Browsing by Author "Vilgalys, Rytas J"

Symbioses have shaped our modern world, providing for the air we breathe; for the plant and animal diversity we celebrate; and for the functioning of ecosystems from the tops of mountains to the ocean floor. Here I study symbiosis using fungal bacterial interactions as a model for understanding symbiotic dynamics. In this dissertation I present interpretations of experimental data about fungal bacterial interactions that lend insight into dynamics of symbiotic establishment and consequences of long-term endosymbiosis. More specifically, I examine the interactions of a plant-associated zygomycete, Mortierella elongata, and its interactions with several Betaproteobacteria in the Burkholderiales. I used genome sequencing, comparative genomics, physiological assays, and time-lapse microfluidic videography to ask the following questions; How are bacterial fungal symbioses initiated? How do bacteria and fungi communicate? What resources do these microbes share? Are long-term symbioses essential for one or both partners? What are the impacts of removing long-term endosymbionts for fungal host physiology? What are the effects long-term fungal endosymbiosis on bacterial genome content?

In chapter 1 I present lessons learned from genome sequencing of fungus Mortierella elongata and its primary resident endosymbiont, Mycoavidus cysteinexigens. I tested the hypothesis that genome reduction is a commonality of eukaryotic endosymbionts, and that characteristic genes and pathways are impacted by gene loss and inactivation in endosymbionts. I found that compared to its free-living relatives, M. cysteinexigens has a highly reduced genome and has lost genes coding for the biosynthesis of amino acids and intermediates of glycolysis, among other metabolic pathways. I describe a method for clearing fungi of endosymbionts using antibiotics. I report comparative physiological data for the cleared and uncleared strains and draw conclusions about the nature of their interactions based on the behavior of the fungal host lacking the endosymbiont. I tested the hypothesis that sharing of fungal fatty acids underpins this symbiosis, as suggested by the genome sequences of both microbes. I found that when cleared of endosymbionts, M. elongata grows more rapidly and accumulates fatty acids that are likely used by M. cysteinexigens when present.

In chapter 2 I investigate the transcriptional control of fungal-endosymbiont phenotypes. I continued working with the cleared and uncleared strains developed in chapter 1 and quantified transcript abundance in each isolate. I assigned functions to differentially expressed genes by identifying homologues in the fungal genetic model organism Saccharomyces cerevisiae. I layered on transcriptional data to the patterns that emerged from comparative analyses in chapter 1to better understand fungal response to endosymbiosis. I showed that differential expression of conserved genes underpin the increases in growth and altered metabolism in M. elongata when cleared of M. cysteinexigens. I found that endosymbiont presence is associated with toggling of metabolic programs that result in resources more or less bioavailable to M. cysteinexigens based on metabolic capability predicted by genome annotation. I found that genes with homologues in mating pheromone perception pathways are differentially regulated in cleared isolates of M. elongata, and that this aspect of clearing is shared by other isolates of M. elongata when cleared of their bacterial endosymbionts.

In chapter 3 I examine dynamics of pre-symbiotic signaling events between fungi and bacteria using Mortierella elongata and a free-living bacterium, Burkholderia BT03. Using microbial growth assays and a suite of conditioned medias I showed that growth stimulation is mutual for fungi and bacteria, and that signaling leading up to symbiotic phenotypes involves multiple bi-directional signal exchanges. I designed and used a microfluidic platform along with plate based and liquid culture systems to compare fungal growth rates in response to conditioned medias. By extrapolating rates from microbial growth assays including M. elongata, Burkholderia BT03 and related microbes, I inferred directionality, order, conditionality, specificity, and nature of signal exchange leading to microbial growth stimulation in this system.

