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AimA detailed, comparative DNA-barcoding and morphospecies based overview of the vertical and horizontal distribution of Lake Ohrid endemicGammarusspecies-flock. Re-evaluation of the origin of the species-flock dating, identification of events that putatively influenced diversification patterns in the species-flock.LocationLake Ohrid: a deep and ancient lake of tectonic origin, biosphere reserve, UNESCO World Heritage Site, located on the Macedonia/Albania border.TaxonGammarusspecies-flock (Amphipoda, Crustacea)MethodsExtensive sampling and DNA barcoding of 600 individuals were carried out. DNA sequences were analysed using species delimitation methods, haplotype network reconstructions, Bayesian molecular dating and demographic analysis. The COI-based delimitation results were validated with nuclear 28S RNA data.ResultsThe species flock distribution has weak horizontal but clear vertical structure. The diversity across bathymetric gradients correlates with temperature and salinity; and the highest diversity with sublittoral and springs of lake’s shore. Two new MOTUs representing putatively new species are revealed and supported also by the nuclear marker. The time of flock radiation overlaps with the time of lake formation. The COI gene shows signs of positive selection and an acceleration in substitution rate. The demographic changes of the flock happened during the last ky.Main conclusionsDistribution of theGammarusspecies-flock is vertically structured, reflecting habitat zonation. Parapatric speciation as one of the mechanisms in flock’s diversification is suggested. Detection of new MOTU suggests that the flock’s diversity is still not fully revealed. Nevertheless, failure to recover three other MOTUs suggests the loss of gammarid diversity in the lake. This represents,together with the current threats to the lake ecosystem (i.e. climate changes, development of tourism), a clear call for conservation efforts. The speciation events and demographic changes within the flock relate presumably to glacial and postglacial water level changes and to colonisation of new depth ranges and the associated springs.

Research focusing on ‘stout-bodied’ typhlotanaids collected from several sites around Iceland and adjacent northern seas has resulted in the description of 15 species new to science, as well as the creation of eight new genera. Typhlotanais eximius Hansen, 1913 was 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 Typhlotanaidae and its serious underestimation was emphasized. Molecular analysis revealed that the ’stout-bodied‘ Typhlotanaidae are monophyletic. Depth, temperature, and salinity were identified as the main environmental parameters determining the distribution of this group of Typhlotanaidae. Several species were clearly associated with 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 around Iceland.

We conducted a molecular survey on microsporidian diversity in different lineages (operational taxonomic units = OTUs) of

The regions of the Black, Caspian, and Azov seas are known for being both (i) the place of extensive crustacean radiation dated to the times of Paratethys and Sarmatian basins, and (ii) present donors of alien and invasive taxa to many areas worldwide. One amphipod morphospecies,Dikerogammarus haemobaphes, is known both as native to rivers draining to the Black and Caspian seas as well as a successful invader (nicknamed demon shrimp) in Central and Western European rivers. Based on mitochondrial (COI and 16S) and nuclear (28S) datasets and 41 sampling sites, representing both the native (19) and the invaded (22) range, we assessed cryptic diversity, phylogeography and population genetics of this taxon. First, we revealed the presence of two divergent lineages supported by all markers and all species delimitation methods. The divergence between the lineages was high (18.3% Kimura 2-parameter distance for COI) and old (ca. 5.1 Ma), suggesting the presence of two cryptic species withinD. haemobaphes. Lineage A was found only in a few localities in the native range, while lineage B was widespread both in the native and in the invaded range. Although genetic divergence within lineage B was shallow, geographic distribution of 16S and COI haplotypes was highly heterogeneous, leading us to the definition of four Geo-Demographic Units (GDUs). Two GDUs were restricted to the native range: GDU-B1 was endemic for the Durugöl (aka Duruşu) Liman in Turkey, whereas GDU-B2 occurred only in the Dniester River. GDU-B3 was both present in several localities in the native range in the Black Sea drainage area and widespread in Central and Western Europe. The GDU-B4 was found exclusively in the Moskva River in Russia. Extended Bayesian Skyline Plot indicated steady growth of GDU-B3 population size since 30 ka, pointing to the rather old history of its expansion, first in the late Pleistocene in the native range and nowadays in Central and Western Europe. The analysis of haplotype distribution across the present distribution range clearly showed two invasion routes to Central and Western Europe. The first one, originating from the lower Dnieper, allowed the demon shrimp to colonize Polish rivers and the Mittellandkanal in Germany. The second one, originating from the Danube delta, allowed to colonize the water bodies in the upper Danube basin. The UK population has originated from the Central Corridor, as only a haplotype found exclusively along this route was recorded in the UK. Population genetics analysis showed that the invasion of the demon shrimp along the Central Corridor was not associated with the loss of genetic diversity, which might contribute to the success of this invader in the newly colonized areas.

