Evolutionary and biogeographical patterns within Iberian populations of the genus Squalius inferred from molecular data

Origins and biogeography of the chub, genus Squalius (formerly within the genus Leuciscus), in the Iberian Peninsula were inferred from comparison between patterns of geographic distribution and phylogenetic relationships among populations belonging to 14 European Squalius species. The phylogeny recovered was based on the complete sequence of the mitochondrial cytochrome b gene. Squalius species were grouped into three major clades. The basal clade included species distributed across the ancient Paratethys Sea. The second clade included species from Central and East Europe and the northern areas of the Mediterranean basin towards Minor Asia. The third clade included species from the Mediterranean Peninsulas (Iberian, Italy, and Balkans). The Iberian Squalius species do not constitutes a monophyletic group. Our data indicate that the Iberian Peninsula was colonized at least twice by two different monophyletic lineages, a meridional group and a Central Europe group. The amount of species diversity found in the Iberian Peninsula and the phylogenetic relationships among these species, together with their geographic distribution, suggest that the Central Europe lineage colonized the Iberian Peninsula at a latter time. Our data indicate that the northeastern Iberian lineage is phylogenetically close to Greek populations of Squalius cephalus, while the second lineage formed a monophyletic group including Squalius pyrenaicus, Squalius carolitertii, Squalius aradensis, and Squalius torgalensis. The speciation process that generated these species and the geographic structure of their populations, principally in S. pyrenaicus, can be attributed to paleogeographical events like the ancient endorrheism and the development of hydrographic basins.     

A natural hybrid of Leuciscus cephalus (L.) and Chalcalburnus chalcoides (Güldenstädt)(Osteichthyes‐Cyprinidae) from Lake Tödürge (Sivas, Turkey)

Hybridization between Leuciscus cephalus and Chalcalburnus chalcoides was identified from biometrical features in 55 fish caught from Lake Tödürge, Sivas, Turkey, between April 1994 and November 1997. The hybrid index V _h for metric and meristic characters was intermediate ( V _h = 45·6) compared with their parents ( L. cephalus V _h = 0 or negative and C. chalcoides V _h ≥ 100). Morphological, metric and meristic features of L. cephalus  ×  C. chalcoides also resembled those of the parents. The majority presented an intermediate position which was best shown by the shape of mouth, caudal fin, gill rakers and pharyngeal teeth, and also by the caudal peduncle depth, head and interorbital width, predorsal and preanal distance, height of anal fin, length of anal base and other characters.     

Dispersal and survival of stocked cyprinids in a small English river: comparison with wild fishes using a multi-method approach

Mark–recapture and fixed-station passive integrated transponder (PIT) telemetry were used to compare movements, distribution and survival of stocked juvenile chub Leuciscus cephalus and roach Rutilus rutilus with those of wild conspecifics. Daily activity of wild fish activity was affected by a combination of river flow and temperature, whereas stocked fishes were not influenced by environmental factors. PIT telemetry recorded exploratory movements of stocked L. cephalus immediately after stocking, a substantial number of stocked fish moved both downstream and upstream during periods of elevated flow, and proportionally more stocked fish moved during the first 6 weeks after release than later on. Proportionally more stocked fish than wild fish moved through PIT antennae, stocked L. cephalus moved greater distances than wild L. cephalus and were more widely distributed than wild fish. Minimum estimates of survival after 5 months were 50·5% for stocked R. rutilus and 28·0% for stocked L. cephalus . Ultimately, stocked cyprinids appeared to be able to cope with elevated flows and most remained in the river section local to the stocking location.     

Genetic and morphological variation in a common European cyprinid, Leuciscus cephalus within and across Central European drainages

Genetic variability and differentiation of the common European cyprinid Leusciscus cephalus was investigated within and across the drainages of the rivers Rhine, Elbe and Danube using starch gel electrophoresis and morphological characters. Eleven of 24 presumptive enzyme loci showed some allozymic variation. Compared to other freshwater fish, genetic variability within all sites was high ( H _e=0·074–0·113). This was explained by the wide geographical and ecological range of the chub. In contrast, genetic and morphological differentiation between sites and drainages was low (F_ST=0·15), which may be due to the high dispersal ability of the chub and translocations of spawn by waterfowl. The phylogeographic structure of Leuciscus cephalus in north-eastern Bavaria showed low congruence with Cottus gobio populations sampled in the same area.     

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.     

