Differential patterns of hybridization and introgression between the swallowtails Papilio machaon and P. hospiton from Sardinia and Corsica islands (Lepidoptera, Papilionidae).

Proportions of hybridization and introgression between the swallowtails Papilio hospiton, endemic to Sardinia and Corsica, and the holarctic Papilio machaon, were characterized using nine fully diagnostic and two differentiated allozyme loci and a mitochondrial DNA marker. Very low frequencies of F1 hybrids were detected in both Sardinia (0-4%, average 1.4%) and Corsica (0-3%, average 0.5%), as well as of first generation backcrosses (B1). No F2 were observed, in agreement with the hybrid breakdown detected in laboratory crosses. In spite of this minimal current gene exchange, specimens carrying introgressed alleles were found in high proportions in P. machaon but in lower proportions in P. hospiton. Introgression apparently occurred through past hybridization and repeated backcrossing, as evidenced by hybrid index scores and Bayesian assignment tests. Levels of introgression were low (0-1%) at two sex-linked loci and mitochondrial DNA, limited (0.4-2%) at three autosomal loci coding for dimeric enzymes, and high (up to 43%) at four autosomal loci coding for monomeric enzymes. Accordingly, selective filters are acting against foreign alleles, with differential effectiveness depending on the loci involved. The low levels of introgression at sex-linked loci and mitochondrial DNA are in agreement with Haldane's rule and suggest that introgression in P. machaon proceeds mainly through males, owing to a lower fitness of hybrid females. Papilio machaon populations showed higher levels of introgression in Sardinia than in Corsica. The role of reinforcement in the present reproductive isolation between P. machaon and P. hospiton is examined, as well as the evolutionary effects of introgressive hybridization between the two species.

Genetic evidence for two sibling species within Contracaecum ogmorhini Johnston & Mawson, 1941 (Nematoda: Anisakidae) from otariid seals of boreal and austral regions.

Genetic variation of Contracaecum ogmorhini (sensu lato) populations from different otariid seals of the northern and southern hemisphere was studied on the basis of 18 enzyme loci as well as preliminary sequence analysis of the mitochondrial cyt b gene (260 bp). Samples were collected from Zalophus californianus in the boreal region and from Arctocephalus pusillus pusillus, A. pusillus doriferus and A. australis from the austral region. Marked genetic heterogeneity was found between C. ogmorhini (sensu lato) samples from the boreal and austral region, respectively. Two loci (Mdh-2 and NADHdh) showed fixed differences and a further three loci (Iddh, Mdh-1 and 6Pgdh) were highly differentiated between boreal and austral samples. Their average genetic distance was D(Nei) = 0.36 at isozyme level. At mitochondrial DNA level, an average proportion of nucleotide substitution of 3.7% was observed. These findings support the existence of two distinct sibling species, for which the names C. ogmorhini (sensu stricto) and C. margolisi n. sp., respectively, for the austral and boreal taxon, are proposed. A description for C. margolisi n. sp. is provided. No diagnostic morphological characters have so far been detected; on the other hand, two enzyme loci, Mdh-2 and NADHdh, fully diagnostic between the two species, can be used for the routine identification of males, females and larval stages. Mirounga leonina was found to host C. ogmorhini (s.s.) in mixed infections with C. osculatum (s.l.) (of which C. ogmorhini (s.l.) was in the past considered to be a synonym) and C. miroungae; no hybrid genotypes were found, confirming the reproductive isolation of these three anisakid species. The hosts and geographical range so far recorded for C. margolisi n. sp. and C. ogmorhini (s.s.) are given.

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)

Genetic divergence and reproductive isolation between Anisakis brevispiculata and Anisakis physeteris (Nematoda: Anisakidae)s.