The severe climatic changes during the Pleistocene ice ages have shaped the genetic structure and distribution of biota in Europe. We aimed to reveal in detail the genetic diversity, geographical population structure, historical and present demography, migration patterns, and the presence of possible glacial refugia within the nominative subspecies Asellus aquaticus aquaticus. We analysed DNA sequences of the mitochondrial cytochrome c oxidase subunit I gene and nuclear noncoding internal transcribed spacer II region, from populations inhabiting post‐glacial Europe (from the British Isles and Scandinavia to the northern Mediterranean and Black Sea coasts). The origin of the taxon, including establishment in the Dinaric Western Balkans, dates to the Middle/Late Pliocene, but most of its genetic diversity emerged during the Middle/Late Pleistocene before the Last Glacial Maximum. Despite the general absence of spatial genetic structure with population growth, we discovered two different phylogeographic stories across 11 clusters revealed by a coalescent approach. Firstly, the periglacial cluster group—spatially restricted mainly to the northern Balkans, Pannonian Basin, and Pontic Region—is older and more divergent. It apparently retained a relatively stable population size during the glacial‐interglacial cycles. Conversely, the proglacial cluster group—widely distributed in areas close to the glacier margins (north of the Alps, Sudetes, Carpathians) and in the Pannonian Basin—is younger and composed of closely related individuals. It originated in Pleistocene and lasted continuously through the Last Glacial Maximum in numerous high latitude refugia. This was probably due to the vast network of proglacial lakes and rivers, which played a crucial role in the maintenance of genetic diversity, population growth, and high dispersal rate. The evolutionary history of A. a. aquaticus reveals unexpected patterns and is an important lesson when making predictions for other aquatic taxa. Our results suggest that we should stop perceiving the proglacial habitats as lifeless ice desert.

We conducted a molecular survey on microsporidian diversity in different lineages (operational taxonomic units = OTUs) ofAsellus aquaticusfrom 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, 247A. aquaticusspecimens 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 4–17 host individuals) while the remaining 12 MICMOTUs were “rare” and found only in 1–3 host individuals. No obvious spatio-genetic pattern could be observed. The MICMOTUs predominantly belonged to Nosematida and Enterocytozoonida. The present study shows that microsporidians inA. aquaticusare abundant and diverse but do not show obvious patterns related to host genetic lineages or geography.

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.

European mountains are important areas regarding biodiversity of the continent, and they also harbour diverse freshwater fauna, which is critically endangered in terms of the current species extinction. However, sufficient knowledge of this valuable part of European biota is no longer possible without molecular data. This study focuses on the genetic diversity and distribution patterns of the classical representative of the mountain freshwater fauna, caddisfly Rhyacophila tristis, in the Western Carpathians. Based on the COI mitochondrial marker, two genetic lineages (separate BINs) were identified. BIN_W was found in 16 localities in the western part of the study area, BIN_E in 44 eastern localities. The data obtained indicate that BIN_W occurs in a significantly narrower altitude range, BIN_E is more closely related to the Balkan populations than to co-occurring BIN_W, and that the contact zone of the lineages passes through the Western Carpathians. The study revealed phylogeographic and demographic differences between lineages, supporting hypothesis of their evolutionary independence and specific ecological preferences. The obtained genetic data shifted our knowledge on the populations of the studied caddisfly and suggested patterns that could be common to other freshwater species. This could help us to protect unique freshwater ecosystems and preserve European biodiversity.

