Inferring predator-prey interaction in the subterranean environment: a case study from Dinaric caves.

Predator-prey interactions are among the most important biotic interactions shaping ecological communities and driving the evolution of defensive traits. These interactions and their effects on species received little attention in extreme and remote environments, where possibilities for direct observations and experimental manipulation of the animals are limited. In this paper, we study such type of environment, namely caves of the Dinarides (Europe), combining spatial and phylogenetic methods. We focused on several species of Niphargus amphipods living in phreatic lakes, as some of them use the dorsal spines as putative morphological defensive traits. We predicted that these spines represent a defense strategy against the olm (Proteus anguinus), a top predator species in the subterranean waters. We tested for spatial overlap of the olm and Niphargus species and showed that spined species live in closer proximity to and co-occur more frequently with the olm than non-spined species. Modeling of the evolution of the spines onto Niphargus phylogeny implies coevolution of this trait in the presence of olm. We conclude that these spines likely evolved as defensive traits in a predator-prey arms race. Combining multiple analyses, we provide an example for a methodological framework to assess predator-prey interactions when in-situ or laboratory observations are not possible.

Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus.

Europe's obligate cave-dwelling amphibian Proteus anguinus inhabits subterranean waters of the north-western Balkan Peninsula. Because only fragments of its habitat are accessible to humans, this endangered salamander's exact distribution has been difficult to establish. Here we introduce a quantitative real time polymerase chain reaction-based environmental DNA (eDNA) approach to detect the presence of Proteus using water samples collected from karst springs, wells or caves. In a survey conducted along the southern limit of its known range, we established a likely presence of Proteus at seven new sites, extending its range to Montenegro. Next, using specific molecular probes to discriminate the rare black morph of Proteus from the closely related white morph, we detected its eDNA at five new sites, thus more than doubling the known number of sites. In one of these we found both black and white Proteus eDNA together. This finding suggests that the two morphs may live in contact with each other in the same body of groundwater and that they may be reproductively isolated species. Our results show that the eDNA approach is suitable and efficient in addressing questions in biogeography, evolution, taxonomy and conservation of the cryptic subterranean fauna.

Population-specific behavioral electrosensitivity of the European blind cave salamander, Proteus anguinus.

In nine salamanders from different Slovenian populations of the urodele Proteus anguinus, including three specimens of its 'black' variety, P anguinus parkelj, thresholds of an overt avoidance response to electrical field stimuli were estimated as a function of frequency (continuous sine-waves in water). Thresholds down to 0.3V/cm (ca 100 nA/cm2) and up to 2 mV/cm (670 nA/cm2), at 'best frequencies' of around 30 Hz were found. Sensitivity covered a total frequency range of below 1 Hz, excluding DC, up to 1-2 kHz with up to 40 dB higher thresholds. Thresholds and tuning curves are compared with those of a Proteus population raised in captivity for more than 35 years. The biological significance and the apparently still ongoing evolution of the electrical sense in urodeles, ie in the genus Proteus, are interpreted in terms of comparative sensory physiology and ethological ecology as a result of more recent evolutionary diversification during and since glaciation in the Pleistocene.