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Long-term effects of nitrogen and phosphorus fertilization on ectomycorrhizal diversity of 18-year-old… Wright, Shannon Heather Ann 2006

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L O N G - T E R M EFFECTS OF NITROGEN A N D PHOSPHORUS FERTILIZATION ON ECTOMYCORRHIZAL DIVERSITY OF 18-YEAR-OLD WESTERN H E M L O C K (TSUGA HETEROPHYLLA) ON NORTHERN V A N C O U V E R ISLAND by S H A N N O N H E A T H E R A N N W R I G H T B . S c , Trinity Western University, 2002 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E F A C U L T Y OF G R A D U A T E STUDIES (Botany) T H E U N I V E R S I T Y OF BRIT ISH C O L U M B I A December 2006 © Shannon Wright, 2006 ABSTRACT This thesis explores the diversity of ectomycorrhizae of western hemlock (Tsuga heterophylla) in nutrient-poor environments and the role of fertilization on the ectomycorrhizal fungal community. In Chapter One, the thesis provides an introduction to mycorrhizal fungi and to my research site, a cedar-hemlock ecosystem on northern Vancouver Island where forest regeneration has been problematic. The first chapter continues with a review of previous studies of mycorrhizae from the same research site and of western hemlock. Chapter Two presents the results of my study on the effects of nitrogen and phosphorus fertilization on ectomycorrhizal species composition and diversity. Nitrogen fertilization typically reduces ectomycorrhizal diversity within the first two years of its application. Less is known about the long-term influence of fertilization. In this research, ectomycorrhizal diversity and community composition were compared among three fertilization treatments in plots of 18-year old western hemlock from northern Vancouver Island, Canada. O f nine plots, three were unfertilized controls. Six plots were fertilized in 1987 and 1997; three with 300 kg/ha urea (N); and three with the kg/ha N plus 100 kg/ha P (N + P). Four sets of 100 ectomycorrhizal root tips were sampled per plot and used for random clone libraries of amplified ITS regions using fungal specific primers ITS IF and TW13. Fungal species were identified from sequenced clones using fast parsimony analysis. Assuming that clones with > 97% identity were conspecific, 99 species were detected among 1004 clones sequenced. Fungal diversity was high and not significantly different across treatments. Species composition differed significantly in N + P plots compared to control plots or plots that received N alone. The presence of Cenococcum geophilum splO and Dermocybe cinnamomea were correlated with control and N plots, whereas the presence of Cenococcum geophilum sp8 and sp9, and Cortinarius spl8 and sp l9 were correlated with N + P plots. Chapter Three of this thesis discusses limitations of a molecular approach to detecting fungal diversity, and ideas for future research to help elucidate the ecological roles of ectomycorrhizal fungi. This thesis contributes new knowledge about the diversity of western hemlock's ectomycorrhizal fungi and shows that N + P fertilization used in forest management has resulted in a long-lasting change in the ectomycorrhizal fungal community composition. TABLE OF CONTENTS Abstract i i Table of Contents i i i List of Tables vi List of Figures v i i List of Symbols, Abbreviations, or Other v i i i Acknowledgements ix Dedication x i Co-Authorship Statement x i i C H A P T E R 1 General introduction and literature review 1 1.1 Thesis theme and objectives 1 1.2 Ectomycorrhizae 1 1.3 Problematic hemlock regeneration in a salal-dominated ecosystem and the Salal Cedar Hemlock Integrated Research Project (SCHIRP) 4 1.4 Ericaceous mycorrhizal contribution to the competitive ability of salal 5 1.5 Current knowledge of hemlock ectomycorrhizal fungal diversity 7 1.6 Diversity of ectomycorrhizal communities and role in plant performance 9 1.7 References 11 C H A P T E R 2 The effect of fertilization on the below-ground diversity and community composition of ectomycorrhizal fungi associated with western hemlock 16 2.1 Introduction 16 2.1.1 Molecular characterization of below-ground diversity of ectomycorrhizal fungi 18 2.1.2 Hypothesis 18 iii 2.2 Materials and methods 19 2.2.1 Site description 19 2.2.2 Sampling design 20 2.2.3 Amplif icat ion and cloning of fungal ITS r D N A 21 2.2.4 Sequence and phylogenetic analyses 22 2.2.5 Diversity measures and statistical analyses 23 2.2.6 Multivariate analyses 24 2.3 Results 26 2.3.1 Patterns of fungal diversity among clone libraries 26 2.3.2 Multivariate analyses o f species composition of fungi associated with hemlock roots from different fertilization histories 27 2.4 Discussion 31 2.4.1 Species composition in N + P plots differ from control and N plots 31 2.4.2 Three OTUs of Cenococcum and three of Cortinarius were correlated with plot fertilization history 31 2.4.3 Other studies have also found Cortinarius to be sensitive to nitrogen 32 2.4.4 Delimiting biologically relevant species 33 2.4.5 Regardless of fertilization treatment, diversity was high and communities were extremely heterogeneous 34 2.4.6 Increased host vigor and changes in site quality from fertilization could also be driving changes in the mycorrhizal fungal community 34 2.4.7 Diversity of fungal species associated with western hemlock broadened.... 3 6 2.5 Tables and figures 37 2.6 References 63 C H A P T E R 3 Discussion and conclusions 68 3.1 Heterogeneity o f ectomycorrhizal communities provides a challenge for sampling effort 68 3.2 Molecular identification of ectomycorrhizal populations 69 3.3 Unknown roles of hemlock fungal associates related to ericaceous mycorrhizae.70 3.4 Functional roles of ectomycorrhizal fungi in C H ecosystems 71 3.5 Correlation between tree performance and ectomycorrhizal community structure 72 3.6 References 75 iv C H A P T E R 4 Appendices 4.1 Multivariate community analyses LIST OF T A B L E S Table 2.1 Summary of mean values and standard error for Shannon's and Simpson's diversity indices, evenness, observed richness and richness estimates 37 Table 2. 2 Summary of variance explained of ectomycorrhizal fungal species composition by N M S axes 38 Table 2. 3 Pearson correlations for tree performance variables indirectly correlated with N M S axes 38 Table 2. 4 Canonical axis summary statistics 38 Table 2. 5 C C A trials for different working species definition 39 Table 2. 6 Summary table of M R P P results for significant differences in ectomycorrhizal presence/absence in the fertilization treatment groups 40 Table 2. 7 Indicator species values 41 Table 2. 8 Ectomycorrhizal fungi associated with western hemlock 42 Table 4.1 Multi-Response Permutations Procedures summary statistics 79 Table 4. 2 Pearson correlations with Non-metric Multidimensional Scaling ordination axes 80 Table 4. 3 Pearson correlations with Canonical Correspondence Analyses ordination axes 81 vi LIST OF FIGURES Figure 2.1 Dermocybe species phylogeny 50 Figure 2. 2 Cenococcum geophilum group phylogeny 51 Figure 2. 3 Pie chart showing the relative abundance of clones by taxonomic group..52 Figure 2. 4 Relative frequency of occurrence of O T U s in Cortinarius (including Cortinarius subgenus Dermocybe) in samples from each fertilization treatment 53 Figure 2. 5 Relative frequency of occurrence of fungal OTUs from ectomycorrhizal genera other than Cortinarius from samples from each fertilizer treatment. 54 Figure 2. 6 Relative frequency of occurrence of fungal OTUs from unidentified genera or genera not known to be ectomycorrhizal 55 Figure 2. 7 Rank abundance curves of ectomycorrhizal fungi detected in each of the fertilizer treatments 56 Figure 2. 8 Diversity of ectomycorrhizal OTUs was similar in control, N fertilized, and N + P fertilized plots. Measures used were: (a) Shannon's indices, (b) Simpson's indices, (c) evenness 57 Figure 2. 9 Species richness of ectomycorrhizal fungi was not significantly different across fertilizer treatment 58 Figure 2.10 Mean Coleman collector curves for each fertilization treatment 59 Figure 2.11 N M S ordination of community composition of ectomycorrhizal fungi 60 Figure 2.12 C C A ordination of community composition of ectomycorrhizal fungi 61 Figure 2.13 Venn diagram of fungal ectomycorrhizal diversity of western hemlock 62 VII LIST OF SYMBOLS, ABBREVIATIONS, OR OTHER C C A Canonical Correspondence Analyses D B H Diameter at Breast Height D N A Deoxynucleic Ac id I S A Indicator Species Analyses ITS Internal Transcribed Spacer of Ribosomal D N A M R P P Multi-Response Permutations Procedures N Nitrogen N M S Non-metric Multidimensional Scaling O T U Operational Taxonomic Unit P Phosphorus P C R Polymerase Chain Reaction R C L Random Clone Library R F L P Restriction Fragment Length Polymorphism S C H I R P Salal Cedar Hemlock Integrated Research Project viii ACKNOWLEDGEMENTS A great amount of thanks and gratitude goes to my supervisor Dr. Mary Berbee for guidance and assistance for this research project and enthusiasm for industry relevant projects. I would also like to thank Dr. Suzanne Simard for her co-supervision, especially during my first year at U B C . I would like to thank my committee members for their advice at several stages in this project. Dr. Cindy Prescott suggested that I study plots fertilized with both nitrogen and phosphorus in addition to control plots and nitrogen-fertilized plots. Dr. Suzanne Simard suggested that I analyze tree performance data along with the ectomycorrhizal fungal communities. Dr. Anthony Glass provided discussion on the competitive ability of salal. I greatly appreciated the assistance with editing from Dr. Mary Berbee, Dr. Cindy Prescott, and Dr. Suzanne Simard. Dr. Gary Bradfield helped with multivariate analyses and discussion on ecological topics. Dr. Clement Tsui provided guidance and discussion on fungal ecology. I would also like to thank Carol Wright for being my unpaid and oddly wi l l ing field assistant in a challenging ecosystem, SeaRa L i m for her assistance with the molecular work, Sarah Leckie for assisting with sampling and locating the Fertilization Demo Plots, Paul Kroeger for instruction on processing root tips and techniques for culturing fungi from mycorrhizal roots, and undergraduate students Matt Denis, Hannah Raphael, Emma Harrower, Laura Super for assisting with lab work. I would like to thank James Wright for writing a computer program that automated the merging of D N A sequence text files into single F A S T A format documents. Much of the molecular work would not have been completed in a timely manner i f Dr. Patricia Schulte had not generously shared lab equipment, especially P C R machines and centrifuge. Research funding was provided by FII and F IA Forest Science Program. Western Forest Products Ltd. sponsored an N S E R C Industrial Postgraduate Scholarship, provided accommodation at the study site, and staff at Port McNe i l l provided maps of the area and assisted with locating research sites. I am very grateful for Annette van Niejenhuis' assistance at Western Forest Products Ltd. and Ray Jacob's field guidance at the SCHIRP ix Installation. Advanced Systems Integrators generously provided a P C computer and software for various analyses. x DEDICATION I would like to dedicate this work first of all to my parents and my older brother James for the many years of inspiration to study and explore the natural world around us, to have a keen, endless interest and curiosity of the many mysteries in science, and for their encouragement and support all along the way. M y pursuit of this research was greatly attributed to several of my undergraduate professors at T W U who inspired me to pursue various areas in biology — Dr. Jake Hintz, whose guidance and patience turned a failing first-year biology student to someone enthusiastic and motivated to excel in biological sciences; Dr. David Clements and Dr. Paul Brown for nurturing a much greater appreciation and interest in botany; and Dr. Richard Paulton for creating a strong aspiration to pursue applied microbial research. Many thanks go to Dr. Mary Berbee, Dr. Gary Bradfield, Dr. Suzanne Simard, and Dr. Clement Tsui for all their guidance, help, and mentorship at U B C . xi CO-AUTHORSHIP STATEMENT This research project was initiated by Dr. Mary Berbee. The author of this thesis, Shannon Wright, was responsible for lab work, field work, and data analyses. Sampling design and writing of this paper were accomplished with the supervision and help of Dr. Mary Berbee. SeaRa L i m assisted with the molecular lab work. xii CHAPTER 1. General introduction and literature review 1.1. Thesis theme and objectives In my thesis, I investigated the below-ground diversity of ectomycorrhizal fungi associated with western hemlock in 18-year-old SCHIRP regeneration plots on northern Vancouver Island, B C , Canada. I used molecular techniques to detect fungal species from random clone libraries from pooled ectomycorrhizal tips. With phylogenetic analyses and comparisons with sequences from identified fungal species, I identified the sequenced clones and then compared the fungal community composition among fertilization treatments. The hypothesis tested was whether a history of fertilization with nitrogen and phosphorus influenced ectomycorrhizal fungal diversity and community composition. 1.2. Ectomycorrhizae Mycorrhizae are symbiotic association that form between the roots of plants and fungi (Harley, 1959; Marks and Kozlowski , 1973; Smith and Read, 1997). In mycorrhizal associations, the host plant provides the fungi with photosynthates, and the fungi provide the plant with nutrients and water from the surrounding soil (Smith and Read, 1997). Mycorrhizae are classified as either ectomycorrhizae or endomycorrhizae, depending on the morphological relationship with their host. Endomycorrhizal fungal hyphae penetrate into the root cells of their host plant, forming intracellular arbuscules or coils, whereas ectomycorrhizae grow in between the host root cells (Smith and Read, 1997). Ectomycorrhizal root tips characteristically have a mantle of fungal tissue surrounding the exterior of the root tip and a Hartig net consisting of hyphae growing between the epidermal and cortical cells. The Hartig net is probably the site of nutrient exchange between the host and the fungus. From the mantle, emanating mycelia extend beyond the root tips into the soil. The presence of a mantle and Hartig net, along with the absence of intracellular fungal penetration of host cells, distinguish ectomycorrhizae from other types of mycorrhizal relationships (Smith and Read, 1997). Ectomycorrhizal roots 1 receive more of a plant's photosynthate than do non-mycorrhizal roots. The direction of carbon flow is controlled by the sink strength of mycorrhizal roots (Buscot et al., 2000). Plants can compensate for the increased demand for carbohydrates caused by their ectomycorrhizal partners by increasing their rates of net photosynthesis (Lynch and Whipps, 1990). Ectomycorrhizae form mainly with woody perennials and about 3% of seed plant species have this association (Smith and Read, 1997). Although 3% is a small percentage of plant diversity, many of the plants dependent on ectomycorrhizal associations are the major or dominant components of forest ecosystems. As an example, the trees in the Pinaceae dominate boreal forests of the western hemisphere and almost all are ectomycorrhizal (Smith and Read, 1997). In a mutualistic relationship, ectomycorrhizae may function by taking over the nourishing of the host plant (Jones and Smith, 2004). The ectomycorrhizal fungal mantle completely surrounds the active rootlets, and so nutrients can only reach the host root cells v ia the fungi (Jones and Smith, 2004). The fungal hyphae extend into the soil beyond the plant's root system and zones of nutrient depletion, and so the association allows plants to access nutrients from a larger volume of soil (Bowen, 1973; Harley and Smith, 1983; Jones and Smith, 2004). Certain species of ectomycorrhizal fungi are also capable of mobil izing otherwise inaccessible nutrients in soils, such as complex organic forms of nitrogen (Bending and Read, 1996; Tibbett et al., 1999; Li l leskov et al., 2002; Hagerberg et al., 2003). Fungal hyphae translocate nutrients faster then nutrients could diffuse through the soil (Jones and Smith, 2004). Benefits to the plant are not only associated with increased nutrient uptake (Smith and Read, 1997; Buscot et al., 2000), but can also include resistance to pathogens (Sinclair et al., 1982; Perrin and Garbaye, 1983; Duchesne et al., 1989; Branzanti et al., 1999; Sen, 2001), improved water retention and drought tolerance (Lehto, 1992), and reduced uptake of heavy metals (Jones and Smith, 2004). Ectomycorrhizal fungi can modify root structure and growth, which in turn can affect nutrient uptake and vegetative growth (Jones and Smith, 2004). Nitrogen fertilization typically reduces ectomycorrhizal diversity within the first two years of its application. From the perspective of plants, ectomycorrhizal symbiosis is an adaptation to nutrient-poor conditions (Wallenda and Kottke, 1998). Positive growth 2 response of plants colonized by ectomycorrhizae typically disappear once available nutrients in soil reach concentrations that no longer limit plant growth, often in association with a reduction in mycorrhizal colonization (Jones and Smith, 2004). In nursery-grown seedlings, high nutrient levels and waterlogged planting media limit ectomycorrhizal development and root branching (Roth and Berch, 1992). Depending on the fungal and host plant genotypes and on environmental conditions, mycorrhizal associations can range from mutualistic to parasitic (Jones and Smith, 2004). Ectomycorrhizal fungal species differ in their ability to influence (either by promoting or depressing) plant nutrient uptake and growth (Burgess et al., 1993; Burgess et al., 1994). Mycorrhizal fungal species also vary in their ability to take up N . Jones et al. (2004) found that non-mycorrhizal Picea engelmannii seedlings absorbed more 1 5 N from nitrate, whereas seedlings colonized with Amphinema or Mycelium radicis atrovirens accumulated less (Jones and Smith, 2004). Some ectomycorrhizal fungi are capable of penetrating senesced parts of the root axis, becoming weak pathogens when the nutrient balance of the symbiosis is disturbed (Smith and Read, 1997). Ectomycorrhizal communities are extremely diverse. The diversity of ectomycorrhizal fungi may be high because available ecological niches offer different opportunities for resource partitioning, and diversity may depend on competition, disturbance, and various interactions with other organisms (Bruns, 1995). Even within single species forests, a high diversity of ectomycorrhizal fungi can be detected in small sites around a single host tree (Bruns, 1995). Byrd et al. (2000) detected an average of 8.3 species per core (392.5 cm ) and a total richness of 106 species when they used molecular identification to compare ectomycorrhizae of clear cut versus undisturbed lodgepole pine sites. Many fungal species can form ectomycorrhizal associations with the same species of plant (Bergero et al. , 2000) and the root system of a host plant is generally colonized by several different fungi (Smith and Read, 1997). Based on field observations of associations between hosts and sporocarps, an estimated 6000 species of fungi form ectomycorrhizal associations, of which 4500 are epigeous and the remainder are hypogeous (Smith and Read, 1997). Smith and Read (1997) caution that many of these fungi have not had their mycorrhizal status rigorously tested. O f the fungal species for which ectomycorrhizal status has been confirmed, certain species of are restricted to a 3 single plant, whereas others are generalists, forming mycorrhizal associations with a number of different plant species (Massicotte et al., 1994; Simard et al., 1997; Smith and Read, 1997; Bergero et al., 2000; Vralstad et al., 2002). Most ectomycorrhizal fungal species are basidiomycetes, a significant number are ascomycetes, and a few are zygomycetes in the genus Endogone (Smith and Read, 1997). Most basidiomycetes form ectomycorrhizal associations as dikaryons and have limited or restricted ability to form and maintain mycorrhizal associations as monokaryons (Smith and Read, 1997). 