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.

BackgroundLeuciscinae is a subfamily belonging to the Cyprinidae fish family that is widely distributed in Circum-Mediterranean region. Many efforts have been carried out to deciphering the evolutionary history of this group. Thus, different biogeographical scenarios have tried to explain the colonization of Europe and Mediterranean area by cyprinids, such as the "north dispersal" or the "Lago Mare dispersal" models. Most recently, Pleistocene glaciations influenced the distribution of leuciscins, especially in North and Central Europe. Weighing up these biogeographical scenarios, this paper constitutes not only the first attempt at deciphering the mitochondrial and nuclear relationships of Mediterranean leuciscins but also a test of biogeographical hypotheses that could have determined the current distribution of Circum-Mediterranean leuciscins.ResultsA total of 4439 characters (mitochondrial + nuclear) from 321 individuals of 176 leuciscine species rendered a well-supported phylogeny, showing fourteen main lineages. Analyses of independent mitochondrial and nuclear markers supported the same main lineages, but basal relationships were not concordant. Moreover, some incongruence was found among independent mitochondrial and nuclear phylogenies. The monophyly of some poorly known genera such as Pseudophoxinus and Petroleuciscus was rejected. Representatives of both genera belong to different evolutionary lineages. Timing of cladogenetic events among the main leuciscine lineages was gained using mitochondrial and all genes data set.ConclusionsAdaptations to a predatory lifestyle or miniaturization have superimposed the morphology of some species. These species have been separated into different genera, which are not supported by a phylogenetic framework. Such is the case of the genera Pseudophoxinus and Petroleuciscus, which real taxonomy is not well known. The diversification of leuciscine lineages has been determined by intense vicariant events following the paleoclimatological and hydrogeological history of Mediterranean region. We propose different colonization models of Mediterranean region during the early Oligocene. Later vicariance events promoted Leuciscinae diversification during Oligocene and Miocene periods. Our data corroborate the presence of leuciscins in North Africa before the Messinian salinity crisis. Indeed, Messinian period appears as a stage of gradually Leuciscinae diversification. The rise of humidity at the beginning of the Pliocene promoted the colonization and posterior isolation of newly established freshwater populations. Finally, Pleistocene glaciations determined the current European distribution of some leuciscine species.

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Parasites exhibiting a high degree of host specificity are expected to be intimately associated with their hosts. Therefore, the evolution of host-specific parasites is at least partially shaped by the evolutionary history and distribution of such hosts. Gill ectoparasites of Dactylogyrus (Monogenea) are specific to cyprinid fish. In the present study, we investigated the evolutionary history of 47 Dactylogyrus species from the Balkan Peninsula, the Mediteranean region exhibiting the highest cyprinid diversity in Europe, and from central European cyprinids. Phylogenetic analyses revealed four well-supported clades of endemic and non-endemic Dactylogyrus spp. with four basal taxa. Endemic cyprinids with a limited distribution range were parasitized by endemic Dactylogyrus species, but some of them shared several Dactylogyrus species with central European cyprinids. Species delimitation analyses based on molecular data suggest that Dactylogyrus diversity is higher than that defined from morphology. Some endemic cyprinid species harboured Dactylogyrus species of different origins, this probably resulting from multiple host switching. Our results support the view that the evolution of Dactylogyrus in the Balkans has been influenced not only by the historical dispersion and distribution of their cyprinid hosts, but also by recent contacts of non-native cyprinid species with endemic cyprinid fauna in this region.

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