Lichens are composite, symbiotic associations of fungi, algae, and bacteria that result in large, anatomically complex organisms adapted to many of the world’s most challenging environments. How such intricate, self-replicating lichen architectures develop from simple microbial components remains unknown because of their recalcitrance to experimental manipulation. Here, we report a metagenomic and metatranscriptomic analysis of the lichen Xanthoria parietina at different developmental stages. We identified 168 genomes of symbionts and lichen-associated microbes across the sampled thalli, including representatives of green algae, three different classes of fungi, and 14 bacterial phyla. By analyzing the occurrence of individual species across lichen thalli from diverse environments, we defined both substrate-specific and core microbial components of the lichen. Metatranscriptomic analysis of the principal fungal symbiont from three different developmental stages of a lichen, compared with axenically grown fungus, revealed differential gene expression profiles indicative of lichen-specific transporter functions, specific cell signaling, transcriptional regulation, and secondary metabolic capacity. Putative immunity-related proteins and lichen-specific structurally conserved secreted proteins resembling fungal pathogen effectors were also identified, consistent with a role for immunity modulation in lichen morphogenesis.

In sexual organisms, inheritance of new mutations is highly dependent on the timing of germline definition. Here, we used the fairy ring–forming fungus Marasmius oreades to challenge the general assumption of a late germline separation in the Fungi. We collected mushrooms from different parts of rings over a 7-year period and identified new mutations in different tissues by whole-genome sequencing. We found evidence that fertile and sterile tissues had accumulated different mutations, suggesting that the germ line, destined for spore production, is already defined in the mycelium in this species. Moreover, the germ line carried fewer mutations than sterile tissues, indicating a lower mutation rate. Our findings suggest that early germline sequestration is more widespread than previously considered across multicellular life.

Sexual compatibility in the Basidiomycota is governed by genetic identity at one or two loci, resulting in compatibility systems called bipolar and tetrapolar. The loci are known as HD and P/R, encoding homeodomain transcription factors and pheromone precursors and receptors, respectively. Bipolarity is known to evolve either by linkage of the two loci or by loss of mating-type determination of either the HD or the P/R locus. The ancestor to basidiomycete fungi is thought to have been tetrapolar, and many transitions to bipolarity have been described in different lineages. In the diverse genus Marasmius (Agaricales), both compatibility systems are found, and the system has been shown to follow the infrageneric sections of the genus, suggesting a single origin of bipolarity. Here, we tested this hypothesis using a comprehensive phylogenetic framework and investigated the mode by which bipolarity has evolved in this group. We utilized available genomic data and marker sequences to investigate evolution of sexual compatibility in Marasmius and allied genera. By generating a concatenated multilocus phylogeny, we found support for a single transition to known bipolarity within Marasmius. Furthermore, utilizing genomic data of the bipolar species Marasmius oreades, we found that the HD and P/R loci likely have remained unlinked through this transition. By comparing nucleotide diversity at the HD and P/R loci in Ma. oreades, we show that the HD locus has retained high diversity, and thus likely the function of determining sexual identity, as similarly in other bipolar mushroom-forming fungi. Finally, we describe the genomic architecture of the MAT loci of species of both sexual compatibility systems in Marasmiaceae and related families.

