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

Summary

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 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 ectomycorrhizal interactions.

I. Introduction

The ectomycorrhizal (ECM) symbiosis formed between plant roots and soil fungi is characterized by the obligate exchange of plant derived carbohydrates for fungal scavenged nutrients (Smith & Read, 2010). These cross-kingdom mutualisms provide both direct and indirect impacts on plant growth, ranging from increased mineral nutrient access to improved abiotic and biotic stress tolerance (Smith & Read, 2010; Branco et al., 2022). These benefits have long been recognized as critical to the health of both natural and managed forest systems, with ECM fungal inoculation regularly employed by the nursery and forestry industries to enhance seedling growth (Torres & Honrubia, 1994; Cripps & Antibus, 2011; Mateos et al., 2017). ECM fungi also offer a multitude of ecosystem services, playing fundamental roles in the functioning and maintenance of community composition, water relations, and global carbon and nutrient cycling (Smith & Read, 2010; Branco et al., 2022). In recent years, these attributes have attracted increased interest from diverse fields including ecosystem ecology, evolutionary biology, biogeochemistry, global change biology, and sustainability studies.

Despite the recognition of their importance and the growing interest in ECM fungi, there are still significant gaps in our understanding of how ECM fungi function and interact with their plant partners, microbial communities, and environments. The cryptic belowground nature of ECM fungi, coupled to high species diversity, variable tractability under laboratory conditions, lack of standardized protocols, as well as technical challenges associated with the isolation, sequencing, and manipulation of host-associated dikaryotic species, have hindered the advancement of the field. Many questions remain unanswered, including how ECM fungi evolve at different biological scales, the mechanisms facilitating interactions between ECM fungi and their hosts, and how ECM communities will be affected by environmental change (Plett et al., 2024; Dauphin & Peter, 2024).

While recent technological advances, such as the ability to generate and analyze extensive genomic datasets, promise to improve our ability to tackle some of the aforementioned questions, our understanding of ECM mutualism remains limited due a scarcity of well-developed model systems. Establishing ECM models that are diverse, ecologically relevant, and amenable to experimentation and in vitro manipulation is critical to advancing mycorrhizal research. The establishment of models allows the scientific community to build capacity around common systems, increasing the breadth and utility of core methodologies, improving reproducibility by using a common set of strains and procedures, and creating discourse and momentum around novel findings and approaches.

Model systems are often defined at the species level, with each system offering unique advantages and limitations. Mycologists have built capacity around the development of model species such as Coprinopsis cinerea for studying fruit body development (Kamada et al., 2010; Plaza et al., 2014), Schizosaccharomyces pombe for probing fundamental molecular and cellular biology (Hoffman et al., 2015), Candida albicans for investigating human fungal pathogenesis (Kabir et al., 2012), Neurospora crassa for understanding gene silencing and circadian rhythms (Selker et al., 1987; Roche et al., 2014), and Saccharomyces cerevisiae for elucidating biological processes from neurodegenerative disorders to biological aging (Miller-Fleming et al., 2008; Murakami & Kaeberlein, 2009; Karathia et al., 2011). The development of these systems has resulted in the creation of numerous tools, and invaluable insights into eukaryotic biology. The concerted effort towards developing ECM model systems for fungal ecological and evolutionary research will foster rapid tool development and the critical mass necessary to cultivate effective collaborations and propel the field of mycorrhizal research into the future.

Several ECM models are in development including Laccaria bicolor, Hebeloma cylindrosporum, and Pisolithus tinctorius. These species have produced fundamental insights into the ecological, physiological, and molecular mechanisms of ECM-host interactions (Cairney & Chambers, 1997; Marmeisse et al., 2004; Martin et al., 2008). To date, these taxa have been primarily used as species-level models, but are rapidly accumulating genomic resources at the genus level. As of February 2024, the MycoCosm web portal (https://mycocosm.jgi.doe.gov/) now includes two species of Laccaria (including 15 genomes of L. bicolor), two species of Hebeloma, and 10 species of Pisolithus. Other ECM fungal genera quickly accumulating sequenced genomes include Tuber (10 species) and Lactarius (12 species). The choice of which ECM lineages should be targeted for model development should take into consideration diversity and tractability, trait variation, reproducibility, accessibility and availability, ecological relevance, conservation/invasion status, applicability and extensibility to other systems, as well as community interest and momentum. Fungi in the genus Suillus fulfill of many of these considerations, including laboratory and field tractability, high ecological relevance, species and trait diversity, accessibility and abundance, and an engaged and rapidly growing research community. To this end, we have been developing Suillus into a genus-level system to characterize the ecology and evolution of ECM fungal symbioses.

Suillus is a speciose genus with most species displaying strong partner specificity with members of the plant family Pinaceae (Kretzer et al., 1996; Nguyen et al., 2016b). Suillus produces abundant fruit bodies, and are some of the most frequently encountered ECM mushrooms in northern temperate and boreal forest systems. The genus includes pioneer species that are instrumental for forest establishment across both natural and managed systems, where they have facilitated both the success of forestry plantations, and extensive ecological damage as invasive host-symbiont pairs encroach into introduced ranges and disrupt local communities (Policelli et al., 2019). The genus includes species that span a diversity of ecological gradients such as stress-tolerance and stress-sensitivity, enabling investigation into environmental adaptation of both ECM fungi and their associated plant partners (Colpaert et al., 2011; Bazzicalupo et al., 2020; Zhang et al., 2021). Finally, relative to other ECM fungi, Suillus is highly tractable to laboratory manipulation, including but not limited to, the ability to be isolated axenically and grown apart from their hosts with specific nutritional supplementation, long term in vitro culture viability, and the ability to colonize host plants via both long-lived reactive spores or mycelial extension (Nguyen et al., 2012).

Here, we present Suillus as an emerging model genus for ECM fungal ecology and evolution. Our goals are 1) to provide a primer on Suillus that synthesizes our current understanding of the biology and eco-evolutionary theory relevant to this unique genus, and 2) to standardize and disseminate the protocols developed to study Suillus both in the field and the laboratory. This primer is not intended to be systematic literature review, but rather a broad introduction to the genus and its potential to serve as a model system. Specifically, we introduce the vision and goals of the International Suillus Consortium (a working group of over 20 independent laboratories collectively dedicated to the development of the Suillus system, http://www2.hawaii.edu/ñn33/suillus), consolidate the latest findings in Suillus biology, ecology, and evolution, and compile molecular and experimental protocols. Sections II-VII cover the development of Suillus as a model genus, its taxonomy and evolution, significant ecological interactions, abiotic environmental adaptations, the biology and implications of introductions and invasions, and a look forward at outstanding questions, current challenges, and concentrations areas for system development. Section VIII outlines a series of protocols for working with the genus which we have included as a series of standalone supplementary documents (see Notes S1-S7).

II. Suillus as a model genus

II.i: The power of genus-level model systems

Unlike model systems that are based on individual species, the strength of the Suillus system lies at the genus level. There are multiple benefits to leveraging genus-level systems. First, they provide the biological and ecological diversity needed to ask questions and test hypotheses across broad scales, from the individual to the ecosystem level. Second, they offer a comparative framework that allows researchers to investigate eco-evolutionary questions about speciation, adaptation, and how closely related taxa respond to environmental perturbation. Third, they allow the generalization of research findings beyond individual species to identify commonalities, trends, and general biological principles that apply to close relatives as well as other organisms occupying the same guild, lifestyle or ecological niche. Finally, there are experimental and research community advantages, such as the development of shared tools that can be used by multiple research groups with minimal protocol optimization. Having shared genomic and computational resources also allows different research teams to tackle diverse questions and minimize experimental overlap while increasing collaborative opportunities. The benefits of using model clades (at the genus level or above) for ecological and evolutionary research are well recognized in other systems, driving the development of model genera such as the bivalve Mytilus for ecotoxicology and climate change research (Leopold et al., 2019; Ribeiro et al., 2019), Mimulus plants for studying adaptation and speciation (Wu et al., 2008; Twyford et al., 2015) and Anolis lizards for studying adaptive radiation and ecomorphology (Sanger & Kircher, 2017).

II.ii: Suillus is an excellent model for ECM ecology and evolution

The genus Suillus is diverse (Fig. 1) and displays a range of host associations and a gradient of partner specificity responses, possesses unique traits with high ecological relevance, and is both accessible and tractable, making it an ideal model for studying ECM ecology and evolution. Generally, members of the genus have strong partner specificity with the plants in the family Pinaceae (Kretzer et al., 1996), making it a favorite target of fungal partner specificity research. In natural environments Suillus typically associates with three of the four subfamilies of the Pinaceae (Pinoideae, Laricoieae, and Piceoideae), allowing researchers to study host-symbiont associations across different taxonomic levels, including among host genera, subgenera, and species. Suillus also facilitates the study of host switching (Lofgren et al., 2021; Zhang et al., 2022), both between typical hosts and outside of these host groups, thanks to several species that defy the canonical partner specificity patterns demonstrated by most species (Lofgren et al., 2018; Pérez-Pazos et al., 2021).

Figure 1: Morphological and phenotypic variation across Suillus.

A-H) Representative members of the major clades within the Suillus phylogeny: Larix-associated species A) S. ampliporus displaying the radiating pores characteristic most members of the genus. B) S. clintonianus displaying the copious viscus slime often produced by Suillus fruit bodies. C) S. grisellus highlighting morphological variation present within Larix-associated species. D) Pseudotsuga-associated S. lakei (Rocky Mountain form) highlighting the significant carbon allocation needed to form Suillus fruit bodies, which here exceed 15 cm in diameter. Pinus associated species: E) S. subalutaceus representing species with longer stipes. F) S. luteus, the type species of the genus, displaying a prominent veil, a diagnostic feature for some species. G) S. spraguei, displaying distinctive red scales on the cap cuticle. H) S. tomentosus with robust fruiting bodies and variable bluing reactions of the trama tissue. I-P) Other diagnostic and morphologically important characteristics of Suillus I) A young specimen of S. weaverae showing milky droplets and clearly punctate stipe as a result of glandular cells. J) An old specimen of S. caerulescens, strongly staining blue, with insect larvae tunnels. K) Insect larvae quickly consume Suillus fruit bodies and can ruin an attempt at making spore prints. L) A spore print of S. lakei on aluminum foil for spore collection. M) A portion of an S. lakei fairy ring with large fruit bodies which require substantial carbon allocations from the host. N) A mycorrhizal root of S. paluster with rhizomorphs at the base, typically found in Suillus mycorrhizae. O) The mycoparasitic Chroogomphus vinicolor (left) fruiting next to its host, a senescing S. pungens (right). P) Hyphae of C. vinicolor, stained dark violet with Melzer’s reagent, within a mycorrhizal root of S. pungens.

Suillus contains species that demonstrate a variety of unique ecological attributes: they occur as pioneers (Peay et al., 2012) and as introduced species (Thompson et al., 2022), demonstrate variable stress tolerance (Branco et al., 2022; Erlandson et al., 2022), and associate with hosts across different life stages, ranging from seedlings to mature forests (Rineau et al., 2016). Suillus serve as effective symbionts that assist in host seedling establishment and development (Jenkins et al., 2018). Further, Suillus can act as hosts themselves for a diversity of fungi and bacteria, including related fungi in the genera Gomphidius and Chroogomphus (Olsson et al., 2000) (Fig. 1 O-P) and bacteria that both associate with living hyphae and mycorrhizas (Izumi et al., 2006; Timonen & Hurek, 2011), and actively decompose dead mycelia (Maillard et al., 2022, 2023). Together, these traits offer exciting opportunities to help solve a broad range of ecological and evolutionary questions.

