Isolation and characterization of microsatellite loci from chub Leuciscus cephalus (Pisces: Cyprinidae)

Twelve microsatellite markers were isolated from chub Leuciscus cephalus Linné, 1766 (Cyprinidae), a freshwater fish widely distributed in Europe. We assessed the level of genetic diversity for these loci in 24 individuals sampled in the Rhone river watershed. Nine loci were polymorphic, displaying from two to 13 alleles per locus with expected heterozygosity ranging from 0.160 to 0.887. Simultaneous migration of different polymerase chain reaction products was developed for routine analysis of L. cephalus populations.     

Genetic variation and divergence of Leuciscus pyrenaicus and L. carolitertii (Pisces, Cyprinidae)

The taxonomy of the genus Leuciscus in the Iberian Peninsula has been presented on the basis of morphological analysis. Two species are believed to occur in Portuguese inland waters, L. carolitertii , the northern basins chub, and L. pyrenaicus , which was described for the Tejo and southern basins. Since only slight differences in the meristic and morphometric characters are observed, the distinction between the two species has been made previously mainly on the basis of osteology. In order to investigate the patterns of genetic differentiation between these species, 24 sites were sampled for both species, and examined electrophoretically for the study of variation at 27 presumptive loci. The results pointed to different levels of population differentiation among drainages and support the recognition of the species level for L. pyrenaicus and L. carolitertii. Our results also suggested that the distribution area of L. pyrenaicus is probably restricted to Tejo and to the small drainages near Tejo, while L. carolitertii seems to have a widespread distribution area, including Lima, Douro, Vouga, Mondego, Guadina and Sado drainages. Moreover, concerning the southern populations, Mira and Arade, the surprisingly high level of differentiation observed at several loci, supports the existence of a further species occurring in these small drainages. The evolutionary aspects related to that differentiation are discussed.     

Identifying main evolutionary mechanisms shaping genetic variation of Leuciscus cephalus L. 1758 (Cyprinidae) in western Greece: discordance between methods.

Numerous methods can be used in intraspecific phylogeographic studies to infer the evolutionary mechanisms that shaped observed genetic variation in populations. However, these methods are scarcely used jointly, and the evolutionary outcomes they could propose are not fully compared. In this study, using a chub (Leuciscus cephalus; Cyprinidae) mitochondrial DNA data set (13 populations in Western Greece, 14 haplotypes), we compare three distinct 'historical' methods that could possibly infer relative importance of basic evolutionary mechanisms (isolation vs migration) shaping genetic variation: the nested clade analysis, the psi-test and the 'mismatch distributions'. Taking together, interpretations of these analyses allow to draw a picture of the evolutionary history of chub in Western Greece based on isolation and genetic drift for higher clades. However, results issued each method can differ for low differentiated clades. We discuss such differences and suggest that methods should be used jointly in phylogeographic studies for a better evaluation of the evolutionary mechanisms that shaped genetic variation.     

Nested clade and phylogeographic analyses of the chub, Leuciscus cephalus (Teleostei, cyprinidae), in Greece: implications for Balkan Peninsula biogeography

Phylogenetic relationships among Greek populations of the chub, Leuciscus cephalus, were investigated using 600 bp of the cytochrome b gene. The aim of this study was to test the assumption that the main difference in ichthyological composition between both sides of the Balkan Peninsula is directly linked to differences in the dispersion mechanisms used by fish in order to extend their distribution range. Phylogenetic and nested clade analyses clearly showed that populations in Greece are significantly differentiated. Greek populations were found to descend from three lineages in three geographical provinces: Western, Central, and Eastern Greece. The chub reached Western Greece at the beginning of the Pleistocene and Eastern Greece during the mid-Pleistocene. Chub dispersion occurred mainly by river confluence due to sea level lowering and river capture in Western Greece and sea dispersal with low-salinity conditions within the Aegean Sea in Eastern Greece. However, in Central Greece, the original mtDNA lineage has presumably been lost owing to a genetic introgression following a second invasion from the Danube during the final stage of the last glaciation. This study provides new elements for a better understanding of the composition of the contemporary ichthyofauna in Greece and highlights possible evolutionary mechanisms responsible for the high endemism rate in the Western Greek biogeographic province.     

