Combining geometric morphometrics with molecular genetics to investigate a putative hybrid complex: a case study with barbels Barbus spp. (Teleostei: Cyprinidae)

This integrative study examined the morphological and genetic affinities of three endemic barbel species from Italy (brook barbel Barbus caninus, Italian barbel Barbus plebejus and horse barbel Barbus tyberinus) and of putative hybrid specimens to their species of origin. Two of the species frequently occur together with the non‐native barbel Barbus barbus. DNA barcoding indicates that mitochondrial (mt) haplotypes often do not match the species expected from morphology. Linear distance measurements and meristics are not informative for discrimination of the species and putative hybrids, but a discriminant analysis of principal components (DAPC) of geometric landmark data produces reassignments largely in congruence with mt and nuclear genetic data. Cyto‐nuclear conflicts confirm the presence of hybridization in B. plebejus and B. tyberinus and identify additional introgressed specimens. A comparison between mixed genotypes and their morphology‐based assignment reveals no predictable pattern. The finding that most individuals of the morphologically similar B. plebejus and B. tyberinus have very high assignment probabilities to their respective species suggests that the presented approach may serve as a valuable tool to distinguish morphologically very similar taxa.     

Asymmetrical introgression in a freshwater fish hybrid zone as revealed by a morphological index of hybridization

Two cyprinid species, Barbus barbus and Barbus meridionalis , form a hybrid zone in the River Lergue (southern France). Hybrids were distinguished morphologically by discriminant function analysis based on samples from the two parental taxa using only three measurements. This morphological hybridization index showed a strong asymmetry in introgression between the two taxa. B. meridionalis can live in the habitats typical of B. barbus and also in habitats where the latter cannot (small upstream tributaries), while remaining essentially unaffected by the hybridization process. By contrast, B. barbus is strongly introgressed because its habitat is invaded at least sporadically by B. meridionalis migrants. There is no behavioral barrier to hybridization, suggesting that an asymmetrical gene flow is probably partly responsible for the asymmetry in introgression.     

A behavioural study of hybridization between Barbus barbus and Barbus meridionalis

The reproductive behaviour of two species of barbel ( Barbus barbus and Barbus meridionalis ) was investigated by means of aquarium experiments in several matings of six combinations. The reproductive behaviour of the two species appears to be similar and hybridizations were observed, showing that no complete behavioural barrier blocks the mating of the two species.     

Probable direction of the postglacial colonization of rivers on northern slopes of the Carpathian Ridge by Barbus carpathicus (Teleostei: Cyprinidae) evidenced by cline of genetic variation

A cline in microsatellite diversity appeared among populations of Barbus carpathicus in four adjacent river basins on the northern slopes of the Carpathian Ridge, with western populations more variable than eastern ones ( H _S equals 0·45, 0·32, 0·23 and 0·13, respectively). Overall genetic variation was low, likely reflecting population bottlenecks during postglacial expansions through the Danube–Vistula watershed and eastward into Carpathian tributaries of the Vistula River. Genetic similarities between populations in the westernmost tributary of the Dniester (Strwiąż River) and those in the adjacent Vistula tributary (San River) suggest that at least the western part of the Dniester basin was colonized from the Vistula River system.     

Natural hybridization of two species of tetraploid barbels: Barbus meridionalis and Barbus barbus (Osteichtyes, Cyprinidae) in southern France

Natural hybridization of Barbus barbus and B. meridionalis has been demonstrated in southern France. A genetical study (isozyme electrophoresis) of these species and their hybrids revealed the characteristics of hybridization. Ten enzyme markers and five morphological parameters were used to distinguish between the two parent species. Enzymatic divergence between the parent species included fixed allelic differences at certain loci and reduction in enzymatic activity, including the silencing of certain genes. A morphological study revealed a good correlation between the isozyme markers and morphological characters. Backcrosses were observed and this raises the question of the integrity of the parent species.     