Suillus is also an accessible and tractable genus that is amenable for study by a diverse array of research specialties. Species in the genus tend to fruit abundantly, are commonly encountered, and are relatively easy to identify in the field (Notes S1). While many ECM taxa are difficult or impossible to culture independently from their hosts (e.g., Cortinarius, Russula, Lactarius, Tomentella) (Palmer, 1971; Brundrett et al., 1996; Nygren et al., 2007), obtaining in vitro axenic cultures of Suillus is a straightforward process across the genus (Notes S2). Cultures of Suillus can be grown on solid media, in liquid culture, or somewhere in between (for example, on glass beads partially submerged in liquid media) and are amenable to long-term storage. Suillus mushrooms produce ample long-lived spores, and mycorrhization can be carried out rapidly (in as little as one month) using either spore-based or mycelial-based inoculation, providing flexibility for either high-throughput colonization or the ability to control the provenance of specific strains. The ability to generate mycelial biomass quickly in culture allows for manipulative studies (such as nutrient use and stress tolerance) either alone, with a plant partner, or with third-party community members such as other fungi or bacteria. Suillus species have relatively small genomes (~50 Mbp) that are comparatively easy to sequence and assemble. Through collaborations with the Joint Genome Institute, the International Suillus Consortium research teams have sequenced and assembled 46 draft genomes to date, spanning the taxonomic diversity of the genus. Sequencing efforts have also resulted in over 300 hundred shallow-depth genomes for particular species of interest such as S. luteus, S. brevipes, and S. salmonicolor (cothurnatus). The depth and breadth of sequencing across the genus provides genomic tools for the community to ask questions that can span the spectrum of systematics, ecology, and evolution. These traits have enabled the development of many Suillus optimized protocols (outlined in section VIII) and make the genus ideal for the development of manipulative and -omics based approaches, such as functional-omics (both in vitro and in situ), and gene editing (discussed in section VII).

II.iii: The SuilluScope database

While genus-level models are powerful systems for addressing questions that span ecological gradients, linking response variables back to genomic variability remains a grand challenge for most biological systems. Conducting genotype-to-phenotype analysis would be greatly aided by coupling genomic resources to the generation of diverse, reproducible, phenotypic data, and the broad availability of this data to the community. The Suillus Genome Strain Culture Collection constitutes a living library of fungal isolates used for whole-genome sequencing projects. To help the community take full advantage of both the genomic resources available for Suillus, and the phenotypic and genotypic diversity across the genus, we present a new interactive Suillus database, SuilluScope (v1.0Beta) available at www.SuilluScope.com. The initial release of the database contains phenotype information on growth rate, optimal growth temperatures, and culture images on multiple media types for isolates in the Suillus Genome Strain Culture Collection. The goal of the database is to act as a centralized and accessible community repository and analysis platform for phenotype assays conducted on the same set of genome-sequenced strains. This resource will allow the community to identify optimal growth conditions for specific strains, and predict and interpret response variables across the genus to prioritize questions for downstream analysis. Subsequent releases will include additional phenotypic variables, and new genome strains will be added as they become available. The Suillus Genome Strain Culture Collection has been integrated into the USDA ARS NRRL culture collection and all strains are available to the community without restriction. Strain accession numbers and detailed isolate information are available as a living document in the SuilluScope database, and will be updated as new assays are conducted, and new genome strains are integrated into the collection.

One example currently offered by the database is the selection of optimal growth conditions for specific species and strains. Many species of Suillus exhibit symptoms of stress on rich media including slower growth and increased pigment production (Fig. 2 A-P). This is a particularly important consideration for preparing material for sequencing and molecular work, as pigmentation is often associated with decreased extraction efficiency. For this reason, rich media preparations such as Modified Melin-Norkrans (MMN) and Hagem’s (Notes S3) are often prepared with a 50% reduction in carbon (glucose, malt extract, or yeast extract depending on the formulation). However, total carbon content is not the only determinant of pigment production, and individual species (or strains) often display unique requirements for pigment reduction, such varying the availability of organic and inorganic nitrogen, media pH, or temperature. Media type has additional wide-ranging effects on culture phenotype, including growth rate, topology, and the production of aerial hypha (Fig. 2 A-H). Similarly, while most species of Suillus will sustain growth at temperatures between 10-30 °C, growth rates vary among Suillus species, and optimal growth temperatures for a given species can be identified using the database. For example, maximum growth rates peak between 19 and 33 days after media transfer and vary between 29mm²/day (S. spraguei EM44) to 137mm²/day (S. quiescens FC197) when grown at room temperature (Fig. 3).

Figure 2: Representatives of the Suillus Genome Srain Culture Collection on four media types.

Cultures were grown in 9 cm petri plates on four media types including (A, E, I, M) Modified Melin-Norkrans (MMN), (B, F, J, N) Modified Fries Media (Fries), (C, G, K, O) Modified Hagem’s Agar (Hagem’s), and (D, H, L, P) Pachlewski’s Media (Pachlewski’s). See Notes S3 for Suillus optimized media recipes. All media types were prepared at their full respective carbon concentrations and adjusted to pH 6 prior to autoclaving. Cultures were grown for 28 days, at room temperature, in the dark, prior to being photographed. Strains represented here are (A-D) S. weaverae FC27, (E-H) S. pungens FC27, (I-L) S. fuscotomentosus FC203, and (M-P) the monokaryotic strain S. hirtellus EM16. These four strains are part of the Suillus Genome Strain Culture Collection, and the Suillus phenotype database, SuilluScope. To access phenotype information and NRRL accession numbers for the full strain collection, please visit www.SuilluScope.com.

Figure 3: Total colony area over time for representatives of the Suillus Genome Strain Culture Collection.

Cultures grown in 9 cm petri plates and started by placing 3mm agar plugs on Modified Melin-Norkrans (MMN) media, adjusted to pH 6 prior to autoclaving. Cultures were grown in temperature adjustable incubators, in the dark, for a total of 33 days (at n=4 replicates per species per temperature treatment). Starting on day 8, colony area was recorded twice per week over the course of the assay by marking the colony margin on the back of each petri dish with a fine-tip sharpie. After 33 days of growth, the back of the petri plates were imaged using a flatbed scanner, and colony area was calculated for each time point using the program imageJ. The data presented here is for replicates grown at room temperature (20°C). To access growth data at other temperatures, and for the full Suillus Genome Strain Culture Collection please visit www.SuilluScope.com.

III. Taxonomy and evolution

III.i: Taxonomy and species divergence

In 1821 S.F. Gray first proposed splitting Suillus from the genus Boletus, which Linnaeus had originally defined to comprise all pored fungi (Murrill, 1909). The genus Suillus was formally sanctioned by Elias Magnus Fries (1821-[1832]), but was not widely accepted until the 1950s (Dahlberg & Finlay, 1999). Perhaps due to the distinct morphological characters typically associated with Suillus mushrooms, the genus Suillus Gray has undergone surprisingly little revision over the years outside of new species additions and occasional species splitting, often associated with the recognition of taxa occurring in distinct geographic regions. To date, there are 112 species of Suillus recognized as valid in the Catalog of Life Database (https://www.catalogueoflife.org/), a number we expect to increase over the coming decade, particularly as new taxa are characterized from understudied areas such as Asia and the Indian subcontinent (Nguyen et al., 2016b). The Suillus phylogeny is broadly divided into 3-5 major phylogenetic sections, and grouped into corresponding subgenera, including Boletinus, Spectabilis, Larigini, Douglasii, and Suillus (Zhang et al., 2022). However, this intrageneric classification has not been formally recognized and the exact number of subgenera remains controversial.

Current classification schemes place Suillus in the order Boletales, in the suborder Suillineae, which contains the genera Truncocollumella, Rhizopogon, Gomphidius, Chroogomphus, and the monotypic genus Psiloboletinus (Besl & Bresinsky, 1997; Binder & Hibbett, 2006; Wu et al., 2020). The Suillineae are colloquially referred to as suilloid fungi, but represent multiple families including the Suillaceae (Suillus, Truncocolumella, and Psiloboletinus), Rhizopogonaceae (Rhizopogon), and the Gomphidiaceae (Gomphidius and Chroogomphus). The Suillineae are thought to be the first clade in the Boletales to have independently evolved the ECM lifestyle (Sato & Toju, 2019), a trait consistent across all families in the Suillineae, except the Gomphidiaceae which contains at least some mycotrophic species (Fig. 1 O-P).

Suillus diverged from other suilloid fungi between 40.2 and 71.1 MYA (Lepage et al., 1997; Zhang et al., 2022) (Fig. 4). Within Suillus, the earliest diverging clades are associated with the hosts Larix and Pseudotsuga. The most significant diversification in the genus occurred during the switch onto Pinus and it is within this Pinus-associated lineage that we find evidence for the active divergence of species complexes. However, the pattern of host switching within Pinus (especially between the host subgenera Pinus and Strobus) is convoluted and not yet fully resolved (Zhang et al., 2022). We find notable cases of ongoing evolution associated with host-switching across the genus, both in clades that are actively diverging and in those that appear relatively stable. For example, S. brunnescens diverged from within the apparently stable S. luteus clade, concurrently switching from subgenus Pinus to subgenus Strobus.

Figure 4: Associations between Suillus and their Pinaceae hosts.

Time calibrated phylogenies of proposed Suillus subgenera and Pinaceae genera and subgenera, displaying distinct topologies and no indication of cospeciation. Molecular clock dating supports the earliest divergence of Suillus between 40.2 and 71.1 MYA depending on the method of fossil calibration used. Dashed lines represent host associations, and ancestral host-switching events, highlighting ancestral associations with Larix and the host switch of Suillus subgenus Suillus from the host subgenera Strobus to the subgenus Pinus, which is thought to have occurred at least four times independently. Figure redrawn with permission from Zhang et al. (2022).

The three most notable actively diverging clades in Suillus are the /albivelatus, /flavidus and /placidus species complexes (Nguyen et al., 2016b). Within these clades, similar morphological forms are often difficult to distinguish among the members within each complex. For example, the /albivelus clade contains a group of Western North American species (S. albivelatus, S. pseudobrevipes, S. volcanalis, and S. watsatchicus) that are difficult to tell apart morphologically. Likewise, the members of the large-pored species within the /flavidus clade (S. umbonatus, S. megaporinus, S. flavidus, and S. helenae) are morphologically similar. Conversely, the /placidus clade contains a group of species (S. placidus, S. subalpinus, S. anomalus and S. punctatipes) which, although morphologically distinct, are not (yet) supported as monophyletic group (Nguyen et al., 2016b). Among these clades are species such as S. punctatipes, which has the ability to associate with alternative hosts such as Picea and Abies (Pérez-Pazos et al., 2021), although it should be noted that species outside of these clades such as S. glandulosus and S. subaureus also have the ability to associate with alternative hosts. The active evolution within (and outside of) these species complexes provide novel information that can be leveraged to understand the molecular and evolutionary mechanisms of host association and host-switching among plant-associated fungi.

III.ii: Atypical morphological features

Secotioid and gasteroid fruit bodies of Suillus-like fungi have occasionally been observed and were once classified as belonging to the genus Gastrosuillus. However, Gastrosuillus was dissolved after molecular evidence showed that these fungi were polyphyletic in Suillus (Baura et al., 1992; Kretzer & Bruns, 1997). Transitions in fruit body form can happen rapidly and are likely controlled by a small number of genes (Bruns et al., 1989). It is currently unknown whether the examples of secotioid/gasteroid Suillus found to date represent ongoing speciation or variants of known species. Such variants could be the result of spontaneous genetic mutations, malformations resulting from environmental stress during development, or ecological plasticity in fruit body form in response to environmental conditions. These scenarios are not mutually exclusive, and it may well be the case that while some secotioid/gasteroid fruit bodies are aberrations (as is likely the case for the secotioid examples of S. grevillei (Kretzer & Bruns, 1997)), others have undergone speciation and are yet to be typified, or have previously been misclassified. This is likely the case for Rhopalogaster cf. transversarius, a morphologically distinct secotioid species currently considered a member of the Rhizopogonaceae, but likely to be reclassified in Suillus, as molecular studies have found it to be nested deeply in the genus (Smith et al., 2018). As the rapid evolution of truffle-like basidiocarps is thought to be adaptive in environments with low moisture availability (as seen in the sister genus Rhizopogon) (Bruns et al., 1989), and intimately connected to differences in dispersal strategy (animal vs. wind), secotioid/gasteroid Suillus represent an intriguing system for future work. This is particularly true given the ongoing interest into Suillus drought responses, and differential dispersal capacity across biotic and abiotic variables (such as the presence or absence of animal vectors typically associated with secotioid/gasteroid dispersal) (Wang et al., 2021; Caiafa et al., 2021; Erlandson et al., 2022; Castaño et al., 2023).