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 shown 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 G. stasiuki sp. nov. and another we redescribe as G. tatrensis (S. Karaman, 1931).

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 cladeevolved 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.

The freshwater ecosystems in the Republic of North Macedonia are considered as biodiversity hotspot on the European level since they provide diverse habitats that correspond with the complex of ecological preferences that many species require. This specially applies to the freshwater crustaceans that, unfortunately, have never been in the focus of a continuous research. In R. North Macedonia, freshwater crustaceans usually inhabit ecosystems exposed to negative anthropogenic impact. Thus, some species may become extinct presenting an irreversible loss for the Macedonian natural heritage. Although DNA barcoding, as a highly effective tool for fast species detection, is already a routine protocol in many taxonomical studies all over the world, there is still no official national DNA barcoding initiative in Republic of North Macedonia. This study employs DNA barcoding based on the ca. 650-bp long standard fragment of the mt COI gene of Astacus astacus, Austropotamobius torrentium, Potamon fluviatile and Potamon ibericum previously identified based on morphological characters, collected from different parts in R. North Macedonia. The ability of the DNA barcoding to rapidly identify all species has been proven. Our research presents the first comprehensive study that employs DNA barcoding as a molecular tool in decapod taxonomy in Republic of North Macedonia, giving the first attempt to establish DNA barcode reference library for freshwater decapod species in the country. We hope that further application of this approach will lead to the construction of DNA barcode reference library for different aquatic biota in the country. Such a library will find purpose in effective and modern bioassessment protocols as well as in phylogenetic research detecting interpopulation genetic variability.

BackgroundDragonflies 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.MethodsThis 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.ResultsOver 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 inCordulia 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.

Lake Ohrid, located on the Balkan Peninsula at the Albanian-Macedonian border, is the oldest European lake (1.3-1.9 My old) and one of the world’s smallest ancient lakes. Taking into account the size of the lake and its biodiversity, it harbors the highest level of endemism, especially within amphipod crustaceans (ca. 90%) with the endemic Gammarus species flock. Our previous studies upon this flock have shown a substantial decoupling between molecular and morphological diversity, existence of cryptic species and puzzling speciation history. In order to explore sources of observed diversity, in the current study we are investigating ecological preferences of the species within the flock, based on their distribution in depth gradient, in relation to molecular diversity based on DNA barcoding. In the study over 200 barcodes were generated and combined with 173 previously published. The specimens were collected from all depth ranges of Lake Ohrid as well as from springs located on or near the banks of the lake. Within the species flock, 13 BIN’s were identified, 12 previously known and one newly recognized, representing separate lineage and putatively a new species. Two of the flock species were found only in the springs: G. sketi and G. cryptosalemaai. G. sketi, previously found only in springs on the southern banks of Lake Ohrid, has now also been discovered in springs in its north-eastern part. Both species show low haplotypic diversity. All remaining species were recorded from the depth between 20 and 60 meters, that is characterized by the highest ecological diversity with different types of substrates: stones, macrophytes, abundant Dreissena shells as well as sand and silt. Among them G. sywulai, G. macedonicus, G. cryptoparechiniformis, G. lychnidensis, G. ochridensis, G. parechinifromis were found exclusively within this depth range. The three latter species represent single BIN and share haplotypes, at the same time this BIN has the highest number of haplotypes in comparison to others. The remaining species found on this depth represent separate BINs with different levels of haplotype diversity. Only G. lychnidensis, G. stankokaramani and G. solidus were found below the depth of 60 meters, in a quite homogenous environment dominated by silt. In the deepest parts of the lake, between 260 and 290 meters, only G. solidus was found. This species is represented only by three haplotypes while G. stankokaramani is characterized by multiple haplotypes partially shared with G. lychnidensis. The shared haplotype represents the only G. lychnidensis occurrences on the depths below 60 meters. Summarizing, the highest abundance of BINs, species and haplotypes was recovered from the most ecologically diversified depth range of the lake (20 to 60 meters). This suggests that ecological heterogeneity could be the main driver of Gammarus species flock diversification in the ancient Lake Ohrid. Due to the complex pattern of morphological diversity, DNA barcoding proved to be the best if not the only method in identification of the species flock diversity.