In order to assess the taxonomic status of Anisakis brevispiculata Dollfus, 1966 population samples of this taxon from central and south-eastern Atlantic ocean were compared at 22 enzymatic loci with samples belonging to Anisakis physeteris Baylis, 1923 from the Mediterranean sea and central-eastern Atlantic ocean. Very low interpopulational genetic divergence was observed both within A. brevispiculata (average D(Nei) = 0.008) and within A. physeteris (D(Nei) = 0.009) despite the geographic distance among the samples, indicating high levels of gene flow in both taxa. On the other hand, the average genetic distance between A. brevispiculata and A. physeteris was found to be D(Nei) = 0.80, a value generally observed between well differentiated congeneric species. The reproductive isolation between A. brevispiculata and A. physeteris is indicated by the following observations: (1) no F(1) hybrids or recombinant genotypes were until now observed; and (2) the two Anisakis species do not seem to share their definitive hosts. The main definitive host of A. brevispiculata is the pygmy sperm whale (Kogia breviceps), while for A. physeteris it is the sperm whale (Physeter catodon). Only adult males differ slightly in spicule length, while females and larval stages are not differentiated morphologically. Both A. brevispiculata and A.physeteris show a type II larva. The correct recognition of A. brevispiculata from A. physeteris and from other Anisakis species studied, in either sexes and at any life stage, is made easy by allozyme markers (e.g. Icdh, Gapdh, Sod-1, Np, Aat-2, Adk-2, fEst-2, PepB, PepC-2, Mpi). Diagnostic keys, which can be used for routine identification in the field of these Anisakis worms, based on genetic markers, are given.

Pseudoterranova decipiens species A and B (Nematoda, Ascaridoidea): nomenclatural designation, morphological diagnostic characters and genetic markers.

Five genetically distinct and reproductively isolated species have been detected previously within the morphospecies Pseudoterranova decipiens from the Arctic-Boreal, Boreal and Antarctic. Morphological analysis was carried out on male specimens identified by genetic (allozyme) markers, allowing the detection of significant differences at a number of characters between two members of the P. decipiens complex, namely P. decipiens A and B. On the basis of such differences, the nomenclatural designation for the two species is discussed. The names Pseudoterranova krabbei n. sp. and P. decipiens (sensu stricto) are proposed for species A and B, respectively. Morphological and genetic differentiation between the two species is shown using multivariate analysis. Allozyme diagnostic keys for routine identification of the four members of the P. decipiens complex, namely P. decipiens (s.s.), P. krabbei, P. bulbosa and P. azarasi, irrespective of sex and life-history stage, are provided.

Two new members in the Contracaecum osculatum complex (Nematoda, Ascaridoidea) from the Antarctic.

The genetic structure of adults and larvae of Contracaecum osculatum (sensu lato) from the Antarctic is analyzed on the basis of 24 enzyme loci. Significant deviations of genotype frequencies from the Hardy-Weinberg equilibrium were found, even in samples recovered from the same host. These data indicate that two distinct, reproductively isolated species coexist in C. osculatum (sensu lato) samples from the Antarctic. They were provisionally designated C. osculatum D and E, as they do not correspond to any of the three species previously detected in this complex from the Atlantic Arctic Boreal region (C. osculatum A, B and C). An allozyme diagnostic key for the identification of the five members of the C. osculatum complex, at the larval and adult stage and in both sexes, is given. Species D and E were found to be genetically quite variable: average P99 = 84.3, A = 3.3 and He = 0.23. Both showed high values of intraspecific gene flow: Nm = 4.6 and 6.1 respectively; similar values were found for the Arctic-Boreal C. osculatum A, B and C. The most related members of the complex are the Antarctic species E and the Arctic-Boreal species A (DNei = 0.21), while the most differentiated ones are the Arctic-Boreal species B and C (DNei = 0.76). The evolutionary divergence of C. osculatum C started more than 3 million years ago, in a Pliocene refugium (Baltic Sea). As to the other C. osculatum species, their evolutionary divergence took place during Pleistocene, when this complex achieved a bipolar distribution. This process involved two distinct colonizations of the marine Antarctic region by ancestors of the northern hemisphere, about 1.5 and 1 million years ago, giving origin to C. osculatum D and E respectively.

