Extreme overall mushroom genome expansion in Mycena s.s. irrespective of plant hosts or substrate specializations.

Mycena s.s. is a ubiquitous mushroom genus whose members degrade multiple dead plant substrates and opportunistically invade living plant roots. Having sequenced the nuclear genomes of 24 Mycena species, we find them to defy the expected patterns for fungi based on both their traditionally perceived saprotrophic ecology and substrate specializations. Mycena displayed massive genome expansions overall affecting all gene families, driven by novel gene family emergence, gene duplications, enlarged secretomes encoding polysaccharide degradation enzymes, transposable element (TE) proliferation, and horizontal gene transfers. Mainly due to TE proliferation, Arctic Mycena species display genomes of up to 502 Mbp (2-8× the temperate Mycena), the largest among mushroom-forming Agaricomycetes, indicating a possible evolutionary convergence to genomic expansions sometimes seen in Arctic plants. Overall, Mycena show highly unusual, varied mosaic-like genomic structures adaptable to multiple lifestyles, providing genomic illustration for the growing realization that fungal niche adaptations can be far more fluid than traditionally believed.

Snowball: a novel gene family required for developmental patterning of fruiting bodies of mushroom-forming fungi (Agaricomycetes).

The morphogenesis of sexual fruiting bodies of fungi is a complex process determined by a genetically encoded program. Fruiting bodies reached the highest complexity levels in the Agaricomycetes; yet, the underlying genetics is currently poorly known. In this work, we functionally characterized a highly conserved gene termed snb1, whose expression level increases rapidly during fruiting body initiation. According to phylogenetic analyses, orthologs of snb1 are present in almost all agaricomycetes and may represent a novel conserved gene family that plays a substantial role in fruiting body development. We disrupted snb1 using CRISPR/Cas9 in the agaricomycete model organism Coprinopsis cinerea. snb1 deletion mutants formed unique, snowball-shaped, rudimentary fruiting bodies that could not differentiate caps, stipes, and lamellae. We took advantage of this phenotype to study fruiting body differentiation using RNA-Seq analyses. This revealed differentially regulated genes and gene families that, based on wild-type RNA-Seq data, were upregulated early during development and showed tissue-specific expression, suggesting a potential role in differentiation. Taken together, the novel gene family of snb1 and the differentially expressed genes in the snb1 mutants provide valuable insights into the complex mechanisms underlying developmental patterning in the Agaricomycetes. IMPORTANCE: Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are complex multicellular structures, with a spatially and temporally integrated developmental program that is, however, currently poorly known. In this study, we present a novel, conserved gene family, Snowball (snb), termed after the unique, differentiation-less fruiting body morphology of snb1 knockout strains in the model mushroom Coprinopsis cinerea. snb is a gene of unknown function that is highly conserved among agaricomycetes and encodes a protein of unknown function. A comparative transcriptomic analysis of the early developmental stages of differentiated wild-type and non-differentiated mutant fruiting bodies revealed conserved differentially expressed genes which may be related to tissue differentiation and developmental patterning fruiting body development.

ContScout: sensitive detection and removal of contamination from annotated genomes.

Contamination of genomes is an increasingly recognized problem affecting several downstream applications, from comparative evolutionary genomics to metagenomics. Here we introduce ContScout, a precise tool for eliminating foreign sequences from annotated genomes. It achieves high specificity and sensitivity on synthetic benchmark data even when the contaminant is a closely related species, outperforms competing tools, and can distinguish horizontal gene transfer from contamination. A screen of 844 eukaryotic genomes for contamination identified bacteria as the most common source, followed by fungi and plants. Furthermore, we show that contaminants in ancestral genome reconstructions lead to erroneous early origins of genes and inflate gene loss rates, leading to a false notion of complex ancestral genomes. Taken together, we offer here a tool for sensitive removal of foreign proteins, identify and remove contaminants from diverse eukaryotic genomes and evaluate their impact on phylogenomic analyses.

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria.

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.

Genomes of fungi and relatives reveal delayed loss of ancestral gene families and evolution of key fungal traits.

