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Europe’s obligate cave-dwelling amphibian  Proteus anguinus  inhabits subterranean waters of the north-western Balkan Peninsula. Because only fragments of its habitat are accessible to humans, this endangered salamander’s exact distribution has been difficult to establish. Here we introduce a quantitative real time polymerase chain reaction-based environmental DNA (eDNA) approach to detect the presence of  Proteus  using water samples collected from karst springs, wells or caves. In a survey conducted along the southern limit of its known range, we established a likely presence of  Proteus  at seven new sites, extending its range to Montenegro. Next, using specific molecular probes to discriminate the rare black morph of  Proteus  from the closely related white morph, we detected its eDNA at five new sites, thus more than doubling the known number of sites. In one of these we found both black and white  Proteus  eDNA together. This finding suggests that the two morphs may live in contact with each other in the same body of groundwater and that they may be reproductively isolated species. Our results show that the eDNA approach is suitable and efficient in addressing questions in biogeography, evolution, taxonomy and conservation of the cryptic subterranean fauna.

Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus