Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Hume, John
Master of Science - MSc
University of British Columbia
Despite diminished health and richness of ectomycorrhizal (EcM) fungal communities compared with those in undisturbed environments, the drivers of diversity in urban EcM fungal communities are poorly understood. Studies of diversity in mycorrhizal fungal communities often overlook fundamental ecological theories used to explain diversity in plant and animal communities. In this study, I used Illumina next-generation sequencing to characterize EcM fungal communities associated with a common urban tree species, Carpinus betulus, in three British Columbian cities, Vancouver, Victoria, and Kelowna. Species diversity was compared across the three cities and related to ecological theory to investigate mechanisms that modulate diversity. Furthermore, soil chemical properties were analyzed at each site to identify specific soil characteristics associated with changes in community structure. I found that gamma diversity of the fungal communities was not positively correlated with environmental heterogeneity, as would be predicted by the habitat-heterogeneity hypothesis. Community composition, however, was significantly distinct in each of the three cities. A small number of species were found to be indicative of fungal communities in each city. Soil chemical properties were significantly associated with changes in community structure, with soil acidity and nitrogen content being the primary associated variables. No significant correlations were found, however, between individual soil properties and the most abundant taxa. While more research is required to determine the driving forces of EcM fungal diversity, this study serves as an example of applying ecological theory to understand diversity in urban EcM fungal communities.
ECTOMYCORRHIZAL FUNGAL COMMUNITIES ASSOCIATED WITH CARPINUS BETULUS TREES ACROSS URBAN AREAS OF BRITISH COLUMBIA by John Hume B.Sc., University of British Columbia Okanagan, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) February 2022 © John Hume, 2022 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Ectomycorrhizal fungal communities associated with urban Carpinus betulus trees across urban areas of British Columbia submitted by John Hume in partial fulfilment of the requirements of the degree of Master of Science . Dr. John Klironomos, Irving K. Barber Faculty of Science Supervisor Dr. Daniel Durall, Irving K. Barber Faculty of Science Supervisor Dr. Melanie Jones, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Jason Pither, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Richard Hamelin University Examiner iii Abstract Despite diminished health and richness of ectomycorrhizal (EcM) fungal communities compared with those in undisturbed environments, the drivers of diversity in urban EcM fungal communities are poorly understood. Studies of diversity in mycorrhizal fungal communities often overlook fundamental ecological theories used to explain diversity in plant and animal communities. In this study, I used Illumina next-generation sequencing to characterize EcM fungal communities associated with a common urban tree species, Carpinus betulus, in three British Columbian cities, Vancouver, Victoria, and Kelowna. Species diversity was compared across the three cities and related to ecological theory to investigate mechanisms that modulate diversity. Furthermore, soil chemical properties were analyzed at each site to identify specific soil characteristics associated with changes in community structure. I found that gamma diversity of the fungal communities was not positively correlated with environmental heterogeneity, as would be predicted by the habitat-heterogeneity hypothesis. Community composition, however, was significantly distinct in each of the three cities. A small number of species were found to be indicative of fungal communities in each city. Soil chemical properties were significantly associated with changes in community structure, with soil acidity and nitrogen content being the primary associated variables. No significant correlations were found, however, between individual soil properties and the most abundant taxa. While more research is required to determine the driving forces of EcM fungal diversity, this study serves as an example of applying ecological theory to understand diversity in urban EcM fungal communities. iv Lay Summary Urban trees are of growing importance in an urbanizing world, and they convey many benefits and services within cities. Mutualist fungi are crucial for the health and resilience of their plant hosts; however, urban trees are faced with diminished communities of fungal partners along with other difficulties unique to urban spaces. Many ecological theories purport to explain biodiversity; however, these theories are usually applied to plant and animal communities. This research applied ecological theory to study diversity of fungal communities associated with the roots of urban hornbeam trees while also exploring relationships between these fungi and specific soil characteristics. I found that a prominent ecological theory was unable to explain patterns in fungal diversity, and that soil acidity was associated with changes in the structure of these fungal communities. v Table of Contents Abstract .................................................................................................................................... iii Lay Summary ........................................................................................................................... iv Table of Contents ...................................................................................................................... v List of Tables .......................................................................................................................... viii List of Figures .......................................................................................................................... ix List of Abbreviations .............................................................................................................. xii Acknowledgements ............................................................................................................... xiii Dedication .............................................................................................................................. xiv Chapter 1: Introduction ............................................................................................................ 1 1.1 Ectomycorrhizal fungi ............................................................................................... 1 1.2 Ectomycorrhizal fungal communities ......................................................................... 3 1.2.1 Urban ectomycorrhizal fungal communities ..................................................... 5 1.3 Ecological theories of diversity .................................................................................. 6 1.3.1 Ecological theory in mycorrhizal systems .......................................................11 1.4 Impact of diversity on ecosystem functioning ...........................................................13 1.5 Carpinus betulus ......................................................................................................15 1.6 Amanita phalloides ..................................................................................................16 1.8 Thesis objectives and hypotheses ...........................................................................17 1.8.1 Objective 1: Species diversity and community composition ................................18 1.8.2 Objective 2: Soil chemical properties and associations with community composition ...................................................................................................................19 Chapter 2: Ectomycorrhizal fungal diversity and environmental correlates ...................... 21 2.1 Synopsis ..................................................................................................................21 2.2 Materials and methods ............................................................................................22 2.2.1 Sampling and experimental design ....................................................................22 vi 2.2.2 Soil analysis .......................................................................................................25 2.2.3 Fungal DNA extraction and Illumina sequencing ................................................26 2.2.3 Sequencing analysis and bioinformatics ............................................................27 2.2.4 Statistical analysis ..............................................................................................28 2.2.4.1 Gamma and alpha diversity ........................................................................28 2.2.4.2 Beta diversity and community composition .................................................29 2.2.4.3 Relationships between community composition and soil properties ............30 2.3 Results ....................................................................................................................31 2.3.4 Overview of sequencing results .........................................................................31 2.3.1 Soil chemical properties .....................................................................................35 2.3.1 Gamma and Alpha diversity ...............................................................................37 2.3.2 Beta diversity and community composition.........................................................41 2.3.3 Relationships between community composition and soil properties ...................45 2.4 Discussion ...............................................................................................................49 2.4.1 Mock community ................................................................................................49 2.4.1 Gamma diversity of fungal communities is not associated with environmental heterogeneity.................................................................................................................49 2.4.2 Composition of ectomycorrhizal fungal communities differed across cities .........51 2.4.3 Soil chemical properties are associated with ectomycorrhizal fungal community structure ........................................................................................................................55 Chapter 3: Conclusion ............................................................................................................ 59 3.1 Summary .................................................................................................................59 3.2 Novelty of work ........................................................................................................62 3.3 Future research .......................................................................................................63 Bibliography ............................................................................................................................ 65 Appendices ............................................................................................................................. 79 vii Appendix A: Sampling locations ........................................................................................79 Appendix B: Soil data ANOVA and PERMDISP results .....................................................80 Appendix C: Richness ANOVA ..........................................................................................82 Appendix D: Beta diversity PERMDISP .............................................................................83 Appendix E: RDA overall ANOVA ......................................................................................84 viii List of Tables Table 2.1: Summary of relevant climate data for the study locations. Data retrieved from en.climate-data.org. Averages were calculated from data collected between 1999-2019. .........24 Table 2.2: Total number of reads and mean number of reads per sample of ectomycorrhizal fungal communities associated with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), Victoria (n = 30), and in total (n = 90). ................................................................................32 Table 2.3: Mean values of soil chemical properties in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). Superscripts denote statistically significant differences between cities determined by ANOVA and Tukey HSD tests, based on α = 0.05. ............................................36 Table 2.4: All ectomycorrhiza fungal taxa identified via Illumina sequencing in association with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30) ordered by the abundance of read numbers in each city. .......................................................................38 Table 2.5: PERMANOVA results of the species composition of EcM fungal communities associated with European hornbeams in Kelowna, Vancouver and Victoria based on 999 unrestricted permutations of Hellinger transformed data. D.f. = degrees of freedom .................42 Table 2.6: Multilevel Pattern Analysis of ectomycorrhizal fungal ASVs in communities associated with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). Each taxon presented was deemed a significant indicator species for its city based on α = 0.05. ..........................................................................................................................................45 Table 2.7: ANOVA like permutation tests for significance of individual constrained variables on the structure of ectomycorrhizal fungal communities associated with Carpinus betulus trees (n = 90). Bolded values represent statistically significant results. Results are based on 999 free permutations of Hellinger transformed