Gene variation and gene flow inOrchis morio (Orchidaceae) from Italy

Data are presented on genetic variation at 27 enzyme loci of the Green-Winged orchid,Orchis morio, in 18 population samples from Italy. The existence in Italy of two subspecies, i.e. subspp.morio andpicta, is not supported by allozyme data. No genetic heterogeneity was found betweenmorio-like andpicta-like samples and specimens. Moreover, morphological transition between the two forms was observed in different Italian populations. The parameters of genetic variability estimated forO. morio populations are consistent with those found among monocotyledon plants, and among those outcrossing, animal-pollinated and with wind-dispersed seeds. Genetic diversity of ItalianO. morio is mostly within populations. Correspondingly, low values of interpopulational genetic distance were found. This appears to be due to high levels of gene flow, which were estimated with different methods. The lack ofO. longicornu from Italian samples, as well as of any hybrid withO. morio (F₁, backcrossed or recombinant individuals) is demonstrated on the basis of genetic data. It is concluded that recurrent reports ofO. longicornu from Italy are due to confusion withO. morio or with otherOrchis species.     

Hybrid zones between two genetically differentiated forms of the pond frog Rana lessonae in southern Italy

Two distinct population groups of the pond frog Rana lessonae were detected in peninsular Italy and Sicily by multilocus electrophoresis: one group inhabits the peninsula down to northern Calabria, the second occurs in southern Calabria and on Sicily. Fixed alternative alleles distinguish the two groups at 5 of the 25 loci examined; marked allele frequency differences were observed at two additional loci. On average, the two groups differ by a Nei's standard genetic distance of 0.4. A wide hybrid zone (about 120 km) occurs between the two groups, with high genotypic diversity and absence of pure parental genotypes in central Calabria. Patterns of allozyme variation suggest that at least two distinct contact and hybridization events occurred, one in the Catanzaro, the other in the Crati-Sibari plains, about 70 km to the north. Geological evidence indicates that these areas correspond to two main marine-flooded grabens that would have repeatedly interrupted or reduced genetic exchange during Plio-Pleistocene times. The finding of a fixed difference at the Mdhp-1 locus between Sicilian and Calabrian R. lessonae witnesses their continuing differentiation following their last separation by definitive opening of the Strait of Messina, about 50 000 years ago. The wide hybrid zone, the diversity of genotypes and the agreement with Hardy-Weinberg expectations suggest complete hybrid fertility. Different patterns of introgression were observed at the various loci. The pattern of allelic variation at loci in R. lessonae is paralleled by the pattern of variation in lessonae genomes of the sympatric hemiclonal hybrid Rana esculenta, into which lessonae genomes are introduced each generation as a result of hybridogenesis.     

Observations on the taxonomic status of Anopheles sicaulti

Field and laboratory studies were carried out on populations of Anopheles sicaulti and An. labranchiae from the Moroccan province of Tetouan, in order to evaluate the hypothesis of a specific rank for the former taxon, recently suggested by White (1978). The egg morphology, and particularly the exochorion pattern and the number of float ribs, showed a complete range of variation, from typical sicaulti to typical labranchiae. Females laying "intermediate" eggs are those prevailing in the study area (60.5% of the eggs collected in the Tetouan province were of this type); in some localities (Ben Karriche and Restinga) only such females were observed. The latter finding would indicate a polygenic control of the egg pattern, as the two supposed parental forms do not seem to segregate from the "intermediate" one. No pure labranchiae populations were observed in the study area, while a pure sicaulti sample was found at Kantara. More widespread appears to be the coexistence of the "intermediate" form either with sicaulti (at Mdiq and Beni Yder, in both cases with a ratio of about 1.7:1), or with both sicaulti and labranchiae (at Souk Khemis and Tatoufet, with a ratio of about 2:1:1 in the two cases). These data seem to indicate that in the considered area sicaulti interbreeds freely with labranchiae. Crossing experiments failed to evidence post-mating barriers between the two taxa; fertile hybrids were obtained in the expected numbers. The polytene chromosome studies showed no differences of diagnostic value between sicaulti and labranchiae, that have the same banding pattern. Also the genetic structure, analyzed by means of starch-gel electrophoresis on the basis of 16 gene-enzyme systems appears to be quite similar in the two forms: allele frequencies at the polymorphic loci do not show significant differences. The average Nei's genetic distance found between sicaulti and labranchiae is exceedingly low (D = 0.014); values of the same magnitude were observed between conspecific populations both in An. labranchiae and An. atroparvus (average D = 0.016 in the two cases). The polymorphic loci in the Tatoufet population, that includes the three forms: sicaulti, "intermediate" and labranchiae, are in Hardy-Weinberg equilibrium, indicating that it represents a panmictic unit. The overall picture does not favour a specific rank for sicaulti, that seems rather a local variant of An. labranchiae. The need is stressed for further investigations involving populations living in the Rabat area, from where sicaulti was originally described.     

