Revision of the Schistura cincticauda species group (Teleostei, Nemacheilidae) using molecular and morphological markers.

To approach the taxonomy of large and complex animal groups it is of advantage to focus on species groups with shared derived character state. We investigate the composition, morphological characteristics and relationships of and within the Schistura cincticauda species group, whose members are small freshwater fishes that inhabit streams and rivers in eastern Myanmar and western and southern Thailand. A phylogenetic analysis using molecular genetic markers demonstrated the monophyly of this group; a combined genetic and morphological analysis revealed the inclusion of at least twelve species. They share the presence of a pair of black marks on the lower lip, one on each side of the median interruption (these marks may be reduced to few melanophores or even missing in some individuals). Additionally, all species share a small body size (max. 60 mm SL), an incomplete lateral line reaching at most to vertical through anal-fin base, and the absence of sexual dimorphism. Each of the 12 species is diagnosed by a unique combination of character states in fin ray numbers, anus position, presence/absence of an axillary pelvic lobe, and colour pattern. The distribution areas of several species overlap and five cases of syntopic occurrence are known. Five unnamed species are described herein.

Sequence capture: Obsolete or irreplaceable? A thorough validation across phylogenetic distances and its applicability to hybrids and allopolyploids.

As whole-genome sequencing has become pervasive, some have suggested that reduced genomic representation approaches, for example, sequence capture, are becoming obsolete. In the present study, we argue that these techniques still provide excellent tools in terms of price and quality of data as well as in their ability to provide markers with specific features, as required, for example, in phylogenomics. A potential drawback of the wide-scale application of reduced representation approaches could be their drop in efficiency with increasing phylogenetic distance from the reference species. While some studies have focused on the degree and performance of reduced representation techniques in such situations, to our knowledge, none of them evaluated their applicability to inter-specific hybrids and polyploids. This highlights a significant gap in current knowledge since there is increasing evidence for the frequent occurrence of natural hybrids and polyploids, as well as for the major importance of both phenomena in evolution. The main aim of the present study was to carry out a thorough validation of SEQcap applicability to (1) a set of non-model taxa with a wide range of phylogenetic relatedness and (2) inter-specific hybrids of various ploidies and genomic compositions. Considering the latter point, we especially focused on mechanisms causing allelic bias and consequent allelic dropout, as these could have confounding effects with respect to the evolutionary genomic dynamics of hybrids, especially in asexuals, which virtually reproduce as a frozen F1 generation.

Using Species Groups to Approach the Large and Taxonomically Unresolved Freshwater Fish Family Nemacheilidae (Teleostei: Cypriniformes).

Large animal families with unresolved taxonomy are notoriously difficult to handle with respect to their biodiversity, systematics, and evolutionary history. We approach a large and taxonomically unresolved family of freshwater fishes (Nemacheilidae, >600 species) by proposing, on the basis of morphologic data, a species group within the family and study its phylogeny with conclusions regarding its diversity, taxonomy, and biogeographic history. Phylogenetic analyses of two mitochondrial and three nuclear genes of 139 specimens, representing about 46 species (17 candidate species from the proposed species-group, plus 29 comparative species), revealed that the proposed species group does not form a distinct monophyletic lineage, but that the candidate and comparative species mixed in three different lineages. However, the results revealed more than 20% of undescribed species within the ingroup and showed that species do not cluster according to the presently recognised genera. At least one of the genetic clades shows signs of an eastward range expansion during the second half of Miocene from north India via Myanmar into Laos, western China, and western Thailand. We conclude that the approach of picking monophyletic lineages to study biodiversity, systematics, and evolutionary history helps to open the door to large animal families.

One northward, one southward: Contrasting biogeographical history in two benthic freshwater fish genera across Southeast Asia (Teleostei: Cobitoidea: Nemacheilus, Pangio).

