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.

Inter- and intra-specific metal tolerance variation in ectomycorrhizal fungal Suillus species.

Soil metal contamination can affect growth, metabolism, and reproduction of organisms, and can lead to death. However, some fungi have evolved metal tolerance and are able to live in contaminated soils. Species in the ectomycorrhizal genus Suillus from Europe and Asia display variation in metal tolerance, yet it is unknown whether this is a widespread trait in the genus and whether it occurs in North America. Here we investigate cadmium (Cd) and zinc (Zn) tolerance in S. brevipes and S. tomentosus isolates collected from sites in the Rocky Mountains of Colorado displaying different metal content. In line with previous findings for other Suillus species, we hypothesized (1) S. brevipes and S. tomentosus to display intra-specific metal tolerance variation, (2) Zn and Cd tolerance to be correlated to soil metal content, and (3) tolerant isolates to show lower metal tissue content compared to sensitive isolates (due to increased metal exclusion). We found ample intra- and inter-specific Zn and Cd tolerance variation in both S. brevipes and S. tomentosus, but no correlation between soil metal content and tolerance. There was a negative correlation between tolerance level and Zn uptake, indicating an exclusion-based Zn tolerance strategy. Sensitive and tolerant isolates showed no difference in Cd accumulation, indicating that Cd tolerance in these species is likely not dependent on exclusion. Our study sets the groundwork for further investigation into the genetic basis of Suillus metal tolerance and whether and how it impacts pine mycorrhizal partners.

Determining Gene Order Patterns in the Suillus and Boletales through Comparative Analysis of Their Mitogenomes.

Suillus is one of the most important genera of ectomycorrhizal fungi. As a model for studying host specificity, its molecular fragments and nuclear genome have been analyzed. However, its mitochondrial genome has not yet been reported. In this study, we assembled five mitogenomes of Suillus and analyzed and compared their basic characteristics. Owing to the large number of introns as well as intergenic regions, the mitogenomic lengths of species of Suillus were greater than those of other species of Boletales. We identified two main patterns of gene order arrangement in the members of the order Boletales. The Ka/Ks values of 15 protein-coding genes were <1 for the mitochondrial genes of 39 Boletales species, indicating their conserved evolution. Phylogenetic trees, reconstructed using the mitogenomes, indicated that the genus Suillus was monophyletic. Phylogenetic results based on the internal transcribed spacer region and mitogenome were used to confirm the distribution of Suillus placidus in China. The results showed that the mitogenome was superior in distinguishing species compared with a single molecular fragment. This is the first study to investigate the mitogenome of Suillus, enriching the mitogenome information and providing basic data for the phylogeny, resource conservation, and genetic diversity of this genus.

Ectomycorrhizal fungi enhance pine growth by stimulating iron-dependent mechanisms with trade-offs in symbiotic performance.

Iron (Fe) is crucial for metabolic functions of living organisms. Plants access occluded Fe through interactions with rhizosphere microorganisms and symbionts. Yet, the interplay between Fe addition and plant-mycorrhizal interactions, especially the molecular mechanisms underlying mycorrhiza-assisted Fe processing in plants, remains largely unexplored. We conducted mesocosms in Pinus plants inoculated with different ectomycorrhizal fungi (EMF) Suillus species under conditions with and without Fe coatings. Meta-transcriptomic, biogeochemical, and X-ray fluorescence imaging analyses were applied to investigate early-stage mycorrhizal roots. While Fe addition promoted Pinus growth, it concurrently reduced mycorrhiza formation rate, symbiosis-related metabolites in plant roots, and aboveground plant carbon and macronutrient content. This suggested potential trade-offs between Fe-enhanced plant growth and symbiotic performance. However, the extent of this trade-off may depend on interactions between host plants and EMF species. Interestingly, dual EMF species were more effective at facilitating plant Fe uptake by inducing diverse Fe-related functions than single-EMF species. This subsequently triggered various Fe-dependent physiological and biochemical processes in Pinus roots, significantly contributing to Pinus growth. However, this resulted in a greater carbon allocation to roots, relatively reducing the aboveground plant carbon content. Our study offers critical insights into how EMF communities rebalance benefits of Fe-induced effects on symbiotic partners.