Another interesting morphological feature demonstrated by some species of Suillus and Rhizopogon is the formation of tubercles. These structures are characterized by dense clusters of mycorrhized root tips surrounded in a hyphal rind (Randall & Grand, 1985). The biological and ecological function of tubercles is poorly understood, but as they bear resemblance to the bacterial nodules formed by Rhizobium and Bradyrhizobium on legumes or Frankia on Alnus, it has long been thought that they may play a role in nitrogen fixation. Paul et al. (2007) used an acetylene reduction assay to assess potential N-fixation in S. tomentosus tubercles and found evidence of nitrogenase activity up to 25098·8 nmol C₂H₄ g⁻¹ per tubercle over a 24 hour period, suggesting that the ability to form tubercles could play a significant role in N-fixation. Follow up work from the same group later identified multiple species of diazotrophic bacteria in association with the interior tissue of S. tomentosus tubercles (Paul et al., 2012), as well as isotopic evidence for N-transfer to the host tree (Chapman & Paul, 2012). Future work is needed to assess the diversity and frequency of tuberculate forming suilloid fungi, better characterize the bacterial communities associated with these structures, and quantify the contribution of tubercle-associated nitrogenase activity to host nitrogen budgets.

III.iii: Suillus vs. Rhizopogon

The genus Rhizopogon is closely related to Suillus and shares many of the traits that make Suillus an attractive model for studying ecology and evolution. Like Suillus, Rhizopogon species are obligately ECM, easily isolated, and speciose, currently including 225 recognized species which are divided into approximately five subgenera (Grubisha et al., 2002). Rhizopogon exhibits high to moderate partner specificity and typically associates with host trees in the Pinaceae, primarily Pinus and Pseudotsuga (Molina et al., 1997). Rarely, primary host associations have also been observed with Larix (Miyamoto et al., 2019), and secondary colonization is occasionally reported on non-Pinaceae hosts such as Arbutus and Arctostaphylos (Massicotte et al., 1994; Kennedy et al., 2012). Unlike the complex history of host-switching and reversion observed in Suillus, partner specificity in Rhizopogon is primarily reflected by fungal subgeneric classification (e.g., the subgenus Villosuli associates exclusively with Pseudotsuga, whereas the subgenus Amylopogon has a more generalist host range (Grubisha et al., 2002)).

Unlike Suillus, Rhizopogon exclusively forms truffle-like hypogeous fruit bodies, an adaptation which likely evolved from pileate-stipitate (mushroom) ancestors (Bruns et al., 1989; Sánchez-García et al., 2020). Like most hypogeous fungi, Rhizopogon spp. largely depend on animals to excavate their fruit bodies and distribute their spores (Bradshaw et al., 2022). The clear differentiation in fruit body forms between Rhizopogon and Suillus has been cited as evidence of strong selection against intermediate forms, putatively driven by differences in dispersal mechanisms (Bruns et al., 1989) (but see section III.ii for exceptions). Like Suillus, Rhizopogon species form long-lived spore-banks in the soil (Shemesh et al., 2023) and are often associated with early successional colonization, including seedlings and invasion fronts (Policelli et al., 2019), but often demonstrate poor competitive ability outside of these scenarios (Bruns et al., 2002).

The hypogeous and cryptic nature of Rhizopogon fruit bodies makes them more difficult to survey and sample compared to mushroom-forming fungi like Suillus. Similarly, quantifying the percent of individual root tips colonized by Rhizopogon for field or in-vitro assays can be challenging due to their typically coralloid morphology (Kennedy & Peay, 2007). For fruit bodies, morphological differentiation of Rhizopogon is notoriously complicated by a lack of variable diagnostic characters, which confound field identification, and contribute to the instability of species concepts developed in the pre-molecular era (Bidartondo & Bruns, 2002; Bubriski & Kennedy, 2014; Koukol et al., 2022; Karlsen-Ayala et al., 2022). Although Rhizopogon was long thought to be sister to Suillus, multiple molecular studies have identified fungi in the family Gomphidiaceae as more closely related to Suillus (Grubisha et al., 2002; Wu et al., 2020). However, given that the Gomphidiaceae are putatively nonmycorrhizal, and the Suillaceae genera Truncocolumella and Psiloboletinus while ECM, together contain only five recognized species (Wu et al., 2020), these genera have a distinct, but limited, use as comparative genus-level systems. In contrast, work in Rhizopogon has contributed a rich independent body of research to our understanding of ECM ecology and evolution and is the most ecologically relevant genus for comparative studies with Suillus. In addition to many similarities, Rhizopogon also demonstrates traits which are unique from Suillus, including a high propensity to be parasitized by mycoheterotrophic plants (Bidartondo & Bruns, 2002; Dowie et al., 2012; Grubisha et al., 2014), and the potential for not only heat tolerance, but growth stimulation in response to fire (Bruns et al., 2019). As we continue to build Suillus into a model genus for ecology and evolution, the concurrent development of other groups with contrasting ecological and life history strategies, including but not limited to other suilloid fungi, will complement our understanding of the mechanisms and ecological drivers at play in ECM forest systems.

III.iv: Population genetics

Because genetic divergence can often be detected before morphological divergence, deep sampling of individual species can provide a wealth of information into microevolutionary processes. To this end, member labs of International Suillus Consortium have focused efforts on continental-to-global sampling of S. luteus (the type species of Suillus), as well as S. brevipes, S. salmonicolor (cothurnatus), S. pungens, and S. quiescens. The deep sampling of these species across both native and introduced ranges has facilitated our ability to define population structure, characterize diversification events, and identify genetic bottlenecks associated with global introductions, as well as other microevolutionary processes that can drive divergence (Branco et al., 2017; Pildain et al., 2021; Ke et al., 2023).

Population genetics, the study of intraspecific variation and the forces that result in evolutionary changes in species over time (Wills, 2007), is a critical tool for understanding fungal evolution. However, the cryptic nature of fungi makes population studies challenging. Specifically, accurately counting and characterizing fungal individuals, as well as delimiting and describing fungal populations and their respective allele pools is notably more complicated in fungi than for plants or animals. Suillus is no exception to this generalization, and technical challenges to studying microevolutionary changes have historically complicated our understanding of how populations are structured and distributed, the patterns and processes that drive gene flow, and how environmental change affects genetic diversity.

Despite these challenges, Suillus fungi have been targeted for population genetic analysis since the early 1990s, capitalizing on the new availability and affordability of DNA sequencing. These studies contributed key insights about genet size (Dahlberg & Stenlid, 1994; Bonello et al., 1998; Zhou et al., 1999), population genetic structure (Dahlberg & Stenlid, 1990; Muller et al., 2007; Burchhardt et al., 2011), and gene flow (Zhou et al., 2001; Burchhardt et al., 2011) but were limited in scope regarding the number of individuals, the number of populations, and the number of markers used for population differentiation. Recent population-level sequencing efforts have facilitated major contributions to the understanding of fungal population genetics, including key insights relevant to both Suillus evolution and to ECM fungal evolution in general. Many of these advances have been facilitated by the ability to sequence whole genomes and recent efforts to assemble and annotate high quality reference strains (see Notes S4 - S6 for sequencing protocols for both DNA and RNA). For example, work on S. brevipes showed that the species is highly outcrossing and includes several distinct populations across North America that often display restricted gene flow at the regional scale (Branco et al., 2015, 2017). These studies also revealed that climate and soil chemistry play important roles in S. brevipes population structure. In contrast, the European species S. luteus displays an absence of population structure at small spatial scales, including across highly contrasting environments (Bazzicalupo et al., 2020).

III.v: Mating systems

Whether fungi produce progeny by selfing, inbreeding, or outcrossing impacts the evolutionary trajectory of species by influencing genetic diversity, deviation from Hardy–Weinberg equilibrium, and the relative level of linkage disequilibrium (Nieuwenhuis et al., 2013). The breeding systems of basidiomycetes are categorized based on the number of loci controlling mating type, where two functional MAT loci are present in tetrapolar breeding systems, one functional MAT locus is present in bipolar breeding systems, and a lack of any self-incompatibility loci results in homothallic (self-fertile) breeding systems (Nieuwenhuis et al., 2013). Generally, basidiomycete mating compatibility and mating types are governed by two self-incompatibility loci, the HD MAT locus and P/R MAT locus (Heitman et al., 2013). Two or more self-incompatible allele types at each locus dictate the mating types and the mating compatibility between gametes (Wang & Mitchell-Olds, 2017). Genomic analysis has shown that in Suillus, the P/R MAT locus contains three pheromone receptors and three or more pheromone precursors (Mujic et al., 2017) and the HD MAT locus contains a pair of homeodomain proteins flanked by genes encoding mitochondrial intermediate peptidase (MIP), as well as a gene known as the ‘beta-flanking gene’; a syntenic arrangement that appears to be conserved across most basidiomycetes (Ke et al., 2023).

In Suillus, breeding systems can be studied by both genomic methods and by constructing crosses. Crossing experiments require monokaryotic strains, most frequently produced via the germination of basidiospores (discussed in Notes S2). Crossing experiments in Suillus indicate that S. luteus, S. variegatus, S. granulatus, and S. bovinus all have bipolar, multi-allelic mating systems (Fries & Neumann, 1990; Fries & Sun, 1992; Fries, 1994). In these species, incompatibility between individuals is rare, implying that there are a high number of mating types maintained at the population level. The highest diversity of mating types recorded to date are in the species S. luteus where eight mating types were recovered from four individuals, and all mating types were found to be unique (Fries & Neumann, 1990). Although these studies have added significantly to our understanding of ECM fungal genetics, Suillus presents several challenges to constructing and confirming laboratory crosses. For example, clamp connections are not a consistent trait among dikaryotic strains of Suillus, and therefore the presence of clamps cannot be used as a proxy for monokaryon compatibility as in other groups (Fries & Neumann, 1990). Further, the secondarily homothallic binucleate basidiospores produced by some species of Suillus complicate both the determination of breeding systems and crossing experiments (Jacobson & Miller, 1994). Although the incidence of binucleate spore production by Suillus appears to be low (Horton, 2006), the population frequency and fecundity of these binucleate spores has yet to be resolved.

Insights into the evolution of breeding systems in the genus have also been supported by genome-based analyses. In agreement with studies employing laboratory crosses, comparative genomic based analysis of pheromone receptors suggests that S. brevipes has a bipolar breeding system (Mujic et al., 2017). Allelic analysis of Suillus genomes suggest that many species have a long-term multi-allelic state, and a trans-specific polymorphism at the HD MAT locus (Ke et al., 2023). This result is in agreement with the high diversity of mating types previously identified in crossing experiments, supporting HD MAT as the primary locus contributing to the bipolar mating system observed in Suillus. The closely related species Rhizopogon rubescens, also has a bipolar, multi-allelic breeding system (Kawai et al., 2008). This fact, in combination with the recognition of the wide-spread bipolar, multi-allelic mating systems observed in multiple species of Suillus, suggests that a bipolar multi-allelic breeding system is likely the ancestral state for this group.

Population-level research suggests that Suillus generally displays high levels of outcrossing. For example, studies using molecular markers in S. pungens (Bonello et al., 1998) and S. spraguei (Burchhardt et al., 2011) have highlighted an excesses of heterozygosity at the population level, suggesting that outcrossing is more predominant than random mating. Similarly, population genomic analysis of S. brevipes has shown a sharp decay in linkage disequilibrium, which also indicates a high level of outcrossing (Branco et al., 2017). The mechanisms associated with the extensive outcrossing observed in Suillus have not been tested experimentally but may be the result of self-incompatibility and heterosis. Crossing studies have found mixed results for the presence of a functional somatic incompatibility system, which may be present only in certain species such as S. luteus (Fries & Neumann, 1990). Identifying the molecular mechanisms responsible for the extremely high levels of outcrossing observed in the genus, including characterizing the identity and frequency of self-incompatibility systems, is a key future direction for understanding Suillus genetics.

