Transcriptome Profile Analyses of Head Kidney in Roach (Rutilus rutilus), Common Bream (Abramis brama) and Their Hybrids: Does Infection by Monogenean Parasites in Freshwater Fish Reveal Differences in Fish Vigour among Parental Species and Their Hybrids?

Hybrid generations usually face either a heterosis advantage or a breakdown, that can be expressed by the level of parasite infection in hybrid hosts. Hybrids are less infected by parasites than parental species (especially F1 generations) or more infected than parental species (especially post-F1 generations). We performed the experiment with blood-feeding gill parasite Paradiplozoon homoion (Monogenea) infecting leuciscid species, Abramis brama and Rutilus rutilus, their F1 generation and two backcross generations. Backcross generations tended to be more parasitized than parental lines and the F1 generation. The number of differentially expressed genes (DEGs) was lower in F1 hybrids and higher in backcross hybrids when compared to each of the parental lines. The main groups of DEGs were shared among lines; however, A. brama and R. rutilus differed in some of the top gene ontology (GO) terms. DEG analyses revealed the role of heme binding and erythrocyte differentiation after infection by blood-feeding P. homoion. Two backcross generations shared some of the top GO terms, representing mostly downregulated genes associated with P. homoion infection. KEGG analysis revealed the importance of disease-associated pathways; the majority of them were shared by two backcross generations. Our study revealed the most pronounced DEGs associated with blood-feeding monogeneans in backcross hybrids, potentially (but not exclusively) explainable by hybrid breakdown. The lower DEGs reported in F1 hybrids being less parasitized than backcross hybrids is in line with the hybrid advantage.

The parasites of a successful invader: monogeneans of the Asian topmouth gudgeon Pseudorasbora parva, with description of a new species of Gyrodactylus.

Monogenean parasites are often co-introduced with their fish hosts into novel areas. This study confirmed co-introduction of two dactylogyrids, Dactylogyrus squameus Gusev, 1955 and Bivaginogyrus obscurus (Gusev, 1955), and a newly described gyrodactylid species, Gyrodactylus pseudorasborae n. sp. into Europe along with their fish host, the invasive topmouth gudgeon Pseudorasbora parva (Temminck & Schlegel) from East Asia. All three species were observed in the lower Dnieper and middle Danube basin regions and had slightly larger haptoral hard parts than the same parasites in their native range. While dactylogyrids occurred sporadically, we recorded regular infection by G. pseudorasborae n. sp. at relatively high prevalence and abundance. This latter species was observed in both the native and non-native range of topmouth gudgeon, and resembles Gyrodactylus parvae You et al., 2008 recently described from P. parva in China. Both species were distinguished based on genetic analysis of their ITS rDNA sequence (6.6% difference), and morphometric differences in the marginal hooks and male copulatory organ. Phylogenetic analysis of dactylogyrid monogeneans showed that B. obscurus clustered with Dactylogyrus species parasitising Gobionidae and Xenocyprididae, including D. squameus, supporting recent suggestions of a paraphyletic origin of the Dactylogyrus genus. In addition to co-introduced parasites, topmouth gudgeon was infected with a local generalist, G. prostae Ergens, 1964, increasing the number of monogeneans acquired in Europe to three species. Nevertheless, monogenean infections were generally lower in non-native host populations, potentially giving an advantage to invading topmouth gudgeon.

