, effects of abundance on communities, and other cross-taxon effects. As a distributed database network, FISHNET has some characteristics of which users should be aware (and which are detailed on The Species Analyst website). The information provided is only as good as the information associated with the specimens and its manifestation in the database. Different collections and different parts of a single collection may vary in quality of taxonomic identifications associated with specimens. Some collections are extensively geo-referenced, others are not, and some geographic references can be erroneous. FISHNET data are no replacement for thorough systematic and taxonomic study of a group, nor are they a replacement for looking at specimens. The Species Analyst and FISHNET Research The Species Analyst permits investigators access to specimen occurrence data. In this basic form, the data can be used to prepare occurrence maps (“spot maps”) for species or groups of species. As a partner in the Ocean Biogeographic Information System (OBIS: http://marine.rutgers.edu/OBIS/), The Species Analyst and the FishNet partnership enable users to access an on-line atlases of distributions of fishes on a world-wide basis for both the marine and freshwater environments. Instructions of how to use The Species Analyst are available from this site. Information about the FishNet partnership is also available (http://www.speciesanalyst.net/fishnet). Once abundant distributional information is in hand, in particular via The Species Analyst, diverse opportunities open for synthetic analyses. Although these possibilities have yet to be explored in aquatic systems, the abundant rewards that have been reaped in terrestrial systems suggest that their application to fish and other aquatic organisms will be rewarding. A brief outline of some possibilities, along but one line of investigation (ecological niche modeling) follows. The basic procedure is one of using known occurrence points (Peterson et al. 1998) and their relationship to features of the ecological and environmental landscape to create a model of ecological requirements of the species. This ecological niche model can be projected back onto the landscape to produce a prediction of a potential geographic distribution for the species (Chen & Peterson 2000). Tests of the accuracy of these predictions have yielded encouraging results: about 90% of species can be modeled with high degrees of accuracy (Peterson et al. 2002, Sánchez-Cordero et al. 2000, Peterson 2001, Peterson et al. 2002). These models are robust to reasonable densities of environmental coverages (Peterson & Cohoon 1999), as well as to sample sizes in the range of 20-50 locality points (Stockwell & Peterson 2002). With accurate predictions of geographic distributions in hand, sets of species can be assembled, and examined with regard to conservation strategies (Sánchez-Cordero et al. 2000). For instance, Egbert et al. (1998) and Peterson et al. (2000) modeled the geographic distributions of bird and mammal species endemic to eastern Mexico, and were able to identify a set of just seven areas that would protect all species analyzed. Other studies of this sort include Godown & Peterson (2000), who analyzed the geographic distributions

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of U.S. endangered bird species, and several others in preparation. Peterson et al. (2000) extended this methodology further, using both distributional predictions and complementarily algorithms to optimize conservation strategies. In the realm of basic biology, Peterson et al. (1999) used ecological niche modeling approaches to test the conservative nature of ecological niche evolution in 37 species pairs of birds, mammals, and butterflies. They concluded that ecological niches evolve slowly, indeed more slowly than speciation acts; this result suggests that niches are in general stable over evolutionary time periods. Stability of ecological niches over such time periods makes possible additional inferences. For instance, Peterson & Vieglais (2001) successfully predicted geographic dimensions of species invasions, and Peterson et al. (2002) predicted species’ distributions in the face of global climate change, by projecting ecological niche models onto alternative ecological/environmental landscapes. This summary should serve to illustrate the diversity of analytical possibilities that springs from the application of but one tool to biodiversity data such as that served by the Species Analyst. Further investigation and experimentation will doubtless open still additional doors, and the array of tools available for insightful analysis of biodiversity data will be further enhanced. Addendum The Species Analyst web site is undergoing revisions and some functions mentioned in the text are not currently available. References CHEN G. & A.T. PETERSON. 2000. A new technique for predicting distributions of terrestrial vertebrates using inferential modeling. Zoological Research 21:231-237. EGBERT S.L., PETERSON A.T., SÁNCHEZ-CORDERO V. & K.P. PRICE 1998. Modeling Conservation Priorities in Veracruz, Mexico. In Morain S. (ed.), GIS in Natural Resource Management: Balancing the Technical-Political Equation. High Mountain Press, Santa Fe, New Mexico, pp. 55-63. GODOWN M.E. & A.T. PETERSON. 2000. Preliminary distributional analysis of U.S. endangered bird species. Biodiversity and Conservation 9:1313-1322. KRISHTALKA L. & P.S. HUMPHREY. 1998. Fiddling while the Planet Burns: The Challenge for U.S. Natural History Museum. Museum News 77(2): 29-35. KRISHTALKA L. & P.S. HUMPHREY 2001. Can Natural History Museums Capture the Future? BioScience 50(7): 611-617. LYNCH C. 1997. The Z39.50 Information Retrieval Standard. D-LIB Magazine, April 1997. URL: http://www.dlib.org/dlib/april97/04lynch.html. NISO 1995. Information Retrieval Protocol (Z39.50): Application Service Definition and Protocol Specification. NISO Press, Bethesda, MD. (Available in electronic form at the Z39.50 Maintenance Agency (http://lcweb.loc.gov/z3950/agency). PCAST 1998. Teaming with Life: Investing in science to understand and use America’s living capital. President’s Committee of Advisors On Science and Technology: Panel on Biodiversity and Ecosystems. 86p.

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PETERSON, A. T. 2001. Predicting species’ geographic distributions based on ecological niche modeling. Condor 103: 599-605. PETERSON A.T., BALL L.G. & K.C. COHOON 2002. Predicting distributions of tropical birds. Ibis 144: e27-e32. PETERSON A.T. & K.P. COHOON 1999. Sensitivity of distributional prediction algorithms to geographic data completeness. Ecological Modelling 117: 159-164. PETERSON A.T., EGBERT S.L., SÁNCHEZ-CORDERO V. & K.P. PRICE. 2000. Geographic analysis of conservation priorities using distributional modelling and complementarity: Endemic birds and mammals in Veracruz, Mexico. Biological Conservation 93:85-94. PETERSON A.T., NAVARRO-SIGÜENZA A.G. & H. BENÍTEZ-DÍAZ. 1998. The need for continued scientific collecting: A geographic analysis of Mexican bird specimens. Ibis 140: 288-294. PETERSON A.T., SOBERÓN J. & V. SÁNCHEZ-CORDERO. 1999. Conservatism of ecological niches in evolutionary time. Science 285: 1265-1267. PETERSON A.T. & D.A. VIEGLAIS 2001. Predicting species invasions using ecological niche modeling. BioScience 51: 363-371. SÁNCHEZ -CORDERO V., PETERSON A.T. & P. PLIEGO-ESCALANTE 2000. Modelado de la distribución geográfica de las especies y conservación de la diversidad biológica. In Enfoques Contemporáneos en el Estudio de la Diversidad Biológica. Instituto de Biología, UNAM and Academia Mexicana de Ciencias. México, D.F., pp. 359-379. STOCKWELL D.R.B. & I.R. NOBLE. 1991. Induction of sets of rules from animal distribution data: a robust and informative method of data analysis. Mathematics and Computers in Simulation 32: 249-254. STOCKWELL D.R.B. & D.G. PETERS. 1993. Artificial Intelligence predicts biodiversity. ERINYES 18. STOCKWELL D.R.B. & A.T. PETERSON. 2002. Controlling bias in biodiversity data. In J.M. Scott, P.J. Heglund, & M.L. Morrison, (eds.) Predicting Species Occurrences: Issues of Scale and Accuracy. Island Press, Washington, D.C., pp. 537-546.

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Remedies for the Taxonomic Impediment in Zoology 627 The New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 627-630, 2003

Remedies for the Taxonomic Impediment in Zoology F.D. Por Hebrew University of Jerusalem, 91904 Jerusalem, Israel. E-mail: [email protected]

At the first International Congress in Paris in 1889, Raphael Blanchard initiated the intensive preoccupation of the Congresses with the nomenclature rules for a rapidly increasing inventory of described animal species. All the following congresses, till 1972, dealt with the need to keep pace with the incoming amounts of zoological material. In the last two decades just as the recognition dawned on us that there are probably ten times more species out there than it was previously thought, the numbers of available taxonomists started to spiral down. Already half a century ago, the problems facing the taxonomists in the modern world and their numerical demise, was signalled (Hedgpeth et al. 1953). In 1992 Gaston and May alerted on the decline in the taxonomist positions in Britain. Feldman and Manning (1992) broadened this sad panorama. After the Rio de Janeiro Conference of 1992, as the care for the sustained biodiversity of the globe became unanimously recognised, it became more than obvious that the king has no clothes: The taxonomists disappeared and disappear at a more accelerated rate than the very species they are meant to study. Most of the monitoring projects and ecological studies are being carried-out presently without a proper taxonomic identification of the study material. To amend this situation is in most of the cases already an unrealistic desideratum because the available taxonomists either do not exist anymore or are retired persons. The number of orphaned taxa, taxa with no active knowledgeable specialist in the world, is increasing by the year. In many areas the “secrets” of the trade have been lost and a new person will have to start painfully from zero. The problems facing taxonomic training have been discussed in a British House of Lords Committee and as a response the Linnean Society organised in 1995 a workshop in London (Blackmore & Watson 1995). Butman and Carlton (1995) spoke of a “Taxonomic Predicament”. IUBS/Diversitas called it “Taxonomic Impediment”. More data on the declining numbers of taxonomists were given by Cotterill (1995). A “White Paper” by ASC first defined and detailed the problem (Hoagland 1996). The Taxonomic Impediment has been analysed in detail also at the Montreal Meeting of CBD’s SBSTTA in 1996 (Practical Approaches for Capacity Building for Taxonomy. Note by the Secretariat. SBSTTA Second Meeting, Montreal, September 1996), intensively discussed at the Darwin Workshop (The Darwin Declaration. Australian Biological Resource Study, Environment Australia and National Museum of Natural History, Smithsonian Institution, 1998) and

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finally the Global Taxonomic Initiative was launched in London in 1998 (The Global Taxonomy Initiative; Using Systematic Inventories to Meet Country and Regional Needs. The Center for Biodiversity and Conservation, American Museum of Natural History, UNESCO, IUBS, UNEP Diversitas, Systematic Agenda 2000 International). My estimate is that 70% of the 7000 actively publishing taxonomists in all the kingdoms of nature are over the age of 50. Many of the 2-3000 active zoo-taxonomists are furthermore engaged in monographic revision work, in cladistic analyses and in molecular taxonomy. This reduces even more the possibility to respond to the needs of the day-by-day taxonomy in the field. The fear of being classified as an old-fashioned “classical, descriptive zoologist” has demoralised many taxonomists. The pressure to deal with sophisticated and more timeconsuming molecular taxonomy, though highly justified in basic scientific terms, has driven away many colleagues from the old morphological taxonomy. As an added unhappy, though basically important recognition, the “splitters” have definitively won the epic battle against the “lumpers” and many old descriptions have to be re-submitted to more discerning methods. The “solid” basis of around 1.5 million described species (probably some 10% of what exists out there) needs to be corrected upwards too. Since the training of a young research taxonomist needs more than the 2-3 years allowed by most PhD programs and research grants, the putative profession of “parataxonomists” was proposed by the “Systematic Agenda 2000” project (Systematics Agenda 2000: Charting the Biosphere. Technical Report, NY American Museum of Natural History 1994). To my knowledge such people exist only in the Costa Rican experiment. If applied with much care, their existence could alleviate and accelerate the identifying of the endangered animal diversity around us. Finally, the blame for the public devaluation of taxonomy is often put on the door of the taxonomists themselves. With every DNA sequencing duly priced, each hour of computer consult paid, every inch of lab-space overheaded, and the cost of each page of publication budgeted, the taxonomists behave like gracious chivalrous old-timers. Our money-dominated science does not appreciate volunteering. As sad as it is, it even ridicules it. The taxonomists have to proclaim openly their own value and this is measured in pricing as well as in publishing. The recently established GTI (Global Taxonomy Initiative) of the CBD (the international Convention on Biological Diversity) intends to remedy the problem of the Taxonomic Impediment on the level of the different signatory states, or on a regional basis. Furthermore, although with due reservation, GTI intends to prioritise taxonomic groups that have presently evident importance in the ecosystems and have a presently recognised economic value. Differently, the renascent Zoological Congresses are an across-theboundaries professional association. Our interests will be even-handed, considering all the animal organisms, small or big, keystone species or just small stage-hands. Suggestions coming from the International Congress of Zoology can be considered by the Clearing House Mechanism of CBD as a complementary tool coming from an associated organisation. Furthermore, if, as expected, there will be a continuity of the congresses and a permanent global zoological presence on the web will be activated, we shall be able to put in place an advisory organism for CBD, GTI and for other future global efforts.

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I shall list now, for our discussion, two separate sets of proposals. One meant directly for CBD-GTI and the other to be carried-out as an inter-congress activity of our web-site (http://www.globalzoology.org). Proposals for CBD- GTI: • To stimulate universities to re-establish doctoral programs in taxonomy, by a system of 5 year study programs ( instead of the usual 2-3 years. The same for grants for specialisation in taxonomy coming from national and otherwise public research entities. • To organise regional specialised taxonomic sorting centres and around them to organise courses for parataxonomists. To define the academic parameters of this new profession. • To call upon the governments to emphasise the need for involving and budgeting trained active taxonomists in the environmental impact statements required by the law as well as in all the environmental programs. • To approach granting agencies, scientific journals and publishers world wide to require a taxonomic affidavit, i.e. professional taxonomic identification to species level of the treated biota and designation of the material repository, for all the final reports and/or publications dealing with diversity assessing and ecological research. • To stimulate on the different national levels the continued research activity of retired taxonomists and of high-level amateurs, and to find the adequate academic framework for this activity. • While stimulating electronic-produced identification sheets and species lists, to ask producing durable and library deposited hard copies and furthermore to promote the care for collections of vital old literature. • Suggest to budget separately the necessary, but time-consuming routine activity of computerising historical collections, from the support for the taxonomic work of the curators. A permanent congress organisation with its own interactive web-site, will be able to carry out the following activities and supply the following services: • Assemble urgently a list of orphaned taxa (family level for Arthropoda; above-family for others) and of “endangered” taxa, for which there are single and/or old-aged specialists. • Establish a centralised web-contact with all the zoological taxon-organisations and their respective newsletters. • Establish a central web site of all the national zoological societies • Based on the feedback received from the taxon organisations and individuals, to obtain and publicise taxonomic state of arts reports of the different major entities of the animal world. • Serve as a portal for the active zoological collections • Serve as a portal for information on existing electronic taxonomic literature

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• Build, with the help of the existing programs, a world-list of active zootaxonomists, their specific interests and availability to remunerated activity. . • Serve as a clearing house for sorting and identification of zoological specimens, on the basis of an accepted price-table. • Inform and orient the funding agencies and the journal editors, which want to find reviewers to give a taxonomic affidavit to relevant sections of the reports and manuscripts. • Advertise job opportunities, training courses, collecting programs, etc. References BLACKMORE S. & E. WATSON 1995. Priorities in Systematic Research and Training. Report of a workshop held at the Linnean Society of London. BUTMAN C.A & J.T. CARLTON 1995. Marine biological diversity: Some important issues and critical research needs. U.S. Nat. Rept. to Internat. Union of Geodesy & Geophysics 19911994, Contributions in Oceanography AGU, Reviews in Geophysics Supplement: 1201-1209. COTTERILL F.P.D. 1995. Systematics, biological knowledge and environmental conservation. Biodiversity and Conservation 4: 183-205. HEDGPETH J.W., MENZIES R.J., HAND C.H. & M.D. BURKENROAD 1953. On certain problems of a taxonomist. Science 117: 17-18. FELDMAN R.M. & R.J. MANNING 1992. Journal of Paleontology 66: 157-158. GASTON K.I. & R.M. MAY 1992. Taxonomy of taxonomists. Nature 336: 281-282. HOAGLAND K.E. 1996. White Paper. The Taxonomic Impediment and the Convention on Biodiversity. Association of Systematic Collections.

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The Global Taxonomy Initiative (GTI) and the International Congress on Zoology – a perspective on the role of the Convention on Biological Diversity and UNESCO I.D. Cresswell1 & P.B. Bridgewater2 1. Australian Biological Resources Study, GPO Box 787, Canberra, ACT, 2601, Australia. (Formerly at Secretariat of the Convention on Biological Diversity, Montreal, Quebec Canada). E-mail: [email protected] 2. Man and the Biosphere Programme, UNESCO, 1 rue Miollis, Paris, 75015 France. E-mail: [email protected]

Abstract Taxonomy has been discussed since the first meeting of the Convention’s Subsidiary Body on Scientific, Technical and Technological Advice (SBSTTA) in Paris in 1995, and the governments of the world who recognize the CBD have acknowledged the existence of a taxonomic impediment to sound management of biodiversity. Some of the key reasons for the importance of taxonomy include an understanding of key organisms that enable; 1. 2. 3. 4. 5.

development of food security, the promotion of health, identification and control of disease vectors, identification and control of vectors of ecosystem dysfunction, a scientific basis for conservation management and planning.

The Global Taxonomy Initiative (GTI) is all about removing or ameliorating this socalled taxonomic impediment – that is the gaps of knowledge in our taxonomic system (including knowledge gaps associated with genetic systems), the shortage of trained taxonomists and curators, and the impact these deficiencies have on our ability to conserve, use and share the benefits of our biological diversity. There is a broad consensus on the major elements of the GTI, but what is now required is agreement on the priorities for immediate action. This paper will present the decisions from the fifth and sixth Conferences of the Parties (the political body of the Convention), and illustrate the way forward, including how the ICZ can help implement the GTI.

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Introduction The Convention on Biological Diversity (CBD, 1992) recognizes the “general lack of information and knowledge regarding biological diversity and the urgent need to develop scientific, technical and institutional capacities to provide the basic understanding upon which to plan and implement appropriate measures” The lack of a sound taxonomic information base for conservation and sustainable use of biological diversity has been discussed since the first meeting of the Scientific Subsidiary Body of the CBD – held in UNESCO in 1995. Numerous meetings since have also affirmed that a taxonomic impediment is a rate-limiting step to progress in implementing biodiversity programmes. The Conference of the Parties to the CBD has been making decisions on how to improve the taxonomic base for the conservation and sustainable use of biodiversity since 1996. In May 2000 the fifth meeting of the Conference of the Parties (COP V) urged Parties, Governments and relevant organizations to undertake a range of activity, including: • Establishing a coordination mechanism for the Global Taxonomy Initiative; • Identification of national and regional priority taxonomic information requirements; • Assessments of national taxonomic capacity to identify and, where possible, quantify national and regional-level taxonomic impediments and needs, including the identification of taxonomic tools, facilities and services required at all levels, and mechanisms to establish, support and maintain such tools, facilities and services; • Establishment or consolidation of regional and national taxonomic reference centres; and • The building of taxonomic capacity, in particular in developing countries, including through partnerships between national, regional and international taxonomic reference centres, and through information networks (CBD 2000). In April 2002 the sixth meeting of the Conference of the Parties (COP VI) emphasised that taxonomy was a priority for the implementation of the CBD, and agreed to a comprehensive Programme of Work for the GTI, and urged Parties, Governments, international and regional organizations, and other relevant organizations to promote, and, as appropriate, carry out, the programme of work (CBD 2002). This GTI Programme of Work has five major operational objectives: 1. Assess taxonomic needs and capacities at national, regional and global levels for the implementation of the Convention 2. Provide focus to help build and maintain the systems and infrastructure needed to obtain, collate and curate the biological specimens that are the basis for taxonomic knowledge. 3. Facilitate an improved and effective infrastructure/system for access to taxonomic information; with priority on ensuring countries of origin gain access to information concerning elements of their biodiversity.

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4. Include, within the major thematic work programmes of the Convention, key taxonomic objectives to generate information needed for decision-making in conservation and sustainable use of biological diversity and its components. 5. Include, within the work on cross-cutting issues of the Convention, key taxonomic objectives to generate information needed for decision-making in conservation and sustainable use of biological diversity and its components (CBD 2002). Our current taxonomic system is a long way from perfect, as we still don’t know all the components of biodiversity, and indeed for some components, like most micro-organisms, we are not even able to properly describe them. Recent estimates of species numbers suggest that somewhere in the vicinity of 12 million species live on Earth, with approximately 1.76 million described (Hammond 2000). Furthermore, current description rates when averaged out over decades, for all taxon groups except birds, shows remarkably stable trends in the number of species described, neither major decreases or increases (Hammond 2000). In the face of this, the trend for the number of practising taxonomists in major institutions is decreasing, and the overall trend shows the average age of taxonomists is increasing, and that within 10-15 years we will face a major loss of expertise as many current experts retire. Against these negative trends we find that the demand for up-to-date complete taxonomic information is greater than ever, with the need for reliable taxonomy to underpin ecological inventory and assessment at a level never seen before. And so we have the quandary, what should we do first? How do we prioritise the taxon groups that should be completed now? How do we utilise the information that already exists in museums and herbaria, but is inaccessible in its current form? The Global Taxonomy Initiative has been set up to provide answers to these questions, and more, in order that the Convention on Biological Diversity can answer its mandate of coordinating the conservation and sustainable use of biological diversity. Critical for conservation and sustainable use is an understanding of what the elements of biodiversity are, and how they depend on each other, in order to truly adopt a proactive ecosystem approach, as required by the CBD mandate. Throughout the world, and especially in the case of developing countries, existing taxonomic information is often not organized in a way that the CBD objectives can be achieved. The Global Taxonomy Initiative (GTI) The GTI is a global policy response to help remove or ameliorate the taxonomic impediment, to help in the identification and filling of the gaps in our taxonomic system, be they data or human and institutional capacity. A major component of the GTI is aimed at addressing the needs of developing countries in capacity building, which will need to happen in conjunction with taxonomic institutions world-wide. GTI activities must be broadly-based on the needs of the Convention and linked with on-going activities with related scientific initiatives eg, Global Biodiversity Information Facility (GBIF), Association of Systematics Collections (ASC), BioNET INTERNATIONAL, Species 2000, DIVERSITAS and IUBS.

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Ways and Means One effective way to assist recipient countries overcome the taxonomic impediment and promote CBD objectives of conservation, sustainable use and benefit sharing would be to support efforts to organize available taxonomic information in electronic databases, especially geo-referenced data. But we can’t build databases simply for taxonomic information alone, they must be clearly designed to also support a broader understanding of ecological systems. Global information systems must provide easy access to taxonomic information now often lodged in herbaria, museums and libraries of countries of the north; rather than repatriation of physical specimens since the latter would call for much greater effort and financial investments (as well as being politically difficult). The Global Biodiversity Information Facility (GBIF) will provide a major platform for this to be achieved (GBIF 2002). Such efforts will also benefit from the development of easily searchable meta-databases (i.e., databases of existing databases). Another exercise in the management of information would be to include along with taxonomic information, popular names and names in different local languages. Assistance will also be needed not only to build taxonomic capacity, but capacity in the use of information technology. The taxonomic impediment cannot be resolved without developing the human and institutional capacities in developing countries. Examples of success are INBio (INBio 2000) and SABONET (SABONET 2000), and the programmes of BioNET INTERNATIONAL (BioNET 2000). UNESCO also recognizes the importance of an effective taxonomic base in the environmental sciences is essential. UNESCO’s main involvement with taxonomic products is through developing effective management of biological diversity through its’ World Network of Biosphere Reserves. This network covers a representative - and growing - sample of the major biomes and ecological regions of the earth (In June 2002 408 Biosphere Reserves in 94 countries). They are linked by scientific exchanges and sharing of experience. Biosphere Reserves are recognized areas of representative environments, which have been internationally designated within the framework of UNESCO’s MAB Program for their value to conservation through providing the scientific knowledge, skills and values to support sustainable development. But to achieve these objectives an effective taxonomic base is needed, especially in developing countries. A particular initiative, which will interact with the GTI, is the Biosphere Reserve Integrated Monitoring programme (BRIM). This initiative includes MAB Flora and Fauna – a series of databases detailing the major taxa known from Biosphere Reserves. UNESCO is also undertaking an effort to integrate traditional taxonomies with more recent approaches, so as to link cultural and biological diversity. More generally, UNESCO sees the need for a considerable increase in taxonomic capacity in order to provide: • • • •

A scientific basis for action under CBD; Food security, Identification and control of human disease vectors, Identification and control of vectors contributing to ecosystem dysfunction, &

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• An understanding of preventative and curative agents found in biodiversity. The role of the Global Environment Facility (GEF) The Scientific and Technical Advisory Panel (STAP) of the GEF considered the issue of taxonomy in July 1999, and produced some key guidance for GEF to pursue in ensuring the role of taxonomy in its work: • How management and use of taxonomic information can help the Conventions address the issues of conservation, management, benefit sharing and sustainable use of biodiversity; • Needs and priorities assessment at the national and regional level; • Mechanisms at the national and regional level for capacity building required to address the taxonomic impediment; • Taxonomy related products such as electronic identification keys and monitoring; • Linking institutions in North-South and South-South with collaborative activities. Yet there has been little effect from all these fine words and ideas, perhaps now the Parties to the CBD have agreed on a clear workplan for the GTI we can develop new, and just as importantly expand existing, initiatives and projects, to fulfil GEF requirements to clearly meet the needs of the Convention when funding taxonomic projects. What taxonomists will need to do much better than ever before is to demonstrate how taxonomy can help in the implementation of the work programmes of Conventions like the CBD. Discussion Maintaining and improving the existing taxonomic infrastructure is a costly business, and new strategies are required to maximise our past investments, while minimising the costs and maximising the benefits of future investments. The Conference of the Parties of the CBD has urged countries to jointly undertake a massive rebuilding of taxonomic capacity. We need to explore globally how we can achieve the best possible outcomes for improving taxonomic capacity regionally. Perhaps at first glance some efforts will appear to run counter to commonly held wisdom, such as provision of funds from developing countries to maintain collections in the established Institutions in the North, while at the same time opening up much easier access to the data and expertise associated with these collections. Importantly though taxonomists need to improve overall performance in terms of • • • •

publication rates, broader use of taxonomic information simplicity of product use, and understanding the imperatives faced by policy-makers and managers in the biodiversity field.

It is clear this can happen: now we must MAKE it happen! The best way is through a consolidated voice on behalf of taxonomists. For example, in February 1998 a group of

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experts gathered in Darwin, Australia and discussed “Removing the taxonomic impediment”. The Suggestions for Action from that meeting (ABRS 1998a) were taken to the 4th meeting of the Conference of the Parties of the CBD in Bratislava and became the basis for definitive actions that the governments of the world should undertake (CBD 1998). Similarly, DIVERSITAS organized meetings in September and October 1998 that further defined key actions to improve taxonomic knowledge (ABRS 1998b, AMNH 1998), and many of the recommendations from those meetings are now part of the decision from the 5th meeting of the Conference of the Parties in Nairobi (CBD 2000), and are an integral part of the GTI Progamme of Work (CBD 2002). We must not lose sight of the value of joining together to seek solutions to problems that individually seem insurmountable. The ICZ must also play its part in helping to remove the taxonomic impediment and to promote better awareness of the role (at all levels of the biological hierarchy) of biological diversity to human society. Future Congresses should include specific sessions that provide a forum for zoologists to demonstrate their vital role in fulfilling these new biodiversity policy initiatives, like GTI. It is up to all of us to work together to actually implement the fine words and lofty ideals that is the GTI, and to then promote these at local, national, regional and global levels. References ABRS 1998a. http://www.anbg.gov.au/abrs/flora/webpubl/darwinw.htm ABRS 1998b. http://www.anbg.gov.au/abrs/flora/webpubl/london.htm AMNH 1998. http://research.amnh.org/biodiversity/acrobat/gti2.pdf BioNET INTERNATIONAL 2000. http://www.bionet-intl.org/ CBD 1992. http://www.biodiv.org/convention/articles.asp CBD 1998. http://www.biodiv.org/decisions/default.asp?lg=0&m=cop-04 CBD 2000. http://www.biodiv.org/decisions/default.asp?lg=0&m=cop-05&d=09 CBD 2002. http://www.biodiv.org/decisions/default.asp?lg=0&m=cop-06&d=08 GBIF 2002. http://www.gbif.org/ HAMMOND P. 2000. Address to the XXIst International Congress on Entomology (ICE), Iguassu Falls, Brazil, http://www.embrapa.br/ice/ INBIO 2000. http://www.inbio.ac.cr/ SABONET 2000. http://www.sabonet.org

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Taxonomic impediment in the study of The marine invertebrates 637 New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 637-642, 2003

Taxonomic impediment in the study of marine invertebrates K. Fauchald Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington DC, 20560-0163, USA

A primary element of the taxonomic impediment is access to information. We must make available what information already has been published or is present as records in museum collections. Once that has been done, the next step will be to encourage the development of new information where it is missing. An example may illustrate the situation. The collections of the Department of Invertebrate Zoology at the National Museum of Natural History, Smithsonian Institution includes more than 31 million cataloged or accessioned specimens and probably about the same number of specimens not accounted for in any fashion as unsorted benthic or pelagic samples. We are currently making information about these 31 million specimens available in the form of a new cataloging system, which will be mirrored on the web. Even this, relatively simple task, is expensive and labor intensive, since we are doing basic data cleanup as we go. We know that much more information exists since we have the material, but this half of the job will require considerably more work than the first. Museum collections and the catalogs are used to retrieve specimens; each collection represents a historical record of the development of zoology. The scientific names in the catalogs were appropriate the last time the specimens were examined and identified. We keep a record of every re-identification of the specimens since future scientists will be able to examine the exact specimens used by earlier authors, making interpretations much easier. However, unless a collection has been re-examined recently, the scientific names for each record represents a point in time in the 200+ years of progress in descriptive biology. Unless an easy-to-use cross-reference system is available, retrieving all information about a species described early is simply impossible without reexamination of the specimens. Such examination requires the attention of a scientist with expertise in the group in question. “Invertebrates” are members of every metazoan phylum. Phylogeneticists studying invertebrates, whether through morphological or molecular means, are generally focused on reconstructing links between the major clades (e.g., Nielsen 1995); some of us are somewhat more narrowly focused, keeping our attention on a single phylum or class and attempting to resolve issues of relationships within a more easily defined taxon (e.g., Rouse & Fauchald 1997). Traditionally students of invertebrate zoology have

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worked without much collaboration, since the number of shared features of taxonomic importance from one phylum to another is minimal. The terminology, especially as it concerns the morphology, has developed largely independently in each major group studied by a distinct group of scientists. Most phyla of marine invertebrates remain poorly studied; consequently the number of newly described taxa per systematic-taxonomic paper is high and can be expected to remain high for the foreseeable future. For example, in the Annelida new taxa are sometimes subfamilies, but more usually genera and species (for documentation see Zoological Records for the last several years). Only rarely are new families or any higher taxa newly described and those that are newly named, are often lumped back into one or another of the previously known taxa once a study of shared derived similarities has been performed (e.g., Pogonophora and Vestimentifera, see Rouse & Fauchald 1997). In the Annelida, as in many other phyla, there are two distinct levels of problems. One is related to the relationships among the highest included taxa, the other is linked closely to the terminal taxa, species, if you will, as documented by the presence of unresolved polytomies in trees such as those presented by Rouse and Fauchald (1997). The problematic taxonomic situation has distinct consequences for non-systematic studies. The lack of detailed knowledge, especially of terminal taxa, means that analytic studies, such as for example syn-ecological studies are hampered by a lack of consistent information. In addition, in most standard benthic investigations only a fraction of the fauna can be identified and enumerated; other organisms may be recognized to the phylum level. In many benthic sampling programs a large, but variable number of specimens is never accounted for at all, but are tossed out as unrecognizable organic material, either because they are too fragile to sample well, or because the treatment of the sample was too rough. I do not want to name any particular benthic investigation for documentation of these statements; I would suggest studying the tables of materials identified in a few published benthic investigations. I would especially suggest that a list of the phyla identified to species be compared to a list of material identified to phylum but also to a list of all phyla potentially present in the area. In part these abbreviated lists are a necessity; it is simply impossible to find scientists capable of getting the animals collected identified in a timely fashion. In turn this is due to an absolute lack of qualified scientists, in part is due to a lack of comprehensive and readily available studies that could be used as aids in identification by less well trained staff. In other fields, such as physiology or biochemistry, comparative studies of an appropriate scale have been undertaken for very few terminal taxa within each phylum. Species called by the same name from widely scattered areas often do not belong to the same terminal taxon. Taxonomic traditions have developed in the use of a given name for any of the better studied areas so you may find 100 years worth of physiological studies for example, from a given area, with a completely incorrect name applied to it. The number of taxa yet to be described has been calculated. Even the most conservative ones demonstrate that we have an overwhelming task in front of us. While in the best studied phyla, only few taxa may remain to be described, in most about half of the taxa are currently known; in poorly studied groups we really have no good base for making any kind of calculations, such as for example for marine free-living nematodes (W. Duane Hope, personal communication).