III.vi: Genomics

While light-coverage genome sequencing has added important insights about population-level processes in Suillus, light-coverage sequencing is not adequate to capture nuanced information about gene and genome diversity. To this end, member labs of the International Suillus Consortium have spearheaded large-scale initiatives to sequence and annotate a phylogenetically and ecologically diverse set of Suillus fungi, using deep-coverage, whole genome sequencing of both DNA and RNA, with an emphasis on long-read technologies. Many of these efforts have been carried out in collaboration with the US Department of Energy’s Joint Genome Institute, resulting in a genome set comprising more species from a single genus than any other ECM group to date. Currently, this genome set includes 46 Suillus genomes, representing approximately 40 distinct species. Many of these genomes have been released publicly on the MycoCosm Web Portal at https://mycocosm.jgi.doe.gov (Grigoriev et al., 2014), facilitating community access and further work into Suillus biology, such as the ability to compare and contrast Suillus with other fungal groups (Lofgren et al., 2019; Miyauchi et al., 2020; Wu et al., 2022).

Comparative genomic studies using these genomes have revealed the extent of genetic diversity in the genus, identifying a dynamic genomic landscape involving many gene families, including a higher number of rapid gene family expansions and a higher number of rapid gene family contractions than any other ECM group investigated to date (Lofgren et al., 2021). These analyses have identified several genomic features that appear to be characteristic of the genus, including a diversity of gene clusters involved in secondary metabolism, and genes involved in the processing of reactive oxygen species (Lofgren et al., 2021). The ecological roles that these genes play in Suillus biology is an active area of research. While secondary metabolic clusters likely function in a variety of biological processes in Suillus, the genus-wide expansion of terpene and non-ribosomal peptide synthetase (NRPS)-like clusters hint at roles in interspecific interactions such as microbial and host communication and are ideal targets for investigating the remarkable partner specificity displayed by the genus. Recently, LC/MS based untargeted metabolomic analysis of three genome-sequenced strains of Suillus identified hundreds of unique secondary metabolites, including 116 putative terpenes (Mudbhari et al., 2023). The most abundant terpene classes were identified as terpene lactones, sesquiterpenes and di-terpenes, including several compounds induced exclusively when grown in co-culture with other species of Suillus. These results further suggest that these metabolites play a role in interactions between Suillus and other community members, but further work is needed to characterize these compounds and their ecological functions. Similarly, the processing of reactive oxygen species is known to be an important part of host colonization (Liao et al., 2016), as well as mitigating host-mediated stress responses such as the oxidative bursts characteristic of plant drought stress (Zou et al., 2021), or the unique ability of Suillus to withstand high levels of heavy metal exposure and to facilitate metal sequestration (Bazzicalupo et al., 2020). Future work linking the genomic landscape of Suillus to the functions displayed by the genus will be key to developing a deeper mechanistic understanding of Suillus biology.

Efforts to leverage the benefits of both deep-coverage whole genome sequencing and population-level sampling are underway, with projects designed to characterize genome variation in target species such as S. quiescens, S. luteus, S. brevipes, S. salmonicolor, S. pungens, and S. tomentosus. We expect these projects to yield novel insights into intraspecific variation in Suillus, including genome structure, noncoding and repeat regions, and improved frameworks for linking genotypes to phenotypes. For example, comparative genomic analysis including 12 strains of S. brevipes found that rDNA copy number varied from 72 to 156 within the species, only slightly less variation than was estimated across the genus as a whole (44 to 198 copies) (Lofgren et al., 2019). Leveraging functional-omics technologies on genome-sequenced strains will produce further insights into natural variation, and the ecology of Suillus-community interactions. Continuing efforts to produce more reference-quality Suillus genomes, the increased sampling breadth of target species for pangenomic analysis, improved pipelines for de novo assembly and annotation of unique strains, and an increase in the use of long read sequencing, will all propel and direct future insights into ECM fungal genomics.

IV. Ecological interactions

The unique intrinsic traits discussed above have enabled many studies on ecological interactions between Suillus and their surrounding communities. There has long been a focus on Suillus in the context of ECM partner specificity as well as many investigations of Suillus species as both competitors of other fungi and symbionts mediating plant responses to changing environmental conditions (see Notes S7 on bioassay construction). Below we outline some of the major findings that have emerged from these ecologically focused studies.

IV.i: Partner specificity

Generally, co-occurring ECM host species exhibit large overlaps in ECM species associations, suggesting that partner specificity is low for most ECM taxa (Peay et al., 2015; Peay, 2016; Arraiano-Castilho et al., 2021). While recent work has highlighted the possibility that cryptic species may inflate estimates of host generalism by artificially combining taxa that are actually distinct, a phenomena known as cryptic specificity (Sato et al., 2007; Wilson et al., 2017; van der Linde et al., 2018), how often this occurs in practice is unclear. Importantly, partner specificity is not a binary classification of specialists versus generalists, but rather a spectrum of interactions, ranging from fungal associations with a single host species to those spanning hundreds of host species. While it is likely that some genera such as Laccaria, Thelephora and Russula, are largely composed host generalists (Roy et al., 2008; Smith et al., 2009; Cho et al., 2021), others such as Lactarius and Strobilomyces probably exhibit gradients of partner specificity or host preference (unequal colonization when multiple host taxa are available) that warrant more nuance then they have been historically afforded (Looney et al., 2018; Tang et al., 2021).

Early on, it was recognized that a small number of ECM fungal groups exhibited remarkable and conspicuously high partner specificity with certain host taxa. These specialists include Alnicola, Alpova and other basidiomycetes associated with the actinorhizal host genus Alnus (Tedersoo et al., 2009; Rochet et al., 2011), the genus Leccinum with various species of broad-leaf trees (Den Bakker et al., 2004), and the genera Suillus and Rhizopogon with the plant family Pinaceae (Dahlberg & Finlay, 1999). Each of the major host genera (Pseudotsuga, Larix, and Pinus) are associated with a suite of Suillus species that appear to form mycorrhizas almost exclusively with that host (Table S1, Nguyen et al., 2016b). While partner specificity in ECM fungi is most well recognized at the level of host genus (Molina et al., 1992), Suillus species tend to be specific to host subgenera, particularly within the genus Pinus. The most detailed account of this level of specificity was presented by Liao et al. (Liao et al., 2016), who combined seedling bioassays with metatranscriptomics to critically assess specific plant-fungus associations. The authors compared colonization and gene expression patterns across 5 species of Suillus and 10 species of Pinus and showed that several Suillus species, including S. americanus, S. granulatus, and S. spraguei only formed compatible associations with Pinus species in a single subgenus. This subgenus-scale specificity appeared to be associated with the expression of similar genes and metabolic pathways previously identified in pathogenic plant-fungal interactions.

While close pairings of particular Suillus species with specific hosts is the most common framing of this ECM symbiosis, recent studies indicate that patterns of Suillus partner specificity are strongly mediated by ecological context. Within the plant host family Pinaceae, multiple tree genera have long been considered non-hosts of Suillus, including Picea (a genus in the subfamily Piceoideae nested within a clade of known Suillus host genera; (Gernandt et al., 2016) as well as Abies and Tsuga (in the subfamily Abietoideae, a clade that does not contain other Suillus host genera) (Fig. 4). Based on sporadic field reports of Suillus mushrooms being collected in Picea or Abies/Tsuga forests with no known Suillus host genera present (Doudrick et al., 2011), Pérez-Pazos et al. (Pérez-Pazos et al., 2021) combined field root tip sampling and experimental seedling bioassays to assess the partner specificity of two Suillus species; S. glandulosus and S. punctatipes. The authors found that Picea, Abies, and Tsuga root tips were colonized by Suillus species in the field and that both Suillus species were capable of colonizing alternative hosts in the laboratory. Importantly, however, the colonization of alternative hosts only occurred when a known host (a species of Pinus or Pseudotsuga) was co-planted in the same container. This outcome matched prior studies of ECM partner specificity (Massicotte et al., 1994), where it was shown that certain ‘primary’ host species are required to trigger spore germination, but that additional ‘secondary’ hosts could be colonized once the ECM fungus was growing as mycelium. Even greater host phylogenetic breadth was shown for S. subaureus by Lofgren et al. (2018), who demonstrated through a combination of field sampling and seedling bioassays that this species was capable of colonizing both Pinus and Quercus hosts. This unusual association with Quercus (an angiosperm) appeared to be associated with the local extirpation of Pinus hosts during intensive logging decades earlier, resulting in a unique ecological legacy effect. Differences in host range between primary and secondary colonization such as those identified for S. glandulosus, S. punctatipes and S. subaureus may seem like curious exceptions but could have significant consequences for ecosystems. Secondary colonization is likely responsible for reports of Suillus on other non-canonical hosts, including Betula (Nara, 2006), Cupressus, and Ceratonia (Avital et al., 2022), and could influence the succession dynamics of plant communities, particularly if carbon transfer from secondary hosts is sufficient to produce fruit bodies or if resources are transferred via exudates or common mycelial networks. As plant host ranges shift due to a changing climate and human introductions, understanding ECM partner specificity is key to predicting the establishment of these interactions in novel ecological contexts.

IV.ii: Competition

Like all ECM fungal species, members of the genus Suillus compete to colonize host root tips as their main source of carbon in natural settings (Baldrian, 2009). Prior to molecular identification of ECM root tips, the relatively high annual production of sporocarps by most Suillus species suggested this genus was a dominant part of the ECM fungal community in many conifer forests (Dahlberg & Finlay, 1999). However, as molecular-based root tip surveys proliferated in the 1990s and 2000s, it became apparent that most Suillus species colonize only a limited number of root tips compared to other ECM genera such as Tomentella and Russula (Gardes & Bruns, 1996; Jonsson et al., 1999; Peay et al., 2007). However, in early successional forests, non-native environments, and post-fire environments Suillus often deviate from this pattern and dominate root tip communities (Visser, 1995; Chapela et al., 2001; Hayward et al., 2015; Miyamoto et al., 2021; Thompson et al., 2022). This pattern of root-tip dominance can also be seen in some specialized habitats such as hummocks present in Larix systems (accumulations of organic matter that exist directly above the waterline), where Suillus can occupy a significantly higher proportion of the ECM fungal community than is typical (Kennedy et al., 2018). In general, however, Suillus is rarely the most dominant genus colonizing ECM root tips in conifer forest soils where it is endemic.

Given the aforementioned pattern, one might speculate that Suillus species are relatively weak competitors for host root tips or easily excluded by other ECM fungi during fungal community succession. Support for a limited competitive ability has been observed in multiple seedling bioassay studies involving Suillus and other ECM fungi. For example, Kennedy et al. (2011) found that S. pungens was outcompeted by two species of Rhizopogon when colonizing Pinus muricata seedlings from mycelia. Similar results were found by Moeller and Peay (2016) who demonstrated that S. pungens was also outcompeted by both Thelephora terrestris and R. occidentalis when colonizing P. muricata seedlings from spore. Collectively, these experimental findings, in conjunction with root tip abundance patterns in field studies, suggest that Suillus species may indeed be relatively weak competitors when faced with either spore- or mycelial-based competition from other ECM fungi. It should be noted, however, that Rhizopogon and Thelephora are aggressive colonizers of seedlings, and studies across a much wider range of Suillus and competitor species are needed to have greater confidence in this pattern. The pattern of low root tip abundance, increased plant health, and large and abundant fruit bodies suggests an effective carbon trading balance where only a limited number of root-tips are needed to achieve advantageous outcomes for both hosts and symbionts.

