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.

A new species of Serpentirhabdias Tkach, Kuzmin et Snyder, 2014 (Nematoda: Rhabdiasidae) parasitic in the herald snake, Crotaphopeltis hotamboeia (Laurenti) (Reptilia: Serpentes: Colubridae) in South Africa.

Serpentirhabdias mamlambo n. sp. is described from the lung of the herald snake, Crotaphopeltis hotamboeia (Laurenti) in KwaZulu-Natal Province, South Africa. The new species is characterised by the small lips, the absence of the buccal capsule, the large excretory glands (1.5-2.3 times longer than the oesophagus), and the numerous eggs in uteri. Serpentirhabdias mamlambo n. sp. differs from other Serpentirhabdias spp. in the presence of M-shaped cuticular folds on dorsal and ventral sides of apical surface, the eye-shaped internal labial papillae, and the distinct dilatation of the oesophagus at its mid-length. Morphological differentiation of S. mamlambo n. sp. from its congeners is supported by molecular evidence based on cox1 mitochondrial gene, and ITS and partial 28S region of nuclear rDNA. Serpentirhabdias mamlambo n. sp. is the first species of the genus Serpentirhabdias described from the Afrotropical Realm.

Rhabdias glaurungi sp. nov. (Nematoda: Rhabdiasidae), parasite of Scinax gr. ruber (Laurenti, 1768) (Anura: Hylidae), from the Brazilian Amazon.

The genus Rhabdias Stiles & Hassal, 1905 includes about 83 species of nematodes parasitic in amphibians and reptiles worldwide. Herein, we describe Rhabdias glaurungi sp. nov. from the hylid frog Scinax gr. ruber (Laurenti, 1768) in the Gunma Ecological Park, Santa Bárbara municipality, state of Pará, Brazil. This species has six small lips, an inflated cuticle along the entire body and a cup-shaped buccal capsule with smooth internal surface of its anterior part and irregularly folded internal surface of its posterior part in apical view. From the 17 valid species recognized in the Neotropical realm, the new species can be distinguished by the number of lips, the morphology and size of its buccal capsule, as well as the extent and shape of its cuticular inflation; in addition, there are molecular differences. Sequences of the mitochondrial Cytochrome c Oxidase subunit I gene strongly support the status of this form as a separate species. Molecular phylogenetic analysis shows R. glaurungi sp. nov. nested within the R. pseudosphaerocephala Kuzmin, Tkach & Brooks, 2007 species complex. Rhabdias glaurungi sp. nov. is the second species of the genus described from hosts of the family Hylidae in the Neotropical realm. We conclude that the diversity of Rhabdias within the Neotropics is likely largely underestimated.

Description, molecular characterization and life cycle of Serpentirhabdias mussuranae n. sp. (Nematoda: Rhabdiasidae) from Clelia clelia (Reptilia: Colubroidea) in Brazil.

Serpentirhabdias mussuranae n. sp. is described from the lungs of the mussurana, Clelia clelia (Daudin, 1803), from vicinities of Lábrea, Amazonas State, Brazil. The species is characterized by the triangular oral opening, the presence of teeth (onchia) in the oesophastome, the excretory glands longer than the oesophagus and the tail abruptly narrowing in its anterior half and gradually tapering in posterior half. Among the Neotropical representatives of the genus, three species are known to possess the onchia in the oesophastome: S. atroxi, S. moi and S. viperidicus. Serpentirhabdias mussuranae n. sp. differs from S. atroxi and S. viperidicus by its triangular shape of the oral opening and the oesophastome in apical view, vs. round in the latter two congeners. Additionally, S. viperidicus has a larger oesophastome, 13-22 micrometers wide and 13-23 micrometers deep. The new species has relatively longer excretory glands than S. moi. The new species is morphologically and genetically close to S. atroxi, S. moi and S. viperidicus, all parasitic in Brazilian snakes, based on the presence of onchia and the comparison of nucleotide sequences of nuclear ribosomal DNA and mitochondrial cox1 gene (differences varied between 3.8% and 7.1%). Data on the life cycle of S. mussuranae n. sp. is provided, and the life cycle is typical of the genus Serpentirhabdias, with the combination of direct development and heterogony. Free-living larval stages and the adults of amphimictic free-living generation are described. The results of molecular phylogenetic analysis based on nuclear ribosomal internal transcribed spacer (ITS) + partial 28S region and partial mitochondrial cox1 gene are provided.

Description, biology and molecular characterisation of Serpentirhabdias moi n. sp. (Nematoda: Rhabdiasidae) from Chironius exoletus (Serpentes: Colubridae) in Brazil.

