Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.

Legume-nodulating rhizobia are widespread in soils and plants across the island of O'ahu, Hawai'i.

Legumes and their interaction with rhizobia represent one of the most well-characterized symbioses that are widespread across both natural and agricultural environments. However, larger distribution patterns and host associations on isolated Pacific islands with many native and introduced hosts have not been well-documented. Here, we used molecular and culturing techniques to characterize rhizobia from soils and 24 native and introduced legume species on the island of O'ahu, Hawai'i. We chose two of these isolates to inoculate an endemic legume tree, Erythina sandwicensis to measure nodulation potentials and host benefits. We found that all rhizobia genera can be found in the soil, where only Cupriavidus was found at all sites, although at lower abundance relative to other more common genera such as Rhizobium (and close relatives), Bradyzhizobium, and Devosia. Bradyrhizobium was the most common nodulator of legumes, where the strain Bradyrhizobium sp. strain JA1 is a generalist capable of forming nodules on nine different host species, including two native species. In greenhouse nursery inoculations, the two different Bradyrhizobium strains successfully nodulate the endemic E. sandwicensis; both strains equally and significantly increased seedling biomass in nursery inoculations. Overall, this work provides a molecular-based framework in which to study potential native and introduced rhizobia on one of the most isolated archipelagos on the planet.

Genomic determination of breeding systems and trans-specific evolution of HD MAT genes in suilloid fungi.

Studying the signatures of evolution can help to understand genetic processes. Here, we demonstrate how the existence of balancing selection can be used to identify the breeding systems of fungi from genomic data. The breeding systems of fungi are controlled by self-incompatibility loci that determine mating types between potential mating partners, resulting in strong balancing selection at the loci. Within the fungal phylum Basidiomycota, two such self-incompatibility loci, namely HD MAT locus and P/R MAT locus, control mating types of gametes. Loss of function at one or both MAT loci results in different breeding systems and relaxes the MAT locus from balancing selection. By investigating the signatures of balancing selection at MAT loci, one can infer a species' breeding system without culture-based studies. Nevertheless, the extreme sequence divergence among MAT alleles imposes challenges for retrieving full variants from both alleles when using the conventional read-mapping method. Therefore, we employed a combination of read-mapping and local de novo assembly to construct haplotypes of HD MAT alleles from genomes in suilloid fungi (genera Suillus and Rhizopogon). Genealogy and pairwise divergence of HD MAT alleles showed that the origins of mating types predate the split between these two closely related genera. High sequence divergence, trans-specific polymorphism, and the deeply diverging genealogy confirm the long-term functionality and multiallelic status of HD MAT locus in suilloid fungi. This work highlights a genomics approach to studying breeding systems regardless of the culturability of organisms based on the interplay between evolution and genetics.

Fungal Hyphosphere Microbiomes Are Distinct from Surrounding Substrates and Show Consistent Association Patterns.

Mat-forming fungi are common in forest and grassland soils across the world, where their activity contributes to important soil ecological processes. These fungi maintain dominance through aggressive and abundant hyphae that modify their internal physical and chemical environments and through these modifications select for what appears to be a suite of mycophilic bacteria. Here, the bacteria associated with the fungal mats of Leucopaxillus gentianeus and Leucopaxillus albissimus from western North America are compared to adjacent nonmat substrates. Within the mats, the bacterial richness and diversity were significantly reduced, and the community composition was significantly different. The bacterial community structure between the two fungal hosts was marginally significant and indicated a shared set of bacterial associates. The genera Burkholderia, Streptomyces, Bacillus, Paenibacillus, and Mycobacterium were significantly abundant within the fungal mats and represent core members of these hypha-rich environments. Comparison with the literature from fungal mat studies worldwide showed that these genera are common and often significantly found within fungal mats, further reinforcing the concept of a mycophilic bacterial guild. These genera are incorporated into a synthesis discussion in the context of our current understanding of the nature of fungal-bacterial interactions and the potential outcomes of these interactions in soil nutrient cycling, plant productivity, and human health. IMPORTANCE Fungi and bacteria are the most abundant and diverse organisms in soils (perhaps more so than any other habitat on earth), and together these microorganisms contribute to broad soil ecosystem processes. There is a suite of bacteria that appears consistently within the physical space called the hyphosphere, the area of influence surrounding fungal hyphae. How these bacteria are selected for, how they are maintained, and what broader ecological functions they perform are subjects of interest in this relatively new field-the cross-kingdom interactions between fungi and bacteria. Understanding their cooccurrence and their interactions can open new realms of understanding in soil ecological processes with global consequences.

A ridge-to-reef ecosystem microbial census reveals environmental reservoirs for animal and plant microbiomes.

Microbes are found in nearly every habitat and organism on the planet, where they are critical to host health, fitness, and metabolism. In most organisms, few microbes are inherited at birth; instead, acquiring microbiomes generally involves complicated interactions between the environment, hosts, and symbionts. Despite the criticality of microbiome acquisition, we know little about where hosts' microbes reside when not in or on hosts of interest. Because microbes span a continuum ranging from generalists associating with multiple hosts and habitats to specialists with narrower host ranges, identifying potential sources of microbial diversity that can contribute to the microbiomes of unrelated hosts is a gap in our understanding of microbiome assembly. Microbial dispersal attenuates with distance, so identifying sources and sinks requires data from microbiomes that are contemporary and near enough for potential microbial transmission. Here, we characterize microbiomes across adjacent terrestrial and aquatic hosts and habitats throughout an entire watershed, showing that the most species-poor microbiomes are partial subsets of the most species-rich and that microbiomes of plants and animals are nested within those of their environments. Furthermore, we show that the host and habitat range of a microbe within a single ecosystem predicts its global distribution, a relationship with implications for global microbial assembly processes. Thus, the tendency for microbes to occupy multiple habitats and unrelated hosts enables persistent microbiomes, even when host populations are disjunct. Our whole-watershed census demonstrates how a nested distribution of microbes, following the trophic hierarchies of hosts, can shape microbial acquisition.

Fungal Community Shift Along Steep Environmental Gradients from Geothermal Soils in Yellowstone National Park.

Geothermal soils offer unique insight into the way extreme environmental factors shape communities of organisms. However, little is known about the fungi growing in these environments and in particular how localized steep abiotic gradients affect fungal diversity. We used metabarcoding to characterize soil fungi surrounding a hot spring-fed thermal creek with water up to 84 °C and pH 10 in Yellowstone National Park. We found a significant association between fungal communities and soil variable principal components, and we identify the key trends in co-varying soil variables that explain the variation in fungal community. Saprotrophic and ectomycorrhizal fungi community profiles followed, and were significantly associated with, different soil variable principal components, highlighting potential differences in the factors that structure these different fungal trophic guilds. In addition, in vitro growth experiments in four target fungal species revealed a wide range of tolerances to pH levels but not to heat. Overall, our results documenting turnover in fungal species within a few hundred meters suggest many co-varying environmental factors structure the diverse fungal communities found in the soils of Yellowstone National Park.

Comparative genomics reveals dynamic genome evolution in host specialist ectomycorrhizal fungi.

While there has been significant progress characterizing the 'symbiotic toolkit' of ectomycorrhizal (ECM) fungi, how host specificity may be encoded into ECM fungal genomes remains poorly understood. We conducted a comparative genomic analysis of ECM fungal host specialists and generalists, focusing on the specialist genus Suillus. Global analyses of genome dynamics across 46 species were assessed, along with targeted analyses of three classes of molecules previously identified as important determinants of host specificity: small secreted proteins (SSPs), secondary metabolites (SMs) and G-protein coupled receptors (GPCRs). Relative to other ECM fungi, including other host specialists, Suillus had highly dynamic genomes including numerous rapidly evolving gene families and many domain expansions and contractions. Targeted analyses supported a role for SMs but not SSPs or GPCRs in Suillus host specificity. Phylogenomic-based ancestral state reconstruction identified Larix as the ancestral host of Suillus, with multiple independent switches between white and red pine hosts. These results suggest that like other defining characteristics of the ECM lifestyle, host specificity is a dynamic process at the genome level. In the case of Suillus, both SMs and pathways involved in the deactivation of reactive oxygen species appear to be strongly associated with enhanced host specificity.

Community RNA-Seq: multi-kingdom responses to living versus decaying roots in soil.

