Complete rDNA of Diplodiscus japonicus and Diplodiscus mehrai reveals IGS as a promising marker for intraspecific genetic variation.

Diplodiscus japonicus (Yamaguti, 1936) and Diplodiscus mehrai (Pande, 1937) (Trematoda: Diplodiscidae) are parasitic trematodes infecting wood frogs (Anura: Ranidae) in Heilongjiang Province, China. In this study, we amplify the complete rDNA of five D. japonicus and five D. mehrai. The assembled sequences were processed using DNASTAR and MEGA X to evaluate the sequence length and genetic divergence of the 18S, ITS1, 5.8S, ITS2, 28S rRNA, and IGS regions. To quantitatively assess the intraspecific genetic polymorphism, the nucleotide diversity (π) and haplotype diversity (Hd) for each rDNA region were calculated using DnaSP. Tandem repeats within the rDNA arrays were identified via REPuter. Maximum likelihood, maximum parsimony, and Bayesian inference phylogenetic trees were reconstructed based on these rDNA markers, with Glanitaenia osculata (Proteocephalidea: Proteocephalidae) and Gyrodactylus salaris (Gyrodactylidea: Gyrodactylidae) designated as outgroups. The results showed that complete rDNA lengths of D. japonicus ranged from 7,226 bp to 7,268 bp, with 18S, ITS, 28S and IGS measuring 1,954 bp, 909 bp, 3,527 bp, and 836 to 878 bp, respectively. For D. mehrai, the full-length rDNA varied from 7,266 bp to 8,534 bp, with 18S, ITS, 28S and IGS corresponding to 1,991 bp, 930 bp, 3,504 bp, and 841 to 2,109 bp, respectively. Comparative analyses revealed that length polymorphisms occurred mainly in the IGS region, which exhibited significantly higher intraspecific variations than other ribosomal regions. Calculation results of π and Hd further verified that the IGS region possessed far higher genetic polymorphism indices than all other rDNA segments; specifically, the π value of IGS in D. japonicus was dozens to hundreds times higher than that of coding and ITS regions, and the IGS of D. mehrai also displayed remarkably elevated nucleotide diversity compared with other rDNA regions. Fifty forward and inverted repeat sequences were found in the complete rDNA of both species. The phylogenetic trees based on 18S, ITS, 28S and IGS sequences supported their utility as genetic markers at different levels. Notably, the IGS-based phylogeny recovered monophyletic clades for Opisthorchiidae, Paramphistomidae, Prosthogonimidae, Dicrocoeliidae, Echinostomatidae, and Schistosomatidae, and conspecific sequences clustered robustly together. This study provides the complete rDNA data for the D. japonicus and D. mehrai, offering valuable molecular resources for taxonomic, population genetic, and systematic studies of Diplodiscus species. Furthermore, our findings reinforce that IGS region represents a promising potential and novel genetic marker for investigating intraspecific genetic variation in Diplodiscidae.