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.

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.

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.

The concept of molecular clock was established more than half a century ago. Since then it has played animportant role in studies dealing both with the description of evolutionary processes and with the estimation of timeperiods at which evolutionary lineages diverged. The molecular clock measures the time passed between subsequentevolutionary events, based on the assumed model of evolution (substitution rate). Decades of studies have shownthat the substitution rate is not universal for all organisms. It differs among taxa and this heterogeneity is relatedto a number of factors, such as: body size, generation time, and metabolic rate. Uncovering causes and correlatesof the rate variation have induced development of many new models of molecular clock, taking into account thevariation of substitution rate along the branches in the phylogenetic tree. Proper calibration of molecular clockplays a major role in inferring time-calibrated phylogeny. There are several calibration methods and their applicationdepends on the availability of calibration points for the studied organisms. The main aim of our paper is tofamiliarize the reader with both the theoretical and practical issues related to the idea of molecular clock and itspractical application.

The fresh waters of the Baltic German and Polish lowlands are inhabited by several Gammarus species. One of them, Gammarus fossarum, a common inhabitant of lowland and submontane waters in western ...

The Balkan Lake Skadar used to be recognized as potentially ancient. Recent studies revealed that, although the present lake is of post-Pleistocene origin, its hydrological system is of at least Pliocene origin. The basin of this lake is characterized by rich biodiversity and high endemism, particularly in the local spring areas. Our study upon freshwater shrimps from the family Atyidae conducted in the Skadar Lake as well as adjacent springs and rivers led to the description of a new species Atyaephyra vladoi, on the basis of morphological features and molecular data. Phylogenetic investigation using mitochondrial COI gene sequences revealed that this new species is most closely related to the south-Balkan A. thyamisensis, from which it diverged in Late Miocene. Population of A. vladoi has expanded both in demographic and spatial terms during the last major interglacial period before the Holocene, i.e. Marine Isotope Stage 5 or Eemian. Combining this evidence with occurrence of A. vladoi mainly in the areas under influence of spring and riverine waters, we can conclude that the species is probably a relic of the old marshlands occupying the basin before the present lake. This supports the supposedly ancient, pre-Holocene, origins of Lake Skadar hydrological system.

The molecular population structure of eight benthic Foraminifera with the same or similar morphotypes in shallow waters of West Antarctica and southern Patagonia was analyzed based on the SSU rDNA. The data suggest recent gene flow across the Drake Passage for the foraminiferal species Pullenia subcarinata as well as for a molecular operational taxonomic unit (MOTU) of Micrometula but not for the remaining majority of taxa/MOTUs. The molecular population structures of different taxa shown by haplotype networks is highly variable, indicating different dispersal histories. Antarctic species/MOTUs show star-like topologies reflecting post-glacial demographic and/or spatial expansion from limited refugia on the Antarctic continental shelf. Some Patagonian species/MOTUs show reticulate topologies suggesting more steady and prolonged evolution, while others show star-like topologies that could reflect impact of South American glaciations. The time-calibrated Bayesian phylogenetic reconstruction suggests that the isolation between Antarctic and Patagonian species/MOTUs post-dates the Eocene/Oligocene boundary. Large differences in divergence times between the examined Foraminifera support the hypothesis that separation of ecosystems on the opposite sides of the Drake Passage was a gradual process that started >30 Ma. It seems that the shallow-water monothalamids were the first to be impacted. Among the calcareous forms, the majority of the divergence ages were at 15 Ma or younger, suggesting that the mid-Miocene was the most important period concerning the separation between Antarctic and Patagonian shallow-water benthic biota.

