Diversity under a magnifier lens: the case of Typhlotanaidae (Crustacea: Tanaidacea) in the N Atlantic.

Research focusing on 'stout-bodied' typhlotanaids collected from several sites around Iceland and adjacent N Atlantic region has resulted in the description of 15 species new to science, as well as the creation of eight new genera. Typhlotanais eximius Hansen, 1913 is redescribed and transferred to a new genus, while Typhlotanais crassus and Peraeospinosus adipatus are transferred to the genus Larsenotanais. The morphological and the molecular data were combined to consolidate and confirm the validity of the results obtained from both approaches. The polyphyletic nature of the Typhlotanaidae and its serious of its taxonomic diversity are emphasized, although molecular analysis reveals that the 'stout-bodied' Typhlotanaidae form monophyletic clade. Depth and temperature are identified as the main environmental parameters determining the distribution of this group of Typhlotanaidae. Several species are clearly associated with the shelf and upper bathyal of Iceland. The Greenland-Iceland-Faroe Ridge is shown to be a distinct zoogeographical barrier for typhlotanaids inhabiting the deeper slope and abyssal regions around Iceland.

Microsporidian diversity in the aquatic isopod Asellus aquaticus.

We conducted a molecular survey on microsporidian diversity in different lineages (operational taxonomic units = OTUs) of Asellus aquaticus from 30 sites throughout Europe. Host body length was determined, and DNA was extracted from host tissue excluding the intestine and amplified by microsporidian-specific primers. In total, 247 A. aquaticus specimens were analysed from which 26.7% were PCR-positive for microsporidians, with significantly more infections in larger individuals. Prevalence ranged between 10 and 90%. At 9 sites, no microsporidians were detected. A significant relationship was found between the frequency of infected individuals and habitat type, as well as host OTU. The lowest proportion of infected individuals was detected in spring-habitats (8.7%, n = 46) and the highest in ponds (37.7%, n = 53). Proportion of infected individuals among host OTUs A, D and J was 31.7, 21.7 and 32.1%, respectively. No infections were detected in OTU F. Our results are, however, accompanied by a partially low sample size, as only a minimum of 5 individuals was available at a few locations. Overall, 17 different microsporidian molecular taxonomic units (MICMOTUs) were distinguished with 5 abundant isolates (found in 4­17 host individuals) while the remaining 12 MICMOTUs were "rare" and found only in 1­3 host individuals. No obvious spatio-genetic pattern could be observed. The MICMOTUs predominantly belonged to Nosematida and Enterocytozoonida. The present study shows that microsporidians in A. aquaticus are abundant and diverse but do not show obvious patterns related to host genetic lineages or geography.

DNA barcodes evidence the contact zone of eastern and western caddisfly lineages in the Western Carpathians.

The region of the Western Carpathians is, among other aspects, very important for survival and diversity of European freshwater fauna due to the presence of a large number of (sub)mountain springs and streams. However, these ecologically and faunistically diversified habitats are still understudied in the context of genetic diversity and population structure of their inhabitants. This study focuses on genetic diversity and distribution patterns of the caddisfly Rhyacophila tristis, common and widespread representative of mountain freshwater fauna. Analysis of the COI mitochondrial marker revealed presence of the western and eastern lineages, with samples from both lineages being grouped in BOLD (Barcode of Life Data System) into separate BINs (Barcode Index Numbers). Our data indicates that eastern lineage (BIN_E) is more closely related to the Balkan populations than to co-occurring western lineage (BIN_W), and that the contact zone of the lineages passes through the W Carpathians. The study revealed phylogeographic and demographic differences between lineages, supporting hypothesis of their evolutionary independence and specific ecological preferences. The obtained genetic data of the R. tristis population from W Carpathians improved our knowledge about population genetics of this aquatic species and can contribute to understanding the state and evolution of biodiversity of freshwater ecosystems in Europe.

First Glimpse at the Diverse Aquaporins of Amphipod Crustaceans.