Microsatellites from the vairone Leuciscus souffia (Pisces: Cyprinidae) and their application to closely related species

Eleven microsatellites were isolated from the vairone Leuciscus souffia (Risso 1826), an endangered fish that inhabits river systems in and around the Alps in Europe. The level of genetic diversity was assessed in 29 individuals of the subspecies L. s. souffia, and their variability was further estimated in seven individuals of a different subspecies, L. s. muticellus. Eight of these microsatellite loci were also applied to seven closely related cyprinid species. Availability of the reported microsatellite loci will facilitate the investigation of population genetic structure of these species with applications for the development of conservation strategies and phylogeographical approaches.     

Comparative analysis of mitochondrial DNA variation in four species of Far Eastern redfins of the genus Tribolodon (Pisces, Cyprinidae)

The variability of mtDNA in four Far Eastern endemic species of the genus Tribolodon was studied in the significant part of their areas. There was no close relationship between the lifetime and the level of mtDNA variability. The relation between the population genetic structure and the level of amphidromic species was revealed. The presence of mtDNA phylogroup differed in three of the four species shows that there was a long period of independent intraspecific divergent evolution in the history of these species. This period falls on aproximately the same historical age, and, apparently, is caused by one and the same factors. The period was not enough long and ended in the integration of genetically independent units into a single species.     

Isolation and characterization of microsatellites in Leuciscus cephalus (Cypriniformes,Cyprinidae) and cross‐species amplification within the family Cyprinidae

Thirteen polymorphic microsatellites were isolated from Leuciscus cephalus, a widespread cyprinid species with great ecological tolerance. Together with the cross‐species amplification of six additional loci originally published for three cyprinid fish species, we optimized a multiplex panel for L. cephalus allowing the genotyping of 19 polymorphic loci. Number of alleles and heterozygosity per locus in a sample of 20 fish individuals ranged from two to 16 and from 0.05 to 0.90, respectively. These primers will be useful in determining the population structure of L. cephalus. In addition, successful cross‐amplification was obtained for several species of Cyprinidae.     

Analysis of mitochondrial DNA: Taxonomic and phylogenetic relationships in two fish taxa (Pisces: Mugilidae and Cyprinidae)

To solve some systematic questions as well as to study genetic variability and evolutionary relationships in two groups of fish belonging to the Mugilid (Mugilidae) and Cyprinid (Cyprinidae) families, we have used restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) fragments amplified in polymerase chain reaction. The analysis of three mtDNA fragments of 7220 bp total length of six Mugilid species has shown that Mediterranean Liza aurata, L. ramada, L. saliens, and Chelon labrosus form a common cluster, L. aurata and C. labrosus being the closest relatives, whereas L. haematocheilus (syn. C. haematocheilus) of the Sea of Japan forms a sister group to the Mediterranean cluster. It was found that Chelon and Liza genera are paraphyletic, and therefore their division into two genera is unnatural and they should be synonymized. According to priority, Liza species should be ascribed to Chelon genus. Mugil cephalus is the most distant compared to the rest of the species studied. The level of genetic divergence between allopatric samples of M. cephalus from the Sea of Japan and the Mediterranean Sea has proved to be very high—4.5% of nucleotide substitutions. The analysis of four mtDNA fragments of 9340 bp total length of six Cyprinid species has shown that L. waleckii is the most genetically distant. Pseudaspius leptocephalus is a sister group to Tribolodon species. All Tribolodon species form a common cluster with T. sachalinensis as a root. The remaining species form two branches, one of which includes T. nakamurai and T. brandtii, another one combines T. hakonensis and a new form of Tribolodon revealed that is close to T. hakonensis by its mtDNA (2.4% of nucleotide substitutions). This new form might be an independent species. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 12, pp. 1651–1658.     

Social grouping and genetic variation in common shiners,Notropis cornutus (Pisces,Cyprinidae)

Synopsis Heterogeneous gene frequencies of Est-1 across groups ofNotropis cornutus provide evidence of behaviourally imposed restrictions on stock structuring. Positive fixation indices (F_1S = 0.056 and F_1T = 0.085) were reflected by a deficiency of heterozygotes for pooled groups. The degree of subdivision ofN. cornutus stocks cannot be evaluated with the present evidence. but it is likely that their schooling behaviour is associated with significant genotypic structuring of the species.     

