title: Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus.
abstract: 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.
authors.affiliation: Society for Cave Biology, Tular Cave Laboratory, Oldhamska cesta 8a, 4000 Kranj, Slovenia.
authors.forename: Špela
authors.initials: Š
authors.lastname: Gorički
authors.affiliation: Society for Cave Biology, Tular Cave Laboratory, Oldhamska cesta 8a, 4000 Kranj, Slovenia.|Department of Animal Sciences, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia.|Department of Life Sciences, University of Trieste, Via Licio Giorgieri 5, Trieste 34127, Italy.
authors.forename: David
authors.initials: D
authors.lastname: Stanković
authors.affiliation: Department of Animal Sciences, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domžale, Slovenia.
authors.forename: Aleš
authors.initials: A
authors.lastname: Snoj
authors.affiliation: Institute of Biology, Scientific Research Centre, Slovenian Academy of Sciences and Arts, Novi trg 2, 1000 Ljubljana, Slovenia.
authors.forename: Matjaž
authors.initials: M
authors.lastname: Kuntner
authors.affiliation: Department of Biology, University of Maryland, College Park, MD 20742, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Jeffery
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Peter
authors.initials: P
authors.lastname: Trontelj
authors.affiliation: Biospeleological Society of Montenegro, Cara Lazarja 22, 81000 Podgorica, Montenegro.
authors.forename: Miloš
authors.initials: M
authors.lastname: Pavićević
authors.affiliation: Scientific Research Society Versus, Vitina bb, 88326 Vitina, Bosnia and Herzegovina.
authors.forename: Zlatko
authors.initials: Z
authors.lastname: Grizelj
authors.affiliation: Society for Cave Biology, Tular Cave Laboratory, Oldhamska cesta 8a, 4000 Kranj, Slovenia.|University of Bucharest Research Institute, ICUB, Transdisciplinary Research Centre Landscape - Territory - Information Systems, CeLTIS, 91-93, Splaiul Independentei, 050095 Bucharest, Romania.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Năpăruş-Aljančič
authors.affiliation: Society for Cave Biology, Tular Cave Laboratory, Oldhamska cesta 8a, 4000 Kranj, Slovenia.
authors.forename: Gregor
authors.initials: G
authors.lastname: Aljančič
mesh_terms: D000818:Animals; D059552:Caves; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D011483:Proteidae / Q000145:classification / Q000235:genetics* / Q000502:physiology