Roots are a primary source of organic carbon input in most soils. The consumption of living and detrital root inputs involves multi-trophic processes and multiple kingdoms of microbial life, but typical microbial ecology studies focus on only one or two major lineages. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis ("community RNA-Seq") and simultaneously assess the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in 13CO2-labeled microcosms amended with 15N-root litter to identify the preferences of rhizosphere organisms for root exudates (13C) versus decaying root biomass (15N) using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group's more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving complex interactions, and our methodology can be used to track the trajectory of carbon through multi-kingdom soil food webs.

Fungal heavy metal adaptation through single nucleotide polymorphisms and copy-number variation.

Human-altered environments can shape the evolution of organisms. Fungi are no exception, although little is known about how they withstand anthropogenic pollution. Here, we document adaptation in the mycorrhizal fungus Suillus luteus driven by soil heavy metal contamination. Genome scans across individuals from recently polluted and nearby unpolluted soils in Belgium revealed low divergence across isolates and no evidence of population structure based on soil type. However, we detected single nucleotide polymorphism divergence and gene copy-number variation, with different genetic combinations potentially conferring the ability to persist in contaminated soils. Variants were shared across the population but found to be under selection in isolates exposed to pollution and located across the genome, including in genes involved in metal exclusion, storage, immobilization and reactive oxygen species detoxification. Together, our results point to S. luteus undergoing the initial steps of adaptive divergence and contribute to understanding the processes underlying local adaptation under strong environmental selection.

Does fungal competitive ability explain host specificity or rarity in ectomycorrhizal symbioses?

Two common ecological assumptions are that host generalist and rare species are poorer competitors relative to host specialist and more abundant counterparts. While these assumptions have received considerable study in both plant and animals, how they apply to ectomycorrhizal fungi remains largely unknown. To investigate how interspecific competition may influence the anomalous host associations of the rare ectomycorrhizal generalist fungus, Suillus subaureus, we conducted a seedling bioassay. Pinus strobus seedlings were inoculated in single- or two-species treatments of three Suillus species: S. subaureus, S. americanus, and S. spraguei. After 4 and 8 months of growth, seedlings were harvested and scored for mycorrhizal colonization as well as dry biomass. At both time points, we found a clear competitive hierarchy among the three ectomycorrhizal fungal species: S. americanus > S. subaureus > S. spraguei, with the competitive inferior, S. spraguei, having significantly delayed colonization relative to S. americanus and S. subaureus. In the single-species treatments, we found no significant differences in the dry biomasses of P. strobus seedlings colonized by each Suillus species, suggesting none was a more effective plant symbiont. Taken together, these results indicate that the rarity and anomalous host associations exhibited by S. subaureus in natural settings are not driven by inherently poor competitive ability or host growth promotion, but that the timing of colonization is a key factor determining the outcome of ectomycorrhizal fungal competitive interactions.

Changes in soil bacterial community diversity following the removal of invasive feral pigs from a Hawaiian tropical montane wet forest.

Nonnative, invasive feral pigs (Sus scrofa) modify habitats by disturbing soils and vegetation, which can alter biogeochemical processes. Soil microbial communities drive nutrient cycling and therefore also play important roles in shaping ecosystem structure and function, but the responses of soil microbes to nonnative ungulate removal remains poorly studied. We examined changes in the soil bacterial community over a ~25 year chronosequence of feral pig removal in tropical montane wet forests on the Island of Hawai'i. We extracted bacterial eDNA from soil samples collected inside and outside of ungulate exclosures along this chronosequence and sequenced the eDNA using the Illumina platform. We found that ungulate removal increased diversity of soil bacteria, with diversity scores positively correlated with time since removal. While functional and phylogenetic diversity were not significantly different between pig present and pig removed soils, soil bulk density, which decreases following the removal of feral pigs, was a useful predictor of dissimilarity among sites and correlated to changes in functional diversity. Additionally, increases in soil porosity, potassium, and calcium were correlated to increases in functional diversity. Finally, sites with greater mean annual temperatures were shown to have higher scores of both functional and phylogenetic diversity. As such, we conclude that feral pigs influence overall bacterial community diversity directly while influencing functional diversity indirectly through alterations to soil structure and nutrients. Comparatively, phylogenetic differences between communities are better explained by mean annual temperature as a climatic predictor of community dissimilarity.

Rhizopogon olivaceotinctus increases its inoculum potential in heated soil independent of competitive release from other ectomycorrhizal fungi.