Does resource exchange in ectomycorrhizal symbiosis vary with competitive context and nitrogen addition?

Ectomycorrhizal symbiosis is essential for the nutrition of most temperate forest trees and helps regulate the movement of carbon (C) and nitrogen (N) through forested ecosystems. The factors governing the exchange of plant C for fungal N, however, remain obscure. Because competition and soil resources may influence ectomycorrhizal resource movement, we performed a 10-month split-root microcosm study using Pinus muricata seedlings with Thelephora terrestris, Suillus pungens, or no ectomycorrhizal fungus, under two N concentrations in artificial soil. Fungi competed directly with roots and indirectly with each other. We used stable isotope enrichment to track plant photosynthate and fungal N. For T. terrestris, plants received N commensurate with the C given to their fungal partners. Thelephora terrestris was a superior mutualist under high-N conditions. For S. pungens, plant C and fungal N exchange were not coupled. However, in low-N conditions, plants preferentially allocated C to S. pungens rather than T. terrestris. Our results suggest that ectomycorrhizal resource transfer depends on competitive and nutritional context. Plants can exchange C for fungal N, but coupling of these resources can depend on the fungal species and soil N. Understanding the diversity of fungal strategies, and how they change with environmental context, reveals mechanisms driving this important symbiosis.

Traits and tradeoffs among non-native ectomycorrhizal fungal symbionts affect pine seedling establishment in a Hawaiian coinvasion landscape.

Pine invasions lead to losses of native biodiversity and ecosystem function, but pine invasion success is often linked to coinvading non-native ectomycorrhizal (EM) fungi. How the community composition, traits, and distributions of these fungi vary over the landscape and how this affects pine success is understudied. A greenhouse bioassay experiment was performed to test the effects of changes in EM fungal community structure from a pine plantation, to an invasion front to currently pine-free areas on percent root colonization and seedling biomass. Soils were also analysed by qPCR to determine changes in inoculum and spore density over distance for a common coinvading EM fungus, Suillus pungens. Percent colonization increased with distance from the plantation, which corresponded with an increase in seedling biomass and stark changes in EM fungal community membership where Suillus spp. dominated currently pine-free areas. However, there was a negative relationship between S. pungens inoculum potential versus root colonization over distance. We conclude that the success of pine invasions is facilitated by specific traits of Suillus spp., but that the success of Suillus is contingent on a lack of competition with other ectomycorrhizal fungi.

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.

Are southern African solitary mole-rats homeothermic or heterothermic under natural field conditions?

Abandoning of a stable body temperature (Tb), a phenomenon known as heterothermy, is an adaptation to cope mainly with a lack of food and water, especially in species inhabiting daily or seasonally variable environments. There is increasing evidence that African mammals avoid adverse conditions by heterothermy and eventually by entering torpor. Members of subterranean rodent family, the African mole-rats (Bathyergidae), are suitable candidates to study both phenomena, because of the diversity of their strategies in respect of maintaining stable Tb ranging from homeothermic species to a mammal with the most labile Tb, the naked mole-rat. Currently, there are field data on daily and seasonal Tb in one social species only and such information are lacking for any solitary mole-rat. In our study, we recorded yearly Tb in two solitary bathyergids, the Cape mole-rat Georychus capensis and the Cape dune mole-rat Bathyergus suillus from South Africa using intraperitoneally implanted dataloggers. Since this region is characterised by changing ecological characteristics, we expected either decreases of Tb within 24 h indicating daily torpor and/or longer-term decreases of Tb, which would indicate multiday torpor. Although we found seasonally phase shifted low amplitude daily Tb cycles, we did not find any remarkable and regular daily and/or seasonal Tb deviations, likely showing an absence of torpor in both species. Due to absence of this energy saving mechanism, we may speculate that both species could be vulnerable to ongoing global climatic change.

