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

Tentatively dated, the Plio‐/Pleistocene origin of the ancient Lake Ohrid on the Balkan Peninsula makes it the oldest ancient lake in Europe. Given the surface area of the lake and the adjusted endemicity rate, it may be also defined as the most diverse of all the ancient lakes in the world. From all the animal groups endemic to this lake, gammarids are amongst the most scarcely known in terms of their diversity and phylogenetic relationships. Partial DNA sequences of two mitochondrial genes, cytochrome oxidase subunit I (cox1) and 16S ribosomal RNA (16S rRNA) of eight known endemic Gammarus species from the Lake Ohrid valley were analysed. Phylogenetic analyses showed that endemic Gammarus species comprise an ancient species flock, with Gammarus sketi from the feeder springs being their sister taxon outside the lake. Amongst the species inhabiting the lake, Gammarus solidus and Gammarus salemaai are morphologically and molecularly well defined. By contrast, Gammarus ochridensis, Gammarus parechiniformis, Gammarus lychnidensis, and Gammarus stankokaramani revealed high discrepancy between morphological and genetic data. None of these morphospecies form a monophyletic clade and a significant degree of apparent gene flow occurs between them. This could be caused by incomplete lineage sorting and/or hybridization events. Two novel mtDNA lineages were found within the lake, possibly constituting two new species (Gammarus sp. 1 and Gammarus sp. 2). Molecular clock analysis showed that the split between G. sketi and the Gammarus species flock from the lake occurred approximately 5–7 Mya, whereas within the flock there were at least two intralacustrine radiations: one estimated at 2–3 Mya and the second at less than 1 Mya. The first one could be associated with the origin of the lake and the second with the lake water‐level fluctuations during Pleistocene. © 2013 The Linnean Society of London

Quantifying patterns of genetic diversity and differentiation among populations of Arctic birds is fundamental for understanding past and ongoing population processes in the Arctic. However, the genetic differentiation of many important Arctic species remains uninvestigated. Here, phylogeography and population genetics were examined in the worldwide population of a small seabird, the little auk (dovekie, Alle alle)-the most numerous avian species of the Arctic ecosystem. Blood samples or feathers were collected from 328 little auks (325 from the nominate subspecies and 3 from the A. a. polaris) in nine main breeding aggregations in the northern Atlantic and one location from the Pacific Ocean. The mtDNA haplotypes of the two subspecies were not segregated into separate groups. Also, no genetic structure was found within the nominate race based on microsatellite markers. The level of genetic differentiation among populations was low yet significant (mean F ST = 0.005). Some pairwise F ST comparisons revealed significant differences, including those involving the most distant Pacific colony as well as among some Atlantic populations. Weak population differentiation following the model of isolation by distance in the little auk is similar to the patterns reported in other highArctic bird species, indicating that a lack of distinct genetic structure is a common phenomenon in the Arctic avifauna.

Bacterial endosymbionts of aquatic invertebrates remain poorly studied. This is at least partly due to a lack of suitable techniques and primers for their identification. We designed a pair of non-degenerate primers which enabled us to amplify a fragment of ca. 500 bp of the 16S rRNA gene from various known bacterial endosymbiont species. By using this approach, we identified four bacterial endosymbionts, two endoparasites and one uncultured bacterium in seven, taxonomically diverse, freshwater crustacean hosts from temporary waters across a wide geographical area. The overall efficiency of our new WOLBSL and WOLBSR primers for amplification of the bacterial 16S rRNA gene was 100%. However, if different bacterial species from one sample were amplified simultaneously, sequences were illegible, despite a good quality of PCR products. Therefore, we suggest using our primers at the first stage of bacterial endosymbiont identification. Subsequently, genus specific primers are recommended. Overall, in the era of next-generation sequencing our method can be used as a first simple and low-cost approach to identify potential microbial symbionts associated with freshwater crustaceans using simple Sanger sequencing. The potential to detected bacterial symbionts in various invertebrate hosts in such a way will facilitate studies on host-symbiont interactions and coevolution.

Ancient lakes as places of extensive speciation processes have been characterized by a high degree of endemicity and biodiversity. The most outstanding European ancient lake is the oligotrophic and karstic Balkan Lake Ohrid. The lake is inhabited by a number of endemic species, but their evolutionary history is largely unresolved. in the present study, the genetic structure, gene genealogy and demographic history of the representatives of the Ohridian endemic Proasellus species were studied using both biparentally (allozyme loci) and maternally (partial mitochondrial cytochrome oxidase subunit I gene) inherited markers. Both data sets gave similar results and supported discrepancies among genetic differentiation, the current morphology-based taxonomy and bathymetric segregation. Horizontal distribution of endemic Proasellus species (Lake Ohrid vs adjacent feeder springs) within the lake presumably promote parapatric speciation whereas the main role of vertical barriers into diversification processes was not fully supported. The analyses of demographic history suggested the decline of endemic isopod populations. The radiation of endemic Proasellus populations within the lake could have started from the sublittoral/profundal zone towards the littoral or in the opposite direction — from the littoral to the profundal. Our analyses did not exclude both possibilities.