Rhizopogon olivaceotinctus is a rarely collected ectomycorrhizal fungus that has been found primarily in California and southern Oregon. Prior work has shown that it (i) is common in soil spore banks associated with pine forests from these areas; (ii) is rare or absent on trees in undisturbed forests in these same areas; (iii) exhibits an increased abundance on pine seedlings following fire or experimental soil heating; and (iv) has spores that are more resistant to heat than those of several other ectomycorrhizal species tested to date. Here, we reject the hypothesis that the increased abundance of the species following soil heating is caused only by reduced competition with other ectomycorrnizal fungi and show instead that heating alone significantly increases the inoculum potential of its spores. We argue that this is likely caused by heat stimulation of the spores, a process that has precedent in saprotrophic fungi and plant seeds. This result, in combination with those of previous studies, shows that Rhizopogon olivaceotinctus is well adapted to fire.

Molecular basis of flowering under natural long-day conditions in Arabidopsis.

Plants sense light and temperature changes to regulate flowering time. Here, we show that expression of the Arabidopsis florigen gene, FLOWERING LOCUS T (FT), peaks in the morning during spring, a different pattern than we observe in the laboratory. Providing our laboratory growth conditions with a red/far-red light ratio similar to open-field conditions and daily temperature oscillation is sufficient to mimic the FT expression and flowering time in natural long days. Under the adjusted growth conditions, key light signalling components, such as phytochrome A and EARLY FLOWERING 3, play important roles in morning FT expression. These conditions stabilize CONSTANS protein, a major FT activator, in the morning, which is probably a critical mechanism for photoperiodic flowering in nature. Refining the parameters of our standard growth conditions to more precisely mimic plant responses in nature can provide a powerful method for improving our understanding of seasonal response.

Longevity of light- and dark-colored basidiospores from saprotrophic mushroom-forming fungi.

Fungi can produce resistant propagules that may last for decades. Basidiospores from ectomycorrhizal fungi had been experimentally shown to last for at least 6 yr, but there are few reports on the longevity of saprotrophic members of mushroom-forming fungi. Here, the author shows evidence of spore longevity of these fungi by collecting, drying, storing, and germinating these spores over time. Results showed that dark-colored spores have a much-extended longevity as compared to light-colored spores. Dark-colored spores of some species are viable to at least 2.8 yr, whereas light-colored spores are generally viable for a much shorter period of time. The author proposes that mushroom-forming basidiomycete fungi employ two different ecological strategies: one with extended longevity that allows for long-distance dispersal, and the other takes advantage of optimal conditions that support both mushroom formation, basidiospore dispersal, and germination locally.

Synergy among Microbiota and Their Hosts: Leveraging the Hawaiian Archipelago and Local Collaborative Networks To Address Pressing Questions in Microbiome Research.

Despite increasing acknowledgment that microorganisms underpin the healthy functioning of basically all multicellular life, few cross-disciplinary teams address the diversity and function of microbiota across organisms and ecosystems. Our newly formed consortium of junior faculty spanning fields such as ecology and geoscience to mathematics and molecular biology from the University of Hawai'i at Manoa aims to fill this gap. We are united in our mutual interest in advancing a new paradigm for biology that incorporates our modern understanding of the importance of microorganisms. As our first concerted research effort, we will assess the diversity and function of microbes across an entire watershed on the island of Oahu, Hawai'i. Due to its high ecological diversity across tractable areas of land and sea, Hawai'i provides a model system for the study of complex microbial communities and the processes they mediate. Owing to our diverse expertise, we will leverage this study system to advance the field of biology.

Ecological responses to forest age, habitat, and host vary by mycorrhizal type in boreal peatlands.