Southeast Asia is one of the world's biodiversity hotspots, and the high level of diversity and endemism was reached by colonisation events as well as internal diversification. We investigate the phylogenetic relationships and biogeographic history of the loach genus Nemacheilus, which is widely distributed and common across freshwaters of Southeast Asia. In addition we present the ancestral range reconstruction of the related loach genus Pangio that commonly occurs in the same region as Nemacheilus. Our results reveal that the species currently classified as Nemacheilus in fact are a polyphyletic assemblage; most species are now retaining in a monophyletic Nemacheilus sensu stricto and five species belong to different lineages. We further indicate the existence of hidden diversity within Nemacheilus in the form of several undescribed species. Three major clades (Selangoricus, Masyae and Ornatus) are found within the genus Nemacheilus sensu stricto. These clades generally correspond to the species groups formerly defined on the basis of their pigmentation pattern. The biogeographic analyses show that Nemacheilus most likely originated in mainland Southeast Asia and subsequently expanded in a southward direction to Borneo, Sumatra and Java and the southern Malay Peninsula. In contrast, the genus Pangio originated in Sundaland, from where it extended several times northwards into Indochina and to northern India. Our results demonstrate that small freshwater fishes with restricted dispersal ability are very helpful for the reconstruction of biogeographic history. The contrasting biogeographic history of these two groups of small, benthic and related fish show how complex and case-specific the processes that lead to the biodiversity richness of Southeast Asia are.

Resolving an unnoticed diversity within the Schistura robertsi species complex (Teleostei: Nemacheilidae) using molecules and morphology.

The Schistura robertsi species complex is a group of freshwater fish inhabiting streams in southeast Myanmar as well as in western and southern Thailand. In southern Thailand, the distribution exceeds the biogeographically important 'Surat Thani - Krabi line'. The complex is believed to include five described and one undescribed species, but monophyly and systematics of the group have never been studied explicitly. The present study aims to resolve the number of species within the Schistura robertsi group as well as their distribution areas and phylogenetic relations. We analysed mitochondrial and nuclear sequence data of 86 specimens from 47 localities and 18 morphological characters of 193 specimens. The phylogenetic analyses revealed the S. robertsi complex to be monophyletic and to be composed of ten major lineages. Six of them correspond to the known described or undescribed species, but another four newly identified clades reveal the existence of an overlooked diversity within the group. All genetic lineages are statistically highly supported and all are morphologically diagnosable, suggesting that they represent distinct species. The distribution areas of several clades overlap, the cases of direct co-occurrence show no sign of hybridisation.

Sea water shaping the freshwater biota: Hidden diversity and biogeographic history in the Paracanthocobitis zonalternans species complex (Teleostei: Nemacheilidae) in western Southeast Asia.

Western Southeast Asia is hosting one of the world's most diverse faunas, and one of the reasons for this huge diversity is the complex geologic past of the area, increasing the frequency of isolation and expansion events over evolutionary time scale. As an example case, the present study reveals the phylogeny and biogeographic history of the Paracanthocobitis zonalternans species complex, small benthic freshwater fish (Teleostei: Nemacheilidae) that are commonly occurring across western Southeast Asia (from central Myanmar through western and southern Thailand to northern Malaysia). The group is particularly interesting since it occurs in three biogeographic subdivisions (Indian, Indochinese, Malay/Sundaic) and across all of the major biogeographic barriers in the region. Basing on mitochondrial and nuclear sequence data of 93 samples from about 50 localities we found six major clades, most with exclusive geographic distribution. Divergence time dated the origin of the P. zonalternans species complex to early Miocene (17.8 MYA) and a biogeographic analysis identified the Tenasserim region as the ancestral region. From this region the fish spread during periods of lowered global sea level, particularly during late Miocene (11-8 MYA) northwards into all Burmese river basins and southwards into south Thailand and northern Malaysia. Besides lowered global sea level periods, local stream capture events allowed the complex to expand, e.g. into the Mae Klong basin. Strong fragmentations during periods with elevated sea level during the Pliocene and Pleistocene repeatedly restricted populations to refuges and shaped the observed major lineages. Our results document a higher diversity within the P. zonalternans species complex than formerly believed and a strong impact of global sea level on its evolutionary history. Low sea levels promoted dispersal and elevated sea levels fragmentation events. A very similar impact of sea level changes can be expected in all stationary fauna (freshwater and terrestrial) in all non-mountainous coastal regions worldwide.

Dynamics of tandemly repeated DNA sequences during evolution of diploid and tetraploid botiid loaches (Teleostei: Cobitoidea: Botiidae).

