Genetic structure of species complex of the spined loach Cobitis auc. (Cypriniformes: Cobitidae) in Severskiĭ Donets river basin

Biochemical, genetic, cytometric and morphological analyses of spined loaches of the middle stream of Severskiy Donetz river revealed 3 bisexual species: Cobitis taenia s.l. (68% of the sample); C. melanoleuca (11%); Sabanejewia aurata (9%) and 2 hybrid forms: triploid C. taenia(2)-sp. (9%) and diploid C. taenia-melanoleuca (3%). Distinctive features of genetic structure of polyploid hybrids C. taenia(2)-sp. as well as taxonomic identity of diploid C. taenia s.l. of Severskiy Donetz river were discussed in regard to the Dnieper population ones.     

Phylogenetic relationships and historical biogeography of spined loaches (Cobitidae, Cobitis and Sabanejewia) as indicated by variability of mitochondrial DNA

For sequence analysis of the 12S rRNA gene in spined loaches, specimens of the following taxa were used: Sabanejewia balcanica, Cobitis paludica, C. bilineata, C. fahireae, C. elazigensis, C. elongata , two different sub-populations of C. taenia and four different sub-populations of C. turcica . Phylogenetic relationships among taxa were estimated using parsimony, neighbor-joining, and maximum likelihood algorithms. The calculation of the transition-tranversion ratio indicated that the taxa analysed were rather distantly related. Our analyses using the genus Sabanejewia as an outgroup suggest that C. bilineata is separated from the subgenus Cobitis sensu stricto and placed together with C. elongata as basal to all other species of the genus Cobitis . Support for the placement of C. paludica as basal to both the subgenera Bicanestrinia and Cobitis s. s. (without C. bilineata ) is given by outcomes from three independent methods of phylogenetic reconstruction. A sister-taxon relationship for the subgenera Bicanestrinia and Cobitis s. s. without C. bilineata was consistently found. C. elazigensis was closely related to the population of C. turcica from Lake Beysehir in Turkey. Both formed a sister-group to the remaining populations of C. turcica , while C. fahireae was basal to C. taenia . A molecular clock was calculated based on sequence divergence values and palaeogeographical data. This suggests that different historical colonization routes must have been used by different clades of spined loaches.     

Diversity of European spined loaches (genus Cobitis L.): an update of the geographic distribution of the Cobitis taenia hybrid complex with a description of new molecular tools for species and hybrid determination

Although the unique features of asexual reproduction and hybridization among European spined loaches (genus Cobitis ) have recently attracted the attention of conservation biologists, faunists and evolutionary biologists, the research has suffered from uncertain identification of specimens and their genomes because of the extreme morphological similarity of all the species within the hybrid complex. In this article, a Europe-wide study is reported, which was performed on samples collected by several research teams. Several complementary methodologies, such as allozyme analysis, karyotyping, flow cytometry and DNA sequencing allowed us to confirm or reject the existence of all previously reported species and their hybrids as well as to uncover several new hybrid biotypes. The biogeography of all the known biotypes, that is, parental species and hybrid biotypes, has been summarized here and the taxonomic position of two undescribed putative species mentioned in previous publications has been established. New polymerase chain reaction restriction fragment length polymorphism markers for species determination have further been developed and applied, which would allow the unambiguous identification of parental species and their genomes in the known hybrid biotypes within the complex.     

Ice age cloning--comparison of the Quaternary evolutionary histories of sexual and clonal forms of spiny loaches (Cobitis; Teleostei) using the analysis of mitochondrial DNA variation

Recent advances in population history reconstruction offered a powerful tool for comparisons of the abilities of sexual and clonal forms to respond to Quaternary climatic oscillations, ultimately leading to inferences about the advantages and disadvantages of a given mode of reproduction. We reconstructed the Quaternary historical biogeography of the sexual parental species and clonal hybrid lineages within the Europe-wide hybrid complex of Cobitis spiny loaches. Cobitis elongatoides and Cobitis taenia recolonizing Europe from separated refuges met in central Europe and the Pontic region giving rise to hybrid lineages during the Holocene. Cobitis elongatoides due to its long-term reproductive contact with the remaining parental species of the complex--C. tanaitica and C. spec.--gave rise to two clonal hybrid lineages probably during the last interglacial or even earlier, which survived the Würmian glaciation with C. elongatoides. These lineages followed C. elongatoides postglacial expansion and probably decreased its dispersal rate. Our data indicate the frequent origins of asexuality irrespective of the parental populations involved and the comparable dispersal potential of diploid and triploid lineages.     