Despite covering vast areas of boreal North America, the ecological factors structuring mycorrhizal fungal communities in peatland forests are relatively poorly understood. To assess how these communities vary by age (younger vs. mature), habitat (fen vs. bog), and host (conifer trees vs. ericaceous shrub), we sampled the roots of two canopy trees (Larix laricina and Picea mariana) and an ericaceous shrub (Ledum groenlandicum) at four sites in northern Minnesota, USA. To characterize the specific influence of host co-occurrence on mycorrhizal fungal community structure, we also conducted a greenhouse bioassay using the same three hosts. Root samples were assessed using Illumina-based high-throughput sequencing (HTS) of the ITS1 rRNA gene region. As expected, we found that the relative abundance of ectomycorrhizal fungi was high on both Larix and Picea, whereas ericoid mycorrhizal fungi had high relative abundance only on Ledum. Ericoid mycorrhizal fungal richness was significantly higher in mature forests, in bogs, and on Ledum hosts, while ectomycorrhizal fungal richness did not differ significantly across any of these three variables. In terms of community composition, ericoid mycorrhizal fungi were more strongly influenced by host while ectomycorrhizal fungi were more influenced by habitat. In the greenhouse bioassay, the presence of Ledum had consistently stronger effects on the composition of ectomycorrhizal, ericoid, and ericoid-ectomycorrhizal fungal communities than either Larix or Picea. Collectively, these results suggest that partitioning HTS-based datasets by mycorrhizal type in boreal peatland forests is important, as their responses to rapidly changing environmental conditions are not likely to be uniform.

Ectomycorrhizal host specificity in a changing world: can legacy effects explain anomalous current associations?

Despite the importance of ectomycorrhizal (ECM) fungi in forest ecosystems, knowledge about the ecological and co-evolutionary mechanisms underlying ECM host associations remains limited. Using a widely distributed group of ECM fungi known to form tight associations with trees in the family Pinaceae, we characterized host specificity among three unique Suillus-host species pairs using a combination of field root tip sampling and experimental bioassays. We demonstrate that the ECM fungus S. subaureus can successfully colonize Quercus hosts in both field and glasshouse settings, making this species unique in an otherwise Pinaceae-specific clade. Importantly, however, we found that the colonization of Quercus by S. subaureus required co-planting with a Pinaceae host. While our experimental results indicate that gymnosperms are required for the establishment of new S. subaureus colonies, Pineaceae hosts are locally absent at both our field sites. Given the historical presence of Pineaceae hosts before human alteration, it appears the current S. subaureus-Quercus associations represent carryover from past host presence. Collectively, our results suggest that patterns of ECM specificity should be viewed not only in light of current forest community composition, but also as a legacy effect of host community change over time.

Ectomycorrhizal fungal response to warming is linked to poor host performance at the boreal-temperate ecotone.

Rising temperatures associated with climate change have been shown to negatively affect the photosynthetic rates of boreal forest tree saplings at their southern range limits. To quantify the responses of ectomycorrhizal (EM) fungal communities associated with poorly performing hosts, we sampled the roots of Betula papyrifera and Abies balsamea saplings growing in the B4Warmed (Boreal Forest Warming at an Ecotone in Danger) experiment. EM fungi on the root systems of both hosts were compared from ambient and +3.4 °C air and soil warmed plots at two sites in northern Minnesota. EM fungal communities were assessed with high-throughput sequencing along with measures of plant photosynthesis, soil temperature, moisture, and nitrogen. Warming selectively altered EM fungal community composition at both the phylum and genus levels, but had no significant effect on EM fungal operational taxonomic unit (OTU) diversity. Notably, warming strongly favored EM Ascomycetes and EM fungi with short-contact hyphal exploration types. Declining host photosynthetic rates were also significantly inversely correlated with EM Ascomycete and EM short-contact exploration type abundance, which may reflect a shift to less carbon demanding fungi due to lower photosynthetic capacity. Given the variation in EM host responses to warming, both within and between ecosystems, better understanding the link between host performance and EM fungal community structure will to clarify how climate change effects cascade belowground.

Continental-level population differentiation and environmental adaptation in the mushroom Suillus brevipes.

Recent advancements in sequencing technology allowed researchers to better address the patterns and mechanisms involved in microbial environmental adaptation at large spatial scales. Here we investigated the genomic basis of adaptation to climate at the continental scale in Suillus brevipes, an ectomycorrhizal fungus symbiotically associated with the roots of pine trees. We used genomic data from 55 individuals in seven locations across North America to perform genome scans to detect signatures of positive selection and assess whether temperature and precipitation were associated with genetic differentiation. We found that S. brevipes exhibited overall strong population differentiation, with potential admixture in Canadian populations. This species also displayed genomic signatures of positive selection as well as genomic sites significantly associated with distinct climatic regimes and abiotic environmental parameters. These genomic regions included genes involved in transmembrane transport of substances and helicase activity potentially involved in cold stress response. Our study sheds light on large-scale environmental adaptation in fungi by identifying putative adaptive genes and providing a framework to further investigate the genetic basis of fungal adaptation.