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Comparative and pan-genomic analyses of the endophytic fungus Pezicula neosporulosa(Helotiales, Ascomycota) from needles of the relict fir, Abies beshanzuensis, showed expansions of carbohydrate metabolism and secondary metabolite biosynthetic genes characteristic for unrelated plant-beneficial helotialean, such as dark septate endophytes and ericoid mycorrhizal fungi. The current species within the relatively young Pliocene genus Peziculaare predominantly saprotrophic, while P. neosporulosalacks such features. To understand the genomic background of this putatively convergent evolution, we performed population analyses of 77 P. neosporulosaisolates. This revealed a mosaic structure of a dozen non-recombining and highly genetically polymorphic subpopulations with a unique mating system structure. We found that one idiomorph of a probably duplicated mat1-2gene was found in putatively heterothallic isolates, while the other co-occurred with mat1-1locus suggesting homothallic reproduction for these strains. Moreover, 24 and 81 genes implicated in plant cell-wall degradation and secondary metabolite biosynthesis, respectively, showed signatures of the balancing selection. These findings highlight the evolutionary pattern of the two gene families for allowing the fungus a rapid adaptation towards endophytism and facilitating diverse symbiotic interactions.

Understanding how organisms adapt to extreme living conditions is central to evolutionary biology. Dark septate endophytes (DSEs) constitute an important component of the root mycobiome and they are often able to alleviate host abiotic stresses. Here, we investigated the molecular mechanisms underlying the beneficial association between the DSE Laburnicola rhizohalophilaand its host, the native halophyte Suaeda salsa, using population genomics. Based on genome-wide Fst (pairwise fixation index) and Vst analyses, which compared the variance in allele frequencies of single-nucleotide polymorphisms (SNPs) and copy number variants (CNVs), respectively, we found a high level of genetic differentiation between two populations. CNV patterns revealed population-specific expansions and contractions. Interestingly, we identified a ~20?kbp genomic island of high divergence with a strong sign of positive selection. This region contains a melanin-biosynthetic polyketide synthase gene cluster linked to six additional genes likely involved in biosynthesis, membrane trafficking, regulation, and localization of melanin. Differences in growth yield and melanin biosynthesis between the two populations grown under 2% NaCl stress suggested that this genomic island contributes to the observed differences in melanin accumulation. Our findings provide a better understanding of the genetic and evolutionary mechanisms underlying the adaptation to saline conditions of the L. rhizohalophila–S. salsasymbiosis.

Fungal evolutionary biology is impeded by the scarcity of fossils, irregular life cycles, immortality, and frequent asexual reproduction. Simple and diminutive bodies of fungi develop inside a substrate and have exceptional metabolic and ecological plasticity, which hinders species delimitation. However, the unique fungal traits can shed light on evolutionary forces that shape the environmental adaptations of these taxa. Higher filamentous fungi that disperse through aerial spores produce amphiphilic and highly surface-active proteins called hydrophobins (HFBs), which coat spores and mediate environmental interactions. We exploited a library of HFB-deficient mutants for two cryptic species of mycoparasitic and saprotrophic fungi from the genus Trichoderma(Hypocreales) and estimated fungal development, reproductive potential, and stress resistance. HFB4 and HFB10 were found to be relevant for Trichodermafitness because they could impact the spore-mediated dispersal processes and control other fitness traits. An analysis in silico revealed purifying selection for all cases except for HFB4 from T. harzianum, which evolved under strong positive selection pressure. Interestingly, the deletion of the hfb4gene in T. harzianumconsiderably increased its fitness-related traits. Conversely, the deletion of hfb4in T. guizhouenseled to the characteristic phenotypes associated with relatively low fitness. The net contribution of the hfb4gene to fitness was found to result from evolutionary tradeoffs between individual traits. Our analysis of HFB-dependent fitness traits has provided an evolutionary snapshot of the selective pressures and speciation process in closely related fungal species.

