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https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] MOLECULAR SYSTEMATICS OF RECENT AND PLEISTOCENE BRACHIOPODS Kazuyoshi Endo Thesis submitted for the degree of Doctor of Philosophy Department of Geology and Applied Geology University of Glasgow March 1992 © Kazuyoshi Endo 1992 ProQuest Number: 11011458 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 11011458 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 For my father, Juitsu Endo (1917-1988) iii Acknowledgements This project was funded by NERC, the Royal Society of London, and the University of Glasgow. I also gratefully acknowledge the financial support of a postgraduate scholar ship from Glasgow University, the ORS award, the Ikueikai scholarship (Japan), the Systematics Association (U.K.), and the Shimadzu Foundation (Japan). Even more than most Ph.D theses written by overseas students, this thesis owes its very existence to the help and encouragement of many people, and I would like to thank them all. Special thanks are due to Dr. Gordon B. Curry, for his supervision and constant intellectual support, and also to Dr. G. B. Curry and Mrs. Gillian Curry for all their kindness outside the department from my very first day in the U.K. I would like to thank the staff and technicians of the Department of Geology and Applied Geology, with thanks to Professor Bernard E. Leake for the use of facilities and to Mr. Roddy Morrison for technical support. Many thanks are due also to the staff and technicians of the Department of Genetics, Glasgow University, for facilitating the work on Lingula DNA. I am es pecially grateful to Dr. Howard T. Jacobs for allowing me to use his laboratory facilities, and for his excellent advice and suggestions. It is a privilege to thank Sir Alwyn Williams for his encouragement and for generously allowing me to occupy one of his visitor’s offices, which was essential for the preparation of this thesis. Mrs. Sue Ogden is thanked for facilitating the work in that office. I am grateful to Dr. Rosalind Quinn for the preparation of antisera and for her advice on immunological experiments. Many thanks to Dr. Matthew Collins and Dr. Gerard Muyzer who provided a number of brilliant ideas, technical advice, and brachiopod antisera. I would like to thank Professor Peter Westbroek, who is the source of stimulating ideas, for his encouragement. I am indebted to Dr. Maggie Cusack for her advice on every aspect of biochemical experiments, to Derek Walton for conducting amino acid analysis, and to both of them for critically reading the draft of this thesis. Thanks also to Mrs. Ikbal Al-Rikabi for her help in electrophoresis experiments, and to Stephen Ewing for some brachiopod antisera. I am grateful to Suzanne Forbes and Sandra McCormack for technical support. Thanks are due to the people in the ‘Howy lab’, especially Dr. Sohail Qureshi and Dr. Abdellatif El-Meziane for their patience in instructing molecular biology from scratch to a geologist. I would like to thank Professor Itaru Hayami, Professor Kiyotaka Chinzei, Dr. Kazushige Tanabe, Dr. Katsumi Abe, and Dr. Tatsuo Oji for their encouragement and advice throughout my career both in Glasgow and in Tokyo. iv At the Natural History Museum, Smithsonian Institution, U.S.A., I would like to thank Dr. Richard Grant and Dr. Rex Doescher, for their help and hospitality during the month of my working stay there, and for providing specimens for immunology. I am indebted to Dr. Tomowo Ozawa, Dr. Daphne Lee, Mr. Eiji Tsuchida, and Mr. Masahiro Kiuchi for the donation of brachiopod samples. My appreciation goes also to many other researchers who have kindly provided samples for the Molecular Palaeontology laboratory. I am grateful to Dr. David MacKinnon for the access to his unpublished translation of Russian literature, and for constructive discussions. At the sampling trip in New Zealand, I express my gratitude to every member of the caravan brigade, Dr. G. B. Curry, Dr. Spaff Ackerley, Derek Walton, and Heather Clegg, for the memorable month. I would like to thank my mother Shiho Endo, Mr. Kenichi Maeda, Mrs. Hagiko Maeda and my brothers for their constant support and encouragement. Finally, very special thanks to Shuko Endo and Wakana Endo who provided