Leuciscus cephalm (Linnaeus), with records of fingerling adult males, Leuciscus plemobipunctatus (Stephanidis) and their hybrids from western Greece

The spawning season of chub, Leuciscus cephalus , in western Greece starts in January-February when males are aged at least 2 + and females 3 + (rarely 2 +) years. In the R. Ermitsa (Achelos R. basin) a population is reported with fingerling adult males with developed gonads and breeding tubercles at a size of 42 72 mm s.l . Leuciscus pleurobipunctatus breeds between February and the first half of May, when males are aged at least 1 + and females 2 + (sometimes also 1 +); their life span is about 4 years. L. cephalus × L. pleurobipunctatus hybrids were found in the R. Ermitsa (five specimens) and R. Louros (one specimen). In the former the contemporary presence of adult fingerling male chub, the smallest adults of L. pleurobipunctatus , and hybrids between the two species, may be related to channel width and the human interference which has resulted in considerable disturbance of the habitat.     

A natural hybrid of Leuciscus cephalus macedonicus X Chalcalburnus chalcoides macedonicus (Pisces, Cyprinidae) from Lake Volvi (Macedonia, Greece)

A massive hybridization between Leuciscus cephalus macedonicus and Chalcalburnus chalcoides macedonicus has been recognized and studied by examining 67 specimens from Lake Volvi (Macedonia, Greece). The majority of meristic and other characters present an intermediate position in the hybrid (total hybrid index Vh = 38.37). Nevertheless, the best expression of intermediacy is shown most clearly by the numbers of anal fin soft rays, lateral line scales and gill rakers, and also by the height of the dorsal fin and the length of the base of anal fin. Of the examined hybrid specimens. 92.5% were females. It seems that hybridization takes place during the reproduction period in the main streams flowing into the lake under special conditions which hinder the migration of the majority of the population of C. chalcoides from the lake to the breeding sites. It is suggested that individuals of C. chalcoides , which manage to arrive in these sites, mate with L. cephalus , the normal inhabitant of the streams, thus producing the hybrid.     

Local and global multivariate analysis of geographical mitochondrial DNA variation in Leuciscus cephalus L. 1758 (Pisces: Cyprinidae) in the Balkan Peninsula

Geographical variation of cytochrome b mitochondrial DNA (mtDNA) in chub (Leuciscus cephalus L.) was analysed in 31 samples from 20 different river basins in the Balkan Peninsula and Danube catchment. Multivariate methods of ordination were used to analyse variation of the data sets. The results were interpreted in the context of the proposed ichthyogcographic districts separating the Balkan Peninsula into two main ichthyogeographic divisions (Eastern Greece/Ponto-Acgcan and Western Greece/South Adriatic-Ionian). Boundary detection supported these two ichthyogeographic districts for L. cephalus , revealing a boundary that ran from north to south through the Balkan Peninsula and the middle of Greece. The results also revealed the existence of a third division in Central Greece. The results of ordination techniques on homogeneous zones and analysis of the molecular variance confirmed the results obtained in studying local variability (boundaries). They also allowed us to test the existence of possible subdivisions proposed by different authors inside the two main ichthyogeographic districts. These subdivisions were not supported. The multivariate methods used in this study allowed us to propose a coherent picture of chub ichthyogeographic districts in terms of boundary detection and maximal autocorrelation between populations and to explain the patterns of chub mtDNA variation. A complete interpretation of results concerning L. cephalus requires careful consideration of both boundary analysis and autocorrelative approach. Results from an the autocorrclativc approach alone could lead to substantial misinterpretations.     

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.     

Evaluation of VIE and PIT tagging methods for juvenile cyprinid fishes

Retention and mortality associated with visible implant elastomer (VIE) and passive integrated transponder (PIT) tagged juvenile chub [ Leuciscus cephalus (L.)], dace [ Leuciscus leuciscus (L.)] and roach [ Rutilus rutilus (L.)] were evaluated. PIT tag retention (96.6–100%) was higher than VIE over the 6-month duration of the experiment. VIE retention was significantly better in the head (96.3–98.8%) than in the fins (78.8–90.9%) the first month after tagging, but the opposite was found after 6 months (head = 21.5–57.5%; fins = 77.2–88.8%). Survival was not significantly different from controls for any treatment, except dace tagged with 23-mm PIT (significantly influenced by mass of fish at tagging) and sham PIT tagged dace, because of initial losses. PIT tags are recommended as the most suitable method for tagging individual juvenile chub, dace and roach based on high retention and survival. VIE implantation in the head (studies < 30 days) and fins (studies > 30 days) could provide a cheap, batch-marking alternative, provided retention rates are monitored.     

