Previous studies on Pleistocene phylogeography of European taxa are biased towards (i) vertebrates, (ii) terrestrial taxa, (iii) single species, and (iv) taxa that survived the Pleistocene in southern refugia. Relatively little is known about whether evolutionary patterns of vertebrate and terrestrial taxa are also applicable to freshwater invertebrates, whether cold-adapted freshwater species could survive in extensive permafrost areas without retreating into refugia, and whether Pleistocene phylogeographical patterns are influenced by phylogeny. Here, the widespread and species-rich European spring snail genus Bythinella Moquin-Tandon, 1856 is utilized in an attempt to mitigate this bias. These strongly cold-adapted freshwater animals mostly occur in springs--highly isolated habitats that are relatively unaffected by anthropogenic influences. Phylogenetic and phylogeographical analyses based on mitochondrial DNA and nuclear DNA sequence data were conducted in 458 specimens from 142 populations occurring throughout Europe. The study provides evidence that most Bythinella spp. survived the Pleistocene in restricted northern glacial refugia that largely correspond to refugia previously recognized for other European biota. However, survival of Bythinella spp. in extensive permafrost areas outside of refugia can likely be rejected. Low dispersal ability and the isolation and fragmentation of spring habitats, as well as the distribution of perennial springs within permafrost regions, may account for this result. Tests involving a total of 29 nominal species showed that phylogenetically closely related Bythinella species did not occupy similar refugia. This lack of phylogenetic concordance could possibly be explained by the stochasticity of survival and dispersal in spring snails.

Abstract. Ancient Lake Ohrid on the Balkan Peninsula is considered to be the oldest ancient lake in Europe with a suggested Plio-/Pleistocene age. Its exact geological age, however, remains unknown. Therefore, molecular clock data of Lake Ohrid biota may serve as an independent constraint of available geological data, and may thus help to refine age estimates. Such evolutionary data may also help unravel potential biotic and abiotic factors that promote speciation events.Here, mitochondrial sequencing data of one of the largest groups of endemic taxa in the Ohrid watershed, the leech genus Dina, is used to test whether it represents an ancient lake species flock, to study the role of potential horizontal and vertical barriers in the watershed for evolutionary events, to estimate the onset of diversification in this group based on molecular clock analyses, and to compare this data with data from other endemic species for providing an approximate time frame for the origin of Lake Ohrid.Based on the criteria speciosity, monophyly and endemicity, it can be concluded that Dina spp. from the Ohrid watershed, indeed, represents an ancient lake species flock. Lineage sorting of its species, however, does not seem to be complete and/or hybridization may occur. Analyses of population structures of Dina spp. in the Ohrid watershed indicate a horizontal zonation of haplotypes from spring and lake populations, corroborating the role of lake-side springs, particularly the southern feeder springs, for evolutionary processes in endemic Ohrid taxa. Vertical differentiation of lake Correspondence to: T. Wilke (tom.wilke@allzool.bio.uni-giessen.de) taxa, however, appears to be limited, though differences between populations from the littoral and the profundal are apparent. Molecular clock analyses indicate that the most recent common ancestor of extant species of this flock is approximately 1.99 ± 0.83 million years (Ma) old, whereas the split of the Ohrid Dina flock from a potential sister taxon outside the lake is estimated at 8.30 ± 3.60 Ma. Comparisons with other groups of endemic Ohrid species indicated that in all cases, diversification within the watershed started ≤2 Ma ago. Thus, this estimate may provide information on a minimum age for the origin of Lake Ohrid. Maximum ages are less consistent and generally less reliable. But cautiously, a maximum age of 3 Ma is suggested. Interestingly, this time frame of approximately 2-3 Ma ago for the origin of Lake Ohrid, generated based on genetic data, well fits the time frame most often used in the literature by geologists.