data. ..............................................................................47 ix List of Figures Figure 2.1: Sampling location maps. Red dots represent individual Carpinus betulus trees from which roots were sampled. Thirty samples were taken from each city, resulting in ninety samples total for genetic and chemical analyses. ......................................................................23 Figure 2.2: Schematic diagram of experimental design including visualization of sampling approach and how each diversity type is defined. .....................................................................31 Figure 2.3: Relative abundance of ectomycorrhizal fungal taxa associated with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). Each taxon represents a single ASV. ...............................................................................................................................33 Figure 2.4: Community composition of the Illumina EcM fungal mock community sample (left) (n=1) compared to the expected composition (right) of the mock community based on Sanger sequencing of individual fungi before being combined and Illumina sequenced. Failed (1) and Failed (2) are fungi that were unsuccessfully Sanger sequenced before being added to the Illumina mock community sample. Each taxon in the mock column represents a single ASV. ...35 Figure 2.5: Principal coordinates analysis (PCoA) ordination plot of soil chemical properties at sites in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Red dots represent sites in Kelowna. Green triangles represent sites in Vancouver. Blue boxes represent sites in Victoria. Standard errors are represented by colour coded ellipses while black lines indicate the distance from the centroid for each site. ..................................................................................................37 Figure 2.6: Boxplots showing comparisons of Shannon (left) and Simpson’s (right) diversity indices of ectomycorrhizal fungal communities associated with individual Carpinus betulus trees in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Boxes represent the 1st to 3rd quartiles; thick horizontal lines represent group means and whiskers extend to 1.5 x IQR. Black dots represent outlier observations. Means were not deemed significantly different based on Kruskal-Wallis tests (α = 0.05). .................................................................................................40 x Figure 2.7: Boxplot showing comparisons of species richness of ectomycorrhizal fungal communities associated with individual Carpinus betulus trees in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Boxes represent the 1st to 3rd quartiles; thick horizontal lines represent group means and whiskers extend to 1.5 x IQR. Black dots represent outlier observations. Floating horizontal lines and stars indicate significant differences between cities. ANOVA and Tukey HSD tests indicated that communities in Kelowna had significantly lower richness than in Vancouver and Victoria (α = 0.05). ..................................................................41 Figure 2.8: Principal coordinates analysis (PCoA) ordination plot of Hellinger transformed ectomycorrhizal fungal communities associated with Carpinus betulus trees in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Red dots represent sites in Kelowna. Green triangles represent sites in Vancouver. Blue boxes represent sites in Victoria. Standard errors are represented by colour coded ellipses while black lines indicate the distance from the centroid for each site. ..................................................................................................................................43 Figure 2.9: Abundance heatmap of the ten most abundant individual ectomycorrhizal fungal ASVs associated with Carpinus betulus trees in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Colouring is based on the log10 of % abundance. ...........................................44 Figure 2.10: Canonical redundancy analysis (RDA) ordination biplot of ectomycorrhizal fungal communities associated with Carpinus betulus trees in Kelowna (red, n = 30), Vancouver (green, n = 30) and Victoria (blue, n = 30). Black arrows represent constrained environmental variables used in the model. The direction and length of the arrows are proportional to the explained variation in the communities. Moisture refers to the moisture concentration of the soil. Organic refers the soil organic matter. Nitrogen represents extractable inorganic nitrogen and phosphorus represents extractable phosphorus. .......................................................................46 Figure 2.11: Spearman correlation heatmaps displaying relationships between the ten most abundant ectomycorrhizal fungal taxa associated with European hornbeams in Kelowna (n=30), Vancouver (n=30) and Victoria (n=30) and measured environmental variables. Positive xi correlations appear blue and negative correlations appear red. The colour gradient indicates the strength of the correlation. Significant correlations after Bonferroni correction are shown with an asterisk (α = 0.05). “nitrogen” refers to the concentration of KCl-extractable nitrate and ammonium. “phosphorus” refers to the concentration of Bray-P1 extractable phosphorus. “organic” refers to the % of soil organic matter. .........................................................................48 xii List of Abbreviations NGS Next Generation Sequencing QIIME2 Quantitative Insights into Microbial Ecology 2 ITS Internal Transcribed Spacer IGS Intergenic Spacer PCoA Principle Coordinate Analysis ASV Amplicon Sequence Variant ANOVA Analysis of Variance PERMDISP Permutational Test of Homogeneity of Multivariate Dispersions RDA Redundancy Analysis DNA Deoxyribonucleic Acid RNA Ribonucleic Acid EcM Ectomycorrhizal AMF Arbuscular Mycorrhizal Fungi PCR Polymerase Chain Reaction DBH Diameter at Breast Height KCl Potassium Chloride VIF Variance Inflation Factor xiii Acknowledgements I would like to thank my supervisors, Dr. Daniel Durall and Dr. John Klironomos for the immense support and guidance I have received throughout my honours and master’s research projects. I want to thank them for inspiring a lifelong passion for mycology in me beginning with the field mycology course they instructed in 2017, and for encouraging me to pursue research in the field. I would also like to thank my committee members Dr. Melanie Jones and Dr. Jason Pither for the invaluable knowledge they have provided throughout my years in graduate school. I also owe thanks to all members of the Durall, Klironomos, and Jones labs for the knowledge and support they have provided. In particular, I would like to thank Naomi Yamaoka for her support in the lab, and Maddi Arnold for her abundant assistance throughout the bioinformatics and statistical analysis stages of my thesis. Finally, thank you to the UBC Okanagan Biology department, to NSERC for funding my time at UBCO, and to staff at the Integrated Microbiome Resource at Dalhousie University for support in Illumina sequencing. xiv Dedication To my parents and Zoë, I don’t know where I would be without your endless love and support. 1 Chapter 1: Introduction 1.1 Ectomycorrhizal fungi A mycorrhizal relationship is a symbiosis between a fungus and the roots of a plant host (Smith and Read, 2010). In such symbioses, the fungi often provide several benefits to the plant host in return for carbon in the form of photosynthate. The primary benefits to the plant host are an increased range for the uptake of nutrients such as phosphorus and nitrogen, as well as increased drought and disease resistance (Smith and Read, 2010). The exchange of nutrients occurs where the hyphae of the fungus make physical contact with the root tissue of the plant host (Smith and Read, 2010). An extensive study found that 92% of surveyed land plant species are mycorrhizal and argued that a continuous phylogenetic distribution of mycorrhizas in land plants suggests that the interactions began at the origin of land plants (Wang and Qiu, 2006). Ectomycorrhizal (EcM) fungi are a group of mycorrhizal fungi whose namesake refers to the observation that their hyphae do not penetrate beyond the cell wall of the host. Ectomycorrhizae can be characterized by a few notable features including a dense concentration of hyphae surrounding the host root known as the sheath or mantle, an inward growth of hyphae surrounding the host’s cortical and epidermal cells called the Hartig net, and an external mycelium that spreads throughout the surrounding environment and links the host to the soil and the mushroom fruiting bodies (Smith and Read, 2010). Ectomycorrhizal fungal species can be found in both the Basidiomycota and the Ascomycota and many of these species form mushrooms as fruit bodies (Tedersoo et al., 2010). 2 EcM fungi associate primarily with woody perennial plants (Smith and Read, 2010). More specifically, EcM fungi tend to associate with plants in nutrient-poor environments such as temperate forests (Wang and Qiu, 2006). Ectomycorrhizal fungi display great variation in host specificity (Molina et al., 1992). One study analyzed the possible benefits to an EcM fungal species of specializing on a small number of hosts (Bruns et al., 2002). The authors suggested that specialist species can derive more resources from the host than would be available to generalist species. On the other hand, generalist species benefit from having a greater diversity of potential hosts (Bruns et al., 2002). Both generalist and specialist species have the potential to associate with multiple hosts at once, establishing mycorrhizal networks. Mycorrhizal networks serve to optimize the exchange of resources between the fungus and its hosts and to facilitate the transfer of resources between plants in the network. An individual fungus can span more than 30 m underground and contact the roots of multiple hosts, thereby forming extensive links (Beiler et al 2010). The linking of multiple trees by a network allows the fungus to mediate the transfer of resources between plants (Selosse et al., 2006). This means that a fungus may redirect incoming photosynthate to another plant in its network as opposed to incorporating the carbon into its own mycelium. Mycelial networks have also been found to incorporate developing seedlings, which are too immature to meet the carbon demands of the fungus (Nara, 2006). This is reinforced by studies that have found increased growth in seedlings connected to a mycorrhizal network including mature trees compared to isolated seedlings (Cline et al., 2005). The facilitation of seedling development can be thought of as an investment for the fungus in return for a future carbon source. In some 3 cases, seedlings derive no benefit or even a negative effect from mycorrhizal networks (Van der Heijden and Horton, 2009). The more salient point is that these dynamics, which can have significant effects on forest ecosystems, are ultimately mediated by mycorrhizal fungi. 1.2 Ectomycorrhizal fungal communities Ectomycorrhizal fungi play important roles in the functioning of many terrestrial ecosystems. For example, they are movers of important plant nutrients such as nitrogen and phosphorus throughout the soil. Ecosystems are often inhabited by diverse assemblages of EcM fungal species organized in complex communities. Water availability and temperature can have a significant influence on the survival of EcM fungal species and, therefore, alter community composition (Erland and Finlay, 1992; Pigott, 1982). Soil chemical composition can also have a significant impact on EcM fungal community composition (Horton et al., 2013). The structure and composition of communities can also vary based on environmental gradients such as nitrogen availability or spatial gradients such as elevation. (Cox, 2010; Geml et al., 2017). Although the consequence of changes in fungal communities to ecosystem functioning is poorly understood, it is generally accepted that species differ in their ability to confer advantages to host plants. For example, fungal species secrete different enzyme profiles allowing for differentiation in the uptake efficiency of various substrates in the soil (Courty et al., 2005). Networks created by various EcM fungal species also differ in their ability to support tree seedlings (Nara, 2006). Therefore, having high diversity within communities can potentially lead to increased resiliency within ecosystems. 4 Although there is experimental evidence showing that EcM fungal species differ in their ability to confer host advantage, the importance of diversity remains mostly suggested, as generating diverse mycorrhizal associations in an experimental setting is complex and difficult to achieve (Ishida et al., 2008). One example of such a study found that more diverse fungal communities conveyed only modest benefits to host birch trees, such as increased nitrogen and phosphorus concentration in the host tissue and increased mycorrhizal root biomass at the expense of shoot biomass (Baxter and Dighton, 2001). The effect of EcM fungal diversity on ecosystem functioning remains poorly understood. Another important concept that influences EcM fungal community structure is competition. The role of competition in fungal communities has been studied from an agricultural perspective since the 1950s (Garrett, 1956). Competition among saprotrophic and arbuscular fungi have also received considerable attention in the mycological community since the early 2000s (Boddy, 2000; Lekberg, 2007). EcM fungal researchers have been slow to catch up due to the previously mentioned difficulties associated with studying EcM fungal communities. A review by Kennedy (2010) on the effects of competition on community structure found that in multiple pair-wise interactions, competition is rarely negative for both EcM fungal species. It was asserted that a larger number of studies would be necessary to better understand these interactions. The review also found that competitive effects are environmentally context-dependent. For example, temperature could affect whether a species was a weak or superior competitor. The remaining conclusions were that field distributions of EcM fungi 5 often reflect competitive interactions, and the timing of colonization influences competitive success (Kennedy, 2010). 