Contracaecum gibsoni n. sp. and C. overstreeti n. sp. (Nematoda: Anisakidae) from the Dalmatian pelican Pelicanus crispus (L.) in Greek waters: morphological and genetic evidence

Two new species of Contracaecum Railliet & Henry, 1912, previously referred to as C. multipapillatum sp. A and C. multipapillatum sp. B by Nascetti et al. (1990) from the Dalmatian pelican Pelecanus crispus (L.) in the Ambracian Gulf off Greece, are described as C. gibsoni n. sp. and C. overstreeti n. sp., respectively. Morphological analysis and the differential diagnosis of genetically recognised male specimens of C. gibsoni and C. overstreeti with respect to C. multipapillatum (von Drasche, 1882) (sensu lato) from Egretta alba (L.) in northern Colombia and other morphologically related Contracaecum spp. enabled the detection of differences between the two species in a number of characters, including spicule length and the shape of its tip, and the arrangement of the proximal and distal papillae on the male tail. Accordingly, formal descriptions are presented for C. gibsoni n. sp. and C. overstreeti n. sp. from P. crispus. The genetic characterisation of the two taxa is based on 20 allozyme loci and sequence analyses (519 bp) of the mtDNA cox2 gene. Reproductive isolation was demonstrated between these two taxa, which sympatrically infect the same definitive host, and fixed allele differences between the two species were found at some (Aat-2, PepC-1, PepC-2 and Pgm-1) of the 20 allozyme loci analysed. Their genetic divergence, estimated at the allozyme level, was D (Nei) = 0.31. The genetic relationships of C. gibsoni and C. overstreeti with respect to C. multipapillatum (s. l.) collected from E. alba in Colombia, as well as with other congeners from fish-eating birds which had previously been genetically characterised using the same genetic markers, i.e. C. rudolphii A and C. rudolphii B of Bullini et al. (1986), C. septentrionale Kreis, 1955, C. microcephalum (Rudolphi, 1809), C. bioccai Mattiucci et al., 2008, C. pelagicum Johnston & Mawson, 1942 and C. micropapillatum (Stossich, 1890), were inferred from mtDNA cox2 sequence analysis. The MP, NJ and BI trees obtained were congruent in depicting C. gibsoni and C. overstreeti as closely related species but quite distinct from each other and forming a subclade with specimens of C. multipapilllatum (s. l.) from E. alba (L.) in Colombia. This subclade was also found to be distinct from the remainder of the taxa considered.