Recombination suppression often evolves around sex-determining loci and extends stepwise, resulting in adjacent regions with different levels of divergence between sex chromosomes, called evolutionary strata. In Ascomycota fungi, evolutionary strata around the mating-type (MAT) locus have been reported only in pseudo-homothallic species, which have a diploid-like life cycle with mycelia carrying nuclei of both mating types. In contrast, no recombination suppression has been observed in heterothallic fungi, where colonies contain only a single mating type. Here, we investigated the evolution of recombination suppression in a clade of dung fungi encompassing 16 pseudo-homothallic and three heterothallic sibling species from the Schizothecium genus (Ascomycota, Sordariales). The analysis of genetic divergence based on genome sequencing indicated recombination suppression around the MAT locus in all 13 pseudo-homothallic species examined. The nonrecombining region ranged from 600 kb to 1.6 Mb and harbored multiple evolutionary strata, varying in size and number among species. The clustering of alleles according to mating type in gene genealogies, the high linkage disequilibrium, and an inversion in one species supported the lack of recombination in the MAT-proximal region in pseudo-homothallic species. The overall lack of trans-specific polymorphism suggested multiple independent recombination suppression events or occasional recombination/genic conversion. In heterothallic species, progeny analyses showed that recombination occurs in regions at physical distances from the MAT locus similar to those in which it is lacking in the pseudo-homothallic species. We thus revealed here multiple, likely independent evolutionary strata, associated with an extended diploid-like stage in Schizothecium fungi.

Analyses of the genetic diversity of well-studied fungi of the Sordariales, such as Neurospora spp. and Podospora anserina (syn. Triangularia anserina), have shown that the species classically defined by morphology are often complexes of cryptic species. Here, we report on the species delimitation among 76 strains producing mycelium and sexual reproductive structures identical to those of the pseudo-homothallic Sordariales species Schizothecium tetrasporum (syn. Neoschizothecium tetrasporum). Their whole genomes were sequenced as well as those of six strains closely related to Schizothecium tetrasporum but producing eight-spored asci instead of four-spored ones. The clustering based on the Average Nucleotide Identity (ANI) between the genomes identified eighteen species grouped into three clades, which were further supported by a phylogenetic tree constructed with whole genome Single Nucleotide Polymorphisms (SNPs). Based on their contrasting breeding systems and their large evolutionary distances, we considered the three clades as distinct species complexes. Indeed, two of them, the Schizothecium tetrasporum and Schizothecium pseudotetrasporum complexes, contain pseudo-homothallic species producing fourspored asci, whereas the third one, which we named Schizothecium octosporum, contains heterothallic species producing eight-spored asci. Surprisingly it was nestled between the two complexes of pseudo-homothallic species. Our data thus reveals a huge genetic diversity of the Schizothecium tetrasporum morpho-species and a convergent evolution of pseudo-homothallism or reversion to heterothallism within the complexes. An epitype for Schizothecium tetrasporum sensus stricto is defined and the seventeen new Schizothecium species are formally described.

Podospora anserina (Rabenh.) Niessl is a model fungus that was shown to belong to a complex of seven species with morphologically indistinguishable sexual fruiting bodies. Through the morphological analyses and ITS (Intergenic Transcribed Spacer) sequences of 86 newly-isolated strains, as well as the genome sequences of five strains, we show that members of the P. anserina species complex present different distribution ranges in metropolitan and overseas France. Podospora anserina is present all-over metropolitan France, including Corsica, while P. comata Milovtz. is restricted to the Northern part and P. pauciseta (Ces.) Traverso to the Southern part of continental France. The Guadeloupe hosts P. pseudoanserina C.Boucher, T.S.Nguyen & P.Silar and La R & eacute;union island a species new to science, described here as Podospora reunionensis Silar, sp. nov. This species is closely related to P. comata, but exhibit clear morphological and biological differences.