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.

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., This paper is a deliverable of the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219) Working Group 1, led by Torbjørn Ekrem and Fedor Čiampor. Thanks to the University of Minho and University of Pécs for hosting workshops and working group meetings. We also thank staff at National Environment Agencies and others that provided national checklists of taxa used in biomonitoring, and otherwise assisted with checklist proof-reading: Jarmila Makovinská and Emília Mišíková Elexová (Slovakia); Steinar Sandøy and Dag Rosland (Norway); Mišel Jelič (Croatia); Marlen Vasquez (Cyprus); Adam Petrusek (Czech Republic); Kristel Panksep (Estonia); Panagiotis Kaspiditis (Greece); Matteo Montagna (Italy); Marija Katarzyte (Lithuania); Ana Rotter (Slovenia); Rosa Trabajo (Spain); Florian Altermatt (Switzerland); Kristian Meissner (Finland), Rigers Bakiu (Albania), Valentina Stamenkovic and Jelena Hinic (Macedonia); Patricia Mergen (Belgium); Gael Denys & the French Biodiversity Agency (France); Mary Kelly-Quinn (Ireland); Piotr Panek and Andrzej Zawal (Poland); Cesare Mario Puzzi (Italy); Carole Fitzpatrick (United Kingdom); Simon Vitecek (Austria); Ana Filipa Filipe (Portugal); Peter Anton Stæhr & Anne Winding (Denmark); Michael Monaghan (Germany); Alain Dohet, Lionel L'Hoste, Nora Welschbillig & Luc Ector (Luxembourg), Lujza Keresztes, (Romania). The authors also want to thank Dirk Steinke for providing the original European ERMS list for marine taxa and Florian Malard for comments on the manuscript. The preparation of the AMBI checklist was carried out in the scope of a Short-term Scientific Mission (ECOST-STSM-CA15219-150217- 082111) granted to SD visiting AZTI, Spain. ZC was supported by grants EFOP-3.6.1.-16-2016-00004 and 20765-3/2018/FEKUTSTRAT. TE was supported by the NorBOL-grant (226134/F50) from the Research Coun cil of Norway. BR, FL and MFG contributed through support from the GBOL project, which is generously funded by the German Federal Min istry of Education and Research (FKZ 01LI1101 and 01LI1501). MG contributed through support of the Polish National Science Centre, grants N N303 5794 39 and 2014/15/B/NZ8/00266. SF was funded by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER).

The Balkan Peninsula is recognized to be one of the most important biodiversity hotspots worldwide. However, biogeographic patterns of its freshwater fauna are poorly understood due to the lack of basic data on distribution and ecology, particularly for endemic taxa. We studied the distribution, habitats and conservation status of two endemics, Echinogammarus acarinatus and Fontogammarus dalmatinus , in the Dinaric karst rivers. Altogether, 711 sites were sampled from 2000 to 2014 and supplemented by literature data. Although previously known only from rivers of the Adriatic Sea (AS) basin, both species were also found in the Black Sea (BS) basin. E. acarinatus has a mosaic distribution extending from the Una River to the delta of the Neretva River, particularly in the rivers Vrbas and Bosna (BS). Its presence in the AS basin rivers, Krka and Neretva, could result from river capture events by which former tributaries of the Una and Bosna (BS) were directed to the AS basin. Two subspecies of F. dalmatinus are distributed in the Una, Zrmanja and Krka Rivers. F. dalmatinus dalmatinus occurs in upper and middle parts of the Zrmanja (AS), and in the upper part of the Una (BS), while F. dalmatinus krkensis was found exclusively in springs and upper parts of the Krka (AS). Distribution of both subspecies in the Zrmanja and Krka indicates former connection between these rivers. The ecology and conservation status of all the studied taxa are presented and discussed. This study illustrates the importance of including ecological and historical interpretations of species’ distributions for managing conservational issues in the case of freshwater endemics with narrow ranges.

