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Ectomycorrhizal communities of Douglas-fir and paper birch along a gradient of stand age following clearcutting… Twieg, Brendan David 2006

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Ectomycorrhizal Communities of Douglas-fir and Paper Birch Along a Gradient of Stand Age Following Clearcutting and Wildfire in the Interior Cedar-Hemlock Zone, Southern British Columbia by B R E N D A N DAVID TWIEG B.Sc, Humboldt State University, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In The Faculty of Graduate Studies • (Forestry) The University of British Columbia June 2006 © Brendan David Twieg}2006 Abstract Ec tomyco r rh i z a l ( E C M ) commun i t i e s o f Doug las- f i r and paper b i rch were character ized in 5-, 26-, 65-, and 100-year-old stands us ing E C M root t ip m o r p h o l o g y and ITS reg ion D N A sequences. Stands d isturbed by w i l d f i r e (a l l age classes) and c learcut t ing ( two youngest age classes) were studied (4 repl icates per stand type) in the Interior Ceda r-Hemlock forests o f southern B r i t i sh C o l u m b i a . E C M c o m m u n i t y species r ichness on Douglas-f i r d i f fe red w i th stand age, be ing three t imes h igher in 65- and 100-yr-old stands than 5-yr-old stands; 26-yr-old stands were intermediate. In the 5-yr-old stands, the E C M c o m m u n i t y o f paper b i r ch had - 7 0 % h igher species r ichness than Douglas-f i r . Roots o f sprout ing paper b i r ch stumps may support mycor rh i zae or i nocu l a that persist through disturbance. O v e r a l l E C M d ivers i t y increased substant ia l ly f r om 5- to 26-yr-old stands, but changed little w i th further stand age increase. E C M c o m m u n i t y compos i t i on and structure shif ted f r o m 5- to 26-yr-old stands and cont inued to change f r om 26- to 65-yr-old stands, i n c l ud ing increases in Russula and Piloderma re lat ive abundances. S im i l a r E C M commun i t i e s occurred on 65-and 100-yr-old stands. Cortinarius and Hebeloma were near ly absent in 5-yr-oid stands and peaked in relat ive abundance in 26-yr-old stands. Gene ra l l y , patterns in relat ive abundance o f funga l taxa w i t h stand age para l le led those in f requency . Host-spec i f i c E C M fung i were most dominant in the youngest stands, par t i cu lar ly Rhizopogon vinicolor-type on Douglas-f i r and Lactariuspubescens on paper b i r ch . Host-general ists were more abundant on paper b i rch than Doug las- f i r at younger ages, suggest ing that paper b i rch may be important in the establ ishment o f these fung i on Douglas-f i r . Paper b i rch also had an important pos i t i ve in f luence on E C M funga l d ivers i ty in 5-yr-old stands, w h i c h shou ld be cons idered in forest management . There was no d i f ference in E C M d ivers i ty between c learcut and w i l d f i r e o r i g i n sites, a l though c o m m u n i t y structure d i f fe red s l ight l y between these disturbances in 5-yr-old stands. W i t h i n the range o f ages studied, it appears that the E C M funga l c o m m u n i t y changed l itt le after 65 years f o l l o w i n g disturbance. A v a i l a b l e P was correlated w i th abundance o f two dominant taxa, but ava i lab le N and P, m ine ra l i zab le N , and organic P were not related to E C M d ivers i ty or c o m m u n i t y structure. ii Table of Contents Abstract ii Table of Contents iii List of Tables v List of Figures. vii Acknowledgements xi Dedication xii Chapter 1 Introduction and Literature Review 1 Introduction 1 Literature Review 3 Fungal Identification 3 E C M Ecology 4 Study Overview, Objectives, and Hypotheses 7 References 9 Chapter 2 Below-ground ectomycorrhizal community succession on Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyriferd) in southern interior British Columbia 13 Introduction 13 Materials and methods 16 Site description and study design 16 Sampling for Ectomycorrhizae 17 Sorting of Root Tips 18 Morphological and Molecular Identification of E C M Root Tips 19 Data analyses 23 Results : 27 E C M from Soil Samples 27 E C M from Seedling Samples 32 Discussion 34 Identification of E C M Fungi and Taxonomic Diversity 34 E C M Community Composition and Structure 36 Network Potential between Host Species 39 Conclusions 40 Figures 41 Tables 61 References 67 Chapter 3 Do soil properties and tree cover variables explain variation in ectomycorrhizal fungal community diversity and structure along a forest chronosequence? 73 Introduction 73 Materials and Methods 75 iii Data Collection and Soil Analyses 75 Data Analysis 76 Results 78 Discussion 81 Conclusions '. 84 Figures 85 Tables 92 References 98 Chapter 4 Conclusions 101 Ectomycorrhizal Diversity and Communities 101 Effects and Likely Causes 101 Implications 102 Succession Models and Fungal Strategies 103 Study Shortcomings 105 Future Directions '. 106 References 108 Appendix A 110 Appendix B 137 Appendix C 144 Appendix D 147 Appendix E 148 List of Tables Table 2.1 Site locations and characteristics. Interior Cedar-Hemlock biogeoclimatic subzones are defined in Lloyd et al. (1990) 61 Table 2.2 Analysis of Variance table for split-plot analyses, n = 4 replicates 63 Table 2.3 Statistical analysis table for differences among stand type means of E C M fungal diversity variables and relative abundances. Fd = Douglas-fir; Ep = paper birch. SP = split-plot; K - W = Kruskal-Wallis. See associated figures for further details 64 Table 2.4 Mean diversity measurements calculated from frequency data for Douglas-fir, paper birch, and combined E C M communities. Numbers in parentheses are one standard error of the mean 65 Table 2.5 Summary of generalized linear models, estimated by maximum likelihood, of taxon abundances and frequencies. Abundance models are based on per core abundances and the negative binomial distribution. Frequency models are based on occurrences per site and the Poisson distribution. SA = stand age; SBZ = biogeoclimatic subzone (ICHmw or ICHmk). "ICHmk effect" column details the effect of this subzone in comparison to the ICHmw subzone. Significant variables gave p < 0.05 for likelihood ratio tests 66 Table 3.1 Tree cover variables by site; Fd = Douglas-fir, Ep = paper birch 92 Table 3.2 Mean soil properties by stand age and initiation type (n = 4; standard error of mean in parentheses). Means followed by the same letter (within one soil layer and variable) are not significantly different (p > 0.05) by multiple comparisons. § = difference among treatment means, but no significant differences found in pairwise mean comparisons. F-ratios and p-values are from one-way A N O V A s 93 Table 3.3 Mean relative abundances (respective of host) of dominant taxa by soil layer. Fd = Douglas-fir; Ep = paper birch. Significant differences have p-values in bold (paired t-test) 94 Table 3.4 Summary of stepwise regression analyses predicting E C M diversity variables. R 2 values in bold correspond to the model that contains all significant predictor variables; those not in bold correspond to model including that predictor variable and those above it for the corresponding dependent variable 95 Table 3.5 Summary of stepwise regression models predicting relative abundances (%) of dominant E C M taxa. R 2 values as in Table 3.4 96 Table 3.6 Details of C C A ordination of sites based on E C M fungi frequency in the combined community and environmental variables. Correlations of environmental variables to ordination axes are Pearson's r, and those of 0.5 or above are in bold. R 2 is the proportion of Chi-square distance among sites that is explained by ordination axes. P-values for Monte Carlo test of ordination structure represent the proportion of randomized sets of the real data giving ordination axes with axis eigenvalues greater than or equal to the real data. Those for tests of inter-matrix correlation represent the proportion of randomized data sets giving correlations between E C M species matrices and the environmental variables equal to or greater than the real data vi List of Figures Figure 2.1 Taxa-sample unit curves for the combined community (a) site-level, including all sites; (b) site-level, excluding the 26-yr-old burned ICHmk sites and two oldest age classes, and (c) cumulative for each stand type. Filled circles = 5-yr-old clearcuts; open circles = 26-yr-old burned sites; filled triangles = 26-yr-old clearcuts; open triangles = 65-yr-old burned sites; and filled squares = 100-yr-old burned stands 41 Figure 2.2 Rank abundance plots for E C M communities with (a) both hosts combined; (b) Douglas-fir; and (c) paper birch. Filled circles = 5-yr-old clearcuts; open circles = 26-yr-old burned sites; filled triangles = 26-yr-old clearcuts; open triangles = 65-yr-old burned sites; and filled squares = 100-yr-old burned stands 42 Figure 2.3 E C M community diversity variables (a-d) by stand type (cc = clearcut, b — burned); bars represent means (n = 4) and error bars represent one standard error of the mean. Black bars = Douglas-fir; light grey bars = paper birch; dark grey bars = combined community. Means within host species (i.e. with the same bar colour) that share the same letter do not differ significantly (p > 0.05). Bars without any letters indicate no significant difference found among stand type means for that host. * indicates a significant difference between host species within that stand type (from multiple comparisons). Significant stand type by host species interactions were detected for all analyses (split-plot A N O V A ) . Combined communities were analysed by separate one-way ANOVAs 43 Figure 2.4 Mean (n = 4) 1 s t (black bars) and 2 n d (grey bars) order jackknife estimates of species richness by stand type (n = 4); cc = clearcut; b = burned. Error bars represent one standard error of the mean. Not statistically tested 44 Figure 2.5 Observed site scores on first principal component axis from PCA on 13 E C M diversity variables (circles = ICHmw sites; triangles = ICHmk sites) and values predicted by the model (line) for the ICHmw subzone 45 Figure 2.6 Mean percentage (n = 4) of E C M root tips colonised by fungi observed on both hosts by stand type. Black bars = Douglas-fir community; light grey bars = paper birch community; dark grey bars = combined community (expressed as average values of Douglas-fir and paper birch communities); cc = clearcut; b = burned. Combined community means with the same letters are not significantly different (p > 0.05). No significant stand type by host species interaction found (split-plot A N O V A ) . Host species effect was significant (p < 0.05), but multiple comparisons between hosts within each stand type detected no significant differences 46 Figure 2.7 N M S ordinations of sites based on the combined E C M fungal community of both hosts using (a) species frequency; (b) frequency of species lumped into genera; and (c) species abundance. Filled circles = 5-yr-old clearcut; open vii circles = 26-yr-old burned sites; filled triangles = 26-yr-old clearcut sites; open triangles = 65-yr-old burned sites; and squares — 100-yr-old burned sites. R 2 values represent the proportion of total variation in Relative Sorensen distance among sites explained by ordination axes. Correlations of stand age to ordination axes are Pearson's r 47 Figure 2.8 NMS ordinations of sites based on E C M fungal communities on Douglas-fir using (a) species frequencies; (b) frequency of species lumped into genera; and (c) by species abundance. R 2 values represent the proportion of total variation in Relative Sorensen distance among sites explained by ordination axes. Correlations of stand age to ordination axes are Pearson's r. Symbols are as in Figure 2-7 48 Figure 2.9 N M S ordinations of sites based on E C M fungal communities on paper birch using (a) species frequencies; (b) frequency of species lumped into genera; and (c) by species abundance. R 2 values represent the proportion of total variation in Relative Sorensen distance among sites explained by ordination axes. Correlations of stand age to ordination axes are Pearson's r. Symbols are as in Figure 2-7 49 Figure 2.10 Mean relative abundances (n = 4) of fungal taxa that occurred on both hosts by stand type (a-h; cc = clearcut, b = burned). Black bars = Douglas-fir; light grey bars = paper birch; and dark grey bars = combined community. Error bars represent one standard error of the mean. Means within host species (i.e. with the same bar colour) that share the same letter do not differ significantly (p > 0.05). * indicates a significant difference between host species within that stand age-initiation type (p < 0.05). Split-plot A N O V A used for a-c; no stand type by host species interactions found; (a) no stand type effect; (b) no host species effect; c) no stand type effect; host species effect significant (p < 0.05), but not in mean comparisons. Kruskal-Wallis test used for d-h; (d) and (e) showed significant stand type effect; (f)-(h) showed no significant stand type effect 51 Figure 2.11 Mean relative abundances (n = 4) of two host-specific fungi on Douglas-fir by stand age-initiation type (n = 4); cc = clearcut, b = burned. Error bars represent one standard error of the mean. Means with the same letter are not significantly different (p > 0.05). R. vinicolor-type tested by one-way A N O V A ; S. lakei showed significant stand type effect by Kruskal-Wallis test (p<0.05) 52 Figure 2.12 Mean relative abundances (n = 4) of two host specific fungi on paper birch by stand type. Error bars represent one standard error of the mean. No significant stand type effect for either species (one-way A N O V A ; p > 0.05)..53 Figure 2.13 Maximum likelihood models (curves) for Cortinarius and Hebeloma spp. in the combined community. Predicted relative abundances were estimated by dividing predictions of per soil sample taxon abundance by the average number of E C M root tips per soil sample. Points are site-level values of relative abundance and number of occurrences (frequency) 54 Figure 2.14 Maximum likelihood models (curves show predictions for ICHmw only) for Piloderma spp. in the combined community (a-b). Predicted relative abundances were estimated by dividing predictions of per soil sample taxon abundance by the average number of total E C M root tips per soil sample. V l l l Points are site-level values of relative abundance and number of occurrences (frequency) 55 Figure 2.15 Maximum likelihood models (curves) for Russula spp. and R. nigricans in the combined community (lines show predictions for ICHmw only in (a) and (b)). Predicted relative abundances were estimated by dividing predictions of per soil sample taxon abundance by the average number of total E C M root tips per soil sample. Points are site-level values of relative abundance and number of occurrences (frequency) 56 Figure 2.16 Maximum likelihood models (curves) for two host specific E C M species on Douglas-fir. Predicted relative abundances were estimated by dividing predictions of per soil sample taxon abundance by the average number of Douglas-f ir E C M root tips per soil sample. Points are site-level values of relative abundance and number of occurrences (frequency). In (d), Lines 1 and 2 represent predictions for burned and clearcut sites, respectively, from the best model. Line 3 represents the second-best model, in which stand initiation type was not a predictor 57 Figure 2.17 Maximum likelihood models (curves) for two host specific E C M species on paper birch. Predicted relative abundances were estimated by dividing predictions of per soil sample taxon abundance by the average number of birch E C M root tips per soil sample. Points are site-level values of relative abundance and number of occurrences (frequency). In (a), Lines 1 and 2 represent the best and second-best models, respectively 58 Figure 2.18 Comparison of mean E C M community diversity measures (n = 4) for Douglas-fir seedlings between stand initiation types in 5-yr-old stands. Error bars represent one standard error of the mean. Tested by two-sample t-test (two-tailed): (a) t = -1.9, p = 0.11; (b) t = -2.1, p = 0.08; (c) t = -2.1, p = 0.08; (d) t = -2.1, p = 0.08 59 Figure 2.19 Comparison of mean relative abundance (n = 4) of Rhizopogon rudus and R. vinicolor-type on Douglas-fir seedlings between 5-yr-old burned and clearcut stands. Black bars = wildfire origin; grey bars = clearcut origin. Error bars represent one standard error of the mean. Not statistically tested 60 Figure 3.1 Mean forest floor thickness (n = 4) by stand age-initiation type; cc = clearcut; b = burned. Error bars represent one standard error of the mean. N o differences were detected by one-way A N O V A (F = 1.26, p = 0.29) 85 Figure 3.2 Mean E C M fungal species richness of the community of both hosts in mineral soil and forest floor layers. Error bars represent one standard error of the mean. Significant difference detected by paired t-test (n = 20, t = -3.02, p = 0.007) 86 Figure 3.3 Frequency histogram of number of E C M rot tips available per soil sample from mineral soil (black bars) and forest floor (grey bars) (to max. of 100 tips). 0 = 0 tips examined; 1 = 1-10 tips; 2 = 11-20 tips ...; 10 = 91-100 tips examined 87 Figure 3.4 Mean ratio of number of root tips examined from the forest floor to the number examined from the mineral soil per site (n = 4). Error bars represent one standard error of the mean. No significant differences detected by one-way A N O V A (F = 1.9, p = 0.17) 88 ix Figure 3.5 Maximum likelihood model (using Poisson distribution) prediction of E C M fungal species richness per soil sample by stand age. Number of root tips examined from the forest floor was also a significant predictor variable in this model (see Results). Stand age was a significant predictor (likelihood ratio test; p < 0.05).. : 89 Figure 3.6 Scatterplot of principal component axis 1 site scores (from PCA on 13 E C M diversity variables) against forest floor organic P; filled circles = 5-yr-old clearcuts, open circles = 26-yr-old wildfire origin, filled triangles = 26-yr-old clearcuts, open triangles = 65-yr-old wildfire origin, squares = 100-yr-old wildfire origin 90 Figure 3.7 C C A ordination of sites based on frequency of E C M fungi in the combined community of both hosts and environmental variables (stand age, site index, and soil variables). Filled circles = 5-yr-old clearcut; open circles = 26-yr-old burned sites; filled triangles = 26-yr-old clearcut sites; open triangles = 65-yr-old burned sites; and squares = 100-yr-old burned sites. X = axis eigenvalue; R 2 = proportion of variance in Chi-squared distance among sites explained by ordination axes 91 Acknowledgements First, I'd like to thank my supervisors, Dr. Dan Durall and Dr. Suzanne Simard, for the significant amount of energy they put into this project. They provided the basis for the study design and were very patient and supportive. Dr. Melanie Jones also helped considerably towards the betterment of the study and in the logistics of its implementation. Dr. Gary Bradfield put in valuable suggestions throughout the project as well. All four of these UBC professors were thorough and thoughtful in the editing of this document and deserve special recognition for their parts in assuring its quality. Funding for this project came from several sources, and I would like to thank all of them: NSERC for the operating grant held by Dr. Dan Durall, which funded a large portion of the project, and NSERC also funded a portion of soil analyses through a Discovery grant held by Dr. Melanie Jones; the Forest Inventory Investment (FII) for a long term grant held by Dr. Suzanne Simard (Principal Investigator), which funded a substantial portion of the molecular work; UBC, which supported me with scholarships and a Graduate Fellowship; and the Edward R. Bassett Memorial Fund, which provided a generous scholarship. This project would have been impossible for me to finish without the help of many individuals, all of whom worked very hard and were a pleasure to be around. Lenka Kudrna and Bill Clark were responsible for a large amount of the molecular work, and Tanis Gieselman, Danielle Larsen, and Allana Leverrier also helped with this part. Tanis, Lenka, Danielle, Ben Chester, Kristen MacKay, Nicole Bergh, Chelsea Ricketts, Julie Brown, Tanya Seabacher, and Denise Brooks all were wonderful in the field and endured rain, heat, and lots of mosquitos. Chelsea did additional lab work, as well. Thank you all! Several others provided valuable contributions to the project and/or aided academically. Jean Roach of Skyline Forestry chose most of the research sites and provided expertise in forest ecology. Dr. Tony Kozak, Francois Teste, and Brock Simons were extremely helpful with statistics. The B.C. Ministry of Forests aided in protecting the research sites from damage, as did the timber companies holding tenures at the sites: Riverside Lumber, Canoe Lumber, LP Engineering Malakwa, and Tolko Lumber. All were friendly and cooperative, particularly Kevin New at Canoe Lumber. Last, I'd like to give my warmest thanks to Lydia Stepanovic, my fiancee, for amazing support, as well as lots of efficient field help and computer expertise. I couldn't have done it without you! xi Dedication I wish to dedicate this work to my family, who are just as supportive and loving as one could ever hope a family to be, and to Dr. David Largent, who sparked my earliest interest in fungi and taught me an awful lot about them. xii Chapter 1 Introduction and Literature Review Introduction E c t o m y c o r r h i z a l ( E C M ) fung i are an ext remely d iverse g roup o f organ isms compr i sed m a i n l y o f species in the B a s i d i o m y c o t a , w i th a smal le r number o f species in the A s c o m y c o t a . These fung i exh ib i t a w ide var iety o f above-ground reproduct ive structures, i n c l ud ing mush rooms , cups (apothecia) , po lypores , and resupinate fo rms (appressed to the surface) on dead w o o d . M o s t fung i w i t h hypogeous (below-ground) f ru i t ing bodies , k n o w n as truff les or false t ruf f les , are also e c tomyco r rh i za l . It has been est imated that over 5000 funga l species are in the E C M group ( M o l i n a et al, 1992), and the commun i t i e s o f E C M fung i are often several t imes more d iverse than associated commun i t i e s o f vascu lar plants. The vast major i ty o f f ine roots o f a l l con i fe r species in the P inaceae, as w e l l as some broad lea f genera such as Betula , Quercus, and Fagus, f o r m a s y m b i o t i c ' re la t ionship w i th these f ung i . O ther broad lea f hosts, such as those in the Sa l icaceae, f o r m symbioses w i th both E C M and arbuscular m y c o r r h i z a l ( A M ) fung i . M o s t E C M fung i f o rm an obv ious mant le (sheath o f t issue) a round the termina l and penul t imate root segments, thus inter fac ing between f ine roots and so i l . T h e y a lso f o r m a hart ig net, a co l l ec t i ve o f r ami f i ed funga l ce l l s in the interce l lu lar space o f the root ep idermis and cortex, w h i c h is a lways present even though a mant le is somet imes not. The term " m y c o r r h i z a " has often been appl ied on l y to mutua l i s t i c symbioses ( A l l e n , 1991). H o w e v e r , some myco r rh i z a l f ung i , i n c l ud ing E C M fung i , may act as parasites, mutual is ts , or somewhere in between, dependent upon the context (e.g. Jonsson et al., 2 0 0 1 ; Hash imo to & H y a k u m a c h i , 2 0 0 1 ; Johnson et al., 1997). Jones & Smi th (2004) , in their thorough r e v i ew o f this top i c , conc luded that " m y c o r r h i z a s shou ld be def ined on a structural or deve lopmenta l basis and any requirement to demonstrate mutua l i sm must be e l im ina t ed " . A central p rob lem in the f i e l d o f myco r rh i z a l research, and one that led Jones & Sm i th (2004) to this conc l u s i on , is that E C M effects on plant host f i tness cannot be studied under natural cond i t ions in the f i e ld . Here , I use the structural de f in i t ion for the term " e c t o m y c o r r h i z a l " ; i.e. an E C M root is one that has a har t ig net, funga l mant le , or both. ' The term "symbiosis" is used here to mean two organisms living in intimate physical contact; there is no implication of mutualism in this term. 1 Science is continually discovering new below-ground ECM relationships between fungi and plants. It provides insight into the range of ECM fungal habitats and a fundamental reference for more detailed studies of their specific functions in ecosystems. Although some ECM fungi can grow in culture without a host plant association, it is widely accepted that most are obligate symbionts in nature and require connections to hosts in order to grow vegetatively (Fleming, 1984) and sexually reproduce (Lamhamedi et al, 1994). Many fungi can associate with a variety of host plants, but a substantial proportion of them are also restricted to a particular host species or genus. Thus, the ecological niches for many species of ECM fungi are largely defined by the range of their hosts. Maintenance of tree diversity is therefore essential for maintaining ECM fungal diversity (Massicotte et al., 1999). Mycorrhizal research dates back to the late 1800's, and great advances have been made in the field since then. Describing effects of ECM fungi on host plants has depended on isolating the fungi in pure culture and using them to inoculate hosts in controlled experiments. Researchers have inoculated host trees with a variety of ECM fungi to examine their abilities to take up nutrients from natural substrates and model compounds, as well as to transport nutrients and carbon to and between different host trees. Such studies have been carried out mostly in artificial conditions. Meanwhile, mycologists and ecologists have described host species and habitats these fungi associate with in nature, and how they are affected by disturbance. The field of plant ecology is somewhat ahead of that of ECM ecology because organisms that live mostly underground are difficult to study. However, recent advances in molecular biology have allowed researchers to characterize below-ground ECM communities more accurately and more easily. As a result, ECM community ecology is increasingly being related to plant and soil ecology to provide more thorough understanding of ecosystem structure and function. 2 Literature Review Fungal Identification M o r p h o l o g i c a l M e t h o d s E C M fungi are most readily identified by their sporocarps (sexual reproductive structures). Although fungal ecology studies based on sporocarps provide valuable information, they are not always representative of E C M fungal community structure on root tips (Jonsson et al., 1999b; Durall et al, 1999; Gardes & Bruns, 1996; Visser, 1995). The advantage to observing the E C M community on root tips is that one can directly link each fungal species with its host plant, which is particularly helpful in mixed stands. Agerer (1987) and Ingleby et al. (1990) produced the first comprehensive guides for identification of E C M fungi based on morphology and anatomy of mycorrhizal root tips. Their identification was based on tracing fungal mycelia from sporocarps to root tips, and (or) by regular co-occurrence of particular sporocarp species and E C M root tip morphotypes. These guides are useful for identifying most E C M root tips to family or genus, and in the few cases where morphology is particularly distinctive, to species. Many useful studies have used morphotyping exclusively to characterize below-ground E C M communities in nature and in bioassays (e.g. Kranabetter, 1999; Durall et al., 1999; Goodman & Trofymow, 1998a; Harvey et al., 1997). However, morphotyping alone is usually limited in identification to E C M fungal species. This is partly because of the wide range of root tip morphology displayed by a single E C M species on different hosts, in different environments, and at different developmental stages. Furthermore, the suite of morphological characters available for examination on E C M root tips is often inadequate to discern species within difficult taxonomic groups, such as Cortinarius, Inocybe, Russula, and Tomentella, all of which are important E C M genera. In diverse E C M systems, molecular methods must be employed to accurately determine the taxonomic placement of E C M fungi. Another more recent guide to E C M descriptions (Goodman et al., 1996) incorporates molecular identification information in addition to detailed morphological descriptions. M o l e c u l a r M e t h o d s Since the early 1990s, researchers have been identifying fungi by using PCR to amplify known regions of fungal DNA from E C M root tips. The most commonly amplified region is the internal transcribed spacer (ITS) region, consisting of two ITS segments between the nuclear DNA coding for 18s, 5.8s, and 28s ribosomal subunits. While combined analyses of other DNA loci are often necessary to 3 establish well-supported phylogenies at higher taxonomic levels (Froslev et al, 2005; Bruns & Shefferson, 2004), the ITS region is adequate to distinguish species by RFLP or sequence analysis because of high interspecific and low intraspecific variation (Horton & Bruns, 2001; Karen et al, 1997). Horton & Bruns (2001), however, note that taxonomic affinity of fungi on E C M roots often remains unknown in studies using RFLPs because central databases for sporocarp RFLP patterns are not available and a variety of primers are used to amplify the region. Furthermore, there are several examples of different species within a genus giving the same RFLP pattern (Durall et al, 2006; Peter et al, 2001b; Karen et al, 1997), as well as different RFLP patterns coming from a single species due to a single nucleotide difference in a restriction enzyme binding site (Kretzer et al, 2003b). Matching RFLP patterns is also sometimes difficult due to error associated with resolution of agarose gels (Dickie et al, 2003). Recent studies have used RFLPs or T-RFLPs of the ITS region to group samples first, then sequence unique RFLP types to determine taxonomic affinity (e.g. Horton et al, 2005; Cline et al, 2005; Izzo et al, 2005; Tedersoo et al, 2003; Sakakibara et al, 2002). It is common practice to accept that a similarity of at least 97% or 98% between an unknown sample sequence and a database (e.g. GenBank) sequence constitutes a species-level match. Izzo et al. (2005) and Cline et al. (2005) have also recently used ITS DNA sequences to group unknown genotypes of multiple E C M root tip samples with each other using sequence similarity or phylogenetic trees. E C M Ecology E C M and Disturbance Response of forest E C M communities to disturbance has been a central topic in recent years. Many researchers have examined how E C M communities differ between young and mature forests, or how they are affected by natural disturbances or forest management practices. Clearcutting has consistently resulted in dramatic changes to E C M community composition and structure (Jones et al, 2003). Wildfire effects have been weaker where fires are patchy and of low intensity (Jonsson et al, 1999b), but intense stand-replacing fires can change community composition radically (Grogan et al, 2000). Fire may especially reduce E C M fungi proliferating in the forest floor (Stendell et al, 1999), but some species may readily recolonise after wildfire because they have resistant spores or vegetative propagules (Taylor & Bruns, 1999; Baar et al, 1999). These propagules may successfully colonise post-fire because of their resistance to heat and desiccation, or because they tend to be distributed lower in the soil profile and thus avoid heat from fires. 