Title: Les parasites d'un envahisseur à succès : les monogènes du goujon asiatique Pseudorasbora parva, avec description d'une nouvelle espèce de Gyrodactylus. Abstract: Les parasites monogènes sont souvent co-introduits avec leurs hôtes poissons dans de nouvelles zones. Cette étude a confirmé la co-introduction de deux Dactylogyridae, Dactylogyrus squameus Gusev, 1955 et Bivaginogyrus obscurus (Gusev, 1955), et d'une espèce de Gyrodactylidae nouvellement décrite, Gyrodactylus pseudorasborae n. sp. en Europe, en même temps que leur hôte poisson, le goujon envahissant Pseudorasbora parva (Temminck & Schlegel) qui vient d'Asie de l'Est. Les trois espèces ont été observées dans les régions du bas Dniepr et du bassin moyen du Danube et avaient des parties sclérifiées haptorales légèrement plus grandes que les mêmes parasites dans leur aire de répartition d'origine. Alors que les Dactylogyridae étaient rares, nous avons enregistré une infection régulière par G. pseudorasborae n. sp. à une prévalence et une abondance relativement élevées. Cette dernière espèce a été observée à la fois dans l'aire de répartition indigène et non indigène du goujon asiatique et ressemble à Gyrodactylus parvae You et al., 2008 récemment décrit chez P. parva en Chine. Les deux espèces ont été distinguées sur la base de l'analyse génétique de leur séquence ITS de l'ADNr (différence de 6,6 %) et des différences morphométriques dans les crochets marginaux et l'organe copulateur mâle. L'analyse phylogénétique des monogènes Dactylogyridae a montré que B. obscurus se regroupait avec des espèces de Dactylogyrus parasitant les Gobionidae et les Xenocyprididae, y compris D. squameus, soutenant les suggestions récentes d'une origine paraphylétique du genre Dactylogyrus. En plus des parasites co-introduits, le goujon asiatique était infecté par un généraliste local, G. prostae Ergens, 1964, portant le nombre de monogènes acquis en Europe à trois espèces. Néanmoins, les infections par les monogènes étaient généralement plus faibles dans les populations d'hôtes non indigènes, donnant potentiellement un avantage à l'invasion du goujon asiatique.

Monogeneans in intergeneric hybrids of leuciscid fish: Is parasite infection driven by hybrid heterosis, genetic incompatibilities, or host-parasite coevolutionary interactions?

BACKGROUND: Several hypotheses have been proposed to explain parasite infection in parental species and their hybrids. Hybrid heterosis is generally applied to explain the advantage for F1 generations of hybrids exhibiting a lower level of parasite infection when compared to parental species. Post-F1 generations often suffer from genetic incompatibilities potentially reflected in the higher level of parasite infection when compared to parental species. However, the presence of specific parasites in an associated host is also limited by close coevolutionary genetic host-parasite associations. This study focused on monogenean parasites closely associated with two leuciscid fish species-common bream and roach-with the aim of comparing the level of monogenean infection between parental species and hybrids representing two F1 generations with different mtDNA and two backcross generations with different cyto-nuclear compositions. RESULTS: Monogenean infection in F1 generations of hybrids was lower when compared to parental species, in line with the hybrid heterosis hypothesis. Monogenean infection in backcross generations exhibited similarities with the parental species whose genes contributed more to the backcross genotype. The distribution of monogeneans associated with one or the other parental species showed the same asymmetry with a higher proportion of roach-associated monogeneans in both F1 generations and backcross generation with roach in the paternal position. A higher proportion of common bream-associated monogeneans was found in backcross generation with common bream in the paternal position. CONCLUSIONS: Our study indicated that cyto-nuclear incompatibilities in hybrids do not induce higher monogenean infection in backcross generations when compared to parental species. However, as backcross hybrids with a higher proportion of the genes of one parental taxon also exhibited high level of this parental taxon-associated parasites, host-parasite coevolutionary interactions seem to play an obvious role in determining the level of infection of host-specific monogeneans in hybrids.

Diversity of MHC IIB genes and parasitism in hybrids of evolutionarily divergent cyprinoid species indicate heterosis advantage.

The genes of the major histocompatibility complex (MHC) are an essential component of the vertebrate immune system and MHC genotypes may determine individual susceptibility to parasite infection. In the wild, selection that favors MHC variability can create situations in which interspecies hybrids experience a survival advantage. In a wild system of two naturally hybridizing leuciscid fish, we assessed MHC IIB genetic variability and its potential relationships to hosts' ectoparasite communities. High proportions of MHC alleles and parasites were species-specific. Strong positive selection at specific MHC codons was detected in both species and hybrids. MHC allele expression in hybrids was slightly biased towards the maternal species. Controlling for a strong seasonal effect on parasite communities, we found no clear associations between host-specific parasites and MHC alleles or MHC supertypes. Hybrids shared more MHC alleles with the more MHC-diverse parental species, but expressed intermediate numbers of MHC alleles and positively selected sites. Hybrids carried significantly fewer ectoparasites than either parent species, suggesting a hybrid advantage via potential heterosis.

Vigour-related traits and immunity in hybrids of evolutionary divergent cyprinoid species: advantages of hybrid heterosis?

