Luciano Bullini

Electrophoretic studies on the Anisakis simplex complex (Ascaridida:Anisakidae) from the Mediterranean and North-East Atlantic.

Abstract Nascetti G., Paggi L., Orecchia P., Smith J.W., Mattiucci S. and Bullini L. 1986. Electrophoretic studies on the Anisakis simplex complex (Ascaridida: Anisakidae) from the Mediterranean and North-East Atlantic. International Journal for Parasitology 16: 633–640. The genetic variation of the sibling species Anisakis simplex A and A. simplex B was investigated by electrophoretic analysis of 22 gene-enzyme systems. The two species are reproductively isolated and no gene flow takes place between them. Three loci, Sod, Adk-2 and Lap-1, show distinct alleles in A. simplex A and A. simplex B, allowing their reliable identification both at the larval and adult stages. A fourth locus, Got, appears to be diagnostic at the 95% level. The value of Neis genetic distance found between A. simplex A and A. simplex B is 0.28. Parameters of genetic variability (He, P, A) are given for both species. The geographic distribution of A. simplex A and A. simplex B appears to be mainly Mediterranean for the former, and mainly North Atlantic for the latter. Several paratenic hosts (fish and squid) and one cetacean definitive host are identified for each of the two species. The names A. pegreffii and A. simplex are tentatively proposed for A. simplex A and A. simplex B respectively.

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).

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 Neis 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 Neis 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 Neis 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.

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 Neis 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.

Evidence for a new species of Anisakis 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 DNei=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 INei=0.68, between the sibling species of the A. simplex complex, to INei=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 indciated 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 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.

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 Haldanes 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.

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.

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.

A new species of Anisakis Dujardin, 1845 (Nematoda, Anisakidae) from beaked whales (Ziphiidae): allozyme and morphological evidence

Larvae and adults of Anisakis, recovered from the beaked whales Mesoplodon layardii and Ziphius cavirostris from the Mediterranean Sea and South African waters, were analysed morphologically and by molecular markers (allozymes). A new Anisakis species was identified, showing fixed allele differences at a number of loci from the other Anisakis spp. tested (A. simplex complex, A. physeteris). The lack of hybrid or recombinant genotypes in mixed infections with A. pegreffii, A. simplex C and A. physeteris, as well as the high values of genetic distance (average DNei = 1.65 from the members of the A. simplex complex, and DNei = 3.09 from A. physeteris) showed that the new species is reproductively isolated. This new Anisakis species is morphologically different from the other Anisakis retained by Davey (1971) as either good species or species inquirendae. The name Anisakis ziphidarum n. sp. is proposed for the new species.