Phylogeography of Altai osmans (Oreoleuciscus sp., Cyprinidae, Pisces) based on sequence variation of the mitochondrial DNA cytochrome b gene

Representatives of the genus Oreoleuciscus (Altai osmans) from the water bodies of Western Mongolia were examined for sequence polymorphism of the mitochondrial DNA fragment containing the cytochrome b (Cyt-b) gene. A total of 17 sequence variants (haplotypes) were discovered, which formed two clusters, A and B, with nonoverlapping geographic localization. Cluster A included haplotypes of Altai osmans from the Valley of Lakes water bodies. Cluster B consisted of two subclusters, the first of which (B1) united sequence variants of the populations from Hollow of the Great Lakes and the lakes of the Great Altai Range. The second subcluster (B2) was formed by the haplotypes originating from the Hollow of the Lake Uvs, some isolated lakes of the Northwestern Khangay region, and the water bodies of the Arctic Ocean Basin (basins of Selenga and Orchon rivers). Based on the genetic divergence estimates and the radiation time of genetic geographic groups identified, the existence of three allopatric species of Altaic osmans in the genus Oreoleuciscus was substantiated.     

The fine structure of muscle fibres of roach, Rutilus rutilus (L.), and chub, Leuciscus cephalus (L.), Cyprinidae, Teleostei: interspecific differences and effects of habitat and season

Red and white axial muscle fibres from roach and chub were investigated by electron microscopy. Fish from three different localities were compared. Qualitative and quantitative analysis of myofibrils, mitochondria, lipid and subsarcolemmal cytoplasm with regard to muscle fibre type, species, season and habitat were made. Muscle fibre types differ significantly with the exception of the subsarcolemmal cytoplasm in roach. Within-species lipid content of red fibres differs between seasons. However, the most marked effect on red muscle fibres within species and season as regards volume density of lipid and mitochondria can be attributed to the different localities. The results are discussed in relation to mode of life and differences in habitat.     

Leaflet morphometric variation of service tree (Sorbus domestica L.) in the Balkan Peninsula

Abstract

In most European countries, the service tree (Sorbus domestica L.) is a rare and threatened species and its conservation has been recognised as a priority. The aim of this study was to asses its morphologic variation in the western and central part of the Balkan Peninsula and in southern Central Europe. Three populations were analysed: one in Serbia, one in Bosnia and Herzegovina and one in Slovenia. In each population 30 trees were selected, and from each tree 30 leaves were collected for morphometric analysis based on nine leaflet morphological traits. Univariate (ANOVA) and multivariate (MANOVA) analysis of variance were used to estimate the variation within- and between populations and a discriminant analysis was performed to examine the structure of the between-population differences. The values of particular morphological traits found in our study did not differ considerably from the values reported elsewhere. The results revealed significant within- and between population variation. Variation within populations was highly significant for all the scored leaf morphological traits, while variation between populations was significant for all the studied traits except for the leaflet length. The discrimination between the three populations was significant. High percentages of correctly classified samples demonstrate good discriminating employability of the analysed leaf morphological traits and indicate differentiation of the analysed populations.     

The evolutionary history of the allopolyploid Squalius alburnoides (Cyprinidae) complex in the northern Iberian Peninsula

Understanding the population structure, population dynamics and processes that give rise to polyploidy and helps to maintain it is central to our knowledge of the evolution of asexual vertebrates. Previous studies revealed high genetic diversity and several reproductive pathways in the southern populations of the Squalius alburnoides hybrid complex. In contrast, lower genetic variability and the associated limited chance of introducing new genetic combinations may threaten the survival of the northern Mondego populations. We analysed the genetic diversity and structure of nine populations of S. alburnoides in the Iberian Peninsula using microsatellite loci to provide further insights on the evolutionary history of this complex. Special attention was given to the less-studied northern populations (Mondego and Douro basins). Marked population structure, a high frequency of private alleles and a high diversity of some biotypes in the Douro basin indicate that some northern populations may not be at high risk of extinction, contrary to what was expected. The genetic diversity found in the northern Douro populations contradicts the general trend of remarkable genetic impoverishment northwards that occurs in other species and regions. The results indicate the possible existence of a glacial refugium in the Rabaçal River, corroborating findings in other species of this region. Historical events seem to have affected the geographical patterns of genetic variability found among and within the northern and southern populations of this complex and contributed to different patterns of genome composition. Therefore, historical events might have a major role in the long-term persistence of some polyploid hybrid taxa.     