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Over the last several years the numbers of scientists involved with primary systematic studies of invertebrates has decreased with anywhere from 20-50%, depending on taxon and geographic area. At the same time we know that for example the deterioration of the coral reefs have accelerated, partly due to human interference, partly due to climatic shifts (Wilkinson 2000). If we are going to describe the diversity we clearly need to increase the number of taxonomists but just as important is the increase in the efficiency of each of us. Many practical problems influence the situation and make it difficult for us to make strategic investment of time and effort. Primary descriptions of new taxa in many groups are fairly routine and can be undertaken by mid-level trained persons. These descriptions represents the data-points needed for phylogenetic, geographic, ecological and all other large-scale comparative studies. Ph.D-level systematists ought to be relieved of primary descriptive work whenever possible; they should be expected to develop the major theoretical and practical concepts that inform research and forms the knowledge background for integrated information systems. The systematists should create the means wherewith systematics is done: The phylogenetic analyses, the keys, the monographs and the complex computerized applications. This in turn leads to material making it possible for all scientists to trust the use of a particular scientific name. We need a cadre of taxonomists to give us competent assistance in sorting, primary identifications and primary descriptive work. Taxonomists must be able to identify the bulk of the taxa in well-studied areas; even in poorly studied areas, they should be able to sort out the material that needs detailed treatment. The rest can be cataloged in suitable chunks in our collections, for example, by genus or family. Such a process, the details do not matter and must be considered local options, can limit the work of the systematists to taxa where their training and knowledge is needed. Cataloging by higher taxon, consequently making it available also by locality, will make the available material much more easily retrievable and that in itself will save time and increase efficiency: We will be able to find all material of a higher taxon available, not only the often much smaller fraction that has been fully identified. The presence of the taxonomists will lead to a restructuring of administration of collections and in personnel practices of museums worldwide. The taxonomist career must be at least as long as that of a systematist, in that the taxonomist will probably have a Master’s Degree level of education, compared to the PhD and post-doctoral experience very nearly required of systematists in major positions. Thus, the taxonomists are going to be at least five years younger when hired than will the systematists. The problem with communicating results of systematic studies can be divided into two distinct sets of problems: The very slow speed of publications and the lack of comprehensive dispersal of research results. Both can be helped with current technology. Traditional keys, monographs and papers are inadequate; for example, most published keys are usable only for experts who do not need them. The keys in my study of the genus Eunice is a characteristic example (Fauchald 1990). However, we have the means to streamline and rationalize taxonomic work making descriptions and illustrations available so that newly recognized taxa can be readily incorporated into overall schemes. Web-based, integrated data systems must be used to store, structure and transmit information. The formatting of the information must be sufficiently simple so that any

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person capable of reading and handling a computerized database, can use the information and when needed have on-line access to primary training. Integrated systems must contain interactive, heavily illustrated keys, necessary references to paperpublications, preferably with all early original descriptions directly available (as scanned from the original publications), and linked to the presence of specimens in collections. Links to basic training material could suitably contain invertebrate texts, or lectures notes covering invertebrate zoology in general. The applications must not be linked to a specific software or hardware: Future developments in computer technology will leave us with even more rapid and readily retrievable applications; linking us to a specific technology will inevitably slow down the process of dispersal. The change in publication pattern to an electronic format will change systematic zoology as a conceptual domain but it will also have practical consequences. For one, the Code of Zoological Nomenclature will have to be modified again, to take the new situation into account. In addition, hiring and promotion of taxonomists and systematists will have to credit the new kinds of publications.. There are complex problems associated with internationally shared systems; these problems must be resolved in international fora of various sorts (e.g., GBIF, GTI). However, the magnitude of the taxonomic impediment, in the more limited sense of this phrase, cannot really be estimated before we have taken advantage of all the technological means we have for sharing and streamlining the available information. However, there is no doubt that we are not training enough systematists. Ten years ago a committee in the Department calculated if we would be likely to find competent scientists who were US citizens to fill positions in the Department by the year 2000. We came to the conclusion that we would have available to us no more than five or six morphology-based systematists and a similar number of molecular based students as applicants for positions to cover the 30+ phyla of non-insect invertebrates. At the time we had a staff of 18 scientists plus four active emeriti; of these 22, 10 could be expected to have departed by 2000. To be minimally qualified for a position at the Museum, we assumed that a student would start graduate studies by 1992-1993 and requested information about plans for new students from our colleagues around the country. For most invertebrate phyla we were unable to find any candidates at all; only for the really species-rich phyla did we find more than one or two. Thus the supply of candidates looked bleak since we are expected to hire only US citizens. The ten years since our estimate has borne out our predictions; we have now 14 active curators, of which the oldest is 81; we have no scientists younger than 45 and only one emeritus is left. Thus we now have a grand total of 15 scientists working on the invertebrates, in contrast to 10 years ago when we had 22. We are not expecting to be able to increase the permanent staff soon, but may be able to get sufficient temporary funding to hire scientists on contract for specific projects. We are actively studying various options. The distribution of invertebrate zoologists appears to have shifted, at least in the group I study; there are now relatively more polychaetologists in many other countries than in the United States, including many relatively poor nations. We are clearly and obviously short trained people. In the United States very few systematists are trained. The reasons are not difficult to find; over the last 60-100 years systematics and especially taxonomy have been considered second-rate sciences;

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handmaidens to the experimental fields. Relatively little money and attention has gone into the study of comprehensive comparative biology, another phrase which describes systematic zoology well. One obvious reason is that a focus on human health is much more likely to get the attention of the general public than a focus on marine worms would in a world in which the study of both is considered part of the same field. In part we have ourselves to thank for this development. We have focused intently on the orderliness of the naming system. This focus on nomenclature has to a considerable extent become our defining feature as far as other scientists and the general public are concerned. The Code which is absolutely necessary to maintain an orderly information flow, has also become a millstone in its focus on precedent and prior publication. In the future we must no longer focus our attention on names, but on organisms. One of the greatest boons to the study of comparative biology would be a moratorium on looking up old names, especially when no specimens could be linked to the names. We are comparative biologists, all observations we can make that can be linked to the genome, are grist for our mills. The discussion of analytic methods, such as among the cladists, is also somewhat of a distraction: We get pre-occupied with algorithms and the issue of what is “best” under all circumstances. The methods we use are not ideal, and will certainly be improved as time goes by, but a search for the ideal analytic method is a chimaera; it does not take much philosophical acumen to demonstrate that no single method can resolve all problems. Early taxonomists could get away with just a sentence or two in their descriptions; we are lucky if we can get away with a page or two of text. This does not mean that we are describing the animals completely any more than they did: None of us will ever fully describe any organism, since there is an infinite amount of observations that can be made. Observations that appear trivial at this point may later turn out to be important. I believe that comprehensive retrieval systems for ALL information about a taxon is to be preferred to one that is limited to what is now considered to be taxonomically important features. A well developed database should allow us to make ecological and physiological predictions at appropriate hierarchical levels, making it possible for scientists to spend less time on identification and more on the appropriate questions. The most recent development in systematic theory appears to be at odds with this demand. A cladogram is a clearly hierarchical structure, but the numbers of branching points between the terminal taxa and the rather arbitrary taxonomic level considered a phylum varies enormously and the level of predictability at any given level varies enormously. The use of specifically named hierarchical levels tempts scientists to compare membership of each of the levels across large-scale groups, making the assumption that a “family” or “class” has some characteristic property independent of the group to which it has been assigned. These kinds of comparisons cannot be accommodated, but integrated information will be available at various levels within each clade. Clearly the standard hierarchical system lacks sufficient levels to account for the branching in most areas; however, each of the branching point can be named as appropriate or needed without references to levels.

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What we need, in addition to an increased number of scientists, is a much more flexible and comprehensive way of getting information issued and distributed. A differentiated staff of systematists, taxonomists and parataxonomists (sensu Janzen) to develop the necessary information would probably also increase the information flow. Resolving the impediment must be an international effort, one in which all the inter-governmental aspects of the interactions can be taken care of in a reasonable manner and one in which the scientists can increase the efficiency of their ongoing collaborations, without having to worry unduly about unrelated issues. References FAUCHALD K. 1990 A Review of the Genus Eunice (Polychaeta: Eunicidae) Based upon Type Material. Smithsonian Contributions to Zoology 523: 1- 422. NIELSEN C. 1995. Animal Evolution - Interrelationships of the living Phyla. Oxford University Press, Oxford, IX + 467p. ROUSE G.W. & K. FAUCHALD 1997. Cladistics and polychaetes. Zoologica Scripta 26(2): 139-204 WILKINSON C.R. (ed.) 2000. Status of Coral Reefs of the World: 2000. Global Coral Reef Monitoring Network, Australian Institute of Marine Science, Townsville, Qld, XII and 363p.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) A “Taxonomic Affidavit”. it is needed? The Why New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 643-646, 2003

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A “Taxonomic Affidavit”. Why it is needed? F.D. Por Department of Evolution, Systematics and Ecology, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel. E-mail: [email protected]

Nobody would question the basic requirement that every research paper in the experimental fields should clearly state its material and methodology in order to allow for others to repeat and check the results obtained. Although there are always isolated cases of unintentional sloppiness and even of intentioned fraud, the very structure of modern experimental science is based on the principle reproducibility. If the same material is used and the methodology is flawless, the influence of subjective factors and circumstance is minimal and, what is more important, can be sorted-out in the eventual repetition of the research. Good experimental science is by definition based on replicable results. Natural history, in its classical sense, is in a far worse situation. Since studies in this field are necessarily anti-reductionist, the complexities of the live objects and the inevitable background noise of the environment, reduce the chances of replicability of the results. Statistics comes to the help and if the uncertainties are inevitable, at least a high probability in reaching the same results can be expected. By repeating the same methodology in the laboratory or in the field, and taking the environmental factors into account, we can reasonably expect to obtain results which are consistent with the original ones, if the objects of the study are identical. The main point of weakness is therefore the identity of the live objects of the field researches, or of the specimens collected in the field for experimental purposes. This identity should be unmistakably established. Without the possibility to clearly identify the species on which the work has been done, the whole project is fatally flawed. Its results cannot be validated and cannot serve as a basis for further research. In practical terms, good taxpayer money is being often spent on dubious results. One cannot avoid he fact that each living individual is in principle different from every other living individual at present and even more certainly so, in the past. Conversely, each quantum of force, each chemical element and even every organic molecule, complex as it might be, is strictly identical to its correctly measured and identified peer. Near-identity can be achieved among the individuals of strains of laboratory organisms., or among cloned organisms. However, these laboratory bred individuals, supremely useful in basic biochemical, physiological and biomedical research are no

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paragons of the biological beings bred naturally in the environments and freely interacting with their living surroundings. They ceased to be specimens of a biological species in the strictest sense of the word. A cloned frog, genetically frozen in time, can compare only to a limited extent with a “natural” frog born of amphimyxis and developing under its specific microclimatic conditions. Even extremely wide-ranging and expensive experimental projects can suffer of sloppy taxonomy. This was for instance the case with the genome project of Caenorhabditis ellegans. Special care is being taken now in order not to mix Arabidiopsis thaliana material with that of its different congeneric species. Research done on natural populations is vulnerable to the flaw of irreproducibility, first of all, because the taxonomic uncertainty. Does this mean that identification of the species on which the study is done can be entirely neglected as an unrealistic requirement and a thankless complication? We encounter here a dangerous vicious circle. Among the non-taxonomist biologists there is a growing lack of confidence about taxonomic identification. The frequent changes in the taxonomic position of a certain organism bred impatience on the side of the nontaxonomists, often bordering with ridiculing. One can often meet biologists who will try to convince that taxonomic identification is impossible because the taxonomists themselves are uncertain of their decisions and that nomenclature with its long list of synonymies is unintelligibly esoteric. The conclusion, as it filtrated up to the granting levels, was that taxonomy is not worth investing in. In a sense, those who would be more in need for taxonomic services are joined up against taxonomy itself. Happily, this was the situation before Rio de Janeiro, but the concrete results in the field are late in materializing. There are many research projects that are based on complete disregard for the need of basic taxonomic information. It would be of course embarrassing to give concrete example, but everybody can easily find many such cases in his own field of specialty. There are for instance such cases like those in which chironomids are all treated like “detritivores” because the classical Chironomus plumosus. is one. Or cyclopoids are collectively treated as “plankton”, although very many of them are substrate-bound. In other cases the faunal lists of an environment receive differentiated treatment: The vertebrates are identified to the species level, the small invertebrates are treated as OTU’s (Observable Taxonomic Units), and dabbed “cylopoid 1”, “cyclopoid 2” and at the end of the list appear “Ostracoda spp.” and “Nematoda spp.”, cases in which even the higher taxonomic ranks are left uncertain. In many cases identifying to the generic level is considered as sufficient. This does not prevent the authors from calculating similarity indexes between the different biota, in which each item receives the same statistical value and to construct nice branching graphics. Still in other cases the authors prefer to use vernacular names, such as “the lobster” “the mud-puppy”, leaving to the reader the task of finding the correct name of the treated species. To add more complication, the vernacular nomenclature does not respect the strict rules of Linnean nomenclature and, perhaps with the exception of the Japanese vernacular names, there is an uncontrolled liberty to use different “popular” names for the same species. Most bewildering are the vernacular trinominal names, such as those of the birds. And what does, for example a Russian reader make out of American vernacular names?

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Already on the more serious side, there are cases in which old identifications are used for decades in successive field-studies and the authors are not aware that the original status of the species has changed in the meantime. The brine shrimp Artemia salina is a classical example of such a stubborn survivor in literature. The originally held “cosmopolitic” species has been long since split-up into many congeners. In a worse scenario it may happen that unawares, the original species was even replaced in the ecosystem by a superficially similar looking invading species, or by a species that corresponds more to the changed environmental conditions. Taxonomy has advanced much in the last decades, both by using micro-morphological criteria and especially by the use of molecular methods. Very many of the old-time species of our environments have to be submitted to revision. In many cases they turnout to be complex sibling species, each with different physiological requirements and ecological behavior. This makes things more difficult for the impatient field researchers, but since when was Science a simple matter? The criticized authors can rightfully answer that it is difficult and time wasting to find a specialist who is ready to identify the species, or at least to confirm the older identification. They are often right in this and here we meet a second vicious circle. For instance there are in the world today only about 10 people who are able to identify and if needed, to describe new species of marine Copepoda Harpacticoida It will be difficult to ask one of them for a collegial help and wait for accurate identifications. Nevertheless, on one square meter of reef or one grab sample of mud, might contain as many as 30 species of such harpacticoids. These need to be identified. If the sample happens to be from fresh waters, there is practically no specialist in attendance in the whole world, to identify freshwater Harpacticoida. The world list of taxonomists is getting shorter by the year. There is an increasing number of “orphaned” taxa, groups of animals for which there is no specialist anymore. Among the Coleoptera, the most numerous order of the animal world, there are around a dozen of families without a living specialist. Not only species and environments disappear, so do taxonomists. With less and less funds going to basic taxonomic research, this trend seems to be unavoidable. Does this mean that future field research is doomed to be less and less accurate, less and less valid? This basic quandary of the biodiversity research, the global demise of taxonomy, called “ taxonomic impediment”, cannot be solved by one single measure and of course not rapidly. I am proposing here one of the modalities, one treatment for the cure of the impediment. Briefly it sounds like this: Each paper and research report dealing with naturally occurring biological species should present a “Taxonomic Affidavit”. Under the heading of the Materials and Methods, the accurate identification of the species concerned should be mentioned with the identifying taxonomist acknowledged, as well as the location of the museum collection in which the studied material has been deposited. Requirement of a repository should be made also for unsorted “mother samples”, or for unidentified material. In this way at least, future researchers will be able to refer to

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a comprehensive baseline situation presented in the previously published article, and to complete the lacking taxonomic information contained in the treated material. Identified species that are reposited in specific museum collections should be accessed or obtained on loan for eventual future validation or taxonomic revision. If the two requirements of the “Taxonomic Affidavit” will be accepted and required by publishers and granting agencies, just as they request other methodological information, the vicious circle can be broken. Adequate budget allowance for taxonomic services and for the repository services will become an integral part of every research proposal dealing with biota. The accuracy of the identifications should be subject to peer revision, just like any other aspects of the manuscripts. With more research funds available for taxonomic work, more taxonomists will be able to receive a proper financial support for their specialty. Universities interested in capturing research fund money will stimulate and prepare young researchers to specialize in supplying taxonomic services in a wide array of animal (and plant) taxa. This would be one of the avenues to remedy the present world-crisis in trained taxonomists. Museums will be requested to increase not only their staff of taxonomists, but also develop sorting programs for unsorted field samples and of course radically increase their repositing services. All this might sound like a utopia. But the alternative is the increasing volume of less and less reproducible and controllable field results despite the astronomic sums of money squandered on them. At least some of the money should find its way to the laboratory of the taxonomists. Good science will profit.

A “Taxonomic Affidavit”. Why it is needed?

The New International Code of Zoological Nomenclature and Related Issues

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT PublishersZoological nomenclature after the publication of theThe Fourth EditionofofAnimal ... Evolution 649 New Panorama Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 649-658, 2003

Zoological nomenclature after the publication of the Fourth Edition of the Code A. Minelli Department of Biology, University of Padova, Via Ugo Bassi 58 B, I 35131 Padova, Italy. E-mail: [email protected]

Abstract The fourth edition of the Code marks a few sensible changes in the way the scientific names of animals are produced and used, but more momentous changes may be waiting ahead. Some prospective advances, e.g. the possible adoption of registration procedures, will still be within the scope of Linnaean nomenclature but the latter, perhaps, will not be able to accommodate for all kinds of names required by zoologists. The frequent use of formulae rather than Linnaean binomens, e.g. in phylogeographic studies, exemplifies conceptual and practical requirements beyond current species concepts and species names. Furthermore, the formal ranks of the Linnaean classification are at odd with the principles of phylogenetic systematics. No doubt, future zoological nomenclature will be influenced by the concepts and requirements of evolutionary biology. Linnaean and not-Linnaean classifications will be perhaps developed side-by-side. Others may wish to replace the Linnaean classification altogether but I strongly recommend, instead, dialogue and co-ordination of efforts. To ignore our historical heritage would be not less disastrous than to ignore the conceptual limits of Linnaean nomenclature. Rules may require evolving but history teaches that viable codes follow and consolidate practice, they do not establish it from scratch.

Congresses, Commission, Code Sessions on Zoological Nomenclature have been a prominent feature of all previous editions of the International Congresses of Zoology, starting with the very first Congress (Paris, 1889). However, it was at the 3rd Congress, held in Leiden in 1895, that an International Commission on Zoological Nomenclature (ICZN) was first established, under the leadership of Raphael Blanchard. This Commission eventually produced the Règles de la Nomenclature Zoologique that were adopted by the 5th Congress (Berlin, 1901) but only published in 1905. Discussions developed at later Congresses lay also behind the adoption of the first document actually known as an International Code of Zoological Nomenclature. This was published in 1961 and reissued in 1964 in a slightly revised second edition. Twenty-one years were to elapse before a third edition was issued, but

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in the meantime the zoological community had since long ceased to gather at its International Congresses. In fact, the 17th Congress, held in Monaco in 1972, seemed to be the last in the series. During that congress, responsibility for future Codes, as well as the Commission, was transferred to the International Union of Biological Sciences (IUBS). A third edition of the International Code of Zoological Nomenclature was published in February 1985, but the need for an fourth edition was becoming apparent quite soon. A new Editorial Committee was eventually appointed in 1988. The prospective new edition of the Code was discussed at open sessions during the Fourth International Congress of Systematic and Evolutionary Biology (ICSEB) at the University of Maryland in July 1990 and at the Section on Zoological Nomenclature during the IUBS General Assembly in Amsterdam in September 1991. A critical stage in the preparation of the new edition was a meeting of the Editorial Committee held near Hamburg in October 1993. After further revision, the resulting Discussion Draft was issued to the zoological community at large in May 1995. Many zoologists from all over the world contributed comments on this draft: more than 800 pages of documents from some 500 people or groups were received within 12 months from the first public circulation of the document. All these papers were discussed at a week-long meeting of the Editorial Committee held in Vicenza (Italy) in June 1996. Careful evaluation of this very extensive collection caused the Committee to redraft many provisions; some of the proposals in the Discussion Draft (such as the mandatory “registration” of all new names and the abandonment of gender agreement between generic and specific names) were abandoned, because of practical difficulties and/or because they were not acceptable to a sufficiently wide consensus of zoologists. The Committee’s conclusions were discussed by the Commission on the occasion of the Fifth ICSEB Congress (Budapest, August 1996), and its agreement on all major points was then endorsed by a meeting of the IUBS Section on Zoological Nomenclature. The main features of the new Code were publicized on the World Wide Web. In 1997 the major changes in the Code were accepted in an indicative postal ballot of the whole Commission. The Editorial Committee proceeded further with the task of polishing and checking the text. In the meantime, a team based at the Muséum National d’Histoire Naturelle, Paris, started working on the French text of the Code. This proved of major importance for the final polishing of the English text too. In October 1998 the Code was circulated to the Commission for the definitive three-month vote to adopt it as the fourth edition. At the same time the new Code was made available to the Executive Committee of IUBS, and this has ratified it on behalf of the Union’s General Assembly. This Fourth Edition of the International Code of Zoological Nomenclature was published in August 1999 and has taken effect from 1 January 2000. Since then, official versions in language other than English and French have been published (Spanish) or are about to appear (Russian, Japanese, German). The Fourth Edition I am confident that the new text is quite better than the preceding versions. David Ride, former President of the Commission and Chairman of the Editorial Committee for this fourth edition, will tell you in more details about the qualities of this document.

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To be sure, many of the changes adopted will hardly be of interest to people other than professional taxonomists: I mean, the changes affecting neotypes, or lectotype designation, or the way a new name must be proposed. Other changes however, although still technical, are certainly more understandable and conspicuous: for instance, those affecting publication (a difficult topic, where the Commission has clearly shown an open mind in respect to the new electronic technology), and those through which the principle of priority has been cautiously relaxed in order to facilitate the preservation of names in demonstrably established usage but potentially threatened by senior synonyms unused for one century or more. I will discuss later another set of new provisions, those enabling the establishment of official Lists of Available Names. All these more or less conspicuous changes – I do not mention, of course, the literally hundreds of minor textual improvements embodied in the new edition – do not mean that both the Commission and the zoological community at large may regard the 4th Edition of the Code as the last word for many years ahead. Quite to the contrary, there are lots of questions left to be addressed in the immediate future. Registration Some of these questions are the direct legacy of the lengthy discussion on the many drafts of the Fourth Edition. The first drafts – in particular, the document publicly circulated in May 1995 – contained many more radical changes than those that were eventually adopted. The corresponding issues, however, are far from definitely settled. I will only mention here one of these issues, that is, registration. To an outside observer – but also to many of us in the trade – it appears unbelievable that up to now, despite the protested universality of science and the current availability of cheapest electronic ways for the immediate release of information word-wide, we still do not have a single official site or source where to find a list of all existing names of animals. All practising taxonomists know very well how much precious time is spent in browsing through a more or less scattered literature, both old and recent, in the search for papers dealing with the group of one’s interest, with the usual additional worry, that one is never sure not to have missed a few names published in some obscure book or journal not to be found in any major library and never recorded by the international abstracting services. To move away from this traditional state of affairs we must provide an easy and reliable access to the names already present in the literature as well to the newly published ones. It is widely known that both aspects of the problem have been resolved by bacteriologists, for the not too many thousands of names they have to deal with. First, they fixed a point in time (January 1st, 1976), as the starting point of a new treatment of taxonomic literature. An international committee was appointed to screen the whole literature for generic and specific names proposed before that date, so as to provide an official list of accepted names, that was eventually published in 1980 (Skerman et al. 1980, see also Hill et al. 1984). With the publication of that list, there was no need for searching further for old names. That is, the list provided a closed initial pool of names available to the systematic bacteriologist. As for the names published thereafter,

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the radical solution adopted by bacteriologists consists in officially acknowledging privileged media were to publish. All new names of bacteria published between 1976 (the starting point of the “new” nomenclature) and 1980 (publication of the Approved Lists of Bacterial Names) were retained as valid only if published in the International Journal of Systematic Bacteriology. That is, for the years 1976-80 bacteriologists adopted the principle of compulsory publication in one “registered journal.” From 1980 on, they moved however to the principle of “registration of names;” that is, a researcher is free to describe new taxa in any adequate journal, but the proposed names only receive nomenclatural validation when “registered” through a short notice published in the International Journal of Systematic Bacteriology (Sneath 1986). Accordingly, the International Journal is the only bibliographic source a bacteriologist must look for, to be aware of the whole nomenclaturally relevant literature. No additional name is to be found elsewhere. It must be stressed, that registration is not a way to exercise any censorship on the taxonomic evidence or rationale behind the introduction of new names but it is, in the essence, a way to obtain full and timely awareness of the actual existence of the names and other relevant nomenclatural acts. What matters most, however, is that unregistered names are as much as unpublished. This is a critical difference between the official registration of names and the already existing, precious but not official services traditionally provided, for instance, by the Zoological Record. Joan Thorne, however, will tell you later how the Zoological Record’s recording scheme has been recently improved and what its role could be, in the context of a registration policy possibly to be adopted by the Commission in the future. As it is already apparent in the bacteriologists’ example, two different approaches to registration are potentially available: registration of names and registration of journals. Registration of names means, that a new name may be published virtually everywhere, as now, but only takes validity at the time an adequate bibliographic reference to its publication is registered at one official centre (or network, or journal). Registration of journals, instead, would mean that valid publication requires the new name to appear in any of a limited set of officially acknowledged “registered journals.” Why not to apply some procedures for registration to the names of animals? To be sure, in regard to the number of old names to be listed and the new names to be annually registered, zoology is not bacteriology. Neither is botany. In this respect, it will be of interest to us zoologists to listen to Werner Greuter’s talk on an experiment on registration of names recently developed by botanists. Let me add, however, that botanists have very good comprehensive lists of all their generic names and also many good lists of specific epithets, whereas zoology suffers from a distinct lack of comprehensive and updated nomenclators, not only at the specific, but also at the generic level. Things are possibly changing, however. The recent monumental Catalog of Fishes produced by William Eschmeyer (1998) is an excellent example of the kind of work we need as a sound starting point for the future of zoological nomenclature. The International Commission on Zoological Nomenclature begun working towards the adoption of some form of registration as soon as the Editorial Committee for the fourth edition of the Code was appointed. Eventually, however, it proved too difficult to identify a workable and “politically” acceptable solution to the problem.

