Integrative taxonomy of the genus Pseudoacanthocephalus (Acanthocephala: Echinorhynchida) in China, with the description of two new species and the characterization of the mitochondrial genomes of Pseudoacanthocephalus sichuanensis sp. n. and Pseudoacanthocephalus nguyenthileae.

Background: Acanthocephalans (thorny headed worms) of the genus Pseudoacanthocephalus mainly parasitize amphibians and reptiles across the globe. Some species of the genus Pseudoacanthocephalus also can accidentally infect human and cause human acanthocephaliasis. Current knowledge of the species composition of the genus Pseudoacanthocephalus from amphibians and reptiles in China is incomplete. An insufficiency of genetic data on species of the genus Pseudoacanthocephalus, including the complete mitochondrial genomes, has limited the use of molecular-based methods to better define the taxonomy and phylogeny of the genus Pseudoacanthocephalus. A more rigorous molecular phylogeny with broader representatives of the genus Pseudoacanthocephalus is required to further clarify the systematic status of the family Pseudoacanthocephalidae.

Methods: Many specimens of the genus Pseudoacanthocephalus collected from toads and frogs in China were identified to species level using integrated morphological methods (light and scanning electron microscopy) and molecular approaches (sequencing different nuclear and mitochondrial genetic markers). The Assemble Species by Automatic Partitioning (ASAP) and Bayesian inference (BI) methods were applied for species delimitation. The complete mitochondrial genomes of two Pseudoacanthocephalus species were also sequenced and annotated to enrich the body of mitogenomic data on acanthocephalans. Additionally, phylogenetic analyses based on the amino acid sequences of 12 protein-coding genes (PCGs) of mitochondrial genomes of acanthocephalans using maximum likelihood (ML) and BI were performed to further investigate the phylogenetic position of the family Pseudoacanthocephalidae in the order Echinorhynchida.

Results: Three Pseudoacanthocephalus species, including P. sichuanensis sp. n., P. previatesticulus sp. n. and P. nguyenthileae were described. The results of ASAP and BI analyses based on the cytochrome c oxidase subunit 1 and subunit 2 (cox1, cox2) and 12S ribosomal RNA (12S) sequences supported the separation of P. sichuanensis and P. previatesticulus from the congeneric species. The results of BI inference using the internal transcribed spacer (ITS), cox1, cox2 and 12S sequence data indicated that P. sichuanensis and P. nguyenthileae have a closer relationship than P. previatesticulus and P. bufonis in Pseudoacanthocephalus. The complete mitogenomes of P. sichuanensis and P. nguyenthileae have 15,812 and 13,701 bp, respectively, with both including 36 genes and two non-coding regions. Phylogenetic results based on mitogenomic data demonstrated that the two families Pseudoacanthocephalidae and Arhythmacanthidae have a sister relationship in the order Echinorhynchida.

Conclusions: Two new species of the genus Pseudoacanthocephalus, namely P. sichuanensis sp. n. and P. previatesticulus sp. n., were identified based on integrated evidence. This is the first report of P. nguyenthileae in China. A revised key for the species of the genus Pseudoacanthocephalus was provided. Molecular analyses revealed that the mitochondrial cox1, cox2 and 12S genes as genetic markers seem to be more suitable for species delimitation of Pseudoacanthocephalus than the nuclear ITS region. BI results suggested a close affinity between P. sichuanensis and P. nguyenthileae. The mitochondrial genomic data of P. sichuanensis and P. nguyenthileae are provided for the first time. Mitogenomic phylogenetic results further confirmed the validity of the family Pseudoacanthocephalidae.