基于线粒体CO 1基因序列的DNA条形码在鲤科鲌属鱼类物种鉴定中的应用

本研究探讨了线粒体CO1基因作为DNA条形码对鲌属鱼类进行物种鉴定的可行性。研究中获得了鲌属4种鱼类共32个个体长度为816bp的CO1基因序列。利用MEGA软件计算鲌属鱼类种间及种内遗传距离,利用邻接法、最大简约法、最大似然法和Bayesian方法分别构建分子系统树。结果显示,鲌属鱼类的种间遗传距离显著大于种内遗传距离。在系统树中,鲌属鱼类每一物种的个体分别形成各自独立的分支。基于CO1基因的DNA条形码在识别鲌属鱼类物种方面和传统形态学基本一致,而且该基因可以探讨鲌属鱼类种间的系统发育关系。本研究表明以CO1基因作为鲌属鱼类DNA条形码进行物种鉴定具有一定的可行性。     

Phylogeography of Altai osmans fishes (Oreoleuciscus sp., Cyprinidae, Pisces) inferred from nucleotide variation of the mitochondrial DNA cytochrome b gene

In this study, analyses based on cytochrome b gene (Cyt-b) mtDNA sequences were undertaken to clarify phylogenetic and phylogeographical relationships among populations Oreoleuciscus sp. (Pisces: Cyprinidae) from Western Mongolia. Two major clusters, designated clusters A and B, and minor clusters were discriminated. Cluster A comprised specimens of Oreoleuciscus from the Valley of Lakes. The cluster B consists of two subclusters; one of them (B1) including populations Oreoleuciscus of the Hollow of the Big Lakes and lakes Big Altai Range. Second subclusters (B2) combines specimens of Oreoleuciscus from Hollow of the Lake Uvs, some isolated lakes of the North-Western Khangay and waterbodies of the Arctic Ocean Basin (Selenge and Orchon basins). On the basis of estimations of sizes genetic and time of a divergence of the revealed genetikal-geographical groups existence of three allopatric species of the within genera Oreoleuciscus is proved.     

Differentiation and levels of genetic variation in northern European lynx (Lynx lynx) populations revealed by microsatellites and mitochondrial DNA analysis

The European lynx (Lynx lynx) hasexperienced significant decline in populationnumbers over large parts of its formerdistribution area in central and northernEurope. In Scandinavia (Sweden and Norway), thespecies has been subject to intense hunting and inthe early 20th century the population size mayhave been as low as about 100 animals. Duringthe rest of the century there have beenalternating periods of restricted hunting andtotal protection. Future management of theScandinavian lynx population will requireinsight into what effects demographicbottlenecks may have had on genetic variabilityand structure. For this purpose, 276 lynxesfrom Sweden, Norway, Finland, Estonia andLatvia were analysed for polymorphism at 11feline microsatellite loci and at themitochondrial DNA (mtDNA) control region.Scandinavian lynxes were found to be fixed fora single mtDNA haplotype, while this and threeadditional haplotypes were seen in Finland andthe Baltic States (Estonia and Latvia); thehaplotypes were all very similar, onlydiffering at 1–4 sites within a 700 bp regionsequenced. Microsatellite variability wasmoderate (H_e = 0.51–0.62) with lowerheterozygosity and fewer alleles in Scandinaviathan in Finland and the Baltic States together,though significant so only for the latter.Heterozygosity data in Scandinavia wereconsistent with a recent population bottleneck.Various analyses (e.g. F_st, individual-basedtree, assignment test) revealed distinctgenetic differentiation between Scandinavianlynxes and animals from Finland and the BalticStates. Some structure was evident withinScandinavia as well, suggesting an isolation bydistance. The observed partition of geneticvariability between Scandinavia and the easterncountries thereof indicates that lynxpopulations from the two regions may need to beseparately managed. We discuss what factors canhave contributed to the population geneticstructure seen in northern European lynxpopulations of today.     