4 Two important factors that contribute to ECM colonisation of new hosts after disturbance are spatial extent of the area disturbed and distance of new hosts from mature trees with established ectomycorrhizae. Durall et al. (1999) found a marked decrease in ECM sporocarp richness with increasing size of cutblocks, particularly larger than 900 m2, but this decrease was not mirrored as strongly in the below-ground community. Hagerman et al. (1999b) found no effect of cutblock size on below-ground ECM communities. Studies show, however, that ECM diversity decreases and (or) community structure changes drastically with increasing distance from intact forest (Hagerman et al., 1999a; Durall et al, 1999) or isolated mature trees left after logging (Cline et al, 2005; Kranabetter, 1999) . This distance effect, in addition to seedling isolation effects (Simard et al., 1997b; Fleming, 1984) suggests that vegetative parts of ECM fungi attached to live trees are important inoculum sources. However, there is also evidence that soil biology and chemistry contribute to differences between ECM communities in mature and recently disturbed stands (reviewed in Jones et al., 2003). E C M and the Soil Environment The biotic and abiotic complexity of the soil environment is astounding, and physiological processes and interactions that occur underground are difficult to study. Nutrient cycling is a critical ecosystem function that is dependent on many factors including amounts and types of available substrates (e.g. Prescott et al, 2000; Frazer et al, 1990), soil moisture and pH (e.g Barg & Edmonds, 1999), soil microbial community composition (e.g. Houston et al., 1998), and soil fauna (e.g. Forge & Simard, 2000) . ECM fungi play a key role in nutrient cycling by transforming and translocating chemicals in the soil. While transfer of available mineral nutrients from soil to plants is well known (see Smith & Read, 1997), the ability of ECM fungi to acquire nutrients from soil organic compounds has been explored more recently (Read & Perez-Moreno, 2003). Generally, ECM fungi are important to plant nutrition when available nutrients are limiting, but ECM colonisation and diversity decrease when nutrients are readily available (Jones & Smith, 2004). Amounts and forms of nutrients vary widely with forest type, making it difficult to generalize about ECM functions, but nitrogen is usually limiting in northern coniferous forests. Some of the factors that control nutrient cycling also help determine ECM community structure. For instance, higher diversity of available substrates may result in higher ECM diversity. While some fungi prefer decaying wood (Smith et al., 2000; Goodman & Trofymow, 1998b), others have affinity to particular forest floor layers or mineral soil horizons (Rosling et al, 2003; Nelville et al, 2002). Soil 5 moisture (O'Dell et al, 1999; Gehring & Whitman, 1994) and available soil nitrogen (Avis et al, 2003; Li l leskov et al, 2002; Peter et al, 2001a) also affect E C M community diversity and composition above-and below-ground over long gradients. Plant Community Dynamics and Ectomycorrhizae Plant communities in E C M forests are constantly changing because of variable disturbances, spatial structure, and plant competition. This state of f lux has implications for E C M fungi. First, occurrence of tree hosts change, as do tree sizes and availability of roots. The two common models of plant succession, "relay floristics", where species groups establish sequentially over time, and "initial floristics", where all species establish soon after disturbance, are evident to varying degrees in temperate forests (Oliver & Larson, 1996). The pattern of succession that occurs in a given forest depends on pre-disturbance conditions, disturbance type, environmental conditons, and plant physiology and tolerances to environmental factors (Agee, 1993). Avai labi l i ty and dispersal of propagules are also important. Parallel models of E C M succession in forests have also been proposed, as discussed in detail in Chapter 2. 6 Study Overview, Objectives, and Hypotheses This study was conducted in serai stands of paper birch and Douglas-fir the Interior Cedar-Hemlock biogeoclimatic zone (ICH) of southern interior British Columbia. The ICH zone is characterized by mild winters, when most of the precipitation falls as snow, and warm summers with scattered rains in May-June and sometimes in August. Seasonal drought can occur during July-August. Clearcutting has been the most common cutting method since the 1970's, but variable retention has been applied extensively over the past decade. Up to 13 conifer and broadleaf tree species grow in mixture in the ICH zone. The overarching goal of my research was to re-evaluate existing theories concerning successional roles of E C M fungi. To do this, I studied E C M communities along a chronosequence of mixed stands of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and paper birch (Betvdapapyrifera Marsh.). I also examined relationships between E C M and site attributes, and compared wildfire with clearcutting effects on the E C M community. The specific hypotheses tested in this study were based on previous chronosequence studies of forest E C M fungal communities, studies of the effects of disturbances on E C M fungal communities, and studies of relationships between E C M fungal communities and environmental attributes. The fol lowing study objectives and hypotheses, along with their associated backgrounds and contexts, are addressed in Chapters 2 and 3: OBJECTIVE 1: To characterize successional patterns of the below-ground E C M community with increasing age of mixed Douglas-fir — paper birch forests (Chapter 2) • HYPOTHESES 1. E C M fungal species richness, diversity and evenness increase with stand age. 2. E C M communities and diversity differ between clearcut and burned forests. 3. Previously described "early stage" fungi (those forming E-strain mycorrhizae, and Thelephora terrestris) decrease in abundance and frequency with increasing stand age, while "late stage" fungi (Cortinarius, Prtoderma, and Russula spp.) increase, and "multi stage" fungi (Cenococcum geophUum and Inocybe spp.) have no distinctive pattern. 4. The proportion of the E C M community comprised of E C M fungi shared between Douglas-fir and paper birch decreases with increasing stand age. 7 OBJECTIVE 2: To examine relationships between soil properties and ECM fungal community measures along a chronosequence of mixed forest stands that were similar in vegetation composition and site quality (Chapter 3) • HYPOTHESES 1. Soil N and P availability and mineralizable N decrease with stand age, while the C:N ratio increases 2. Soil variables explain a substantial degree of variation in ECM diversity that is not accounted for by stand age; namely, inorganic N and P availability are negatively correlated with ECM fungal diversity, while organic P and C:N ratio are positively correlated 3. These soil variables are related to ECM community composition and structure. 8 References A g e e J K . 1 9 9 3 . Fire Ecology of Pacific Northwest Forests. Washington D.C., USA: Island Press. A g e r e r R. 1987. Colour Atlas of Ectomycorrhizae . Schwabisch Gmiind, Germany: Einhorn. A l l e n M F . 1 9 9 1 . The ecology of mycorrhizae. Cambridge, U K : Cambridege University Press. A v i s P J , M c L a u g h l i n D J , D e n t i n g e r B C , R e i c h P B . 2 0 0 3 . Long-term increase in nitrogen supply alters above-and below-ground ectomycorrhizal communities and increases the dominance of Russula spp. in a temperate oak savanna. New Phytologist 1 6 0 : 239-253. 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S a k a k i b a r a S M , J o n e s M D , G i l l e s p i e M , H a g e r m a n S M , F o r r e s t M E , S i m a r d S W , D u r a l l D M . 2 0 0 2 . A comparison of ectomycorrhiza identification based on morphotyping and PCR-RPLP analysis. Mycological Research 1 0 6 : 868-878. S i m a r d S W , P e r r y D A , S m i t h J E , M o l i n a R. 1 9 9 7 . Effects of soil trenching on occurrence of ectomycorrhizas on Pseudotsuga menziesii seedling grown in mature forests of Betulapapyrifera and Pseudotsuga menziesii. New Phytologist 1 3 6 : 327-340. S m i t h J E , M o l i n a R, H u s o M M P , L a r s e n M J . 2 0 0 0 . Occurrence of Pilodermafallax in young, rotation-age, and old-growth stands of Douglas-fir {Pseudotsuga menziesii) in the Cascade Range or Oregon, U.S.A. Canadian Journal of Botany 7 8 : 995-1001. 11 Smith SE, Read DJ. 1997. Mycorrhizal Symbioses. San Diego, USA: Academic Press. Stendell ER, Horton TR, Bruns TD. 1999. Early effects of prescribed fire on the structure of the ectomycorrhizal fungus community in a Sierra Nevada ponderosa pine forest. Mycological Research 103: 1353-1359. Taylor DL, Bruns TD. 1999. Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities. Molecular Ecology 8: 1837-1850. Tedersoo L, Koljalg U, Hallenberg N, Larsson K-H. 2003. Fine scale distribution of ectomycorrhizal fungi and roots across substrate layers including coarse woody debris in a mixed forest. New Phytologist 159: 153-165. Visser S. 1995. Ectomycorrhizal fungal succession in jack pine stands following wildfire. New Phytologist 129: 389-401. 12 Chapter 2 Below-ground ectomycorrhizal community succession on Douglas-fir {Pseudotsuga menziesii) and paper birch {Betula papyrifera) in southern interior British Columbia1 Introduction Ectomycorrhizal symbioses are ubiquitous in northern forest ecosystems and essential for tree host productivity. Because reciprocal feedbacks occur continuously between ectomycorrhizal ( E C M ) fungal species and tree hosts, E C M fungal species composition patterns should integrate with floristic succession of forested stands. In British Columbia, forested landscapes are frequently disturbed by fire, insects, disease, wind and humans, resulting in a dynamic mosaic of stands. Patterns of above-ground vegetation succession fol lowing wildfire and clearcutting have been fairly well characterised in forested systems (Oliver & Larson, 1996; Agee, 1993; Shugart, 1984), but patterns in E C M fungal succession have not (Jones et al, 2003). E C M fungal succession fol lowing afforestation by birch was first described in Britain over two decades ago, and E C M fungi were categorized as either "early-stage" or "late-stage", depending on infection timing, year of fruiting, and other fungal characteristics (Fleming, 1984; Mason etal., 1983; Deacon & Donaldson, 1983; Fleming, 1983; Fox, 1983). However, this work was performed in young plantations on agricultural soils, which were not comparable to natural stands. Researchers continue to use "early-stage" and "late-stage" terminology (Bergemann & Mi l ler, 2002; Visser, 1995). For example, some specific "early-stage" fungi, such as fungi forming E-strain mycorrhizae, fungi in the M R A complex, Thelephora terrestris, and Amphinema byssoides, dominate seedlings regenerating soon after disturbance, (Kranabetter, 2004; Hashimoto & Hyakumachi, 2000; Jones et al, 1997). "Early-stage" and "late-stage", however, are general descriptive terms that do not adequately reflect the complexity of fungal succession patterns in ecosystems. Nonetheless, describing E C M succession patterns in relation to their environments may be fundamental to understanding their life-history strategies and functions. ' A version of this chapter will be submitted for publication. Twieg BD, Durall D M , Simard S W, Jones M D . Below-ground ectomycorrhizal community succession on Douglas-fir {Pseudotsuga menziesii) and paper birch (Betula papyrifera) in southern interior British Columbia. 13 Chronosequences representing forest succession after wildfire or clearcutting have recently been used to study E C M fungal sporocarp and root-tip communities (Kranabetter etal, 2005; Smith etal, 2002; Visser, 1995). Although effects of stand age on E C M diversity varied among these studies, some general trends in E C M fungal species composition and community structure have emerged. In North American forests, for example, "multi-stage" fungi (e.g. Cenococcum geophilum, Inocybe spp.) that occur in all stand ages have been described (Visser, 1995). As stands age, "multi-stage" fungi are augmented, rather than replaced, by a community of several "late-stage" fungi (e.g., Cortinarius spp., Russula spp., Piloderma byssinum, and Tricholoma spp.). In the jack pine forests studied by Visser (1995), only a few "early-stage" fungi (Thelephora and fungi forming E-strain mycorrhizae) were present in young stands but did not occur in older stands. Smith et al. (2002) found no strong differences in cumulative species richness of E C M sporocarps among stand ages, but observed increases in sporocarp biomass of Cortinarius, Ramaria, Russula, Elaphomyces, and Rhizopogon with increasing stand age. Kranabetter et al. (2005) found increased E C M sporocarp richness with western hemlock forest age, including increased frequency of some Cortinarius and Russula species, as well as overall increased richness of Tricholoma and Craterellus species. Visser (1995) and Kranabetter et al. (2005) both found that E C M fungal diversity was low in young, open stands but was significantly higher in the youngest stand ages sampled that had closed canopies. Both studies also found diversity did not change much with further stand development. Host specificity of E C M fungi is also important in the succession of mixed-ECM host forests. Generalist fungal taxa comprise substantial proportions of the E C M community in mixed coniferous/broadleaf forests (Kennedy et al, 2003; Mol ina et al, 1992). The same is true in forests with two conifer hosts (Cull ings et al, 2000; Horton & Bruns, 1998). Older stands may, however, accumulate host-specific fungi with age, and some researchers believe that cl imax tree species associate with host generalist fungi to the greatest degree during their establishment period because it is important for them to achieve maximal colonisation by fungi present in the existing mycorrhizal networks of serai tree species (Horton et al, 2005; Kropp & Trappe, 1982). Where the dominant tree species in a forest are infected by a high proportion of generalist fungi, there exists good potential for common mycorrhizal networks (CMNs) to connect trees underground (Simard et al, 2002). These C M N s can facilitate carbon transfer between tree species, possibly moderating interspecific competitive interactions (Simard et al, 1997a). Certain E C M fungi appear to require established connections to trees in order to colonise new hosts (Hagerman etal, 1999b; Kranabetter, 1999; Dural l etal, 1999; Kranabetter & Wy lie, 1998; Simard etal, 1997b; Fleming, 1984). This may be particularly true for fungi forming mycelial strands and/or rhizomorphs, which appear to function in vegetative spread (Cline et al, 2005; Agerer, 2001; Newton, 14 1992). Several studies have shown that conspecific mature trees contribute to the establishment of seedling E C M communities (Jonsson et al, 1999a; Kranabetter, 1999). Some shrub and herbaceous perennials also harbour E C M fungal species common to coexisting conifer hosts, contributing to the establishment of their E C M communities (Dickie et al, 2004; Hagerman et al, 2001; Horton et al, 1999). Jones et al. (1997) found a high proportion of shared fungi on one and two-year-old Douglas-fir and paper birch seedlings, and Simard et al. (1997c) found that the richness of shared fungi on one-yr-old Douglas-fir increased when it was grown in mixture with paper birch. How patterns of colonisation by host-specific and host-general ist E C M fungi change with stand age in succeeding mixed conifer/broadleaf forests has yet to be studied. Ectomycorrhizal communities have been well studied on conifer seedlings regenerating within ten years of a stand-replacing disturbance, such as wildfire or clearcutting. These studies show that E C M fungal community composition differs between regenerating and mature stands, even though species diversity and colonisation often do not differ (Jones et al, 2003). Few E C M community comparisons have been made between stands of different disturbance histories. Lazaruk et al. (2005) found fewer active roots in the soil immediately fol lowing burning of a Picea glauca (Moench) Voss stand than after clearcutting, as well as lower E C M fungal species richness and diversity, but these differences were slight. Mah et al. (2001) also found E C M fungal community composition differences between clearcut versus clearcut and broadcast burned sites, but no differences in diversity or colonisation. The objective of this study was to characterize successional patterns of the below-ground E C M community with increasing age of mixed Douglas- f i r— paper birch forests, and to improve on earlier studies of E C M succession by using molecular identification of E C M fungi and a well-replicated study design. The hypotheses addressed were: 1) E C M species richness, diversity and evenness increase with stand age; 2) E C M communities and diversity differ between forest of clearcut and wildfire origin; 3) previously described "early stage" fungi (E-strain-forming fungi and Thelephora terrestris) decrease in abundance and frequency with increasing stand age, while "late stage" fungi {Cortinarius, Piloderma, and Russula spp.) increase, and "multi stage" fungi {Cenococcum geophilum and Inocybe spp.) have no distinctive pattern; and 4) the proportion of the E C M community comprised of E C M fungi shared between Douglas-fir and paper birch decreases with increasing stand age. These hypotheses were tested by morphological and molecular characterization of E C M communities across a chronosequence of mixed Douglas-fir — paper birch forests in southern interior British Columbia. 15 Materials and methods Site description and study design The study sites were located in the Thompson Moist Warm (!CHmw3), Shuswap Moist Warm (ICHmw2), and Thompson Moist Cool (ICHmk2) Interior Cedar-Hemlock variants of southern interior British Columbia (Lloyd et al, 1990). Following wildfire, Douglas-fir, paper birch and lodgepole pine (Pinus contorta var. latifolia Doug. Ex Loud.) dominate serai ICH forests to approx. 120-150 years. Shade tolerant conifers establish immediately post-disturbance or slowly ingress with time (Agee, 1993), and form climatic climax stands comprised of western redcedar (Thujaplicata (Donn ex D. Don) Spach) and either western hemlock (Tsuga heterophylla (Raf.) Sarg.) in the moist warm variants or hybrid spruce (Picea engelmannii Engelm x Picea glauca (Moench) Voss) in the moist cool variants ( Lloyd et al, 1990). Maturing serai stands of Douglas-fir are often logged at between 120 and 150 years of age in the study area. Single cohort stands were selected to represent a chronosequence following stand-replacing fire and clearcutting. Four stand age classes were chosen to represent important stages (Oliver & Larson, 1996) of mixed stand development: 4- to 6-years-old (stand initiation stage, where Douglas-fir naturally seeds in or is planted, and paper birch naturally seeds-in or sprouts from existing root stocks), 21- to 30-years-old (canopy closure followed by stem exclusion stage), 60- to 70-years-old (stand re-initiation stage, where birch starts to die out of the stand, creating canopy gaps) , and 90- to 103-years-old (stand re-initiation stage, where Douglas-fir is dominant over remaining paper birch). For brevity, these age classes will be referred to as 5-, 26-, 65-, and 100-yr-old stands, respectively, and stands of wildfire origin will be referred to as burned. For the two youngest age classes, clearcut and burned stands were selected, but only burned stands were used for the oldest two age classes. Clearcut stands in the two oldest age-classes were not available in the study area, precluding a complete factorial study design. Four replicate sites were selected for each of the six stand (age class/initiation-origin) types. The only sites not located in the ICHmw variants were two 26-yr-old burned sites, which were located in the ICHmk2 variant due to difficulty finding appropriate sites in the ICHmw variants. All burned sites of 20-years-old and older naturally regenerated to Douglas-fir and paper birch, whereas clearcut sites and 5-yr-old burned sites had been planted to Douglas-fir with natural regeneration of paper birch. Paper birch trees from both seed and stump-sprout origin were present at both clearcut and burned 5-yr-old sites. Stand ages were determined from B.C. forest cover maps (B.C. Ministry of Forests; Victoria, B.C., Canada) and, for naturally regenerated sites, by coring five Douglas-fir trees. For 16 plantations in the youngest age class, age represents the number of years since planting; naturally regenerated Douglas-fir were absent from these stands. Site selection was based on the fol lowing criteria: 1) Douglas-fir and paper birch comprised at least 75% of the total canopy cover of E C M host tree species in a 40 m by 40 m area (Table 2.1), 2) moisture regime was submesic to mesic and site series was zonal ( L loyd et al., 1990), and 3) distance to other replicate stands was at least one kilometre. Careful excavation of Douglas-fir seedlings prior to sampling in 5-yr-old sites showed that their root systems rarely extended beyond 40 cm of their bole, and tracing of other conifers' root systems outward indicated little chance that their root systems overlapped those of the Douglas-fir seedlings. Once all sites were selected, one 30 m by 30 m plot was established at each site. Buffer zones of 10 m (5-yr-old sites), 15 m (26-yr-old sites) 25 m (65- yr-old sites), and 30 m (100-yr-old sites) were delineated to eliminate edge effects. Midpoints of plot edges were marked to delineate quadrants. Sampling for Ectomycorrhizae Soils were sampled for ectomycorrhizae twice in 2004: late May to early June (spring) and late September to early October (fall). For each sampling period at each site, eight separate pairs of trees, including one Douglas-fir and one paper birch each, were selected inside the plot (two each per plot quadrant). The maximum inter-tree sampling distance for greatest l ikelihood of obtaining roots from both tree species was 3 m in 5-yr-old stands, 5 m in 26-yr-old stands, 7.5 m in 65-yr old stands, and 10 m in 100-yr-old stands. One soil sample was taken from within a 0.5 m-wide transect between the two trees in each pair (one sample x eight locations x 2 sampling seasons = 16 samples taken per site). In 5-yr-old sites, samples were taken within 30 cm of the Douglas-fir boles due to the limited radial extent of their root systems, and preliminary samples showed ample birch roots were also available in these sampling locations. In all other age classes, samples were taken at the midpoint between the two trees of each pair. The minimum distance between any two sampling locations was 2 m. Soils were sampled far enough from other E C M host trees that there was little chance that soil samples included their roots. This was an issue mostly in 26-yr-old stands, where black cottonwood (Populus trichocarpa Torr. & Gray), trembling aspen {Populus tremuloides Michx.), wi l low (Salix) spp., and hybrid spruce occurred in the canopy, and western hemlock occurred in the understorey. Where sampling locations were less than 5 m from these trees, their presence was noted. A t each sampling location, forest floor and mineral soils were removed in a 9 cm X 9 cm area using a machete and trowel (soils were too stony to use corers). The forest floor depth was recorded, and the L F H layers placed 17 together in a plastic bag. Mineral soil was then removed to 20 cm depth from the bottom of the forest floor and bagged separately. Samples were stored on ice in a cooler until transfer later the same day to a 4 °C walk-in cooler. Soil samples collected in the spring from 5-yr-old burned sites yielded insufficient root tips. For fall sampling, three Douglas-fir seedlings were therefore removed from plot buffer zones in these sites and 5-yr-old clearcut sites. Seedlings were excavated to a depth of 30 cm and 25 cm radius around the bole, their stems removed, and the soil blocks transported in garbage bags the same day to the 4° walk-in cooler. Core-type soil samples and seedlings were taken in fall from 5-yr-old clearcut sites, but only seedlings were taken from 5-yr-old burned sites. This meant that 5-yr-old burned stands could be compared statistically only to 5-yr-old clearcut stands, and only based upon the E C M communities of the excavated Douglas-fir seedlings. Sorting of Root Tips One soil sample from one site of each stand type was processed in a rotation, with the site changed in each successive rotation to mitigate potential confounding effects from samples remaining in the cold room for different amounts of time. Forest floor (when present) and mineral soil samples were processed separately. Forest floor samples were first soaked in distilled water for a few hours. A l l samples were washed gently with tap water over 4-mm and 2-mm stacked sieves. A l l woody and fine roots were removed with forceps from both sieves and placed in distilled water in a glass baking dish. Root segments were cut into 1.5 cm segments and gently mixed, and obvious E C M tubercles were sliced in half due to their dumpiness. Segments were randomly selected over a numbered 2 cm grid for viewing under a dissecting microscope. Viabi l i ty of root tips was determined based on colour and turgidity (Harvey et al, 1976), and by whether or not the stele was intact and not easily broken. Ectomycorrhizal status was determined by the presence or absence of a fungal mantle. For tips lacking an obvious mantle but showing E C M characteristics (e.g., inflated shape, branching, lack of root hairs), a representative was examined for presence of a Hartig net at 400X magnification under a compound microscope. Douglas-fir and paper birch roots were differentiated by the size of root tips and, to a lesser extent, by colour and texture of larger roots. When these characteristics were ambiguous, E C M cross sections were viewed under a compound microscope. Mycorrhizal paper birch root tips were differentiated from Douglas-fir by their radially elongated epidermal cells. It is possible that roots of other broadleaf trees were counted as birch roots and those of other conifer species counted as Douglas-fir, so error in host tree identification was 18 estimated from root tips using molecular methods. Primers trnLc and trnLd were used to amplify host DNA from the same root tip samples used for ECM fungal identification, and PCR products were digested with Hinfl and Taq I as described in Brunner et al. (2001). Ten samples of Douglas-fir root tips and fifteen samples of paper birch root tips were identified in this manner from samples taken within 5m of other ECM host trees, and fifteen more samples of each host species were identified from randomly selected samples. Successive root segments were examined until 50 live Douglas-fir and 50 live paper birch root tips were counted from each soil layer per sample. Percent ECM colonisation was determined from the ratio of live mycorrhizal to total live tips. Segments with ectomycorrhizae were then further cut into 5-10 mm segments (without cutting ECM tips) and 25 tips were randomly selected from a 5 mm grid for morphotyping (2 hosts x 2 soil layers x 25 tips =100 root tips per soil sample). Where less than 50 root tips of a particular host occurred in a soil sample, root tips of the other host species were sampled to bring the total number of ECM tips to 100 per soil sample. Similarly, if one soil layer from a soil sample did not contain enough roots, or if no forest floor layer was present for that sample, then additional roots were sampled from the other soil layer. Roots of seedlings from 5-yr-old sites were washed, clipped from the bole, cut into 1.5 cm pieces, and randomly sampled until 400 root tips were counted and classified as mycorrhizal or non-mycorrhizal. Morphotypes were determined for 200 randomly selected tips. Paper birch roots present in the samples were similarly subsampled and morphotyped where available. Morphological and Molecular Identification of E C M Root Tips Morphotyping All subsamples were examined according to Goodman et al. (1996). Branching pattern, system and root tip sizes, mycelial strands, and emanating hyphae were examined under a dissecting microscope. Emanating elements and inner and outer mantle patterns of mantle peels were examined at 400X and 1000X under a compound microscope. Chemical tests were used for taxonomic affinity to family or genus as described in Kernaghan et al. (2003) and Agerer, (1987). Notes were compiled on colour, branching pattern, system dimensions, emanating elements, cell dimensions, mantle patterns, hyphal junctions and anastomoses, cystidia, etc., and photographs were taken of important features (Appendix A) using a DMX 12000 digital camera mounted to a SMZ 1000 dissecting microscope and Eclipse 800 compound microscope outfitted with Differential Interference Contrast (Nikon, Melville, New York, USA). Up to three subsamples, where available, per morphotype per host in each soil sample were set 19 aside for potential molecular identification. For each subsample, 1-5 root tips were placed in 100 pi of sterilised millicue water in a microcentrifuge tube, freeze-dried, and stored at -80°C until DNA extraction. Subsamples of morphotypes were neither mixed within a soil sample nor between soil samples. Soil samples were processed over six months for each sampling period. DNA Extraction and PCR Amplification One subsample per morphotype per soil sample was subject to DNA extraction and PCR amplification of the fungal ITS region of nuclear rDNA. About 540 of these samples had ITS regions DNA sequenced. In addition, 2-3 samples each of 15 morphotypes were analysed by RFLP or DNA sequencing to check accuracy of within-soil sample morphotype sorting. Seven of these 15 morphotype selections contained subsamples from both host species in the same soil samples. ECM root tip samples were pulverized in 400 u.1 of CTAB buffer (3% CTAB; 100 m M Tris-Cl pH 8.0; 1.4 M NaCl; 20 m M EDTA; 2 % PVP; 0.2% (3-mercaptoethanol) with a ceramic bead in a FastPrep beater machine (Qbiogene, Irvine, CA, USA) for 45 sec. and were then incubated at 65°C for 90 minutes. DNA was isolated with two repetitions by adding of an equal volume of chloroform-isoamyl alcohol (24:1), mixing by inverting for one minute, centrifugation at 13 000 rpm for 10 min., and removal of the aqueous phase. DNA was precipitated overnight in 2/3 volume of isopropanol, followed by two washes with 70% ethanol. The pellet was dried in a speed-vac for 10 minutes, and resuspended in 100 pi of low-EDTA TE buffer (10 m M Tris-Cl pH 8.0; 0.1 m M EDTA). PCR reactions were carried out in 25 or 50 u.1 reactions with the following concentrations: 1.5 m M MgCl 2, 1.6 mg of1 BSA, 0.2 m M dNTP's, 0.48 pm each primer, and 0.25 units pi"' of Ampli-Taq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA). Primers were synthesized by Nucleic Acid and Protein Services (NAPS) at UBC, Vancouver. Template DNA was added in the amount of 1 pi per 50 pi reaction. DNA was diluted tenfold for samples that did not amplify the first time. A PTC-200 Thermocycler (MJ Research Inc., Waltham, M A , USA) and a GeneAmp 2700 Thermocycler (Applied Biosystems) were used. An initial cycle of 10 min. at 95 °C was used to activate the polymerase, followed by 34 cycles of the following: 45 sec. denaturation at 94 °C, 45 sec. of annealing, and 1 min. of extension at 72 °C. A final extension step of 7 min. was added. Annealing temperatures varied with the primers and thermocyclers used. Optimum annealing temperatures were determined on the PTC-200 by running a gradient in 0.5 °C increments above and below the T M calculated by NAPS, and on the GeneAmp 2700 by running the annealing temperature at 1 °C below the T M determined by the thermocycler's calculator. 