1.2.1 Urban ectomycorrhizal fungal communities EcM fungi face unique challenges in urban areas compared to their naturalized counterparts. For example, they encounter high levels of heavy metal pollutants, and their hosts suffer restricted volumes of soil (Lindsey and Bassuk, 1992; Wei and Yang, 2010). Early research into urban EcM fungal communities, which utilized morphotyping to characterize the EcM fungi colonizing roots, found that so-called early successional species persisted for decades around urban spruce trees (Danielson and Pruden, 1989). Another study that utilized morphological methods found that arbuscular mycorrhizal fungi (AMF) colonization of host trees was significantly lower in urban areas compared to rural areas (Bainard et al., 2011). Similar results have been found in EcM fungal communities using molecular methods (Karpati et al., 2011; Schmidt et al., 2017). In addition to lower levels of host colonization, research has also shown that urban EcM fungal communities associated with oak trees have lower species richness than those associated with rural individuals (Baxter et al., 1999). It is still unclear exactly which factors diminish the success of urban EcM fungal communities. Although it stands to reason that urban fungal communities would abide by similar driving forces to those in undisturbed environments, there have been relatively few studies on this topic. One study of fungal communities associated with urban linden trees found that soil pH and moisture were primary drivers of urban EcM fungal community composition (Van Geel et al., 2018). Interestingly, the study found that high concentrations of heavy metals 6 associated with urban soils did not have a significant impact on EcM fungal community composition and therefore does not explain reduced colonization of trees in urban areas (Van Geel et al., 2018). Another study of EcM fungal communities associated with urban linden trees found that soil moisture was a significant driver of community composition (Alzetta et al., 2012). 1.3 Ecological theories of diversity Before expanding on theories of diversity, it is important to understand the different types of diversity that are studied in community ecology: alpha, beta, and gamma diversity. Alpha diversity refers to the diversity of a single site or community. Alpha diversity can be quantified by richness, the number of unique species, or a diversity index that considers richness and evenness, such as the Shannon-Wiener index. Gamma diversity is the total diversity of a region, which is calculated by enumerating unique species from all sites or communities in a region. Beta diversity is a measure of change in diversity from one environment to another. Ecologists have long been interested in determining how diversity is modulated. In other words, why are some ecosystems more diverse than others? Furthermore, why does diversity exist at all? Why is it not the case that one species can outcompete all others and dominate entire ecosystems? Vellend (2010) developed a framework that breaks down the processes involved in community ecology, which provides context to the various theories attempting to explain the diversity of communities. The primary processes that drive patterns of diversity are selection, speciation, drift, and dispersal (Vellend, 2010). Most theories of diversity can be unified within this framework; the 7 differences lie in the relative contribution of each of these processes regarding their impact on community assembly. Many theories emphasize the contribution of either niche-based processes or neutral processes. Niche-based processes are those that are impacted by differences in traits possessed by species that alter their fitness in an environment and represent the role of selection in Vellend’s (2010) framework. Broadly speaking, Niche-based processes can be either bottom-up or top-down in nature. So-called bottom-up theories of diversity posit that diversity is related to the availability of resources in an environment. An early example that relates to succession is the resource-ratio hypothesis of plant succession (Tilman, 1985). Though the resource-ratio hypothesis pertains to plant succession specifically, it outlines a concept that has important implications for diversity. This hypothesis suggests that species thrive in different niches that are composed of certain ratios of key resources. For example, a given plant may thrive in an environment with relatively high sunlight and relatively low nitrogen. The ratio between these two resources represents a niche that a given species may be particularly well adapted to inhabit. This helps to explain primary successions because, as these key resources change in availability throughout a succession, so too do the ratios present in the environment, which are accompanied by a subsequent shift in dominant species (Tilman, 1985). In addition to primary succession, bottom-up theories also apply to ecosystems as a whole. The habitat-heterogeneity hypothesis is rooted in a similar idea that species occupy niches that are related to the resources available to them (MacArthur and MacArthur, 1961). Given this, environments that are more heterogeneous in resources should also be more diverse as the variation in resources would result in more unique 8 niches. Many studies have found empirical evidence for this relationship (Douda et al., 2012.; Hamm and Drossel, 2017; Tews et al., 2004). Although ecologists may disagree on details relating to how resource availability impacts diversity, there is much support for the idea that diversity is modulated through bottom-up mechanisms. Some more recent theories, however, have taken a different approach to survey the drivers of diversity. Just as hypotheses exist for diversity being influenced by bottom-up mechanisms, some ecologists argue that top-down effects play the dominant role in modulating diversity. One example is the trophic theory of diversity outlined by Terborgh (2015). Terborgh (2015) argues that the bottom-up approach to species diversity relies on several assumptions that have yet to be empirically verified. An example of one such assumption is that species are at or near carrying capacity in nature; therefore, competition for resources dictates the relative frequencies of populations. Terborgh (2015) argues that because the true carrying capacity of a given species is difficult to estimate, it is a significant assumption to base a hypothesis of diversity on competition for resources. Terborgh suggests that predation regulates densities of most species to levels far below carrying capacity; therefore, predation is the dominant modulator of diversity. Many examples of this phenomenon are cited to support the claim that predation increases diversity. One example cited by Terborgh (2015) was the experimental removal of starfish from rocky intertidal zones in Washington state that led to a dominant competitor, the mussel, which displaced many other species and decimated diversity (Paine, 1966). This case is an example of a keystone predator, where the starfish has a preference for 9 preying on the dominant competitor, keeping its density low enough to allow other species to flourish (Paine, 1966). Despite the strength and simplicity of this example, Terborgh (2015) argues that ecologists have largely failed to see the generality of it. An important caveat is that just as predation can promote diversity, too much predation can also decrease diversity drastically. This phenomenon is abundantly clear in cases of novel super predators. One such example is the introduction of the Burmese python in the Florida everglades, leading to decimation in small mammal populations. With this in mind, Terborgh (2015) clarifies that intermediate levels of predation are key to maximizing diversity. This leads to a closely related hypothesis, the intermediate disturbance hypothesis, which suggests that intermediate levels of disturbance in general, as opposed to predation specifically, serve to maximize diversity in an ecosystem (Connell, 1978). The argument presented is that diversity of competing species is highest when the frequency or intensity of disturbance or environmental change broadly is at an intermediate level (Connell, 1978). A study by Molino and Sabatier (2001) sought to validate this hypothesis with a case study of tropical rainforests in French Guiana. The authors compared levels of tree diversity to the frequency of heliophilic and pioneer plant species indicative of disturbance. These comparisons produced a “hump-back” shaped relationship, which was interpreted as maximum diversity at intermediate disturbance. It is important to mention that this hypothesis has received significant criticism for a lack of empirical evidence and a weak theoretical basis (Fox, 2013). Another approach to understanding diversity is through the lens of neutral or stochastic processes. In stark contrast to niche-based theories, neutral macroecology 10 assumes no differences between species and how they interact with their environments and instead suggests that diversity can be explained simply by the random initial composition of species in an environment and rates of migration and dispersal (Bell, 2001). This hypothesis came about when improved modelling capabilities uncovered the fact that patterns observed in macroecology that were previously attributed to differences in the traits of species were, in fact, indistinguishable from those observed in community models where all individuals had identical demographic properties (Bell 2001). It is important to note that these neutral models exclude important interactions such as those between predator and prey, parasite and host, and mutualist relationships (Bell, 2001). For this reason, neutral macroecology applies more narrowly to competition rather than trophic interactions and diversity as a whole. Several hypotheses have been discussed in this section and on the surface, they appear mutually exclusive. For example, it is difficult to reconcile the concept of neutral macroecology in some contexts such as primary successions, where the adaptations of specific species seem to be critical in explaining where and when they dominate in a community. This begs the question: which of these proposed mechanisms is correct? Each of the hypotheses introduced seem to have a logical theoretical basis and at least some empirical evidence to support the theory. With this in mind, it may be more valuable to question which theory is most appropriate to apply in a given context. The model being studied may have a significant impact on the relevance of a given ecological theory of diversity. 11 1.3.1 Ecological theory in mycorrhizal systems Although competition for resources can be found up and down the food chain, there is at least some evidence that it impacts some trophic levels more than others. As early as 1960, Hairston et al. (1960) noted that primary producers and decomposers are more likely to be resource-limited than herbivores, which are more likely to be limited by predation. The barrier presented by dispersal also varies by trophic level. Several studies have provided support that dispersal rates and distances of microorganisms far exceeds those of macroorganisms, indicating that microbes are less likely to be dispersal limited and therefore impacted by neutral processes (Finlay, 2002; Finlay and Clark, 1999; Wilkinson et al., 2012). If the assumption is accepted that soil fungi populations are limited by competition for resources, then it stands to reason that bottom-up theories of diversity such as the habitat-heterogeneity hypothesis are suitable in making predictions about diversity in soil fungal communities (MacArthur and MacArthur, 1961). Research has shown that the advantage that arbuscular mycorrhizal fungi (AMF) convey to their hosts can serve as evidence for the resource-ratio hypothesis in plant communities. Direct evidence for a bottom-up influence on diversity within fungal communities is sparse, however. Despite the observations that microorganisms are ubiquitous and therefore unlikely to be dispersal limited, several studies have found evidence that neutral processes indeed play a role in EcM fungal community assembly. Peay (2010) found that EcM fungal communities associated with isolated host trees have reduced richness compared to those associated with mainland (non-isolated) hosts. This provided evidence that decreased dispersal of EcM fungi to isolated host trees could lead to 12 reduced richness in the presence of stochastic processes and local extinction events (Peay, 2010). Peay’s (2010) work demonstrated the role of island biogeography in impacting EcM fungal diversity, which may be highly relevant to EcM fungal communities in urban environments due to the unique isolation of urban host trees compared to those in undisturbed environments. Research by De-Wei (2005) provides context for why, unlike in microorganisms broadly, dispersal may be limiting for EcM fungal species. Despite the production of huge numbers of wind-dispersed spores, less than 5% of these spores disperse more than 5 m from the basidiocarp in one EcM fungal species (De-Wei, 2005). Although this topic is not well researched, it calls into question previous notions that fungal spores are truly ubiquitous. Additional studies also call into question the importance of niche-based processes in modulating EcM fungal diversity by establishing the existence of priority effects in EcM fungi (Kennedy and Bruns, 2005; Kennedy et al., 2009). These studies found that the timing of colonization had a significant impact on the long-term competitive advantage