Updated molecular phylogenetic data for Opisthorchis spp. (Trematoda: Opisthorchioidea) from ducks in Vietnam

Background: An opisthorchiid liver fluke was recently reported from ducks (Anas platyrhynchos) in Binh Dinh Province of Central Vietnam, and referred to as "Opisthorchis viverrini-like". This species uses common cyprinoid fishes as second intermediate hosts as does Opisthorchis viverrini, with which it is sympatric in this province. In this study, we refer to the liver fluke from ducks as "Opisthorchis sp. BD2013", and provide new sequence data from the mitochondrial (mt) genome and the nuclear ribosomal transcription unit. A phylogenetic analysis was conducted to clarify the basal taxonomic position of this species from ducks within the genus Opisthorchis (Digenea: Opisthorchiidae). Methods: Adults and eggs of liver flukes were collected from ducks, metacercariae from fishes (Puntius brevis, Rasbora aurotaenia, Esomus metallicus) and cercariae from snails (Bithynia funiculata) in different localities in Binh Dinh Province. From four developmental life stage samples (adults, eggs, metacercariae and cercariae), the complete cytochrome b (cob), nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and near-complete 18S and partial 28S ribosomal DNA (rDNA) sequences were obtained by PCR-coupled sequencing. The alignments of nucleotide sequences of concatenated cob + nad1 + cox1, and of concatenated 18S + 28S were separately subjected to phylogenetic analyses. Homologous sequences from other trematode species were included in each alignment. Results: Phylogenetic trees were inferred from concatenated (cob + nad1 + cox1) nucleotide sequences and combined 18S + 28S nucleotide sequences of five Opisthorchis sp. BD2013 samples and additional reference taxa. Both trees demonstrated the anticipated clustering of taxa within the superfamily Opisthorchioidea, the paraphyly of the genus Opisthorchis and the sister-species relationship of Opisthorchis sp. BD2013 with O. viverrini. Conclusions: While it is likely that Opisthorchis sp. BD2013 is distinct from O. viverrini, it is clearly a sister taxon of O. viverrini within the limited number of Opisthorchis species for which appropriate sequence data are available. The new sequences provided here will assist the diagnosis and the taxonomic clarification of the opisthorchiid species.