ABSTRACTFourteen Trichoderma(Hypocreales) species were identified during a survey of the genus in South Africa. These include T. afroharzianum, T. asperelloides, T. asperellum, T. atrobrunneum, T. atroviride, T. camerunense, T. gamsii, T. hamatum, T. koningii, T. koningiopsis, T. saturnisporum, T. spirale, T. virens, and T. viride. Ten of these species were not known to occur in South Africa prior to this investigation. Five additional species were novel and are described here as T. beinartii, T. caeruleimontis, T. chetii, T. restrictum, and T. undulatum. These novel Trichodermaspecies display morphological traits that are typical of the genus. Based on molecular identification using calmodulin, endochitinase, nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2), RNA polymerase II subunit B, and translation elongation factor 1-α gene sequence data, T. beinartii, T. caeruleimontis, and T. chetiiwere found to belong to the Longibrachiatumclade, whereas T. restrictumis a member of the Hamatumclade. Trichoderma undulatumoccupies a distinct lineage distantly related to other Trichodermaspecies. Strains of T. beinartiiand T. chetiiwere isolated previously in Hawaii and Israel; however, T. caeruleimontis, T. restrictum, and T. undulatumare so far known only from South Africa.

ABSTRACTSpecies of the fungal genus Trichoderma(Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichodermapolyketide synthase (PKS)-encoding genes, the pks4from T. reeseiwas shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusariumspp. In this study, we show that deletion of this gene in T. reeseiresults in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reeseiagainst other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.

The European species Hypocrea epimyces(Hypocreales, Ascomycota, Fungi) is redescribed based on the holotype including the drawing on its envelope by Saccardo and freshly collected material. The holomorphs of two closely related species, H. alniand H. brunneoviridis, are described as new species of the genus. They are characterized with morphological and molecular methods, including culture studies and phylogenetic analyses with internal transcribed spacers 1 and 2 as a part of the ribosomal RNA gene cluster, calmodulin, endochitinase, intron 4 of the translation elongation factor 1-alpha gene, and a part of the RNA polymerase II subunit B gene as phylogenetic markers. All species described here have green ascospores. Although phylogenetically closely related to H. lixii, they form reddish brown instead of green to black stromata. Except for H. brunneoviridis, forming nearly gliocladium-like conidiophores, the anamorphs of these species are similar to each other but vary in the angles of conidiophore branches and phialides, in phenotypic arrangement of conidiation on growth plates and in growth rates of cultures.

Species of the mycoparasitic fungal genus Hypocrea/Trichoderma are prominent producers of peptaibols, a class of small linear peptides of fungal origin. Some of these peptaibols have been shown to act synergistically with cell-wall-degrading enzymes in the inhibition of the growth of other fungi in vitro and in vivo. Here we present the structure of the Hypocrea atroviridis peptaibol synthetase gene (pbs1), deduced from the genome sequence of H. atroviridis. It consists of 19 typical peptide synthetase modules with the required additional modifying domains at the N and C termini. Phylogenetic and similarity analyses of the individual amino acid-activating modules is consistent with its ability to synthesize atroviridins. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of surface-grown cultures of H. atroviridis showed that no peptaibols were formed during vegetative growth, but a microheterogenous mixture of atroviridins accumulated when the colonies started to sporulate. This correlation between sporulation and atroviridin formation was shown to be independent of the pathway inducing sporulation (i.e., light, mechanical injury and carbon starvation, respectively). Atroviridin formation was dependent on the function of the two blue light regulators, BLR1 and BLR2, under some but not all conditions of sporulation and was repressed in a pkr1 (regulatory subunit of protein kinase A) antisense strain with constitutively active protein kinase A. Conversely, however, loss of function of the G-protein GNA3, which is a negative regulator of sporulation and leads to a hypersporulating phenotype, fully impairs atroviridin formation. Our data show that formation of atroviridin by H. atroviridis occurs in a sporulation-associated manner but is uncoupled from it at the stage of GNA3.