me with most support and encouragement. Declaration The material presented in this thesis summarises the results of three years of research carried out in the Department of Geology and Applied Geology of Glasgow University under the supervision of Dr. Gordon B. Curry. This study is based on my own independent research and any previously published or unpublished results of other researchers used in this thesis have been given full acknowledgement in the text. Kazuyoshi Endo March 1992 VI Contents Title page i Dedication ii Acknowledgements iii Declaration v Contents vi List of figures x List of tables xi Abbreviations xiii Units xiv Three letter abbreviations for common amino acids xiv Definitions of immunological terms xv Summary xvi Outline of the thesis xviii Chapter 1 Molecular approaches in Palaeontology 1 1.1 Introduction 1 1.2 Molecular fossils 2 1.3 Fossil proteins 3 1.4 Fossil DNA 5 1.5 Molecular Systematics 7 1.6 Conclusions 9 Chapter 2 The Brachiopoda 11 2.1 Introduction 11 2.2 Origin of Brachiopoda 11 2.3 Main divisions of Brachiopoda 13 2.4 Morphology of terebratulide brachiopods 15 2.4.1 General comments 15 2.4.2 External features 15 2.4.3 Internal features 16 2.4.4 Shell structure 20 2.5 Phylogeny and taxonomy of terebratulides based on morphology 20 2.6 Previous molecular studies on the Brachiopoda 26 Chapter 3 Materials and methods 29 3.1 Materials 29 3.1.1 Chemicals and equipment 29 3.1.2 Brachiopod samples 30 VXI 3.1.3 Antisera 30 3.2 Methods 37 3.2.1 Isolation of shell intracrystalline macromolecules 37 3.2.2 Immunisation protocol 37 3.2.3 Isolation of serum from blood 38 3.2.4 Enzyme-linked immunosorbent assay (ELISA) 38 3.2.5 Titre determination 39 3.2.6 ELISA with Sodium metaperiodate treated antigens 39 3.2.7 Inhibition ELISA 39 3.2.8 Calculation of immunological reactivity 40 3.2.9 Liquid chromatography 41 3.2.10 Amino acid analysis 44 3.2.11 Morphometries on cancellothyridoid species 45 Chapter 4 Purification of shell intracrystalline macromolecules 46 4.1 Introduction 46 4.2 Gel filtration 46 4.3 Reverse phase chromatography 47 4.4 Ion exchange chromatography 51 4.5 Hydrophobic interaction chromatography 56 4.6 Discussion and conclusions 56 Chapter 5 Sero-taxonomy of Recent articulate brachiopods 62 5.1 Introduction 62 5.2 Materials and methods 63 5.3 Results 63 5.4 Discussion 79 5.4.1 Nature of the immunological data 79 5.4.2 Framework of the articulate serotaxonomy 82 5.4.3 Interpretation of the immunological data 85 5.4.4 Terebratulide evolution 91 5.5 Conclusions 91 Chapter 6 Relationships among the living Cancellothyridoidea 94 6.1 Introduction 94 6.2 Materials and methods 94 6.3 Results 95 6.4 Discussion 99 6.4.1 Subgroups of the living Terebratulina 99 _________________________________________________________________________________________________ viii 6.4.2 Phylogenetic relationships between the subgroups of Terebratulina 102 6.4.3 Affinity of Cnismatocentrum, Cancellothyris and Chlidonophora to the Terebratulina species 105 6.5 Conclusions 105 Chapter 7 Preservation of intracrystalline macromolecules in fossil Terebratulina shells 107 7.1 Introduction 107 7.2 Materials and methods 107 7.2.1 Materials 107 7.2.2 Methods 108 7.3 Results and discussion 109 7.3.1 Intracrystalline macromolecules of Recent Terebratulina unguicula 109 7.3.2 Preservation of intracrystalline macromolecules in the Pleistocene Terebratulina species 110 7.3.3 Immunological responses from Pleistocene brachiopods 118 7.4 Conclusions 124 Chapter 8 An immunological investigation of the phytogeny of an extinct Pleistocene Terebratulina species 125 8.1 Introduction 125 8.2 Materials and methods 126 8.2.1 Samples 126 8.2.2 Methods 127 8.3 Morphometry 127 8.4 Results of the immunological assays 128 8.5 Discussion 134 8.6 Estimation of the divergence time using ‘quasi-immunological distances’ 135 8.7 Conclusions 136 Chapter 9 General discussion and suggestions for further study 138 9.1 Serotaxonomy of living brachiopods 138 9.2 Mitochondrial DNA of Lingula 140 9.3 Characterisation of living brachiopod intracrystalline proteins 141 9.4 Study of fossil intracrystalline proteins 143 9.5 Concluding remarks 144 ix References 145 Appendix I Morphometric data of the cancellothyridoid samples 167 Appendix II Publications 174 Appendix II-1 Endo, K.