20 Several primer pairs were used to amplify fungal DNA from root tips. After many initial amplification attempts with primer NL6Btnun for intended RFLP analyses, it was determined that its success rate (about 50%) was unacceptable, and its use was discontinued. For samples slated for DNA sequencing, primers NSI1 and NLC2 (Martin & Rygiewics, 2005) were attempted first because they showed the highest success in a comparison of several primer pairs on a set of random samples. Initial results showed poor amplification of Rhizopogon and Suillus mycorrhizae with these primers. Subsamples fitting those morphotypes were thereafter amplified with ITS If and ITS4 because these primers have been used successfully with these taxa before (Kretzer et al, 2003b; Bruns et al, 2002). Other basidiomycete morphotypes that did not amplify well with NSI 1 and NLC2 were attempted with ITS1 and 1TS4B (Gardes & Bruns, 1993). PCR products were visualized on 1.5 or 2% agarose gels made with ethidium bromide. Gels were photographed with a Kodak Gel Logic® 440 gel documentation system. Single-band PCR products were cleaned with a Charge Switch PCR Cleanup Kit (Invitrogen, Carlsbad, CA, USA). For products that showed multiple bands and ample amounts of DNA, 20-40 ul of PCR product were separated on 1.5% gels. If bands were sufficiently far apart, they were excised from gels on a UV transilluminator, and their DNA was purified with a QIAquick Gel Extraction kit (Qiagen Inc., Valencia, CA, USA). R F L P Analysis It was decided that using RFLPs as the main method for grouping samples would be inadequate because of the problems discussed in Chapter 1. Therefore, RFLPs of the fungal ITS region were used in this study mainly to check on accuracy of within-soil sample morphotype sorting. Primers ITS1 and NL6Bmun were used to amplify DNA for these RFLPs. Several tuberculate Rhizopogon samples were also checked by amplification with ITS If and ITS4B and restriction digestion with/l/w I, as Kretzer et al. (2003b) showed this to be an easy and effective way of distinguishing species of Rhizopogon vinicolor-like ECM. Restriction digests were performed on PCR products and RFLPs visualized on gels as described by Hagerman et al. (1999b). Kodak ID software (Kodak Instruments, Rochester, New York, USA) was used to estimate fragment lengths, and GERM software to suggest matches within the acceptable error suggested by Dickie et al. (2003). DNA Sequence Analysis Samples amplified with primers NSI1 and NLC2 were sequenced with primers ITS1 and NLB4 (Martin & Rygiewics, 2005) using the Big Dye Terminator Kit (Applied Biosystems). Those initially 21 amplified with ITS1 and ITS4B were sequenced with ITS1 and ITS4. Sequencing was performed on a 3730S capillary sequencer (Applied Biosystems) at N A P S or on a 3130x1 capillary sequencer (Applied Biosystems) at U B C Okanagan Fragment Analysis D N A Sequencing Services (FADSS) . Forward and reverse sequences were aligned, manually corrected, and trimmed in Sequencher 4.2 (GeneCodes, Ann Arbor, MI, USA) . Consensus sequences were B L A S T searched (Altschul et al, 1997) through N C B I (National Center for Biotechnology Information, 2006) and UN ITE (http://unite.zbi.ee) (Koljalg et al, 2005) websites to suggest taxonomic affinities of the samples. A taxon was considered a proper species match to a root tip sample i f their sequences had 98% or greater similarity and aligned over at least 450 base pairs. Samples that sequenced poorly in one direction were B L A S T searched with a single-pass sequence. These samples were considered proper matches at 97% similarity or better due to error rates of single-pass sequencing (Izzo et al, 2005). The number of bases aligned and percent similarity were checked for the ten highest-scoring matches. When matches were less than 98% (97% for single pass) and/or under 450 bp, it was deemed relatively unimportant which B L A S T match was "best" by any objective measure, since these matches did not solely determine the taxonomic placement of samples. Preference was given to matches to identified sporocarps. If no proper species match was made to a sample, then the taxonomic placements of the ten top-scoring matches were checked. If they consistently fell in the same family or genus, then the unknown sample was placed into that group. After general taxonomic placement of samples by B L A S T searching, a separate multiple alignment file of root tip sample sequences and sporocarp sequences from Durall et al. (2006) was made for each of the fol lowing groups: Cortinarius, Hebeloma, Inocybe, Lactarius, Piloderma, Russula and non-Lactarius Russulaceae, and Thelephoraceae. Mult iple alignments were performed in ClustalX (Thompson et al, 1997) for each group using the IUB algorithm, and corresponding sequence similarity matrices were created in the D N A D I S T program in a current version (3.63) of P H Y L I P (Felsenstein, 1989). Pairwise and multiple alignment gap penalty parameters were adjusted as suggested by Hal l (2004), and the resulting alignments were visually compared for quality. Since the ITS region is so variable due to common indels (Horton & Bruns, 2001), biologically reasonable alignments usually resulted from setting gap penalties somewhat low (gap opening at a value of 12 and gap extension at a value of 1). Similarity calculations ignored gaps and missing data. The same criteria for species matching as used with B L A S T searching were used to match unknown samples to each other. After matches were determined, multiple alignments were again scrutinised in order to detect potential spurious matches due to alignment flaws. Samples with ambiguous matching were excluded from analyses. 22 W h i l e the o r ig ina l intent was to obta in D N A in fo rmat ion on one subsample per morphotype per so i l sample , lack o f t ime and f und ing necessitated that we use some m o r p h o l o g i c a l data a long w i t h D N A in fo rmat ion fo r the f ina l c l ass i f i ca t ion . N o attempt was made to analyse mo lecu l a r data for Cenococcum geophilum, because Sakak iba ra et al. (2002) con f i rmed w i th mo lecu l a r methods ( ITS region) that morpho log i c a l ident i f i cat ion was re l iable for this species. D o u h a n & R i z z o (2005) a lso showed that genet ic d ivers i ty was as h igh between C. geophilum samples f r o m the same so i l core as it was between samples f r om dif ferent parts o f the U . S .A . They state that this mul t i- locus genetic d ivers i t y may s ign i f y sympat r i c c rypt i c spec iat ion. G i v e n l im i t ed project resources, it was dec ided that attempts to def ine di f ferent species in the C. geophilum c o m p l e x w o u l d be imposs ib l e . Data analyses E C M from Soil Samples Stat ist ical analyses were carr ied out us ing S A S vers ion 9.1 ( S A S Institute Inc., C a r y , N C , U S A ) unless otherwise noted. Least-squares means o f percent E C M co lon i sa t ion o f Doug las- f i r and b i r ch were ca lcu lated for stand types, due to the unequal numbers o f so i l samples con ta in ing target root t ips f r o m dif ferent sites. L o g i s t i c regress ion was used to determine whether t ime o f so i l sample refr igerat ion affected mo lecu l a r results. F o r this ana lys is , mo lecu l a r " s u c c e s s " was def ined as a sequence that was useable and consistent w i th the morphotype . " F a i l u r e s " had consistent double-peaks, were matched w i t h saprotrophic taxa , o r were otherwise inconsistent w i t h the morphotype . Taxa-sample unit curves were created us ing the r e samp l i ng procedure in P C - O R D ve rs ion 4 ( M c C u n e & M e f f o r d , 1995-2002) fo r eva luat ing whether the sample s ize was suf f i c ient to character ize the c o m m u n i t y at each site. One set o f curves represents the average o f curves for the four repl icates per stand type, and a second set was created by p o o l i n g the four repl icates o f each stand. Rank-abundance plots were also generated for each stand type and host, us ing stand type species abundance data poo led fo r the four repl icates. Separate ca lcu la t ions and analyses were per formed for site-level E C M funga l abundance data (i.e. number o f root t ips co lon i sed b y each taxon) and f requency data (i.e. the number o f so i l samples in w h i c h each funga l t axon was found) . Roo t t ip abundance o f E C M funga l taxa is more in format i ve about c o m m u n i t y structure than their f requency a lone. H o w e v e r , abundance may g ive a skewed representat ion o f E C M c o m m u n i t y structure because o f spatial patchiness o f E C M species (C l i ne et al., 2005) . P C - O R D was used to calculate site-level species r ichness, evenness, and Shannon-Weaver and S i m p s o n d ivers i t y (1-D, the comp lement o f S i m p s o n ' s o r ig ina l index) ind ices . These data were ca lcu la ted for Doug las- f i r 23 and paper birch E C M communities separately and for their combined communities. 1 s t and 2 n d order jack-knife estimators of species richness were also calculated, based on the combined community, for each site. Two-way analyses of variance ( A N O V A ) were used to test for mean differences in species richness, evenness, diversity (Shannon-Weaver and Simpson indices), and relative abundance of shared E C M fungal species (i.e., species on both hosts) among stand types (main treatment effect) and between' host species (subplot effect) using a split-plot completely randomised design (CRD) (Table 2.2). One-way A N O V A for a C R D was used to compare the whole E C M fungal community (of both hosts combined) between stand types. PROC G L M was used for the A N O V A s and TDIFF for pairwise mean comparisons, with a Bonferroni adjusted significance level of a = 0.05, unless otherwise noted. Normality was checked with P R O C U N I V A R I A T E , using a p > 0.05 criterion for Anderson-Darling and Shapiro-Wilks tests. Homogeneity of variance was checked by examination of residual vs. predicted plots and by Bartlett's test. Principal Components Analysis was performed on the multivariate dataset of species richness, evenness, Shannon-Weaver diversity, and Simpson diversity of the Douglas-fir, paper birch, and combined communities, plus the 1 s t order jackknife estimate for the combined community (i.e. 13 variables). Site scores for the first principal component were regressed, using least-squares, against all possible combinations of the fol lowing predictor variables that included at least one age variable: stand age, stand age squared, 1/stand age, initiation type (dummy variable) and subzone (dummy variable). Akaike 's Information Criterion (AIC) adjusted for small sample size (AICc) was used to select the best model from the set of candidates (Burnham & Anderson, 2002). Since the ICHmk has a different cl imax tree community and a shorter growing season than the ICHmw, regressions were performed on E C M diversity data of the Douglas-fir community and the combined community to check for effects of subzone. The same five predictor variables were included as were used in the regression of the diversity principal component. However, three model selection methods were used in PROC R E G instead of A ICc for each diversity variable: stepwise, backward, and forward. Significance of a = 0.1 was used for the criterion of variable retention in models. Sites were ordinated according to E C M fungal communities using nonmetric multidimensional scaling (NMS) . Separate N M S ordinations were performed on abundance data and frequency data in PC-O R D using the Relative Sorensen distance measure. With frequency data,.another ordination was done with species grouped at the genus level because some community trends can be more obvious at this 24 level. This is true because genera like Russula, Cortinarius, and Tomentella are very speciose and E C M communities are often comprised of many rare species and only a few, i f any, frequently found species (Taylor, 2002). Ordinations with all three types of input data were run for the community on Douglas-fir, the community on paper birch, and their combined community. For these ordinations, "autopilot mode" was employed from a random starting configuration (McCune et al, 2002). This mode chooses the best solution for each dimensionality (up to six) from 40 runs on the real data and provides a Monte Carlo test for significance of real data runs by comparing them to 50 randomizations on each dimension. Abundance and frequency of particular E C M fungal taxa were examined more closely. These taxa were chosen because they had correlations of at least 0.6 (absolute value) to at least one of the two axes with the highest R 2 values from an ordination, or because they ranked as one of the ten fungal taxa of highest mean relative abundance in at least one of the stand types. Two statistical approaches were taken. First, mean relative abundances of taxa occurring on only one host were tested for stand type differences with one-way A N O V A s . Mean relative abundances of taxa that occurred on both hosts were also tested for differences among stand types and between host species using two-way (split-plot) A N O V A as above. Data not meeting normality assumptions were arcsine-square-root transformed; where this did not help, data were analysed using Kruskal-Wall is tests. The second approach for examining individual species occurrence patterns was to use P R O C G E N M O D for modelling taxa abundance and frequency using count data (Cameron & Trivedi, 1998; McCul lagh & Nelder, 1983). A l l possible combinations of the variables stand age, 1 /stand age, subzone, and stand initiation type were used as predictors. The reciprocal of stand age was included as a predictor because earlier chronosequence studies (see Introduction) showed that relationships of E C M fungal abundances to stand age likely are not linear. Extra soil samples previously excluded from site-level analyses for removing sampling-effort differences between sites were included in abundance models because these models were based on abundance data from individual soil samples. A l l models were run with the Poisson and negative binomial distributions. Whi le the Poisson distribution is often used to model count data, clumpy spatial distribution of species can result in data better fit to the negative binomial distribution (Krebs, 1999). Clumpy spatial distributions of E C M fungi within sites are well documented (Tedersoo et al, 2003), but patchiness among geographically distinct stands is not wel l studied. Fit to the two distributions was compared as suggested by Cameron & Trivedi (1998). A IC was used to select the best model from abundance models, and A I C C was used to select the best frequency models. Likel ihood ratio tests of predictor variables were checked, and Type III tests were used in models with more than one predictor variable. Predicted values from abundance models were relativised to average root tips per core for the appropriate host(s). 25 E C M from Seedlings Shannon-Weaver and Simpson diversity indices, as well as species richness and evenness were calculated for both initiation types of 5-yr-old stands based on abundance data from seedlings. Each o f these four measurements, plus percent E C M colonisation, was tested for a difference between initiation types with two-tailed t-tests assuming equal variance. N M S ordinations were run with frequency and abundance data as described for the E C M community from soil samples. 26 Results E C M from Soil Samples E C M Colonisation and Distribution Mean ECM colonisation was at least 97% for all stand types. This was based on the following average sample characteristics per site: 6 soil samples containing 50 root tips each of Douglas-fir and paper birch, 2 soil samples containing 100 root tips of only Douglas-fir, and 2 soil samples containing only 100 paper birch tips. One important exception was one 26-yr-old burned site that yielded only 5 soil samples with Douglas-fir root tips and one 65-yr-old site that yielded only 9 total soil samples with sufficient ECM root tips. Molecular methods showed that identification of Douglas-fir root tips was correct for all of the 25 samples selected. However, for paper birch, 2 out of 15 samples taken within 5 m of other ECM broadleaf host trees gave RFLPs matching the genus Salix (Brunner et al., 2001). One out of 15 samples taken more than 5 m from other ECM broadleaf host species also matched to the genus Salix. This genus had the greatest presence in 26-yr-old stands, with scattered occurrences in 5-yr-old stands and only one other occurrence in a 65-yr-old stand. However, the 15 samples checked accounted for 83% of the total examined samples that were taken within 5 m of another broadleaf species in 26-yr-old stands, so the overall error in birch identification was likely quite low. Identification of E C M Fungi On average, 476 Douglas-fir and 462 paper birch ECM root tips were identified per site with sufficient morphological and/or DNA support. Out of 541 DNA sequences analysed, 83% were of sufficient quality and length to place into genotypes, but 13% of these were identified by BLAST search as non-target, co-occurring fungi that were preferentially amplified in the PCR. About 30% of the non-target sequences were either from fungi in either the MRA complex (mostly Phialocephala fortinii) or matched fungal sequences amplified from roots of Ericaceous plants. Several samples that were obviously colonised by Leccitmm, Suillus, and Lactarius spp. from morphotyping produced sequences that grouped well with Rhizoctonia or Inocybe spp. when amplified with ITS4B. Other non-target species included saprotrophic Ascomycetes and Basidiomycetes. The remaining 17% of sequences could not be analysed due to double sequence peaks. Logistic regression analysis showed that the probability of PCR products succeeding to produce useful target sequences was significantly negatively related to the amount of time their respective soil samples were stored in refrigeration. The predicted decrease in the 27 probability of success decreased from about 80% initially to 58% after samples had been stored for 6 months (p = 0.02 for likelihood ratio test for variable of time stored; A I C of model including time in storage variable= 649.9; A IC of model with intercept only = 653.3). In total, 105 unique E C M genotypes (hereafter referred to as "species"), all with genetically-determined taxonomic affinity appropriate to their respective morphotypes, were used for analyses in this part of the study. The average useable sequence length was 706 base pairs (std. dev. = 148). Proper matches to query sequences that aligned over at least 450 bp were mostly unambiguous, with the exception of samples matching Tomentella ramosissima in the N C B I B L A S T search, which also matched well to T. lapida in the UN ITE B L A S T search. In the genus Cortinarius, one sample gave a D N A sequence of 467 base pairs, but matched 98%) or above with several obviously distinct Cortinarius genotypes from this study. This sample was thus excluded from analyses, and probably matched spuriously because the 467 base pairs included the whole 5.8s ribosomal D N A sequence. E C M fungal species and their corresponding B L A S T search results and mean relative abundances by stand type are listed in Appendix B. Generally, morphotypes deemed unique due to obvious macro- and microscopic characteristics were supported as being distinct species by D N A evidence. However, distinctions by morphotyping within and among the genera Cortinarius and Hebeloma, among most species of the genus Russula, and within the families Thelephoraceae and Sebacinaceae were not possible due to morphological similarity of different species. Therefore, only samples of distinctive morphotypes that were well-supported by D N A sequences, as well as samples that were sequenced, were included in analyses. Exceptions to this rule were two Lactarius morphotypes for which D N A amplification attempts were unsuccessful, but which had very distinct morphology and anatomy in comparison to other Lactarius species identified by molecular methods. A l l RFLPs and sequences of morphotype subsamples taken within the same soil samples indicated that within-soil sample morphotype groupings were accurate. Rhizopogon vinicolor and R. vesiculosus (sensu Kretzer et al, 2003b), tuberculate species difficult to distinguish by morphology, were encountered frequently. We could not analyse D N A from all observations of these sister species, but all D N A sequences that were analysed for this tuberculate morphotype matched one of these two species. Some ambiguity was encountered in the morphological features separating the species (Kretzer et al, 2003b). Since both species were identified by D N A analysis for numerous observations in all stand types, they were lumped as Rhizopogon vinicolor-type for analyses. The NSI1 -NLC2 primer pair seemed to preferentially amplify Ascomycetes co-inhabiting tuberculate Rhizopogon samples, as over 50% of sequences from these samples matched Ascomycete 28 sequences. There were also two cryptic species in the Piloderma fallax-Vike morphotype as determined by D N A sequence analysis, but their high frequency also did not permit sequence analysis of many samples. Hence, these two species were lumped as Piloderma spp. in the analyses. E C M Community Diversity Mean species-sample unit curves showed that sampling was inadequate for all stand types (Fig. 2.1a). Sampling inadequacy appeared least serious in the 5-yr-old clearcuts and 26-yr-old burned sites, but curves for the 26-yr-old burned sites were more similar to 26-yr-old clearcuts when the two burned ICHmk sites were removed from the analysis (Fig. 2.1b). The overall pattern of curves generated by grouping all four replicates for each stand type was similar to that of site-level curves (Fig. 2.1c). The rank abundance graph for the combined community shows that evenness was lower in the 5-yr-old clearcuts than the older types (Fig. 2.2a). In the Douglas-fir community, 5-yr-old stands displayed a much steeper abundance curve than older stands (Fig. 2.2b). Species abundance patterns in the paper birch community were similar among age classes (Fig. 2.2c). Analyses of variance on richness, evenness, and diversity all detected significant stand type by host species interactions because differences among stand types were more extreme in the Douglas-fir E C M community than that of paper birch (see Table 2.3 for A N O V A results). Mean richness of the Douglas-fir E C M community was significantly lower in the 5-yr-old clearcuts than all other stand types except the 26-yr-old burned type (Fig. 2.3a). The two oldest stand types were also significantly richer than the 26-yr-old burned type. Richness on paper birch tended to be lower in 5-yr-old clearcuts than the other sites, and this difference was significant for 26-yr-old clearcut and 65-yr-old stand comparisons. The maximum difference in richness between stand types was much larger for Douglas-fir than birch, and the birch E C M community was significantly richer than the Douglas-fir community in 5-yr-old clearcuts and 26-yr-old burned sites. E C M species diversity and evenness on Douglas-fir were lower in the 5-yr-old clearcuts than all other stand types, but these measures for paper birch did not differ among stand types (Figs. 2.3b-d). Diversity was about 3 times as high for paper birch as Douglas-fir in the 5-yr-old clearcuts. The jack-knife richness pattern for the combined community was similar to the patterns in Shannon-Weaver diversity for the Douglas-fir and combined communities (Fig. 2.4). Using frequency for diversity estimates yielded the same patterns as abundance (Table 2.4). 29 The first principal component representing a combination of species diversity variables accounted for 74% of the total variation. The best AICc-selected regression model of site scores for this principal component included 1/stand age and subzone as predictor variables (R2 = 0.65; both predictors significant at a = 0.05). It predicted a sharp difference between 5- to 26-yr-old stands, and slight differences with among the older ages (Fig. 2.5). Subzone was significant (at a = 0.1, sometimes at a = 0.05) in regression models predicting species richness and diversity indices, except for evenness of the Douglas-fir or combined communities, and Simpson's diversity of the Douglas-fir community. Adjustment for subzone in the models removed the difference between clearcut and wildfire initiation types. Of the 105 ECM fungal species observed in soil samples, 42 occurred on both hosts (i.e., were shared ECM fungi), 23 occurred only on Douglas-fir, and 40 only on paper birch. Overall mean relative abundance of shared ECM fungi was significantly higher on paper birch than Douglas-fir (by 75%). Shared ECM fungi on Douglas-fir were over five times more abundant in 65- and 100-yr-old stands than 5-yr-old stands (Fig. 2.6). E C M Community Composition and Structure Cenococcum geophilum was the most frequently encountered species, followed by Rhizopogon vinicolor-type, Piloderma spp., and Leccinum scabrum. R. vinicolor-type was the most abundant ECM type on Douglas-fir in every stand type (mean relative abundance was 23-82%). Paper birch root tips were dominated (in terms of abundance) by C. geophilum, except in 5-yr-old clearcuts, where Lactarius pubescens was most abundant, and in 100-yr-old stands, where Piloderma was dominant. Second-most dominant on Douglas-fir was Rhizopogon rudus in 5-yr-old clearcuts, Suillus lakei in both 26-yr-old stand types, and Piloderma in 65- and 100-yr-old stands. Although 22 species in Thelephoraceae were found, most were rare, and their combined relative abundance in all sites averaged only 5%. However, seven species in this family occurred at one 100-yr-old site and totalled 20% relative abundance at that site. Amphinema byssoides and MRA mycorrhizae were found in all stand types, but were relatively infrequent and low in abundance. Laccaria spp. were found only in 5-yr-old stands, but were neither frequent nor abundant. All NMS ordinations were significant by Monte Carlo test except for the ordination based on ECM fungal species abundance on Douglas-fir. All ordinations were best represented by three axes, of which two explained 59- 85% of the total variation. Correlations of species to axes and R2 values of axes are summarized in Appendix C. In all ordinations, stand age was well correlated with at least one of the two axes that had the highest R2 values. These ordinations generally showed strong grouping of 5-yr-old 30 sites along both axes, while the older sites were more diffusely positioned along axes to which age was not a strong correlate (Figs. 2.7a-c, 2.8a-c, and 2.9a-c). The ordination based on E C M species frequency on Douglas-fir showed better grouping of sites by stand type than did the ordination based on E C M species abundance. Russula and Piloderma were consistently strongly correlated, and in the same direction, to axes to which stand age was well correlated. Rhizopogon vinicolor-type, Rhizopogon as a genus, Leccinum scabrum, and Lactarius pubescens were consistently correlated to axes in the opposite direction to which stand age was correlated. Cenococcum geophilum relative abundance was unaffected by stand type, but was about four times higher on paper birch than Douglas-fir (17% vs. 4.4%). This difference was significant only in 26-yr-old clearcuts and 65-yr-old burned stands (Fig. 2.1 Oa). Mean relative abundance of Russula spp. did not differ between host species, but was higher in the oldest than all other stand types, except the 65-yr-old stands (Fig. 2.10b). The 100-yr-old stands had eighteen times more Russula than 5-yr-old stands, and three times more than 26-yr-old stands. Russula was also more abundant in 65- than 5-yr-old stands. Lactarius scrobiculatus was more abundant on paper birch than Douglas-fir (6.2% vs. 1.3%, respectively, particularly at the 26-yr-old burned sites) (Fig. 2.10c). Relative abundance of Cortinarius and Piloderma spp. differed among stand types (Figs. 2.1 Od and e, respectively), but that of Hebeloma spp., Inocybe spp., and Russula nigricans did not (Figs. 2.10f-h, respectively). Rhizopogon vinicolor-type was more abundant on Douglas-fir in 5-yr-old clearcuts than all other stand types (Fig. 2.1 la). Site-level relative abundance of Rhizopogon vinicolor-type was negatively related to richness and Shannon-Weaver diversity of the Douglas-fir E C M community (R 2 = 0.73 and 0.82, respectively). Adding stand age improved the R 2 of the diversity model by only 0.02. Suillus lakei was most abundant and frequent in 26-yr-old stands (Fig. 2.1 lb), but it was absent from 5-yr-old clearcut soils, and occurred in only one soil sample from 65- and 100-yr-old stands. S. lakei data differed among stand types. Lactarius pubescens was the most abundant E C M on paper birch in 5-yr-old clearcuts (mean relative abundance = 27%), and was generally absent from all other sites. Leccinum scabrum reached its highest abundance in 5-yr-old stands (Fig. 2.12a), and was also a strong component of 26-yr-old clearcuts (p = 0.08 for stand type effect). Although Thelephora terrestris was found in low abundance on Douglas-fir seedlings, it was found only on paper birch in soil samples, and only on 5-yr-old clearcut sites, where its mean relative abundance was 6%. Lactarius torminosus did not differ statistically among stand types, but was absent in soil samples from 5-yr-old sites (Fig. 2.12b). 