Seasonal activities of Barbus barbus: effect of temperature on time-budgeting

Surgically implanted activity-circuit radio transmitters (40 MHz) were used to study the seasonal activities of 21 adult (males: 23 to 35 cm f.l. and females 38 to 55 cm f.l. ) Barbus barbus (Pisces, Cyprinidae) in the River Ourthe (Southern Belgium) in 1989–1991. During the autumnal thermal transition (water temperature 9 to 10° C), the typical dusk and dawn pattern observed in summer turned to a trimodal pattern with the emergence of a diurnal phase. The auroral then crepuscular and finally diurnal activity periods progressively vanished as water temperature decreased down to the thermal limit for activity (4.0° C), when barbel entered a dormancy period. An opposite progressive shift was observed during the spring thermal transition. Daily activity budgets ranged from 0 to 720 min—on the annual cycle and were significantly ( r ²=0.686, P <0.05, d.f. = 36) dependent on water temperature and on morphodynamic unit size, while fish size was non-significant. Although the dusk and dawn rhythm pattern was consistent throughout summer, water temperature significantly ( P <0.05) interfered with the respective duration of crepuscular and auroral activities ( r ²=0.586, d.f. = 57 and r ²=0.692, d.f. = 55). The precise timing of activities was also thermal-related and the activities of small male barbel were proportionally more nocturnal than those of large female barbel (ANCOVA, F =80.61, d.f. = 31 and F =4.s5, d.f. = 23, at dusk and dawn respectively), possibly due to predation pressure on small fish. It is concluded that the seasonal variations of activity budgets, rhythm patterns and timings in B. barbus correspond to a form of time-budgeting partly to achieve thermal homeostasis in a variable environment.     

Updating the geographical range of Terek barbel <Emphasis Type="Italic">Barbus ciscaucasicus</Emphasis> Kessler, 1877 (Cyprinidae) using the cytochrome <Emphasis Type="Italic">b</Emphasis> molecular marker

An analysis of the mtDNA marker allowed expanding the geographical range of the Terek barbel Barbus ciscaucasicus Kessler. The new findings expand the geographic range to more than 100 km southwards down to the Pirsaat River basin in Azerbaijan. It has been suggested earlier that the Pirsaat River was inhabited by the Kura barbel Barbus cyri De Filippi. The population of the Terek barbel in the Pirsaat River is isolated genetically from the barbels of the main geographic area of this species.     

HYBRID ZONES AND HOST-PARASITE RELATIONSHIPS: EFFECT ON THE EVOLUTION OF PARASITIC SPECIFICITY

In southern France, Diplozoon gracile (Monogenea, Polyopisthocotylea), parasitizes four sympatric cyprinids. One of these host species, Barbus meridionalis, naturally hybridizes with another species of barbel, Barbus barbus, which is never parasitized by D. gracile under natural conditions. This hybridization has previously been studied and described as an introgression of B. barbus by B. meridionalis. The hybrids are parasitized by D. gracile, and parasite prevalence increases in proportion to the introgression rate, i.e., the percentage of B. meridionalis genes. The causes for this preferential distribution of the parasite in the hybrid population are analysed on the basis of ecological and ethological differences between the two parent species.     

Phylogeography of the barbel (Barbus barbus) assessed by mitochondrial DNA variation

Using the phylogeographic framework, we assessed the DNA sequence variation at the mitochondrial cytochrome b gene across the distribution range of the barbel Barbus barbus, a widely distributed European cyprinid. Reciprocal monophyly of non-Mediterranean European and Balkan/Anatolian populations is taken as evidence for a long-term barrier to gene flow, and interpreted as a consequence of survival of the species in two separate refugia during several later glacial cycles. Lack of profound genealogical divergence across Europe from western France to the northwestern Black Sea basin is consistent with recent colonization of this area from a single glacial refuge, which was probably located in the Danube River basin. This may have occurred in two steps: into the Western European river basins during the last interglacial, and throughout the Central European river basins after the last glacial. The populations from the Balkans and Anatolia apparently did not contribute mitochondrial DNA to the post-Pleistocene colonization of non-Mediterranean Europe. Lack of detectable variation within the Balkans/Anatolia is attributed mainly to recent expansion throughout these regions, facilitated by the freshwater conditions and seashore regression in the Black Sea during the Late Pleistocene and Early Holocene.     