European colonization by the spined loach (Cobitis taenia) from Ponto-Caspian refugia based on mitochondrial DNA variation

In the last 20 years, new species, asexual reproduction, polyploidy and hybridization have all been reported within the genus Cobitis. An understanding of the current distribution and baseline phylogeographical history of 'true' nonhybrid Cobitis species is crucial in order to unravel these discoveries. In the present work, we investigated the phylogeography of the spined loach, Cobitis taenia, using 1126 bp of the mitochondrial cytochrome b gene from 174 individuals collected at 47 sites. In total, 51 haplotypes that differed at 49 positions (4.35%) were detected. We deduce that C. taenia survived European glaciations in at least three refugees in the Ponto-Caspian area. Two of these refugees each provided a major lineage that recolonized Europe in separate directions: one westward to England and the other spreading north into Russia before moving west. A third (minor) lineage that contributed little to the recolonization of Europe was also revealed--remaining near its Black Sea refuge. However, more recent history was difficult to resolve with colonization from a more western refugium during the last glacial maximum (LGM) a distinct possibility. Nested clade analysis indicates a pattern of restricted gene flow with isolation by distance at the first two levels and overall. Unlike many other European freshwater fish species, the Danube is not part of the current distribution of C. taenia, nor was it used as either a refuge or a source of colonization of Europe. Low genetic diversity within C. taenia suggests that its colonization of Europe is relatively recent. Demographic analyses revealed a history of recent expansion and isolation by distance.     

Molecular differentiation of three loach species (Pisces, Cobitidae) based on the nuclear 5S rDNA marker

The diversity of the 5S rDNA fragment among three loach species: Cobitis taenia, C. elongatoides and Sabanejewia aurata was investigated using universal PCR primers for this gene. Three amplification products were obtained: 220 bp length for C. taenia and C. elongatoides, and 330 bp for S. aurata. Two amplicons with the same length (in Cobitis) were digested with TaqI restriction endonuclease. This enzyme found one restriction site T/CGA in the C. elongatoides fragment, while in the case of C. taenia no cleavage effect was observed. On this basis we constructed an easy and cheap method for loach species discrimination. It seems adequate for effective support of conservation initiatives for endangered loaches.     

Comparative morphology and histology of the ovaries of the spined loach Cobitis taenia L. and natural allopolyploids of Cobitis (Cobitidae)

The maturity of ovaries, gonado-somatic index and size of oocytes in particular developmental stages during the reproductive period of spined loach Cobitis taenia and allotriploid and allotetraploid Cobitis were examined. The ploidy levels of all the loaches were determined according to karyotypes. All the investigated females spawned multiple times and the spawning period was from the end of May (water temperature c. 18·5° C) to mid- or the end of July. Some of the triploids had a longer spawning period which lasted until the end of August. Each female could lay several portions of eggs which differed in size and number during the spawning season. The relative proportion of oocytes of each stage in the ovaries during the reproductive period changed. The sizes of oocytes at the particular stages of triploids were statistically significantly larger than oocytes in the respective stages in C. taenia (diploid). The sizes of the oocytes (each stage) of C. taenia from both populations were not significantly different.     

Development of nine polymorphic microsatellite loci in the spined loach, Cobitis taenia, and cross-species amplification in the related species C. elongatoides, C. taurica and C. tanaitica

Nine polymorphic microsatellite loci were developed for the spined loach, Cobitis taenia (Teleostei: Cobitidae). The loci were validated using 50 individuals from a population in Belgium. Moderate to high levels of polymorphism were detected (two to 11 alleles). In addition, most markers amplified successfully in three closely related taxa that are known to hybridize with C. taenia: C. elongatoides, C. taurica and C. tanaitica. Some of the loci are most likely diagnostic among species. These markers will be valuable for the study of the historical and contemporary interactions within C. taenia and the Cobitis species complex.     

