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.     

Phylogeography of the vairone (Leuciscus souffia,Risso 1826) in Central Europe

The vairone Leuciscus souffia is a cyprinid fish that inhabits river systems in and around the Alps. The complete mitochondrial DNA control region (945 bp) was sequenced in 295 vairone from 22 populations in Central Europe. A total of 51 haplotypes were identified with a maximum pairwise distance between haplotypes of 5.6%. Phylogenetic analyses revealed two major clades in L. souffia, an ‘Italian’ clade, and an ‘Alpine’ clade. Two hybrid zones exist, in the Mediterranean Alps and in the Soca basin. The position of the sister species of L. souffia, L. turskyi, to the ‘Alpine’ and the ‘Italian’ clade could not be resolved unambiguously. However, a linearized tree analysis indicated that L. turskyi represents a third lineage, that originated at the same time as the ‘Alpine’ and the ‘Italian’ clades of L. souffia. In the ‘Alpine’ clade two groups were resolved, a subclade with haplotypes from the Rhône and Var basins and a cluster with haplotypes from the Danube and Rhine systems. Our data suggest a long history of the vairone in Central Europe, predating Pleistocene glacial cycles. Two main refugia during glaciations must have existed, one in Italy and another one most probably in the Danube system. However, age estimates based on molecular clock calibrations suggest the survival of ‘Alpine’ haplotypes in several drainages during the last glaciation cycles. The Rhine system was only recently colonized.     

Structure of the seminal pathway in the European chub, Leuciscus cephalus (Cyprinidae); Teleostei

The testicular efferent duct system of Leuciscus cephalus (Cyprinidae), is described for three phases of testicular development. Testicular main ducts were analyzed by means of conventional histology and transmission electron microscopy. Additional techniques were applied for lectin histochemistry to determine secretory activity, as well as immunohistochemistry for cell proliferation activity and for muscle actin to demonstrate the distribution and amount of contractile cells. The contribution of the main ducts' epithelia and of degenerating spermatocytes to seminal fluid composition was confirmed, with the former being a source of carbohydrates and the latter that of phospholipids. The apical glycocalyx of epithelial cells, which is important in cell recognition and potentially involved in sperm storage, was marked by RCA I, LCA, and WGA lectin. Higher numbers of proliferating epithelial cells were ascertained during spawning phase compared to pre- and postspawning phases. In the ducts' stroma, a large number of cells expressed muscle actin and tropomyosin, indicating the ducts' contractile potential for the transport of seminal fluid towards release. Adjacent to these contractile cells, numerous nerves were found, indicating neuronal control of sperm fluid flow.     

Growth and morphological development of chub, Leuciscus cephalus (L.), during the first year of life

The growth and the morphological development of larvae and juveniles of chub, Leuciscus cephalus (L.), from a small stream of western Greece is described, based on field samples and laboratory-raised fish. In the laboratory, size hierarchy was developed and morphological differentiation occurred at a younger age in the fastest growing larvae. Growth was faster in the field and differentiation of fins and scales began at a larger size, but apparently at a younger age, than in the laboratory.     

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.     

Phylogeography of the bigeye chub Hybopsis amblops (Teleostei: Cypriniformes): early Pleistocene diversification and post‐glacial range expansion

The bigeye chub, Hybopsis amblops, is a member of the Central Highlands ichthyofauna of eastern North America. Phylogenetic analyses of the H. amblops species group based on a 1059 bp fragment of the mitochondrial DNA cytochrome b gene did not recover a monophyletic group. The inclusion of Hybopsis hypsinotus in the species complex is questionable. Within H. amblops, five strongly supported clades were identified; two clades containing haplotypes from the Ozark Highlands and three clades containing haplotypes from the Eastern Highlands and previously glaciated regions of the Ohio and Wabash River drainages. Estimates of the timing of divergence indicated that prior to the onset of glaciation, vicariant events separated populations east and west of the Mississippi River. East of the Mississippi River glacial cycles associated with the blocking and rerouting of the Teays River system caused populations to be pushed southward into refugia of the upper Ohio River. Following the most recent Wisconsinan glaciation, populations expanded northward into previously glaciated regions and southward into the Cumberland River drainage. In the Ozarks, west of the Mississippi River, isolation of clades appears to be maintained by the lack of stream capture events between the upper Arkansas and the White River systems and a barrier formed by the Arkansas River.     

