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.     

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.     

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.     

Species and hybrid richness in spined loaches of the genus Cobitis (Teleostei: Cobitidae), with a checklist of European forms and suggestions for conservation

Until recently, one widespread species of the genus Cobitis was thought to be present in Europe, the common spined loach Cobitis taenia . Recent studies have shown that the diversity of spined loaches is considerably higher due to (1) presence of several species as well as C. taenia and (2) presence of hybrid biotypes, living together with species. Here knowledge about distribution and diversity of spined loaches in Europe is summarized by combining original with published data. In Southern Europe, a number of distinct species occur allopatrically, while in Central and Eastern Europe a few species have wide distribution areas. Hybrid biotypes were found exclusively in Central and Eastern Europe. They were not restricted to the contact areas between the parental species, but occur in almost the entire area. In total, 15 hybrid biotypes with different genomic compositions were recorded. In the most diverse complex, four hybrid biotypes were associated with one species. In general, a syntopic occurrence of different species of Cobitis can be considered as exceptional, while the association of species with hybrid biotypes is the rule in Central and Eastern Europe. The composition of complexes seems to be most strongly influenced by the kind of associated species and by local history. Implications for conservation are (1) not to disturb the natural patterns of diversity, (2) to consider the known richness in legislation, and (3) to consider the evolutionary significance of hybrid biotypes.     

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.     

Allozyme variation of African and Iberian populations of the genus Cobitis

African and Iberian populations of the genus Cobitis exhibit high morphological polymorphism and their taxonomy is unclear. In seven such populations the percentage of polymorphic loci ranged between _ P =0.08–0.48 and mean heterozygosity per locus H =0.005–0.073. The lowest Nei genetic distance between African and Iberian populations of Cobitis was D =0.250. The populations separated with the formation of the Gibraltar strait 5 million years ago. C. calderoni is a well-differentiated branch from the rest of the Iberian populations of the genus. Our analysis supports the concept that C. paludica populations and C. maroccana are referable to the subgenus Iberocobitis . These African and Iberian populations represent two taxa, C. maroccana Pellegrin, 1929 and C. paludica De Buen, 1930. C. maroccana and C. paludica populations are monophyletic and the African population is the most differentiated genetically. The Arrago population of C. paludica is genetically the most differentiated of the Iberian populations. Our allozyme data do not support the previous consideration of the Albufera lagoon population as a different species from C. paludica .     

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.     

Vicariance, colonisation, and fast local speciation in Asia Minor and the Balkans as revealed from the phylogeny of spined loaches (Osteichthyes; Cobitidae)

We reconstruct the phylogeny of the morphologically diagnosable subgenera Bicanestrinia, Beysehiria, and Cobitis sensu stricto of the genus Cobitis from Asia Minor and the Balkans. We used the complete cytochrome b gene of 65 specimens in order to infer their evolutionary history in this zoogeographically interesting area. Our phylogeographic analysis did not evidence the previously suggested monophyly of the Bicanestrinia subgenus but revealed five monophyletic lineages in the area: the lineages Bicanestrinia I-IV including all species of Bicanestrinia plus the lineage Cobitis s. str. The monotypic subgenus Beysehiria from Lake Beysehir in Anatolia was closely related to the syntopic population of C. turcica and nested inside the lineage Bicanestrinia III. The strictly allopatric distribution of the four lineages of Bicanestrinia suggests that vicariance has played a major role in the diversification of Bicanestrinia. All analysed species of Cobitis s. str. from Asia Minor and Balkans were closely related to Cobitis s. str. from Central Europe, the Danube basin and the Caucasus, indicating at least two colonisation events into Asia Minor and the Balkans. A third, recent colonisation event led to the presence of C. strumicae, generally restricted to the Aegean Sea drainage, in the Danube basin. Besides the evidences of vicariance and colonisation events in the phylogenetic history of the genus Cobitis in Asia Minor and the Balkans, our analysis suggested also a rapid morphological evolution of C. bilseli in a lacustrine environment. Application of Cobitis mitochondrial cytochrome b clocks of 0.68% sequence divergence per million years (MY) suggest that the split between the five major lineages happened approximately 12.4-17.6 MYA, and according to the lack of basal resolution of this monophyletic group probably the split of all lineages happened within a narrow time window.     