Hybrid advantage, described as the superiority of hybrids in some traits over their parents and termed the "heterosis effect," is widely documented in the case of reciprocal crosses of parental species (i.e., hybrids representing the F1 generation). In fish, high survival, fast growth and better health status have been widely documented in F1 hybrids. Nonetheless, the effects of interspecific hybridization on vigour, physiology and immunity-related traits in fish are largely unknown, especially concerning native systems of coexisting parental and hybrid genomes in the same habitat. The present study examined the potential physiological and immune aspects of hybrid heterosis by comparing condition status (measured especially by indexes), haematological profile, glucose concentration and selected parameters of non-specific and specific immunity between the evolutionarily divergent non-congeneric cyprinoid species Abramis brama and Rutilus rutilus and their hybrids representing the F1 generation, all of them caught in nature. Clear differences were documented for vigour-related, physiological and immune parameters between the two divergent species. Hybrids generally tended to express intermediate characters of the measured traits, likely generated by the evolutionary divergence of the hybridizing species; nonetheless, for some traits, hybrids exhibited a character that was more similar to one parental species than to the other. This was interpreted as the heterozygote advantage for F1 hybrids. It is suggested that a maternally inherited genetic background may potentially influence the expression of some branches of non-specific immunity or other aspects related to the fish health status.

Distribution of host-specific parasites in hybrids of phylogenetically related fish: the effects of genotype frequency and maternal ancestry?

BACKGROUND: Host specificity is one of the outputs of the coevolution between parasites and their associated hosts. Several scenarios have been proposed to explain the pattern of parasite distribution in parental and hybrid genotypes ranging from hybrid resistance to hybrid susceptibility. We hypothesized that host-parasite co-adaptation limits the infection of host-specific parasites in hybrid genotypes even under the condition of the high frequency of hybrids. The experimental monogenean infection in pure breeds of Blicca bjoerkna and Abramis brama and cross-breeds (the F1 generation of hybrids) under the condition of similar frequencies of pure and hybrid genotypes was investigated. We also examined the potential effect of the maternal origin of hybrids (potential co-adaptation at the level of mitochondrial genes) on monogenean abundance. METHODS: Pure breeds of two cyprinids and two cross-breeds (one with B. bjoerkna, the next with A. brama in the maternal positions) were exposed to infection by monogeneans naturally occurring in B. bjoerkna and A. brama. The experiment was run under similar frequencies of the four breed lines. RESULTS: We showed similar levels of monogenean infection in B. bjoerkna and A. brama. However, each species harboured specific monogenean fauna. Hybrids harboured all monogenean species specifically infecting one or the other species. Monogenean infection levels, especially those of Dactylogyrus specific to A. brama, were lower in hybrids. For the majority of host-specific parasites, there was no effect of the maternal origin of hybrids on monogenean abundance. Asymmetry was found in the distribution of specific parasites in favour of specialists of B. bjoerkna in the monogenean communities of hybrids. CONCLUSIONS: Our results indicate that the maternal mtDNA of hybrids is not an important predictor of host-specific monogenean infection, which may suggest that mitochondrial genes are not strongly involved in the coadaptation between monogeneans and their associated hosts. The asymmetry of species-specific parasites suggests similarity between the molecular components of the immune mechanisms in hybrids and B. bjoerkna. Our results revealed a difference between the degree of host-parasite coadaptation in specific parasites of A. brama and the degree of host-parasite coadaptation in specific parasites of B. bjoerkna and their associated hosts.

A Long Temporal Study of Parasitism in Asexual-Sexual Populations of Carassius gibelio: Does the Parasite Infection Support Coevolutionary Red Queen Dynamics?

Carassius gibelio is an extraordinary cyprinid species exhibiting both sexual and asexual reproduction. We hypothesized that parasitism selection is one of the potential mechanisms contributing to the coexistence of the two reproductive forms of C. gibelio living in the same habitat. We performed a four-year study to investigate the dynamics of parasite infection in C. gibelio. According to the Red Queen prediction, the asexual form is a target of parasite adaptation due to its low genetic variability. Both sexual and gynogenetic forms of C. gibelio exhibited similar levels of prevalence, with monogeneans being the most frequently observed parasite group. We observed the temporal dynamics of parasite infection in the last year of investigation, when both forms were more strongly parasitized. The sexual form was more parasitized by ectoparasites in the first and last years and less parasitized by nematodes in the last year when compared to the gynogenetic form. We found no trend of high parasite infection in gynogenetic mtDNA haplotypes. We conclude that Red Queen dynamics is not the mechanism driving parasite infection in sexual-gynogenetic C. gibelio over a long time scale. Alternatively, we suggest that the dynamics of parasite infection in this complex may be generated by multiple mechanisms.