Phylogeography and postglacial dispersal of arctic charr Salvelinus alpinus in North America

Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) was used to reconstruct postglacial dispersal routes of arctic charr Salvelinus alpinus in North America. Twelve of 35 restriction enzymes detected polymorphisms among representative populations, revealing two distinct lineages with an estimated nucleotide divergence of 1.32%. Subsequent screening of 869 fish from 54 populations with four diagnostic restriction enzymes showed that these lineages have largely allopatric distributions, suggesting their dispersal from separate northern and eastern glacial refugia. In addition, geographical and genetic structure among eastern populations suggested the existence of a second eastern refuge. Among the three lineages, the most divergent (Arctic) lineage occurred from Alaska east to northern Labrador. Quebec, New Brunswick, and New England were colonized by a second (Laurentian) lineage, and Labrador by a third group. Contact between refugial groups was only detected in two Labrador populations. The Arctic lineage was highly differentiated from eastern North American and European haplotypes, and probably diverged during the early Pleistocene. By contrast, the Laurentian and Labrador groups were similar to Old World charr, suggesting a shared ancestry during the mid-Pleistocene. In addition, the close relationship between Labrador and Laurentian charr indicates their probable divergence during the Wisconsinan glaciation.     

Phylogeography of bleaks Alburnus spp. (Cyprinidae) in Italy, based on cytochrome b data

Sequence variation of a fragment of the mitochondrial DNA encoding for the cytochrome b gene was used to reconstruct the phylogeography of the two species of bleaks occurring in Italy: the alborella Alburnus arborella in northern Italy and the vulturino Alburnus albidus in southern Italy. The study includes four populations of the alborella and 14 populations of the vulturino. A total of 57 haplotypes were identified; these could not be sorted into two reciprocally monophyletic clusters. Multiple phylogenetic methods and nested clade phylogeographical analysis consistently retrieved three well-supported clades, two of which contained both Northern and Southern Italian haplotypes. A third clade is limited to southern Italy. This clade is tentatively assigned to the vulturino. The placement in the same clade of northern and southern Italian haplotypes is explained in light of the introductions of fishes operated from northern to central and southern Italy. The origin of the vulturino dates back to the last two million years. This divergence time estimate identifies the Pleistocene confluences between adjacent river basins along the Adriatic slope of the Italian peninsula and their subsequent isolation as the cause that triggered the diversification of the genus in the area. The existence of a clade endemic to southern Italy supports the recognition of the area as a new peri-Mediterranean ichthyogeographic district, the borders of which correspond to the northern and southern edges of the vulturino range.     

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.     

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.     

Phylogeography of red deer (Cervus elaphus) in Europe

Aim To investigate the phylogeographical patterns of red deer (Cervus elaphus) in Europe, and to disentangle the influence of ancient (e.g. Pleistocene ice ages) from more recent processes (e.g. human translocations). Location Europe. Methods In this study we provide by far the most extensive analysis of genetic structure in European red deer, based on analyses of variation at two mitochondrial markers (cyt b and D‐loop) in a large number of individuals from 39 locations. Relationships of mitochondrial DNA haplotypes were determined using minimum spanning networks and phylogenetic analyses. Population structure was examined by analyses of molecular variance. Historical processes shaping the present patterns were inferred from nested clade analysis and nucleotide diversity statistics. Results Within Europe, we detected three deeply divergent mitochondrial DNA lineages. The three lineages displayed a phylogeographical pattern dividing individuals into western European, eastern European and Mediterranean (Sardinia, Spain and Africa) groups, suggesting contraction into three separate refugia during the last glaciation. Few haplotypes were shared among these three groups, a finding also confirmed by F_ST values. Calculations of divergence times suggest that the groups probably split during the Pleistocene. Main conclusions The observed pattern is interpreted to result from isolation in different refugia during the last glaciation. The western and eastern European lineages could be linked to an Iberian and Balkan refugium, respectively. The third lineage might originate from a Sardinian or African refugium. We link local phylogeographical patterns observed in Europe to the post‐glacial recolonization process, shaped by the geographical localization of refugia and barriers to gene flow. Regardless of the importance of red deer as a game species and the tradition of translocating red deer in Europe, we detected few individuals that did not match the trichotomous pattern, suggesting that translocations have occurred mainly at smaller spatial scales.     

