Fungi of Forests: Examining the Diversity of Root-associated Fungi and Their Responses to Acid Deposition

Global importance of forests is difficult to overestimate, given their role in oxygen production, ecological roles in nutrient cycling and supporting numerous living species, and economic value for industry and as recreational zones. Fitness of the forest-forming trees strongly depends on microbial communities associated with tree roots. In particular, fungi impact tree fitness: mycorrhizal species provide water and nutrients for the trees in exchange for C, endophytic fungi play key roles in host defense against pathogenic organisms, and saprotrophic fungi decompose dead organic matter and facilitate nutrient cycling. In addition, pathogenic fungal species strongly affect forest fitness. Despite their importance, fungal communities associated with forest trees are largely unknown because the typical morphological assay takes into consideration a scarce portion of fungal diversity: species that produce visible fruiting bodies at relatively frequent intervals. A more accurate assessment of fungal diversity in forests has become possible with the development of next-generation sequencing, where fungal species are being identified based on the presence of their DNA in the sample. In this work, DNA-metabarcoding was utilized to assess the diversity of fungi associated with roots of forest-forming trees within the families Fagaceae and Betulaceae (Europe), and Fagaceae, and Juglandaceae (North America). The data obtained provided unprecedented insight into hidden richness of root-associated fungi, which approached 1756 OTUs (a proxy for species) in the European dataset, and 2769 – in the North American dataset. Variation in fungal community composition was largely explained by geographical location (ca. 30%). However, the effect of host specificity (ca. 9-15% of variation) was significant as well. DNA-based data revealed strong positive and negative patters in fungal co-occurrence (e.g., a positive relationship was observed between Cenococcum geophilum and species of Russulaceae), which could indicate interactions between fungal species. In addition to diversity assays, fungal responses to acid precipitation were quantified, and revealed strong declines in fungal richness and abundance, including ectomycorrhizal species. I conclude that compositional shifts in root-associated fungal communities could be particularly suitable for monitoring of forest ecosystems, given an optimal response time in fungi (not too slow as in wooded plants and not as high as in bacteria). Acknowledgements Only a few times in life, I think, do we pause and take a moment to appreciate our accomplishments and the people who helped to make them possible. For me, this document, and the work it represents, is one of the most valued accomplishments of my life. There are many to whom I owe a debt of gratitude and thanks for their help along the way, and I’d like to take the time to recognize them here. First, I would like to extend my sincere gratitude for the guidance and mentorship of my academic mentors during my undergraduate studies at Minnesota State University, Mankato, for without them I would have been unable to find my way through the trials of undergraduate and graduate education. My thanks go out to Dr. John Krenz, who taught me as an undergraduate what research entails by guiding me in my first research project. I appreciate very much the willingness of Dr. Alison Mahoney to mentor me in my desire to pursue independent studies in mycology as an undergraduate. Thanks go out to Dr. Brad Cook for teaching me that a university education is about so much more than grades. I am also thankful for the continued friendship and occasional advice proffered by Dr. Christopher Ruhland. I would also like to thank the professors who guided me through my masters research at Louisiana State University: Dr. Cathy Aime, Dr. Meredith Blackwell, Dr. Chris Clark, and Dr. Marc Cohn. Their contribution to my development as a scientist and student made it possible to continue on to this point. All of which brings me to my current studies and the people who have been very important and influential in the development of this work. First and foremost I need to thank my advisor, Dr. Steve Stephenson. A long time ago, before I had any experience in mycology, Dr. Stephenson took a chance on me and gave me the opportunity to research fungal biodiversity abroad. It is because of his mentorship, advice, and willingness to trust in my abilities that I have been able to progress to the point of conducting independent research in pursuit of a PhD. I owe a debt of gratitude to my committee members as well, without which none of this would have been possible. Thank you to Dr. Burt Bluhm for believing in my desire to do good research, and for the logistical support that made it possible for me to actually do it. Thank you to Dr. Fred Spiegel for providing lively critical debate and friendly conversation about my research goals and the academic lifestyle, as well as the opportunity to assist with the mycology course. Thank you also to Dr. Ravi Barabote, for excellent advice about much of the molecular and bioinformatic components of my research project. A very big thank you to my lab mates: Isadora Cuelho, Geoff Zahn, Laura Walker, Karen Willard, Sittie Macabago, Brandy Garrett-Kluthe, Eric Hearth, Rajaa Al-Anbagi, and Autumn Lynn Coffey Olsen. Being surrounded by your good company throughout graduate school was a valuable thing during some very stressful times. The same can be said of my fellow graduate students in the department. A thank you to the following for being a laborer, an ear, a shoulder, or sharing a coffee or beer: Anant Deshwal, Brian Becker, Auriel Fournier, Brad Austin, Hal Halvorson, Laura Berryman-Williams, Melissa Welch, and so many others I’m not sure I can list