The molecular systematics and biogeography of the European cobitids based on mitochondrial DNA sequences

Phylogenetic inference regarding the biogeography and evolution of the family Cobitidae depends in large part on the correct interpretation of transitions between the morphological states of secondary sexual characters (e.g., the scale of Canestrini or lamina circularis). Here, we use the complete mitochondrial ATP synthase 8 and 6 and cytochrome b genes to provide an independent assessment of systematics and biogeographic relationships of species in the genus Cobitis, including geographic and subgeneric sampling of species with Canestrini's scale present, duplicated, or absent. The mtDNA-based phylogeny for the genus Cobitis provides the first formal hypothesis for the group and permits a phylogenetic-based assessment of the morphological transitions demonstrated by Canestrini's scale. Our data confirm the monophyly of the genus Cobitis and indicate that European Cobitis comprise six evolutionarily independent lineages. These lineages were defined by nucleotide synapomorphies permitting bootstrapped confidence estimates of 95% or greater and mtDNA genetic distances greater than 4.5% and correspond with moderate fidelity to the Cobitis groups defined by Bacescu (1962, Rev. Roum. Biol. 4, 435-448). The Caucasian lineage, C. cf. sibirica, represents the basal sister species of the genus Cobitis, supporting an eastern Asiatic origin of the European Cobitis: Cobitis sensu stricto, Acanestrinia, Bicanestrinia, Iberocobitis, and Cobitis calderoni. Phylogenetic relationships among Cobitis subgenera and species indicate that the ancestral condition of one scale of Canestrini was duplicated once at the origin of the Bicanestrinia lineage and has been independently lost by C. calderoni and C. elongata. The absence of the scale of Canestrini is the synapomorphy defining the subgenus Acanestrinia, but the mtDNA phylogeny indicates that Acanestrinia is not a natural group and places C. calderoni as the sister lineage to the subgenus Iberocobitis, a finding that is also geographically parsimonius.     

Length–weight relationships of six species of the genera Cobitis and Sabanejewia (Cobitidae) in Iranian river systems

Length‐weight relationships (LWRs) were estimated for six spined loach species from Iran. Data were collected between 2010 and 2015. These represent the first reports of length–weight data for five of the species (Cobitis avicennae, Cobitis faridpaki, Cobitis keyvani, Sabanejewia aurata and Sabanejewia caspia) while also providing additional data on the remaining species (Cobitis linea). Five of these species are endemic and the remaining S. caspia, is native to the country. Estimated b values obtained for the studied species ranged from 2.381 for S. aurata to 3.411 for C. keyvani.     

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.     

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.     

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 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.     

Occurrence of parasites in Cobitidae from Croatian rivers draining into two different watersheds

Members of the Cobitidae family are not economically important, thus few studies have been made on their pathology. A survey of parasitic fauna of some representatives of the loaches, such as Cobitis elongata , C. elongatioides, and the recently described C. bilineata, was carried out during the spring and summer of 2006. Fish samples were collected from four Croatian rivers: the Sava, Drava and Kupa draining into the Danubian system, and the Zrmanja draining into the Adriatic system. Specimens were of varying size, weight and age. Ectoparasitological examination revealed infections by the ciliate parasites Trichodina nigra (61; 29%), Epistylis sp. (12; 9%), Chilodonella cyprinid (22; 58%), Ichthyophthyrius multifiliis (81; 81%), dinoflagellate Piscioodinium pillulare (6; 45%) and monogenean Gyrodactylus cobitis (22; 22%). Histopathological examination of haematoxylin-eosin stained tissues showed the presence of Sarcocystis sp. in the muscle of one specimen. Dissections revealed an isolated finding of plerocercoid of Ligula colymbi in a Drava river specimen. A more comprehensive survey is necessary to determine the actual threat of the parasitoses to endangered species of the Cobitidae family.     

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.     

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.     

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.     

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.