Effects of handling and electrofishing on plasma glucose and whole blood lactate of Leuciscus cephalus

Chub Leuciscus cephalus exposed to simulated pulsed direct current electrofishing operations exhibited rapid elevations in plasma glucose and blood lactate levels. Plasma glucose levels were significantly higher 0·5 h after simulated electrofishing operations, and peaked 2 h after treatment. Glucose levels remained high for up to 4 h. No changes in plasma glucose were evident following handling. Simulated electrofishing operations and handling induced an immediate lactacidosis in chub. Initial responses to both treatments were similar except that blood lactate was significantly higher in fish exposed to simulated electrofishing operations than in handled fish 5 and 15 min after treatment. Blood lactate remained elevated in fish exposed to simulated electrofishing operations for 2 h, while blood lactate of handled fish returned to levels similar to those in the control fish within 0·5 h post‐treatment.     

Reproductive biology of the threatened golden galaxias Galaxias auratus Johnston and the influence of lake hydrology

Golden galaxias Galaxias auratus (31–235 mm fork length, L _F) were collected monthly from littoral habitats in Lakes Crescent and Sorell, Tasmania, Australia, between July 2000 and December 2002. Spawning habitats were identified and monitored in both lakes, and surveyed in Lake Crescent. Trends in gonado-somatic indices and reproductive stages of development indicated that gonad development in both sexes begins in midsummer and peaks in late autumn to early winter. Males mature at smaller sizes (50% at 52 mm L _F) than females (50% at 76 mm L _F), larger individuals are predominately females (95% of fish ≥138 mm L _F), and overall male to female ratios are female biased ( c . 1:2). Spawning occurs late autumn to early spring (water temperatures = 1·4–9·7° C) with peaks in spawning activity in winter (mean water temperatures <5° C). Demersal adhesive eggs ( c. 1·5 mm diameter) were found on cobble substrata ( c. 20–250 mm diameter) in littoral areas ( c. 0·2–0·6 m deep) and fecundity of fish 71–181 mm L _F ranged from 619 to 14 478 eggs. The rate of change in water level over the 20 days prior to monthly sampling was important in explaining the occurrence of spent fish and this accounted for temporal differences in spawning between the populations. Lake hydrology influences the reproductive cycle of G. auratus by possibly providing a stimulus for spawning and it controls the availability of spawning habitat in Lake Crescent. Seasonal hydrological cycles ( i.e. rises during late autumn to winter) and a minimum water level of 802·20 m Australian Height Datum in Lake Crescent during autumn (above which littoral areas of cobble substratum are inundated) are critical to G. auratus populations.     

Acute effects of the sodium salt of 2,4-D on the early developmental stages of bleak, Alburnm alburnus

The acute effects of a herbicide based on 2,4-D on embryos and larvae of bleak, Alburnus alburnus L. exposed to concentrations of 25–3200 mg 1⁻¹ for 12–48 hours were investigated. The 2,4-D delayed or stopped the development of embryos in their early stages, caused behavioural changes and morphological alterations. The L.C.₅₀ values for embryos varied between 12–9 and 15·9–4mg 1⁻¹ and for larvae from 51·6 to 111·2mg 1⁻¹ according to the time of exposure. The level of 2,4-D in Lake Balaton, or similar shallow lakes, without substantial injury of the littoral fish fauna should not surpass 0·5–1·0 mg 1⁻¹.     

Spatial and temporal variations in fish populations in the upper Thames estuary

Between February 1989 and August 1990, the upper Thames estuary contained 23 species of fish. Fish numbers were higher and relatively constant in the uppermost part of the estuary. Number of species was augmented in summer from fresh water and from downstream, coinciding with high temperature, low flow and high salinity. The eight most abundant species contributed to 98·5% of the total number. Flounder Pleuronectes flesus , dace Leuciscus leuciscus and perch Perca fluviatilis , recruited from May to August, and common goby Pomatoschistus microps , roach Rutilus rutilus and chub Leuciscus cephalus , from August to November. The upper estuary (salinity 0·34–2·96 p.s.u.) formed a species transition area between the freshwater but salinity-resistant roach, chub, and gudgeon Gobio gobio upstream, and the estuarine eurhyhaline common goby and flounder downstream. The three-spined stickleback Gasterosteus aculeatus and cyprinids were more abundant at upstream while perch was more abundant at downstream sites. High abundances of gudgeon, chub and roach were associated with high transparency and dissolved oxygen and low salinity, while high abundances of perch were associated with high salinity and low transparency. Dace and three-spined stickleback were associated with high dissolved oxygen and low pH, and common goby with high pH. Flounder showed no clear preferences.     

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.     

Spawning migrations of the chub in the River Spree, Germany

Out of 52 chub Leuciscus cephalus tagged with internal radio‐transmitters, 46 migrated to spawning grounds in the River Spree, Germany. All chub started their first spawning migration on 21–22 May in both years of study, 1995 and 1996. They arrived at three different spawning grounds within a day and left after 1 to 6 days to return to their original sites. On 17 and 18 June 1995 and 1996 chub started their second spawning migration with the same pattern as the first. About 60% of all migrating chub moved between 1 and 13 km upstream to the nearest spawning ground. Some exceptions were observed: a few chub did not use the nearest spawning ground, some chub used different spawning grounds at first and second spawning or moved downstream to a spawning ground. Spawning grounds were characterized by 0·4 m s⁻¹ current velocity, shallow depth (0·1–0·8 m) and a stony bottom, where most eggs adhered.     