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Nevertheless, this fourth edition of the Code has already taken a significant step in this direction, by including provisions for the development and adoption of List(s) of Available Names in Zoology, i.e. documents equivalent, in meaning and force, to the Approved Lists of Bacterial Names. This will enable zoologists to settle their requirements with past names. The problem remains, however, of adopting a workable way for registering the new names. Names and formulae With the issue of registration we are still within the framework of the traditional Linnaean nomenclature, but, are we sure that the latter can satisfy all conceptual and practical requirements of today’s zoologists? Just browsing through the literature seems to suggest that it is really not so. I am not speaking, of course, of the current practice of simply calling C. elegans a little nematode whose generic name Caenorhabitis is often never quoted in extenso in the papers; neither do I point my finger against the not less common practice of calling Drosophila what is specifically meant to be Drosophila melanogaster. In such cases, there is clearly no conflict between science (e.g. genetics, developmental biology, molecular biology) and nomenclature, but simply an unpleasant disregard for nomenclature. Things are quite different, however, with the widespread use of formulae, rather than Linnaean binomens, to label the samples used e.g. in phylogeographic studies. The fact that this use is very common in a journal whose title is Systematic Biology (Systematic Zoology until 1991), only strengthens the case – that journal’s authors and editors are hardly unaware of the Code or, at least, of the basic rules of the Linnaean nomenclature. Why are those authors using formulae rather than Latin binomens, o trinomens? The reason is, that the populations sampled in those studies seem very often to defy a formal classification as species, or subspecies. And authors seem often to suspect, that the difficulty they experience in giving them a formal species or subspecies status derives from the populations themselves, rather than from the researchers’ ignorance. The formal ranks for which the Code provides naming rules are sometimes too narrow, too rigid, to accommodate the actual diversity of natural populations. The finer a phylogeographic study becomes, the more difficult it is to arrange the findings within the simple hierarchical scheme of classification we assume for granted as the taxonomic backbone of the Code. To the best of my knowledge, no effort has been produced until now, in order to standardize these formulae, where generic names, specific epithets, alphanumerical codes and geographical data are promiscuously used in very different ways; neither do I wish to recommend the development of a Code of Formulae. The existing (and increasing) practice, however, must invite a reflection on the basic proposition of the Code’s Preamble, that none of the Code’s provisions and recommendations “restricts the freedom of taxonomic thought or actions.” What should we do, when neither the species nor the subspecies level seems to be adequate to describe the entity we are studying? If the choice is, either to straightjacket the facts, thus labelling our populations in accordance to the Code, or to use informal but uncommitted formulae, outside the Code’s rules – I would not hesitate to embrace this second alternative. But, at the same time, I would immediately start discussing, whether there may be a way to

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improve the Code, so to allow these clusters of biological diversity to get names formally co-ordinated with those of the more conventional Linnaean taxa. Rankless nomenclature – the PhyloCode Other contentious issues stem from an increasingly widespread dissatisfaction with the Linnaean hierarchy. This is, in essence, a consequence of the spreading of phylogenetic systematics. In fact, in a tree of lower taxa nested within higher taxa, there is absolutely no criterion for assigning ranks: there are no genera, indeed, no families, no orders, no classes. Just taxa. De Queiroz & Gauthier (1990, 1992, 1994) were the first to suggest developing a rankless system of biological nomenclature (see also de Queiroz 1992, 1996,1997a, b, Rowe & Gauthier 1992, Bryant 1994, 1996, 1997, Sundberg & Pleijel 1994, Schander & Thollesson 1995, Lee 1996a, b, Cantino et al. 1997, Kron 1997, Härlin 1998, Härlin & Sundberg 1998, Schander 1998). More recently, prospects for the development of a code of nomenclature dispensing with Linnaean ranks have been rapidly developing, as Kevin de Queiroz will illustrate in his talk. After two years of lively discussion, a group of zoologists and botanists has produced the draft PhyloCode now available on the web. Prospects for its actual implementation, however, are still open to dispute. The next few years may prove critical in this respect. The species problem Possibly but not necessarily related to the issue of phylogenetic nomenclature is the rejection of the species as a biologically meaningful concept and, therefore, as the fundamental unit of biological classification. A few authors have developed what I once defined (Minelli 1993) as a nihilistic approach to the species problem. Nelson (1989) was possibly the first to explicitly claim that species are simply the smallest diagnosable units, i.e. the tips, of the nested pattern of lower taxa within higher taxa. Until recently, however, these views only remained in the realm of theory and abstract definitions, with the conspicuous exception of Cracraft’s (1992) paper who, disregarding the traditional distinctions between species and subspecies, raised from 40-42 to about 90 the number of existing (an named) “species” of birds of paradise. More recently, Pleijel (1999) has attempted to describe new taxa of polychaete annelids in a way that formally rejects the concept as well as the name of species. Pleijel’s descriptive units, for which he provides a uninominal nomenclature, rather than traditional binomens, are called by Pleijel & Rouse (2000) LITUs, that is, least-inclusive taxonomic units – something quite similar to the species defined according to Nelson’s nihilistic approach. I am obviously aware of the theoretical and practical problems we may have with all kinds of species definitions, either biological, or phylogenetic, or evolutionary, or otherwise. I have myself pointed my finger against the widespread mistake, especially common amongst ecologists, biogeographers and palaeontologists, to operationally regard as equivalent all those “objects” for which a Linnaean binomen has been provided: these seem to be all the same kind of things, right because to all of

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them a species name has been given according to the zoological (or botanical) code (Minelli 1995, 2000)! This is a real problem, but is it resolved by substituting the neuter (simply operational, hence definitely subjective) term LITU for the theory-laden (and often perhaps misapplied) term species? Will this really help describing biodiversity in a more meaningful way? I do not think so. Behind many recent efforts to reform nomenclature there is the desire to keep names apace with the changing views and concepts of biology. Up to a degree, this seems to be desirable. We do not object to our vocabulary expanding daily, to accommodate for terms describing the new concepts, objects and operations increasingly filling our private and professional life. To be sure, new concepts and new working tools are now taking stance in biological systematics, and this affects the traditional notion of species not less than other basic concepts of our business. I am not convinced, however, that calling LITUs, rather than species or subspecies, the least-inclusive taxonomic units, and providing these with a completely new nomenclature, would help more than would do simply declaring that we must be extremely critical of any special (or, at least, unique) biological sense we might have attributed in the past to the entities christened with Linnaean binomials. I think that most users of zoological nomenclature will nevertheless hope and expect that all these entities, conventionally called species, will continue to be named as in the past, for the sake on continuity with two centuries and half of scientific and applied literature. A plea for dialogue One hundred and five years since it was first established at the Leiden International Congress of Zoology, the International Commission on Zoological Nomenclature is now attentively examining what its future role should be. To be sure, the Commission’s duties are clearly written in Chapter 17 of the Code, as the Code’s raison d’être is clearly written in the Code’s Preamble. Nobody, however, can escape the perception that many things are changing, in zoology as well as in the society in which we operate. In the past, no problem would have arisen from the proposition, that the Code deals with the published names of animals, but it has become increasingly difficult to say what “published” may mean, or what an “animal” may be. Accordingly, operational definitions of “publication” have been introduced and progressively updated in the different editions of the Code, and the fourth edition includes also an operational definition of “animal.” Stability and universality of nomenclature are the obvious aims of all the Code’s provisions, but our current views on how to achieve them are not exactly the same as at the time the Commission was established. I reminded before, that according to the Code’s Preamble none of the Code’s provisions and recommendations “restricts the freedom of taxonomic thought or actions.” In the past, freedom of taxonomic thought or action could mean, that the Code should enable taxonomists to use adequate i.e. stable and universal names for all taxa involved, irrespective of subjective taxonomic opinions, that is, irrespective of whether the nominal

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taxa A and B are regarded as two good species to be placed in the same genus, or two different species belonging to different genera, or two subspecies of one and the same species, or even two subjective synonyms. Today, however, freedom of taxonomic thought may also mean, that none of the traditional species concepts is fully adequate to represent the taxonomic diversity within a given group, or that all traditional supraspecific ranks, such as genus, family, order, class, and phylum, are best disposed of as irreducibly arbitrary. All these perspectives would clearly represent a challenge to the Linnaean nomenclatural tradition, that is, to the actual text of the present Code, but this is a challenge that the Commission, as such, must be ready to accept. I am not suggesting that the Commission should be the active proponent of any radical change. I firmly contend, however, that it must always be an active partner in any discussion of possible changes, either small or large. My personal view is, that the conventional Linnaean hierarchy will possibly not survive alone. From the perspective of phylogenetic systematics, our traditional nomenclature is too prescriptive and too permissive at the same time. Too prescriptive, in so far as it forces all taxa (and their names) to fit into the arbitrary ranks of the hierarchy; too permissive, in so far as it can be equally applied to paraphyletic as to monophyletic groups. New proposals, such as the PhyloCode, are therefore expected. But even in the perspective of new developments, I believe that it will never be possible or desirable to dispose of 250 years of Linnaean taxonomy and nomenclature. One should always keep in mind that an important function of classifications is information retrieval. The Linnaean tradition will be perhaps supplemented, rather than replaced, by new semantic and lexical tools. As I recently said, by presenting the new Code from the columns of Trends in Ecology in Evolution, “It is time to open the debate, to avoid either party to go astray: Linnaeanstyle taxonomists on one side, patiently continuing to produce names that others may be unwilling to use, and phylogenetists on the other, perhaps too ready to change the rules. It took one century from Linnaeus to the Strickland Code, and another sixty years to the Règles. Let’s talk one another. Rules can still evolve but a code, historically, follows and consolidates practice. It does not establish it from scratch” (Minelli 1999). References BRYANT H.N. 1994. Comments on the phylogenetic definitions of taxon names and conventions regarding the naming of crown clades. Syst. Biol. 43: 124-130. BRYANT N.H. 1996. Explicitness, stability, and universality in the phylogenetic definition and usage of taxon names: A case study of the phylogenetic taxonomy of the Carnivora (Mammalia). Syst. Biol. 45: 174-189. BRYANT N.H. 1997. Cladistic information in phylogenetic definitions and designated phylogenetic contexts for the use of taxon names. Biol. J. Linn. Soc. 62: 495-503. CANTINO P.D., OLMSTEAD R.G. & S.J. WAGSTAFF 1997. A comparison of phylogenetic nomenclature with the current system: a botanical case study. Syst. Biol. 46: 313-331. CRACRAFT J. 1992. The species of the birds-of-paradise (Paradisaeidae): Applying the phylogenetic species concept to a complex pattern of diversification. Cladistics 8: 1-43.

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DE QUEIROZ K. 1992. Phylogenetic definitions and taxonomic philosophy. Biol. Philos. 7: 295-313. DE QUEIROZ K. 1996. A phylogenetic approach to biological nomenclature as an alternative to the Linnean systems in current use. In Reveal J.L. (ed.), Proceedings of a mini-symposium on biological nomenclature in the 21st century. University of Maryland, http://www.life.umd.edu/ bees/96sym.html. DE QUEIROZ K. 1997a. The Linnean hierarchy and the evolutionization of taxonomy, with emphasis on the problem of nomenclature. Aliso 15: 125-144. DE QUEIROZ K. 1997b. Misunderstandings about the phylogenetic approach to biological nomenclature: a reply to Lidén and Oxelman. Zool. Scripta 26: 67-70. DE QUEIROZ K. & J. GAUTHIER 1990. Phylogeny as a central principle in taxonomy: Phylogenetic definitions of taxon names. Syst. Zool. 39: 307-322. DE QUEIROZ K. & J. GAUTHIER 1992. Phylogenetic taxonomy. Ann. Rev. Ecol. Syst. 23: 449-480. DE QUEIROZ K. & J. GAUTHIER 1994. Toward a phylogenetic system of biological nomenclature. Trends Ecol. Evol. 9: 27-31. ESCHMEYER W.N. (ed.) 1998. Catalog of Fishes. California Academy of Sciences, San Francisco, 2905p. HÄRLIN M. 1998. Taxonomic names and phylogenetic trees. Zool. Scripta 17: 381-390. HÄRLIN M. & P. SUNDBERG 1998. Taxonomy and philosophy of names. Biol. Philos. 13: 233-244. HILL L.R., SKERMAN V.B.D. & P.H.A. SNEATH (eds) 1984. Corrigenda to the approved lists of bacterial names. Int. J. syst. Bacteriol. 34: 508-511. INTERNATIONAL COMMISSION ON ZOOLOGICAL NOMENCLATURE 1905. Règles internationales de la nomenclature zoologique. International rules of zoological nomenclature. Internationale Regeln der zoologischen Nomenklatur, Rudeval, Paris, 57p. INTERNATIONAL COMMISSION ON ZOOLOGICAL NOMENCLATURE 1999. International Code of Zoological Nomenclature. Fourth Edition, The International Trust for Zoological Nomenclature, London. xxix+306p. KRON K.A. 1997. Exploring alternative systems of classification. Aliso 15: 105-112. LEE M. 1996a. The phylogenetic approach to biological taxonomy: practical aspects. Zool. Scripta 25: 187-190. LEE M.S.Y. 1996b. Stability in meaning and content of taxon names: an evaluation of crownclade definitions. Proc. R. Soc. London B 263: 1103-1109. MINELLI A. 1993. Biological systematics: the state of the art. Chapman & Hall, London. MINELLI A. 1995. The changing paradigms of biological systematics: new challenges to the principles and practice of biological nomenclature. Bull. zool. Nomencl. 52: 303-309. MINELLI A. 1999. The names of animals. Trends Ecol. Evol. 14: 462-463. MINELLI A. 2000. The ranks and the names of species and higher taxa, or, a dangerous inertia of the language of natural history. In Ghiselin M.T. & Leviton A.E. (eds.) Cultures and institutions of natural history. Essays in the history and philosophy of science. San Francisco. California Academy of Sciences Memoir 25: 339-351. NELSON G.J. 1989. Species and taxa. Systematics and evolution. In: Otte D. & Endler J.A. (eds.) Speciation and its consequences. Sinauer Associates, Sunderland, Ma., pp. 60-81. PLEIJEL F. 1999. Phylogenetic taxonomy, a farewell to species, and a revision of Heteropodarke (Hesionidae, Polychaeta, Annelida). Syst. Biol. 48: 755-789. PLEIJEL F. & G. ROUSE 2000. Least-inclusive taxonomic unit: a new taxonomic concept for biology. Proc. R. Soc. London B 267: 627-630.

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ROWE T. & J.A. GAUTHIER 1992. Ancestry, paleontology and definition of the name Mammalia. Syst. Biol. 41: 372-378. SCHANDER C. 1998. Mandatory categories and impossible hierarchies – a reply to Sosef. Taxon 47: 407-410. SCHANDER C. & M. THOLLESSON 1995. Phylogenetic taxonomy - some comments. Zool. Scripta 24: 263-268. SKERMAN, V.B.D., MCGOWAN, V. & P.H.A. SNEATH 1980. Approved lists of bacterial names. Int. J. syst. Bacteriol. 30: 225-420. SNEATH P.H.A. 1986. Nomenclature of Bacteria. In: Ride D.L. & Younès, T. (eds), Biological nomenclature today. A review of the present state and current issues of biological nomenclature of animals, plants, bacteria and viruses. IUBS Monograph series No.2. Eynsham, Oxford, IRL Press, pp. 36-48. SUNDBERG P. & F. PLEIJEL 1994. Phylogenetic classification and the definition of taxon names. Zool. Scripta 23: 19-25.

© PENSOFT Publishers Sofia - Moscow

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Zoological Record – a bibliographic service andThe taxonomic resource 659 New Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 659-663, 2003

Zoological Record – a bibliographic service and taxonomic resource J.M Howcroft & M J. Thorne BIOSIS UK, 54 Micklegate, York YO1 6WF, UK

Abstract Zoological Record (ZR) is an index to the world’s zoological literature, covering all aspects of animal biology and specializing in systematics. Since volume 1 in 1864 ZR has provided a printed current awareness index and a nomenclatural archive, while more recently ZR has also been made available online, on CD-ROM and through the web. New tools, derived from ZR, are described including the internet-based Index to Organism Names, which allows users to determine the existence of names indexed in ZR and track their use. Initially restricted to names indexed since 1978, the prospect of extending the coverage of ION back to 1864, is discussed. Issues relating to the development of a formal register of zoological nomenclature are considered, and suggestions made on how ZR and ION might be used to develop a central listing of names to which all could refer. Links with projects such as Species 2000, ITIS and GBIF are also explored. Future developments relating to the provision of information contained in ZR in new, non-bibliographic, formats are explained.

INTRODUCTION Zoological Record (ZR) is an annual index to the world’s zoological literature, covering all aspects of animal biology, recent and fossil, but specializing in systematics and nomenclature. For most of the 136 years of its existence it has been published by a learned society - the Zoological Society of London (ZSL). However, since 1980 it has been a joint publication of the Zoological Society and BIOSIS - an American ‘not for profit’ organization based in Philadelphia, and producer of a variety of information tools for life scientists. Compilation of ZR is done by the 40 or so staff of BIOSIS UK, a wholly owned subsidiary of BIOSIS, located in York, with a small unit of staff at the Natural History Museum in London. Volume 1 of ZR, covering the literature of 1864, was published in 1865 with the object of giving in an annual volume, ’reports on, abstracts of, and an index to the various zoological publications which have appeared in the preceding year; to acquaint zoologists with the progress of every branch of their science in all parts of the globe; and to form a repertory which will retain its value for the student of future years’. This objective holds

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good today - serials published in over 100 different countries are indexed in ZR, and there are subscribers/users from over 50 countries. In addition, 135 volumes later, ZR provides a substantial archival tool as well as a current awareness service. Each recent volume of ZR contains reference to some 72,000 publications, selected from about 4,500 serials, and between 1,200 and 1,500 books. To cover the content of these publications requires the creation of over 400,000 separate index entries, including individual records for some 20,000 new names, and 8,000 changes in combination or of synonymy between existing names. From this it will be clear that systematics, in the strictest sense, constitutes only a small proportion of ZR’s coverage, though it takes significantly more than a small proportion of total indexing effort to record. Until the early 1980s, ZR, in common with most other secondary services, was available only as a printed publication. However, in 1982 the first electronic version, ZR Online, was launched, covering 1978 and later published data. This was followed in 1993 with the launch of ZR on CD, and more recently still, in 1998, users were offered access to ZR on the web. As yet, this is only through a third party, though developing an ‘in house’ web version of ZR is planned as resources become available in the next year or so. ZR and nomenclature In addition to its traditional role as a systematic archive, over the years ZR has developed a variety of more specialized nomenclatural tools, several of which are available on the BIOSIS web site. Probably the most relevant in the context of this paper is the Index to Organism Names (ION). ION is an online nomenclator which allows any user to check on the existence of names and track their use in ZR. Like ZR on CD, ION contains names data from 1978 forwards, and it can be accessed at http://www.york.biosis.org/free_resources/ ion.html. This tool was developed in response to the International Commission on Zoological Nomenclature’s proposal, in the draft 4th edition of the Code, to change the criteria of availability of names by making it a requirement that names must be indexed in ZR. ION was a mechanism for allowing zoologists to check, free of charge, that a name had been indexed in ZR. While the proposed change to the Code did not receive sufficient support to be adopted, having developed ION it was decided to continue to make it freely available. ION now accesses a database of over one million animal names, at all taxonomic ranks, reported from the scientific literature of the last 20+ years, and is probably the most complete central record of recently used zoological names available. It offers basic nomenclatural and taxonomic hierarchy information, and ZR volume occurrence counts which reflect the use of animal names in the literature. It also includes some 400,000 names of non animal organisms provided by collaborating organizations: mosses from the Missouri Botanical Garden; fungi from CABI Bioscience/USDA Systematic Botany & Mycology Laboratory; and seaweeds from the Martin Ryan Marine Science Institute. From this it will be clear that ION is not just a resource for zoologists, but is an aid to the general bioscience community. ION is maintained as resources allow and additional features e.g. more frequent, eventually perhaps daily, updates, interface improvements, and additional non animal names will be added as programming time becomes available. A further improvement

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would be the addition of data from earlier volumes, but extending the coverage of ION back to1864 is a huge task, and one which is beyond our current resources. However, the American Museum of Natural History is showing interest in digitizing this older material, so there is a possibility that the older names might be made available through this route – see later in this paper. Two other nomenclatural tools which have been developed are TRITON and TITAN. TRITON, the Taxonomy Resource & Index To Organism Names, is the full system from which ION queries return a small subset of data. It was developed as a prototype to provide subscription based access to full index and bibliographic data for all newly published and changed animal names, including all new combinations and new synonyms, reported in ZR since 1978. Currently the TRITON database refers to over a million names, 420,000 of which are newly described, and the idea is to update the database as sectors of the current ZR volume in production are completed, adding about 20,000 names of newly described animal taxa each year. However, it is important to note that TRITON will not deliver a list of valid organism names, this is the intention of the Species 2000 project – see later in this paper. Instead, for animal names, it offers a view of how the names have been used in the scientific literature, and what changes have been formally proposed for them. For other organism groups, it provides links and/or direct access to other servers where nomenclatural data can be found. The full TRITON system is not yet publicly available, but controlled access can be given to anyone interested in commenting on its usefulness and future development. Details are available on our web site at http://www.york.biosis.org/TRITON/TRITON.htm. In addition to the name files available to anyone who accesses the BIOSIS web site, we have also developed files for internal use, in particular, TITAN, The Index to Taxonomic Animal Names. This file of over 320,000 largely generic names has been built over the years from names used in ZR, and from various authority sources. The file is used in the ZR production process to spell check and classify names, and ensure their consistent placement in ZR. ZR and ‘registration’ It seems that many in the community believe that a single central source of new animal names would be a valuable tool, and we would agree. Furthermore, we believe that ZR already provides an informal centralized list of names, and we would suggest that this could be a sound basis for developing a more formal list or ‘register’. However, when such a proposal was made by the Commission, the community rejected it. There were three main concerns. Accessibility: it was assumed that users would have to pay to check whether or not a name had been indexed. This was never the case; ION was, and still is, a free tool. A user does not have to subscribe to ZR to use it. Omissions: these were thought to be unacceptably high. However, in studies done with Philippe Bouchet of the Muséum national d’Histoire naturelle in Paris, using mollusc name data from the period 1980-1992, it was estimated that ZR was some 90% complete in its coverage of all new generic names. More recently, following improvements in coverage procedures, we believe that our current success rate is even higher.

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Accuracy: in the study with Bouchet, an average of one new mollusc name/year was found to be spelt incorrectly, i.e. less than 1% of misspellings/year; these are attributable to simple human error. We acknowledge that ZR is not perfect. However we are over 90% of the way to providing the community with a comprehensive source of new names, and could easily become 100% comprehensive if recording in ZR were made compulsory. Links with other projects ZR staff play an active role in several names related activities. Species 2000 – a project which aims to provide an approved list of names for all organisms. Global checklists of species names, compiled by experts in different taxonomic fields, are being linked to provide seamless access to approved names. We have actively supported this project since its inception, and our role, in addition to general support for the concept, is to offer a link to ION, something which would be particularly useful in the short term to fill any gaps in the global checklists. We have also provided the project with a workable, skeleton classification scheme for all organisms. Probably the single most important point to be made about the relationship between ZR and Species 2000 is that ZR makes no judgements on the validity of names, it simply reflect the literature, while Species 2000 seeks to provide a validated list of names. ITIS – the Integrated Taxonomic Information System. This is a US partnership of federal agencies whose goal is to ‘create an easily accessible database with reliable information on species names and their hierarchical classification’. Currently ITIS staff are gathering new names data themselves, a direct duplication of our work, and so there is great interest in how we might collaborate. The GBIF project – the Global Biodiversity Information Facility. This is an initiative proposed by the Organization for Economic and Community Development. It recommends that OECD members should ‘collaborate with each other and other countries and entities to build and sustain a distributed Global Biodiversity Information Facility, which would be accessible to all countries and all people’. We have had preliminary discussions on the possibility of using ZR new names and other nomenclatural information in this project. TDWG – The International Working Group on Taxonomic Databases. The mission of this group of the International Union for Biological Sciences, is to provide an international forum for biological data projects, developing and promoting the use of standards to facilitate data exchange. BIOSIS hosts and maintains the TDWG web site. The future Historically, BIOSIS has seen its role as one of collecting, analyzing and filtering the primary literature so that it is manageable by the individual researcher. However, to

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keep up with the accelerating trend towards electronic resources in distributed form, changes are needed. BIOSIS needs to expand its traditional role to include support for navigation and interconnectivity of the new, often disparate, life science resources. A good example of what is being done is the Internet Resource Guide to Zoology on the web. This is a navigation aid, which provides a starting point for finding sites of educational, scientific and academic interest, primarily in zoology, taxonomy, and related areas. It is the most heavily used area of the BIOSIS web site with around 40,000 accesses per week, and provides a virtual hub for zoologists. It guides users through the vast realm of web information, using categorized and regularly updated lists of links, and hosts information provided by organizations and individuals who do not have their own web sites. Its url is http://www.york.biosis.org/free_resources/ resource_guide.html ZR has well established working thesauri of zoological terminology, which have been used to successfully organize information within the ZR product. These form a ready resource, which could be more widely used in other systems. Currently under consideration is a proposal by the American Museum of Natural History (AMNH) Digital Library Project for collaboration between BIOSIS, the ZSL and the AMNH. The proposal is to use the ZR system to organize various forms of digitized data at the AMNH, and to digitize back (pre-electronic) volumes of ZR. The ZR system provides an already existing and well tested framework for access to the large body of AMNH scientific publications. Adaptation of this framework is seen to provide an elegant solution to the problem of integrating highly disparate digitized information resources. A preliminary proof of concept study is underway. Conclusion ZR continues to serve the community as a current awareness service and nomenclatural archive, but it is also responding to recent advances in information technology. New web based tools have been developed. Some of these assist with navigating the scientific content of the web, others are specialized nomenclatural tools. The Index to Organism Names (ION) gives access to all names recorded in ZR since 1978. With the assistance of the community, gaps in new name coverage could be filled to make it effective as a formal list of new names in zoology, freely accessible to all. Collaboration with other organizations with common interests is regarded as the best means of using the limited resources available to the life science community.

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Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers Biological nomenclature in A. the electronic era: chances, challenges, risks 665 The New Panorama of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 665-672, 2003

Biological nomenclature in the electronic era: chances, challenges, risks W. Greuter Botanic Garden and Botanical Museum Berlin-Dahlem, Free University of Berlin, Germany.

Abstract The way in which nomenclature operates under the current Codes is an impediment to taxonomic research. The fact that taxonomists on average spend almost 20 % of their research time on sterile nomenclatural routine is intolerable, because it is avoidable. Unless they adapt to the rapidly changing environment in which they operate, the current systems of biological nomenclature risk being dismantled and replaced. Based on the botanical experience, a threefold strategy is recommended, no part of which has so far been implemented. (1) The names of the past should, in so far as they are currently required for use, be compiled into lists specifying their essential nomenclatural parameters, on the basis of current knowledge, and should then be stabilised and protected against any competing unlisted names; in the new edition of the zoological Code, the “List of available names” offers such an option, but the procedures for implementing it are cumbersome. (2) Currently established names should undergo registration in order to become available, so that information on them is made generally, immediately and reliably accessible. Botanists have promoted, successfully tested and then nevertheless rejected such a system; zoologists have envisaged but not yet adopted it. (3) In order to simplify the work of taxonomists of future generations, who promise to proliferate in the developing world in particular, the publication of abridged editions of the botanical and zoological Codes might be envisaged, concentrating on the normal nomenclatural routines and foregoing the complexities that overburden the official editions. These complexities (which in the long term may become obsolete thanks to the development of stabilised lists of names) can be left to the experienced specialists working at the major museums. If for nothing else, the “Draft BioCode” (which is unlikely to be approved in the near future, if ever) might serve as a model for such abridged editions of the Codes.

Introduction Two and a half years ago, I took part in a workshop in the city of Darwin, Northern Territory of Australia, which was devoted to a significant topic: “Removing the taxonomic impediment”. “Taxonomic impediment” was then still a novel phrase (it has not lost

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any of its appropriateness since): it spells out the fact that lack of knowledge in the field of biological taxonomy and systematics, and shortage of human resources qualified to fill that gap, are a major hindrance to the development of rational policies for the conservation and sustainable management of the biological diversity of our planet. The significance of biotaxonomy for the implementation of the Convention on Biological Diversity (CBD), and vice versa, is a fascinating topic, but lies outside the scope of this talk. I mention it here because at the Darwin workshop I was given the opportunity to introduce a then novel concept: the “nomenclatural impediment” (Greuter 1998), the hindrance that biological nomenclature, with its inadequate concepts and mechanisms, constitutes for taxonomic work, and thus for removal of the “taxonomic impediment”. My ideas were so well received that the essential conclusions were embodied as part of the “Darwin Declaration”, the influential statements and recommendations resulting from the Darwin workshop, which were subsequently endorsed by the Conference of Parties to the CBD. The Nomenclatural Impediment All practising taxonomists know in fact the “nomenclatural impediment” from firsthand experience. Many (honestly, I cannot exclude myself altogether) do indeed relish that impediment. For them nomenclatural work is something basically positive. If asked, they will likely qualify it as a refreshing and highly educational exercise of the mind, or even as a fascinating intellectual challenge; and they are, incidentally, the people who largely govern the present and future shape of the nomenclatural rules (Ride et al. 1999, Greuter et al. 2000a). Yet, if pressed, all are likely to agree that nomenclature is not a science but a technique, not a goal in itself but an instrument. An important instrument, for sure, as it provides the language by which we communicate any and all information relative to organisms, not only in biology but in all fields of pure and applied learning, such as law, economy, engineering, and the social and medical sciences. However, the way in which nomenclatural work has been done for well over a century, and is still done today, is a luxury in which we may no longer indulge. In a most informative survey, Hawksworth (1992) has assessed the amount of time and money that goes into that work: for botanists in the UK alone, it sums up to 52 full-time research positions, or £1.3 Million per year (disregarding inflation). Isn’t this intolerable? Can we afford to discard the option of a 20 % increase of cost-effectiveness of taxonomic work, if there is such an option? Just think of how much of that 20 % of working time is now spent: painstaking, often random searches for elements required for making a decision; controversy, often in print, over doubtful cases, where either the facts (think of publication dates) or the application of the rules to the facts (the inventiveness of biologists in finding ways to create ambiguous situations being unlimited) are unclear; and whatever new facts or interpretations may result from such searches are likely to destabilise the previously used nomenclature, may require further work by committees to undo the damage, or failing this, may result in changes that are at best unpalatable and will add little credit to taxonomy in the eyes of its customers and sponsors. Worse still: all this work, however carefully done, serves for nothing in the end: it will have to be redone by the next to work in the same area, in an endless cycle. Each taxonomist has

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the moral obligation to check the nomenclatural facts for him- or herself from scratch; there is no guarantee that previous work, however carefully done, is either fully accurate or complete. Recipes to overcome the “nomenclatural impediment” have been developed and proposed, initially by bacteriologists who applied them as early as 1980. The number of known bacterial species was then c. 2000; it is more than 100 times higher in botany, and an additional factor of 10 applies for animal species. Progress in electronic data processing and databasing that has been achieved since then compensate easily these numerical disparities as far as the operational background is concerned. Numbers of specialists involved can, I believe, make up for the different size scales for labour. New options of electronic data transfer and online information access multiply the usefulness of the bacteriologists’ approach. During the last decade, botanists have been spearheading progress toward more rational nomenclatural procedures, with the primary aim of increasing the ease, speed, and security of taxonomic work. The task, as I envision it, is vast and complex. Let me divide it into three main fields that are complementary but independent of each other. I will refer to them concisely, if not fully appropriately, by the catchwords “names in current use” (NCU), “Registration”, and “BioCode”. The past: protecting Names in Current Use The first of these fields concerns the names of the past, those already in existence. In so far as they are at all needed, they should be stabilised once and forever on the basis of available knowledge – the stabilisation to concern all nomenclaturally relevant parameters: source and date of establishment, type, authorship, spelling, and if appropriate, gender. To that effect, lists of all names that are currently used (NCU), or likely to be required for use, are to be drawn up, their types established and verified to make sure that they correspond to the taxon to which the name is currently applied, and after due wide circulation, vetting and correction the listed names would then be declared protected with all their relevant nomenclatural properties, as set out in the list. The proposed approach was carefully defined to allow for maximum flexibility (e.g., as to coverage in terms of taxa, ranks, period of time, and nomenclatural parameters) and also for ease of implementation. Sample lists were prepared for extant plant family names, excepting algae (Greuter 1993a), for names in four selected families (Greuter 1993b), for all names on non-fossil plant genera (Greuter et al. 1993), and for one special parameter, nomenclatural types, of Linnaean generic names (Jarvis et al. 1993). Feasibility and usefulness of the principle was thus adequately demonstrated, but probably at too short notice to sway the botanists’ opinion. Even so, at the XV International Botanical Congress (IBC) in 1993 the NCU principle was all but accepted (by a 55 % positive majority but not by the required qualified majority of 60 %). At the last IBC in 1999, however, it was dismissed without serious consideration. Zoologists have now taken a lead over botanists with respect to their means of dealing with the names of the past. The new edition of their Code (Ride et al. 1999) provides for the piecemeal adoption of a “List of Available Names in Zoology” that embodies exactly the same stabilising features of zoological names as botanists had envisaged through

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their approved NCU lists. The easy conclusion is that my present sermon is addressing the wrong audience. Perhaps this is true, and certainly the makers of the new zoological Code have my admiration for the progress they have achieved. Let me however point out two differences between the zoological model and the defunct botanical NCU approach. First, zoologists propose to list all names ever established in a given group, even those that are long dead, or never came to be alive, or cannot be properly interpreted: this signifies an immense additional work load for little practical gain, except that it makes it possible to declare all unlisted names to be unavailable (whereas under the NCU model, unlisted names would not lose their status, but only their potential precedence against listed names). Second, the approval of any part of the List is subject to severe procedural conditions, resulting in a dreadful overhead in terms of bureaucracy, labour, and time. It is a safe guess that, upon its approval, no part of the list will include any name established less than 10 years before. While this price had presumably to be paid in order to make the idea of the List acceptable at all, it will make it hard to avoid a gap between the approved parts of the List and any future system to deal with currently established names. The present: registration of new names New brands, trademarks, botanical cultivar names in definite groups, all have to be registered in order to achieve recognition. Scientific names of organisms must not. Is it because they have a venerable tradition rooting in times when registration was unknown? Perhaps this is so. Certainly, if biological nomenclature were invented today, nothing would appear more natural than having names registered in order to enter the “market”. Registration is, as a matter of course, required by the draft versions of the prospective “PhyloCode”. So far, names in many groups of organisms are indexed. Some of this indexing functions pretty well, has a fairly good coverage, an adequate infrastructure, a regular output at a satisfactory speed. Features of BIOSIS’s “Zoological Record” and “Index to Organism Names” are being presented in the next subsequent paper (Dadd et al. 2000). The new International Plant Name Index (IPNI; Croft et al. 1999), which, contrary to what its name promises, covers flowering plants only, is a model of how well indexing can function when it makes full and rational use of today’s technology. Indexing occurs decentrally, resulting in a distributed yet fully congruent database; indexed entries are released in a timely way; and free online access to the information is provided to all Internet users. Indexing, however, won’t do. Alone, it cannot solve the combined problems of defining “publication” in a changed technological environment, of coping with a flood of “grey literature” meeting all traditional standards of “publication”, and of keeping abreast with the explosion of taxonomic activity all over the world, inclusive of countries with scant tradition in such research yet with enormous potential for taxonomic output. The registration principle is simple enough. By placing the responsibility for making a new name known and acknowledged on the “producer” (the author or, by delegation, the publisher) rather than on the “consumer” assisted by the indexers, registration will on the one hand greatly reduce and speed up the labour of indexers, thus contributing to

