University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2015 The olecM ular Evolution of Non-Coding DNA and Population Ecology of the Spiny Softshell Turtle (Apalone spinifera) in Lake Champlain Lucas Edward Bernacki University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biology Commons, Genetics and Genomics Commons, and the Natural Resources and Conservation Commons Recommended Citation Bernacki, Lucas Edward, "The oM lecular Evolution of Non-Coding DNA and Population Ecology of the Spiny Softshell Turtle (Apalone spinifera) in Lake Champlain" (2015). Graduate College Dissertations and Theses. 289. https://scholarworks.uvm.edu/graddis/289 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. THE MOLECULAR EVOLUTION OF NON-CODING DNA AND POPULATION ECOLOGY OF THE SPINY SOFTSHELL TURTLE (APALONE SPINIFERA) IN LAKE CHAMPLAIN A Dissertation Presented by Lucas E. Bernacki to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Biology January, 2015 Defense Date: August 29, 2014 Dissertation Examination Committee: C. William Kilpatrick, Ph.D., Advisor J. Ellen Marsden, Ph.D., Chairperson Alison Brody, Ph.D. Lori Stevens, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Spiny softshell turtles (Apalone spinifera) occur at the northwest limit of their range in Lake Champlain. This species, although widespread across North America, is listed as threatened in Vermont due to habitat destruction and disturbances of anthropogenic origin. The population of spiny softshell turtles in Lake Champlain is isolated from other North American populations and is considered as an independent management unit. Efforts to obtain information on the biology of spiny softshell turtles in Lake Champlain precede 1936 with conservation measures being initiated in 1987. Methods of studying spiny softshell turtles in Lake Champlain have included direct observation, mark-recapture, nest beach monitoring, winter diving, and radio telemetry. Each of these approaches has provided some information to the sum of what is known about A. spinifera in Lake Champlain. For example major nesting beaches, hibernacula, and home range size have been determined. Currently spiny softshell turtles primarily inhabit two areas within Lake Champlain, Missisquoi Bay and the mouth of the Lamoille River. However, the population structure and gene flow between spiny softshell turtles inhabiting the Lamoille and Missisquoi regions remained unknown. A GIS model was created and tested in order to identify additional nesting beaches used by spiny softshell turtles along the Vermont shores of Lake Champlain. Although some additional small potential nesting beaches were found, no additional major nesting sites were found. The GIS model identified the mouth of the Winooski River (the site of a historical population) as potentially suitable nesting habitat; however, no evidence of spiny softshell turtle nesting was found at this site. A series of methods developed for collecting molecular and population genetic data about spiny softshell turtles in Lake Champlain are described, including techniques for DNA extraction of various tissue types and the design of new primers for PCR amplification and sequencing of the mitochondrial control region (mtD-loop). Techniques for circumventing problems associated with DNA sequence alignment in regions of a variable numbers of tandem repeats (VNTRs) and the presence of heteroplasmy within some individuals are also described. The mtD-loop was found to be a suitable marker to assess the genetic structure of the Lake Champlain population of spiny softshell turtles. No significant genetic sub- structuring was found (FST=0.082, p=0.223) and an indirect estimate of the migration rate between Lamoille and Missisquoi regions of Lake Champlain was high (Nm>5.576). In addition to consideration of A. spinifera in Lake Champlain, the mtD-loop was modeled across 46 species in 14 families of extant turtles. The primary structure was obtained from DNA sequences accessed from GenBank and secondary structures of the mtD-loop were inferred, (from thermal stabilities) using the program Mfold, for each superfamiliy of turtles. Both primary and secondary structures were found to be highly variable across the order of turtles; however, the inclusion of an AT-rich fold (secondary structure) near the 3’ terminus of the mtD-loop was common across all turtle families considered. The Cryptodira showed conservation in the primary structure at regular conserved sequence blocks (CSBs), but the Pluerodira displayed little conservation in the primary structure of the mtD-loop. Overall, greater conservation in secondary structure than primary structure was observed in turtle mtD-loop. The AT-rich secondary structural element near the 3’ terminus of the mtD-loop may be conserved across turtles due to it serving a functional role during mtDNA transcription. ACKNOWLEDGEMENTS First, I would like to thank Dr. Bill Kilpatrick, my advisor, who has been indispensable during my development as a scientist. He has coaxed, coached and, at times, forced me to be objective and true to the scientific method. I would also like to thank my committee members, Dr. Ellen Marsden, Dr. Lori Stevens, and Dr. Alison Brody, who have patiently guided me through a challenging project over the past several years. Dr. Nick Gotelli has been a valuable teaching mentor. Steve Parren has generously shared his expertise in the field as well as the entire collection of turtle tissue, without which the completion of this dissertation would not have been possible. Dr. JoeSchall kindly allowed me to use his lab and equipment. Additionally, I would like to thank my fellow graduate students Isaac Chellman, Nate Newman, Laura Caputo Newman, Chris Gray, Dr. Laura Farrell, Nelish Pradhan, Dr. Laura Hill, Dr. Heather Axen, and Nabil Nasseri all of whom have aided me during my time as a graduate student in various ways. Finally, I would like to thank my family, all of whom have been immensely supportive. Dr. Bern and Mary Bernacki, my parents, have instilled in me a thirst for knowledge and a devotion to education from an early age. My brothers Dr. Matt Bernacki and Jon Bernacki have offered their individual talents. Matt has shared much of his knowledge and experience as a professor and former graduate student, and Jon has kept me grounded with constructive distractions and our shared appreciation for the outdoors. Katharine Cahoon has been a model of strength, patience, and charity. Katharine’s encouragement and generosity throughout the past few years have been vital to the process of completing my dissertation. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ ii LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii CHAPTER 1. Spiny Softshell Turtle (Apalone spinifera) Life History and GIS Modeling of Nesting Beaches in Lake Champlain, Vermont ..............................................................1 ABSTRACT ....................................................................................................................1 INTRODUCTION ..........................................................................................................1 MATERIALS AND METHODS ...................................................................................6 Orthophotos ..................................................................................................................6 GIS Analysis .................................................................................................................6 Ground-Truthing ..........................................................................................................8 RESULTS .......................................................................................................................8 Orthophotos .................................................................................................................8 GIS Model and Ground-Truthing ................................................................................9 Missisquoi Bay Region .........................................................................................10 Champlain Islands Region ....................................................................................10 River Region .........................................................................................................11 Southernmost Region ............................................................................................12 DISCUSSION ...............................................................................................................12 Winooski River Mouth and Mallets Bay Area ...........................................................13 Lamoille and Missisquoi Area ..................................................................................15