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

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades-primarily plant-associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

Small genes (40,000 small-gene families in ∼2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000 families encode predicted anti-CRISPR proteins, and more than 500 families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.

Fungi of the Conidiobolus group belong to the family Ancylistaceae (Entomophthorales, Entomophthoromycotina, Zoopagomycota) and include over 70 predominantly saprotrophic species in four similar and closely related genera, that were separated phylogenetically recently. Entomopathogenic fungi of the genus Batkoa are very close morphologically to the Conidiobolus species. Their thalli share similar morphology, and they produce ballistic conidia like closely related entomopathogenic Entomophthoraceae. Ballistic conidia are traditionally considered as an efficient tool in the pathogenic process and an important adaptation to the parasitic lifestyle. Our study aims to reconstruct the phylogeny of this fungal group using molecular and genomic data, ancestral lifestyle and morphological features of the conidiobolus-like group and the direction of their evolution. Based on phylogenetic analysis, some species previously in the family Conidiobolaceae are placed in the new families Capillidiaceae and Neoconidiobolaceae, which each include one genus, and the Conidiobolaceae now includes three genera. Intermediate between the conidiobolus-like groups and Entomophthoraceae, species in the distinct Batkoa clade now belong in the family Batkoaceae. Parasitism evolved several times in the Conidiobolus group and Ancestral State Reconstruction suggests that the evolution of ballistic conidia preceded the evolution of the parasitic lifestyle.

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

Within the forest community, competition and facilitation between adjacent-growing conspecific and heterospecific plants are mediated by interactions involving common mycorrhizal networks. The ability of plants to alter their neighbors microbiome is well documented, but the molecular biology of plant-fungal interactions during competition and facilitation has not been previously examined. We used a common soil-plant bioassay experiment to study molecular plant-microbial interactions among rhizosphere communities associated with Pinus taeda (native host) and Populus trichocarpa (non-native host). Gene expression of interacting fungal and bacterial rhizosphere communities was compared among three plant-pairs: Populus growing with Populus, Populus with Pinus, and Pinus with Pinus. Our results demonstrate that heterospecific plant partners affect the assembly of root microbiomes, including the changes in the structure of host specific community. Comparative metatranscriptomics reveals that several species of ectomycorrhizal fungi (EMF) and saprotrophic fungi exhibit different patterns of functional and regulatory gene expression with these two plant hosts. Heterospecific plants affect the transcriptional expression pattern of EMF host-specialists (e.g., Pinus-associated Suillus spp.) on both plant species, mainly including the genes involved in the transportation of amino acids, carbohydrates, and inorganic ions. Alteration of root microbiome by neighboring plants may help regulate basic plant physiological processes via modulation of molecular functions in the root microbiome.

Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

Mortierella and Ilyonectria genera include common species of soil fungi that are frequently detected as root endophytes in many plants, including Populus spp. However, the ecological roles of these and other endophytic fungi with respect to plant growth and function are still not well understood. The functional ecology of two key taxa from the P. trichocarpa rhizobiome, M. elongata PMI93 and I. europaea PMI82, was studied by coupling forest soil bioassays with environmental metatranscriptomics. Using soil bioassay experiments amended with fungal inoculants, M. elongata was observed to promote the growth of P. trichocarpa. This response was cultivar independent. In contrast, I. europaea had no visible effect on P. trichocarpa growth. Metatranscriptomic studies revealed that these fungi impacted rhizophytic and endophytic activities in P. trichocarpa and induced shifts in soil and root microbial communities. Differential expression of core genes in P. trichocarpa roots was observed in response to both fungal species. Expression of P. trichocarpa genes for lipid signaling and nutrient uptake were upregulated, and expression of genes associated with gibberellin signaling were altered in plants inoculated with M. elongata, but not I. europaea. Upregulation of genes for growth promotion, downregulation of genes for several leucine-rich repeat receptor kinases, and alteration of expression of genes associated with plant defense responses (e.g., jasmonic acid, salicylic acid, and ethylene signal pathways) also suggest that M. elongata manipulates plant defenses while promoting plant growth.

We identified two poplar (Populus sp.)-associated microbes, the fungus, Mortierella elongata strain AG77, and the bacterium, Burkholderia strain BT03, that mutually promote each other's growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.

Phylogenetic taxon definitions (PTDs) are explicit, phylogeny-based statements that specify clades. PTDs are central to the system of rank-free classification that is governed by the PhyloCode, but they can also be used to clarify the meanings of ranked names. We present PTDs for four major groups: Fungi, Dikarya, Ascomycota, and Basidiomycota.

