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Distribution and Conservation Genetics of the Cow Knob Salamander, Plethodon Punctatus Highton (Caudata: Plethodontidae) Matthew R

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Distribution and Conservation Genetics of the Cow Knob Salamander, Plethodon Punctatus Highton (Caudata: Plethodontidae) Matthew R Marshall University Marshall Digital Scholar Theses, Dissertations and Capstones 1-1-2007 Distribution and Conservation Genetics of the Cow Knob Salamander, Plethodon punctatus Highton (Caudata: Plethodontidae) Matthew R. Graham [email protected] Follow this and additional works at: http://mds.marshall.edu/etd Part of the Cell Biology Commons, Other Animal Sciences Commons, and the Population Biology Commons Recommended Citation Graham, Matthew R., "Distribution and Conservation Genetics of the Cow Knob Salamander, Plethodon punctatus Highton (Caudata: Plethodontidae)" (2007). Theses, Dissertations and Capstones. Paper 174. This Thesis is brought to you for free and open access by Marshall Digital Scholar. It has been accepted for inclusion in Theses, Dissertations and Capstones by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected]. Distribution and Conservation Genetics of the Cow Knob Salamander, Plethodon punctatus Highton (Caudata: Plethodontidae) Thesis submitted to The Graduate College of Marshall University In partial fulfillment of the Requirements for the degree Master of Science Biological Sciences by Matthew R. Graham Thomas K. Pauley, Committee Chairman Victor Fet, Committee Member Guo-Zhang Zhu, Committee Member April 29, 2007 ii Distribution and Conservation Genetics of the Cow Knob Salamander, Plethodon punctatus Highton (Caudata: Plethodontidae) MATTHEW R. GRAHAM Department of Biological Sciences, Marshall University Huntington, West Virginia 25755-2510, USA email: [email protected] Summary Being lungless, plethodontid salamanders respire through their skin and are especially sensitive to environmental disturbances. Habitat fragmentation, low abundance, extreme habitat requirements, and a narrow distribution of less than 70 miles in length, makes one such salamander, Plethodon punctatus, a species of concern (S1) in West Virginia. To better understand this sensitive species, day and night survey hikes were conducted through ideal habitat and coordinate data as well as tail tips (10 to 20 mm in length) were collected. DNA was extracted from the tail tips and polymerase chain reaction (PCR) was used to amplify mitochondrial 16S rRNA gene fragments. Maximum parsimony, neighbor-joining, and UPGMA algorithms were used to produce phylogenetic haplotype trees, rooted with P. wehrlei. Based on our DNA sequence data, four disparate management units are designated. Surveys revealed new records on Jack Mountain, a disjunct population that expands the known distribution of the species 10 miles west. In addition, surveys by Flint verified a population on Nathaniel Mountain, WV and revealed new records on Elliot Knob, extending the known range several miles south. DNA sequencing of 24 individuals revealed 8 haplotypes. 16 individuals from the main population on Shenandoah Mountain all had the same haplotype, suggesting low genetic variability. Conversely, each individual from all other areas possessed a unique haplotype. Most importantly, a haplotype from Nathaniel Mountain, WV was deeply divergent and has probably been isolated since the early Pleistocene, making the population a conservation priority. It is hoped that this new genetic data will increase the efficacy of Cow Knob salamander conservation efforts by providing the means to implement management plans that conserve intraspecific genetic diversity. iii “Nature uses only the longest threads to weave her patterns, so each small piece of her fabric reveals the organization of the entire tapestry.” Richard Feynman The Character of Physical Law (1965) “When we begin to plan how to use a piece of Appalachia, we must set out from the start to save the full complement of native species. Nothing less will do.” George Constantz Hollows, Peepers & Highlanders (2004) iv DEDICATIONȱ This work is dedicated to my parents, George and Penny Graham, who have remained supportive of my sometimes crazy ventures in academia. Whether it was collecting venomous scorpions from cactus-littered desert floors, cross-country trips in exponentially colder weather than they’re used to, or listening to me whine about an organic chemistry exam, they stood by. Mom and Dad, I couldn’t have done it without you. This thesis is dedicated, with love, to you. v ACKNOWLEDGEMENTSȱ First of all I want to thank Dr. Thomas Pauley for the opportunity to be a part of the legacy that is his lab. Dr. Pauley’s knowledge of herpetology, conservation, and overall biology contributed greatly to this thesis and to me as a scientist. Next, I wish to thank Dr. Victor Fet. As an undergraduate, I received an invitation to come study with him at Marshall. Before