As a whole this thesis explores how comparative microbial genomics and phenotypic assays can provide mechanistic insight into symbiotic establishment and the effects of long-term symbioses. The results presented here provide novel insights into biotic and abiotic factors dictating symbiotic establishment. Second, they suggest long-term endosymbionts of eukaryotic cells experience convergent gene loss. Lastly they emphasize that long-term endosymbionts strongly impact host metabolism, and that host-microbe metabolic intertwining is a commonality of many symbioses. The use of a systems biology approach to generate comparative genomics data on multiple levels that enable insight into the consequences of fungal bacterial symbioses is a novel contribution for the field.

The mycorrhizal mutualism is one of the earliest and most influential of all terrestrial symbioses. As the primary method used by most plants to acquire nutrients from the soil, mycorrhizal fungi help to shape the structure and composition of many ecosystems. Ectomycorrhizal (EM) fungi play an especially significant role because most EM fungi prefer a limited number of host taxa, and EM plant species likewise associate with only a fraction of the available EM fungi. This host preference issue, combined with the high diversity of EM fungi in forest ecosystems, complicates interspecies competition both among fungi and among plants, because these plant and fungal communities interact.

Despite recent attempts at documenting mycorrhizal fungi in the context of ecological succession, many questions remain about the underlying causal relationships among EM fungi, soil conditions, and plant community assembly. The succession of mycorrhizal fungi often mirrors the succession of plants, and ectomycorrhizal (EM) community composition may affect the outcome of competition among trees during succession. In a pine-oak seral system, we tested the ability of Pinus taeda and Quercus alba seedlings to associate with EM fungi when planted under both conspecific and heterospecific adults. We found that EM communities under pine and oak canopy were distinct regardless of seedling identity, indicating that the fungal associations of adult trees determine which EM species are available in the soil. In addition, pine seedlings planted under oak canopy showed decreased mycorrhization and growth compared to those planted under pine canopy, while oak seedlings showed no negative effects of heterospecific planting. This impaired ability of pine seedlings to associate with the EM community established under oaks may deter pine recruitment and facilitate the late-seral replacement of pines with oaks.

While EM fungal communities correlate with the dominant species of host tree, soil properties do as well, making it difficult to establish causality among these three variables. Soil was collected from oak- and pine-dominated stands and dried to kill off mature mycelium, leaving only the spore bank as a source of inoculum for pine and oak seedlings. EM root tips were collected for molecular identification of fungal species based on ITS barcoding, and soil samples from field and laboratory conditions were analyzed for fungal diversity using 454 sequencing. We found a reduced influence of canopy type and a more pronounced influence of seedling identity when compared to the EM communities on seedlings planted in the field, suggesting that adult trees do alter the availability of fungi by directly promoting the growth of their preferred EM associates.

The availability of EM fungi can also affect seedlings at the interface between EM- an AM-dominated forest. We tested the hypothesis that seedlings of Dicymbe corymbosa which recruit outside of monodominant stands have limited access to EM symbionts compared with those which recruit inside D. corymbosa stands. EM root tips and rhizosphere soil were collected from seedlings along two transects inside monodominant stands and three transects in the transition zone into mixed forest dominated by AM associates. Seedlings inside monodominant stands yielded both a greater quantity of mycorrhized root tips and a higher diversity of EM species than transition zone seedlings. Of the fungal families commonly found on adult roots, the Boletaceae were notably underrepresented on all seedlings. In the transition zones, high-throughput sequencing of soil also detected a decrease in EM diversity with distance from the parent tree.

Seedlings of D. corymbosa may benefit from recruiting within monodominant stands by tapping into common mycorrhizal networks (CMNs) to acquire low-cost nitrogen and, potentially, photosynthates produced by conspecific adults. Leaves of stand adults, stand seedlings, and mixed-forest seedlings were collected for stable isotope analysis to track the transfer of nitrogen and carbon through CMNs. The δ13C and δ15N results contradicted each other, suggesting that more complicated interactions may be playing out among adults, seedlings, and fungi.