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maintain the viability of current indexing systems; and on the other hand, it will ensure that the indexes, by becoming registers, are ipso facto complete and fully reliable, adding enormously to their practical value. The XV IBC, in 1993, had approved registration in principle, subject to a successful trial phase and to reconfirmation by the next subsequent IBC. The first of these conditions was met. Under the auspices of the International Association for Plant Taxonomy (IAPT) a registration secretariat was set up at the Botanic Garden and Botanical Museum BerlinDahlem (BGBM), which was supposed to work in close co-operation with the main indexing centres: the International Mycological Institute (IMI) in Egham for fungi and the Royal Botanic Gardens, Kew, for flowering plants. IMI enthusiastically agreed, but not Kew, so except for the fungi the actual work was done in Berlin. The trial phase (Borgen et al. 1997, Greuter & Raab-Straube 1998, Raab-Straube 1999) lasted one year and a half, from the end of 1997 to mid-1999 (it was extended up to the end of 1999 for the algae and fossil plants) and was a complete success. Starting to become operational on 1 January 1998, it registered over 10,000 new plant names (excluding fungi, but including new combinations and rank transfers), which on average were released on the Internet by means of a searchable database within less than a week after receipt of the printed matter in Berlin. In the same time, the text and images accompanying each new name upon publication (the “protologue”, as botanists call it) were scanned optically, to be available on-screen for further internal checking (and could in the future, if copyright questions could be settled, be released on the Internet as well). The literature scanned for new names included over 200 scientific journals that accepted to participate by sending free copies of each issue immediately upon publication, plus items submitted directly by the authors. In addition, books and journals received at the BGBM library were scanned ex-officio for new names and combinations. The whole operation, including correspondence with the 38 national registration offices that had agreed to co-operate, was handled by two half-time employees and cost the IAPT less than US$100,000 in total, including the cost of software design. The second condition for making registration a mandatory requirement sadly failed. The St Louis IBC did not accept the corresponding proposals submitted by the IAPT Officers (Borgen et al. 1998); on the contrary, it decided to expunge any mention of a future registration option from the botanical Code. I consider this decision, which in effect scrapped a fully functional and affordable system, as the last century’s single major failure of botany’s nomenclature system to its duties toward the scientific community. Nevertheless, that decision was taken democratically by a substantial majority of those entitled to vote, so it cannot be challenged on that account. At worst, it demonstrates how democratic decision processes can be manipulated and corrupted. Powerful and well orchestrated Internet campaigns, massive lobbying and mobbing ahead of and during the Congress were novel phenomena in botanical nomenclature, as was the fact that purely emotional, factually wrong arguments played a key role in the discussion and swayed the decision (see Greuter et al. 2000b). Weeping over the past is of no use. Defeat in one battle is not a lost war. Registration will come, must come in the end, even in botany. Meanwhile, my advice to zoologists is: take the lead. If nothing else, the botanical experience tells that setting up a functional registration system, even on such a large scale as zoology requires it, is technically feasible

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and fundable, and that the result is eminently useful. But beware of letting emotions take over, be prepared to invest in information campaigns and in efficient, professionally designed PR actions; use modern communication means such as the Internet to your own ends; and argue convincingly that zoologists around the world, but those in the developing world more than any others, will profit from a functional system of registration. The future: a common BioCode? Although together with John McNeill I am its main inventor, I have never considered the “Draft BioCode” (Greuter et al. 1998) with stubborn seriousness. It was in my view an exercise of style, meant to demonstrate that a common biological code of nomenclature for the future is feasible and can become reality if and when the need for it is felt. Ride (in Ride et al. 1999: XXIII), referring to the prospective “BioCode”, writes: “Looking ahead to the future, if progress in all disciplines continues towards developing acceptable systems for registering new names, and officially listing all extant available names, so that the rules protecting them can become a thing of the past, a single code will become a possibility”. Come to think of it, the number of those who have a vital interest in common rules for all organisms is rather small, and they have no strong lobby backing them. Biology teachers come to mind, when they teach across the current codes’ domains; to most of them, having a uniform terminology and nomenclature at the higher ranks might do. Database managers dislike inter-kingdom homonyms (with which they will however have to live) and disparate or absent standardisation of spelling. Specialists of the socalled “ambiregnal” organisms, variously treated under different codes by different authors, are the worst affected; let them as a first step choose by consensus, or by majority vote, the Code under which they want to be placed. All these problems are certainly serious for each of these groups of people, and they are likely difficult to solve (think of debates on orthography!), but it is not clear that solving them requires a common “BioCode”. Once a dialogue between zoologists, bacteriologists and botanists has been established, it might be a better idea, and no more difficult, to introduce concerted changes as they are needed into the individual codes. Why, then, not simply forget the “BioCode” exercise? Well, the “Draft BioCode” has one feature to it that has never been properly appreciated, perhaps not even clearly mentioned, but which for the common user of the Code is probably paramount: simplicity. This is an incidental by-product of the fact that the “BioCode” was deliberately designed for names and nomenclature of the future. By its simplicity and ease of handling, the “BioCode” is tailored for the needs of the innumerable taxonomists we need and will hopefully get in the next generation, most of them in the developing world – and don’t tell me that translating the botanical or zoological Code into, say, Portuguese makes it easily understood in Brazil, when few taxonomists outside of the respective editorial committees do fully understand the French or English version. So my advice would be: forget the “BioCode” as such, but use it as a model to write a simplified zoological (or botanical) Code for use by “common” taxonomists. Tell its users: if you describe and name a new taxon, you must (or should) ...; in applying existing

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names to a known taxon, when there is a choice [and taking for granted that the names are all available, their date and author known, their type and spelling fixed], choose as follows ... Ridded of its historical complexities and refinements, which cannot anyway be complied with outside of the major museums and libraries in the industrialised countries, such a “core Code” will be a handy, logical document of perhaps one third of the Code’s present size. The space so gained could then perhaps be used to add cookingbook recipes on formation and spelling of new names, to stem the tide of barbarism and illiteracy in that domain; on state-of-the-art formats for synonymies and (type) specimen citation; and on other matters that are nomenclaturally relevant and useful for the practising taxonomist. Bottom line The rules governing biological nomenclature adapt but timidly to the new environment in which science operates today, and sometimes they refuse to adapt at all. Half a dozen years ago, botanists were way ahead of zoologists in their apparent willingness to take new ideas on board by reaping the benefits of new technologies for an expedient, secure and meaningful system for naming their respective organisms. Times have changed and the pendulum has swung back. The Nomenclature Section of the 1999 IBC in Saint Louis can only be qualified as reactionary in its decisions and irrationally emotional in the way they were reached. Zoologists may learn from botanists, and botanists have to learn for themselves, that major changes of the rules need time to become acceptable and accepted, and that trying to force the pace may well result in failure. Viewed from the outside, the isolated cosmos of organismic nomenclature may appear to be dominated by inveterate cracks who have little to gain from innovative change, which at best will force them to adapt and at worst may threaten their intellectual monopoly of the subject. We know that such a view is a caricature of reality, but good caricatures always have an element of truth in them. Nomenclature has so far survived by playing the role of a “black box”, which those suffering from its results grudgingly accept as a kind of arcane, inescapable doom. But this may not go on indefinitely. It is a question of time: if the process of adapting the traditional naming procedures to the ever more rapidly changing environment in which they operate takes too long, those frustrated by this apparent inertia may well be tempted to throw tradition over board in a much more radical way than we now can think of. References BORGEN L., GREUTER W., HAWKSWORTH D.L., NICOLSON D.H. & B. ZIMMER 1997. Announcing a test and trial phase for the registration of new plant names (1998-1999). Taxon 46: 811-814. BORGEN L., GREUTER W., HAWKSWORTH D.L., NICOLSON D.H. & B. ZIMMER 1998. (8895) Proposals to implement mandatory registration of new names. Taxon 47: 899-906. CROFT J., CROSS N., HINCHCLIFFE S., NIC LUGHADHA E., STEVENS P.F., WEST J.G. & G. WHITBREAD 1999. Plant names for the 21st century: the International Plant Names Index: a distributed data source of general accessibility. Taxon 48: 317-324.

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DADD M.N., HOWCROFT J.M. & J. THORNE 2000. Zoological Record – a bibliographic service and taxonomic resource. In: Anonymous (ed.), XVIII International Congress of Zoology, Athens, Greece, 28/08 - 02/09/2000. Book of abstracts. Hellenic Zoological Society, Athens, p. 163. GREUTER W. (ed.) 1993a. NCU-1. Family names in current use for vascular plants, bryophytes, and fungi. Regnum Veg. 126. GREUTER W. (ed.) 1993b. NCU-2. Names in current use in the families Trichocomaceae, Cladoniaceae, Pinaceae, and Lemnaceae. Regnum Veg. 128. GREUTER W. 1998. The nomenclatural impediment. SABONET News 3(1): 21-24. GREUTER W., BRUMMITT R.K., FARR E., KILIAN N., KIRK P.M. & P.C. SILVA 1993. NCU-3. Names in current use for extant plant genera. Regnum Veg. 129. GREUTER W., HAWKSWORTH D.L., MCNEILL J., MAYO M.A., MINELLI A., SNEATH P.H.A., TINDALL B.J., TREHANE P. & P. TUBBS 1998. Draft BioCode (1997): the prospective international rules for the scientific names of organisms. Taxon 47: 127-150. GREUTER W., MCNEILL J., BARRIE F.R., BURDET H.-M., DEMOULIN V., FILGUEIRAS T.S., NICOLSON D.H., SILVA P.C., SKOG J.E., TREHANE P., TURLAND N.J. & D.L. HAWKSWORTH (eds) 2000a. International Code of Botanical Nomenclature (Saint Louis Code) adopted by the Sixteenth International Botanical Congress, St Louis, Missouri, July-August 1999. Regnum Veg. 138. GREUTER W., MCNEILL J., HAWKSWORTH D.L. & F.R. BARRIE 2000b. Report on botanical nomenclature – Saint Louis 2000. XVI International Botanical Congress, St Louis: Nomenclature Section, 26 to 30 July 2000. Englera 30. GREUTER W. & E. von RAAB-STRAUBE 1998. Registration progress report, 1. Taxon 47: 497-502. HAWKSWORTH D.L. 1992. The need for a more effective biological nomenclature for the 21st century. Bot. J. Linn. Soc. 109: 543-567. JARVIS C.E., BARRIE F.R., ALLAN D.M. & J.L. REVEAL 1993. A list of Linnaean generic names and their types. Regnum Veg. 127. RAAB-STRAUBE E. von 1999. Registration progress report 2, with a comment on censorship. Taxon 48: 407-412. RIDE W.D.L., COGGER H.G., DUPUIS C., KRAUS O., MINELLI A., THOMPSON F.C. & P.K. TUBBS (eds) 1999. International Code of Zoological Nomenclature, ed. 4. International Trust for Zoological Nomenclature, London.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

The International Code of Zoological Nomenclature, 4th Edition ... Evolution 673 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 673-682, 2003

The International Code of Zoological Nomenclature, 4th Edition - What Next? W.D.L. Ride Department of Geology, Department of Geology, Australian National University, Canberra, Australia, 0200 Chairman, Editorial Committees of the 3rd and 4th Editions of The I.C.Z.N. E-mail: [email protected]

Abstract Zoological Nomenclature, as a process, has developed since Linnaeus established the binominal system for animal names in 1758. The outcome of that development is represented in the evolved Linnaean System. It is in universal use today with rules and conventions provided by the International Code of Zoological Nomenclature. The 4th Edition of the Code came into force in January 2000. However, the process of further evolution continues and pressures for other changes can be foreseen. The three most significant of these are examined, discussed, and suggestions made as to how the Code might meet them. The first pressure is from changes in information technology which open new possibilities for wide and rapid dissemination of information and which raise the possibility that electronic publication of new names, and acts affecting nomenclature, might become possible, supplementing, and even replacing traditional methods of publication. The second is the need for change to meet difficulties resulting from advances in gene technology in relating nomenclatural types (as name bearers) to taxa. The third issue is whether there is any need for the Code to be modified to meet the concerns expressed by some phylogenetic taxonomists that there are insurmountable shortcomings in the binominal system and it must be abandoned.

Introduction Zoological nomenclature has one purpose - to provide unambiguous names for animals that can be used universally, with stability, and consistent with taxonomy. To achieve this aim without infringing upon the freedom of taxonomists to describe and delimit taxa, the Code provides rules (Articles) through which zoologists may determine the correct and unique name for them - irrespective of the processes used in postulating the taxa.

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Zoological nomenclature is not practised in a vacuum. Therefore its principles and rules represent the products of interaction between various factors over many years – a process which is active today. The principles on which the modern rules are based have proved to be robust. Since the latter part of the 18th Century, when the Linnaean binominal system became adopted universally, there have been major shifts in the climate of scientific opinion and in taxonomic theory (see Cain 1959), including the shift during the second half of the 19th Century from a static, non-evolutionary view of living things that the system was initially designed to reflect, to evolutionary biology. Today, the underlying principles of the Code (ICZN 1999) face no significant challenge (these principles are common to all other biocodes that had evolved by the second decade of the 20th Century – The Maxims of Nomenclature, (Ride 1988: 336,7) – except that there is currently an opinion expressed by some phylogeneticists (e.g., Cantino et al. 1999, Pleijel 1999, Cantino & de Queiroz 2000) that they are inadequate. That, in requiring ranks to be assigned to taxa, Linnaean nomenclature conflicts fundamentally with the naming of the evolutionary continua (clades) which can be inferred from observed nature, and of which the taxa form only short artificial discontinuities (segments, clusters of segments, and terminating “leastinclusive taxonomic units”) within the continuum. By contrast with the very stable principles which achieved full form in 1913, the rules which determine the way in which Zoological Nomenclature satisfies those principles, have changed quite rapidly since the mid-20th Century. Some of the changes have been made to improve presentation and to reduce ambiguity. But the most far-reaching have been made to meet the demands of shifting needs and opinions of practitioners and the zoological public generally in a rapidly changing scientific environment. These latter changes have reflected such social elements as increasing demands for nomenclatural stability and the widening of decision making power for individual zoologists, as well as such practical factors as movements in printing technology, the need to eliminate difficulties in identifying types (both generic and specific) and shrinking comprehension of Latin in the scientific community. Changes made recently to the present edition of the Code reflect these elements (see Ride 1999) Background to the Principles The binominal system of nomenclature which Linnaeus adopted for the Animal Kingdom in 1758 soon became accepted universally but, universality in names that Linnaeus achieved was soon lost and newly introduced names represented numerous philosophical, scholastic, personal and national viewpoints of authors. But reform followed when biologists recognized that universality and stability in zoological names could only be achieved if there was to be general acceptance of a set of propositions for naming. Firstly, to restrain the amenders of names and, secondly, to enable names to be independent of any philosophical considerations that would affect their form or acceptance (Strickland 1838). The main propositions advanced (Strickland et al. 1843) were that every scientific name must be published and widely accessible, that it should meet certain orthographic and form criteria (and not be amendable if it did), and, most importantly, that the precedence of competing names that met these criteria (whether synonyms or

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homonyms) must be determined by their dates of first publication. They also insisted that the application of each generic name must be determined only by its typical species – whatever its original circumscription. They saw that only by reference to their types, and the precedences of their names, could the same generic name be retained by the principal part of each genus, no matter how generic limits might shrink or merge in different classifications. This last element, which led to the development of the Principle of Typification, has, more than any other, given the Linnaean nomenclatural system its robustness. As a result, names became independent of concepts - but applicable to them. Strickland saw very clearly that the binominals, when applied to species, could only become universal and “permanent” (i.e., stable) if they functioned as no more than labels, independent of any competing classificatory theories on which their taxa were based. Subsequently, the type system was also extended to species and rules were formulated for the discernment of type specimens for previously proposed names (Ride 1988: 341-3). Following the international adoption of the Règles (ICZN 1905), agreement was reached in 1913 to adopt an additional principle to limit the Law of Priority when its operation conflicted with stability or universality. Also, building on the Stricklandian proposition that a subfamily be given the same name as the family, based upon the name of a contained genus, and differing only in suffix (Strickland et al. 1843: 272,3, Prop. B), the Règles contained rules (Arts 4, 7, 12) that names proposed within each of three parts of the nomenclatural hierarchy (Family, Genus, and Species) would be nomenclaturally co-ordinate (i.e., that the establishment of a name at any rank within a group, automatically established a name with the same precedence, based upon the same type, and available for use at any other). While the named (i.e., “ranked”) categories within the genus and species-groups remain at two each (species and subspecies, genus and subgenus – although additional names may be interpolated if needed, Art. 6.2). In the case of family-group names, the provision now allows for the naming of as many categories as a phylogenetic taxonomist may need to describe a lineage, whether formally ranked or not. The term “rank” is not biologically meaningful above the species-group. Its use in the Code is only to name those categories in the hierarchy at which the form of the name is prescribed (for prescribed ranks in the family group, see ICZN 1999, Art. 29.2); for the taxonomic significance of names at the ranks of species and subspecies, see standard texts of taxonomy and systematics such as Mayr, Linsley and Usinger, 1953). To make it easier for names in common use to be retained in their accustomed meanings and to further reduce the need for historical research, the latest edition of the Code enables lists of names that are available and potentially valid, to be compiled by international bodies of zoologists and adopted by the International Commission on Zoological Nomenclature. Unlisted names within the major taxa covered by adopted lists lose their availability by this process. Thus, as well as fixing orthography and the precedence of names listed, the adopted lists will have the effect of producing a fresh start to the nomenclature of any group of animals so listed (see Ride 1991: 112-15, 119-121, Ride 1999). While these changes will have potentially eliminated the displacement of names in common use by “forgotten names”, neither the Code nor the Commission can or should provide a legislated remedy to preserve “familiar names” when these are threatened by changes in taxonomy. The solution can only reside with taxonomists themselves. For

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instance, many of the generic limits now imposed as a result of increased phylogenetic understanding are unnecessarily restrictive and conceal affinities by being so. In practice, and without infringing upon monophyly, taxonomists can do much themselves to reduce instability by restraining themselves from adopting narrow nomenclatural generic limits (see De Smet 1974: 13-14, 59, Ride 1988: 347). Pressures for Change Information technology During the last century, progressive changes were made in the Code to keep up with developments in printing and publishing technology. Thus, the production of multiple copies by a range of processes, that have supplemented typeset printing with ink on paper, have all been dealt with, progressively, after each came into general use, by amending the Articles that define what is and what is not “publication” (see Ride 1988: 345). The latest of these changes has been to allow publication in durable electronically produced works such as CD-ROM, providing that copies in the form in which they are published have been deposited in designated libraries (ICZN 1999, Arts 8 and 9). But today it is patently clear that this process of redefinition cannot continue in this electronic age and registration of new names has been proposed as an alternative. Despite wide awareness and acceptance of the need for reform (see Ride 1988, 1991), to date there has not been the public support that would make such a radical change possible (Ride 1999: xxiv, xxv). Today, developments in information technology, in combination with electronic data handling and analysis, which can greatly increase efficiency of identification, are also revolutionizing the way in which taxonomists work. As a result, taxonomists now have new and efficient tools to make rapid and effective progress in describing the huge and mostly unknown world fauna. In this endeavour, the formal requirements of nomenclature must not become an impediment. Already, practice is moving ahead of the requirements of the Code. Some major taxonomic and nomenclatural works are moving wholly into electronic form. For instance, in Australia, the government ministry responsible for the Australian Biological Resources Study has decided that the definitive Zoological Catalogue of Australia (the basic work on the taxonomic arrangement and nomenclature of the complete Australian fauna) will be published henceforth only in electronic form. Future electronic versions of this work, and nomenclatural changes in it, will not be “available” in the sense of the Code. The challenge to the Code of these developments, and in particular of the internet, is to discover how to use them to improve the machinery of nomenclature. We must not regard them as presenting difficulties to be overcome by procedures designed to maintain its present requirements. A radically new approach is needed to processes of establishing new names and acts affecting nomenclature, that will enable us to move forward from the traditional machinery. We must now re-evaluate the purposes of publication in the Code and, then, develop a new modus operandi to achieve them by taking advantage of what is now possible. In the current Code, the purpose achieved by publishing is to ensure wide, open, and unrestricted public circulation and preservation of two sorts of information. These are,

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firstly, the primary nomenclatural data of the name itself, its precedence, its correct original spelling, its type, its authorship and its place of publication and, secondly, the supporting taxonomic information which may extend beyond the bare requirements of the Code for a description and diagnosis. This usually consists of an extended description of the new taxon, of its type, justification that it is new, a statement of its affinities and inferred phylogeny, and information on its geographical distribution, etc. To these basic aims, a third aim – of quality assurance - has become increasingly valued as a benefit that results from pre-publication scrutiny by referees and editorial actions taken during publication. Therefore, while electronic technology now provides rapid and wide consultation and discussion, and enables the definitive outcomes of that discussion to be distributed more rapidly and widely than ever before, two elements are lacking: (1) the process must retain the benefits of rigorous scrutiny by peers before adoption and (2) it must lead to a durable record of the outcome. Today, an indication of the way forward is provided in part in web sites established by museums on the World Wide Web where zoologists are “publishing” records of taxonomic research and attracting comments on them. See, for example, the “Sea Slug Forum” on the Australian Museum web site (http://austmus.gov.au/seaslugs/ ). In that example, the extensive correspondence between interested specialists which has been generated in response to papers presented electronically by W.B. Rudman (e.g., Rudman 2000, Siraius immonda and Diniatys dentifer), demonstrates that part of the purpose of publication has been served. As a result of this exposure, Rudman will have obtained wide and open circulation and exposed his nomenclatural and taxonomic intentions to a wider group of his peers for review than he would have by current refereeing. If he were then to decide that formal establishment of an act (such as a new name) was justified as an outcome, all that would be needed would be for him to formalize the action. Currently, this would have to be done by some method of publication that would satisfy Articles 8 and 9 of the Code. However, it is not difficult to envisage the process being completed electronically (i.e., without further delay) if, following a prescribed consultative period, the new name or act was confirmed by its author as from the date of its original electronically published proposal (the earlier date being necessary to circumvent “piracy”) and registered electronically with an appropriate registering body (such as the Zoological Record, Howcroft & Thorne, this volume). By following such a process very wide circulation and exposure to peer review would have been achieved and a permanent accessible record of the primary data made. But, the aim of providing a permanent consultable record of the taxonomic data would have to be achieved by other means, possibly by commitment by libraries of designated and cooperating museums and similar institutions, to down-load and preserve definitive hard copy. As long as traditional methods of publication remain available, there is no reason why both the traditional and electronic processes should not run concurrently but, clearly, it would be desirable for authors publishing traditionally, to record new names with the registering authority to enable these to be included in the same information base (rather than rely wholly on the Zoological Record to find and list them – as it does currently).

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Up to now, the zoological community has consistently rejected the proposal that the criterion of publication be replaced by a registration procedure. I accept that zoologists have been extremely chary of placing the future of zoological nomenclature in the hands of any one bureaucratic system. I also accept that there is a danger of censorship and interference with the rights of individuals to name new taxa. But the very capacity of electronic systems to develop distributed databases can be used advantageously if combined with appropriate safeguards. I am sure that the time has now been reached when there is a willingness to re-examine the matter and to develop an electronic alternative to “publication”. Microbiologists (with an admittedly far fewer named species than zoology or botany) have accepted the proposition to register names, and have put it into effect. The time has come for us to move forward. The practical benefits will be very great, and, in combination with listing of all potentially valid names (as the Code now provides) a new era could commence in zoological nomenclature comparable with those that opened in 1758, 1843, and 1905. Classificatory technology Genetics - “ancillary types” A number of cases have come before the Commission in recent years (e.g., Smith et al. 1998) that reveal a need to accommodate the impact on type specimens of new technology, especially genetic technology, that enables taxonomic discrimination to be made at a level previously unknown. For instance, in the case of certain “sibling species” (and populations of subspecies), in which individuals can only be identified by genetic criteria the names potentially available for them cannot be allocated with certainty because of the state of preservation of their types in which the information is no longer observable. The same difficulty applies when populations can only be separated on the basis of their behaviour, or ecology, and the relevant information on the types is lacking. Under the present rules (Art. 75.5) such impasses can only be resolved by a cumbersome process of requesting the Commission under its plenary power to replace the type with a neotype (possible only when stability and universality are demonstrably threatened). Predictably such situations will become more common. I suggest that it is now time for zoologists to accept a means of adding the missing information to the type. To achieve this aim, the Commission would need to establish a new provision in the Code to enable (under the First Reviser principle) fixation of an “ancillary type” that would add to the type the information it lacks, and which would have the same nomenclatural status as though it were derived from the type itself. Phylogenetic Taxonomy Criticism has been levelled by some taxonomists practising phylogenetic taxonomy (especially cladistic methodology) that the hierarchical system provided by the Code does not meet the needs of phylogenetic systematics because they hold that it is designed to serve a taxonomy which requires (and implies) discontinuities between taxa.

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Phylogenetic taxonomy has as its aim the elucidation of evolutionary continua defined only by monophyly (clades) and the terminal segments of them that emerge from final branching points (“litu” = least inclusive taxonomic units). The proponents of litu (Pleijel & Rouse 2000a) hold that, however defined, species are “an extravagant extrapolation that has no place in science”. Moreover, they hold that because species have to be assigned also to a taxon at the rank of genus under the binominal system, and because “rank” within the supraspecific continuum is philosophically offensive to “tree thinking”, binomina should be dispensed with as species names. They like other critics claim that binomina are inherently nomenclaturally unstable because of the requirement to preserve grammatical integrity (see Cantino et al. 2000). A solution, recently proposed in a draft PhyloCode (Cantino et al. 2000), is to dispense with binominal nomenclature for species and to define clades by several methods, each of which requires the clade name to be tied to the circumscription of the clade instead of to a type. The proposed PhyloCode has drawn strong criticism (Benton 2000) on the primary grounds that it is based on a fundamental misunderstanding of the utilitarian difference between phylogeny and classification. I agree and, in particular, consider that the most serious consequence to phylogenetic classification is the proposal to reject the principle of nomenclatural types. The PhyloCode has a fundamental tenet that names are names of taxa (or clades) rather than being applicable to taxa by means of types. The present system has developed and used nomenclatural types since 1843 as being fundamental to continuity in names of taxa and nomenclatural stability (see “Background”, above). The history of nomenclature demonstrates that to return to Linnean “circumscription nomenclature”, whether circumscriptions are stem based, node based, or apomorphy based, would rapidly take the nomenclature of phylogenetic systematics to the chaotic state of the early 19th Century when continuity and universality became lost. Taxonomy as a science attempts to represent true phylogeny (the tree of life and its branches) but, irrespective of the methodology employed, its conclusions are hypotheses liable to falsification and replacement. These, in turn, are dependent upon the quality of the data and on the principles employed in their interpretation. Ultimately, outcomes are determined by taxonomic judgement of homology and polarity, see Eldredge 1979, pp. 170-173 for an outline of principles used in interpretation, and Gingerich 1979 for specific comments on the application of stratigraphic data, Campbell & Barwick 1990 for comments on and references to functional complexes, and the applicability of parsimony analysis, and Schultze & Marshall 1993 for a comparison between the uses of functional complexes and parsimony in phylogeny reconstruction. Moreover, experience now shows that when cladistic methodology is employed (and especially when fossils are involved), numbers of contemporaneous, equally plausible, but inherently unstable, cladograms are liable to result; yet fossil data must be used in combination with those of modern forms when they are available (Hennig 1979: 16369). Ultimately, selection is made between competing cladograms by tests which judge their plausibility (e.g., by boot strapping, etc.; see Schultz & Marshall 1993:212). Therefore, although it is true to assert philosophically that there is only one tree of life, in practice our cladograms and trees are far from the objective, stable, namable units of evolution that stability and universality require of a system of nomenclature. The system of

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nomenclature we follow must make allowances for phylogenetic uncertainty so that the names applied to real organisms we place in the taxa vacillate as little as possible as the boundaries of taxa containing them vary. A classification is a word picture representing a phylogeny (whether a cladogram or a tree, Eldredge 1979) The question, therefore, is whether the words and structures enabled by the modern Code reflecting the evolved Linnaean system of nomenclature can meet the requirements of phylogenetic classification as well as providing names that can be applied to individual organisms in nature. Conclusion The challenges to nomenclature presented by phylogenetic classification are not different from those of traditional evolutionary classification, i.e., to provide names that are unambiguous, informative and as stable and universal in usage as possible for organisms found in nature and for the stages of their evolutionary pathways. Classifications are biological hypotheses and to expect to produce stability by basing names on the concepts of categories is a pipe dream. To fulfil both purposes of phylogenetic systematics, names must be provided for organisms that represent two fundamentally different classes of information – the inferred historical pattern of descent with modification leading to the organism, and the features of the organism that reflect its uniqueness as an operational unit of evolution (Wiley 1979: 212-4, and 1981: 74-6). The evolved Linnaean system does both. The binominals of the species-group names indicate the start points of the clades as well as indicating their evolved terminals. In each binomen the first supraspecific (generic) name does the former and, in turn, provides a linkage through the historical sequence of supraspecific names leading to it. The second specific epithet does the latter. Accordingly, I have concluded that no change in the Code is necessary to provide for the needs of phylogenetic classification (or “tree thinking”). Without changing the rules, some additional conventions may be used to make classifications more informative (such as the use of the term “sensu …”. The six conventions for use in phylogenetic classification summarized by Wiley (including additional qualifying phrases such as sedis mutabilis, incertae sedis, and indenting - Wiley 1981: 205-13) would require no changes to the Code. Whether or not the PhyloCode comes into use will depend upon the degree to which its names are found to be informative, simple to apply and memorable - and it is found to have benefits to phylogenetic classification not possessed by the evolved Linnaean system and its binomina. I hope that I have demonstrated that the Zoological Code is equally applicable. And to those intending to try to use the PhyloCode, for the sake of their colleagues needing to be able to apply names to organisms under the existing codes, I can only ask the users of that code that they, when naming new taxa, will ensure that any new names proposed are also available (or legitimate) for use in Linnaean taxonomy. To do otherwise (see, for example capricornia in Pleijel & Rouse 2000) is to force later users into usurping their rightful authorship.

