Morphology, complete mitochondrial genome, and molecular phylogeny of Rhabdias macrocephalum n. sp. (Nematoda: Rhabdiasidae) from Diploderma splendidum (Reptilia: Agamidae).

Species of the genus Rhabdias Stiles & Hassall, 1905 are common parasitic nematodes occurring in the lungs of amphibians and reptiles worldwide. In the present study, Rhabdias macrocephalum n. sp. is described using integrated morphological methods (light and scanning electron microscopy) and molecular approaches (sequencing of the nuclear 28S and ITS regions, and mitochondrial cox1, cox2, and 12S genes) based on specimens collected from the green striped tree dragon Diploderma splendidum (Barbour & Dunn) (Reptilia: Agamidae) in China. The complete mitochondrial genome of R. macrocephalum n. sp. was sequenced and annotated: it is 14,819 bp in length, including 12 protein coding genes (missing atp8), 22 tRNA genes, 2 rRNA genes and three non-coding regions. The gene arrangement of R. macrocephalum n. sp. is different from all of the currently available mitogenomes of nematodes and represents a novel type of mitochondrial gene arrangement reported in Nematoda. Molecular phylogenetic results based on the ITS + 28S data support the monophyly of Entomelas, Pneumonema, Serpentirhabdias, and Rhabdias, and showed R. macrocephalum n. sp. forming a most basal lineage in Rhabdias.

Title: Morphologie, génome mitochondrial complet et phylogénie moléculaire de Rhabdias macrocephalum n. sp. (Nematoda : Rhabdiasidae) de Diploderma splendidum (Reptilia : Agamidae). Abstract: Les espèces du genre Rhabdias Stiles & Hassall, 1905 sont des nématodes parasites courants présents dans les poumons des amphibiens et des reptiles du monde entier. Dans cette étude, Rhabdias macrocephalum n. sp. est décrit à l'aide de méthodes morphologiques intégrées (microscopie optique et électronique à balayage) et d'approches moléculaires (séquençage des régions nucléaires 28S et ITS et des gènes mitochondriaux cox1, cox2 et 12S) basées sur des spécimens collectés chez le lézard Diploderma splendidum (Barbour & Dunn) (Reptilia : Agamidae) de Chine. Le génome mitochondrial complet de R. macrocephalum n. sp. a été séquencé et annoté : il a une longueur de 14 819 pb, dont 12 gènes codants pour des protéines (atp8 manquant), 22 gènes d'ARNt, 2 gènes d'ARNr et trois régions non codantes. L'arrangement génétique de R. macrocephalum n. sp. est différent de tous les mitogénomes de nématodes actuellement disponibles et représente un nouveau type d'arrangement de gènes mitochondriaux signalé chez les nématodes. Les résultats phylogénétiques moléculaires basés sur les données ITS + 28S ont soutenu la monophylie d'Entomelas, Pneumonema, Serpentirhabdias et Rhabdias, et ont montré que R. macrocephalum n. sp. forme la lignée la plus basale chez Rhabdias.

Molecular phylogeny of the family Rhabdiasidae (Nematoda: Rhabditida), with morphology, genetic characterization and mitochondrial genomes of Rhabdias kafunata and R. bufonis.

BACKGROUND: The family Rhabdiasidae (Nematoda: Rhabditida) is a globally distributed group of nematode parasites, with over 110 species parasitic mainly in amphibians and reptiles. However, the systematic position of the family Rhabdiasidae in the order Rhabditida remains unsolved, and the evolutionary relationships among its genera are still unclear. Moreover, the present knowledge of the mitochondrial genomes of rhabdiasids remains limited. METHODS: Two rhabdiasid species: Rhabdias kafunata Sata, Takeuchi & Nakano, 2020 and R. bufonis (Schrank, 1788) collected from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) in China, were identified based on morphology (light and scanning electron microscopy) and molecular characterization (sequencing of the nuclear 28S and ITS regions and mitochondrial cox1 and 12S genes). The complete mitochondrial genomes of R. kafunata and R. bufonis were also sequenced and annotated for the first time. Moreover, phylogenetic analyses based on the amino acid sequences of 12 protein-coding genes (PCGs) of the mitochondrial genomes were performed to clarify the systematic position of the family Rhabdiasidae in the order Rhabditida using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic analyses based on the 28S + ITS sequences, were also inferred to assess the evolutionary relationships among the genera within Rhabdiasidae. RESULTS: The detailed morphology of the cephalic structures, vulva and eggs in R. kafunata and R. bufonis was revealed using scanning electron microscopy (SEM) for the first time. The characterization of 28S and ITS regions of R. kafunata was reported for the first time. The mitogenomes of R. kafunata and R. bufonis are 15,437 bp and 15,128 bp long, respectively, and both contain 36 genes, including 12 PCGs (missing atp8). Comparative mitogenomics revealed that the gene arrangement of R. kafunata and R. bufonis is different from all of the currently available mitogenomes of nematodes. Phylogenetic analyses based on the ITS + 28S data showed Neoentomelas and Kurilonema as sister lineages, and supported the monophyly of Entomelas, Pneumonema, Serpentirhabdias and Rhabdias. Mitochondrial phylogenomic results supported Rhabdiasidae as a member of the superfamily Rhabditoidea in the suborder Rhabditina, and its occurrance as sister to the family Rhabditidae. CONCLUSIONS: The complete mitochondrial genome of R. kafunata and R. bufonis were reported for the first time, and two new gene arrangements of mitogenomes in Nematoda were revealed. Mitogenomic phylogenetic results indicated that the family Rhabdiasidae is a member of Rhabditoidea in Rhabditina, and is closely related to Rhabditidae. Molecular phylogenies based on the ITS + 28S sequence data supported the validity of Kurilonema, and showed that Kurilonema is sister to Neoentomelas. The present phylogenetic results also indicated that the ancestors of rhabdiasids seem to have initially infected reptiles, then spreading to amphibians.