Examining patterns of genetic variation in Canadian marine molluscs through DNA barcodes by Kara Layton A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Integrative Biology Guelph, Ontario, Canada © Kara Layton, January, 2012 ABSTRACT Examining patterns of genetic variation in Canadian marine molluscs through DNA barcodes Kara Layton Advisor: University of Guelph, 2013 Professor P.D.N Hebert In this thesis I investigate patterns of sequence variation at the COI gene in Canadian marine molluscs. The research presented begins the construction of a DNA barcode reference library for this phylum, presenting records for nearly 25% of the Canadian fauna. This work confirms that the COI gene region is an effective tool for delineating species of marine molluscs and for revealing overlooked species. This study also discovered a link between GC content and sequence divergence between congeneric species. I also provide a detailed analysis of population structure in two bivalves with similar larval development and dispersal potential, exploring how Canada’s extensive glacial history has shaped genetic structure. Both bivalve species show evidence for cryptic taxa and particularly high genetic diversity in populations from the northeast Pacific. These results have implications for the utility of DNA barcoding both for documenting biodiversity and broadening our understanding of biogeographic patterns in Holarctic species. Acknowledgements Firstly, I would like to thank my advisor Dr. Paul Hebert for providing endless guidance and support during my program and for greatly improving my research. You always encouraged my participation in field collections and conferences, allowing many opportunities to connect with colleagues and present my research to the scientific community. I am also incredibly grateful for the invaluable feedback and input I received from my committee members, Dr. Elizabeth Boulding and Dr. André Martel. Your enthusiasm for this project always kept me motivated and I thank you for teaching me all about the wonderful world of malacology. I would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC), the International Barcode of Life project and Genome Canada through the Ontario Genomics Institute for funding this research. I am also grateful to Aboriginal Affairs and Northern Development Canada for providing me with a Northern Scientific Training Program grant that aided in field collections in Churchill, Manitoba. Lastly, I thank the Canadian Centre for DNA Barcoding (CCDB) for help in sequence acquisition. Research teams at the Kasitsna Bay Lab, the Churchill Northern Studies Centre and the Huntsman Marine Science Centre provided logistical support for field collections and I am extremely grateful for their help. I would also like to extend my gratitude to Sarah Hardy, Katrin Iken, Suzanne Dufour, Barry McDonald, Robert Frank, Nicholas Jeffrey and Paolo Pierrossi for aid in specimen collections. A sincere thank you to all graduate students in the Hebert, Adamowicz, Smith, Crease, Hajibabaei and Gregory labs for providing input into my project and contributing greatly to my happiness during this program. I especially want to thank Christy Carr for her advice, patience and compassion- you truly are a fantastic scientist. Finally, I need to thank my close friends and family, particularly my parents and Ryan, who not only provided unconditional love but have shaped the person I am today. iii Table of Contents Abstract……………………………………………………………………………………………………ii Acknowledgements……………………………………………………………………………………….iii List of Tables……………………………………………………………………………………………...vi List of Figures……………………………………………………………………………………………vii General Introduction………………………………………………………………………………...........1 Chapter 1: Patterns of DNA barcode variation in Canadian marine molluscs………………….........3 Abstract…………………………………………………………………………………………….........3 Introduction……………………………………………………………………………………………...4 Methods…………………………………………………………………………………………….........5 Specimen collection and data scrutiny………………………………………………………………..5 DNA extraction, amplification and sequencing……………………………………………………….5 Data analysis…………………………………………………………………………………………..6 Results…………………………………………………………………………………………………..7 Sequence recovery…………………………………………………………………………….............7 COI variation in molluscs……………………………………………………………………………..7 Variation in nucleotide composition…………………………………………………………………..8 Distribution of indels…………………………………………………………………………………..8 Discussion………………………………………………………………………………………………8 Sequencing success in Mollusca………………………………………………………………………8 Patterns of sequence variation…………………………………………………………………….......9 Insertions and deletions in COI……………………………………………………………………...10 Patterns of nucleotide