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Acknowledgements I gratefully acknowledge the help and interest of my friends who have read drafts, commented on my presentation in Athens, and provided references to examples that underpin statements in it. These are Walter Bock, Ken Campbell, Chris Glasby, John Kirsch, Sandro Minelli, Philip Tubbs and Richard Willan. My wife Margaret provided constant support throughout its gestation and delivery. But beyond these immediate debts, I express my underlying gratitude to Arthur Cain who, 50 years ago, as my friend, colleague, tutor and supervisor, made me think, planted the seeds, and started me on this track. References BENTON M.J. 2000. Stems, nodes, crown clades, and rank-free lists: is Linnaeus dead? Biol. Revs 75(4):633-648. CAIN A.J. 1959. Deductive and inductive methods in post-Linnaean taxonomy. Proc. Linn. Soc., Lond. 170: 185-217. CAMPBELL K.S.W. & R.E. BARWICK 1990. Paleozoic dipnoan phylogeny: functional complexes and evolution without parsimony. Paleobiol. 16: 143-169. CANTINO P.D., BRYANT H.N., DE QUEIROZ K., DONOGHUE M.J., ERIKSSON T., HILLIS D.M. & M.S.Y. LEE 1999. Species names in phylogenetic nomenclature. Syst. Biol. 48(4): 790-807. CANTINO P.D. & K. DE QUEIROZ 2000. PhyloCode: A Phylogenetic Code of Biological Nomenclature. http://www.ohiou.edu/phylocode/ DE SMET W.M.A. 1974. An Introduction to New Biological Nomenclature (NBN). Kalmthout, Belgium. ELDREDGE N. 1979. Cladism and common sense. In Cracraft J. & N. Eldredge (eds), Phylogenetic Analysis and Paleontology. Proceedings of a symposium entitled “Phylogenetic Models,” convened at the North American Paleontological Convention II, Lawrence, Kansas, August 8, 1977. Columbia University Press, New York, pp. 165-198. GINGERICH P.D. 1979. Stratophenetic approach to phylogeny reconstruction. In Cracraft J. & N. Eldredge (eds), Phylogenetic Analysis and Paleontology. Proceedings of a symposium entitled “Phylogenetic Models,” convened at the North American Paleontological Convention II, Lawrence, Kansas, August 8, 1977. Columbia University Press, New York, pp. 41-77. HENNIG W. 1979. Phylogenetic Systematics. Translated by D. Dwight Davis and Rainer Zangerl. Foreword by Donn E. Rosen, Gareth Nelson and Colin Patterson. University of Illinois Press, Urbana, 263p. HOWCROFT J.M. & M.J.THORNE 2003. Zoological Record – a bibliographic service and a taxonomic resource. – Legakis et al. (eds.). The new panorama of animal evolution. Proc. 18th Int. Congr. Zoology. Athens, Greece, 28.08-02.09.2000. Pensoft Publishers, Sofia-Moscow, pp. 659663. ICZN 1905. Règles internationales de la nomenclature zoologique. International Rules of zoological nomenclature. Internationale regeln der zoologischen Nomenclatur, Ridevcal, Paris, 57p. ICZN 1999. The International Code of Zoological Nomenclature (4th Edn). The International Trust for Zoological Nomenclature, London, 305p. MAYR, E., LINSLEY, E.G., & USINGER, R.L. 1953. Methods and Principles of Systematic Zoology. McGraw-Hill Book Company, Inc., New York, 336p.

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PLEIJEL F. 1999. Phylogenetic taxonomy, a farewell to species, and a revision of Heteropodarke (Hesionidae, Polychaeta, Annelida). Syst. Biol. 48(4): 755-789. PLEIJEL, F. & G.W. ROUSE 2000. A new taxon, capricornia (Hesionidae, Polychaeta), illustrating the LITU (“Least-Inclusive Taxonomic Unit”) concept. Zoologica Scripta 29: 157-168. PLEIJEL F. & G.W. ROUSE 2000a. Least-inclusive taxonomic unit: a new taxonomic concept for biologists. Proc. R. Soc. Lond. B 267: 627-30. RIDE W.D.L. 1988. Towards a unified system of biological nomenclature. In Hawksworth D.L. (ed.), Prospects in Systematics. The Systematics Association. Clarendon Press, Oxford, pp. 332-353. RIDE W.D.L. 1991. Justice for the living: A review of Bacteriological and Zoological initiatives in nomenclature. In Hawksworth D.L. (ed.), Improving the Stability of Names: Needs and Options. [Regnum Vegetabile No.123]. Koeltz Scientific Books, Konigstein, pp.105-122. RIDE W.D.L. 1999. Introduction, pp. xix – xxix, of The International Code of Zoological Nomenclature (4th ed). The International Trust for Zoological Nomenclature, London. 305p. RUDMAN W.B. 2000. Siraius immonda (Risbec, 1928) and Diniatys dentifer (A.Adams, 1850) in Sea Slug Forum. http://www.seaslugforum.net/dinident.htm SMITH H.M., BROWN L.E., CHISZAR D., GRISMER L.L., ALLEN G.S., FISHBEIN A., HOLLINGSWORTH B.D., MCGUIRE J.A., WALLACH V., STRIMPLE P. & E.A. LINER 1998. Crotalus ruber Cope, 1892 (Reptilia, Serpentes): proposed precedence of the specific name over that of Crotalus exsul Garman, 1884. Bull. zool. Nomencl. 55(4): 229-231. STRICKLAND H.E. 1838. On the inexpediency of altering established terms in natural history. Mag. Nat. Hist. (n.s., Charlesworth) 1: 127-31. STRICKLAND H.E., PHILLIPS J., RICHARDSON J., OWEN R., JENYNS L., BRODERIP W.J., HENSLOW J.S., SHUCKARD W.E., WATERHOUSE G.R., YARRELL W., DARWIN C. & J.O. WESTWOOD 1843. Series of Propositions for rendering the Nomenclature of Zoology uniform and permanent, being the report of a Committee for the consideration of the subject appointed by the British Association for the Advancement of Science. Ann. Mag. Nat. Hist. 11: 259-75. WILEY E.O. 1979. Ancestors, species and cladograms – remarks on the symposium, In Cracraft J. & N. Eldredge (eds), Phylogenetic Analysis and Paleontology. Proceedings of a symposium entitled “Phylogenetic Models,” convened at the North American Paleontological Convention II, Lawrence, Kansas, August 8, 1977. Columbia University Press, New York, pp. 211-225. WILEY E.O. 1981. Phylogenetics. The Theory and Practice of Phylogenetic Systematics. John Wiley & Sons, New York. 439p.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.)

Still Desiderata: Scientific Names for Domestic Animals and Their ... Evolution 683 The New Panorama of Animal Proc. 18th Int. Congr. Zoology, pp. 683-697, 2003

Still Desiderata: Scientific Names for Domestic Animals and Their Feral Derivatives T.W. Wyrwoll J. W. Goethe-Universität Frankfurt am Main, Albert-Schweitzer-Str. 52, 60437 Frankfurt am Main, Germany. E-mail: [email protected]

Abstract This paper is a proposal of an easy and consistent nomenclature system for domestic (sensu domesticated) and feral animals. These types of taxa are not sufficiently governed by the regulations of the present Code, for they cannot be equalised with the hithertoregulated species-group taxa. As a result, there are no commonly-accepted scientific names for them, and thus neither they nor the in many cases depending scientific designations of their wildforms, i.e. their wild “ancestral” relatives from the stock of which they once were derived, are yet unanimously agreed upon. Hitherto approaches towards domesticanimal nomenclature do not meet the requirements of the field. A new alternative naming system is introduced, based on the first name given to a wildform and the addenda forma domesticata (= f. dom.) and forma efferata (= f. eff.), respectively, and recommendations on its application are made. Some suggestions to amend the general zoological nomenclature system – inter alia by the use of the terms et, sive and vulgo – are provided, too.

1. Introduction The scientific naming of domestic animals still remains an unresolved problem. Whilst the issue has been debated for more than 60 years,1 as yet no commonly-accepted result is available. Consequently, the International Code of Zoological Nomenclature (International Commission on Zoological Nomenclature 1999) still does not include any particular regulations for domesticates. They are only mentioned in its Article 1.2.1, that states the scope of the Code also includes “names based on domestic animals”. However, no further guidance how to treat them is provided, at least not explicitly. Before the current version of the Code came into power, even the inclusion of domesticates within nomenclature could have been doubted, e.g. for in their Article 1, 1 In particular cf. the contributions of Pohle (1935), Bohlken (1958), (1961), Dennler de la Tour (1968), Groves (1971), (1995), Clutton-Brock et al. (1976), Corbet (1978), Corbet & Clutton-Brock (1984), Odening (1979), and Uerpmann (1993), in which different solutions were suggested.

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the Second and Third Editions of the Code (International Commission on Zoological Nomenclature 1964, 1985) expressively limited its scope as to “animals known to occur in nature”. However, the current situation is not satisfactory either, for – as will be shown below in some more detail – domesticates cannot be equalised with those taxa yet regulated by the Code, such as subspecies or species. Any identification with these taxa necessarily leads to inconsistencies, and consequently several severe problems to apply the Code do arise. The result of this unsettled situation is a considerable variance in nowadays naming approaches. Whilst many biologists regard names given to domesticates as not applicable, or only to a limited extent, some others fully accept them, though yet mostly for reasons of convention. However, at present the majority of researchers understand names given to domesticates as invalid. The solutions suggested up to this time by this latter group of zoologists are different in their intention to be based on the Code or not – some researchers even explicitly place theirs outside the Code, e.g. Groves (1971) and Uerpmann (1993). However, in view of the new definition of the Code’s scope (see above) an inclusion of domesticate names into the provisions of the Code seems to be advisable. Although this is not the proper place to discuss those proposals which intend to expand the Code or to modify the names based on its application, for our purpose it may be said they all exhibit some inconsistencies, or at least certain complications.2 Most “schools” of researchers working on domestic animals meanwhile developed their own nomenclatoric approach, that in some cases changed over time, and occasionally has been adopted to a different extent by scholars outside these schools, but probably in most cases inconsistently. The use of different nomenclatoric systems resulting from this situation currently leads to a considerable confusion, both in sciences and in the public, and has caused a variety of negative effects. This unnecessary problem should be solved. Since there is no special taxonal group for the domesticates provided for by the regulations of the Code, the question to which of the taxa currently governed they may be equalised with is fundamental – and not only for the domesticates themselves. If domesticates are regarded as equivalents of natural taxa, as is conventionally the case, this too becomes important for the naming of most of their wildforms, i.e. the natural taxon from which they once were derived.3 Due to the principle of priority, many names of domesticates which antedate those of their wildforms would become the types of their respective taxa. In other cases, domesticate names are contemporary or even identical with those of wildforms. Here the Code, as hitherto understood, does not automatically provide a solution how to proceed either. For example, the scientific names of wild species such as wolf, cattle (= urus), horse4, ass, goats and ibexes, sheep, 2

However, it may be remarked that the explanation in a recent application to the Commission by Gentry et al. (1996) is largely incorrect and neither describes these systems properly nor their pitfalls to any extent. Some comments on Bohlken’s approach can be found below (Chapter 4).

3

The English expression “wild type” must be regarded as unclear in terms of systematics, so here I prefer to use the word “wildform” instead.

4

The question of the earliest available scientific name for a wildform of the horse has been debated for quite a long time and could not yet be unanimously settled. Equus ferus or Equus equiferus as of Pallas, 1811 do not exist, for Pallas used these terms merely in a descriptive way (e.g. mostly referring to „equiferi”), and thus not as scientific designations – a misinterpretation that also applies to many other supposedly

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waterbuffalo, yak, gaur, the Old and New World camels5, wildcat, polecat, rabbit, coypu, and others depend on the solution of these nomenclatoric problems. The present paper is a proposal of a new naming system for domesticates and feral animals. It also embraces a short discussion of the above nomenclatoric questions and concludes they can solved by a consistent application of the Code. However, in order to clarify certain points, some suggestions to amend the Code are made, too. 2. Domesticates are neither subspecies nor species If the Code is expected to be used for names given to domesticates, one very fundamental problem arises: To which of the taxa dealt with by the Code domesticates may be equalised? As I will demonstrate here none of the available taxonal groups – only subspecies and species will have to be considered – is qualified for this purpose. In order to explain this statement, I may initially try to give a useful definition of the term “domestication” that can be subscribed by virtually all researchers working in zoological and anthropozoological disciplines: Domestication is the process of genetic changes in animal populations that occurs due to human control of their breeding, leading to an alteration of their genetic and bodily conformation. Animals resulting from such a process are considered domestic, unless they live in established free-ranging populations independent of human control, which are regarded as feral. These definitions may now help us to understand why domesticates are taxa not yet sufficiently regulated by the Code.6 scientific names which were originally published in Latin texts. In any case it is predated by Equus ferus Boddaert, 1785 that forms the earliest available name for wild horses. Although Boddaert’s general description would neatly fit with a tarpan-type of horse, it may be doubted whether some of his literature sources indeed would not comprise other taxa, too. For this reason, I suggest a redefinition of Boddaert’s type. In addition, the name of the Mongolian wildhorse, Equus przewalskii Polyakov, 1881, and possibly also Equus sylvestris de Brincken, 1828 are available as names for (sub-) recent Eurasian wildhorses (with if at all so but a minor impact of domestic blood, that in any such case should be neglected in the interest of appropriately-stable nomenclature). 5 Both New World forms are included here for the vicuna could be a (further) ancestor (at least) of the domestic alpaca. However, the question of the alpaca’s origin is not yet finally settled. The Old World camels, according to my point of view, should be regarded as forming one species. There are clear indications that during the Holocene, roughly speaking, wild one-humped camels thrived in North Africa and on the Arabian Peninsula, whereas wild two-humped camels could be found in cum grano salis Central Asia. Most probably, both of these groups comprised different subspecies, which however could not be described so far. To me, the known archaeological record clearly suggests that independent domestication processes of Old World camels took place in Africa, Arabia, and mainland Asia. Some genetic exchange between these domestic populations must be expected even for the distant past, and is well-known from the breeding of “tulu” and other types of hybrids between dromedaries and Bactrian camels, both in historic times and nowadays. The naming of the wild Old World camel may cause some debate: The commonly favoured name Camelus ferus as based on Przewalski’s description is preoccupied, but in future may be made available by decision of the International Commission on Zoological Nomenclature. Otherwise there already exists the name thomasii for a North African find, that provides the presently valid (see below) species name based on a wildform: Camelus thomasii. 6 A further group of animals that if often incorrectly referred to as “domesticates” are animals which are highly adopted to man-made or man-influenced environments, and which do currently either occur largely or entirely in such habitats, or meanwhile became real domesticates. These animals are still or were in the past in particular commensals or other types of synanthropics. I strongly suggest such populations

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— Domesticates derived from a wild species are not a subspecies of the latter since they are not based on one shared population origin and since the traits separating them from closely-related wild populations are not inherited due to natural selection. Thus these traits are different in their evolutionary function from those of wild populations, i.e., they are not adapted to fit with natural environmental conditions. Of course, domestic animals also do not thrive in a geographic range (or in an ecological niche) of their own that is not inhabited by conspecifics, so basic taxonomic concepts such as the terra typica cannot be applied on them. Also the differentiating pressures on domesticates are not uniform, but substantially different in their direction within various populations. Above that, these populations are by no means stable in their structure, for their constitution is changed time after time by humans for man’s specific needs. Therefore, domesticates of one species do not form an evolving or at least: evoluted unit as do naturally-defined subspecies. Second, and more a technical argument, the differentiating traits of domesticates consequently usually differ to a substantial degree between various breeds and races, and mostly even within one such taxon. Differences in the natural origin between the diverse domestic populations do further contribute to this end, too. Therefore, the limited degree of uniformity within the domesticates of one species does not allow to define them morphologically, i.e. by their similarity with each other, as one subspecies (assuming the respective traits could be used for systematic purposes).7 A however not practicable approach to overcome these latter problems would be to regard every race or breed of domesticates separately as a subspecies of its own. However, the above fundamental philosophical arguments in every case to at least some extent would also apply to any single breed, even though its origin, development and morphology necessarily would be narrower than in the entirety of the domesticates of its species, and even in some but rare cases certain of the above counterarguments might well not work. Certainly, in nomenclature any approach to separately name the breeds would lead to a plethora of names for rather arbitrary forms and various undesirable discussions about a variety of issues, including e.g. their priority. The result would certainly diminish the worth of nomenclature considerably. Fortunately, this approach is not en vogue, and, at least largely, has not been so during previous decades. which differentiated from other taxa of their kind due to adaptations to a human-mould environment should be understood as subspecies. For these animals do not fulfil the above definition of domesticates, their acceptance as a member of this special type of taxon would be misleading. In order to point out their particular origin, the zoologist might be recommended to add the term anthropogaia (which means a “human-generated landscape”) in brackets after the species or subspecies name, e.g. “Mus musculus brevirostris (anthropogaia)”. This term generally cannot be mistaken for a taxon name, and its Greek origin also contrasts the Latin-derived naming proposal for domesticates below, thus highlighting the systematic differences between these two groups. Where populations are addressed which are only a part of a larger taxon and which are not named separately, the terms “synanthropic population” (or “commensal population” etc.) might be placed after the name in brackets. Whilst the general proposals made in this paper should be formally incorporated into the Code, the above suggestions do not need a formal approval. However, they might preferably be included with the Code in form of a recommendation. 7

Systematists traditionally expect the majority of the members of one subspecies to be identical in a certain set of characters not shared by other subspecies of their species. There should be no reason to give up this claim in the case of domesticates.

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Since the peculiar traits of domesticates did not come into being and still do not develop on the basis of evolutionary and collective processes which govern those of real subspecies, these two groups cannot be equalised for fundamental reasons. — Domesticates are also not species since there are no genetically-fixed traits which would avoid mating between them and their wild “ancestral species”. Thus the domesticates do not exhibit the very fundamental character of a species and so necessarily they belong to the same species as their wild ancestors. Domesticates are therefore entities of their own merits. They are neither species nor subspecies, nor of course any taxon of higher systematic rank, i.e., they do not fit with any of the categories yet fully regulated by the Code. 3. Domesticates are hybrids In the previous chapter, I explained why domesticates are taxa of their own and why they cannot be dealt with in the same way as natural taxa. However, even if one would accept domesticates as representatives of natural taxa, their vast majority would not be suitable to become a nominal taxon of their wildform. The reason for this is that almost all “traditional” domesticates are either known or most likely to be descendants of more than one subspecies. Some of them may have even received some (however minor) genetic impact from other species, i.e. per definitionem taxa they would not normally interbreed with under natural conditions, too. This has principal implications as for their use in nomenclature. It means that even if one would regard domesticates as appropriate to be considered according to the Code, their names could never become valid: Being hybrids in origin, on the one hand their names were available, whether they were originally known to be hybrids or not (Article 17.2), but on the other hand as such these names were not valid (Article 23.8). The latter article says that “A species-group name established for an animal later found to be a hybrid … must not be used as the valid name for either of the parental species, even if it is older than all available names for them.” For logical reasons, for a consistent interpretation of the Code, i.e., for a proper definition of the term “hybrid” in its regulations, that likewise can be found in the Code’s Glossary, and to allow the typebased nomenclature system to function, the term “parental species” in this sentence necessarily has to be interpreted as “parental species-group taxa”, i.e. it should include the subspecies, too.8 This reading is essential for the entire applicability of the Code: Due to the regulations on nominotypical taxa, nominotypical subspecies are the types of their species, these the types of their genera, and so on, and thus in consequence the nominotypical subspecies becomes the very type of any taxon higher in rank (cf. in particular Articles 46.1, 47.1, 61 and 72.8 of the Code). As a result, any formal acceptance 8 This inaccuracy here is based on a rather misleading definition of “hybrid” within the Glossary of the former Third Edition of the Code that also formed the basis of certain of its articles (International Commission on Zoological Nomenclature 1985; see also below) and should be corrected by the Commission. I also suggest to reconsider the wording of this article in view of its differences with Article 17.2, to which it is both historically and factually connected, for the latter now includes taxa which were already known to be hybrids when described.

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of subspecies hybrids as subspecies, may they be domesticates or not, would eventually jeopardize the use of the entire Code. Thus the meaning of Article 23.8 as for the majority of domesticates is obvious: First, Linné and other authors who described them did certainly not consider them hybrids, so this article has to be applied.9 Second, if one understands them in their relation to those taxa which are already regulated by the Code, i.e. here (at least primarily), as nowadays we can see, to subspecies, they are indeed subspecies hybrids. Their names therefore cannot be valid for either of their parental subspecies, and consequently the same goes for their entire species, too. A consistent application of the current Code thus excludes the majority of names originally given to domesticates from becoming valid names of their entire species, i.e., also of their respective “ancestral” wildform. Accepting this simple conclusion, the long-debated hitherto naming inconsistencies are already remedied.10 It may be remarked that the above, and zoologically very appropriate, reading of the Code was not formally possible before the Code’s current Fourth Edition. The Second Edition (International Commission on Zoological Nomenclature 1964) in its Articles 1 and 17 only stated that scientific names based on hybrids were available if not given to hybrids as such. The Code’s Third Edition (International Commission on Zoological Nomenclature 1985) repeats the above points, however in Article 23 (h) – as does the present Code (see above) – clarifies that names of hybrids “must not be used as the valid name of either of the parental species.” Unfortunately, and this chiefly accounts for the improper reference to “species” in this sentence, the Glossary as an integral part of that Code explicitly states that subspecies hybrids “are not hybrids” (op. cit., p. 256). This of course is a misleading conception, for it severely hinders the use of the type concept on which the Code is so essentially based, even though it probably was meant to stabilise names of higher taxa where indeed this is not necessary, and fortunately it has been changed meanwhile. The now senseless relic wording in Article 23.8 as seen above should consequently be regarded as irrelevant, for it is an obstacle to the Code’s intended very meaning and positive content, however still would have to be corrected. In any case, such an act of correction would ultimately support the above-given interpretation of the validity of names based on subspecies hybrids, including the domesticates. The only exceptions from the above rule are those traditional domesticates being, monosubspecific in origin, i.e. the domestic Japanese quail that descends from Coturnix coturnix japonica Temminck and Schlegel, 1849, the wild Japanese quail, and possibly too the Guinea pig that some zoologists (e.g. Herre & Röhrs 1990) believe to be derived from Cavia aperea tschudii Fitzinger, 1867.11 However, for the name of the Japanese subspecies of the quail clearly postdates the Linnaean valid species name, it does not at all affect the problem dealt with here. Therefore, there is only one single species within the entire realm of traditionally discussed naming ambiguities where its earlier-named domesticates are possibly derived from one wild subspecies. Theoretically, for some more exotic domesticates 9

However, in case the referred to passage of Article 23.8 would be changed as suggested in the footnote above, of course the following considerations still would maintain their validity.

10 11

A formal solution to identically treat the nomenclature of all domesticates is suggested below.

However, the breeding history of the Guinea pig is still far from being sufficiently understood, and the systematics of the wild cavies may well need some revision, too.

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such an origin might also be discussed, however until nowadays has not been done so to any major extent, and there is likely no reliable proof for such viewpoints anyway.12 Even if there might be further examples of monosubspecific domesticates to be detected some future day, most probably there are and will remain different and changing opinions on their natural ancestors. Considering these domesticates nomenclatorically as types of their natural taxa would thus certainly result in different names to be used for one taxon, and thus contribute to nomenclatoric instability. Would it be decided to use different naming systems for domesticates with a monosubspecific and polysubspecific origin, respectively, most probably the identification of both groups would be hampered. All these problems strongly contradict a practical solution as aimed for by the Code. So it must be concluded that scientific names for natural taxa based on the original names of domesticates of monosubspecific origin are by no means desirable, too. For all these reasons, domesticates obviously are not suitable types for natural taxa such as subspecies or species. 4. The forerunner: Bohlken’s system The probably most-widely accepted system to name domesticates yet available is that of the German zoologist Herwart Bohlken (1961). It replaces an earlier suggestion of the same author (Bohlken 1958), in which domesticates were referred to by using a trinomen, i.e. as subspecies, based on the species name being the first scientific name given to their wildform, and a subspecies name taken from the first scientific name ever applied to the domesticate; where such a subspecies name did not exist, Bohlken instead added the word domesticus in Italics. His modified system (Bohlken 1961) is commonly referred to as the one of the “Kieler Schule” and is used particularly amongst European zoologists, but likewise has been adopted by many other researchers worldwide. As in his original proposal, Bohlken’s newer approach is to use the first available name given to a wild animal as the species name of the entire species (i.e. for both the wild and the domestic animals). In order to denote a domesticate, in those cases where an earlier domesticate name (e.g. authored by Linné) exists, it adds the word “forma” in normal characters and the subspecies name as above in Italics (or shortly “f. subspecies name”) after the species name. Alternatively, where there is no such domesticate name in existence, the words “forma domestica” (or shortly: “f. domestica”) are added. The term “forma”13 is not written in Italics for it is not considered part of the scientific name itself. Its addition is to explicitly indicate that domesticates are not “natural” subspecies. The author and date are given as those of the domesticate form’s name. 12 Otherwise, only neo-domesticates are in their entirety of monosubspecific origin, such as Mesocricetus auratus and Chinchilla chinchilla (of C. c. boliviana Brass, 1911). 13

Bohlken’s use of the word forma seems to be to some extent a result of a discussion with Prof. Erich Martin Hering, Berlin, cf. Bohlken (1961), p. 109, footnote 1. This term may possibly have been debated earlier for the same purpose at the XVth International Congress of Zoology in London, to which Otto Kraus refers in a letter to Bohlken (op. cit., p. 109, footnote 3). In his letter, Kraus already uses a Bohlken-styled naming system, however, with the addenda to the species name not written in Italics and not accepted as subspecies names. Unfortunately, the origin and the development of these ideas do not become clear from Kraus’ letter.

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A logical inconsistency of this system is that names originally given to domesticates initially were neglected and eventually reappear. The problem is even more puzzling for according to Bohlken (1961, pp. 108 – 109) these names are indeed, and therefore also were treated as (see above), subspecies names. Such a biased treatment strongly contradicts the basal provisions of the Code, e.g. Article 47 on nominotypical taxa.14 A further major problem of Bohlken’s approach is that he regards domesticates as subspecies. By inference from his short discussion of the nature of speciation processes (Bohlken 1961, pp. 108 – 109), one sees that he defines domesticates as “unnatural ecological subspecies”, i.e. subspecies different from other subspecies due to their humaninduced development. In Bohlken’s view, they have to be accepted as subspecies simply for they are subgroups of the species, for which there would be no other type of taxon available in nomenclature (op. cit., p. 109). A further explanation is not provided. Therefore, in commenting this point I may simply refer to my above discussion of the topic that basically contradicts Bohlken’s opinion: Domesticates are not subspecies, and they should also not be equalised with them. Also Bohlken does not provide conclusive arguments why domesticates were, as he treats them, nomenclatorically inferior taxa in comparison to wildforms. In his earlier proposal, Bohlken (1958) refers to a historical argument in order to subordinate names of domesticates: “Since in nature the wildform was primary, it would not be logic to name the species after the domesticate.” (op. cit., p. 168; translation T.W.). In his second proposal (Bohlken 1961), he repeats his former point (p. 109), and additionally cites from a letter send to him by Otto Kraus, member of the International Commission on Zoological Nomenclature, saying that “the (sic) scientific names of domesticates in future”, resulting from recently passed amendments of the thence Rules, would have to be regarded as “infrasubspecific in status” and therefore were not to constitute a subject of zoological nomenclature (p. 109, footnote 3; translation T.W.). Both of these opinions are neither explained nor discussed by Bohlken at all, and in my point of view they do not provide any real argument in favour of his approach. First, an earlier occurrence within organismal history cannot be taken as an argument for nomenclature, for the one has nothing to do with the other. Should one intend to include such a misleading argumentation within the Code, for example any name given to a Holocene specimen of a species would have to be replaced when a name for a Pleistocene specimen of the same species becomes available. This cannot be in the interest of nomenclatoric stability. Second, domesticates do certainly not have a biologically infrasubspecific status (with the exception of but a few cases of possibly a monosubspecific origin; see above). If one intends to give them a status based on their taxonal origin, they would have to be placed somewhere in between that of those subspecies from which they once were derived, i.e. maybe the category 14

This may also be the reason why Bohlken did not ask the Commission for an amendment of the Code, as he initially intended (Bohlken 1961, pp. 109 – 110). In his proposal, Bohlken explains that antedated (sic) names (contemporary names are not referred to here) of wildforms should be placed on the list of Nomina conservata, whereas domesticate names should be indicated, i.e., suppressed (loc. cit.). This too contradicts with his system, for as described above here these latter names are nevertheless allowed to form the subspecies names for the domesticates. A better way to formally avoid names of domesticates to become valid species names will be suggested below.

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of the subspecies, on the one and their entire species on the other hand – a positioning that bears no whatsoever nomenclatoric advantage. The wording of Kraus’ letter, and this in fact to me seems the most likely interpretation, would allow to understand his remark as referring to the use of the word “forma” when recently applied to domesticates, i.e. within Bohlken’s or a similar system. This is exactly what the then decided new Rules said on the use of “form” after 1960 (International Commission on Zoological Nomenclature 1964, Articles 1 and 45 (e) ii). However, this neither says domesticates were biologically infrasubspecific, nor would it have any meaning as for the availability of those names originally applied to domesticates by Linné and others. It would only point out that such a system is an approach outside the scope of the new Rules. In sum, therefore, Bohlken did not provide any substantial argument why the earlier or contemporary domesticates’ names would have to be excluded from zoological nomenclature. In addition, one might say one should more properly express the domesticated status – “domestica” is not an appropriate designation, because it has a rather broad literal and zoological meaning,15 for it likewise addresses animals living in other kinds of a companionship together with humans (cf. e.g. its equivalents as given by Diefenbach 1857 or Georges 1959), though of course it designates domesticated animals, too (e.g., Werner 1972, Niemeyer 1976). Also the term occurs in scientific names for wild animals, so it may cause some confusion, e.g. when modern search engines for bibliographic databases are used. And finally, the co-existence of earlier subspecies names and “domestica” to designate the formae makes it unnecessarily difficult to immediately recognise domesticates as such by their names, in particular for the zoological layman. An easy recognition of the domestic status, however, is certainly desirable. No matter how many are the problems of Bohlken’s approach, there are very considerable advantages in it: (1) It is an extension of species names according to the Code’s type of names by an addendum to indicate the domestic status. The relatedness to the “ancestral species” thus can be easily recognised. (2) The designation of the domesticates as a forma of their species makes it clear that they are on the one hand a subgroup of the species, however on the other hand not a natural subspecies.16 This of course neatly fits with the zoological facts. Therefore, in my viewpoint Bohlken established a naming system that can be rightly used as a basic model for a solution of our problem, and hereafter I will propose such a system. 15 The term means “house- or family-related”, as does its English successor, more specifically may stand for a member or friend of the family, and in a figurative sense denotes meanings like private, personal, personspecific, native, local, national, compatriot, home, civil, etc. In relation to animals, it refers to all kinds of house-related animals alike, be they domesticates or somehow controlled wild animals. 16 However, to Bohlken they were indeed “unnatural ecological subspecies”, a viewpoint that I cannot subscribe (see above). For this reason, I here suggest a re-definition of Bohlken’s use of forma, i.e. as a category different from that of the subspecies. One may only question whether forma would be too close to general non-Latin zoological language, i.e. vernacular words derived from this expression, so the term might be better replaced by an alternative, such as aspectus. However, any solution of this terminological issue is solely based on convention, so I feel there is no real problem as with Bohlken’s word. In addition, forma is a very convenient term familiar to many zoologists. It is quite short and further can be easily abbreviated as “f.”, whereas other suitable terms would be longer, both in full and when abbreviated in a recognisable manner. Therefore, I think forma is a good candidate to be used in future time, too.

