Abstract
A multi-marker nuclear genotyping approach was performed on larval and adult specimens of Anisakis spp. (N = 689) collected from fish and cetaceans in allopatric and sympatric areas of the two species Anisakis pegreffii and Anisakis simplex (s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1 α-1 nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N = 678) were identified as the parental species [i.e. A. pegreffii or A. simplex (s. s.)], whereas a smaller portion (N = 11) were recognized as F1 hybrids. Discordant results were obtained when using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of 'hybrids' both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parental Anisakis and their hybrid categories derived from the application of that single marker (i.e. PCR-RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1 α-1 nDNA markers, has demonstrated that hybridization between A. pegreffii and A. simplex (s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species.
Highlights
Ecological and epidemiological studies require that actual parasite species can be accurately defined (Criscione et al 2005)
The present study has demonstrated that single molecular markers on their own based on the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) were not always able
Based on the present results, the use of other nuclear markers permitted the clear distinction of specimens belonging to the two cryptic species A. simplex (s. s.) and A. pegreffii, and enabled the distinguishing of current hybridization and introgressive hybridization events
Summary
Ecological and epidemiological studies require that actual parasite species can be accurately defined (Criscione et al 2005). Molecular methodologies provide a valuable source of additional markers in such cases of heterogeneity and fill the gap in delimiting populations and species of parasites. It has been frequently reported that sympatric populations of closely related parasite species might interbreed (Agatsuma et al 2000; Steinauer et al 2008; Detwiler and Criscione, 2010), including parasitic nematodes (Anderson, 2001; Criscione et al 2007; Dunams-Morel et al 2012). Molecular/genetic markers, especially the nuclear ones, are making these analyses accessible via a large number of tested individuals
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