ABSTRACTSpecies of the mycoparasitic fungal genus Hypocrea/Trichodermaare prominent producers of peptaibols, a class of small linear peptides of fungal origin. Some of these peptaibols have been shown to act synergistically with cell-wall-degrading enzymes in the inhibition of the growth of other fungi in vitro and in vivo. Here we present the structure of the Hypocrea atroviridispeptaibol synthetase gene (pbs1), deduced from the genome sequence of H. atroviridis. It consists of 19 typical peptide synthetase modules with the required additional modifying domains at the N and C termini. Phylogenetic and similarity analyses of the individual amino acid-activating modules is consistent with its ability to synthesize atroviridins. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of surface-grown cultures of H. atroviridisshowed that no peptaibols were formed during vegetative growth, but a microheterogenous mixture of atroviridins accumulated when the colonies started to sporulate. This correlation between sporulation and atroviridin formation was shown to be independent of the pathway inducing sporulation (i.e., light, mechanical injury and carbon starvation, respectively). Atroviridin formation was dependent on the function of the two blue light regulators, BLR1 and BLR2, under some but not all conditions of sporulation and was repressed in a pkr1(regulatory subunit of protein kinase A) antisense strain with constitutively active protein kinase A. Conversely, however, loss of function of the Ga-protein GNA3, which is a negative regulator of sporulation and leads to a hypersporulating phenotype, fully impairs atroviridin formation. Our data show that formation of atroviridin by H. atroviridisoccurs in a sporulation-associated manner but is uncoupled from it at the stage of GNA3.

The new species Hypocrea crystalligena(Hypocreales, Ascomycota, Fungi) is described as a holomorph and characterized based on an integrated phenotypic and phylogenetic approach, using teleomorph and anamorph morphologies, culture studies and analyses of phylogenetic markers including internal transcribed spacer 1 and 2 (ITS1 and 2), two last introns of the translation elongation factor 1-alpha encoding gene (tef1), and a portion of the rpb2gene, encoding the second largest RNA polymerase subunit. Stromata of H. crystalligenashow similarities with those of species from Trichodermasect. Trichodermabut differ in several respects, including color, presence of white crystals on the surface and small ascospores. Colonies on CMD appear distinct, form colorless to white crystals on isolation, a yellowish to brown pigment and an anamorph with hyaline conidia exhibiting verticillium-like to gliocladium-like structural elements. ITS1 and 2 sequences exhibit all genus-specific features but also contain several unique hallmarks permitting development of a species-diagnostic barcode. Based on the analyses of partial rpb2and tef1sequences, H. crystalligenaconstitutes a separate evolutionary lineage with H. megalocitrinaand H. psychrophilaas its nearest neighbors. All these species form one phylogenetic clade with the H. pulvinata/H. citrinanode.

The type species of the genus Hypocrea(Hypocreaceae, Hypocreales, Ascomycota, Fungi), H. rufa, is re-defined and epitypified using a combination of phenotype (morphology of teleomorphs and anamorphs, and characteristics in culture) and phylogenetic analyses of the translation-elongation factor 1α gene. Its anamorph, T. viride, the type species of Trichoderma, is re-described and epitypified. Eidamia viridescensis combined as Trichoderma viridescensand is recognised as one of the most morphologically and phylogenetically similar relatives of T. viride. Its teleomorph is newly described as Hypocrea viridescens. Contrary to frequent citations of H. rufaand T. viridein the literature, this species is relatively rare. Although both T. virideand T. viridescenshave a wide geographic distribution, their greatest genetic diversity appears to be in Europe and North America. Hypocrea vinosais characterised and its anamorph, T. vinosumsp. nov., is described. Conidia of T. vinosumare subglobose and warted. The new species T. gamsiiis proposed. It shares eidamia-like morphology of conidiophores with T. viridescens,but it has smooth, ellipsoidal conidia that have the longest L/W ratio that we have seen in Trichoderma. Trichoderma scalesiae, an endophyte of trunks of Scalesia pedunculatain the Galapagos Islands, is described as new. It only produces conidia on a low-nutrient agar to which filter paper has been added. Additional phylogenetically distinct clades are recognised and provisionally delimited from the species here described. Trichoderma neokoningii, a T. koningii-like species, is described from a collection made in Peru on a fruit of Theobroma cacaoinfected with Moniliophthora roreri.