Fungi are ecologically important heterotrophs that have radiated into most niches on Earth and fulfil key ecological services. Despite intense interest in their origins, major genomic trends of their evolutionary route from a unicellular opisthokont ancestor to derived multicellular fungi remain poorly known. Here we provide a highly resolved genome-wide catalogue of gene family changes across fungal evolution inferred from the genomes of 123 fungi and relatives. We show that a dominant trend in early fungal evolution has been the gradual shedding of protist genes and the punctuated emergence of innovation by two main gene duplication events. We find that the gene content of non-Dikarya fungi resembles that of unicellular opisthokonts in many respects, owing to the conservation of protist genes in their genomes. The most rapidly duplicating gene groups included extracellular proteins and transcription factors, as well as ones linked to the coordination of nutrient uptake with growth, highlighting the transition to a sessile osmotrophic feeding strategy and subsequent lifestyle evolution as important elements of early fungal history. These results suggest that the genomes of pre-fungal ancestors evolved into the typical filamentous fungal genome by a combination of gradual gene loss, turnover and several large duplication events rather than by abrupt changes. Consequently, the taxonomically defined Fungi represents a genomically non-uniform assemblage of species.

Hagnosa longicapillata, gen. nov., sp. nov., a New Sordariaceous Ascomycete in the Indoor Environment, and the Proposal of Hagnosaceae fam. nov.

Hagnosa longicapillata, gen. nov., sp. nov, is described and illustrated from wooden building materials collected in Hungary and from pure culture. This species has been collected exclusively from indoor environments, where it was quite common. The ascocarps develop in a thick layer of brown, woolly mats of mycelia. The ostiolar region of the perithecia is ornamented with a five-lobed, flower-shaped crown. Asci are four-spored; ascospores are dark brown, smooth, muriform, not constricted at the septa, and liberated mostly through crackings of the thin ascomatal wall. Apparently, ascospores are dispersed by the mechanical disturbance of the mycelial web. In the phylogenetic tree, Hagnosa samples were placed as a basal lineage, independently from the other family of Sordariomycetidae, with high support. To place Hagnosa in Sordariales, the new family, Hagnosaceae, is proposed.

Gene age shapes the transcriptional landscape of sexual morphogenesis in mushroom-forming fungi (Agaricomycetes).

Multicellularity has been one of the most important innovations in the history of life. The role of gene regulatory changes in driving transitions to multicellularity is being increasingly recognized; however, factors influencing gene expression patterns are poorly known in many clades. Here, we compared the developmental transcriptomes of complex multicellular fruiting bodies of eight Agaricomycetes and Cryptococcus neoformans, a closely related human pathogen with a simple morphology. In-depth analysis in Pleurotus ostreatus revealed that allele-specific expression, natural antisense transcripts, and developmental gene expression, but not RNA editing or a 'developmental hourglass,' act in concert to shape its transcriptome during fruiting body development. We found that transcriptional patterns of genes strongly depend on their evolutionary ages. Young genes showed more developmental and allele-specific expression variation, possibly because of weaker evolutionary constraint, suggestive of nonadaptive expression variance in fruiting bodies. These results prompted us to define a set of conserved genes specifically regulated only during complex morphogenesis by excluding young genes and accounting for deeply conserved ones shared with species showing simple sexual development. Analysis of the resulting gene set revealed evolutionary and functional associations with complex multicellularity, which allowed us to speculate they are involved in complex multicellular morphogenesis of mushroom fruiting bodies.

Evolutionary Morphogenesis of Sexual Fruiting Bodies in Basidiomycota: Toward a New Evo-Devo Synthesis.

The development of sexual fruiting bodies is one of the most complex morphogenetic processes in fungi. Mycologists have long been fascinated by the morphological and developmental diversity of fruiting bodies; however, evolutionary developmental biology of fungi still lags significantly behind that of animals or plants. Here, we summarize the current state of knowledge on fruiting bodies of mushroom-forming Basidiomycota, focusing on phylogenetic and developmental biology. Phylogenetic approaches have revealed a complex history of morphological transformations and convergence in fruiting body morphologies. Frequent transformations and convergence is characteristic of fruiting bodies in contrast to animals or plants, where main body plans are highly conserved. At the same time, insights into the genetic bases of fruiting body development have been achieved using forward and reverse genetic approaches in selected model systems. Phylogenetic and developmental studies of fruiting bodies have each yielded major advances, but they have produced largely disjunct bodies of knowledge. An integrative approach, combining phylogenetic, developmental, and functional biology, is needed to achieve a true fungal evolutionary developmental biology (evo-devo) synthesis for fungal fruiting bodies.