Perhaps to compensate for limited interspecific competitive ability, members of Suillus appear to have specialized in reproductive traits that favor rapid and abundant dispersal (Policelli et al., 2019), which likely favors their ability to colonize root tips that may have few other ECM fungi present. This has been best evidenced in the quantitative estimates of ECM fungal spore dispersal at increasing distances from established forests, which typically reveal that Suillus represent an increasing proportion of ECM spores at the farthest distances (Peay et al., 2012; Thompson et al., 2022). Similar tradeoffs in competitive versus colonization ability have been routinely observed in plant communities (Tilman, 1994) and Suillus is arguably one of the best documented examples of this phenomenon within ECM fungi (Peay et al., 2007).

Studies focused on congeneric competition among Suillus species have also revealed interesting ecological trends. Working with a set of species that are commonly associated with Pinus strobus, Kennedy et al. (2020) found a clear competitive hierarchy of S. americanus > S. subaureus > S. spraguei. The outcome of the competitive interactions appeared to be largely the result of differences in spore germination timing, with S. americanus being the first and most consistent to colonize P. strobus seedling root tips, followed by S. subaureus, and then S. spraguei. The early colonization by one species precluding the establishment of subsequent colonizers, also known as a priority effect, has been observed consistently in a variety of other ECM study systems as well (Kennedy, 2010; Bogar & Kennedy, 2013). Interestingly, S. subaureus, is a much rarer species than either S. americanus or S. spraguei, which suggests that other factors besides competitive ability are also likely important in shaping abundance in ECM fungal communities.

To investigate the extent to which ECM host plants influence ECM fungal competitive outcomes, Bogar et al. (2019) conducted a pair of seedling bioassays using the Larix-associated species; S. clintonianus, S. grisellus, and S. spectabilis. The authors used a split root design in which competing Suillus species were spatially isolated when colonizing a shared host (eliminating the role of direct fungal-fungal interactions). They found that colonization by two of the three species was not changed by a second species being present on a different portion of the root system, but that the colonization by S. spectabilis was significantly lower if a second Suillus species was present. This suggested that plants can differentiate amongst different Suillus species and can potentially mediate root tip colonization to privilege preferred species. Further evidence for differential plant investment was found in the second bioassay, in which seedlings allocated greater amounts of carbon to the portion of the split-root system that provided the greatest amount of nitrogen. Intriguingly, seedlings independently colonized by S. spectabilis were not significantly lower in needle nitrogen content than those colonized by S. grisellus or S. clintonianus, so exactly why the Larix host plants were preferentially colonized by the latter two species was not clear. In natural habitats, S. spectabilis can only be found with mature host plants, whereas the other two species can be found with younger hosts, suggesting a host age-dependent context of interactions. This study, along with additional studies manipulating soil nitrogen levels (Bogar et al., 2022), highlight that competitive outcomes among ECM fungi may be influenced by host plant tendencies toward specific taxa.

IV.iii: Soil nutrient cycling

Similar to other ECM fungi, Suillus are thought to have a limited capacity to decompose complex plant cell wall polymers (Kohler et al., 2015). As the vast majority of ECM fungi evolved from saprotrophic ancestors (Tedersoo et al., 2010) many have retained the ability to decompose less complex polymers such as protein and chitin, which could account for observed patterns of organic matter turnover (up to 30%) in introduced Pinus plantations (Chapela et al., 2001). Protein decomposition appears to be dependent on forest age, with Suillus species in younger forests having lower protein decomposition activity (Rineau et al., 2016), which could be advantageous given that more mature forests contain higher levels of organic nitrogen. Genomic and functional assays have demonstrated that S. luteus also uses endochitinases to decompose complex chitin polymers (Maillard et al., 2023). Initial work suggests that in low carbon soils, S. salmonicolor (cothurnatus), like some other ECM species, can prime decomposition by saprotrophic fungi (Bhatnagar et al., 2021). These pieces of evidence indicate that although limited in their capabilities to decompose complex plant cell wall polymers, Suillus can substantially contribute to decomposition of soil organic matter, and thus nutrient cycling, especially nitrogen cycling in soil.

Suillus themselves can be consumed, decomposed, and returned to the soil as labile sources of carbon and nutrients. Members of this this genus often produce large aggregations of fruit bodies, estimated to be upward of 63.9kg ha⁻¹ year⁻¹ in dry weight for S. variegatus (Ohenoja & Koistinen, 1984), to one metric ton ha⁻¹ year⁻¹ dry weight for S. luteus (Hedger, 1986; Chapela et al., 2001). Notably, these estimates did not include hyphae and rhizomorphs, which likely represent a substantial additional pool of tissue available for decomposition. These high nutrient resources are rapidly consumed by insects, in particular phorid and mycetophilid flies (Bruns, 1984). The fruit bodies (Gohar et al., 2022) and mycelium (Brabcová et al., 2016) can also be decomposed by bacteria and fungi that appear to have specificity towards these fungal-derived resources (Nguyen, 2023). Enabled by the ready abundance of fruit bodies and the easy culture of Suillus mycelium Fernandez et al. (2019) and Maillard et al. (2022) used S. grisellus to conduct manipulative experiments characterizing the contributions of Suillus necromass to soil nutrient cycling as well as identifying the microbial members involved in this process. This area of research is currently experiencing a renaissance as modern tools and techniques provide exciting opportunities to dissect the molecular mechanisms and community interactions that facilitate the degradation of fungal necromass (Fernandez & Kennedy, 2018; Kennedy & Maillard, 2023) and will be influential in elucidating the contributions of Suillus (and other high-biomass producing fungal species) to soil nutrient cycling.

V. Environmental (abiotic) adaptation

Studies in the genus Suillus have enabled significant advances in our knowledge of fungal abiotic adaptation. In this section, we outline recent progress in our understanding of how the genus impacts, and is impacted, by environmental variation. Specifically, we highlight studies assessing climate change, ionic radiation, metal pollution, and enzymatic activity and degradation. The availability of public protocols and genomic resources are expected to catalyze further research in these areas and assist in the development of new strategies to remediate and recover degraded habitats.

V.i: Climate change

Climate change, characterized by elevated temperatures and shifts in patterns of precipitation, is increasingly impacting forest ecosystems, and influencing symbiotic relationships between fungi and their host plants (Baldrian et al., 2023). Recent research has identified Suillus as a key player in enhancing host plant resilience to environmental stress, and advanced our understanding of how ECM fungi respond and adapt to changing environmental conditions (Malcolm et al., 2008; Branco et al., 2017; Li et al., 2021; Qi & Yin, 2022; Hou et al., 2022; Erlandson et al., 2022).

As a result of shifting climatic conditions, there has been a global increase in the frequency and severity of drought in recent years (Dai, 2012), posing a significant threat to the resilience of forest ecosystems (Brodribb et al., 2020). Suillus exhibit considerable drought tolerance, which potentially sustain symbiotic relationships and enhance plant stress tolerance under drought conditions. The long-distance rhizomorphs of Suillus are well known to aid water transport (Duddridge et al., 1980), which may help host plants to maintain positive water relations during times of abiotic stress. Support for this hypothesis was recently demonstrated by Castaño et al. (2023), who found that Pinus pinaster seedlings exposed to experimental drought in Spain were enriched in ECM fungal taxa with long-distance rhizomorphs. In particular, they found that S. variegatus was an indicator species of drought (i.e. was more relatively abundant under the drought treatment), suggesting that some species of Suillus may be better able to tolerate low water availability than other ECM taxa. At the same time, this study also reported that the abundance of S. bovinus declined significantly in the drought treatment, indicating that not all Suillus species are drought resistant. This result is consistent with the findings of Erlandson et al. (2022), who showed that long-term water reduction dramatically decreased root tip colonization by S. pungens on Pinus muricata seedlings. This study found that while short-term water reduction led to major changes in the expression of functional genes associated with water acquisition, gene expression under long-term water stress was very similar to that of well-watered controls. This suggests that ECM fungi may be capable of significant acclimation under prolonged stress, which may aid in maintaining symbiotic functioning under challenging environmental conditions (Palumbi et al., 2014). Erlandson et al. (2022) further found that drought-exposed S. pungens was able to maintain cellular integrity by up-regulating genes related to fungal cell wall synthesis.

Significantly, Suillus may pass drought tolerance benefits onto their host trees. Several studies have found evidence for this effect and have proposed several potential mechanisms including osmoregulation, oxidative protection, and the preferential relocation of plant resources. For example, osmoprotective compounds such as proline and trehalose have been shown to contribute to plant osmoregulation by providing protection against drought-induced damage, and appear to be upregulated in Suillus during drought stress (Li et al., 2021; Erlandson et al., 2022). Osmoregulation may also occur by increasing host water uptake via the induction of water-channel proteins such as aquaporins (Lee et al., 2010; Xu & Zwiazek, 2020). While multiple studies have indicated that S. tomentosus can improve host tolerance to salt exposure via aquaporin-mediated water transport (Lee et al., 2010; Calvo-Polanco & Zwiazek, 2011), researchers have been unable to provide direct evidence that Suillus regulates host-water movement under drought conditions. As such, future research is needed to confirm the induction of water-channel proteins as a drought tolerance mechanism in the genus.

A second proposed mechanism for Suillus mediated drought tolerance is via oxidative defense. Li et al. (2021) found that roots colonized by S. placidus appeared to trigger antioxidant defense against drought-induced oxidative stress through the activation of antioxidant enzymes. These results suggest that some species of Suillus may be capable of counteracting the negative impacts of drought-induced oxidative damage, thereby promoting plant health and survival.

Finally, Suillus may mediate drought responses by altering the physiology of their hosts. For example, Qi and Yin (2023) found that under drought conditions S. luteus altered host root traits including diameter, length, surface area, and volume, improving water and nutrient uptake efficiency. A similar pattern was detected in S. pungens-inoculated mycorrhizal roots which responded to drought by up-regulating genes involved in energy production, signal transduction, metabolism, and the transport of amino acids, lipids, and carbohydrates (Erlandson et al., 2022). Despite the inhibitory effects of drought stress on photosynthesis, plant development, and growth, Suillus appears to assist in mitigating these effects by enhancing photosynthetic performance and increasing stomatal area and density (Li et al., 2021; Qi & Yin, 2022). Taken together, these results suggest that Suillus may confer drought tolerance to host plants by increasing the production of photosynthetically-fixed carbon that is then preferentially reallocated to belowground structures, which in turn support mycorrhizal root growth and hyphal water uptake.

V.ii: Ionizing Radiation

High levels of radiation are a major concern for human health and ecosystem function. Suillus has provided important insights into how ionizing radiation affects the fungal kingdom. Irradiation results from radionuclides, unstable isotopes that release radiation when they break down to more stable elements. While these elements occur naturally, they are primarily concerning when produced artificially and subsequently enter the environment in high concentrations, typically via industrial waste or the detonation of nuclear weapons (Hu et al., 2010). Exposure to high levels of ionizing radiation has wide ranging negative effects, including genetic and epigenetic changes, decreased reproductive ability, abnormal morphology, and mortality (Hinton et al., 2007).

The devastating accidents that occurred in Chernobyl (Ukraine) in 1986 and in Fukushima (Japan) in 2011 heavily impacted vast areas with radio-active fallout. These events have been widely studied to determine how chronic irradiation affects species and ecosystems. The genus Pinus is extremely sensitive to irradiation (Geras’kin et al., 2008), and the nuclear accidents that occurred in Japan and Ukraine affected large swaths of pine-dominated forest (Vinichuk & Johanson, 2003; Pumpanen et al., 2016; Bondarenko et al., 2023). Studies from these areas suggest that some fungi, including Suillus, are relatively tolerant towards ionizing radiation (Dighton et al., 2008). These fungi act as ¹³⁷Cs radionuclide sinks, effectively removing them from the soil. This process may influence the cycling of radionucleotides across soil compartments, as both S. luteus and S. variegatus accumulate high levels of ¹³⁷Cs in fruit body tissue (Nikolova et al., 1997; Ronda et al., 2022). Similarly, Vinichuk and Johanson (2003) found that an astounding 19% of total soil ¹³⁷Cs were transported into S. variegatus mycelium. Importantly, these effects appeared to be species-specific, as not all Suillus species accumulated ¹³⁷Cs to the same extent. The ability of some Suillus species to act in the bioaccumulation of ¹³⁷Cs raises the question of whether Suillus impacts ¹³⁷Cs uptake in host trees. In plants, ¹³⁷Cs uptake is mediated by K transporters and K channels (Zhu & Smolders, 2000). In Suillus however, the concentration of ¹³⁷Cs is not correlated with K concentration (Dighton et al., 2008; Vinichuk et al., 2011), suggesting that in fungi, ¹³⁷Cs uptake may be independent of K pathways. Interestingly, there seem to be differences in irradiation effects and molecular responses among fungal species that differently impact growth, melanin production, and ROS scavenging enzymes. For example, Kothamasi (2019) found that while multiple S. luteus isolates displayed reduced biomass upon irradiation exposure, S. bovinus was unaffected. Similarly, while all Suillus isolates showed melanin accumulation in response to irradiation, the response of ROS decomposing enzymes varied both between and within species.

