Five New Tolypocladium Species from Lichen Thalli in the Tibet Autonomous Region with Antifungal and Cytotoxic Activities

Endolichenic fungi associated with lichen thalli remain insufficiently documented. During a survey of lichens from the Tibet Autonomous Region of China, twelve Tolypocladium isolates were recovered from eight lichen species. Preliminary ITS screening indicated low sequence similarity to previously described species, prompting detailed taxonomic investigation. Multilocus phylogenetic analyses based on ITS, LSU, rpb2 and tef1-α sequences resolved these isolates into five well-supported, genealogically distinct lineages representing five novel species: Tolypocladium crinitum sp. nov., T. ellipsoideum sp. nov., T. orientalis sp. nov., T. parapustulatum sp. nov. and T. xizangense sp. nov. Species delimitation was supported by consistent monophyly across loci, numerous fixed nucleotide substitutions and diagnostic morphological features. In preliminary bioactivity assays, selected isolates showed at least 50% inhibition of two plant pathogens and different levels of cytotoxicity against H460 and A549 cells at the tested concentrations. These results are preliminary, as most isolates were tested at a single concentration, and IC50 values and effects on normal cells were not determined. This study increases the known diversity of Tolypocladium from lichen habitats and provides baseline taxonomic and biological data for future work on endolichenic fungi from alpine environments in China.