31 In all AlC-chosen models in which one stand age predictor variable was significant by likelihood ratio test, the other predictor variables (i.e. subzone, initiation type, and/or a transformation of stand age) were also significant, with the exception of models for two species described below. A l l abundance data for individual taxa in soil samples were severely overdispersed compared to the Poisson distribution, so models using the negative binomial were used. The associated dispersion parameter k, estimated by maximum likelihood, is listed for each modelled taxon on its respective figure (Figs. 2.13-2.17). Models are summarised in Table 2.5. Model selection by A IC and A I C C was generally unambiguous, with selection criteria values of the best models in most cases being at least 0.75 lower (better) than the second-best model. Exceptions were Suillus lakei and Lactarius torminosus (Table 2.4), for which model selection by A IC was less obvious. The best model of S. lakei frequency included initiation type as a predictor variable (Fig. 2.16d). The second-best model included only age variables, and its A I C C value was only 0.34 higher than the best model. The best model was overdispersed compared to the Poisson (chi 2/df = 2.9), and its predictors were not significant by likelihood ratio test. The second-best model was better fit to the distribution (chi 2/df = 1.6), and its predictors were significant. Two models of abundance of Lactarius torminosus on paper birch had A IC values that were only separated by 0.2. Both models are included in Figure 2.17a. E C M from Seedling Samples Mean E C M colonisation of Douglas-fir was 96% and 98% on burned and clearcut 5-yr-old sites, respectively. There was a tendency for greater E C M richness, evenness, and diversity on burned than clearcut sites, but differences between stand types were not significant (Fig. 2.18a-d). Neither ordinations based on abundance nor frequency data showed significant structure in the seedling E C M communities (Monte Carlo test; p = 0.21 and 0.25, respectively), nor did they group sites according to initiation type. Rhizopogon vinicolor-type was more abundant in clearcuts, while R. rudus was more dominant in burned stands (Fig. 2.19). Mean relative abundance of E C M fungal taxa on burned and clearcut 5-yr-old sites are listed in Appendix D. Four unexpected taxa were found on seedlings from 5-yr-old clearcuts: Lactarius rubrilacteus, Phallales 1, Piloderma fallax and Russula nigricans. There were no occurrences of Piloderma spp. on either host, nor were there any occurrences of any Russula spp. on Douglas-fir roots, in soil samples taken from the 5-yr-old sites. On the single seedling where it occurred, Russula nigricans colonised 66% of the root tips. Lactarius rubrilacteus was also found in several soil samples from older stands, and Phallales 1 from several in the 26-yr-old stands, but neither occurred in 5-yr-old clearcut soil 32 samples. Paper birch roots of sufficient quantity were found in soil sampled with only one Douglas-fir seedling from each of two 5-yr-old burned stands (see Appendix E for E C M on birch roots). 33 Discussion Identification of E C M Fungi and Taxonomic Diversity R igo rous m o r p h o t y p i n g c o m b i n e d w i t h D N A sequence ana lys is p rov ided strong support for ident i f i ca t ion o f E C M funga l taxa. The extensive use o f D N A sequenc ing a l l owed unambiguous p lacement o f most samples into un ique t axonomic group ings , as w e l l as detect ion o f non-target fung i c o -ex i s t ing on root t ips not necessar i ly recognisable by R F L P s . The ma in disadvantage to the sequenc ing approach was the c o m m o n occurrence o f double-peaks, l i k e l y resu l t ing f r om the sens i t iv i ty o f cyc losequenc ing react ions to non-mycor rh iza l fung i co-ampl i f i ed f r o m E C M root t ips ( Izzo et al, 2005) . Deta i l ed morpho t yp ing methods a l l owed re l iable separation o f taxa w i th i n cores that appeared s im i l a r under a d issec t ing m i c roscope (e.g., separation o f wh i te Piloderma spp. var iants f r om Pha l la les 1 (Hysterangium-Mke); or Russula spp. that do not bear cys t id i a f r om Lactarius spp. and other taxa , etc.). T h e y were also useful in detect ing sequence matches f r om non-target f ung i . Howeve r , the negat ive re la t ionship between mo lecu l a r success and length o f so i l sample refr igerat ion suggests that a s l ight l y less-detailed morpho t yp ing approach w o u l d have substant ia l ly increased mo lecu l a r success. Cons ide rab le t ime cou ld have been saved by not e x a m i n i n g in detai l a l l m i c ro s cop i c features in d i f f i cu l t species groups. Rather, a more rap id morpho logy-based p lacement to genus or f a m i l y w o u l d have been suf f i c ient for most samples. Never the less , v i e w i n g mantle peels and/or emanat ing elements in appropriate stains under a c o m p o u n d m ic roscope is st i l l often necessary to achieve proper t axonomic p lacement and corroborate mo lecu l a r results. Th i s study underest imated the total number o f species. H o w e v e r , our success was s im i l a r to other studies (e.g. C l i n e et al, 2005) , and is understandable g i ven the d i f f i cu l t y in adequate ly s amp l i ng E C M funga l commun i t i e s (Tay lo r , 2002) . T h e number o f E C M funga l species found here is comparab le to several other recent studies o f above- and be low-ground E C M fung i (Du ra l l et al, 2 0 0 6 ; Hor ton et al, 2 0 0 5 ; Kranabetter et al, 2 0 0 5 ; Sm i th et al, 2 0 0 2 ; O ' D e l l et al, 1999). A l t h o u g h s amp l i ng occur red o n l y over one g r o w i n g season in this study, Izzo et al. (2005) found E C M funga l c o m m u n i t y compos i t i on var ied more between plots w i t h i n one year than w i th i n plots across years. Hence , it is un l i ke l y that E C M funga l trends ident i f ied in the current study w o u l d have been great ly d i f ferent had s amp l i ng been done over addi t iona l years. N o attempt was made in the current study to account fo r d i f ferences between spr ing and fa l l E C M funga l commun i t i e s because the number o f t ips sampled in one season was p robab ly gross ly insuf f i c ient for accurate c o m m u n i t y representat ion. 34 The data supported our first hypothesis that ECM species richness, diversity and evenness increase with stand age. The greatest increase in richness occurred from the 5- to 26-yr-old age class, a period corresponding with tree canopy closure, and increasing only slightly thereafter, agreeing with the results of Visser et al. (1995) and Kranabetter et al. (2005). At canopy closure, tree growth rates are rapid and leaf area maximal (Simard et al, 2004), with correspondingly high potential for carbon allocation to roots and mycobionts. ECM species richness and diversity tended to increase at a lower rate from 26- to 65-years, and then level off in older age classes. The ECM community of paper birch was richer and more even than that of Douglas-fir in young stands, and increased less dramatically with stand age. Paper birch roots may remain intact and healthy following cutting or burning of shoots, providing a large carbon source and ECM legacy for stump sprouts, as well as large ECM inoculum potential for seedlings germinating nearby. By contrast, Douglas-fir does not sprout from old stumps, and seedlings are often not replanted until a few years after logging, requiring inoculation of seedlings from other plants, hyphae or spores. Jones et al. (1997) found that richness and evenness of the ECM fungal community on Douglas-fir was actually higher than that of paper birch at four months after outplanting, but was no different at sixteen or twenty-eight months. In that study, sites had been destumped prior to planting of both host species, supporting that birch stump sprouting could have been important in maintaining higher ECM fungal diversity on birch in the current study. Durall et al. (2006) found no difference in epigeous ECM sporocarp diversity among recently planted birch, Douglas-fir, and mixed stands. However, studies commonly show a strong discrepancy between above- and below-ground ECM community composition and structure (Peter et al, 2001b; Durall et al, 1999; Gardes & Bruns, 1996). An important cause of this discrepancy is likely the exclusion of, or difficulties in sampling, hypogeous and resupinate fruiting bodies of ECM species in sporocarp studies. Our second hypothesis, that ECM communities differ between clearcut and burned forests, was rejected in this study. ECM community diversity was similar among 5-yr-old stands regardless of whether they originated after fire or clearcutting, and there was no grouping of 5-yr-old sites by initiation type in NMS ordinations based on their ECM fungal communities. These results suggest that fungal inoculum was not limiting on these sites, even though the fires were intense (based on forest floor observations and extent of disturbance) and would have likely reduced inoculum of many ECM fungal species (Lazaruk et al, 2005; Bruns et al, 2002; Grogan et al, 2000; Taylor & Bruns, 1999). Although ECM richness and diversity tended to be lower in 26-yr-old burned than clearcut stands, this may have resulted from half the burned sites occurring in the ICHmk rather than the ICHmw subzone, where ECM communities were generally more diverse. 35 E C M Community Composition and Structure ECM community composition varied with stand age, particularly for the Douglas-fir and combined communities, supporting our third hypothesis regarding patterns of fungal succession. Our results show that some fungal succession patterns are clearer at the genus than species taxonomic level. For example, Russula, one of the three most speciose genera in this study, increased in abundance and frequency with stand age. This is consistent with the other recent chronosequence studies of ECM fungal communities (Kranabetter et al, 2005; Smith et al, 2002; Visser, 1995). Patterns for individual Russula species, however, were more variable, probably because they occurred rarely. These fungi likely have patchy distributions, as suggested by the high dispersion parameter for R. nigricans. R. brevipes, R. aeruginea, and R. roseipes were similar, with infrequent occurrence but high root tip abundance where found. Patchy distributions, both horizontally as well as vertically in soil profiles, are generally expected for ECM fungi (Lilleskov E.A. etal, 2004; Rosling et al, 2003; Tedersoo etal, 2003; Dickie etal, 2002; Bidartondo et al, 2000). Russula brevipes occurred only at the 100-yr-old sites, supporting Bergemann et al's (2002) reference to it as a "late-stage" fungus. Russula species were absent from young stands on Douglas-fir roots, except for extensive colonisation of a single Douglas-fir seedling by R. nigricans. New hosts may be infected by Russula species primarily from existing fungal networks, but the spread and patchy distribution may also result from within-stand spore dispersal (Redecker et al, 2001). Spore germination and survival of new mycelia may, however, be sensitive to soil conditions and the presence of other soil microorganisms. Piloderma also increased in frequency and abundance with stand age, agreeing with patterns observed by Visser (1995) and Smith et al. (2000). Smith et al. (2000) found that Piloderma mycelia and mycelial cord occurrence also increased with abundance of well-decayed coarse woody debris. Species of Cortinarius also tended to increase in frequency and abundance with stand age after 5 years. Cortinarius species were not found in this study as frequently as they were in ICH sporocarp studies (Durall etal, 2006; Kranabetter et al, 2005). Again, above- and below-ground views of ECM communities are often quite different. In this case, patchy distribution of ECM tips may have caused lower observation frequency of Cortinarius spp., as they were often abundant in the cores in which they were found despite their infrequence. Rhizopogon vinicolor-type was considerably more dominant on Douglas-fir in 5-yr-old than older stands, both from soil and seedling samples. Other studies also show that Rhizopogon species are common following disturbance, with high frequency on seedlings grown in disturbed areas or in bioassay soils taken from wildfires or clearcuts (Grogan etal, 2000; Baar et al, 1999; Simard et al, 1997c; Jones 36 et al, 1997). Douglas-fir seedlings in this study were dominated by R. vinicolor-type more than in other nearby studies. For example, the relative abundance of R. vinicolor-Uke on 28-month-old field-grown Douglas-fir seedlings was only 37% in Jones et al. (1997), roughly half of the average for the 5-yr-old stands in this study. In that study, fungi forming E-strain mycorrhizae and Thelephora occupied considerable portions of the E C M fungal community on Douglas-fir, but they did not in the current study. These fungi are often considered to be "early-stage" (e.g. Visser, 1995) and commonly colonise seedlings in greenhouses, but they may not compete effectively with fungi like Rhizopogon after a few years of growth in nature. There are several plausible explanations for why Rhizopogon vinicolor-type dominated the mycorrhizal community of Douglas-fir in 5-yr-old stands. Rhizopogon spores are known to persist as viable E C M inocula for long periods of time, and can be abundantly and uniformly distributed even in soils where their E C M hosts are not present (Horton et al, 1998), so it is not surprising that they survived where Douglas-fir roots were only patchily distributed. The rhizomorphs of R. vinicolor and R. vesiculosus may be particularly advantageous for infecting seedling roots (Simard et al, 1997b), particularly after spores and inocula of many E C M fungi have declined , which usually happens within two years after logging (Hagerman et al, 1999a). R. vesiculosus appears to spread vegetatively to several hosts more readily than R. vinicolor (Kretzer et al, 2003a), but these species could not be analyzed separately in this study. Environmental conditions in young stands may also be well-suited to the physiology of R. vinicolor and R. vesiculosus (e.g., nutrient and moisture uptake and transfer), or young Douglas-fir may select for these host-specific fungi in this environment. Species interactions with other E C M fungi are l ikely important in determining community structure as well (Koide et al, 2005; Jonsson etal, 2001; Wu etal, 1999). Rhizopogon rudus may have been more dominant in burned than clearcut 5-yr-old stands because its spores may survive fire better than those of R. vinicolor and R. vesiculosus. Soil moisture availability may also have played a role in colonisation patterns; soils are often dry after wildfire because moderate to severe burns cause hydrophobicity of the uppermost soil layers (Certini, 2005), and this effect has been found to persist for almost two years fol lowing severe wildfire (Huffman et al, 2001). Baar et al. (1999) found greater colonisation by R. olivaceotintus than R. ochraceorubens (= R. occidentalis; (Kjoller & Bruns, 2003)) in dry bioassay soil, but not in moist soil. In the present study, R. rudus displayed a preference for mineral soil over the forest floor (see Chapter 3), so it was likely of higher relative abundance in burned stands because forest floor was generally present in clearcut stands but absent from burned stands in the 5-yr-old age class. Timing of colonisation may also explain why different Rhizopogon species dominate in certain environments (Kennedy & Bruns, 2005). Cl ine et al. (2005) 37 found nursery grown Douglas-fir to be already heavily colonised with R. rudus contaminants when outplanted. The higher relative abundance of R. rudus on young burned than clearcut sites in the current study therefore could have simply originated from nursery stock colonisation differences. Two birch-specific fungi, Lactarius pubescens and Leccinum scabrum, were very abundant in 5-yr-old stands, paralleling Rhizopogon stand age patterns on Douglas-fir. These results contrast with Mason et al. (1983), who refer to strand-forming Lactarius pubescens and Leccinum as "late-stage" fungi. The "late-stage" description arises from bioassay studies (Fox, 1983; Deacon & Donaldson, 1983), which demonstrated that these two fungal taxa do not readily inoculate birch from spores or mycelium dislocated from live hosts. This suggests that vegetative spread was important in young stands after limited initial colonisation by spores or fragmented mycelia, or that these fungi were legacies of pre-disturbance birch roots from which sprouts arose. It is not surprising that the dominant fungi on both hosts in 5-yr-old stands were strand-forming, as host roots are less abundant and separated by greater distances than in older stands. These same researchers that described Leccinum as "late-stage" also found that Hebeloma crustuliniforme and Hebeloma sacchariolens were early colonisers, but Hebeloma velutipes and H. incarnatulum in the current study were generally absent from 5-yr-old stands and frequent in older stands. Similar to this study, Kranabetter (2005) also found that prevalence of different Lactarius species varied between young and mature stands. These results suggest that generalisations about E C M succession are sometimes only possible at the species level and cannot always be extrapolated to an entire genus. Cenococcum geophilum was ubiquitous in this study, occurring at every site. Although it was not consistently dominant in root tip abundance, it was unparalleled in frequency, occurring in 51% of soil samples. Such an apparently uniform distribution may be a result of colonisation from many separate inoculum sources, as suggested by high genetic diversity of C. geophilum across fine spatial scales (Douhan & Rizzo, 2005). C. geophilum colonised birch more than Douglas-fir, which is consistent with Kernaghan et a/.'s finding (2003) that it associated more with hardwoods than conifers in mixed boreal forests. However, Durall et al. (1999) found overall C. geophilum relative abundance on western hemlock to be near 30%, similar to its highest site-level relative abundances on birch in this study. Dural l et al. (1999) also found C. geophilum relative abundance was less than 10% on lodgepole pine co-occurring with the western hemlock. Whi le this fungus is certainly a host generalist, it may associate with some hosts more readily than others. 38 Network Potential between Host Species The results of this study do not support the hypothesis that the proportion of E C M root tips colonised by shared fungi decreases with increasing stand age. In contrast, there was a lower proportion of shared E C M fungi in 5 yr-old than older stands because of the high relative abundance of a few host-specific fungi. Shared fungi nevertheless occupied a significant proportion of birch roots in young stands. Douglas-fir seedlings initially may have been dominated by shared species as suggested by Jones et al. (1997) and Simard et al. (1997c), but not when they are 4-6-years-old as in this'study. While these results suggest that C M N s are likely to form in young stands, they also show that they should be considerably more extensive in older stands. The proportion of root tips colonised by shared fungi increased significantly with stand development, and was greatest on Douglas-fir in the oldest age classes. C M N s in older stands may facilitate direct transfer of carbon and nutrients from dying roots of senescent birch trees to Douglas-fir. The few host-specific fungi dominating young stands in this study might tend to be categorized as ruderal species. However, Rhizopogon vinicolor-type fungi also accounted for a substantial proportion of the community in older stands, and therefore might be better categorized as competitive ("C") strategists, as described by Grime (1977) than r-selected (ruderal). Leccinum scabrum was also not restricted to young stands and therefore should not be thought of as a ruderal fungus. A "C"-type strategy would hold under the assumption that the soil environment in young sites fol lowing disturbance is generally less stressful than older sites. It appears that competition from other E C M fungi is likely lower in young stands, perhaps because inoculum is more limiting for other E C M species. However, soil nitrogen and phosphorus were not found to be more available in young regenerating sites than in older sites (see Chapter 3). Other unstudied ecological factors, such as microbial community structure and processes, may more strongly affect E C M community structure. 39 Conclusions Stand age clearly affected diversity and structure of E C M communities in this study. Paper birch appeared important in maintaining E C M fungal diversity during stand initiation. However, its potential to form C M N s with Douglas-fir in this stand development stage was relatively low in comparison to older stands. This does not, however, preclude the indirect effects that birch and the diversity of its E C M community have on soils from playing a role in productivity and E C M colonisation of establishing Douglas-fir. Stand initiation type (i.e. wildfire or clearcut) did not appear to affect E C M diversity, community composition, or structure within the age range of stands studied. Our ability to detect differences was diminished by the short time spans represented in the two age classes for which stands of both initiation types were studied. This study revealed some strong E C M community trends with stand development, and some were clearer at the genus level while others were apparent at the species level. Overall E C M fungal diversity increased mainly from 5- to 26-yr-old stands, but community composition and structure continued to change from 26- to 65-yr-old stands. It was not possible to draw general conclusions about relationships between fungal life-history strategies or fungal species composition with forest stand development. However, ecological traits of individual fungal species are difficult to characterise in a community study. Further autecological and population genetics studies are needed to improve our understanding of the links between plant and fungal succession in forest communities. 40 Figures F i g u r e 2.1 T a x a - s a m p l e u n i t c u r v e s f o r t he c o m b i n e d c o m m u n i t y (a) s i t e- l eve l , i n c l u d i n g a l l s i t e s ; (b ) s i te-l e v e l , e x c l u d i n g the 2 6 - y r - o l d b u r n e d I C H m k si tes a n d t w o o l d e s t age c l a sses , a n d (c) c u m u l a t i v e f o r e a c h s t a n d t y p e . F i l l e d c i r c l e s = 5-yr-old c l e a r c u t s ; o p e n c i r c l e s = 2 6 - y r - o l d b u r n e d s i t e s ; f i l l e d t r i a n g l e s = 26-yr-o l d c l e a r c u t s ; o p e n t r i a n g l e s = 6 5 - y r - o l d b u r n e d s i t e s ; a n d f i l l e d s q u a r e s = 1 0 0 - y r - o l d b u r n e d s t a n d s . 41 F i g u r e 2.2 R a n k a b u n d a n c e p l o t s f o r E C M c o m m u n i t i e s w i t h (a) b o t h hos ts c o m b i n e d ; (b ) D o u g l a s - f i r ; a n d (c) p a p e r b i r c h . F i l l e d c i r c l e s = 5-yr-o ld c l e a r c u t s ; o p e n c i r c l e s = 2 6 - y r - o l d b u r n e d s i t e s ; filled t r i a n g l e s = 26-y r - o l d c l e a r c u t s ; o p e n t r i a n g l e s = 6 5 - y r - o l d b u r n e d s i t e s ; a n d filled s q u a r e s = 1 0 0 - y r - o l d b u r n e d s t a n d s . 42 a) m 25 in a> c .n 20 -o i tn cie 15 -a) a w •o 10 ffi se se 5 .D o 0 *I ab i I be I b e I c a b 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ab I b : : 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type C) 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type 1.0 Q 0.8 c o </) a . in 0.2 * b ab _ b e , , i t 0.0 5-cc 26-b 26-cc 65-b 100-b Stand Age-lnititation Type Figure 2.3 E C M community diversity variables (a-d) by stand type (cc = clearcut, b = burned); bars represent means (n = 4) and error bars represent one standard error of the mean. Black bars = Douglas-fir; light grey bars = paper birch; dark grey bars = combined community. Means within host species (i.e. with the same bar colour) that share the same letter do not differ significantly (p > 0.05). Bars without any letters indicate no significant difference found among stand type means for that host. * indicates a significant difference between host species within that stand type (from multiple comparisons). Significant stand type by host species interactions were detected for all analyses (split-plot ANOVA) . Combined communities were analysed by separate one-way ANOVAs . 43 60 -If) (/> a c 50 si o br 40 -Q) ' o <D 30 • a in •a a 20 • ro E *^  10 -tu 0 Combined Community I I 5-cc 26-b 26-cc 65-b 100-b S t a n d A g e - I n i t i a t i o n T y p e Figure 2.4 Mean (n = 4) 1 s t (black bars) and 2nd (grey bars) order jackknife estimates of species richness by stand type (n = 4); cc = clearcut; b = burned. Error bars represent one standard error of the mean. Not statistically tested. 44 0 10 20 30 40 50 60 70 80 90 100 Stand Age F i g u r e 2.5 O b s e r v e d s i te s co res o n f i r s t p r i n c i p a l c o m p o n e n t a x i s f r o m P C A o n 13 E C M d i v e r s i t y v a r i a b l e s ( c i r c l e s = I C H m w s i t es ; t r i a n g l e s = I C H m k s i tes ) a n d v a l u e s p r e d i c t e d b y t h e m o d e l ( l i ne ) f o r t he I C H m w s u b z o n e . 45 100 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type Figure 2.6 Mean percentage (n = 4) of E C M root tips colonised by fungi observed on both hosts by stand type. Black bars = Douglas-fir community; light grey bars = paper birch community; dark grey bars = combined community (expressed as average values of Douglas-Fir and paper birch communities); cc = clearcut; b = burned. Combined community means with the same letters are not significantly different (p > 0.05). No significant stand type by host species interaction found (split-plot ANOVA) . Host species effect was significant (p < 0.05), but multiple comparisons between hosts within each stand type detected no significant differences. 46 a) b) V • V o • I -1.0 -0.5 0.0 0.5 1.0 1.5 Axis 1; R2 = 0.23; r (stand age) = - 0.31 CM O 1.5 -I O II aT 1.0 • D l ro •a £ ro 0.5 • 51 0.0 • co T — o II -0.5 • D i is 3; -1.0 • X < • o • • o -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 Axis 2; R2 = 0.67; r (stand age) = 0.84 C) CD co ro •a c CO u> II ol a. CO (ft '5 < 1.5 ~ 1.0 0.5 r - o.o -0.5 -1.0 -1.5 •o • V -1.0 -0.5 0.0 0.5 1.0 Axis 2; R2 = 0.22; r (stand age) = - 0.16 F i g u r e 2.7 N M S o r d i n a t i o n s o f s i tes b a s e d o n the c o m b i n e d E C M f u n g a l c o m m u n i t y o f b o t h hos ts u s i n g (a) spec i es f r e q u e n c y ; (b ) f r e q u e n c y o f spec i es l u m p e d i n t o g e n e r a ; a n d (c) spec i es a b u n d a n c e . F i l l e d c i r c l e s = 5-y r - o l d c l e a r c u t ; o p e n c i r c l e s = 2 6 - y r - o l d b u r n e d s i t e s ; f i l l e d t r i a n g l e s = 2 6 - y r - o l d c l e a r c u t s i t e s ; o p e n t r i a n g l e s = 6 5 - y r - o l d b u r n e d s i t e s ; a n d s q u a r e s = 1 0 0 - y r - o l d b u r n e d s i tes . R 2 v a l u e s r e p r e s e n t t he p r o p o r t i o n o f t o t a l v a r i a t i o n i n R e l a t i v e S o r e n s e n d i s t a n c e a m o n g s i tes e x p l a i n e d b y o r d i n a t i o n axes . C o r r e l a t i o n s o f s t a n d age to o r d i n a t i o n axes a r e P e a r s o n ' s r. 47 a) b) o> CN O I I 1.5 • age) 1.0 • (stand 0 .5 • .47; ri 0 .0 • o I I - 0 . 5 -C M OL n - 1 . 0 • II) < - 1 . 5 • o # o o • • - 1 . 0 - 0 . 5 0 .0 0 . 5 1.0 Axis 2; R2 = 0.22; r (stand age) = 0.62 o I I aT u> ro TJ c o I I n cn 1.5 1.0 0 . 5 0 . 0 - 0 . 5 - 1 . 0 - 1 . 5 v • o • • - 1 . 0 - 0 . 5 0 .0 0 .5 1.0 Axis 2; R = 0.27; r (stand age) = - 0.75 C) o o f • .0 - 0 . 8 - 0 . 6 - 0 . 4 - 0 . 2 0 .0 0 .2 0 . 4 0 . 6 0 .8 Axis 2; R2 = 0.25; r (stand age) = - 0.46 F i g u r e 2.8 N M S o r d i n a t i o n s o f s i tes b a s e d o n E C M f u n g a l c o m m u n i t i e s o n D o u g l a s - f i r u s i n g (a) spec i es f r e q u e n c i e s ; (b ) f r e q u e n c y o f spec ies l u m p e d i n t o g e n e r a ; a n d (c) b y spec i es a b u n d a n c e . R 2 v a l u e s r e p r e s e n t t he p r o p o r t i o n o f t o t a l v a r i a t i o n i n R e l a t i v e S o r e n s e n d i s t a n c e a m o n g s i tes e x p l a i n e d b y o r d i n a t i o n axes . C o r r e l a t i o n s o f s t a n d age to o r d i n a t i o n axes a r e P e a r s o n ' s r. S y m b o l s a r e as i n F i g u r e 2-7. 