Precisely how irradiation affects ECM fungi, if ECM fungi are involved in plant tolerance to ionizing radiation, and if so, by what mechanisms, are active areas of research (Ladeyn et al., 2008). Given the extensive areas affected by irradiation and the abundance and relevance of Suillus species in these ecosystems, Suillus is an ideal model to investigate the impacts of radiation, and a prime candidate for the development of bioremediation systems. To this end, members of the International Suillus Consortium are currently building a Suillus culture collection from Fukushima radionuclide-contaminated soil to investigate both the effects of irradiation in ECM systems and develop urgently needed bioremediation strategies.

V.iii: Metal tolerance

Soil metal contamination through anthropogenic activity can be extremely detrimental to ecosystems, negatively impacting metabolism, growth and reproduction (Branco et al., 2022). Metal tolerance in Suillus is a widely documented phenomenon and several species are routinely found in metal contaminated soils, including sites near mining and metal manufacturing facilities, decommissioned smelters and mills, and sites with heavy ore enrichment (Colpaert & van Assche, 1987; Leski et al., 1995; Blaudez et al., 2000; Adriaensen et al., 2005). This finding has driven multiple studies focused on characterizing metal tolerant isolates with the goal of assessing whether the presence of metal contamination has selected for metal tolerant traits and led to local adaptation (Colpaert & van Assche, 1987; Bazzicalupo et al., 2020).

Early studies demonstrated that metal tolerance in Suillus not only varies across species, but also across isolates within the same species. For example, S. luteus, S. variegatus and S. granulatus display interspecific variation in tolerance to Cd, Pb, Sb and Zn (Hartley et al., 1997), while both S. luteus and S. bovinus display intraspecific variation in tolerance to Cd, Cu, Ni and Zn (Colpaert & Van Assche, 1992; Blaudez et al., 2000; Adriaensen et al., 2005). Interestingly, although Suillus isolated from metal contaminated sites generally display higher levels of metal tolerance, metal sensitive isolates have also been collected from metal contaminated soils (Blaudez et al., 2000; Fomina et al., 2005; Adriaensen et al., 2005). Fomina et al. (2005) proposed that this observation could be explained by the heterogeneous nature of soil, which could lead to uneven metal concentrations across a single soil patch, allowing metal sensitive isolates to persist. However, metal tolerant Suillus have also been discovered in non-contaminated areas (Leski et al., 1995; Hartley et al., 1997; Colpaert et al., 2004), suggesting that the fitness costs to metal tolerance may be inconsequential, or that there may be benefits to retaining metal tolerance traits apart from metal exposure. While Colpaert (2004) suggested that gene flow could occasionally bring tolerant genes into sensitive populations, genetic analysis of S. luteus from contaminated and non-contaminated sites did not support the presence of separate populations (Bazzicalupo et al., 2020).

Metal tolerance in Suillus appears to result from the ability to maintain metal homeostasis through a combination of metal immobilization, exclusion, and detoxification. These mechanisms rely on a large number of genes, including transmembrane transporters (which move metal into, out of and around the cell), chelators (that bind and immobilize ions both within and outside the cell), and antioxidants that counteract the effects of metal toxicity (Bazzicalupo et al., 2020; Branco et al., 2022). Analysis of S. luteus demonstrated that metal tolerance is likely achieved through genetic variants arising from standing genetic variation (Bazzicalupo et al., 2020). In a recent study, Smith et al. (2023) conducted a transcriptomic analysis of Zn-tolerant and Zn-sensitive S. luteus isolates exposed to high Zn concentrations and confirmed that Zn tolerance in this species is polygenic. This study showed that different isolates achieve tolerance through distinct mechanisms and that Zn-tolerant isolates are largely constitutively tolerant regardless of metal exposure. However, the authors also identified two Zn related genes for which expression was affected by Zn concentration, suggesting that Zn tolerance in S. luteus also includes an environmental component.

Studies investigating the genetics and physiology of Suillus metal tolerance have demonstrated that metal accumulation in cells is a limiting factor for tolerance (Colpaert & van Assche, 1987). For example, Colpaert et al. (2005) found that Zn uptake is negatively correlated with tolerance in S. luteus, and that the exclusion of Zn from cells allows tolerant isolates to persist when exposed to high Zn concentrations. Cadmium tolerance in S. luteus may similarly rely on a metal exclusion mechanism, as tolerant isolates have been shown to take up less metal into the mycelium than sensitive isolates (Krznaric et al., 2009). Cadmium tolerant S. luteus isolates have also been shown to express antioxidant transcripts at much lower levels than Cd sensitive isolates, suggesting Cd tolerance is grounded in exclusion, and that Cd tolerant Suillus likely avoid cellular damage by preventing Cd uptake in the first place (Ruytinx et al., 2011).

There has been substantial progress in illuminating the mechanisms of metal exclusion and metal binding in Suillus. Multiple transmembrane transporters have been identified including Cation Diffusion Facilitator (CDF) family transporters and Zrt-, Irt-Like Protein (ZIP) transporters. Whereas CDF transporters are membrane bound proteins that transport a variety of metals (Kolaj-Robin et al., 2015), ZIP transporters are Zn pumping proteins that are typically localized to the plasma membrane. These transporters act to bring external Zn into the cells and to draw Zn from vacuolar stores to prevent toxicity (Coninx et al., 2017), and have been characterized in S. bovinus (Ruytinx et al., 2013). In S. luteus there are four CDF proteins predicted to be Zn transporters. Two of these (SIZnT1 and SIZnT2), have been functionally characterized, and S1ZnT1 has been shown to confer Zn tolerance using heterologous expression in yeast (Ruytinx et al., 2017). S. luteus also has four predicted ZIP transporters, two of which have been described. SIZRT1 is a plasma membrane transporter heavily involved in maintaining cellular Zn homeostasis (Coninx et al., 2017) and SIZRT2 is localized to the plasma membrane and the perinuclear region and also appears to play a role in Zn uptake (Coninx et al., 2019).

Metal binding is also an important mechanism of metal tolerance in Suillus. In S. luteus, there are two known metallothioneins (small proteins that can bind and immobilize metals) involved in Cu homeostasis (SlMTa and SlMTb). These genes are upregulated in excess Cu, but not in excess Zn or Cd. Heterologous expression of these genes in a Cu sensitive yeast mutant has been shown to restore tolerance (Nguyen et al., 2017). Metallothionein homologues with a role in Cu homeostasis have also been identified in S. himalayensis. These genes were shown to be induced by excess Cu, but not by Cd and heterologous expression in yeast confirmed a role in Cu tolerance, along with possible roles in Cd and Zn homeostasis (Kalsotra et al., 2018).

Notably, Suillus metal tolerance appears to have significant downstream effects on host plant fitness (Adriaensen et al., 2005). When associated with plant roots exposed to high metal concentrations, some Suillus isolates can reduce metal transfer and accumulation in host plant tissues while still efficiently transferring nutrients to the host (Adriaensen et al., 2006; Colpaert, 2008; Krznaric et al., 2010; Colpaert et al., 2011; Branco et al., 2022). While metal-tolerant and metal-sensitive Suillus seem to be equally effective mycorrhizal partners regarding nutrient uptake and transfer, there are clear differences in plant nutrient uptake and nutrient status when exposed to high metal concentrations (Adriaensen et al., 2004; Krznaric et al., 2010). For example, Andriaensen et al. (2005) demonstrated that inoculation with Cu-tolerant isolates of S. luteus significantly improved plant phosphorus and nitrogen uptake in Cu-contaminated soil, while Cu-sensitive isolates reduced nutrient uptake and transfer. Suillus colonization can also facilitate metal tolerance indirectly, for example, Liu et al. (2020, 2021) demonstrated that S. luteus activates multiple plant stress pathways, enhancing plant fitness during metal exposure. Similarly, metal exclusion mechanisms in tolerant Suillus isolates have been shown to reduce metal transfer to the host (Krupa & Kozdrój, 2004; Zhang et al., 2021). For example, when associated with Pinus roots under high external Zn, S. luteus accumulates Zn in the hyphal mantle, preventing Zn accumulation in host tissues (Zhang et al., 2021).

VI. Biological introductions and invasions

The strong ecological linkages between Suillus and the Pinaceae help explain the key role of the genus in the spread and establishment of Pinaceae into novel habitats. While this ability has long been beneficial for the forestry industry, enabling the wide-spread establishment and success of tree plantations, it has also facilitated the invasion of introduced host taxa into native plant communities around the world, displacing species and disrupting ecosystems (Read, 1998; Policelli et al., 2019). While many Suillus host species have been introduced across the world, not all introductions have resulted in biological invasions. The variables determining invasion capacity, along with the ecological impacts of Suillus-associated host-tree establishment in non-native environments are important areas of research.

VI.i: Introduction via forestry

While trees in the plant family Pinaceae are native to the northern hemisphere, they have been planted globally for shade, shelter and the establishment of softwood plantations. Exotic Pinus plantations currently cover over five million hectares, and their global effects on primary productivity, biogeochemistry, and biodiversity represent some of the most widespread and impactful introductions on Earth (Simberloff & Von Holle, 1999; Hoeksema et al., 2020).

Most early attempts to introduce Pinaceae into the southern hemisphere failed due to the lack of availability (and knowledge) of suitable ECM fungi (Mikola, 1970). As foresters became aware of ECM and their role in forest nutrition, inoculating seedlings with ECM fungi became common practice. Historically, the most commonly used inoculum source was transplanted soil from established Pinaceae plantations (Marx, 1992; Marx et al., 2002). This practice led to the successful establishment of plantations across both the northern and southern hemispheres, along with the widespread dissemination of Pinaceae-associated ECM fungi. Early on, it was recognized that the ECM species that established themselves in exotic Pinaceae plantations represented only a fraction of native ECM biodiversity, and overwhelmingly consisted of early colonizers such as Suillus and Rhizopogon (Theodorou, 1967; Theodorou & Bowen, 1973). In areas where Pinaceae have been introduced, this process of ecological filtering has resulted in as few as 1-5 species of ECM fungi in New Zealand Pinus plantations (Chu-Chou & Grace, 1988), and as few as one species (S. luteus) in Pinus plantations in Chile (Hayward et al., 2015). This reduction in diversity is also evident for other introduced members of the Pinaceae. For example, Larix and Pseudotsuga have been widely planted in the southern hemisphere, where they exhibit strong partner specificity with Suillus, including S. cavipes with introduced Larix and S. lakei with Pseudotsuga (McNabb, 1968; Moeller et al., 2015; Pietras et al., 2018).

