The effect of decayed or downed wood on the structure and function of ectomycorrhizal fungal communities at a high elevation forest by Jennifer Karen Marie Walker B.Sc., The University of Northern British Columbia, 2003 M.Sc., The University of Northern British Columbia, 2006 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The College of Graduate Studies (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) March 2012 !Jennifer Karen Marie Walker, 2012 Abstract Shifts in ectomycorrhizal (ECM) fungal community composition occur after clearcut logging, resulting in the loss of forest-associated fungi and potential ecosystem function. Coarse woody debris (CWD) includes downed wood generated during logging; decayed downed wood is a remnant of the original forest, and important habitat for ECM fungi. Over the medium term, while logs remain hard, it is not known if they influence ECM fungal habitat. I tested for effects of downed wood on ECM fungal communities by examining ECM roots and fungal hyphae of 10-yr-old saplings in CWD retention and removal plots in a subalpine ecosystem. I then tested whether downed and decayed wood provided ECM fungal habitat by planting nonmycorrhizal spruce seedlings in decayed wood, downed wood, and mineral soil microsites in the clearcuts and adjacent forest plots, and harvested them 1 and 2 years later. I tested for differences in the community structure of ECM root tips (Sanger sequencing) among all plots and microsites, and of ECM fungal hyphae (pyrosequencing) in forest microsites. I assayed the activities of eight extracellular enzymes in order to compare community function related to nutrient acquisition. The retention of CWD caused a shift in ECM root tip fungal species composition on saplings at the plot scale within 12 years of clearcutting. Decayed wood and hard downed wood also provided habitat for some ECM fungal species. Abiotic ii conditions in decayed wood and near downed wood on clearcuts were most similar to forest soils, but I did not detect a shift in ECM root tip or ECM hyphae community composition or function among microsites. Instead, ECM fungus community structure and enzyme activity differed most between clearcut and forest plots, and among forest plots. I could not determine if ecosystem function, in terms of soil macromolecule breakdown by ECM fungi, was maintained in clearcuts. Amphinema byssoides, Thelephora terrestris, and Tylospora asterophora were consistently the most abundant ECM taxa at Sicamous Creek. With pyrosequencing of fungal DNA, I was able to identify more ECM fungal taxa than in my previous experiments at this site. I concluded that CWD on clearcut blocks provides habitat for ECM fungi. iii Preface A version of Chapter 2 has been accepted by Applied Soil Ecology (February 22, 2012): Walker, J. K. M., Ward, V., Paterson, C., Jones, M.D. Coarse woody debris retention in subalpine clearcuts affects ectomycorrhizal root tip community structure within fifteen years of harvest. The original experimental design was part of a Forest Science Program grant written by M.D. Jones. I was responsible for harvesting the mesh bags, additional molecular identification of fungi on root tips and all molecular work on mesh bags, culturing and cloning of Alloclavaria purpurea, data analysis of all results but those related to A. purpurea, and writing the manuscript. Collection of the sapling roots, construction and burial of the mesh bags, morphotyping and molecular identification of some of the root tips, was performed by Valerie Ward. Courtney Paterson and Melanie Jones were responsible for all but the culturing and cloning portion of the of A. purpurea experiment. Melanie Jones contributed substantially to editing of the manuscript. I have retained the ‘we’, ‘us’, and ‘our’ throughout Chapter 2 to reflect the language used in the manuscript for work done in this collaboration. In all references to the work done in subsequent chapters, I have used ‘I’, ‘me’, and ‘my’. Field work for Chapter 3 required the help of many people. Specifically, Valerie Ward, Fawn Ross, Maryann Olson, and Brendan Twieg assisted in the planting iv of hundreds of seedlings; Corey Anderson, Kate Sidlar, Ayla Fortin, and Natasha Lukey assisted in their harvesting. Lab work for Chapter 3 also required additional personnel. Specifically, Valerie Ward and Natasha Lukey assisted in performing the enzyme assays. I designed and implemented this experiment, and parts of it were used for Chapter 4. I was responsible for growing, planting, and harvesting the seedlings, installing, maintaining, and downloading the dataloggers, morphotyping and molecularly identifying the root tips for community analysis and enzyme assays, performing the enzyme assays, all data analysis, and writing the chapter. Jason Pither contributed crucial comments on the data analysis, while Melanie Jones contributed to editing of the chapter. I was accompanied during seedling and substrate collection for Chapter 4 by Cynthia Wonham, Bailey Nicholson, Jeremy Bougoure, and Lori Ann Phillips. Lori Ann Phillips optimized lab protocols for soil assays and pyrosequencing, and analysed the carbon fraction of substrate samples. I was responsible for seedling and substrate collection, drying, grinding and preparing substrate samples for chemical analysis, performing pH tests, all DNA extraction and sample preparation for pyrosequencing, substrate enzyme assays, all data analysis, and writing the chapter. Melanie Jones contributed to editing of the chapter, and valuable input was added to this and all chapters by committee members Jason Pither, Louise Nelson, Craig Nichol, and Dan Durall. v Table of Contents Abstract.............................................................................................................................. ii! Preface.............................................................................................................................. iv! Table of Contents............................................................................................................ vi! List of Tables..................................................................................................................... x! List of Figures ................................................................................................................. xv! Acknowledgements.....................................................................................................xviii! 1 Introduction .................................................................................................................... 1! 1.1 Experimental context based on the current literature...................................... 1! 1.2 Site description, experimental design, and sampling scheme ....................... 7! 1.3 Chapter objectives and hypotheses ................................................................. 14! 1.3.1 Coarse woody debris retention in subalpine clearcuts affects the community structure of ectomycorrhizal fungi within fifteen years of harvest (Chapter 2)...................................................................................... 14! 1.3.2 Ectomycorrhizal root tip community structure and enzyme activity varies among forest and clearcut plots, but not among decayed wood, downed wood, and mineral soil microsites (Chapter 3). ............ 15 1.3.3 The community composition and enzymatic activity of fungal hyphae colonizing decayed wood and mineral soil microsites differs among forest plots (Chapter 4). ................................................................. 23 2 Coarse woody debris retention in subalpine clearcuts affects the community structure of ectomycorrhizal fungi within fifteen years of harvest ....................... 24! 2.1 Synopsis................................................................................................................ 24! 2.2 Methods ................................................................................................................ 26! 2.2.1 Site description and experimental design ................................................ 26! 2.2.2 Root tip sampling and molecular identification of fungi from ectomycorrhizae ........................................................................................... 27! 2.2.3 Sampling and molecular identification of fungal hyphae within mesh bags................................................................................................................ 29! vi 2.2.4 Sequence processing and phylogenetic-based naming ........................ 31! 2.2.5 Analysis of ECM community structure...................................................... 33! 2.2.6 Frequency and abundance of individual ECM fungal species.............. 35! 2.2.7 Testing the trophic status of Alloclavaria purpurea................................. 36! 2.3 Results .................................................................................................................. 39! 2.3.1 ECM fungal communities on root tips ....................................................... 39! 2.3.2 ECM fungal communities occurring as extramatrical hyphae............... 41! 2.3.3 Effects of CWD retention of the structure of ECM fungal communities .................................................................................................. 43! 2.3.4 Comparison of ECM fungi occurring on roots and as hyphae in mesh bags....................................................................................................