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.     

Biochemical genetic evidence for a hybrid origin of spined loach, Cobitis taenia taenia, in Japan

Starch gel electrophoresis and isoelectric focusing were performed on sarcoplasmic proteins from three taxa of cobitid fish in Japan, Cobitis biwae , C. taenia striata and C. l. taenia . These taxa are hardly distinguishable from each other by external appearance or morphological characters. Electrophoretic patterns of sarcoplasmic proteins from white muscle suggested fixed allelic differences between C. biwae and C. t. striata at two protein-coding loci (parvalbumin and creatine kinase). The third taxon, C. t. taenia , showed composite banding patterns with respect to the above two proteins, suggesting C. t, taenia originated from hybridization between C. biwae and C, t. striata . Previously, C. t, taenia and C. t. striata were believed to have shared a common ancestor subsequent to their divergence from C. biwae .     

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.     

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.     

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.     

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.     

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.     

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 microsatellite markers for the red tide‐forming harmful species Chattonella antiqua,C. marina,and C. ovata (Raphidophyceae)

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide‐forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.     

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.     

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.     

Taxonomic study of the Iberian Cobitis (Osteichthyes, Cobitidae), with description of a new species

Morphometric and electrophoretic variation are analysed among Iberian and African populations of Cobitis (Cobitidae). Three discrete morphotypes are distinguished, showing a remarkable genetic differentiation among them. Each of the three morphotypes is judged to represent a species, one being described as a new species. Both approaches agree on the separate status of the Iberian populations from the Alagón drainage, Tagus basin of west central Spain. This population is described as a new species, Cobitis vettonica sp. nov., a sexually dimorphic spined loach was assigned to the subgenus Iberocobitis Bacescu, 1961. Morphometric and electrophoretic data placed this new species phenetically close to Cobitis maroccana Pellegrin, 1929 and Cobitis pahdica De Buen, 1930, but differs from these species in overall shape, male coloration pattern, shorter barbels and fins, posterior lamina of the cleithrum, seven branched rays on the dorsal fin and in having a unique alleles at sAAT-2*, EST-1* and MPI* loci. The new species range described in this paper is more restricted than that of C. pahdica. Both inhabit the Tagus basin but have not been reported to be sympatric.     

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.     

Microsatellite markers for the hybridizing tiger swallowtails, Papilio glaucus and P. canadensis, and their applicability to historic specimens and congeneric species

We report the development of microsatellite markers for the sister species Papilio glaucus and P. canadensis (Papilionidae: Lepidoptera). All 16 markers displayed a high degree of variation in both species, ranging from eight to 24 alleles per locus. Substantial heterozygote deficits were observed for several loci, indicating the presence of null alleles. All markers were successfully used to genotype dried samples from a historical collection. Cross-species amplification with six additional Papilio species showed that most loci can be used to study genetic variation in other closely related species of tiger swallowtails.     

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.     

New details of the ultrastructure (TEM, SEM) of taste buds in fishes

Quantitative distribution of the taste buds (TB) in different parts of the body and the fine structure of the TB components are described in Cobitis taenia L. No evidence of synaptic contacts between any cellular components in the TB has been found. The afferent synapses have been recognized only at the gustatory cells and at the basal cell. The microvillar processes on the upper side of the basal cell are demonstrated for the first time in fishes. These processes resemble those at the basal cells in the TB of tetrapods and at the Merkel cells, scattered in the epidermis of all vertebrates. Since the basal cells of fish TB correspond also to other criteria of the Merkel cells, its mechanoreceptive function in the TB is discussed. A relatively large number of atypical gustatory cells has been found in the studied material. As the examined specimens of C. taenia lived for a long time in aquarium conditions, it may be supposed that these gustatory cells are damaged by pollutants introduced mainly with food such as Tubifex. For purposes of comparison, a related species, Misgurnus fossilis L., was used in the study.     

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.     

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 .     

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.     

Feeding habits and diet in two populations of spined loach, Cobitis taenia (L.)

SUMMARY. The feeding habits of Cobitis taenia were studied by means of monthly samples taken over a 2-year period, October 1972-October 1974, from two sites, one on the River Great Ouse at Newport Pagnell and the other at some neighbouring gravel pits. Dietury components, seasonal variation in these components, and seasonal and diurnal variation in feeding intensity were examined. The ecological niche of the food items and their size range are discussed, since both these factors appeared important in governing the micro-habitat of the fish.
The feeding activity followed a seasonal cycle with little or no feeding in the winter and a high level of feeding activity in mid-summer.
The diet showed a predominance of small food items, such as chydorids. copepods and rhizopods. These items varied in occurrence during the feeding period which appeared to be related to their own abundance, pointing to non-seleclive feeding by the fish. The small size and benthic habits of the food items, many of which are closely associated with surface layers of mud and weed, indicated a preference by Cobitis taenia (L.) for feeding in this type ot habitat and on this type of food, in contrast to the stone loach. Noemacheilus barbatulus (L.), which is believed to inhabit stony areas and take larger food items.