Genetic markers in the study of Anisakis typica (Diesing, 1860): larval identification and genetic relationships with other species of Anisakis Dujardin, 1845 (Nematoda: Anisakidae)

Genetic variation at 21 gene-enzyme systems was studied in a sample of an adult population of Anisakis typica (Diesing, 1860) recovered in the dolphin Sotalia fluviatilis from the Atlantic coast of Brazil. The characteristic alleles, detected in this population, made it possible to identify as A. typica, Anisakis larvae with a Type I morphology (sensu Berland, 1961) from various fishes: Thunnus thynnus and Auxis thazard from Brazil waters, Trachurus picturatus and Scomber japonicus from Madeiran waters, Scomberomorus commerson, Euthynnus affinis, Sarda orientalis and Coryphaena hippurus from the Somali coast of the Indian Ocean, and Merluccius merluccius from the Eastern Mediterranean. Characteristic allozymes are given for the identification, at any life-stage and in both sexes, of A. typica and the other Anisakis species so far studied genetically. The distribution of A. typica in warmer temperate and tropical waters is confirmed; the definitive hosts so far identified for this species belong to delphinids, phocoenids and pontoporids. The present findings represent the first established records of intermediate/paratenic hosts of A. typica and extend its range to Somali waters of the Indian Ocean and to the Eastern Mediterranean Sea. A remarkable genetic homogeneity was observed in larval and adult samples of A. typica despite their different geographical origin; interpopulation genetic distances were low, ranging from D _Nei=0.004 (Eastern Mediterranean versus Somali) to D _Nei=0.010 (Brazilian versus Somali). Accordingly, indirect estimates of gene flow gave a rather high average value of Nm = 6.00. Genetic divergence of A. typica was, on average, D _Nei=1.12 from the members of the A. simplex complex (A. simplex s.s, A. pegreffii, A. simplex C) and D _Nei=1.41 from A. ziphidarum, which all share Type I larvae; higher values were found from both A. physeteris (D _Nei=2.77)     

Evidence for a new species of <Emphasis Type="Italic">Anisakis</Emphasis> Dujardin, 1845: morphological description and genetic relationships between congeners (Nematoda: Anisakidae)

In the present study, a new biological species of Anisakis Dujardin, 1845, was detected in Kogia breviceps and K. sima from West Atlantic waters (coast of Florida) on the basis of 19 (nuclear) structural genes studied by multilocus allozyme electrophoresis. Fixed allele differences at 11 enzyme loci were found between specimens of both adults and larvae of the new species and the other Anisakis spp. tested. Reproductive isolation from A. brevispiculata Dollfus, 1968 was demonstrated by the lack of hybrid or recombinant genotypes in mixed infections in K. breviceps. Genetic distance of the new species from its closest relative, A. brevispiculata, was D(Nei)=0.79. The new species is morphologically different from the other species which have been genetically characterised and from the other Anisakis retained by Davey (1971) as valid or as species inquirendae: the name of Anisakis paggiae n. sp. is proposed for the new taxon. Anisakis Type II larvae (sensu Berland, 1961) from the European hake Merluccius merluccius in the northeastern Atlantic Ocean (Galician coast) and from the scabbard fish Aphanopus carbo in Central Atlantic waters (off Madeira), were identified as A. paggiae n. sp. Its genetic relationships with respect to the seven species previously characterised (A. simplex (Rudolphi, 1809) sensu stricto), A. pegreffii Campana-Rouget & Biocca, 1955, A. simplex, (A. typica (Diesing, 1860), A. ziphidarum Paggi et al., 1998, A. physeteris Baylis, 1923 and A. brevispiculata) were also inferred. Overall, a low genetic identity was detected at allozyme level between the eight Anisakis species. Interspecific genetic identity ranged from I(Nei)=0.68, between the sibling species of the A. simplex complex, to I(Nei)=0.00 (no alleles shared at the considered loci) when A. physeteris, A. brevispiculata and the new species were compared with the other species of the genus. Concordant topologies were obtained using both UPGMA and NJ tree analyses for the considered species. In both analyses, A. paggiae n. sp. clustered with A. brevispiculata. They also indicated two main clades, the first including A. physeteris, A. brevispiculata and A. paggiae n. sp., the second containing all of the remaining species (i.e. A. simplex (s.s.), A. pegreffii, A. simplex, A. typica and A. ziphidarum). A deep separation between these two main Anisakis clades, also supported by high bootstrap values at the major nodes, was apparent. This is also supported by differences in adult and larval morphology, as well as with respect to their main definitive hosts. A morphological key for distinguishing adult A. paggiae n. sp., A. physeteris and A. brevispiculata is presented. Allozyme markers for the identification of any life-history stage of the Anisakis spp. so far studied, as well as ecological data on their definitive host preferences and geographical distribution, are updated.     