48 a) b) 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 Axis 1; R2 = 0.33; r (stand age) = - 0.69 1.5 -I d ll age) 1.0 -:and 0.5 • in v . 0.0 -d I I -0.5 -0Z is 2; -1.0 • X < V o -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 Axis 1; R2 = 0.29; r (stand age) = - 0.67 C) IO CM 1.0 -I d n age) 0.5 -(stand 0.0 -0.31; -0.5 • I I a: is 3; -1.0 • X < -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 Axis 2; R2 = 0.42; r (stand age) = - 0.70 F i g u r e 2.9 N M S o r d i n a t i o n s o f s i tes b a s e d o n E C M f u n g a l c o m m u n i t i e s o n p a p e r b i r c h u s i n g (a) spec i es f r e q u e n c i e s ; (b ) f r e q u e n c y o f spec ies l u m p e d i n t o g e n e r a ; a n d (c) b y spec i es a b u n d a n c e . R 2 v a l u e s r e p r e s e n t t he p r o p o r t i o n o f t o t a l v a r i a t i o n i n R e l a t i v e S o r e n s e n d i s t a n c e a m o n g s i tes e x p l a i n e d b y o r d i n a t i o n axes . C o r r e l a t i o n s o f s t a n d age t o o r d i n a t i o n axes a r e P e a r s o n ' s r. S y m b o l s a r e as i n F i g u r e 2-7. 49 a) b) Cenococcum geophilum 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type 50 g. 40 CD u c TO T3 c < a > ro 1 1 0 30 20 Russula spp. ab 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type C) d) Lactarius scrobiculatus J L 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type 20 o c ro •D | 10 < > ro 5 CD Cortinarius spp. I 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type 50 e) 25 £ 20 e o c (0 •o c 3 < > 15 10 co Piloderma s p p . 5-cc 26-b 26-cc 65-b 100-b Stand Age-lnititation Type 30 25 c 20 n SI < CO > '-3 _ro co a; 15 10 Hebeloma s p p . I I r [ I T T 1 s 1 J I i ik 5-cc 26-b 26-cc 65-b 100 b Stand Age-Initiation Type g) 14 -=• 12 1 8 I 6 > 1 4 CO Ct 2 Inocybe s p p . 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type h) 20 0 15 o c CO 1 10 < CO > « 5 CD or Russula nigricans ill 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type Figure 2.10 Mean relative abundances (n = 4) of fungal taxa that occurred on both hosts by stand type (a-h; cc = clearcut, b = burned). Black bars = Douglas-fir; light grey bars = paper birch; and dark grey bars = combined community. Error bars represent one standard error of the mean. Means within host species (i.e. with the same bar colour) that share the same letter do not differ significantly (p > 0.05). * indicates a significant difference between host species within that stand age-initiation type (p < 0.05). Split-plot ANOVA used for a-c; no stand type by host species interactions found; (a) no stand type effect; (b) no host species effect; c) no stand type effect; host species effect significant (p < 0.05), but not in mean comparisons. Kruskal-Wallis test used for d-h; (d) and (e) showed significant stand type effect; (f)-(h) showed no significant stand type effect. 51 a) Rhizopogon vinicolor-type 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type b) 40 CD O c ro •a c 3 .Q < CD > i 10 or 30 20 Suillus lakei 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type F i g u r e 2.11 M e a n r e l a t i v e a b u n d a n c e s (n = 4 ) o f t w o hos t - spec i f i c f u n g i o n D o u g l a s - f i r b y s t a n d a g e - i n i t i a t i o n t y p e (n = 4 ) ; c c = c l e a r c u t , b = b u r n e d . E r r o r b a r s r e p r e s e n t o n e s t a n d a r d e r r o r o f the m e a n . M e a n s w i t h t h e s a m e l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t ( p > 0.05) . R. vinicolor-type t e s t ed b y o n e - w a y A N O V A ; S. lakei s h o w e d s i g n i f i c a n t s t a n d t y p e e f fec t b y K r u s k a l - W a l l i s test ( p < 0.05) . 52 a) b) Leccinum scabrum h n : 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type 30 25 0) o c (0 •o . c 3 . Q < > 20 15 10 Lactarius torminosus 5-cc 26-b 26-cc 65-b 100-b Stand Age-lnitation Type F i g u r e 2.12 M e a n r e l a t i v e a b u n d a n c e s (n = 4) o f t w o hos t s p e c i f i c f u n g i o n p a p e r b i r c h b y s t a n d t y p e . E r r o r b a r s r e p r e s e n t o n e s t a n d a r d e r r o r o f t h e m e a n . N o s i g n i f i c a n t s t a n d t y p e e f f ec t f o r e i t h e r spec i es ( one-way A N O V A ; p > 0.05) . 53 F i g u r e 2.13 M a x i m u m l i k e l i h o o d m o d e l s ( c u r v e s ) f o r Cortinarius a n d Hebeloma s p p . i n t he c o m b i n e d c o m m u n i t y . P r e d i c t e d r e l a t i v e a b u n d a n c e s w e r e e s t i m a t e d b y d i v i d i n g p r e d i c t i o n s o f p e r s o i l s a m p l e t a x o n a b u n d a n c e b y t h e a v e r a g e n u m b e r o f E C M r o o t t i p s p e r s o i l s a m p l e . P o i n t s a r e s i t e- leve l v a l u e s o f r e l a t i v e a b u n d a n c e a n d n u m b e r o f o c c u r r e n c e s ( f r e q u e n c y ) . 54 Figure 2.14 Maximum likelihood models (curves show predictions for ICHmw only) for Piloderma spp. in the combined community (a-b). Predicted relative abundances were estimated by dividing predictions of per soil sample taxon abundance by the average number of total E C M root tips per soil sample. Points are site-level values of relative abundance and number of occurrences (frequency). 55 / a) b) 3 0 ] 0 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 Stand Aqe 14 ! 1 2 10 1 8 6 4 2 0 4H Russula s p p . • I C H m w A I C H m k 0 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 Stand Age F i g u r e 2 .15 M a x i m u m l i k e l i h o o d m o d e l s ( c u r v e s ) f o r Russula s p p . a n d R. nigricans i n t h e c o m b i n e d c o m m u n i t y ( l i nes s h o w p r e d i c t i o n s f o r I C H m w o n l y i n (a) a n d (b)) . P r e d i c t e d r e l a t i v e a b u n d a n c e s w e r e e s t i m a t e d b y d i v i d i n g p r e d i c t i o n s o f p e r s o i l s a m p l e t a x o n a b u n d a n c e b y the a v e r a g e n u m b e r o f t o t a l E C M r o o t t i p s p e r s o i l s a m p l e . P o i n t s a r e s i te- leve l v a l u e s o f r e l a t i v e a b u n d a n c e a n d n u m b e r o f o c c u r r e n c e s ( f r e q u e n c y ) . 56 a) b) 100 80 -I 60 40 20 Rhizopogon vinicolor-type; k = 5.2 0 10 20 30 40 50 60 70 80 90 100 Stand Age 10 O ° c CD 5 6 o O 4— 0) n £ 2 2 Rhizopogon vinicolor-type 0 10 20 30 40 50 60 70 80 90 100 Stand Age F i g u r e 2.16 M a x i m u m l i k e l i h o o d m o d e l s ( c u r v e s ) f o r t w o hos t s p e c i f i c E C M spec i es o n D o u g l a s - f i r . P r e d i c t e d r e l a t i v e a b u n d a n c e s w e r e e s t i m a t e d b y d i v i d i n g p r e d i c t i o n s o f p e r s o i l s a m p l e t a x o n a b u n d a n c e b y the a v e r a g e n u m b e r o f D o u g l a s - f i r E C M r o o t t i p s p e r s o i l s a m p l e . P o i n t s a r e s i te- leve l v a l u e s o f r e l a t i v e a b u n d a n c e a n d n u m b e r o f o c c u r r e n c e s ( f r e q u e n c y ) . In (d ) , L i n e s 1 a n d 2 r e p r e s e n t p r e d i c t i o n s f o r b u r n e d a n d c l e a r c u t s i tes , r e s p e c t i v e l y , f r o m the bes t m o d e l . L i n e 3 r e p r e s e n t s t h e s e cond-bes t m o d e l , i n w h i c h s t a n d i n i t i a t i o n t y p e w a s n o t a p r e d i c t o r . 57 F i g u r e 2 .17 M a x i m u m l i k e l i h o o d m o d e l s ( c u r v e s ) f o r t w o hos t s p e c i f i c E C M spec i es o n p a p e r b i r c h . P r e d i c t e d r e l a t i v e a b u n d a n c e s w e r e e s t i m a t e d b y d i v i d i n g p r e d i c t i o n s o f p e r s o i l s a m p l e t a x o n a b u n d a n c e b y the a v e r a g e n u m b e r o f b i r c h E C M r o o t t i p s p e r s o i l s a m p l e . P o i n t s a r e s i te- leve l v a l u e s o f r e l a t i v e a b u n d a n c e a n d n u m b e r o f o c c u r r e n c e s ( f r e q u e n c y ) . In (a) , L i n e s 1 a n d 2 r e p r e s e n t t h e best a n d s e cond-bes t m o d e l s , r e s p e c t i v e l y . 58 a) b) 8 -, in in ne 6 -.c o 5 in a> 4 o a> Q . CO c ra 2 a> S 0 -B u r n e d C l e a r c u t Initiation Type 1.0 0.8 in in a E 0.6 0.4 0.2 0.0 B u r n e d C l e a r c u t Initiation Type C) d) B u r n e d C l e a r c u t Initiation Type 1.0 in * 0.8 O s c o in a. E 0.6 0.2 ] 0.0 B u r n e d C l e a r c u t Initiation Type F i g u r e 2 .18 C o m p a r i s o n o f m e a n E C M c o m m u n i t y d i v e r s i t y m e a s u r e s (n = 4) f o r D o u g l a s - f i r s e e d l i n g s b e t w e e n s t a n d i n i t i a t i o n t ypes i n 5-yr-o ld s t a n d s . E r r o r b a r s r e p r e s e n t o n e s t a n d a r d e r r o r o f t he m e a n . T e s t e d b y t w o - s a m p l e t-test ( t w o - t a i l e d ) : (a) t = -1 .9 , p = 0 . 1 1 ; (b ) t = - 2 . 1 , p = 0 . 0 8 ; (c) t = - 2 . 1 , p = 0 . 0 8 ; (d ) t = - 2 . 1 , p = 0 .08 . 59 100 R. rudus R. vinicolor-type Total Rhizopogon Species Figure 2.19 Comparison of mean relative abundance (n = 4) of Rhizopogon rudus and R. vinicolor-type on Douglas-fir seedlings between 5-yr-old burned and clearcut stands. Black bars = wildfire origin; grey bars clearcut origin. Error bars represent one standard error of the mean. Not statistically tested. 60 Tables T a b l e 2.1 Site locations and characteristics. Initiation ICH Elevation Latitude/ Site Age Type Subzone (meters) Longitude Inter ior C e d a r - H e m l o c k biogeoclimatic subzones are defined in L l o y d et al. (1990). Tree Species Composition2 Soil Crown Other ECM Closure Texture Douglas-fir Paper birch broadleaf spp. 19MR AL BC WL 1DA1 IDA2 1DA3 IDA4 EDI ED2 MAI MA2 4 5 5 30 30 24 24 clearcut clearcut clearcut clearcut wildfire wildfire wildfire wildfire wildfire wildfire wildfire wildfire mw3 mw2 mw2 mw3 mw2 mw2 mw2 mw2 mk2 mk2 mw3 mw3 700 600 750 700 1100 950 650 500 1200 1000 930 975 N 50° 58' 42" W 118°35'40" N 50° 32' 43" W 118° 52'49" N 50° 38' 48" W 118° 45'36" N 50° 53' 51" W 119° 16' 27" N 50° 38' 54" W 119° 18' 18" N 50° 39' 29" W 119° 18' 03" N 50° 40' 00" W 119° 18'57" N50°39'-42" W 119° 19' 57" N 50° 44' 19" W 119° 23'09" N50°44' 12" W 119° 22' 17" N 50° 55' 20" W 118° 50'41" N 50° 55' 3" W 118° 50' 56" 50% 45% 65% 50% 30% 25% 35% 30% 80% 85% 70% 70% SiL SL SiL fSL SCL SL SL SL SiL SiL SL SL 10% 25% 5% 10% 15% 10% 7.5% 5% 35% 35% 40% 45% 65% 50% 85% 70% 60% 65% 80% 80% 40% 45% 45% 40% 2.5% 7.5% 5% 2.5% 10% 5% 2.5% 2.5% 12.5% 5% 10% 7.5% Tree Species Composition2 Initiation ICH Elevation Latitude/ Crown Soil Site Age Type Subzone (meters) , Longitude Closure Texture1 Other ECM Douglas-fir Paper birch broadleaf spp. DISC 27 NM SRC ZP BA SL 4WD ACR BBP WAP 21 22 25 63 MARA 71 RR 68 98 101 90 clearcut clearcut clearcut clearcut wildfire wildfire wildfire wildfire 103 wildfire wildfire wildfire wildfire mw2 mw2 mw2 mw2 mw2 mw2 mw2 mw2 mw2 mw2 mw2 mw2 600 550 900 650 700 600 800 700 550 600 750 650 N 50° 32' 31" W 118° 52' 33" N 50° 36' 15" W 118° 40' 47" N 50° 43' 04" W 119° 06' 54" N 50° 36' 34" W 118° 39' 47" N 50° 34' 03" W 118° 50' 50" N50°39' 28" W 119° 03'49" N 50° 41' 55" W 118° 46'07" N 50° 22* 07" W 118° 32' 03" N 50° 36' 47" W 118° 50" 26" N50°37' 25" W 118° 46'06" N 50° 27' 17" W 118° 49' 30" N 50° 45' 1" W 118°34' 12" 65% 60% 75% 70% 85% 80% 75% 90% 90% 80% 80% 80% SL SL vfSL SL SL SL SL SL SL LS SL SL 40% 40%. 47.5% 40% 45% 45% 40% 45% 50% 55% 40% 50% 60% 45% 50% 40% 50% 50% 60% 45% 45% 30% 40% 40% 0% 5% 2.5% 10% 0% 0% 0% 2.5% 0% 0% 0% 0% SiL = silty loam; SL = sandy loam; SCL = sandy clay loam; LS = loamy sand; f = fine; vf =very fine. Tree species compositional percentages were estimated as each species' or group's proportion of the total estimated canopy cover. T a b l e 2.2 A n a l y s i s o f V a r i a n c e t a b l e f o r s p l i t - p l o t a n a l y s e s , n = 4 r e p l i c a t e s . Degrees of Critical F-value (alpha = 0.05); Variation Source Freedom F-test Denominator Stand-type (age/initiation type) 4 3.06; Error 1 Error 1 15 Host Species 1 4.54; Error 2 Stand-type * Host species 4 3.06; Error 2 Error 2 15 Total 39 63 Table 2.3 Statistical analysis table for differences among stand type means of E C M fungal diversity variables and relative abundances. Fd = Douglas-fir; Ep = paper birch. SP = split-plot; K-W = Kruskal-Wallis. See associated figures for further details. Variable Host Statistical Test F-ratio or x2 value: effect of stand type P-value F-ratio: effect of host species P-value F-ratio: stand type x host sp. interaction P-value Associated figure Species richness Both SP A N O V A 8.45 0.0009 36.14 O.0001 3.89 0.0231 2.3a Shannon diversity Both SP A N O V A 7.67 0.0014 44.65 O.0001 6.86 0.0024 2.3b Evenness Both SP A N O V A 4.68 0.0118 25.69 0.0001 7.00 0.0022 2.3c Simpson diversity Both SP A N O V A 19.2 O.0001 162.06 O.0001 20.47 O.0001 2.3d Relative Abundances Shared E C M species Both SP A N O V A 9.49 0.0005 23.34 0.0002 0.63 0.6454 2.6 Cenococcum geophilum Both SP A N O V A 2.02 0.1438 37.18 <0.0001 1.17 0.3635 2.10a Russula spp. Both SP A N O V A 7.25 0.0019 <0.01 0.9638 0.52 0.7203 2.10b Lactarius scrobiculatus Both SP A N O V A 1.28 0.3200 9.85 0.0068 1.64 0.2162 2.10c Cortinarius spp. Both K-W 9.96 0.0410 N / A N / A N / A N / A 2.10d Piloderma spp. Both K-W 11.99 0.0174 N / A N / A N / A N / A 2.10e Hebeloma spp. Both K-W 4.94 0.2939 N / A N / A N / A N / A 2.10f Inocybe spp. Both K-W 6.37 0.1508 N / A N / A N / A N / A 2.10g Russula nigricans Both K-W 5.13 0.2379 N / A N / A N / A N / A 2.10h Rhizopogon vinicolor-type Fd One-way A N O V A 11.51 0.0002 N / A N / A N / A N / A 2.11a Suillus lakei Fd K-W 10.89 0.0279 N / A N / A N / A N / A 2.11b Leccinum scabrum Ep One-way A N O V A 2.58 0.0804 N / A N / A N / A N / A 2.12a Lactarius torminosus Ep One-way A N O V A 1.36 0.2946 N / A N / A N / A N / A 2.12b T a b l e 2.4 M e a n d i v e r s i t y m e a s u r e m e n t s c a l c u l a t e d f r o m f r e q u e n c y d a t a f o r D o u g l a s - f i r , p a p e r b i r c h , a n d c o m b i n e d E C M c o m m u n i t i e s . N u m b e r s i n p a r e n t h e s e s a r e o n e s t a n d a r d e r r o r o f the m e a n . Host species; Mean Shannon- Mean Simpson Douglas-fir 5-yr-old clearcut 0.756 (0.058) 1.022 (0 25) 0.529 (0.106) 26-yr-old burned 0.903 (0.023) 1.719(0 07) 0.788(0.016) 26-yr-old clearcut 0.935 (0.012) 2.155 (0 12) 0.862 (0.015) 65-yr-old burned 0.932 (0.007) 2.376 (0 11) 0.886 (0.015) 100-yr-old burned 0.935 (0.014) 2.379 (0 15) 0.880 (0.023) Paper birch 5-yr-old clearcut 0.920 (0.021) 2.054 (0 17) 0.841 (0.035) 26-yr-old burned 0.890 (0.010) 2.156(0 12) 0.846 (0.015) 26-yr-old clearcut 0.930 (0.009) 2.450 (0 08) 0.896 (0.008) 65-yr-old burned 0.901 (0.019) 2.386 (0 10) 0.871 (0.015) 100-yr-old burned 0.955 (0.009) 2.446 (0 14) 0.897 (0.013) Both Hosts 5-yr-old clearcut 0.898 (0.016) 2.233 (0 15) 0.862 (0.023) 26-yr-old burned 0.906 (0.011) 2.459 (0 08) 0.892 (0.009) 26-yr-old clearcut 0.941 (0.006) 2.844 (0 10) 0.929 (0.007) 65-yr-old burned 0.923 (0.007) 2.920 (0 04) 0.929 (0.004) 100-yr-old burned 0.939 (0.008) 2.829 (0 11) 0.927 (0.008) 65 T a b l e 2.5 S u m m a r y o f g e n e r a l i z e d l i n e a r m o d e l s , e s t i m a t e d b y m a x i m u m l i k e l i h o o d , o f t a x o n a b u n d a n c e s a n d f r e q u e n c i e s . A b u n d a n c e m o d e l s a r e b a s e d o n p e r c o r e a b u n d a n c e s a n d the n e g a t i v e b i n o m i a l d i s t r i b u t i o n . F r e q u e n c y m o d e l s a r e b a s e d o n o c c u r r e n c e s p e r s i te a n d the P o i s s o n d i s t r i b u t i o n . S A = s t a n d a g e ; S B Z = b i o g e o c l i m a t i c s u b z o n e ( I C H m w o r I C H m k ) . " I C H m k e f f e c t " c o l u m n d e t a i l s t he e f f e c t o f t h i s s u b z o n e i n c o m p a r i s o n to the I C H m w s u b z o n e . S i g n i f i c a n t v a r i a b l e s g a v e p < 0.05 f o r l i k e l i h o o d r a t i o tests . A b u n d a n c e M o d e l F r e q u e n c y M o d e l Variables Significant ICHmk included variables effect Fig. Variables included Significant variables ICHmk effect Fig. B o t h H o s t s Cortinarius spp. Hebeloma spp. Piloderma spp. Russula nigricans 1/SA 1/SA N / A 2.13a 1/SA SA, 1/SA SA, 1/SA N / A 2.13c SA, 1/SA 1/SA, SBZ 1/SA, SBZ 2.14a 1/SA, SBZ by ~ 50% 1/SA SA, 1/SA 1/SA, SBZ N / A 2.13b N / A 2.13d reduced by= 2.14b 50% Russula spp. 1/SA, SBZ 1/SA, SBZ 1/SA 1/SA reduced 2 ] 5 a 1 / S A S B Z l /SA, SBZ by = 65% N / A 2.15c 1/SA 1/SA reduced by = 70% 2.15b N / A 2.15d D o u g l a s - f i r Rhizopogon vinicolor- 1/SA type 1/SA Suillus lakei SA, 1/SA SA, 1/SA N / A 2.16a 1/SA 1/SA N / A 2.16b N / A 2.16c 1/SA, IT N ° " e , ( s e e N / A 2.16d Results) P a p e r b i r c h 1/SA SRZ Lactarius SA, 1/SA, " ^ v R e d u c e d SA, 1/SA, SA, 1/SA, Reduced torminosus SBZ _ l s e f . to = 0 ' / a SBZ SBZ to = 0 Results) Saturn S A S A N / A 2 - , 7 c S A S A N / A 2 - 1 7 d 66 References A g e e J K . 1993 . 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T a y l o r A F S . 2 0 0 2 . Fungaf diversity in ectomycorrhizal communities: sampling effort and species detection. Plant and Soil 2 4 4 : 19-28. T a y l o r D L , B r u n s T D . 1999 . Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities. Molecular Ecology 8 : 1837-1850. T e d e r s o o L , K o l j a l g U , H a l l e n b e r g N , L a r s s o n K - H . 2 0 0 3 . Fine scale distribution of ectomycorrhizal fungi and roots across substrate layers including coarse woody debris in a mixed forest. New Phytologist 1 5 9 : 153-165. T h o m p s o n J D , G i b s o n T J , P l e w n i a k F, J a n m o u g i n F, H i g g i n s P G . 1 9 9 7 . The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality control analysis tools. Nucleic Acids Research 2 4 : 4876-4882. V i s s e r S. 1 9 9 5 . Ectomycorrhizal fungal succession in jack pine stands following wildfire. New Phytologist 1 2 9 : 389-401. 71 Wu B, Nara K, Hogetsu T. 1999. Competition between ectomycorrhizal fungi in colonizing Pinus densiflora. Mycorrhiza 9: 151-159. 72 Chapter 3 Do soil properties and tree cover variables explain variation in ectomycorrhizal fungal community diversity and structure along a forest chronosequence?1 Introduction Ectomycorrhizae are prevalent in coniferous forests and play an important role in trees' uptake of water and nutrients and resistance to environmental stresses (Smith & Read, 1997). There is evidence that increasing diversity in ectomycorrhizal ( ECM) communities has positive effects on host tree productivity under some conditions (Baxter & Dighton, 2001; Jonsson et al., 2001), which is probably because different E C M fungi differ in functional abilities. The realized niches of E C M fungi are determined in part by their dispersal abilities, specificity to host species, uptake and resistance capabilities, competitive abilities, and response to other environmental factors. There are myriad biotic and abiotic factors which influence E C M communities. The most obvious are E C M tree community composition and structure, which have been shown to be more important in determining E C M community diversity and composition than other environmental variables, such as soil properties and understorey vegetation (Kernaghan et al, 2003; Nantel & Neumann, 1992; Vil leneuve et al, 1989). However, Nantel & Neumann (1992) stress that many E C M fungi occur only within a certain range of abiotic factors for a given host range. Soil properties also affect E C M communities. Nitrogen (N) and phosphorus (P) acquisition and translocation to plant hosts are especially important functions of E C M fungi. E C M communities may vary over natural gradients of these nutrients. Recent work has shown, for example, that E C M diversity generally decreases and community composition changes over long gradients of increasing available N (Avis etal, 2003; Li l leskov et al, 2002; Petered/ . , 2001a; Taylor et al, 2000). Douglas etal. (2005), in contrast, found no strong evidence that soil properties, including available N and P, correlated with E C M community differences among lodgepole pine and mixed conifer stands. They examined E C M communities in both forest floor and mineral soil layers, however, and measured soil chemical properties ' A version of this chapter will be submitted for publication. Twieg BD, Durall D M , Simard SW, Jones M D . Do soil properties and tree cover variables explain variation in ectomycorrhizal fungal community diversity and structure along a forest chronosequence? 73 only of mineral soil. Kernaghan et al. (2003) also found no effects of N or P on E C M communities in boreal mixed forests, but did f ind reasonable correlations between soil exchangeable cations and Russula and Cenococcum abundance. Ectomycorrhizae are often concentrated in the fermentation layer of the forest floor (Perez-Moreno & Read, 2000), probably because of its high nutrient availability. Many E C M fungal taxa also occur in, and even show preference for, mineral horizons (Rosling et al, 2003; Dickie et al, 2002; Taylor & Bruns, 1999). Whi le N and P can be acquired in mineral form by E C M fungi, it is now recognized that some E C M fungi may also have saprotrophic abilities and take up or transform organic nutrients before translocation to host plants (Read & Perez-Moreno, 2003). Conn & Dighton (2000) showed that litter types affect N and P immobilization and E C M fungal community composition. Conn & Dighton (2000) also found that E C M fungal types differed in their acid phosphatase activities, and greater activity was correlated to litter types with greater P immobilization. This is further evidence that E C M fungi and their functions are strongly linked to soil properties. Stand age strongly affects E C M communities (see Chapter 2) (Smith et al, 2002; Visser, 1995), but has variable effects on soil properties. Available N and N mineralization can be higher in young stands than in older stands (Bradley et al, 2002; Thibadeau et al, 2000; Prescott, 1997; Bauhus, 1996), but this is not always the case (Kranabetter & Coates, 2004; Griffiths & Swanson, 2001; Barg & Edmonds, 1999; Houston et al, 1998). Organic phosphorus can also be more abundant in recently disturbed than mature stands (Quails et al, 2000), or may not differ (Kranabetter & Coates, 2004). The C:N ratio of the forest floor can decrease after clearcutting (Olsson et al, 1996), which is l ikely to affect nutrient cycl ing (Prescott, 2002). Although models of total organic matter in forest floors often predict losses from the time o f forest harvest up to about 20 years of age, the presence and extent of this loss varies substantially (Yanai et al, 2003). The objective of this study was to examine relationships between soil properties and E C M community measures along a chronosequence of mixed forest stands that were similar in vegetation composition and site quality. The fol lowing hypotheses were tested: 1) soil N and P availability and mineralizable N decrease with stand age, while the C:N ratio increases; 2) soil variables explain a substantial degree of variation in E C M diversity that is not accounted for by stand age; namely, inorganic N and P availability are negatively correlated with E C M fungal diversity, while organic P and C:N ratio are positively correlated; and 3) these soil variables are related to E C M community composition and structure. 74 Materials and Methods Data Collection and Soil Analyses Site attributes and methods for characterising the E C M community are detailed in Chapter 2. Six stand types dominated by Douglas-fir and paper birch were selected to represent important serai stand development stages in southern Interior Cedar-Hemlock forests of British Columbia; these were wildfire-origin sites that were in 5-, 26-, 65-, and 100-yr-old age classes, and clearcut-origin sites that were in 5-and 26-yr-old age classes. There were 4 replicate sites selected for each of these six stand types. Total crown cover, and cover of tree species or groups, was estimated along four parallel transects, spaced approx. 5-6 m apart. Canopy cover was estimated for the fol lowing groups: 1) Douglas-fir; 2) paper birch; 3) other E C M conifer hosts; 4) other E C M broadleaf hosts; and 5) western redcedar. Percent cover of understorey E C M conifers and western redcedar was also estimated along the transects. Soil samples were collected in August, 2004. Approximately 1 kg of mineral soil and 300 g of forest floor were removed from each of eight sampling locations at each site. Soil sampling locations were adjacent to four spring and four fall E C M sampling locations. A t the 5-yr-old wildfire sites, however, soils were sampled next to eight randomly selected Douglas-fir seedlings located within 3 m of a paper birch tree. No forest floor samples were taken from these burned sites due to the near absence of forest floor layers. Mineral and forest floor samples were bulked separately for each site, separated into three subsamples, sealed in polyethylene bags, and transported on ice in coolers to the lab. Forest floor thickness was measured at each soil sampling location for ectomycorrhizae in spring and fall (see Chapter 2), yielding 16 observations per site. Mineral soil samples were sieved to 2 mm, air-dried, and sent to the B C Ministry of Forests (MOF) Analytical Chemistry Laboratory in Victoria, B C for all soil analyses except organic phosphorus. Forest floor samples were air-dried and then milled prior to analysis. Total C and N were determined by combustion elemental analysis, using a Leco CHN-600 Elemental Analyser (Leco Corp., St. Joseph, MI , USA ) (method described in instrument instructions). Available ammonium and nitrate were extracted by shaking for 2 hours in 2N K G (Bremner, 1996) and their concentrations measured on a Technicon AutoAnalyser II. Potentially mineralizable ammonium and nitrate were estimated by anaerobic incubation (Waring & Bremner, 1964), done in waterlogged conditions at 30° C for two weeks. Ammonium and nitrate were extracted as above, and initial available N values were subtracted from post-incubation values to estimate mineralizable N. Available P was determined using the Bray-1 method. Organic phosphorus of the forest floor was estimated from the difference in sulphuric acid-extractable phosphorus between post-ignited and pre-ignited soil samples, as described in Page et al. (1982). 75 Measurements were averaged f rom f i ve readings on the spectrophotometer for each sample . O r g a n i c phosphorus ana lys is was per formed at the Un i ve r s i t y o f B r i t i sh C o l u m b i a , Okanagan . Data Analysis Stat ist ical analyses were carr ied out in S A S v. 9.1 ( S A S Institute, Ca rey , N Y , U S A ) and s ign i f i cance fo r a l l statistical tests was set at a = 0.05 unless otherwise noted. O n e w a y analyses o f var iance ( A N O V A s ) were used to test for d i f ferences a m o n g stand age-init iat ion types for a l l so i l var iab les . These analyses were per fo rmed on averages o f the three subsamples per repl icate site. Stand type means were separated us ing Bon fe r ron i mu l t ip le compar i son tests. A N O V A assumpt ions were checked as descr ibed in Chapter 2, and where necessary, data were t ransformed by the natural log . F ive-year-old burned stands were omi t ted f r om the rest o f analyses because ec tomycor rh izae were sampled d i f ferent ly . E C M funga l species r ichness and evenness were ca lcu lated in P C - O r d v. 4 ( M c C u n e & M e f f o r d , 1995-2002) for forest f l oo r and minera l so i l layers separately, and at both the ind i v i dua l so i l sample- and site-levels. Pa i r ed t-tests were used to compare site-level species r ichness and evenness between the forest f l oo r and minera l so i l . Gene ra l i zed l inear mode l s , based on m a x i m u m l i k e l i hood and the Po i sson d i s t r ibu t ion , were used to predict per so i l sample E C M species r ichness. A l l poss ib le combina t ions o f the f o l l o w i n g pred ic tor var iables were used: stand age, 1/stand age, number o f tips ident i f ied f r om the forest f l oo r and minera l so i l (separately) , and forest f l oor th ickness . A I C was used to select the best m o d e l f r o m the resu l t ing set o f candidate mode l s ( B u r n h a m & A n d e r s o n , 2002) . Re la t i ve abundance o f the most abundant taxa (see Chapter 2) were ca lcu la ted fo r minera l and forest f l oo r layers by d i v i d i n g the total number o f t ips o f each taxon in each layer by the total number o f E C M tips examined f r o m each site on the appropriate host(s). Two-ta i l ed paired t-tests were used to determine whether taxa had h igher relat ive abundance in one o f the two layers. Sites that had no occurrences o f the concerned taxon were removed f r om this ana lys is because lack o f d i f ferences between so i l layers is un in format i ve in c i rcumstances where a taxon is not detected. S tepwise least-squares mu l t ip l e regress ion was used to exp lo re re la t ionships between so i l var iab les and E C M divers i ty . T o do this , site scores for the f i rst P r i nc ipa l C o m p o n e n t o f a P r i nc ipa l Componen t s A n a l y s i s ( P C A ) on E C M funga l d ivers i ty var iab les (see Chapter 2) , species r ichness in the c o m b i n e d c o m m u n i t y (i.e. both hosts), and species r ichness on Doug las- f i r were predicted f r o m the f o l l o w i n g var iables for minera l so i l and forest f loor : C : N rat io, ava i lab le N , potent ia l ly m ine ra l i zab l e N , and ava i lab le P. Stand age, b i ogeoc l ima t i c subzone , forest f l oo r o rgan i c P, and site index were a lso used 76 as predictor variables. The criteria for entry and retention in the regression models were 0.15 and 0.1 for F-test and partial F-test significance, respectively. Stepwise regressions were also used to predict relative abundance of Cenococcum geophilum, Rhizopogon vinicolor-type, and Russula spp. using the same methods and predictor variables as for site-level ECM diversity models. These taxa were chosen because of their high frequency and abundance, and because their relative abundance data met normality and homoscedasticity assumptions. It was originally intended that ECM variables would be regressed against tree cover variables, but each had 7-10 zero values out of the 20 sites, rendering these data inappropriate for multiple regression analyses. The ranges of values in tree cover variables were also fairly low (see Table 3.1) because the sites were intentionally selected to be relatively pure mixtures of Douglas-fir and paper birch. A Mantel Test was therefore used to determine whether site similarity in the ECM community was related to site similarity in tree cover variables. Frequency of ECM fungal species in the combined community was used in one matrix, and the tree cover variables presented in Table 3.1 were used in the other matrix. The Relative Sorensen distance measure was chosen for the ECM community matrix and Sorensen distance for tree cover, and tests based on both Monte Carlo randomizations (1000) and Mantel's Asymptotic z Approximation were checked for significance of the intermatrix correlation. This analysis was performed both with and without 5-yr-old sites included because root systems of other tree species in these stands were much less likely to overlap those of target hosts and were therefore less likely to affect their ECM fungal communities. Another analysis was done with Douglas-fir and paper birch cover removed from the tree variables matrix to get an idea of how much other trees were related to ECM communities on the target hosts. Canonical Correspondence Analysis (CCA) in PC-ORD was used to ordinate sites and ECM fungal species based on species frequencies (the number of soil samples each species occurred in per site). The same soil variables used as independent variables in regressions predicting diversity PCA scores were used as the environmental matrix. Only species that occurred in more than two sites were included. Monte Carlo randomisations (1000) were used to test significance of site ordination and correlation between the environmental variables and ECM communities. Stepwise regression analysis was also used with stand age and soil variables as predictors for site scores from a well-structured NMS ordination of the entire ECM community (see Chapter 2). 