Monitoring of Proteus distribution by environmental DNA sampling


 eDNA Proteus

(Photo: Gregor Aljančič)


Tular Cave Laboratory (Society for Cave Biology) & partners developed a research tool to overcome the critical problem when determining Proteus occurrence – inaccessibility of its subterranean habitat. This highly efficient, non-invasive and innovative molecular genetic tool helps to detect traces of Proteus environmental DNA (eDNA) by filtering water samples from groundwater (karst springs, wells or caves). Coupled with GIS analysis, the method was implemented to test for the presence of Proteus in new potential localities, regardless of whether or not the animal has been observed there, as well as to verify selected old data on its distribution.

The results of this study offered a complete picture of Proteus distribution within the study area and its most vulnerable parts. Findings were published on 27th March2017 in the journal Scientific Reports: "Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus" (Read open access article).


Main results of the study (read more in PR announcement)

  • The most extensive survey of Proteus distribution ever conducted: 56 karst springs, wells or caves in Slovenia, Bosnia & Herzegovina and Montenegro were sampled and analysed (95 visited).
  • A likely presence of Proteus confirmed at seven new sites along the southern limit of its known range.
  • First significant extension of Proteus range after 1929: the presence of Proteus documented for the first time in Montenegro.
  • A highly specific method was developed to detect the eDNA of the black Proteus morph, which occurs only on 30 km2 in Southeastern Slovenia.
  • A major increase in the number of known black Proteus sites since its discovery in 1986: the black Proteus was detected at five new sites, which more than doubled the number of sites of this extremely vulnerable population – basis for an efficient conservation management.
  • The eDNA of both black and white Proteus was discovered together in one of the springs, which represents the first evidence that these two populations may be in contact with each other. 


The study was organized and conducted by the Tular Cave Laboratory (Society for Cave Biology, Slovenia), in partnership with the Department of Animal Science (Biotechnical Faculty, University of Ljubljana, Slovenia), the Evolutionary Zoology Laboratory (Institute of Biology at the Scientific Research Centre of the Slovenian Academy of Sciences and Arts, Slovenia), the Jeffery Lab (Department of Biology, University of Maryland, USA) and the Department of Life Sciences (University of Trieste, Italy), field work was supported by the Biospeleological Society of Montenegro, the Scientific Research Society Versus (Bosnia & Herzegovina) and Center for Karst and Speleology (Bosnia & Herzegovina).

The study was part of the project “A survey of the distribution of Proteus anguinus by environmental DNA sampling”, co-financed by the Critical Ecosystem Partnership Fund, BirdLife International and DOPPS (CEPF GEM No. 45), and the project “With Proteus we share dependence on groundwater”, financed by the EEA Financial Mechanism and the Norwegian Financial Mechanism 2009-2014 (SI03-EEA2013/MP-17).



Monitoring of Proteus anguinus by environmental DNA sampling

Project cofinanced by Critical Ecosystem Partnership Fund, BirdLife International and DOPPS, 2013–2014

Final project completion report (PDF, 3.89MB)


Finding and protecting hidden species: environmental DNA

Amongst cold, dark caves of dripping stalagtites, there is a diverse community of highly-adapted subterranean species. A scientific breakthrough is helping ensure they are monitored and protected in the future. (Read more)

CEPF Top articles: Cave-Dwelling Salamander Sheds Light on Drinking Water Resources

BirdLife International: Scientific breakthrough reveals evidence of ‘human fish’ locked away in cave system




Hutovo Blato photo Gregor Aljancic a

Tular logo Rodica M cepf PARTNERJI1                                                    cepf logos1


Conserving the Biodiversity of the Balkans: A Long-Term Strategic Vision for Europe’s natural jewel

Proteus is the flagship species of the Dinaric karst groundwater and its endangered cave fauna. As such, Proteus represents our joint aspiration to raise scientific and public attention needed to preserve them in the future!

Take a visual journey through the Mediterranean Basin Biodiversity Hotspot - explore its StoryMap!

"This network is not just 93 civil society organisations supported by CEPF. It is also 93 organisations who work together more and more as a team to preserve the amazing biodiversity of this region. Despite facing a lot of difficulties today and in the future, civil society organisations are finding inspiration in the work of their fellow CEPF partners from other countries in the hotspot." , said Pierre Carret, Grant Director, CEPF (read more).


Vulnerability of Proteus and groundwater