Fungi have NOD-Like receptors (NLRs), homologous to the innate immune receptors found in animals, plants and bacteria. Fungal NLRs are characterized by a great variability of domain organizations, but the identity of the nucleotide-binding domains, the genomic localization, and the factors associated with variation in the composition of repertoires of fungal NLRs are not yet fully understood. To better understand the variability of fungal NLR repertoires and the underlying determinants, we conducted a thorough analysis of genome data from the ascomycete order Sordariales. Using similarity searches based on hidden Markov models profiles for canonical N-terminal, nucleotide-binding, or C-terminal domains, we characterized 4613 NLRs in 82 Sordariales taxa. By examining the Helical Third section of the nucleotide-binding domains, we substantially improved their annotation. We demonstrated that fungi have NACHT domains of both NAIP-like and TLP1-like types, similar to animals. We found that the number of NLR genes was highly variable among Sordariales families, and independent of the stringency of defense mechanisms against genomic repeat elements. NLRs were organized in clusters in the majority of taxa, and the strong correlation between the number of NLRs and the number of NLR clusters suggested that organizing in clusters may contribute to repertoire diversification. Our work highlights the similarity of fungal and animal NLRs in terms of nucleotide-binding domain types, and between fungal and plant NLRs in terms of genomic organization in clusters. Our findings will aid in the comparative analysis of the patterns and processes of diversification of NLR repertoires in various lineages of fungi and between the different kingdoms and domains of life.

Fungi are a highly diverse group of organisms, of which only a small subset has been taken into cultivation for application in biotechnology and food industry. Accordingly, outside of a few model species, there is a lack of knowledge about the isolation and cultivation of fungi. In this study, we isolated 17 wild strains of 14 different species of edible, mushroom-forming fungi growing in Swedish nature. We documented their growth rates under different temperatures, investigated their fruiting characteristics, and compared the results to data obtained from common laboratory strains. Our results show that the strains from commercially cultivated species have a higher mycelial growth rate and tend to grow faster at higher temperatures than strains from less frequently cultivated species. The fruiting experiments led to successful fruiting of four newly collected wild strains, belonging to the species Hericium coralloides, Pleurotus pulmonarius, and Schizophyllum commune. Although some strains fruited on potato dextrose agar (PDA), more specific substrates such as straw or birch pellets indicated more potential for mushroom production. All newly isolated strains of this study have been deposited at the Westerdijk Fungal Biodiversity Institute (CBS) collection and are thereby made available for further studies and/or use in application in the food industry or biotechnology. Two species isolated in this study are entirely novel to widely used culture collections, and for nine species no Swedish strain has been deposited previously. The description of the mycelial growth and fruiting of the isolated strains in this study is a first step on their way to further use.

Here we present a study on amino acid composition, codon usage bias (CUB), and levels of selection driving codon usage in Sordariales fungi. We found that GC ending codons are used more often than AT ending codons in all Sordariales genomes, but the strength of CUB differs amongst families. The families Podosporaceae and Sordariaceae contain relatively low genome-wide levels of CUB, while the highest levels of CUB are found in Chaetomiaceae and the “BLLNS”-group, a monophyletic group of five other Sordariales families. Based on genomic clustering, we show that Podosporaceae and Sordariaceae are more similar to each other than either of them are to any of the other groups. Comparatively, the Chaetomiaceae and BLLNS show increased natural selection driving use of specific codons, resulting in higher genome-wide CUB. We hypothesize that the higher levels of CUB in Chaetomiaceae genomes might have been caused by ecological niche specialization, versus the more generalist nature of many Sordariaceae and Podosporaceae species.

NOD-like receptors (NLRs) are intracellular immune receptors that detect pathogen-associated cues and trigger defence mechanisms, including regulated cell death. In filamentous fungi, some NLRs mediate heterokaryon incompatibility, a self-/non-self-recognition process that prevents the vegetative fusion of genetically distinct individuals, reducing the risk of parasitism. The het-d and het-e NLRs in Podospora anserina are highly polymorphic incompatibility genes (het genes) whose products recognize different allelic variants of the HET-C protein via a sensor domain composed of WD40 repeats. These repeats display unusually high sequence identity maintained by concerted evolution. However, some sites within individual repeats are hypervariable and under diversifying selection. Despite extensive genetic studies, inconsistencies in the reported WD40 domain sequence have hindered functional and evolutionary analyses. Here, we confirm that the WD40 domain can be accurately reconstructed from long-read sequencing (Oxford Nanopore and PacBio) data, but not from Illumina-based assemblies. Functional alleles are usually formed by 11 highly conserved repeats, with different repeat combinations underlying the same phenotypic het-d and het-e incompatibility reactions. AlphaFold 3 structure models suggest that their WD40 domain folds into two 7-blade β-propellers composed of the highly conserved repeats, as well as three cryptic divergent repeats at the C-terminus. We additionally show that one particular het-e allele does not have an incompatibility reaction with common het-c alleles, despite being 11-repeats long. Finally, we present evidence that the recognition phenotypes of het-e and het-d arose through convergent evolution. Our findings provide a robust foundation for future research into the molecular mechanisms and evolutionary dynamics of het NLRs, while also highlighting both the fragility and the flexibility of β-propellers as immune sensor domains.

