Špela GORIČKI et al.: Searching for the black Proteus with the help of eDNA / »SOS PROTEUS« - FIELD NOTE
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Searching for the black Proteus with the help of eDNA
Iskanje črnega močerila s pomočjo okoljske DNA
Špela GORIČKI1, David STANKOVIC1'2, Magdalena ALJANČIČ1, Aleš SNOJ3, Matjaž KUNTNER4, Tajda GREDAR1, Luka VODNIK1, Gregor ALJANČIČ1 1Tular Cave Laboratory, Society for Cave Biology, Oldhamska cesta 8a, SI-4000 Kranj, Slovenia E-mail: goricki.spela@gmail.com 2Department of Life Sciences, University of Trieste, Via L. Giorgeri 5, 34127 Trieste, Italy 3Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, SI-1230 Domžale, Slovenia 4Institute of Biology, Scientific Research Centre of the Slovenian Academy of Sciences and Arts, Novi trg 2, SI-1000 Ljubljana, Slovenia
Proteus is a specialized, blind and depigmented subterranean salamander inhabiting groundwaters of the Dinaric Karst. In Bela krajina, Slovenia, a unique black population is known from only four springs in the proximity of a closely related white, troglomorphic population (Aljančič et al. 1986, Sket 1997, Ivanovič 2012, Škedelj Petrič et al. 2014). In the face of the scarcity of the distributional information, potentially very limited distribution and impending threat due to heavy fertilizer load, establishing the detailed distribution of these populations was the focus of our work. Because its subterranean habitat is inaccessible, several techniques were employed at springs, including traps and visual observation in the dark using night goggles, but the survey predominantly aimed to introduce a novel approach - forensic analysis of traces of Proteus DNA released in water (environmental DNA or eDNA).
To detect Proteus eDNA in samples of spring water and to discriminate between the black and white populations, we developed specific TaqMan probes and PCR primers, homologous to variable regions of Proteus mtDNA (Gorički 2006, Gorički & Trontelj 2006). Of the 19 spring water samples collected and filtered, six were positive for Proteus DNA, five of which were also positive for black Proteus. All five are new localities, where Proteus has neither previously nor during the survey been sighted or
otherwise detected. Along with detection of Proteus eDNA in spring and cave water in southern Herzegovina and Montenegro (Aljančič et al. 2014, Stankovic et al. 2016), this survey represents the first successful application of the eDNA approach in detection of a subterranean organism. The methodology developed and tested herein is the only quick and accurate method available for determining its presence and is fully appropriate for monitoring the distribution of this rare and endangered stygobiont in Bela krajina.
Acknowledgments
Andrej Hudoklin^ Nataša Toplak, Valerija Zakšek, Ida Djurdjevic, Žiga Fišer, Tjaša Lokovšek, Julija Benko Miller and Peter Trontelj are thanked for their help and support during the field survey or laboratory work. The research was performed during the project »With Proteus we share dependence on groundwater«, financially supported by the EEA Financial Mechanism and the Norwegian Financial Mechanism 2009-2014 (SI03-EEA2013/MP-17). eDNA analyses were carried-out at the facilities of the Department of Animal Science, Biotechnical Faculty, University of Ljubljana and at the Institute of Biology ZRC SAZU.
References
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