Mushroom-forming fungi (Agaricomycetes) have the greatest morphological diversity and complexity of any group of fungi. They have radiated into most niches and fulfil diverse roles in the ecosystem, including wood decomposers, pathogens or mycorrhizal mutualists. Despite the importance of mushroom-forming fungi, large-scale patterns of their evolutionary history are poorly known, in part due to the lack of a comprehensive and dated molecular phylogeny. Here, using multigene and genome-based data, we assemble a 5,284-species phylogenetic tree and infer ages and broad patterns of speciation/extinction and morphological innovation in mushroom-forming fungi. Agaricomycetes started a rapid class-wide radiation in the Jurassic, coinciding with the spread of (sub)tropical coniferous forests and a warming climate. A possible mass extinction, several clade-specific adaptive radiations and morphological diversification of fruiting bodies followed during the Cretaceous and the Paleogene, convergently giving rise to the classic toadstool morphology, with a cap, stalk and gills (pileate-stipitate morphology). This morphology is associated with increased rates of lineage diversification, suggesting it represents a key innovation in the evolution of mushroom-forming fungi. The increase in mushroom diversity started during the Mesozoic-Cenozoic radiation event, an era of humid climate when terrestrial communities dominated by gymnosperms and reptiles were also expanding.

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.

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.

Fungi evolved complex fruiting body (‘mushroom’) morphologies as adaptations to efficient spore dispersal in terrestrial habitats. Mushroom-forming fungi (Agaricomycetes) display a graded series of developmental innovations related to fruiting body morphology, however, how these evolved is largely unknown, leaving the functional biology and evolutionary principles of complex multicellularity in the third largest multicellular kingdom poorly known. Here, we show that developmental innovations of mushroom-forming fungi that enclose the spore-producing surface (hymenophore) in a protected environment display significant asymmetry in their evolution and are associated with increased diversification rates. ‘Enclosed’ development and related tissues (partial and universal veils) evolved convergently and became a widespread developmental type in clades in which it emerged. This probably mirrors increased fitness for protected fruiting body initials in terrestrial habitats, by better coping with environmental factors such as desiccation or predators, among others. We observed similar patterns in the evolution of complex hymenophore architectures, such as gills, pores or teeth, which optimize biomass-to-propagule number ratios and were found to spur diversification in mushrooms. Taken together, our results highlight new morphological traits associated with the adaptive radiation of mushroom-forming fungi and present formal phylogenetic testing of hypotheses on the reproductive ecology of a poorly known but hyperdiverse clade.

Contamination of genomes and sequence databases is an increasingly recognized problem, however, efficient tools for removing alien sequences are still sparse and the impact of impure data on downstream analyses remains to be fully explored. Here, we present a new, highly sensitive tool, ContScout, for removing contamination from genomes, evaluate the level of contamination in 844 published eukaryotic genomes and show that contaminating proteins can severely impact analyses of genome evolution. Via benchmarking against synthetic data, we demonstrate that ContScout achieves high specificity and sensitivity when separating sequences of different high level taxa from each other. Furthermore, by testing on manually curated data we show that ContScout by far outperforms pre-existing tools. In the context of ancestral genome reconstruction, an increasingly common approach in evolutionary genomics, we show that contamination leads to spurious early origins for gene families and inflates gene loss rates several fold, leading to false notions of complex ancestral genomes. Using early eukaryotic ancestors (including LECA) as a test case, we assess the magnitude of bias and identify mechanistic bases of the estimation problems. Based on these results, we advocate the incorporation of contamination filtering as a routine step of reporting new draft genomes and caution against the outright interpretation of complex ancestral genomes and subsequent gene loss without accounting for contamination.

Jelen doktori munka során a termőtestfejlődés kezdeti és végső fejlődési lépéseit vizsgáltuk, különös tekintettel a konzervált, ismeretlen funkcióval bíró és annotálatlan génekre. Két különböző aspektusból vizsgáltuk meg a termőtestfejlődésben résztvevő géneket. Egyfelől megvizsgáltuk a feltehetően sejtfelszíni receptorokat kódoló, a termőtest iniciációja során felülexpresszált konzervált géneket, másfelől RNS-szekvenálással feltártuk a spóraképzés posztmeiotikus lépéseit. A háromból két prediktált 7-transzmembrán doménnel rendelkező fehérjét (p7TMP) kódológ gének deléciós mutánsai kifejezetten érdekes morfológiai elváltozásokat mutatott. Ezek közül a p7TMP79 deléciós mutáns törzs a szakirodalomban eddig le nem írt morfológiai jellegekkel rendelkező termőtesteket hozott létre. A fehér, szférikus primordiumokban a differenciáció szinte teljes egészében elmaradt. A deléciós mutáns hógolyó-szerű fenotípusa miatt a kiütött gént az angol snowball (hógolyó) után snb1-nek neveztük el. A Δsnb1 törzs belső szövetekkel nem rendelkező termőtestei lehetővé tették a szöveti differenciációban résztvevő gének azonosítását. Filogenetikai vizsgálatok alapján az SNB1 ortocsoport egy eddig nem ismert, új géncsaládot alkot. Korábbi, a sporulációval kapcsolatos kutatások elsősorban a haploid magokat létrehozó meiózis lépéseit vizsgálták. Kutatásunk során célunk volt a spóraképzés meiotikus lépései mellett a posztmeiotikus folyamatok jellemzése is. A transzkriptomikai analízis ezen folyamatok feltárásán túl alkalmasnak bizonyult olyan génjelöltek azonosítására, amik deléciójával a spóraképzés elakadhat, így értékes forrása lehet ipari spóramentes gombatörzsek fejlesztésére is. Három konzervált, annotálatlan vagy funkcionálisan kevésbé ismert gént ütöttünk ki Coprinopsis cinerea-ban, amik deléciójával spóraszegény és teljesen spóramentes törzseket tudtunk előállítani, amik nem szenvedtek más, látható morfológiai defektusoktól.