Patterns of parasite distribution in the hybrids of non-congeneric cyprinid fish species: is asymmetry in parasite infection the result of limited coadaptation?

Hybrids and their parasite diversity represent interesting models for evolutionary ecology. The modified immune response, shifted ecology, inheritance, and maternal ancestry of hybrid host fish are supposed to affect the diversity of their parasite communities. The pattern of metazoan parasite distribution in non-congeneric cyprinids - common bream (Abramis brama) and roach (Rutilus rutilus) (species with different morphology and ecology, and harbouring different specific parasites) - and their hybrids was analysed. Four static alternative scenarios based on parasite infection levels in hybrids and parental taxa are known. The hybrid resistance scenario predicts that hybrids are more resistant than parental taxa, resulting in low parasite infection in hybrids. This scenario is principally consistent with hybrid heterosis advantage. In accordance with this prediction, metazoan parasite abundance and prevalence were higher in parental species when compared with their hybrids. Alternatively, the dynamic Red Queen scenario of infection in hybridising systems predicts parasite adaptation to common hosts. Temporal (six sampling events) and spatial (two sampling sites) aspects as possible factors influencing parasite distribution were analysed. We found no support for this hypothesis, i.e. no changes in the frequency of hybrids or their parental species and no changes in parasite infection in parental species or hybrids were found in the different time periods. The effect of maternal ancestry on infection level was evident; hybrids exhibiting common bream mtDNA were more strongly parasitized by digeneans and crustaceans than hybrids exhibiting roach mtDNA. Hybrids harboured a majority of the specific parasites of both parental species; however, the level of infection of common bream-specific parasites (especially monogeneans) in hybrids was low. Such an asymmetrical distribution of parental species-specific parasites in hybrids may suggest the limited inheritance of protective immunological mechanisms from one parental species and reveal stronger coadaptation between common bream and its specific parasites.

A multilocus assessment of nuclear and mitochondrial sequence data elucidates phylogenetic relationships among European spirlins (Alburnoides, Cyprinidae).

The phylogenetic relationships and taxonomy of the spirlins in the genus Alburnoides are examined by comparative sequencing analysis of mitochondrial and nuclear markers. Molecular analyses revealed 17 Eurasian lineages divided into two main clades, termed the Ponto-Caspian and European in accordance with the lineage distribution. The indel diagnostics of ß-actin and S7 markers and translation of cyt b to the amino acid chain were evaluated as a reliable identifying tool for most of the recognised lineages. Lineage richness is closely connected with the existence of known glacial refugia in most cases. The underestimation of species richness in the genus Alburnoides is confirmed: the genetic analyses support the validity of 11 morphologically accepted species; apart from them, four phylogenetic lineages requiring descriptions as separate species were revealed. The distribution area of the nominotypical species A. bipunctatus s. stricto is newly defined. Two diverging phylogenetic lineages, A. ohridanus, and A. prespensis complex, were observed in the Southeast Adriatic Freshwater Ecoregion, confirmed as a hotspot of endemic biodiversity. A. ohridanus demonstrates high divergence from the A. prespensis complex, represented by three similar mitochondrial lineages with the same nuclear haplotypes and sympatric occurrence. The range restricted endemism was confirmed for at least seven species. The Albanian river systems, as well as the wider Ponto-Caspian basin exhibit complications among definite species delineations and gaps in understanding of microevolutionary processes; these areas require further investigations.

Physiological and condition-related traits in the gynogenetic-sexual Carassius auratus complex: different investments promoting the coexistence of two reproductive forms?