1.3. Problematic hemlock regeneration in a salal-dominated ecosystem and the Salal Cedar Hemlock Integrated Research Project (SCHIRP) Ectomycorrhizal fungi seemed likely to be important in nutrient relations at my study site, on which comprised of nutrient-limited hemlock regeneration plots in northern Vancouver Island. The plots are located in the Coastal Western Hemlock very wet maritime biogeoclimatic subzone (Pojar et al., 1991). Approximately 60% of this subzone is described as a SI ecosystem in the Lewis Classification (Lewis, 1982), and is characterized by western redcedar, western hemlock, amabilis fir, and salal (Fraser et al., 1995). This ecosystem includes two phases -the hemlock-amabilis fir (HA) phase and the cedar-hemlock (CH) phase. The H A phase is the younger forest (less than 100 years), and is dominated by hemlock and amabilis fir. The H A forests have dense canopies, a sparse salal understory, and high productivity. The H A phase probably resulted from a wind-throw event in 1908 and the consequent disturbance and soil mixing. The C H phase, on the other hand, was not affected by wind-throw and, until harvest, consisted of old-growth (more than 100 years old) western redcedar and western hemlock with an open crown, dense salal understory, and relatively low annual productivity (Lewis, 1982; Fraser et al., 1995). After clear-cutting at the C H sites, salal dominated the regeneration sites. Both naturally regenerated and planted conifer seedlings were performing poorly and western hemlock in particular had slow growth and chlorosis (Prescott and Weetman, 1994; Fraser etal . , 1995). To identify and understand the underlying cause of growth check in regeneration of C H sites, and to develop methods for improving tree growth, a long-term research project called the Salal Cedar Hemlock Integrated Research Project (SCHIRP) was 4 established. Deficiencies of N and P were identified as the cause of conifer growth check (Messier, 1993) and fertilization improved tree growth (Prescott and Weetman, 1994; Blevins and Prescott, 2002). L o w nutrient availability in the forest floor prior to clear-cutting, along with interference from salal, were leading causes of the N and P deficiencies (Messier, 1993; Fraser et al., 1995; Mal l ik and Prescott, 2001). The forest floor in the C H phase is wet and the cedar litter is both low in N and resistant to decomposition, resulting in low N mineralization (Prescott and Weetman, 1994). Litter inputs from salal are rich in phenolic acids, which further interferes with N mineralization by inhibiting decomposition enzymes (Cairney and Meharg, 2003). 1.4. Ericaceous mycorrhizal contribution to the competitive ability of salal Western hemlock and its ectomycorrhizal fungi are part of a larger community of organisms, and interactions with other fungal or plant species may influence the nutritional status of hemlock. On the C H sites, where regenerating unfertilized hemlock were stunted and chlorotic, the woody shrub salal was vigorous. Salal and hemlock roots are both located within the top organic layer (Bennett et al., 2002) but salal seems to be a stronger competitor for available nutrients (Fraser et al., 1995). The reason for the difference between the two species is unclear. It may result from intrinsic physiological differences between species. Host species may differ in sink strength for nutrients and salal may be a stronger sink especially for nitrogen (personal communication, Anthony Glass, U B C Botany). Alternatively, the difference in vigour may be related to the activities of mycorrhizal fungi. Earlier studies at the same sites investigated the role of ericoid mycorrhizal fungi in promoting growth of salal and possibly restricting growth of hemlock. The ericoid mycorrhizal fungi seemed to inhibit growth of ectomycorrhizal fungi. Using in vitro tests, (Xiao, 1994) examined the interaction between ericoid mycorrhizal isolates from salal roots (Acremonium strictum, Oidiodendron griseum and two unknown non-sporulating species) and ectomycorrhizae from hemlock roots (Pisolithus tinctorius, Rhizopogon semireticulatus and Suillus lakei). A l l interspecific 5 pairings between the ericoid mycorrhizae and the ectomycorrhizae resulted in inhibition of the ectomycorrhizal growth (Xiao, 1994). Ericoid mycorrhizae are well adapted to harsh edaphic environments (Perotto et al., 2002; Cairney and Meharg, 2003) and they produce a broad range of hydrolytic and oxidative enzymes to obtain nutrients from organic complexes (Xiao and Berch, 1999; Cairney and Ashford, 2002; Cairney and Meharg, 2003). Proteases and chitinases provide access to nitrogen from proteins or from chitin. Carbohydrolase and phenol-oxidizing activities may be important in mobil izing nitrogen and phosphorus from complexes found in dead plant cell walls (Bending and Read, 1996). The phenol-oxidizing enzymes produced by ericoid mycorrhizae may improve host root growth by increasing N availability, by detoxifying phenolic compounds in the soil, and facilitating protease access to proteins complexed with phenolic substrates (Bending and Read, 1996; Cairney and Ashford, 2002; Cairney and Meharg, 2003). Some recent studies suggest that ericoid mycorrhizal fungi may also form ectomycorrhizae when partnered with ectomycorrhizal host species (Perotto et al., 2002; Cairney and Meharg, 2003). Using ITS1 sequences for molecular identification, Vralstad et al. (2000) found that the ectomycorrhizal fungi that produced the Piceirhiza bicolorata morphotype were genetically related to the ericoid mycorrhizae, Rhizoscyphus ericae (formerly known as Hymenoscyphus ericae). However, in inoculation trials, only isolates of'Rhizoscyphus ericae of ectomycorrhizal origin, and not isolates of ericoid origin, were able to form the Piceirhiza bicolorata morphotype under axenic conditions (Vralstad et al., 2002). Ericoid mycorrhizal isolates, Rhizoscyphus ericae and a Variable White Taxon, did form mycorrhizal associations with Pxcea mariana under axenic conditions, however, these differed from typical ectomycorrhizae in having intra as wel l as intercellular colonization of both epidermal and cortical root cells (Piercey et al., 2002). Fungi in the family Sebacinaceae have been detected in ectomycorrhizae of Picea abies, Tilia sp., Eucalyptus oblique, Corylus avellana, Carpinus betulus (Urban et al., 2003), achlorophyllous and chlorophyllous orchids (Warcup, 1988), and have also been detected in ericoid mycorrhizal roots of salal (Berch et al., 2002). Bergero et al. (2000) detected ericoid fungi that could form root associates with Quercus ilex, however, under axenic conditions these fungi did not produce true ectomycorrhizae, but rather exhibited a poorly 6 developed mantle, epidermal cell penetration, or both. If the same fungal individuals can simultaneously participate in partnerships as ericoid mycorrhizae in salal and as ectomycorrhizae with hemlock, then their mycelia could serve as bridges for nutrient exchange between plant species. 1.5. Review of previous studies of hemlock ectomycorrhizal fungal diversity From previous studies, more than 200 species have been listed as possible ectomycorrhizal fungi on western hemlock (Table 2.8 in Chapter 2; (Trappe, 1962; Mol ina, 1980; Kropp, 1981a, 1981b; Kropp and Trappe, 1982; Kropp, 1982; Mol ina and Trappe, 1982; Kropp et al., 1985; Kranabetter and Wyl ie, 1998; O'Del l et al., 1999; Kranabetter and Kroeger, 2001; Kranabetter and Friesen, 2002; Trappe, 2004; Kranabetter et al., 2005) and I anticipated finding some of the same species in our investigation. Most of earlier studies were sporocarp surveys. From forests of western hemlock mixed with lodgepole pine in northwestern Brit ish Columbia, 130 ectomycorrhizal species were detected from mushroom surveys over a 3-year period (Kranabetter et al., 2005). In another northwestern Brit ish Columbia forest where western hemlock was mixed with western redcedar, Kranabetter and Kroeger (2001) identified 70 species in sporocarp surveys. They estimated that at least 30 additional unidentified species of Cortinarius were also present. O 'De l l et al. (1999) identified sporocarps of 150 ectomycorrhizal species in the Tsuga heterophylla zone in Oregon, U S A , in mixed forest ecosystems dominated with western hemlock and Pseudotsuga menziesii. Most diversity studies have not included experimental tests of whether the fungal species can form mycorrhizae. However, several earlier investigations by Mol ina (1980), Mol ina and Trappe (1982), and Kropp and Trappe (1982) did involve isolating pure fungal cultures from identified sporocarps that were associated with western hemlock. They used re-synthesis experiments to test the mycorrhizal status of isolates that grew in pure culture. From the sporocarp identifications, over 100 fungal species were suggested as likely ectomycorrhizal associates of western hemlock. O f the species that were cultured, 50 formed mycorrhizal associations in pot experiments or under axenic conditions (Molina, 1980; Kropp and Trappe, 1982; Mol ina and Trappe, 1982). 7 Surveys of sporocarps from hemlock forests typically detected higher ectomycorrhizal fungal diversity than surveys of morphotypes (types of ectomycorrhizal root tips distinguishable by morphology). This has probably been largely because morphotypes have too few diagnostic characters to allow identification of the fungal partner to species (Gi l l and Lavender, 1983; Kernaghan et al., 1995; Kranabetter and Wyl ie, 1998; Horton, 2002; Kranabetter and Friesen, 2002). Kropp (1982), for example, detected 11 morphotypes, without identifying the fungal partners to species. Kranabetter and Friesen (2002) used morphotyping of root tips from transplanted western hemlock seedlings and identified 16 species from among the 38 different morphotypes. Kranabetter and Wyl ie (1998) detected 44 ectomycorrhizal morphotypes from 144 excavated western hemlock seedlings and identified most of the fungi to genus. Using morphotyping, Kernagahn et al. (1995) detected Cenococcum geophilum as the most abundantly occurring morphotype (60%). A mantleless morphotype was the next most frequently encountered morphotype (20%) and 10 % of the root tips were colonized with a heterogeneous combination of Russula spp., Mycelium radicis atrovirens, Lactarius scrobiculatus, Thelephora terrestris, and rhizomorphic morphotypes. Using pot experiments containing natural soil collected from ericaceous dominated ecosystems, Smith et al. (1995) detected Thelephora, Cenococcum, Wilcoxina, Rhizopogon, Lactarius, Byssoporia, Laccaria, and Tuber types as well as Mycelia radicis myrtillus ( M R A ) as the most frequent mycorrhizae of western hemlock. Durall et al. (1999) detected 18 morphotypes on seedlings, most identified to fungal genus, whereas in their sporocarp survey in a neighboring forest, they detected 115 species along a 300 m of transect. Interestingly, molecular surveys of ectomycorrhizal fungal diversity of tree species other than western hemlock have shown just as much diversity below ground as above ground (Dahlberg et al., 1997; Horton, 2002). The DNA-based surveys can detect species present on roots having inconspicuous or hypogeous fruiting bodies, as well as species that lack fruiting bodies. Molecular approaches detect different species that produce similar morphotypes. For western hemlock, Horton et al. (2005) detected 26 morphotypes from excavated hemlock seedling root tips from Douglas-fir - western hemlock late serai stage forests. By matching D N A restriction length polymorphism 8 patterns (RFLPs) from sporocarps of voucher collections with R F L P s from root tips, only 8 species could be identified. Sequences of individual root tips revealed 45 fungal species among the morphotypes. The number of potential hemlock mycorrhizal species based on sporocarp surveys remains much greater than the numbers of species found to date in association with roots. The mycorrhizal status of the species known from sporocarps would be better supported i f the same species were found in association with roots, especially i f associated with a particular mycorrhizal morphotype. 1.6. Diversity of ectomycorrhizal communities and role in plant performance Whether the diversity of ectomycorrhizal fungi influences plant productivity remains an open question. Theoretically, it is possible that that it does (Cairney, 1999). It has been shown that the diversity of vesicular arbuscular mycorrhizal fungi influences the plant biodiversity and productivity (van der Heijden et al., 1998). Similarly, because of the ecological importance and the high degree of physiological variability of ectomycorrhizal fungal species, maintaining ectomycorrhizal diversity may be important for preserving critical ecosystem processes (Lazaruk et al., 2005). Different fungal species vary in their functional ability to explore different soil layers, access forms of nutrients, and outperform competing ectomycorrhizal species (Newton, 1992; Buscot et al., 2000; Agerer, 2001). Potentially, a high diversity of fungal partners could promote plant growth by broadening the plant's range of nutrient uptake capabilities and optimizing its foraging and mobil izing of nutrients (Buscot et al., 2000; Agerer, 2001). Some ectomycorrhizal fungal species may be functionally redundant, able to fulf i l l similar ecological functions (Anderson and Cairney, 2004). However, some studies of ectomycorrhizal fungal communities suggest that species succession takes place and that species may be portioned into guilds, like "early-stage" and "late-stage" fungi (Newton, 1992; Cairney, 1999). For the succession of fungal species to fol low a pattern, some species must have different ecological niches. Understanding the relationship between ectomycorrhizal fungal diversity and plant productivity in natural habitats would require reliable estimates of fungal species composition combined with data on function of the ectomycorrhizal fungal partners in 9 different combinations. However, reliable estimates of species composition, where D N A -based identifications are backed by sporocarp or morphotype surveys, are available for very few sites. Quantifying the structure and dynamics of fungal communities depends on repeated estimates of species composition over time, a time-consuming and expensive project that has yet to be undertaken to any great extent. Functional diversity is even less well understood than species diversity. Very few ectomycorrhizal species from natural populations are available in pure culture for experimentation and most experimental studies use only a few isolates (Burgess et al., 1993; Burgess et al., 1994). Designing experiments to test the roles of multiple fungal partners of the same plant w i l l be challenging. 10 1.7. References Agerer R (2001) Exploration types of ectomycorrhizae: A proposal to classify ectomycorrhizal mycelial systems according to their patterns of differentiation and putative ecological importance. Mycorrhiza 11: 107 - 114 Anderson IC, Cairney JWG (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environmental Microbiology 6: 769-779 Bending GD, Read DJ (1996) Nitrogen mobilization from protein-polyphenol complex by ericoid and ectomycorrhizal fungi. 