The importance of aquaporins (AQPs) in the transport of water and solutes through cell membranes is well recognized despite being relatively new. To date, despite their abundance, diversity, and presence in disparate environments, amphipods have only been mentioned in studies about the AQPs of other animals and have never been further investigated. In this work, we aimed to recover from public data available AQPs of these crustaceans and reconstruct phylogenetic affinities. We first performed BLAST searches with several queries of diverse taxa against different NCBI databases. Then, we selected the clades of AQPs retrieving the amphipod superfamily Gammaroidea as monophyletic and ran phylogenetic analyses to assess their performances. Our results show how most of the AQPs of amphipods are similar to those of other crustaceans, despite the Prip-like displayed different paralogs, and report for the first time a putative Aqp8-like for arthropods. We also found that the candidate genes of Prip-like, Bib-like, Aqp12-like, and Glp-like help solve deeper relationships in phylogenies of amphipods while leaving uncertainties in shallower parts. With our findings, we hope to increase attention to the study of amphipods as models for AQP functioning and evolution.

Finding Aquaporins in Annelids: An Evolutionary Analysis and a Case Study.

Aquaporins (AQPs) are a family of membrane channels facilitating diffusion of water and small solutes into and out of cells. Despite their biological relevance in osmoregulation and ubiquitous distribution throughout metazoans, the presence of AQPs in annelids has been poorly investigated. Here, we searched and annotated Aqp sequences in public genomes and transcriptomes of annelids, inferred their evolutionary relationships through phylogenetic analyses and discussed their putative physiological relevance. We identified a total of 401 Aqp sequences in 27 annelid species, including 367 sequences previously unrecognized as Aqps. Similar to vertebrates, phylogenetic tree reconstructions clustered these annelid Aqps in four clades: AQP1-like, AQP3-like, AQP8-like and AQP11-like. We found no clear indication of the existence of paralogs exclusive to annelids; however, several gene duplications seem to have occurred in the ancestors of some Sedentaria annelid families, mainly in the AQP1-like clade. Three of the six Aqps annotated in Alitta succinea, an estuarine annelid showing high salinity tolerance, were validated by RT-PCR sequencing, and their similarity to human AQPs was investigated at the level of "key" conserved residues and predicted three-dimensional structure. Our results suggest a diversification of the structures and functions of AQPs in Annelida comparable to that observed in other taxa.

Fuzzy species borders of glacial survivalists in the Carpathian biodiversity hotspot revealed using a multimarker approach.

The Carpathians are one of the key biodiversity hotspots in Europe. The mountain chain uplifted during Alpine orogenesis and is characterised by a complex geological history. Its current biodiversity was highly influenced by Pleistocene glaciations. The goal of the current study was to examine the phylogenetic and demographic history of Gammarus balcanicus species complex in the Carpathians using multiple markers as well as to delimit, using an integrative approach, and describe new species hidden so far under the name G. balcanicus. Results showed that divergence of the studied lineages reaches back to the Miocene, which supports the hypothesis of their survival in multiple micro refugia. Moreover, the increase of their diversification rate in the Pleistocene suggests that glaciation was the driving force of their speciation. The climatic changes during and after the Pleistocene also played a major role in the demography of the local Carpathian lineages. Comparison of diversity patterns and phylogenetic relationships of both, the mitochondrial and nuclear markers, provide evidence of putative hybridisation and retention of ancient polymorphism (i.e., incomplete lineage sorting). The morphological examination supported the existence of two morphological types; one we describe as a G. stasiuki sp. nov. and another we redescribe as a G. tatrensis (S. Karaman, 1931).

Mitochondrial Genomes, Phylogenetic Associations, and SNP Recovery for the Key Invasive Ponto-Caspian Amphipods in Europe.

