No abstract

The formation of continental Europe in the Neogene was due to the regression of the Tethys Ocean and of the Paratethys Sea. The dynamic geology of the area and repetitious transitions between marine and freshwater conditions presented opportunities for the colonization of newly emerging hydrological networks and diversification of aquatic biota. Implementing mitochondrial and nuclear markers in conjunction with a large-scale sampling strategy, we investigated the impact of this spatiotemporal framework on the evolutionary history of a freshwater crustacean morphospecies. The Gammarus balcanicus species complex is widely distributed in the area previously occupied by the Paratethys Sea. Our results revealed its high diversification and polyphyly in relation to a number of other morphospecies. The distribution of the studied amphipod is generally characterized by very high local endemism and divergence. The Bayesian time-calibrated reconstruction of phylogeny and geographical distribution of ancestral nodes indicates that this species complex started to diversify in the Early Miocene in the central Balkans, partially in the shallow epicontinental sea. It is possible that there were several episodes of inland water colonization by local brackish water lineages. Subsequent diversification within clades and spread to new areas could have been induced by Alpine orogeny in the Miocene/Pliocene and, finally, by Pleistocene glaciations. The present distribution of clades, in many cases, still reflects Miocene palaeogeography of the area. Our results point out that investigations of the historical aspect of cryptic diversity in other taxa may help in a general understanding of the origins of freshwater invertebrate fauna of Europe.

BackgroundThe Balkans are a major worldwide biodiversity and endemism hotspot. Among the freshwater biota, amphipods are known for their high cryptic diversity. However, little is known about the temporal and paleogeographic aspects of their evolutionary history. We used paleogeography as a framework for understanding the onset of diversification in Gammarus roeselii: (1) we hypothesised that, given the high number of isolated waterbodies in the Balkans, the species is characterised by high level of cryptic diversity, even on a local scale; (2) the long geological history of the region might promote pre-Pleistocene divergence between lineages; (3) given that G. roeselii thrives both in lakes and rivers, its evolutionary history could be linked to the Balkan Neogene paleolake system; (4) we inspected whether the Pleistocene decline of hydrological networks could have any impact on the diversification of G. roeselii.Material and MethodsDNA was extracted from 177 individuals collected from 26 sites all over Balkans. All individuals were amplified for ca. 650 bp long fragment of the mtDNA cytochrome oxidase subunit I (COI). After defining molecular operational taxonomic units (MOTU) based on COI, 50 individuals were amplified for ca. 900 bp long fragment of the nuclear 28S rDNA. Molecular diversity, divergence, differentiation and historical demography based on COI sequences were estimated for each MOTU. The relative frequency, geographic distribution and molecular divergence between COI haplotypes were presented as a median-joining network. COI was used also to reconstruct time-calibrated phylogeny with Bayesian inference. Probabilities of ancestors’ occurrence in riverine or lacustrine habitats, as well their possible geographic locations, were estimated with the Bayesian method. A Neighbour Joining tree was constructed to illustrate the phylogenetic relationships between 28S rDNA haplotypes.ResultsWe revealed that G. roeselii includes at least 13 cryptic species or molecular operational taxonomic units (MOTUs), mostly of Miocene origin. A substantial Pleistocene diversification within-MOTUs was observed in several cases. We evidenced secondary contacts between very divergent MOTUs and introgression of nDNA. The Miocene ancestors could live in either lacustrine or riverine habitats yet their presumed geographic localisations overlapped with those of the Neogene lakes. Several extant riverine populations had Pleistocene lacustrine ancestors.DiscussionNeogene divergence of lineages resulting in substantial cryptic diversity may be a common phenomenon in extant freshwater benthic crustaceans occupying areas that were not glaciated during the Pleistocene. Evolution of G. roeselii could be associated with gradual deterioration of the paleolakes. The within-MOTU diversification might be driven by fragmentation of river systems during the Pleistocene. Extant ancient lakes could serve as local microrefugia during that time.

100Effective identification of species using short DNA fragments (DNA barcoding and DNA 101 metabarcoding) requires reliable sequence reference libraries of known taxa. Both 102 taxonomically comprehensive coverage and content quality are important for sufficient 103 accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are 104 particularly important if molecular identification tools are to be implemented in biomonitoring 105 and reports in the context of the EU Water Framework Directive (WFD) and the Marine 106Strategy Framework Directive (MSFD). We analysed gaps in the two most important 107 reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a 108 focus on the taxa most frequently used in WFD and MSFD. Our analyses show that 109 coverage varies strongly among taxonomic groups, and among geographic regions. In 110 general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, 111 caddisflies and vascular plants) are well represented in the barcode libraries, while others 112 have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also 113 found that species monitored in several countries often are represented by barcodes in 114 reference libraries, while species monitored in a single country frequently lack sequence 115 records. A large proportion of species (up to 50%) in several taxonomic groups are only 116represented by private data in BOLD. Our results have implications for the future strategy to 117 fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the 118 monitoring of European aquatic biota under the WFD and MSFD. For example, missing 119 species relevant to monitoring in multiple countries should be prioritized. We also discuss 120 why a strategy for quality control and quality assurance of barcode reference libraries is 121 needed and recommend future steps to ensure full utilization of metabarcoding in aquatic 122 biomonitoring. 123 124

Summary Pleistocene glaciations affected the present‐day distribution and genetic diversity of animal species in Europe. Deep genetic subdivisions observed in European populations of the widespread freshwater isopod morphospecies, Asellus aquaticus, suggest the presence of putative cryptic species. We used the DNA barcodes of the cytochrome c oxidase subunit 1 (COI) gene combined with distance‐ and tree‐based methods of species delimitation as a rapid tool for assessing the number of distinct operational taxonomic units (OTUs) representing potential cryptic species. The spatial and demographic aspect of A. aquaticus distribution was also analysed. We generated a tentative temporal framework for diversification within the morphospecies provided by the molecular clock approach. Altogether, our study included 603 COI sequences from 147 populations from all over Europe and Asia Minor, including the already published data deposited in GenBank. The mtDNA‐based phylogenetic and OTU delimitation pattern was assessed with results of the nuclear data set analysis including the sequence data derived from this study and those previously submitted in GenBank. In total, 16 haplotypes of 28S rDNA were used representing all COI‐based OTUs and 53 localities. Our results show that A. aquaticus is a conglomerate of genetically distinct COI OTUs. One of the OTUs seems to correspond to the nominative subspecies of A. aquaticus aquaticus, recently redescribed from Sweden, and another with the recently described A. kosswigi. Most of the OTUs are probably of pre‐Pleistocene origin and have narrow ranges in southern Europe. A recent expansion, in both demographic and spatial terms, was revealed in one OTU, which is widely distributed in Europe and represents A. aquaticus aquaticus. This may be explained by the post‐glacial recolonisation processes. According to our data, this OTU probably emerged and initially diversified in the west Balkans in the Middle/Late Pliocene with several lineages surviving and diversifying through the Pleistocene glaciations and expanding during the interglacials. In some cases, our 28S data support the COI‐based OTUs and provide ample evidence for the existence of distinct OTUs, especially in mountainous and karst areas. However, other COI OTUs are not reciprocally monophyletic with respect to nuclear marker. This phylogenetic pattern can be interpreted predominantly as a result of incomplete sorting of nuclear lineages, potentially indicating an ongoing speciation process, but also as an effect of introgression resulting from secondary contact of formerly peripatric or allopatric mitochondrial lineages.