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.

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

1. 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.2. 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).3. 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. 4. 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. 5. 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. K E Y W O R D S Asellus aquaticus, Europe, phylogeography, Pleistocene, post-glacial colonisation | 1029 SWOROBOWICZ et al. Whole Data Set N = 740, H = 263, S = 185, K = 16.27, G = 11 Proglacial area N = 352, H = 132, S = 129, K = 10.51, G = 9 Periglacial area N = 388, H = 138, S = 162, K = 18,95, G = 11 Whole N = 81, H = 22, S = 62, K = 8.93, G = 7 Whole N = 158, H = 59, S = 90, K = 10.52, G = 7 Whole N = 113, H = 63, S = 82, K = 10.24, G = 5 Whole N = 156, H = 81, S = 130, K = 15.60, G = 8 Alps N = 23, H = 6, S = 23, K = 6.34, G = 3 N = 208, H = 54, S = 105, K = 18.82, G = 6 Pontic Province Whole N = 172, H = 39, S = 93, K = 17.51, G = 6 SE Plains N = 36, H = 18, S = 56, K = 14.32, G = 2 NE Plains N = 54, H = 32, S = 62, K = 10.76, G = 4 Hungarian Lowlands N = 101, H = 59, S = 88, K = 10.24, G = 5 Central Highlands N = 39, H = 10, S = 37, K = 10.59, G = 5 Central Plains N = 107, H = 48, S = 79, K = 10.08, G = 7 Ireland N = 5, H =...

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.