Phylogeography, Population History and Conservation Genetics of the Endangered Green and Golden Bell Frog (Litoria aurea) Emma Louise Burns Ph.D. School of Biological, Earth and Environmental Sciences University of New South Wales December 2004 For Ben …without you this would never have been possible. I hereby declare that this submission is my own work and to the best of my knowledge it contains no material previously published or written by an other person, nor material which to a substantial extend has been accepted for the award of any other degree or diploma at UNSW or any other education institution, except where due acknowledgement is made in this thesis. Any contribution made to the research by other, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged. Signed: Emma Burns Preface This thesis consists of four stand-alone papers (chapters two to five) that have been published, submitted, or prepared for submission, to international journals of high standing. The publication details of each paper and the contribution of co-authors are detailed at the end of each chapter. To prevent unnecessary duplication a single reference list is provided at the end of the thesis. Chapter one, a general introductory chapter, describes the study species (Litoria aurea) and provides background information on conservation genetics. Chapter two provides a description of the isolation and characterisation of microsatellite markers, which are employed in chapter three. Chapter three provides a detailed examination of the genetic variability and population structure in L. aurea using microsatellite markers. Chapter four, examines the concept of a ‘bell frog species group’ using mitochondrial DNA and tests for the sister taxon to L. aurea, to use as an out group taxon for analyses in the proceeding chapter. Chapter five examines the intraspecific phylogeography of L. aurea using mitchonchondrial DNA. The concluding chapter, chapter six, provides a synthesis of the preceding chapters and a discussion of the conservation management of L. aurea based on the findings of this study. i Acknowledgements This has been a long journey and there are numerous people that I need to acknowledge and thank. Firstly, I would like to thank my husband Ben Noyen whose constant support and encouragement has made getting to the finish line a reality. Ben, for all the times I contemplated giving up when the obstacles just seemed too big, thank you for talking me out of it! To my family, Mum, Dad, Fiona, Ashley and Steve, thank you for all your support and for not giving up on me. You never once encouraged me to walk away and that was what I needed. Mum, thanks for the company, fun and great frog catching on the Victorian field trip. You are wonderful. To the Noyen family, Jo, Di, Summa and Jason, thank you for your support. I know at times you found it difficult to understand why I needed to finish this when it all seemed too hard but thank you for understanding. Summa, thanks for your help formatting and Jas thanks for letting me use your wiz bang computer in the dying days, it made life that little bit easier. To my old boss at AMBS, Jayne Tipping and my current supervisor Kerry Doyle at MSMR, a huge thank you to you both. Working full time for the last two and a half years whilst trying to complete my thesis has been made easier with your support. Now, moving on to the many people who have helped make this project happen. Starting at the beginning, to my initial supervisor Bronwyn Houlden, thank you for inspiring me to do ii research. Your commitment to me during this journey has made this all possible. Though I thought my world was slipping away when you first left UNSW, it did not happen. Thank you for continuing to read manuscripts and provide advice after you left. Rob Brooks, thank you for ‘adopting me’ and taking on my supervision. You have been fantastic and thank you for always encouraging me to “just get it done”. Your faith in me helped make this a reality, and your support made sure I did not “slip through the cracks”. Now to my two saving ‘knights’, Mark Eldridge and Darren Crayn, without your help mid way through the game, the end would of never been achievable. Thank you for helping me complete my thesis and manuscripts and Darren without you I am sure PAUP and I would never have gotten along! A special thank you to Bill Sherwin whose door was always open to me and Jo Zuccarello who is a complete wiz in the lab and was always ready to help and listen. A big thanks also to Hal Cogger for providing the title page photo and for all the talks and support you gave me during our habitat inspections etc at Sydney Olympic Park whilst I worked at AMBS. A special mention now for my fellow students who made this journey so fun; Meg, Ange, Shaun, Michael, Deirdre and Megan, you guys are great. I have not only achieved a degree (and hopefully a career) through this process but I have also made life long friends. Also a big, big thank you to all those who helped with field work and sample collection. A project of this magnitude could not succeed without the help of numerous people in the field and there are heaps of people to thank. I would like to start with a very special thank iii you to Ross Wellington and Arthur White, both of you made yourselves readily available to me and shared your knowledge of the green and golden bell frog which enabled me to plan successful field trips, well mostly, but it is an endangered species so I shan’t complain. I would also like to thank Graham Pyke, Garry Daly, Andrew Hamer, Simon Lane, Mike Mahony, Rod Pietsch, Graeme Gillespie, John Clulow, Scott Filmer, Rebecca Rudd, Candice Webb, Dion Hobcroft, Kerry Darcovich, Glenn Muir and many field volunteers and property owners for aid in the collection of tissue samples. All of you helped make this project happen, thank you. Thank you, Steve Donnellan and Terry Bertozzi of the South Australian Museum and Denis O’Meally and Don Colgan of the Australian Museum for providing tissue from their collections for Chapter four. Steve Donnellan was also generous enough to review an earlier draft of chapter four and provide helpful comments, thank you. A special thank you to Alaxandra Schulmeister and Gianfrancesco Ferrari for their volunteer work in the laboratory. Francesco, it was a joy spending time with you and I dearly hope we get to meet again. Thank you Eli Geffen for advice with some statistical analysis in Chapter three and Eric Harley for supplying the program AGARst. Finally, on a more official note, this project could never have happened without financial support from an Australian Postgraduate Research Award and the following sources: University of New South Wales-URSP, ARC small grant, Zoological Parks Board of New South Wales, Roads and Traffic Authority, National Parks and Wildlife Service, Mary Ethel Read Research Grant, the Joyce W. Vickery Scientific Research Fund, and the W.V. iv Scott Foundation. Also, this study was conducted under University of New South Wales Animal Care and Ethics Committee approval (ACEC 99/39), Zoological Parks Board of NSW approval (ACEC 3a/06/99), New South Wales National Parks and Wildlife Service scientific permits (A2608, B2022), State Forests of New South Wales special purpose permit (05449) and Victorian Department of Natural Resources and Environment scientific permit (10000851). v Table of Contents PREFACE............................................................................................................................... I ACKNOWLEDGEMENTS ........................................................................................................ II LIST OF FIGURES..................................................................................................................X LIST OF TABLES................................................................................................................XIII THESIS ABSTRACT.............................................................................................................XV CHAPTER ONE: INTRODUCTION AND BACKGROUND INFORMATION.......... 1 STUDY SPECIES ................................................................................................................... 1 Species description ......................................................................................................... 1 Species history and conservation status......................................................................... 4 CONSERVATION GENETICS .................................................................................................. 7 Previous molecular studies of frogs ............................................................................... 9 THESIS OBJECTIVES........................................................................................................... 11 CHAPTER TWO: ISOLATION AND CHARACTERISATION OF MICROSATELLITE LOCI IN THE GREEN AND GOLDEN BELL FROG (LITORIA AUREA)............................................................................................................. 12 ABSTRACT......................................................................................................................... 12 INTRODUCTION.................................................................................................................