77 Results Mineral soil C:N ratio differed among stand types, mostly due to higher C content in 26-yr-old clearcut stands (Table 3.2). C:N ratio of the forest floor was 40% higher in 5-yr-old clearcut stands than 100-yr-old stands because of lower total N in the 5-yr-old stands. Total C content of the forest floor was similar among stand types (range 34.4-38.5%). Stand types differed in available nitrate of the mineral soil and available ammonium of the forest floor, but multiple comparison tests revealed no pairwise differences. Mineral soil mineralizable nitrate in 26-yr-old sites was about twice as high as in 65- and 100-yr-old sites, but overall, mineralizable nitrate averaged only 5% of mineralizable ammonium levels (range 3.1-10.5%). Stand types did not differ in any of the other soil parameters measured. Forest floor thickness was similar among stand types (Figure 3.1). The total number of root tips included in site-level analyses was also similar for all sites (see Chapter 2), as were the cumulative numbers of ECM fungal species detected in the mineral soil and forest floor layers (73 and 82, respectively). Mean site-level species richness, however, was 27% higher in the forest floor than the mineral soil (Fig. 3.2). The average number of root tips examined from the forest floor was also about 30% higher than the number examined from mineral soil. A frequency distribution comparing the number of tips examined per core between soil layers shows that more mineral soil samples had fewer than 50 ECM root tips than did forest floor samples (141 vs. 99, respectively; Fig. 3.3). The ratio of the number of root tips examined from the forest floor layer to mineral soil varied considerably, but was not related to site-level diversity, nor did it differ among stand types (Fig. 3.4). Site-level evenness of the ECM community did not differ between soil layers. The best models predicting per soil sample ECM richness of Douglas-fir and the combined community included stand age (Fig. 3.5) and the number of root tips examined from the forest floor; neither the number of tips from the mineral soil nor forest floor thickness was a significant predictor. Including forest floor root tip number increased soil sample-level prediction of ECM species richness on Douglas-fir and the combined community by about one species per 10 root tips. Fifty-five of the 105 identified species of ECM fungi were found in only one of the two soil layers, but these species were too infrequent to evaluate their soil layer preference. Several frequently observed ECM taxa occurred in both layers, but showed preference for one layer. Leccinum scabrum, Rhizopogon rudus, and Suillus lakei were more abundant in the mineral soil, while Lactarius torminosus and the genera Cortinarius, Hebeloma, and Piloderma were more abundant in the forest floor (Table 3.3). 78 Cenococcum geophilum, Lactarius pubescens, Lactarius scrobiculatus, and the genus Russula did not differ in relative abundance between soil layers. Models predicting site-level E C M richness and diversity are summarised in Table 3.4. A s detailed in Chapter 2, stand age and sometimes biogeoclimatic subzone were significant predictors of E C M species richness and diversity. Forest floor organic P was positively related to E C M diversity P C A axis one scores and E C M diversity on Douglas-fir. Mineral soil available P was negatively related to diversity on Douglas-fir. However, when one site that had the lowest E C M fungal richness and diversity and second-lowest organic P value (555 mg/kg soil) was removed from these analyses, no soil variables were significant predictors, but stand age and biogeoclimatic subzone remained significant. This site was an outlier, being the only site with richness and diversity values for the E C M fungal community more extreme than two standard deviations from overall mean values. Diversity is plotted against organic P in Figure 3.6, including the outlier site. There were some significant relationships between soil variables and relative abundances of dominant E C M taxa. Mineral soil available N and P and forest floor available P were significant in models predicting Cenococcum geophilum relative abundance, and mineral soil available N and P were also significant in predicting Rhizopogon vinicolor-type relative abundance (Table 3.5). Out of the variables that were significant in predicting Cenococcum geophilum and Rhizopogon vinicolor-type relative abundances from both soil layers combined, only forest floor available P remained significant in regressions predicting abundances from each soil layer separately. Tree cover variables (% canopy cover of Douglas-fir, paper birch, other E C M broadleaves, other E C M conifers, and western redcedar, and understorey cover of other E C M conifers and western redcedar) were fairly well correlated with E C M community. The relationship was stronger with 5-yr-old stands included (Mantel's Standardized r = 0.44; p = 0.00001 and 0.001 for Mantel 's Asymptotic Approx. and Monte Carlo tests, respectively) than when they were removed (r = 0.25; p = 0.016 and 0.007). Correlation values (r = 0.39 with and 0.30 without 5-yr-old sites) and significance were similar with Douglas-fir and paper birch cover removed from the analysis. Roughly half of the E C M fungal species occurred in two or fewer sites, and were removed for C C A . Sites grouped strongly by stand type in the ordination (Fig. 3.7), but ordination structure was better than random permutations of the data on only one axis (Table 3.6). Whi le site index and mineral soil C:N ratio and available P had moderate correlations to one axis each, their correlations were not nearly as strong as that of stand age. Overall correlations between species and environmental matrices were not 79 significant. Similarly, no soil variables were correlated to axis scores from a well-structured N M S ordination of the combined community, nor was there a significant correlation between the E C M community and soil variable matrices (Mantel's Standardized r = 0.10; p-values = 0.32 and 0.18 for Mantel's Asymptotic Approx. and Monte Carlo tests, respectively). 80 Discussion The data did not support the first hypothesis that mineral forms of N and P are more available, and C:N ratio lower, in younger stands. Indeed, general patterns in C, N , P, or forest floor depth across stand ages were not evident. Although N mineralization is commonly higher in recent clearcuts or forest gaps than in mature forests, this effect is most pronounced in the first year following disturbance and subsequently tapers off over the next few years (Prescott, 2002; Bauhus & Barthel, 1995). In this study, an early N flush would have been missed because the youngest age class sampled was 5 years old. Consistent with this, Kranabetter & Coates (2004) found no difference in soil available N and P, or organic P between mature ICH stands and 10-yr-old plantations. Forge & Simard (2000), however, found that mineral N was lower in 10 yr-old plantations than adjacent mature ICH forests, probably because of uptake by the lush herbaceous vegetation layer that had developed after harvest. Forest floor mineralizable N levels found by Forge & Simard (2000) were generally lower than, but not outside of the range of, those found in this study. The overall difference is surprising, given the geographical proximity of these two studies, but it may have been caused by sampling at different times of year; Forge & Simard (2000) sampled in June and September, while samples were taken in August in the current study. Soil N and P values in this study are similar to Kranabetter & Coates (2004), who examined western hemlock-dominated sites of consistent quality in the ICH of northern British Columbia. There was considerable variation in soil properties among the sites, even though all were mesic (zonal site series; (Lloyd et al, 1990)). Our data did not strongly support the second hypothesis that soil nitrogen, phosphorus, and C:N ratio explain a high degree of the variation in ECM diversity. Relationships between diversity and both available and organic P were largely the result of one site that was at the low end of the range of organic P levels encountered in this study. A study concentrating on more sites within the lower range of organic P values would be necessary to confirm or refute this relationship. In this study, there were only two sites with levels of organic P that were below two standard deviations of the overall mean, and both sites were from the youngest age class. Soil N was not related to diversity. Although Lilleskov et al. (2002) found that below-ground ECM diversity was negatively related to extractable (available) mineral N in the forest floor, their nitrogen gradient was extreme compared to this study. Avis et al. (2003) and Peter et al. (2001 a) both found that nitrogen addition affected species richness and diversity of the ECM sporocarp communities more than root tip communities. Lilleskov & Bruns (2001) suggest that nitrogen fertilization may simply reduce the amount of resources allotted to fruiting without having a prominent effect on below-ground community structure. 81 The hypothesis that soil nutrients are related to ECM community structure was supported by some specific taxa in this study. Although soil variables did not greatly improve relative abundance models, two out of three dominant ECM fungal taxa were positively related to available forest floor phosphorus. This study cannot determine whether higher available P caused higher ECM abundance of certain taxa or vice-versa, but it is known that ECM fungi can access organic P and make it available to plants (Sawyer et al, 2003) and that ECM fungi can reduce levels of organic P more substantially than available P from forest floor material (Perez-Moreno & Read, 2000). It is somewhat surprising that soil N was not strongly related to abundance of dominant ECM taxa. Cenococcum relative abundance from both soil layers combined was positively related to available N of the mineral soil only, but this relationship was not apparent when only its abundance in the mineral soil was considered. This was not surprising given available N was much higher in the forest floor than mineral soil, that Cenococcum was unrelated to forest floor N, and that this taxon showed no mineral soil versus forest floor preference. Nilsen et al. (1998) also found no effect of N on Cenococcum relative abundance on root tips. Nevertheless, available N in the mineral soil could have some effect on colonisation in both soil layers by influencing exploratory patterns of Cenococcum hyphae, or indirectly by influencing plant and microbial communities. Although below-ground Russula abundance has been shown to decrease (Peter et al, 2001a) or increase (Avis et al, 2003) following N-fertilization, it was not related to N levels in this study. Changes in Russula abundance in these two studies were largely due to one Russula species each, and effects of higher N availability in fertilized treatments in the study of Avis et al. (2003) was somewhat confounded by simultaneously higher available P. While ECM community structure was reasonably correlated with tree cover variables, there was generally no relationship between ECM community structure and soil variables. Mantel correlations suggested that tree cover of species besides Douglas-fir and paper birch was less correlated to ECM communities of these two hosts in 5-yr-old stands than in older stands. This is not surprising, given that tree root systems are far more developed in older stands and are therefore more likely to interact with each other. CCA ordinations of sites based on ECM community and environmental variables only showed a strong correlation of ECM communities and stand age. NMS ordination (see Chapter 2) accounted for much more variation in ECM community structure than did CCA. This may be attributed partly to the fact that the implicit Chi-square distance measure in CCA gives higher weight to rare species than the Relative Sorensen distance measure that was applied in NMS (McCune et al, 2002), but also indicates that the measured soil variables were not related to ECM communities in predictable ways. The fact that these soil variables were not significantly related to site scores on NMS axes or significantly 82 correlated to ECM communities is further evidence that they had little effect on ECM community structure. Although total ECM root tips in the mineral soil and forest floor were not directly counted, they appeared denser and more regularly distributed in the forest floor than mineral soil. The overall preference of ectomycorrhizae for the forest floor over the mineral soil was particularly apparent because a much higher volume of mineral soil than forest floor was examined on average per sample, and yet there were many more mineral soil samples than forest floor samples with fewer than fifty ECM root tips. ECM tips have previously been shown to be concentrated more in the forest floor than mineral soil (Nelville et al, 2002; Erland & Taylor, 2002), which probably reflects greater resource availability and heterogeneity in the forest floor. Site-level ECM richness was not substantially higher in the forest floor than the mineral soil, although richness was significantly higher in the forest floor at the soil sample-level. The high ECM fungal diversity and preference of some ECM fungal species for mineral soil suggests that both forest floor and mineral soil require sampling to accurately characterise ECM communities. 83 Conclusions While soil properties were not related to E C M community structure at the site level in this study, they may affect E C M composition at very small spatial scales (Schimel & Bennett, 2004). As suggested by recent studies, total soil organic content and mineral forms of N and P are likely not as closely related to ectomycorrhizae as are organic nutrient complexes. Other soil attributes, such as pH, soil moisture variation overtime, and micronutrients may also play important roles in E C M community structure. Differences in inoculum availability and non-ECM plant community structure were likely responsible for much of the variation in E C M diversity and community composition that were not accounted for by stand age. 84 Figures -in 5-cc 26-b 26-cc 65-b 100-b Stand Age-Initiation Type F i g u r e 3.1 M e a n f o r e s t f l o o r t h i c k n e s s (n = 4 ) b y s t a n d a g e - i n i t i a t i o n t y p e ; cc = c l e a r c u t ; b = b u r n e d . E r r o r b a r s r e p r e s e n t o n e s t a n d a r d e r r o r o f t he m e a n . N o d i f f e r e n c e s w e r e d e t e c t e d b y o n e - w a y A N O V A ( F = 1.26, p = 0 .29) . 85 12 10 Mineral Forest Floor Soil Layer F i g u r e 3.2 M e a n E C M f u n g a l spec ies r i c h n e s s o f t h e c o m m u n i t y o f b o t h hos t s i n m i n e r a l s o i l a n d f o r e s t f l o o r l a y e r s . E r r o r b a r s r e p r e s e n t o n e s t a n d a r d e r r o r o f t h e m e a n . S i g n i f i c a n t d i f f e r e n c e d e t e c t e d b y p a i r e d t-test (n = 2 0 , t = -3 .02 , p = 0 .007 ) . 86 50 1 I 0 1 2 3 4 5 6 7 8 9 10 Root Tips Examined per Core by Classes Figure 3.3 Frequency histogram of number of E C M rot tips available per soil sample from mineral soil (black bars) and forest floor (grey bars) (to max. of 100 tips). 0 = 0 tips examined; 1 = 1-10 tips; 2 = 11-20 tips ...; 10 = 91-100 tips examined. 87 4 5-cc 26-b 26-cc 65-b 100-b S t a n d Age- In i t ia t ion T y p e F i g u r e 3.4 M e a n r a t i o o f n u m b e r o f r o o t t i p s e x a m i n e d f r o m the f o r e s t f l o o r t o the n u m b e r e x a m i n e d f r o m the m i n e r a l s o i l p e r s i te (n = 4 ) . E r r o r b a r s r e p r e s e n t o n e s t a n d a r d e r r o r o f t h e m e a n . N o s i g n i f i c a n t d i f f e r e n c e s d e t e c t e d b y o n e - w a y A N O V A ( F = 1.9, p = 0.17) . 88 a. co 2.0 ± . 1 . . . . , , , 1 5 15 25 35 45 55 65 75 85 95 105 Stand Age F i g u r e 3.5 M a x i m u m l i k e l i h o o d m o d e l ( u s i n g P o i s s o n d i s t r i b u t i o n ) p r e d i c t i o n o f E C M f u n g a l spec i es r i c h n e s s p e r s o i l s a m p l e b y s t a n d age . N u m b e r o f r o o t t i p s e x a m i n e d f r o m the f o r e s t f l o o r w a s a l s o a s i g n i f i c a n t p r e d i c t o r v a r i a b l e i n th i s m o d e l (see R e s u l t s ) . S t a n d age w a s a s i g n i f i c a n t p r e d i c t o r ( l i k e l i h o o d r a t i o t es t ; p < 0.05) . 89 V • V T V O O I o 200 400 600 800 1000 1200 1400 Forest Floor Organic P (mg kg 1 soil) F i g u r e 3.6 S c a t t e r p l o t o f p r i n c i p a l c o m p o n e n t a x i s 1 s i te s c o r e s ( f r o m P C A o n 13 E C M d i v e r s i t y v a r i a b l e s ) a g a i n s t f o r e s t f l o o r o r g a n i c P ; filled c i r c l e s = 5-yr-old c l e a r c u t s , o p e n c i r c l e s = 2 6 - y r - o l d w i l d f i r e o r i g i n , filled t r i a n g l e s = 2 6 - y r - o l d c l e a r c u t s , o p e n t r i a n g l e s = 6 5 - y r - o l d w i l d f i r e o r i g i n , s q u a r e s = 1 0 0 - y r - o l d w i l d f i r e o r i g i n . 90 1.5 v • • o o • -1.0 -0.5 0.0 0.5 1.0 Axis 1; k= 0.38; R2 = 0.062 F i g u r e 3.7 C C A o r d i n a t i o n o f s i tes b a s e d o n f r e q u e n c y o f E C M f u n g i i n t he c o m b i n e d c o m m u n i t y o f b o t h hos ts a n d e n v i r o n m e n t a l v a r i a b l e s ( s t a n d age , s i te i n d e x , a n d s o i l v a r i a b l e s ) . F i l l e d c i r c l e s = 5-yr-o ld c l e a r c u t o p e n c i r c l e s = 2 6 - y r - o l d b u r n e d s i t e s ; f i l l e d t r i a n g l e s = 2 6 - y r - o l d c l e a r c u t s i t e s ; o p e n t r i a n g l e s = 6 5 - y r - o l d b u r n e d s i t e s ; a n d s q u a r e s = 100-y r-o ld b u r n e d s i tes . k = a x i s e i g e n v a l u e ; R 2 = p r o p o r t i o n o f v a r i a n c e i n C h i -s q u a r e d d i s t a n c e a m o n g s i tes e x p l a i n e d b y o r d i n a t i o n a x e s . 91 Tables Table 3.1 Tree cover variables by site; Fd = Douglas-fir, Ep = paper birch. Site Age Percent Canopy Cover % Understorey Cove r -Othe r E C M Conifers % Understorey Cover - Western redcedar Fd Ep Other E C M Conifers 1 Other E C M Broadleaves 2 Western redcedar 19MR 6 5 33 8 1 4 0 0 A L 6 11 23 8 3 0 0 0 B C 4 3 55 3 3 0 0 0 W L 5 5 35 9 1 1 0 0 EDI 30 28 32 6 10 0 8 8 ED2 30 30 38 9 4 0 3 3 M A I 24 28 32 4 7 0 13 8 M A 2 24 32 28 5 5 0 8 3 DISC 27 26 39 0 0 0 0 0 N M 21 24 27 0 3 6 13 8 SRC 22 36 38 0 2 0 0 3 ZP 25 28 28 7 7 0 8 3 B A 63 38 43 4 0 4 0 3 M A R A 71 36 40 0 0 4 3 8 RR 61 30 45 2.5 0 0 8 0 SL 68 41 41 5 2 2 0 8 4WD 103 45 41 0 0 5 0 0 A C R 98 44 24 6 0 6 0 13 BBP 101 32 32 5 0 8 8 3 W A P 90 40 32 0 0 8 3 8 ' Includes hybrid spruce, western hemlock (canopy in 5-year-old stands only; understorey in older stands), western white pine {Pinus monticola Dougl. Ex D. Don in Lamb.), and lodgepole pine 2 Includes black cottonwood, trembling aspen, and willow spp. T a b l e 3.2 M e a n s o i l p r o p e r t i e s b y s t a n d age a n d i n i t i a t i o n t y p e (n = 4 ; s t a n d a r d e r r o r o f m e a n i n p a r e n t h e s e s ) . M e a n s f o l l o w e d b y t h e s a m e l e t t e r ( w i t h i n o n e s o i l l a y e r a n d v a r i a b l e ) a r e no t s i g n i f i c a n t l y d i f f e r e n t (p > 0.05) b y m u l t i p l e c o m p a r i s o n s . § = d i f f e r e n c e a m o n g t r e a t m e n t m e a n s , b u t n o s i g n i f i c a n t d i f f e r e n c e s f o u n d i n p a i r w i s e m e a n c o m p a r i s o n s . F- ra t i os a n d p-va lues a r e f r o m o n e - w a y A N O V A s . S t a n d A g e a n d I n i t i a t i o n T y p e C / N R a t i o A v a i l a b l e A m m o n i u m A v a i l a b l e N i t r a t e M i n e r a l i z a b l e A m m o n i u m M i n e r a l i z a b l e N i t r a t e O r g a n i c P A v a i l a b l e P M i n e r a l S o i l 5-yr-old Burned 23.2 (0.55)a 2.60 (0.42) 0.18 (0.06) § 18.3 (1.3) 0.678 (0.149)ab N / A 143 (24) 5-yr-old Clearcut 29.9(1.48)ab 1.97 (0.18) 0.33 (0.03) 15.3 (1.8) 0.730 (0.145)ab N / A 159(19) 26-yr-old Burned 29.6(1.94)ab 2.23 (0.51) 0.38 (0.06) 14.4 (2.8) 0.974 (0.128)b N / A 208 (49) 26-yr-old Clearcut 35.0 (1.13)b 2.44 (0.38) 0.28 (0.04) 9.2 (3.2) 0.971 (0.111)b N / A 95 (16) 65-yr-old Burned 24.6 (0.56)a 2.18(0.33) 0.38 (0.02) 14.8(2.7) 0.490 (0.114)a N / A 139(18) 100 yr-old Burned 25.3 (0.79)ab 2.02 (0.27) 0.30 (0.02) 14.1 (1.3) 0.442 (0.128)a N / A 209 (24) F-ratio 4.11 0.45 3.00 1.68 5.28 N / A 0.88 P-value 0.0115 0.8108 0.0386 0.1914 0.0037 N / A 0.5162 F o r e s t F l o o r 5-yr-old Clearcut 47.0 (2.55)b 11.7 (2.5)§ 0.39(0.23) 230 (29) 1.111 (0.193) 685 (121) 101 (11) 26-yr-old Clearcut 38.3 (1.17)ab 27.1 (3.5) 0.95 (0.22) 375 (22) 1.570 (0.344) 973 (108) 133 (16) 26-yr-old Burned 34.1 (1.80)ab 22.8 (2.3) 0.58 (0.14) 440 (34) 1.660 (0.310) 980 (39) 150(7.4) 65-yr-old Burned 35.2 (1.58)ab 18.4 (3.8) 0.83 (0.34) 309 (29) 1.512(0.246) 765 (55) 112 (8.6) 100-yr-old Burned 33.7 (1.05)a 26.9 (4.8) 1.0 (0.39) 411 (48) 1.363 (0.219) 954 (63) 102 (6.2) F-ratio 3.09 3.40 0.93 1.94 0.54 2.67 1.24 P-value 0.0485 0.0362 0.4717 0.1557 0.7807 0.0729 0.3357 T a b l e 3.3 M e a n r e l a t i v e a b u n d a n c e s ( r e s p e c t i v e o f hos t ) o f d o m i n a n t t a x a b y s o i l l a y e r . F d = D o u g l a s - f i r ; E p = p a p e r b i r c h . S i g n i f i c a n t d i f f e r e n c e s h a v e p-va lues i n b o l d ( p a i r e d t-test). E C M T a x o n H o s t T r e e N u m b e r o f S i t es (n) M i n e r a l S o i l R e l a t i v e A b u n d a n c e (% ) F o r e s t F l o o r R e l a t i v e A b u n d a n c e (% ) P - v a l u e : P a i r e d T-test Rhizopogon vinicolor-type Fd 20 18.0 23.5 0.266 Rhizopogon rudus Fd 6 8.95 2.39 0 .026 Suillus lakei Fd 9 14.9 2.86 0 .016 Lactarius pubescens Ep 5 8.84 7.48 0.759 Lactarius torminosus Ep 10 2.74 12.1 0 .033 Leccinum scabrum Ep 16 8.70 1.58 0.012 Cenococcum geophilum Both 20 4.11 6.15 0.059 Cortinarius spp. Both 16 0.91 4.40 0 .030 Hebeloma spp. Both 11 1.43 4.79 0 .003 Lactarius scrobiculatus Both 11 1.96 4.24 0.245 Piloderma spp. Both 14 0.89 7.68 0 .002 Russula spp. Both 18 7.08 6.66 0.881 94 T a b l e 3.4 S u m m a r y o f s t e p w i s e r e g r e s s i o n a n a l y s e s p r e d i c t i n g E C M d i v e r s i t y v a r i a b l e s . R 2 v a l u e s i n b o l d c o r r e s p o n d to the m o d e l t h a t c o n t a i n s a l l s i g n i f i c a n t p r e d i c t o r v a r i a b l e s ; t h o s e n o t i n b o l d c o r r e s p o n d t o m o d e l i n c l u d i n g t h a t p r e d i c t o r v a r i a b l e a n d those a b o v e i t f o r t he c o r r e s p o n d i n g d e p e n d e n t v a r i a b l e . S l o p e o r P a r t i a l M o d e l Y - v a r i a b l e / R a n g e o f R a n g e o f I n t e r c e p t F-test F-test x-variables Y - v a r i a b l e X - v a r i a b l e E s t i m a t e P-va lue R 2 P-va lue D i v e r s i t y P r i n c i p a l C o m p o n e n t -6.92 to 4.35 0.0001 1/stand age .009 to 0.17 -27.407 0.0004 0.51 Subzone 0 (ICHmw); 1 (ICHmk) -4.148 0.0074 0.65 Forest Floor Organic P 412 to 1166 0.0043 0.0590 0.72 Intercept N / A 2.054 0.3703 R i c h n e s s o f C o m b i n e d C o m m u n i t y 9 to 27 0.0010 1/stand age See above -46.397 0.0006 0.40 Subzone See above -6.44 0.0246 0.56 Intercept N / A 21.991 O.0001 R i c h n e s s o f D o u g l a s - f i r C o m m u n i t y 2 to 16 0.0001 Stand age 4 to 103 -0.0885 O.0001 0.52 Forest Floor Organic P See above 0.00771 0.0177 0.62 Mineral Soil Available P 58 to 484 -0.0131 0.0294 0.72 Intercept N / A 0.9157 0.7279 95 T a b l e 3.5 S u m m a r y o f s t e p w i s e r e g r e s s i o n m o d e l s p r e d i c t i n g r e l a t i v e a b u n d a n c e s (% ) o f d o m i n a n t E C M t a x a . R 2 v a l u e s as i n T a b l e 3.4. S l o p e o r P a r t i a l M o d e l Y - v a r i a b l e / x- R a n g e o f R a n g e o f I n t e r c e p t F-test F-test variables Y - v a r i a b l e X - v a r i a b l e E s t i m a t e P-va lue R 2 P-va lue Cenococcum geophilum 2.9 to 18.8% 0.0005 Mineral Soil Available N 1.55 to 3.94 4.383 0.0005 0.41 Forest Floor Available P 45 to 195 0.0563 0.0100 0.51 Mineral Soil Available P 58 to 484 -0.0200 0.0152 0.66 Intercept N / A -4.367 0.1696 Rhizopogon vinicolor-type 5.3 to 89.1% O.0001 1/stand age .009 to 0.17 295.11 O.0001 0.64 Forest Floor Available P See above 0.1570 0.0695 0.74 Mineral Soil Available P See Above 0.0568 0.0770 0.79 Intercept N / A -4.09 0.6898 Russula spp. 0 to 37.8% O.0001 Stand age See above 0.2749 O.0001 0.68 Intercept N / A 0.6974 0.7787 96 T a b l e 3.6 D e t a i l s o f C C A o r d i n a t i o n o f s i tes b a s e d o n E C M f u n g i f r e q u e n c y i n t h e c o m b i n e d c o m m u n i t y a n d e n v i r o n m e n t a l v a r i a b l e s . C o r r e l a t i o n s o f e n v i r o n m e n t a l v a r i a b l e s to o r d i n a t i o n axes a r e P e a r s o n ' s r, a n d those o f 0.5 o r a b o v e a r e i n b o l d . R 2 is t he p r o p o r t i o n o f C h i - s q u a r e d i s t a n c e a m o n g s i tes t h a t is e x p l a i n e d b y o r d i n a t i o n axes . P-va lues f o r M o n t e C a r l o test o f o r d i n a t i o n s t r u c t u r e r e p r e s e n t t he p r o p o r t i o n o f r a n d o m i z e d sets o f the r e a l d a t a g i v i n g o r d i n a t i o n axes w i t h a x i s e i g e n v a l u e s g r e a t e r t h a n o r e q u a l t o t h e r e a l d a t a . T h o s e f o r tests o f i n t e r - m a t r i x c o r r e l a t i o n r e p r e s e n t t he p r o p o r t i o n o f r a n d o m i z e d d a t a sets g i v i n g c o r r e l a t i o n s b e t w e e n E C M spec i es m a t r i c e s a n d the e n v i r o n m e n t a l v a r i a b l e s e q u a l to o r g r e a t e r t h a n the r e a l d a t a . A x i s 1 A x i s 2 A x i s 3 Eigenvalue 0.378 0.302 0.257 R 2 0.062 0.184 0.015 P (Monte Carlo test): Ordination structure 0.008 0.122 0.360 P (Monte Carlo test): Inter-matrix correlation 0.057 0.515 0.243 C o r r e l a t i o n s Stand Age -0.919 -0.201 -0.234 Site Index -0.358 -0.019 -0.558 M i n e r a l S o i l C:N ratio 0 .533 0.187 0.249 Available N 0.066 -0.026 -0.356 Mineralizable N -0.080 0.254 -0.283 Available P -0.242 0 .523 0.003 F o r e s t F l o o r C:N ratio 0.438 0.274 0.374 Available N -0.185 -0.366 -0.220 Mineralizable N -0.018 -0.441 -0.236 Available P 0.023 0.063 0.218 Organic P -0.088 -0.248 0.103 97 References A v i s P J , M c L a u g h l i n D J , D e n t i n g e r B C , R e i c h P B . 2 0 0 3 . Long-term increase in nitrogen supply alters above-and below-ground ectomycorrhizal communities and increases the dominance of Russula spp. in a temperate oak savanna. New Phytologist 1 6 0 : 239-253. B a r g A K , E d m o n d s R L . 1 9 9 9 . 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Y a n a i R D , C u r r i e W S , G o o d a l e C L . 2 0 0 3 . Soil carbon dynamics after forest harvest: an ecosystem paradigm reconsidered. Ecosystems. 