The asexual reproduction cycle of the cheese-ripening fungus Penicillium camemberti is typical for a filamentous ascomycete fungus. It involves the production of conidia by successive mitotic divisions from specialized cells called conidiophores. Conidia are used for inoculating the fungus onto French soft cheeses and are produced industrially by submerged fermentation. However, the impact of mycelium macromorphology on conidial production for this type of fermentation has been little investigated. By studying the physiological effect of different sources and concentrations of carbohydrates at the laboratory scale, it was observed that the fungus takes on different morphologies associated with varying ability to produce conidia. Especially, a dispersed morphology was not always associated with production of conidia. Through RNA-seq transcriptomic analyses, we followed the expression profiles of P. camemberti genes during the time course of liquid cultures made in the presence of two concentrations of glucose, one of sucrose and one with a mix of glucose and fructose, which are conditions for which P. camemberti presents varying morphologies and efficiencies to produce conidia. This led to confirm that, like other ascomycetes, P. camemberti likely uses the conidiation pathway first described in the model fungus Aspergillus nidulans. It also enabled the identification of a potential conidiation-inhibiting transcription factor, specific to Eurotiales and only upregulated in conditions without conidia production. Functional studies of its ortholog in A. nidulans, for which the conidiation pathway has been extensively studied, should allow to verify whether this factor indeed plays a role in the asexual cycle.

Fungal species delimitation and phylogeny will likely rely in the future upon whole genome sequence comparison, as the costs of such sequences are rapidly decreasing. Average Nucleotide Identity (ANI) between genomes is a convenient metric that can be rapidly calculated for species delimitation. However, there is presently no easy-to-use program calculating the ANI between two fungal genomes and providing easy-to interpret results that can be help mycologists having limited access to bioinformatic facilities. Here, we present FungANI, a customizable BLAST-based program that calculate ANI between genomes. The program primarily targets Linux workstations or servers but it can be run on the latest Windows, macOS and Linux 64-Bit operating systems as a standalone desktop application. It was tested with various publicly-available genomes from species belonging to the Sordariales order. It proved efficient to differentiate closely related species and retrace their possible phylogenetic relationships. However, FungANI did not perform well for phylogenetic reconstruction on a broader evolutionary scale such as inferring relationships between distant genera. The program is freely available at https://github.com/podo-gec/fungani.

The centromere of the eukaryotic chromosome is necessary for the accurate segregation during cell division. Yet, centromeric DNA is highly variable and rapidly evolving. In fungi, centromeres range from point- to regional centromeres, some of which are hundreds of thousands of base pairs long and filled with transposable elements. As fungi have evolved several specialized defense mechanisms against transposable elements, these regional centromeres are intriguing sites for investigating the connection between genome defense and centromere evolution. Here, we investigated the structure of the centromeres of seven species of the Podospora anserina species complex, which is made up of closely related filamentous ascomycetes that diverged less than 1 MYA. We discovered that one species in the complex, P. pseudocomata, lacks the genomic signature of the specialized genome defense mechanism called Repeat Induced Point mutations (RIP). We identified the centromeric regions in P. anserina and P. pseudocomata using chromatin immunoprecipitation targeting the centromere-specific histone variant cenH3, and using comparative genomics we inferred the size of centromeric regions in the other species. We found that while the centromere structure in the complex is generally well conserved, the centromeric regions of P. pseudocomata has gone through a rapid change. Specifically, the size of the centromeres in P. pseudocomata are 35-46 kb, which is significantly smaller than those of the other species (44-90 kb), and the DNA-transposon discoglosse is the most abundant TE family instead of the typical LTR-retrotransposon crapaud . Taken together, our data strongly indicates a link between genome defense and centromere evolution in fungi.