The Piedmont region of the southeastern United States has undergone considerable land-use change since settlement by Europeans and Africans. Forests were cleared for agriculture, followed centuries later by land abandonment. Following abandonment, natural recruitment, plantings for erosion control, and plantation forestry have resulted in a large area of the region covered by loblolly pine, Pinus taeda. Today, the Piedmont is a mosaic of farm fields, pastures, pine forests, and relic woodlots. The Calhoun Experimental Forest, located in Union County, SC, has provided a unique history of land use change's alteration of soil properties and processes, the ability of reforestation to restore or deplete soil fertility, and provided insights into the effects this change has on biological diversity.

In this work, the diversity of fungi living in soil is examined in the context of land-use change and soil biogeochemical change in and around the Calhoun Forest. This study uses molecular tools to identify fungal species from soil and to identify mycorrhizal associates of loblolly pine in a bioassay of propagule diversity, and proposes a novel use of quantitative PCR to quantify the relative abundance of major fungal families affected by land-use change.

Fungal diversity in soils is high in all land uses, but fungal communities shift from agricultural field communities largely comprised of unicellular ascomycetes and basal lineages to forest communities dominated by saprophytic and symbiotic basidiomycetes. In addition to this shift across a land use gradient, fungal communities are also responding to changes in carbon quantity and quality, biologically available nitrogen and phosphorus, pH, acidity and texture.

ECM propagule communities also differ across a land use gradient of cultivated fields, grasslands, pine forests, and mixed hardwood stands. There are few ECM propagules able to associate with loblolly pine in cultivated and grassland soils. There is a trend towards higher ECM diversity in the hardwood and pine soils, and both of those soil communities are distinct from each other as well as from soils from field treatments.

Quantitative PCR, coupled with a nested set of taxon-specific, fungal primers, is a potential way to estimate the abundance of the given taxon relative to all fungi in an environmental DNA. Primers specific to several taxonomic level of fungi were tested to confirm amplification in PCR, then were tested for taxonomic specificity by generating clone libraries with environmental DNA. Several of the successful primers were tested with soil DNA extracts in QPCR and the calculated ratios of fungal abundance varied widely by method of analysis. The results suggest that many repeated measurements and many replicates are required for a robust estimate of the relative abundance of a specific taxon.

Human migrations over many centuries have resulted in considerable movements of organisms beyond their natural ranges. Study of these exotic introductions can help us to understand their ecology and biology as well as provide clues for the management of biological invasions. Suillus luteus is one of the most abundant globally introduced symbiotic fungi associated with exotic pine afforestation. I studied the population genomics of native and introduced populations of this ectomycorrhizal fungus to understand its evolution and genetics. Whole genomes of 274 S. luteus strains along with strains of closely related sister species were sequenced using the Illumina sequencing platform. These strains represent native populations of S. luteus from its native range across Europe and Asia as well as from five introduced populations in North America, South America, Australia, New Zealand, and Africa. Taking advantage of this rich dataset, I analyzed and compared the genetic relationships, population genetic parameters, and signatures of selection across populations to understand the natural history of S. luteus, the processes of its introductions, and the evolutionary consequences in the introduced populations.Using the sister species S. brunnescens as an outgroup, phylogenomic, multi-species coalescent model, and admixture analyses of SNP data revealed three genetically divergent clades in the native range, which likely represent at least two cryptic species within the single morphological species S. luteus. Phylogenomic analyses showed that all introduced populations belong to the Central Europe clade, and multiple independent introductions for exotic populations in Australia, New Zealand, South America, Africa, and North America. North American introduced populations were found to be the least differentiated from the Central Europe population, implying a more recent introduction and/or limited sexual reproduction since the introduction. The introduced population from South America was the most isolated from others, with little evidence of gene flow with other populations. All introduced populations experienced different levels of loss in genetic diversity and had more limited numbers of mating types, which suggests those exotic population experienced genetic bottlenecks during the introductions. Demographic analyses revealed significant expansion of population size for introduced populations in Western Australia and Argentina. Higher inbreeding coefficients were observed in all introduced populations, likely as a result of smaller founder population sizes. However, high rates of outcrossing in the introduced populations and selfing only observed in one introduced population indicate the changes in mating system of S. luteus is not common in introduction contexts. I examined the occurrence of local adaptation in each population by scanning genome regions of high divergence and selective sweeps. Several genes associated with regulatory functions were observed to have shared signatures of selections among multiple introduced populations, implying their important roles in adaptation for diverse exotic environments. The genomics approach used to study the mating type loci of S. luteus was applied to genomes of other Suillus and Rhizopogon species to understand the evolution of the self-incompatibility system in suilloid fungi. I employed a novel method of de novo assembly to assemble the highly divergent self-incompatibility loci and then recovered haplotypes from the genomes. Strong patterns of trans-specific polymorphisms and high sequence divergence in the HD MAT locus suggest that this locus is under strong balancing selection and confirm that this locus functions to control mating types in suilloid fungi. Phylogenetic analyses of HD MAT haplotypes showed that HD MAT loci were multiallelic in suilloid fungi and that the origins of some mating types predated the split between Rhizopogon and Suillus. Population genomics of native and introduced populations provides new insight into the evolution of S. luteus, the process and consequences of introduction, and the evolution associated with global exotic introductions. This study also demonstrates how the genomics and theories of molecular evolution can be used to study the evolution and genetics of fungi albeit the difficulties in ecological survey and limited genetic methods in ectomycorrhizal fungi.

