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.

Summary1. The recognition of management units (MUs) that respect interpopulation distinctions in management needs is central to many biological applications addressing species conservation, biological invasions and ecosystem processes. 2. We present a methodological approach for the evaluation of population-level differences in the host compatibility of natural populations of affiliate (dependent) species. Two experiments were performed to diagnose the sources of variability in the relationships between an endangered freshwater mussel Unio crassus and its host fish species in a fragmented river system in Central Europe. 3. Despite the common approach applied to the conservation of U. crassus throughout the study area, we identified differences in the ability of U. crassus to infest particular host fish species between nearby and recently isolated mussel populations. These differences could strongly influence their reproductive success under a constant conservation regime. 4. The observed differences were accompanied by genetic and morphometric distinctions, as revealed by microsatellite, mtDNA and elliptic Fourier descriptor analyses. The genetic data indicated substantial reproductive isolation, which could have resulted in adaptive or random changes responsible for the differences in host compatibility. 5. We showed that experimental testing of physiological host compatibility can be effectively used for the detection of different MUs of U. crassus, with direct implications for the management of host populations. In combination with traditional genetic and morphometric techniques, this approach may provide a general framework for the recognition and care of management units of affiliate species. 6. Synthesis and applications. Population-level evaluations of host compatibility should be utilized in management unit recognition and the formulation of management targets for affiliate species. We demonstrated that small-scale cross-compatibility testing can be effectively used to diagnose the sources of variability in host relationships with direct management implications. This approach can enhance the targeting of management actions in many biological applications addressing species conservation, biological invasions and ecosystem processes.

Aim Lake Ohrid is the oldest existing lake in Europe, dating back to the Tertiary. Given its surface area and the adjusted endemism rate, it seems to hold the greatest biodiversity of any ancient lake. Of all the animal groups endemic to this lake, gammarids form one of the largest species flocks. The goal of our study was (1) to develop the phylogenetic framework for the Ohridian endemic Gammarus species flock and place it within a regional palaeobiogeographical context, and (2) to interpret the data with respect to the putative origin of the Lake Ohrid fauna. Location Lake Ohrid, Balkan Peninsula. Methods Sequences of two mitochondrial genes (cytochrome c oxidase subunit I and 16S ribosomal RNA) and one nuclear gene (28S ribosomal RNA) of the Ohridian endemic Gammarus species and of other Balkan gammarids from 69 localities were analysed. The phylogenetic relationships of the group were studied using Bayesian methods. The biogeographical history and the chronology of diversification events were investigated using a relaxed molecular clock with two calibration points. Results It appears that the Lake Ohrid gammarids derive from an old local lineage of the Gammarus balcanicus complex. This lineage arrived from the northern part of the Proto‐Balkans, after its emergence during the Tethys/Paratethys regression, and differentiated during the last 18 Myr of the Dinaric uplift (Alpine orogeny). It is also closely affiliated with the biota endemic to the neighbouring area of the former Miocene lake system and to lineages from the upper Vardar and Črni Drim river systems. Main conclusions The Lake Ohrid endemic fauna is closely affiliated to the local biota and its roots are more ancient than the lake itself.

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

The cytochrome c oxidase subunit I (cox1) gene is the main mitochondrial molecular marker playing a pivotal role in phylogenetic research and is a crucial barcode sequence. Folmer’s “universal” primers designed to amplify this gene in metazoan invertebrates allowed quick and easy barcode and phylogenetic analysis. On the other hand, the increase in the number of studies on barcoding leads to more frequent publishing of incorrect sequences, due to amplification of non-target taxa, and insufficient analysis of the obtained sequences. Consequently, some sequences deposited in genetic databases are incorrectly described as obtained from invertebrates, while being in fact bacterial sequences. In our study, in which we used Folmer’s primers to amplify COI sequences of the crustacean fairy shrimp Branchipus schaefferi (Fischer 1834), we also obtained COI sequences of microbial contaminants from Aeromonas sp. However, when we searched the GenBank database for sequences closely matching these contaminations we found entries described as representatives of Gastrotricha and Mollusca. When these entries were compared with other sequences bearing the same names in the database, the genetic distance between the incorrect and correct sequences amplified from the same species was c.a. 65%. Although the responsibility for the correct molecular identification of species rests on researchers, the errors found in already published sequences data have not been re-evaluated so far. On the basis of the standard sampling technique we have estimated with 95% probability that the chances of finding incorrectly described metazoan sequences in the GenBank depend on the systematic group, and variety from less than 1% (Mollusca and Arthropoda) up to 6.9% (Gastrotricha). Consequently, the increasing popularity of DNA barcoding and metabarcoding analysis may lead to overestimation of species diversity. Finally, the study also discusses the sources of the problems with amplification of non-target sequences.