Molecular Evidence on the Evolutionary and Biogeographical Patterns of European Cyprinids

The phylogenetic relationships of 106 European cyprinid taxa were determined based on the complete nucleotide sequence (1140 bp) of the mitochondrial cytochrome b gene. The molecular phylogeny was used (1) to revise the current systematics of European cyprinids, (2) to establish the phylogenetic utility of traditional morphological characters that are widely used in Cyprinidae systematics, and (3) to discuss alternative hypotheses on the biogeography of the family in Europe. The age of the major lineages within European cyprinids was tentatively estimated with a molecular clock and showed full agreement with the fossil record of the group. Moreover, the results provided unambiguous evidence for a close phylogenetic affinity of some Caucasian and Greek endemic cyprinid taxa (e.g., B. capito and B. brachycephalus and Leuciscus keadicus, Barbus graecus, and B. albanicus, respectively) to Iberian and North African, but not Central European, cyprinids. The existence of such unexpected phylogenetic relationships refutes the classical hypothesis on the biogeography of European cyprinids, which assumes a dispersal of the cyprinid fauna from central Europe to southern Europe and northern Africa during the Miocene (and, hence, predicts a close phylogenetic relationship of all Caucasian, Greek, Iberian, and North African cyprinids to central European taxa). Instead, the existence of a Mediterranean realm independent of the central European route seems plausible based on the molecular evidence. It is likely that the new biogeographical scenario proposed here might apply to other primary freshwater European animals with low dispersal abilities, including fish, amphibians, and invertebrates. Received: 2 February 1999 / Accepted: 16 March 1999     

Mitochondrial phylogeny and biogeographic history of the Greek endemic land-snail genus Codringtonia Kobelt 1898 (Gastropoda, Pulmonata, Helicidae)

The aim of this work was to infer the phylogeny of the Greek endemic land-snail genus Codringtonia Kobelt 1898, estimate the time frame of the radiation of the genus, and propose a biogeographic scenario that could explain the contemporary distribution of Codringtonia lineages. The study took place in the districts of Peloponnese, Central Greece and Epirus of mainland Greece. Sequence data originating from three mtDNA genes (COI, COII, and 16S rDNA) were used to infer the phylogeny of the eight nominal Codringtonia species. Furthermore, the radiation time-frame of extant Codringtonia species was estimated using a relaxed molecular clock analysis and mtDNA substitution rates of land snails. The phylogenetic analysis supported the existence of six Codringtonia lineages in Greece and indicated that one nominal species (Codringtonia neocrassa) might belong to a separate genus distantly related to Codringtonia. The time frame of differentiation of Codringtonia species was placed in the Late Miocene-Pleistocene epoch. The dispersal-vicariance analysis performed indicated that most probably Codringtonia exhibited a north-to-south spread with the ancestral area being that of central Greek mainland, accompanied with duplication (speciation) and vicariance events.     

Evidence for independent origin of two spring‐spawning ciscoes (Salmoniformes: Coregonidae) in Germany

Combined analyses of mitochondrial DNA (mtDNA) and microsatellite loci were performed to assess the genetic differentiation of two spring‐spawning ciscoes from each other and from sympatric Coregonus albula in two German lakes. Polymorphism was screened at six microsatellite loci and mtDNA for a total of 247 and 94 ciscoes, respectively. Microsatellite data showed a weak differentiation between spring‐spawning Coregonus fontanae and sympatric C. albula in Lake Stechlin ( F _ST = 0–0·008), whereas a significant differentiation was observed between spring‐spawning Coregonus lucinensis and sympatric C. albula in Lake Breiter Luzin ( F _ST = 0·013–0·039). A more pronounced genetic difference was observed between both spring‐spawning species ( F _ST = 0·05–0·128). Shared mtDNA haplotypes among sympatric species within both Lake Stechlin and Lake Breiter Luzin were observed, whereas no haplotype was shared between C. fontanae and C. lucinensis . These results suggest an independent origin for spring‐spawning ciscoes in each lake. Evidence is also provided for mtDNA introgression of Coregonus sardinella into C. lucinensis and C. albula in Lake Breiter Luzin. Postglacially, this species or at least a population which showed mtDNA introgression has colonized the Baltic Sea basin up to the glacial margin that was located between Lakes Stechlin and Breiter Luzin.