The morphological concept of Trichoderma koningiiis found to include several species that differ from each other in details of phenotype (including conidium morphology, growth rate) and biogeography. Phylogenetic analysis utilizing partial sequences of the translation-elongation factor 1 alpha (tef1), as well as fragments of actin and calmodulin genes, indicate that phenotypic characters typical of T. koningiievolved independently in three well-separated main lineages. Combined molecular and phenotype data lead to the development of a taxonomy with the recognition of twelve taxonomic species and one variety within the three lineages. These lineages include: (1) T. koningiiand T. ovalisporumand the new species T. caribbaeumvar. caribbaeum, T. caribbaeumvar. aequatoriale, T. dorotheae, T. dingleyae, T. intricatum, T. koningiopsis, T. peterseniiand T. taiwanense; (2) the new species T. rogersoniiand T. austrokoningii, and (3) the new anamorph T. stilbohypoxyli.

The holomorph of the new species Hypocrea voglmayrii(Hypocreales, Ascomycota, Fungi) is described by a combined approach, using morphology of the teleomorph, morphology of the anamorph, culture studies and phylogenetic analyses of ITS1 and 2, ech42and rpb2gene sequences. Its anamorph Trichoderma voglmayriiis described as a new anamorph species. Unlike most other species of Hypocreathe teleomorph of H. voglmayriioccurs on dry standing trunks and exhibits well defined black ostioles. Although exclusively collected at higher altitudes, this species grows at 35 C in culture. Hypocrea voglmayriidevelops pale yellowish to greenish conidia, a yellowish pigment and a coconut-like odor on CMD. Phylogenetically, H. voglmayriiforms a distinct, isolated branch between the section Trichodermaand the H. pachybasioidesclade but does not associate with any of these clades in different gene trees.

We describe or redescribe species of Hypocrea/Trichoderma(Ascomycetes, Hypocreales) having hyaline ascospores and pachybasium-like conidiophores. Teleomorphs are reported for Trichoderma minutisporum(Hypocrea minutisporasp. nov.) and T. polysporum(H. pachybasioides). Hypocrea pilulifera/T. piluliferumis redescribed. Trichoderma croceumis synonymized with T. polysporum.The new species H. parapilulifera, H. stellataand H. lacuwombatensisare described. All of these species fall within the morphological concept of Trichodermasect. Pachybasiumand within the phylogenetic group pachybasium B5 of Kullnig-Gradinger et al (2002). Parsimony analysis of nucleotide sequences from three unlinked loci—ITS1 and 2, endochitinase (ech42) and translation elongation factor 1-alpha (tef1)—detects two distinct phylogenetic lineages within the group pachybasium B5. One comprises H. pachybasioides/T. polysporum, H. pilulifera/T. piluliferum, H. parapiluliferaand H. stellata; this group, the “polysporum” lineage, is characterized by having conidia that are white in mass and is the only lineage within Hypocreacharacterized by such conidia. The second group includes the green conidial T. minutisporumand H. lacuwombatensis.The partition homogeneity test reveals significant recombination within the “polysporum” lineage but not within the “minutisporum” lineage.

True fungi (Fungi) and fungus-like organisms (e.g. Mycetozoa, Oomycota) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.

Background: Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma. Results: Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride(teleomorph Hypocrea atroviridis) and Trichoderma virens(formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei(teleomorph Hypocrea jecorina). These three Trichodermaspecies display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reeseior other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reeseiand T. virensare derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichodermaancestor but were subsequently lost in T. reesei. Conclusions: The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

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