Phylogenetic relationship of Prohemistomum vivax to other trematodes based on the internal transcribed spacer region and mitochondrial genes

Prohemistomum vivax is a zoonotic small cyathocotylid trematode that inhabits the intestines of fish-eating birds and mammals. Here, we amplified the internal transcribed spacer (ITS) sequence and six mitochondrial protein-coding genes (PCGs) from P. vivax . The ITS region was 1389 base pairs long and had a partial 18S ribosomal RNA gene, a full ITS1, 5.8S rRNA, and ITS2 sequence, and a partial 28S rRNA gene. The ITS region of P. vivax showed a minimum pairwise distance (0.3–0.6%) from the ITS sequences of Cyathocotylidae sp. 1 and 2 metacercariae from Clarias gariepinus . This result suggests that these metacercariae belong to P. vivax metacercariae. We first amplified mitochondrial genes from P. vivax , including cytochrome c oxidase subunit III ( cox 3) partial sequence; tRNA-His, cytochrome b ( cyt b), and NADH dehydrogenase subunit 4L ( nad 4L) complete sequences; and NADH dehydrogenase subunit 4 ( nad 4), cytochrome c oxidase I ( cox 1), and NADH dehydrogenase subunit 5 ( nad 5) partial sequences. P. vivax was most closely related to Cyathocotyle prussica (NC_039780) and Holostephanus sp. (OP082179), with cox 1, cox 3, and cyt b genes conserved among the three trematodes. The ML phylogenetic tree of ITS sequences supports the order Diplostomida, divided into two main clades (the superfamily Diplostomoidea and Schistosomatoidea). The phylogeny of concatenated amino acid sequences of P. vivax six PCGs revealed that diplostomoids and Clinostomum sp. evolved in a clade with Plagiorchiida members, away from Schistosoma species. These results may yield ribosomal and mitochondrial genetic markers for molecular epidemiological investigations of cyathocotylid intestinal flukes.