THE POLYCHAETA OF CANADA: EXPLORING DIVERSITY AND DISTRIBUTION PATTERNS USING DNA BARCODES A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by CHRISTINA M. CARR In partial fulfilment of requirements for the degree of Master of Science April, 2010 © Christina M. 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The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada ABSTRACT THE POLYCHAETA OF CANADA: EXPLORING DIVERSITY AND DISTRIBUTION PATTERNS USING DNA BARCODES Christina M. Carr Advisor: University of Guelph, 2010 Dr. P.D.N. Hebert This thesis investigates the diversity and distribution of polychaetes from Canadian marine waters employing both morphological (literature reports) and molecular (mtDNA) approaches. Collation of species reports from the literature indicated that nearly 15% of the 1,023 Canadian polychaete species occur in all three of Canada's oceans. In contrast, DNA barcode analysis of these widespread species indicated that most were species complexes. Barcode analysis of 333 provisional polychaete species revealed 40 times more sequence divergence between than within species, and extensive cryptic diversity in 36 morphospecies. Further examination of genetic discontinuities between Pacific and Atlantic-Arctic sister taxa indicated that periodic ice retreats during the Pleistocene enabled multiple migrations of polychaete species across the Bering Strait, ultimately increasing polychaete diversity in Canada. This thesis demonstrates the utility of DNA barcoding as a tool for species identification in polychaetes and for illuminating evolutionary and biogeographic patterns among species. ACKNOWLEDGEMENTS / have grown so much during the two and a half years of this Masters degree and owe several people thanks. First and foremost I thank my advisor, Paul Hebert, for allowing me to choose my own path. He never told me no and was genuinely excited about everything I pursued. There was never-ending infrastructure andfieldwork support, allowing me to sequence every polychaete (and amphipod) I could collect, and to travel to various coasts of Canada and Russia. I learned the value of each specimen, and how to smuggle them across borders. I thank the 1999-2001 collectors who traveled to and dove in Resolute and Igloolik, NU. Your collections inspired me to pursue research in polychaete worms, a group in which I took an immediate and lasting personal interest. The acquisition of over 2000 specimens was a team effort. I thank my co-workers and friends for help in the field: Brandon Laforest, Erin Corstorphine, Jayme Sones, Jeremy deWaard, Lauren Wallace, Martin Brummell, Nick Jeffery, Paola Pierossi, Sandra McCubbin, and the Saunders lab. The "zodiak of adventure " and running tactfully from polar bears will never be forgotten. I thank the researchers and staff at the Churchill Northern Studies Centre for providing great accommodations and delicious meals. I thank my advisory committee, Alex Smith and Jonathan Witt, for fruitful discussions and guidance. I thank my lab mates for teaching me everything I know about being a graduate student. I am forever grateful that Erin chose to work on echinoderms, and not moths, so we could travel together. Beth Clare was always there to train me in the lab, to edit my drafts, and to bounce ideas off Kevin Kerr, John Wilson, and Vazrick Nazari i were great lab mates, willing to listen and take time out of their day to give advice. My office mates, Taika von Konigslow and Chandni Kher, provided many laughs and distractions along the way; thanks! Thank you to several BIO people for helping me with numerous tasks along the way, including sequencing bone tissue from a potential Lake Okanogan Ogopogo. I thank Alex Borisenko, Jay Cossey, Justin Schonfeld, Liuqiong Lu, Maria Kuzmina, Megan Milton, Natalia Ivanova, Rick Turner, Rob Dooh, and Sujeevan Ratnasingham for help and support with programming, imaging, lab protocols, software, and specimen curation. Thanks to Susan Mannhardt for keeping our lives in order, and to Sarah Adamowiczfor providing thoughtful feedback and inspiration along the way. Several organizations and grants supported this research. Funding to Paul Hebert supporting DNA barcode analysis include a NSERCIPYgrant, NSERC Discovery grant, NSERC Strategic Network grant to the Canadian Healthy Oceans Network, and grants through Genome Canada. The Churchill Northern Studies Centre, the Census of Marine Life, the Northern Scientific Training Program, and the Ontario Graduate Scholarship Program provided additional support for my research. I owe great thanks to those who helped with identification of specimens: Tara Macdonald, Ruth Barnich, Dieter Fiege, Torkild Bakken, and Myriam Schuller. Thanks also to Sarah Mincks Hardy and Tanya Brown for providing valuable specimens. Finally, I thank my family for support, encouragement, and always believing in me more than I did myself. Especially I thank Mark, without whom I would not have made it through this thesis in one piece. I will never forget his guidance, help, and endless patience. ii TABLE OF CONTENTS LIST OF TABLES vii LIST OF FIGURES viii LIST OF APPENDICES ix GENERAL INTRODUCTION 1 CHAPTER 1: Polychaete diversity and distribution patterns in Canadian marine waters 5 Abstract 6 Introduction 7 Methods 10 Geographic regions 10 Data collection 11 Similarity indices 12 Faunal distributions 12 Results 13 Sampling effort and taxonomy 13 Species diversity 13 Faunal distributions 14 Discussion 14 A checklist for Canadian polychaetes 14 Species diversity in Canadian waters 15 iii Geographic distributions of Canadian polychaete species 17 Boreal distributions 17 Boreal-arctic distributions 17 Amphiboreal and amphiboreal-arctic distributions 18 Conclusions 19 CHAPTER 2: DNA barcoding in Canadian polychaetes 28 Abstract 29 Introduction 30 Methods 33 Specimen collection and taxonomy 33 DNA isolation, extraction, and amplification 34 Species delineation 35 Diversity and regional similarity indices 36 Nucleotide and amino acid composition 37 Statistical analyses 37 Results 38 Amplification and sequence success 38 Species diversity and genetic distances 39 Diversity and regional similarity indices 40 Nucleotide and amino acid composition 41 Discussion 41 A DNA barcode reference library for Canadian polychaetes 42 Species diversity and divergence 42 iv BINs as biological species 44 Comparison of morphological and molecular diversity 47 Geographic distributions 48 Conclusions 50 CHAPTER 3: High divergence rates and staggered Pleistocene migrations in trans- Arctic polychaetes 68 Abstract 69 Introduction 70 Methods 72 Specimens 72 Sequence divergence 73 Saturation 74 Molecular clock tests 74 Results 75 Sequence divergence 75 Saturation 75 Molecular clock tests 76 Divergence rates and divergence times of sister taxa 76 Discussion 77 Conclusions 84 SUMMARY AND CONCLUSIONS 89 Chapter summary 89 v Future directions 90 LITERATURE CITED 92 APPENDIX 1.1 123 APPENDIX 3.1 229 APPENDIX 3.2 236 VI LIST OF TABLES CHAPTER 1 Table 1 Biogeographic distributions of Canadian polychaetes 21 CHAPTER 2 Table 1 List of primers 51 Table 2 Faunal composition of polychaete species with DNA barcodes 52 Table 3 Thirty-six polychaete morphospecies split into multiple provisional species by DNA barcodes 54 vii LIST OF FIGURES CHAPTER 1 Figure 1 Map of collection sites from the reference literature 22 Figure 2 Sampling effort vs. number of species in five ocean regions 23 Figure 3 Reports of polychaete species from Canadian waters 24 Figure 4 Dates of first descriptions for polychaete species in Canada 25 Figure 5 Faunal composition of polychaetes in five ocean regions of Canada 26 Figure 6 Similarity plot of