Thanks to next-generation sequencing (NGS) technologies, the diversity of fungi can now be investigated through the analysis of their genome sequences. Naviculisporaceae is a family within the Sordariales, whose diversity is not well-known, with only one genome sequence published for this family. Here, we report on the isolation and cultivation of 20 new strains of Naviculisporaceae. Their genome sequences, as well as those of the five commercially available strains, were determined, thus providing complete genome sequences for 25 new Naviculisporaceae strains. Species delimitation was conducted using a combination of (1) ITS + LSU phylogenetic analysis of the new isolates along with other known species of the family, (2) comparisons between DNA barcode sequences of the new strains with those of the known species, and (3) average genome-wide nucleotide identity calculation. We built a phylogenomic tree and studied the organization of the mating-type locus. In vitro fruiting was obtained for 16 strains, enabling the definition of seven new species, namely Pseudorhypophila gallica, Pseudorhypophila guyanensis Rhypophila alpibus, Rhypophila brasiliensis, Rhypophila camarguensis, Rhypophila reunionensis and Rhypophila thailandica, as well as two new combinations, namely Pseudorhypophila latipes and Pseudorhypophila oryzae. Eight strains for which in vitro fruiting was not obtained may belong to additional new species. These results expand the known diversity of the Naviculisporaceae and greatly enlarge the genomic data available for the family.

Major role of peroxidases in plant biomass degradation is well-established in the white rot basidiomycetes. On the contrary, peroxidases are not used for this purpose by brown rot basidiomycetes, which use instead a non-enzymatic mechanism. In the case of the ascomycetes, not much is known although these fungi have peroxidase genes. Here, we identify and characterize the peroxidase genes of Podospora anserina (Rabenh.) Niessl, an ascomycete used to study development and lignocellulose degradation. We show that this fungus has one class II peroxidase, one hybrid B peroxidase, one haloperoxidase, four functional aromatic peroxygenases, one glutathione peroxidase, one cytochrome C peroxidase and one alkyl peroxidase, but lacks a dye peroxidase. We show that potentially secreted peroxidases (i.e., the class II, hybrid B, haloperoxidase and aromatic peroxygenase peroxidases) present a patchy phylogenetic distribution compatible with an accessory role in finely adapting the different fungal species to their ecological niche, rather than being involved in fundamental roles in fungal biology. Accordingly, targeted gene deletions of the different P. anserina peroxidase genes identified only one phenotype, seemingly an alteration of the timing of ascospore maturation at intermediate concentration of vanillic acid. However, direct measure of peroxidase activity did not show drastic loss of activity in the tested mutants, suggesting compensation between the enzymes. Hence, in P. anserina peroxidases appears to have a minor role in biomass degradation, unlike what has been described in white rot fungi, and thus in this regard appears to be similar to the brown rot fungi.

The ascomycete Podospora anserina is a heterothallic filamentous fungus found mainly on herbivore dung. It is commonly used in laboratories as a model system, and its complete life cycle lasting eight days is well mastered in vitro. The main objective of our team is to understand better the global process of fruiting body development, named perithecia, induced normally in this species by fertilization. Three allelic mutants, named pfd3, pfd9, and pfd23 (for “promoting fruiting body development”) obtained by UV mutagenesis, were selected in view of their abilities to promote barren perithecium development without fertilization. By complete genome sequencing of pfd3 and pfd9, and mutant complementation, we identified point mutations in the mcm1 gene as responsible for spontaneous perithecium development. MCM1 proteins are MADS box transcription factors that control diverse developmental processes in plants, metazoans, and fungi. We also identified using the same methods a mutation in the VelC gene as responsible for spontaneous perithecium development in the vacua mutant. The VelC protein belongs to the velvet family of regulators involved in the control of development and secondary metabolite production. A key role of MCM1 and VelC in coordinating the development of P. anserina perithecia with gamete formation and fertilization is highlighted.

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.

