Hybridization and introgression of the mitochondrial genome between the two species Anisakis pegreffii and A. simplex (s.s.) using a wide genotyping approach: evolutionary and ecological implications

Abstract

Anisakis pegreffii and A. simplex (s.s.) are the two zoonotic anisakids infecting cetaceans as well as pelagic/demersal fish and squids. In European waters, A. pegreffii prevails in the Mediterranean Sea, while A. simplex (s.s.) in the NE Atlantic Ocean. Abiotic conditions likely play a significant role in shaping their geographical distribution. The Iberian Atlantic and Alboran Sea waters are sympatric areas of the two species. A total of 429 adults and L3 stage from both sympatric and allopatric areas were studied by a wide nuclear genotyping approach (including newly and previously found diagnostic single nucleotide polymorphisms (SNPs) at nuclear DNA (nDNA) and microsatellite DNA loci) and sequenced at mitochondrial DNA (mtDNA) cox2. Admixture between the two species was detected in the sympatric areas studied by STRUCTURE Bayesian analysis; NEWHYBRIDS revealed different categories of hybridization between the two species, representing approximately 5%. A tendency for F1 female hybrids to interbreed with the parental species at the geographical distribution limits of both species was observed. This finding suggests that hybridization occurs when the two parental species significantly differ in abundance. Mitochondrial introgression of A. simplex (s.s.) in A. pegreffii from Mediterranean waters was also detected, likely as a result of past and/or paleo-introgression events. The high level of genetic differentiation between the two species and their backcrosses indicates that, despite current hybridization, reproductive isolation which maintains evolutionary boundaries between the two species, exists. Possible causes of hybridization phenomena are attempted, as well as their evolutionary and ecological implications, also considering a sea warming scenario in European waters.

Keywords: Anisakis; DNA microsatellites; SNPs; hybridization; mitochondrial introgression; mtDNA cox2.

Graphical abstract

Figure 1.

Collecting sites of A. simplex (s.s.) and A. pegreffii analysed in the present study, mapped onto the geographical range of the two parasite species (coloured in red and green, respectively). ADS, Adriatic Sea; TYR, Tyrrhenian Sea; IOS, Ionian Sea; TOR, Torrevieja (Mediterranean Spain coast); ALB, Alboran Sea; CDZ, Cadíz (Atlantic Spain Coast); HUE, Huelva (Atlantic Spain Coast); VIG, Vigo (Atlantic Spain Coast); BSB, Bay of Biscay; SCO, Seas West of Scotland; NWS, Norwegian Sea; NZE, New Zealand coast.

Figure 2.

Chromatograms at the diagnostic SNPs in Nas10, EF1 α-1, Sod and Adk nDNA loci and electropherograms of the 5 diagnostic and partially diagnostic SSR loci (Anisl 7, Anisl 22, Anisl 15, Anisl 4 and Anisl 10535), showing the heterozygous genotype having a double peak between A. simplex (s.s.) and A. pegreffii exhibited by admixed specimens between the two species.

Figure 3.

(A) Alignment of the region of the Sod nDNA, showing the 100% diagnostic positions: 6 SNPs (black triangles) between A. simplex (s.s.) and A. pegreffii. (B) Alignment of the region of the Adk nDNA, showing the 100% diagnostic positions: 8 SNPs (black triangles) between A. simplex (s.s.) and A. pegreffii.

Figure 4.

Distribution of allele frequencies at the partially diagnostic and 100% diagnostic SSR loci between A. pegreffii (green) and A. simplex (s.s.) (red).

Figure 5.

(A) Percentage contribution (Q) of each specimen of A. pegreffii and A. simplex (s.s.) to each cluster following a Bayesian clustering analysis implemented in STRUCTURE on 17 diagnostic SNPs at nDNA gene loci (EF1 α-1, Nas10, Sod and Adk) and at the SSR loci (i.e. Anisl 10535 and Anisl 05784; sex-linked loci were excluded to include male specimens in the elaboration), with K = 2. (B) The posterior probability of assignment to 1 of 5 genotype classes following a Bayesian clustering analysis implemented in NEWHYBRIDS. Those specimens with evidence of admixture were assigned to pure A. pegreffii and A. simplex (s.s.), F1 hybrids or backcrossed categories with a 100% posterior probability value of assignment.

Figure 6.

Statistical parsimony haplotype network (TCS) among mtDNA cox2 haplotypes showed by pure specimens of the two parental species A. pegreffii and A. simplex (s.s.) and by their hybrid individuals, plus the specimens from ATYS, ADS and EMS sea waters showing mitochondrial introgression. Circles suggest the two different haplogroups represented by the parental species (A. pegreffii: light green, A. simplex (s.s.): light red).

Figure 7.

Spatial distribution and relative proportions of the F1 hybrids, backcrosses and mitochondrial introgressed specimens detected, in the present study, by multi-markers approach according to their nuclear genotype (A) as inferred from STRUCTURE and NEWHYBRIDS (see FIGURE 5), and mithocondrial genes (B).