Intraspecific variation and phylogeographic patterns of Fagus crenata (Fagaceae) mitochondrial DNA

Mitochondrial (mt) DNA variation in Japanese beech, Fagus crenata (Fagaceae), was studied in 17 populations distributed throughout the species' range. Total genomic DNA of samples from single trees representing each of 12 populations were digested with 18 restriction enzymes and hybridized with three probes containing coxI, coxIII, and atpA gene sequences. Thirty-four of the 54 enzyme/probe combinations showed polymorphisms and all the individuals were subsequently analyzed with six combinations of three probes and two enzymes. Restriction fragment length polymorphisms were evident around all three genes, allowing the identification of eight distinct haplotypes. Haplotype diversity within the populations was found to be very low (HS = 0.031), but population differentiation to be much higher (GST = 0.963). The mtDNA variation was strikingly different from allozyme variation (HS = 0.209; GST = 0.039). Gene flow for maternally inherited mtDNA should be restricted to seed dispersal while nuclear gene flow occurs by both seed and pollen dispersal. Therefore, the difference in the variation between mtDNA and allozymes may be largely a result of the much higher rate of gene flow associated with pollen dispersal than with seed dispersal. The mtDNA variation displayed strong geographic structure, which may reflect the species' distribution in the last glacial maximum and subsequent colonization, and probably also reflects intraspecific phylogeography of the species.     

Age, growth and reproductive characteristics of chub, Leuciscus cephalus (L., 1758) in the İkizcetepeler dam lake (Balikesir), Turkey

In this study, the population structure, growth and reproduction characteristics of 414 chub (Leuciscus cephalus L., 1758) from the ?kizcetepeler dam lake were investigated monthly between January and December 2000. Age groups ranged between I and VI for this species in the reservoir, with the second and third year‐classes dominating. Sex ratio was 1 : 1.4 (M : F), corresponding to 58.4% males and 41.6% females. Females attained greater size and age than males. The largest female captured was 24.8 cm FL, the largest male was 24.1 cm FL, both age VI. The von Bertalanffy growth equations and length–weight relationships were found as: L_t = 28.89[1 ?e^{?0.224(t+1.55)}] for females, L_t = 26.71[1 ? e^{?0.259(t+1.55)}] for males; W_t = 347.386[1?e^{?0.224h (t+1.55)}]^2.86 for females, W_t =286.48[1?e^{?0.259 (t+1.55)}]^2.92 for males; W = 0.0227 × L^2.87 for females and W = 0.0194 × L^2.92 for males. Significant statistical differences in condition factors between age classes and sexes were not found (P > 0.05, t‐test). Spawning period of this species in the lake was between April and May.     

Genetic divergence and phylogenetic independence of Far Eastern species in subfamily Leuciscinae (Pisces: Cyprinidae) inferred from mitochondrial DNA analyses

Classification of freshwater fish in the subfamily Leuciscinae, Cyprinidae is hampered by complexity or lack of morphological diversity. In this study, analyses based on mtDNA sequences were undertaken to clarify phylogenetic relationships among Far Eastern, North American and European species in the Leuciscinae. Evolutionary rate in cytochrome b gene (Cyt-b) and D-loop sequences appear to be almost constant in Leuciscinae. The topology of phylogenetic trees generated by neighbor-joining (NJ) and maximum likelihood (ML) methods based on Cyt-b gene and D-loop sequences was similar. Five major clades, designated clades 1-5, and a minor clade were discriminated. Most of the Far Eastern, North American and European species were included in the major clades. Clade 1, comprised almost entirely of Far Eastern phoxinins, is monophyletic and greatly diverged from the other species of Leuciscinae. From the present phylogenetic relationships and the previous studies, we present the following hypotheses with respect to the evolutionary history of the Far Eastern phoxinins. The Far Eastern species should be classified into Far Eastern-specific genera, although ichthyologists have still insisted that the species should be included in the European genera. The Far Eastern clade 1 consists of two subclades, including genera Pseudaspius-Tribolodon and Far Eastern Phoxinus species. According to our phylogenetic analyses, Pseudaspius leptocephalus and Tribolodon species should be reclassified into the same genus. On the basis of evolutionary rate in Cyt-b gene in Cyprinidae, it is estimated that the Far Eastern lineage diverged approximately 10-14 million years ago (mya) from the common ancestor of Leuciscinae. It is deduced that speciation of the Far Eastern species occurred until approximately 4 mya, in relation to the formation of the Sea of Japan and the Japanese Islands.