David STANKOVIC et al.: Application of environmental DNA for detection of Proteus/ »SOS PROTEUS« - FIELD NOTE 55
Application of environmental DNA for detection of Proteus
Uporaba okoljske DNA pri iskanju človeške ribice
David STANKOVIC1'2'3, Špela GORIČKI1, Magdalena ALJANČIČ1, Aleš SNOJ3, Matjaž KUNTNER4, Gregor ALJANČIČ1 1Tular Cave Laboratory, Society for Cave Biology, Oldhamska cesta 8a, SI-4000 Kranj, Slovenia E-mail: david.stankovic@guest.arnes.si 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
The rare and highly endangered olm (Proteus anguinus) is the only obligate subterranean vertebrate of Europe. It inhabits subterranean waters of the Dinaric Karst in the north-western Balkan Peninsula (Sket 1997). As only small fragments of its subterranean habitat are accessible, the basic but important question of its exact distribution has been difficult to address. Its presence can only rarely be confirmed by classical survey methods such as trapping and visual encounters. For this reason, alternative methods are required to test for its presence in new potential localities. Detection of species-specific DNA released into the aquatic environment (environmental DNA or eDNA) has already been shown as an appropriate approach to monitoring the distribution of vertebrates from surface waters. In aquatic environments rapid diffusion of eDNA from its source means that the presence of a specific animal could be detected anywhere within such water body and not just at its point of origin, thus making this approach particularly useful for those species that are difficult to detect using conventional methods or are very rare (Rees et al. 2014). We developed an adjusted eDNA approach for filtering water samples from karst springs, wells and caves and provide two specific primer sets that can be used to amplify short conserved fragments of P. anguinus mitochondrial DNA (Gorički 2006, Gorički & Trontelj 2006) by real-time PCR based on SYBR chemistry. The specificity of
the assay was first tested on trout, crested newt and human DNA. In controlled conditions at the Tular Cave Laboratory the minimum density at which its DNA could still be detected corresponded to one animal per 256 m3 of standing water, when sampling 20 L of water. The method, tested at three Slovenian field test sites occupied by different lineages of P. anguinus, was 100% effective. Subsequently, a pilot survey of its distribution was conducted along the southern limit of its known range in Herzegovina and Montenegro. Using DNA-based identification, we unequivocally established the presence of P. anguinus at four sites, and found its likely traces at additional eight sites - most of them new localities for this species. Even though the SYBR chemistry-coupled real-time PCR approach was shown to be very successful and time-efficient method for detection and monitoring of P. anguinus that can be applied with fidelity anywhere within its known range of occurrence, detectability can still be increased using TaqMan probes (Gorički et al. 2016).
Acknowledgments
Zlatko Griželj, Miloš Pavičevič, Ivan Bebek, Jasminko Mulaomerovič, William Jeffery, Andrej Renčelj and Ana Lazar are thanked for their help and support during the field survey or laboratory work. The research was performed during the project »A survey of the distribution of Proteus anguinus by environmental DNA sampling« and was cofinanced by the Critical Ecosystem Partnership Fund, BirdLife International and DOPPS - BirdLife Slovenia.
References
Gorički Š. (2006): Filogeografska in morfološka analiza populacij močerila (Proteus anguinus). Ph.D. Dissertation, University of Ljubljana, 76 pp.
Gorički Š., Stankovič D., Aljančič M., Snoj A., Kuntner M., Gredar T., Vodnik L., Aljančič G. (2016): Searching for black Proteus with the help of eDNA. Nat. Slo. 18(1): 57-58.
Gorički Š., Trontelj P. (2006): Structure and evolution of the mitochondrial control region and flanking sequences in the European cave salamander Proteus anguinus. Gene 378: 31-41.
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56 David STANKOVIC et al.: Application of environmental DNA for detection of Proteus / »SOS PROTEUS« - FIELD NOTE
Rees H.C., Maddison B.C., Middleditch D.J., Patmore H.R.M., Gough K.C. (2014): The detection of aquatic animal species using environmental DNA - a review of eDNA as a survey tool in ecology. J. Appl. Ecol. 51: 1450-1459.
Sket B. (1997): Distribution of Proteus (Amphibia: Urodela: Proteidae) and its possible explanation. J. Biogeogr. 24: 263-280.
NATURA SLOVENIAE 18(1): 55-56