A new species of the genus Serpentirhabdias Tkach, Kuzmin et Snyder, 2014, S. moi n. sp., is described from a colubroid snake Chironius exoletus from Caxiuanã National Forest, State of Pará, Brazil. The species is characterised by having a triangular oral opening, absence of the buccal capsule, presence of six minute onchia in the oesophastome, and excretory glands of approximately the same length as the oesophagus. These qualitative morphological characters, as well as some measurements, differentiate the new species from other Neotropical and Nearctic Serpentirhabdias spp. The morphological description of parasitic adults of S. moi n. sp. is complemented by the description of free-living stages including males, females, and infective larvae. Comparative analysis of partial sequences of cox1 and 12S mitochondrial genes strongly supported the status of S. moi n. sp. as a new species. Molecular phylogeny based on sequences of the nuclear DNA region spanning the 3' end of the 18S nuclear rRNA gene, ITS region (ITS1 + 5.8S + ITS2) and 5' end of the 28S gene supported monophyly of all rhabdiasid genera included in the analysis and placed the new species into the Serpentirhabdias clade as sister taxon to S. fuscovenosa.

Two new species of Rhabdias Stiles et Hassall, 1905 (Nematoda: Rhabdiasidae) from anuran amphibians in Pará, Brazil.

Two new lung-dwelling nematode species of the genus Rhabdias Stiles et Hassall, 1905 were discovered in Caxiuanã National Forest, Pará state, Brazil. Rhabdias galactonoti sp. n. was found in a dendrobatid frog Adelphobates galactonotus (Steindachner). The species is characterised by the regularly folded inner surface of the anterior part of the buccal capsule seen in apical view, flask-shaped oesophageal bulb and narrow, elongated tail. Rhabdias stenocephala sp. n. from two species of leptodactylid frogs, Leptodactylus pentadactylus (Laurenti) (type host) and L. paraensis (Heyer), is characterised by a narrow anterior end that is separated from the remaining body by a constriction. Both species possess six small but distinct lips, a cuticle that is inflated along the whole body, a doliiform buccal capsule separated into a longer anterior and a shallow, ring-shaped posterior part, lateral pores in the body cuticle and zones of spermatogenesis in the syngonia. Rhabdias galactonoti sp. n. is the first species of the genus found in Dendrobatidae; R. stenocephala sp. n. is the second species described from Leptodactylidae in eastern Amazonia.

Molecular insight into systematics, host associations, life cycles and geographic distribution of the nematode family Rhabdiasidae.

Rhabdiasidae Railliet, 1915 is a globally distributed group of up to 100 known species of nematodes parasitic in amphibians and reptiles. This work presents the results of a molecular phylogenetic analysis of 36 species of Rhabdiasidae from reptiles and amphibians from six continents. New DNA sequences encompassing partial 18S rDNA, ITS1, 5.8S rDNA, ITS2 and partial 28S rDNA regions of nuclear ribosomal DNA were obtained from 27 species and pre-existing sequences for nine species were incorporated. The broad taxonomic, host and geographical coverage of the specimens allowed us to address long-standing questions in rhabdiasid systematics, evolution, geographic distribution, and patterns of host association. Our analysis demonstrated that rhabdiasids parasitic in snakes are an independent genus sister to the rest of the Rhabdiasidae, a status supported by life cycle data. Based on the combined evidence of molecular phylogeny, morphology and life cycle characteristics, a new genus Serpentirhabdias gen. nov. with the type species Serpentirhabdias elaphe (Sharpilo, 1976) comb. nov. is established. The phylogeny supports the monophyly of Entomelas Travassos, 1930, Pneumonema Johnston, 1916 and the largest genus of the family, Rhabdias Stiles and Hassall, 1905. DNA sequence comparisons demonstrate the presence of more than one species in the previously monotypic Pneumonema from Australian scincid lizards. The distribution of some morphological characters in the genus Rhabdias shows little consistency within the phylogenetic tree topology, in particular the apical structures widely used in rhabdiasid systematics. Our data suggest that some of the characters, while valuable for species differentiation, are not appropriate for differentiation among higher taxa and are of limited phylogenetic utility. Rhabdias is the only genus with a cosmopolitan distribution, but some of the lineages within Rhabdias are distributed on a single continent or a group of adjacent zoogeographical regions. Serpentirhabdias, Entomelas and Pneumonema show rather strict specificity to their host groups. The evolution of the Rhabdiasidae clearly included multiple host switching events among different orders and families of amphibians as well as switching between amphibians and squamatan reptiles. Only a few smaller lineages of Rhabdias demonstrate relatively strict associations with a certain group of hosts.

Rhabdias paraensis sp. nov.: a parasite of the lungs of Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

The nematode parasites of Rhinella marina include species of the genus Rhabdias (Rhabdiasidae: Rhabditoidea). The present study describes Rhabdias paraensis sp. nov., which parasitizes the lungs of R. marina in Brazilian Amazonia. Of the more than 70 known species of this genus, 18 are parasites of bufonids, of which, eight are Neotropical. The new species described here is similar to Rhabdias alabialis in the absence of lips is different by the presence of conspicuous cephalic papillae. We describe details of the four rows of pores, which are distributed equally along the whole of the length of the body and connected with hypodermal cells, using histology and scanning electron microscopy. Other histological aspects of the internal structure of this nematode are also described.