Glycerol uptake as a carbon source has been investigated in a few fungi, mainly in Saccharomyces cerevisiae Meyen ex E.C.Hansen, Aspergillus spp., and Neurospora crassa Shear & B.O.Dodge. In the present study, we aimed at understanding glycerol use as food source in another fungus, the model Podospora anserina (Rabenh.) Niessl. Gene deletion is easy in this ascomycete and it has been used to study various biological phenomena including biomass degradation. We show that P. anserina is unable to use glycerol as a sole carbon source to fuel its vegetative growth; glycerol is even toxic to the fungus. However, P. anserina is able to use glycerol during perithecium maturation, albeit inefficiently. Genome mining identified possible glycerol uptake and catabolic pathways of P. anserina, and the two genes coding for enzymes of the glycerol-3-phosphate pathway were deleted. Deletions resulted in a lack of perithecium production on glycerol media, but not on optimal media containing dextrin as a carbon source. Intriguingly, the presence of bacteria in co-culture with the fungus greatly helps P. anserina to use glycerol both during vegetative growth and perithecium production. This offers new perspectives for improved biotransformation of glycerol into high-value products.

The regulation of ascospore germination in filamentous fungi has been poorly investigated so far. To unravel new genes involved in this regulation pathway, we conducted a genetic screen in Podospora anserina , and we isolated 57 mutants affected in ascospore germination.

AbstractThe filamentous fungusPodospora anserinais 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 thatP. anserinais a member of a complex of seven, closely related species. In addition toP. anserina, high-quality genomic resources are available for two of these taxa. Here we provide chromosome-level annotated assemblies of the four remaining species of the complex, as well as a comprehensive dataset of annotated assemblies from a total of 28Podosporagenomes. We find that all seven species have genomes of around 35 Mbp arranged in seven 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.SignificanceHere we provide a dataset of 28 annotated genomes from theP. anserinaspecies complex, including chromosome-level assemblies of four species that lacked a reference genome. With this dataset in hand, biologists can take advantage of the molecular tools available forP. anserinato study evolutionary dynamics at the interphase between micro- and macroevolution, with particular emphasis on trait evolution, genome architecture, and speciation.

Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i.e. the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma. However, it is unclear whether this intriguing association holds in other species. We show here that Schizothecium tetrasporum, a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina. The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.47 Mb around the mating-type locus. We detected three evolutionary strata, indicating a stepwise extension of recombination suppression. The three strata displayed no rearrangement or transposable element accumulation but gene losses and gene disruptions were present, and precisely at the strata margins. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association between pseudo-homothallism and recombination suppression across lineages and the presence of gene disruption at the strata limits are consistent with a recently proposed mechanism of sheltering deleterious alleles to explain stepwise recombination suppression.

Penicillium species are ubiquitous in all kinds of environments, and they are of industrial, agricultural and clinical importance. In this study, soil fungal diversity in Southwestern China was investigated, and that of Penicillium turned out to be unexpectedly high. The survey included a total of 179 cultures of the genus isolated from 33 soil samples. Three-locus phylogenetic analyses and morphological comparisons were carried out. The examinations revealed that they belonged to two subgenera (Aspergilloides and Penicillium), 11 sections (Aspergilloides, Canescentia, Citrina, Exilicaulis, Fasciculata, Gracilenta, Lanata-Divaricata, Penicillium, Ramosum, Robsamsonia, and Sclerotiorum), 25 series, and 74 species. Forty-three species were discovered as new to science, and a new series, Simianshanica, was established in sect. Aspergilloides. Additionally, 11 species were recorded for the first time in China. Species isolation frequency and distribution of the group were also discussed.

The present study aimed to discover good lignocellulolytic enzyme (LCE) producers from Thailand’s tropical forest and then examine their multiple LCE production (including carboxymethyl cellulase (CMCase), xylanase, and laccase) using agricultural wastes as substrate. The total collection was 50 fungi, mainly from the Polyporales, Agaricales, and Xylariales orders. During primary screening by qualitative method and secondary screening by quantitative method, two potential fungi were proposed for multiple LCE production, including Auricularia auricula-judae 088 and Pseudolagarobasidium acaciicola TDW-48. Under solid-state fermentation (SSF) using agricultural wastes as substrates, P. acaciicola TDW-48 performed as a good producer that highly secreted simultaneous CMCase, xylanase, and laccase. In the next stage, the simplex lattice mixture design assessed the interaction of agricultural waste substrates and their effects on P. acaciicola TDW-48’s enzyme production. The results indicated that agricultural waste has different influences on CMCase, xylanase, and laccase production: orange peel showed a positive effect on both CMCase and xylanase activity, but a negative effect on laccase. In contrast, wheat bran positively influenced laccase, while it limited CMCase and xylanase. However, the combination of these substrates in the mixture showed synergic effects and improved enzyme activity. Through numerical optimization, a ternary mixture of wheat bran (1.27 g), orange peel (1.53 g), and rice husk (0.2 g) was identified as the most appropriate formulation for simultaneous multiple LCE production, reaching 20.96 U/g substrate for CMCase, 23.94 U/g substrate for xylanase, and 27.55 U/g substrate for laccase. These results provided a promising candidate for LCE production with high applicability in lignocellulose bioconversion and successfully demonstrated the relationship between the agricultural waste substrate and multiple LCE production that supported the enzyme production following the environmentally friendly and economical approach.

