Diversity and novel lineages of black yeasts in Chaetothyriales from freshwater sediments in Spain

Black yeasts comprise a group of Ascomycota of the order Chaetothyriales with highly variable morphology, a great diversity of ecological niches and lifecycles. Despite the ubiquity of these fungi, their diversity in freshwater sediments is still poorly understood. During a survey of culturable Ascomycota from river and stream sediments invarious sampling sites in Spain, we obtained 47 isolates of black yeasts by using potato dextroseagar supplemented with cycloheximide. Apreliminary morphological study and sequence analyses of the internal transcribed spacerregion(ITS) and the large subunit (LSU) of the nuclear rDNA revealed that most of the isolates belonged to the family Herpotrichiellaceae. We have confidently identified 30 isolates representing the following species: Capronia pulcherrima, Cladophialophora emmonsii, Exophiala equina, Exophiala pisciphila, Exophiala radicis, and Phialophora americana. However, we encountered difficulty in assigning 17 cultures to any known species within Chaetothyriales. combining phenotypic and multi-locus phylogenetic analyses based on the ITS, LSU,β-tubulin (tub 2) and translation elongation factor1 - α( tef 1-α) genemarkers, we propose the new genus Aciculomyces in the Herpotrichiellaceae to accommodate the novel species Aciculomyces restrictus. Othernovel species in this family include Cladophialophora denticulata, Cladophialophora heterospora, Cladophialophora irregularis, Exophiala candelabrata, Exophiala dehoogii, Exophiala ramosa, Exophiala verticillata and Phialophora submersa. The new species Cyphellophora spiralis, closely related to Cyphellophora suttonii, is described, and the phylogeny of the genus Anthopsis in the family Cyphellophoraceae is discussed. By utilizing these four markers, we were able to strengthen the phylogeneticre solution and provide more robust taxonomic assessments within the studied group. Our findings indicate that freshwater sediments may serve as are servoir for intriguing black yeasts, which warrant further investigation to address gaps in phylogenetic relationships, particularly within Herpotrichiellaceae.