Genetic heterogeneity, reproductive isolation and host preferences in mealy aphids of the Hyalopterus pruni complex (Homoptera, Aphidoidea)

Three taxa were detected by allozyme markers within the mealy aphids of the Hyalopterus pruni complex, having different cultivated Prunus species as main primary hosts. The genetically closer H. pruni and H. amygdali A ( D _Nei= 0.10) never share primary hosts, whereas H. amygdali A and B ( D _Nei= 0.32) may occasionally share them, producing few F₁ hybrids. The three species proved reproductively isolated in the field, with no gene exchange. Their speciation seem to have occurred long before the agricultural revolution, crop colonization representing a host range expansion rather than a host shift, as in sympatric speciation.     

Allozyme and morphological identification of shape Anisakis,Contracaecum and Pseudoterranova from Japanese waters (Nematoda,Ascaridoidea)

Allozyme markers were used to identify anisakid nematodes from marine Japanese waters, morphologically assigned to three species complexes: Anisakis simplex (s. l.), Contracaecum osculatum (s. l.) and Pseudoterranova decipiens (s. l.). Samples assigned to A. simplex (s. l.) were found to correspond genetically to A. simplex sensu stricto, those of C. osculatum (s. l.) to C. osculatum A. No morphological characters are yet available to distinguish sibling species of these two complexes. As to the P. decipiens complex, two distinct species were detected: the first corresponded to P. decipiens C, previously recovered in the northern Atlantic, the second to P. decipiens D from Japan. The two species are genetically well differentiated, with five of the 19 loci tested showing distinct fixed alleles. Their reproductive isolation was proved by the lack of hybrids or recombinants in sympatric samples recovered from the same definitive host, Erignathus barbatus. P. decipiens D was found to correspond morphologically to Porrocaecum azarasi, previously considered a synonym of P. decipiens. Accordingly, the name Pseudoterranova azarasi (Yamaguti & Arima, 1942) n. comb. is proposed for P. decipiens D. Similarly, P. decipiens C fits in general morphology, type-locality and host with Ascaris bulbosa, also previously considered a synonym of P. decipiens. The name Pseudoterranova bulbosa (Cobb, 1888) n. comb. is proposed for P. decipiens C.     

Origin and evolution of animal hybrid species

The increasing number of hybrid species, discovered in both vertebrates and invertebrates by the combined use of chromosome, allozyme and molecular markers, calls for a reevaluation of hybrid speciation and reticulate evolution In animals. The array of reproductive strategies recently detected In phylogenetically related stick Insects allows us to Investigate, using a comparative approach, questions such as the relationship between hybridization and unisexuality, and short- versus long-term evolutionary success of hybrid species. Unexpected similarities are now apparent in hybrid evolution of animals as varied as insects, snails, fish, frogs and lizards. Hybrid species may combine, to some extent, the main advantage of sex (genetic diversity) with those of clonal reproduction. This explains why these species are often so successful, and indicates a potential use of some hybrid species in experimental biology and resource management (e.g. mass production of animal proteins).