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.

Suilloid fungi as global drivers of pine invasions.

Belowground biota can deeply influence plant invasion. The presence of appropriate soil mutualists can act as a driver to enable plants to colonize new ranges. We reviewed the species of ectomycorrhizal fungi (EMF) that facilitate pine establishment in both native and non-native ranges, and that are associated with their invasion into nonforest settings. We found that one particular group of EMF, suilloid fungi, uniquely drive pine invasion in the absence of other EMF. Although the association with other EMF is variable, suilloid EMF are always associated with invasive pines, particularly at early invasion, when invasive trees are most vulnerable. We identified five main ecological traits of suilloid fungi that may explain their key role at pine invasions: their long-distance dispersal capacity, the establishment of positive biotic interactions with mammals, their capacity to generate a resistant spore bank, their rapid colonization of roots and their long-distance exploration type. These results suggest that the identity of mycorrhizal fungi and their ecological interactions, rather than simply the presence of compatible fungi, are key to the understanding of plant invasion processes and their success or failure. Particularly for pines, their specific association with suilloid fungi determines their invasion success in previously uninvaded ecosystems.

Plant-mediated partner discrimination in ectomycorrhizal mutualisms.

Although ectomycorrhizal fungi have well-recognized effects on ecological processes ranging from plant community dynamics to carbon cycling rates, it is unclear if plants are able to actively influence the structure of these fungal communities. To address this knowledge gap, we performed two complementary experiments to determine (1) whether ectomycorrhizal plants can discriminate among potential fungal partners, and (2) to what extent the plants might reward better mutualists. In experiment 1, split-root Larix occidentalis seedlings were inoculated with spores from three Suillus species (S. clintonianus, S. grisellus, and S. spectabilis). In experiment 2, we manipulated the symbiotic quality of Suillus brevipes isolates on split-root Pinus muricata seedlings by changing the nitrogen resources available, and used carbon-13 labeling to track host investment in fungi. In experiment 1, we found that hosts can discriminate in multi-species settings. The split-root seedlings inhibited colonization by S. spectabilis whenever another fungus was available, despite similar benefits from all three fungi. In experiment 2, we found that roots and fungi with greater nitrogen supplies received more plant carbon. Our results suggest that plants may be able to regulate this symbiosis at a relatively fine scale, and that this regulation can be integrated across spatially separated portions of a root system.

Metal induction of two metallothionein genes in the ectomycorrhizal fungus Suillus himalayensis and their role in metal tolerance.

Metallothioneins (MTs) are small proteins with highly conserved cysteine residues and are involved in metal homeostasis and metal detoxification. Two metallothionein genes ShMT1 and ShMT2 from the ectomycorrhizal fungus Suillus himalayensis were characterised for their potential role in heavy metal detoxification. The response of these MTs to the exogenous concentrations of copper and cadmium was studied by qPCR analysis. The exogenous copper but not the cadmium at the tested concentrations induced the expression of the MT genes. The functional role of ShMTs was validated by expressing the two genes through functional complementation in yeast mutant strain cup1Δ (copper-sensitive), ycf1Δ (cadmium- sensitive) and zrc1Δ (zinc-sensitive). The mutant strain successfully expressed the two genes resulting in wild-type phenotype restoration of copper, cadmium and zinc tolerance. The present study shows that the ectomycorrhizal fungus S. himalayensis encodes two metallothionein genes (ShMT1 and ShMT2) which are more inducible by copper than cadmium and could play an important role in their detoxification.

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.

Current and potential distribution of the ectomycorrhizal fungus Suillus lakei ((Murrill) A.H. Sm. & Thiers) in its invasion range.