BACKGROUND: Carassius auratus complex is an extraordinary species complex including the diploid and polyploid forms exhibiting asexual and sexual reproduction modes. The coexistence of both forms in the same habitats is currently reported. The stable coexistence of asexual and sexual forms assumes some disadvantages for asexuals that balance the costs of sex. In our study, we hypothesized and tested the differences in physiological (including heamatological and immunological), growth-related, condition-related, and fitness-related traits between gynogenetic females and sexuals. RESULTS: Our results revealed similar growth performance in gynogenetic females and sexuals measured by body size and weight, or expressed by condition factor. The energy allocation in reproduction measured by the relative size of gonads revealed no difference between gynogenetic and sexual females; in addition, both females in spawning expressed the same estradiol levels in blood plasma. We found a gender specific trade-off between investment in reproduction and immunocompetence (measured by the spleen-somatic index). Higher aerobic performance expressed by the heart index and higher oxygen-carrying capacity were found in sexual males, with increasing values before and during spawning. Our study evidenced significantly lower aerobic performance but higher oxygen-carrying capacity per erythrocyte in gynogenetic females when compared to sexuals. IgM production differed between gynogens and sexuals of C. auratus complex. CONCLUSIONS: Our study indicates that a similar amount of energy is invested by both gynogenetic and sexual females of C. auratus complex in reproductive behaviour. We suggest that lower aerobic performance in gynogens may represent their physiological disadvantage balancing the cost of sexual reproduction. A trade-off between the number of erythrocytes and the oxygen-carrying capacity per erythrocyte in sexual males and gynogenetic females may contribute to the coexistence of gynogenetic and sexual forms. In addition, the differences in specific immunity between gynogens and sexuals may also reduce the evolutionary disadvantage of sexual reproduction. In conclusion, we propose that several mechanisms contribute to the coexistence of the gynogenetic-sexual C. auratus complex.

MHC genes and parasitism in Carassius gibelio, a diploid-triploid fish species with dual reproduction strategies.

BACKGROUND: The gibel carp is a fish species with dual reproduction modes, gynogenesis and sexual reproduction, coexisting in mixed diploid-polyploid populations. Following the Red Queen (RQ) assumption, asexual organisms are, due to their low genetic diversity, targets for parasite adaptation. Because MHC polymorphism is maintained by selection from parasites and sexual selection, MHC genes are considered as a suitable candidate for testing the RQ hypothesis. In this study, we investigated MHC variability and the selection pressure acting on MHC genes in sexual diploids and asexual triploids. In addition, we tested whether the asexual form of gibel carp suffers from higher parasite loads than the sexual form. RESULTS: At the population level, genotype and allelic diversity of MHC were reduced in gynogenetic triploids when compared to sexual diploids. Different patterns in positively selected sites (PSS) between gynogens and sexual gibel carp were also found. A weak difference in parasite species richness was found between sexual fish and gynogens. However, two common clones of gynogens were significantly more parasitized than sexual diploids or other gynogens with rare MHC genotypes. At the individual level, the higher number of alleles was not associated with higher parasitism in either sexual diploids or gynogens. CONCLUSIONS: The differences in MHC diversity between gynogenetic triploids and sexual diploids are in accordance with the hypothesis of sexually-mediated selection increasing MHC diversity and fulfil a prerequisite of the Red Queen hypothesis. The different patterns in PSS between gynogens and sexual gibel carp also suggest the potential role of sexual selection and supports parasite-mediated selection maintaining MHC diversity. We showed that the most common MHC genotypes of gynogenetic triploids are the target of parasite selection. Our results suggest that the MHC genotype in gibel carp is more important than allelic number for immunocompetence.

Does interspecies hybridization affect the host specificity of parasites in cyprinid fish?