Polyploidization has played an important role in the evolution of vertebrates, particularly at the base of Teleostei-an enormously successful ray-finned fish group with additional genome doublings on lower taxonomic levels. The investigation of post-polyploid genome dynamics might provide important clues about the evolution and ecology of respective species and can help to decipher the role of polyploidy per se on speciation. Few studies have attempted to investigate the dynamics of repetitive DNA sequences in the post-polyploid genome using molecular cytogenetic tools in fishes, though recent efforts demonstrated their usefulness. The demonstrably monophyletic freshwater loach family Botiidae, branching to evolutionary diploid and tetraploid lineages separated >25 Mya, offers a suited model group for comparing the long-term repetitive DNA evolution. For this, we integrated phylogenetic analyses with cytogenetical survey involving Giemsa- and Chromomycin A3 (CMA3)/DAPI stainings and fluorescence in situ hybridization with 5S/45S rDNA, U2 snDNA and telomeric probes in representative sample of 12 botiid species. The karyotypes of all diploids were composed of 2n = 50 chromosomes, while majority of tetraploids had 2n = 4x = 100, with only subtle interspecific karyotype differences. The exceptional karyotype of Botia dario (2n = 4x = 96) suggested centric fusions behind the 2n reduction. Variable patterns of FISH signals revealed cases of intraspecific polymorphisms, rDNA amplification, variable degree of correspondence with CMA3+ sites and almost no phylogenetic signal. In tetraploids, either additivity or loci gain/loss was recorded. Despite absence of classical interstitial telomeric sites, large blocks of interspersed rDNA/telomeric regions were found in diploids only. We uncovered different molecular drives of studied repetitive DNA classes within botiid genomes as well as the advanced stage of the re-diploidization process in tetraploids. Our results may contribute to link genomic approach with molecular cytogenetic analyses in addressing the origin and mechanism of this polyploidization event.

Leptobotia micra, a new species of loach (Teleostei: Botiidae) from Guilin, southern China.

Leptobotia micra, new species, is described from the upper Li River (Pearl River basin) around Guilin in Guangxi province, southern China. The new species is evidently the smallest species of Leptobotia, with females of 45‒46 mm SL bearing oocytes. It can be distinguished from all other species of Leptobotia by a combination of the following characters: no dark bars or dorsal saddles on body, a row of white dots along dorsal midline, 4+34 vertebrae, a predorsal distance of 58.1‒59.0% SL, eye diameter 1.8‒2.0 % SL, pelvic fins not reaching anus, an emarginated caudal fin (length of median rays 1.3‒1.4 times in length of lower lobe) and the anus positioned distinctly closer to anal-fin origin than to pelvic-fin base.

The complete mitochondrial transcript of the red tail loach Yasuhikotakia modesta as assembled from RNAseq (Teleostei: Botiidae).

Here, we present the first complete mitochondrial genome of the Red Tailed Loach Yasuhikotakia modesta (Teleostei: Botiidae) from Thailand, assembled from next-generation transcriptome sequencing data. The assembled transcript corresponds to the full length mitochondrial genome of Y. modesta, which measured 16,865 bp in length, and contained 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. A slight A + T bias was observed in the mitogenome of Y. modesta with an overall base composition of 32.2% A, 25.8% T, 26.4% C, and 15.4% G, and a GC content of 41.8%. The gene arrangement was identical to that of previously described loach mitogenomes.

Leptobotia bellacauda, a new species of loach from the lower Yangtze basin in China (Teleostei: Cypriniformes: Botiidae).

A new species, Leptobotia bellacauda is described from the lower Yangtze River basin. The new species is distinguished from all other species of Leptobotia by a combination of the following characters: body plain brown, prominent black bar in caudal fin, dorsal half of head dusky black, ventral half of head cream colour, eye well developed, lobes of caudal fin rounded, origin of pelvic fins beneath or anterior to dorsal-fin origin, and pectoral fin in adult males enlarged, with numerous tubercles.

A Ploidy Difference Represents an Impassable Barrier for Hybridisation in Animals. Is There an Exception among Botiid Loaches (Teleostei: Botiidae)?

