Cryptic species ofDidymobothrium rudolphii(Cestoda: Spathebothriidea) from the sand sole,Solea lascaris, off the Portuguese coast, with an analysis of their molecules, morphology, ultrastructure and phylogeny

Abstract

S U M M A R YDidymobothrium rudolphii (Cestoda : Spathebothriidea) was collected seasonally from the sand sole, Solea lascaris, off the northern, central and southern areas of the Portuguese coast. Morphological and molecular analyses were conducted in order to examine the possible existence of cryptic species and to facilitate the circumscription of their morphological boundaries. Data were compared between D. rudolphii specimens from each of the 3 geographical areas and 4 seasons, and principal components analysis of 18 morphological characters was used to detect differences. Two distinct genotypes were present with sequence divergences of 1 . 9% and 2 . 1% in the large subunit (lsrDNA) and second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA), respectively. The less common ' central' genotype was present only off the central area from summer to winter, whereas the ' common ' genotype was present throughout the year off the northern and southern areas, but only during spring in the central area. No sequence variation was found within each genotype. The presence of 2 distinct genetic entities was supported by morphological analyses, which showed the ' central ' genotype specimens to be more slender and elongate, although morphometric ranges overlapped considerably for most characters. Molecular phylogenetic analysis of 4 of the 5 known genera of the Spathebothriidea showed Spathebothrium to be the earliest branching lineage and the 2 genotypes of Didymobothrium formed a sister group to Cyathocephalus. The concordance of genetic differences with variation in host diet according to season and locality could account for sympatric speciation occurring in the central region of the Portuguese coast.

Ultrastructure of the intrauterine eggs of Didymobothrium rudolphii (Monticelli, 1890) (Cestoda, Spathebothriidea, Acrobothriidae) and its phylogenetic implications

“…All specimens examined correspond to the 'common form' of D. rudolphii, as defined by Marques et al (2007). The worms were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 20 days at 5°C.…”

“…As mentioned in the introduction, Burt and Sandeman (1969) considered both Didymobothrium and Diplocotyle as synonyms of Bothrimonus; decisions rejected by Gibson (1994) and apparently not supported by molecular studies (e.g. Marques et al 2007, Olson et al 2008. According to Sandeman and Burt (1972), the suggested life cycle of this parasite is summarised as follows: "Eggs released from gravid worms in the summer adhere to natural objects in water by means of their polar filaments.…”

“…Adding to the complexity of problems concerning the validity of spathebothriidean taxa, it is likely that some or all may comprise a complex of cryptic species, as has been shown by the allozyme studies by Renaud and Gabrion (1988) in which D. rudolphii (referred to as Bothrimonus nylandicus Schneider, 1902) from soleids in the western Mediterranean and eastern Atlantic was found to comprise two sympatric, sibling species. Furthermore, Marques et al (2007) recently demonstrated the presence of two distinct genotypes of D. rudolphii along the Portuguese coast using two molecular markers (large-subunit and ITS rDNA) that correlated with statistically significant differences in the overall length and width of the otherwise cryptic forms.…”

“…To date, there has been no information on the TEM ultrastructure of intrauterine eggs of spathebothriidean cestodes. Information on the intrauterine eggs is restricted to two SEM micrographs of Marques et al (2007); these authors indicated that the eggs of D. rudolphii are 25-42 ×14-22 µm in size, and their SEM observations revealed a rough shell surface and the presence of an operculum at their more rounded pole and a tuft of filaments at their narrower pole. So far, the TEM studies of the eggs of the 'lower' cestodes have been impeded by technical difficulties, including the procurement of eggs with well-fixed contents, the infiltration of embedding media and in the cutting of the thick, hard shells.…”

Ultrastructure of microtriches on the scolex of Cyathocephalus truncatus (Cestoda: Spathe-bothriidea)

“…They are uncommon and most aspects of their biology remain largely unknown (Marques et al 2007). They are polyzoic but lack external segmentation (Gibson 1994).…”