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New Phytologist 126: 677-690 Messier C (1993) Factors limiting early growth of western redcedar, western hemlock and Sitka spruce seedlings on ericaceous-dominated clearcut sites in coastal Brit ish Columbia. Forest Ecology & Management 60: 181-206 Molina R (1980) Ectomycorrhizal inoculation of containerized western conifer seedlings. In. U S D A Forest Service Research Note PNW-357, Pacific Northwest Forest and Range Experiment Station, Corvall is, Oregon, pp 1-10 Molina R, Trappe J M (1982) Patterns of ectomycorrhizal host specificity and potential among Pacific northwest conifers and fungi. Forest Science 28: 423-458 Newton A C (1992) Towards a functional classification of ectomycorrhizal fungi. Mycorrhiza 2: 75-79 13 O'Dell TE, Ammirati JF, Schreiner EG (1999) Species richness and abundance of ectomycorrhizal basidiomycete sporocarps on a moisture gradient in the Tsuga heterophylla zone. Canadian Journal of Botany 77: 1699-1711 Perotto S, Girlanda M, Martino E (2002) Ericoid mycorrhizal fungi: some new perspectives on old acquaintances. Plant & Soil 244: 41-53 Perrin R, Garbaye J (1983) Influence of ectomycorrhizae on infectivity of Pythium-infested soils and substrates. Plant & Soil 71: 345-351 Piercey M, Thormann M, Currah R (2002) Saprobic characteristics of three fungal taxa from ericalean roots and their association with the roots of Rhododendron groenlandicum and Picea mariana in culture. Mycorrhiza 12: 175-180 Pojar J, Klinka K, Dermarchi DA (1991) Coastal western hemlock zone. In D Meidinger, J Pojar, eds, Ecosystems of Brit ish Columbia. Ministry of Forests, Victoria, B C , p p 9 5 - l l l Prescott CE, Weetman G (1994) Salal Cedar Hemlock Integrated Research Program: A Synthesis. In. University British Columbia, Vancouver, B C Roth AL, Berch SM (1992) Ectomycorrhizae of Douglas-fir and western hemlock seedlings outplanted on eastern Vancouver Island. Canadian Journal of Forest Research 22: 1646-1655 Sen R (2001) Multitrophic interactions between a Rhizoctonia sp. and mycorrhizal fungi affect Scots pine seedling performance in nursery soil. New Phytologist 152: 543-553 Simard SW, Molina R, Smith JE, Perry DA, Jones MD (1997) Shared compatibility of ectomycorrhizae on Pseudotsuga menziesii and Betula papyrifera seedlings grown in mixture in soils from southern Brit ish Columbia. Canadian Journal of Forest Research 27: 331-342 Sinclair WA, Sylvia DM, Larsen AO (1982) Disease suppression and growth promotion in Douglas-fir seedlings by the ectomycorrhizal fungus Laccaria laccata. Forest Science 28: 191-201 Smith JE, M o l i n a R, Perry DA (1995) Occurrence of ectomycorrhizas on ericaceous and coniferous seedlings grown in soils from Oregon Coast Range. New Phytologist 129: 73-81 Smith SE, Read DJ (1997) Mycorrhizal Symbiosis, Ed 2nd. 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New Phytologist 145: 549-563 Vralstad T, Schumacher T, Taylor AFS (2002) Mycorrhizal synthesis between fungal strains of the Hymenoscyphus ericae aggregate and potential ectomycorrhizal and ericoid hosts. New Phytologist 153: 143-152 Wallenda T, Kottke I (1998) Nitrogen deposition and ectomycorrhizas. New Phytologist 139: 169-187 Warcup JH (1988) Mycorrhizal associations of isolates of Sebacina vermifera. New Phytologist 110: 227-231 Xiao G (1994) The role of root-associated fungi in the dominance of Gaultheria shallon. PhD. University of Brit ish Columbia, Vancouver Xiao G, Berch SM (1999) Organic nitrogen use by salal ericoid mycorrhizal fungi from northern Vancouver Island and impacts on growth in vitro of Gaultheria shallon. Mycorrh iza9: 145-149 15 CHAPTER 2. The effect of fertilization on the below-ground diversity and community composition of ectomycorrhizal fungi associated with western hemlock 2.1. Introduction Most studies of the response of the ectomycorrhizal fungal community to fertilizer have focused on the effects of fertilizer within a year or two of application, or the effects of regular and repeated fertilizer application. We were interested in finding out whether fertilizer applied early in stand regeneration altered the diversity or community structure of ectomycorrhizal fungi several years after application. In this paper, we compare ectomycorrhizal diversity and community composition of 18-year-old western hemlock among three fertilization treatments that had been previously applied on northern Vancouver Island. We sampled the controlled, replicated plots of the Salal Cedar Hemlock Integration Research Project (SCHIRP). The SCHIRP plots had been established to test whether nitrogen and phosphorus fertilizer would mitigate growth check of regenerating western hemlock (Tsuga heterophylla) on northern Vancouver Island (Mehmann et al., 1995; Mal l ik and Prescott, 2001). Plots had been fertilized in 1987 and 1997 with 300 kg/ha urea (N); with 300 kg/ha N plus 100 kg/ha P (N + P); or left unfertilized as controls. Last fertilized seven years before the beginning of our study, these plots offered an excellent opportunity to evaluate whether fertilization had a long-term effect on the ectomycorrhizal fungal community. Several studies have shown that within a year or two of application, fertilizer influences mycorrhizal populations and diversity of western hemlock. The early responses of ectomycorrhizal fungi were death and a reduction in number during the first 9 months after an application of urea fertilization (Gi l l and Lavender, 1983). Eighteen months after application, the number of mycorrhizal tips from the fertilized and control plots did not differ, however, the relative abundance of species had been altered (Gi l l and Lavender, 1983). Ectendomycorrhizae increased significantly, black mycorrhizae decreased, and white and brown mycorrhizae responded variously, depending on the site. 16 Similarly, within 18 months of fertilization, Kernagan et al. (1995) found no significant change in the population structure of ectomycorrhizal fungal morphotypes, although they noted a trend towards reduction in the proportion of Cenococcum geophilum and mycorrhizal types lacking a mantle. Even though these studies grouped ectomycorrhizal root tips into morphotypes that included a mixture of fungal species, they both suggested that fertilizer could affect species composition. Studies of ectomycorrhizae of other forest types have also found changes in species composition after fertilization. Since fungal species differ both in their ability to use various forms of N and in their response to increased N availability (Ek, 1997; Bidartondo et al., 2001; Li l leskov et a l , 2002; Li l leskov and Bruns, 2003), short-term increases in N may alter the dominance structure of the ectomycorrhizal fungal community because differential sensitivities influence competitive interactions among fungal species (Wallenda and Kottke, 1998; Edwards et a l , 2004). Av is et al. (2003) found that fertilization changed species composition, as assessed by a combination of morphotypes and molecular identification of root tips, in an oak savanna with annual inputs of nitrogen. Similarly, in a pine forest, ectomycorrhizal diversity was significantly lower in N-fertil ized plots; changes in community structure in N plots included a loss of Lactarius theiogalus (a dominant species in control plots) and increase in Piloderma sp. (making it the dominant species of the N fertilized plots) (Frey et al., 2004). Applications of N often reduce sporocarp production (Wallenda and Kottke, 1998; Bidartondo et al., 2001). However, sporocarp production does not necessarily reflect the complete mycorrhizal community below ground, nor does it reflect the abundance of the species found on root tips (Gardes and Bruns, 1993; McCa ig et al., 1999; Li l leskov and Bruns, 2001; Peter et al., 2001; Av is et al., 2003). Given equivalent numbers of mycorrhizal tips, some fungal species can produce large numbers of fruiting bodies, while others might fruit sparingly. Possibly, when given nitrogen fertilizer, the fungi can reduce allocation to fruiting bodies even without change in the community structure below ground (Lil leskov and Bruns, 2001). 17 2.1.1. Molecular characterization of below-ground diversity of ectomycorrhizal fungi Unt i l the development of molecular techniques, mycorrhizal diversity could only be determined based on sporocarp production and root tip morphology (Horton and Bruns, 2001). The above-ground fungal diversity measured by sporocarp production was usually several times higher than the below-ground diversity as measured by morphotype frequencies (Horton, 2002). Different species of fungi can share similar morphotypes, and so the species counts from morphotypes probably underestimate diversity (Egger, 1995; Mehmann et al., 1995). With the application of P C R and sequencing, many genera, and in some cases species can be determined using fungal specific primers and phylogenic analysis of the sequences obtained (Horton and Bruns, 2001). Whole soil communities can now be studied by sequencing ribosomal D N A s that are first amplified using the polymerase chain reaction (PCR) and then separated using random clone libraries. This method, which was used first for determining bacterial community structure (Head et al., 1998; McCa ig et al., 1999), has been applied to studying the fungal communities in roots (Vandenkoornhuyse et al., 2002; A l len et al., 2003; Neubert et al., 2006) and soil samples (Smit et al., 1999; Chen and Cairney, 2002; Anderson et al., 2003; Jumpponen, 2003; Landeweert et al., 2003; Anderson and Cairney, 2004; Hunt et al., 2004; Neubert et al., 2006). Compared with morphotype surveys, molecular surveys of fungi can dramatically increase the numbers of species detected as associates of roots. Surveys based on molecular clone libraries can complement sporocarp and morphotype surveys in contributing to an understanding of which species are most prevalent or sensitive to changes in environmental conditions. 2.1.2. Hypothesis The purpose of this research project was to study the effect of earlier N and P fertilization on the ectomycorrhizal fungal community of western hemlock, by comparing fungal species detected as D N A sequences. We hoped to contribute basic information about which fungal species were frequent, and possibly important, partners of western hemlock. Our null hypothesis was that the diversity and community composition of 18 ectomycorrhizal fungi of western hemlocks from fertilized plots and unfertilized plots did not differ. The alternative hypothesis was that fertilization history changed the ectomycorrhizal fungal diversity and community. 2.2. Materials and methods 2.2.2. Site description This study took place on the 'Plot Fertilization Trials' of the SCHIRP research site on northern Vancouver Island, located between Port McNeil and Port Hardy, British Columbia, Canada (50° 60'N, 127° 35'W). The site is in the Coastal Western Hemlock very wet maritime biogeoclimatic subzone (Pojar et al., 1991). Soils are moderately to imperfectly drained Duric Humo-ferric Podzols with a reddish brown mineral B f horizon (Lewis, 1982). This area receives 1900 mm of precipitation annually (Negrave, 2004), with a low of 50 mm from May to August and a high of 150-250 mm during October to February (Lewis, 1982). Most of the annual precipitation is in the form of rainfall. The average temperature maximum of just over 17°C occurs during July and August, and the minimum of just below 0°C occurs during October to February (Lewis, 1982). The ecosystem of this site was identified as the S1 C H phase in the classification system developed by T. Lewis (1982) for Western Forest Products Ltd. the major species comprising forest vegetation are Tsuga heterophylla, Thuja plicata, Gaultheria shallon, Vaccinium parvifoliutn, Vaccinium ovalifolium, Rubus spectabilis, Cornus canadensis, Anaphalis margaritaceae, Blechnum spicant, Dryopteris expansa, and in some areas, Pinus contorta. This site is operated by Western Forest Products Ltd as part of Tree License 25 Block 4. In 1987, the site was clearcut and slashburned prior to planting. Plots 25 x 25 m in size with a 2.5 m buffer zone were established to study the effects of fertilizer treatments for achieving crown closure. Tree age was 7-years at time of plot establishment. Initial fertilizer treatments in twelve different combinations of nitrogen and/or phosphorus were applied in spring 1987. Three replicates of each treatment were randomly assigned to plots (Blevins and Prescott, 2002). Broadcast treatments were re-19 applied in 1997 as per the original prescriptions for each plot (personal communication, Annette Vaniejenhuis, Western Forest Products Ltd., 2004). The height, diameter, and volume of the hemlock trees were significantly increased with fertilization treatments. Foliar nutrient concentrations of nitrogen and phosphorus also improved with fertilization (raising nitrogen concentrations to adequate levels), whereas levels in the control plots remained inadequate (Blevins and Prescott, 2002). 2.2.2. Sampling design A total of nine plots were sampled at this site; three plots unfertilized, three plots fertilized with 300 kg/ha nitrogen (applied as urea), and three plots fertilized with 300 kg/ha nitrogen in combination with 100 kg/ha phosphorus (applied as triple superphosphate). Samples were obtained using a soil auger (diameter of 4.5 cm, to a depth of 10 cm) from four arbitrarily selected trees per plot. Under ideal circumstances, four cores were extracted at the crown edge of each tree in the four cardinal directions and then combined into a single sample. However, because of high coverage of slash, soil in the four cardinal directions was often not accessible due to large stumps and logs that could not be moved. In these cases, the available surface area of the soil along the crown edge was divided into four sections from which cores were collected. Hemlocks in close proximity to the one P. contorta tree in the study area were not used in order to avoid sampling pine roots by mistake. Diameter at breast height (DBH) and distance to crown edge were measured for each sample tree; distances among trees sampled were also measured. Samples were collected in October 2004, November 2004, and February 2005. One replicate plot per treatment was sampled during each collecting trip. Samples were stored at 4°C until processed. Sampling effort was staggered to allow for immediate processing of samples within a week after collection to ensure that saprophytic species did not overgrow samples and that D N A did not degrade due to senescence. Changes in ectomycorrhizal colonization during the winter season are typically minimal (Majdi et al., 2001; Cheng and Bledsoe, 2002; de Roman and de Miguel , 2005). The samples were washed over a 0.5mm sieve and mycorrhizal root tips were extracted from rinsed pieces 20 of the core under a dissecting microscope at magnifications of 20-40X. From each core, 50 root tips were pooled together with root tips from the three other cores of the same tree, resulting in 200 root tips per tree (50 root tips per core multiplied by four cores per tree). This typically depleted root tips from entire cores. Some root tips of each crude morphotype were examined for the presence of a Hartig net and for healthy cortical cells in cross-sections under 40X magnification. Senesced root tips with many tannin-filled cells were not used. Counts of mycorrhizal versus non-mycorrhizal hemlock root tips were not necessary, since all samples had 100% mycorrhizal fungal colonization on root tips. Composite samples of 200 root tips were divided into two duplicate samples of 100 root tips. These samples of 100 root tips were then placed into 1.5ml microcentrifuge tubes, snap-frozen in l iquid nitrogen, and then lyophilized for D N A extraction. Lyophi l ized samples were weighed to determine dry root-tip biomass and then stored at -20°C until D N A extraction. 2.2.3. Amplif icat ion and cloning of fungal ITS r D N A One sample of 100 root tips per tree was used to create a random clone library. Duplicate samples were set aside as a back-up in case difficulties arose during D N A extraction. Root-tip tissue was ground in l iquid nitrogen using a mortar and pestle. D N A was extracted from the composite samples of root tips using the Q I A G E N DNeasy Plant M a x i D N A kit (model 68163, Q I A G E N , Mississauga Ontario, 2004) following the manufacture's protocol. Only the first eluate from the D N A extraction was used for amplification. The P C R was used to amplify the Internal Transcriber Spacer (ITS) regions ITS1 and ITS2, the 5.8S ribosomal unit and the 5' end of the 28S large ribosomal subunit, using the fungal specific primers ITS IF and TW13 (Bruns, http://plantbio.berkeley.edu/~bruns/primers.html) to permit detection of the widest possible range of mycorrhizal fungal species. Initial amplification was accomplished using PuReTaq Ready-To-Go P C R Beads (Amersham Biosciences Lmt.) fol lowing manufacture's protocol. Final volume of the P C R mixture was 25 ul, containing 0.5 u M of each primer and a 1:20 dilution of D N A extract. The reaction parameters included an 2 1 initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 10 seconds, annealing at 55°C for 20 seconds, and elongation at 72°C for 30 seconds plus 4 additional seconds per cycle and then a final extension at 72°C for 7 minutes. P C R products were checked by electrophoresis on a 1% SeaKem (Cambrex B io Science Rockland, Inc.) agarose gel run at 100V for 1 hour, stained with ethidium bromide, and photographed under U V . To create clone libraries, the mixture of fungal r D N A fragments produced by P C R was sorted by cloning with chemically competent E-coli cells, using the T O P O T A Cloning kit from Invitrogen, following manufacture's instructions. One random clone library was created for each of the composite root samples, resulting in a total of 36 clone libraries (4 composite samples of 100 root tips per plot, 3 replicate plots per treatments, 3 treatments). Prior to cloning, P C R products were purified using E Z N A Cycle-Pure K i t (Omega Bio-tek, Inc, Doravil le U S A ) following manufacture's protocol. Two hundred colonies were randomly selected from each R C L and, rather than isolating plasmids as per kit instructions, the inserts were amplified from recombinant clones using M 1 3 R and M13F (primers specifically designed for sites on the T A vector flanking the D N A insert). Final volume of P C R mixture was 25 ul, containing 0.5 u M of each primer, 0.75 units Fermentas Taq D N A Polymerase (Fermentas Li fe Science), 0.2 m M dNTP (Invitrogen), and 1.75 m M M g C ^ . The reaction parameters included an initial denaturation at 94°C for 3 minutes, followed by 32 cycles of denaturation at 94°C for 20 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 30 seconds plus 4 additional seconds per cycle and then a final extension at 72°C for 7 minutes. P C R products were again checked on an agarose gel to verify that clones contained an insert and to screen for false positives. From each library, 28 amplified clones were arbitrarily selected and sent to Macrogen Inc. (South Korea) for sequencing using the primer ITS IF (28 clones per library, 36 libraries, resulting a total of 1008 clones sequenced). 