of Rhizopogon species. While these studies also provide evidence that abiotic factors play a lesser role in modulating EcM fungal diversity, the process was studied only within a single genus and the competition was between hypha of the original colonizer and spores of the secondary colonizer, which may be less relevant in nature (Kennedy et al., 2009). It is difficult to disentangle the relative contribution of niche-based and neutral processes on EcM fungal diversity, however, insights from Yang et al. (2015) may provide some clarity on the matter. Yang et al. (2015) found that the impact of environmental heterogeneity on community richness depends on the degree of severity within the environment. The analysis found that 13 extreme levels of severity were associated with a greater contribution of heterogeneity in modulating diversity (Yang et al., 2015). While environmental severity may be difficult and subjective to quantify, it is relevant to note that neutral processes may play a larger role in EcM fungal community assembly when resource availability is moderate. Another possible reason that it is difficult to determine the drivers of diversity in EcM fungal communities is the added complexity of host interactions. It is impossible to ignore the role that host tree diversity plays in an ecosystem when studying communities of mycorrhizal fungi, especially when some species are known to specialize on a small number of host species (Bruns et al., 2002). One way around this dilemma is to analyze communities of EcM fungi associated with one specific host species. Although this removes host diversity from the equation, neighbouring plants can still have a detectable influence on the composition of a tree’s associated fungal community (Mony et al., 2021). Although this phenomenon is better studied in AMF, the theoretical basis extends to EcM fungi that the pool of fungal species available to a focal host is directly influenced by fungal communities associated with neighbouring plants, especially considering that many EcM fungal species are generalists that can associate with many plant host species (Bruns et al., 2002). 1.4 Impact of diversity on ecosystem functioning When studying biodiversity, it is important to question what impact diversity has on an ecosystem. It is often taken for granted that diversity is positive for ecosystem functioning; however, it is important to consider what empirical evidence exists in the literature. This is a rich topic that has received much attention from ecologists. Entire 14 books have been devoted to this relationship (Schulze and Mooney, 2012). For this reason, only a small fraction of the literature will be referenced here. One point that is important to mention is that our understanding of ecosystem functioning and how to measure it is constantly evolving and improving. Many studies, especially earlier ones, emphasize plant primary productivity as a proxy for ecosystem functioning; however, it should be noted that this is not a comprehensive substitute for the complex array of functions taking place in nature. Early experimental work on the relationship between diversity and ecosystem functioning by Naeem et al. (1994) utilized microcosms in controlled growth chambers with varying levels of diversity across 4 trophic levels to evaluate whether diversity alone could improve measures of ecosystem functioning. While some metrics of ecosystem functioning, such as primary productivity, did increase with diversity, others showed no significant change (Naeem et al., 1994). A later study by Tilman (1996) evaluated plant primary productivity along a species richness gradient, this time in a field experimental setting. This study yielded similar results whereby experimental plots with greater plant richness exhibited higher levels of primary productivity (Tilman, 1996). One limitation to consider in each of these studies is that the increase in plant productivity in richer communities could potentially be attributed to those communities having a higher likelihood of hosting a so-called super-producer species rather than diversity itself being causal in increased productivity. Following the uptick of interest in the relationship between diversity and ecosystem functioning that took place in the mid-1990s, Bengtsson (1998) published a theoretical paper that can be summarized as arguing that diversity alone is far too 15 simplistic a measure to utilize when investigating relationships with ecosystem functioning. Rather, it is more beneficial to study links between specific species or functional groups and ecosystem functioning rather than diversity broadly. A more recent review of studies that consider the relationship between tree diversity and primary productivity in forests came to a similar conclusion that species identity may be more influential for ecosystem functioning than species diversity (Nadrowski et al., 2010). In mycorrhizal fungi, this relationship has been more well studied in AMF. In AMF, research has shown a positive relationship between fungal diversity and plant primary productivity (Van Der Heijden et al., 1998). A similar relationship has been observed in EcM fungi; however, the benefit to host productivity depended on the host species and substrate fertility (Jonsson et al., 2001). Another study found that more diverse EcM fungal communities provided only an increase in root biomass and nutrient uptake rather than primary productivity (Baxter and Dighton, 2001). Though the benefits of EcM fungal diversity for host productivity has proved difficult to measure, more research is emerging that provides support for the importance of diverse EcM fungal symbionts. More recent research has identified benefits of EcM fungal diversity to host Norway spruce seedlings by way of increased nutrient acquisition from diversified exoenzymes (Velmala et al., 2014). EcM fungal diversity has also been shown to increase phosphorus uptake in European beech saplings (Köhler et al., 2017). 1.5 Carpinus betulus Carpinus betulus, known commonly as European hornbeam, is common and widespread throughout most of Europe, where it is native (Coart et al., 2005). European 16 hornbeams have been managed for construction material and firewood in Europe for centuries due to their physical properties and their ability to be pollarded and coppiced (Rowe and Williamson, 2013). European hornbeams have become popular in Pacific Northwestern cities of North America for a couple of key reasons. Firstly, hornbeams are well suited for the climate of the Pacific Northwest and can tolerate heavy snowfall without damage due to the density of the wood (A. Hunsberger, personal communication, April 25, 2019). Secondly, the columnar “Fastigiata” cultivar produces a narrow column-like crown that is well suited for tight quarters such as city boulevards. Unlike many other common ornamental trees in Pacific Northwestern cities such as maple and cherry, European hornbeams require colonization by EcM fungi (Harley and Harley, 1987). One infamous EcM fungus known as the death cap (Amanita phalloides) has been found to commonly associate with European hornbeams in Vancouver and Victoria, British Columbia. (Berch et al., 2016). 1.6 Amanita phalloides Amanita phalloides, known commonly as the death cap is an ectomycorrhizal fungus native to Europe that produces a deadly poisonous mushroom. The death cap is responsible for the highest number of fatal mushroom-related poisonings on record and has been gaining attention in the Pacific Northwest, where it is invasive. The genus Amanita is commonly recognized by a few notable characteristics including white or pale gills and spores, a universal veil resulting in a volva at the base of the stipe, and often the presence of a partial veil resulting in an annulus or ring (Arora, 1991). The genus Amanita contains many of the world’s most poisonous mushrooms, including the 17 death cap. Amanita phalloides can be distinguished from other members of its genus by its brownish-olive cap that is free from universal veil remnants (Arora, 1986). Amanita phalloides was most likely introduced to North America in the form of ectomycorrhiza on the roots of imported saplings (Pringle et al., 2009). In the Pacific Northwest, the death cap is found frequently in urban areas in association with trees belonging to the following genera: Fagus (beech), Castanea (sweet chestnut), Picea (spruce), Carpinus (hornbeam), Betula (birch), and Tilia (linden). On the east coast of North America, the fungus associates almost exclusively with members of the Pinaceae. Unlike its distribution on the West Coast, A. phalloides does not appear to be spreading in the east and is, instead, primarily confined to exotic tree plantations. In its native range in Europe, A. phalloides associates most frequently with Quercus and Fagus species (Wolfe and Pringle, 2012). Throughout most of its range in North America, A. phalloides fruits exclusively in disturbed habitats in proximity to its historic European host trees. Northern California represents the sole location where A. phalloides has managed to significantly penetrate undisturbed ecosystems and become naturalized alongside native flora (Wolfe et al., 2010). 1.8 Thesis objectives and hypotheses The overall objective for this thesis was to explore urban EcM fungal communities associated with C. betulus trees in British Columbia and utilize ecological theory to gain insight into how diversity is modulated within these communities. Additionally, specific soil characteristics were investigated to determine which chemical properties are associated with patterns observed in community composition. This 18 research stems from previous studies that investigated plant and animal communities to understand mechanisms that promote diversity that have largely been neglected in EcM fungal ecological research. Furthermore, this research took place in an urban setting in an attempt to obtain knowledge that may benefit urban trees, which are gaining importance in an urbanizing world. 1.8.1 Objective 1: Species diversity and community composition To compare diversity of EcM fungal communities associated with C. betulus trees in three British Columbian cities, Kelowna, Vancouver, and Victoria, and investigate whether patterns conform to bottom-up based ecological theory. Additionally, to investigate and compare community composition of sites within each city. Hypothesis 1a: Gamma diversity will be highest in the city with the greatest degree of environmental heterogeneity. Rationale: Though many ecological theories exist that can be utilized to make predictions about diversity, bottom-up niche-based theories, such as the habitat-heterogeneity hypothesis, have a strong theoretical basis considering microbes and decomposer communities are less likely to be limited by predation or disturbance and are often more ubiquitous compared to plant and animal species (Hairston et al., 1960; MacArthur and MacArthur, 1961; Finlay, 2002). 19 Hypothesis 1b: Composition of EcM fungal communities will differ significantly among cities. Rationale: Despite relative geographic proximity, the three cities sampled display considerable variation in annual temperature and precipitation, which are known to affect EcM fungal community composition (Tedersoo et al., 2012). Furthermore, sites within each city will likely be exposed to different neighbouring plants due to the breadth of exotic species that exist in urban landscapes, and distinctions in native plants that inhabit different biogeoclimatic zones of British Columbia. Neighbouring plant species may alter the species pool present at each site (Mony et al., 2021). 1.8.2 Objective 2: Soil chemical properties and associations with community composition To determine which soil chemical properties are significantly associated with differences in EcM fungal community structure and investigate which properties are associated with variation in the abundance of specific taxa in each city. Hypothesis 2: The measured soil chemical properties will be significantly associated with differences in EcM fungal community structure. Soil pH will be more highly associated with variation in community structure than the other measured soil chemical properties. 20 Rationale: Soil acidity has been shown to be a dominant driver of EcM fungal community structure in communities associated with European temperate host species in both undisturbed and urban environments (Suz et al., 2014; Van Geel et al., 2017). 