Subterranean biodiversity in Europe is spectacularly rich, with the Western Balkans being home to about 400 cave species, representing the highest number of species per area worldwide []. Nonetheless, cave fishes, which are the most commonly found vertebrates in underground habitats [], have not been described from Europe so far []. Here, we report the first European record of a cave fish population, a loach of the genus Barbatula ( Figure 1 ), found in the Danube–Aach system, an underground karst water system in Southern Germany []. The fish exhibit traits typically observed in organisms adapted to subterranean life including reduced eyes and pale body coloration []. The newly discovered population also represents globally the northernmost cave fish found so far. The geological history of the region implies that the Danube–Aach system was colonized post-glacially. A recent origin of the cave fish is supported by genetic analyses, because the subterranean population shares COI gene haplotypes with adjacent surface stone loach (Barbatula barbatula) populations ( Figure 1 D). Nonetheless, population genetic analyses based on microsatellites indicated that cave fish are genetically isolated from populations in surface habitats ( Figure 1 E) and exhibit reduced genetic variability. Hence, the newly discovered European cave loaches do not represent individuals displaced from surface populations, but they follow a unique evolutionary trajectory towards cave life.

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4 Hötzl H. Origin of the Danube-Aach system. 6 Abel, T., Hinderer, M., and Sauter, M. (2002). Karst genesis of the Swabian Alb, South Germany, since the Pliocene (Climate changes - The Karst Record II, Czech Republic, Poland and Slovakia, July 27-August 9, 2000). Acta Geologica Polonica 52, 43–54. 2 underground karst water system embedded in the limestone formation of the White Jura known as the ‘Danube–Aach system’, which formed 400,000–450,000 years ago [ 4 Hötzl H. Origin of the Danube-Aach system. 4 Hötzl H. Origin of the Danube-Aach system. 7 Espinasa L.

Jeffery W.R. A troglomorphic sculpin population: Geography, morphology and conservation status. The new cave fish occur in Southern Germany, near the Swabian Alb, home to an elaborate karst region that includes many famous caves and has formed mainly since the Late and Middle Pliocene and during the major glaciation of the Riss-period []. Cave fish were found in the 250 kmunderground karst water system embedded in the limestone formation of the White Jura known as the ‘Danube–Aach system’, which formed 400,000–450,000 years ago []. The recent glacial history (20,000 to 16,000 years ago) indicates several cave-forming processes and the opening of the Aach spring by the retreating Alpine glacier which allowed for first colonization of the cave system with fish []. The Aach spring today is the major outcrop of water, percolating from the upper Danube where water sinks underground at several sinkholes and re-appears in the Aach spring. From there, it flows through the river Radolfzeller Aach to Lake Constance and finally into the Rhine (Supplemental Information). A comparison with the global distribution of subterranean fishes showed that the newly detected cave fish found at 47° north latitude represent the northernmost cave fish known to date and are living closest to a recently glaciated area (Supplemental Information). Aach cave loaches live 760 km further north than Pennsylvanian cave sculpins [], which contradicts the suggestion that Pleistocene glaciations may have prevented cave fish from colonizing numerous subterranean habitats north of 41° latitude. While this may be true for the persistence of older taxa, our finding exemplifies that young lineages of cave fishes may nonetheless evolve following the retreat of the glaciers.

Figure 1 Cave loaches adapted to life in constant darkness are of recent origin but genetically divergent from surface populations residing in the same drainage. Show full caption (A) Two cave loaches in their natural habitat. (B) Adult male loach with typical adaptations to living in caves: reduced eyes, enlarged barbels and pale body coloration. (C) Typical epigean loach from the surface population in the Danube. (D) Neighbour-joining tree of Cytochrome oxidase subunit1 (COI) sequences for European stone loaches. European loaches form three clusters comprising sequences from Rhine, Danube and Elbe drainages. Loaches from the Danube–Aach cave system (AC, highlighted in orange) share haplotypes with individuals from the southern lineage sampled in the upper Rhine and Danube drainage, individuals from GenBank identified by their accession numbers. Numbers at major nodes indicate bootstrap values (>50%, 1000 replicates). (E) Graphical representation of an analysis of genetic population structure. Inferred genomic ancestry in three genetic clusters (y-axis) is depicted by red, green and blue colors for all individuals (x-axis). With prior information on the sample location of individuals, three clearly distinct genetic clusters separate cave loaches from both surface populations upstream (Danube) and downstream (Radolfzeller Aach) in the same drainage. Without prior information on the sample location of individuals, cave fish are grouped with the upstream but clearly separated from the downstream loach population. The cave fish were first observed and photographed in August 2015 by cave divers in the Danube–Aach system ( Figure 1 A) entering the cave from the Aach spring and diving approximately 550 m northwards (Supplemental information). During 15 trips (equivalent of approximately 45 diving hours) between August 2015 and November 2016, several individuals of various size classes were observed, and five cave fish were caught by hand netting. Two were adult fish (body lengths: 82 mm and 65 mm standard length (SL)), and the remaining three were small juveniles (<30 mm SL). The presence of different age classes already indicated the presence of a self-sufficient population of cave loaches in the system. For comparison with surface populations, we collected stone loaches from the adjacent rivers Danube (n = 36) and Radolfzeller Aach (n = 25; Supplemental Information). The small number of cave loaches available to date limited quantitative phenotypic analyses (Supplemental Information). Hence, we considered only the most striking morphometric differences that appear to distinguish the cave fish from epigean populations. Measurements of the head were calculated as percentage of the lateral head length (% HL). The maxillary barbel length was enlarged in the cave fish (32–39% HL vs. 25–31% HL in epigean fish), the eye size was reduced (horizontal eye diameter 6.6–9.7% HL vs. 16–19% HL), and the posterior nare was enlarged (nare diameter 8.0–10.8% HL vs. 5.3–7.5% HL). Furthermore, the cave loaches completely lacked scales, while scales were present in the epigean populations on the flank behind the dorsal-fin origin. Cave loaches also exhibited a shortened lateral line that extended posteriorly to a base vertical above the anal-fin, compared to a complete lateral line reaching to the caudal-fin base in the epigean fish. Finally, all cave loaches lacked the color pattern of large blotches typical of epigean populations ( Figure 1 C). Instead, they showed a more or less dense uniform coverage of individual dark brown pigment cells, which were very sparse in the largest individual ( Figure 1 B), but denser in the other fish.