The truffle genus Tuber (Ascomycota, Pezizales, Tuberaceae) produces underground mushrooms widely sought as edible fungi. Tuber species are distributed throughout Northern hemisphere forests and form obligate ectomycorrhizal symbiosis with trees within the Pinaceae, Fagaceae, Betulaceae, and Juglandaceae.

The transition to a truffle form (from an epigeous form) has occurred independently, multiple times in both the Ascomycetes and Basidiomycetes. One instance has given rise to the Tuberaceae, which is composed entirely of obligate ectomycorrhizal species. Attempts to cultivate European truffle species T. melanosporum, T. aestivum, and T. borchii are underway in North America and other parts of the world and have been met with mixed success.

The overarching goal of my dissertation is to address the systematics, ecology, and biogeography of Tuber within a phylogenetic framework. Multiple loci were sequenced from Tuber ascoma collected worldwide including ectomycorrhizae, though an emphasis was placed on sampling taxon within North American. Maximum likelihood, maximum parsimony, and Bayesian inference were used for phylogenetic reconstructions.

A taxonomic and phylogenetic overview of the family Tuberaceae is presented in Chapter 1. Tuber is resolved as monophyletic. In Chapter 2, through greater taxon sampling including epigeous and hypogeous Helvellaceae outgroups and related South American taxa, a resolved multi-gene phylogeny of the Tuberaceae and putative epigeous ancestor of Tuber is presented. A previously unknown South American lineage that contains both epigeous and hypogeous taxa is resolved as sister to the Tuberaceae. Chapter 3 is focused on issues of cryptic speciation and taxonomy within the Tuber gibbosum clade. The four species resolved in the Gibbosum clade appear to be endemic to the Pacific Northwest and associated primarily with Gymnosperms. Chapter 4 is a meta-analysis of all known Tuber ITS rDNA sequences (e.g. from Genbank and generated from herbarium collections) available at the time. These were placed within the Tuber phylogeny to assess species diversity, long-distance dispersal, and host associations. In total, 120 phylotypes were detected (based on a 96% similarity criterion). Tuber shows high levels of continental endemism. I hypothesize that species shared between continents and having low ITS variability (<1%) are the result of recent human-mediated introduction events. Chapters 5 and 6 are focused on the ectomycorrhizal ecology of the economic truffle T. lyonii, which is native to Eastern and Southern North America. There is a phenomenon of Tuber lyonii fruiting in pecan orchards. Pecans (Carya illinoinensis) are in the Juglandaceae, an understudied ectomycorrhizal plant family. I sampled the ectomycorrhizal communities of pecan orchards (associated with the production of the North American truffle species Tuber lyonii). In Chapter 5 I discuss four Tuber taxa discovered in these pecan orchards, their abundance and haplotype diversity. Chapter 6 examines the ectomycorrhizal communities across the five pecan orchards sampled. I show that multiple Tuber species, including Tuber lyonii, are dominant in the ectomycorrhizal community. Chapters 7 and 8 focus on black truffles in the Melanosporum clade. In Chapter 7 I document that Tuber indicum has been introduced into North America multiple times, and through ectomycorrhizal synthesis I demonstrate that this Asian species can associate readily with angiosperm and gymnosperm hosts endemic to North American. In Chapter 8 I describe a quick and reliable method for the determination of Tuber melanosporum. The method is based on direct PCR and species-specific primers and is very useful for rapid diagnostics. I have adapted this approach for other truffle and mushroom species.