2.2.4. Sequence and phylogenetic analyses 2 2 Sequence chromatograms were examined for errors and ambiguities, corrected manually using Autoassembler D N A software (Applied BioSystems Inc, Perkin Elmer Corporation), and converted into F A S T A format with Factura (Applied BioSystems Inc). Sequences were assigned to preliminary groups B L A S T searches. Chimeras were recognized by visual inspection of pairwise alignments from the blastn results. Often, chimeras could be identified by alignment of ITS 1 with one genus and alignment of ITS2 with another genus. Clones were exempt from exclusion i f ambiguous sequences originating from clones of independent libraries were found to have same sequence. Alignments were created using ClustalX and manually adjusted. Motifs for the flanking regions of the ITS1, 5.8S, ITS2, and 28S were identified and the ends of the 18S and 28S subunit D N A were excluded from phylogenetic analyses. Fast parsimony analyses were accomplished in P A U P * version 4.0b 10 (Swofford, 2003). Using a combination of the best match in GenBank and fast parsimony analyses, the sequences were identified to Operational Taxonomic Units (OTU). Except in the genus Cortinarius sect. Dermocybe, each O T U consisted of sequences that were 97% identical in the ITS1, 5.8S, and ITS2 regions. Sequences in Dermocybe that were 99% or more identical were identified as being from the same O T U . Sequences are available in GenBank (accession numbers DQ474311-DQ474385, DQ474392-DQ474757, and DQ481670-DQ482029). 2.2.5. Diversity measures and statistical analyses To examine how treatments affect ectomycorrhizal fungi, species composition and richness were determined from the frequencies of D N A recovered from the random clone libraries, though not true measures of individuals since P C R biases are not known. Shannon's FT ( H ' = -zZPimPi) a n d Simpson's D (D = 1 - £ p i ) diversity indexes (where p, is the proportion of individual species in a random clone library) and abundance measures based on Shannon's index were calculated. Random clone libraries were pooled for each plot and diversity measures were calculated and then averaged among plots of each treatment. The resulting indices for each treatment were tested for normality prior to comparing treatment effects on measurements of diversity, abundance 23 and richness. Data were tested for normality using the Shapiro-Wilk test, and for homogeneity of variance using Bartlett's test. Most data sets did not meet either the criterion of normality or of equal variance, so non-parametric Kruskal-Wall is tests were used to test for differences among treatments. Richness was estimated using Chaol and abundance-based coverage estimators ( A C E ) using Estimates software version 7.5.0 (Colwel l , University of Connecticut, U S A ) and 100 randomizations for each test. These are non-parametric richness estimators based on mark-release-recapture methods and they work by considering the proportion of species that have been observed more than once relative to the proportion that has been observed only once (Hughes et al., 2001). Analyses were run for individual random clone libraries as wel l as for random clone libraries pooled together for each plot. 2.2.6. Multivariate analyses Non-parametric multivariate analyses were applied to study treatment effect on species composition. Each random clone library was treated as a single sample and only presence/absence data of each species within a random clone library was used. A l l multivariate analyses were accomplished using P C O R D statistical software (version 4.34, M j M Software Design, Oregon, U S A ) . Non-metric Multidimensional Scaling (NMS) was used as an ordination technique to determine and display the overall structure of species presence/absence data among samples. It was also used as both a method for determining the dimensionality of the data set and to detect whether the structure in the response data was stronger than expected by chance. Sorenson's distance measure was used with the automatic settings of P C O R D (McCune and Mefford, 1999). This included a maximum number of iterations of 400, the starting number from a random seed, a starting number of axes of 6, and with 40 real runs and 50 randomized runs. The final instability criterion was 0.00001. Overlays of treatments were performed on the N M S ordinations to detect patterns between treatments. Biplot overlays used measured tree response variables, including D B H , distance to crown edge, and root-tip biomass. 24 Canonical correspondence analysis ( C C A ) was used to find correlations among variance in species composition, tree size, and tree performance. Statistical output from the C C A include: correlations among tree performance variables, eigenvalue of each canonical axis, cumulative variation explained by the first three axes, intraset correlations, and scores for dependent variables. Ordinations of the plots shown for the first two C C A axes were based on the linear combination (LC) scores of response variables (i.e. species composition) and included biplot values for vectors of tree performance variables. Axes were scaled using 'Centered with Unit Variance' and a compromise of species and sites. The significance of the relationships were determined via the Monte Carlo permutation test, based on 99 random trials and a significance level of 0.05. Multi-response Permutation Procedures (MRPP) , a nonparametric statistical method for testing the null hypothesis of no difference between groups (McCune and Grace, 2002), was used with Sorenson's distance measure to determine whether species composition among treatments were significantly different. Indicator Species Analyses were performed to determine whether particular species were significantly correlated with treatment. Monte Carlo test were run to determine whether indicator values were likely to occur by chance alone. 25 2.3. Results 2.3.1. Patterns of fungal diversity among clone libraries We identified 99 OTUs from among 928 unambiguous and non-chimeric sequences of ectomycorrhizal fungal species and fungal root associates of western hemlock. O f the 99 OTUs , 16 each corresponded to sequences from a single identified species in GenBank and 57 clustered with genera. Though 26 did not match an identified species or genus in GenBank, 11 of these did match unidentified environmental sequences. In most cases, O T U s identified using the criterion of shared sequence identity of 97% or higher were congruent with identifications of species in GenBank. However, the "Dermocybes " and C. geophilum were the two exceptions. Clones that matched GenBank sequences of sporocarps from five different, morphologically distinct species of Dermocybe (formally known as a subgenus of Cortinarius) were 99% identical, but were distinguished as separate monophyletic groups and were treated as separate O T U s (Figure 2.1). B y using the working definition of 97% sequence similarity, we found 8 different O T U s , all o f which had sequences identified as " C . geophilum" in GenBank as their best matches (Figure 2.2). The vast majority (91.6%) of the clone sequences were from species in ectomycorrhizal genera (Figure 2.3). Many OTUs were detected infrequently and 55% of the total clones sequenced represented 93 OTUs , each of which accounted for less than 5% of the total number o f clones sequenced. O f these OTUs , 52 occurred as singletons. The remaining 45% of clones represented six ectomycorrhizal OTUs , Pilodermafallax, Lactarius pseudomucidus, Lactarius s p l , Craterellus tubaeformis, Cenococcum geophilum sp8 and Cenococcum geophilum splO. Each of these 6 O T U s was responsible for 5-13%) of the clone sequences. Piloderma fallax and L. pseudomucidus occurred in high frequency among al l clone libraries of all treatments. Numbers o f clones of a species were not always uniform across samples (Figures 2.4, 2.5, and 2.6). Craterellus tubaeformis and Lactarius s p l , occurred infrequently among random clone libraries, but when they were present, they were extremely abundant. Cenococcum geophilum sp8 was 26 most abundant in samples from N + P plots and C. geophilum splO was most abundant in control and N plots. The remaining 8.4% of the clones sequenced were comprised of 32 species of uncertain or unlikely ectomycorrhizal status. Five were most closely related to dark septate endophytes, which included Phialophora finlandia and Rhizoscyphus. O f the sequences, 11 were 97-99%) similar to sequences in GenBank that were obtained from ectomycorrhizal root tips from environmental samples, 15 did not match any other GenBank sequence, and one was from the saprophytic ascomycete Chaetosphaeria. A l l plots and all treatments showed high diversity and heterogeneity in root associated fungi. Rank abundance curves showed little evidence of species dominance in any of the treatments (Figure 2.7) and the slopes of the curves were not significantly different, as determined by A N C O V A (F ratio 1.60, P-value 0.21). Shannon's FT, Simpson's D, and Evenness did not differ significantly among treatments at either the mean random clone library level or the mean plot level. A l l treatment groups had relatively high diversity and evenness (Figure 2.8, Table 2.1). Similarly, relative observed richness and estimated richness did not differ significantly among treatments. For all treatment groups, the total richness determined by the C h a o l , A C E , and Jack richness estimations was almost double the observed richness at both the random clone library and plot level (Figure 2.9, Table 2.1). Most of the Coleman collector curves for random clone libraries or for plots did not plateau, indicating that sampling effort was stil l insufficient to achieve saturation at either random clone library level or at the plot level (Figure 2.10). The few individual curves for random clone libraries that did reach saturation were typically dominated by C. tubaeformis or Lactarius s p l . 2.3.2. Multivariate analyses of species composition of fungi associated with hemlock roots from different fertilization histories Using N M S , we found structure within the community of fungal species, and detected both indirect correlations between fungal communities and tree performance, and an influence of fertilization history on fungal species composition. A 3-dimensional solution (final instability of 0.00007 after 400 iterations), determined by the autopilot mode of P C - O R D , best represented the variation in species composition. The ordination 2 7 axes accounted for a small amount of the total variance (Table 2.2). The final stress for the N M S solutions was 19.55. According to Clarke's rule of thumb, a final stress between 10-20 corresponds to a usable picture, but too much reliance should not be placed on the details of the ordination (McCune and Grace, 2002). Monte Carlo test results (with 50 runs of random data) indicated that the probability of a similar stress found by chance alone was 0.0196. Vectors in the biplot overlay indicated indirect correlations between tree size and root biomass. Distance to crown edge and D B H both had strong positive correlations with axis 1 (Figure 2.11, Table 2.3). A treatment overlay within the N M S ordination showed some separation of the N + P plots from the control and N plots on the first axis (Figure 2.11). A s seen in the ordination, the community structure of the N + P plots also grouped towards the right of the axis, indirectly revealing a correlation between the species composition with the larger tree size found in the N + P plots. A small but significant amount of variation in fungal species presence/absence could be accounted for by tree performance variables, judging by the C C A results (Table 2.4). A stable C C A solution was achieved with tolerances of l .OxlO" 1 3 . Monte Carlo test results for the first axis in each C C A were significant for both the eigenvalue (p-value = 0.003) and the species-tree performance correlation (p-value = 0.016). These results allowed for the rejection of the null hypothesis of the C C A Monte Carlo test of no relationship between the tree response variables and the environmental variables. The first canonical axis was most strongly positively correlated with D B H (r-value 0.885) and distance to crown edge (r-value 0.725), whereas root mass had the strongest negative correlation with the second axis (r-value -0.997) (Figure 2.12). As would be expected, inter-correlations among tree performance variables were moderate to strong ( D B H and crown edge had a correlation of 0.76, whereas root biomass had a value of 0.55 with D B H and 0.27 with crown edge). Total variance ("inertia") in tree response data was 6.488, which indicated high heterogeneity in the data. The large number of fungal OTUs and their low frequencies of occurrence presented a problem in C C A and contributed to stress in N M S . A l l C C A analyses were run with singlets removed, which reduced the richness to 47 OTUs. However, in general, when running a C C A , the number of cases should not exceed the number of variables because when the number of variables increases relative to the 2 8 number of cases, the results may become misleading, often indicating strong relationships even i f the predictors are random numbers (McCune and Grace, 2002). Although there were only three variables as a measure of tree performance, the number of fungal OTUs exceeded the number of cases even with the singlets removed. We next tried to reduce the number of variables by clustering related OTUs or by excluding infrequent OTUs and then running additional C C A and N M S analyses. By retaining the original species definition, but further reducing the number of taxa to 21 by excluding OTUs unless they occurred more than three times, the final stress in N M S analyses was further reduced to 14 and higher amount of variation was accounted for in C C A for the first axis (Table 2. 5). Less helpfully, when OTUs were clumped into genera, patterns and structure within fungal communities were largely lost as determined by the N M S stress and only minimal variation could be accounted for in C C A (Table 2.5). Though significant P-values for testing whether there is a correlation with tree performance variables was detected in the C C A when data was pooled at the genera level (Table 2.5), it was l ikely the symptom of having more variables than samples, since there was no structure within the species data. Multi-response Permutations Procedure (MRPP) was used to test for significant differences in fungal communities from different treatment histories and to measure heterogeneity of species data. M R P P comparison of treatment groups indicated a significant difference in species composition among treatments (Table 2.6). The chance-corrected within-group agreement values for all comparisons were less than or equal to 0.02, which indicates that heterogeneity within groups was almost equal to that expected by chance. Mult iple pair-wise comparisons between treatments indicated that fungal communities in N + P plots were significantly different from control and N plots, but those in control and N plots were not significantly different from each other (Table 2.6). Using Indicator Species Analysis, the presence of a total of six indicator species were significantly correlated with a particular treatment (Table 2.7). The presence of Cenococcum geophilum splO and Cortinarius cinnamomeus were correlated with control and N plots, whereas the presence of C. geophilum sp8 and sp9, and Cortinarius spl8 and spl9 were correlated with N + P plots. Because both treatment overlays in N M S ordinations (Figure 2.11) and M R P P results (Table 2.6) indicated that N and control plots 29 did not significantly differ in species composition, control and N plots were pooled in the Indicator Species Analysis. Interestingly, all indicator species were highly correlated with N M S ordination axes. 30 2.4. Discussion 2.4.1 Species composition in N + P plots differ from control and N plots Our study for the first time showed that a history of fertilization changed mycorrhizal fungal species composition in western hemlock. Even though plots had not been fertilized since 1997, species composition was significantly different in N + P plots versus the control or N plots as detected using M R P P . G i l l and Lavendar, (1983) in an earlier study of western hemlock, did see a significant difference in ectomycorrhizal morphotypes within the first 18 months of urea fertilization, but they did not investigate changes in subsequent years. Kernaghan et al. (1992) did not detect a change in community structure within 11 months of fertilization. Differences in ectomycorrhizal fungal composition between N + P communities compared to control and N plots were probably driven largely by presence and absence of a few indicator species that were directly correlated with plot type, based on Monte Carlo tests. The cumulative effects of differences in presence and absence of other species, which were indirectly correlated in N M S ordinations with plot treatment history, may also have contributed to the significant differences detected by M R P P . 