The microhabitat distribution of two Dactylogyrus species parasitizing the gills of the barbel, Barbus barbus

The microhabitat distribution of two congeneric species Dactylogyrus carpathicus and D. malleus (Monogenea) parasitizing the gills of the barbel (Barbus barbus L.) was investigated. We tested whether congeneric species exhibited microhabitat preference and whether interspecific interactions could be attributed to the microhabitat segregation of congeners. The outlying mean index method was used to evaluate species microhabitats. Gill variables (different microhabitats within gills) were used as environmental factors characterizing the gills. When abundances of both species were highest, and no significant difference was found between the abundance of the two species, the gill segments and gill areas were the most important factors segregating the Dactylogyrus species on the gills. Niche overlap was low within each of the four gill arches, and parasites were segregated in the same microhabitats within each gill arch. When abundances of both species were low, each monogenean species was segregated at the level of the gill arches. When abundances of both species increased, the niche and overlap between species increased. The distribution of both congeneric species confirmed microhabitat preference within the gills. The results suggest that microhabitat preference is dependent on species abundances, species being segregated in the case of low abundance, possibly to increasing mating opportunities. Both niche and overlap between species increased with species abundance. In the case of the high abundance of both species, microhabitat preference seems to be related to interspecific interactions between monogenean species, as previously found for endoparasitic species.     

Polymorphism of white muscle myosin and parvalbumins in the genus Barbus (Teleostei: Cyprinidae)

Muscle proteins were investigated in two large European barbels, Barbus barbus and B. meridionalis , and in four small tropical barbels native to SE Asia: B. conchonius , B. tetrazona , B. sachsi and B. titteya . Polyacrylamide gel electrophoresis was used to analyse myosin heavy and light chains and parvalbumin isotypes from white trunk muscle. Each species could be biochemically identified. The myosin subunit and parvalbumin isotype patterns obtained for the two European barbels were similar. The Asian barbels, on the other hand, not only differed from the European species but displayed a greater diversity within their group. These biochemical results are largely in agreement with morphological and genetic data, but fail to substantiate suggested close relationships between Asian barbel species.     

Does the stock density of stillwater catch and release fisheries affect the growth performance of introduced cultured barbel?

In comparison to barbel Barbus barbus in their natural, riverine environment, the growth performance of cultured barbel, following introduction into stillwater environments, was depressed when the extant stock density was above 500 kg ha⁻¹ and common carp Cyprinus carpio were present.     

Incidences of gross morphological disorders in barbel (Barbus barbus) in three rivers in southern England

Incidences of gross morphological disorders in barbel ( Barbus barbus ) were investigated in three rivers in southern England. The water quality at the sites chosen for study on the Rivers Teme, Kennet and Lee were classified (according to the criteria of the National Water Council, 1978) as 1A, IB and 2, respectively. Generally, low levels of gross morphological abnormalities were seen in fish caught in the Teme and Kennet, although there were incidences of ocular disorders and extra barbules in 10 and 7%, respectively, of the Kennet fish. No tumours were found in barbel from the River Teme whereas there was a 2% incidence in the Kennet fish. In contrast barbel from the River Lee showed high levels of all the gross morphological disorders, especially haemorrhagic patches on the ventral surface (36%) and ocular disorders (72%). Tumours were seen in 25% of the barbel caught in the River Lee. The incidences of most of these gross morphological disorders did not appear to be age related. The possibility that these disorders were related to water quality is discussed.     

A population study of Barbus barbus (L.) in the River Severn, England

The results of the application of Schnabel (Chapman modification), Schumacher & Eschmeyer, Fisher & Ford, and Jolly capture-recapture techniques on the Barbus barbus (L.) population in four areas of the middle Severn are compared. The survival of tagged fish varied in different areas and fish released in some time periods were less vulnerable to recapture than fish released from other samples. It is suggested that the shoaling behaviour of Barbus barbus resulted in non-random mixing of marked fish. The average population size of Barbus barbus greater than 18 cm in length in each of the four sections for the total period of investigation gave population densities ranging from 0.024/m² to 0.202/m².     

Genetic evidence for asymmetric hybridization between menhadens (Brevoortia spp.) from peninsular Florida

Hybridization between the North American menhadens Brevoortia smithi , Brevoortia tyrannus and Brevoortia patronus has been reported by numerous authors, based primarily on the morphological intermediacy of hybrids relative to each parental type. In this study, genetic characterization of individuals in areas previously designated as hybrid zones validates the presence of F₁ hybrids. Evidence from microsatellite genotypes indicates that B. patronus and B. smithi are parental species of hybrids on both sides of the Florida peninsula, and mitochondrial DNA (mtDNA) haplotypes suggest a maternal contribution by B. smithi in each case. Hybrids between B. tyrannus and B. smithi were not identified in this study. The presence of B. patronus on the Atlantic coast of Florida, coupled with admixture between this species and B. tyrannus , has likely influenced the mixed lineage assortment of mtDNA haplotypes demonstrated in this study and in previous studies.     