16 pages; International audience; 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.

Neocaridina davidi (Bouvier, 1904) is an exotic freshwater shrimp originating from Asia and often kept as a pet in amateur aquarium cultures. Herewith, we report on the second finding of N. davidi in fresh waters of Europe and the first discovery of that species both in Poland and in Central Europe. The species was found in samples collected in 2003, 2013 and 2017 in the thermally polluted canal connected to the River Oder, south of Gryfino, in the vicinity of the Dolna Odra Power Plant. The taxonomic identity of the collected shrimp was confirmed by the standard DNA barcoding procedure, using a 610 bp-long fragment of cytochrome oxidase I (COI). The findings spanning more than a decade suggest that N. davidi may have established a self-reproducing population at this site. Following the finding of Atyaephyra desmarestii (Millet, 1831) in 2000, Neocaridina davidi is the second freshwater shrimp species found in the River Oder and in Poland.

16 pages; International audience; 1. The effects of biological invasions are generally more detrimental in isolated ecosystems than in the interconnected ones and freshwater lakes appear to be particularly fragile. The Ponto-Caspian freshwater amphipod Dikerogammarus villosus (Pontogammaridae), also known as the killer shrimp, is a highly invasive species that can have significant ecological impacts on receiving ecosystems. It has colonised most of the European main inland waterbodies, including at least 12 lakes in the Alps – an area of high conservational priority and, at the same time, heavily affected by anthropogenic changes. Particularly, overland translocations of boats among tourist centres may have contributed to colonisation of Alpine lakes by D. villosus.2. We aimed to answer the following questions: Which of the two genetically differentiated populations invading continental lowland Europe has contributed to colonisation of the Alpine lakes? Is it possible to detect independent founder events in different regions of the Alps and/or secondary spread among European Alpine lakes? Have the lacustrine populations suffered any reduction in genetic variation during colonisation? Twelve populations from the Alpine lakes were genotyped for a portion of mtDNA (COI and 16S) and for seven nuclear microsatellite loci. A wide range of methods was used to analyse the data, including haplotype network, Bayesian clustering and demography tests.3. Our results show that the lowland western route (Danube-Rhine waterway) of the killer shrimp invasion was the source for introduction to European Alpine lakes. Unlike invasive populations in the main lowland rivers, most of the Alpine populations suffered bottleneck and had significantly lower genetic diversity compared to their source population along the western route. We found clear patterns of genetic structure, revealing multiple independent lake colonisations followed by secondary spread between lakes. An overland transport of D. villosus with tourist boats is probably the major spreading vector to and among Alpine lakes. Implementing safety programmes such as the ‘check, clean, and dry’ procedures may stop or slow down spreading of D. villosus among the Alpine lakes, and prevent additional long distance transport.