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5. A new proposal to name domestic animals As seen above, domesticates are, generally speaking, entities of their own, i.e., different from wildforms: They are neither subspecies nor species. As such a separate category they are not dealt with by the provisions of the hitherto Code. Since the majority of domesticates are polysubspecific in origin, taken as representatives of natural taxa according to the Code they are subspecies hybrids, and thus cannot form the basis of valid names. In some, however very rare cases, domesticates may also be the descendants of a single subspecies. However, an acceptance as name-bearing types in these latter cases would result in a plethora of doubtful and unclear nomenclatoric solutions. In sum, the fact that the Code does not contain specific rules how to treat domesticates obviously leads to an undesirable confusion in nomenclature. It is therefore necessary to extend the regulations of the Code. My own proposal intends to avoid the shortcomings of Bohlken’s, but to maintain its major advantages. Its basic idea is to name domesticates by using the first available name given to a wild animal of their species and add the words “forma domesticata”, or shortly, “f. dom.”. For example, “Canis lupus forma domesticata” or alternatively “Canis lupus f. dom.” is a domestic dog. If desired, author and date of the wildform may directly follow its species name, but precede the indication of the forma. Example: Canis lupus L., 1758 f. dom.17 In order to denominate a specific race or breed of domesticates, one may add a conventional nonLatin name for them in brackets, that would not become part of the scientific name. Example: Canis lupus f. dom. (German shepherd). The entire scientific name in my proposal is written in Italics, i.e. here including also the words forma domesticata which are integral parts of the scientific name, in contrast to Bohlken’s approach where the word forma is written in conventional characters and not considered part of the scientific name. ‘domesticata’ is a neo-Latin term here used to express the fact of domestication. Originally meaning “to tame” (e.g., Habel 1959, Niemeyer 1976), domesticare was later used also in the sense of “to domesticate” (e.g., Werner 1972), a fact that is also reflected by the usage of vernacular terms for “domestication” derived from this Latin expression in the majority of Europe’s present-day languages. The lexicological shift towards its modern meaning in Latin taxonomic terminology here should be conventionalised for nomenclatoric purposes, too. Formal descriptions of domesticates or feral animals are regarded as taxonomically irrelevant, for they do not have any typological importance for the entire species, and consequently a scientific name with author and date is not applied to them. A sentence stating “Names established on domesticates or their feral forms must not be used as species-group names.” added to the Code would formally allow a consistent application this system to all domesticates, even in case some of them were not subspecies hybrids. Nomenclatoric stability as aimed for by the Code would also be maintained since names given to domesticates or their feral descendents were still available, even they could not be valid. 17

Given the limited number of and the widespread knowledge on domesticates that has to be taken for granted, normally it would not seem to be necessary to cite their wildforms’ authors and publication dates.

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This proposal is a simple solution, short in its form, denoting all domesticates equally, thus making it easy to recognise them as such to everybody even not familiar with biological issues. Since the addition is consisting of two (or three; see below) words, also being different in style from existing subspecies names, it cannot be mistaken as designating a subspecies. For these reasons I think the new system constitutes a sound basis to introduce domesticates as a new taxon of its own rank into the provisions of the Code.18 Whilst the proposal would easily solve the majority of uncertainties concerning the names of species with named domesticated members, there is but one group of such animals where it does not automatically provide a solution: those entire species where there are no separate names for their domestic and wild fractions. Here one has to think, e.g., of Oryctolagus cuniculus and Anas platyrhynchos named by Linné, and some other species. However, these cases are but limited in their number and may be solved by means of a restriction of their types, or alternatively, if there are no proper types, as is the case in most of these instances, by the designation of neotypes. Such a step should be generally advisable here, even when not addressing the domesticates’ naming, let alone in order to have a fixed type specimen for the natural taxa involved, and therefore to be able to use zoological nomenclature properly. 6. A proposal to name feral animals Analogously to the above proposal for domesticates, I suggest that feral animals should be referred to by the first available name given to a wild animal of their respective species and the addition “forma efferata”, or shortly “f. eff.”. As an example, “Canis lupus f. eff.” would be a feral dog. In the case of feral populations of particular zoological importance, which for this reason are conventionally referred to with supposedly scientific names, an addendum in brackets specifying its geographical range or giving a vernacular name, preferably also including a designation of the range, should be recommended. For example: Ovis ammon f. eff. (Corsica) or Ovis ammon f. eff. (Corsican Mouflon).

18 A proposal to the International Commission on Zoological Nomenclature by Gentry, Clutton-Brock & Groves (1996) intends to achieve the names of some domesticates and those of their later or contemporarily described wildforms being equally valid (stating the latter should “not be invalid”) – a status not yet formally in existence, however. Obviously, their aim is to receive some formal basis for the current usage of some later species names originally applied to wildforms for the latter when accepting both as conspecific, and possibly also (although not stated) as the valid species names of the related domesticates. There are two basic problems with this proposal: Any approval by the Commission would implicate that domesticates and their wildforms are conspecific, what is certainly correct from a zoological point of view, however since it forms a ruling on a systematic question, as such it is conventionally regarded as formally not being within the scope of the Commission’s responsibility. More important is that this approach would not in itself constitute a solution of the problem here addressed. Neither the validity of the two names then being in concurrence with each other would receive any guidance, nor a system to name domesticates appropriately would be founded. Therefore, the proposal of Gentry et al. (1996) does not solve the problem, and so I suggest it to be replaced by the above-described or another however similar in its results solution.

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In general Latin, efferatus means feral, or literally: feralized. This addition cannot be mistaken at all and thus this proposal has the same nomenclatoric advantages as the above suggestion for domesticates. 7. How the new system could be extended The above-proposed system can be easily extended according to the needs of the zoologist. Some suggestions can be found below. In any case, author and date of a wildform or explanations in brackets may be added as suggested above, however are left out here for reasons of easier comprehensibility. - If a domesticate is believed to descend from only one known subspecies, the valid name of that subspecies may be used analogously to the first available species name in the general proposal. Example: “Canis lupus pallipes f. dom.” is a dog believed to be solely a descendant of the pallipes subspecies of the wolf. - If the wild species from which a domesticate originated is not known or doubtful, only the generic name may be used instead of the full species name, with the abbreviation “spec.” for “species” added. Example: “Cavia spec. f. dom.” means the domestic Guinea pig, the natural origin of which has not yet been unanimously settled. - Where a hybrid of a wild and a domestic animal has been identified, it might be labelled as follows: species name, possibly with subspecies name or subspec., et (i.e., and) f. dom. “et” would not be written in Italics, for it is not part of the scientific name. Example: “Canis lupus subspec. et f. dom.” is a hybrid of a wolf and a dog. Hybrids involving feral animals may be designated similarly, as should other types of hybrids, too. Indications of races or important feral populations do as generally follow after the entire scientific name in brackets, for example: Canis lupus f. eff. et f. dom. (Australian Dingo and Foxterrier). However, it must be remarked that the order of forms within the scientific name is not intended to designate the taxonomic affiliation of either the male or the female parent, as did certain regulations within earlier versions of the Rules in similar cases. Such regulations would easily lead to misinterpretations of names where no specifications are intended, and it should be better suggested to indicate such facts in the brackets following the name. Example: Canis lupus f. eff. et f. dom. (father Australian Dingo, mother Foxterrier). Species hybrids19 may be named in a manner similar to subspecies hybrids, but with the post-generic names in full or substituted in order to avoid misinterpretations; e.g. “Canis lupus f. dom et latrans subspec.”

19 I suggest the genus in zoology is to include all taxa of which the individual members under certain conditions are able to produce hybrids with each other by means of natural procreation, be it directly or indirectly, and be that offspring fertile or not. This provides a biological definition of the genus that is similar in its applicability to the “biogenetic” one of the species currently in use (and as in the latter, in this case one would have a temporal scope within the present, too). Of course, natural taxa are evolving units, so that such a definition cannot be an absolute one. However, for I refer to this, say, “biogenetic definition”, I do not see any need to discuss the naming of genus hybrids here, since they would not exist.

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- Should a researcher believe that a particular dog breed is derived from two subspecies, he may state this as follows: species name (first subspecies name et second subspecies name) forma domesticata. Example: “Canis lupus (arabs et pallipes) f. dom.” is a dog based on the arabs and pallipes subspecies of the wolf. - In cases where the domestic status is not clear, an animal may be referred to as: species name, possibly with subspecies name or subspec., sive (i.e., or) f. dom. As above the et, sive does not form a part of the scientific name and is therefore not written in Italics. Examples: “Canis lupus sive f. dom.” is either a wolf or a dog. “Canis lupus pallipes sive pallipes f. dom.” is a pallipes wolf or a dog of pure pallipes ancestry. – In order to let researchers more easily understand a name newly introduced for an otherwise well-known taxon, the subspecies name formerly in use may be included in brackets after the new scientific name, preceded by the word vulgo in normal characters (i.e., not in Italics). Example: Ovis ammon f. eff. (Corsican Mouflon, vulgo musimon).20 Some of the above proposals may also be useful for the naming of wildforms, and therefore may be included with the Code in form of recommendations. For example, zoo lion hybrids might be referred to as “Panthera leo leo et melanochaita”. “Panthera leo leo et al.”, however should refer to a predominance of the leo subspecies, whereas “P. leo leo et subspec.” would describe a population with nominotypical and some further, however unknown in its strength subspecies impact. Highly “complicated hybrids” may additionally better be explained also in vernacular terms. Congo lions of unclear subspecific attribution, e.g., may be addressed to as “Panthera leo azandica sive bleyenberghi”. And finally , “vulgo” may be used to indicate former subspecific names as above. Of course, such a usage of addenda to scientific names needs the approval of the International Commission on Zoological Nomenclature in order to be in accordance with the Code. 8. Concluding remarks The here-proposed new scientific naming system for domestic and feral animals, the amendments of and the recommendations on some more general addenda to the Code are of course but proposals. There may be suggestions for their further improvement or even entirely different proposals to replace them. I intend to explain my ideas in appropriate journals subsequently and do look forward to discuss them with any zoologist interested in this matter. At least for the first-mentioned topic, I am convinced that the here-described system is able to settle all hitherto unresolved problems and 20 Also I suggest to include a recommendation that a questionmark placed after one part of a scientific name is to indicate this part as being doubtful. If a questionmark is used after brackets, the entire content of the brackets would be referred to as doubtful. And finally, in order to show that an entire identification remains obscure, a questionmark may be placed after the scientific name in brackets itself.

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inconsistencies, however, I have no doubt that the mammalogists and many other zoologists dealing with taxonomic issues where domesticates are involved would welcome any proper solution. If this paper would eventually lead to such an end, it would more than perfectly fulfil the intention of its author. Acknowledgements I wish to thank the Convener of this symposium, Professor A. Minelli, for his kind invitation to read this paper, although the congress’ Book of Abstracts still scheduled it as a poster presentation only. The members of the International Commission on Zoological Nomenclature present at the congress are thanked for their invitation to describe the above proposal in their Bulletin of Zoological Nomenclature, that I will gladly follow. I also have to thank the Frankfurt University’s Vereinigung der Freunde und Förderer der J. W. Goethe-Universität Frankfurt am Main for a travel grant, and certainly all the colleagues, in Athens and in many countries around the world, whose efforts made this renewed International Congress of Zoology possible. References BOHLKEN H. 1958. Zur Nomenklatur der Haustiere. Zoologischer Anzeiger 160: 167–168. BOHLKEN H. 1961. Haustiere und Zoologische (sic) Systematik. Zeitschrift für Tierzüchtung und Züchtungsbiologie 76: 107–113. CLUTTON-BROCK J., G.B. CORBET & M. HILLIS 1976. A review of the Family Canidae, with a classification by numerical methods. Bull. British Mus. (NH), Zoology 29: 119-199. CORBET G.B. 1978. The Mammals of the Palaearctic Region: A Taxonomic Review. London: British Museum (NH). CORBET G.B. & J. CLUTTON-BROCK 1984. Appendix: Taxonomy and nomenclature. In Mason I.L. (ed.), Evolution of Domesticated Animals. London: Longman, pp. 434-8. DENNLER DE LA TOUR G. 1968. Zur Frage der Haustier-Nomenklatur (sic). Säugetierkundliche Mitteilungen 16 : 1-20. DIEFENBACH L. 1857. Glossarium Latino-Germanicum mediae et infinae aetatis. Frankfurt am Main. Reprint: Darmstadt 1968: Wissenschaftliche Buchgesellschaft. GENTRY A., CLUTTON-BROCK J. & C.P. GROVES 1996. Case 3010. Proposed conservation of usage of 15 mammal specific names based on wild species which are antedated or contemporary with those based on domestic animals. Bulletin of Zoological Nomenclature 53 (1): 28-37. GEORGES K.E. 1959. Ausführliches Lateinisch-Deutsches Handwörterbuch, Bd. 1 (A – H). Hannover (10): Hahnsche Buchhandlung. GROVES C.P. 1971. Request for a declaration modifying Article 1 so as to exclude names proposed for domestic animals from zoological nomenclature. Z.N. (S.) 1935. Bulletin of Zoological Nomenclature 27, parts 5/6: 269–272. GROVES C.P. 1995. On the nomenclature of domestic animals. Bulletin of Zoological Nomenclature 52 (2), 137–141. HABEL E. (ed.) 1959. Mittellateinisches Glossar. Paderborn (2): Schöningh.

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HERRE W. & M. RÖHRS 1990. Haustiere – zoologisch gesehen. Stuttgart and New York (2): Gustav Fischer Verlag. INTERNATIONAL COMMISSION ON ZOOLOGICAL NOMENCLATURE 1964. International Code of Zoological Nomenclature, 2nd Edition. London: The International Trust for Zoological Nomenclature. INTERNATIONAL COMMISSION ON ZOOLOGICAL NOMENCLATURE 1985. International Code of Zoological Nomenclature, 3rd Edition. London: The International Trust for Zoological Nomenclature. INTERNATIONAL COMMISSION ON ZOOLOGICAL NOMENCLATURE 1999. International Code of Zoological Nomenclature, 4th Edition. London: The International Trust for Zoological Nomenclature. NIEMEYER J.F. 1976. Mediae latinitatis lexicon minus. Leiden: Brill. ODENING K. 1979. Zur Taxonomie und Benennung der Haustiere. Der Zoologische Garten N.F. 49(2): 89-103. POHLE H. 1935. Wie benennt man Haustiere ? In Rümmler H., Bericht über die 8. (sic) Hauptversammlung. Zeitschrift für Säugetierkunde 10: 123-124. UERPMANN H.-P. 1993. Proposal for a separate nomenclature of domestic animals. In Clason A.T., S. Payne & H.P. Uerpmann (eds), Skeletons in her Cupboard. Festschrift for Juliet CluttonBrock. Oxford: Oxbow Books. (= Oxbow Monograph 34), pp. 239–241. WERNER F.C. 1972. Wortelemente lateinisch-griechischer Fachausdrücke in den biologischen Wissenschaften. S.l.: Suhrkamp. (= Suhrkamp Tb. 64).

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers BIOTA/FAPESP - Biodiversity Conservation in SãoThe Paulo State (Brazil) 701 New Panorama of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 701-704, 2003

Neotropical Biodiversity Conservation and Sustainable Use in São Paulo State (Brazil) - BIOTA/FAPESP - The Biodiversity Virtual Institute C.G. Tiago & V.F. Hadel Centro de Biologia Marinha - Universidade de São Paulo Rod. Manoel Hyppólito do Rego, Km 131,5, São Sebastião - SP - Brasil, 11600-000. E-mail: [email protected]

Abstract BIOTA/FAPESP (http://www.biota.org.br) is a research Program created to prevent further loss of biological information in the State of São Paulo, Brazil. Information on the State’s biodiversity, ranging from viruses and bacteria to angiosperms and mammals, will be gathered by means of several projects that aim to confirm what it is already known and to add new data to the existing ones. The identification of the organisms to the species level is one of the goals of the Program, and systematic studies will be encouraged. Twenty-three projects, involving more than five hundred researchers and students, already received funds from the Program and at least twenty-six more are under submission. All biological data will carry their geographical coordinates, obtained by means of a GPS receiver, which will be stored on a Geographical Information System database. In the near future one will be able to seek information on the biodiversity of a municipality or region in the web. The biological material will be deposited in collections at zoological museums, herbariums, germ plasm banks, etc.

The Brazilian territory covers more than 8,500,000 km² of the Atlantic side of South America, with a shoreline of 7,408 km. São Paulo State is located in the Southeast region, the most densely populated of the country, crossed by the Tropic of Capricorn at 23º27' S. São Paulo’s area is about 250,000 km² and it has a shoreline of 622 km, respectively 2.9% and 8.4% of the Brazilian territory and littoral. Despite the great number of researchers working in São Paulo (around 16,000), and accounting for 49% of all scientific publications in Brazil, conservation efforts were quite neglected. Surveys on marine invertebrates, for instance, begun only at the end of the 19th century, and until now, several phyla remain unregistered for the State’s coast. The environmental history of this State is one of severe devastation. The Atlantic rain forest, which covered 82% of its area (almost 250,000 km²), is now reduced to only 5%. Among the causes one can mention urbanization and industrialization. But most of all,

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the spreading of coffee and sugar cane plantations, allied to the great expanses of land destined to stock raising can also be blamed. In order to prevent further loss of biological information a research Program on conservation and sustainable use of São Paulo State’s Biodiversity, the BIOTA/FAPESP, was created (http://www.biota.org.br). BIOTA/FAPESP is a special research Program entirely funded by FAPESP (The State of São Paulo Research Foundation). This Program is the result of a joint effort of this Foundation and the State’s scientific community, and falls into the framework of the Convention on Biological Diversity. The Program goals are to survey the biodiversity in the State of São Paulo, to bring it to the knowledge and to stress its importance to the general public and to enhance the capacity for managing, monitoring and using biodiversity resources by the government, the public and private organizations. An information system was launched in June 1997 as an open, cooperative and integrating network, using an Internet web site as the main route for information exchange and dissemination. This system, along with the personal involvement of several scientists, was a fundamental supporting tool for the workshop “Basis for the Conservation of São Paulo State’s Biodiversity” held in this same year. During this workshop the Program was outlined and some tasks were proposed. The first one was to evaluate the existing knowledge on the different taxonomic groups, from bacteria and viruses to mammals and angiosperms. Approximately 100 researchers were invited and contributed with their knowledge producing documents, all of them available in the Internet. One of these products is a map-based database of biological collecting efforts. It’s a searching tool that allows one to seek out information about fauna and flora by choosing a topic, a keyword (http://sinbiota.cria.org.br) or a municipality from a region on the map of the State (http://sinbiota.cria.org.br/atlas/). One can get, also, several information from a particular collecting effort, such as habitat, methods employed, name of the author, and the reference of the papers eventually published. Another outcome of this workshop was a series of seven volumes titled “Biodiversity of the State of São Paulo, Brazil: Synthesis of the knowledge at the end of the 20th century” (http://www.biota.org.br/publi/livros/). This series, available only in Portuguese, presents a thorough review of the knowledge about several groups of extant organisms known from the State of São Paulo and a profile of the research groups working on each one. It contains the following volumes: V. 1: Microorganisms and viruses; V. 2: Macroscopic fungi and plants; V. 3: Marine invertebrates; V. 4: Freshwater invertebrates; V. 5: Terrestrial invertebrates; V. 6: Vertebrates; V. 7: Infrastructure of conservation in situ and ex situ.. This volumes serve as a basis from which the BIOTA/FAPESP project starts. The full text of those books is accessible on the Internet (http://www.biotasp.org.br/ publi/livros/) and can be searched by choosing a keyword, a particular chapter, or by means of a “classification tree”, where you can search out the groups according to its biological characteristics. It is also possible to choose a particular taxon (http:// sinbiota.cria.org.br/info/info_amb), or a single project among all the Program’s projects (http://www.biotasp.org.br/projeto/index).

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The data obtained by all the projects will be stored for common use on the: “Environmental information system for BIOTA/FAPESP Program - SINBIOTA”. This summing-up project was created in order to gather fragmentary information and to standardize procedures for collecting as well as for a collecting form. It will also provide the data necessary to construct an accurate, updateable, digital cartographic base. At present the SINBIOTA project (http://sinbiota.cria.org.br) already has a database comprising more than 190 taxonomic groups ranging from bacteria and fungi to angiosperms and mammals. To enable all other projects to contribute with new information. a standard form will be used having, among several fields, nine obligatory ones: collector’s name; date/daytime/number of days; taxonomic group; collecting method; geographical coordinates; municipality; river basin; identification of a conservation unit and the category of the ecosystem studied. Those data will be stored on a Geographical Information System (GIS) database, which allows the integration between the data on biological collections, bibliographical references, as well as of the genetic, taxonomic and geographical data. By means of this database one can retrieve statistical data of the Program and of its projects. The GIS database will enable the building of an updated digital cartographic base (scale 1:50,000) including urban settlements, road network, municipality boundaries, river basins, contour lines, climate data, soil data, forestation, remnants of native vegetation, and boundaries of conservation units. Using GPS to plot collecting sites on the maps will secure accuracy of their localization. The identification of the organisms to the species level is one of the goals of the Program and systematic studies will be encouraged. It’s important to point out that all the biological material will be deposited in collections at Zoological Museums, Herbaria, Germ Plasm Banks, etc, being therefore accessible to all those who will need it in the future. The Program has a Coordinating Group of four Brazilian researchers whose responsibility is to ascertain if the submitted projects match its goals, to co-opt researchers and projects dealing with similar subjects, and to stimulate new proposals which will help to fulfill the Program’s purposes. There is also a Scientific Advisory Committee formed by eight foreign researchers, all of them involved in other Biodiversity Programs, who evaluate the BIOTA/FAPESP Program on a yearly basis. In 1999 the budget of the Program was about US$ 4,000,000.00. With those resources eighteen projects were funded, including many fellowships for undergraduate students, for graduate students working on their MSc and PhD projects, and for Post-Doctoral researchers. In 2000 twenty-three projects, involving more than five hundred researchers and students, already received funding from the Program, and at least twenty-six more are being analyzed. Those projects cover areas as diverse as Zoology, Botany, Ecology, Genetics, Microbiology, Biochemistry, Marine Biology, Oceanography, Environmental Education, Environmental Design, and Geographical Information System. We were encouraged in our objectives, and the means employed to obtain them, when after its first year the BIOTA/FAPESP Program was granted the Henry Ford Conservation Award as the Conservation Initiative of the Year.

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Acknowledgements The authors wish to deeply thank Dr Francis Dov Por who initiated, from the beginning, the idea of this paper and the presentation of the BIOTA Program at the XVIIIth ICZ at Athens. We are also grateful to the members of the Organizing Committee of the Congress for their kind support.

A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers BIOTA/FAPESP - Biodiversity Conservation in SãoThe Paulo State (Brazil) 705 New Panorama of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 705-709, 2003

Species 2000 New Zealand: Outcomes of the February Symposium J. St J. S. Buckeridge1 & D. Gordon2 1. Earth & Oceanic Sciences Research Centre, Auckland University of Technology, P.O. Box 92006, Auckland, New Zealand. E-mail: [email protected] 2. National Institute of Water & Atmospheric Research, P.O. Box 14901, Wellington, New Zealand. E-mail: [email protected]

Abstract In February 2000, New Zealand scientists met in Wellington for Species 2000 New Zealand, a symposium on the state of New Zealand’s biodiverisity. The symposium was part of a series of events that will result in a formal publication, listing all described taxa, both living and extinct, along with an estimation of the remaining undescribed species of each group. The impetus for the symposium was a growing concern about the present incomplete knowledge of our biota, and an acceptance of New Zealand’s international obligations under both the 1992 Convention on Biological Diversity and the 1996 Global Taxonomy Initiative. Estimates suggest that there are approximately 80,000 eukaryote species in New Zealand’s marine, freshwater, and land environments, of which about 40% are known. Knowledge of the prokaryotes is incomplete. Although eukaryotic micro-organisms, nematodes and small arthropods comprise the great bulk of the almost 50,000 unknown species, significant numbers of larger organisms, particularly in shelf waters, around submarine highs and hydrothermal vents, are awaiting formal description.

Introduction Images of New Zealand are generally coloured green. This country is perceived as one of the last places in a temperate clime to have been ravaged by development. Indeed, a clean, green image is one of the most important platforms through which New Zealand products are marketed. In light of this, one could view New Zealand as an ecological refuge, a country where land degradation following industrialisation has touched but lightly. This however, is an illusion, as the current green grazed pasture was, until the arrival of man, a dense forest of extraordinarily high endemism: particularly of plants, invertebrates and birds. This was to change with the arrival of humans, probably some time in the 12th or 13th centuries. Since then, much of New Zealand’s natural environment

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has been irrevocably lost, (e.g. 32 % of the endemic land and forest birds, and 18% of endemic seabirds have become extinct since the arrival of humans). Further, there are still a number of species classified as “at risk”, e.g. 58% of vertebrates, and indigenous forest now accounts for only 22% of the land area. There is however, much we can do to preserve what remains, and a key part of this must be to understand the current diversity of New Zealand’s biota (Buckeridge 1997, Taylor & Smith 1997). The Symposium Over the period 1-5th February 2000, one hundred and forty New Zealand scientists, representing the widest possible spectrum of biology and palaeontology, gathered in Wellington to present their findings on the state of New Zealand’s biodiversity. Both living and extinct organisms were considered; with lists of all known species, and estimates of the numbers and types of undescribed taxa, presented. The impetus for the symposium was a growing concern about the present incomplete knowledge of our biota, and an acceptance of our international obligations under both the 1992 Convention on Biological Diversity and the 1996 Global Taxonomy Initiative. It is apparent however, that although we have a Global Taxonomy Initiative, we still lack a co-ordinated Global Taxonomic Strategy. An effective strategy would need to be both centrally coordinated and funded, whilst at the same time operating on a continental or regional basis. However, until such a strategy and structure are implemented, activities must be carried out locally, e.g. national biological reviews, similar to the state biological surveys in the United States of America. Species 2000: New Zealand is such a review, and is apparently the first of its kind at the national level. Species 2000: New Zealand aims to review and inventory the entire New Zealand biota, living and fossil, out to the boundaries of the Exclusive Economic Zone (EEZ). It is a three-stage activity, comprising a symposium, a published volume, and electronic checklists of all species posted on appropriate websites. It is a subset of the global biodiversity agenda Species 2000, with which it became formally linked in July 1999. Symposium Outcomes New Zealand is a challenging place for taxonomists and systematic biologists. It has a fauna and flora distinct from that of other parts of the world, and this is reflected in the high endemism. The terrestrial environment is moderately well known, its uniqueness derived from an absence of mammals (apart from bats). This has led to birds and insects evolving to occupy niches that are “typically mammalian” in most other parts of the world, e.g. large flightless birds like Dinornis grazed grasslands, and giant insects, such as Deinacrida adopted a diet similar to that of small rodents. In the marine environment, the high cost of sampling, and the vast size of the EEZ exacerbate the challenge. New Zealand’s EEZ is the fifth largest in the world, and 15 times the land area. Further, most sampling effort has been to assess fish diversity, rather than that of the more numerous and speciose invertebrates. Recent estimates suggest that there are approximately 80,000 eukaryote species in New Zealand’s marine, freshwater, and land environments, of which about 40% are known. Prokaryote

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systematic taxonomy is often poorly known, e.g. there is very little published on marine Cyanobacteria, although they are known to be significant components of potentially toxic “algal” blooms. Although eukaryotic micro-organisms, nematodes and small arthropods comprise the great bulk of the almost 50,000 unknown species, significant numbers of larger organisms, particularly in shelf waters, around submarine highs and Table 1. Species 2000: New Zealand. Preliminary estimates of marine species diversity for major taxonomic groups. Data pertain to species from within New Zealand’s EEZ.

Taxonomic Group Protista, Algae, Fu ngi, Plantae Invertebrates Vertebrates Totals

D escribed species 2,697

Know n unrecorded species 173

Estimated unknow n species 1,200-2,100

Totals 4,070-4,970

7,702 1,213 11,612

3,122 140 3,474

4,320-5,440 160 5,680-7,700

15,140-16260 16,260 1,513 20,723-22,743

Table 2. Species 2000: New Zealand. Preliminary estimates of marine species richness in the EEZ. At the present rate of discovery and formal recording of new species, it will take at least a century to finish the task of marine inventory.

Taxonomic Group

Protista + Fungi Macroalgae + Plantae Porifera Cnid aria Ctenop hora Platyhelm inthes etc. Bryozoa Entoprocta N em ertinea Annelid a Mollu sca Brachiop od a Chaetognatha N em atod a Crustacea etc. Echinod erm ata H em ichord ata Urochord ata Cephalochord ata Vertebrata All other grou ps Totals

N ew Zealand Species (d escribed ) 2,010 687 410 572 13 155 600 9 18 497 2,414 30 18 130 2,009 588 4 187 1 1,212 48 11,612

N .Z. Species (know n u nd escribed unrep orted ) 95 78 255 395 6 90 310 5 6 244 1,174 0 1 60 381 160 1 50 0 140 23 3,474

Global Species (d escribed )

24,540 5,970 6,000 7,025 80 6,795 5,700 150 1,250 8,335 32,890 405 100 4,200 33,780 6,150 90 1,300 29 13,845 1,705 160,339

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hydrothermal vents, are awaiting formal description. Little is known about the biology of many of these organisms or of their relationship to other species that may be dependent on them, but it is demonstrably clear that they are very susceptible to fishing impact. It can be concluded, with some conviction, that it will take centuries for severely altered habitats to fully recover. Importantly, many of the damaged habitats are composed of foliose and branching growths of cnidarians, bryozoans and poriferans, all of which provide niches and food sources for more mobile organisms. The detailed results of the symposium will not be known until the proceedings are published, in 2001 (International Biodiversity Observation Year - IBOY). The biota will be described systematically, and although there will be some sections with single authorship (e.g. Buckeridge in press), it is anticipated that a large proportion will reflect the complexity and diversity of groups by being multi-authored (e.g. Gordon, & Taylor, in press, Kelly et al., in press). There are, none-the-less, preliminary results, and these offer opportunity for sober reflection; e.g. there are just over 10,000 species of insects described in New Zealand, but there are almost a further 9,000 known to be awaiting description – a high number in a country where the economy is closely linked to the agrarian sector. A more complex analysis has been made of the marine biota data, and this is summarised in the tables 1 and 2 above. It is stressed however, that these results are preliminary. Table 2 provides a more detailed overview of the marine biota, and shows (not surprisingly), that the focus to date has been on vertebrates (fish) protozoans and larger invertebrate groups (molluscs and crustaceans). The future The symposium closed with a widespread acknowledgement of the sheer size of the task awaiting New Zealand’s systematists. There was enthusiasm about the opportunity for cross-disciplinary information exchange, but there was also a realisation that the majority of current systematists are close to retirement, or already in retirement (including several in the 70 – 90 age range); further, there are inadequate numbers of new recruits. This shortage will be compounded by the current lack of systematic biology being taught in New Zealand universities. For without an early exposure to taxonomy, the likelihood of graduates undertaking further study in biodiversity and systematics is severely curtailed. References BUCKERIDGE J.S. 1997. Environmental Engineering Education within the Shadow of New Zealand’s ubiquitous Resource Management Act. Abstracts of the Chambéry ’97 Conference on Multidisciplinarity and International Co-operation in Environmental Education. p. 1-11. Chambéry, France. September 18-20, 1997. BUCKERIDGE J.S. (in press). Goose barnacles, acorn barnacles, wart barnacles and burrowing barnacles – Subclass Cirripedia. In Gordon, D.P. (ed.), The New Zealand Inventory of Biodiversity : A Species 2000 Symposium Review. Canterbury University Press, Christchurch, New Zealand. GORDON D.P. & P.D. TAYLOR (in press). Moss animals, sea mats, and lace corals – Phylum Bryozoa. In Gordon D.P. (ed.), The New Zealand Inventory of Biodiversity: A Species 2000 Symposium Review. Canterbury University Press, Christchurch, New Zealand.

