University of Texas at Tyler Scholar Works at UT Tyler Biology Theses Biology Spring 4-30-2012 Molecular Phylogenetics and Phylogeography of the American Box Turtles (Terrapene SPP.) Bradley T. Martin Follow this and additional works at: https://scholarworks.uttyler.edu/biology_grad Part of the Biology Commons Recommended Citation Martin, Bradley T., "Molecular Phylogenetics and Phylogeography of the American Box Turtles (Terrapene SPP.)" (2012). Biology Theses. Paper 15. http://hdl.handle.net/10950/74 This Thesis is brought to you for free and open access by the Biology at Scholar Works at UT Tyler. It has been accepted for inclusion in Biology Theses by an authorized administrator of Scholar Works at UT Tyler. For more information, please contact [email protected]. MOLECULAR PHYLOGENETICS AND PHYLOGEOGRAPHY OF THE AMERICAN BOX TURTLES (TERRAPENE SPP.) by BRADLEY T. MARTIN A thesis/dissertation submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Biology John S. Placyk, Jr., Ph.D., Committee Chair College of Arts and Sciences The University of Texas at Tyler May 2012 The University of Texas at Tyler Tyler, TX This is to certifu that the Master's Thesis/Doctoral Dissertation of BRADLEY T. MARTIN has been approved for the thesisidissertation requirement on April4,2012 for the Master of Science degree Approvals: Thesis/Dissertation C 'ember: James Chair, Department tlbhrS'o.- D.un r Acknowledgements: I wish to give special thanks to my family for supporting and encouraging me in my academic and life pursuits. It is without a doubt that I would not have made it this far without their support. My brother, Charley Martin, has always been and always will be my best friend. He has kept me sane throughout my academic career by being an amazing person to talk to and an exceptional brother and friend. My parents, Ben and Karla Martin, are the kindest and most understanding, supportive, encouraging, and loving parents imaginable. I also wish to give special thanks to my advisor, John S. Placyk, Jr., for taking me on as his graduate student and always being there when I needed him. Dr. Placyk is an exceptional advisor and person, and he has always been someone I could talk to, whether it be academic or personal matters. He has put forth more than his fair share of effort to support me in my academic endeavors, and I will always consider him a close friend. I wish to give special thanks to the undergraduate and graduate students with whom I have become close friends with during my tenure at the UT-Tyler. Their friendship and support means the world to me. I would like to give special thanks to my committee members for their advice, support, and time spent working with me on my M.S. thesis. Finally, this project would not have been possible without the countless volunteers, institutions, universities, wildlife organizations, and museums throughout much of the United States who kindly provided tissue samples for me so that the scientific community can better understand the genetics of the American box turtles and protect them from further population declines. Table of Contents List of Tables ..................................................................................................................... iii List of Figures ..................................................................................................................... v Abstract .............................................................................................................................. vi Chapter One ........................................................................................................................ 1 Introduction ............................................................................................................. 1 Current classification.................................................................................. 2 Problems with the current Terrapene classification ................................... 3 What is a subspecies? ................................................................................. 4 Conservation implications from resolving Terrapene classification .......... 5 Importance of mitochondrial and nuclear DNA in phylogenetic analyses . 7 Phylogeography .......................................................................................... 8 Summary ..................................................................................................... 9 Objectives .................................................................................................. 10 Literature Cited ..................................................................................................... 10 Chapter Two ..................................................................................................................... 16 Introduction ........................................................................................................... 16 Materials and Methods .......................................................................................... 19 DNA extractions, PCR, and sequencing ................................................... 20 Sequence analysis and phylogenetic inference ......................................... 22 Combined data .......................................................................................... 24 Haplotype networks .................................................................................. 24 AMOVA and SAMOVA analyses ............................................................... 25 A priori AMOVAs ...................................................................................... 26 A posteriori AMOVAs ............................................................................... 27 Cytb molecular clock analysis .................................................................. 28 Results ................................................................................................................... 29 Cytb phylogenetic analysis ....................................................................... 29 T. c. carolina - T. c. triunguis and their associated taxa .......................... 30 T. c. major - T. coahuila ........................................................................... 31 Cytb western clade .................................................................................... 31 GAPD phylogenetic analysis .................................................................... 32 T. c. carolina - T. c. triunguis ................................................................... 32 i Taxa associated with T. c. carolina .......................................................... 33 GAPD western clade ................................................................................. 33 Combined mtDNA and nucDNA phylogeny .............................................. 33 Haplotype networks .................................................................................. 34 Population structure ................................................................................. 35 Molecular clock analysis .......................................................................... 35 Discussion ............................................................................................................. 36 Phylogenetic analyses ............................................................................... 36 Eastern clade ............................................................................................ 37 Western clade ............................................................................................ 37 Combined mtDNA and nucDNA data ....................................................... 38 Polyphyly................................................................................................... 39 Haplotype networks .................................................................................. 41 Population structure ................................................................................. 42 T. carolina population structure ............................................................... 42 SAMOVA analyses .................................................................................... 43 Molecular clock ........................................................................................ 44 T. carolina ................................................................................................. 45 T. c. bauri and T. c. major ........................................................................ 45 T. c. triunguis, T. c. mexicana, and T. c. yucatana ................................... 46 Comparisons with published divergence estimates .................................. 47 Percent divergences .................................................................................. 47 Recommendations for classification revisions .......................................... 48 Conservation implications ........................................................................ 50 Literature Cited ..................................................................................................... 51 Appendices.........................................................................................................................91 Appendix A: A supplementary table listing sampling information included in the analyses for this study..........................................................................91 Appendix B: Haplotype designations for