Summary How cave animals adapt to life in darkness is a poorly understood aspect of evolutionary biology [1]. Here we identify a behavioral shift and its morphological basis in Astyanax mexicanus, a teleost with a sighted surface dwelling form (surface fish) and various blind cave dwelling forms (cavefish) [2–4]. Vibration attraction behavior (VAB) is the ability of fish to swim toward the source of a water disturbance in darkness. VAB was typically seen in cavefish, rarely in surface fish, and advantageous for feeding success in the dark. The potential for showing VAB has a genetic component and is linked to the mechanosensory function of the lateral line. VAB was evoked by vibration stimuli peaking at 35 Hz, blocked by lateral line inhibitors, appeared after developmental increases in superficial neuromast (SN) number and size [5–7], and was significantly reduced by bilateral ablation of SN. We conclude that VAB and SN enhancement co-evolved to compensate for loss of vision and help blind cavefish find food in darkness.

SUMMARY1. Various groundwater habitats have exceptionally high levels of endemism caused by strong hydrographical isolation and low dispersal abilities of their inhabitants. More than 10% of macro-stygobiotic species nevertheless occupy relatively large ranges, measuring from some hundred to over 2000 km in length. These species represent a challenge because their distributions disregard hydrographical boundaries, and their means to disperse and maintain long-term gene flow are unknown. 2. Based on mitochondrial and nuclear gene sequences, we examined the phylogeographic structure of six formally recognised stygobiotic species (Niphargus virei, N. rhenorhodanensis, Troglocaris anophthalmus, T. hercegovinensis, Spelaeocaris pretneri, Proteus anguinus) and searched for cryptic lineage diversity in a genus-wide phylogeny of Niphargus. Using treebased criteria as well as comparative divergence measures, we identified cryptic lineages, which may tentatively be equated with cryptic species. 3. Fourteen analysed nominal stygobiotic species with large ranges emerged as highly diversified, splitting into 51 tentative cryptic lineages. The degree of divergence was within the range or larger than the divergence of other related pairs of sister species. A substantial part (94%) of the cryptic lineages had ranges <200 km in length. One half of them were recorded at single sites only. The largest range recorded was that of a cryptic N. virei lineage (700 km), while none of the very large traditional ranges (e.g. Niphargus aquilex -2300 km, N. tauri -1900 km) could be corroborated. 4. These data suggest that small ranges of macro-stygobionts are the rule, and ranges over 200 km are extremely rare. 5. The implications of this result for groundwater biodiversity assessment and conservation include a considerable increase in overall diversity at the regional and continental scale and a strong decrease in faunal similarities among regions, coupled with greater endemism.

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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.

Boris Sket: Ocene starosti za nekatere podzemeljske taksone in živalske linije na Dinarskem krasu Z uporabo primerjalnega filogeografskega pristopa in neodvisnih molekularnih ur smo predlagali časovni potek evolucije troglobiontov Dinarskega krasa, ki velja za sorazmerno veliko število taksonov. Zdi se, da ključni dogodki pripadajo dvema obdobjema. (1) Glavne razdelitve znotraj holodinarskih taksonov so iz obdobje srednjega miocena. Predstavljajo zgornji potencialni časovni limit za naselitev jam. (2) Regionalna diferenciacija, vključno s speciacijo, ki je lahko vsaj deloma povezana s podzemeljsko fazo, naj bi se zgodila med zgodnjim in srednjim pleistocenom. Ocenjujemo, da se je začela invazija večine proučevanih živalskih linij v podzemlje Dinarskega krasa v obdobju med dvema in petimi milijoni let. Ključne besede: podzemlje, molekularna ura, molekularna filogenija, filogeografija, Dinarski kras. Using a comparative phylogeographic approach and different independent molecular clocks we propose a timescale for the evolution of troglobionts in the Dinaric Karst that is relatively consistent over a wide taxonomic range. Keystone events seem to belong to two age classes. (1) Major splits within holodinaric taxa are from the mid-Miocene. They present the potential upper limit for the age of cave invasions. (2) Regional differentiation, including speciation, which can at least in part be associated with a subterranean phase, took place from early Pliocene to mid-Pleistocene. we suggest two to five million years as the time when most of the analyzed lineages started invading the Dinaric Karst underground.