Abstract
Cave environments represent extreme and underexplored ecosystems wherein fungi play a crucial role in nutrient cycling and ecological dynamics. This study provides the first comprehensive assessment of fungal diversity in air samples from caves across Portugal, with six samples from five locations being assessed through culture-dependent and metabarcoding approaches. From the five bat roosts studied, eleven morphologically distinct fungal colonies were isolated, with genera such as Aspergillus, Penicillium, and Chaetomium identified. Concurrently, Oxford Nanopore sequencing of the internal transcribed spacer (ITS) region of fungal rDNA revealed 286 genera, with Aspergillus, Candida, and Calyptella dominating across the sites. Diversity indices and community composition analyses, including Principal Coordinate Analysis (PCoA) and hierarchical clustering, highlighted distinct fungal profiles influenced by site-specific environmental factors and human activity. The data underscores the dual role of fungi in bat roosts as essential decomposers, emphasizing their adaptability to oligotrophic conditions. These findings advance our understanding of subterranean fungal ecology and emphasize the need for targeted conservation efforts to protect cave ecosystems from anthropogenic impacts.