Cobitis derzhavini sp. nova—a New Spined Loach Species (Teleostei: Cobitidae) Discovered in the Transcaucasia

Journal of Ichthyology - The study of spined loaches from the southwestern Caspian Sea basin revealed one new species. Cobitis derzhavini sp. nova is described from the lower Kura River drainage in...     

Making it on their own: sperm-dependent hybrid fishes (Cobitis) switch the sexual hosts and expand beyond the ranges of their original sperm donors

Interspecific hybridization may result in asexual hybrid lineages that reproduce via parthenogenesis. Contrary to true parthenogens, sperm-dependent asexuals (gynogens and hybridogens) are restricted to the range of bisexual species, generally the parental taxa, by their need for a sperm donor. It has been documented that asexual lineages may rarely use sperm from a non-parental species or even switch a host. The available literature reports do not allow distinguishing, between whether such host switches arise by the expansion of asexuals out of their parental's range (and into that of another's) or by the local extinction of a parental population followed by a host switch. The present study combines new and previously collected data on the distribution and history of gynogenetic spined loaches (Cobitis) of hybrid origin. We identified at least three clonal lineages that have independently switched their sperm dependency to different non-parental Cobitis species, and in cases incorporated their genomes. Our current knowledge of European Cobitis species and their hybrids suggests that this pattern most probably results from the expansion of gynogenetic lineages into new areas. Such expansion was independent of the original parental species. This suggests that sperm dependence is not as restrictive to geographical expansion when compared with true parthenogenesis as previously thought.     

The gynogenetic reproduction of diploid and triploid hybrid spined loaches (<Emphasis Type="Italic">Cobitis</Emphasis>: 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 × 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.     

Contents of Trace Elements as Indicators of Ecological Divergence between Sympatric Spined Loaches (Teleostei,Cobitidae) from the Upper Dnieper

The contents of eight trace elements (Ti, Mn, Ni, Cu, Zn, Sr, Ba, and Pb) in muscles of syntopic sexual and clonal spined loaches (the golden loach Sabanejewia baltica, diploid males and females of Cobitis taenia, and congeneric triploid clonal females) from the upper Dnieper River and in the syntopic spined loaches (males and females of C. melanoleuca and males of C. taenia) from the upper Volga River basin were determined using the X-ray fluorescence spectroscopy technique. The contents of Cu in the clonal triploid Cobitis females and diploid C. taenia females from the Dnieper are different. The intersexual differences in the contents of Pb and Zn between C. taenia males and females, as well as the interspecific differences in the content of Ti between the spined loaches from the Dnieper and Volga basins were revealed. The concentrations of Cu and Pb correlated with the individual fish size. The potential for the use of the revealed differences in the trace element contents as an indicator of the divergence of ecological niches in the syntopic spined loaches is discussed.     

PIT-tagging Italian spined loach (Cobitis bilineata): Methodology,survival and behavioural effects

The Italian spined loach (Cobitis bilineata) is an elongated, small-sized (<12 cm) spined loach native to northern Italy, Slovenia and Croatia. As for loaches in general, little is known about the individual movements of this loach in nature. Passive integrated transponders (PIT-tags) are small (typically 7–32 mm), relatively cheap and allow tracking of individual fish movements and behaviour. A fundamental assumption in animal telemetry is that the performance of a tagged animal does not deviate substantially from its natural performance. Although PIT-tagged fish often display high survival and tag retention, the effect varies between species and contexts, and few studies have looked at behavioural effects of PIT-tagging. Here we demonstrate a PIT-tagging methodology for spined loaches, and compare survival, activity and provoked escape response (maximum swimming speed) between tagged and control fish. We also track tag retention in the tagged fish. Italian spined loaches tagged with 12 mm PIT-tags displayed high tag retention and no extra mortality, and no effects of tagging on activity or maximum swimming speed were observed. The tag-to-fish weight and length ratios in our study ranged from 2% to 5% and from 10% to 16%, respectively, and we conclude that PIT-tagging, within these ratios, appears suitable for Italian spined loach.     