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

Hallmarks of Basidiomycete Soft- and White-Rot in Wood-Decay -Omics Data of Two Armillaria Species.

Wood-decaying Basidiomycetes are among the most efficient degraders of plant cell walls, making them key players in forest ecosystems, global carbon cycle, and in bio-based industries. Recent insights from -omics data revealed a high functional diversity of wood-decay strategies, especially among the traditional white-rot and brown-rot dichotomy. We examined the mechanistic bases of wood-decay in the conifer-specialists Armillaria ostoyae and Armillaria cepistipes using transcriptomic and proteomic approaches. Armillaria spp. (Fungi, Basidiomycota) include devastating pathogens of temperate forests and saprotrophs that decay wood. They have been discussed as white-rot species, though their response to wood deviates from typical white-rotters. While we observed an upregulation of a diverse suite of plant cell wall degrading enzymes, unlike white-rotters, they possess and express an atypical wood-decay repertoire in which pectinases and expansins are enriched, whereas lignin-decaying enzymes (LDEs) are generally downregulated. This combination of wood decay genes resembles the soft-rot of Ascomycota and appears widespread among Basidiomycota that produce a superficial white rot-like decay. These observations are consistent with ancestral soft-rot decay machinery conserved across asco- and Basidiomycota, a gain of efficient lignin-degrading ability in white-rot fungi and repeated, complete, or partial losses of LDE encoding gene repertoires in brown- and secondarily soft-rot fungi.

Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles.

Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. In this Brief Report, we use clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. We find that high expression of non-lipidated Atg8b in the male germline is required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility.

Unmatched Level of Molecular Convergence among Deeply Divergent Complex Multicellular Fungi.

Convergent evolution is pervasive in nature, but it is poorly understood how various constraints and natural selection limit the diversity of evolvable phenotypes. Here, we analyze the transcriptome across fruiting body development to understand the independent evolution of complex multicellularity in the two largest clades of fungi-the Agarico- and Pezizomycotina. Despite >650 My of divergence between these clades, we find that very similar sets of genes have convergently been co-opted for complex multicellularity, followed by expansions of their gene families by duplications. Over 82% of shared multicellularity-related gene families were expanding in both clades, indicating a high prevalence of convergence also at the gene family level. This convergence is coupled with a rich inferred repertoire of multicellularity-related genes in the most recent common ancestor of the Agarico- and Pezizomycotina, consistent with the hypothesis that the coding capacity of ancestral fungal genomes might have promoted the repeated evolution of complex multicellularity. We interpret this repertoire as an indication of evolutionary predisposition of fungal ancestors for evolving complex multicellular fruiting bodies. Our work suggests that evolutionary convergence may happen not only when organisms are closely related or are under similar selection pressures, but also when ancestral genomic repertoires render certain evolutionary trajectories more likely than others, even across large phylogenetic distances.

Novel phylogenetic methods are needed for understanding gene function in the era of mega-scale genome sequencing.

Ongoing large-scale genome sequencing projects are forecasting a data deluge that will almost certainly overwhelm current analytical capabilities of evolutionary genomics. In contrast to population genomics, there are no standardized methods in evolutionary genomics for extracting evolutionary and functional (e.g. gene-trait association) signal from genomic data. Here, we examine how current practices of multi-species comparative genomics perform in this aspect and point out that many genomic datasets are under-utilized due to the lack of powerful methodologies. As a result, many current analyses emphasize gene families for which some functional data is already available, resulting in a growing gap between functionally well-characterized genes/organisms and the universe of unknowns. This leaves unknown genes on the 'dark side' of genomes, a problem that will not be mitigated by sequencing more and more genomes, unless we develop tools to infer functional hypotheses for unknown genes in a systematic manner. We provide an inventory of recently developed methods capable of predicting gene-gene and gene-trait associations based on comparative data, then argue that realizing the full potential of whole genome datasets requires the integration of phylogenetic comparative methods into genomics, a rich but underutilized toolbox for looking into the past.