2.4.2. Three OTUs of Cenococcum and three of Cortinarius were correlated with plot fertilization history Using indicator species analyses and considering OTUs as 'species', the presence of three OTUs of Cortinarius and three OTUs of C. geophilum were significantly correlated with treatment. The presence of C. geophilum splO and C. cinnamomeus were correlated with control and N plots, whereas C. geophilum sp8 and sp9, and Cortinarius spl8 and sp l9 were correlated with N + P plots. Differences in species composition among treatments could be detected only when individual OTUs were the units being analyzed. When congeneric OTUs were pooled, species composition differences were no longer significant (Table 2.5). This result showed that closely related OTUs responded differently to fertilization history, and led us to conclude that species definition may be important to understanding differences in ectomycorrhizal physiology. When studies 31 based on morphotyping have not detected significant differences among treatments, it may be because the species that responded differently had been pooled because of their indistinguishable root tip morphology. It raises the possibility that groups below the level of our OTUs may also respond differently and i f we had more information on the population genetic structure within species, we might have found genotype-specific, within-species differences in response. 2.4.3. Other studies have also found Cortinarius to be sensitive to nitrogen In studies of the effects of fertilization on ectomycorrhizal fungal diversity, Cortinarius spp. have typically been reported as nitrogen-sensitive species that act as indicator species for enhanced N levels (Baar, 1995; Brandrud, 1995; Baum and Makeschin, 2000; Li l leskov et al., 2001; Peter et al., 2001; Li l leskov et al., 2002). In studies with ongoing annual fertilization applications, the overall colonization by Cortinarius species was often reduced or eliminated all together. In both greenhouse and field experiments with pine trees, Cortinarius morphotypes were restricted to low levels of N (Dighton et al., 2004). In an oak savanna, N fertilization caused the overall abundance of Cortinarius spp. to decline both above and below ground, and also caused Cortinarius spp. to fail to produce resistant propagules (Avis et al., 2003; Av is and Charvat, 2005). Three years after the end of an 11-year regime of annual fertization in a poplar plantation, two Cortinarius species were significant indicators of treatment -Cortinarius uliginosus occurred only in control plots, whereas Cortinarius croceocaeruleus occurred only in N plots (Baum and Makeschin, 2000). 32 2.4.4. Delimiting biologically relevant species Unfortunately, species delimitation was the most challenging in Cortinarius and Cenococcum, two important genera that included the indicator OTUs that responded differently to differences in fertilization history. Clones of Cortinarius were the most frequently encountered sequences, occurring among all random clone libraries of all treatments. Cortinarius, represented by 37 OTUs, had the greatest number of different OTUs of any genus in this study. A s one of the largest genera of gilled basidiomycete fungi, Cortinarius includes many undescribed species (Peintner et al., 2004). Our 97% cut-off for O T U definition was arbitrary. In general, Karen et al. (1997) found that the ITS regions were not variable enough to distinguish many ectomycorrhizal species. Some species in Cortinarius in particular had low interspecific variation in the ITS regions (Karen et al., 1997). In our study we believed we found an example of low interspecific variation among species in Dermocybe, a subgenus of Cortinarius. Dermocybe species are usually readily distinguishable based on morphological characters, but their ITS sequences were more than 97% identical. On the assumption that morphological characters represent differences among real species, we treated Dermocybe sequences as being different OTUs unless they were at least 99% identical. However, even a morphologically defined species may include multiple biological species, as was shown to the be case for Cortinarius rotundisporus (Sawyer et al., 1999). As in several earlier studies, we found high genetic variability among sequences that all had 'Cenococcum geophilum'' as their best GenBank match. A s in earlier studies, fungi with different sequence variants also had different ecological preferences (LoBugl io, 1999; Shinohara et al., 1999; Panaccione et al., 2001; LoBugl io and Taylor, 2002). Cenococcum geophilum populations on serpentine vs. non-serpentine sites differed genetically and may have had different physiological and functional characters (Panaccione et al., 2001). Jany et al. (2002) and Doughan and Rizzo (2004) presented evidence from multiple genetic loci that Cenococcum geophilum is composed of cryptic species, which can coexist within a single soil sample. Consistent with both of these studies, we found 8 OTUs among our Cenococcum geophilum sequences. Three of these OTUs had different ecological preferences based on our analyses that showed that one of the OTUs was an indicator of the control and N plots, and two were indicators of the N + 33 P plots. Unl ike the Cortinarius species, Cenococcum geophilum isolates are readily culturable. N o w that genetic tools are available for distinguishing closely related isolates, the Cenococcum geophilum species complex would be a good choice for functional ecological studies, expanding upon earlier studies by LoBugl io and Panaccione. 2.4.5. Regardless of fertilization treatment, diversity was high and communities were extremely heterogeneous High heterogeneity, with only a few common species and many rare species, may be the usual structure of ectomycorrhizal communities (Taylor, 2002). However, diversity as determined by ribosomal D N A sequence does not necessarily account for the biochemical or physiological groups affecting ecological processes and function (Mitchell and Zucarro, 2006). Mitchel l and Zuccaro argue that functional groups and their role in the community need to be accurately identified to explain the extensive diversity, and recommend identifying the protein-coding genes to discern ecologically distinct fungal groups, species, or populations (Mitchell and Zucarro, 2006). 2.4.6. Increased host vigor and changes in site quality from fertilization could also be driving changes in the mycorrhizal fungal community Mycorrhizal symbiosis is influenced by host health, the associated fungi, and the environmental conditions, all of which can alter the functional role of the fungi involved or fungal species associated (Jones and Smith, 2004). In our study a correlation between tree size and fungal community was detected across, but not within, treatments. Ours was a correlative study, and therefore we do not know i f the tree health and vigor caused by the fertilization were driving the changes in the fungal community or i f the fertilization in some way directly affected the mycorrhizal species. Even without a difference in fertilization, and even though all trees were clones grown under the same conditions, growth rates of Norway spruce (Picea abies) trees were correlated with ectomycorrhizal diversity and species composition (Korkama et al., 2006). Either some combinations of ectomycorrhizal fungal species improved tree performance, or trees that performed 34 differently selected for different combinations of ectomycorrhizal species (Korkama et al., 2006). Under conditions of mild nutrient deficiency, ectomycorrhizal associations typically increase nutrient uptake (Harley and Smith, 1983). Fertilization causes an immediate change in nutrient availability and a reduction in carbohydrate allocation to the roots from the host (Champigny, 1995; Wallenda and Kottke, 1998; Buscot et al., 2000). In this study, the fertilization trials were established because of failure of tree growth due to extremely low nutrient availability (Blevins and Prescott, 2002). By correcting a severe nutrient deficiency via fertilization, hemlocks were able to achieve adequate foliar nutrient concentrations, and had increased biomass (Blevins and Prescott, 2002; Negrave, 2004). Optimal levels of fertilization that correct for insufficient nutrient status could in turn result in trees with increased photosynthates available to their mycorrhizal symbionts (Lynch and Whipps, 1990; Wardle et al., 2004). Increased root growth and density would provide increased niche availability for the establishment of mycorrhizal fungi (Bruns, 1995; K ing et al., 2002). Mycorrhizal communities could also be influenced by environmental conditions, including soil properties (Cairney, 1999; Hagerberg et al., 2003; Li l leskov and Bruns, 2003). Other SCHIRP silvicultural trials have shown that long-term improvements in nutritional site quality in C H ecosystems occur with N + P fertilization. Humus from plots fertilized with N + P had increased mineral N , reduced salal leaf tannin concentrations, and increased microbial activity 10-13 years after a single fertilizer application (Bradley et al., 2000; Bennett et a l , 2003). Plots fertilized with N + P also have increased dry mass in the L and F layer of the forest floor (Leckie et al., 2004). In this study, soil structure may have been improved in fertilized plots in which trees had faster growth rates. This could have selected for indicator mycorrhizal fungi with different substrate preference (Harvey et al., 1978; Tedersoo et al., 2003). Future studies using a multivariate approach such as C C A could help identify environmental correlations of fungal community structure with differences in soil properties and different fertilization histories. 35 2.4.7. Diversity of fungal species associated with western hemlock broadened We detected a total of 99 OTUs as defined by 97% sequence similarity. Based on collection curves and richness estimates, the actual number of OTUs which could be detected by P C R and sequencing may have been double the observed number within random clone libraries and among samples of a given plot. Even among collector curves from individual clone libraries, only 7 of the 36 showed signs of reaching sampling saturation and none of the individual curves for plots showed saturation. The total number of fungal species associated with the roots may have been even higher, assuming that P C R amplification biases reduced the chance of detecting some species. This means that the total number of species is high and makes the estimation of the total number of species difficult. Even though we detected fewer than half of the possible OTUs that may have been present, this study still broadens the knowledge of diversity of fungi that may be ectomycorrhizal partners of western hemlock. O f the 99 OTUs detected, only 9 species matched the ectomycorrhizal fungal species previously known to be associated with western hemlock (Figure 2.13, Table 2.8). In conclusion, we found that that species composition, but not diversity, evenness or richness, differed significantly in N + P plots compared to control plots or plots that received N alone, even 7 years after fertilization. Species in the same genus, in Cortinarius and in Cenococcum responded differently to fertilization history. The difference between treatments may be due to the general improvement in tree vigour in N + P fertilized plots compared with control plots or N plots. We found many more species than had previously been detected on western hemlock roots, but not as many as had been reported from sporocarp surveys of hemlock forests. A l l indications are that much of the diversity of ectomycorrhizal fungi of western hemlock remains to be discovered and characterized. 36 2.5. Tables and figures Table 2 . 1 . Summary of mean values and standard error for Shannon's and Simpson's diversity indices, evenness, observed richness and richness estimates. Mean diversity values are shown for random clone libraries (n = 12), plots (n = 3) and per treatment. Fertilizer treatment Kruskal-Wallis Control N N + P P-value Diversity at random clone library (mean no. spp + SE) Evenness 0.72 + 0.06 0.78 + 0.01 0.80 + 0.04 0.32 Shannon's H' 1.45 + 0.22 1.45 + 0.10 1.68 + 0.17 0.46 Simpson's D 0.62 + 0.08 0.69 + 0.02 0.71 + 0.05 0.47 Diversity at plot (mean no. spp + SE) Evenness 0.83 + 0;01 0.80 + 0.02 0.83 + 0.02 0.30 Shannon's H' 2.62 + 0.10 2.43 + 0.04 2.66 + 0.23 0.43 Simpson's D 0.89 + 0.01 0.87 + 0.01 0.90 + 0.02 0.29 Diversity at treatment (across all plots per treatment) Evenness 0.84 0.80 0.78 nd Shannon's H' 3.30 3.10 3.11 nd Simpson's D 0.95 0.93 0.92 nd Richness estimates at random clone library (mean no. spp + SE) Observed richness 7.67 + 1.16 7.00 + 0.85 8.50 + 0.98 0.55 Chad 11.95 + 2.27 15.25 + 5.08 15.74 + 3.71 0.75 ACE 13.42 + 2.81 19.47 + 7.69 15.45 + 2.70 0.76 Jackl 11.28 + 1.83 10.45 + 1.67 12.58 + 1.62 0.68 Jack2 13.49 + 2.22 13.09 + 2.42 14.85 + 2.01 0.77 Richness estimates at plot (mean no. spp + SE) Observed richness 23.67 + 3.18 21 67 + 2.67 25 67 + 3.76 0 72 Chaol 35.53 + 4.50 44 56 + 15.66 57 00 + 16.80 0 50 ACE 31.92 + 5.98 44 00 + 15.05 47 27 + 8.47 0 39 Jackl 32.23 + 5.24 31 87 + 5.99 38 20 + 5.77 0 66 Jack2 36.20 + 6.33 39 10 + 9.41 47 03 + 7.16 0 44 Richness estimates at treatment (across all plots per treatment) Observed richness 51.00 49.00 55.00 nd Chad 67.06 83.57 127.90 nd ACE 67.49 88.37 97.65 nd Jackl 67.95 70.93 81.91 nd Jack2 75.92 85.86 103.77 nd 37 Table 2. 2. Summary of variance explained of ectomycorrhizal fungal species composition (n = 36) by NMS axes. I Axis Variance explained Cumulative 1 0.188 0.188 2 0.244 0.432 3 0.248 0.679 Table 2. 3. Pearson correlations for tree performance variables (n = 36) indirectly correlated with N M S axes. Strongest correlations are indicted with bold font. Variable axis 1 axis 2 axis 3 D B H 0.658 -0.015 -0.029 Canopy edge 0.499 -0.002 0.159 root mass 0.313 0.356 -0.207 Table 2. 4. Canonical axis summary statistics. Testing for relationships between ectomycorrhizal fungal species composition and tree performance variables (n = 36). Total variance ("inertia") in the tree response data = 6.488 axis 1 axis 2 axis 3 Eigenvalue 0.357 0.259 0.137 Variance in species data % of variance explained 5.5 4.0 2.1 Cumulative % explained 5.5 9.5 11.6 Pearson Correlation, Spp-Envt 0.904 0.839 0.748 Kendall (Rank) Corr., Spp-Envt 0.565 0.556 0.644 38 Table 2. 5. C C A trials for different working species definition. Testing for structure within ectomycorrhizal species composition and correlations with tree performance variables using different limits for defining the working operational taxonomic units (n = 36). Significant P-values (P < 0.05) and relatively high amount of variation accounted for by tree performance variables are indicated with bold font. Trial Variation in species data accounted for by tree Number performance variables measured ofOTU's Axis 1 Axis 2 P-value for testing whether there is structure within the species data P-value for testing whether there is significant correlation with tree performance variables Original CCA, data pooled at species level. Rare species defined by only occuring once in the data set were removed 48 5.7 0.003 0.002 Data pooled at genera level. 45 4.7 Data pooled at genera level, 15 8.3 but unkowns removed 4 4.5 0.383 0.021 0.008 0.015 Data pooled at genera level, singlets removed 26 6.2 4.7 0.019 0.018 Data pooled at genera level, singlets removed, C. geophilum grouped by degree of sequence similarity instead of genus name 28 6.2 5.2 0.013 0.033 Data pooled at species level, rare species defined by occuring less that three times within the data set removed 21 10.4 4.5 0.001 0.001 Data pooled at species level, only species detected as indicator species, or strongly correlated with the original NMS axis that showed the separation of the groups were kept 19.6 4.9 0.001 0.001 39 Table 2. 6. Summary table of MRPP results for significant differences in ectomycorrhizal presence/absence in the fertilization treatment groups Significant P-values (P < 0.05) are indicated with bold text. Pair-wise comparison ^ All plots Control vs N Control v sN + P N v s N + P Chance-corrected within-group agreement: A 0.02 -0.01 0.02 0.02 P-value 0.03 0.70 0.02 0.01 40 Table 2. 7. Indicator species values (% of perfect indication, based on combining relative frequency) significantly correlated with a treatment group (P < 0.05), as determined by Monte Carlo test of significance of observed maximum indicator value for species (10000 permutations), are denoted by an asterisk (*) and with bold font. Indicator Value Indicator Value species Control & N N + P species Control & N N + P Ceno 10 51* 1 Derm cin 17* 0 Ceno 11 23 2 Derm ida 3 11 Ceno 2 4 0 Derm mal 8 0 Ceno 3 8 0 Derm sem 8 8 Ceno 4 4 0 Ecto 11 7 3 Ceno 5 4 0 Ecto 110 4 0 Ceno 7 4 4 Ecto 112 4 0 Ceno 8 16 54* Ecto 12 0 8 Ceno 9 0 42* Ecto 13 4 0 Chaeto 4 0 Ecto 14 4 0 Clado 0 8 Ecto 15 4 0 Derm 1 4 0 Ecto 16 4 0 Derm 2 4 0 Ecto 17 4 0 Derm 3 0 8 Ecto 18 4 0 Hymeno 8 0 Ecto 19 4 0 Phialop 4 4 Heb inc 4 0 Thel 1 0 8 Heb sub 4 0 Thel2 0 8 Heb 1 0 8 Tylo 1 21 Heb 2 8 0 Cort 1 4 0 Hem 1 17 0 Cort 10 4 0 Hem 10 1 6 Cort 11 4 0 Hem 12 0 17 Cort 12 7 3 Hem 14 8 0 Cort 13 0 25 Hem 15 4 0 Cort 14 0 8 Hem 17 4 0 Cort 15 4 0 Hem 18 4 0 Cort 16 0 8 Hem 2 1 6 Cort 17 4 0 Hem 3 .0 8 Cort 18 0 33* Hem 4 1 6 Cort 19 0 38* Hem 5 4 0 Cort 2 0 8 Hem 6 7 3 Cort 20 0 8 Hem 7 4 4 Cort 21 0 17 Hem 8 1 6 Cort.22 1 13 Hem 9 0 8 Cort 23 0 8 Lac pseu 16 29 Cort 24 1 6 Lact 1 4 4 Cort 25 0 8 Leo ver 8 0 Cort 26 7 3 Phial fo 7 3 Cort 27 4 0 Phial fi 1 21 Cort 28 4 0 Pilo cro 4 0 Cort 29 0 8 Pilo fal 32 28 Cort 3 4 4 Pilo 1 4 0 Cort 4 4 0 Pilo 2 8 8 Cort 5 4 4 Pilo 3 4 0 Cort 6 8 0 Pilo 4 1 6 Cort 7 4 0 Rus 1 0 8 Cort 8 4 0 Tome sub 1 21 Cort 9 4 0 Trie dry 1 6 Crate 9 14 Trie 1 8 0 Derm aur 15 7 Trie 2 4 0 41 Table 2. 8a. Ectomycorrhizal fungi associated with western hemlock: source and location of identification indicated by asterisk (*), and abbreviated literature citations (full author names and references provided at end of table). In the cases of surveys from forests with mixed ectomycorrhizal host trees, fungal species not confirmed to be associated with western hemlock (possibly an associate of another ectomycorrhizal host tree were excluded from this list). Species detected in this research, defined by B L A S T match or 97% working species definition, are indicated under Wright random clone library (RCL) indicated by dot (•). Species detected in the research that have also been identified as hemlock ectomycorrhizae in other studies are indicated with bold font. Species Identified/detected by ~ ra vt -s o •2 o £ « .