21 Chapter 2: Ectomycorrhizal fungal diversity and environmental correlates 2.1 Synopsis Ectomycorrhizal host trees can be home to rich and complex communities of mutualist fungi. Some of these fungi form easily recognizable mushrooms, such as the red and white fly agaric (Amanita muscaria), while others, such as the genus Amphinema, form crust-like fruiting bodies. Although many of these species coexist throughout cities and even beneath a single tree, ecological theories of diversity are rarely implemented when comparing fungal communities across urban areas. A theory that has gained support, especially among communities of decomposers and microorganisms, is the habitat-heterogeneity hypothesis that suggests that diversity increases with the heterogeneity, or patchiness, of an environment (MacArthur and Macarthur, 1961). Ecological theories derived from plant and animal communities can be difficult to apply to mutualist fungi for several reasons. One reason is the added complexity of host interactions, which can become a large factor in diverse forest ecosystems. While isolating a single EcM host species allows for interesting comparisons of associated fungal communities across abiotic gradients, these types of studies are rarely done in urban areas. Urban EcM host trees and their associated fungal communities are subject to unique hardships including reduced volumes of nutrient-rich soil and increased concentrations of heavy metals and other pollutants. Although the exact mechanism is unknown, urban EcM fungi are less abundant, and their communities are less diverse (Baxter et al., 1999; Karpati et al., 2011; Schmidt et al., 2017). Considering most of the world’s population lives in urban areas, it is important to understand how diversity is modulated in urban EcM fungal communities and which specific soil characteristics may alter community composition. I used Illumina 22 next-generation sequencing to characterize EcM fungal communities associated with Carpinus betulus trees in three British Columbian cities: Kelowna, Vancouver, and Victoria. I examined chemical properties of the soil surrounding these communities to determine if heterogeneity at the city scale was associated with gamma diversity. Furthermore, I utilized redundancy analysis and analyzed individual correlations to determine how EcM fungi were associated with specific soil properties. Gamma diversity of EcM fungal communities was not found to be associated with heterogeneity in soil chemical properties among cities in British Columbia. Species composition of the communities overall, however, was significantly distinct among cities. The measured soil chemical properties were found to be significantly associated with differences in community structure at each site; however, few specific correlations were found between any individual property and abundant taxa. These findings indicate that either the habitat-heterogeneity hypothesis does not adequately explain patterns observed in urban EcM fungal communities associated with C. betulus, or that the soil properties selected for analysis, or the degree of heterogeneity were not sufficient to expect an associated change in gamma diversity across cities. More research is required to determine how diversity is modulated in urban EcM fungal communities. 2.2 Materials and methods 2.2.1 Sampling and experimental design Three British Columbian cities were selected for sampling: Vancouver, Victoria and Kelowna, British Columbia. These locations were selected because of the high degree of variation in environmental conditions, such as average annual precipitation 23 and temperature (Table 2.1), which may contribute to differing soil characteristics. Additionally, sampling locations were limited to within British Columbia due to interprovincial and international travel restrictions associated with the COVID-19 pandemic. Thirty mature hornbeams (DBH > 40 cm) were chosen in each city. Potential host trees were identified using city street tree inventory maps. Non-random sampling was used to select trees that were representative of the various growing conditions across each city (Figure 2.1). The following variables were taken into account to obtain representative sampling: geographic location, setting (park or boulevard), and adjacent road type (arterial road or residential street). Figure 2.1: Sampling location maps. Red dots represent individual Carpinus betulus trees from which roots were sampled. Thirty samples were taken from each city, resulting in ninety samples total for genetic and chemical analyses. 24 Table 2.1: Summary of relevant climate data for the study locations. Data retrieved from en.climate-data.org. Averages were calculated from data collected between 1999-2019. At each tree, a soil probe was used to collect soil cores 30 cm deep and 2.5 cm in diameter. In order to maximize the fungal diversity captured at each tree, each sample was comprised of three subsamples taken 2, 3 and 4 m away from the base of each tree, then subsequently pooled in the field. Subsamples were taken directly south of each tree when possible. When boulevard orientation made sampling to the south impossible, samples were taken to the east of each tree. The soil samples were refrigerated at 4 °C within 2 h of collection and frozen at -20°C within 48 h of collection. Using a 2 mm sieve, roots were then isolated from the soil after thawing. The soil was then frozen for further analysis. All fine root sections that appeared to be colonized by EcM fungi were isolated from the soil using a dissecting scope and rinsed with sterile water before being frozen at -20 °C until DNA extraction. Characteristics used to identify EcM root tips included deviations in colour and texture from root tissue, appearance of hyphae, and increased fine root branching. Due to quarantine measures, the preceding steps were all completed from home using lab equipment obtained from the UBC Okanagan BRAES molecular lab. To summarize, three subsamples were taken and 25 subsequently pooled from the base of 30 trees in each of the three cities. In total, 90 samples were sequenced in this study. 2.2.2 Soil analysis Soil chemical properties had to be quantified to determine which city has the greatest degree of environmental heterogeneity (hypothesis 1a) and also to compare these properties to EcM fungal community structure and individual taxa (hypothesis 2). To measure soil acidity, 10 g of fresh soil from each sample was added to 20 ml of distilled water and the mixture was shaken for 30 minutes on a shaker tray. After sitting for 30 minutes, a glass electrode pH meter was used to determine pH. Moisture content was measured by obtaining the mass loss of water from 10 g of soil from each sample after evaporation of water content at 105 °C for 48 hours. Organic matter was measured by obtaining the mass loss of 10 g of dry soil from each sample after combustion of organic matter at 700 °C. To determine dissolved inorganic nitrogen concentration, nitrogen was extracted from fresh soil using a 2 M KCl solution within 48 hours of sampling. Nitrate and ammonium concentrations were measured from the extract and combined to determine inorganic nitrogen concentration (Gordon et al., 1978; Miranda et al., 2001). To measure phosphorus concentration, dried soil samples were sent to the Analytical Chemistry Services Laboratory (NRL) at the British Columbia Ministry of Environment and Climate Change Strategy in Victoria, BC. At the NRL, available phosphorus was measured using a Bray P-1 extraction. 26 2.2.3 Fungal DNA extraction and Illumina sequencing Illumina NGS sequencing was used to characterize the EcM fungal communities to compare the diversity of these communities to environmental properties (hypotheses 1a and 2) and to each other (hypothesis 1b). After thawing, DNA was extracted from EcM root tips using a Dneasy PowerLyzer PowerSoil Kit (Cat. No. / ID: 12855-50, QIAGEN Inc, Toronto, On, Canada). DNA quantity and quality were then quantified using a NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, De, USA). Library preparation and ITS2 rRNA sequencing were performed at Integrated Microbiome Resource (IMR) at Dalhousie University using Illumina MiSeq sequencing using 2x300 bp paired-end V3 chemistry (Comeau et al., 2017). The ITS86F (GTGAATCATCGAATCTTTGAA) and ITS4R (TCCTCCGCTTATTGATATGC) universal primers were used to amplify the ITS2 rRNA region. A negative control sample was included that was generated by using the DNA extraction kit on a sample without the addition of any fungal tissue. Raw Illumina reads were uploaded to GenBank (Accession: PRJNA808645). A mock community was created by isolating fungal DNA from six common EcM fungi encountered throughout the experimental samples. DNA was extracted from each morphotype independently and the ITS1 and ITS2 regions were amplified via PCR using the ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS4R (TCCTCCGCTTATTGATATGC) universal primers. These samples were then submitted to the Fragment Analysis and DNA Sequencing Services at the University of British Columbia (Kelowna, BC, Canada) for Sanger sequencing on a 3130xl DNA sequencer (Applied Biosystems, Foster City, CA, USA). Two of the six fungi were unsuccessfully 27 sequenced and are therefore referred to as Failed (1) and Failed (2). The sequences of the four remaining fungi were then aligned to the NCBI database using the BLAST tool with default criteria to identify the species identity. The four fungi were identified as Russula sp., Laccaria sp., Amanita muscaria, and Inocybe sp.. The extracted DNA from all six fungal samples were then combined in a staggered fashion where DNA of Russula sp., Failed (1), and Failed (2) were present at half the concentration of the DNA of Laccaria sp., Amanita muscaria, and Inocybe sp. based on NanoDrop DNA concentrations. 2.2.3 Sequencing analysis and bioinformatics Analysis of the Illumina sequencing data began with denoising using the DADA2 ITS pipeline (Callahan et al. 2016) in R (v3.6.1). The forward and reverse primers were removed from the demultiplexed paired-end reads, and the forward and reverse reads were merged. All sequences containing Ns, more than two expected errors, and those shorter than 50 base pairs were removed. Sequences were then dereplicated and paired ends were merged. Finally, all chimeras were removed. The representative sequence file and unique amplicon sequence variants (ASV) table were imported into QIIME2 v2019.10 (Caporaso et al. 2010) for downstream processing. First, the paired-end sequences were aligned using MAFFT (Katoh and Standley 2013) and the highly variable regions were masked to reduce noise. FastTree was then used to make a phylogenetic tree (Price, Dehal, and Arkin 2010). To assign taxonomy, a Naïve Bayes fungal classifier was trained using version 8 of the UNITE fungal database (2020-04-02 general release) at a 99% feature similarity threshold (Abarenkov 28 et al. 2020). The resulting taxonomy table was filtered to only include sequence variants that have been identified to the order level or lower, and ASVs with fewer than 100 reads summed across all samples were removed. The resulting feature table was not rarefied because the sequencing depth required to maintain a large enough sample size in each of the groups would result in a significant loss of sequence data. Before statistical analysis, FUNGuild was used to filter out all reads that were not EcM fungi with a confidence ranking of “possible” or greater (Nguyen et al., 2016). 2.2.4 Statistical analysis Statistical analysis and figure creation were performed using R (v3.6.1), with the exception of the community bar plots, which were made using Microsoft Excel (v2107) (R Core Team, 2020). ANOVA and Tukey HSD tests were performed to determine if individual soil properties differed significantly between cities. Environmental heterogeneity was quantified so it could be compared to gamma diversity to address hypothesis 1a. To determine which city displayed the greatest heterogeneity of chemical properties among locations of sampled trees, a permutation test for homogeneity of multivariate dispersions (PERMDISP) was performed on the soil metadata using the “vegan” package (v2.5.6) (Oksanen et al. 2008). 2.2.4.1 Gamma and alpha diversity Gamma diversity of each city was calculated so that it could be related to environmental heterogeneity to address hypothesis 1a. Overall richness was calculated for each city to represent gamma diversity (Figure 2.2). Next, alpha diversity was 29 quantified to investigate diversity at individual trees (Figure 2.2). Alpha diversity, however, could not be used to test the habitat-heterogeneity hypothesis because heterogeneity was not measured at individual trees. Richness, Shannon index, and Simpson’s index were calculated for each individual tree using the “phyloseq” package (version 1.30.0) (McMurdie and Holmes, 2013). Levene’s test was then used to assess variance using the “car” package (Fox et al., 2007). Shapiro-Wilk tests were performed to assess the normality of the data using the “stats” package. An ANOVA was then performed to determine if mean richness differed significantly among cities based on α = 0.05. For Shannon and Simpson’s index, a non-parametric Kruskal-Wallis test was performed to determine if means differed significantly among cities based on α = 0.05. 2.2.4.2 Beta diversity and community composition Beta diversity was measured to assess whether community composition differed significantly among cities (hypothesis 1b). Beta diversity was visualized on a principal coordinate analysis (PCoA) ordination plot using a Bray-Curtis dissimilarity matrix following Hellinger transformation of the data using the “vegan” package (v2.5.6) (Oksanen et al. 2008). A permutation test for homogeneity of multivariate dispersions (PERMDISP) was performed to evaluate the degree of beta diversity within each city, followed by a permutational multivariate analysis of variance (PERMANOVA) with 999 permutations and a pairwise comparison of cities using an open-source R code to compare beta diversity among cities (Martinez Arbizu, 2020). Significance was determined based on α = 0.05. A heatmap was generated using the “phyloseq” package to visualize dominant taxa in each city (version 1.30.0) (McMurdie and Holmes, 2013). 30 Finally, an indicator species analysis was performed for each city using the “indicspecies” package (v1.7.9) (Caceres and Legendre, 2009). 2.2.4.3 Relationships between community composition and soil properties Canonical redundancy analysis (RDA) was performed to explore relationships between EcM fungal communities and measured soil chemical properties (hypothesis 2). This was done using the “vegan” package in R (v2.5.6). The community matrix was first Hellinger transformed. The measured soil properties underwent model selection to determine which would be selected as possible predictors of fungal diversity by using a permutation test of 1000 permutations for constrained ordination. All five measured properties were selected. Variance inflation factors (VIF) were analyzed to ensure low multicollinearity among properties. To test the significance of the model, an ANOVA-like permutation test was conducted with an α = 0.05. The ordination was then displayed using a biplot containing both colour-coded site scores and arrows to represent the relative contribution of each constrained variable. Finally, Spearman correlation heat maps were constructed to identify any correlations between dominant taxa and individual soil properties using the “corrplot” package in R with an α = 0.05 (v0.84) (Wei et al., 2017). 