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Using Environmental DNA (Edna) Metabarcoding to Assess Aquatic Plant Communities

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Using Environmental DNA (Edna) Metabarcoding to Assess Aquatic Plant Communities Using environmental DNA (eDNA) metabarcoding to assess aquatic plant communities A Thesis Submitted to the Committee on Graduate Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Faculty of Arts and Science TRENT UNIVERSITY Peterborough, Ontario, Canada © Copyright by Stephanie A. Coghlan 2018 Environmental and Life Sciences M.Sc. Graduate Program September 2018 Abstract Using eDNA metabarcoding to assess biodiversity of aquatic plant communities Stephanie A. Coghlan Environmental DNA (eDNA) metabarcoding targets sequences with interspecific variation that can be amplified using universal primers allowing simultaneous detection of multiple species from environmental samples. I developed novel primers for three barcodes commonly used to identify plant species, and compared amplification success for aquatic plant DNA against pre-existing primers. Control eDNA samples of 45 plant species showed that species-level identification was highest for novel matK and pre- existing ITS2 primers (42% each); remaining primers each identified between 24% and 33% of species. Novel matK, rbcL, and pre-existing ITS2 primers combined identified 88% of aquatic species. The novel matK primers identified the largest number of species from eDNA collected from the Black River, Ontario; 21 aquatic plant species were identified using all primers. This study showed that eDNA metabarcoding allows for simultaneous detection of aquatic plants including invasive species and species-at-risk, thereby providing a biodiversity assessment tool with a variety of applications. Keywords: aquatic plants, biodiversity, environmental DNA (eDNA), invasive species, species-at-risk, metabarcoding, high throughput sequencing, Illumina, bioinformatics ii Acknowledgements First and foremost, I would like to thank my phenomenal co-supervisors Drs. Joanna Freeland and Aaron Shafer for their positive attitudes and helpful guidance; my project and thesis were much improved thanks to your knowledge and edits throughout. Joanna, thank you for your patience during the project; your words of encouragement and pep-talks kept me motivated while writing this thesis. Aaron, thank you for welcoming me into the Shafer lab family, for helping me work through too many bioinformatics roadblocks to count, and for reminding me to take breaks. Thank you to my committee member, Sabine McConnell, for providing bioinformatics expertise and helpful feedback. Thank you to Melanie Shapiera and Wil Wegman for taking me out in the field with their team to collect water samples on the Black River, and for providing a list of reference plants common to the area. Thank you to Maria O’Sullivan for collecting plants for my project and to Susan Chow for helping with visual plant identifications. Thank you to my Shafer and Freeland lab mates for listening to many practice talks and helping me troubleshoot with lab and bioinformatics errors. In particular, I would like to thank Alicia Martin for helping me get familiar with many lab procedures, and Charise Currier for helping me troubleshoot primer design issues and training me on multiple eDNA protocols. Thank you to Lindsay Bond for helping me get lab work finished on multiple occasions and for providing friendship and laughter in the lab. I would like to thank my family and friends that motivated me to pursue this degree. My big, wonderful family has supported me through all my years of school and continues to push me to reach my goals. I am fortunate to have the greatest friends that iii inspire me daily with their positive outlooks and who are always there when I need a quick escape from schoolwork and a good laugh. Lastly, thank you Vince for being my rock and helping me through the highs and lows of this project. I am extremely grateful for our shared passion for learning, and for your support and encouragement; I would not have been able to do any of this without you. iv Table of Contents Table of Contents Abstract ................................................................................................................................ ii Acknowledgements ............................................................................................................ iii Table of Contents ................................................................................................................. v List of Figures .................................................................................................................... vii List of Tables ...................................................................................................................... ix List of Appendices ........................................................................................................... xiii Chapter 1: General Introduction .......................................................................................... 1 Chapter 2: Using eDNA metabarcoding to assess aquatic plant communities .................... 5 2.1 Introduction .............................................................................................................. 5 Environmental DNA (eDNA) ........................................................................................................ 5 Barcoding and metabarcoding ..................................................................................................... 7 Illumina sequencing & bioinformatics ....................................................................................... 13 Study rationale ............................................................................................................................ 16 2.2 Methods.................................................................................................................. 19 Rationale ..................................................................................................................................... 19 (a) eDNA marker development and in silico analysis of database sequences ........................... 19 (b) Environmental DNA .............................................................................................................. 26 (c) Library preparation .............................................................................................................. 28 (d) Bioinformatics ....................................................................................................................... 32 2.3 Results .................................................................................................................... 33 (a) eDNA marker development and in silico analysis of database sequences ........................... 33 (b) Control and wild sample metabarcoding and bioinformatics .............................................. 36 Single- versus multi-locus approach .......................................................................................... 41 2.4 Discussion .............................................................................................................. 44 In silico primer design and validation on single species .................................................... 44 Control sample results ........................................................................................................ 48 Positive identifications ............................................................................................................... 48 Missing species identifications ................................................................................................... 53 Erroneous identifications ........................................................................................................... 54 Metabarcoding limitations ................................................................................................. 56 Wild sample results ............................................................................................................ 60 Wild versus control results ................................................................................................. 62 ‘All-or-nothing’ phenomenon ............................................................................................ 64 Conclusion ......................................................................................................................... 65 Chapter 3: General Discussion ........................................................................................... 67 References .......................................................................................................................... 70 Appendices ......................................................................................................................... 82 Appendix A – Primer design .............................................................................................. 82 Appendix B – Sample collection, filtration, and extraction ............................................... 91 v Appendix C – Primer testing and optimization, and single-species amplification ............ 96 Appendix D – eDNA sample library preparation ............................................................ 103 Appendix E – Bioinformatics pipeline information ......................................................... 115 1) ecoPrimers and ecoPCR ...................................................................................................... 115 2) Quality-checking .................................................................................................................
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