The amphipod Dikerogammarus villosus has colonized most of the European main inland water bodies in less than 20 years, having deteriorating effect on the local benthic communities. Our aim was to reveal the species phylogeography in the native Black Sea area, to define the source populations for the colonization routes in continental Europe and for the newly established UK populations. We tested for the loss of genetic diversity between source and invasive populations as well as along invasion route. We tested also for isolation by distance. Thirty three native and invasive populations were genotyped for mtDNA (COI, 16S) and seven polymorphic nuclear microsatellites to assess cryptic diversity (presence of deeply divergent lineages), historical demography, level of diversity within lineage (e.g., number of alleles), and population structure. A wide range of methods was used, including minimum spanning network, molecular clock, Bayesian clustering and Mantel test. Our results identified that sea level and salinity changes during Pleistocene impacted the species phylogeography in the Black Sea native region with four differentiated populations inhabiting, respectively, the Dnieper, Dniester, Danube deltas and Durungol liman. The invasion of continental Europe is associated with two sources, i.e., the Danube and Dnieper deltas, which gave origin to two independent invasion routes (Western and Eastern) for which no loss of diversity and no isolation by distance were observed. The UK population has originated in the Western Route and, despite very recent colonization, no drastic loss of diversity was observed. The results show that the invasion of the killer shrimp is not associated with the costs of loosing genetic diversity, which may contribute to the success of this invader in the newly colonized areas. Additionally, while it has not yet occurred, it might be expected that future interbreeding between the genetically diversified populations from two independent invasion routes will potentially even enhance this success.

Background: The Balkans are a major biodiversity and endemism hotspot, worldwide. 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 inGammarus 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 thatG. roeseliithrives 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 ofG. 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 thatG. roeseliiincludes 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 ofG. roeseliicould 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.

Background: Epipactis helleborineis an Eurasian orchid species which prefers woodland environments but it may also spontaneously and successfully colonise human-made artificial and disturbed habitats such as roadsides, town parks and gardens. It is suggested that orchids colonising anthropogenic habitats are characterised by a specific set of features (e.g., large plant size, fast flower production). However, as it is not well known how pollinator diversity and reproductive success ofE. helleborinediffers in populations in anthropogenic habitats compared to populations from natural habitats, we wanted to compare pollinator diversity and reproductive success of this orchid species between natural and anthropogenic habitat types.Methods: Pollination biology, reproductive success and autogamy in populations ofE. helleborinefrom anthropogenic (roadside) and natural (forest) habitats were compared. Eight populations (four natural and four human-disturbed ones) in two seasons were studied according to height of plants, length of inflorescences, as well as numbers of juvenile shoots, flowering shoots, flowers, and fruits. The number and diversity of insect pollinators were studied in one natural and two human-disturbed populations.Results: Reproductive success (the ratio of the number of flowers to the number of fruits) in the populations from anthropogenic habitats was significantly higher than in the natural habitats. Moreover, plants from anthropogenic habitats were larger than those from natural ones. In both types of populations, the main insect pollinators were Syrphidae, Culicidae, Vespidae, Apidae and Formicidae. With respect to the type of pollinators’ mouth-parts, chewing (39%), sponging (34%) and chewing-sucking (20%) pollinators prevailed in anthropogenic habitats. In natural habitats, pollinators with sponging (55%) and chewing mouth-parts (32%) dominated, while chewing-sucking and piercing-sucking insects accounted for 9% and 4% respectively.Discussion: We suggest that higher reproductive success ofE. helleborinein the populations from anthropogenic habitats than in the populations from natural habitats may result from a higher number of visits by pollinators and their greater species diversity, but also from the larger size of plants growing in such habitats. Moreover, our data clearly show thatE. helleborineis an opportunistic species with respect to pollinators, with a wide spectrum of pollinating insects. Summarising,E. helleborineis a rare example of orchid species whose current range is not declining. Its ability to make use of anthropogenically altered habitats has allowed its significant spatial range expansion, and even successful colonisation of North America.

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 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.