= 60 " -5 V P-£ o e o o ^ 00 t/) S S Oregon  r o Washington » Interior BC 3 Reference(s) Albatrellus flettii * * * Kropp & Trap (1982), Kran et al. (2005) Alpova alexsmithii * * Kropp & Trap (1982) Amanita aspera * * * Trap (1962); Kropp & Trap (1982) Amanita fulva Kropp & Trap (1982) Amanita gemmata * Trap (1962); Kropp & Trap (1982) Amanita muscaria * * * * Trap (1962); Kropp & Trap (1982), Kran et al. (2005), Mol & Trap (1982) Amanita pantherina * * * Kropp & Trap (1982) Amanita porphyria * * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Amanita smithiana * * Kropp & Trap (1982) Amanita vaginala * * Trap (1962) Amphinema byssoides * * Kran & Fries (2002), Kran & Wyl (1998) Amphinema sp * * Dur et al. (1999) Astraeus pteridis * Mol & Trap (1982) Barssia oregonensis * Kropp & Trap (1982) Boletopsis subsquamosa * * Kran et al. (2005), Kran & Kroeg (2001) Boletus appendiculatus * Kropp & Trap (1982) Boletus edulis * * * Trap (1962); Kropp & Trap (1982), Mol & Trap (1982) Boletus fragrans * * Kropp & Trap (1982) Boletus mirabilis * * * Kran et al. (2005), Kran & Kroeg (2001), Thiers (1975a); Kropp & Trap (1982), Dur et al. (1999), O'Dell et al. (1999) Boletus porosporus * * Kropp & Trap (1982) Boletus pulverulentus * * Trap (1962) Boletus subtomentosus * * Trap (1962); Kropp & Trap (1982) Boletus zelleri * * * Trap (1962); Kropp & Trap (1982), O'Dell et al. (1999) Byssocortium sp. * * * Hort et al. (2005) Byssoporia terresiris * * * Zak (1969), Zak & Larsen (1978), Kropp (1982a) Camarophyllus borealis * * Kropp & Trap (1982) Cantharellus cibarius * * Trap (1962); Kropp & Trap (1982) Cantharellus infundibuliformis * * Kran & Kroeg (2001), Dur et al. (1999) Cantharellus tubaeformis * * * Kropp & Trap (1982), O'Dell et al. (1999) 42 Table 2. 8a. Continued Species Identified/detected by « £• .a & 2 o 1 3 .c OB C 5 O P-^ o n c/5 S S Oregon o Washington » 5" Interior BC Reference(s) Cenococcum geophilum a * * * * * * Kran & Fries (2002), Kran & Wyl (1998), Kropp et al. (1985), Hort et al. (2005), Kropp & Trap (1982), Kropp (1982a), Mol & Trap (1982), Boullard (1968), Kropp (1982b, 1982c), Mol (1980), Trap (1962); Kropp & Trap (1982), Dur et al. (1999) Cenococcum geophilum (9 species) • Chamonixi caespitosa * * Kropp & Trap (1982) Chroogomphus rutilus * * Kran et al. (2005), Dur et al. (1999) Chroogomphus tomentosus * * » * Trap (1962), Miller (1981); Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Clavulinoid * * * Hort et al. (2005) Cortinarius alboviolaceus * * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Cortinarius armillatus * * Kran & Kroeg (2001), Dur et al. (1999), Kran et al. (2005) Cortinarius aurantiobasis • Cortinarius boulderensis * * Kran & Kroeg (2001), O'Dell et al. (1999) Cortinarius cinnamomeus • * * Kran et al. (2005), Kran & Kroeg (2001), Kran & Fries (2002), Dur et al. (1999) Cortinarius delibutus * * * Kropp & Trap (1982) Cortinarius glaucopus * * Kran & Kroeg (2001), Dur et al. (1999) Cortinarius griseoviolaceus * * * Kropp & Trap (1982), O'Dell et al. (1999) Cortinarius idahoensis • * * O'Dell et al. (1999) Cortinarius malicoria • * * O'Dell et al. (1999) Cortinarius mucosus * * Kropp & Trap (1982) Cortinarius mutabilis * * * * * Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Cortinarius semisanguineus • * * * * Kran et al. (2005), Kran & Kroeg (2001), Kran & Fries (2002), Dur et al. (1999), O'Dell et al. (1999) Cortinarius sp. * * * Hort et al. (2005) Cortinarius (32 species) • Cortinarius subscaurus * * Kran et al. (2005), Kran & Kroeg (2001) Cortinarius traganus * * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Cortinarius vanduzerensis * * * Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Cortinarius vibratilis * * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) 43 Table 2. 8a. Continued Species Wright RCL E Sporocarp § Synthesis S: Molecular §• CL Morphotype <?" Oregon o Washington g. Interior BC Reference(s) Cortinarius zakii * * Kropp & Trap (1982) Craterellus tubaeformis s * * * * * Kran et al. (2005), Trap (2004) E strain * * Kran & Wyl (1998), Dur et al. (1999) Elaphomyces granulatus * * * Kropp & Trap (1982), North & Greenberg (1998) Elaphomyces muricatus * Kropp & Trap (1982) Entoloma spp. * * Kran & Kroeg (2001) Gastroboletus subalpinus * * Kropp & Trap (1982) Gastroboletus turbinatus * Kropp & Trap (1982) Gautieria monticola * * Kropp & Trap (1982) Genea gardneri * * Kropp & Trap (1982) Genea harknessii * * Kropp & Trap (1982) Gomphidius glutinosus * * Kran et al. (2005), Kran & Kroeg (2001) Gomphus floccosus * * * Trap (1962), O'Dell et al. (1999) Gymnophilus terrestris * * Kran et al. (2005), Kran & Kroeg (2001) Hebeloma crustuliniforme * * * * Trap (1977), Kran & Fries (2002), Kran et al. (2005), Kran & Kroeg (2001) Hebeloma cf. incarnatulum • Hebeloma mesophaeum * * * Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Hebeloma sacchariolens * * Kran & Kroeg (2001), Dur et al. (1999) Hebeloma cf. subsaponaceum • Hebeloma (2 species) • Hebeloma spl * Dur et al. (1999) Hydnotrya cubispora * * Kropp & Trap (1982) Hydnotrya variiformis * * Kropp & Trap (1982) Hydnum fuscoindicum * * Kropp & Trap (1982) Hydnum umbilicatum * * * * * Hort et al. (2005), O'Dell et al. (1999) Hygrophorus bakerensis * * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Hygrophorus camarophyllus * * * Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), O'Dell et al. (1999) Hygrophorus erubescens * * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Hygrophorus piceae * * Kran et al. (2005), Kran & Kroeg (2001), Dur etal. (1999) Hygrophorus purpurascens * * Kropp & Trap (1982) Hygrophorus saxatilis * * * Kran et al. (2005), Kran & Fries (2002), Kran & Kroeg (2001), Dur et al. (1999) Hygrophorus tephrolfucus * * Kran & Kroeg (2001), Dur et al. (1999) Hymenogaster parksii * * Kropp & Trap (1982) Hysterangium crassum * * Kropp & Trap (1982) Hysterangium separabile * * * Kropp & Trap (1982), Mol (1981a) lnocybe albodisca * * Kropp & Trap (1982) 44 Table 2. 8a. Continued Species Wright RCL S a Sporocarp S; O. Synthesis S: CD Molecular Morphotype vf Oregon o Washington S Interior BC Reference(s) lnocybe calamistrata * * Kropp & Trap (1982) Inocybe fastigiata * * Kran & Kroeg (2001) lnocybe geophylla * * » Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Inocybe lanuginosa * * * Kran et al. (2005), Kran & Kroeg (2001), O'Dell et al. (1999) Inocybe sororia * * * Kropp & Trap (1982), O'Dell et al. (1999) Laccaria amethysteo-occidentalis * * Kran & Kroeg (2001), O'Dell et al. (1999) Laccaria bicolor * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Laccaria laccata * * * * * Mol (1980), Trap (1962, 1977); Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), Kran & Fries (2002), Dur et al. (1999) Lactarius alniocla * * Kran & Kroeg (2001) Lactarius deliciosus * * * * Trap (1962); Kropp & Trap (1982), Mol & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Lactarius fallax var. concolor * Kropp & Trap (1982), O'Dell et al. (1999) Lactarius glutigriseus * * Hesler& Smith (1979) Lactarius glyciosmus * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Lactarius kauffmanii * * * * Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), O'Dell et al. (1999) Lactarius kauffmanii var. sitchensis * * Kropp & Trap (1982) Lactarius pallescens * * * Kropp & Trap (1982), Kran et al. (2005), O'Dell et al. (1999) Lactarius pseudomucidus , » * * * * Hort et al. (2005), Kran & Fries (2002), Kran & Kroeg (2001), Kran et al. (2005), Dur et al. (1999), O'Dell et al. (1999) Lactarius resimus * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Lactarius rubrilacteus * * * * Kropp & Trap (1982), O'Dell et al. (1999) Lactarius rufus * * * * Kropp & Trap (1982), Kropp (1982a), Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Lactarius scrobiculatus * * * Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Lactarius spl • Lactarius substriatus * * * Trap (1962); Kropp & Trap (1982), O'Dell et al. (1999) Lactarius subviscidus * * Kropp & Trap (1982) Lactarius torminosus * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) 45 Table 2. 8a. Continued Species Wright RCL S Sporocarp Sj a. Synthesis S: Molecular S a. Morphotype <?" Oregon o Washington » 5' Interior BC Reference(s) Lactarius trivialis * * * Kropp & Trap (1982), Kran et al. (2005) Leccinum aurantiacum * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Leccinum manzanitae * * M o l & Trap (1982) Leccinum scabrum * * Kran et al. (2005), Kran & Kroeg (2001) Leomicola verrucosa • Leucogaster microsporus * * Kropp & Trap (1982) Leucogaster rubescens * Kropp & Trap (1982) Leucophleps magnata * * Kropp & Trap (1982) Macowanites chlorinosmus * * Kropp & Trap (1982) Macowanites iodiolens * Kropp & Trap (1982) Marlellia maculata * * Kropp & Trap (1982) Martellia parksii * * Kropp & Trap (1982) Martellia subfulva * * Kropp & Trap (1982) Martellia subochracea * Kropp & Trap (1982) Martellia vesiculosa * Kropp & Trap (1982) Melanogaster eulyspermus * * Kropp & Trap (1982) Melanogaster intermedius * * * M o l (1980a), Kropp & Trap (1982) Mycelium radicis atrovirens * * Kran & Fries (2002), Kran & Wy l (1998), Dur et al. (1999) Paxillus involutus * * * * * Laiho (1970), M o l (1980), M o l & Trap (1982), Kran & W y l (1998), Kran & Fries (2002), Kran & Kroeg (2001), Dur et al. (1999) Phaeocollybia kauffmanii * * Kropp & Trap (1982) Picoa carthusiana * * Kropp & Trap (1982) Piloderma croceum # * * * * * Kropp (1982a), Kropp (1982c), Kran & W y l (1998) Piloderma fallax , * * * * * Hort et al. (2005), Kran & Fries (2002) Piloderma (4 species) • Pisolithus tinclorius * * * Trap (1977), Kropp & Trap (1982), M o l & Trap (1982) Polyozellus multiplex * * Kropp & Trap (1982) Ramaria celerivirescens * * * * Hort et al. (2005) Ramaria subbotrytis * * Kropp & Trap (1982) Rhizopogon abietis * * * M o l (1980a) Rhizopogon atroviolaceus * * Kropp & Trap (1982) Rhizopogon colossus * * * M o l & Trap (1982), Kropp & Trap (1982) Rhizopogon cusickensis * * * M o l & Trap (1982) Rhizopogon ellenae * * * M o l & Trap (1982) Rhizopogon hawkeri * * * M o l (1980a), Kropp & Trap (1982) Rhizopogon liui * * * M o l & Trap (1982) Rhizopogon ochraceisporus * * * Kropp & Trap (1982), M o l & Trap (1982) 4 6 Table 2. 8a. Continued Species Wright RCL S Sporocarp S; Synthesis S: Molecular S a. Morphotype •<* Oregon o Washington g. 5' Interior BC Reference(s) Rhizopogon parks ii * * * * Mol & Trap (1982), Kropp & Trap (1982), North & Greenberg (1998) Rhizopogon pseudovillosulus * * Kropp & Trap (1982) Rhizopogon rubescens * * * Trap (1962), Mol & Trap (1982) Rhizopogon semireticulatus * * * Mol (1 980a) Rhizopogon subcaerulescens * * * Mol(l 980a) Rhizopogon subcinnamomeus * * * Kropp & Trap (1982), Mol & Trap (1982) Rhizopogon subclavitisporus * * * Mol & Trap (1982) Rhizopogon subgelatinosus * * * Kropp & Trap (1982), Mol & Trap (1982) Rhizopogon villescens * * * Mol & Trap (1982) Rhizopogon villosulus * * * Mol & Trap (1982), Kropp & Trap (1982) Rhizopogon vinicolor * * * Kropp & Trap (1982), Mol & Trap (1982) Rhizopogon vulgaris * * * Mol & Trap (1982) Rozites caperata * * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Russula aeruginea * * * Kran et al. (2005), Kran & Kroeg (2001), Kran & Fries (2002) Russula albonigra * * Kran & Kroeg (2001) Russula atrata * * Kropp & Trap (1982) Russula brevipes » * * * Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), O'Dell et al. (1999) Russula cascadensis * * * * Kropp & Trap (1982), O'Dell et al. (1999) Russula claroflava * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Russula crassotunicata * * * Kran & Kroeg (2001), O'Dell et al. (1999) Russula decolorans * * * Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Russula dissimulans * * Kropp & Trap (1982) Russula emetica * * * Trap (1962), Kran & Kroeg (2001), Dur et al. (1999) Russula fragilis * * * * * Hort et al. (2005), Kran et al. (2005), Dur etal. (1999) Russula fragrantissima * * Trap (1962) Russula hertophylla » * * * Hort et al. (2005) Russula lutea * * Kran & Kroeg (2001) Russula maculata * * Kran & Kroeg (2001) Russula nigricans * * Kropp & Trap (1982), Kran & Kroeg (2001) Russula occidentalis * * * * * * Hort et al. (2005), Kran et al. (2005), Kran & Kroeg (2001), Kran & Fries (2002), Dur et al. (1999), O'Dell et al. (1999) Russula silvicola * * Kran et al. (2005), Kran & Fries (2002) Russula spl • 47 Table 2. 8a. Continued Species Wright RCL S 3 Sporocarp S; a. Synthesis 2: Molecular £ Morphotype <<* Oregon Washington » Interior BC 3 Reference(s) Russula xerampelina * * * * * * Hort et al. (2005), Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Sarcodon imbricatus * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Scleroderma hypogaeum * * * Mol & Trap (1982) Sebacinoid * * * Hort et al. (2005) Suillus brevipes * * Kran et al. (2005), Kran & Kroeg (2001) Suillus cavipes * * * Mol & Trap (1982) Suillus granulatus * * * * Kropp & Trap (1982), O'Dell et al. (1999) Suillus imitatus * * Thiers (1975b) Suillus lakei * * * * Mol & Trap (1982), O'Dell et al. (1999) Suillus ponderosus * * Kropp & Trap (1982) Suillus tomentosus * * * * Kropp & Trap (1982), Kran et al. (2005), Kran & Kroeg (2001) Suillus umbonatus * * * Kropp & Trap (1982) Thelephora americana * * Kropp (1982c); Kropp & Trap (1982) Thelephora terrestris * * * * * Trap (1977), Kropp & Trap (1982), Kran & Fries (2002), Kran & Wyl (1998), Dur et al. (1999) Thellephora (2 species) • Tomentella sublilacina • Tricholoma cf. dryophilum • Tricholoma flavovirens * * * * Mol (1980a), Kran et al. (2005), Kran & Kroeg(2001) Tricholoma focale * * * Kran et al. (2005), Kran & Kroeg (2001), O'Dell et al. (1999) Tricholoma imbricaum * * * Kropp & Trap (1982) Tricholoma pardinum * * Kran & Kroeg (2001), Dur et al. (1999) Tricholoma pessundatum * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999) Tricholoma platyphyllum » * Kran & Kroeg (2001), Dur et al. (1999) Tricholoma ponderosum * * Kinugawa & Goto (1978), Ogawa (1979) Tricholoma portentosum * * * * * Hort et al. (2005), O'Dell et al. (1999) Tricholoma saponaceum * * * Kran et al. (2005), Kran & Kroeg (2001), Dur et al. (1999), O'Dell et al. (1999) Tricholoma sejunctum * * Kran et al. (2005), Kran & Kroeg (2001) Tricholoma (2 species) • Tricholoma virgatum * * * Kran & Kroeg (2001), Kran et al. (2005), O'Dell et al. (1999) Tuber sp. * * Kran & Fries (2002), Dur et al. (1999) Tylopilus pseudoscaber * * Kropp & Trap (1982) Tylospora spl • Zelleromyces gilkeyae * * * Mol & Trap (1982), Kropp & Trap (1982) 48 Table 2. 8b. Fungi associated with western hemlock roots: Species detected in this research, defined by B L A S T match or 97% working species definition, are indicated under Wright random clone library (RCL) indicated by dot (•). Mycorrhizal status has not been confirmed. Species Wright RCL E Sporocarp S; Synthesis S: Molecular S re Morphotype <?" Oregon o Washington 8 Interior BC Reference(s) cf. Chaetospaeria • cf. Cladophialophora • cf. Hymenoscyphus • Phialocephela fortinii • Phialophora cf. finlandia • cf. Phialophora • Hem. root associate (26 species) • List of abbreviated authors: Dur et al. (Durall et al., 1999), Hort et al. (Horton et al., 2005), Kran & Fries (Kranabetter and Friesen, 2002), Kran & Kroeg (Kranabetter and Kroeger, 2001), Kran & Wyl (Kranabetter and Wylie, 1998), Kran et al. (Kranabetter et al., 2005), Kropp & Trap (Kropp and Trappe, 1982), Mol (Molina, 1980), Mol & Trap (Kropp and Trappe, 1982; Molina and Trappe, 1982), Nor & Gre (North and Greenberg, 1998), O'Dell et al. (O'Dell et al., 1999), Trap (Trappe, 1962), Trap 2 (Trappe, 2004) List of authors cited in Kropp and Trappe 1982: Kinugawa & Goto 1978, Ogawa 1979, Laiho 1970, Hesler& Smith 1979, Theirs 1975, Boullard 1968, Zak 1969, Zak & Larsen 1978 (Kropp and Trappe, 1982) 49 U5«3Q C.parjcaMls LISSOM C. ecrftM* uWOS c. eamniaut C iwibillCilLW C.mwaoput AF323H3 D.«nra«2 uSKOi: C. dktui U«033 C. •jibitun D. • i/Brwmm* U»K>;a C.Hmcnlu* UMB27 t mmrmi -AF3235B3 D. •plenflcM i | 0 l 0. mmmmm U580SO 0 0Hu»e*O|**» 904* If* a— 1-5CG0E D. idahoensis urns sues AJ2340CO D. MHWuImM U560S7 D. HMUMI U500«0 D. lanpjinm I [ i f K M ! Vffl i D. qmwnaTi*e 9 D crr\«man»e £>, cinnsmomoQ V AJ238030 D "USO03B 0 d> USfiQ3? 0. mMwt'Ktimin 0. aurantiobasis LAJS3a361 D 1. mu .rgi.fi nr. 2C2B 1 U 3(3067 D wmlMnjMlnflJt .AF4302W ftatn^MniinM U»063 0 MMMWMUI UWO*) D. MfnlMngu incus jL5<d&s6$& D. KfrtittTrSUlllCVB phoericea U38073 D ip JFA1110B H rl/5SCM7 D.maylanfiffrisis 1 T.Li560« D jnMmii 4UM033 D. pfroClicca 0. semisanguinea UW0S2 •. IHlJUJUiLH T— 1156071 D. »p JFASDK. L-USS0S4 D. plwrtc«« -^U90O4I D. fmtoi 1^ " 5cf,B 4C10C - L: D nulcona D. mofcoria - USB03- C .aw -5ctiangQ£ Figure 2. 1. Dermocybe species phylogeny. Phylogenetic affinities of Dermocybe (a subgenus of Cortinarius) clone sequences amplified from ectomycorrhizal western hemlock roots. The tree shown was derived by fast parsimony bootstrap analysis of the ITS1, ITS2, and 5.8S regions of r D N A clone sequences along with sequences from their highest-scoring B L A S T hits of from GenBank. Identities of O T U s are indicated to the right. 50 3>xf .ft:. A 5C46C 4c11F 2a; _ AY 3MB! 3 C. gr<( i.u< 24*537 C.B**Nluii Z4S5M C.ptCoNlun 2c10 3c3F IS &18 5c34A 3c2r 3c6G 5c43F C. geophilum sp 11 C. geophilum sp 10 2*527 C. ueqiHIun AV39M14 C G>«Nk. 1 5C55D 5c6lE 3c61Q -4c3H 5e42A 5C2-U' 5o42B 1C4F '1 r5c68A MS 5 ue * Y « » M 8 C. acqsMlLin 6WC 4c13H 1 d U t» 0-10*19 C. gtcyHlun 4C17E 4C13C1 5o44D W C. geophilum sp 9 C. geophilum sp 8 3C56D 5C69E 104 C5 LSe31D L?s5C 5G20H 3c20C 4c3C T 3o42C C. geophilum sp 7 |l3 57F C. geophilum sp 8 — 2cSF • C. geophilum sp 5 i C. geophilum sp 4 ' C. geophilum sp 3 C. geophilum sp 2 - "lOtfianqes F i g u r e 2 . 2 . Cenococcum geophilum group phylogeny. Phylogenetic affinities of Cenococcum geophilum clone sequences amplified from ectomycorrhizal western hemlock roots. The tree shown was derived by fast parsimony bootstrap analysis of the ITS1, ITS2, and 5.8S regions of r D N A clone sequences along with sequences from their highest-scoring B L A S T hits of from GenBank. Identities of OTUs are indicated to the right. 51 Figure 2 .3 . Pie chart showing the relative abundance of clones by taxonomic group. 31.9% of the clones (37 OTUs) were from Cortinarius including Cortinarius subgenus Dermocybe. 15.3% of the clones (8 OTUs) were from the Cenococcum geophilum species complex. 44.2% of the clones (21 OTUs) represented other known ectomycorrhizal fungal genera in the Basidiomycota. 