Some Like It Rock ‘N’ Cold: Speleomycology of Ravništarka Cave (Serbia)

Caves and other subterranean ecosystems are characterized by stable, low temperatures, high humidity, and limited nutrient input, creating unique environments for extremophilic microorganisms. Among them, fungi play key roles in organic matter degradation, mineral interactions, and biogeochemical cycling, yet the diversity and adaptations of cold-adapted fungi in cave habitats remain insufficiently explored. This study investigated psychrophilic and psychrotolerant fungi inhabiting the stone surfaces of Ravništarka Cave in Eastern Serbia. Biofilm samples were collected from nine sites and analyzed using culture-based isolation on both nutrient-rich and diluted media, followed by incubation at 10 °C, 25 °C, and 37 °C. Fungal identification combined morphological characteristics with molecular analyses of the ITS region and <i>BenA</i> gene, while ecological roles were assigned using FUNGuild. A total of 41 fungal species were documented, spanning Ascomycota (53.1%), Basidiomycota (43.7%), and Mortierellomycota (3.1%) phyla. The genus <i>Penicillium</i> exhibited the greatest species richness, with 14 taxa documented, including <i>P. chrysogenum</i>, <i>P. glandicola</i>, and <i>P. solitum</i>, all previously associated with cold or oligotrophic environments. The psychrotolerant species <i>Mortierella alpina</i> was the only representative of Mortierellomycota. Ecological guild assignment revealed fungi functioning at different trophic levels, highlighting their multifunctional ecological roles in extreme subterranean habitats.