Three major findings emerge from my dissertation research: 1) Tuber is more diverse than previously realized; 2) Tuber exhibits high levels of regional and continental endemism; 3) Taxonomic issues remain in many species complexes worldwide (including the Tuber candidum complex in North America, the Tuber excavatum complex in Europe, the Tuber indicum complex in Asia). Taxonomic challenges also remain regarding species known only from ectomycorrhizal or anamorphic states. The discovery of additional Tuber species is expected as the truffle flora of undersampled regions become better studied and incorporated into the Tuberaceae phylogeny.

This dissertation is an investigation of the systematics, phylogeography, and ecology of a globally distributed fungal family, the Elaphomycetaceae. In Chapter 1, we assess the literature on fungal phylogeography, reviewing large-scale phylogenetics studies and performing a meta-data analysis of fungal population genetics. In particular, we examined the possible effects of asexuality, trophic niche, dispersal method, and ocean barriers on population structure. In Chapter 2, we examine the systematics and phylogeography of the Elaphomycetaceae, a family consisting of the truffle genus Elaphomyces and the stalked genus Pseudotulostoma, hypothesizing that the mammal-dispersed truffle would show evidence of dispersal limitation. Using DNA sequence data, we determined that Pseudotulostoma is derived from a lineage of Elaphomyces, indicating that Elaphomyces as currently defined is paraphyletic. The distribution of each subgenus of Elaphomyces is nearly global; representative species have been found on every continent save Africa and Antarctica. This biogeographic pattern does not follow the pattern expected by a scenario of continental vicariance. Dating analysis in BEAST confirmed that broadly distributed clades are, in most cases, too young for this pattern to be explained by continental vicariance, indicating that occasional long-distance dispersal has been a significant component in the biogeographic history of the Elaphomycetaceae. This finding contradicts our initial hypothesis that the mammal-dispersed truffles would be dispersal- limited. In Chapter 3, we investigate the role of Elaphomyces as a host for the fungal parasite Elaphocordyceps, a parasite derived from insect pathogens that attacks both insect larvae and Elaphomyces, its only fungal host. We examined the biogeography of Elaphocordyceps isolated from Elaphomyces specimens in order to test whether it, like its host, showed recent connections between the Southern and Northern Hemispheres. We also evaluated the pathogenicity of infection as determined by a visual rubric for the truffle gleba, the phylogenetic distribution of Elaphocordyceps species on its host, testing for seasonal, climate, and host-parasite effects. In Chapter 4, based on the phylogeographic pattern seen in Elaphomyces that resembles that of some air-dispersed fungi, we used theoretical and experimental methods to test whether Elaphomyces could be dispersed by air. We tested the capacity for air dispersal with an experimental test of passive air dispersal on the powdery spores of Elaphomyces morettii and found that these large spores could disperse over a short distance (10 m) in comparable numbers with the spores of the giant puffball Calvatia cyathiformis, which is known to be air-dispersed. The major findings of this thesis are that 1) fungi in general show high dispersal ability, but that trophic niche and dispersal mode may affect population structure, 2) that Pseudotulostoma, a stalked genus, is derived from the truffle Elaphomyces, 3) that the Elaphomycetaceae have experienced frequent long-distance dispersal despite 4) that the fitness of Elaphomyces as indicated by glebal development varies with host-parasite interactions based on species identity, but not with climate or season, and that 5) Elaphomyces spores, should they be released into the air, can remain in the air long enough to be dispersed long distances by the wind. The overall conclusion of this thesis is that, while Elaphomyces is clearly reliant on animal vectors for excavation and dispersal, its past history of long-distance dispersal and current spore trajectories indicate it can be passively air-dispersed as well.

