ABSTRACT USE OF DNA SEQUENCING TO IDENTIFY THE ORIGIN OF NORTHWESTERN AND SOUTHWESTERN POND TURTLES IN CAPTIVE BREEDING PROGRAMS Captive breeding is a critical management strategy in the recovery and preservation of certain threatened and endangered species. Programs that implement captive breeding must maintain a balance between preserving the genetic purity and genetic diversity of a population in order to build a viable group of individuals for future reintroduction. This may be difficult due to a limited number of extant wild individuals for establishment of founder populations, leading to an increased risk of inbreeding or outbreeding depression in subsequent generations. In this study, I worked in collaboration with 24 zoos, museums, and aquariums through the Western Pond Turtle Species Survival Plan Sustainability Project to aid in conservation of two threatened and endangered freshwater turtle species through captive breeding. As individuals of the two different species can appear morphologically identical, I used DNA sequencing to identify wild-bred Northwestern (Actinemys marmorata) and Southwestern Pond Turtles (Actinemys pallida) to build captive brood stock of both species. Relatively few studies have assessed conservation efforts in consideration of two genetically distinct species within the genus Actinemys as the majority of research on this clade was done before the discovery of a second species. Here, I analyzed the nicotinamide adenine dehydrogenase subunit four (ND4) mitochondrial gene to identify species and geographic origin for 133 captive pond turtles including 71 members of A. marmorata and 62 A. pallida individuals. Results of this study were used to inform captive breeding program collaborators so that husbandry is managed in consideration of species, geographic origin, and the subsequent risks of outbreeding and inbreeding depression. Rachel L. Lopez December 2019 USE OF DNA SEQUENCING TO IDENTIFY THE ORIGIN OF NORTHWESTERN AND SOUTHWESTERN POND TURTLES IN CAPTIVE BREEDING PROGRAMS by Rachel L. Lopez A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology in the College of Science and Mathematics California State University, Fresno December 2019 APPROVED For the Department of Biology: We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree. Rachel L. Lopez Thesis Author Joshua Reece (Chair) Biology Rory Telemeco Biology Rodney Olsen Biology For the University Graduate Committee: Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION OF MASTER’S THESIS X I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship. Permission to reproduce this thesis in part or in its entirety must be obtained from me. Signature of thesis author: ACKNOWLEDGMENTS I would like to sincerely thank my primary advisor, Dr. Joshua Reece, for his continuous support, instruction, and constructive criticism throughout the final year of my undergraduate program and my entire graduate program. I would like to thank Rodney Olsen as a friend, dedicated instructor, and committee member for the direction, instruction, and advice he provided me as an undergraduate student, and his continuous support and constructive comments throughout my graduate program. I would like to thank Dr. Rory Telemeco for his advice and constructive comments throughout the development and completion of this project, and his inspiration as a passionate instructor and herpetologist. I would like to thank Mark Halvorsen and Scott Barton for their contribution to the initiation and continuation of this project. Zoos and all other animal holding facilities that participated in this project were recruited through the Director of Conservation at San Francisco Zoo, Jessie Bushell, who has made an incredible contribution to conservation through her work with the Association of Zoos and Aquariums. I would like to thank collaborators and wildlife managers at each of the following animal holding facilities that participated in this project: The Fresno Chaffee Zoo, Micke Grove Zoo, the Woodland Park Zoo, the Washington Department of Fish and Wildlife, the Oregon Zoo, the High Desert Museum, the Detroit Zoological Society, the Sequoia Park Zoo, Turtle Bay Exploration Park, the Sacramento Zoo, Lindsay Wildlife Experience, the Oakland Zoo, the California Living Museum, the Aquarium of the Bay, Randall Museum, the San Francisco Zoo, Curi Odyssey, the Palo Alto Junior Museum, the Monterey Bay Aquarium, the Santa Barbara Zoo, the California Science Center, the Santa Ana Zoo, the Living Desert, and the San Diego Zoo. Funding for this study was provided through the Graduate Net Initiative Fellowship Award, the Tokalon Alumnae Award, the Graduate Research and Creative Activity Support Award, and the Financial Support for Student Research Award through California State University Fresno. I would v v like to thank all of the students in the Reece Lab who participated in this project and supported me throughout the process. I would like to specifically thank Chris Jorgensen and Shelby Moshier for their continuous support, encouragement, and assistance throughout the development and completion of this project. My family was a crucial support in my education. I would like to thank my husband, Jakob Lopez, and my immediate family, David, Marta, and Emily Morrow for helping me to discover and pursue my passions throughout every stage of my life. I am grateful for all of the incredible professors that have helped me through my undergraduate and graduate degrees, and would like to thank Hawkins Dowis for his support and encouragement throughout the years that I have known him and had the privilege of assisting him and Rodney Olsen in instruction at Fresno City College. I would also like to thank Mark Schreiber for his assistance and advice throughout the development and completion of this project. TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix INTRODUCTION .............................................................................................................. 1 LITERATURE REVIEW ................................................................................................... 6 Species Classification ................................................................................................. 6 Native Range ............................................................................................................... 7 Natural History and Conservation Efforts .................................................................. 8 Conservation Status .................................................................................................. 10 PROJECT DESIGN .......................................................................................................... 12 Sample Collection ..................................................................................................... 12 Mitochondrial DNA Extraction, Amplification, and Isolation ................................. 12 Gene Sequencing ...................................................................................................... 14 Species and Geographic Origin Inference ................................................................ 14 RESULTS ......................................................................................................................... 16 Sample Collection ..................................................................................................... 16 Mitochondrial DNA Data and Clade Membership ................................................... 16 DISCUSSION ................................................................................................................... 23 CONCLUSION ................................................................................................................. 27 REFERENCES ................................................................................................................. 28 APPENDICES .................................................................................................................. 37 APPENDIX A: INFERRED SPECIES AND GEOGRAPHIC ORIGIN FOR INDIVIDUALS HELD IN ANIMAL HOLDING FACILITIES PARTICIPATING IN THE WESTERN POND TURTLE SSP SUSTAINABILITY PROJECT ............................................................................ 38 vii vii Page APPENDIX B: HAPLOTYPES OF MEMBERS OF ACTINEMYS MARMORATA (BEGINNING WITH AM) AND MEMBERS OF ACTINEMYS PALLIDA (BEGINNING WITH AP) .................................................................................... 48 APPENDIX C: NUMBER OF CAPTIVE MEMBERS OF ACTINEMYS MARMORATA AND ACTINEMYS PALLIDA IN EACH COLLABORATING ANIMAL HOLDING FACILITY IN RELATION TO NATIVE DISTRIBUTION OF EACH SPECIES FROM NORTHWEST WASHINGTON TO BAJA CALIFORNIA ...................................................................................................... 50 APPENDIX D: ANIMAL HOLDING FACILITIES PARTICIPATING IN THE