On karst, which represents 40% of the surface in Slovenia and is an important source of drinking water, most of the surface water disappears underground. Using high‐throughput rRNA gene amplicon sequencing (bacterial and archaeal 16S rRNA, eukaryotic 18S rRNA and fungal ITS region), we analysed the microbial community of various karst waters in Slovenia and the effect of total DNA pre-amplification on the detected microbial diversity and community composition. There were no major differences between the waters of the various karst caves. The archaeal community was dominated by the orders Woesearchaeales, Nitrososphaerales and Nitrosopumilales. The genera Limnohabitans, Rhodoferax, Flavobacterium and Pseudomonas represented an important proportion of the bacterial communities. Identified fungal genera represented important proportions in only one or two samples, while in others they were absent or much rarer. These were the genera Stereum, Betamyces, Trametes, Rhizophydium and Mortierella. Among other eukaryotic groups, ciliates (Ciliophora) and golden-brown algae (Chrysophyceae) were common. We also detected arthropods (Arthropoda), namely the Cyclopoida order. When pre-amplification of total DNA was used, microbial richness was lower. The same applies to the diversity, except in the case of the eukaryotic community, where diversity was higher in the samples with pre-amplified DNA. Statistically significant differences were observed only in species richness and diversity of the fungal community and diversity of the archaeal community (p ⡤ 0.05).
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