BACKGROUND: Host specificity varies among parasite species. Some parasites are strictly host-specific, others show a specificity for congeneric or non-congeneric phylogenetically related host species, whilst some others are non-specific (generalists). Two cyprinids, Cyprinus carpio and Carassius gibelio, plus their respective hybrids were investigated for metazoan parasites. The aim of this study was to analyze whether interspecies hybridization affects host specificity. The different degrees of host specificity within a phylogenetic framework were taken into consideration (i.e. strict specialist, intermediate specialist, and intermediate generalist). METHODS: Fish were collected during harvesting the pond and identified using meristic traits and molecular markers. Metazoan parasite species were collected. Host specificity of parasites was determined using the following classification: strict specialist, intermediate specialist, intermediate generalist and generalist. Parasite species richness was compared between parental species and their hybrids. The effect of host species on abundance of parasites differing in host specificity was tested. RESULTS: Hybrids harbored more different parasite species but their total parasite abundance was lower in comparison with parental species. Interspecies hybridization affected the host specificity of ecto- and endoparasites. Parasite species exhibiting different degrees of host specificity for C. carpio and C. gibelio were also present in hybrids. The abundance of strict specialists of C. carpio was significantly higher in parental species than in hybrids. Intermediate generalists parasitizing C. carpio and C. gibelio as two phylogenetically closely related host species preferentially infected C. gibelio when compared to C. carpio, based on prevalence and maximum intensity of infection. Hybrids were less infected by intermediate generalists when compared to C. gibelio. CONCLUSIONS: This finding does not support strict co-adaptation between host and parasite genotypes resulting in narrow host specificity, and showed that hybrid genotypes are susceptible to parasites exhibiting host specificity. The immune mechanisms specific to parental species might represent potential mechanisms explaining the low abundance of parasites in C. gibelio x C. carpio hybrids.

The effect of ploidy and temporal changes in the biochemical profile of gibel carp (Carassius gibelio): a cyprinid fish species with dual reproductive strategies.

The basic biochemical profile of gibel carp (Carassius gibelio), diploid-triploid cyprinid species complex with dual reproduction modes, was investigated. The aim of the study was to test for the effect of fish group (which combines the effects of ploidy and sex) and temporal changes on indicators of the biochemical profile of blood in gibel carp. We showed that the basic biochemical profile of blood is significantly influenced by season and fish group. Triploid gynogenetic females had a better condition measured by total protein concentration than sexual diploids. The higher concentrations of triacylglycerols and cholesterol in triploid females when compared to diploids indicate a higher metabolic rate and higher energy intake during the whole year, which may represent a disadvantage for triploids. Differences in the concentrations of blood biochemical analytes between triploid females and diploids suggest a potential difference in the regulation of a wide range of physiological and biochemical processes in which these analytes participate. The biochemical profiles of two forms of C. gibelio could at least partially explain the changes of character of its naturally occurring populations.

Permanent genetic resources added to molecular ecology resources database 1 April 2012 - 31 May 2012.

This article documents the addition of 123 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Brenthis ino, Cichla orinocensis, Cichla temensis, Epinephelus striatus, Gobio gobio, Liocarcinus depurator, Macrolophus pygmaeus, Monilinia vaccinii-corymbosi, Pelochelys cantorii, Philotrypesis josephi, Romanogobio vladykovi, Takydromus luyeanus and Takydromus viridipunctatus. These loci were cross-tested on the following species: Cichla intermedia, Cichla ocellaris, Cichla pinima, Epinephelus acanthistius, Gobio carpathicus, Gobio obtusirostris, Gobio sp. 1, Gobio volgensis, Macrolophus costalis, Macrolophus melanotoma, Macrolophus pygmaeus, Romanogobio albipinnatus, Romanogobio banaticus, Romanogobio belingi, Romanogobio kesslerii, Romanogobio parvus, Romanogobio pentatrichus, Romanogobio uranoscopus, Takydromus formosanus, Takydromus hsuehshanesis and Takydromus stejnegeri.

Molecular phylogeny of the genus Gobio Cuvier, 1816 (Teleostei: Cyprinidae) and its contribution to taxonomy.

The phylogenetic relationships among gudgeons that represent most nominal taxa within Gobio gobio sensu lato were examined by mitochondrial and nuclear genome sequencing. The molecular analyses confirmed the separate generic status of Gobio as a monophyletic group and revealed 15 Eurasian lineages divided into two main clades, the Northern European and the Ponto-Caspian. The validity of eleven nominal taxa as distinct species was confirmed, gudgeons from the Volga River basin were described as a new species G. volgensis, and three revealed phylogenetic lineages were submitted for a comprehensive revision as "species-in-waiting". The species G. gobio showed a wide range extending from the British Isles to the Black Sea coast and overlapped the areas of several other species. Four pure lineages were detected in the middle Danube River basin. The Crimean Peninsula was found to be a region with the occurrence of individuals of hybrid origin. This region will require special investigation to define species participating in hybridization events, and to establish further steps for the conservation of endemic native gudgeon species. A simple diagnostic method, based on different lengths of the PCR products, called "S7indel diagnostics" is presented for further taxonomic investigations in the genus Gobio.