DIETARY ANALYSIS AND CONSERVATION GENETICS OF THE ENDANGERED OZARK BIG-EARED BAT CORYNORHINUS TOWNSENDII INGENS By DANYELLE NICOLE LEE Bachelor of Science in Biology Angelo State University San Angelo, Texas 2007 Master of Science in Biology Angelo State University San Angelo, Texas 2009 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY July, 2013 DIETARY ANALYSIS AND CONSERVATION GENETICS OF THE ENDANGERED OZARK BIG-EARED BAT, CORYNORHINUS TOWNSENDII INGENS Dissertation Approved: Dr. Ronald A. Van Den Bussche Dissertation Advisor Dr. Meredith J. Hamilton Dr. Anthony A. Echelle Dr. Loren K. Ammerman Dr. David M. Leslie, Jr. Outside Committee Member ii ACKNOWLEDGEMENTS I would like to first thank my advisor, Dr. Ronald A. Van Den Bussche. Through the years, he has provided me with opportunities to grow as a student, teacher, researcher, and person. He has allowed me to go through graduate school in a manner which best suited my priorities, and I thank him for that. I am also grateful to Dr. Meredith J. Hamilton, Dr. David M. Leslie, Jr., and Dr. Loren K. Ammerman for their kind words of support whenever I struggled or felt unsure about something. Their encouragement always came at just the time when I needed it the most. I thank Dr. Anthony A. Echelle for editorial comments, which have made me a more concise writer. I have been able to take a variety of courses at Oklahoma State University so I would like to thank the faculty for expanding my interests and increasing my abilities. I appreciate the staff of the Zoology department and Oklahoma Cooperative Fish and Wildlife Research Unit for administrative support for the past four years, the Zoology Graduate Student Society for financial assistance for professional meeting travel, and Dr. Jerry and Winona Wilhm for their contributions to the Zoology Department Teaching Assistant Award fund. In terms of my specific dissertation project, I have many people to acknowledge, and I have done so at the end of the appropriate chapters. I am very appreciative of funding support from Bat Conservation International and the Oklahoma Department of Wildlife Conservation. I am grateful to the many graduate students at Oklahoma State University for their iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. support and companionship. I especially thank my labmates, Justin Lack, Michael Schwemm, Megan Trope, Jeremy Wilkinson, and Craig Stanley for their friendship. We have had many laughs together and I will miss each of them. Finally, I thank my family members and friends for their love and encouraging words. I thank William and Tiffany Westbo and David and Rhonda Cassel for babysitting so that I may have time to write. I most sincerely thank my husband, Richard Dolman. He is my biggest supporter and helped make all my professional and personal dreams come true. I could have never finished this dissertation without him. I love you and our Mason dearly. iv Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: DANYELLE NICOLE LEE Date of Degree: JULY, 2013 Title of Study: DIETARY ANALYSIS AND CONSERVATION GENETICS OF THE ENDANGERED OZARK BIG-EARED BAT, CORYNORHINUS TOWNSENDII INGENS Major Field: ZOOLOGY Abstract: Populations of endangered Ozark big-eared bats (Corynorhinus townsendii ingens) are restricted to 19 essential maternity caves or hibernacula in Oklahoma and Arkansas and total only 1,600–1,800 individuals. Effective conservation and management should encompass many aspects of the species natural history and genetics. This dissertation first presents a list of insect species consumed by Ozark big- eared bats which can be used to better understand habitat selection if influenced by prey availability. Traditionally, diet studies have relied on morphological analysis of prey body parts found in fecal material, but that method can overlook species because key features used in identification might be damaged by chewing, digestion, or decomposition after excretion. I used molecular analysis of cytochrome c oxidase gene from prey DNA found in bat fecal material to identify 20 insect species from nine families and two orders. Fifteen of these were unknown prey items for this taxon. Notodontids were found in the majority of samples (64.3%) and were consumed in greater proportion than their abundance in the environment. The fragmented nature of populations of Ozark big-eared bats make them susceptible to reduced levels of genetic variation, inbreeding, or fixation of deleterious alleles from genetic drift. For my second chapter, I developed 15 polymorphic nuclear microsatellites to be used in the third chapter with mitochondrial D-loop DNA sequence to characterize the population genetics of these bats and determine conservation units. Molecular data suggested that genetic mixing is occurring at swarming sites among geographically close colonies within Oklahoma but does not include an Arkansas population. I recommend colonies in eastern Oklahoma and western Arkansas be managed as two separate units to protect unique alleles but with protection of surrounding habitat to facilitate low levels of gene flow. Additional genetic analysis of isolated populations in Marion Co., Arkansas is warranted because they may be a separate management unit. Finally, comparison of genetic characteristics of current populations to those 10 years earlier suggested considerable change in gene frequencies, likely as a result of genetic drift. Decreases in genetic variation could potentially affect popoulaitons of Ozark big-eared bats so I strongly recommend continued genetic monitoring. v TABLE OF CONTENTS Chapter Page I. MOLECULAR ANALYSIS REVEALS NEW PREY ITEMS FOR ENDANGERED OZARK BIG-EARED BATS (CORYNORHINUS TOWNSENDII INGENS).........1 Abstract ....................................................................................................................1 Introduction ..............................................................................................................2 Methods....................................................................................................................5 Study site description .........................................................................................5 Sample collection and DNA isolation................................................................5 Identification of bat species ...............................................................................6 Identification of prey species .............................................................................7 Selection of families ........................................................................................10 Results ....................................................................................................................10 Discussion ..............................................................................................................12 Acknowledgements ................................................................................................19 References ..............................................................................................................19 Figure Caption .......................................................................................................28 II. DEVELOPMENT AND CHARACTERIZATION OF 15 POLYMORPHIC TETRANUCLEOTDIE MICROSATELLITE LOCI FOR TOWNSEND’S BIG- EARED BAT (CORYNORHINUS TOWNSENDII) AND CROSS AMPLIFICATION IN RAFINESQUE’S BIG-EARED BAT (CORYNORHINUS RAFINESQUII) ...31 Abstract ..................................................................................................................31 Introduction ............................................................................................................32 Methods..................................................................................................................33 Results ....................................................................................................................34 Discussion ..............................................................................................................35 Acknowledgements ................................................................................................36 References ..............................................................................................................36 III. IDENTIFICATION OF CONSERVATION UNITS AND TEMPORAL VARIATION IN ENDANGERED OZARK BIG-EARED BATS (CORYNORHINUS TOWNSENDII INGENS) .......................................................................................46 Abstract ..................................................................................................................46 vi Chapter Page Introduction ............................................................................................................47 Methods..................................................................................................................51 Current Population Characteristics ..................................................................51 Sampling ....................................................................................................51 Mitochondrial sequencing ..........................................................................51 Microsatellite genotyping