One of the most efficient mechanisms to keep animal lineages separate is a difference in ploidy level (number of whole genome copies), since hybrid offspring from parents with different ploidy level are functionally sterile. In the freshwater fish family Botiidae, ploidy difference has been held responsible for the separation of its two subfamilies, the evolutionary tetraploid Botiinae and the diploid Leptobotiinae. Diploid and tetraploid species coexist in the upper Yangtze, the Pearl River and the Red River basins in China. Interestingly, the species 'Botia' zebra from the Pearl River basin combines a number of morphological characters that otherwise are found in the diploid genus Leptobotia with morphological characters of the tetraploid genus Sinibotia, therefore the aim of the present study is to test weather 'B.' zebra is the result of a hybridisation event between species from different subfamilies with different ploidy level. A closer morphological examination indeed demonstrates a high similarity of 'B.' zebra to two co-occurring species, the diploid Leptobotia guilinensis and the tetraploid Sinibotia pulchra. These two species thus could have been the potential parental species in case of a hybrid origin of 'B.' zebra. The morphologic analysis further reveals that 'B.' zebra bears even the diagnostic characters of the genera Leptobotia (Leptobotiinae) and Sinibotia (Botiinae). In contrast, a comparison of six allozyme loci between 'B.' zebra, L. guilinensis and S. pulchra showed only similarities between 'B.' zebra and S. pulchra, not between 'B.' zebra and L. guilinensis. Six specimens of 'B.' zebra that were cytogenetically analysed were tetraploid with 4n = 100. The composition of the karyotype (18% metacentric, 18% submetacentric, 36% subtelocentric and 28% acrocentric chromosomes) differs from those of L. guilinensis (12%, 24%, 20% and 44%) and S. pulchra (20%, 26%, 28% and 26%), and cannot be obtained by any combination of genomes from L. guilinensis and S. pulchra. Phylogenetic reconstructions based on sequence data of the mitochondrial cytochrome b gene and the nuclear RAG-1 gene invariably places 'Botia' zebra as sister species to S. pulchra, while L. guilinensis is only distantly related. The presented combination of genetic data demonstrates that 'B.' zebra is not the result of a hybridisation, but a species of tetraploid genus Sinibotia with a striking morphological evolution towards an enormous similarity with a co-occurring, but not directly related species. The complete lack of knowledge of the ecology of these species, their main predators or their ecological interactions hampers any conclusion regarding the evolutionary advantage of such adaptation.

Karyotype differentiation in 19 species of river loach fishes (Nemacheilidae, Teleostei): extensive variability associated with rDNA and heterochromatin distribution and its phylogenetic and ecological interpretation.

BACKGROUND: Loaches of the family Nemacheilidae are one of the most speciose elements of Palearctic freshwater ichthyofauna and have undergone rapid ecological adaptations and colonizations. Their cytotaxonomy is largely unexplored; with the impact of cytogenetical changes on this evolutionary diversification still unknown. An extensive cytogenetical survey was performed in 19 nemacheilid species using both conventional (Giemsa staining, C- banding, Ag- and Chromomycin A3/DAPI stainings) and molecular (fluorescence in situ hybridization with 5S rDNA, 45S rDNA, and telomeric (TTAGGG)n probes) methods. A phylogenetic tree of the analysed specimens was constructed based on one mitochondrial (cytochrome b) and two nuclear (RAG1, IRBP) genes. RESULTS: Seventeen species showed karyotypes composed of 2n = 50 chromosomes but differentiated by fundamental chromosome number (NF = 68-90). Nemachilichthys ruppelli (2n = 38) and Schistura notostigma (2n = 44-48) displayed reduced 2n with an elevated number of large metacentric chromosomes. Only Schistura fasciolata showed morphologically differentiated sex chromosomes with a multiple system of the XY1Y2 type. Chromomycin A3 (CMA3)- fluorescence revealed interspecific heterogeneity in the distribution of GC-rich heterochromatin including its otherwise very rare association with 5S rDNA sites. The 45S rDNA sites were mostly located on a single chromosome pair contrasting markedly with a pattern of two (Barbatula barbatula, Nemacheilus binotatus, N. ruppelli) to 20 sites (Physoschistura sp.) of 5S rDNA. The cytogenetic changes did not follow the phylogenetic relationships between the samples. A high number of 5S rDNA sites was present in species with small effective population sizes. CONCLUSION: Despite a prevailing conservatism of 2n, Nemacheilidae exhibited a remarkable cytogenetic variability on microstructural level. We suggest an important role for pericentric inversions, tandem and centric fusions in nemacheilid karyotype differentiation. Short repetitive sequences, genetic drift, founder effect, as well as the involvement of transposable elements in the dispersion of ribosomal DNA sites, might also have played a role in evolutionary processes such as reproductive isolation. These remarkable dynamics of their genomes qualify river loaches as a model for the study of the cytogenetic background of major evolutionary processes such as radiation, endemism and colonization of a wide range of habitats.