31 Figure 2.2: Schematic diagram of experimental design including visualization of sampling approach and how each diversity type is defined. 2.3 Results 2.3.4 Overview of sequencing results In total, 4,371,830 reads were detected after the bioinformatics pipeline. Zero reads were detected in the negative control after filtering and denoising, indicating that no contamination was introduced during DNA extracting and library preparation. After filtering out all non-EcM fungal taxa using FUNGuild, 3,791,049 reads were recovered across all 90 samples, with similar total numbers from each city (Table 2.2) Reads obtained per sample ranged from 5,919 to 152,511. In total, 33 unique EcM fungal ASVs were detected across all three cities. Of the EcM fungal reads, 17.17% belonged to the phylum Ascomycota, while the other 82.83% belonged to the Basidiomycota. Many of the most abundant taxa were found in similar proportions in 32 each city, however, a small number of taxa can be seen to vary widely in abundance among cities (Figure 2.3). Table 2.2: Total number of reads and mean number of reads per sample of ectomycorrhizal fungal communities associated with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), Victoria (n = 30), and in total (n = 90). 33 Figure 2.3: Relative abundance of ectomycorrhizal fungal taxa associated with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). Each taxon represents a single ASV. When comparing the results of the mock community to what was expected, a few notable distinctions were found. Two ASVs were detected in the Illumina sample that are suspected to belong to the two fungal species that were unsuccessfully sequenced (Failed (1) and Failed (2)). An important caveat is that it is also possible that these ASVs were derived from other sample(s) and do not represent Failed (1) and Failed (2). These two ASVs were classified as Tylospora sp. and Heliotails sp.. Six ectomycorrhizal 34 fungal taxa were detected in the Illumina mock community sample; however, the proportions at which they were detected were different from those expected based on the concentrations of DNA added to the mock sample (Figure 2.4). With the staggered approach, Russula sp., Failed (1), and Failed (2) were expected to be approximately half as abundant as the other three fungi in the mock community. However, Russula sp., Tylospora sp., and Helotiales sp. presented closer to one-tenth the number of reads compared to Laccaria sp., Inocybe sp., and Amanita muscaria. 35 Figure 2.4: Community composition of the Illumina EcM fungal mock community sample (left) (n=1) compared to the expected composition (right) of the mock community based on Sanger sequencing of individual fungi before being combined and Illumina sequenced. Failed (1) and Failed (2) are fungi that were unsuccessfully Sanger sequenced before being added to the Illumina mock community sample. Each taxon in the mock column represents a single ASV. 2.3.1 Soil chemical properties Levels of pH, nitrogen, and phosphorus differed among soils collected from different cities (ANOVA, P < 0.01) (Appendix B). Soil pH was significantly higher in 36 Kelowna compared to Vancouver and Victoria (Table 2.3). Concentrations of nitrogen and phosphorus were both significantly lower in Vancouver compared with Kelowna and Victoria (Table 2.3). Table 2.3: Mean values of soil chemical properties in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). Superscripts denote statistically significant differences between cities determined by ANOVA and Tukey HSD tests, based on α = 0.05. Note. Inorganic N represents the sum of nitrate and ammonium concentrations measured from a KCl extract of fresh soil Significant differences in the dispersion of chemical properties were found among cities, indicating differences in heterogeneity, or patchiness, of soil resources (PERMDISP. P < 0.01) (Appendix B). The median distance to the centroid was significantly larger in Kelowna (0.2392) compared to Vancouver (0.1409) and Victoria (0.1679), indicating there is greater heterogeneity of soil chemical properties in Kelowna (Tukey HSD, P < 0.05) (Figure 2.5). This result provides support for Kelowna having the 37 greatest degree of environmental heterogeneity, meaning that it should be accompanied by the highest gamma diversity according to hypothesis 1a. Figure 2.5: Principal coordinates analysis (PCoA) ordination plot of soil chemical properties at sites in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Red dots represent sites in Kelowna. Green triangles represent sites in Vancouver. Blue boxes represent sites in Victoria. Standard errors are represented by colour coded ellipses while black lines indicate the distance from the centroid for each site. 2.3.1 Gamma and Alpha diversity Of the 33 total EcM fungal taxa detected, 23 were found in Kelowna, 26 in Vancouver, and 27 in Victoria. Tomentella sp. was the most abundant ASV in Kelowna 38 and Victoria. Amanita muscaria was the most abundant ASV detected in Vancouver and overall (Table 2.4). Table 2.4: All ectomycorrhiza fungal taxa identified via Illumina sequencing in association with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30) ordered by the abundance of read numbers in each city. 39 Although tests for statistical significance could not be performed on gamma diversity due to each city representing a single replicate, Kelowna appears to have the lowest gamma diversity based on richness (23), whereas Victoria displayed the highest gamma diversity (27), though the difference among cities was minor. This result was inconsistent with hypothesis 1a, which stated that gamma diversity would be highest in Kelowna. A similar result was found when comparing the mean alpha diversity of individual trees among cities (Figure 2.6). The mean richness of communities associated with individual hornbeams in Kelowna was found to be significantly lower than in Vancouver and Victoria (Figure 2.7) (Appendix D). 40 Figure 2.6: Boxplots showing comparisons of Shannon (left) and Simpson’s (right) diversity indices of ectomycorrhizal fungal communities associated with individual Carpinus betulus trees in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Boxes represent the 1st to 3rd quartiles; thick horizontal lines represent group means and whiskers extend to 1.5 x IQR. Black dots represent outlier observations. Means were not deemed significantly different based on Kruskal-Wallis tests (α = 0.05). 41 Figure 2.7: Boxplot showing comparisons of species richness of ectomycorrhizal fungal communities associated with individual Carpinus betulus trees in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Boxes represent the 1st to 3rd quartiles; thick horizontal lines represent group means and whiskers extend to 1.5 x IQR. Black dots represent outlier observations. Floating horizontal lines and stars indicate significant differences between cities. ANOVA and Tukey HSD tests indicated that communities in Kelowna had significantly lower richness than in Vancouver and Victoria (α = 0.05). 2.3.2 Beta diversity and community composition Significant differences in community composition were observed among cities with communities in each city significantly distinct from one another (PERMANOVA, P = 0.001) (Table 2.5). The cities did not differ significantly in levels of beta diversity among sites within each city (PERMDISP, P = 0.476). Overall, these findings indicate that while the degree of beta diversity among sites within each city is similar, the overall 42 composition of communities differs significantly between cities, which supports hypothesis 1b. Despite significant PERMANOVA results, Figure 2.8 shows little clear separation or grouping of sites between cities. Table 2.5: PERMANOVA results of the species composition of EcM fungal communities associated with European hornbeams in Kelowna, Vancouver and Victoria based on 999 unrestricted permutations of Hellinger transformed data. D.f. = degrees of freedom 43 Figure 2.8: Principal coordinates analysis (PCoA) ordination plot of Hellinger transformed ectomycorrhizal fungal communities associated with Carpinus betulus trees in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Red dots represent sites in Kelowna. Green triangles represent sites in Vancouver. Blue boxes represent sites in Victoria. Standard errors are represented by colour coded ellipses while black lines indicate the distance from the centroid for each site. Despite similar levels of alpha and gamma diversity and little to no obvious separation in a PCoA ordination plot, the heatmap displays notable compositional differences in the EcM fungal communities across cities (Figure 2.9). Several of the most abundant taxa stand out as contributors to the observed beta diversity between cities. Russula sp., which was prevalent in Vancouver and Victoria, was almost non-44 existent in Kelowna. Scleroderma sp., prevalent in Kelowna and Vancouver, was found at very few trees in Victoria. Tylospora sp. was detected at nearly every site in Victoria but much less frequently in Kelowna and Vancouver. Cortinarius sp. was detected almost exclusively in Vancouver. Amanita phalloides appeared to occur along a gradient where it was highly abundant in Victoria, infrequent in Vancouver, and nearly absent from hornbeams in Kelowna. Results of the indicator species analysis are consistent with the trends displayed in Figure 2.9 (Table 2.6). Amanita phalloides and Tylospora sp. were deemed indicator species in Victoria. Tuber sp. was found to be indicative of communities in Kelowna. Cortinarius sp. was found to be indicative of communities in Vancouver. Figure 2.9: Abundance heatmap of the ten most abundant individual ectomycorrhizal fungal ASVs associated with Carpinus betulus trees in Kelowna (n=30), Vancouver (n=30), and Victoria (n=30). Colouring is based on the log10 of % abundance. 45 Table 2.6: Multilevel Pattern Analysis of ectomycorrhizal fungal ASVs in communities associated with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). Each taxon presented was deemed a significant indicator species for its city based on α = 0.05. 2.3.3 Relationships between community composition and soil properties All five measured soil chemical properties were selected as constrained variables in the RDA. Low variance inflation factor scores indicated that there was a low degree of multicollinearity among variables (VIF >2.4). The constrained environmental variables overall were significantly redundant with variation in EcM fungal community structure (ANOVA like permutation test, P = 0.001) (Appendix E). Of the individual soil properties, pH had the strongest association with community structure, followed by nitrogen content (Table 2.7, Figure 2.10) (Appendix E). The overall findings of the RDA were consistent with hypothesis 2. Despite this, the measured soil properties described only a small portion of the EcM fungal community variation in the model (Adjusted R-Square, 4.4%). 46 Figure 2.10: Canonical redundancy analysis (RDA) ordination biplot of ectomycorrhizal fungal communities associated with Carpinus betulus trees in Kelowna (red, n = 30), Vancouver (green, n = 30) and Victoria (blue, n = 30). Black arrows represent constrained environmental variables used in the model. The direction and length of the arrows are proportional to the explained variation in the communities. Moisture refers to the moisture concentration of the soil. Organic refers the soil organic matter. Nitrogen represents extractable inorganic nitrogen and phosphorus represents extractable phosphorus. 47 Table 2.7: ANOVA like permutation tests for significance of individual constrained variables on the structure of ectomycorrhizal fungal communities associated with Carpinus betulus trees (n = 90). Bolded values represent statistically significant results. Results are based on 999 free permutations of Hellinger transformed data. Note. Inorganic N represents the sum of nitrate and ammonium concentrations measured from a KCl extract of fresh soil Few correlations of note were identified between soil chemical properties and abundant individual taxa within each city. The only significant correlation identified was a positive relationship between soil phosphorus content and a Scleroderma sp. in Kelowna (Figure 2.11). Although a similar relationship was observed in Vancouver and Victoria, it was not statistically significant. A Russula sp. appeared also to be positively correlated with phosphorus content in Vancouver and Victoria; however, this result was 48 also not significant. Overall, no meaningful correlations were found across cities between individual dominant taxa and soil chemical properties. Figure 2.11: Spearman correlation heatmaps displaying relationships between the ten most abundant ectomycorrhizal fungal taxa associated with European hornbeams in Kelowna (n=30), Vancouver (n=30) and Victoria (n=30) and measured environmental variables. Positive correlations appear blue and negative correlations appear red. The colour gradient indicates the strength of the correlation. Significant correlations after Bonferroni correction are shown with an asterisk (α = 0.05). “nitrogen” refers to the concentration of KCl-extractable nitrate and ammonium. “phosphorus” refers to the concentration of Bray-P1 extractable phosphorus. “organic” refers to the % of soil organic matter. 49 2.4 Discussion 2.4.1 Mock community The Illumina results of the mock community sample suggest that while our methods are accurate in detecting the richness of a sample, they may be inaccurate in approximating the abundance of some EcM fungal taxa, especially less abundant ones. Although the relative abundance of taxa was found to be inaccurate, this inaccuracy appeared to be consistent across taxa with similar expected read numbers. This indicates a lack of primer bias for individual taxa in the sequencing process. It is impossible to rule out the possibility of primer bias toward or against individual taxa in the experimental samples, however, because the mock community consisted of only six taxa compared to 33 ASVs that were identified across all experimental samples. 