VI.ii: Suillus-Pinaceae co-invasions

Successful afforestation and introduction of Pinaceae across the southern hemisphere has inevitably resulted in the escape of introduced trees that interact with and sometimes displace native plant communities (Richardson et al., 1994; Simberloff & Von Holle, 1999; Nuñez et al., 2009). Burdon and Chilvers (1977) were the first to report the process of escape and invasion by wilding Pineaceae trees. The study detailed P. radiata invasion from plantations into adjacent Eucalyptus forests near Canberra, Australia, resulting in the formation of a new type of hybrid forest community. Follow up studies monitoring these forests over the subsequent 14 years, found that the growth of P. radiata was strongly affected by the presence of the other plant species (Chilvers & Burdon, 1983; Burdon & Chilvers, 1994), leading to the eventual coexistence between Pinus and native vegetation. Within Australia, the impact of Pinus invasions seems to be relatively small with most wilding tree escapes occurring after periodic disturbance or fire (Williams & Wardle, 2007; van Etten et al., 2020). This is in contrast to Pinus introductions on other continents, which have resulted in active invasion fronts, tremendous loss of native biodiversity, and habitat alteration. The negative impacts of these invasions have been greatest in regions where host trees are able to grow and shade out other plant species, such as in the Cape Region of South Africa where P. radiata now threatens native Fynbos vegetation (Richardson et al., 1994), and in Brazil, Argentina, and Chile, where invasive Pinus have escaped into native grasslands (Nuñez et al., 2009). To date, efforts to eradicate invasive Pinaceae hosts and restore these ecosystems have achieved only limited success.

The concept that ECM fungi had a role in facilitating host tree invasions was first suggested by Richardson et al. (1994) and Read et al. (1998), and later confirmed using molecular approaches (Nuñez et al., 2009; Dickie et al., 2010; Hynson et al., 2013; Moeller et al., 2015; Horton, 2017). This pattern of invasion has been termed “Linked Plant-Fungal Co-invasion” (Dickie et al., 2017). Today, plantation trees are inoculated with ECM fungi as a matter of course, typically using soil-free spore-based methods or mycelial slurries where strain provenance can be controlled (Marx et al., 2002). Contemporary commercial inoculum is produced using many ECM taxa, including Pisolithus tinctorius, Laccaria spp., Rhizopogon spp. Hebeloma spp., and Suillus spp. (Marx et al., 2002; Charya & Garg, 2019). Whether due to innate trait-based effects, or legacy effects from soil-based inoculation, Suillus seem to have disproportionate invasion potential: in almost every study conducted to date on linked plant-fungal co-invasions, Suillus species have been identified as keystone community members. While tree plantations are the best studied source of Suillus introductions into non-native ranges, it is important to note that ECM fungi can be translocated along with any compatible host tree or introduced intentionally or inadvertently with the movement of soil. This underscores the importance of knowing mycorrhizal host status and the identity of the ECM fungi associated with introduced trees for applications such as forest restoration, shelterbelts (windbreaks), and ornamental plantings.

To generate an up to date assessment of Suillus introductions, we used the Global Database of Alien Macrofungi (Monteiro et al., 2020). This database includes the 770 ECM distribution records originally assembled by Vellinga et al. (Vellinga et al., 2009), but is extended to include multiple guilds, and studies completed since 2009, for a total of 1,966 observations. We pruned this dataset to exclude taxa with uncertain species-level assignments, and those annotated as having established from uncertain geographic origins. To identify ECM species, guild was assigned across the database using FUNGuild (Nguyen et al., 2016a), retaining only species that could be confidently identified as ECM and excluding mixed-guild types, resulting in 946 observations, spanning 55 genera and 241 species. Out of these 55 ECM genera, Suillus made up the vast majority of observed introductions, with a total of 193 records, followed by Scleroderma at 91, and Rhizopogon at 72 (Fig. 5A). While the dominance of Suillus is striking, as noted by Vellinga et al. (Vellinga et al., 2009), occurrence records of species such as Scleroderma and Rhizopogon are likely underestimated due to the relative difficulty of locating their fruit bodies. These results broaden our understanding of genus-level ECM invasion frequency, and emphasize the ongoing prevalence of Suillus in ECM introductions as reported 15 years ago in Vellinga et al. (Vellinga et al., 2009). In total, there were 24 species of Suillus in introduced ranges (Fig. 5B). Largely in agreement with previous studies (Read, 1998; Dunstan et al., 1998; Policelli et al., 2019), the most common species were the European Pinus associated taxa S. luteus (44 observations) and S. granulatus (34 observations), followed by the larch associate S. grevillei (18) and Douglas Fir associate S. lakei (15). It should be noted that the western north American species S. quiescens (typified in 2010) is also an important introduced species (Policelli et al., 2019) but was absent from the database (likely combined with the morphologically similar S. brevipes).

Figure 5: Biological introduction records.

Records from the Global Database of Alien Macrofungi (Monteiro et al., 2020) were pruned to exclude taxa with uncertain species-level assignments, and those annotated as having established from uncertain geographic origins. Guild was assigned across the database using FUNGuild retaining only species that could be confidently identified as Ectomycorrhizal (ECM), resulting in 946 observations. A) Out of 55 genera of ECM fungi with introduction records, Suillus accounted for the majority of observed introductions, totaling 193 records. B) In total, 24 species of Suillus have introduction records, with the most common species identified as S. luteus (44 observations) and S. granulatus (34 observations).

The dominance of Suillus in ECM introductions is likely the result of both the historical frequency of using soil-based inoculations, and the confluence of several traits exhibited by the genus. Policelli et al. (2019) identified several ecological traits which help to make Suillus strong facilitators of host tree invasions. These include 1) long-distance dispersal capacity, 2) the ability to generate a long-lived resistant spore bank, 3) the establishment of positive biotic interactions with mammals, 4) the rapid colonization of host roots, and 5) the long-distance exploration type of Suillus extraradical mycelium.

Within their exotic range, and in the absence of competition from other fungi, Suillus species often fruit especially prolifically, further contributing to their proliferation and spread in these habitats. Hedger (1986) and Chapela (2001) estimated that S. luteus fruit body production in introduced Ecuadoran P. radiata forests, could amount to over one metric ton of fungal tissue per hectare (dry weight): an impressive yield by any measure. These fruit bodies produce enormous numbers of basidiospores which are widely dispersed well beyond the forest boundary. Once dispersed, Suillus spores become part of a long-lived drought and fire-resistant soil spore-bank, and are able to wait many years for a wilding Pineaceae seed to land beside them (Bruns et al., 2010).

In addition to wind dispersal, mycophagy is recognized as an important dispersal mechanism and likely contributes to the establishment of Suillus in invasive ranges (Wood et al., 2015; Caiafa et al., 2021; Elliott et al., 2022). Numerous studies have documented fruit body consumption by both native and non-native animals including rodents, marsupials, deer, pigs, rabbits and potentially birds, which can translocate spores over great distances and disperse them through their excrement (Ashkannejhad & Horton, 2006; Nuñez et al., 2013; Wood et al., 2015; Horton, 2017; Aguirre et al., 2021; Caiafa et al., 2021; Policelli et al., 2022).

Perhaps the strongest factor linking Suillus to host tree invasions is the fact that Suillus species colonize young host seedlings so readily. Once established, Suillus can rapidly spread to nearby root systems by the production of the abundant rhizomorphs that are a hallmark of this clade. This type of extramatrical mycelium, which Agerer (2001) termed the “long-distance exploration-type”, not only enables mycelial-based colonization of neighboring host trees, but likely helps to facilitate the success of Pinaceae across exotic ranges by enabling better water withdrawal, increased access to resources patches, and increased acquisition of organic nitrogen (Hobbie & Agerer, 2009; Koide et al., 2014). Although Suillus are often thought of as weak competitors, new plantation environments are typically depauperate in ECM symbionts capable of colonizing the introduced host trees, resulting in reduced competition (Hedger, 1986; Chapela et al., 2001). In addition, novel environments offer a high likelihood of ‘enemy escape’ from co-evolved pathogens, which may also contribute to establishment across introduced ranges (Dickie et al., 2010).

VI.iii: Downstream effects and unresolved questions

The strong obligate symbiosis between Suillus and the Pinaceae is an example of a positive plant-soil feedback. Plant-soil feedbacks are often associated with biological invasions, and have numerous downstream effects on ecosystems (Simberloff & Von Holle, 1999; Simberloff, 2006). Pinus plantations are known to greatly alter soil biogeochemistry by increasing soil acidity and litter production, and depleting soil carbon (Chapela et al., 2001). This feedback is thought to be one of the primary factors preventing effective ecological restoration (Hoeksema et al., 2020). Even after host removal, the enzymatic function of soil can be slow to recover (Sapsford & Dickie, 2023). As primary colonizers of introduced Pinaceae forests, Suillus spp. likely contribute to the biogeochemical changes associated with plantation forestry. The precise role that Suillus fungi play in altering soil chemistry and the mechanisms involved in this phenomenon are active areas of research.

The combined impacts of simultaneous co-invasion can have disastrous consequences on native biodiversity, a process which has been termed “invasional meltdown” by Simberloff and Von Holle (1999). Whether facilitated directly by the fungus, or via host-mediated mechanisms, early evidence points toward the capacity of Suillus (and also likely Rhizopogon) to displace native fungal communities at least when the density of introduced hosts is high (Sapsford et al., 2022; Mujic et al., 2023). In linked plant-fungal co-invasions, it is difficult to uncouple the invasive capacity of the fungus from that of the host, complicating the question of whether Suillus should be treated as invasive species in their own right. Generally, ECM species are primarily constrained to their introduced hosts (Vellinga et al., 2009; Policelli et al., 2020). While the high partner specificity exhibited by Suillus should reduce overall invasion potential compared to host generalist species that are more likely to establish on novel hosts after introduction, this benefit seems to be overwhelmed by the influence of the early-successional life history traits exhibited by Suillus (Vlk et al., 2020).

Previous work has shown no phylogenetic signal for informing which species of Suillus are likely to be introduced (Vellinga et al., 2009), and while phylogenetically diverse Suillus species are capable of establishing in introduced ranges, occurrence records are dominated by a small number of species. Interesting questions remain as to what traits and qualities make these species invasive, while the majority of Suillus species have never been associated with introduction or linked plant-fungal co-invasions.

VII. Conclusions and future directions

Here, we have shown that Suillus fungi play essential roles in natural and managed ecosystems, have greatly added to our understanding of fungal evolution, the ecology of fungal-host and fungal-fungal interactions, the stress responses of ECM fungi to both biotic and abiotic factors, as well as to population genetics, comparative genomics, and invasion biology. Suillus holds immense potential to serve as a model genus to advance our understanding of the ecology and evolution of ECM systems. Additionally, the combination of experimental tractability, the availability of experimental protocols, the flexibility of a well-defined genus-level system, a large set of publicly available genomes and respective annotations, and the cultures associated with these genomes, make Suillus one of the few well-established ECM fungal models for addressing questions that can scale from genes to ecosystems.

VII.i: Outstanding questions and research priorities

Research leveraging Suillus over the last decade has helped to define and prioritize outstanding questions in the field. First, phylogenetic analysis to define and place taxa from under sampled areas will help to fully delineate taxonomic boundaries and clarify the evolutionary history of host switching and diversification in the genus. While the strength of the Suillus system is based at the genus level, selecting target species for deep investigation will allow researchers to leverage both intra- and interspecific comparisons to derive the true impacts of fungal trait variation. Currently, the most well-studied species of Suillus is S. luteus, driven largely by the fact that it was the first species in the genus to undergo whole genome sequencing (Kohler et al., 2015). However, current efforts to develop other species including S. brevipes, S. quiescens, S. salmonicolor, S. pungens, and S. tomentosus will soon provide more insight into trait and genome variation and provide a framework for testing specific hypotheses that have so far been difficult to address. These efforts include both population-level sampling, and chromosome-level reference assemblies, enabling a deeper understanding of genomic diversity, and the influence of genome structure on ecological traits.

Another research priority is to define the mechanisms facilitating partner specificity and partner switching in Suillus. This line of research will not only enhance our fundamental understanding of these ecological phenomena but also has practical implications for effective ecosystem management and conservation, particularly in the face of climate change-induced shifts in host tree distributions.

Ongoing research efforts using Suillus should aim to quantify key aspects of the ECM symbiosis such as realized nutrient trading dynamics. For instance, employing emerging techniques such as quantum dots and new advances in stable isotope tracing will help to address a multitude of both ecological and mechanistic questions related to nutrient cycling. In addition to quantifying carbon and nitrogen trading across species and environmental variables, defining the role and importance of non-canonically traded macro and micro-nutrients should be a priority for gaining a comprehensive understanding of metabolic interactions.