Origin, radiation, dispersion and allopatric hybridization in the chub Leuciscus cephalus

The phylogenetic relationships of 492 chub (Leuciscus cephalus) belonging to 89 populations across the species' range were assessed using 600 base pairs of cytochrome b. Furthermore, nine species belonging to the L. cephalus complex were also analysed (over the whole cytochrome b) in order to test potential allopatric hybridization with L. cephalus sensu stricto (i.e. the chub). Our results show that the chub includes four highly divergent lineages descending from a quick radiation that took place three million years ago. The geographical distribution of these lineages and results of the nested clade analysis indicated that the chub may have originated from Mesopotamia. Chub radiation probably occurred during an important vicariant event such as the isolation of numerous Turkish river systems, a consequence of the uplift of the Anatolian Plateau (formerly covered by a broad inland lake). Dispersion of these lineages arose from the changes in the European hydrographic network and, thus, the chub and endemic species of the L. cephalus complex met by secondary contacts. Our results show several patterns of introgression, from Leuciscus lepidus fully introgressed by chub mitochondrial DNA to Leuciscus borysthenicus where no introgression at all was detected. We assume that these hybridization events might constitute an important evolutionary process for the settlement of the chub in new environments in the Mediterranean area.     

Population dynamics of Barbus barbus plebejus (Valenciennes) and Leuciscus cephalus cabeda (Risso) in the middle River Po (Italy)

Scales from 427 Barbus barbus plebejus and 406 Leuciscus cephalus cabeda from the middle River Po were examined. The last annulus was formed between April and May 1977, except the 0–1 age-group of L.c. cabeda which developed its first year-ring between March and April 1977. The survival (S) and annual mortality (A) rates were equivalent in both fish populations; the same was true of both sexes of Leuciscus, but S in Barbus males was lower than in females. The sex ratio showed a preponderance of females in both species.Growth rate from June to September 1976 was higher than in the rest of the year. Annual growth in Barbus females was higher than in males, whereas both sexes in Leuciscus showed equivalent growth rates; growth rate was higher in Barbus than in Leuciscus. The P/B ratio of both species was higher in the young age-groups.Presented at the XLVII Congress of the Italian Zoological Union, Bergamo (Italy), September 1979.Presented at the XLVII Congress of the Italian Zoological Union, Bergamo (Italy), September 1979.     

Phylogeography of the tidewater goby, Eucyclogobius newberryi (Teleostei, Gobiidae), in coastal California

The tidewater goby, Eucyclogobius newberryi, inhabits discrete, seasonally closed estuaries and lagoons along approximately 1500 km of California coastline. This species is euryhaline but has no explicit marine stage, yet population extirpation and recolonization data suggest tidewater gobies disperse intermittently via the sea. Analyses of mitochondrial control region and cytochrome b sequences demonstrate a deep evolutionary bifurcation in the vicinity of Los Angeles that separates southern California populations from all more northerly populations. Shallower phylogeographic breaks, in the vicinities of Seacliff, Point Buchon, Big Sur, and Point Arena segregate the northerly populations into five groups in three geographic clusters: the Point Conception and Ventura groups between Los Angeles and Point Buchon, a lone Estero Bay group from central California, and San Francisco and Cape Mendocino groups from northern California. The phylogenetic relationships between and patterns of molecular diversity within the six groups are consistent with repeated, and sometimes rapid, northward and southward range expansions out of central California caused by Quaternary climate change. Plio-Pleistocene tectonism, Quaternary coastal geography and hydrography, and historical human activities probably also influenced the modern geographic and genetic structure of E. newberryi. The phylogeography of E. newberryi is concordant with phylogeographic patterns in several other coastal California taxa, suggesting common extrinsic factors have had similar effects on different species. However, there is no evidence of a phylogeographic break coincident with a biogeographic boundary at Point Conception.     

