Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule-coalescent (GMYC) model-based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA-barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large-scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.

A phylogeographic analysis of mitochondrial DNA variation was performed in order to test the hypothesis of a postglacial recolonization of mid- and north-European rivers from a Danubian refuge. Over 345 chub specimens from European rivers covering most of the species' native range were investigated using 600 bp of the cytochrome b gene. Chub in European rivers belong to four highly divergent mitochondrial groups (lineages) differing by mean divergence estimates from 5.2% to 7.89%. These four lineages have a largely allopatric distribution, implying four geographical sets: two Mediterranean, and two north-European sets. This pattern provided strong evidence for: (i) the eradication of this species from most of Europe during maximum ice extent; (ii) its survival in four refugia (Adriatic side of the Balkans, eastern Greece (Aegean Rivers), southern tributaries of the Danube, and periphery of Black and Caspian Seas); (iii) a differential postglacial recolonization of mid- and northern Europe from the last two refugia only; (iv) the occurrence of this recolonization in two steps for the Danubian (western) lineage that entered western Europe (Rhine-Rhone-Loire drainages) during the Riss-Würm interglacial period and survived the last glaciation there before colonizing Garonne, UK and German drainages up to the Elbe during the Holocene; and (v) the occurrence of this recolonization in a single step for the Ponto-Caspian (eastern) lineage that entered the Baltic area as far as the Oder in the Holocene. Both lineages came into contact in the River Elbe without evident mixing.

The complete mitochondrial DNA (mtDNA) control region (1043 base pairs) and 162-bp of flanking transfer RNA genes were sequenced in 316 European grayling, Thymallus thymallus, from 44 populations throughout the Western European range of the species. A total of 58 haplotypes were revealed with pairwise divergence ranging from 0.001 to 0.038. An inferred intraspecific phylogenetic tree revealed two well-supported clades within the Danube basin, one highly divergent clade in the Adriatic basin, and one large, diverse group representing most other populations. A deeply divergent haplotype fixed in the Loire basin in central France, more groups of haplotypes from distinct Danubian tributaries, and a relatively ancestral haplotype fixed in former tributaries of the Elbe in Denmark all suggest a complex pattern of interglacial and postglacial expansions originating from disjunct refugia throughout central Europe. Despite some evidence of human-mediated stock transfers, parsimony-network-based nested-clade analysis (NCA) supported specific inferences relating to corridors of postglacial expansion such as the lower Rhine (Moselle) and Elbe systems (Danish populations) serving as sources for expansion into the Baltic to the north as well as the upper Rhine and Danube to the south; and specific Rhine populations (Doller, Orbe and Reuss) serving as sources for colonization of the Rhone. The multiple divergent clades representing populations in the upper Danube, as well as the deeply divergent haplotypes found in the Adriatic and Loire basins (> 5% divergence from Asian outgroups) support the theory that European grayling have had a long history in Western Europe, pre-dating Pleistocene glacial cycles. The patterns of mtDNA divergence shown here support a perspective of rich inter- and intrabasin genetic diversity that should be protected from current trends to translocate brood stocks for rearing and release in response to declining populations, especially in southern European basins.