The entire transcribed sequences (from the 5' terminus of 18S to the 3' terminus of 28S rRNA genes) of the ribosomal transcription units (rTU*) of five Asian Paragonimus species were obtained and characterized. The rTU* length was 7661bp for P. heterotremus (LC strain, Vietnam), 7422bp for P. iloktsuenensis (Amami strain, Japan), 6932bp for P. skrjabini miyazakii (OkuST1 strain, Japan), 7422bp for P. ohirai (Kino strain, Japan), and three strains of P. westermani: 8616bp (Megha strain, India), 7292bp (Bogil strain, South Korea), and 7052bp (QT2 strain, Vietnam) without intergenic spacer region (IGS). All seven Asian Paragonimus strains' genetic characteristics were described, including the length of individual genes/regions, repeat polymorphism, base composition, and skewness. To investigate the superfamilial relationships in the Xiphidiata, with a focus on the Troglotrematoidea and its associated superfamilies, we used the PhyML software package to create three comprehensive maximum-likelihood phylogenies. The datasets used were 83 concatenated 28S + 18S, 83 single complete 18S, and 157 single, partial 28S rDNA sequences, respectively, from entire rTUs and/or accessible ribosomal sequences of the same species from the suborders Xiphidiata, Echinostomata, and Haplosplanchnata, with a Schistosoma sequence as an outgroup. Three phylogenetic trees revealed that Echinostomata and Haplosplanchnata are monophyletic, while Xiphidiata is polyphyletic and contains the monophyletic Troglotrematoidea. The concatenated 18S + 28S and single 18S phylogenies revealed well-bootstrap supported seven superfamilies (Troglotrematoidea, Haploporoidea, Gorgoderoidea, Brachycladioidea, Microphalloidea, Plagiorchioidea, and Opecoeloidea) that are monophyletic in the Xiphidiata. The Haploporoidea was a basal superfamily nested close to the Gorgoderoidea and Troglotrematoidea and was not supported as a distinct suborder Haploporata. Six of seven xiphidiatan superfamilies were monophyletic in the partial 28S phylogeny, with the exception of Opecoeloidea, which was separated into two different subclades: Opecoelidae and Stenakridae/Zdzitowieckitrematidae. The monophyletic Haploporoidea/Haploporata was separated from the Gorgoderoidea associates and placed in a marginal group in Xiphidiata. There were two notable clusters in the Paragonimidae: mixed-Paragonimus, which included a fairly compact group of P. heterotremus strains, and P. westermani/siamensis, which was divided into geographical/country strain groups. In conclusion, combined ribosomal rDNA sequences were more effective than single rDNA markers in resolving interfamilial and familial relationships. The ribosomal datasets presented here will be useful for taxonomic reassessment, as well as evolutionary and population genetics research in the Troglotrematoidea and other superfamilies in the Xiphidiata and the class Trematoda.
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