DNA BARCODING OF ECHINODERMS: SPECIES DIVERSITY AND PATTERNS OF MOLECULAR EVOLUTION A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by ERIN A. CORSTORPHINE In partial fulfillment of requirements for the degree of Master of Science April, 2010 © Erin A. 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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. ••I Canada ABSTRACT DNA BARCODING OF ECHINODERMS: SPECIES DIVERSITY AND PATTERNS OF MOLECULAR EVOLUTION Erin A. Corstorphine Advisor: University of Guelph, 2010 Professor P.D.N. Hebert This thesis investigates species diversity and patterns of molecular evolution in the phylum Echinodermata. The first chapter tests and confirms the utility of DNA barcoding for species identification in 131 species of echinoderms. The impact of larval development and dispersal on intraspecific divergence is examined for trans-oceanic and putative cryptic species. The second chapter investigates the association between rates of molecular evolution and developmental mode by employing phylogenetically independent comparisons between species with contrasting modes of larval development (e.g., planktotrophy vs. maternal brood-protection). The results show that species with nonpelagic development have accelerated rates of evolution when compared to those with pelagic development. However, further investigation is required to determine the factors responsible for this trend. These results suggest that reproductive mode is an important factor in the establishment and maintenance of patterns and rates of genetic divergence in echinoderms. ACKNOWLEDGEMENTS This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) graduate student scholarship to myself, and a NSERC research grant to my advisor Paul Hebert. I would like to thank members of my advisory committee (Teri Crease, Andreas Heyland and Paul Hebert) for their guidance and support. In particular I am deeply grateful to Paul Hebert for providing me with the opportunity to pursue my academic goals and for providing encouragement throughout my degree. My time in the field at Churchill, St. Andrews and Bamfield, provided me with greater perspective on my work and equipped me with valuable field experience. For that, I am also thankful. I would also like to acknowledge by defense committee (Teri Crease, Elizabeth Boulding, Paul Hebert and Pat Wright) for providing thoughtful comments and revisions for the finished thesis. I would like to thank everyone who made this research possible by either contributing specimens or aiding in field collections including: Christina Carr, Sandra McCubbin, Robert Frank, Victoria Frank, Jeremy deWaard, Tanya Brown, Tom Sheldon, Jim Boutillier, Claudia Hand, Katy Hind, and Bridgette Clarkston. In particular, I would like to thank Christy, for being my partner in the field. Learning to drive boats, fishing for dogfish, scouring the barren intertidal in Churchill, digging for critters while up to our knees in mud, not to mention, the countless hours processing, photographing and organizing specimens; none of this would have been the same without you. To Bob and Victoria, thank you for teaching me the art of drysuit diving, for providing me with equipment, great company, lobster dinners and of course, comic relief. i I am particularly grateful to Kelly Sendall for providing access to the extensive echinoderm collection at the Royal British Columbia Museum and to Phil Lambert for his taxonomic advice. I would also like to thank my external collaborators: Peter Smith, Doug Eernisse, Megumi Strathmann and Chris Mah for including me in their work and providing me with opportunities to further my knowledge and involvement in echinoderm research. A heartfelt thanks to my labmates: Beth, Christy, John, Kevin, Taika and Vazrick, for providing advice on all aspects of the graduate student experience and for keeping things interesting. In particular, thank you to Beth and Christy for helping me with lab techniques, clarifying concepts and providing helpful comments on early renditions of this thesis. Thank you to the many staff at the Biodiversity Institute of Ontario, for providing support at all stages of the process. A special thank you to Natalia Ivanova for aid with laboratory protocols, Luiqiong Lu for speedy processing, Dirk Steinke for helpful advice, Alex Borisenko and Jayme Sones for support with specimen collections and Susan Mannhardt for her support in all aspects of the administrative process. I thank all my friends, near and far, for their encouragement and friendship. I would especially like to thank Mike for helping me take the leap and for providing unwavering encouragement and support - you were always there when I needed you most, whether I needed to ramble off ideas, vent my frustrations, cry in despair or celebrate even the smallest of achievements. Thank you. Finally I would like to thank my mom (Norma), dad (Wayne), sister (Lindsay), brother (Jamie), grandfather (Ed) and extended family, including all the Fitz's, for their love and support. ii TABLE OF CONTENTS LIST OF TABLES vi LIST OF FIGURES vii LIST OF APPENDICES viii GENERAL INTRODUCTION 1 Marine biodiversity and consequences of larval dispersal 1 Present study 2 CHAPTER 1: Exploring Canadian echinoderm biodiversity through DNA barcoding 4 Abstract 4 Introduction 5 Methods 9 Specimen collection and taxonomy 9 DNA extraction, COI amplification and sequencing 10 Sequence analysis 12 Species delineation 12 Results 13 Sequence recovery 13 Taxonomic issues 14 Genetic distances within and among taxa 16 Divergence across oceans 18 Discussion 18 Species delineation 19 in Intraspecific divergence between allopatric populations 19 Intraspecific divergence in sympatry 21 Patterns of divergence in a species-rich genus 22 Effects of distribution on interpretation of barcode data 23 Future work 25 Conclusion 27 CHAPTER 2: Patterns of molecular evolution in the COI gene in the Echinodermata 43 Abstract 43 Introduction 44 Methods 48 Taxon choice, developmental mode and independent comparisons 48 Sequence acquisition 49 Nucleotide diversity 49 Relative rate tests 50 Tests for lineage-specific selection 50 Meta-analysis 52 Results 52 Nucleotide diversity 52 Relative rate tests 52 Lineage-specific selection 53 Discussion 53 Possible explanations for elevated rates in nonpelagic lineages 54 IV Study design 57 Future work 58 Conclusion 59 GENERAL DISCUSSION 68 Summary of results 68 DNA barcoding: applications and patterns of diversity 69 Implications of rate heterogeneity 71 REFERENCES 72 APPENDICES 93 v LIST OF TABLES CHAPTER 1 Table 1: Primer combinations, length and location of amplicon and primer references 28 Table 2: Echinoderm species with maximum intraspecific distance > 2% 29 Table 3: Echinoderm species sampled from at least two of Canada's three oceans 30 CHAPTER 2 Table 1: List of phylogenetically independent comparisons for pelagic and nonpelagic lineages 61 Table 2: Nucleotide diversity estimates 62 Table 3: Results of relative rate tests 63 Table 4: Ratios of rates of nonsynonymous (dN) to synonymous (dS) substitutions 64 VI LIST OF FIGURES CHAPTER 1 Figure 1: Map of Canada indicating collection locations and sample sizes 31 Figure 2: Class-level neighbour-joining trees indicating number of species and sample sizes 32 Figure 3: Frequency distribution for intra- and interspecific pairwise comparisons 37 Figure 4: Neighbour-joining tree for Henricia 38 Figure 5: Neighbour-joining trees and collection maps for three species with variable intraspecific divergence associated with geographic separation 40 Figure 6: Neighbour-joining trees and collection maps for