Influence of salinity on early development in the spined loach

Spined loach Cobitis taenia developed successfully between 0·12 and 4·80‰ salinity. At 6·00‰, net production was strongly reduced, and development failed at or above 7·20‰. Below 0·12‰ S, net production became variable, indicating restrictive effects. In comparison with other primary freshwater fish species C. taenia has a low sensitivity to salinity. The upper limit for early development was equal to the highest salinity under which C. taenia adults are found along the Baltic coast. Therefore, salinity should not limit early development within the brackish habitats of spined loach.     

Genetic diversity, origin and patterns of geographic distribution of polyploid spiny loach (Cypriniformes, Cobitidae, Cobitis) in Ukraine

Only 16 biotypes from 28 possible ones, which could be generated during hybridization of C. elongatoides, on the one hand, and representatives of superspecies C. taenia s. lato (C. taenia, C. tanaitica, C. species-1), on the other hand, were discovered in water areas of Ukraine. Furthermore. two biotypes, presumably C. aff. melanoleuca-tanaitica, were discovered in the north-western regions. Polyploids have compounded 65% of investigated individuals of Cobitis genus, the average quantity of males has compounded less than 1% and it was equal among triploids and tetraploids. Absence of amphidiploids and also lack of sufficiently large number of individuals with recombined genotype and abnormal electrophoretic spectra should be stressed. Two loci of polyploid biotypes formation are marked out: southern (the Lower Danube) and northern (the Upper Danube, Oder and Rhine). C. taenia and non-specified C. species, typical of the north-western basins of Ukraine, participate in formation of polyploids in the last mentioned river alongside with C. elongatoides and C. tanaitica. It is established that in spite of clearly evident expansion capacity, so-called southern biotypes (C. 2 (3) elongatoides-tanaitica, C. elongatoides--2 (3) tanaitica) in comparison with the northern ones, which genome includes chromosomal complement C. taenia or C. species-1, are sharply limited in their spread to the East. The reason of such a situation is unbalanced gynogenetic crossing between females of the southern biotypes and C. taenia males, accompanied by introgressions, genetic instability and reduced posterity viability.     

Evolutionary history of asexual hybrid loaches (Cobitis: Teleostei) inferred from phylogenetic analysis of mitochondrial DNA variation

Reconstruction of the evolutionary history of asexual lineages undermines their suitability as models for the studies of evolutionary consequences of sexual reproduction. Using molecular tools we addressed the origin, age and maternal ancestry of diploid and triploid asexual lineages arisen through the hybridization between spiny loaches Cobitis elongatoides, C. taenia and C. tanaitica. Reconstructions of the phylogenetic relationships among mitochondrial DNA (mtDNA) haplotypes, revealed by sequence analyses, suggest that both hybrid complexes (C. elongatoides-taenia and C. elongatoides-tanaitica) contained several asexual lineages of independent origin. Cobitis elongatoides was the exclusive maternal ancestor of all the C. elongatoides-tanaitica hybrids, whereas within the C. elongatoides-taenia complex, hybridization was reciprocal. In both complexes the low haplotype divergences were consistent with a recent origin of asexual lineages. Combined mtDNA and allozyme data suggest that the triploids arose through the incorporation of a haploid sperm genome into unreduced ova produced by diploid hybrids.     

Phylogenetic relationships among the Ibero-African cobitids (Cobitis, cobitidae) based on cytochrome b sequence data