Mountainous areas are characterized by substantial biodiversity and endemicity due to their complex geological history and habitat fragmentation. Hence, it can be assumed that particularly high species richness can be found in organisms with limited dispersal capabilities that inhabit mountain streams. A number of scientific papers focus on molecular phylogeography or traditional taxonomy of species or species groups inhabiting such habitats. However, there is a lack of studies that integrate morphological and molecular data to identify and delineate cryptic species. For practical reasons, uncovering cryptic diversity is crucial in taxa used in biomonitoring. Distinct species, hard to separate based on morphology only, may have different tolerance ranges towards a variety of factors. Thus, our goal is to combine the two approaches to reveal potential patterns of diversification within a species widely distributed across European mountains: the amphipod crustacean Gammarus balcanicus. The data were obtained from 13 populations spread across the range of the species. Individuals were initially ascribed to G. balcanicus based on conventional fauna key morpho-anatomical diagnostic features and were further analysed for 23 additional features to explore any putative diversification. Morphometric data were analysed with use of the multiple correspondence analysis and anova. Molecular distances were calculated for 551-bp-long COI sequences. Test for isolation by distance was performed for both morphological and molecular data. The morphometric studies showed that some of the analysed features differed significantly between populations, although there was only a weak correlation between the morphological divergence and the between-population geographical distances. Moreover, high morphological diversity was present within sites. A set of 42 COI haplotypes was identified among the 135 individuals sequenced. No haplotype was shared among populations. The molecular p-distances within the nine localities presenting more than one haplotype were either almost null (ca. 0.14), similar as between other well-defined species of the genus Gammarus. Surprisingly, G. balcanicus appears to be polyphyletic based on topology of the neighbour-joining tree. The level of genetic distance between localities was not correlated with their geographical proximity. Globally, combining spatial patterns of morphological versus molecular divergence indicates a high level of cryptic diversity within a species conventionally defined based upon fauna key morphological features. In this context, the name G. balcanicus should be applied only to the population from locus typicus, while the other populations represent a number of putative distinct species. We may expect that such phenomenon would apply also to other animal taxa with conserved morphology, which are widespread over different mountain ranges in Europe.

Although deep sea has become a subject of intense studies in the recent years, our knowledge of the diversity of benthic organisms, especially at the molecular level, is far from complete. In the present study we focused on Amphipoda, known to be an abundant and diverse component of the marine zoobenthos. COI barcoding of an extensive collection of abyssal and hadal amphipods from the Kuril-Kamchatka Trench (KKT) area revealed 133 Molecular Operational Taxonomical Units (MOTUs). The richness of species varied between stations, with the highest observed in the areas where high organic carbon (Corg) content was recorded. Among the recognized MOTUs as many as 42% were singletons and only 28 were found at three or more stations. Their distribution was variable with seven taxa occurring at the most distant stations situated across the KKT. This shows that the trench does not constitute a barrier for the distribution of certain species. However, it may limit the gene flow in the case of others. The most common MOTUs were divided into three bathymetric categories: typically abyssal and hadal species as well as eurybathic ones inhabiting both the abyssal and hadal depth zones.

Gammarus varsoviensis is morphologically close to G. lacustris, with which it is often misidentified. Geographic range of G. varsoviensis includes Germany, Poland, Lithuania, Latvia, Belarus and Ukraine. Such a distribution pattern led us to the assumption that the species might have originated in the Black Sea drainage area. From there, as early as the nineteenth century, it could possibly have migrated to the Baltic basin through the Pripyat-Bug canal. Thus, the goals of this study are: (1) to indicate the level of genetic divergence of G. varsoviensis from the morphologically closest species – G. lacustris and (2) to investigate the possibility of the Pontic origin of G. varsoviensis and its range expansion across the Black Sea/Baltic Sea watershed to Central Europe through the artificial canal network. Altogether 128 partial 16S rDNA sequences of Gammarus varsoviensis from 19 localities were gained. They were analysed in conjunction with a sequence of G. lacustris obtained in this study from the Dnieper system and sequences of G. lacustris, G. pulex and G. fossarum available in GenBank in order to estimate the relationships among the species. GenBank accession numbers for all the haplotypes defined within this study are: G. varsoviensis – from JN641868 to JN641875; G. lacustris – JN641876. The genetic distance within and between the species was calculated, as well as phylogenetic relationships among haplotypes, which were inferred with Neighbor-Joining method. The haplotype relationships were analysed with the Minimum Spanning Network. Also mismatch distribution of the haplotypes were tested under sudden expansion model and sequence deviations from selective neutrality. Neighbor-Joining analysis revealed that G. varsoviensis and G. lacustris haplotypes formed separate well defined clades. Mean genetic diversity between the two species was ca. 15× higher than the intraspecific distance and similar to the value obtained for G. fossarum/G. pulex species pair. Therefore, G. varsoviensis can be definitely considered as a distinct species from G. lacustris. Among 128 sequences obtained from G. varsoviensis, 8 haplotypes were identified and grouped into two clades: one found only in the lower Dnieper (two haplotypes) and the second one encompassing the rest of haplotypes observed in the upper Dnieper and Baltic Sea basin. Only one haplotype was found in the Baltic Sea drainage area. A mismatch distribution curve as well as selective neutrality tests demonstrated sudden expansion model. Our findings suggest that G. varsoviensis is an alien gammarid that originated in the Pontic area. Its expansion in Central Europe apparently started soon after the opening of the artificial waterways joining the Black and the Baltic Sea drainage basins.

