Microbiome analysis of groundwaters in the Slovenian Dinaric Karst, a recognized biodiversity hotspot

Groundwater is the most important source of drinking water supply, irrigation, and industrial use, as well as a habitat for a diverse range of (micro)organisms. Some of the first studies of underground environments were carried out in the Dinaric Karst region of Slovenia, which harbors one of the highest subterranean biodiversities in the world. Despite their ecological importance, groundwater microbial communities remain underexplored. In this study, we conducted a comprehensive microbiome analysis of karst groundwater, investigating the abundance and diversity of viruses, archaea, bacteria, fungi, and protozoa. We detected a high diversity of both prokaryote- and eukaryote-infecting viruses, indicating the connection of subterranean environments with surface waters. In the archaeal community, seven different phyla were identified, dominated by members of "Candidatus Woesearchaeota" and Nitrososphaerota (syn. Thermoproteota). In the bacterial community of majority of the samples, the Pseudomonadota was the most abundant phylum, followed by Bacteroidota, Actinomycetota, and Verrucomicrobiota. Fungal abundance and diversity differed greatly between cave water samples, with the Ascomycota families Cladosporiaceae, Didymellaceae, Aspergillaceae and saprotrophic Basidiomycota detected in all samples. Besides fungi, microscopic eukaryotic diversity consisted of ciliates, copepods and golden-brown algae present in all samples. Differences in microbial communities between the sampling sites highlight the need for tailored site-specific groundwater conservation and monitoring strategies. Moreover, the identification of microbes associated with anthropogenic activities underscores their potential as source-specific indicators of groundwater pollution. As the first holistic overview of microbial diversity of subterranean water, the study establishes a valuable baseline for future research and conservation of these unique karst ecosystems.