Molecular Species Delimitation and Biogeography of Canadian Marine Planktonic Crustaceans by Robert George Young A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Integrative Biology Guelph, Ontario, Canada © Robert George Young, March, 2016 ABSTRACT MOLECULAR SPECIES DELIMITATION AND BIOGEOGRAPHY OF CANADIAN MARINE PLANKTONIC CRUSTACEANS Robert George Young Advisors: University of Guelph, 2016 Dr. Sarah Adamowicz Dr. Cathryn Abbott Zooplankton are a major component of the marine environment in both diversity and biomass and are a crucial source of nutrients for organisms at higher trophic levels. Unfortunately, marine zooplankton biodiversity is not well known because of difficult morphological identifications and lack of taxonomic experts for many groups. In addition, the large taxonomic diversity present in plankton and low sampling coverage pose challenges in obtaining a better understanding of true zooplankton diversity. Molecular identification tools, like DNA barcoding, have been successfully used to identify marine planktonic specimens to a species. However, the behaviour of methods for specimen identification and species delimitation remain untested for taxonomically diverse and widely-distributed marine zooplanktonic groups. Using Canadian marine planktonic crustacean collections, I generated a multi-gene data set including COI-5P and 18S-V4 molecular markers of morphologically-identified Copepoda and Thecostraca (Multicrustacea: Hexanauplia) species. I used this data set to assess generalities in the genetic divergence patterns and to determine if a barcode gap exists separating interspecific and intraspecific molecular divergences, which can reliably delimit specimens into species. I then used this information to evaluate the North Pacific, Arctic, and North Atlantic biogeography of marine Calanoida (Hexanauplia: Copepoda) plankton. My work provides essential information on the molecular data and analysis tools necessary to conduct rapid biodiversity surveys on marine plankton, facilitating the exploration of unknown faunas using DNA barcoding and providing baseline data against which future comparisons can be made. Acknowledgements First and foremost, I would like to thank my two co-advisors Dr. Sarah Adamowicz and Dr. Cathryn Abbott for their support through my program. The guidance from both of you gave me the confidence to pursue my own scientific interests. Cathryn, I very much appreciate your thoughtful advice in assisting me with my study designs and molecular work. Sally, I would not have been able to complete this work without your help. I very much appreciate all of our conversations which helped me work through my project. I would like to also say a special thank you to you Sally, for all of your assistance in preparing my thesis. Your help in editing and revising my message to its current state was instrumental in getting me to the finishing line. I would also like thank Dr. Thomas Therriault for his helpful discussions giving me insight into my study designs and results. To Dr. Elizabeth Boulding, I would like to say thank you for your thoughtful comments on my thesis and help in navigating the university process. I would like to also thank all of my friends that I have had during my time here at the University of Guelph. In particular, I would like to say thank you to Gillian Martin, Anne Chambers, and Fatima Mitterboeck for their great conversations and humour. Thank you to Alex Chen, Tooba Khan, Jessmyn Niergarth, Rebeca Orue Herrera, Michelle Pyle, and Jenna Snelgrove for their assistance with specimen sorting and photography. I would also like to thank Jinal Pandya for her excellent work in helping me with sorting and molecular work. And thank you to the rest of the Adamowicz/Hebert lab members over the last four years. During my program, I was very fortunate to have been able to participate in a collection trip to the Arctic region. My time spent in both Deception Bay and Steensby Inlet was educational and rewarding. My inclusion on this trip was generously offered by Kim Howland, and I would like to thank Kim, the rest of the collections team, and my advisors and committee for supporting my decision to go. I would also like to acknowledge expert identifiers Carol Cooper, Mary Greenlaw, Rebecca Milne, and Lou Van Guelpen. I would also like to recognize the Advanced Analysis Centre at the University of Guelph for their sequencing support. Thank you to Niels Van Steenkiste, Magalie Castelin, Geoff Lowe, and Sujeevan Ratnasingham for helpful discussions. I would like to thank the staff of the Canadian Centre for DNA Barcoding at iii the Biodiversity Institute of Ontario, University of Guelph, for their help with molecular troubleshooting and analysis. I would like to acknowledge my funding agencies. This research was made possible through funding by the Natural Sciences and Engineering Research Council of Canada (NSERC) through the Second Canadian Aquatic Invasive Species Network (CAISN II) grant to Hugh MacIsaac and colleagues and by an NSERC Discovery Grant to Sarah Adamowicz. The vouchered barcode collection was generated with partial support from Canada’s Genomics Research and Development Initiative (Quarantine and Invasive Species Project), including DNA sequencing by the Applied Genomics facility of the National Research Council of Canada. I am also grateful to the CAISN collection teams for obtaining the specimens used in this study. Last, but certainly not least, I would like to most importantly thank my family. The help that my parents, parents-in-law, and siblings provided were invaluable. But the patience and support of my loving wife has been the most important on my journey. Without Kimberley I would not have been able to complete this process. Kimberley was always supportive through family losses, house renovations, and pregnancy, even while I was 4,400 km across the country. And Kimberley was still patient while I spent hours and hours writing while carrying baby number two. Because of these and so, so, so, many other things thank you very much Kim, George, and baby two, I love you all. iv Table of Contents Abstract .............................................................................................................................. ii Acknowledgements .......................................................................................................... iii Table of Contents ...............................................................................................................v List of Tables .................................................................................................................... ix List of Figures .....................................................................................................................x List of Appendices ............................................................................................................ xi Chapter 1: General introduction to Canadian marine zooplankton biodiversity ...........................1 Introduction ...............................................................................................................1 The known versus expected diversity of Canadian marine biota ...................4 Canadian glacial cycles and biological landscape ........................................5 Understanding common species concepts applied in marine biogeography and biodiversity research ..........................................................................7 The taxonomy of Canadian marine planktonic crustaceans ........................10 DNA barcoding: applications and challenges when studying marine plankton...................................................................................................11 Thesis Overview ......................................................................................................15 Chapter 2 .............................................................................................................16 Objective .......................................................................................................16 Questions ......................................................................................................16 Significance ..................................................................................................16 Conclusion ....................................................................................................17 Chapter 3 .............................................................................................................17 Objective .......................................................................................................17 Questions ......................................................................................................17 Significance ..................................................................................................18 Conclusion ....................................................................................................18 v Chapter 4 .............................................................................................................18 Objective .......................................................................................................18 Questions ......................................................................................................19 Significance ..................................................................................................19