Branchial osteogenetic neoplasm in barbel Barbus barbus plebejus.

A branchial osteogenetic neoplasm affecting a barbel Barbus barbus plebejus (Valenciennes, 1829) is described. The osteoblasts' pleomorphism, the lack of a well-developed and complete separation, the presence of eccentric, terminal proliferative edges infiltrating the lining tissues and the abundant tumour matrix suggest a histopathological diagnosis of a 'productive osteoblastic osteosarcoma'. The occurrence of eosinophilic granule cells (EGCs) scattered among neoplastic tissue is discussed in relation to the neoplastic growth and the inflammatory reaction, with reference to recent discoveries in mammalian mast cell biology.     

Spatially varying natural selection in a fish hybrid zone

A capture-mark-recapture (CMR) study in a hybrid zone between two species of barbel, Barbus barbus and Barbus meridionalis , in the Lergue River (southern France) failed to reject the null hypothesis of no difference in survival among phenotypes. Power calculations indicated that very intensive fieldwork should be carried out if this hypothesis was to be investigated again. The study demonstrated, however, that there were marked differences in survival between upstream and downstream sites. It is suggested that there is an environmental gradient of selection along the hybrid zone (extrinsic component of natural selection).     

Phylogeny and evolution of the genus Barbus in the Iberian Peninsula as revealed by allozyme electrophoresis

Variation of 14 enzyme systems encoded by 31 presumptive loci in different barbel species was studied using starch gel electrophoresis. Eighteen samples representing 11 Barbus tetraploid taxa were taken, including 10 populations from the Iberian Peninsula, six from other parts of western and southern Europe, one from northern Africa and one diploid species as outgroup from Asia Minor. The genetic analysis reassessed of the taxonomic status of the Iberian barbels into two distinct species groups. The first group included B. bocagei, B. comiza, B. graellsii, B. gulraonis, B. microcephalus , and B. sclateri , that aligned with B. callensis from northern Africa and with B. apoensis from Asia Minor; the other group included B. haasi and B. merldlonahs that was related to the European species, B. barbus, B. plebejus and B. peloponnesius . These groups are probably not monophyletic. It is suggested that the isolation of the Iberian Peninsula from Europe since the Oligocene-Miocene may explain the genetic affinities of the Iberian barbels with those of North African rather than with the European group.     

Hybridization and reproductive isolation among syntopic populations of the topminnows Fundulus notatus and F. olivaceus

Fundulus notatus and Fundulus olivaceus are two closely related topminnow species that exhibit similar ecological niches and broad, largely overlapping, North American ranges extending throughout much of the Mississippi River drainage as well as the coastal drainages of the Gulf of Mexico. Previous studies have suggested that these two species are reproductively compatible despite cytogenetic differences and will hybridize when syntopic. We used nuclear and mtDNA loci to assess levels of hybridization and test for introgression in syntopic populations of these two species in four drainages in southern Illinois. Although hybridization was detected in all syntopic populations, an assessment of the proportion of hybrid individuals indicated a deficiency of hybrids relative to expectations under random mating. We determined that, although mtDNA introgression was prevalent and extended beyond the zones of contact, evidence of nuclear introgression was limited to the zone of sympatry.     

Introgressive hybridization in fishes: the biochemical evidence

Biochemical methods can detect variation at individual genetic loci, making possible the direct assessment of natural hybridization and introgression between fish populations. Protein electro-phoresis has been used to confirm and extend knowledge of many situations where species hybrids have been detected by morphological analyses. New cases of natural hybridization, including some at the subspecies level, have also been identified. Biochemical studies have provided the first conclusive evidence of natural post F₁ hybrids and of introgression between fish taxa. The strongest cases for introgression have used a combined analysis of nuclear protein genes and taxaspecific maternally inherited mitochondrial DNA variation. Information on the significance of introgression as a source of gene flow between taxa, particularly below the species level where sympatric subspecies and sibling species are involved, should expand in the future as the numbers and types of nuclear and mitochondrial DNA loci which can be assayed for variation increase. The full importance of introgressive hybridization in speciation may then be understood.