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

ABSTRACT Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i . e ., the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma . However, it is unclear whether this intriguing convergent association holds in other species. We show here that Schizothecium tetrasporum , a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina . The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.3 Mb around the mating-type locus. We detected three evolutionary strata, displaying a stepwise extension of recombination suppression, but no rearrangement or transposable element accumulation in the non-recombining region. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association is consistent with a recently proposed mechanism of deleterious allele sheltering through recombination suppression around a permanently heterozygous locus. AUTHOR SUMMARY Recombination allows faster adaptation and the purging of deleterious mutation but is often paradoxically lacking in sex chromosomes. It has been recently recognized that recombination can also be suppressed on fungal mating-type chromosomes, but the evolutionary explanation and the proximal mechanism of this phenomenon remain unclear. By studying here the sexual biology of a poorly studied mold living in rabbit dung, we reveal a striking convergence in three distant fungal lineages of an independently evolved association between the production of self-fertile sexual spores (carrying two nuclei with opposite mating types), a particular segregation of the mating-type locus and the lack of recombination on mating-type chromosomes, having evolved stepwise. Such a convergent association suggests causal relationships and will contribute to unveil the evolutionary causes of recombination suppression. Graphical summary

The Crippled Growth (CG) cell degeneration of the model ascomycete Podospora anserina (strain S) is controlled by a prion-like element and has been linked to the self-activation of the PaMpk1 MAP kinase cascade. Here, we report on the identification of the "86-11" locus containing twelve genes, ten of which are involved either in setting up the self-activation loop of CG or in inhibiting this loop, as demonstrated by targeted gene deletion. Interestingly, deletion of the whole locus results only in the elimination of CG and in no detectable additional physiological defect. Sequence comparison shows that these ten genes belong to four different families, each one endowed with a specific activity: two encode factors activating the loop, a third one encodes a factor crucial for inhibition of the loop and the fourth one participates in inhibiting the loop in a pathway parallel to the one controlled by the previously described PDC1 gene. Intriguingly, a very distant homologue of this "86-11" locus is present at the syntenic position in Podospora comata (strain T) that do not present Crippled Growth. Introgression of the P. comata strain T locus in P. anserina strain S and the P. anserina strain S in P. comata strain T showed that both drive CG in the P. anserina strain S genetic background, but not in the genetic background of strain P. comata T, indicating that genetic determinants outside the twelve-gene locus are responsible for lack of CG in P. comata strain T. Our data question the role of this twelve-gene locus in the physiology of P. anserina.

Melanins are pigments used by fungi to withstand various stresses and to strengthen vegetative and reproductive structures. In Sordariales fungi, their biosynthesis starts with a condensation step catalyzed by an evolutionary-conserved polyketide synthase. Here we show that complete inactivation of this enzyme in the model ascomycete Podospora anserina through targeted deletion of the PaPks1 gene results in reduced female fertility, in contrast to a previously analyzed nonsense mutation in the same gene that retains full fertility. We also show the utility of PaPks1 mutants for detecting rare genetic events in P. anserina, such as parasexuality and possible fertilization and/or apomixis of nuclei devoid of mating-type gene.