Fungal pathogens likely play an important role in regulating populations of tree seedlings and preserving forest diversity, due to their ubiquitous presence and differential effects on survival. Host-specific mortality from natural enemies is one of the most widely tested hypotheses in community ecology to explain the high biodiversity of forests. The effects of fungal pathogens on seedling survival are usually discussed under the framework of the Janzen-Connell (JC) hypothesis, which posits that seedlings are more likely to survive when dispersed far from the parent tree or at low densities due to pressure from host-specific pathogens (Janzen 1970, Connell 1971). One of the key challenges to assessing the importance of JC effects has been to identify and quantify the effects of the large numbers of potential pathogens required to maintain host diversity. The primary objectives of this research were to (1) characterize the fungi associated with seedling disease and mortality for a number of important southeastern US forest tree species; and (2) determine if these associations are consistent with the Janzen-Connell hypothesis in terms of differential effects on seedling survival.

Culture-based methods and ribosomal DNA (rDNA) sequencing were used to characterize the fungal community in recently dead and live seedlings of thirteen common tree species in a temperate mixed hardwood forest (North Carolina, USA), with the goal of identifying putative seedling pathogens. Cultures were initially classified and grouped into 130 operational taxonomic units (OTUs) using 96% internal transcribed spacer (ITS) sequence similarity; 46% of all OTUs were found only once. Using rarefaction, it was concluded that the richness of the system was not fully sampled and likely included over 200 taxa (based on non-parametric richness estimators). Species richness did not differ between sampling sites or among the five most common hosts sampled. The large ribosomal subunit (LSU) region of rDNA was then sequenced for representative samples of common OTUs and refined identifications using a constrained maximum likelihood phylogenetic analysis. Phylogenetic placement verified strong BLAST classifications, and allowed for placement of unknown taxa to the order level, with many of these unknowns placed in the Leotiomycetes and Xylariales (Sordariomycetes).

Next, a hierarchical Bayesian model was developed to predict the effects of multiple putative fungal pathogens on individual seedling survival, without forcing the effects of multiple fungi to be additive. The process of disease was partitioned into a chain of events including incidence, infection, detection, and survival, and conditional probabilities were used to quantify each component individually, but in the context of one another. The use of this modeling approach was illustrated by examining the effects of two putative fungal pathogens, Colletotrichum acutatum and Cylindrocarpon sp. A, an undescribed species of Cylindrocarpon, on the survival of five seedling hosts in both a maximum likelihood and Bayesian framework.

Finally, the model was used to assess the impacts of these fungi on seedling survival, alone and in combination, using data on five potential fungal pathogens and five hosts. Multi-host fungi had differential effects on seedling survival depending on host identity, and multiple infections may impact survival even when single infections do not. Evaluating these interactions among multiple plant and fungal species generates a set of targeted hypotheses of specific plant-fungal combinations that could help us better understand pathogen-driven diversity maintenance at larger scales than previously possible. Building on these results, some recommendations are provided as to how the Janzen-Connell hypothesis can be re-evaluated with respect to host specificity, pathogen distribution, and environmental context.