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Soil and sediment host microorganisms are able to survive in extremely resource-limited environments. Therefore, more and more attention is being paid to cave sediments as a reservoir of microbiota. The aim of this study is the speleomycological evaluation of the culturable soil and sediment fungal communities in the Brestovská Cave. To explore the origins of fungi, speleomycological studies were conducted both inside and outside the cave under investigation. Additionally, two incubation temperatures (5 and 24 °C) were used to increase the species spectrum of isolated fungi. To achieve the most accurate species identification, we combined an assessment of morphological characteristics of the isolates with molecular sequencing (ITS, internal transcribed spacer). Twenty different species were found and the most frequent was Penicillium commune, followed by Trichosporiella cerebriformis and Pseudogymnoascus pannorum. To our knowledge, our study has enabled the first identification of fungal species such as Penicillium swiecicki, Cephalotrichum hinnuleum, Cosmpospora berkeleyana, Lecythophora hoffmannii, Ambomucor seriatoinflatus, and Mortierella minutissima in underground sites. Our data showed that the abundance and composition of the fungal community varied between the indoor and outdoor samples and thus from the entrance and less visited sites deeper in the cave.

Despite speleomycological research going back to the 1960s, the biodiversity of many specific groups of micromycetes in underground sites still remains unknown, including keratinolytic and keratinophilic fungi. These fungi are a frequent cause of infections in humans and animals. Since subterranean ecosystems are inhabited by various animals and are a great tourist attraction, the goal of our research was to provide the first report of keratinophilic and keratinolytic fungal species isolated from three caves in Tatra Mts., Slovakia (Brestovská, Demänovská Ľadová and Demänovská Slobody). Speleomycological investigation was carried out inside and outside the explored caves by combining culture-based techniques with genetic and phenotypic identifications. A total of 67 fungal isolates were isolated from 24 samples of soil and sediment using Vanbreuseghem hair bait and identified as 18 different fungal species. The study sites located inside the studied caves displayed much more fungal species (17 species) than outside the underground (3 species), and the highest values of the Shannon diversity index of keratinophilic and keratinolytic fungi were noted for the study sites inside the Demänovská Slobody Cave. Overall, Arthroderma quadrifidum was the most common fungal species in all soil and/or sediment samples. To the best of our knowledge, our research has allowed for the first detection of fungal species such as Arthroderma eboreum, Arthrodermainsingulare, Chrysosporiumeuropae, Chrysosporiumsiglerae, Keratinophytonwagneri, and Penicillium charlesii in underground sites. We also showed that the temperature of soil and sediments was negatively correlated with the number of isolated keratinophilic and keratinolytic fungal species in the investigated caves.