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The Detection and Quantification of Aquatic Reptilian Environmental DNA Clare Isabel Ming-Ch'eng Adams Iowa State University

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The Detection and Quantification of Aquatic Reptilian Environmental DNA Clare Isabel Ming-Ch'eng Adams Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2017 The detection and quantification of aquatic reptilian environmental DNA Clare Isabel Ming-Ch'eng Adams Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biodiversity Commons, Ecology and Evolutionary Biology Commons, Genetics Commons, Natural Resources and Conservation Commons, and the Natural Resources Management and Policy Commons Recommended Citation Adams, Clare Isabel Ming-Ch'eng, "The detection and quantification of aquatic reptilian environmental DNA" (2017). Graduate Theses and Dissertations. 15481. https://lib.dr.iastate.edu/etd/15481 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The detection and quantification of aquatic reptilian environmental DNA by Clare Isabel Ming-ch’eng Adams A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Ecology and Evolutionary Biology Program of Study Committee: Fredric Janzen, Major Professor Julie Blanchong John Nason Kevin Roe Michelle Soupir Iowa State University Ames, Iowa 2017 Copyright © Clare Isabel Ming-ch’eng Adams, 2017. All rights reserved. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ......................................................................................... iv ABSTRACT ............................................................................................................... vi CHAPTER 1. INTRODUCTION .......................................................................... 1 CHAPTER 2. COMPARISON OF PAINTED TURTLE (CHRYSEMYS PICTA) ENVIRONMENTAL DNA EXTRACTION TECHNIQUES IN A LENTIC POND SYSTEM ......................................................................................................... 17 Introduction ......................................................................................................... 18 Materials and Methods ......................................................................................... 21 Results ......................................................................................................... 25 Discussion ......................................................................................................... 27 CHAPTER 3. ESTIMATING AQUATIC REPTILE DENSITY UNDER FIELD CONDITIONS USING ENVIRONMENTAL DNA................................................. 49 Introduction ......................................................................................................... 50 Materials and Methods ......................................................................................... 52 Results ......................................................................................................... 57 Discussion ......................................................................................................... 60 CHAPTER 4. SUMMARY AND CONCLUSIONS ............................................. 88 iii ACKNOWLEDGMENTS I would like to thank my major professor, Fredric Janzen, and my committee members, Julie Blanchong, John Nason, Kevin Roe, and Michelle Soupir for their guidance and support throughout the course of this research. Fred, I thank you for giving me the chance and support to become a scientist. I hope to one day view science as you do and develop the skills to see the “big picture” in turtle research, ecology, evolutionary biology, and life. Secondly, I want to thank Julie for being my Preparing Future Faculty mentor and taking the time to sit down and painstakingly work on writing with me. Without your generosity I wouldn’t be where I am now. Lastly, I want to thank Dr. Reg for being my diversity mentor. I arrived here not knowing how to navigate this space and you are the reason I am still here and able to complete my degree. I will miss our chats and your one- liners that helped me get through the day, week, and years. In addition, I would also like to thank the Janzen Lab. Luke, you are way too kind to me and overly generous with your time. These projects would not be where they are currently without your input, guidance, and help. Rebecca, thank you for looking out for me in the lab. I appreciate that you took me under your wing and helped me navigate the Janzone. Morgan, you are the best undergrad I have ever had. I cannot wait to see how you grow. I would be nowhere without my friends, especially Andrew and Jermaine. Andrew, thanks for putting up with me for a whole entire year and being an ever-supportive friend. I miss our late-night chats about life overlooking the “skyline” of Ames. Jermaine, words cannot describe how much you mean to me. Thanks for always having my back, yo. You are my emotional lighthouse in the dark. Tori, Monica, Amy G, Hilary – It has been fantastic getting to know iv you and I wish you the best. I wish our time together had been longer, but on to the next great adventure. Thank you Jack Gallup, the department faculty, and staff for making time and space for me at Iowa State University. Jack, I wish we had met sooner. Additionally, I want to thank my mom and dad for supporting me all the way, especially when times have been tough. Your ability to adapt to my needs is amazing and I am super thankful I got you as my parents. Zach, Kiran, Bradley, thank you for being my oasis whenever I went back to the East Coast. I miss you so much. I also want to offer my appreciation to turtles, without whom, this thesis would not have been possible. Too often we forget that how we treat nature, in your case – turtles, is a reflection upon humanity itself. Turtles are the best! v ABSTRACT Monitoring individuals of a population is aided by sampling techniques for determining organism presence. However, invasive sampling methods may harm target and non-target individuals, not capture a fully representative population demographic, or may be difficult to use due to secretive and seasonally active species. Recent developments in non- invasive technology propose environmental DNA (eDNA) as a solution to mitigate some of these challenges. Environmental DNA is DNA captured from target organisms that is extracted from environmental samples such as water, soil, or air. Although this technique has been widely explored for fish and amphibian species, it is used less often for aquatic reptiles. This thesis attempts to create an eDNA methodology for an imperiled reptilian taxa, turtles, for future monitoring use. We set up four experimental ponds with varying numbers of turtles (0, 11, 23, 38) and sampled once every three days throughout the spring field season to determine effects of painted turtle (Chrysemys picta) density and time on eDNA technique utility. The first chapter compares two common eDNA methodologies, filtration and sodium acetate precipitation in a Midwestern lentic semi-natural environment. The second chapter uses the more efficient filtration to quantify eDNA based upon turtle abundance in the same environment. We conclude that eDNA may not currently be an effective monitoring method for aquatic turtles. Overall, filtration was a more effective approach to capturing turtle eDNA than sodium acetate precipitation but visual surveys of turtles in our experimental setup led to an even higher rate of detection. Despite developing a sensitive qPCR protocol, we were unable to amplify turtle eDNA sufficiently to distinguish it from the negative control. We vi nonetheless identified a rank-order trend positively correlated with turtle density despite not obtaining large amounts of species-specific turtle eDNA. Furthermore, we found that total eDNA concentration did not follow a linear pattern throughout the field season. While we cannot recommend eDNA for aquatic turtles at this time, we believe this method could be refined with further technological advances such as better inhibition removers. 1 CHAPTER 1 INTRODUCTION Population ecology addresses the dynamics of how a group of individuals in the same species interacts with other biotic and environmental factors (Wells and Richmond, 1995). Part of population ecology attempts to describe how groups of individuals in a certain species vary demographically – such as in abundance or density (Hutchinson, 1991; Turchin, 1999). The factors that shape population numbers may be environmental or density dependent (MacArthur, 1958). For example, extreme environmental events such as a heat wave may cause population decline, whereas rate of growth depends on previous population abundance (Kendall, 1949; Garrabou et al., 2009). The need to predict changes drives the importance in studying these factors; too much growth or decline in numbers of individuals within a population can disrupt an ecosystem or forewarn extinction (Brown et al., 1995; Brooks et al., 2002). Yet, to make the most accurate predictions, ecologists must have some knowledge of a species’ presence and abundance. One factor in population growth and decline is population abundance itself. Besides density-independent environmental events, populations depend upon reproductively mature adults to prosper (Frankham, 1995). Temporary decline in population abundance can result in demographic changes causing Allee effects or population
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