Using Barcode Similarity Groups to Organize Cortinarius Sequences by Emma Harrower A thesis submitted in conformity with the requirements for the degree of Master of Science Ecology and Evolutionary Biology University of Toronto © Copyright by Emma Harrower 2010 Using Barcode Similarity Groups to Organize Cortinarius Sequences Emma Harrower Master of Science Name of Graduate Ecology and Evolutionary Biology University of Toronto 2010 Abstract To improve fungal identification using a single DNA sequence, I introduce the Barcode Similarity Group (BSG) defined as a cluster of sequences that share greater than or equal to a threshold amount of genetic similarity with each other. As a test case, I created 393 BSGs from 2463 Cortinarius ITS sequences using a 94% similarity cut-off value in DOTUR. Some BSGs may contain multiple species. The BSG database was used to label environmental sequences, find misidentified or mislabeled sequences, and find potential cryptic species and novel species. Expert taxonomists will be needed to perform detailed morphological and phylogenetic studies to identify the individual species within each BSG. The main advantage of using BSGs is that it clusters together sequences using total genetic relatedness and does not rely on any taxonomy for identification. A website was created where the RDP Classifier is used to classify a query sequence into a BSG. ii Acknowledgments I would like to thank Dr. Jean-Marc Moncalvo for his guidance, and Simona Margaritescu for her help in the lab. I would like to thank Dr. Nicholas Provart and Hardeep Nahal for their help with providing me with Perl scripts to use, for providing me an account on the BAR server and for helping set up a MySQL account for me. I would like to thank Bryn Dentinger for his understanding of the difficulties I faced and for his encouragement. I would like to thank my family and friends for their support and encouragement. The National Science and Engineering Research Council provided funding for this research. iii Table of Contents ACKNOWLEDGMENTS...............................................................................................................................................III TABLE OF CONTENTS ................................................................................................................................................ IV LIST OF ABBREVIATIONS......................................................................................................................................... VI LIST OF FIGURES ........................................................................................................................................................VII LIST OF TABLES ........................................................................................................................................................VIII LIST OF APPENDICES ................................................................................................................................................. IX CHAPTER 1: INTRODUCTION .................................................................................................................................1 FUNGAL DIVERSITY AND ECOLOGICAL ROLES .................................................................................................................1 SPECIES CONCEPTS IN FUNGI ............................................................................................................................................2 DIFFICULTIES IN IDENTIFYING FUNGAL SPECIMENS.........................................................................................................3 MOLECULAR IDENTIFICATION ..........................................................................................................................................4 GENBANK AND BLAST....................................................................................................................................................4 THE DNA BARCODING INITIATIVE...................................................................................................................................5 WHAT IS A BARCODE SIMILARITY GROUP?.....................................................................................................................7 THE GENUS CORTINARIUS (AGARICALES, BASIDIOMYCOTINA) ......................................................................................8 MOLECULAR IDENTIFICATION OF CORTINARIUS...............................................................................................................9 OBJECTIVES .....................................................................................................................................................................10 CHAPTER 2: THE CREATION OF ITS-RDNA BARCODE SIMILARITY GROUPS FOR THE GENUS CORTINARIUS (AGARICALES, FUNGI)...................................................................................................................12 INTRODUCTION................................................................................................................................................................12 MATERIALS AND METHODS............................................................................................................................................15 Obtaining sequences .................................................................................................................................................15 Creating Barcode Similarity Groups .......................................................................................................................15 Matching a query sequence to a BSG ......................................................................................................................16 Web access to the BSG database..............................................................................................................................17 RESULTS ..........................................................................................................................................................................18 ITS sequence diversity and taxonomic accuracy in public databases ...................................................................18 Creating the BSG Database......................................................................................................................................19 Finding misidentified sequences ..............................................................................................................................20 Finding novel and cryptic species............................................................................................................................21 Collecting and labeling environmental sequences and matching them to voucher specimens............................21 Metadata retrieved from BSGs.................................................................................................................................21 The BSG Classifier and website ...............................................................................................................................22 iv DISCUSSION .....................................................................................................................................................................23 CHAPTER 3: GENERAL DISCUSSION AND PERSPECTIVES FOR FUTURE STUDIES........................28 TAXONOMIC DELIMITATION AND CLASSIFICATION ......................................................................................................28 Delimitation of Species .............................................................................................................................................28 Classification tools....................................................................................................................................................30 Fungal identification tools........................................................................................................................................32 BARCODE SIMILARITY GROUPS......................................................................................................................................33 How are BSGs an improvement on the current taxonomic database model?.......................................................33 How the 94% similarity value was chosen ..............................................................................................................33 Uses of BSGs .............................................................................................................................................................34 FUTURE DEVELOPMENTS................................................................................................................................................38 CONCLUSION ...................................................................................................................................................................40 LITERATURE CITED ....................................................................................................................................................41 FIGURES............................................................................................................................................................................50 TABLES..............................................................................................................................................................................58