Anthropogenic Nitrogen Deposition and Decomposer Fungi: Altered Composition and Function Fosters Greater Soil Carbon Storage by Elizabeth Mae Entwistle A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Natural Resources and Environment) in the University of Michigan 2016 Doctoral committee: Professor Donald R. Zak, Chair Associate Professor Gregory Dick Associate Professor Inés Ibáñez Associate Professor Timothy Y. James ACKNOWLEDGMENTS This research was funded with support from the Department of Energy’s Office of Biology & Environmental Research and the National Science Foundation Long-term Research in Environmental Biology program. I also wish to acknowledge the Rackham Graduate School, the School of Natural Resources & Environment, and the Undergraduate Research Opportunities Program for their support of my research. There are many people whose contributions were essential to making this research and my progress towards completing my PhD possible. First and foremost, I’d like to thank my advisor, Dr. Don Zak, for his immense support and mentorship throughout my years as a graduate student. I am grateful for the opportunities I have had to grow as a scientist and also for his unwavering belief in me over my years as a graduate student in his lab. I’d also like to thank my committee members, Dr. Tim James, Dr. Inés Ibáñez, and Dr. Greg Dick, for their time, thoughtfulness, and assistance over the course of my degree. There are many current and former Zak lab members to whom I owe a debt of gratitude. Rima Upchurch has had a hand in making this research possible at every step in the process; her invaluable and immense assistance over the years, with everything from setting up a field experiment by flashlight to helping me troubleshoot complicated bioinformatics problems, is appreciated beyond measure. Without the contributions of my colleagues William Argiroff and Karl Romanowicz, the research in Chapters 3 and 4 could not have happened; I thank them for their skill, dedication, and persistence in the face of the challenges presented by these projects. I’d like to thank Dr. Zachary Freedman for his assistance with troubleshooting and his willingness to talk through and offer advice at so many steps along the way. I thank Dr. Lauren Cline for her support, counsel, and comraderie in the realms of fungal ecology and graduate school. I also appreciate the intellectual contributions of Sarah Eisenlord, who helped influence ii some of the research directions pursued in this dissertation, and the mentorship of Dr. Ivan Edwards. I’d like to thank all my current and former colleagues in the lab over the years for their feedback as my project developed. Finally, I’d like to thank my husband, Michael Gray, for his understanding, patience, and support throughout this process. iii TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………………………………………...ii LIST OF TABLES…………………………………………………………………………….......v LIST OF FIGURES……………………………………………………………………………..viii LIST OF APPENDICES…………………………………………………………………………..x ABSTRACT……………………………………………………………………………………...xi CHAPTER 1. Introduction………………………………………………………………………..1 2. Long-term experimental nitrogen deposition alters the composition of the active fungal fommunity in the forest floor……………………………………………..22 3. Anthropogenic N deposition increases soil C storage by reducing the relative abundance of lignolytic fungi…………………………………………………....63 4. Anthropogenic N deposition alters the composition, but not the diversity of expressed class II fungal peroxidases…………………………………………..123 5. Conclusions……………………………………………………………………..172 APPENDICES………………………………………………………………………………….180 iv LIST OF TABLES TABLE 1.1. Climatic, floristic, and edaphic properties of two northern hardwood forests receiving experimental N deposition……………………………………………………………….20 2.1. Climatic, floristic, and edaphic properties of two northern hardwood forests receiving experimental N deposition……………………………………………………………….52 2.2. Chao1 richness for Dikarya 28S rRNA cDNA libraries from two hardwood forest stands (B and D) under ambient and experimental N deposition………………………..53 2.3. Inverse Simpson diversity for Dikarya 28S rRNA cDNA libraries from two hardwood forest stands (B and D) under ambient and experimental N deposition.…......54 2.4. Shannon diversity for Dikarya 28S rRNA cDNA libraries from two hardwood forest stands (B and D) under ambient and experimental N deposition………….