2.4.1 Gamma diversity of fungal communities is not associated with environmental heterogeneity Despite having the greatest degree of environmental heterogeneity, Kelowna’s EcM fungal communities did not display greater levels of gamma diversity compared to those in Vancouver and Victoria. In fact, Kelowna had the lowest total species richness, though this was not statistically significant. This finding was inconsistent with hypothesis 1a, which stated that gamma diversity would be highest in the city with the greatest degree of environmental heterogeneity. This finding can be interpreted in several ways. It is possible that EcM fungal communities associated with urban C. betulus trees do not conform to the habitat-heterogeneity hypothesis (MacArthur and MacArthur, 1961). If this were the case, diversity would likely be modulated by neutral processes such as dispersal limitation, or potentially some other mechanism. Another possibility is that 50 diversity is controlled by the relative availability of key soil resources but the degree of variation in environmental heterogeneity across cities observed in this study was not sufficient to result in different levels of gamma diversity in the sampled fungal communities. Although PERMDISP analysis showed that differences in the degree of dispersion of soil chemical properties were statistically significant, it is not clear what amount of variation of environmental heterogeneity would be biologically significant to EcM fungi. There is also evidence that the degree of severity in the environment can impact the role that environmental heterogeneity plays in modulating diversity (Yang et al., 2015). Severity was not quantified in this study, so it is not clear whether this was a factor in the lack of gamma diversity differences among cities. It is important to point out a limitation in the design of this study that makes the relationship between diversity and heterogeneity more difficult to analyze. Because environmental heterogeneity was quantified at the city level, it could only be directly compared to the gamma diversity of EcM fungal communities in each city. Therefore, diversity could not be compared statistically among cities regarding the relationship with heterogeneity. At the site level, there was not a statistically significant difference in the mean of alpha diversity indices across cities; however, this could not be related to environmental heterogeneity because heterogeneity in soil chemical properties was not measured at each tree. A study that incorporates measures of environmental heterogeneity at both the ecosystem and site levels would be more suitable for addressing the habitat-heterogeneity hypothesis in EcM fungal communities. Illumina sequencing of mycorrhizal root tips presented unique challenges for estimating richness in fungal communities. Many diversity indices, such as the Chao1 51 index, rely on singletons or rare taxa to estimate the true richness of sampled communities. Simply by selecting roots for extraction based on the presence of fungal mantle tissue, all rare EcM fungal taxa are effectively filtered out (Chao, 1984). This resulted in Chao1 richness values exactly equal to observed richness. Some Indices that estimate the true richness of communities may be inaccurate without rare taxa and therefore should be avoided when comparing diversity of different ecosystems if sequence data is derived from colonized root tips (Patil and Taillie, 1982). This represents a limitation in using EcM colonized root tips as a proxy for EcM species present in an environment; however, it is necessary for studying communities associated with a particular tree because extraradical hyphae in the soil may not be derived from individual fungi associated with the tree of interest. 2.4.2 Composition of ectomycorrhizal fungal communities differed across cities Of the 33 total EcM fungal taxa identified, 17 were present in all three cities. The 15 most abundant taxa were found in all three cities. Only 8 taxa were confined to a single city. Based on these findings, there is a striking similarity in the EcM fungal communities across cities. Despite being subjected to different climates and relative availability of soil resources, the most abundant taxa occurred in all cities sampled. A factor that could contribute to the similarities of the communities in each city is the source nursery or nurseries from which the hornbeams came. Unfortunately, there are no easily accessible records of the source nurseries for the host trees in Vancouver and Victoria, so it is unknown whether the trees share a common source. There is one indication that the source nurseries may be impacting the composition of the EcM fungal 52 communities that are associated with the mature trees. For example, root tips belonging to Amanita phalloides, a species native to Europe, were found in each city. This could indicate that nurseries are harbouring fungal species from the native range of introduced ornamental tree species such as C. betulus. Because A. phalloides fruiting bodies have become common in Vancouver and Victoria in the last couple of decades, it is also possible that the presence of A. phalloides on the roots of sampled hornbeams in Vancouver and Victoria is a result of natural spore dispersal and therefore reflective of the local species pool rather than the source nursery (Berch et al., 2016). A. phalloides has gained notoriety in British Columbia recently after the fatal poisoning of a child in Victoria in 2016 (Johnson, 2016). Although the exact number of poisonings annually in the province is unknown, it is responsible for the most mushroom related poisoning worldwide, and its range on the west coast is expanding (Diaz, 2018; Pringle et al., 2009). While fruit bodies have been found frequently in Vancouver and Victoria, I found that EcM root tips belonging to A. phalloides are much more abundant in Victoria compared to Vancouver (Berch et al., 2016), so much so that A. phalloides was found to be an indicator species for Victoria’s EcM fungal communities. Root tips colonized by A. phalloides were found associated with 22 of the 30 hornbeams sampled in Victoria, compared to only six of the 30 sampled in Vancouver. A. phalloides is known to associate with several other introduced host species in Vancouver and Victoria, so it is possible that this pattern is not universal among hosts. Though present only at very low frequencies, A. phalloides root tips were also found to be associated with four C. betulus trees in Kelowna. Despite fruit bodies never being documented in Kelowna, previous research has identified A. phalloides root tips associated with a C. betulus 53 individual at one site in the city (Planz et al., 2021, unpublished). These findings indicate that this invasive and deadly mushroom may become a concern in the province’s interior. It is unclear whether the presence of A. phalloides root tips will necessarily lead to the production of fruit bodies, so monitoring these sites will be critical in assessing the potential for death cap poisonings in Kelowna. Despite the aforementioned similarities, PERMANOVA results revealed that significant beta diversity existed between EcM fungal communities in each city, indicating that the compositions of these communities are distinct across cities (Table 2.5). This finding supports hypothesis 1b, which stated that community composition would differ significantly among cities. Considering the 15 most abundant taxa were present in all three cities, it appears likely that the local environment played a significant role in shaping the EcM fungal community structure compared to the local species pools. In the instance of taxa found in only one city, it is unclear what is preventing these species from persisting in the other urban areas. For example, Clavulina sp. is the tenth most abundant taxa in EcM fungal communities associated with C. betulus trees in Kelowna; however, it is completely absent from communities in Vancouver and Victoria. It is not clear whether abiotic factors in Vancouver and Victoria are inconsistent with the survival of this species, or if it is simply absent from the area due to a lack of other suitable host species, be it native or introduced. Due to the high diversity of introduced host plant species in urban settings, it is difficult to quantify the impacts of the local species pool on urban fungal communities; however, emerging research is showing that neighbouring plants have a significant influence on the composition of these communities (Mony et al., 2021). Another factor that could contribute to the observed 54 differences in EcM fungal communities across cities is host tree maturity. Successional patterns are known to occur in EcM fungal communities, so the observed beta diversity may be a result of communities shifting to later successional stages (Visser, 1995). Unfortunately, the ages of the C. betulus individuals were not reliably recorded in Vancouver and Victoria so it was not possible to analyze this potential link. Judging by the DBH values and age estimates from Victoria and Kelowna as well as anecdotal reports from Vancouver, the trees sampled are estimated to be 40-80 years old. The largest and therefore likely the most mature trees were located in Vancouver while the smallest trees were located in Kelowna. There was, however, significant variation in tree maturity within each city. The most abundant taxon identified throughout all cities was Tomentella sp.. Species in this genus have been found to occupy early successional stages of EcM host trees in naturalized settings (Leski and Rudawska, 2012; Twieg et al., 2007; Visser, 1995). The sixth most abundant taxon, Inocybe sp. found in all three cities, has also been shown to occupy early successional stages of EcM fungal communities (Nara et al., 2003; Twieg et al., 2007). This finding is consistent with research that has found that mature urban EcM host trees are commonly associated with early successional EcM fungal species (Danielson and Pruden, 1989). Although the exact mechanism for this phenomenon is unclear, it adds further support to the notion that urban EcM hosts and fungi face unique hardships compared to those in undisturbed environments, leading to long term persistence of early successional EcM fungal species. Though less abundant, species considered to occupy late successional stages, such as Russula sp. and 55 Cortinarius sp. were also common, especially in Vancouver and Victoria where the hornbeams are suspected to be more mature (Nara et al., 2003; Visser,1995). Despite the statistical significance of beta diversity among cities, there was very little obvious grouping of points by city visible in the PCoA ordination plot (Figure 2.8). This may be caused by a large overlap in the species present in each city and a relatively small number of species that differed largely in abundance across cities. This is supported by the results of the indicator species analysis that identified a single species indicative of EcM fungal communities associated with hornbeams in Kelowna and Vancouver, and two species indicative of communities in Victoria. Indicator species represent taxa that are characteristic of a particular environment, or in this case, a city. This is determined by the frequency and abundance of species in each city compared to one another (Cáceres and Legendre, 2009). Tuber sp. was selected as an indicator species for Kelowna. Cortinarius sp. was selected as an indicator species for Vancouver. Considering species of this genus have been shown to occupy late successional stages (Visser, 1995), this finding is consistent with the observation that Vancouver is suspected to have the oldest host trees. The taxa that were selected as indicator species in Victoria were Tylospora sp. and Amanita phalloides. 