8.6% of the clones (comprised of 33 species) were unknowns, from saprotrophs, or closest to ericoid mycorrhizal fungal sequences. 52 Cortinarius (= Dermocybe) cinnamomeus Cortinarius (= Dermocybe) aurantiobasis Cortinarius sp26 Cortinarius sp12 Cortinarius (= Dermocybe) semisanguineus Cortinarius sp5 Cortinarius sp3 Cortinarius (= Dermocybe) malicoria Cortinarius sp6 Cortinarius sp24 Cortinarius sp31 Cortinarius sp32 Cortinarius sp28 Cortinarius sp27 Cortinarius sp17 Cortinarius sp11 Cortinarius sp4 Cortinarius spl Cortinarius sp15 Cortinarius sp10 Cortinarius sp9 Cortinarius sp8 Cortinarius sp7 Cortinarius sp19 Cortinarius sp18 Cortinarius sp13 Cortinarius sp21 Cortinarius sp22 Cortinarius (= Dermocybe) cf. idahoensis Cortinarius sp33 Cortinarius sp29 Cortinarius sp25 Cortinarius sp23 Cortinarius sp20 Cortinarius sp16 Cortinarius sp14 Cortinarius sp2 ^^^^ • Control • N + P 0 10 20 30 40 50 60 70 80 90 100 Relative frequency (%) of each species occuring within samples for a treatment Figure 2. 4. Relative frequency of occurrence of OTUs in Cortinarius (including Cortinarius subgenus Dermocybe) in samples from each fertilization treatment. Asterisks designate indicator species that correlated significantly with treatment. 53 Piloderma cf. fallax Cenococcum geophilum sp11 Cenococcum geophilum sp3 ] Cenococcum geophilum sp7 Lactarius spl Tricholoma cf. dryophilum Piloderma sp4 Cenococcum geophilum sp5 ' Piloderma spl ' Piloderma cf. croceum 1 Cenococcum geophilum sp10 '^j^wX'Xii'.iX\<.\\\ Craterellus tubaeformis ^^^^^L^™ Piloderma spl Leomicola verrucosa Tricholoma spl Hebeloma sp2 Hebeloma cf. subsaponaceum Hebeloma cf. incarnatulum Cenococcum geophilum sp4 Cenococcum geophilum sp2 Tricholoma sp2 Piloderma sp3 Cenococcum geophilum sp8 Lactarius pseudomucidus Cenococcum geophilum sp9 cf. Tylospora spl gggy Tomentella sublilacina Russula spl Hebeloma spl cf. Thellephora sp2 cf. Thellephora spl CD Control • N + P 0 10 20 30 40 50 60 70 80 90 100 Relative frequency (%) of each species occuring within samples for a treatment Figure 2. 5. Relative frequency of occurrence of fungal OTUs from ectomycorrhizal genera other than Cortinarius from samples from each fertilizer treatment. Asterisks designate indicator species that correlated significantly with treatment. 54 Hemlock root associated fungus clone sp6 cf. Phialophora cf. Hymenoscyphus Hemlock root associated fungus clone sp7 Hemlock root associated fungus clone sp14 Hemlock root associated fungus clone sp10 Hemlock root associated fungus clone sp2 Hemlock root associated fungus clone sp18 Hemlock root associated fungus clone sp17 Hemlock root associated fungus clone sp15 Hemlock root associated fungus clone sp5 Ectomycorrhizal fungus clone sp9 Ectomycorrhizal fungus clone sp3 Hemlock root associated fungus clone spl Ectomycorrhizal fungus clone spl Phialocephela fortinii Hemlock root associated fungus clone sp8 Hemlock root associated fungus clone sp4 Ectomycorrhizal fungus clone sp12 Ectomycorrhizal fungus clone sp10 Ectomycorrhizal fungus clone sp8 Ectomycorrhizal fungus clone sp7 Ectomycorrhizal fungus clone sp6 Ectomycorrhizal fungus clone sp5 Ectomycorrhizal fungus clone sp4 cf. Chaetospaeria Phialophora cf. finlandia Hemlock root associated fungus clone sp12 Hemlock root associated fungus clone sp9 Hemlock root associated fungus clone sp3 Ectomycorrhizal fungus clone sp2 cf. Cladophialophora 0 Control S N • N + P 0 10 20 30 40 50 60 70 80 90 100 Relative frequency (%) of each species occuring within samples for a treatment Figure 2. 6. Relative frequency of occurrence of fungal OTUs from unidentified genera or genera not known to be ectomycorrhizal. These OTUs were similar to various sequences from ericoid mycorrhizal fungi, from saprotrophic species, or from hemlock root associates, or they lacked sequence matches in GenBank. 55 100 N + P Control N 0.1 I 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 Rank Figure 2. 7. Rank abundance curves of ectomycorrhizal fungi detected in each of the fertilizer treatments. 56 • m 03 Control E3 N • N + P R C L Rot (a) 1.00 5 0 .80 0.60 0.40 0.20 0.00 E3 Control E3 N • N + P RCL Rot (b) n Control ra N • N + P R C L Rot (C) Figure 2. 8. Diversity of ectomycorrhizal OTUs was similar in control, N-fertil ized, and N + P fertilized plots. Measures used were: (a) Shannon's indices, (b) Simpson's indices, (c) evenness. Mean diversity among random clone libraries (RCL) and among plots are not significantly different. Bars represent 1 SE. 57 RCL Plot Treatment Figure 2. 9. Species richness of ectomycorrhizal fungi was not significantly different across fertilizer treatments. Average richness is plotted for each fertilizer treatment, first for random clone libraries (RCL) , and second for plots. The last three histograms show total richness per treatment. For each bar, the observed richness and the Jack 1 and Chao estimated richness are given. Error bars are 1 SE for each mean value of observed and estimated richness values. 58 12 10 0 • 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Number of clones sequenced per random clone library 30 : Number of clones sequenced per plot Figure 2 .10. Mean Coleman collector curves for each fertilization treatment. The number of ectomycorrhizal fungal species detected with increasing the number of clones sequenced do not reach saturation, either for (a) random clone libraries or (b) plots. Bars represent 1 SE . 59 NMS A Treatment • A Control • N • N + P A - A • @ Canopy Edge A • • . A A A © • A • • • 0 Axis 1 NMS 80 • • A A Treatment A Control • N • N + P Axis 2 NMS o • A . ® i A A@ Canopy Edge m * • DBH I Treatment A Control • N • N + P • © • • A A A A f A Axis 1 Figure 2 .11 . N M S ordination of community composition of ectomycorrhizal fungi. Fertilization treatments are indicated by biplot overlay. Vectors indicate the strongest indirect correlations of species composition with the tree performance variables of diameter at breast height and distance to crown edge. 60 CCA A O A ^ m Treatment A Control © N • N + P . D B H foot mas Axis 1 Figure 2 .12. C C A ordination of community composition of ectomycorrhizal fungi. Ordination is based on linear combination scores of species composition in space defined by tree response variables. Fertilization treatments are indicated by biplot overlay. Vectors indicate the strongest indirect correlations of species composition with the tree performance variables diameter at breast height and distance to crown edge on axis 1 and root biomass on axis 2. Axes are scaled by optimizing tree performance data. 61 Figure 2 .13. Venn diagram of fungal ectomyorrhizal diversity of western hemlock, showing the number of ectomycorrhizal species previously identified as sporocarps of ectomycorrhizal fungi of western hemlock (201 species total), the number of sequence types detected in random clone libraries in this study (99 OTUs total), and the number of overlapping species (9). 62 2.6. References Allen TR, Millar T, Berch SM, Berbee M L (2003) Culturing and direct D N A extraction find different fungi from the same ericoid mycorrhizal roots. New Phytologist 160: 255-272 Anderson IC, Cairney JWG (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environmental Microbiology 6: 769-779 Anderson IC, Campbell CD, Prosser JI (2003) Potential bias of fungal 18S r D N A and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. 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New Phytologist 139: 169-187 Wardle DA, Bardgett RD, Klironomos JN, Setala H, van der Putten WH, Wall DH (2004) Ecological linkages between aboveground and below ground biota. Science 304: 1629-1633 67 C H A P T E R 3. Discussion and conclusions M y research, presented in this thesis, contributed to knowledge of the below-ground diversity of ectomycorrhizal fungi associated with western hemlock, and provided insight into the effects that a history of fertilization can have on community composition of ectomycorrhizae. In this concluding chapter, I relate my results to the ectomycorrhizal field of study and discuss possible further research. 3.1. Heterogeneity of ectomycorrhizal communities provides a challenge for sampling effort Consistent with earlier DNA-based studies of below-ground microbial and fungal diversity (Horton and Bruns, 2001; Anderson and Cairney, 2004; O'Brien et al., 2005), I observed high diversity among ectomycorrhizal fungi and the estimated species richness was about twice as high as the observed richness. Even individual clone libraries had many more species than could be sequenced, based on estimates of their richness. The large number of low-frequency species in ectomycorrhizal communities complicates the statistical analysis of richness and community structure (Dunbar et al., 1999; Hughes et al., 2001; Anderson and Cairney, 2004; Neubert et al., 2006). The most accurate estimates of species richness result when individuals are easy to count and when most species are fairly common. Mycorrhizal fungal communities, however, typically have few common species and many rare species, and species generally show patchy distributions (Dahlberg, 2001; Morris et al., 2002; Taylor, 2002). Few spatial studies have been done to determine the influence of spatial autocorrelation, and therefore the distance necessary to achieve a random sampling of the community is also unknown (Taylor, 2002; Boerner et al., 2004; Li l leskov et al., 2004). In fungal communities, defining individuals can be difficult because the fungi exist as filamentous mycelia in soil and the same genet can only be recognized by genetic analysis. Estimating species' biomass is also challenging. Numbers of root tips colonized, which can be counted, are not necessarily proportional to the overall biomass of mycelium, which is difficult to 68 determine (Taylor, 2002). The high diversity of ectomycorrhizal fungi and lack of information about their spatial distribution may limit the reliability of estimates of ectomycorrhizal diversity, not only from this study but also from earlier studies that used other detection techniques. 3.2. Molecular identification of ectomycorrhizal populations Limitations of molecular techniques can further influence the ability to detect species. During P C R , D N A that matches primer sequences perfectly is amplified preferentially compared with D N A that has mismatches with the primers. The P C R product from fungal species that have D N A mismatches with primers may not be abundant enough for detection (Glen et al., 2001; Anderson et al., 2003; Anderson and Cairney, 2004; Mitchel l and Zucarro, 2006). I used the ITS region for recognition of OTUs because high similarity in these regions often indicates conspecificity. However, the cut-off for O T U recognition (97% identity in the ITS region) was arbitrary. This level has been used in other studies as well because it is considered to be within the range of intraspecific variation (O'Brien et al., 2005; Neubert et al., 2006), however, some researchers have used a level of 99% sequence similarity to define their OTUs (Anderson et al., 2003; Landeweert et al., 2003). The amount of interspecific and intraspecific genetic variation expected in the ITS regions has yet to be rigorously studied across a wide range of fungi, but it probably differs depending on the fungal groups (Karen et al., 1997; Buscot et al., 2000; Gomes et al., 2002; Horton, 2002). Karen et al . (1997) detected polymorphisms in the ITS region ranging from 5 to 15 bp within Cortinarius species. Recently evolved species might not have accumulated enough ITS characters to be distinguishable (Mitchell and Zucarro, 2006). A n example would be Armillaria species, which are indistinguishable by ITS regions (K im et al., 2006). Bruns cautions that lack of variation in the ITS region is not sufficient evidence for conspecificity and that other loci and multi-gene phylogenies may be useful to better distinguish species (Bruns, 2001; Mitchel l and Zucarro, 2006). I depended on the genetic database GenBank for identifications of clone sequences. Identifications were only possible when the species that produced the clone 69 sequences matched sequences available from previously identified fungi. M y finding that 26 sequences of different fungal species could not be identified even to genus is not surprising given that genetic databases are under-represented for many fungal groups (Mitchell and Zucarro, 2006). GenBank only contains sequences of a small subsample of the species that have long been known based on morphology, and the unidentified sequences in this study may represent species known to science but not yet included in databases. Some of my unidentified sequences may also represent species new to science, but this can be determined only through careful morphological comparison of the corresponding sporocarps with known species. Several new sequences from my study were identified only as "hemlock root associates." Other sequences from my work matched sequences from an earlier study, which were annotated only as "hemlock ectomycorrhizal root tips" in GenBank. A potential source of error in identifications were the estimated 20% of sequences in GenBank which may be taxonomically inaccurate (Bruns, 2001; Mitchel l and Zucarro, 2006). When possible, I used multiple sequences of the same species from GenBank and phylogenetic comparisons to avoid being misled by these errors in sequence identity. The sequences that were most similar to mine based on B L A S T searches were usually identified as being from the same species. However, further taxonomic identification of fungal taxa and expansion of the sequence databases are important for better characterizing the molecular diversity. Inferences about whether species were mycorrhizal depended on previous studies. Experimental testing of isolated hemlock fungal root associates in axenic synthesis and pot experiments would be useful to confirm their association and provide further insight into their potential roles as endophytes or ectomycorrhizae of western hemlock. 3.3. Unknown roles of hemlock fungal associates related to ericaceous mycorrhizae Sequences from fungi similar to known ericoid mycorrhizal fungi were detected from western hemlock roots. This may mean that the same fungal species can serve both as an ericoid mycorrhizal partner of an ericaceous plant like salal, and as an ectomycorrhizal partner of hemlock. However, whether or not the same fungus forms 70 both kinds of mycorrhizae requires testing through culturing and mycorrhizal re-synthesis experiments of the same isolate on both hosts. One of the ericoid mycorrhizal species possibly shared by western hemlock and salal, Cadophora finlandia, is a plant root endophyte, one of several species grouped informally together as 'dark septate endophytes'. Dark septate endophytes are among the most commonly observed endophytes in roots, however, the total diversity and identity of the types of fungi forming these associations is unknown and the roles of these fungi are also uncertain. Possibly, the dark septate endophytes play a role either as accessory mutualists, contributing to some degree in nutrient and water acquisition, or as weak pathogens (Jumpponen and Trappe, 1998; Griinig et al., 2002). Interestingly, associations other than ectomycorrhizal relationships have been detected in hemlock seedlings from salal-dominated ecosystems. Plant roots are habitat for a heterogeneous group of fungal endophytes that can colonize either inter or intracellularly (Bergero et al., 2000). Vesicular arbuscular mycorrhizal colonization in young seedlings by Glomus-Xype endophytes was detected in hemlock seedlings obtained from field studies (Cazares and Trappe, 1993) and also in hemlock seedlings grown in greenhouse bioassays with soil collected from the Oregon Coast Range (Cazares and Smith, 1996). Dual colonization of root tips was occasionally observed, with vesicles occurring concurrently with ectomycorrhizal colonization (Cazares and Trappe, 1993), or ectomycorrhizal colonization along with dark septate endophytes (Cazares and Trappe, 1993; Cazares and Smith, 1996). In my study, vesicular arbuscular mycorrhizal fungi were not detected among the clone sequences that were identified to genus. 3.4. Functional roles of ectomycorrhizal fungi in CH ecosystems Studies of functional diversity of ectomycorrhizal fungi are in their infancy, due largely to the high diversity of ectomycorrhizal species, many of which have yet to be identified, much less cultured or characterized experimentally (Zak and Visser, 1996; Anderson and Cairney, 2004). In general, the few studies of function of ectomycorrhizal fungi have concentrated on a small number of isolates (Burgess et al., 1993; Burgess et al., 1994; Cairney, 1999; Guidot et al., 2004). Past SCHIRP studies of mycorrhizae have 71 focused on ericoid mycorrhizae (Xiao and Berch, 1992; Xiao and Berch, 1995; X iao and Berch, 1996; Monreal et al., 1999; Berch et al., 2002; A l len et al., 2003) and their functional roles (Prescott and Weetman, 1994; X iao and Berch, 1999). Er icoid mycorrhizae may contribute to salal's ability to out-compete hemlock on S C H I R P sites. Ericoid mycorrhizal fungi produce enzymes to access different nutrient forms (Xiao and Berch, 1999; Cairney and Meharg, 2003). They can also inhibit ectomycorrhizal growth, based on in vitro tests of their interactions with three ectomycorrhizal cultures that were not, however, isolated from the C H ecosystems (Xiao, 1994). To understand the functional role of hemlock ectomycorrhizal fungi in C H ecosystems, ectomycorrhizal isolates from the S C H I R P sites could be tested for their effect on seedling growth, and for their abilities to compete with ericoid mycorrhizae and to access nitrogen from various organic complexes. Different ectomycorrhizal species may differ in the enzymes they secrete and the nutrients that they take up and perhaps, pass on to the trees. Fungi growing in rotten wood may be common ectomycorrhizal associates of western hemlock (Kropp, 1981a, 1981b, 1982). Ectomycorrhizal fungi in the genera Piloderma, Tomentella, Tomentellopsis, and Tylospora have homologues to genes required for lignin decomposition, including lignin peroxidases and manganese peroxidases, and the corresponding proteins may help these fungi to access nutrients by breaking down wood (Read and Perez-Moreno, 2003). 