The Ponto-Caspian region is the main donor of invasive amphipods to freshwater ecosystems, with at least 13 species successfully established in European inland waters. Dikerogammarus spp. and Pontogammarus robustoides are among the most successful, due to their strong invasive impact on local biota. However, genomic knowledge about these invaders is scarce, while phylogeography and population genetics have been based on short fragments of mitochondrial markers or nuclear microsatellites. In this study, we provide: (i) a reconstruction of six mitogenomes for four invasive gammarids (D. villosus, D. haemobaphes, D. bispinosus, and P. robustoides); (ii) a comparison between the structure of the newly obtained mitogenomes and those from the literature; (iii) SNP calling rates for individual D. villosus and D. haemobaphes from different invasion sites across Europe; and (iv) the first time-calibrated full mitogenome phylogeny reconstruction of several Ponto-Caspian taxa. We found that, in comparison to other gammarids, the mitogenomes of Ponto-Caspian species show a translocation between the tRNA-E and tRNA-R positions. Phylogenetic reconstruction using the mitogenomes identified that Ponto-Caspian gammarids form a well-supported group that originated in the Miocene. Our study supports paraphyly in the family Gammaridae. These provided mitogenomes will serve as vital genetic resources for the development of new markers for PCR-based identification methods and demographic studies.

Cenozoic climatic changes drive evolution and dispersal of coastal benthic foraminifera in the Southern Ocean.

The Antarctic coastal fauna is characterized by high endemism related to the progressive cooling of Antarctic waters and their isolation by the Antarctic Circumpolar Current. The origin of the Antarctic coastal fauna could involve either colonization from adjoining deep-sea areas or migration through the Drake Passage from sub-Antarctic areas. Here, we tested these hypotheses by comparing the morphology and genetics of benthic foraminifera collected from Antarctica, sub-Antarctic coastal settings in South Georgia, the Falkland Islands and Patagonian fjords. We analyzed four genera (Cassidulina, Globocassidulina, Cassidulinoides, Ehrenbergina) of the family Cassidulinidae that are represented by at least nine species in our samples. Focusing on the genera Globocassidulina and Cassidulinoides, our results showed that the first split between sub-Antarctic and Antarctic lineages took place during the mid-Miocene climate reorganization, probably about 20 to 17 million years ago (Ma). It was followed by a divergence between Antarctic species ~ 10 Ma, probably related to the cooling of deep water and vertical structuring of the water-column, as well as broadening and deepening of the continental shelf. The gene flow across the Drake Passage, as well as between South America and South Georgia, seems to have occurred from the Late Miocene to the Early Pliocene. It appears that climate warming during 7-5 Ma and the migration of the Polar Front breached biogeographic barriers and facilitated inter-species hybridization. The latest radiation coincided with glacial intensification (~ 2 Ma), which accelerated geographic fragmentation of populations, demographic changes, and genetic diversification in Antarctic species. Our results show that the evolution of Antarctic and sub-Antarctic coastal benthic foraminifera was linked to the tectonic and climatic history of the area, but their evolutionary response was not uniform and reflected species-specific ecological adaptations that influenced the dispersal patterns and biogeography of each species in different ways.

Coverage and quality of DNA barcode references for Central and Northern European Odonata.

BACKGROUND: Dragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches. METHODS: This study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD. RESULTS: Over 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation in Cordulia aenea (Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database are highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.

Evolution of reproductive strategies in the species-rich land snail subfamily Phaedusinae (Stylommatophora: Clausiliidae).

Most of the present knowledge on animal reproductive mode evolution, and possible factors driving transitions between oviparity and viviparity is based on studies on vertebrates. The species rich door snail (Clausiliidae) subfamily Phaedusinae represents a suitable and unique model for further examining parity evolution, as three different strategies, oviparity, viviparity, and the intermediate mode of embryo-retention, occur in this group. The present study reconstructs the evolution of reproductive strategies in Phaedusinae based on time-calibrated molecular phylogenetics, reproductive mode examinations and ancestral state reconstruction. Our phylogenetic analysis employing multiple mitochondrial and nuclear markers identified a well-supported clade (including the tribes Phaedusini and Serrulinini) that contains species exhibiting various reproductive strategies. This clade evolved from an oviparous most recent common ancestor according to our reconstruction. All non-oviparous taxa are confined to a highly supported subclade, coinciding with the tribe Phaedusini. Both oviparity and viviparity occur frequently in different lineages of this subclade that are not closely related. During Phaedusini diversification, multiple transitions in reproductive strategy must have taken place, which could have been promoted by a high fitness of embryo-retaining species. The evolutionary success of this group might result from the maintenance of various strategies.

