Assessing Belowground Plant Diversity in Wetland Soil through DNA Metabarcoding: Impact of DNA Marker Selection and Analysis of Temporal Patterns by Nicole Allison Fahner A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Integrative Biology Guelph, Ontario, Canada © Nicole Fahner, December, 2015 ABSTRACT ASSESSING BELOWGROUND PLANT DIVERSITY IN WETLAND SOIL THROUGH DNA METABARCODING: IMPACT OF DNA MARKER SELECTION AND ANALYSIS OF TEMPORAL PATTERNS Nicole Allison Fahner Advisor: University of Guelph, 2015 Professor Mehrdad Hajibabaei This thesis is an investigation of the DNA metabarcoding approach to biodiversity assessment of vascular plant diversity. Specifically, the investigation focused on DNA metabarcoding of environmental DNA extracted from unsorted soil samples. There were two main research goals: to evaluate the suitability of four established DNA marker regions – matK, rbcL, ITS2, and the P6 loop of the trnL intron – for biodiversity assessment of vascular plants and to examine community turnover in total belowground vascular plant diversity. Based on the relative annotation, resolution and recovery ability of the DNA markers, rbcL and ITS2 were recommended for future biodiversity assessments. Annual variability in belowground diversity was consistent in magnitude with previous aboveground observations suggesting that accumulation of plant tissues is not a major restriction for soil-based biodiversity assessments. Finally, an interaction between DNA marker and observed community turnover was identified and positively correlated with length of DNA marker. iii Contents LIST OF TABLES ..................................................................................................................................................... IV LIST OF FIGURES ..................................................................................................................................................... V ACKNOWLEDGEMENTS ......................................................................................................................................... VI GENERAL INTRODUCTION ...................................................................................................................................... 1 CHAPTER ONE - RELATIVE PERFORMANCE OF FOUR DNA MARKERS FOR SURVEYING VASCULAR PLANT DIVERSITY FROM SOIL ENVIRONMENTAL DNA ........................................................................................................................ 4 ABSTRACT ..................................................................................................................................................................... 4 INTRODUCTION .............................................................................................................................................................. 5 MATERIALS AND METHODS ............................................................................................................................................ 10 Study Site ............................................................................................................................................................ 10 In silico – Analysis of Database Sequences ......................................................................................................... 10 In situ – Analysis of Soil Cores ............................................................................................................................. 13 RESULTS ..................................................................................................................................................................... 17 In silico – Analysis of Database Sequences ......................................................................................................... 17 In situ – Analysis of Soil Cores ............................................................................................................................. 18 DISCUSSION ................................................................................................................................................................ 24 In silico – Analysis of Database Sequences ......................................................................................................... 24 In situ – Analysis of Soil Cores ............................................................................................................................. 29 Conclusions ......................................................................................................................................................... 34 TABLES AND FIGURES .................................................................................................................................................... 36 CHAPTER TWO – DNA METABARCODING ASSESSMENT OF TEMPORAL VARIABILITY IN BELOWGROUND PLANT DIVERSITY IN A DELTAIC WETLAND ...................................................................................................................... 42 ABSTRACT ................................................................................................................................................................... 42 INTRODUCTION ............................................................................................................................................................ 43 Hypotheses and Predictions ................................................................................................................................ 46 METHODS ................................................................................................................................................................... 46 Statistical Methods ............................................................................................................................................. 48 RESULTS ..................................................................................................................................................................... 50 DISCUSSION ................................................................................................................................................................ 54 FIGURES ..................................................................................................................................................................... 63 GENERAL CONCLUSIONS ...................................................................................................................................... 67 LITERATURE CITED ............................................................................................................................................... 70 APPENDIX A – METABARCODING METHODOLOGY .............................................................................................. 75 Sample Collection ................................................................................................................................................ 75 Subsampling ........................................................................................................................................................ 75 DNA Extraction .................................................................................................................................................... 75 PCR Amplification ................................................................................................................................................ 76 Library Preparation and Sequencing ................................................................................................................... 78 Sequence Processing ........................................................................................................................................... 79 APPENDIX B – DATABASE COVERAGE .................................................................................................................. 87 APPENDIX C – SEQUENCING PROCESSING OUTPUT .............................................................................................. 92 APPENDIX D – TAXONOMIC ASSIGNMENT DATA ............................................................................................... 100 APPENDIX E – STATISTICAL OUTPUT SUMMARY TABLES .................................................................................... 112 iv List of Tables Chapter 1 Table 1 Database coverage by DNA marker 36 Table 2 Total numbers of taxa observed across the four PAD sites 37 Appendix A Table 3 Primer sequences and expected amplicon sizes 82 Table 4 Optimized PCR conditions used for first round amplification 83 Table 5 Thermocycler programs 84 Table 6 Optimized PCR conditions for amplification with Illumina tailed primers 85 Table 7 Search criteria used to build reference databases 86 Appendix B Table 8 List of previously observed taxa and associated sequence database coverage 87 Appendix C Table 9 Sequence processing and filtering output for the OTU pipeline 92 Table 10 Sequencing processing and filtering output for the taxonomy pipeline 96 Appendix D Table 11 Taxonomic assignments passing all filters at order, family and genus levels 100 Appendix E Table 12 Statistical test output for nearest neighbour distance comparison 112 Table 13 Statistical test output for comparison of DNA marker sequence recovery 113 Table 14 Statistical test output for comparison of DNA marker taxonomic resolution 115