3.5. Correlation between tree performance and ectomycorrhizal community structure At another SCHIRP site referred to as the SCHIRP Installation, fertilization of hemlock on C H plots resulted in a growth response of the trees similar to unfertilized hemlock on H A plots (Wright, unpublished results). This raises the question as to whether similarities exist between ectomycorrhizal communities associated with the different fertilization histories and the different ecosystems or stand ages. Characterizing mycorrhizal communities of uncut C H and H A ecosystems would provide insight as to whether different fertilization histories in C H regeneration sites influence the development of mycorrhizal communities into those more reflective of old-growth C H or second-growth H A ecosystems. 72 Western hemlock has shown a lack of uniformity in its response to nitrogen fertilizers, from good to minimal or even negative growth responses (DeBel l et al., 1975; Radwan and DeBel l , 1989; Radwan et al., 1990; Radwan et al., 1991; Radwin, 1992). Catovsky and Bazzaz (2000) found that hemlock seedlings had greatest performance, as measured by emergence, survival, and growth, in soils that were not amended by nitrogen. At numerous sites in B .C. , productivity of western hemlock increased with relatively low nitrogen levels (Kayahara et al., 1995). This response may have been in part due to the effect of nitrogen fertilizer on the ectomycorrhizae of hemlock (Kernaghan et a l , 1992, 1995). Fertilization in some situations does not improve plant growth, possibly because it reduces ectomycorrhizal associations. Increases in N and P reduce extramatrical mycelium and fungal biomass of ectomycorrhizal roots (Wallander et al., 1994; Wallenda and Kottke, 1998). In hardwood and pine stands, Frey et al. (2004) found that chronic nitrogen amendment reduced the active fungal biomass and lowered phenol oxidase activity (a lignin-degrading enzyme produced by white-rot fungi). The reduction of mycorrhizal formation with increased nitrogen availability may result from the decreased photoassimilate allocation to the roots and therefore, to the fungi. Higher N availability increases the shoot:root ratio in most plant species (Wallenda and Kottke, 1998). Adding N to nitrogen-deficient plants shifts the partitioning of photosynthetic carbon rather than increasing photosynthesis (Champigny, 1995). Surplus of nitrogen can divert photoassimilates away from storage and transport carbohydrates, and direct them into amino acid and protein synthesis and towards host glycolysis rather than sucrose formation (Buscot et al., 2000). Reduction of carbohydrates to mycorrhizal roots in turn can reduce fungal biomass (Buscot et al., 2000). Many of these physiological responses of the plant would use its energy for its own growth, and growth does usually improve with fertilization. However, roots with reduced mycorrhizal colonization due to N inputs can suffer from nutrient imbalances, as fungal hyphae no longer contribute as effectively to the acquisition of other nutrients. Plants with reduced mycorrhizal colonization can become more susceptible to drought and root-pathogen attack (Buscot et al . , 2000). B y using molecular techniques to test for differences in ectomycorrhizal communities, measuring plant response variables, and evaluating correlations using multivariate 73 statistical approaches, it may be possible to identify the reasons for inconsistent tree response to fertilizer, and to predict where fertilizer would be most beneficial. 74 3.6. References Allen T R , Mi l lar T , Berch S M , Berbee M L (2003) Culturing and direct D N A extraction find different fungi from the same ericoid mycorrhizal roots. N e w Phytologist 160: 255-272 Anderson I C , Cairney J W G (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environmental Microbiology 6: 769-779 Anderson I C , Campbell C D , Prosser JI (2003) Potential bias of fungal 18S r D N A and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. Environmental Microbiology 5: 36-47 Berch S M , Allen T R , Berbee M L (2002) Molecular detection, community structure and phylogeny of ericoid mycorrhizal fungi. Plant & Soil 244: 55-66 Bergero R, Perotto S, Gir landa M , Vidano G , L u p p i A M (2000) Ericoid mycorrhizal fungi are common root associates of a Mediterranean ectomycorrhizal plant (Quercus ilex). Molecular Ecology 9: 1639-1649 Boerner R E J , DeMars B G , Leicht P N (2004) Spatial patterns of mycorrhizal infectiveness of soils long a successional chronosequences. Mycorrhiza 6: 79-90 Bruns T D (2001) ITS Reality. Inoculum 52: 2-3 Burgess T I , Dell B , Malajczuk N (1994) Variation in mycorrhizal development and growth stimulation by 20 Pisolithus isolates inoculated on to Eucalyptus grandis W. H i l l ex Maiden. New Phytologist 127: 731-739 Burgess T I , Malajczuk N , Grove T S (1993) The ability of 16 ectomycorrhizal fungi to increase growth and phosphorous uptake of Eucalyptus globulus Labi l l . and E. diversicolor F. Muel l . Plant & Soil 153: 155-164 Buscot F , M u n c h J C , Charcosset J Y , Gardes M , Nehls U , H a m p p R (2000) Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems. F E M S Microbiology Reviews 24: 601-614 Cairney J W G (1999) Intraspecific physiological variation: implications for understanding functional diversity in ectomycorrhizal fungi. Mycorrhiza 9: 125-135 Cairney J W G , Meharg A A (2003) Ericoid mycorrhiza: a partnership that exploits harsh edaphic conditions. European Journal of Soi l Science 54: 735-740 Catovsky S, Bazzaz F A (2000) The role of resource interactions and seedling regeneration in maintaining a positive feedback in hemlock stands. Journal of Ecology 88: 100-112 Cazares E , Smith J E (1996) Occurrence of vesicular-arbuscular mycorrhizae in Pseudotsuga menziesii and Tsuga heterophylla seedlings grown in Oregon Coast Range soils. Mycorrhiza 6: 65-67 Cazares E , Trappe J M (1993) Vesicular endophytes in roots of Pinaceae. Mycorrhiza 2: 153-156 Champigny M - L (1995) Integration of photosynthetic carbon and nitrogen metabolism in higher plants. Photosynthesis Research 46: 117-127 75 Dahlberg A (2001) Community ecology of ectomycorrhizal fungi: an advancing interdisciplinary field. New Phytologist 150: 555-562 DeBell DS, Mallonee EH, Lin JY, Strand RF (1975) Fertilization of western hemlock: A summary of existing knowledge. In. Crown Zellerbach Corp. Center Research Department, Washington State Dunbar J, Takala S, Barns SM, Davis JA, Kuske CR (1999) Levels of bacterial community diversity in four arid soils compared by cultivation and 16S r R N A gene cloning. Appl ied & Environmental Microbiology 65: 1662-1669 Frey SD, Knorr M, Parrent JL, Simpson RT (2004) Chronic nitrogen enrichment affects the structure and function of the soil microbial community in temperate hardwood and pine forests. Forest Ecology & Management 196: 159-171 Glen M, Tommerup IC, Bougher NL, O'Brien PA (2001) Specificity, sensitivity and discrimination of primers for P C R - R F L P of larger basidiomycetes and their applicability to identification of ectomycorrhizal fungi in Eucalyptus forests and .plantations. Mycological Research 105: 138-149 Gomes EA, Kasuya M C M , de Barros EG, Borges AC, Araujo EF (2002) Polymorphism in the internal transcribed spacer (ITS) of the ribosomal D N A of 26 isolates of ectomycorrhizal fungi. Genetics & Molecular Biology 25: 477-483 Griinig CR, Sieber TN, Rogers SO, Holdenrieder O (2002) Spatial distribution of dark septate endophytes in a confined forest plot. Mycological Research 106: 832-840 Guidot A, Debaud J-C, Effosse A, Marmeisse R (2004) Below-ground distribution and persistence of an ectomycorrhizal fungus. New Phytologist 161: 539-547 Horton TR (2002) Molecular approaches to ectomycorrhizal diversity studies: variation in ITS at a local scale. Plant & Soil 244: 29-39 Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology 10: 1855-1871 Hughes JB, Heilmann JJ, Ricketts TH, Bohannan BJM (2001) Counting the uncountable: statistical aproaches to estimating microbial diversity. Appl ied & Environmental Microbiology 67: 4399-4406 Jumpponen A, Trappe JM (1998) Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi. New Phytologist 140: 295-310 Karen O, Hogberg N, Dahlberg A, Jonsson L, Nylund J-E (1997) Inter- and intraspecific variation in the ITS region of r D N A of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytologist 136: 313-325 Kayahara GJ, Carter RE, Klinka K (1995) Site index of western hemlock (Tsuga heterophylla) in relation to soil nutrient and foliar chemical measures. Forest Ecology & Management 74: 161-169 Kernaghan G, Berch SM, Carter R (1992) Effect of applied nitrogen on the mycorrhizae of western hemlock. Northwest Environmental Journal 8: 173-174 Kernaghan G, Berch SM, Carter R (1995) Effect of urea fertilization on ectomycorrhiza of 20-year-old Tsuga heterophylla. Canadian Journal of Forest Research 25: 891-901 Kim M-S, Klopfenstein NB, Hanna JW, McDonald GI (2006) Characterization of North American Armil lar ia species: genetic relationships determined by ribosomal D N A sequences and A F L P markers. Forest Pathology 36: 145-164 76 Kropp BR (1981a) Fungi from decayed wood as ectomycorrhizal symbionts of western hemlock. Canadian Journal of Forest Research 12: 36-39 Kropp BR (1981b) Rotten wood as mycorrhizal inoculum for containerized western hemlock. Canadian Journal of Forest Research 12: 428-431 Kropp BR (1982) Formation of mycorrhizae on nonmycorrhizal western hemlock outplanted on rotten wood and mineral soil. Forest Science 28: 706-710 Landeweert R, Leeflang P, Kuyper TW, Hoffland E, Rosling A, Wernars K, Smit E (2003) Molecular identification of ectomycorrhizal mycelium in soil horizons. Appl ied Environmental Ecology 69: 327-333 Lilleskov EA, Bruns TD, Horton TR, Taylor DL, Grogan P (2004) Detection of forest stand-level spatial structure in ectomycorrhizal fungal communities. F E M S Microbiology Ecology 49: 319-332 Mitchell JI, Zucarro A (2006) Sequences, the environment and fungi. Mycologist 20: 62-74 Monreal M, Berch SM, Berbee M (1999) Molecular diversity of ericoid mycorrhizal fungi. Canadian Journal of Botany 77: 1580-1594 Morris CE, Bardin M , Berge O, Frey-Klett P, Fromin N, Girardin H, Guinebretiere M-H, Lebaron P, Thiery JM, Troussellier M (2002) Microbial biodiversity: approaches to experimental design and hypothesis testing in primary scientific literature from 1975 to 1999. Microbiology & Molecular Biology Reviews 66: 592-616 Neubert K, Mendgen K, Brinkmann H, Wirsel SGR (2006) Only a few fungal species dominate highly diverse mycofloras associated with the common reed. Appl ied & Environmental Microbiology 72: 1118-1128 O'Brien HE, Parrent JL, Jackson JA, Moncalvo J-M, Vilgalys R (2005) Fungal community analysis by large-scale sequencing of environmental samples. Appl ied & Environmental Microbiology 71: 5544-5550 Prescott CE, Weetman G (1994) Salal Cedar Hemlock Integrated Research Program: A Synthesis. University Brit ish Columbia, Vancouver, B C . http://www.forestry.ubc.ca/schirp/reports.htm Radwan MA, DeBell DS (1989) Effects of different urea fertilizers on soil and trees in a young thinned stand of western hemlock. Soil Science Society of America Journal 53: 941-946 Radwan MA, DeBell DS, Wilcox. JE (1990) Influence of family and nitrogen fertilizer on growth and nutrition of western hemlock seedlings. In Res. Pap. P N W - R P -426. U.S. Department of Agriculture, Forest Service, Pacific Northwest Research Station, Portland, OR, p 10 Radwan MA, Shumway JS, DeBell DS, Kraft JM (1991) Variance in response of pole-size trees and seedlings of Douglas-fir and western hemlock to nitrogen and phosphorus fertilizers. Canadian Journal of Forest Research 21: 1431-1438 Radwin MA (1992) Effect of forest floor on growth and nutrition of Douglas-fir and western hemlock seedlings with and without fertilizer. Can. J . For. Res. 22: 1222-1229 Read DJ, Perez-Moreno J (2003) Mycorrhizas and nutrient cycling in ecosystems - a journey towards relevance? New Phytologist 157: 475-492 77 Taylor AFS (2002) Fungal diversity in ectomycorrhizal communities: sampling effort and species detection. Plant & Soi l 244: 19-28 Wallander BH, Nylund J-E, Sundberg B (1994) The influence of I A A , carbohydrate and mineral concentration in host tissue on ectomycorrhizal development on Pinus sylvestris L. in relation to nutrient supply. New Phytologist 127: 521-528 Wallenda T, Kottke I (1998) Nitrogen deposition and ectomycorrhizas. N e w Phytologist 139: 169-187 Xiao G (1994) The role of root-associated fungi in the dominance of Gaultheria shallon. PhD. University of Brit ish Columbia, Vancouver Xiao G, Berch SM (1992) Ericoid mycorrhizal fungi of Gaultheria shallon. Mycologia 84: 470-471 Xiao G, Berch SM (1995) The ability of known ericoid mycorrhizal fungi to form mycorrhizae with Gaultheria shallon. Mycologia 87: 467-470 Xiao G, Berch SM (1996) Diversity and abundance of ericoid mycorrhizal fungi of Gaultheria shallon on forest clearcuts. Canadian Journal of Botany 74: 337-346 Xiao G, Berch SM (1999) Organic nitrogen use by salal ericoid mycorrhizal fungi from northern Vancouver Island and impacts on growth in vitro of Gaultheria shallon. Mycorrhiza 9: 145-149 Zak JC, Visser S (1996) A n appraisal of soil fungal biodiversity: the crossroads between taxonomic and functional biodiversity. Biodiversity & Conservation 5: 169-183 7 8 CHAPTER 4. Appendices 4.1. Multivariate community analyses Table 4.1. Multi-Response Permutations Procedures summary statistics. Pair-wise comparison all plots Control vs N Control vs N + P N vs N + P Test statistic: T -2.08 0.60 -2.29 -2.84 Observed delta 0.81 0.83 0.80 0.80 Expected delta 0.82 0.82 0.82 0.82 Variance of delta 4.10E-05 4.95E-05 4.86E-05 4.31 E-05 Skewness of delta -0.50 -0.69 -0.62 -0.63 Chance-corrected within-group agreement: A 0.02 -0.01 0.02 0.02 P-value 0.03 0.70 0.02 0.01 7 9 Table 4.2. Pearson correlations with Non-metric Multidimensional Scaling ordination axes. Operational taxonomic units with strongest r-values are indicated with bold font. Species axis 1 axis 2 axis 3 Species axis 1 axis 2 axis 3 Ceno 1 -0.576 -0.449 0.353 Derm cin -0.474 -0.167 -0.225 Ceno 11 -0.026 -0.166 0.048 Derm ida 0.064 0.326 0.458 Ceno 2 -0.151 0.045 0.315 Derm mal -0.206 0.223 0.114 Ceno 3 -0.193 -0.118 -0.170 Derm sem -0.043 -0.345 -0.362 Ceno 4 -0.115 0.084 0.085 Ecto 11 -0.060 -0.015 0.314 Ceno 5 -0.029 -0.174 0.058 Ecto 110 -0.046 -0.110 -0.016 Ceno 7 0.226 -0.138 -0.330 Ecto 112 -0.020 -0.229 0.237 Ceno 8 0.637 -0.458 -0.080 Ecto 12 0.232 -0.076 0.136 Ceno 9 0.545 -0.126 0.007 Ecto 13 -0.144 -0.184 0.046 Chaeto -0.020 -0.229 0.237 Ecto 14 -0.020 -0.229 0.237 Clado 0.206 0.028 -0.103 Ecto 15 -0.020 -0.229 0.237 Derm 1 -0.193 0.016 -0.166 Ecto 16 -0.020 -0.229 0.237 Derm 2 -0.029 -0.174 0.058 Ecto 17 -0.020 -0.229 0.237 Derm 3 0.255 -0.117 -0.126 Ecto 18 -0.020 -0.229 0.237 Hymeno 0.023 0.337 -0.165 Ecto 19 0.037 -0.144 -0.253 Phialop 0.164 0.037 0.038 Heb inc -0.020 -0.229 0.237 Thel 1 0.233 0.064 0.004 Heb sub -0.151 0.045 0.315 Thel2 0.206 0.028 -0.103 Heb 1 0.206 0.028 -0.103 Tylo 0.388 -0.121 -0.108 Heb 2 -0.384 0.207 0.233 Cort 1 0.000 0.177 0.466 Hem 1 -0.006 0.003 -0.284 Cort 10 0.069 -0.187 0.113 Hem 10 0.025 0.244 0.129 Cort 11 -0.077 -0.181 -0.071 Hem 12 0.315 -0.034 0.024 Cort 12 -0.379 -0.210 -0.352 Hem 14 0.090 -0.082 -0.308 Cort 13 0.173 -0.302 -0.056 Hem 15 -0.144 -0.184 0.046 Cort 14 0.123 0.159 -0.026 Hem 17 -0.029 -0.174 0.058 Cort 15 -0.384 0.244 0.009 Hem 18 0.050 0.136 -0.162 Cort 16 0.272 -0.118 -0.034 Hem 2 0.066 0.340 -0.051 Cort 17 0.050 0.136 -0.162 Hem 3 0.182 0.016 0.143 Cort 18 0.485 -0.053 -0.047 Hem 4 0.193 0.033 -0.024 Cort 19 0.550 -0.238 0.062 Hem 5 -0.006 0.002 0.089 Cort 2 0.041 0.338 0.091 Hem 6 0.001 -0.178 0.176 Cort 20 0.255 -0.117 -0.126 Hem 7 0.079 0.268 -0.273 Cort 21 0.297 -0.043 0.200 Hem 8 0.219 -0.276 0.110 Cort 22 0.111 0.108 0.253 Hem 9 0.097 -0.241 -0.142 Cort 23 0.255 -0.117 -0.126 Lac pseu 0.286 0.673 -0.129 Cort 24 -0.008 0.023 -0.016 Lact 1 -0.022 0.046 0.493 Cort 25 0.233 0.064 0.004 Leo ver 0.035 -0.299 0.251 Cort 26 -0.271 -0.264 -0.247 Phial fo -0.090 -0.279 0.318 Cort 27 0.037 -0.144 -0.253 Phial fi 0.325 -0.226 0.197 Cort 28 -0.193 0.016 -0.166 Pilo cro -0.006 0.002 0.089 Cort 29 0.041 0.338 0.091 Pilo 1 0.037 -0.144 -0.253 Cort 3 0.085 0.483 -0.064 Pilo 2 0.079 -0.047 0.196 Cort 4 -0.332 -0.108 0.024 Pilo 3 0.041 -0.023 -0.388 Cort 5 0.075 -0.018 -0.123 Pilo 4 0.144 0.022 -0.010 Cort 6 -0.379 0.043 0.040 Rus 1 0.237 -0.197 0.041 Cort 7 -0.384 0.244 0.009 Tome sub 0.368 0.023 -0.236 Cort 8 -0.384 0.244 0.009 Trie dry -0.002 0.014 -0.238 Cort 9 -0.189 -0.136 0.183 Trie 1 -0.114 0.054 0.252 Derm aur 0.045 -0.102 -0.612 Trie 2 -0.151 0.045 0.315 80 Table 4.3. Pearson correlations with Canonical Correspondence Analyses ordination axes. Operational taxonomic units with strongest r-values are indicated with bold font. Species axis 1 axis 2 Species axis 1 axis 2 Ceno 1 -0.274 0.478 Derm sem 0.018 0.026 C e n o l l 0.059 0.355 Ecto 11 0.084 0.232 Ceno 3 -0.071 0.178 H e b 2 -0.168 0.065 Ceno 7 0.074 0.199 Hem 1 0.065 0.155 Ceno 8 0.322 -0.018 Hem 10 -0.225 -0.165 Ceno 9 0.641 -0.113 Hem 12 0.460 -0.017 Hymeno -0.175 -0.085 Hem 14 -0.150 0.188 Phialop 0.156 0.127 Hem 2 -0.266 -0.286 Tylo 0.588 0.095 Hem 4 0.171 0.110 Cort 12 -0.117 0.008 Hem 6 -0.011 0.233 Cort 13 0.234 -0.222 Hem 7 -0.177 -0.210 Cort 18 0.694 -0.066 Hem 8 0.066 0.184 Cort 19 0.509 -0.059 Lac pseu -0.043 -0.346 Cort 21 0.515 0.136 Lact 1 0.159 0.206 Cort 22 0.198 -0.147 Leo ver -0.135 0.153 Cort 24 0.134 0.112 Phial fo 0.140 0.240 Cort 26 -0.220 0.087 Phial fi 0.488 0.278 Cort 3 -0.226 -0.317 Pi lo fal 0.011 0.086 Cort 5 0.177 0.227 Pi lo 2 0.311 0.232 Cort 6 -0.281 0.051 Pi lo 4 0.105 -0.042 Derm aur -0.002 0.083 Tome sub 0.323 -0.457 Derm cin -0.285 0.126 Trie dry 0.137 -0.143 Derm ida 0.015 -0.157 Trie 1 -0.099 0.202 Derm mal -0.195 0.028 81 

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