100 Chapter 4 Conclusions Ectomycorrhizal Diversity and Communities Effects and Likely Causes The 5-year-old c learcuts supported less d iverse E C M commun i t i e s than o lder stands, w h i c h is consistent w i th many other studies (Du ra l l et al, 2 0 0 6 ; Hage rman et al, 2 0 0 1 ; B y r d et al, 2 0 0 0 ; Hagerman etal, 1999a; D u r a l l et al, 1999; Kranabetter & W y l i e , 1998). Reduc t ions in d ivers i ty after d isturbance are part ly caused by poor inocu la t ion o f seedl ings es tab l i sh ing in iso la t ion o f mature trees o r forest (see C l i n e et al, 2 0 0 5 ; Hage rman et al, 1999a). There was l itt le ev idence that l ower E C M divers i t y in y o u n g stands was associated w i th ava i lab le fo rms o f N or P, organ ic P, or C : N rat io. A l t h o u g h h igh m y c o r r h i z a l d ivers i t y is often corre lated w i t h l o w nutrient ava i l ab i l i t y , th is d i d not appear to be the cause o f h igher E C M d ivers i t y in o lder stands. W h i l e effects o f n o n - E C M plants on ec tomycor rh izae were not examined here, y o u n g stands were domina ted by shade intolerant shrubs and herbs rather than E C M coni fers . A g e o f host trees also affects E C M commun i t i e s (Jones et al, 2003) . The rare occurrence in y o u n g stands o f some fung i c o m m o n in o lder stands suggests that some o f these fung i (e.g. Piloderma fallax, Russula nigricans, Lactarius rubrilacteus, and Pha l la les 1) are p h y s i o l o g i c a l l y able to f o r m myco r rh i zae w i th very y o u n g trees, but do not compete w e l l in the c learcut env i ronment . A s suggested by Kranabetter & Fr iesen (2002) , these funga l species may require connect ions to mature trees; a l though lack o f mature trees may not prec lude them f rom estab l i sh ing on seedl ings, greater ava i lab le carbon f r o m o lder trees may enhance their compet i t i ve ab i l i t ies on seed l ing root t ips. The greater E C M c o m m u n i t y d ivers i t y on paper b i rch than Doug las- f i r in the youngest stands was s t r ik ing . The stump sprout ing habit o f b i r ch a l l ows many o f its roots to cont inue l i v i n g after stems have been cut, p r o v i d i n g pre-disturbance i nocu lum for es tab l i sh ing seedl ings . Re ta in ing l i v i n g b i r ch stumps w o u l d therefore mit igate i n o c u l u m loss that occurs f o l l o w i n g c learcut t ing (Hagerman et al, 1999b; Hagerman et al, 1999a) and w i l d f i r e (G rogan et al, 2 0 0 0 ; Baar et al, 1999). In m y study, it is l i ke l y that some Douglas-f i r i n o c u l u m was lost between the t ime o f c learcut t ing and p lant ing . Fur thermore , E C M c o m m u n i t y compos i t i on o f paper b i rch in y o u n g stands was st i l l d i f ferent f r o m o lder 101 stands. Clearcutting affects more than just inoculum levels, and results in changes to host age, non-ECM host plant communities, microbial communities, and environmental conditions and resources, all which can affect the ECM of establishing hosts. Implications Although I could not determine the cause of lower diversity in young stands, I feel there are clear land management implications from this study. Based on the taxa-sampling unit curves, it appears that the site-level difference in diversity between the 5-year-old and older stands was underestimated, and that landscape-level richness is probably several times lower in an equivalent area of 5-year-old forest than in mature forest. There was ample inoculum in young stands to facilitate complete colonisation of seedlings, but lower ECM diversity in young stands will reduce the diversity of inoculum available on a landscape level if a large proportion of young forest is maintained. While this reduction in diversity of available inocula may not cause a reduction in species diversity in the short term, there is high potential for loss of genetic diversity. Another obvious implication is that removal of paper birch from young stands will decrease stand-level ECM diversity. Although the effect of stump sprouts on ECM diversity was not directly tested in this study, my results suggest that living roots of sprouting stumps link one forest generation to the next by providing inoculum for newly establishing hosts. Conversely, removal of birch stumps after logging for mitigation of Armillaria ostoyae root disease or for reduction of competition with conifers may reduce ECM diversity during stand initiation. Despite the beneficial contribution of paper birch to total ECM diversity in young stands, it appears unlikely that it contributed to Douglas-fir ECM communities because of the dominance of host-specific fungi on Douglas-fir in 4- to 6-year-old stands. This was probably not due to more limited contact between roots of paper birch with those of Douglas-fir, because roots of both hosts were commonly found in the same soil samples in the 5-yr-old stands as well as the old stands. However, compared with Douglas-fir, paper birch accumulated a higher proportion of ECM species compatible with both hosts in earlier age classes. Hence, it likely played an important role in determining ECM community structure on Douglas-fir over the chronosequence. 102 Succession Models and Fungal Strategies The E C M fungal patterns observed in this study are only partially consistent with historical models recognizing only two or three categories ("early-, multi-, and late-stage") of E C M fungi in forest succession (Visser, 1995; Mason et al, 1983; Fleming, 1983). M y results do not support the early dichotomous classification system (Mason et al, 1983), in which "early-stage" fungi dominate young stands, but are replaced by "late-stage" fungi in older stands. Lactarius pubescens was the only E C M fungus that was dominant in young stands but absent in older ones, consistent with Visser's (1995) finding of few "early-stage" fungi in young stands. However, earlier research categorized L. pubescens as a "late-stage" fungus (Fleming, 1983; Fox, 1983), which appears to disagree with plant and fungal succession patterns in ICH forests. Small gap disturbances, which are common in mature ICH forests, can also result in localized dominance of fungi common in early stand initiation (Kranabetter & Friesen, 2002). Thus, the use of "early-stage/late-stage" terminology should be applied carefully when describing E C M community dynamics in ICH forests. Fungal species that dominated the youngest stands were not necessarily ruderal strategists because they were previously shown to have substantially reduced inoculation potential when disconnected from parent trees (Simard et al, 1997b; Fleming, 1984). Rhizopogon vinicolor-type fungi and L. pubescens, for example, l ikely expend substantial energy inoculating new E C M root tips via vegetative spread rather than concentrating on formation of spores in response to reductions in host availability. It is difficult to fit E C M fungi into plant strategies proposed by Grime (1977) because little is known about which resources (e.g. host roots, soil nutrients and moisture) E C M fungi are competing most strongly for. Low availability of host roots and associated scarcity of carbon in 5-yr-old stands could be the most important resource stress imposed on E C M fungi among the stands studied. Thelephora terrestris, often referred to as an "early-stage" or ruderal fungus, occurred only in 5-year-old sites, but its frequency and abundance was low. Wilcoxina rehmii, part of the group of fungi forming E-strain mycorrhizae that also tends to proliferate immediately after disturbance, occurred at very low frequency and abundance at al l forest ages. These fungi may indeed act as true ruderal strategists, establishing well from spores in the wake of disturbance but apparently lacking the ability to compete after other fungi establish. This study does support the grouping of several fungi as "multi-stage", as described by Visser (1995) and supported by Smith et al. (2002) and Kranabetter et al. (2005). Fungi that fall into this category include Rhizopogon vinicolor, R. vesiculosus, Amphinema byssoides, Cenococcum geophilum, Tuber 1, and some Inocybe and Tomentella species. Fungi that augmented the community in stands older 103 than the 5-years were not necessarily "late-stage". For instance, Russula and Piloderma were nearly absent in 5-year-old stands, then increased to comprise a moderate proportion of the community in 26-year-old stands, and continued to increase in frequency and relative abundance with increasing stand age. Therefore, at the genus level, Russula and Piloderma fall somewhere between "multi-stage" and "late-stage". Other fungi, such as Lactarius scrobiculatus, were mostly absent from 5-year-old stands, but were abundant to similar degrees in all other age classes. Parallels can be drawn between plant-community succession models and ECM community successional patterns. Examples consistent with both "relay floristics" and "initial floristics" models were observed in this study. Lactarius pubescens was largely replaced by L. torminosus in older stands, which is consistent with the "relay floristics" concept, but L. torminosus was also found once in a 5-year-old site. It is likely that many other taxa common to the older age classes, such as Russula and Piloderma species, were also present in 5-year-old stands but not detected due to their very low frequency and abundance. This is more consistent with the "initial floristics" model. It seems that no single model is adequate to describe the complexity of successional patterns of ECM fungi. Using forest stand development stages to help characterize ECM community succession patterns may be more useful than classifying fungi into their own successional categories or trying to fit them into simplified plant succession models. Sites in the stand initiation stage (5-years-old) had distinctive ECM community composition and structure as well as low ECM diversity. Stem exclusion stage sites (26-years-old) had higher ECM diversity, but community structure was intermediate between 5-year-old sites and older sites. For instance, Rhizopogon vinicolor-type was much less dominant and Lactarius pubescens was almost absent in this age class, while Russula and Piloderma were significant components of the community. However, other fungi, like Suillus lakei, Hebeloma spp., and Cortinarius spp., were prominent in 26-year-old stands, but were largely supplanted by Russula and Piloderma in older stands. Sites in the stand re-initiation stage (65- and 100-year-old) were similar to each other, more so than to other age classes, in ECM diversity and community composition and structure. 104 Study Shortcomings The most important shortcoming of this study was its use of a chronosequence of sites as a proxy for observing succession on the same sites through time. Variation in E C M communities that was unaccounted for by stand age may have been partially due to the inherent flaws in chronosequences (Simard & Sachs, 2004), and this may have hindered the ability to detect relationships between E C M fungal parameters and soil properties. It cannot be assumed that the history of all replicate stands within the same disturbance type were the same. There were also slight differences in the management regime of clearcuts (e.g. nursery stock, conifer mixtures replanted) because different forest companies were responsible for managing different sites. It was necessary to select replicate stands that were far enough apart to avoid spatial autocorrelation; this may have increased between-site variability. In spite of these shortcomings, chronosequence studies provide a useful assessment of succession without waiting lifetimes for forests to develop. I found that combining morphotyping and D N A analysis was highly successful at fungal taxon identification. However, several samples could not be included in analyses because more than one fungus was present on a single root tip. In addition, the cost of molecular analysis was too high for analysis of all samples. The morphology of only a small percent of the E C M fungal species that occurred in this study had been previously described, so some subjectivity was necessary in combining molecular and morphological data to arrive at the final data set for analyses. However, a cautious approach was taken, and samples with ambiguous taxonomic placement were omitted from the analyses. In the future, a less rigorous morphotyping approach would allow more resource allocation to D N A analyses, although examination of E C M tips under a compound microscope is still recommended to reduce sorting error. A more intensive soil sampling approach would have enhanced our ability to correlate soil parameters with E C M parameters, but most project resources went to proper identification of ectomycorrhizae. Given our resources, it was not possible to measure soil properties and ectomycorrhizae within the same core, with many cores sampled over an experimental unit. This might have been useful, since both sets of factors vary at the microsite level. Nevertheless, our sampling intensity for environmental variables allowed us to characterize each study site. 105 Future Directions A better assessment o f forest management effects on E C M commun i t i e s requires study a l ong a longer age trajectory, f r om post-disturbance to c l imat i c c l i m a x stands. Re l a t i ng E C M c o m m u n i t y dynamics to forest success ion w o u l d also be best done over a larger scale so that each deve lopmenta l stage was represented in propor t ion to its occurrence over a landscape. A d d i t i o n a l l y , accurate representations o f total funga l c o m m u n i t y structure w o u l d incorporate a l l g rowth fo rms o f E C M fung i i n c l ud ing sporocarps, root t ips, and extramatr ica l hyphae. T e c h n o l o g y to pe r fo rm such deta i led studies does exist , and w i th t ime this comprehens ive approach w i l l become more feas ib le and cost e f f i c ient . Be low-ground funga l popu la t ion genetics is a c r i t i ca l f i e ld o f study to understanding w h y certa in E C M fung i dominate in certain forest stages, and this f i e ld is b e c o m i n g more access ib le w i th app l i ca t ion o f microsate l l i te techniques. M i c rosa te l l i t e s w i l l a lso be useful to determine whether Doug las- f i r and paper b i rch can be l inked by a c o m m o n E C M i nd i v i dua l . Thus far, funga l i nd i v idua l s have been ident i f ied ma in l y f r om sporocarps , but their ephemera l nature and myster ious f ru i t ing habits prec lude r igorous spatial ana lys is o f funga l d is t r ibut ions. It was su rpr i s ing and interest ing that host-speci f ic E C M fung i were dominant in y o u n g stands. Perhaps a study compa r i ng commun i t i e s o f y o u n g stands in w h i c h var ious proport ions o f either host tree were removed w o u l d be in format ive . If dominance by host-speci f ic fung i was af fected by interspec i f i c tree compet i t i on , then the relat ive abundance o f host-specif ic fung i w o u l d tend to decrease upon r emova l o f one tree species. H o w e v e r , such a decrease cou ld also result f r om changes to l itter inputs, the so i l m i c roc l ima te , or so i l food web , w h i c h cou ld also be exp lo red by man ipu la t i ve f i e ld tr ia ls . It w o u l d also be he lp fu l to study E C M commun i t i e s o f other con i fe r and broad lea f species f r o m the same sites to see i f their proport ions o f host-speci f ic fung i were s imi la r . The effect o f site env i ronment on E C M parameters remains poo r l y understood. N o n - E C M vascu lar and nonvascu lar plants, so i l chemis t ry ( i n c l ud ing more deta i led analys is o f organ ic compounds ) , and so i l m i c rob i a l commun i t i e s and their phys io l ogy may a l l affect E C M commun i t i e s . A s ment ioned above, it is l i k e l y 1 d id not sample so i l nutrients at a smal l enough spatial scale to see re lat ionships w i th E C M var iables . D e v e l o p i n g these re la t ionships f r om more intensive s amp l i ng might p rov ide a basis fo r func t iona l studies on spec i f i c E C M taxa. 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Below-ground ectomycorrhizal community structure in a recently burned bishop pine forest. Journal of Ecology 8 8 : 1051-1062. H a g e r m a n S M , J o n e s M D , B r a d f i e l d G E , G i l l e s p i e M , D u r a l l D M . 1 9 9 9 a . Effects of clear-cut logging on the diversity and persistence of ectomycorrhizae at a subalpine forest. Canadian Journal of Forest Research 2 9 : 124-134. H a g e r m a n S M , J o n e s M D , B r a d f i e l d G E , S a k a k i b a r a S M . 1 9 9 9 b . Ectomycorrhizal colonization of Picea engelmannii x Picea glauca seedlings planted across cut blocks of different sizes. Canadian Journal of Forest Research 2 9 : 1856-1870. H a g e r m a n S M , S a k a k i b a r a S M , D u r a l l D M . 2 0 0 1 . The potential for woody understory plants to provide refuge for ectomycorrhizal inoculum at an interior Douglas-fir forest after clear-cut logging. Canadian Journal of Forest Research 3 1 : 711-721. J o n e s M D , D u r a l l D M , C a i r n e y J W G . 2 0 0 3 . Ectomycorrhizal fungal communities in young forest stands regenerating after clearcut logging. New Phytologist 1 5 7 : 399-422. K r a n a b e t t e r J M , F r i e s e n J . 2 0 0 2 . Ectomycorrhizal community structure on western hemlock seedlings transplanted from forests into openings. Canadian Journal of Botany 7 6 : 189-196. K r a n a b e t t e r J M , F r i e s e n J , G a m i e t S, K r o e g e r P. 2 0 0 5 . Ectomycorrhizal mushroom distribution by stand age in western hemlock - lodgepole pine forests of northwestern British Columbia. Canadian Journal of Forest Research 3 5 : 1527-1539. K r a n a b e t t e r J M , W y l i e T . 1 9 9 8 . Ectomycorrhizal community structure across forest openings on naturally regenerated western hemlock seedlings. Canadian Journal of Botany 7 6 : 189-196. 108 M a s o n P A , W i l s o n J , L a s t F T . 1 9 8 3 . The concept of succession in relation to the spread of sheathing mycorrhizal_ fungi on inoculated tree seedlings growing in unsterile soils. Plant and Soil 7 1 : 247-256. S i m a r d S W , P e r r y D A , S m i t h J E , M o l i n a R. 1 9 9 7 . Effects of soil trenching on occurrence of ectomycorrhizas on Pseudotsuga menziesii seedling grown in mature forests of Betula papyrifera and Pseudotsuga menziesii. New Phytologist 1 3 6 : 327-340. S i m a r d S W , S a c h s D L . 2 0 0 4 . Assessment of interspecific competition using relative height and distance indices in an age sequence of serai interior cedar-hemlock forests in British Columbia. Canadian Journal of Forest Research 3 4 : 1228-1240. S m i t h J E , M o l i n a R, H u s o M M P , L u o m a D L , M c K a y D , C a s t e l l a n o M A , L e b e l T , V a l a c h o v i c Y . 2 0 0 2 . Species richness, abundance, and composition of hypogeous and epigeous ectomycorrhizal fungal sporocarps in young, rotation-age, and old-growth stands of Douglas-fir {Pseudotsuga menziesii) in the Cascade Range of Oregon, U.S.A. Canadian Journal of Botany 8 0 : 186-204. V i s s e r S. 1 9 9 5 . Ectomycorrhizal fungal succession in jack pine stands following wildfire. New Phytologist 1 2 9 : 389-401. 109 Appendix A D e s c r i p t i o n s a n d p h o t o g r a p h s o f a f e w c o m m o n l y e n c o u n t e r e d e c t o m y c o r r h i z a e f o l l o w ; these a r e i n t e n d e d to p r o v i d e a n e x a m p l e o f t he r i g o r o u s m o r p h o t y p i n g a p p r o a c h u n d e r t a k e n i n t h i s s t u d y . D e s c r i p t i o n s g e n e r a l l y f o l l o w f o r m a t a n d p r o t o c o l o f G o o d m a n et al. ( 1996 ) . E a c h d e s c r i p t i o n is f o l l o w e d b y o n e o r t w o pages o f p h o t o s d e t a i l i n g i m p o r t a n t f e a t u r e s , w h i c h w e r e t a k e n o n d i s s e c t i n g a n d c o m p o u n d m i c r o s c o p e s as d e t a i l e d i n C h a p t e r 2. E C M f u n g a l spec ies f o u n d o n b o t h hos t s s h o w e d d i f f e r e n t s y s t e m a n d t i p s izes w i t h hos t ( i .e. l a r g e r o n D o u g l a s - f i r ) , b u t m i c r o s c o p i c f e a t u r e s w e r e o f s i m i l a r s i ze o n b o t h hos t s . W h i l e m o s t p h o t o s t a k e n o n t he c o m p o u n d m i c r o s c o p e w e r e t a k e n i n b l a c k a n d w h i t e , p h o t o s t a k e n o n the d i s s e c t i n g s c o p e w e r e t a k e n i n c o l o u r . T h e r e f o r e , s o m e i m p o r t a n t d e t a i l s w i l l be los t i n b l a c k a n d w h i t e p r i n t e d v e r s i o n s . Lactarius pubescens o n p a p e r b i r c h (pg . 1) DISTINGUISHING FEATURES: smooth tips with a network of laticifers visible on the mantle surface; tips often white to yellowish with purple apices; outer mantle a net prosenchyma surrounded by a gelatinous matrix; mycelial strands common, with an inner core of laticifers; oleiferous cells present in inner mantle which appear similar to laticifers but do not react in sulphovanillin MORPHOLOGY (Dissection Microscope): E C T O M Y C O R R H I Z A L S Y S T E M : S h a p e a n d d i m e n s i o n s : (Figs. 1 to 3) monopodial pinnate to pyramidal systems 4 (2-8) mm long by 2.5 (1-4) mm wide; tips 1.5 (0.3-3) mm long by 300 (150-500) urn wide C o l o u r a n d t e x t u r e : (Figs. 1 to 4) White to cream to yellow to pinkish to dark purple, smooth, shiny, host not visible through mantle E M A N A T I N G E L E M E N T S : M y c e l i a l S t r a n d s : (Figs. 2 to 4 and 10 to 11) common, white, round in cross section, rarely branched, often running along non-ectomycorrhizal portions of birch roots to which ectomycorrhizal systems are attached; attachments are restricted points at bases of systems H y p h a e : none no Lactarius pubescens on paper birch (pg. 2) ANATOMY (Compound Microscope): M A N T L E IN P L A N V I E W : thick mantle, Hartig net present O u t e r L a y e r : a net prosenchyma (Figs. 5 to 7) with gelatinous matrix; cells 35 (10-50) urn long by 3 (2-5) pm wide, smooth, hyaline; laticifers common, 5 (3-7) pm wide; junctions common, 90-120°; anastomoses common, H-shaped Inner L a y e r : a net synenchyma (Figs. 8 to 9); cells 20 (10-45) pm long by 3 (1-5) pm wide; contact anastomoses common; junctions rare, variable; oleiferous cells common in some samples but absent in others, 4 (2-6) pm wide M Y C E L I A L S T R A N D S IN P L A N V I E W : differentiated (Figs. 10 tol l ) ; outer hyphae thick walled, 40 (10-80) pm long by 2.5 (1-3.5) pm wide; inner hyphae are laticifers, 4 (3-5.5) pm wide, length not determined; no junctions or anastomoses seen in either layer E M A N A T I N G H Y P H A E : none seen C Y S T I D I A : none seen OTHER FEATURES: S C L E R O T I A A N D M I C R O S C L E R O T I A : none seen C H L A M Y D O S P O R E S : none seen A U T O F L O U R E S C E N C E O F W H O L E T I P S : not tested C H E M I C A L R E A C T I O N S : laticifers dark blue to black in sulphovanillin; no reaction to 15% KOH or Melzer's; hyphae turn only slightly blue in toluidine blue D N A : ITS-region DNA sequences run for several samples A D D I T I O N A L C H A R A C T E R S : none observed Lactarius pubescens o n p a p e r b i r c h ( p g . 3) ADDITIONAL INFORMATION: C O L L E C T I O N A N D I D E N T I F I C A T I O N : collected by B.D. Twieg; identified by B.D. Twieg, initially by comparison with description in Ingleby et al. (1990), but the current description differs in that oleiferous cells were found in the inner mantle, and photos are more detailed. Identification was confirmed by DNA sequence comparison to online databases (see Appendix B). E C O L O G Y : found many times in 5-yr-old stands R E F E R E N C E S : Ingleby K, Mason PA, Last FT, Fleming LV. 1990. Identification of ectomycorrhizae. ITE Research Pulication No. 5. London: Her Majesty's Stationery Office. 112 Latarius pubescens on paper birch (pg. 4) Fig. 3 Older purple tips and mycelial strand running Fig. 4 Restricted point attachment of mycelial along main root axis strand to base of system (black and white photo) 113 Lactarius pubescens on paper birch (pg. 5) Fig. 9 Inner mantle showing oleiferous cells Fig. 10 Outer layer of mycelial strand, showing slightly thickened walls Fig. 11 Mycelial strand showing laticifers of inner layer and oleiferous cell in sulphovanillin 114 Lactarius torminosus o n p a p e r b i r c h (pg . 1) DISTINGUISHING FEATURES: large systems with a network of laticifers visible on the mantle surface; systems white with pink-purple patches when young and orange-pink when older; outer mantle a net synenchyma with large intracellular oil bodies; mycelial strands common, with an inner core of laticifers MORPHOLOGY (Dissection Microscope): ECTOMYCORRHIZAL SYSTEM: Shape and dimensions: (Figs. 1 to 3) monopodial pinnate, 7 (2.5-12) mm long by 5 (1.5-7) mm wide; tips 2.5 (1-6) mm long by 350 (250-500) pm wide Colour and texture: (Figs. 1 to 3) White to pink-purple to pink to orange, smooth, shiny, host not visible through mantle EMANATING ELEMENTS: Mycelial Strands: (Figs. 1 and 3) common, white, round in cross section, rarely branched, often running along non-ectomycorrhizal portions of birch roots to which ectomycorrhizal systems are attached; attachments are restricted points at bases of systems (Fig. 3) Hyphae: none ANATOMY (Compound Microscope): MANTLE IN PLAN VIEW: thick mantle, Hartig net present Outer Layer: a net synenchyma (Fig. 4) with abundant intracellular oil bodies; cells 15 (10-35) urn long by 4 (2.5-7) pm wide, smooth, hyaline, swellings to 10pm around septa; laticifers common (Fig. 5), 6.5 (4.5-8) pm wide; junctions rare, variable; anastomoses not seen Inner Layer: a net synenchyma (Fig. 6); cells 30 (10-60) pm long by 3.5 (1.5-6.5) pm wide; contact anastomoses common; junctions common, 90-120°; septa common, with rare clamp connections 115 Lactarius torminosus o n p a p e r b i r c h (pg . 2) M Y C E L I A L S T R A N D S IN P L A N V I E W : differentiated (Figs. 7 to 8); outer hyphae thick walled, 70 (15-150) pm long by 3.5 (2-4) pm wide; inner hyphae are laticifers, 6.5 (5-8) pm wide, at least 20 pm long; no junctions or anastomoses seen in either layer E M A N A T I N G H Y P H A E : none seen C Y S T I D I A : none seen OTHER FEATURES: S C L E R O T I A A N D M I C R O S C L E R O T I A : none seen C H L A M Y D O S P O R E S : none seen A U T O F L O U R E S C E N C E O F W H O L E T I P S : not tested C H E M I C A L R E A C T I O N S : laticifers dark blue to black in sulphovanillin; no reaction to 15% KOH or Melzer's; mantle hyphae turn slightly blue in toluidine 1 blue D N A : ITS-region DNA sequences run for several samples A D D I T I O N A L C H A R A C T E R S : none observed ADDITIONAL INFORMATION: C O L L E C T I O N A N D I D E N T I F I C A T I O N : collected by B.D. Twieg; identified by B.D. Twieg. Identification was made by DNA sequence comparison to online databases (see Appendix B). E C O L O G Y : found many times in 26- to 100-yr-old stands 116 Lactarius torminosus on paper birch (pg. 3) Fig. 1 System showing monopodial pinnate branching and orange to pink color Fig. 2 Closeup of young tips showing network of laticifers on the mantle surface E l • . / ; J t ... - . " ML, . ' Fig. 3 Closeup showing restricted point attach- Fig. 4 Outer mantle showing large intracellular ments of mycelial strand at bases of tip sytems oil bodies Fig. 5 Laticifers on mantle surface P. ' \ /• Fig. 6 Inner mantle V * "i I \ A 117 Lactarius torminosus on paper birch (pg. 4) Fig. 7 Surface of mycelial strand showing slightly Fig. 8 Inner core of mycelial strand, composed of thickened cell walls laticifers 118 P h a l l a l e s 1 (Hysterangium-\ike) o n D o u g l a s - f i r a n d p a p e r b i r c h (pg . 1) DISTINGUISHING FEATURES: white systems with abundant white mycelial strands that often have orange to red colour in isolated areas; large, circular ornaments (composed of radiating elements) abundant on mycelial strands and associated hyphae; inflated hyphae common in outer mantle MORPHOLOGY (Dissection Microscope): ECTOMYCORRHIZAL SYSTEM: Shape and dimensions: (Figs. 1 to 5) monopodial pinnate, Douglas-fir systems 7 (4-10) mm long by 3 (1.5-5) mm wide, tips 1 (0.4-5) mm long by 500 (300-700) p m wide; paper birch systems 5 (2-7) mm long by 2.5 (1-4.5) mm wide, tips 0.6 (0.3-1.5) mm long by 350 (250-500) p m wide Colour and texture: (Figs. 1 to 5) white felty mantle with patches of host tissue sometimes visible; refractive patches sometimes present EMANATING ELEMENTS: Mycelial Strands: (Figs. 1 to 5) common, white, flattened in cross section, frequently branched; flat angle attachment Hyphae: patches of cottony hyphae commonly emanate from strands, and sometimes from mantle ANATOMY (Compound Microscope): MANTLE IN PLAN VIEW: variable in thickness, Hartig net present Outer Layer: a felt prosenchyma (Fig. 8); cells at least 50pm long by 2.5 (1.5-3.5) pm wide, smooth, hyaline, swellings to 6 pm around septa; septa common, undamped; contact anastomoses common; junctions common, 90-120° Inner Layer: a net synenchyma(Fig. 9), not regularly distributed; cells 40 (50-80) p m long by 2.5 (1.5-3.5) pm wide; contact anastomoses common; junctions common, variable, septa common, undamped MYCELIAL STRANDS IN PLAN VIEW: (Fig. 6) undifferentiated; hyphae 2.5 (2-3.5) urn wide, length undetermined, surface covered in globular ornaments that are 3 (1.5-5) p m wide, protrude up to 2.5 pm, and are composed of radiating elements; septa rare, clamped; 119 Phallales 1 (Hysterangium-Wke) on Douglas-fir and paper birch (pg. 2) E M A N A T I N G H Y P H A E : (Fig. 7) same as hyphae of mycelial strands C Y S T I D I A : none seen OTHER FEATURES: S C L E R O T I A A N D M I C R O S C L E R O T I A : none seen C H L A M Y D O S P O R E S : none seen A U T O F L O U R E S C E N C E O F W H O L E T I P S : not tested C H E M I C A L R E A C T I O N S : no reaction to KOH or Melzer's; other chemicals not tested D N A : ITS region DNA sequences run for several samples A D D I T I O N A L C H A R A C T E R S : none observed ADDITIONAL INFORMATION: C O L L E C T I O N A N D I D E N T I F I C A T I O N : collected by B.D. Twieg; identified by B.D. Twieg. Morphotype matched well to Hysterangium crassirachis (Agerer, 1987), but sequence aligned to various Ramaria and Gautieria species over only short (~150 bp) segments in web searches. Identification was made by DNA sequence comparison to online databases (see Appendix B). E C O L O G Y : found many times in 26- to 65-yr-old stands R E F E R E N C E S : Agerer R. 1987. Colour Atlas of Ectomycorrhizae . S c h w a b i s c h GmCind, Ge rmany : E inhorn. 120 Phallales 1 (Hysterangium-like) on Douglas-fir and paper birch (pg. 3 ) F i g . 1 Doug las- f i r systems s h o w i n g patchy mant le F i g . 2 Doug las- f i r system s h o w i n g m o n o p o d i a l and redd ish hue i n some m y c e l i a l strands pinnate b ranch ing F i g . 3 C l o seup o f Doug las- f i r system s h o w i n g fe l ty F i g . 4 C l o s e u p o f Doug las- f i r system s h o w i n g f lat mant le and redd ish co lo r on m y c e l i a l strands angle m y c e l i a l strand attachment F i g . 5 Paper b i rch system F i g . 6 M y c e l i a l strand surface s h o w i n g heavy crysta l encrustat ion ( f r om paper b i r ch t ip) 121 P l i a I Ui les 1 (Hysterangium-tike) on Douglas-fir and paper birch (pg. 4) Fig . 7 Hyphae emanating from mycelial strand F ig . 