Phylogeography of the olive sea snake, Aipysurus laevis (Hydrophiinae) indicates Pleistocene range expansion around northern Australia but low contemporary gene flow

Pleistocene sea-level fluctuations profoundly changed landmass configurations around northern Australia. The cyclic emergence of the Torres Strait land bridge and concomitant shifts in the distribution of shallow-water marine habitats repeatedly sundered east and west coast populations. These biogeographical perturbations invoke three possible scenarios regarding the directions of interglacial range expansion: west to east, east to west, or bidirectional. We evaluated these scenarios for the olive sea snake, Aipysurus laevis, by exploring its genetic structure around northern Australia based on 354 individuals from 14 locations in three regions (Western Australia, WA; Gulf of Carpentaria, GoC; Great Barrier Reef, GBR). A 726-bp fragment of the mitochondrial DNA ND4 region revealed 41 variable sites and 38 haplotypes, with no shared haplotypes among the three regions. Population genetic structure was strong overall, phiST=0.78, P<0.001, and coalescent analyses revealed no migration between regions. Genetic diversity was low in the GBR and GoC and the genetic signatures of these regions indicated range or population expansions consistent with their recent marine transgressions around 7000 years ago. By contrast, genetic diversity on most WA reefs was higher and there were no signals of recent expansion events on these reefs. Phylogenetic analyses indicated that GBR and GoC haplotypes were derived from WA haplotypes; however, statistical parsimony suggested that recent range expansion in the GBR-GoC probably occurred from east coast populations, possibly in the Coral Sea. Levels of contemporary female-mediated gene flow varied within regions and reflected potential connectivity among populations afforded by the different regional habitat types.     

Phylogeography of the plains killifish, Fundulus zebrinus

Drainage systems of the Great Plains and western Gulf Slope underwent substantial changes through diversions and stream captures during the Pleistocene, either as the result of the glacial advances or through independent geologic processes. The distributions of a variety of fishes that range across west-central North America, such as the plains killifish (Fundulus zebrinus), are thought to be the product of this Pleistocene influence. We examined the geographic pattern of genetic variation in F. zebrinus using three allozyme loci (n = 793), mitochondrial DNA restriction fragment length polymorphisms (RFLPs, n = 352), and sequencing of the mitochondrial cytochrome oxidase I (COI, n = 23) in an attempt to understand the roles of dispersal and vicariance. The phylogeographic patterns were concordant between the allozyme and mitochondrial data with the exception of the population in the North Canadian River. The populations fell into three geographic assemblages, which we designated as northern, central, and southern. A large phylogenetic break (average Roger's D = 0.702; average sequence divergence in RFLPs = 4.6%; average sequence divergence in COI = 5.5%) separated the northern/central and southern assemblages. The northern region was likely colonized sometime during the mid-Pleistocene. Fish in the Brazos and Pecos Rivers probably reached these drainages through stream captures of the Red River. The large phylogenetic break between the northern/central and southern clades supports previous attempts to recognize two species of plains killifish: F. zebrinus and F. kansae.     