Genome differentiation in a species pair of coregonine fishes: an extremely rapid speciation driven by stress-activated retrotransposons mediating extensive ribosomal DNA multiplications.

BACKGROUND: Sympatric species pairs are particularly common in freshwater fishes associated with postglacial lakes in northern temperate environments. The nature of divergences between co-occurring sympatric species, factors contributing to reproductive isolation and modes of genome evolution is a much debated topic in evolutionary biology addressed by various experimental tools. To the best of our knowledge, nobody approached this field using molecular cytogenetics. We examined chromosomes and genomes of one postglacial species pair, sympatric European winter-spawning Coregonus albula and the local endemic dwarf-sized spring-spawning C. fontanae, both originating in Lake Stechlin. We have employed molecular cytogenetic tools to identify the genomic differences between the two species of the sympatric pair on the sub-chromosomal level of resolution. RESULTS: Fluorescence in situ hybridization (FISH) experiments consistently revealed a distinct variation in the copy number of loci of the major ribosomal DNA (the 45S unit) between C. albula and C. fontanae genomes. In C. fontanae, up to 40 chromosomes were identified to bear a part of the major ribosomal DNA, while in C. albula only 8-10 chromosomes possessed these genes. To determine mechanisms how such extensive genome alternation might have arisen, a PCR screening for retrotransposons from genomic DNA of both species was performed. The amplified retrotransposon Rex1 was used as a probe for FISH mapping onto chromosomes of both species. These experiments showed a clear co-localization of the ribosomal DNA and the retrotransposon Rex1 in a pericentromeric region of one or two acrocentric chromosomes in both species. CONCLUSION: We demonstrated genomic consequences of a rapid ecological speciation on the level undetectable by neither sequence nor karyotype analysis. We provide indirect evidence that ribosomal DNA probably utilized the spreading mechanism of retrotransposons subsequently affecting recombination rates in both genomes, thus, leading to a rapid genome divergence. We attribute these extensive genome re-arrangements associated with speciation event to stress-induced retrotransposons (re)activation. Such causal interplay between genome differentiation, retrotransposons (re)activation and environmental conditions may become a topic to be explored in a broader genomic context in future evolutionary studies.

Synthesis of clonality and polyploidy in vertebrate animals by hybridization between two sexual species.

Because most clonal vertebrates have hybrid genomic constitutions, tight linkages are assumed among hybridization, clonality, and polyploidy. However, predictions about how these processes mechanistically relate during the switch from sexual to clonal reproduction have not been validated. Therefore, we performed a crossing experiment to test the hypothesis that interspecific hybridization per se initiated clonal diploid and triploid spined loaches (Cobitis) and their gynogenetic reproduction. We reared two F1 families resulting from the crossing of 14 pairs of two sexual species, and found their diploid hybrid constitution and a 1:1 sex ratio. While males were infertile, females produced unreduced nonrecombinant eggs (100%). Synthetic triploid females and males (96.3%) resulted in each of nine backcrossed families from eggs of synthesized diploid F1s fertilized by haploid sperm from sexual males. Five individuals (3.7%) from one backcross family were genetically identical to the somatic cells of the mother and originated via gynogenesis; the sperm of the sexual male only triggered clonal development of the egg. Our reconstruction of the evolutionary route from sexuality to clonality and polyploidy in these fish shows that clonality and gynogenesis may have been directly triggered by interspecific hybridization and that polyploidy is a consequence, not a cause, of clonality.

Phylogeny of the Southeast Asian freshwater fish genus Pangio (Cypriniformes; Cobitidae).

The genus Pangio is one of the most species-rich of the loach family Cobitidae and widespread across South and Southeast Asia. Its species diversity has never been studied under a clear phylogenetic approach, but four 'species-groups' were proposed according to the most obvious morphological characters. We present here phylogenetic analyses of the genus Pangio based on sequence data of the mitochondrial cytochrome b gene, the nuclear recombination-activating gene 1 (RAG 1) and a combined dataset of 109 specimens from 18 morphologically identified species across the whole distribution area of the genus. Our data reveal the existence of three major lineages within Pangio. Two of our major lineages were congruent with formerly proposed species-groups, the remaining two species-groups together formed the third major lineage; herein we refer to the lineages as to anguillaris-group, kuhlii-oblonga group and shelfordii-group. The application of a molecular clock dated the age of the three lineages to 33-29 million years. At the species level, our data suggest about 30 distinct lineages, indicating that there is a high number of undescribed species within Pangio. The use of Pangio to address biogeographic questions is demonstrated with the example of the shelfordii-group, which is distributed across Sundaland.

