The aim of this study was to determine the presence of the hyperparasitic fungus Arthrorhynchus nycteribiae and to analyze its spatio-temporal pattern in the two bat flies (Penicillidia conspicua and P. dufourii) parasitizing on bats. We collected 612 samples of bat flies from 400 bats in 20 caves in the Central Balkans. Hyperparasite was identified based on morphological and molecular analyses of rDNA genes (LSU and SSU). A. nycteribiae was reported for the first time in Bosnia and Herzegovina and Montenegro, and confirmed in Serbia. Of the 20 sites examined, we found A. nycteribiae at 11 sites. The prevalence of A. nycteribiae infection in the bats examined was approximately 17%. Miniopterus schreibersii harbored the highest number of bat flies and was the only bat species hosting the infected bat flies of the species P. conspicua. Our results showed significant differences in infection patterns during the different seasons: the highest prevalence of bat flies with hyperparasitic fungi was found in the summer season (23%) and the lowest in spring (2%). Female bat fly hosts showed a significantly higher prevalence of infection than male bat flies. This study makes an important contribution to the knowledge of the distribution of A. nycteribiae and to the understanding of complex parasite-host relationships in the poorly studied areas of the Central Balkans.

Cave ecosystems represent environmentally constrained habitats that host diverse and highly specialized fungal communities. Many cave-dwelling fungi act as decomposers, transient colonizers, or cave fauna symbionts. During a mycological survey of Sesalačka cave (Serbia), a previously undescribed species was isolated from the skin of a Miniopterus schreibersii. The aim of this study was to characterize this isolate using an integrative taxonomic approach combining morphology, physiology, and multilocus phylogenetics. The fungus was cultured on different media under and its morphophysiological traits were recorded. DNA sequences of ITS, LSU, SSU, and TEF1α were compared with existing Gamsia species and phylogenetic analysis placed the isolate within the Gamsia clade, forming a well-supported lineage the most closely related to G. aggregata, but differing from it by 8–12 base pairs across loci. Distinctive morphological features of this species include obovoid to pyriform polyblastic conidia, hyaline to pale-brown annelloconidia, and reduced conidiophores, clearly separating the species from described congeners. It is psychrotolerant and does not grow at 37 °C, suggesting it is a cave-associated saprobe rather than a mammalian pathogen. This study expands the known diversity of Gamsia species and contributes to the growing evidence that subterranean habitats harbor numerous undescribed fungal kingdom members.