Aim We analyse patterns of biodiversity in the spring snail genus Bythinella, a group of highly isolated and stenotopic freshwater species. We aim to test: (1) whether there are European areas of increased diversity (i.e. 'hotspots'), (2) whether the potential hotspots inferred show qualitative differences in biodiversity characteristics such as endemicity, distinctiveness of taxa, age of lineages or degree of fragmentation, and (3) whether these hotspots match the Pleistocene refugia of Bythinella spp.Location Europe, Asia Minor. MethodsThe analyses are based on genetic data from 717 Bythinella specimens sampled at 194 sites. We used haplotypes as operational units in all analyses. To test hypothesis 1, mean pairwise genetic distances between Bythinella populations within each 1°· 1°geographical grid cell sampled in Europe were calculated. Within individual mountain ranges, grid cells with high diversity were grouped with neighbouring ones and hotspots were identified based on pre-defined criteria. Then, to test hypothesis 2, different biodiversity indices of these regions were calculated and compared. Finally, to test hypothesis 3, the spatial distribution of the identified hotspots was compared with the known Pleistocene refugia of Bythinella spp.Results Five areas showed increased levels of genetic diversity: the Massif Central/Pyrenees, the western and eastern Alps, and the western and eastern Carpathians. These regions showed qualitative differences in biodiversity, with the eastern Carpathians holding the highest number of (endemic) haplotypes, the oldest and most distinct lineages and the highest degree of fragmentation. Only three of the five detected hotspots matched previously identified Pleistocene refugia for Bythinella spp.Main conclusions The genetic diversity of Bythinella spp. is not randomly distributed throughout Europe. Some of the hotspots we identify coincide with those found in other freshwater taxa; others have not previously been reported. Thus, spring organisms may reflect a unique evolutionary history that is distinct from lentic and lotic taxa. Our findings may be useful for conservation purposes even though the species-level taxonomy of the genus is still under discussion.

No abstract

Ancient Lake Ohrid on the Balkan Peninsula is considered to be the oldest ancient lake in Europe with a suggested Plio-Pleistocene age. Its exact geological age, however, remains unknown. Therefore, molecular clock data of Lake Ohrid biota may serve as an independent constraint of available geological data, and may thus also help to refine age estimates. Such evolutionary data may also help unravel potential biotic and abiotic factors that promote speciation events.

Here, mitochondrial sequencing data of one of the largest groups of endemic taxa in Lake Ohrid, the leech genus Dina, is used to test whether it represents an ancient lake species flock, to study the role of horizontal and vertical barriers in Lake Ohrid for evolutionary events, to estimate the onset of intralacustrine diversification in this group based on molecular clock analyses, and to compare this data with data from other endemic species for providing an approximate time frame for the origin of Lake Ohrid.

Based on the criteria speciosity, monophyly and endemicity, it can be concluded that Lake Ohrid Dina, indeed, represents an ancient lake species flock. Lineage sorting of its species, however, does not seem to be complete. Analyses of population structures of Dina spp. in the Ohrid watershed indicate a horizontal zonation of haplotypes from spring and lake populations, corroborating the role of lake-side springs, particularly the southern feeder springs, for evolutionary processes in endemic Ohrid taxa. Vertical differentiation of lake taxa, however, appears to be limited, though differences between populations from the littoral and the profundal are apparent. Molecular clock analyses indicate that the most recent common ancestor of extant species of this flock is approximately 1.99±0.83 Ma old, whereas the split of the Lake Ohrid Dina flock from a potential sister taxon outside the lake is estimated at 8.30±3.60 Ma. Comparisons with other groups of endemic Ohrid species indicated that in all cases, intralacustrine diversification started ≤2 Ma ago. Thus, this estimate may provide information on a minimum age for the origin of Lake Ohrid. Maximum ages are less consistent and generally less reliable. But cautiously, a maximum age of 3 Ma is suggested. Interestingly, this time frame of approximately 2–3 Ma for the origin of Lake Ohrid, generated based solely on evolutionary data, well fits the time frame most often used in the literature by geologists. Future studies must show whether this concurrence holds true