Molecular data suggest multiple origins and diversification times of freshwater gammarids on the Aegean archipelago.

Our main aim was to investigate the diversity, origin and biogeographical affiliations of freshwater gammarids inhabiting the Aegean Islands by analysing their mtDNA and nDNA polymorphism, thereby providing the first insight into the phylogeography of the Aegean freshwater gammarid fauna. The study material was collected from Samothraki, Lesbos, Skyros, Evia, Andros, Tinos and Serifos islands as well as from mainland Greece. The DNA extracted was used for amplification of two mitochondrial (COI and 16S) and two nuclear markers (28S and EF1-alpha). The multimarker time-calibrated phylogeny supports multiple origins and different diversification times for the studied taxa. Three of the sampled insular populations most probably represent new, distinct species as supported by all the delimitation methods used in our study. Our results show that the evolution of freshwater taxa is associated with the geological history of the Aegean Basin. The biogeographic affiliations of the studied insular taxa indicate its continental origin, as well as the importance of the land fragmentation and the historical land connections of the islands. Based on the findings, we highlight the importance of studying insular freshwater biota to better understand diversification mechanisms in fresh waters as well as the origin of studied Aegean freshwater taxa.

Continental-scale patterns of hyper-cryptic diversity within the freshwater model taxon Gammarus fossarum (Crustacea, Amphipoda).

Traditional morphological diagnoses of taxonomic status remain widely used while an increasing number of studies show that one morphospecies might hide cryptic diversity, i.e. lineages with unexpectedly high molecular divergence. This hidden diversity can reach even tens of lineages, i.e. hyper cryptic diversity. Even well-studied model-organisms may exhibit overlooked cryptic diversity. Such is the case of the freshwater crustacean amphipod model taxon Gammarus fossarum. It is extensively used in both applied and basic types of research, including biodiversity assessments, ecotoxicology and evolutionary ecology. Based on COI barcodes of 4926 individuals from 498 sampling sites in 19 European countries, the present paper shows (1) hyper cryptic diversity, ranging from 84 to 152 Molecular Operational Taxonomic Units, (2) ancient diversification starting already 26 Mya in the Oligocene, and (3) high level of lineage syntopy. Even if hyper cryptic diversity was already documented in G. fossarum, the present study increases its extent fourfold, providing a first continental-scale insight into its geographical distribution and establishes several diversification hotspots, notably south-eastern and central Europe. The challenges of recording hyper cryptic diversity in the future are also discussed.

The tale of springs and streams: how different aquatic ecosystems impacted the mtDNA population structure of two riffle beetles in the Western Carpathians.

The Western Carpathians are a particularly interesting part of the Carpathian Arc. According to recent molecular data upon aquatic and terrestrial taxa, this mountain area is an important biodiversity hotspot of Europe. Moreover, the W Carpathians include rich systems of karst springs inhabited by specific fauna, where molecular diversity and phylogeographic patterns are yet to be fully explored. Our study aims to compare population genetic structure and molecular diversity of two related and commonly co-occurring riffle beetles, Elmis aenea (PWJ Müller, 1806) and Limnius perrisi (Dufour, 1843) in the springs and streams of the W Carpathians using the mitochondrial DNA barcoding fragment of the cytochrome c oxidase subunit I gene (COI). The relatively stable thermal and chemical conditions of springs throughout unfavourable climatic settings make these highly specific lotic systems potentially ideal for a long-term survival of some aquatic biota. Populations of both elmid species were relatively homogeneous genetically, with a single dominant haplotype. However, we revealed that E. aenea significantly dominated in the springs, while L. perrisi preferred streams. Relative isolation of the springs and their stable conditions were reflected in significantly higher molecular diversity of the E. aenea population in comparison to L. perrisi. The results of Bayesian Skyline Plot analysis also indicated the exceptional position of springs regarding maintaining the population size of E. aenea. On the other hand, it seems that streams in the W Carpathians provide more effective dispersal channels for L. perrisi, whose population expanded much earlier compared to E. aenea. Present study points out that different demographic histories of these two closely related elmid species are manifested by their different habitat preference and molecular diversity.