Phylogenomic approaches have the potential to improve confidence about the inter-relationships of species in the order Mucorales within the fungal tree of life. Rhizopus species are especially important as plant and animal pathogens and bioindustrial fermenters for food and metabolite production. A dataset of 192 orthologous genes was used to construct a phylogenetic tree of 21 Rhizopus strains, classified into four species isolated from habitats of industrial, medical and environmental importance. The phylogeny indicates that the genus Rhizopus consists of three major clades, with R. microsporus as the basal species and the sister lineage to R. stolonifer and two closely related species R. arrhizus and R. delemar A comparative analysis of the mating type locus across Rhizopus reveals that its structure is flexible even between different species in the same genus, but shows similarities between Rhizopus and other mucoralean fungi. The topology of single-gene phylogenies built for two genes involved in mating is similar to the phylogenomic tree. Comparison of the total length of the genome assemblies showed that genome size varies by as much as threefold within a species and is driven by changes in transposable element copy numbers and genome duplications.

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

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 (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions. [ABSTRACT FROM AUTHOR]

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

Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed.

Ecologists have long acknowledged the importance of seed banks; yet, despite the fact that many plants rely on mycorrhizal fungi for survival and growth, the structure of ectomycorrhizal (ECM) fungal spore banks remains poorly understood. The primary goal of this study was to assess the geographic structure in pine-associated ECM fungal spore banks across the North American continent. Soils were collected from 19 plots in forests across North America. Fresh soils were pyrosequenced for fungal internal transcribed spacer (ITS) amplicons. Adjacent soil cores were dried and bioassayed with pine seedlings, and colonized roots were pyrosequenced to detect resistant propagules of ECM fungi. The results showed that ECM spore banks correlated strongly with biogeographic location, but not with the identity of congeneric plant hosts. Minimal community overlap was found between resident ECM fungi vs those in spore banks, and spore bank assemblages were relatively simple and dominated by Rhizopogon, Wilcoxina, Cenococcum, Thelephora, Tuber, Laccaria and Suillus. Similar to plant seed banks, ECM fungal spore banks are, in general, depauperate, and represent a small and rare subset of the mature forest soil fungal community. Yet, they may be extremely important in fungal colonization after large-scale disturbances such as clear cuts and forest fires.

Identifying the ecological processes that structure communities and the consequences for ecosystem function is a central goal of ecology. The recognition that fungi, bacteria, and viruses control key ecosystem functions has made microbial communities a major focus of this field. Because many ecological processes are apparent only at particular spatial or temporal scales, a complete understanding of the linkages between microbial community, environment, and function requires analysis across a wide range of scales. Here, we map the biological and functional geography of soil fungi from local to continental scales and show that the principal ecological processes controlling community structure and function operate at different scales. Similar to plants or animals, most soil fungi are endemic to particular bioregions, suggesting that factors operating at large spatial scales, like dispersal limitation or climate, are the first-order determinants of fungal community structure in nature. By contrast, soil extracellular enzyme activity is highly convergent across bioregions and widely differing fungal communities. Instead, soil enzyme activity is correlated with local soil environment and distribution of fungal traits within the community. The lack of structure-function relationships for soil fungal communities at continental scales indicates a high degree of functional redundancy among fungal communities in global biogeochemical cycles.

Multi‐locus sequence data are widely used in fungal systematic and taxonomic studies to delimit species and infer evolutionary relationships. We developed and assessed the efficacy of a multi‐locus pooled sequencing method using PacBio long‐read high‐throughput sequencing. Samples included fresh and dried voucher specimens, cultures and archival DNA extracts of Agaricomycetes with an emphasis on the order Cantharellales. Of the 283 specimens sequenced, 93.6% successfully amplified at one or more loci with a mean of 3.3 loci amplified. Our method recovered multiple sequence variants representing alleles of rDNA loci and single copy protein‐coding genes rpb1, rpb2 and tef1. Within‐sample genetic variation differed by locus and taxonomic group, with the greatest genetic divergence observed among sequence variants of rpb2 and tef1 from corticioid Cantharellales. Our method is a cost‐effective approach for generating accurate multi‐locus sequence data coupled with recovery of alleles from polymorphic samples and multi‐organism specimens. These results have important implications for understanding intra‐individual genomic variation among genetic loci commonly used in species delimitation of fungi. [ABSTRACT FROM AUTHOR]

The fungus Entomophthora muscae (Entomophthoromycota, Entomophthorales, Entomophthoraceae) is a widespread insect pathogen responsible for fatal epizootic events in many dipteran fly hosts. During epizootics in 2011 and 2012 in Durham, North Carolina, we observed a transition of fungal infections from one host, the plant-feeding fly Delia radicum, to a second host, the predatory fly Coenosia tigrina. Infections first appeared on Delia in the middle of March, but by the end of May, Coenosia comprised 100% of infected hosts. Multilocus sequence typing revealed that E. muscae in Durham comprises two distinct subpopulations (clades) with several haplotypes in each. Fungi from either clade are able to infect both fly species, but vary in their infection phenologies and host-specificities. Individuals of the more phylogenetically diverse clade I predominated during the beginning of the spring epizootic, infecting mostly phytophagous Delia flies. Clade II dominated in late April and May and affected mostly predatory Coenosia flies. Analysis of population structure revealed two subpopulations within E. muscae with limited gene exchange. This study provides the first evidence of recombination and population structure within the E. muscae species complex, and illustrates the complexity of insect-fungus relationships that should be considered for development of biological control methods.