Comparative genomics reveals the origin of fungal hyphae and multicellularity.

Hyphae represent a hallmark structure of multicellular fungi. The evolutionary origins of hyphae and of the underlying genes are, however, hardly known. By systematically analyzing 72 complete genomes, we here show that hyphae evolved early in fungal evolution probably via diverse genetic changes, including co-option and exaptation of ancient eukaryotic (e.g. phagocytosis-related) genes, the origin of new gene families, gene duplications and alterations of gene structure, among others. Contrary to most multicellular lineages, the origin of filamentous fungi did not correlate with expansions of kinases, receptors or adhesive proteins. Co-option was probably the dominant mechanism for recruiting genes for hypha morphogenesis, while gene duplication was apparently less prevalent, except in transcriptional regulators and cell wall - related genes. We identified 414 novel gene families that show correlated evolution with hyphae and that may have contributed to its evolution. Our results suggest that hyphae represent a unique multicellular organization that evolved by limited fungal-specific innovations and gene duplication but pervasive co-option and modification of ancient eukaryotic functions.

Transcriptomic atlas of mushroom development reveals conserved genes behind complex multicellularity in fungi.

The evolution of complex multicellularity has been one of the major transitions in the history of life. In contrast to simple multicellular aggregates of cells, it has evolved only in a handful of lineages, including animals, embryophytes, red and brown algae, and fungi. Despite being a key step toward the evolution of complex organisms, the evolutionary origins and the genetic underpinnings of complex multicellularity are incompletely known. The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under laboratory conditions. We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall remodeling, targeted protein degradation, signal transduction, adhesion, and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, expansin-like proteins, protein kinases, and transcription factors, showed expansions in Agaricomycetes, many of which convergently expanded in multicellular plants and/or animals too, reflecting convergent solutions to genetic hurdles imposed by complex multicellularity among independently evolved lineages. This study provides an entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms.

Phylogenetic overview of the genus Genea (Pezizales, Ascomycota) with an emphasis on European taxa.

We constructed a comprehensive phylogeny of the genus Genea, with new molecular data from samples collected in several countries in temperate and Mediterranean Europe, as well as North America. Type specimens and authentic material of most species were examined to support identifications. The molecular identity of the most common species in Genea was compared with nuc rDNA internal transcribed spacer (ITS), D1-D2 domains of 28S nuc rDNA (28S rDNA) and translation elongation factor 1-α ene (TEF1) profiles of 10 recently proposed taxa, G. brunneocarpa, G. compressa, G. dentata, G. fageticola, G. lobulata, G. oxygala, G. pinicola, G. pseudobalsleyi, G. pseudoverrucosa and G. tuberculata, supporting their status as distinct species. Genea mexicana and G. thaxteri on the one hand and G. sphaerica and G. lespiaultii on the other are closely related. Multiple lineages were recorded for G. verrucosa and G. fragrans, but we found no morphological traits to discriminate among them, so we tentatively interpreted them as cryptic species. A key to species of the genus Genea is provided to facilitate identification. We provide macroscopic images of fresh specimens and of representative spores of most species. Finally, we conducted a molecular analysis of the divergence time for Genea and discuss the implications of our results.

Phylogeny and phylogeography of the Tuber brumale aggr.

The Tuber brumale (winter truffle) is a black truffle reported from most European countries, belonging to the Melanosporum group. Its significance in the economy is ambivalent as the winter truffle has been shown to be a frequent contaminant species in the orchards of the Perigord truffle and occasionally in those of the summer truffle, yet owing to its delicate fragrance, its trade is worthy of note. The phylogeny and phylogeography of economically important truffles are relatively well-explored; however, no thorough research has been published on these aspects of the winter truffle. Therefore, here, we report the first phylogeographic analyses based on samples representing the entire distribution of the species. ITS sequences were used in this survey for haplotype and coalescent analyses, while phylogenetic analyses were based on the ITS, LSU and PKC loci. According to all loci, the samples clustered into two big clades imply the existence of two phylogenetic species. Based on our results, one of these appears to be endemic to the Carpathian Basin. In the other more widespread species, two main phylogeographic groups can be distinguished that show east-west separation with a zone of overlap in the Carpathian Basin, suggesting that they survived the latest glacial period in separate refugia.