Successful post-glacial colonization of Europe by single lineage of freshwater amphipod from its Pannonian Plio-Pleistocene diversification hotspot.

Gammarus roeselii Gervais, 1835 is a morphospecies with a wide distribution range in Europe. The Balkan Peninsula is known as an area of pre-Pleistocene cryptic diversification within this taxon, resulting in at least 13 Molecular Operational Taxonomic Units (MOTUs). The morphospecies diversified there during Neogene and has probably invaded other parts of the continent very recently, in postglacial or even historical times. Thus, the detailed goals of our study were to (1) identify which lineage(s) colonized Central-Western Europe (CWE), (2) determine their possible geographical origin, (3) verify, whether the colonisation was associated with demographic changes. In total, 663 individuals were sequenced for the cytochrome oxidase I (COI) barcoding fragment and 137 individuals for the internal transcribed spacer II (ITS2). We identified two MOTUs in the study area with contrasting Barcode Index Number and haplotype diversities. The Pannonian Basin (PB) appeared to be a potential ice age refugium for the species, while CWE was colonised by a single lineage (also present in PB), displaying low genetic diversity. Our results suggest that G. roeselii is a relatively recent coloniser in CWE, starting demographic expansion around 10 kya.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

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

Europe-wide reassessment of Dictyocoela (Microsporidia) infecting native and invasive amphipods (Crustacea): molecular versus ultrastructural traits.

Microsporidia are common parasites infecting animals and protists. They are specifically common pathogens of amphipods (Crustacea, Malacostraca), with Dictyocoela spp. being particularly frequent and highly prevalent, exhibiting a range of phenotypic and ecological effects. Until now, seven species of Dictyocoela were defined, predominantly based on the genetic distance. However, neither the taxonomic status of this provisionally erected genus (based on eight novel sequences and one micrograph of the spore), nor its internal phylogenetic relationships have been clearly revealed. The formal description of the genus and of most of the putative species are still lacking. Here we aimed to fill this gap and performed both ultrastructural and molecular studies (based on SSU, ITS and partial LSU) using different species delimitation methods. As a consensus of these results and following conservative data interpretation, we propose to distinguish five species infecting gammarid hosts, and to keep the names introduced by the authors of the type sequences: Dictyocoela duebenum, D. muelleri, D. berillonum and D. roeselum. We provide full descriptions of these species. Moreover, thanks to our extensive sampling, we extend the known host and geographic range of these Microsporidia.

First endemic freshwater Gammarus from Crete and its evolutionary history-an integrative taxonomy approach.

The Mediterranean islands are known as natural laboratories of evolution with a high level of endemic biodiversity. However, most biodiversity assessments have focused mainly on terrestrial and marine fauna, leaving the freshwater animals aside. Crete is one of the largest islands in the Mediterranean Basin, with a long history of isolation from the continental mainland. Gammarid amphipods are often dominant in macrozoobenthic communities in European inland waters. They are widely used in biomonitoring and exotoxicological studies. Herein, we describe Gammarus plaitisi sp. nov., endemic to Cretan streams, based on morphological characters and a set of molecular species delimitation methods using mitochondrial cytochrome oxidase subunit I and 16S rRNA genes as well as nuclear 28S rDNA, ITS1 and EF1-alpha genes. The divergence of the new species is strongly connected with the geological history of the island supporting its continental origin.

Neogene paleogeography provides context for understanding the origin and spatial distribution of cryptic diversity in a widespread Balkan freshwater amphipod.

BACKGROUND: The 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 METHODS: DNA 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. RESULTS: We 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. DISCUSSION: Neogene 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.

The legacy of a vanished sea: a high level of diversification within a European freshwater amphipod species complex driven by 15 My of Paratethys regression.

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.