Caves, characterized by enclosed, unique, and stable environments, provide favorable conditions for fungal growth and harbor high levels of fungal diversity. Although Yunnan Province is home to more than 200 caves, studies on cave-associated fungi in this region remain scarce. To address this knowledge gap, we surveyed six accessible karst caves in the eastern, western, and southeastern Yunnan. A total of 34 fungal strains representing 17 species were isolated. Morphological characteristics and multi-gene phylogenetic analyses revealed taxonomic diversity among culturable fungi isolated from the sampled Yunnan caves, including numerous previously undescribed taxa. This study introduces one new genus ( Neoachaetomiella ), 12 novel species ( Aspergillus dehongensis , Cosmospora cavernicola , Neoachaetomiella cavernicola , Pithoascus cavernicola , Trichoderma dehongense , T. hongheense , T. mangshiense , T. neogongcheniae , T. neopyramidale , T. nujiangense , T. yingjiangense , and Trichocladium cavernicola ), and reports five species ( Aspergillus baeticus , Chaetomium subaffine , Daldinia eschscholtzii , Mucor phayaoensis , and Trichoderma obovatum ) as new records for caves in Yunnan, China. Detailed descriptions, illustrations, and phylogenetic analyses of all 17 taxa are provided. Additionally, a global checklist of cave-associated fungi is presented.
The phylogenetic affiliations of anaerobic gut fungi ( Neocallimastigomycota ) are typically evaluated using single-gene markers. However, this approach often fails to resolve relationships between closely related lineages. To address this issue and identify alternative markers, we created a curated database comprising the complete ribosomal operon sequences of 156 isolates, representing 20 of the 22 recognized genera and two new genus-level clades. Using long-read sequencing, we obtained ~9 kbp operon sequences and developed a robust analysis pipeline. Incorporating both coding genes and non-coding regions (excluding IGS1) improved phylogenetic resolution. This phylogenetic approach successfully resolved the Cyllamyces and Caecomyces clades (hard-to-distinguish genetically), as well as seven analysed Piromyces species. We also scanned the operon for markers that are suitable for short-read sequencing platforms, with the aim of enhancing biodiversity and phylogenetic studies. Notably, the ETS1 genetic region also enabled the distinction between these lineages, indicating its phylogenetic value within the ribosomal operon. The resulting database is a valuable resource for expanding and strengthening phylogenetic frameworks.
