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Black yeasts comprise a group of Ascomycota of the order Chaetothyriales with highly variable morphology, a great diversity of ecological niches and life cycles. 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 in various sampling sites in Spain, we obtained 47 isolates of black yeasts by using potato dextrose agar supplemented with cycloheximide. A preliminary morphological study and sequence analyses of the internal transcribed spacer region (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, ß-tubulin (tub2) and translation elongation factor 1-a (tef1-a) gene markers, we propose the new genus Aciculomyces in the Herpotrichiellaceae to accommodate the novel species Aciculomyces restrictus. Other novel 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 phylogenetic resolution and provide more robust taxonomic assessments within the studied group. Our findings indicate that freshwater sediments may serve as a reservoir for intriguing black yeasts, which warrant further investigation to address gaps in phylogenetic relationships, particularly within Herpotrichiellaceae. [ABSTRACT FROM AUTHOR]