Multiple transitions between realms shape relict lineages of Proteus cave salamanders.

In comparison to biodiversity on Earth's surface, subterranean biodiversity has largely remained concealed. The olm (Proteus anguinus) is one of the most enigmatic extant cave inhabitants, and until now little was known regarding its genetic structure and evolutionary history. Olms inhabit subterranean waters throughout the Dinaric Karst of the western Balkans, with a seemingly uniform phenotypic appearance of cave-specialized traits: an elongate body, snout and limbs, degenerated eyes and loss of pigmentation ("white olm"). Only a single small region in southeastern Slovenia harbours olms with a phenotype typical of surface animals: pigmented skin, eyes, a blunt snout and short limbs ("black olm"). We used a combination of mitochondrial DNA and genome-wide single nucleotide polymorphism data to investigate the molecular diversity, evolutionary history and biogeography of olms along the Dinaric Karst. We found nine deeply divergent species-level lineages that separated between 17 and 4 million years ago, while molecular diversity within lineages was low. We detected no signal of recent admixture between lineages and only limited historical gene flow. Biogeographically, the contemporaneous distribution of lineages mostly mirrors hydrologically separated subterranean environments, while the historical separation of olm lineages follows microtectonic and climatic changes in the area. The reconstructed phylogeny suggests at least four independent transitions to the cave phenotype. Two of the species-level lineages have miniscule ranges and may represent Europe's rarest amphibians. Their rarity and the decline in other lineages call for protection of their subterranean habitats.

Toward the massive genome of Proteus anguinus-illuminating longevity, regeneration, convergent evolution, and metabolic disorders.

Deciphering the genetic code of organisms with unusual phenotypes can help answer fundamental biological questions and provide insight into mechanisms relevant to human biomedical research. The cave salamander Proteus anguinus (Urodela: Proteidae), also known as the olm, is an example of a species with unique morphological and physiological adaptations to its subterranean environment, including regenerative abilities, resistance to prolonged starvation, and a life span of more than 100 years. However, the structure and sequence of the olm genome is still largely unknown owing to its enormous size, estimated at nearly 50 gigabases. An international Proteus Genome Research Consortium has been formed to decipher the olm genome. This perspective provides the scientific and biomedical rationale for exploring the olm genome and outlines potential outcomes, challenges, and methodological approaches required to analyze and annotate the genome of this unique amphibian.

Vitellogenin in the European cave salamander, Proteus anguinus: Its characterization and dynamics in a captive female as a basis for non-destructive sex identification.

Vitellogenin (Vtg) is a precursor protein of egg yolk proteins in oviparous and ovoviviparous vertebrates. Except in a case of exposure to estrogenic endocrine disruptors, Vtg is a female-specific protein and could be used as a molecular marker for sex identification. This would be especially useful in the case of the endangered European cave salamander Proteus anguinus in which sexes are indistinguishable according to external morphology, which hinders the establishment of a successful captive breeding program. Here we describe the identification, partial characterization, and purification of Vtg from P. anguinus. Vtg was identified in the plasma of a vitellogenic proteus female with visible oocytes. The identification of this protein was accomplished by mass spectrometry analysis. Two-dimensional gel electrophoresis revealed proteus Vtg as a mix of 190 kDa isoforms with isoelectric points in the pH range 5.3-6.0. Vtg was purified from proteus blood by gel filtration followed by anion-exchange chromatography. Using specific staining of SDS-PAGE gels, the Vtg was found to be phosphorylated and lipidated. Unlike the case in some other aquatic vertebrates, in P. anguinus, Vtg was not present in detectable amounts in cutaneous mucus. Degradation of oocytes in the captive vitellogenic female was accompanied by simultaneous decrease of Vtg concentration. Over a period of 10 months, the concentration of Vtg dropped from maximal to sub-detectable. Our results show that Vtg is a promising molecular marker for sex identification and ovary maturation in P. anguinus, which could contribute to the development of a viable program for captive reproduction of this unique species.

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.

Structure and evolution of the mitochondrial control region and flanking sequences in the European cave salamander Proteus anguinus.

The European cave salamander Proteus anguinus Laurenti 1768 is one of the best-known subterranean animals, yet its evolutionary history and systematic relationships remain enigmatic. This is the first comprehensive study on molecular evolution within the taxon, using an mtDNA segment containing the control region (CR) and adjacent sequences. Two to seven tandem repeats of 24-32 bp were found in the intergenic spacer region (VNTR1), and three, four or six repeats, 59-77 bp each, in the 3' end of the CR (VNTR2). Different molecular mechanisms account for VNTR2 formation in different lineages of Proteus. The overall CR variation was lower than that of the spacer region, the 3' end of the cytb gene, or the tRNA genes. Individual genes and the concatenated non-repetitive sequences produced similar, well resolved maximum likelihood, Bayesian inference and parsimony trees. The numbers of repeat elements as well as the genealogy of the VNTR2 repeat units were mostly inconsistent with the groupings of the non-repetitive sequences. Different degrees of repeat array homogenization were detected in all major groups. Orthology was established for the first and the second VNTR2 elements of some populations. These two copies may therefore be used for analyses at the population level. The pattern of CR sequence variation points to strong genetic isolation of hydrographically separated populations. Genetic separation of the major groups of populations is incongruent with the current division into subspecies.