8 Outer mantle showing inflated hyphae showing clamp connection and ornamentation F i g 9 Inner mantle showing contact anastomoses 122 Russula aeruginea on Douglas-fir and paper birch (pg. 1) DISTINGUISHING FEATURES: tips have a short-spiny surface over at least part of tips, caused by long (average 55 um), bristle-like cystidia with inflated bases; other parts of surface have a warty or frosted appearance; outer mantle is an irregular non-interlocking synenchyma composed of ovoid cells, MORPHOLOGY (Dissection Microscope): ECTOMYCORRHIZAL SYSTEM: Shape and dimensions: (Figs. 1 to 3) monopodial pinnate, Douglas-fir systems 6 (3-8) mm long by 2.5 (1.5-5) mm wide, tips 1.5 (0.25-2.5) mm long by 400 (200-600) pm wide; paper birch systems 4 (2-7) mm long by 2 (1-4.5) mm wide, tips 1 (0.25-2) mm long by 300 (200-500) pm wide on paper birch Colour and texture: (Figs. 1 to 4) light brown to pink to purple with white reflective patches; short spiny in parts and finely warty or frosted-looking in others EMANATING ELEMENTS: Mycelial Strands: none seen Hyphae: none seen ANATOMY (Compound Microscope): MANTLE IN PLAN VIEW: variable in thickness, Hartig net present Outer Layer: (Fig. 5 and 6) a non-interlocking irregular synenchyma; cells smooth, with granular contents, ovoid, 8 (5-13) pm long by 5 (3-7) pm wide; no septa within ovoid cells; septa at bases of ovoid cells undamped; clumps of extra layers of these cells likely give tips their warty or frosted appearance Inner Layer: a net prosenchyma, cells smooth, hyaline, 30 (10-50) pm long by 3 (2-4) pm wide; anastomoses rare, H-shaped; junctions common, 90-120°; septa common, undamped MYCELIAL STRANDS IN PLAN VIEW: none seen EMANATING HYPHAE: none seen 123 Russula aeruginea o n D o u g l a s - f i r a n d p a p e r b i r c h (pg . 2) C Y S T I D I A : abundant; tapering to a point but not thick-walled; hyaline; no contents; 55 (30-70) urn long by 2.5 (1-4) urn wide (5-6 pm wide at base) OTHER FEATURES: S C L E R O T I A A N D M I C R O S C L E R O T I A : none seen C H L A M Y D O S P O R E S : none seen A U T O F L O U R E S C E N C E O F W H O L E T I P S : not tested C H E M I C A L R E A C T I O N S : no reaction to KOH, Melzer's, or sulphovanillin; cystidia strongly purple and outer mantle faintly blue in toluidine blue D N A : ITS region DNA sequences run for several samples A D D I T I O N A L C H A R A C T E R S : none observed ADDITIONAL INFORMATION: C O L L E C T I O N A N D I D E N T I F I C A T I O N : collected by B.D. Twieg; identified by B.D. Twieg. Morphotype matched to Kranabetter & Friesen's (2004) description of R. aeruginea on pine. Identification was made by DNA sequence comparison to online databases (see Appendix B). E C O L O G Y : found several times in 26- to 100-yr-old stands R E F E R E N C E S : Kranabetter M, Friesen J. 2004. Morphotype Descriptions of Common Ectomycorrhizal Fungi. May 2006. 124 Russula aeruginea on Douglas-fir and paper birch (pg. 3) Fig. 1 Douglas-fir system showing monopodial Fig. 2 Closeup of Douglas-fir system showing pinnate branching; mycelial strand does not belong frosted and short-spiny textures of surface Fig. 3 Paper birch system showing clinging Fig. 4 Paper birch root tip showing short spiny mineral soil and reflective patches surface texture Fig. 5 Cystidia and egg-shaped cells of mantle Fig. 6 Cystidia with mantle stained in toluidine surface; from Douglas-fir root tip blue; from paper birch root tip 125 Russula brevipes on Douglas-fir (pg. 1) DISTINGUISHING FEATURES: large systems with a velvety appearance due to abundant and regularly distributed, long cystidia MORPHOLOGY (Dissection Microscope): ECTOMYCORRHIZAL SYSTEM: Shape and dimensions: (Figs. 1 to 3) monopodial pinnate, 9 (5-20) mm long by 7 (4-10) mm wide; tips 4 (1-7) mm long by 600 (400-750) pm wide Colour and texture: (Figs. 2 and 3) light pink to brown to copper with purple patches; texture velvety EMANATING ELEMENTS: Mycelial Strands: none seen Hyphae: none seen ANATOMY (Compound Microscope): MANTLE IN PLAN VIEW: medium-thick, Hartig net present Outer Layer: (Fig. 5) a net prosenchyma Inner Layer: a net prosenchyma, cells smooth, hyaline, 15 (10-30) pm long by 2.5 (2-3) pm wide; anastomoses common, contact-type to H-shaped; junctions common, 120°; septa common, undamped; hyphae often swollen to up to 8 pm around septa MYCELIAL STRANDS IN PLAN VIEW: none seen EMANATING HYPHAE: none seen CYSTIDIA: bottle-shaped, 40 (20-55) pm long by 3 (2-4) urn wide (6-7 pm wide at base), with refractive oily contents; one or two apical buds rarely present 126 Russula brevipes o n D o u g l a s - f i r (pg. 2) OTHER FEATURES: S C L E R O T I A A N D M I C R O S C L E R O T I A : none seen C H L A M Y D O S P O R E S : none seen A U T O F L O U R E S C E N C E O F W H O L E T I P S : not tested C H E M I C A L R E A C T I O N S : no reaction to KOH, Melzer's, or sulphovanillin; cystidia and mantle pink to light purple in toluidine blue D N A : ITS region DNA sequences run for several samples A D D I T I O N A L C H A R A C T E R S : none observed ADDITIONAL INFORMATION: C O L L E C T I O N A N D I D E N T I F I C A T I O N : collected by B.D. Twieg; identified by B.D. Twieg. Identification was made by DNA sequence comparison to online databases (see Appendix B). E C O L O G Y : found several times in 26- to 100-yr-old stands 127 Russula brevipes on Douglas-fir (pg- 3) Fig. 1 System showing beaded tip shape and Fig. 2 Closeup of tips showing velvety surface monopodial pinnate branching. texture and inflated apices. Fig. 5 Outer mantle Fig. 6 Inner mantle 128 Tomentella 10 on paper birch and Douglas-fir (pg. 1) DISTINGUISHING FEATURES: irregularly branched systems with white felty patches and brown smooth patches; thin brown strands usually present; outer mantle a felt prosenchyma ornamented with irregularly-shaped verrucae; short, pointed cystidia present, also covered in verrucae MORPHOLOGY (Dissection Microscope): ECTOMYCORRHIZAL SYSTEM: Shape and dimensions: (Figs. 1 to 3) irregularly branched, paper birch systems 3 (2-6) mm long by 1.5 (1-4) mm wide, tips 1.5 (1-4) mm long by 300 (250-500) urn wide; Douglas-fir systems 6 (4-10) mm long by 5 (1-7) mm wide, tips 5 (3-8) mm long by 500 (400-700) pm wide Colour and texture: (Figs. 2 to 5) white and brown; white patches felty, matte to reflective; brown patches smooth; matte to shiny, with localized dark-blue to black patches EMANATING ELEMENTS: Mycelial Strands: common, brown, round in cross section, commonly branched, wiry-looking, flat-angle attachment to tips Hyphae: none seen ANATOMY (Compound Microscope): MANTLE IN PLAN VIEW: medium-thick, Hartig net present Outer Layer: (Fig. 6) a felt prosenchyma; cells at least 8 pm long (max. length not determined) by 2.2 (1.5-3.5) pm wide, sometimes inflated at middle, heavily verrucose; anastomoses common, H-shaped; junctions common, sometimes inflated, 90-120°; septa common, undamped Inner Layer: (Fig. 7) a net synenchyma, cells smooth, mostly hyaline, 50 (15-70) pm long by 3 (1.5-3.5) pm wide; patches of dark-blue to black granular contents present; anastomoses common, contact to H-shaped; junctions common, inflated, angle variable; septa common, undamped 129 Tomentella 10 on paper birch and Douglas-fir (pg. 2) MYCELIAL STRANDS IN PLAN VIEW: (Fig. 8) differentiated-random hyphae; 35 (20-80) um wide; narrow hyphae smooth, brown, 2.5 (1.5-3) pm wide, septa rare, undamped; wide hyphae heavily verrucose, hyaline, 6 (4-7) pm wide; septa rare, undamped EMANATING HYPHAE: none seen CYSTIDIA: (Fig. 6) sparsely distributed, pointed, verrucose, 10 (7-12) pm long by 3(2.5-4.5) pm long OTHER FEATURES: SCLEROTIA AND MICROSCLEROTIA: none seen CHLAMYDOSPORES: none seen AUTOFLOURESCENCE OF WHOLE TIPS: not tested CHEMICAL REACTIONS: no reaction to Melzer's, or sulphovanillin; dark granular contents of inner mantle slightly greenish in KOH DNA: ITS region DNA sequences run for several samples ADDITIONAL CHARACTERS: none observed ADDITIONAL INFORMATION: COLLECTION AND IDENTIFICATION: collected by B.D. Twieg; identified by B.D. Twieg. Identification was made to genus by comparison of morphological features to manuals (Goodman et al., 1996; Agerer, 1987), and by DNA sequence comparison to online databases (see Appendix B). ECOLOGY: found infrequently in 5-, 26, and 100-yr-old stands; several occurrences of this morphotype on paper birch were found, but it was found only once on Douglas-fir 130 Tomentella 10 on paper birch and Douglas-fir (pg. 3) R E F E R E N C E S : Agerer R. 1987. Colour Atlas of Ectomycorrhizae . Schwabisch Gmund, Germany: Einhorn. Goodman DM, Durall DM, Trofymow JA. 1996. A manual of concise descriptions of North American ectomycorrhizae. Sydney, B.C.: Mycologue. 131 Tomentella 10 on paper birch and Douglas-fir (pg. 4) Fig. 1 Paper birch system showing irregular branching and clinging forest floor material Fig. 2 Paper birch system showing white and brown tip colors Fig. 3 Paper birch system showing felty mantle patches Fig. 4 Paper birch tips showing flat angle mycelial strand attachment Fig. 5 Douglas-fir tip Fig. 6 Outer mantle showing heavily verrucose hyphae and pointed cystidium (center) 132 Tomentella 10 on paper birch (pg. 5) Fig. 7 Inner mantle Fig. 8 Mycelial strand showing thin pigmented hyphae and wide verrucose hyphae 1 3 3 Tuber 1 o n D o u g l a s - f i r (pg . 1) DISTINGUISHING FEATURES: gold to orange-brown to brown systems that are often short-spiny due to characteristic long, thick-walled, awl-shaped cystidia; tips sometimes appear finely grainy due to additional bundles of short cystidia that are sometimes branched MORPHOLOGY (Dissection Microscope): ECTOMYCORRHIZAL SYSTEM: Shape and dimensions: (Figs. 1 to 2) simple to monopodial pinnate, but sometimes corraloid, 6 (3-10) mm long by 6 (2-8) mm wide; tips 3 (0.2-5) mm long by 450 (400-550) pm wide Colour and texture: (Figs. 1 to 3) gold to orange-brown to brown; smooth to short-spiny to finely grainy; matte to shiny; host tissue not visible EMANATING ELEMENTS: Mycelial Strands: none seen Hyphae: none seen ANATOMY (Compound Microscope): MANTLE IN PLAN VIEW: medium-thick, Hartig net present Outer Layer: (Fig. 5) a net prosenchyma usually only one cell layer thick; cells hyaline, 10 (8-20) pm long by 2 (2-8) pm wide, highly branched with pentagonal or hexagonal spaces often seen between cells; junctions common, sometimes inflated, 120°; septa common, undamped, often with an obvious pore Inner Layer: (Fig. 6) an irregular interlocking synenchyma; cells hyaline, 15 (10-20) pm long by 6 (4-12) pm wide, usually smooth but sometimes with a rugose surface; septa, anastomoses, and junctions not seen MYCELIAL STRANDS IN PLAN VIEW: none seen EMANATING HYPHAE: none seen 134 Tuber 1 o n D o u g l a s - f i r (pg . 2) CYSTIDIA: (Fig. 4) two types present: one type is awl-shaped, 55 (40-75) pm long by 2.5 (2-2.75) pm wide, walls thick (~1pm); other type is short, often branched and occurring in bundles, 9 (5-12) pm long by 4 (3-5) pm wide, slightly tapered from base upward OTHER FEATURES: SCLEROTIA AND MICROSCLEROTIA: none seen CHLAMYDOSPORES: none seen AUTOFLOURESCENCE OF WHOLE TIPS: not tested CHEMICAL REACTIONS: no reaction to KOH, Melzer's, or sulphovanillin DNA: ITS region DNA sequences run for several samples ADDITIONAL CHARACTERS: none observed ADDITIONAL INFORMATION: COLLECTION AND IDENTIFICATION: collected by B.D. Twieg; identified by B.D. Twieg. Identification was made to genus by comparison of morphological features to manuals (Agerer, 1987), and by DNA sequence comparison to online databases (see Appendix B). ECOLOGY: found infrequently in all stand types; several occurrences of this species were found on both hosts studied REFERENCES: Agerer R. 1987. Colour Atlas of Ectomycorrhizae . Schwabisch Gmund, Germany: Einhorn. 135 Tuber 1 on Douglas-fir (pg. 3) Fig. 1 System showing monopodial pinnate Fig. 2 System showing subcorraloid branching and branching clinging mineral soil Fig. 3 Closeup showing spiny surface Fig. 4 Cystidia on mantle surface 136 Appendix B T h i s t a b l e is a l i s t o f E C M t a x a a n d w e b - b a s e d a l i g n m e n t / m a t c h i n f o r m a t i o n . * s a r e p r e s e n t i n U N I T E d a t a b a s e a c c e s s i o n n u m b e r s ; t h i s m e a n s to i n s e r t t h r e e z e r o s t o o b t a i n t he c o m p l e t e n u m b e r . O t h e r a c c e s s i o n n u m b e r s a r e f o r N C B I - l i n k e d d a t a b a s e s . M e a n r e l a t i v e a b u n d a n c e s a r e m e a n s o f f o u r r e p l i c a t e s i tes f o r e a c h s t a n d t y p e (age a n d c c = c l e a r c u t , b = b u r n e d ) , a n d a r e r e s p e c t i v e o f t he hos t l i s t e d i n t he H o s t c o l u m n . F i r = D o u g l a s - f i r ; B i r c h = p a p e r b i r c h . Functional "Species" Name Closest NCBI or UNITE BLAST Match Accession Number Total Base Pairs Aligned1 % Sim.2 Host Mean Relative Abundance (%) 5 cc 26 b 26 cc 65 b 100 b Agaricales 1 Entoloma nitidutn AY228340 491 88% Both 0 0 0 0 3.6 Amphinema byssoides Amphinema byssoides AY838271 623 98% Both 1.5 3.4 0.4 2.0 2.5 Atheliaceae 1 Uncultured ECM AF476986 593 99% Birch 0 0 1.0 0 0 Boletus 1 Boletus calopus AJ889928 684 97% Birch 0 1.0 0 0 0 Cenococcum geophilum N/A N/A N/A N/A Both 7.7 11 15 12 7.3 Cortinarius 1 Cortinarius cf. sertipes AJ889969 680 96% Both 0 0 0 0.3 0.1 Cortinarius 2 Cortinarius traganus AF335546 811 96% Both 0 0.3 1.5 0 0.6 Cortinarius 3 Cortinarius traganus AF335546 775 96% Both 0 1.2 0.1 0.2 0.1 Cortinarius 4 Cortinarius traganus AF335546 742 95% Both 0 0 1.6 0.6 0 Cortinarius 5 Cortinarius humicola AY083191 642 91% Both 0 0 0.5 0 0.1 Cortinarius 7 Cortinarius parahumilis AF539731 586 92% Fir 0 0 3.7 0 0 Cortinarius 9 Cortinarius cephelixus AY 174784 748 92% Birch 0 0 0.9 0 0 Functional "Species" Name Closest NCBI or UNITE BLAST Match Accession Number Total Base Pairs Aligned1 % Sim.2 Host Mean Relative Abundance (%) 5 cc 26 b 26 cc 65 b 100 b Cortinarius 12 Cortinarius rapaceus AF289146 564 94% Fir 0 0 1.4 0 0 Cortinarius 13 Cortinarius traganus AF335546 775 95% Fir 0 1.7 0 0 0 Cortinarius 16 Cortinarius heterosporus AF268894 604 94% Fir 0 0 0 1.0 0 Cortinarius armillatus Cortinarius armillatus AF037223 533 98% Birch 0 0 0 3.3 0 Cortinarius balaustinus Cortinarius balaustinus AF389153 586 98% Birch 0 0 0 0 1.5 Cortinarius flexipes Cortinarius flexipes AJ889971 666 98% Birch 0 0 0 0 0.1 Cortinarius gentiles Cortinarius gentiles AF325589 488 98% Fir 0 0 0 1.9 0 Cortinarius hemitrichus Cortinarius hemitrichus Durall3 -800 99% Birch 0 0 0 1.4 0 Cortinarius melliolens Cortinarius melliolens AF389144 510 99% Birch 0 1.5 0 0 0 Cortinarius porphyropus Cortinarius porphyropus AY714854 755 98% Birch 0.3 0 0 0 0 Cortinarius cf. sertipes Cortinarius cf. sertipes AJ889969 738 99% Both 0 0 0 0.1 0.2 Cortinarius umbilicatus Cortinarius umbilicatus U56032 464 100% Both 0 0 0 1.0 0 Cortinarius spp. Total N/A N/A N/A N/A Both 0.2 5.9 6.8 5.8 1.8 Hebeloma 2 Hebeloma incarnatulum AF430291 876 96% Birch 0 0.7 0 0 0 Hebeloma incarnatulum Hebeloma incarnatulum AF430291 828 99% Both 0 5.5 0 0.8 0.6 Hebeloma velutipes Hebeloma velutipes AF430254 656 99% Both 0 4.4 4.2 2.3 1.3 Hebeloma spp. Total N/A N/A N/A N/A Both 0 10 4.2 3.2 1.9 Inocybe 1 Inocybe godeyi AJ889954 368 95% Both 0.4 0 2.3 0.8 2.8 Inocybe 2 Inocybe cf. glabripes AJ889952 295 92% Fir 0 0 0.4 0.9 0 Inocybe 3 Inocybe abietis AY038311 199 98% Both 0 0 0 2.9 0 Functional "Species" Name Closest NCBI or UNITE BLAST Match Accession Number Total Base Pairs Aligned1 % Sim.2 Host Mean Relative Abundance (%) 5 cc 26 b 26 cc 65 b 100 b Inocybe 4 Inocybe cf. glabripes AJ889952 164 100% Birch 0 0 0.9 0 0 Inocybe 5 Inocybe flocculosa AY228354 201 96% Both 0 0 0.5 0 0 Inocybe 6 Inocybe sierraensis AY239025 283 97% Birch 0 0 0 0 0.2 Inocybe 8 cf. Inocybe sp. AY751588 552 88% Birch 0.4 0 0 0 0 Inocybe 9 Inocybe pudica AY228341 475 97% Fir 0 0 0 1.2 0 Inocybe 10 Inocybe abietis AY038311 252 98% Both 0.7 0 0 0 0 Inocybe 11 Inocybe godeyi AF335452 218 97% Fir 0 0 0 0.3 0 Inocybe 12 Inocybe maculata DQ241778 165 100% Birch 0 0 0 0 0.2 Inocybe 13 Inocybe godeyi AJ889954 381 93% Fir 0 0 0 0 2.4 Inocybe nitidiuscula Inocybe nitidiuscula AJ534934 735 100% Birch 0 0 0 •0.3 0 Inocybe spp. total N/A N/A N/A N/A Both 1.2 0 •3.4 5.1 4.1 Laccaria 1 Laccaria amethystia AF539737 766 97% Birch 3.4 0 0 0 0 Laccaria bicolor Laccaria bicolor AY254878 573 98% Both 2.2 0 0 0 0 Laccaria spp. total N/A N/A N/A N/A Both 4.1 0 0 0 0 Lactarius 1 Lactarius uvidus AJ534936 775 96% Birch 1.6 0 0 1.4 0.2 Lactarius 2 Morphotype only N/A N/A N/A Both 0 0 0 0 0.3 Lactarius 3 Morphotype only N/A N/A N/A Birch 2.6 0 0 0 0 Lactarius pallescens Lactarius pallescens Durall3 -650 99% Birch 0 0 0.4 0 0 Lactarius pubescens Lactarius pubescens AY336958 688 99% Birch 27 0 0.4 0 0 Lactarius rubrilacteus Lactarius rubrilacteus Durall3 -700 99% Fir 0 2.1 2.2 8.9 0.8 Functional "Species" Name Closest NCBI or UNITE BLAST Match Accession Number Total Base Pairs Aligned1 % Sim.2 Host Mean Relative Abundance (%) 5 cc 26 b 26 cc 65 b 100 b Lactarius scrobiculatus Lactarius scrobiculatus AF140263 690 98% Both 0.5 8.1 2.2 4.8 2.8 Lactarius torminosus Lactarius torminosus AY336959 686 99% Birch 0 5.6 12 9 11 Lactarius spp. total N/A N/A N/A N/A Both 19 12 9.6 14 8.7 Leccinum scabrum Leccinum scabrum AF454583 628 97% Birch 16 4.9 12 2.9 2.6 MR A Cadophora finlandia AY394885 614 99% Both 1.9 4.5 1.9 1.2 0.1 Phallales 1 Ramaria flavobrunnescens AY 102864 165 100% Both 0 2.3 2.8 0.1 0 Piloderma spp. Piloderma fallax AYO10281 580 99% Both 0 3.5 5.4 9.5 12 Piloderma sp. B22 AJ534903 662 100% Rhizopogon rudus Rhizopogon rudus AF377107 611 98% Fir 6.0 2.6 4.3 0 0.9 Rhizopogon vinicolor-type Rhizopogon vinicolor AF263933 697 99% Fir 82 46 27 23 30 Rhizopogon vesiculosus AF262931 700 99% Rhizopogon spp. total N/A N/A N/A N/A Fir 88 49 31 23 31 Russula 1 Russula delica AY061671 613 95% Both 0 0 0 4.8 0 Russula 2 Russula gracillima AY061678 631 97% Birch 0 6.5 0 0.5 0.9 Russula 3 Russula gracillima AY061678 635 96% Birch 0 0.9 1.8 0 C Russula 5 Russula cf. xerampelina AY228344 244 96% Birch 0 0 1.4 0 ' 0 Russula 6 Russula ilicis AY061682 213 97% Fir 0 0 2.6 0 0 Russula 1 Russula ilicis AY061682 374 94% Fir 0 0 0 0.6 0 Russula 8 Russula adusta AY061652 645 95% Birch 0 0 0 0.6 0 Russula aeruginea Russula aeruginea AF418612 323 99% Both 0 0 1.4 1.5 4.5 Functional "Species" Name Closest NCBI or UNITE BLAST Match Accession Number Total Base Pairs Aligned1 % Sim.2 Host Mean Relative Abundance (%) 5 cc 26 b 26 cc 65 b 100 b Russula brevipes Russula brevipes AF349714 607 99% Both 0 0 0 0 6.0 Russula fragilis Russula fragilis Durall3 -700 100% Fir 0 0 0 1.8 1.7 Russula nigricans Russula nigricans Durall3 -800 99% Both 0 1.7 2.4 4.9 7.1 Russula postiana Russula postiana AF230898 600 98% Both 0 1.0 0.6 4.6 0.2 Russula roseipes Russula roseipes AY061716 655 98% Fir 0 0 0 0 3.6 Russula velenovskyi Russula velenovskyi AY061721 654 98% Birch 0.9 0 0 4.1 0 Russula versicolor Russula versicolor AY061722 634 98% Both 1.0 1.9 1.8 0.9 0 Russulaceae 1 Gymnomyces monticola AY239313 645 93% Both 0 0 0 2.3 6.5 Russula spp. total N/A N/A N/A N/A Both 1.5 8.8 7.9 18 27 Sebacinaceae 1 Sebacina endomycorrhiza AF440648 817 96% Both 0 0 0 1.0 1.1 Sebacinaceae 2 Sebacina endomycorrhiza AF440650 697 99% Birch 0 0 1.2 0 0 Sebacinaceae 3 Uncultured ECM AJ893264 471 97% Birch 0 0 0 0 0.4 Sebacinaceae 4 Sebacina endomycorrhiza AF440651 877 98% Birch 0 0 0.3 0 0 Sebacinaceae spp. total N/A N/A N/A N/A Both 0 0 0.7 1.0 1.3 Suillus lakei Suillus lakei L54086 627 98% Fir 0 14 26 0.7 1.8 Thelephora terrestris Thelephora terrestris U83486 685 99% Both 5.7 0 0 0 0 Thelephoraceae 1 Pseudotomentella tristis UDB*279 419 93% Birch 0 0 1.4 0 0 Thelephoraceae 3 Thelephora terrestris UDB*215 483 91% Fir 0 0 0 0.2 0 Thelephoraceae 4 Tomentella laterita UDB*954 584 89% Fir 0 0 0 0 0.7 Tomentella 1 Tomentella coerula UDB*266 587 93% Both 0 0.1 0 0.1 0.3 Functional "Species" Name Closest NCBI or UNITE BLAST Match Accession Number Total Base Pairs Aligned1 0/ /o Sim.2 Host Mean Relative Abundance (%) 5 cc 26 b 26 cc 65 b 100 b Tomentella 2 Tomentella bryophila UDB*035 607 95% Fir 0 0 0 1.5 0.1 Tomentella 3 Tomentella fuscocinerea UDB*240 584 94% Both 0 0 0 0 0.4 Tomentella 4 Tomentella viridula UDB*261 492 95% Birch 0 0 0.5 0 0.4 Tomentella 5 Tomentella atramentaria UDB*235 587 97% Birch 0 1.1 0 0 0 Tomentella 6 Tomentella atramentaria UDB*235 469 93% Both 0 0 0 0.1 1.8 Tomentella 7 Tomentella subclavigera UDB*259 468 96% Both 0 0.7 2.2 1.2 0.4 Tomentella 8 Tomentella subclavigera UDB*259 559 94% Birch 2.1 0 0 0.6 0.4 Tomentella 9 Tomentella lilacinogrisea UDB*272 556 94% Birch 2.0 0 0 0 0 Tomentella 10 Tomentella lilacinogrisea UDB*272 547 97% Both 0.9 0.6 0.8 0 0.5 Tomentella 11 Tomentella bryophila UDB*035 673 92% Both 0 0 0 0 2.9 Tomentella 12 Tomentella bryophila UDB*035 672 93% Birch 0 0 0 1.1 0 Tomentella 13 Tomentella subclavigera UDB*957 572 94% Birch 0 0 0 0.1 0 Tomentella 14 Tomentella badia UDB*961 578 97% Fir 0 0 0 0.8 0 Tomentella 15 Tomentella bryophila UDB*035 672 92% Birch 0 1.6 0 0 0 Tomentella ramosissima Tomentella ramosissima U83480 615 98% Both 0 0 0 0 0.3 Tomentella lapida UDB*250 582 99% Tomentella terrestris Tomentella terrestris AF272911 582 99% Both 2.9 0 0 0.8 0.7 Thelephoraceae spp. total N/A N/A N/A N/A Both 9.0 2.9 3.6 4.3 8.0 Tricholoma jlavovirens Tricholoma Jlavovirens AF349689 462 98% Birch 0 0 0 0.4 0 Tricholoma scalpaturatum Tricholoma scalpaturatum AF377199 413 99% Birch 0 1.3 0 0 0 Functional "Species" Name Closest NCBI or UNITE BLAST Match Accession Number Total Base Pairs Aligned1 % Sim.2 Host Mean Relative Abundance (%) 5 cc 26 b 26 cc 65b 100 b Truncocolumella citrina Truncocolumella citrina L54097 653 99% Fir 0 0 1.3 1.1 1.1 Tuber 1 Tuber borchii AF106890 476 92% Both 0.8 0,3 1.0 3.5 0.2 Wilcoxina rehmii Wilcoxina rehmii AF266708 536 99% Both 0.7 0.1 0.2 0 0.2 ' Includes only the longest aligned segment for alignments to a single taxon in which unaligned gaps were present. " Percent similarity of sample sequence(s) to database sequence, ignoring gaps and unknown bases; respective of footnote '. J Sequences matched to sporocarps collected and identified in Durall et al. (2006)(see Chapter 2). Appendix C This table lists correlations (Pearson's r) of stand age and species to NMS ordination axes. "Frequency" and "Abundance" denote the type of data input used to generate ordinations. "By Species" ordinations included species groups in which not all occurrences were resolved to a unique genotype (i.e. there were two genotypes each in Rhizopogon vinicolor-type and Piloderma spp). "By Genera" ordinations included species that were the sole representatives of their genera in this study. Only species with a correlation with an absolute value of 0.5 or higher to at least one ordination axis are listed (correlations of 0.5 or larger absolute value are in bold). Both hosts; Frequency; By Species Both hosts; Frequency; By Genera Both hosts; Abundance; By Species Douglas-fir; Frequency; By Species Douglas-fir; Frequency; By Genera Douglas-fir; Abundance; By Species Paper birch; Frequency; By Species Paper birch; Frequency; By Genera Paper birch; Abundance; By Species Axes 1 3 2 3 2 3 2 3 2 3 2 3 1 3 1 2 2 3 R2 0.23 0.46 0.67 0.18 0.22 0.45 0.22 . 0.47 0.27 0.39 0.25 0.19 0.33 0.38 0.29 0.30 0.42 0.31 P (Monte Carlo) 0.02 0.02 0.02 0.02 0.04 0.02 0.06 0.02 0.02 0.02 0.24 0.53 0.02 0.04 0.02 0.02 0.04 0.04 Correlations with axes Stand Age -0.31 0.88 0.84 .02 -0.16 -0.86 0.62 0.29 -0.75 0.27 -0.46 0.25 -0.69 -0.73 -0.67 0.74 -0.70 0.25 Cenococcum geophilum -0.39 -0.16 0.19 -0.14 0.40 -0.11 0.30 0.29 -0.07 0.09 0.20 -0.28 -0.21 -0.03 -0.36 -0.39 -0.54 -0.05 Cortinarius 2 -0.32 -0.13 N/A N/A 0.25 0.05 -0.01 0.17 N/A N/A -0.21 -0.26 0.08 -0.20 N/A N/A -0.09 0.51 Cortinarius 4 -0.30 -0.05 N/A N/A 0.44 0.03 -0.13 0.53 N/A N/A -0.46 -0.19 -0.10 -0.29 N/A N/A 0.11 0.30 Cortinarius 5 0.07 0.30 N/A N/A -0.04 -0.04 0.06 -0.05 N/A N/A -0.33 0.61 -0.02 -0.02 N/A N/A -0.27 -0.03 Cortinarius cf. sertipes -0.02 0.62 N/A N/A -0.34 -0.47 0.06 -0.05 N/A N/A -0.33 0.61 -0.36 -0.12 N/A N/A -0.29 0.03 Cortinarius spp. N/A N/A 0.46 -0.24 N/A N/A N/A N/A -0.78 0.38 N/A N/A N/A N/A -0.41 -0.15 N/A N/A Hebelonia incarnatulum 0.23 0.16 N/A N/A 0.06 0.01 0.06 -0.05 N/A N/A -0.33 0.61 -0.21 0.15 N/A N/A -0.21 -0.33 Hebeloma velutipes -0.69 -0.09 N/A N/A 0.07 -0.05 -0.04 0.45 N/A N/A -0.40 0.11 0.46 -0.39 N/A N/A 0.36 0.36 Both hosts; Frequency; By Species Both hosts; Frequency; By Genera Both hosts; Abundance; By Species Douglas-fir; Frequency; By Species Douglas-fir; Frequency; By Genera Douglas-fir; Abundance; By Species Paper birch; Frequency; By Species Paper birch; Frequency; By Genera Paper birch; Abundance; By Species Axes 1 3 2 3 2 3 2 3 2 3 2 3 1 3 1 2 2 3 R2 0.23 0.46 0.67 0.18 0.22 0.45 0.22 0.47 0.27 0.39 0.25 0.19 0.33 0.38 0.29 0.30 0.42 0.31 P (Monte Car lo) 0.02 0.02 0.02 0.02 0.04 0.02 0.06 0.02 0.02 0.02 0.24 0.53 0.02 0.04 0.02 0.02 0.04 0.04 Correlations with axes Stand A g e -0.31 0.88 0.84 .02 -0.16 -0.86 0.62 0.29 -0.75 0.27 -0.46 0.25 -0.69 -0.73 -0.67 0.74 -0.70 0.25 Inocybe 1 0.06 0.11 N/A N/A -0.05 -0.04 0.21 -0.23 N/A N/A 0.01 0.16 -0.38 -0.09 N/A N/A -0.52 -0.17 Laccaria 1 0.49 -0.27 N/A N/A -0.29 0.40 N/A N/A N/A N/A N/A N/A -0.12 0.50 N/A N/A 0.47 -0.45 Lactarius pubescens 0.71 -0.46 N/A N/A -0.40 .060 N/A N/A N/A N/A N/A N/A 0.11 0.74 N/A N/A 0.51 -0.65 Lactarius rubrilacteus -0.33 0.30 N/A N/A 0.16 -0.37 0.61 0.26 N/A N/A -0.03 -0.34 N/A N/A N/A N/A N/A N/A Lactarius scrobiculatus -0.32 -0.28 N/A N/A 0.66 -0.20 -0.27 0.55 N/A N/A -0.45 -0.33 0.17 -0.01 N/A N/A -0.18 0.52 Lactarius torminosus -0.54 0.03 N/A N/A 0.27 -0.45 N/A N/A N/A N/A N/A N/A 0.25 -0.35 N/A N/A -0.01 0.57 Lactarius spp. N/A N/A -0.33 0.29 N/A N/A N/A N/A 0.01 0.51 N/A N/A N/A N/A 0.57 -0.36 N/A N/A Leccinum scabrum -0.20 -0.40 0.31 -0.37 -0.23 0.46 N/A N/A N/A- N/A N/A N/A 0.69 0.20 0.60 -0.14 0.70 -0.34 M R A -0.23 -0.60 0.46 -0.50 0.35 0.28 -0.21 0.24 0.08 0.23 -0.11 -0.39 0.67 0.20 0.58 -0.47 0.14 -0.38 Phallales 1 -0.53 -0.31 -0.11 -0.69 0.57 0.14 -0.15 0.64 0.15 0.72 -0.41 -0.39 0.28 -0.21 0.14 -0.12 0.11 0.19 Piloderma spp. -0.57 0.82 0.81 0.06 -0.18 -0.78 0.83 0.47 -0.43 0.38 -0.27 -0.48 -0.63 -0.68 -0.62 0.77 -0.57 -0.23 Rhizopogon vinicolor-type 0.69 -0.44 N/A N/A -0.40 0.79 -0.50 -0.64 N/A N/A 0.75 0.19 N/A N/A N/A N/A N/A N/A Rhizopogon spp. N/A N/A -0.52 0.47 N/A N/A N/A N/A 0.55 -0.65 N/A N/A N/A N/A N/A N/A N/A N/A Russula brevipes 0.01 0.55 N/A N/A -0.44 -0.36 0.34 0.02 N/A N/A -0.33 0.60 -0.59 -0.19 N/A N/A -0.50 -0.22 Russula fragilis 0.05 0.60 N/A N/A -0.47 -0.28 0.28 -0.10 N/A N/A -0.32 0.53 N/A N/A N/A N/A N/A N/A Russula nigricans -0.14 0.39 N/A N/A 0.13 -0.26 -0.32 0.01 N/A N/A -0.18 0.01 -0.47 -0.51 N/A N/A -0.57 0.12 Russula roseipes 0.06 0.45 N/A N/A -0.42 -0.39 0.06 -0.05 N/A N/A -0.33 0.61 N/A N/A N/A N/A N/A N/A Russula versiclolor -0.25 -0.25 N/A N/A 0.34 0.16 -0.10 0.52 N/A N/A -0.44 -0.25 0.31 -0.06 N/A N/A 0.19 0.22 Both hosts; Frequency; By Species Both hosts; Frequency; By Genera Both hosts; Abundance; By Species Douglas-fir; Frequency; By Species Douglas-fir; Frequency; By Genera Douglas-fir; Abundance; By Species Paper birch; Frequency; By Species Paper birch; Frequency; By Genera Paper birch; Abundance; By Species Axes 1 3 2 3 2 3 2 3 2 3 2 3 1 3 1 2 2 3 R 2 0.23 0.46 0.67 0.18 0.22 0.45 0.22 0.47 0.27 0.39 0.25 0.19 0.33 0.38 0.29 0.30 0.42 0.31 P (Monte Carlo) 0.02 0.02 0.02 0.02 0.04 0.02 0.06 0.02 0.02 0.02 0.24 0.53 0.02 0.04 0.02 0.02 0.04 0.04 Correlations with axes Stand Age -0.31 0.88 0.84 .02 -0.16 -0.86 0.62 0.29 -0.75 0.27 -0.46 0.25 -0.69 -0.73 -0.67 0.74 -0.70 0.25 Russulaceae 1 -0.21 0.58 N/A N/A -0.57 -0.45 0.26 0.02 N / A N / A -0.42 0.60 -0.11 -0.51 N / A N / A -0.08 0.26 Russula spp. N / A N / A 0.85 -0.30 N / A N / A N / A N / A -0.90 0.27 N / A N / A N / A N / A -0.33 0.50 N / A N / A Sebacinaceae 1 0.01 0.61 N / A N/A -0.10 -0.55 0.42 0.04 N / A N / A 0.16 0.37 -0.71 -0.23 N/A N / A -0.38 -0.26 Suillus lakei -0.44 -0.43 -0.17 -0.59 0.65 0.18 -0.43 0.61 0.12 0.68 -0.50 -0.42 N / A N / A N / A N / A N/A N / A Thelephora terrestris 0.54 -0.43 -0.53 0.36 -0.23 0.55 N / A N / A N / A N / A N / A N / A 0.40 0.54 0.57 -0.22 0.46 -0.36 Tomentella 1 -0.32 0.24 N/A N/A -0.17 -0.22 0.41 0.08 N / A N / A 0.02 -0.19 0.11 -0.47 N / A N / A 0.14 0.39 Tomentella 2 -0.25 0.32 N / A N/A 0.14 -0.33 0.57 0.44 N / A N / A -0.08 -0.28 N / A N / A N / A N / A N / A N/A Tomentella 3 0.37 -0.01 N / A N/A -0.20 -0.12 -0.21 -0.25 N / A N / A 0.30 0:08 -0.50 -0.12 N / A N / A -0.45 -0.15 Thelephoraceae spp. N / A N/A 0.49 0.16 N / A N / A N/A N / A -0.44 -0.03 N / A N / A N / A N / A -0.23 0.62 N / A N / A Truncocolumella citrina -0.32 0.54 0.62 -0.30 -0.17 -0.17 0.34 0.16 -0.76 -0.26 -0.55 0.03 N / A N / A N / A N / A N / A N / A Tuber 1 0.32 -0.27 0.03 -0.20 0.31 -0.24 0.51 0.27 0.02 0.27 0.10 0.32 0.17 0.12 0.32 -0.27 0.22 -0.33 Appendix D This table gives a species list and web-based alignment/match information for E C M community of Douglas-fir seedlings observed in 5-yr-old stands. Base pairs aligned and percent similarity as per Appendix B. Functional Species Closest NCBI or UNITE BLAST Name Match Amphinema byssoides See Appendix A Boletus calopus Boletus calopus Cenococcum N / A geophilum Cortinarius 6 Cortinarius fusisporus Hebeloma 1 Hebeloma testaceum Inocybe 4 See Appendix A Lactarius rubrilacteus See Appendix A M R A See Appendix A Phallales 1 See Appendix A Piloderma fallax See Appendix A Rhizopogon rudus See Appendix A Rhizopogon vinicolor-type See Appendix A Russula nigricans See Appendix A Russulaceae 1 See Appendix A Suillus lakei See Appendix A Thelephora terrestris See Appendix A Thelephoraceae 1 See Appendix A Tomentella 5 See Appendix A Tomentella 10 See Appendix A Truncocolumella citrina See Appendix A Tuber 1 See Appendix A Wilcoxina rehmii See Appendix A Accession Number Base Pairs % ^ligne^^^Jsim^ Mean Relative Abundance (%) Burned Clearcut AJ889928 N / A AY254877 AY320395 654 N / A 266 272 99% N / A 97% 99% 3.9 2.3 0.75 0 0 1.7 0 0 0 0 37 35 0 0 1.1 3.6 2.0 2.3 0.20 4.9 0 2.2 0 0 1.6 1.2 0 3.1 0.16 0.71 0.71 10 67 5.6 2.1 0.2 3.0 0 0 0 1.1 2.0 1.4 147 Appendix E This table gives a species list and web-based alignment/match information for E C M community of paper birch observed in soil associated with seedlings (clearcut and burned) and soil samples from spring (burned only) 5-yr-old stands. Percent similarity as per Appendix B. Functional Species Name Closest NCB1 or UNITE BLAST Match Accession Number Base Pairs Aligned % Sim. Present (P) or Absent (A) Burned Clearcut Boletus I Cenococcum geophilum Cortinarius spilomius Inocybe 7 Lactarius pubescens Lactarius torminosus See Appendix A N / A Cortinarius spilomius Inocybe dulcimara See Appendix A See Appendix A Leccinum scabrum See Appendix A M R A See Appendix A Phallales 1 See Appendix A Russula velenovskyi See Appendix A Tomentella 10 See Appendix A Tomentella terrestris See Appendix A Wilcoxina rehmii See Appendix A Durall et al. -650 99% 2006 UDB0001196 627 96% A P A P P A P A P A P P P P A P P P P P A P P P 148 

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