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.

Parallels between two geographically and ecologically disparate cave invasions by the same species, Asellus aquaticus (Isopoda, Crustacea).

Caves are long-known examples of evolutionary replications where similar morphologies (troglomorphies) evolve independently as the result of strong natural selection of the extreme environment. Recently, this paradigm has been challenged based on observations that troglomorphies are inconsistent across taxa and different subterranean habitats. We investigated the degree of replicated phenotypic change in two independent cave invasions by the freshwater isopod Asellus aquaticus; the first in a sulphidic aquifer in Romania, the second in a sinking river in the Dinaric Karst in Slovenia. Both ancestral surface populations still live alongside the subterranean ones. Phylogenetic analyses show independence of the two colonization events, and microsatellite analysis shows no evidence of ongoing genetic exchange between surface and subterranean ecomorphs. The overall morphology has changed dramatically at both sites (50 of 62 morphometric traits). The amount of phenotypic change did not reflect differences in genetic diversity between the two ancestral populations. Multivariate analyses revealed divergent evolution in caves, not parallel or convergent as predicted by the current paradigm. Still, 18 traits changed in a parallel fashion, including eye and pigment loss and antennal elongation. These changes might be a consequence of darkness as the only common ecological feature, because Romanian caves are chemoautotrophic and rich in food, whereas Slovenian caves are not. Overall, these results show that morphologically alike surface populations can diverge after invading different subterranean habitats, and that only about one-third of all changing traits behave as troglomorphies in the traditional sense.

Using a reference population yardstick to calibrate and compare genetic diversity reported in different studies: an example from the brown bear.

In species with large geographic ranges, genetic diversity of different populations may be well studied, but differences in loci and sample sizes can make the results of different studies difficult to compare. Yet, such comparisons are important for assessing the status of populations of conservation concern. We propose a simple approach of using a single well-studied reference population as a 'yardstick' to calibrate results of different studies to the same scale, enabling comparisons. We use a well-studied large carnivore, the brown bear (Ursus arctos), as a case study to demonstrate the approach. As a reference population, we genotyped 513 brown bears from Slovenia using 20 polymorphic microsatellite loci. We used this data set to calibrate and compare heterozygosity and allelic richness for 30 brown bear populations from 10 different studies across the global distribution of the species. The simplicity of the reference population approach makes it useful for other species, enabling comparisons of genetic diversity estimates between previously incompatible studies and improving our understanding of how genetic diversity is distributed throughout a species range.

Phylogeography of a subterranean amphipod reveals cryptic diversity and dynamic evolution in extreme environments.

Extreme conditions in subsurface are suspected to be responsible for morphological convergences, and so to bias biodiversity assessment. Subterranean organisms are also considered as having poor dispersal abilities that in turn generate a large number of endemic species when habitat is fragmented. Here we test these general hypotheses using the subterranean amphipod Niphargus virei. All our phylogenetic analyses (Bayesian, maximum likelihood and distance), based on two independent genes (28S and COI), revealed the same tripartite structure. N. virei populations from Benelux, Jura region and the rest of France appeared as independent evolutionary units. Molecular rates estimated via global or Bayesian relaxed clock suggest that this split is at least 13 million years old and accredit the cryptic diversity hypothesis. Moreover, the geographical distribution of these lineages showed some evidence of recent dispersal through apparent vicariant barrier. In consequence, we argue that future analyses of evolution and biogeography in subsurface, or more generally in extreme environments, should consider dispersal ability as an evolving trait and morphology as a potentially biased marker.

The colonization of Europe by the freshwater crustacean Asellus aquaticus (Crustacea: Isopoda) proceeded from ancient refugia and was directed by habitat connectivity.

Recent continental-scale phylogeographic studies have demonstrated that not all freshwater fauna colonized Europe from the classic Mediterranean peninsular refugia, and that northern or central parts of the continent were occupied before, and remained inhabited throughout the Pleistocene. The colonization history of the ubiquitous aquatic isopod crustacean Asellus aquaticus was assessed using mitochondrial COI and a variable part of nuclear 28S rDNA sequences. Phylogeographic analysis of the former suggested that dispersion proceeded possibly during late Miocene from the western part of the Pannonian basin. Several areas colonized from here have served as secondary refugia and/or origins of dispersion, well before the beginning of the Pleistocene. Postglacial large-scale range expansion was coupled with numerous separate local dispersions from different refugial areas. Connectivity of the freshwater habitat has played an important role in shaping the current distribution of genetic diversity, which was highest in large rivers. The importance of hydrographic connections for the maintenance of genetic contact was underscored by a discordant pattern of mtDNA and nuclear rDNA differentiation. Individuals from all over Europe, differing in their mtDNA to a level normally found between species or even genera (maximal within population nucleotide divergence reached 0.16 +/- 0.018), shared the same 28S rRNA gene sequence. Only populations from hydrographically isolated karst water systems in the northwestern Dinaric Karst had distinct 28S sequences. Here isolation seemed to be strong enough to prevent homogenization of the rRNA gene family, whereas across the rest of Europe genetic contact was sufficient for concerted evolution to act.

Phylogeography of subterranean and surface populations of water lice Asellus aquaticus (Crustacea: Isopoda).

The water louse Asellus aquaticus is a widespread, euryoecious species, mostly uniform throughout its range. However, six subspecies are known from the Dinaric karst in the northwestern Balkans. They include some specialized subterranean populations. The pattern of genetic variation among subterranean and surface populations in this hydrographically highly fragmented karst region was investigated using a 653 bp fragment of the mitochondrial gene (COI). Sequencing of 168 individuals from 25 localities revealed 72 haplotypes. amova and methods of phylogenetic reconstruction all uncovered hydrographic structuring of genetic variation of the populations. Nested clade analysis pointed out several fragmentation events, along with some range expansions within hydrographical systems. By superimposing the subterranean mode of life on the phylogeographical pattern, three independent cave colonizations could be inferred within a distance of < 100 km. Caves were invaded after the ancestral surface populations became isolated through vicariant fragmentation. A possible scenario of hydrographic history of the region was constructed combining the molecular data with palaeogeographical information.

Random amplified polymorphic DNA diversity among surface and subterranean populations of Asellus aquaticus (Crustacea: Isopoda).

The ecological and evolutionary processes leading to isolation and adaptation of cave animals compared to their surface ancestors are not yet unequivocally understood. In this study the genetic relations of four cave and three surface population of the freshwater crustacean Asellus aquaticus in the Karst region of SW Slovenia and NE Italy were assessed using RAPDs as genetic markers. The results suggest that specialized populations from two caves invaded their subterranean habitat independently, and that their morphological similarity is a result of convergent evolution. Another, less specialized cave population seems to originate from a later colonization of a cave system already inhabited by a more specialized population, but the two populations do not interbreed. This series of temporally and spatially independent invasions has generated a diversity hotspot of non-interbreeding populations of a ubiquitous freshwater crustacean, which is uniform over most of its range. Genetic variability estimated by the percentage of polymorphic RAPD fragments was similar (86-91%) in most cave and surface populations. Substantially lower values (as low as 49%) were found in two cave populations affected by heavy pollution. Two a priori groupings of populations, traditional subspecies and hydrologically connected groups, were rejected as not significant by means of nested analysis of molecular variance (AMOVA). On the other hand, groupings revealed by UPGMA clustering displayed a significant component of among-group variance. An analysis of gene flow between populations using estimated migration rates confirmed these findings.