2.4.3 Soil chemical properties are associated with ectomycorrhizal fungal community structure Though the effect of soil chemical composition on EcM fungal community structure is complex and poorly understood, several studies have shown soil acidity to be a dominant influencer of community structure for EcM fungi associated with European temperate host trees (Suz et al., 2014, Van Geel et al., 2018). Although soil 56 pH is generally not considered to be a resource for EcM fungi in the conventional sense, it is proposed that soil pH works as an integrator of soil nutrient availability making for more efficient uptake of other soil resources (Glassman et al., 2017). pH has also been shown to affect the intensity at which EcM fungal species colonize host roots (Soudzilovskaia et al., 2015). With these mechanisms in mind, along with the empirical evidence cited above, I hypothesized that soil acidity would be significantly associated with the structure of EcM fungal communities associated with C. betulus in British Columbia (hypothesis 2). This hypothesis was supported through canonical redundancy analysis (Table 2.7). Soil pH was found to be most associated with variation in the EcM fungal communities followed by nitrogen content. These two variables were the only ones to show statistical significance in the RDA (Table 2.7). This finding adds more support to the growing literature that suggests that pH is a dominant driver of fungal community structure (Glassman et al., 2017; Tedersoo et al., 2014). Through permutation tests, the RDA was shown to have global significance indicating that, overall, the measured soil chemical properties were significantly related to EcM fungal community structure. However, the constrained variables only accounted for 4.4% of the observed variation in these communities. This indicated that there are likely other variables in the soil contributing to variation in the structure of these communities observed at each site. As mentioned previously, host tree age is a likely contributor to at least a portion of this variation; however, this could not be analyzed due to a lack of adequate records. It has been theorized that heavy metal concentrations present in urban environments caused by pollution could be an explanation for the decreased abundance and diversity of EcM fungi in urban areas: however, research has 57 failed to identify an association between heavy metals and EcM fungal community structure (Van Geel et al., 2018). It is well established that climatic variables such as temperature and precipitation are associated with the structure and richness of fungal communities (Tedersoo et al., 2014; Větrovský et al., 2019). Because the variation of such variables would be negligible between sites within a city, it was not appropriate to include them in the redundancy analysis. It is possible that climatic variables were responsible for a portion of the variation in EcM community structure observed between cities. Since only three cities were sampled in this study, analyzing the impacts of climatic variables would be better suited to meta-analyses and studies that incorporate broad sampling from many climatically variable regions so that an adequate gradient can be established. Despite overall significance in the association of soil chemical properties with EcM fungal community structure, very few correlations of note were identified between individual soil properties and individual taxa. The only statistically significant correlation was a positive relationship between phosphorus and Scleroderma sp. in Kelowna (Figure 2.11). This finding contradicts the assertion the Scleroderma species are considered early successional stage species; however, this correlation was not observed in Vancouver and Victoria, indicating that it is a weak and likely non-causal relationship (Jeffries, 1999; Newton, 1992). It could have been predicted that EcM fungal species considered to be early successional such as those within the genera Inocybe and Tomentella would be correlated with lower levels of soil moisture and nutrients due to their ability to colonize immature host saplings in nutrient-poor environments (Newton, 1992; Twieg et al, 2009; Visser, 1995); however, such 58 correlations were not observed. Conversely, late-successional species such as those within the genera Russula and Cortinarius could have been predicted to be correlated with higher levels of soil resources because they rely on stable and well-established soil conditions surrounding mature host trees (Newton, 1992; Visser, 1995). Again, no such correlations were observed. These findings highlight the complexity of how EcM fungal species interact with their environments, and that the successional stage in which a species is usually found is not necessarily predictive of the environment in which it thrives in a disturbed setting. 59 Chapter 3: Conclusion 3.1 Summary This research aimed to analyze EcM fungal communities associated with Carpinus betulus trees in urban areas across southern British Columbia and to investigate potential drivers of diversity and community structure. To achieve this, EcM fungal communities associated with C. betulus trees in Kelowna, Vancouver, and Victoria were characterized using NGS. The relationship between diversity and environmental heterogeneity was investigated by quantifying multiple soil chemical properties at each tree to investigate whether urban EcM fungal communities conform to the habitat-heterogeneity hypothesis (MacArthur and MacArthur, 1961). The compositions of EcM fungal communities were also compared across cities. In addition to analyzing patterns of diversity, soil chemical properties were related to EcM fungal community structure to determine which specific properties were associated with variation among fungal communities. It was hypothesized that measures of gamma diversity would be highest in the city with the greatest degree of environmental heterogeneity quantified by dispersion of soil chemical properties (hypothesis 1a). Despite significant variation in measures of environmental heterogeneity, little to no difference in levels of gamma diversity were detected across cities. This may be because diversity in urban EcM fungal communities does not conform to the habitat-heterogeneity hypothesis and is instead modulated by another mechanism, or because the variation in levels of environmental heterogeneity was not great enough to be biologically relevant to the EcM fungal species present. The ability to detect a relationship between diversity and environmental heterogeneity was 60 limited in this study by the lack of a measure of soil heterogeneity at the site level. Because ecological theories of diversity commonly applied to plant and animal communities are rarely used when studying the diversity of mutualist fungal communities, the mechanism that modulates diversity in EcM fungal communities remains unclear. Due to differences in climatic variables and assumed variation in regional species pools, it was hypothesized that the species composition of EcM fungal communities would differ significantly across cities (hypothesis 1b). It was determined that despite a large overlap in the specific taxa present in each city, the composition of communities was significantly distinct in each city. The overlap in taxa suggests that environmental variables are likely influencing the structure of EcM fungal communities associated with urban C. betulus trees. It is unclear whether taxa restricted to a single city are filtered by biotic or abiotic forces. A relatively small number of taxa were primarily responsible for the significant beta diversity across cities. This is supported by the small number of indicator species detected. The age and source nursery of host trees may be influential to the composition of urban EcM fungal communities; however, records of this information were unable to be obtained. One indicator that the host’s source nursery may be impacting fungal communities was the presence of A. phalloides, a fungus native to Europe, in all three cities. The death cap fungus was very common in Victoria and has now appeared to have spread to the interior of the province, indicating that the potential for poisonings in British Columbia’s interior may be increasing. The use of NGS to characterize EcM fungal communities allows for the known distribution of many 61 fungal species of interest, including A. phalloides, to be improved in southern British Columbian cities. It was hypothesized that the soil chemical properties measured in this study would be significantly associated with variation in EcM fungal community structure. More specifically, it was hypothesized that soil pH would have the strongest association (hypothesis 2). Through canonical redundancy analysis, it was determined that pH was primarily associated with community structure of the EcM fungal communities associated with urban C. betulus trees. This finding adds support to the growing literature showing that soil acidity is a dominant driver of community structure in soil fungi. The model had overall significance, indicating that all measured soil chemical properties together had a significant association with community structure; however, the chosen variables explained very little of the variation in communities. This may indicate that other variables not measured in this study may be significantly influencing EcM fungal community structure. Climatic variables may play a significant role in influencing community structure; however, this study was not designed to address these variables as the sampling of only three cities results in a weak gradient of variables such as temperature and precipitation. No significant correlations were found between the most abundant taxa and individual soil chemical properties that were consistent across cities, highlighting the complexities of a local environment’s influence on EcM fungal community composition. 62 3.2 Novelty of work This study represents the first characterization of EcM fungal communities associated with C. betulus trees using NGS. It is also one of the very few studies that have analyzed urban EcM fungal communities using NGS and the first to do so in British Columbia. Considering the majority of the world’s population live in urban areas, it may be becoming more relevant to research ecological patterns in urban settings. This study implemented a mock community constructed from taxa present in experimental samples and identified via Sanger sequencing. This approach allowed for a direct comparison of mock community results to experimental results to assess the accuracy of Illumina NGS methods. This approach for mock community utilization may be more appropriate than the use of commercially available mock communities that bear little resemblance to the communities being studied. The use of NGS of EcM root tips allowed us to improve the known distribution of many soil fungal species in southern British Columbia. This is perhaps most notable in the case of A. phalloides, which has been responsible for fatal poisonings in cities on the North American west coast. A. phalloides was found to be highly abundant in association with C. betulus trees in Victoria and was also identified beyond its known range in Kelowna, indicating that the potential for future poisonings may be increasing in the province. Despite the rejection of hypothesis 1a, this study serves as an example of utilizing ecological theory to make predictions about diversity in EcM fungal communities. This study was designed to test a potential mechanism for the modulation of diversity in EcM fungal communities, rather than solely analyzing variables that correlate with diversity. In an attempt to improve the understanding of how individual soil chemical properties influence EcM fungal 63 community structure, associations were studied using both canonical redundancy analysis and individual correlations. 3.3 Future research Addressing the shortcomings of this study’s design would be a promising avenue to determine if EcM fungal communities conform to the habitat-heterogeneity hypothesis. This would include measuring environmental heterogeneity at the site level and sampling from cities or regions with greater variation in levels of environmental heterogeneity. Future research may further improve the understanding of how diversity is modulated in EcM fungal communities by testing other ecological theories using EcM models. This study utilized EcM fungal root tips as a proxy for species associated with a host tree. While this method avoids the potential for accidentally sampling fungal species associated with an unintended host, it also leaves out the analysis of extramatrical mycelium in the soil, which may be highly relevant to nutrient dynamics in the soil. Future research may benefit from using a tandem of both approaches. EcM fungal communities have been characterized using NGS in only a small number of EcM host species. By sampling from more host species, a more complete understanding can be gained of the interactions between the numerous EcM fungal species and their hosts. This study showed that it is possible to utilize sequencing of root tips to identify fungal species well outside of the known ranges of their sporocarps. This strategy may be used to predict the potential range of invasive fungal species such as A. phalloides for the purpose of monitoring and the implementation of mitigation strategies. The use of Illumina MiSeq alongside an experimental sample derived mock community provides a high degree of confidence in the accuracy and precision of taxonomic assignment. A 64 drawback to this method, as opposed to other contemporary technologies such as PacBio sequencing, is the relatively short read lengths obtained. Because of the variability in the length of the ITS region across EcM fungal species, the maximum 600bp sequence length obtained via Illumina sequencing was insufficient to result in all species matching to taxonomic databases to the genus or species level. For this reason, future studies may consider utilizing other sequencing technologies, even if only to draw comparisons. 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The effect of environmental heterogeneity on species richness depends on community position along the environmental gradient. Scientific reports, 5(1), 1-7. 79 Appendices Appendix A: Sampling locations Table 1: Approximate location of individual hornbeam trees sampled corresponding to sample ID. 80 Appendix B: Soil data ANOVA and PERMDISP results Table 1: ANOVA results of pH differences between soils in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). Table 2: ANOVA results of nitrogen concentration differences between soils in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). Table 3: ANOVA results of phosphorus concentration differences between soils in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). 81 Table 4: PERMDISP results of soil data comparing environmental heterogeneity in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). 82 Appendix C: Richness ANOVA Table 1: ANOVA results of mean richness between ectomycorrhizal fungal communities associated with individual Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). 83 Appendix D: Beta diversity PERMDISP Table 1: PERMDISP analysis of ectomycorrhizal fungal communities associated with Carpinus betulus trees in Kelowna (n = 30), Vancouver (n = 30), and Victoria (n = 30). 84 Appendix E: RDA overall ANOVA Table 1: ANOVA like permutation tests for overall significance of constrained variables on the structure of ectomycorrhizal fungal communities associated with Carpinus betulus trees (n = 90).
Thesis/Dissertation
eng
Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Klironomos, John
Durall, Daniel
Ectomycorrhizal fungal communities associated with Carpinus betulus trees across urban areas of British Columbia
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