Three sibling species within Contracaecum osculatum (Nematoda, Ascaridida, Ascaridoidea) from the Atlantic Arctic-Boreal region: reproductive isolation and host preferences.

Genetic variation within and between population samples from 22 locations of the Atlantic Arctic-Boreal region, including 1657 specimens morphologically assigned to Contracaecum osculatum, was electrophoretically analysed at 17 loci. Highly significant deviations from the Hardy-Weinberg equilibrium were found at various loci in several samples, owing to the existence of three distinct gene pools within C. osculatum (sensu lato) from the study area. These gene pools correspond to three biological species (provisionally designated A, B and C), characterized by distinct genotypes at several diagnostic loci. Reproductive isolation between C.osculatum A, B and C is confirmed by the lack of F1, recombinant, or backcross genotypes in sympatric areas, despite the occurrence of multiple infections. Mean heterozygosity per locus is on average 0.11 in species A, 0.10 in B and 0.07 in C. High levels of gene flow were found within each of the three species, the values of Nm (number of migrant individuals) ranging from 3.41 (C. osculatum C) to 5.77 (C. osculatum A). Average Nei's genetic distance is 0.46 between A and B, 0.50 between A and C and 0.77 between B and C. From these values, times of evolutionary divergence from 2 to 4 million years can be estimated. Genetic relationships among populations and species of the C. osculatum complex are illustrated by principal component analysis. The role of both geographical isolation and host preferences in the speciation of C. osculatum (sensu lato) is discussed. A morphological distinction of the three species has not yet been possible (sibling species). However, there is evidence that the name C. osculatum (sensu stricto) should be used for species C, which shows a geographical distribution and definitive host corresponding to the neotype of C. osculatum (sensu stricto). Finally, a comparison is made between the members of the C. osculatum complex from the Atlantic Arctic-Boreal region and those of the Pseudoterranova decipiens complex from the same area, as to: (i) times of evolutionary divergence, (ii) geographical distribution, and (iii) host preferences.

Genetic evidence for three species within Pseudoterranova decipiens (Nematoda, Ascaridida, Ascaridoidea) in the North Atlantic and Norwegian and Barents Seas.

Genetic variation of 1017 specimens of codworm, Pseudoterranova decipiens, collected from fish and seals at 23 sampling locations in the North Atlantic and Norwegian and Barents Seas, was analysed on the basis of 16 enzyme loci. Three reproductively isolated species, provisionally designated P. decipiens A, B and C, were detected, showing distinct alleles at the following loci: Mdh-1, 6Pgdh, Np, Pgm, Est-2 (between species A and B); Mdh-3, 6Pgdh, Np, Sod-1, Adk, Pgm, Est-2, Mpi (between A and C); Mdh-1, Mdh-3, Sod-1, Adk, Pgm, Est-2, Mpi (between B and C). One F1 hybrid was observed between P. decipiens A and B, but this apparently does not lead to any gene exchange between the two species, which do not show any evidence of introgression. No hybrids or introgressed individuals were observed between P. decipiens C and either A or B. Genetic distances among conspecific populations were low (average Nei's D 0.001-0.005), even though they were collected thousands of kilometres apart, indicating high levels of gene flow within each of the three species. The values of Nei's index D were 0.44 between P. decipiens A and B, 0.57 between B and C, and 0.79 between A and C. Estimated evolutionary divergence times, using Nei's formula, range from 2 to 4 million years. Differences between P. decipiens A, B and C were also found with respect to genetic variability, morphology, geographical distribution and hosts. Mean heterozygosity values of 0.08, 0.05 and 0.02 were obtained for P. decipiens A, B and C, respectively. Preliminary morphological examination of adult males, previously identified by multilocus electrophoresis, revealed differences in the relative size and pattern of caudal papillae. P. decipiens B is widespread in the study area, whereas P. decipiens A was found only in the North-East Atlantic and Norwegian Sea. In this area P. decipiens A is most common in the grey seal, Halichoerus grypus, while the common seal, Phoca vitulina, is the main host for P. decipiens B. In Canadian Atlantic waters, where P. decipiens A is apparently absent, P. decipiens B infects both grey and common seals; a few specimens were also found in the hooded seal, Cystophora cristata. The only definitive host so far identified for P. decipiens C is the bearded seal, Erignathus barbatus; P. decipiens C appears to be widespread, occurring in both the North-West Atlantic and Barents Sea.