We estimated the phylogenetic relationships of all Ibero-African spined loaches of the genus Cobitis using the complete mitochondrial cytochrome b gene (1140bp). We analysed 93 individuals of seven cobitid species found in all the principal drainages of the Iberian Peninsula and North Africa. A molecular phylogeny was used to revise current systematics of the Ibero-African Cobitis species and to infer a biogeographical model for Cobitis in the Western Mediterranean area during the Cenozoic period. Phylogenetic analysis provided support for the monophyly of two mtDNA clades: Clade A or Italian Clade with the Italian species (C. bilineata, C. zanandreai), and Clade B or the Ibero-African Clade. The Ibero-African Clade included all species endemic for the Iberian Peninsula (C. vettonica, C. calderoni, and C. paludica) and North Africa (C. maroccana). The species C. paludica does not constitute a natural group, and could be divided into at least four monophyletic mtDNA lineages with moderate to high bootstrap values and posterior probability support. Phylogenetic relationships of the Ibero-African species were not resolved satisfactorily, but in all analyses C. calderoni from Northern Iberian Peninsula was basal. We have re-calibrated a molecular clock for the genus Cobitis (0.68% per million year by pairwise) using populations inhabiting both sides of the Gibraltar Strait. Application of this Cobitis mtDNA clock provides evidence that the Messinian salinity crisis played a primary role in the diversification of some Ibero-African cobitid species. The basal polytomies of the Ibero-African Clade might suggest that all mtDNA lineages diversified rapidly.     

Dynamic Formation of Asexual Diploid and Polyploid Lineages: Multilocus Analysis of Cobitis Reveals the Mechanisms Maintaining the Diversity of Clones

Given the hybrid genomic constitutions and increased ploidy of many asexual animals, the identification of processes governing the origin and maintenance of clonal diversity provides useful information about the evolutionary consequences of interspecific hybridization, asexuality and polyploidy. In order to understand the processes driving observed diversity of biotypes and clones in the Cobitis taenia hybrid complex, we performed fine-scale genetic analysis of Central European hybrid zone between two sexual species using microsatellite genotyping and mtDNA sequencing. We found that the hybrid zone is populated by an assemblage of clonally (gynogenetically) reproducing di-, tri- and tetraploid hybrid lineages and that successful clones, which are able of spatial expansion, recruit from two ploidy levels, i.e. diploid and triploid. We further compared the distribution of observed estimates of clonal ages to theoretical distributions simulated under various assumptions and showed that new clones are most likely continuously recruited from ancestral populations. This suggests that the clonal diversity is maintained by dynamic equilibrium between origination and extinction of clonal lineages. On the other hand, an interclonal selection is implied by nonrandom spatial distribution of individual clones with respect to the coexisting sexual species. Importantly, there was no evidence for sexually reproducing hybrids or clonally reproducing non-hybrid forms. Together with previous successful laboratory synthesis of clonal Cobitis hybrids, our data thus provide the most compelling evidence that 1) the origin of asexuality is causally linked to interspecific hybridization; 2) successful establishment of clones is not restricted to one specific ploidy level and 3) the initiation of clonality and polyploidy may be dynamic and continuous in asexual complexes.     

Genetic structure of a diploid-polyploid complex of the spined loach Cobitis taenia (Cypriniformes: Cobitidae) from the middle Dnieper bassin

Biochemical genetic typing and cytometry showed that polyploid females account for 87% of the spined loach Cobitis taenia population from the middle Dnieper basin. The polyploidy series included triploids, tetraploids, and, possibly, a few pentaploids. A characteristic feature of the genetic structure of polyploids was that their genetic variation was due to the clonal variation in the haploid portion of the genome originating from Cobitis sp. and to polymorphism of the diploid portion originating from C. taenia. The results are discussed with regard to comparative evolution of alloploid complexes in fish and terrestrial vertebrates.     

Age, reproduction and fecundity of the spined loach Cobitis taenia L. (Pisces, Cobitidae) from Lake Klawój (Poland)

This is the first study concerning the features of the reproduction process of the karyologically identified spined loach C. taenia (2n=48). The histology of 71 ovaries, and gonadosomatic index (GSI) of karyologically identified spined loach Cobitis taenia L. from Lake Klawój (Northern Poland) were examined. The absolute and relative fecundity of 25 females was estimated by gravimetric method. The age of fish was determined according to the annual increments of otholits. The spawning of C. taenia from Lake Klawój took place from May to July, at a water temperature exceeding 18.5 degrees C. The GSI values at the beginning of the reproduction period ranged from 7 to 19%. The average absolute fecundity of females was 2078 eggs, with the number ranging from 869 to 3371 eggs. High individual variability in the gonad histology and the GSI values during the reproductive period was observed. Such variability could be the result of beginning the reproduction process in the fish at various times and, probably, due to the various numbers of batches laid and various numbers of eggs per batch.