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Metabarcoding Approach to Identifying Early Life Stages of Great Lakes Fishes

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Metabarcoding Approach to Identifying Early Life Stages of Great Lakes Fishes Metabarcoding approach to identifying early life stages of Great Lakes fishes by Kavishka Gallage A thesis submitted in conformity with the requirements for the degree of Master of Science Ecology and Evolutionary Biology University of Toronto ãCopyright by Kavishka Gallage 2020 Metabarcoding approach to identifying early life stages of Great Lakes fishes Kavishka Gallage Master of Science Ecology and Evolutionary Biology University of Toronto 2020 Abstract Accurately identifying fishes in their early life stages using morphology is challenging, time-consuming, and requires taxonomic expertise. Metabarcoding is a method that can be used to identify species in batch samples (Cruaud et al. 2017). Detection of early life stages of fishes is important for understanding life history patterns and critical spawning habitat. In this study, metabarcoding is used as an identification tool to identify 1119 egg and larva batch samples from Sydenham River and Rondeau Bay. I identified 34 species from Sydenham River and 8 species from Rondeau Bay and the spawning months of these species based on date of capture. I determined the materials and supplies cost of metabarcoding in this study to be $6597.33, compared to $62289.09 for individual-based barcoding. This study shows the potential of metabarcoding as a broad-scale detection and identification method for early life stages of Great Lake fishes. ii Acknowledgements Many people contributed to the completion of my Master’s thesis through academic and emotional support. First, I would like to thank my supervisors Dr. Nicholas Mandrak and Dr. Nathan Lovejoy for their continuous support throughout the duration of my thesis. I am grateful for opportunity to work with you and learn from you. I am grateful to have the opportunity to research and work on a project that I was able to intertwine my passion for molecular biology and fish conservation. I thank you for the guidance and encouragement throughout the countless setbacks during my thesis. I would also like to thank my supervisory committee Dr. Jason Weir and Dr. Roberta Fulthorpe for their helpful comments and questions during my committee meeting, which contributed to the advancement and success of this thesis. Thank you to the staff at Fisheries and Oceans Canada for the time spend completing field work and providing me with my sample set. Also, thank you for the continuous support throughout the project. I would like to thank Alex Van Nynatten for all of your help during the troubleshooting of this project, it was extremely useful to be able to openly communicate my ideas. I thank Nathan Lujan for teaching advance molecular lab techniques and designing this project. I thank other members of Lovejoy lab for their continuous support and enthusiasm during my lab work. I thank Mandrak lab for their continuous support during my conference presentations and the completion of my thesis. You all have been great friends and colleagues to learn from during the duration of my thesis. Finally, I would like to thank Connor Grey, my friends, and my family for all of your emotional support throughout my academic career. I thank my parents for inspiring me to become the person I am today and inspiring me to work in fish conservation through countless days spend snorkeling around coral reefs. I would like to thank Connor for your continuous emotional support throughout my thesis by keeping me focused on my goals and helping me to be the best version of myself. iii Table of Contents Abstract ............................................................................................................... ii Acknowledgments ............................................................................................. iii Table of Contents .............................................................................................. iv List of Tables ..................................................................................................... vii Chapter 2 ........................................................................................................ vii Appendix A ................................................................................................... vii Appendix C ..................................................................................................... viii List of Figures .................................................................................................... ix Chapter 2 ....................................................................................................... ix Appendix A ................................................................................................... ix Appendix B ..................................................................................................... ix List of Appendices ............................................................................................. x Chapter 1 Introduction to metabarcoding and the Great Lakes .......................... 1 Barcoding for Detection ................................................................................ 2 DNA barcoding ......................................................................................... 2 DNA metabarcoding .................................................................................. 4 Abundance in larvae and early detection .................................................. 6 Limitations of metabarcoding ................................................................... 8 Applications of barcoding and metabarcoding .............................................. 8 Barcoding to detect diversity ..................................................................... 8 Barcoding to detect threats ........................................................................ 9 Combining barcoding with High Throughput Sequencing ....................... 10 Possible future applications ....................................................................... 11 Introduction to Laurentian Great Lakes ........................................................ 12 History of changes to the Great Lakes ...................................................... 12 iv Threats to Great Lakes .............................................................................. 13 Species at risk ............................................................................................ 13 Aquatic invasive species ........................................................................... 14 Sydenham River watershed ....................................................................... 15 Rondeau Bay ............................................................................................. 16 Purpose of the study ...................................................................................... 17 Significance of this Study .............................................................................. 17 References ..................................................................................................... 19 Chapter 2 Metabarcoding approach to identifying Great Lake fishes in their early life stages .......................................................................................... 29 Introduction ................................................................................................... 29 Methods ......................................................................................................... 31 Sample collection ...................................................................................... 31 DNA extraction ......................................................................................... 33 Reference Library and Primer design ........................................................ 34 Positive and negative controls ................................................................... 36 Library preparation .................................................................................... 36 Bioinformatics ........................................................................................... 38 Cost-effectiveness analysis ........................................................................ 40 Results ........................................................................................................... 41 Species detection ....................................................................................... 41 Spatial patterns ........................................................................................... 45 Temporal patterns ...................................................................................... 45 Cost-effectiveness analysis ........................................................................ 47 Discussion ..................................................................................................... 47 Species detection ....................................................................................... 48 Spatial patterns ........................................................................................... 49 v Spawning timing ........................................................................................ 51 Detecting species at risk and invasive species .......................................... 52 Conservation relevance ............................................................................. 55 Cost effectiveness ...................................................................................... 56 Conclusion ....................................................................................................
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