Preliminary data on the genetic differentiation of Onchocerca volvulus in Africa (Nematoda: Filarioidea).

Data are reported on the genetic structure of three Onchocerca volvulus populations, respectively from Mali (savanna), Ivory Coast (forest), and Zaire (forest gallery in savanna). Electrophoretic analysis, carried out on 25 gene-enzyme systems, has shown a remarkable genetic heterogeneity existing within O.volvulus. Zaire and West Africa populations appear chiefly differentiated at Mdh-1 and 6Pgdh loci, their average Nei's genetic distance being 0.11. In West Africa Nei's D found between the savanna and forest samples is 0.04. The savanna population from Zaire is more similar to the savanna one from Mali (D = 0.09) than to the forest one from Ivory Coast (D = 0.13). This appears mainly due to the loci Ldh and Hbdh (possibly linked), some alleles of which seem to be selected for in forest populations (Ldh110, Hbdh108), while others in the savanna ones (Ldh100, Hbdh100). The hypothesis that the discrepant epidemiological patterns of human onchocerciasis are related to intrinsic differences in the parasite seems supported by the obtained data. The differences in allele frequencies found at the reported loci appear strong enough to allow biochemical identification of O. volvulus populations from different geographic regions and different habitats.

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 distance between two sibling species of the Aedes mariae complex (Diptera, Culicidae).

Two sibling species of the mariae complex of the Aedes genus have been studied: Ae. mariae and Ae. zammitii. Thee mosquitoes are allopatric and show similar adaptations to rocky mediterranean coasts. The isolating mechanisms between the two species are well studied. A partial sterility of hybrid F1, limited to males, has been found with laboratory cross experiments. Strong pre-mating isolating mechanisms were shown in nature by means of release experiments, the frequency of hybrids never exceeding 2% without evidence of introgression. We analyzed genetic variation at 26 enzyme loci in a population of each species: 35% of loci were polymorphic, with an observed mean heterozygosity of 0.07 in Ae. mariae and 0.06 in Ae. zammitii; 6 loci allow discrimination between the two species at a probability of at least .99. Nei's measures of genetic identity and genetic distance are respectively I = 0.6096 and D = 0.2828. The distribution of genetic identities relative to loci is strongly bimodal, reflecting a different contribution of highly variable and fast evolving loci on one side and conservative and slowly evolving loci on the other. We consider, on the base of various experimental data (see Powell, 1975), in the first class ("fast evolving" loci) both variable substrate and regulatory enzymes, in the second ("slow evolving" loci) non regulatory enzymes. Values of genetic distance calculated separately for the "fast" (54% of the loci studied) and "slow" (46%) evolving loci are respectively Df = 0.41 and Ds = 0.03, showing that the main contribution to genetic distance in the first period of divergence is due to fast evolving loci, whose rate of evolution is about ten times more rapid with respect to slow evolving loci.

Isocitrate dehydrogenase in Aedes aegypti: formal genetics, preliminary linkage data and study of natural populations.

Two loci for isocitrate dehydrogenase (Idh-1 ad Idh-2) are described in Ae. aegypti, both polymorphic with two codominant alleles. Crosses made to test linkage relationships of Idh-1 indicate that this locus is independent from sex (chromosome 1) and from Sod-1 and Hk-1 loci (chromosome 3), while it is linked to Pgm on the second chromosome. Average percent of recombination is 11.37, but significant differences have been found among strains. Data on genetic variability of Idh-1 and Idh-2 in three domestic african field populations are presented.