A Taxonomic and Phylogenetic Investigation of Conifer Endophytes of Eastern Canada by Joey B. Tanney A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Carleton University Ottawa, Ontario © 2016 Abstract Research interest in endophytic fungi has increased substantially, yet is the current research paradigm capable of addressing fundamental taxonomic questions? More than half of the ca. 30,000 endophyte sequences accessioned into GenBank are unidentified to the family rank and this disparity grows every year. The problems with identifying endophytes are a lack of taxonomically informative morphological characters in vitro and a paucity of relevant DNA reference sequences. A study involving ca. 2,600 Picea endophyte cultures from the Acadian Forest Region in Eastern Canada sought to address these taxonomic issues with a combined approach involving molecular methods, classical taxonomy, and field work. It was hypothesized that foliar endophytes have complex life histories involving saprotrophic reproductive stages associated with the host foliage, alternative host substrates, or alternate hosts. Based on inferences from phylogenetic data, new field collections or herbarium specimens were sought to connect unidentifiable endophytes with identifiable material. Approximately 40 endophytes were connected with identifiable material, which resulted in the description of four novel genera and 21 novel species and substantial progress in endophyte taxonomy. Endophytes were connected with saprotrophs and exhibited reproductive stages on non-foliar tissues or different hosts. These results provide support for the foraging ascomycete hypothesis, postulating that for some fungi endophytism is a secondary life history strategy that facilitates persistence and dispersal in the absence of a primary host. This thesis provides an alternate approach to identifying endophytes by considering the whole fungus interacting within its natural habitat. ii Acknowledgements I would first like to thank my supervisor Dr. Keith A. Seifert for providing me with the freedom and support to conduct the research presented in this thesis as well as many other side projects and curiosities. Keith generously provided many opportunities to travel to domestic and international conferences, which made me more aware of the directions of global mycological research and provided the opportunity to meet many of the great mycological heroes and their contemporaries. Our discussions over the last five years have contributed to my development as a more well-rounded and open- minded researcher, professional, and person; his honesty and integrity are qualities that all professionals should aspire to have. I am indebted to Keith for the knowledge, experience, and personal growth I gained over the last five years in his lab. I thank Dr. J. David Miller for providing stimulating discourse, travel and collecting opportunities, and of course the unique opportunity to work on this project, which proved to be an exciting and captivating experience. I hope that we can continue collaborating on this and other projects in the near future. Myron Smith provided support and advice throughout this process. The Carleton University Graduate Administrators Darlene Moss and Lisa Chiarelli also provided assistance. I wish to thank my other committee members and external reviewers (Nicolas Corradi, André Levesque, and George Carroll). Working at the AAFC Ottawa Research and Development Centre has been an exceptional experience. I would like to thank the research scientists John Bissett, Sarah iii Hambleton, André Levesque, Miao Liu, and Scott Redhead for general advice and discussion over the last several years. Scott Redhead has provided stimulating discussion on taxonomy and nomenclature and, along with other mycologists, helped me during my time of need after eating Mexican food in Bangkok. Kelly Babcock, Daniel Frank, and Benoit Goulet facilitated depositing cultures into the CCFC and Jennifer Wilkinson helped with depositing and finding specimens in the DAOM herbarium. Rafik Assabgui, Wayne McCormick, and Lisa Koziol provided advice and excellent sequence services and Satpal Bilkhu provided database and general computer help. Quinn Eggertson provided guidance for extracting DNA and sequencing herbarium specimens. Wen Chen provided guidance and, with Jeni Liu, helped with querying and retrieving bulk endophyte sequences and related data from GenBank. Nicolas Poliquin and Anna Tomczak assisted in sequencing and A.T. also tested primers and preserved many endophyte cultures. I would like to thank past and present members of the KAS lab: Rafik Assabgui, Yuuri Hirooka, Allison Walker, Cobus Visagie, Neriman Yilmaz, and especially Gerry Louis-Seize for giving me my first introduction to molecular methods and Hai Nguyen for being a good office mate and putting up with my cultures. Stephen Clayden and Donald McAlpine (New Brunswick Museum) facilitated collecting in Fundy National Park and provided me with exceptional field collecting opportunities in New Brunswick. David and Louise Malloch graciously hosted me as a guest in their home when I was collecting in New Brunswick and provided memorable discussion and company. iv I have had excellent discourse with many mycologists who have helped with identifications and provided taxonomic discussion on many of the fungi included in this thesis, including Hans-Otto Baral, Adrian Carter, Pedro Crous, Brian Douglas, Andreas Gminder, Ewald Groenewald, Stip Helleman, Tsuyoshi Hosoya, Jason Karakehian, David Malloch, Kadri Pärtel, and many others. I am also grateful to the mycologists of the past for their efforts, insight, and uncommon passion. I am grateful to Dr. Leonard Hutchison for introducing and cultivating my passion for mycology and being a mentor. Life has certainly taken an interesting and more meaningful path since third year Forest Pathology and I cannot imagine what I would be doing if not for Dr. H. I thank my parents, Brent and Janet Tanney, and David and Anne Spizarsky for their support and understanding during this long period of study. It is a cliché to acknowledge partners and their patience, but after so many years of odd and obsessive work hours with meagre income and the constant drone of fungal biology, taxonomy, and nomenclature, I have to thank Helena Spizarsky for her patience, empathy, support, and love and for being a great mother. Our son Myles was born on December 30th, 2016 and has provided much joy and sleep deprivation; I look forward to us collecting discomycetes soon. I must acknowledge my best fiend, Birdie the German Shorthaired Pointer, whose presence marked the beginning of our time in Ottawa and has provided much happiness and frustration. Skijoring, canoeing, and enjoying the changes of the seasons during our walks in the forest are among my best memories. v Lastly, I express my gratitude to the fungi that I have encountered and the forests in which they inhabit, for not only permitting life but enrichening it for those who look. Research for this thesis was conducted within the Biodiversity (Mycology) group at the Agriculture and Agri-Food Canada Ottawa Research and Development Centre from 2012–2016, supported by the NSERC PGSD2-459312-2014 and NSERC CRDPJ 421782-11. My mind was, as it still is, a vagabond easily distracted from one thing to another. And then thought never stops either. The end of the last page is only the beginning of another chapter, and every peak attained is but the spring-board for a new leap. • • • But as yet I have hardly begun. A child is born when he is nine months old, a man when he is thirty. The blossom has come and gone, but the fruit has to ripen before it rots on the tree. Giovanni Papini, Un uomo finito vi Table of Contents List of Tables ................................................................................................................................... xii List of Figures ................................................................................................................................. xiii List of Appendices .......................................................................................................................... xx General introduction ....................................................................................................................... 1 Project background ............................................................................................................ 2 What is an endophyte? ..................................................................................................... 5 Conifer endophytes: ubiquitous, complex, and diverse ................................................... 8 Problems identifying endophytes .................................................................................... 10 How to identify endophytes ............................................................................................ 13 Overview of thesis ............................................................................................................ 15 Chapter 1: Sexual and asexual states of some endophytic Phialocephala species of Picea ...... 17 Abstract ............................................................................................................................ 18 Keywords .........................................................................................................................