Molecular phylogeny of the freshwater fish family Cobitidae (Cypriniformes: Teleostei): delimitation of genera, mitochondrial introgression and evolution of sexual dimorphism.

The family Cobitidae represents a characteristic element of the Eurasian ichthyofauna. Despite diverse features of sexual dimorphism, comparably few morphological characters have been utilized for taxonomic studies resulting in many unresolved puzzles. Here we present the phylogenetic relationships of Cobitidae as inferred from the mitochondrial cytochrome b gene and the nuclear gene RAG-1. Analyses of both markers show a group of eight nominal genera, which all occur in Europe and eastern, northern and western Asia, forming a monophyletic lineage (northern clade) while all other clades inhabit South and Southeast Asia (southern lineages). While all eight southern lineages correspond to genera as defined by morphological studies, only four lineages were reliably recovered within the northern clade, and of these only one (Sabanejewia) corresponds to a formerly considered genus. The genera Cobitis, Iksookimia and Niwaëlla were polyphyletic. A comparison of the two markers shows several incongruities within the northern clade and mitochondrial introgression at least in the genus Misgurnus. Mapping the characters of sexual dimorphism on our cladogram, we identified five character states that are diagnostic for certain lineages. Estimations of the divergence times dated the separation of the northern clade from the southern lineages to the middle Eocene (46 MYA) and the origin of "Cobitis"misgurnoides, the basal taxon of the northern clade, during early Oligocene (30-35 MYA). The geographic distribution of the major clades supports recently developed hypotheses about the river history of East Asia and further suggests that a range expansion of the northern clade in late Miocene (15 MYA) led to the colonisation of Europe by three already distinct genera.

The gynogenetic reproduction of diploid and triploid hybrid spined loaches (Cobitis: Teleostei), and their ability to establish successful clonal lineages--on the evolution of polyploidy in asexual vertebrates.

Polyploidisation is assumed to have played a significant role in the evolution of hybrid asexual lineages. The virtual absence of natural asexual systems in which more than a single ploidy level successfully establishes successful independent clonal lineages is generally explained by the strong effects of polyploidisation on fitness. Experimental crosses were made between diploid and triploid asexual Cobitis elongatoides x C. taenia hybrids (female) and both parental spined loach species (male). Genotyping of the progeny using allozymes and multilocus DNA fingerprinting, along with flow cytometric measurement of ploidy level, demonstrated the occurrence of gynogenetic reproduction in both female biotypes. The incorporation of the sperm genome occurred in some progeny, giving rise to a higher ploidy level, but the rate of polyploidisation differed significantly between the diploid and triploid females. These outcomes are consistent with the existence of developmental constraints on tetraploidy, which determine the rarity of tetraploids in natural populations. No cases of ploidy level reduction were observed. Since diploid and triploid hybrid populations occur where the lack of potential progenitor excludes the possibility of de novo origin, it is probable that both diploid and triploid females can establish successful clonal lineages. Spined loaches represent a unique example, among asexual vertebrates, where more than one ploidy level can establish persistent clonal lineages, which are reproductively independent of one another.

Across Siberia and over Europe: Phylogenetic relationships of the freshwater fish genus Rhodeus in Europe and the phylogenetic position of R. sericeus from the River Amur.

We present the phylogenetic relationships of the freshwater fish genus Rhodeus from 45 localities in Europe and three localities in the Amur basin using the mitochondrial cytochrome b sequence. The study aims to (1) test the colonisation history between Europe and East Asia and (2) to reconstruct the evolutionary history of Rhodeus in Europe. Our analyses revealed five monophyletic clades which correspond to four species: R. sericeus in the Amur basin, R. meridionalis in the River Vardar, R. colchicus in western Caucasus and two sister lineages in most of Europe which together represent R. amarus. The reconstructed colonisation scenario assumes a continuous distribution of Rhodeus from Europe through Siberia until East Asia during Pliocene, depletion of the Siberian population in late Pliocene, isolation of the population in River Vardar during early Pleistocene, refuges during late Pleistocene glaciations in the Danubian basin, in the lowlands of western Black Sea and in western Caucasus Mountains and a post-glacial expansion from the Danubian refuge towards western Europe and from the Black Sea refuge across Eastern Europe.