I knew it, I was flying across the country, naive but ready for something new, and landed in Huntington, WV during a bitter cold January still daunting my beach clothes. Dr. Fet patiently taught me genetics skills that were all so important in formulating, constructing, and carrying out this thesis. Although, I deviated from Dr. Fet’s main area of interest (scorpions), he held no objection when I took a new route and entered the slimy world of salamander biology. Dr. Fet, more than anyone, has influenced my ideas and shaped me into the well-rounded biologist that I am today, and “I look forward to our future collaborations.” I want to thank Dr. Guo-Zhang Zhu for friendly advice regarding molecular techniques and for kindly serving on my committee. Billy Flint of James Madison University provided helpful Cow Knob salamander expertise and contributed tremendously to this project. I owe Billy much gratitude for his enthusiasm and willingness to collaborate, and for many hours of hard work surveying and collecting tail tips. While doing field surveys in the remote mountainous corners of West Virginia, I was fortunate enough to enjoy the company of several assistants. Josh Greenwood, one of my greatest friends in WV, spent many hours camping and hiking to isolated salamander locations with me. Ashley Fisher nearly froze to death on an icy cold and rainy day (perfect salamander conditions) while collecting Cow Knob salamanders on Shenandoah Mountain. My girlfriend Jessica Casto, and our trusty sidekick Fajita, braved the West Virginia woods to assist me as well. Jessica and Fajita also kept me sane during the long hours spent writing this thesis and all the associated papers that go with a project like this. I also owe thanks to Jessica’s parents, Vernon and Cindy Casto for vi graciously letting us use their beautiful cabin in Randolph County, one heck of a field station. Ally Hogsett, contributed a lot of time meticulously carrying out many of the DNA extractions, amplifications, and purifications. Mike Brewer and Cassie York kept me up-to-date on the utility of Dr. Fet’s DNA lab. Mike, a molecular master, always had valuable advice on molecular techniques and problem solving when things went wrong. My friend and roommate Conor Keitzer engaged me in interesting conversation and brainstorming that ultimately contributed significantly to this study. Most of the funding from this project was provided by the West Virginia Division of Natural Resources. Additional support was provided by a gracious donation to the graduate college by Dave Haden. Thank you Dave, your kind contribution made my life a lot easier while working in the field. Last but not least, many thanks of course go out to the Marshall University Herpetology lab. vii Table of Contents ABSTRACT ....................................................................................................................... ii DEDICATION ................................................................................................................. iv ACKNOWLEDGEMENTS.............................................................................................. v TABLE OF CONTENTS ................................................................................................ vii LIST OF FIGURES.........................................................................................................viii LIST OF TABLES............................................................................................................. ix CHAPTER ONE INTRODUCTION....................................................................................................... 1 CHAPTER TWO BACKGROUND 2.1 Plethodon in West Virginia................................................................................. 6 2.2 The Cow Knob Salamander................................................................................ 8 2.3 Distribution of P. punctatus ............................................................................. 10 2.4 Conservation Units ............................................................................................. 13 CHAPTER THREE METHODS 3.1 Geographic and Taxonomic Sampling ........................................................... 25 3.2 DNA Extraction, Amplification, and Purification........................................ 27 3.3 Phylogenetic Analysis........................................................................................ 29 CHAPTER FOUR RESULTS 4.1 Disribution and Surveys ................................................................................... 35 Graham Surveys..................................................................................................... 35 Flint Surveys........................................................................................................... 38 4.2 Phylogenetics......................................................................................................
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