Searching for a Salamander: Distribution and Habitat of the Common Mudpuppy (Necturus maculosus) in Southeast Ohio Using Environmental DNA A thesis presented to the faculty of the Voinovich School of Leadership and Public Affairs of Ohio University In partial fulfillment of the requirements for the degree Master of Science Merri K. Collins August 2017 © 2017 Merri K. Collins. All Rights Reserved. 2 This thesis titled Searching for a Salamander: Distribution and Habitat of the Common Mudpuppy (Necturus maculosus) in Southeast Ohio Using Environmental DNA by MERRI K. COLLINS has been approved for the Program of Environmental Studies and the Voinovich School of Leadership and Public Affairs by Shawn R. Kuchta Associate Professor of Biological Sciences Mark Weinberg Dean, Voinovich School of Leadership and Public Affairs 3 ABSTRACT COLLINS MERRI K., M.S., August 2017, Environmental Studies Searching for a Salamander: Distribution and Habitat of the Common Mudpuppy (Necturus maculosus) in Southeast Ohio Using Environmental DNA Director of Thesis: Shawn R. Kuchta Habitat destruction and anthropogenic drivers have led to a decline of amphibian populations worldwide, but the conservation status of many species remains in question. Environmental DNA is a new monitoring methodology that non-invasively detects the presence of imperiled, rare, and secretive species. Although the use of environmental DNA (eDNA) to detect species presence is increasing, it is not often paired with habitat data. This study focuses on the declining Common Mudpuppy salamander, Necturus maculosus. I conducted both traditional and eDNA field surveys at 10 stream sites located in Southeastern Ohio. I detected the presence of Mudpuppies at 6 of 10 streams using eDNA. In contrast, I only observed individuals at one site using stream surveys Presence was detected in fourhistoric streams, and established at two new locations. I quantified physical and chemical habitat characteristics over six months for each stream using water quality surveys. I recorded measurements of heavy metals, nutrients physical stream habitat, conductivity, pH, temperature, total dissolved solids, and oxygen. Logistic regression analysis determined that QHEI (Qualitative Habitat Evaluation Index) scores for riparian zone was the best predictor of mudpuppy presence. This sampling methodology could be broadly applied to study other aquatic species. 4 DEDICATION This thesis is dedicated to my late grandparents, Russell and Betty Wilkinson, who instilled empathy and compassion in my heart for all wild things, and to my mother, Beverly, for her support and strength that knows no bounds. You are my inspiration. This one’s for you. 5 ACKNOWLEDGMENTS I would first like to thank Crane Hollow for their generous support that made this research possible, for continuing to support the research goals of Ohio University students, and for protecting the beautiful and unique habitat within the preserve. It is truly a remarkable place. A huge thanks to Stephen Spear for his incredible advice and guidance, without you this thesis would be incomplete. To Shawn, for your kindness in taking on a student with little experience, your positivity along the way, and for the blind faith you put in me. To Denim, for her warm and welcoming friendship. To my MSES cohort, I could not have imagined a better group of friends, cheers to you. To my mother, for always believing in me. To my brother for exploring the woods with me as a child and being my partner in adventure, that sense of adventure never died. To my lab mates for your encouragement. To Bill Broach for being a great lab teacher, and Erin Murphy for allowing me to use her equipment. To my field help, those who taught me protocol in the lab, and who edited my work with heart and patience. To the countless friends that I have made on this journey who kept me laughing. To my committee members Natalie and Nancy who pointed me in a direction I never thought I would (or could) go. To everyone who believed in me, thank you all, I would have never succeeded without you. 6 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 4 Acknowledgments............................................................................................................... 5 List of Tables ...................................................................................................................... 7 List of Figures ..................................................................................................................... 8 Chapter 1: Introduction ....................................................................................................... 9 Chapter 2: Materials ad Methods ...................................................................................... 15 Chapter 3: Results ............................................................................................................. 49 Chapter 4: Discussion ....................................................................................................... 61 Literature Cited ................................................................................................................. 69 Appendix A: Myron Results ............................................................................................. 76 7 LIST OF TABLES Page Table 1 Crane Creek eDNA ............................................................................................. 24 Table 2 Pine Creek eDNA ............................................................................................... 27 Table 3 Clear Creek eDNA .............................................................................................. 30 Table 4 East Fork Queer Creek eDNA ............................................................................ 32 Table 5 Raccoon Creek eDNA ........................................................................................ 34 Table 6 Heweet-Fork eDNA ............................................................................................ 36 Table 7 Leading Creek EPA 1993 ................................................................................... 39 Table 8 Leading Creek eDNA ......................................................................................... 39 Table 9 Forked-Run eDNA .............................................................................................. 42 Table 10 Hocking eDNA ................................................................................................. 44 Table 11 Primer and Probe .............................................................................................. 47 Table 12 QHEI Scores ..................................................................................................... 50 Table 13 Contaminants .................................................................................................... 51 Table 14 Nutrients............................................................................................................ 53 Table 15 Mudpuppy Measurements................................................................................. 53 Table 16 DNA Quantity ................................................................................................... 55 Table 17 Logistic Regression........................................................................................... 58 8 LIST OF FIGURES Page Figure 1. eDNA Sites in southeast Ohio. .......................................................................... 21 Figure 2. Crane Creek eDNA............................................................................................ 23 Figure 3. Pine Creek eDNA. ............................................................................................. 26 Figure 4. Clear Creek eDNA ............................................................................................ 29 Figure 5. East Fork Queer Creek eDNA. .......................................................................... 31 Figure 6. Raccoon Creek eDNA ....................................................................................... 33 Figure 7. Hewett-Fork eDNA. .......................................................................................... 35 Figure 8. Leading Creek eDNA ........................................................................................ 38 Figure 9. Forked-Run eDNA. ........................................................................................... 41 Figure 10. Hocking eDNA ................................................................................................ 43 Figure 11. Mudpuppy Egg Nest ........................................................................................ 55 Figure 12. Riparin Zone Score Spline............................................................................... 59 Figure 13. Pearson’s 1....................................................................................................... 59 Figure 14. Pearson’s 2....................................................................................................... 60 Figure 15. Pearson’s 3....................................................................................................... 60 9 CHAPTER 1: INTRODUCTION Globally, even in protected areas amphibian