composition…………………………………………………………………..11 Conclusions…………………………………………………………………………………………..12 Chapter 2: Geographic patterns of mtCOI diversity in two species of Canadian marine bivalves……………………………………………………………………………23 Abstract………………………………………………………………………………………………..23 Introduction……………………………………………………………………………………………24 Methods………………………………………………………………………………………………..25 Specimen collection…………………………………………………………………………………..25 DNA extraction, amplification and sequencing……………………………………………………...26 Data analysis…………………………………………………………………………………………26 Results…………………………………………………………………………………………………27 Sequence recovery and haplotype diversity………………………………………………………….27 iv Patterns of genetic diversity…………………………………………………………………………28 Population structure…………………………………………………………………………………29 Discussion……………………………………………………………………………………………..30 Comparing diversity and structure in two bivalves with planktotrophic larval development……....30 Implications for glacial refugia in the northeast Pacific……………………………………………30 Evidence of sibling species…………………………………………………………………………..31 Conclusions…………………………………………………………………………………………..32 General Conclusions……………………………………………………………………………………..44 Summary of findings…………………………………………………………………………………44 The application of a DNA barcode library for Canadian marine molluscs………………………....44 Gene flow in marine populations…………………………………………………………………….45 Conclusions and implications for conservation……………………………………………………...46 Literature Cited………………………………………………………………………………………….47 Appendix A: Specimen Preservation…...……………………………………………………………....56 Appendix B: Species Identifications…………………………………………………………………....57 Appendix C: Chapter 1 Supplementary Material……………………………………………………..61 Appendix D: Chapter 2 Supplementary Material……………………………………………………..65 Appendix E: R Code……………………………………………………………………………………..74 v List of Tables Chapter 1 ………………………………………………………………………………………………...13 Table 1.1. Parameter settings for each OTU algorithm………………….………...………………….13 Table 1.2. Percent success in recovery of a COI sequence …………....……………………………..13 Table 1.3. The number of COI sequences and BINs, intraspecific and nearest neighbour distances and mean GC content for each of 33 orders………………………………………………...14 Table 1.4. Mean intraspecific divergence, number of genetic clusters, number of individuals sampled and locality information for each potential cryptic species complex in this study……………………………………………………………………………………………15 Table 1.5. Intra and interspecific distances (K2P) for taxonomic groups examined by DNA barcoding in previous literature…………………………………………………...……………15 Chapter 2…………………………………………………………………………………………………33 Table 2.1. Genetic diversity in populations of the bivalve species, Hiatella arctica and Macoma balthica……………………………………………………………………………………..33 Table 2.2. Overall genetic structure measured by AMOVA for Hiatella arctica…...………………..34 Table 2.3. Overall genetic structure measured by AMOVA for Macoma balthica balthica…...…….34 Table 2.4. Overall genetic structure measured by AMOVA for ATL Macoma balthica…..………...35 Table 2.5. FST for populations of Hiatella arctica, Macoma balthica balthica and ATL Macoma balthica……………………………………………………………………………….36 Appendix B……………………………………………………………………………………………….57 Table B.1. References for species identifications………………...…………………………………58 Appendix C………………………..…………………………………………………………………...…61 Table C.1. List of COI primers used for molecular techniques in Chapter 1…………..……………61 Table C.2. List of GenBank specimens used for analysis in Chapter 1……………………………...61 Appendix D……………………………………………………………………………….………………65 Table D.1. Detailed collection information for all 172 Hiatella arctica specimens…………………65 Table D.2. Detailed collection information for all 196 Macoma balthica specimens.........................67 vi List of Figures Chapter 1…………………………………………………………………………………………………16 Figure 1.1. Sampling locations and the number of specimens examined in this study……...…….…16 Figure 1.2. Rarefaction curves for the five classes of Canadian marine mollusc represented in this study……………...……………………………………………………………….17 Figure 1.3. Mean intraspecific divergences (K2P) and nearest neighbour distances for all specimens in this study………………...………………………………………………………18 Figure 1.4. Maximum and mean intraspecific divergences plotted against the number of individuals analyzed for 157 species……………………………………………………………….18 Figure 1.5. Box plots comparing mean nearest neighbour distance with the number of species sampled from each genus with ≥ 2 representative species………………………..19 Figure 1.6. Neighbour-joining trees (K2P), with locality information, for 9 cryptic species complexes in this study……………………………………………………………………….20 Figure 1.7. Mean nearest neighbour distance (K2P) plotted against mean GC content for the 33 orders of