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KELLY M.K., WILKINSON M.R., de GLASBY B.A., COOK S.de C., BERQUIST P.R., CAMPBELL H.J., BUCKERIDGE J.S., REISWIG H.M. & C. VALENTINE (in press). Sponges – Phylum Porifera. In Gordon D.P. (ed.), The New Zealand Inventory of Biodiversity: A Species 2000 Symposium Review. Canterbury University Press, Christchurch, New Zealand. TAYLOR R & I. SMITH 1997. The State of New Zealand’s Environment 1997. The Ministry for the Environment. Government Printer, Wellington. 653p.

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S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT PublishersLarge computer monographsA.inLegakis, zoology - possibilities ... Evolution 711 Theand Newperspective Panorama of Animal Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 711-713, 2003

Large computer monographs in zoology - possibilities and perspective. Demonstration of a test case - “Salticidae (Araneae) of the World” J. Prószy nski ´ Muzeum i Instytut Zoologii, PAN, ul. Wilcza 64, 00-679 Warszawa, Poland. E-mail: [email protected]

Abstract Book-size publications on computer (CD disks, Internet) seem to be a revelation. Inexpensive, fast produced, give complete freedom to Authors inventions, as they can easily accommodate text, drawings, colour photographs and scanning microscope photographs, even movies. Systems of hyperlink switches permit instant connection of various parts of the work (drawings with catalogue, or geographical check lists, photographs, references, works referred to, and any other features the Author may wish, or invent, even e-mail addresses/links to authors concerned). Other features are possibility of comparing several drawings on the same screen. An example of newest version of such monograph (http:/ /spiders.arizona.edu/salticid/main.htm) is demonstrated; particular parts of this monograph were opened in the Internet over 10690 times (Catalogue) and 2567 times (Diagnostic Drawings). Several problems posed by publication in this medium deserve some consideration. 1. Permanency - they are vulnerable to a server failure, or future changes in computer hardware and programs. 2. Computer publications are often discriminated by omissions in references, and other Copyrights privileges. 3. Monopolistic attitudes of some Authors and/or Publishers, which try to reserve their publications for only own Internet use.

Continuation of taxonomic zoology as a science is endangered. Ernst Mayr forecasted in 1969 a rosy future for taxonomic zoology: new programs in ecology will need specialists able to identify species for such research. Unfortunately that did not happen: materials collected during ambitious projects in exotic lands are usually deposited but not studied, in Museums, where they will fade and decay for the next hundred years. Results of such projects, necessary to account for received grants, are often presented in general terms, without even naming species. So there are plenty of grants for exotic

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explorations and almost none for taxonomic studies of the material collected. And so zoological taxonomy is as endangered as animal species it is supposed to study. Failure to identify exotic species is to some extent justified: identification would require a whole library of available publications, and a life long experience of persons involved in studies. If identification procedures would become less demanding and easier, we could perhaps, promote better use of collections of endangered faunas. What we need are easy tools for identification of species from anywhere in world, universally available and inexpensive. In other words, we should make experience and knowledge of leading taxonomists available to everybody and everywhere. There is now a technical possibility for that, by using Internet and/or large memory computer disks. But will there be taxonomists capable to produce such works? Will there be Institutional support for that? We observe now spontaneous spreading of small Internet homepages: usually presentation of individual scientific results, or images of an Institution, sometimes even a single photograph. These are useful, but inadequate to ensure continuous development of taxonomy. I wish to present now an example of a large project, intended to facilitate identification of all world species of a selected group. The reason I wish to discuss it is that I am unsure whether practical use will confirm theoretical expectations. The project I prepared is a computer monograph called “Salticidae (Araneae) of the World” - accessible on both Internet (http://spiders.arizona.edu/salticid/main.htm) and on CD disks. It permits, at present, immediate retrieval of diagnostic characters of over 4000 species of jumping spiders of 5 continents, which is about 3/4 of the total species known, and is interactively connected with a Catalogue to about 4400 nominal species of that group of animals. It is a result of 40 years of my research on family Salticidae, and several years of developing a computer monograph. The importance of this work may be demonstrated by the 10,696 users who opened the Catalogue part within 3 years, and the 2,527 users of the Diagnostic Drawings part. Reaction of specialists to the Monographs seems also to be enthusiastic. A special property of Salticidae is that most important taxonomic characters are presented in a form of drawings, hence the demonstrated Monograph is based primarily on drawings. Drawings of species are interactively connected by links to records of references, and to colour photographs. Check lists of particular continents and countries permit immediate scan of diagnostic drawings of species of these areas. There is a facility for comparison of two different species on a screen, or checking scanner microphotographs. Also using keys to identification one can immediately check the diagnostic drawings of the searched taxa. An additional use of the monograph is a survey of the state of knowledge of particular genera and species in various geographical areas, comparison of methods and standards of various authors, also blueprinting of future research. It would be difficult to list all the advantages of that taxonomic tool, so I propose to the readers to connect now to Internet address http://spiders.arizona.edu/salticid/main.htm, and check it themselves. As an author I may confess, that the biggest pleasure it provides is a freedom for inventing and solving problems - almost effortlessly and instantly - such as making comparison of related species and genera, comparison of faunas of distant areas and checking views of various authors on the same problem. Practically any

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researcher’s whim may be implemented almost instantly. And this is accessible in any place in the World through the Internet or, if one prefers, from a CD disk. Problems requiring considerations There are, however, some problems, requiring further consideration. 1. With progress in computer hardware and programs, a Monograph prepared today may not fit to the future facilities. How to ensure renewal of such works and their permanent adaptation to changing computers and software? 2. Ambitious scientific works may be available on computer disks only if rights of their authors will be full respected: accepted as a part of scientific achievements, quoted in references in other publications, included in authors’ evaluation for further career, and accounted for grants. At present, works in computer are usually even not quoted in references, although subsequent authors often use them liberally. The conditions mentioned above require perhaps some institutional protection. 3. It should be easier to obtain copyright permissions for digitizing drawings for computer Monographs. For example, for half a year now, I cannot receive copyright permission from a small periodical in Karlsruhe, which happened to publish a very important paper. After several letters, my request was rejected: “We are mounting our homepage and will include the drawings in the Arachnid section”. Without questioning Editors’ rights, monopolistic attitudes jeopardize general scientific interest. There should be an easy access to scientific illustrations in the whole scientific literature for preparation of bona fide electronic publication. 4. Excessive proliferation of individual homepages seems to be a temporary feature. Because of inconvenience of searching for a few species among say, 100 homepages, there will follow voluntary aggregation into larger electronic works. However, no monopolistic practices should prevent bona fide, non-commercial scientific creativity.

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Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) © PENSOFT Publishers New data on “satellite” fishA.species and their evolutionary significance 715 The New Panorama of Animal Evolution Sofia - Moscow Proc. 18th Int. Congr. Zoology, pp. 715-724, 2003

New data on “satellite” fish species and their evolutionary significance P.M. Bãnãrescu Institutul de Biologie, Splaiul Independenþei 296, RO-79651 Bucureºti, România

Abstract The term “satellite” species, used for non-predatory lamprey species, each derived from a predatory one, is extended to some bony fishes whose ranges are totally included within those of their widely distributed closest relatives. Some satellite species may have originated through sympatric ecological isolation, but others through geographical isolation: double colonization or secondary overlap without hybridization of geographically and morphologically extreme conspecific populations. The existence of satellite species demonstrates that geographical speciation can take place in quite small areas and suggests that the divergence between a mother species and its satellite may be more recent than the divergence between geographically distant groups of populations of the mother species.

The term “satellite” species has been proposed by Vladykov and Kott (1979) for nonpredatory and non-migratory species of lampreys closely related to a predatory and usually migratory (anadromous) species which lives in the same area and which is assumed to have been its ancestor. The supposition that each non-predatory lamprey species derives from a related predatory one has been asserted first by Zanandrea (1958, 1969) who used the term “paired species”, respectively “specie appaiate”. The term “satellite” is more adequate since it clearly suggests derivation while “paired” can be used for each pair of sisters, allopatric or sympatric as well. Vladykov and Kott list a great number of satellite lamprey species from North America, mentioning the presumed predatory ancestor of each of them; to the predatory Lethenteron japonicum (Mertens) four satellites correspond, two northwestern American and two northeastern Asian. One example listed by Vladykov and Kott cannot be accepted: Lethenteron meridionale from the southern part of the Atlantic watershed cannot be a satellite of the predatory north Pacific L. japonicum (Mertens). According to Vladykov and Kott, the migratory Atlantic and partly Mediterranean Lampetra fluviatilis gave birth to two satellite species: the central European and western Italian L. planeri (Bloch) and the northern Italian L. zanandreai (Vladykov). Actually the latter is presently ascribed to the prevailingly arctic Lethenteron; its ancestor may have

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been an extinct species of this genus, which lived during the Ice Age in the Mediterranean and the Adriatic seas. The origin of the non-predatory species of Eudontomyzon is obscure. Vladykov and Kott consider E. vladykovi Oliva and Zanandrea a satellite of the predatory (but non migratory) E. danfordi Regan; the ranges of both species however are, in a large measure, allopatric. The more eastern not migratory E. mariae Berg may be a satellite of a migratory, not described species that lived in historical quite recent years in the Black Sea. A difficulty rises however from the fact that the genus comprises, besides the four European species (all sedentary, one of them predatory, the others non-predatory) a fifth one in Korea. The ancestor of the genus may hence have been an extinct migratory species from the Arctic Ocean, which gave birth to one or several sedentary satellite species in Siberia, and their descendants dispersed, by continental routes, westwards to Europe and eastwards to Korea. The term “satellite species” has been extended by Bãnãrescu (1996) to a special category of teleost fishes: species with a small range that are totally included within the ranges of their much more widely distributed sister species. He listed four examples from the Danube basin: 1. Sabanejewia romanica (Bãcescu), endemic to an area in the south of this basin, sister to S. aurata (Filippi), widely distributed north to Neman River and east to Transcaucasia and the watershed of the Aral Sea (Fig. 1);

Fig. 1. Range of Sabanejewia aurata (1) and its satellite S. romanica (2).

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2. Telestes polylepis Steindachner, endemic to a restricted area of the system of the river Sava, a secondary tributary of the Danube; its sister, T. souffia (Risso), ranges from the southern France and Italy to the northwest and southwest of the Danube basin, inclusively the entire Sava River system (Fig. 2); 3. Rutilus pigus (La Cépède), endemic to the upper and middle Danube basin, sister of the Euro-Siberian R. rutilus (Linnaeus) (Fig. 4); 4. Vimba elongata (Valenciennes), endemic to the lakes in the upper Danube basin, sister of the central and eastern European V. vimba (Linnaeus) (Fig. 5). A further example can be added: Scardinius racovitzai (Muller), endemic to a thermal pond in the middle Danube basin (western Romania), sister of the widely distributed central, western and eastern European rudd, S. erythrophthalmus (Linnaeus) (Fig. 3). This fish was until recently considered a subspecies of the common rudd. Recent studies have shown that there are not only morphological and physiological, but also behavioral differences between S. racovitzai and its congener (N. Crãciun, unpublished Ph. D. thesis). The widely distributed European and western Asian common chub, Leuciscus cephalus (Linnaeus) has given birth to several satellite species, each of them endemic to a restricted area of its range: - L. lucumonis Bianco in a few rivers on the Adriatic Sea watershed of central Italy; - L. cephaloides Battalgil in a restricted area of northwestern Anatolia; its specific validity is however uncertain;

Fig. 2. Range of Telestes souffia (1) and its satellite T. polylepis (2).

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Fig. 3. Range of Scardinius erythrophthalmus (1) and its satellite S. racovitzai (2).

Fig. 4. Range of Rutilus rutilus (1) and its satellite R. pigus (2).

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Fig. 5. Range of Vimba vimba (1) and its satellite V. elongata (2).

- L. spurius (Heckel) in Orontes River, sympatric with L. cephalus berak (Weckel); - L. lepidus (Heckel) in the Tigris – Euphrates basin, sympatric with L. cephalus orientalis Nordmann; - L. ulanus Günther in lake Orumiyeh or Urmia (northwestern Iran), again within the range of L. cephalus orientalis (Fig. 6); - An undescribed species in the western Balkan rivers Krka and Zrmanja (mentioned by Bianco and Knezeviæ 1985), sympatric with L. cephalus. (The data on the distribution of the species and subspecies of the L. cephalus group in Bãnãrescu, 1996, are in a large measure outdated.) Further examples are furnished by the fish faunas of other continental areas, namely East Asia and Australia: - Saurogobio lissilabris Bãnãrescu and Nalbant, 1973, confined to the central part of the Changjiang or Yangtse River, totally included in the wide range of S. dabryi Bleeker; - Abbottina springeri Bãnãrescu and Nalbant in southwestern Korea and A. guentheri Bãnãrescu and Nalbant in the lower Changjiang River, the ranges of both being included within that of the third species of the genus, A. rivularis (Basilewski) (Fig. 7); - Gadopsis bispinosus Sanger from a few tributary rivers of Murray River in Victoria, Australia; the range of its sister species, G. marmoratus Richardson, encompasses whole southwestern Australia, inclusively the Murray-Darling basin and northern Tasmania (Allen 1989) (Fig. 8).

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Fig. 6. Range of Leuciscus cephalus (1) and its satellites L. lucumonis (2), L. cephaloides (3), L. spurius (4), L.lepidus (5) and L. ulanus (6).

Fig. 7. Range of Abbottina rivularis (1) and its satellites A. springeri (2) and A. guentheri (3).

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Fig. 8. Range of Gadopsis marmoratus (1) and its satellite G. bispinosus (2).

Bãnãrescu (1996) gives a further example from East Asia: Hemiculter nigromarginis Yih and Woo, whose range is included within that of H. leucisculus (Basilewski). Actually, both species may not be sister taxa. Finally, Bãnãrescu lists (after the data of collaborators in Lee et al. 1980) four examples among cyprinid fishes in eastern North America. Speciation takes place, in biparental organisms, usually or even always, through geographic isolation. The ranges of sister species are initially vicariant; partial range overlap results from range extension and if this extension continues, it can finally lead to total sympatry. How, then, to explain the origin of satellite species of teleosts, whose ranges are totally included within those of their closest relatives? One mechanism is the double (or repeated) colonization, the importance of which has been emphasized especially by Mayr (1942, 1963). Satellite species having probably originated through normal double colonization are especially those inhabiting the peripheral area of their closest relative, e.g. Leuciscus lepidus: a number of individuals of the ancestral L. cephalus stock entered, my mean of a short-lasting river capture, the drainage area of the Tigris – Euphrates, where it lives sympatrically with L. lepidus, since in the meantime reproductive isolation mechanism have evolved. There is however also another possibility for the evolution of the satellite species, especially of those inhabiting not the peripheral, but the central part of the area of their relative, e.g. Sabanejewia romanica (Fig. 1), Telestes polylepis (Fig. 2), Leuciscus lucumonis (Fig. 6), Saurogobio lissilabris, Gadopsis bispinosus (Fig. 8).

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This mechanism is “secondary overlap without hybridization of geographically and morphologically extreme conspecific populations” (Mayr 1942, 1963, Bãnãrescu 1996). The classical examples are those of the birds Parus major – cinereus – minor and Larus argentatus – glaucoides – fuscus. Many other examples are known among birds, mammals, amphibians and insects (Mayr 1963). Very suggestive is the case of one fish, Sabanejewia aurata, the sister of the already mentioned S. romanica (Fig. 1). The variability of S. aurata has been very incompletely studied on most of its wide range, except in the drainage area of the middle and lower Danube (Romania and eastern Hungary). Four nominal subspecies have been described from this area (Fig. 9): bulgarica that inhabits the main channel of the Danube and of its large tributary the Tisa (Fig. 9.1), balcanica from the upper and middle reaches of the rivers of Romania flowing west and southwestwards into the Tisa and the Danube (Fig. 9.2), radnensis in the upper reach of Mure¸s River, a tributary of the Tisa (Fig. 9.4) and vallachica in the rivers from southeastern Romania flowing into the lower Danube (Fig. 9.7). A gradual and continuous intergradation between the subspecies balcanica and bulgarica takes place in the lower reaches of the rivers of western and southwestern Romania, except the Mure¸s (Fig. 9.3); the population from the middle and lower Mure¸s catchment area are, morphologically, intermediate

Fig. 9. Distribution of the subspecies and intergrades of Sabanejewia aurata in the drainage area of the Middle and Lower Danube basin: 1. S. aurata bulgarica; 2. S. aurata balcanica; 3. intergrades balcanica bulgarica; 4. S. aurata radnensis; 5. intergrades balcanica radnensis; 6. S. aurata vallachica; 7. intergrades balcanica vallachica.

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between the subspecies radnensis and balcanica (Fig. 9.5) and can be considered intergrades and similarly the populations from the rivers of south-central and northeastern Romania are intermediate between the subspecies balcanica and vallachica (Fig. 9.6). However, the balcanica – radnensis population from the lower Mure¸s does not intergrades with bulgarica contrary to the pure balcanica populations from the other western rivers; at the confluence of the Mure¸s River with the Tisa, S. aurata balcanica – radnensis and S. aurata bulgarica occur sympatrically, like “good” species. A similar situation takes place in southern Romania: there is no intergradation between S. aurata vallachica (or the balcanica – vallachica intergrades) from the rivers and S. aurata bulgarica that ascends from the Danube into the lowermost stretches of the rivers; on the contrary, they live sympatrically, again like “good” species (Fig. 9). Hence, S. aurata balcanica intergradates with the three other subspecies but these, when in (secondary) contact, live sympatrically, without any intergradation or hybridization. Does one consider only the situation in the lower stretches on the Mure¸s or of the southern Romanian rivers, the three subspecies, bulgarica, radnensis and vallachica can be considered valid species that evidently originated through circular overlap of extreme conspecific populations. The satellite non-migratory species of lampreys originated through a rather ecological mechanism; a number of specimens of the migratory ancestor did not return to the sea, remained confined to the breeding area of the species getting reproductive isolation mechanism. Also Scardinius racovitzai, from the mentioned thermal pond, may have originated in a rather ecological manner. The phenomenon of speciation through circular overlap of extreme populations is a strong argument in favor of the possibility of “speciation through distance” (i.e. without any range interruption through a barrier) and a particular case of “double colonization”. The origin of satellite species has several evolutionary implications: - it demonstrates that speciation often is a local phenomenon, that implies only a restricted part of the mother species range, i.e. only a few populations - the divergence between the satellite species and the neighboring populations of the mother species can have a younger age than the divergence between the distant populations of the latter. For example the divergence of Sabanejewia romanica and especially of S. a. vallachica S. a. bulgarica from S. aurata (balcanica) took probably place after the species S. aurata reached its wide range, from Central Europe to the Aral Sea basin. An argument for this assertion is the fact that preliminary, unpublished investigations of Dr. C. Tesio showed that electrophoretically S. aurata vallachica is closer to S. romanica (with which it occurs sympatrically in many rivers) than to S. aurata balcanica, in spite of the occurrence of morphological intergrades between it and the latter subspecies. - According to the W. Hennig school, when a mother species splits into the two daughter species, it ceases its existence. This is not the case with the species having given birth to satellites, especially for those species that have two or more satellites (e.g. Lethenteron japonicum among lampreys, Leuciscus cephalus among bony fishes). Extant closest related species are designed as sisters. In the case of a species from which a satellite originates and of its satellite it is better to use the terms “mother” and “daughter” species.

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The study of satellite species is one of the most interesting in evolutionary ichthyology. Thorough investigations in this field are necessary, especially with the help of molecular methods. References ALLEN G.R. 1989. Freshwater Fishes of Australia. T.H.F. Publ., Neptune, New Jersey. BÃNÃRESCU P. 1995. On satellite species in ichthyology and on speciation through secondary overlap of geographically and morphologically extreme conspecific populations. Evolution and Adaptation (Cluj) 5: 121-143. BÃNÃRESCU P., NALBANT T. & S. CHELMU 1972. Revision and geographical variation of Sabanejewia aurata in Romania and the origin of S. bulgarica and S. romanica (Pisces, Cobitidae). Annot. zool. et botan. (Bratislava) Nr. 75: 1-49. LEE D.S., GILBERT C.R., HOCCUT C.H., JENKINS R.E., MCALLISTER D.E. & J.R. STAUFFER (eds.) 1980. Atlas of North American Fresh Water fishes. North Carolina Mus. of Nat. Hist., Raleigh, New Carolina, 867 pp. MAYR E. 1942. Systematics and the Origin of Species. Columbia University Press, New York, 334 pp. MAYR R. 1963. Animal Species and Evolution. The Belknap Press at Harvard Univ. Press, Cambridge, Massachusets, 797 pp. VLADYKOV V.D. & E. KOTT 1979. Satellite species among the holarctic lampreys (Petromyzonidae). Can. J. Zool. 57(4): 360-367. ZANANDREA G. 1958. Recenti richerche sulle forme “appaiate” di lamprede dell’Italia e del Danubio. Boll. Zool. 26(2): 545-554. ZANANDREA G. 1969. Speciation among lampreys. Nature (London) 184: 380.

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) Participants TheList Newof Panorama of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 725-735, 2003

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List of Participants Ahlberg, Per Erik, Dept. of Paleontology, The Natural History Museum, Cromwell Rd., London SW7 5BD, UK [[email protected]] Akoumianaki, Ioanna, Inst. of Marine Biology of Crete, P.O. Box 2214, 71003 Irakleion, Greece [[email protected]] Almeida Prado Por, M. Scintila, de, Hebrew University of Jerusalem, 91904 Jerusalem, Israel Alves, Orane, Dept. Zoologia, Inst. Biologia, Federal Univ. of Bahia, Ondina, Salvador, BA 40.170110, Brazil [[email protected]] Andreone, F., Lab. of Vertebrate Taxonomy and Ecology, Museo Regionale di Scienze Naturali, Via Giolitti 36, I-10123 Torino, Italy [[email protected]] Anjubault, Elisabeth, Lab. de Biologie Générale, Univ. Catholique de Lyon, 25 rue du Plat, F69288 Lyon, France [[email protected]] Apostolou, Ioanna, 77, Formionos str., 161 21 Athens, Greece Arad, Zeev, Technion, Dept. of Biology, Technion, Haifa 32000, Israel [[email protected]] Arendt, Detlev, European Molecular Biology Lab., Meyerhofstrasse 1, 69012 Heidelberg, Germany [[email protected]] Arizza, Vincenzo, Lab. of Marine Immunology, Dept. of Animal Biology, Univ. of Palermo, via Archirafi 18, 90123 Palermo, Italy [[email protected]] Arnoult, Fabrice, Lab. d’Ecotoxicologie, Univ. de Reims Champagne-Ardenne, UFR Sciences, BP 1039, F-51687 Reims, France [[email protected]] Avise, John, Dept. of Genetics, University of Georgia, Athens, Georgia 30602, USA [[email protected]] Azariah J., Dept. of Zoology, Univ. of Madras, Guindy Campus, Chennai 600 025, India [[email protected]] Bakst, Murray, Germplasm and Gamete Physiology Lab., Agricultural Research Service, US Dept. of Agriculture, Beltsville, MD 20705-2350, USA [[email protected]] Banarescu, Petre-Mihai, Institute of Biology, Univ. of Bucharest, Splaiul Independentei 296, PO.Box 56-58, 79651 Bucharest, Romania. Bartsiokas, Antonis, Democritus University of Thrace, 6 Aiginis str., 166 73 Voula, Greece [[email protected]] Becker, Peter-Renee, Übersee-Museum Bremen, Bremen, Germany [[email protected]] Ben-Eliahu, Nechama, Dept. of Evolution, Systematics and Ecology, The Hebrew Univ. of Jurusalem, 91904 Jerusalem, Israel [[email protected]] Bengtson, Stefan, Dept. of Paleozoology, Swedish Museum of Natural History, Box 50007, 104 05 Stockholm, Sweden [[email protected]] Bergquist, Derk, 208 Mueller Lab., The Pennsylvania State University, State College, PA 16802, USA [[email protected]]

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Bergström, Jan, Dept. of Paleozoology, Swedish Museum of Natural History, Box 50007, 104 05 Stockholm, Sweden [[email protected]] Bock, Walter, Columbia University, 1200 Amsterdam Ave., Mail Box 5521, New York NY 10027, USA [[email protected]] Boufersaoui, Abdelkader, Lab. of Entomology, Inst. des Sciences de la Nature, Univ.de Sciences et Techniques Houari Boumediene, B.P. 32, Bab Ezzouar, 16111 El Alia, Algeria [[email protected]] Bouhadad, Rachid, Inst. des Sciences de la Nature, Univ.de Sciences et Techniques Houari Boumediene, B.P. 32, Bab Ezzouar, 16111 El Alia, Algeria [[email protected]] Bourtzis, Kostantinos, Insect Molecular Genetics Group, Inst. of Molecular Biology & Biotechnology, Vasilika Vouton, P.O. Box 1527, Irakleion, Crete, Greece [[email protected]] Brasier, Martin, Dept. of Earth Sciences, University of Oxford, Parks Road, Oxford OX1 3PR, U.K. [[email protected]] Bridgewater, Peter, UNESCO-MAB, Div. of Ecological Sciences, 1 rue Miollis, F-75015 Paris, France [[email protected]] Briggs, Derek, Dept. of Earth Sciences, Univ. of Bristol, Wills Memorial Building, Queen’s Road, Bristol BS8 1RJ, UK [[email protected]] Brooks, Daniel, Dept. of Zoology, University of Toronto, 25 Harbord str., Toronto ON M5S 3G5, Canada [[email protected]] Brothers, Denis, School of Botany and Zoology, Univ. of Natal, Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa [[email protected]] Buckeridge, John, Earth & Oceanic Sciences Research Centre, Auckland University of Technology, P.O. Box 92006, Auckland, New Zealand [[email protected]] Budd, Graham, Dept. of Earth Sciences, Historical Geology & Paleontology, Univ. of Uppsala, Norbyvaegen 22, Uppsala S-75236, Sweden [[email protected]] Burkhardt, Richard W., Dept. of History, Univ. of Illinois at Urbana-Champaign, 309 Gregory Hall, 810 South Wright Str., Urbana, IL 61801, USA [[email protected]] Camargo Abdalla, Fabio, Inst. de Biocencias de Rio Carlo, UNESP, Rio Carlo, Brazil [[email protected]] Candiani, Simona, Dip. di Biologia Sperimentale, Ambientale e Applicata, Sez. di Neuroendocrinologia e Biologia dello Sviluppo, Univ. di Genova, viale Benedetto XV, 5, 16132 Genova, Italy [[email protected]] Canepa Garcia, Gloria, Decanato de Ciencias Veterinarias, Univ. Central de Venezuela, Caracas, Venezuela [[email protected]] Casartelli, Morena, Dept. of Biology, Univ. of Milan, via Celoria 26, 20133 Milano, Italy [[email protected]] Castaño Meneses, Gabriela, Lab. de Ecologia y Sistematica de Microartropodos, Dpt. de Biologia, Fac. de Ciencias, Univ. Nacional Autonoma de Mexico, 04510 Mexico, Mexico [[email protected]] Chatzaki, Maria, Dept. of Biology, University of Crete, Irakleion, Greece [[email protected]] Chen Guangwen, Dept. of Biology, Henan Normal University, Xinxiang 453002, China [[email protected]] Chinnici, Cinzia, Lab. of Marine Immunology, Dept. of Animal Biology, Univ. of Palermo, via Archirafi 18, 90123 Palermo, Italy [[email protected]]

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Chintiroglou, Chariton, Sect. of Zoology, Dept. of Biology, Aristotle University of Thessaloniki, 540 06 Thessaloniki, Greece [[email protected]] Chu, Ka Hou, Dept. of Biology, The Chinese Univ. of Hong Kong, Hong Kong [[email protected]] Combes, Claude, Lab. de Biologie Animale, Centre de Biologie et d’ Ecologie Tropicale et Mediterranée, Univ. de Perpignan, 66860 Perpignan, France [[email protected]] Cooper, Edwin L., Lab. of Comparative Immunology, Dept. of Neurobiology, School of Medicine, University of California, Los Angeles, CA 90095-1763, USA [[email protected]] Côté, Isabelle, University of East Anglia, Norwich NR4 7TJ, UK [[email protected]] Currie, Philip, Royal Tyrrell Museum of Paleontology, Box 7500, Drumheller, T0J 0Y0 Alberta, Canada [[email protected]] D’Hondt, Jean- Loup, Lab. de Biologie des Invertebrés Marins et Malacologie, Muséum National d’Histoire Naturelle, 57 rue Cuvier, F-75231 Paris cedex 5, France D’Hondt Marie–Jose, Museum National d’Histoire Naturelle de Paris, Paris, France Daguzan, Jacques, Lab. de Zoologie et d’Ecophysiologie, Université de Rennes 1, UMR CNRS 6553, Campus de Beaulieu, Ave. de Géneral Leclerc, 35042 Rennes cedex, France [[email protected]] De Eguileor, Magda, Dept of Structural and Functional Biology, Univ. of Insubria, via J.H. Dunant 3, 21100 Varese, Italy [[email protected]] De Oliveira Abrantes, Isabel Maria, Dept. de Zoologia & Inst. do Ambiente e Vida, Univ. of Coimbra, 3004-517 Coimbra, Portugal [[email protected]] De Queiroz, Kevin, Dept. of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington DC, 20560-0163 USA [[email protected]] Desbruyères, Daniel, Dept. Environnement Profond (URM no 7) – Centre de Brest de l’IFREMER, BP 70, F-29280 Plouzané, France [[email protected]] Dewel, Ruth Ann, Depts. of Biology and Geology, Appalachian State University, Boone, NC 28608, USA [[email protected]] Domingo-Roura, Xavier, Unitat de Biologia Evolutiva, Univ. Pompeu Fabra, Dr. Aiguader 80, 08003 Barcelona, Spain [[email protected]] Duan Enkui, State Key Laboratory of Reproductive Biology, Inst. of Zoology, Academia Sinica, 19 Zhongguancun Lu, Haidian, Beijing 100080, China [[email protected]] Dupuis, Claude, Museum National d’Histoire Naturelle, Paris, France [[email protected]] Eraky, El-Sayed Ali, Plant Protection Department, Fac. of Agriculture, Assiut University, Assiut 71516, Egypt [[email protected]] Eshmeyer, William, California Academy of Sciences, San Francisco CA 94118, USA [[email protected]] Estabel, Jeanne, Lab. de Biologie Générale, Univ. Catholique de Lyon, 25 rue du Plat, F-69288 Lyon, France [[email protected]] Exbrayat, Jean Marie, Lab. de Biologie Générale, Univ. Catholique de Lyon, 25 rue du Plat, F69288 Lyon, France [[email protected]] Eyal-Giladi, Hefzibah, Dept. of Cell and Animal Biology, Inst. of Life Sciences, Hebrew University, 91904 Jerusalem, Israel [[email protected]] Fagerholm, Hans-Peter, Inst. of Parasitology, Dept. of Biology, Abo Akademi Univ., 20520 Abo, Finland [[email protected]] Fagerholm, Gunnel, Abo, Finland

