POPULATION GENETICS AND CONSERVATION OF THE FLORIDA MANATEE: PAST, PRESENT, AND FUTURE By ROBERT KNUDSEN BONDE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1 © 2009 Robert Knudsen Bonde 2 To the perpetuation of mermaid myths 3 ACKNOWLEDGMENTS I would like to thank my committee, especially Dr. Peter McGuire whose ideas, guidance, and direction were a major contributor for all our present day understanding of sirenian genetics. It was through his unselfish determination and fortitude that all of his enthusiastic graduate students, me included, found their strengths. Dr. Roger Reep was the guiding light that led me into graduate school so many years after establishing my career with the Sirenia Project. Roger’s encouragement, perseverance, and compassion helped mold my personality and way of approaching problems too. Dr. Lynn Lefebvre was my mentor and direct-line supervisor for much of my professional career with U. S. Geological Survey (USGS) Sirenia Project. Through her support, I was able to juggle the responsibilities of my job with those required of my academic career. Dr. Tim King of the USGS Leetown Science Center whose expertise on population genetics is rivaled by few in the world; Tim your guidance and advice are ingrained and treasured by all immersed in wildlife genetics today. Finally, Dr. Ruth Francis-Floyd, who joined my committee in the later stages and still provided upbeat enthusiasm, even when times appeared discouraging. Thanks to all. Of particular importance to me and my career is Dr. Maggie Kellogg Hunter (fellow graduate student) who guided me through the final stages of my work and who will help lead future genetic efforts at the USGS Sirenia Project. Other stellar graduate students from UF who shared their knowledge are Dr. Angela Garcia-Rodriguez who designed the first studies and thinking relative to trichechid biogeography and laid the foundation for development of Florida manatee population genetics. Thanks to Dr. Kim Pause Tucker (University of South Florida) who carried the torch further and helped establish our present day understanding of Florida manatee genetics. None of these studies would have been possible without the knowledge and tutoring provided by AnnMarie “Ginger” Clark of the University of Florida (UF) 4 Interdisciplinary Center for Biotechnology Research genetics lab. Ginger is a great friend and resource that we have all become accustomed to use during times of uncertainty. Fellow student, Coralie Nourisson, who is conducting similar studies on manatee genetics in Mexico, also is thanked. Good luck with your research Coralie. I also thank Marta Rodriguez-Lopez from Puerto Rico who developed a pioneering genetic study on Antillean manatees a number of years ago. Dr. Juliana Vianna was also very helpful in sharing her study of manatee populations from throughout their range using mtDNA and microsatellite analysis in Brazil. The critical thinking about evolution by Dr. Daryl Domning of Howard University, who was responsible for much of the formulation leading to the marriage (disparities) between paleontology and genetics, is greatly appreciated. International researchers that have aided the sirenian genetics programs with valuable sample collection include Nicole Auil, Belize; Dr. Miriam Marmontel, Brazil; Dr. Antonio Mignucci-Giannoni, Puerto Rico; Drs. Janet Lanyon and Helene Marsh, Australia; and Dr. Benjamin Morales-Vela, Mexico. A sincere thanks to my colleague and dear friend, Dr. James “Buddy” Powell with Sea to Shore Alliance. All the manatee rescue teams and rehabilitation facilities also are owed a great deal of gratitude for assistance with genetic sample collection, as well as Kit Curtin for her efforts in South Florida. Federal research permits and encouragement was extended through many of our colleagues in management, especially the U.S. Fish and Wildlife Service. Particular thanks are extended to Monica Farris, Nicole Adimey, Dawn Jennings, and Jim Valade (thanks Jim, who purchased my first PCR machine which is still in use today), as well as all the members of the Coordinated Florida Manatee Genetics Working Group. This project would not have been possible without the foresight and stalwart efforts of many dedicated people, especially the staff of friends and colleagues at the USGS, Sirenia 5 Project. Cathy Beck, Jim Reid, Susan Butler, Amy Teague, Gaia Meigs-Friend, Howard Kochman, Dr. Cathy “KB” Langtimm, Dr. Tom O’Shea, Dr. Galen Rathbun, Dr. Lynn Lefebvre, and Dr. Jeff Keay. Thanks to Dr. Kay Briggs (USGS), Dr. Daryl Domning (Howard University), Dr. Bill Farmerie (UF), Sean McCann (UF), and Dr. John Reynolds III (Mote Marine Lab). A special thanks to all our colleagues at the Florida Fish and Wildlife Conservation Commission, especially Michelle Davis, Dr. Charles “Chip” Deutsch, Dr. Martine deWit, Andy Garrett, Tom Pitchford, Dr. Mike Tringali, and Leslie Ward-Geiger. A special thanks to my wife, best friend, and companion during our journey with the manatees, Cathy Beck, and to our children, Michael and Julie, that through their eyes we hope to be able to continue to carry on the pursuit of science and understanding. 6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF FIGURES .......................................................................................................................11 LIST OF ABBREVIATIONS ........................................................................................................13 Organizations ..........................................................................................................................13 General Abbreviations ............................................................................................................13 Laboratory ...............................................................................................................................14 Genetics ..................................................................................................................................14 Genetic Analyses ....................................................................................................................15 ABSTRACT ...................................................................................................................................17 CHAPTER 1 INTRODUCTION ..................................................................................................................19 Project Summary ....................................................................................................................19 The Manatee ...........................................................................................................................20 Sirenian Evolution ...........................................................................................................20 Florida Manatee Biology .................................................................................................21 Overview of Manatee Status, Research, and Management ....................................................24 Why Do We Need to Study Manatee Genetics? .....................................................................29 2 TRICHECHID EVOLUTION ................................................................................................35 Systematic Characters .............................................................................................................35 Manatee Evolution ..................................................................................................................36 Population Expansion .............................................................................................................41 Molecular Status .....................................................................................................................44 Extinction Processes ...............................................................................................................45 3 CRYSTAL RIVER MANATEE FINE-SCALE Population ANALYSIS ..............................53 Introduction .............................................................................................................................53 Materials and Methods ...........................................................................................................58 Sample Collection and DNA Extraction .........................................................................58 DNA Isolation .................................................................................................................58 Microsatellite Analysis ....................................................................................................58 Statistical Analysis ..........................................................................................................59 Neighbor-joining trees ..............................................................................................60 Analysis using STRUCTURE ......................................................................................61 Multivariate analysis ................................................................................................61 7 Results .....................................................................................................................................62