Phylogeography of the cyprinid Squalius aradensis and implications for conservation of the endemic freshwater fauna of southern Portugal

The Iberian cyprinid fauna, characterized by the presence of numerous endemic species, has suffered from significant habitat degradation. The critically endangered Squalius aradensis is restricted to small drainages of southern Portugal, habitats that typically exhibit a characteristic Mediterranean-type heterogeneous hydrological system throughout the year, including alternation of flooding events during winter and complete drought in large river sections during summer. To assess the effect of historical and recent processes on genetic diversity in S. aradensis we examined within- and among-population variability in cytochrome b and six polymorphic microsatellite loci. Estimates of genetic diversity in time and space through the combined use of traditional Phi-/F-statistics, phylogenetic trees, ordination methods and nested clade analysis indicated significant and congruent structuring among populations. Data suggest that the Arade drainage represent the evolutionary centre of the species, with subsequent allopatric fragmentation across drainages. Factors other than isolation by distance strongly affected the within-drainage genetic differentiation observed in these Mediterranean-type drainages, including recent population expansion from a bottleneck event and restricted gene flow imposed by a long-term barrier (brackish water area). Significant correlation was found between S. aradensis allelic diversity and upstream drainage area. The relevance of findings for conservation issues is discussed in relation to local intermittent hydrological conditions, the highly restricted distribution and the critically endangered status of the species.     

Mitochondrial DNA and microsatellite variation in the eider duck (Somateria mollissima) indicate stepwise postglacial colonization of Europe and limited current long-distance dispersal

To unravel the postglacial colonization history and the current intercolony dispersal in the common eider, Somateria mollissima, we analysed genetic variation at a part of the mitochondrial control region and five unlinked autosomal microsatellite loci in 175 eiders from 11 breeding colonies, covering the entire European distribution range of this species. As a result of extreme female philopatry, mitochondrial DNA differentiation is substantial both among local colonies and among distant geographical regions. Our study further corroborates the previous hypothesis of a single Pleistocene refugium for European eiders. A nested clade analysis on mitochondrial haplotypes suggests that (i) the Baltic Sea eider population is genetically closest to a presumably ancestral population and that (ii) the postglacial recolonization progressed in a stepwise fashion via the North Sea region and the Faroe Islands to Iceland. Current long-distance dispersal is limited. Differentiation among colonies is much less pronounced at microsatellite loci. The geographical pattern of this nuclear genetic variation is to a large extent explained by isolation by distance. As female dispersal is very limited, the geographical pattern of nuclear variation is probably explained by male-mediated gene flow among breeding colonies. Our study provides genetic evidence for the assumed prominent postglacial colonization route shaping the present terrestrial fauna of the North Atlantic islands Iceland and the Faroes. It suggests that this colonization had been a stepwise process originating in continental Europe. It is the first molecular study on eider duck populations covering their entire European distribution range.     

Spatial aspects of genetic differentiation of the European chub in the Rhone River basin

Starch-gel electrophoresis was used to examine genetic differentiation at four polymorphic loci in the European chub Leuciscus cephalus from 27 samples and three main areas of the Rhone River basin in France. The Upper Rhone samples appeared to be genetically distinct at locus PGM-2 * from the Lower Rhone and Saone-Grosne samples according to the constant row total-multiple correspondence analysis (CRT-MCA), which allowed the estimation of multivariate F_st equivalents. No difference between Lower Rhone and Saone-Grosne samples was revealed by the global analysis considering the three areas. However, distinct local analyses in the three areas showed that the spatial structure of genetic variability was dissimilar in these last two areas, suggesting a non-equilibrium process under the influence of dams in the Lower Rhone, and isolation by distance in Saone system. Isolation by distance was also suggested for the Upper Rhone samples.     

Hybrids of chub and roach (Leuciscus cephalus and Rutilus rutilus) in English rivers

The occurrence of hybrids between chub, Leuciscus cephalus , and roach, Rutilus rutilus , from three rivers in England is reported. The hybrids are described and illustrated, and means of identification are indicated. Chub X roach hybrids, like those between other leuciscine cyprinid species, can be positively identified by the number and form of the pharyngeal teeth.