Research Article: Surveying Europe’s Only Cave-Dwelling Chordate Species (Proteus anguinus) Using Environmental DNA

Date Published: January 27, 2017

Publisher: Public Library of Science

Author(s): Judit Vörös, Orsolya Márton, Benedikt R. Schmidt, Júlia Tünde Gál, Dušan Jelić, Brian Gratwicke.


In surveillance of subterranean fauna, especially in the case of rare or elusive aquatic species, traditional techniques used for epigean species are often not feasible. We developed a non-invasive survey method based on environmental DNA (eDNA) to detect the presence of the red-listed cave-dwelling amphibian, Proteus anguinus, in the caves of the Dinaric Karst. We tested the method in fifteen caves in Croatia, from which the species was previously recorded or expected to occur. We successfully confirmed the presence of P. anguinus from ten caves and detected the species for the first time in five others. Using a hierarchical occupancy model we compared the availability and detection probability of eDNA of two water sampling methods, filtration and precipitation. The statistical analysis showed that both availability and detection probability depended on the method and estimates for both probabilities were higher using filter samples than for precipitation samples. Combining reliable field and laboratory methods with robust statistical modeling will give the best estimates of species occurrence.

Partial Text

Subterranean ecosystems are among the biomes with the highest number of narrowly distributed and relict taxa [1–3]. This is related to the geographic isolation of subterranean habitats, which facilitate evolutionary drift [4,5]. It is also explained by the lack of Pleistocene glaciations, as these well-buffered habitats were not affected by climatic fluctuations for long periods of time [2,4,6]. Traditionally, compared to terrestrial biomes, subterranean habitats were considered to be less species rich [1]. However, based on the findings of the last few decades and the recently described high incidence of cryptic diversity mostly in invertebrates [4,7–9], this opinion should be revised. While the obligate subterranean fauna is dominated by invertebrates [4,10,11], bony fishes and salamanders were able to successfully colonize these habitats [1,12–14].

Tissue sampling and research on the olms were approved by the Ministry of Environment and Nature protection of Croatia (UP/I-612-07/11-33/0075, 532-08-01-01-01/1-11-02; UP/I-612-07/15-48/119, 517-07-1-1-1-15-04). Krka National Park provided permission for field work.

Even though our target sequence of 64 bp was shorter than the recommended 90–120 bp length [53], its specificity was confirmed using various tests described below. The in silico analysis indicated the specificity of the primers and resulted in no co-amplifying species at three mismatches between the primers and the target sequences. The in vitro specificity of the primers was confirmed as they did not amplify any of the tested co-occuring species (Salamandra salamandra, Bufo bufo, Bombina variegata, Phoxinus lumaireul and Squalius illyricus). The NCBI Blast search resulted in 34 hits with 100–98% identity to Proteus anguinus D-loop sequences.

We successfully developed a non-invasive detection method for the endangered and elusive amphibian species, Proteus anguinus, using environmental DNA. Although DNA metabarcoding is more useful and cost-efficient when detecting several target organisms at the same time [55], because of the need of high specificity and sensitivity to identify P. anguinus DNA from cave water, we opted for a single-species and single-marker detection approach. Previous studies showed that environmental conditions, biomass and production rate of specimens strongly influence detectability of organisms [27,51,56]. Barnes et al [57] reviewed the environmental factors that affect eDNA persistence and showed that abiotic factors, e.g. temperature, ultraviolet radiation and light exposure has negative impact on DNA degradation. Effects of abiotic and biotic factors on detectability in these habitats are unknown because we could not do experiments using our target species and very few studies have investigated eDNA dynamics in caves [58]. Nevertheless, caves inhabited by P. anguinus represent an environment with climate buffered against weather fluctuations, darkness and cold water all year round which may help eDNA to persist for longer than in surface waters.




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