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Fauchald, Kristian, Dept. of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington DC, 20560-0163 USA [[email protected]] Fiandra, Luisa, Dept. of Biology, Univ. of Milan, via Celoria 26, 20133 Milano, Italy [[email protected]] Fisher, Charles, The Pennsylvania State University, 208 Mueller Lab., University Park, PA 16802, USA [[email protected]] Foissner, Wilhelm, Inst. für Zoologie, Univ. Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria [[email protected]] Fokin, Sergei, St.Petersburg State University, 199034 St. Petersburg, Russia [[email protected]] Fortelius, Mikael, Dept. of Geology, University of Helsinki, P.O. Box 11, FIN-00014 Helsinki, Finland [[email protected]] Fortunato, Helena, Smithsonian Tropical Research Institute, P.O.Box 2072, Balboa, Panama [[email protected]] Fraguedakis-Tsolis, Stella, Sect. of Animal Biology, Dept. of Biology, University of Patras, 260 01 Patras, Greece Franzen, Christina, Dept. of Palaeozoology, Swedish Museum of Natural History, Box 50007, 104 05 Stockholm, Sweden [[email protected]] Froese, Rainer, ICLARM, M.C., P.O.Box 2631, 0718 Makati, Philippines [[email protected]] Gäde, Gerd, Zoology Dept., University of Cape Town, Rondebosch 7701, South Africa [[email protected]] Gaill, Françoise, UMR CNRS 7622, Biologie Cellulaire et Moleculaire du Developpement, Université Pierre et Marie Curie, 7 quai St. Bernard, 75252 Paris cedex 5, France [[email protected]] Gallardo, Carlos-Sebastian, Inst. de Zoologia E.F. Kilian, Universidad Austral de Chile, Casilla 567, Valdivia, Chile [[email protected]] Gallardo, Milton, Inst. de Ecología y Evolución, Universidad Austral de Chile, Casilla 567, Valdivia, Chile [[email protected]] Ganzhorn, J.U., Zoologisches Institut, Univ. Hamburg, Martin-Luther-King Platz 3, 20146 Hamburg, Germany [[email protected]] Garey, James R., Dept. of Biological Sciences, College of Arts and Sciences, 4202 East Fowler Ave., SCA 110, University of South Florida, Tampa, Florida, USA [[email protected]] Gentry, Anthea, ICZN, c/o The Natural History Museum, Cromwell Rd., London SW7 5BD, UK [[email protected]] Gernigon-Spychalowicz, Therese, Endocrinology, Inst. des Sciences de la Nature, Univ.de Sciences et Techniques Houari Boumediene, B.P. 32, Bab Ezzouar, 16111 El Alia, Algeria [[email protected]] Giokas, Sinos, Zoological Museum, Dept. of Biology, Univ. of Athens, Panepistimioupolis, 157 84 Athens, Greece [[email protected]] Giordana, Barbara, Dept. of Biology, Univ. of Milan, via Celoria 26, 20133 Milano, Italy [[email protected]] Gist, Daniel, Dept. of Biological Sciences, Univ. of Cincinnati, POB 21006, Cincinnati, Ohio 452210006, USA [[email protected]] Goldsworthy, Graham John, School of Biological and Chemical Sciences, Birkbeck College, Malet str., London WC1E 7HX, UK [[email protected]] Graur, Dan, Dept. of Biology, Tel Aviv University, Ramat Aviv 69978, Israel [[email protected]]

List of Participants

729

Greuter, Werner, Botanischer Garten und Botanisches Museum Berlin-Dahlem, Königin-Luise – Str. 6-8, D-14191 Berlin, Germany [[email protected]] Grimaldi, Annalisa, Dept of Structural and Functional Biology, Univ. of Insubria, via J.H. Dunant 3, 21100 Varese, Italy [[email protected]] Hackstein, Johannes, Dept. of Evolutionary Microbiology, Fac. of Science, University of Nijmegen, NL-6525 ED Nijmegen, The Netherlands [[email protected]] Haim, Abraham, University of Haifa, Mount Carmel, Haifa 31905, Israel [[email protected]] Hamlett, William, South Bend Center for Medical Education, Indiana Univ. School of Medicine, B-10 Haggar Hall, Notre Dame, IN 45556, USA [[email protected]] Heddi, Abdelaziz, UMR INRA/INSA de Lyon, Biologie Fonctionelle, Insectes et Interactions (BF21), Bâtiment 406, 20, Ave. Albert Einstein, 69621 Villeurbanne cedex, France [[email protected]] Heller, Joseph, Hebrew University of Jerusalem, 91904 Jerusalem, Israel [[email protected]] Hellstedt, Paavo, University of Helsinki, P.O. Box 33 (Yliopistonkatu 4), 00014 Helsinki, Finland [[email protected]] Hirsch, Leonard, Office of International Relations, Smithsonian Institution, Washington DC, 20560-0705 USA [[email protected]] Holm, Lena, Dept. of Animal Physiology, Swedish University of Agricultural Science, Box 7045, S-750 07 Uppsala, Sweden [[email protected]] Huchon, Dorothée, Lab. de Paléontologie, Paléobiologie & Phylogénie, Inst. des Sciences et de l’Evolution, UMR 5554 CNRS, Univ. de Montpellier II, Place E. Bataillon, 34095 Montpellier cedex 5, France [[email protected]] Hugot, Jean-Pierre, Museum National d’Histoire Naturelle, Mammifères et Oiseaux, 55, rue Buffon, 75231 Paris cedex 5, France [[email protected]] Ishikawa, Hajime, Dept. of Biological Sciences, Graduate School of Science, Univ. of Tokyo, Hongo-Bunkyo-Ku, Tokyo 113-0033, Japan [[email protected]] Ivovic, Vladimir, Inst. of Medical Research, Dr. Subotica 4, P.O.Box 102, 11129 Belgrade, Yugoslavia [[email protected]] Jax, Kurt, Zentrum für Ethik in der Wissenschaften, Univ. Tübingen, Keplerstr. 17, D-72074 Tübingen, Germany [[email protected]] Jeon, Kwang, Dept. of Biochemistry, Univ. of Tennessee, Knoxville, TN 37996-0840, USA [[email protected]] Kauschke, Ellen, Zoological Institute and Museum, Ernst-Moritz-Arndt Univ. Greifswald, Domstrasse 11, 17487 Greifswald, Germany [[email protected]] Kelmo, Francisco, Coral Reef Ecology, Benthic Ecology Research Group, Dept. of Biological Science, Univ. of Plymouth, Drake Circus, Plymouth PL4 8AA, UK [[email protected]] Klaa, Kamel, Institut National d’ Agronomie, Hacen Badi 16200 El Harrach, Alger, Algeria Koomen, Peter, Naturalis, National Museum of Natural History, Office for Development of Cultural Projects, P.O. Box 9517, NL-2300 RA Leiden, The Netherlands [[email protected]] Koutsoubas, Drosos, Dept. of Marine Sciences, Univ. of the Aegean, Sapfous 5, 811 00 Mytilini, Greece [[email protected]] Kristensen, Reinhardt, Dept. of Invertebrate Zoology, Zoological Museum, Univ. of Copenhagen, Regnskabskontoret Kassen, Postboks 2177, 1017 Copenhagen, Denmark [[email protected]]

730

The new panorama of animal evolution

Lazaridou, Maria, Sect. of Zoology, Dept. of Biology, Aristotle University of Thessaloniki, 540 06 Thessaloniki, Greece [[email protected]] Legakis, Anastasios, Zoological Museum, Dept. of Biology, Univ. of Athens, Panepistimioupolis, 157 84 Athens, Greece [[email protected]] Lehtinen, Pekka, Zoological Museum, University of Turku, 20014 Turku, Finland [[email protected]] Leitz, Thomas, Entwicklungsbiologie der Tiere, Fachbereich Biologie, Univ. Kaiserslautern, Erwin-Schroedinger Str., D-67663 Kaiserslautern, Germany [[email protected]] Lessios, Harilaos, Smithsonian Tropical Research Institute, Box 2072, Balboa, Panama [[email protected]] Liu Baozhong, Inst. of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China [[email protected]] Lohrmann, Karin, Fac. de Ciencias del Mar, Universidad Católica del Norte, Cas. 117, Cocuimbo, Chile [[email protected]] Lourenço, Wilson, Lab. de Zoologie (Arthropodes), Museum National d’ Histoire Naturelle , 61 rue de Buffon, 75231 Paris cedex 5, France [[email protected]] Lugon-Moulin, Nicolas, Inst. d’Ecologie, Lab. de Zoologie et Ecologie Animale, Univ. de Lausanne, CH-1015 Lausanne-Dorigny, Switzerland [[email protected]] Lymberakis, Petros, Natural History Museum of Crete, 157 Knosou Ave., Irakleion, Greece [[email protected]] Magowski, Wojciech Lukas, Dept. of Animal Taxonomy and Ecology, A. Mickiewicz University, Szamarzewskiego 91A, 60-569 Poznan, Poland [[email protected]] Maragou, Panagiota, WWF-Greece, 26 Filellinon str., 105 58 Athens, Greece [[email protected]] Marciniak, Arkadiusz, Inst. of Prehistory, Adam Mickiewicz University of Poznan, Sw. Marcin 78, 60 809 Poznan, Poland [[email protected], [email protected]] Marco, Heather, Zoology Dept., University of Cape Town, Rondebosch 7701, South Africa [[email protected]] Mariani, Stefano, Dept. of Animal and Human Biology, Univ. of Rome “La Sapienza”, viale dell’ Universitá 32, 00185 Rome, Italy [[email protected]] Martens, Jochen, Institut für Zoologie, Johannes-Gutenberg Univ., Saarstrasse 21, D-55099 Mainz, Germany [[email protected]] Martindale, Mark Q., Kewalo Marine Lab., Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii, USA [[email protected]] Masseti, Marco, Istituto di Antropologia, Universitá di Firenze, via del Proconsolo 12, 50122 Firenze, Italy [[email protected]] McFall-Ngai, Margaret, Pacific Biomedical Research Center, University of Hawaii, 41 Ahui Str., Honolulu, Hawaii 96813, USA [[email protected]] McLennan, Deborah, Dept. of Zoology, University of Toronto, 25 Harbord str., Toronto, Ontario M5S 3G5, Canada [[email protected]] McMahon, Brian R, Biological Sciences, University of Calgary, Calgary T2N 1N4, Canada [[email protected]] Megalofonou, Persefoni, Sect. of Zoology-Marine Biology, Dept. of Biology, Univ. of Athens, Panepistimioupolis, 157 84 Athens, Greece [[email protected]] Mesbah, Aicha, Lab. of Entomology, Inst. des Sciences de la Nature, Univ.de Sciences et Techniques Houari Boumediene, B.P. 32, Bab Ezzouar, 16111 El Alia, Algeria [[email protected]]

List of Participants

731

Mindell, David, Dept. of Biology and Museum of Zoology, University of Michigan, Ann Arbor, MI 48109, USA [[email protected]] Minelli, Alessandro, University of Padova, via Trieste 75, 35121 Padova, Italy [[email protected]] Miyazaki, Katsumi, Seto Marine Biological Laboratory, Kyoto University, 459 Shrirahama, Wakayama 649-2211, Japan [[email protected]] Mokady, Ofer, Institute for Nature Conservation Research, Tel Aviv University, Tel Aviv 69978, Israel [[email protected]] Møller, Anders P., Lab. d’Ecologie, Univ. Pierre et Marie Curie, Bat. A, 7, quai St. Bernard, Case 237, F-75252 Paris cedex 5, France [[email protected]] Morand, Serge, Lab. de Biologie Animale, Centre de Biologie et d’ Ecologie Tropicale et Mediterranée, Univ. de Perpignan, 66860 Perpignan, France [[email protected]] Morris, Molly, Dept. of Biological Sciences, Ohio University, Athens, OH 45701, USA [[email protected]] Müller, Werner E.G, Inst. für Physiologische Chemie, Abt. für Angewandte Molekularbiologie, Johannes Gutenberg Universität Mainz, Düsbergweg 6, D-55099 Mainz, Germany [[email protected]] Muñoz Frigola, Marta, Dpt. Ciencies Ambientals, Univ. de Girona, Campus de Montilivi, 17071 Girona, Spain [[email protected]] Nardon, Paul, UMR INRA/INSA de Lyon, Biologie Fonctionelle, Insectes et Interactions (BF21), Bâtiment 406, 20, Ave. Albert Einstein, 69621 Villeurbanne cedex, France [[email protected]] Nasser, Mohamed Abdel-Kareem, Plant Protection Department, Fac. of Agriculture, Assiut University, Assiut 71516, Egypt [[email protected]] Newton de Almeida Santos, Maria Susana, Dept. de Zoologia & Inst. do Ambiente e Vida, Univ. of Coimbra, 3004-517 Coimbra, Portugal [[email protected]] Nicolaidou, Artemis, Sect. of Zoology-Marine Biology, Dept. of Biology, Univ. of Athens, Panepistimioupolis, 157 84 Athens, Greece [[email protected]] Nielsen, Claus, Zoological Museum, University of Copenhagen, Regnskabskontoret Kassen, Postboks 2177, 1017 Copenhagen, Denmark [[email protected]] Nikaido, Masato, Fac. of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan [[email protected]] Novgorodova, Tatyana, Inst. of Animal Systematics and Ecology, Frunze 11, Novosibirsk 630091, Russia [[email protected]] Nylin, Søren, Dept. of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden [[email protected]] Okada, Norihiro, Fac. of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan [[email protected]] Pafilis, Panagiotis, Sect. of Human and Animal Physiology, Dept. of Biology, University of Athens, Panepistimioupolis, 157 84 Athens, Greece [[email protected]] Palo, Jukka, Dept. of Ecology and Systematics, University of Helsinki, Box 17, FIN-00014 Helsinki, Finland [[email protected]] Pancucci-Papadopoulou, Maria-Antonella, National Center for Marine Research, Agios Kosmas, 166 04 Elliniko, Athens, Greece [[email protected]]

732

The new panorama of animal evolution

Papapavlou, Kelli, 15 Artemidos str., 144 52 Metamorfosi, Athens, Greece [[email protected]] Paravas, Vagelis, Natural History Museum of Crete, 157 Knosou Ave., Irakleion, Greece [[email protected]] Parrinello, Nicolo, Lab. of Marine Immunology, Dept. of Animal Biology, Univ. of Palermo, via Archirafi 18, 90123 Palermo, Italy [[email protected]] Pearse, John, Inst. of Marine Sciences, University of California at Santa Cruz, Santa Cruz, CA 95064, USA [[email protected]] Pearse, Vicki, Inst. of Marine Sciences, University of California at Santa Cruz, Santa Cruz, CA 95064, USA [[email protected]] Pennati, Roberta, Dept. of Biology, University of Milano, via Celoria 26, 20133 Milano, Italy Pestarino, Mario, Dip. di Biologia Sperimentale, Ambientale e Applicata, Sez. di Neuroendocrinologia e Biologia dello Sviluppo, Univ. di Genova, viale Benedetto XV, 5, 16132 Genova, Italy [[email protected]] Petrinos, Kostantinos, 23 Spetson str., 153 42 Agia Paraskevi, Athens, Greece [[email protected]] Pianka, Eric, Dept. of Zoology, University of Texas at Austin, Austin, Texas 78712-1064, USA [[email protected]] Planes, Serge , EPHE- ESA CNRS 8046, Univ. de Perpignan, 66860 Perpignan cedex, France [[email protected]] Polymeni, Rosa, Sect. of Zoology-Marine Biology, Dept. of Biology, Univ. of Athens, Panepistimioupolis, 157 84 Athens, Greece [[email protected]] Por, Francis Dov, Hebrew University of Jerusalem, 91904 Jerusalem, Israel [[email protected]] Poss, Stuart, University of Southern Mississippi, Gulf Coast Research Laboratory, P.O. Box 7000, Ocean Springs, MS 39566-700, USA [[email protected]] Poulin, Robert, Dept. of Zoology, University of Otago, P.O. Box 56, Dunedin, New Zealand [[email protected]] Pradillon, Florence, UMR 7622 CNRS, Groupe de Biologie Marine, Université Pierre et Marie Curie, 7 quai St. Bernard, 75252 Paris cedex 5, France [[email protected]] Proszynski, Jerzy, Muzeum i Instytut Zoologii, Ul. Wilcza 64, 00-679 Warszawa, Poland [[email protected]] Queinnec, Eric, Equipe Développement et Evolution, Biologie Moleculaire et Cellulaire dy Développement, Université Paris VI, case 241, 9 quai St. Bernard, F-75252 Paris cedex 05, France [[email protected]] Radojicic, Jelena, Inst. of Zoology, Fac. of Biology, Univ. of Belgrade, Studentski trg 16, 11000 Belgrade, Yugoslavia [[email protected]] Raineri, Margherita, Dip. di Biologia Sperimentale, Ambientale e Applicata, Sez. di Neuroendocrinologia e Biologia dello Sviluppo, Univ. di Genova, viale Benedetto XV, 5, 16132 Genova, Italy [[email protected]] Ramachandra, Mohan, Fishery research Unit, Dept. of Zoology, Bangalore University, Bangalore, Karnataka, 560056 India [[email protected]] Rania, Lisa, Dept. of Biology, Saint Mary’s College, Notre Dame, IN 46556, USA [[email protected]] Ravaux, Juliette, UMR 7622 CNRS, Groupe de Biologie Marine, Université Pierre et Marie Curie, 7 quai St. Bernard, 75252 Paris cedex 5, France [[email protected]]

List of Participants

733

Remerie, Thomas, University of Gent, Gent, Belgium – Blauwesteenstraat 72, 9070 Heusden, Belgium [[email protected]] Revanasiddaiah, H. M., Dept. of Zoology, Bangalore University, Bangalore 560056, India Ride, William David Lindsay, Dept. of Geology, Australian National University, Canberra 0200 ACT, Australia [[email protected], [email protected]] Rieger, Reinhard, Abt. für Ultrastrukturforschung und Evolutionsbiologie, Inst. für Zoologie und Limnologie, Univ. Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria [[email protected]] Rizzo, Carolina, Dept. of Animal Biology, Univ. of Palermo, via Archirafi 18, 90123 Palermo, Italy [[email protected]] Roch, Philippe, DRIM, CNRS-IFREMER, Univ. de Montpellier II, cc 80, Place E. Bataillon, 34095 Montpellier cedex 2, France [[email protected]] Rouland-Lefevre, Corinne, IRD, Lab. de Microbiologie, Dakar, Senegal [[email protected]] Saldarriaga, Juan Fernando, University of British Columbia, 3529-6270 University Boulevard. Vancouver, BC, V6T 1Z4, Canada [[email protected]] Salzet, Michel, Lab. d’Endocrinologie des Annélides, UPRES-A CNRS 8017, SN3, Univ. des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq cedex, France [[email protected]] Santa Isabel, Leda Maria, Dept. de Zoologia, Inst. de Biologia, Universidade Federal da Bahia, Campus Universitario de Ondina, s/n. CEP. 40170-290 Salvador, BA, Brazil [[email protected]] Santini, Francesco, Dept. of Zoology, University of Toronto, 25 Harbord str., Toronto ON M5S 3G5, Canada [[email protected]] Satoh, Nori, Dept. of Zoology, Kyoto University, Sakyo, Kyoto 606-8502, Japan [[email protected]] Schmidt-Rhaesa, Andreas, Zoomorphologie und Systematik, Fak. für Biologie, Univ. Bielefeld, Postfach 100131, 33501 Bielefeld, Germany [[email protected]] Schmitt, Michael, Zoologisches Forschungsinstitut und Museum Alexander König, Adenauerallee 160, D-53113 Bonn, Germany [[email protected]] Scholtz, Gerhard, Inst. für Biologie/Vergleichende Zoologie, Humboldt-Universität zu Berlin, Philippstr. 13, D-10115 Berlin, Germany [[email protected]] Schram, Frederick R., Zoological Museum, Univ. van Amsterdam, P.B. 94766, NL-1090 GT Amsterdam, The Netherlands [[email protected]] Schwenk, Kurt, Dept. of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269-3043, USA [[email protected]] Sellami, Mahdi, Lab. of Ecology of Vertebrates, National Institute of Agronomy, 16200 El-Harrach, Algeria [[email protected]] Sergejeva, Galina, Inst. of Cytology, Russian Academy of Sciences, St.Petersburg, Russia [[email protected]] Seridji, Rabia, Inst. des Sciences de la Nature, Univ.de Sciences et Techniques Houari Boumediene, B.P. 32, Bab Ezzouar, 16111 El Alia, Algeria Sever, David M., Dept. of Biology, Saint Mary’s College, Notre Dame, IN 46556, USA [[email protected]] Sfenthourakis, Spyros, Sect. of Animal Biology, Dept. of Biology., Univ. of Patras, 260 01 Patras, Greece [[email protected]]

734

The new panorama of animal evolution

Shankland, Marty, 140 Paterson Laboratories, C0930, 24th & Speedway, Molecular Cell & Developmental Biology, University of Texas at Austin, Austin TX 78712, USA [[email protected]] Shen Yunfen, Chinese Academy of Sciences, Wuhan, Hubei Province, China [[email protected]] Shields, Graham, University of Ottawa, Ottawa, Ontario, Canada [[email protected]] Sideris, George, Long Island University, University Plaza, Brooklyn, NY 11201, USA [[email protected]] Silva de Jesus, Ana Clara, Dept. de Zoologia, Inst. de Biologia, Universidade Federal da Bahia, Campus Universitario de Ondina, s/n. CEP. 40170-290 Salvador, BA, Brazil [[email protected]] Skoracka, Anna, Dept. of Animal Taxonomy and Ecology, A. Mickiewicz University, Szamarzewskiego 91A, 60-569 Poznan, Poland [[email protected]] Smocovitis, Vassiliki Betty, Dept. of History, Univ. of Florida, Gainesville, FL 32611, USA [[email protected], [email protected]] Solé-Cava, Antonio, Lab. de Biodiversidad Molecular, Dept. de Genética, Inst. de Biologia, Universidade Federal de Rio de Janeiro, Bloco A, CCS-Ilhado Fundão, 21941-490, Rio de Janeiro, Brazil [[email protected]] Song Daxiang, College of Biological Sciences, Hebei University, Baoding, Hebei Province, China [[email protected], [email protected]] Sotiropoulos, Konstantinos, Sect. of Zoology-Marine Biology, Dept. of Biology, Univ. of Athens, Panepistimioupolis, 157 84 Athens, Greece [[email protected]] Stotz, Henrik, Max-Planck-Institute of Chemical Ecology, Carl-Zeiss Promenade 10, 07745 Jena, Germany [[email protected]] Suarez, Susan, Dept. of Biomedical Sciences, T5-006 Veterinary Research Tower, Cornell University, Ithaca, NY 14853, USA [[email protected]] Tettamanti, Gianluca, Dept of Structural and Functional Biology, Univ. of Insubria, via J.H. Dunant 3, 21100 Varese, Italy [[email protected]] Thalmann, Urs, Anthropological Inst. and Museum, Univ. of Zürich, Winterhurerstr. 190, CH8057 Zürich, Switzerland [[email protected]] Thessalou-Legaki, Maria, Sect. of Zoology-Marine Biology, Dept. of Biology, Univ. of Athens, Panepistimioupolis, 157 84 Athens, Greece [[email protected]] Thorne, Joan, BIOSIS UK, 54 Micklegate, York YO1 6WF, UK [[email protected]] Tiago, Claudio Goncalves, Centro de Biologia Marinha, Universidade de São Paulo, Cx Postal 83, São Sebastião, SP, Brasil, 11600-970[[email protected]] Tryjanowski, Piotr, Inst. of Environmental Biology, University Mickiewicza, Fredy 10. PL-61 701 Poznan, Poland [[email protected]] Tsekoura, Nadia, Sect. of Animal Biology, Dept. of Biology, University of Patras, 260 01 Patras, Greece [[email protected]] Tubbs, Philip, I.C.Z.N., c/o The Natural History Museum, Cromwell Rd., London SW7 5BD, U.K [[email protected]] Töpfer- Petersen, Edda, Inst. of Reproductive Medicine, School of Veterinary Medicine, Buenteweg 15, 30559 Hannover, Germany [[email protected]] Uribe, Francesco, Museu de Zoologia de Barcelona, Parc de la Ciutadella, Ap. De correus 593, E08080 Barcelona, Spain [[email protected]] Ürpmann, Hans Peter, Arbeitsbereich Archäobiologie, Abteilung Ältere Urgeschichte und Quartärökologie, Institut für Ur- und Frühgeschichte und Archäologie des Mittelalters, Eberhard-

List of Participants

735

Karls-Universität Tübingen, Eugenstrasse 40, 72072 Tübingen, Germany [[email protected]] Valvassori, Roberto, Dept of Structural and Functional Biology, Univ. of Insubria, via J.H. Dunant 3, 21100 Varese, Italy [[email protected]] Vardala, Evi, Goulandris Museum of Natural History, Levidou 13, 145 62 Kifisia, Greece Vasic, Voislav, Natural History Museum, Njegoseva 51, P.O.Box 401, 11000 Belgrade, Yugoslavia [[email protected]] Vasquez Dominguez, Ella, Dept. of Zoology and Entomology, University of Queensland, St. Lucia 4072, QLD, Australia [[email protected]] Vernet, Guy, Lab. d’Ecotoxicologie, Univ. de Reims Champagne-Ardenne, UFR Sciences, BP 1039, F-51687 Reims, France [[email protected]] Vizzini, Aiti, Dept. of Animal Biology, Univ. of Palermo, via Archirafi 18, 90123 Palermo, Italy [[email protected]] Waloszek, Dieter, Sect. for Biosystematic Documentation, Universität Ulm, Helmholtzstraße 20, D-89081 Ulm, Germany [[email protected]] Wiley, Edward O., Division of Fishes, Natural History Museum, University of Kansas, Lawrence, KS 66045, USA [[email protected]] Wilkens, Barbara, Dip. di Storia, Univ. di Sassari, V.le Umberto 52, 07100 Sassari, Italy [[email protected]] Williams, Suzanne, Smithsonian Tropical Research Institute, Box 2072, Balboa, Panama [[email protected]] Winston, Judith, Virginia Museum of Natural History, 1001 Douglas Ave., Martinsville, VA 24112, USA [[email protected]] Woo, Norman Y. S., Dept. of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China [[email protected]] Wright, William G., Colorado State University, Fort Collins, CO 80523, USA [[email protected]] Wyrwoll, Thomas, Johann Wolfgang Goethe Universität, Albert-Schweizer Str. 52, D-60437 Frankfurt, Germany [[email protected]] Young, Craig, Div. of Marine Sciences, Harbor Branch Oceanographic Institution, 5600 U.S. Hwy. 1 N., Ft. Pierce, FL 34946, USA [[email protected]]

Secretariat Georgiakakis, Panagiotis Germanou, Natasa Legakis, Antonios Merakou, Athina Papaioannou, Vassiliki Papaspyrou, Sokratis Polymeni, Vassiliki

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A. Legakis, S. Sfenthourakis, R. Polymeni & M. Thessalou-Legaki (eds.) The New Panorama Index of Animal Evolution Proc. 18th Int. Congr. Zoology, pp. 737-738, 2003

737

INDEX Ahlberg P.E. 45 Andreone F. 403 Arizza V. 167 Augustin R. 127 Aumelas A. 177 Azariah J. 569 Bakst M.R. 447 Bãnãrescu P.M. 715 Bengtson S. 289 Bergström J. 43, 89 Bosch T.C.G. 127 Bourtzis K. 523 Brasier M. 259 Bridgewater P.B. 631 Brizzi R. 431 Buckeridge J.St.J.S. 19, 561, 581, 705 Budd G.E. 479 Burkhardt R.W. 329 Calas B. 177 Cammarata M. 167 Chavnieu A. 177 Combes C. 227 Cooper E.L. 117, 139, 147, 167 Côté I.M. 219 Cresswell I.D. 631 Currie P.J. 55 d’Hondt J.-L. 345 de Eguileor M. 139 Delsol M. 345 Delussu F. 303 Dewel W.C. 269 Dewel R.A. 269 Emblow C.S. 603 Fauchald K. 637 Foissner W. 243 Fortelius M 61

Fortunato H. 591 Froese R. 611 Gaill F. 513 Ganzhorn J.U. 393 Garey J.R. 503 Génermont J. 345 Goodman S.M. 393 Gordon D. 705 Greuter W. 665 Greven H. 421 Grimaldi A. 139 Hackstein J. 277 Hadel V.F. 701 Hamlett W.C. 421, 439 Heddi A. 521, 527 Hou Xianguang 89 Howcroft J.M. 659 Hugot J. P. 235 Ishikawa H. 535 Jax K. 337 Jeon K.W. 541 Kauschke E. 133 Khalturin K. 127 Kristensen R.M. 467 Kuznetsov S. 127 Lefebvre C. 147 Legakis A. 603 Lourenço W.R. 383, 385 Marciniak A. 309 Martens J. 551 McKinney F.K. 269 Minelli A. 649 Mitta G. 177 Mohrig W. 133 Møller A.P. 199 Morand S. 213 Müller I.M. 99

Müller W.E.G. 99 Nardon P. 521 Noirot Ch. 345 Nylin S. 107 Päckert M. 551 Parrinello N. 167 Pestarino M. 159 Peterson A.T. 619 Pianka E.R 3 Por F. D. 27, 575, 627, 643 Poss S.G. 585 Poulin R. 205 Pradillon F. 513 Prószy´nski J. 711 Rakotondravony D. 393 Rakotosamimanana. B. 393 Ramanamanjato J.-B. 393 Rania L.C. 431 Reyes R. Jr. 611 Ride W.D.L. 673 Rieger R.M. 247 Rinkevich B. 127 Roch Ph. 177 Salzet M. 147, 177 Schindler J. 421 Schmidt-Rhaesa A. 461 Schmitt M. 369 Scholz G. 489 Schram F.R. 359 Schröder J. 127 Sever D.M. 431, 439 Shankland M. 187 Shields G. 243 Smocovitis V.B. 351 Stotz H.U. 127 Suarez S.S. 451 Tasiemski A. 147

738

The new panorama of animal evolution

Tettamanti G. 139 Thalmann U. 409 Thorne M.J. 659 Tiago C.G. 701 Valvassori R. 139

Vandenbulcke F. 177 Vazzana M. 167 Waloszek D. 69 Wiley E.O. 15, 619 Wilkens B. 303

Winston J.E. 321 Wyrwoll T.W. 683 Yang Y.-S. 177 Zbinden M. 513