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.

No abstract

Recent studies have revealed high local diversity and endemism in groundwaters, and showed that species with large ranges are extremely rare. One of such species is the cave shrimp Troglocaris anophthalmus from the Dinaric Karst on the western Balkan Peninsula, apparently uniform across a range of more than 500 kilometers. As such it contradicts the paradigm that subterranean organisms form localized, long-term stable populations that cannot disperse over long distances. We tested it for possible cryptic diversity and/or unexpected evolutionary processes, analysing mitochondrial (COI, 16S rRNA) and nuclear (ITS2) genes of 232 specimens from the entire range. The results of an array of phylogeographical procedures congruently suggested that the picture of a widespread, continuously distributed and homogenous T. anophthalmus was wrong. The taxon is composed of four or possibly five monophyletic, geographically defined phylogroups that meet several species delimitation criteria, two of them showing evidence of biological reproductive isolation in sympatry. COI genetic distances between phylogroups turned out to be a poor predictor, as they were much lower than the sometimes suggested crustacean threshold value of 0.16 substitutions per site. Most results confirmed the nondispersal hypothesis of subterranean fauna, but the southern Adriatic phylogroup displayed a paradoxical pattern of recent dispersal across 300 kilometers of hydrographically fragmented karst terrain. We suggest a model of migration under extreme water-level conditions, when flooded poljes could act as stepping-stones. In the north of the range (Slovenia), the results confirmed the existence of a zone of unique biogeographical conflict, where surface fauna is concordant with the current watershed, and subterranean fauna is not.

Ecologists increasingly rely on molecular delimitation methods (MMs) to identify species boundaries, thereby potentially increasing the number of putative species because of the presence of morphologically cryptic species. It has been argued that cryptic species could challenge our understanding of what determine large‐scale biodiversity patterns which have traditionally been documented from morphology alone. Here, we used morphology and three MMs to derive four different sets of putative species among the European groundwater crustaceans. Then, we used regression models to compare the relative importance of spatial heterogeneity, productivity and historical climates, in shaping species richness and range size patterns across sets of putative species. We tested three predictions. First, MMs would yield many more putative species than morphology because groundwater is a constraining environment allowing little morphological changes. Second, for species richness, MMs would increase the importance of spatial heterogeneity because cryptic species are more likely along physical barriers separating ecologically similar regions than along resource gradients promoting ecologically‐based divergent selection. Third, for range size, MMs would increase the importance of historical climates because of reduced and asymmetrical fragmentation of large morphological species ranges at northern latitudes. MMs yielded twice more putative species than morphology and decreased by 10‐fold the average species range size. Yet, MMs strengthened the mid‐latitude ridge of high species richness and the Rapoport effect of increasing range size at higher latitudes. Species richness predictors did not vary between morphology and MMs but the latter increased the proportion of variance in range size explained by historical climates. These findings demonstrate that our knowledge of groundwater biodiversity determinants is robust to overlooked cryptic species because the latter are homogeneously distributed along environmental gradients. Yet, our findings call for incorporating multiple species delimitation methods into the analysis of large‐scale biodiversity patterns across a range of taxa and ecosystems.

Subterranean species show a distinct morphology, yet the adaptive significance of some traits, like body size and shape, is poorly understood and cannot be explained solely by distinct environmental conditions (darkness, less food). We predicted that in females some morphological changes may have co‐evolved with life history traits, and that co‐evolving life history traits provide at least part of the explanation for evolutionary changes of morphology. Using museum material we tested this prediction on the subterranean amphipod genus Niphargus. We studied six species found in springs and eight species found in cave lakes. We treated them as two ecologically distinct groups, and the major ecological differences between them were the availability of nutrients and the water currents. Cave species were found to be larger and stouter (as inferred from the shape of coxal plates, which are part of the marsupium), they had larger eggs and lower reproductive effort per brood, whereas the egg number and brood volume if corrected for the body size were not different. Using phylogenetic independent contrasts, we found a positive correlation between body shape and egg volume, a positive correlation between body size and egg volume, and a negative correlation between body size and reproductive effort per brood. We tentatively conclude that evolutions of morphology and life histories are functionally connected and that co‐evolving traits contribute to overall selective regime.