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.

Radiation of mushroom-forming fungi correlates with novel modes of protecting sexual fruiting bodies.

The protection of vulnerable developing structures evolved repeatedly in terrestrial organisms and includes, among others, viviparity in animals and the seed in land plants. In mushroom-forming fungi (Agaricomycetes), sexual spores are born on fruiting bodies, the growth of which is a complex developmental process that is exposed to environmental factors (e.g., desiccation, fungivorous animals). Mushroom-forming fungi evolved a series of innovations in fruiting body protection, however, how these emerged is obscure, leaving the evolutionary principles of fruiting body development poorly known. Here, we show that developmental innovations that lead to the spore-producing surface (hymenophore) being enclosed in a protected environment display asymmetry in their evolution and are associated with increased diversification rates. 'Enclosed' development evolved convergently and became a dominant developmental type in several clades of mushrooms. This probably mirrors spore production benefits for species with protected fruiting body initials, by better coping with environmental factors. Our observations highlight new morphological traits associated with mushroom diversification that parallel the evolution of protection strategies in other organisms, such as viviparity or the seed in animals or plants, respectively, but in the context of spore development, highlighting the general importance of protecting vulnerable progeny across the tree of life.

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.

Evolution of opposing regulatory interactions underlies the emergence of eukaryotic cell cycle checkpoints.

In eukaryotes the entry into mitosis is initiated by activation of cyclin-dependent kinases (CDKs), which in turn activate a large number of protein kinases to induce all mitotic processes. The general view is that kinases are active in mitosis and phosphatases turn them off in interphase. Kinases activate each other by cross- and self-phosphorylation, while phosphatases remove these phosphate groups to inactivate kinases. Crucial exceptions to this general rule are the interphase kinase Wee1 and the mitotic phosphatase Cdc25. Together they directly control CDK in an opposite way of the general rule of mitotic phosphorylation and interphase dephosphorylation. Here we investigate why this opposite system emerged and got fixed in almost all eukaryotes. Our results show that this reversed action of a kinase-phosphatase pair, Wee1 and Cdc25, on CDK is particularly suited to establish a stable G2 phase and to add checkpoints to the cell cycle. We show that all these regulators appeared together in LECA (Last Eukaryote Common Ancestor) and co-evolved in eukaryotes, suggesting that this twist in kinase-phosphatase regulation was a crucial step happening at the emergence of eukaryotes.

Megaphylogeny resolves global patterns of mushroom evolution.

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.