Suillus lakei is an ectomycorrhizal fungus native to North America and known in Europe, South America, and New Zealand. This contribution aims to illustrate the worldwide biogeography of S. lakei based on sporocarp records. Species distribution modeling was used to assess the suitable niche distribution of S. lakei, based on the climatic variables as well as distribution of its ectomycorrhizal partner, Douglas fir. In general, distribution of suitable niches of S. lakei greatly overlaps with the distribution of Douglas fir in North America. By spatial distribution modeling, we found that the precipitation of the coldest quarters, isothermality, and annual mean temperature are important factors influencing the potential distribution of S. lakei. Nevertheless, the most crucial factor limiting expansion of S. lakei in its invasion range is Douglas fir occurrence. This factor reached an 86.4% contribution for the S. lakei species distribution model. Additionally, we compare the aboveground and belowground presence of S. lakei based on surveys in the field. Our study shows that even extremely low abundance of ectomycorrhizas can open the possibility of using an ectomycorrhiza survey for their quantification as a good indicator of the presence of S. lakei in field conditions. Both sporocarps and ectomycorrhizas occurred only in gardens, where Douglas fir seedlings were outplanted at the beginning of the 1990s as an ornamental plant. Presumably, international trade of ornamental plants was one possible route of introduction of S. lakei to Poland.

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.

Two new species in the Suillus spraguei complex from China.

Suillus spraguei, synonym S. pictus, has been reported from eastern North America and eastern Asia associated with Pinus subgenus Strobus. Published phylogenetic analyses of rRNA internal transcribed spacer (ITS) sequence and population genetic studies indicated that S. spraguei as currently circumscribed might contain several geographically distinct species. This study examined this possibility through a multigene analysis of S. spraguei specimens from eastern North America and eastern Asia. These specimens were associated with Pinus strobus, P. koraiensis, P. armandii, and P. kwangtungensis. The multigene analysis included three genomic regions: the genes for translation elongation factor 1α (TEF1) and RNA polymerase II largest subunit (RPB1), and the nuc rRNA segments ITS1-5.8S-ITS2 (ITS) and 28S D1-D2 domains (28S). This study confirms that the S. spraguei complex consists of at least three cryptic species: S. spraguei sensu stricto associated with P. strobus in eastern North America; S. phylopictus associated with multiple species in Pinus subgenus Strobus (5-needle pines) throughout China and Japan; and S. kwangtungensis, currently found only in P. kwangtungensis forests in southeastern China. A third new species from Japan and Korea was suggested based on ITS phylogeny. Morphologically, S. spraguei and S. phylopictus resemble each other, whereas S. kwangtungensis is covered with more floccose scales. The new species add to the knowledge of macrofungal diversity in eastern Asia and highlight the necessity of comparing broadly distributed species complexes using morphological, molecular, and ecological data.

Preparative isolation of a cytotoxic principle of a forest mushroom Suillus luteus by sodium dodecyl sulfate based "salting-in" countercurrent chromatography.

In the course of screening new anticancer natural products, an edible forest mushroom Suillus luteus (L. Ex Franch). Gray was found to have potent cytotoxicity against several human cancer cells. However, the lipophilic sample made some countercurrent chromatography solvent systems emulsify, which caused difficulties in the separation of its cytotoxic components. Here, we found that the addition of an organic salt sodium dodecyl sulfate could efficiently shorten the settling time of the mushroom sample solutions by eliminating the emulsification of two-phase solvent systems. Moreover, we found that sodium dodecyl sulfate could play a new "salting-in" role and made the partition coefficients of the solutes decrease with the increased concentrations. Thus, a sodium dodecyl sulfate based salting-in countercurrent chromatography method has been successfully established for the first time for preparative isolation of a cytotoxic principle of the mushroom. The active component was identified as isosuillin. Whole results indicated that sodium dodecyl sulfate could be used as an efficient salting-in reagent for two-phase solvent system selection and targeted countercurrent chromatography isolation. It is very useful for current natural products isolation and drug discovery.