This paper analyses contact zones, range boundaries, and the vertical distribution of three epigean species of Gammarus in the ca. 1200 km long chains of the Sudeten Mountains and the Carpathian Mountains (including the Outer Western and the Central Western Carpathians, as well as the Outer Eastern Carpathians and the Northern Sub-Carpathians), which form the southern border of Poland. The Sudeten Mts. are inhabited exclusively by Gammarus fossarum , up to an altitude of 540 m asl. The species occurs up to 900 m asl in the Outer Western and in the Central Western Carpathians, there often co-occurring with G. balcanicus . The latter species is an altitudinal vicariant to the former. It inhabits also higher localities, and is the sole gammarid species in the Tatra Mts., where it reaches 1400 m asl. In the Outer Eastern Carpathians, G. balcanicus co-occurs with G. leopoliensis up to 920 m asl, replacing it above that elevation and occurring up to at least 1160 m asl. All three species reach the limits of their respective distribution ranges in the area. Localities with G. balcanicus in the Northern Sub-Carpathians form the northwestern border of its range in Europe. G. leopoliensis , restricted to the Eastern Carpathians, is a geographical vicariant to G. fossarum with the meridian 22°E as a quite sharp borderline between the ranges of the two species. The vertical distribution of all three species in the area studied seems to be associated with the altitudinal range of deciduous and mixed forest zones. Possibly, such type of vegetation, through influencing nutrient content and pH conditions in mountain streams, is one of the main factors limiting the vertical distribution of Gammarus spp. Cette publication analyse les zones de contact, l'etendu des limites, et la distribution verticale de trois especes epigees de Gammarus le long des 1200 km de la chaine des montagnes Sudeten et des montagnes des Carpates (incluant le centre et l'extremite ouest des Carpates ainsi que l'extreme est des Carpates et le nord des Sub-Carpates), qui forment la frontiere sud de la Pologne. Les montagnes Sudeten sont habitees exclusivement par Gammarus fossarum , jusqu'a une altitude de 540 m. L'espece est trouvee jusqu'a 900 m dans le centre et l'extremite ouest des Carpates, souvent co-localisee avec G. balcanicus . Cette derniere espece est l'espece remplacante en altitude de G. fossarum . Elle colonise aussi des regions plus elevees et est la seule espece locale des montagnes Tatra, ou elle atteint 1400 m. A l'extremite est des Carpates, G. balcanicus co-habite avec G. leopoliensis jusqu'a 920 m, la remplacant au dessus de cette altitude et presente jusqu'a au moins 1160 m. Ces trois especes atteignent dans cette region les limites respectives de leur distribution. Les zones avec G. balcanicus dans le nord des sub-Carpates sont la limite nord-ouest de sa distribution en Europe. G. leopoliensis , restreinte a l'est des Carpates, est un vicariant geographique de G. fossarum avec le meridien 22°E comme frontiere assez precise entre la distribution des deux especes. La distribution verticale de ces trois especes dans la zone etudiee semble associee avec l'echelonnement altitudinal des zones forestieres decidues ou melangees. Il est possible que ce type de vegetation, par son influence sur le contenu en nutriments et le pH des ruisseaux montagneux, soit l'un des facteurs limitant la distribution verticale de Gammarus spp.

16 pages; International audience; 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.