………….....55 2.5. Significance values for pair-wise Unifrac, weighted Unifrac, and Martin’s P-test for cDNA clone libraries of 28S rRNA for Dikarya fungi from two northern hardwood forests (Sites B and D) receiving ambient and experimental N deposition……………...56 3.1. Summary of results of biochemical analysis of low-lignin, high-lignin, and wood substrates………………………………………………………………………………..110 3.2. Agaricomycete taxa selected as highly lignolytic…………………………………111 3.3. PERMANOVA results for comparisons of fungal communities under ambient and experimental rates of N deposition…………………………………………………….112 v 3.4. SIMPER results for the ten OTUs with the highest average dissimilarity between fungal communities under ambient and experimental N deposition…………………...113 4.1. Observed ranges for richness, Shannon diversity, Shannon evenness, and Simpson diversity of expressed fungal peroxidases……………………………………………...157 4.2. Observed richness, Shannon diversity, Shannon evenness and inverse Simpson diversity of expressed fungal peroxidases analyzed with two-way ANOVA...………..158 4.3. Fungal peroxidases which were abundant under both ambient and experimental N deposition……………………………………………………………………………….160 4.4. Effect of experimental N deposition on composition of expressed fungal peroxidases nucleotide as measured with PERMANOVA….………………………………………161 4.5. Effect of experimental N deposition on composition of expressed fungal peroxidases as measured with PERMDISP………………………………………………………….162 4.6. Effects of N deposition treatment and Site on Unifrac metric (unweighted and weighted) for expressed fungal peroxidase amino acid sequences……………………..163 SUPPLEMENTAL TABLE S2.1. Estimation of Good’s coverage for Dikarya sequences recovered in fungal 28S rRNA cDNA clone libraries from two northern hardwood forest stands under ambient and experimental N deposition…………………………………………………………..59 APPENDIX TABLE B1. Accession numbers and clone library abundance information for 99% sequence similarity OTUs………………………………………………………………………...183 C1. Physiological categories to which we assigned OTUs identified in SIMPER analysis………………………………………………………………………………….192 D1. Additional information on the taxa we included in our compilation of highly lignolytic taxa used in our study………………………………………………………..198 E1. Agaricomycete taxa excluded from our list of “highly lignolytic taxa”…………...202 vi F1. Top ten SIMPER results for each type of sample for each collection date………...221 vii LIST OF FIGURES FIGURE 1.1. Map of four replicate northern hardwood forests part of a long-term N deposition experiment………………………………………………………………………………..21 2.1. Relative abundance (%) of taxa in Dikarya communities under ambient N deposition - -2 -1 and experimental N deposition ( + 3 g NO3 -N m y ) in two northern hardwood forests, a) Site B and b) Site D…………………………………………………………………...57 3.1. Mass loss of wood, high-lignin, and low-lignin substrates under ambient and experimental N deposition after 7 months of decomposition in the field………………114 3.2. The response (% change in relative abundance) of fungi of different physiologies to experimental N deposition……………………………………………………………...115 3.3. Relative abundance of Agaricomycetes and highly lignolytic taxa in fungal communities on a wood , high-lignin (middle), and low-lignin (bottom) substrates which after 7 and 18 months of decomposition in the field…………………………………...116 3.4. Relative abundance of Agaricomycetes and highly lignolytic taxa of fungal communities in forest floor and soil……………………………………………………118 3.5. Relative abundance of highly lignolytic taxa on the high-lignin substrate collected after 7 and 18 months of decomposition under ambient and experimental rates of N deposition……………………………………………………………………………….119 3.6. Fungal abundance as measured by quantitative PCR on decomposing substrates of varying recalcitrance……………………………………………………………………120 3.7. Fungal abundance as measured by quantitative PCR in forest floor and soil……...122 4.1. The location of four northern hardwood forest stands in our long-term N deposition experiment in Michigan, USA………………………………………………………….164 viii 4.2. Richness, diversity, and evenness of fungal peroxidases expressed under ambient and experimental rates of N deposition……………………………………………………..165 4.3. Scaled Venn diagrams for expressed peroxidase OTUs occurring under ambient and experimental rates of N deposition……………………………………………………..166 4.4. Neighbor-joining diagram of 127 OTU sequences occurring under ambient N deposition, experimental N deposition, or both………………………………………...170 SUPPLEMENTAL FIGURE S2.1. Maximum likelihood tree of Dikarya fungi from two northern hardwood forests receiving ambient and experimental N deposition……………………………………….60 S2.2. Rarefaction curve of observed OTU richness in northern hardwood forest stands at Site B (panels A-B) and Site D (panels C-D) receiving ambient (panels A & C) and experimental (panels B & D) rates of N deposition……………………………………..62 ix LIST OF APPENDICES APPENDIX A. Dikarya reference sequences………………………………………………………..180 B. OTU accession numbers and abundances…………………………………………..183 C. Fungal physiological