Ectomycorrhizal fungal communities coinvading with Pinaceae host plants in Argentina: Gringos bajo el bosque.

Coinvasive ectomycorrhizal (ECM) fungi allow Pinaceae species to invade regions otherwise lacking compatible symbionts, but ECM fungal communities permitting Pinaceae invasions are poorly understood. In the context of Pinaceae invasions on Isla Victoria, Nahuel Huapi National Park, Argentina, we asked: what ECM fungi are coinvading with Pinaceae hosts on Isla Victoria; are some ECM fungal species or genera more prone to invade than others; and are all ECM fungal species that associate with Northern Hemisphere hosts also nonnative, or are some native fungi compatible with nonnative plants? We sampled ECMs from 226 Pinaceae host plant individuals, both planted individuals and recruits, growing inside and invading from plantations. We used molecular techniques to examine ECM fungal communities associating with these trees. A distinctive subset of the ECM fungal community predominated far from plantations, indicating differences between highly invasive and less invasive ECM fungi. Some fungal invaders reported here have been detected in other locations around the world, suggesting strong invasion potential. Fungi that were frequently detected far from plantations are often found in early-successional sites in the native range, while fungi identified as late-successional species in the native range are rarely found far from plantations, suggesting a means for predicting potential fungal coinvaders.

Genetic isolation between two recently diverged populations of a symbiotic fungus.

Fungi are an omnipresent and highly diverse group of organisms, making up a significant part of eukaryotic diversity. Little is currently known about the drivers of fungal population differentiation and subsequent divergence of species, particularly in symbiotic, mycorrhizal fungi. Here, we investigate the population structure and environmental adaptation in Suillus brevipes (Peck) Kuntze, a wind-dispersed soil fungus that is symbiotic with pine trees. We assembled and annotated the reference genome for Su. brevipes and resequenced the whole genomes of 28 individuals from coastal and montane sites in California. We detected two clearly delineated coast and mountain populations with very low divergence. Genomic divergence was restricted to few regions, including a region of extreme divergence containing a gene encoding for a membrane Na(+) /H(+) exchanger known for enhancing salt tolerance in plants and yeast. Our results are consistent with a very recent split between the montane and coastal Su. brevipes populations, with few small genomic regions under positive selection and a pattern of dispersal and/or establishment limitation. Furthermore, we identify a putatively adaptive gene that motivates further functional analyses to link genotypes and phenotypes and shed light on the genetic basis of adaptive traits.

Effects of four bolete species on ectomycorrhizae formation and development in Pinus thunbergii and Quercus acutissima.

BACKGROUND: Bolete cultivation is economically and ecologically valuable. Ectomycorrhizae are advantageous for plant development and productivity. This study investigated how boletes affect the formation of Pinus thunbergii and Quercus acutissima ectomycorrhizae using greenhouse-based mycorrhizal experiments, inoculating P. thunbergii and Q. acutissima with four species of boletes (Suillus bovinus, Suillus luteus, Suillus grevillei, and Retiboletus sinensis). RESULTS: Three months after inoculation, morphological and molecular analyses identified S. bovinus, S. luteus, S. grevillei and R. sinensis ectomycorrhizae formation on the roots of both tree species. The mycorrhizal infection rate ranged from 40 to 55%. The host plant species determined the mycorrhiza morphology, which was independent of the bolete species. Differences in plant growth, photosynthesis, and endogenous hormone secretion primarily correlated with the host plant species. Infection with all four bolete species significantly promoted the host plants' growth and photosynthesis rates; indole-3-acetic acid, zeatin, and gibberellic acid secretion increased, and the abscisic acid level significantly decreased. Indole-3-acetic acid was also detected in the fermentation broths of all bolete species. CONCLUSIONS: Inoculation with bolete and subsequent mycorrhizae formation significantly altered the morphology and hormone content in the host seedlings, indicating growth promotion. These findings have practical implications for culturing pine and oak tree species.