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It should be noted, however, that while the maximum span of the black Proteus range to the east, north and south was established in the present study, the extent of its distribution to the west remains unknown. This conclusion is consistent with earlier predictions, when only one 9 or two sites 10 were known. The finding is strongly supported by an existing intermittent hydrogeological connection 17 with Otovski Breg, a nearby site occupied by the white Proteus no.

Rare cases of subterranean syntopic occurrence of closely related lineages are valuable for the study of the poorly understood mechanisms of speciation and differentiation within the subterranean realm e. The distribution of the black and white Proteus eDNA in Bela Krajina is in agreement with the existence of a potential reproductive barrier between these two lineages, at least regarding female mating preferences. Similarly, if inter-lineage mating regularly occurred in the other direction, we would expect black Proteus eDNA to appear in the sample at Otovski Breg, a site which was found to harbour only white Proteus eDNA.

Significantly, despite a low degree of sequence divergence between the two populations observed in the mitochondrial control region 14 , 15 , 45 , none of the comparative studies to date have detected any signs of their interbreeding, e. In combination with these observations, eDNA data suggest that the two populations may represent independent species, but additional analyses are needed to resolve the taxonomic status of the present as well as of other apparently monophyletic groups of Proteus.

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In conclusion, we have demonstrated that the qPCR-based eDNA method can be utilised for a rapid detection of a rare subterranean species inhabiting karst groundwater. Furthermore, as suggested by the results of the Bela Krajina survey in particular, the qPCR-based approach enables us to assess the relative abundance of Proteus eDNA in groundwater over a small spatial scale. Finally, we have shown that the eDNA approach can also be helpful in identifying potentially sympatric populations in the cryptic subterranean environment and therefore can be useful in the study of evolutionary history and taxonomy of subterranean taxa.

Development of our methodology to detect traces of Proteus eDNA in water involved the following steps see Supplementary Information : 1 development of specific oligonucleotides for eDNA detection with qPCR, 2 testing the specificity of the oligonucleotides on tissue samples, 3 testing the lower detection limit of the method in laboratory conditions and 4 testing the performance of the method in nature, at three verified sites in Slovenia SYBR qPCR only. Samples were collected in brand-new 5- or L plastic canisters and stored in a dark cool room until filtration.

Water samples were filtered through sterile 0. Up to four filter membranes were used per sample, depending on the degree of clogging by sediment and other particles in the water. For each template dilution, both target fragments were amplified in triplicate using separate plates for each primer pair.


A single well qPCR plate contained between four to 12 samples, so that individual samples were separated by at least one empty row and column. In addition, each plate included six negative controls double distilled and tap water from outside of Proteus range and two positive controls tissue DNA and eDNA extracted from the laboratory water tanks , for a comparison of melting curves. Samples were scored positive for Proteus eDNA if at least two out of three replicate wells of at least one combination dilution-primer pair were positive by qPCR see Fig. If only one of the three wells was positive by qPCR, the sample was re-analysed using the same primer combination.

Again, if at least two of the three replicate wells were positive in the re-run, the sample was scored positive for Proteus eDNA. If only one of the three wells was positive in the re-run, the sample was considered plausible to contain Proteus eDNA.

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If all wells were negative in the re-run, the presence of Proteus eDNA in the sample was considered uncertain. Finally, samples with all negative wells in the first run were scored negative for Proteus eDNA and were not analysed further. Three primer-probe combinations were designed to first test for the presence of Proteus eDNA in each sample and subsequently to determine whether the eDNA was characteristic for the black or the white Proteus see Supplementary Information for details. As all three probes were FAM-labelled, the three assays were performed in separate reactions.

Each probe-template combination was run in a triplicate. Individual field samples were separated by at least two empty rows and columns. If a specific product was observed in at least three out of six reactions containing either undiluted or diluted sample, the sample was scored positive for the presence of respective eDNA see Fig. On the other hand, samples with all negative wells were scored negative and were not analysed further. If at least two out of three replicates were positive in the repeat, the presence of the respective eDNA marker in the sample was considered plausible.

If none or one of the replicates were positive in the re-run, the presence of the respective eDNA marker in the sample was considered uncertain. The minimal density of Proteus in water at which its eDNA can still be detected i. The authorities of all political entities visited were informed of the purpose of our fieldwork and granted its approval. Samples were collected in three rounds: March 31 — April 8, , April 27 — May 10, and June 19—20, A total of 38 sites were visited karst springs, caves and wells , of which 23 samples were analysed see Supplementary Table S1.

Subterranean Fishes of the World

Because of sample transportation delays during the first field trip, several sites were visited twice. Sampling in Montenegro was also organised in three rounds: October 3—4, , November 16—22, and June 5—10, In total, 15 localities were visited, 11 of which were analysed see Supplementary Table S1. Between July 20—29, , 36 sites were visited, 13 of which were sampled karst springs and a cave; see Supplementary Table S1. After some precipitation, on November 2, , five additional springs were sampled.

Sample collection. Samples were taken at low to medium water levels and during the lowest to average annual discharge rates of individual springs. Field samples were collected and filtered as randomly as possible, i. The investigators performing the qPCR assays were blinded with respect to detailed hydrogeological information pertaining to individual samples.

Rigorous controls for preventing and monitoring contamination were employed throughout the entire procedure see Supplementary Information for details. The Supplement also provides the methods for GIS database construction and mapping of hydrogeological and geological data and information on Proteus sites. Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus.

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Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Sket, B. Distribution of Proteus Amphibia: Urodela: Proteidae and its possible explanation. Distribution of the Olm Proteus anguinus , Laur. Bressi, N. Underground and unknown: updated distribution, ecological notes and conservation guidelines on the Olm Proteus anguinus anguinus in Italy Amphibia, Proteidae. Modena 71 , 55—59 Salvaging the washed-out Proteus.

Natura Sloveniae 18 , 65—67 Bulog, B.

An account of the subterranean (hypogean) fishes with a bibliography from 1436.

Trace element concentrations in the tissues of Proteus anguinus Amphibia, Caudata and the surrounding environment. Water Air Soil Pollut.

Subterranean Fishes of the World - Graham Proudlove

Hudoklin, A. Are we guaranteeing the favourable status of the Proteus anguinus in the Natura network in Slovenia? A black, non-troglomorphic amphibian from the karst of Slovenia: Proteus anguinus parkelj n. Urodela: Proteidae. Grillitsch, H.

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Herpetozoa 7 , — Arntzen, J. Speak of the devil: the taxonomic status of Proteus anguinus parkelj revisited Caudata: Proteidae. Herpetozoa 8 , — Morphometric analysis of black and white European cave salamanders , Proteus anguinus. Structure and evolution of the mitochondrial control region and flanking sequences in the European cave salamander Proteus anguinus. Gene , 31—41 Trontelj, P. A molecular test for cryptic diversity in ground water: how large are the ranges of macro-stygobionts?

N-vestnik 34 , 1—2 Acta Carsol. Novak, D. Guzik, M. Evidence for population fragmentation within a subterranean aquatic habitat in the Western Australian desert. Heredity , — Niemiller, M.

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Effects of climatic and geological processes during the pleistocene on the evolutionary history of the northern cavefish, Amblyopsis spelaea Teleostei: Amblyopsidae. Evolution 67 , — Asmyhr, M.

Fine-scale genetics of subterranean syncarids. Rees, H. Ficetola, G.