Background The genome of the filamentous ascomycete Podospora anserina shows a relatively high abundance of retrotransposons compared to other interspersed repeats. The LTR-retrotransposon family crapaud is particularly abundant in the genome, and consists of multiple diverged sequence variations specifically localized in the 5' half of both long terminal repeats (LTRs). P. anserina is part of a recently diverged species-complex, which makes the system ideal to classify the crapaud family based on the observed LTR variation and to study the evolutionary dynamics, such as the diversification and bursts of the elements over recent evolutionary time.Results We developed a sequence similarity network approach to classify the crapaud repeats of seven genomes representing the P. anserina species complex into 14 subfamilies. This method does not utilize a consensus sequence, but instead it connects any copies that share enough sequence similarity over a set sequence coverage. Based on phylogenetic analyses, we found that the crapaud repeats likely diversified in the ancestor of the complex and have had activity at different time points for different subfamilies. Furthermore, while we hypothesized that the evolution into multiple subfamilies could have been a direct effect of escaping the genome defense system of repeat induced point mutations, we found this not to be the case.Conclusions Our study contributes to the development of methods to classify transposable elements in fungi, and also highlights the intricate patterns of retrotransposon evolution over short timescales and under high mutational load caused by nucleotide-altering genome defense.

The genomes of ectomycorrhizal (ECM) fungi have a reduced number of genes encoding Carbohydrate-Active EnZymes (CAZymes), expansions in transposable elements (TEs) and small secreted proteins (SSPs) compared with saprotrophs. Fewer genes for specific peptidases and lipases in ECM fungi are also reported. It is unclear whether these changes occur at the shift to the ECM habit or are more gradual throughout the evolution of ECM lineages. We generated a genomic dataset of 20 species in the ECM lineage Inocybaceae and compared them with six saprotrophic species. Inocybaceae genomes have fewer CAZymes, peptidases, lipases, secondary metabolite clusters and SSPs and higher TE content than their saprotrophic relatives. There was an increase in the rate of gene family evolution along the branch with the transition to the ECM lifestyle. This branch had very high rate of evolution in CAZymes and had the largest number of contractions. Other significant changes along this branch included expansions in transporters, transposons-related genes and communication genes such as fungal kinases. There is a high concentration of changes in proximity to the transition to the ECM lifestyle, which correspond to the identified key changes for the gain of this lifestyle.

The filamentous fungus Podospora anserina is a model organism used extensively in the study of molecular biology, senescence, prion biology, meiotic drive, mating-type chromosome evolution, and plant biomass degradation. It has recently been established that P. anserina is a member of a complex of 7 closely related species. In addition to P. anserina, high-quality genomic resources are available for 2 of these taxa. Here, we provide chromosome-level annotated assemblies of the 4 remaining species of the complex, as well as a comprehensive data set of annotated assemblies from a total of 28 Podospora genomes. We find that all 7 species have genomes of around 35 Mb arranged in 7 chromosomes that are mostly collinear and less than 2% divergent from each other at genic regions. We further attempt to resolve their phylogenetic relationships, finding significant levels of phylogenetic conflict as expected from a rapid and recent diversification.