Clarifying the roles and mechanisms of non-nutritional partner benefits, such as stress tolerance to abiotic factors like drought, metal exposure, and ionizing radiation will help researchers to understand how Suillus and their hosts will adapt to a rapidly changing world. Understanding these variables will also help to identify the characteristics that contribute to the invasive potential of certain Suillus species, thereby enabling researchers to safeguard native ecosystems while supporting forestry practices.

Finally, research on the interactions between Suillus and other community members beyond the plant-fungal partnership is also vital for understanding the broader ecological context in which the ECM symbiosis operates. Exploring the consequences of these interactions, including those involving other fungi, bacteria, non-host plants, macro- and micro-fauna, will provide insights into the mechanisms of coexistence, competition, and facilitation.

VII.ii: Limitations

While the Suillus system presents numerous advantages, it is important to also acknowledge its limitations within specific research contexts. In fact, some of the very traits that make Suillus an ideal model for certain research objectives render it unsuitable for others. For example, the close association between Suillus and the Pinaceae, makes the genus valuable for studying partner specificity and diversification events associated with host switching, but also confines its applicability primarily to host associations within the Pinaceae (although associations of certain Suillus species with angiosperms may bring different perspectives). Similarly, because Suillus is naturally found only in the northern hemisphere where Pinaceae hosts are native, and in the southern hemisphere only as introduced species, it serves as an excellent system for studying reproductive bottlenecks, dispersal, and invasion biology across introduced ranges. These benefits do not negate the fact that Suillus is non-native on half the planet, limiting its applicability as a model for understanding many systems across the global south, such as understudied tropical ECM forests.

Finally, we emphasize that the advancement of the Suillus system as a model does not preclude the progress of other systems. On the contrary, we hope that formalizing the development of the Suillus model will catalyze researchers to establish similar collaborative working groups on other promising ECM model genera such as Laccaria, Russula, and Pisolithus, which may be better suited for specific research objectives. The ECM lifestyle is thought to have evolved independently at least 78 times over, and represents at least 251 distinct genera, with diverse traits, host-associations, and geographic distributions (Tedersoo & Smith, 2013). With recognition that no single set of taxa can hope to represent the entirety of ECM diversity, concentrating efforts around the development of carefully chosen groups will enable the rapid development of resources needed to address the most pressing questions in ECM ecology and evolution.

VII.iii: Technical challenges and future directions

Fully developing Suillus as a model genus presents some technical challenges that the community is currently working to address. Resolving these issues will soon open up a multitude of possibilities to further use Suillus as a means to understand ECM ecology, biology, and evolution.

First, using Suillus to fully address the mechanisms underlying ECM metabolism, requires the development of systems capable of tracking nutrient dynamics under field conditions, with adult trees, and in complex communities. Bolstered by the availability of a wide array of publicly available Suillus genomes, continual improvements in functional -omics and meta-omics approaches promise to facilitate comprehensive links between genomes and functional traits. These advancements are expected to have significant implications across both laboratory and field settings, facilitating a better understanding of ECM biodiversity across biological scales: linking genes to communities and ecosystems.

Another significant challenge is the complexity of working with heterokaryotic dikaryons; a biological consideration for most ECM basidiomycetes. Optimized protocols to dependably monokaryotize and dikaryotize Suillus strains in culture, along with efficient assays to confirm karyotic state, would go far to facilitate studies in ECM fungal genetics, gene regulation, and nuclear control.

An additional challenge common to most ECM fungi is the inability to dependably produce fruit bodies under laboratory conditions. In theory, this limitation is related to the size of the carbon stores necessary to support the development of reproductive structures. Seedling-based bioassays are favored in ECM studies because of the impracticality of manipulating large trees in the laboratory. This is thought to limit total photosynthetic capacity and the amount of carbon transferred to the fungus, particularly over limited time scales. Circumnavigating this limitation would require a system which increases carbon transfer from the host without disrupting the symbiosis, or a system for triggering fruit body development apart from the host entirely. The development of such a system would facilitate significant advances in our understanding of ECM genetics, enabling gene association studies, and studies in morphogenesis and histology.

Finally, the development of gene editing systems for Suillus will radically improve our ability to manipulatively address the genetic basis of adaptation, evolution, and the role of specific genes in important ecological and biological processes. The International Suillus Consortium considers the development of gene editing tools to be of the highest priority and has formed a working group to address this challenge, making use of multiple gene editing platforms and targeting multiple species in the genus.

VII.iv: Conclusion

Studies in Suillus have already made significant contributions to our understanding of the biology and ecology and evolution of the ECM symbiosis. The availability of tools and protocols optimized for Suillus, including specific approaches for in vitro culturing, plant partner bioassays, high quality DNA and RNA extraction included in Section VIII, along with the large number of publicly available reference genomes will catalyze future research to address long-standing questions in the field. We expect that continued community development of the Suillus system will solidify the genus as a model for understanding the ecology and evolution of plant-fungal mutualisms. We invite anyone interested in collaborating with The International Suillus Consortium to please contact the authors.

VIII. Resources and protocols

Despite the importance of ECM fungi to both natural and managed forests, manipulative work has largely lagged behind other experimental systems due in part to the technical challenges of working with host-associated, dikaryotic species. In an effort to standardize protocols and stimulate future improvements that will build off of these resources, we present a series of protocols for working with Suillus that will allow researchers to circumnavigate many of these challenges. These protocols are available as a series of supplemental Notes files on topics ranging from axenic isolation and long-term storage, to mycorrhizal synthesis and the application of DNA based approaches. Here, we provide instruction for navigating these resources, outline specific use cases, limitations, and recommendations.

VIII.i: Fruit body and spore collection (Notes S1)

One of the benefits of the Suillus system is that fruit bodies of the genus are conspicuous, frequently abundant, and relatively easy to identify. Both amateur and professional mycologists alike have long targeted the collection of charismatic Suillus mushrooms, with over 46,000 vouchered specimens currently digitized in the MyCoPortal collections database (https://www.mycoportal.org/) and over 62,000 observations of Suillus currently in iNaturalist (https://www.inaturalist.org/). Vouchered collections have facilitated phylogenetic reclassifications across the genus (Nguyen et al., 2016b), illuminated the association between species diversification and host shifts (Zhang et al., 2022), clarified species rarity (Kennedy et al., 2020), and helped to identify the biotic and abiotic factors facilitating range shifts and invasion potential (Pietras et al., 2018). The collection of Suillus fruit bodies is the first step to obtaining pure cultures, spores for use in bioassays, and preserved specimens for morphological studies or new species descriptions. Notes S1 details the process of finding, identifying, and collecting Suillus fruit bodies for down-stream applications, and the processing of fruit bodies for spore collection and preservation.

VIII.ii: Isolation (Notes S2)

Unlike many groups of ECM fungi, Suillus can be readily isolated and grown in pure culture. Cultures can be isolated fruit bodies, ECM rootlets, or basidiospores, to flexibly obtain dikaryotic or monokaryotic strains. Notes S2 gives an overview of best practices and specific instructions for obtaining pure cultures of Suillus from all three of these sources.

VIII.iii: Culture conditions and culture storage (Notes S3)

Suillus can accommodate growth on a variety of media types, but are most commonly cultured on Fries Media, Hagems Agar, Modified Melin-Norkrans (MMN) or Pachlewski’s Media (Px). Recipes for all four media types, slightly modified from their original formulations for optimized Suillus growth, are available in Notes S3, along with comments on optimized growth conditions and recommendations for culture storage and long-term culture preservation.

VIII.v: Sequencing (Notes S4-S6)

Whole genome and next-generation sequencing using genomic, transcriptomic, and metatranscriptomic approaches have revolutionized our ability to identify and characterize ECM fungal communities (Bork et al., 2015; Thompson et al., 2017; Quince et al., 2017; Lu & Salzberg, 2020), and helped to shed light on fungal ecology and evolution (Miyauchi et al., 2020; Feurtey et al., 2023; Ali et al., 2023). In Suillus, these technologies have enabled the characterization of high resolution phylogenies, revealed the extent of intrageneric genome diversity, and helped to illuminate the functional mechanisms of important traits like stress tolerance and partner specificity (Liao et al., 2016; Bazzicalupo et al., 2020; Lofgren et al., 2021; Erlandson et al., 2022). However, compared to other ECM groups, Suillus spp. present some unique technical challenges to sample preparation for -omics analysis. The genus is a notorious producer of pigments and extraction inhibitors that can complicate quality control measurements, interfere with PCR amplification, and confound DNA/cDNA library construction. DNA and RNA extraction from mixed biological samples such as mycorrhizal root tips is a critical but often difficult step in preparing material for molecular work. In general, ectomycorrhizae (which are a mix of plant and fungal tissue) contain polysaccharides, hydroxybenzenes, esters, and other secondary metabolites that can confound extraction protocols. Compared to many other ECM groups, Suillus has an expanded secondary metabolite repertoire (Lofgren et al., 2021), that likely further complicates DNA extraction. Additionally, because Suillus associates almost exclusively with plants in the family Pinaceae, which famously produce a wide array of secondary metabolites (Bashalkhanov & Rajora, 2008), the confounding effect of extraction inhibitors are likely compounded across the genus.

To minimize the impact of inhibitors, we have developed and optimized three methods for nucleic acid extraction that cover a broad range of applications including fresh and dried mushrooms, root-tips, cultures, and soil, and include them here as three separate protocols. The NaOH extraction method yields DNA for fast genotyping of cultures or root-tips (Notes S4). The CTAB extraction method is generally used for whole genome sequencing and can be performed either for DNA alone, or as a DNA/RNA co-extraction (Notes S5). The high-molecular weight DNA extraction method is recommended for high-quality long-read sequencing of Suillus from fruit bodies or cultures (Notes S6).

VIII.vi: Host-Suillus bioassays (Notes S7)

The tractability of Suillus spp. to in vitro mycorrhization via both spore inoculation and mycelial extension is one of the primary factors making this genus an excellent experimental system for ecological and evolutionary research. While spore-based inoculation methods are high-throughput and easily incorporate fungal genetic variation into mycorrhizal synthesis studies, mycelial-based methods provide a mechanism to control strain provenance and can be used to construct either mid-throughput pot-based assays, or low-throughput but highly flexible mesocosms that are otherwise axenic, facilitating the ability to control and manipulate the microbial community. All Suillus species we have examined to date are amenable to laboratory mycorrhization using spore and mycelial methods, achieving a high level of colonization (up to 100% of root tips) when paired with the appropriate host (Table S1). In Notes S7, we provide an overview of these procedures and specific protocols for inoculating Suillus onto host trees to conduct bioassays using spore-based, and mycelial-based mycorrhizal synthesis.

VIII.vii: Suillus-host associations (Supplemental Table S1)

Understanding the specificity patterns between different Suillus species and their host trees in natural environments is fundamental to successful experimental design, interpreting high throughput sequence data, and to locating and identifying Suillus fruit bodies. In Table S1, we provide putative host associations for all currently recognized species of Suillus as well as several additional taxa that are yet to be formally typified but clearly represent independent species based on molecular analysis. Citations are provided for all putative host associations. Species with sequenced genomes are noted.

Supplementary Material

Notes S1

Notes S1: Fruit body and spore collection

Notes S2

Notes S2: Isolation

Notes S3

Notes S3: Culture conditions and storage

Notes S4

Notes S4: NaOH extraction

Notes S5

Notes S5: CTAB DNA/RNA co-extraction

Notes S6

Notes S6: High-molecular weight DNA extraction

Tab S1

Table S1: Host-Suillus associations and genome-sequenced species

Notes S7

Notes S7: Host-Suillus bioassays

Data availability

All data used in this manuscript has been made available on the open access, interactive phenotype database SuilluScope (www.SuilluScope.com) released in beta as part of this manuscript. The code used for analysis, as well as all of the code used to create the SuilluScope reactive web interface are publicly available on GitHub at https://github.com/MycoPunk/SuilluScope