DIET ANALYSIS OF MAUMEE RIVER FISHES USING CYTOCHROME C OXIDASE (COI) DNA METABARCODING ― INSIGHTS INTO A CRITICAL TIME OF YEAR Megan G. Shortridge A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 2016 Committee: Jeff Miner, Advisor Daniel Heath R. Michael McKay Christine Mayer © 2016 Megan Shortridge All Rights Reserved iii ABSTRACT Jeffrey Miner, Advisor In recent years, DNA barcoding, the sequencing of a common marker region for taxonomic identification, has become integrated into U.S. agency protocols and procedures. Chapter 1 provides an overview of areas where DNA barcoding is currently being used by U.S. agencies to address questions of management concern; the benefits and limitations of using barcoding in an agency setting are considered, as well as how the technology may evolve in the near future. A diet metabarcoding study was then conducted in Chapter 2, which addressed a question of fisheries management concern, the diet of Maumee River fishes at an important time of year using cytochrome c oxidase (COI) DNA metabarcoding, with a particular focus on detecting predation on early life stages (ELS) of walleye (Sander vitreus). DNA amplified from the homogenized gut contents of fishes captured in the Maumee River during early spring was analyzed using next generation sequencing. Walleye eggs and larvae were present when predators were collected, although at lower densities than previously reported at peak density in the Maumee River. Despite the presence of walleye ELS in the system, the number of fishes with sequences assigned to walleye was lower than initially expected. One female white perch (Morone americana), one male white bass (Morone chrysops), and two emerald shiners (Notropis atherinoides) that were caught in the spawning grounds (Orleans Park) had gut content sequences assigned to walleye. Relatively low density of walleye in the system, the presence of alternative prey items (e.g., chironomids), lower overall feeding intensity by predator fishes near the onset of spawning, and/or turbidity in the Maumee River acting as a predation refuge may explain the lower than expected predation on walleye ELS, however, this requires further investigation and confirmation. Overall, sequences assigned to 7 phyla of metazoans were detected using DNA metabarcoding, including 9 genera of chironomids. Unexpected diet items iv were encountered, including potential predation on the bryozoan, Plumatella casmiana, by emerald shiner. This study reinforced the utility of DNA barcoding in providing insight where morphological identification is difficult as described in Chapter 1, but also points to areas where methods need improvement. v Dedicated to all those who pursue science to better understand our world, gain insight on our past, and prepare for our future. This thesis is dedicated to those with an endless curiosity for the natural world. “More broadly, DNA barcoding allows a day to be envisioned when every curious mind, from professional biologists to schoolchildren, will have easy access to the names and biological attributes of any species on the planet,” (Hebert & Gregory 2005). vi ACKNOWLEDGMENTS Numerous people have been instrumental in making this thesis possible, first of which, are my family. Thanks especially to my mom, dad, brother, aunts, uncles, and grandparents for your endless support and encouragement. Thanks for putting up with gill nets being power- washed and repaired in long driveways, and for even donning waders once or twice to lend a hand. Thank you to my partner, Phil, for your support, love, and endless patience and encouragement. I could not have done it without you. Thank you to my funding sources for making this work possible: Toledo Naturalists' Association and Jeff Schaeffer and the U.S. Geological Survey (USGS). This thesis would not be here without your support and I cannot thank you enough. A big thank you to my lab mates and to undergrads who have helped with this research intellectually and technically. A special thanks to Rich Budnik, Jake Miller, Jamie Justice, Mandy Nourse, Jamie Russell, Dani McNeil, and Kevin Bland for assistance sampling. Also, thanks to my friend and fellow grad student Scott Mastrocinque for your friendship during these years and encouragement. Thank you to my advisor, Jeff Miner, my committee members, and to Scott Rogers for all of your help and advice. Thank you to Kyle Wellband for being such a patient teacher at Windsor, and for showing me techniques that made this study possible. The learning curve was steep, but I learned a lot. Thank you to the Ohio Department of Natural Resources for help in collecting organisms used during this study, and for fun and educational times out on the river. A special thanks to Mike Wilkerson and ODNR District 2. vii TABLE OF CONTENTS Page CHAPTER I: THE APPLIED USE OF DNA BARCODING IN REGULATORY SCIENCE: CURRENT USES, PRESENT PROBLEMS, AND FUTURE APPLICATIONS ................ 1 Abstract…………………………………………. ..................................................... 1 Introduction…………………………………………………………………………… 2 DNA Barcoding Background and Methods…………………………………………… 3 DNA Barcoding Aids in Protection of Public Safety………………………………… 7 DNA Barcoding and Biosecurity .................................................................................. 10 DNA Barcoding in Biomonitoring and Biodiversity………………………………… 19 Challenges and Limitations of DNA Barcoding, and Future Directions .................... 22 Quantitativeness in DNA barcoding studies…………………………………. 22 Portability, affordability, and speed in DNA barcoding ................................... 25 The challenge of incomplete reference databases……………………………. 28 Conclusions ................................................................................................................ 29 CHAPTER II: DIET ANALYSIS OF MAUMEE RIVER FISHES USING CYTOCHROME C OXIDASE (COI) DNA METABARCODING ― INSIGHTS INTO A CRITICAL PERIOD .............................................................................................................. 30 Abstract ...................................................................................................................... 30 Introduction ............................................................................................................... 31 Methods...................................................................................................................... 38 Collection of predators ................................................................................... 38 Timing of larval walleye drift ........................................................................ 40 viii Extraction of predator gut contents ................................................................ 40 DNA extraction, library preparation, and sequencing ................................... 42 Bioinformatics and data analysis ................................................................... 44 Results ........................................................................................................................ 47 Discussion .................................................................................................................. 56 LITERATURE CITED .......................................................................................................... 75 APPENDIX A: IACUC APPROVAL FOR PROTOCOL 14-006 ....................................... 149 APPENDIX B: ACRONYMS AND ABBREVIATIONS USED IN THESIS .................... 150 ix LIST OF FIGURES Figure Page 1.1 The general workflow for taxonomic identification of an organism using DNA barcoding ................................................................................................................... 6 1.2 Amplification curve during quantitative real-time PCR (qRT-PCR) reaction .......... 18 1.3 Applications of DNA Barcoding by agencies ............................................................ 24 2.1 Developmental stages of Sander vitreus .................................................................... 37 2.2 Map of the two fish collection locations on the Maumee River, a major tributary of Lake Erie in Ohio ................................................................................................................ 39 2.3 Processing workflow for analysis of next generation dataset .................................... 46 2.4 Mean daily density of larval walleye ± 1 standard deviation in Orleans Park (OP) and Buttonwood Park (BP) (spawning grounds) on selected dates in 2014 with sample size represented in parentheses ......................................................................................... 53 2.5 Mean density of walleye larvae ± 1 standard deviation in the Rossford Marina (RM) area on selected dates in 2014 with sample size in parentheses ................................................. 54 2.6 Percent of total sequences from each predator species assigned to each metazoan phylum With sample size for each species represented in parentheses .................................. 55 B.1 Sequence length frequency distribution of original Ion Torrent PGM dataset produced using FastQC Report (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) via Galaxy ........................................................................................................................ 124 B.2 Sequence quality score frequency distribution of original Ion Torrent