Integrative taxonomy in the genus Rhabdias Stiles et Hassall, 1905 from anuran in Brazil,description of two new species and phylogenetic analyses

About 20 valid species of the genus Rhabdias are known in the Neotropical region. The present study aimed to describe two new species of Rhabdias parasitizing the lungs of Leptodactylus macrosternum and Leptodactylus podicipinus from Brazil. Distinctive characteristics between these species are numerous and based on body size, size of the buccal capsule, shape and size of the oesophagus, and position of the vulva. Molecular data based on ribosomal genes 28S and ITS region and mitochondrial COI of the two species are presented. Molecular analysis and comparison of the partial mitochondrial COI sequence of Rhabdias matogrossensis n. sp. and Rhabdias guaianensis n. sp. revealed a genetic divergence between these new species and the sequences of Rhabdias spp. previously deposited in GenBank. In the phylogenetic analysis, R. matogrossensis n. sp. was grouped with R. breviensis species complex, and R. guaianensis n. sp. was grouped as a sister group of R. cf. stenochepala. This study contributes to improving the diversity of known species of Rhabdias described in Brazilian anurans.     

Diversity in the genus Rhabdias (Nematoda,Rhabdiasidae): Evidence for cryptic speciation

Lungworms from the genus Rhabdias are common parasites of amphibians and reptiles distributed worldwide. To assess the diversity of Rhabdias spp., we performed molecular analyses of 35 specimens sampled in different regions of Brazil. Molecular analyses were based on the internal transcribed spacer (ITS), large subunit (28S) ribosomal and the cytochrome oxidase I (COI) mitochondrial genes. DNA sequence divergence was compared among ribosomal and mitochondrial genes, analyses using the general mixed Yule‐coalescent (GMYC) method based on the COI gene were used to identify possible cryptic diversity, and phylogenetic analyses using concatenated ITS and 28S ribosomal genes were used to test the monophyly of Rhabdiasidae. We revealed five morphospecies: R. cf. stenocephala, R. breviensis, R. pseudosphaerocephala and two new species, Rhabdias sp.4 and Rhabdias sp.5. DNA sequence levels of divergence among genes ITS, 28S and COI were compared, and the efficiency of the molecular markers to identify species (ITS and COI) and lineages (COI) was tested. GMYC was assigned to 17 well‐supported clades (i.e., 17 species), and cryptic diversity was detected in the Neotropical region as evidenced by the multiple lineages in R. breviensis and R. pseudosphaerocephala. In addition, our results suggest evidence for host–parasite cophylogeny in the R. pseudosphaerocephala complex and dispersal events among their populations. Phylogenetic analyses supported the monophyly of Rhabdiasidae and improved the resolution of main clades. Rhabdias breviensis is closely related to Rhabdias cf. africanus, Rhabdias cf. stenocephala, R. pseudosphaerocephala, Rhabdias sp.4 and Rhabdias sp.5 grouping together in a main clade with Neotropical‐related species. The large geographical distribution appeared to be a phylogenetic pattern among the species of Rhabdias from the neotropics.     

Phylogeography of Rhabdias spp. (Nematoda: Rhabdiasidae) collected from Bufo species in Honshu,Shikoku, and Kyushu,Japan including possible cryptic species

The genus Rhabdias Stiles & Hassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta, we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus, B. japonicus formosus, and B. torrenticola, collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species.     

Rhabdias rhampholeonis n. sp. and Rhabdias mariauxi n. sp. (Nematoda, Rhabdiasoidea), first lung worms from leaf chameleons: Description, molecular evidence and notes on biology

Rhabdias rhampholeonis n. sp. from Rhampholeon (Rh.) spectrum, Cameroon, and Rhabdias mariauxi n. sp. from Rieppeleon brevicaudatus, Tanzania, are the first lung worms from leaf chameleons. The new species are similar to the majority of species parasitic in chamaeleonids by having a long (≥10 mm) and thick body (≥500 µm), long oesophagus (≥800 µm), wide buccal capsule (≥40 µm) and low buccal ratio (<0.5). They most closely resemble Rhabdias chamaeleonis and Rhabdias cristati parasitic in Trioceros spp. from East Africa and Cameroon, respectively. Main distinctive characters are a buccal capsule composed of two segments and the head shape. The dorso-ventrally flattened buccal capsule of R. mariauxi n. sp. is unique in Rhabdias parasitising Chamaeleonidae. Sequences of the 12S rDNA and mitochondrial cytochrome c oxidase subunit 1 (coxI) genes were obtained and compared to those of Rhabdias okuensis, the only sequences published for chamaeleonid lung worms. The smallest nucleotide interspecific distances were found between R. mariauxi n. sp. and the former species of Trioceros from Cameroon. Hermaphroditism in females in the lungs, and R. mariauxi n. sp. free-living stages are like in other species from Chamaeleonidae, but the number of infective larvae produced per free-living female (one or two) was not fixed.     

Morphological and biological study of Rhabdias spp. (Nematoda) from African chameleons with description of a new species

Lung-dwelling females of Rhabdias (Rhabdiasidae), and possible migrating ceolomic young females were searched for in 46 chameleons, belonging to seven species. Rhabdias chamaeleonis, the single species identified to date in Africa, was found in Chamaeleo (Trioceros) johnstoni and C. (T.) hoehnelii, and redescribed; the sizes of the buccal capsule and oesophagus were stable compared to the length of the female parasiles, which varied from 6 mm to 22 mm in length. A second species, R. jarki n. sp., was identified from one C. (T.) johnstoni; it differed from R. chamaeleonis in the shape of anterior region, the longer and slender oesophagus (ratio bulb diameter-body diameter at that level about 1/5 instead of 1/2), the arrangement of the head papillae, the shape of the buccal capsule, and the anatomy of the genital apparatus: one of the ovaries (the anterior or posterior one according to the specimen) had a band of small cells, among larger ovocytes of the synapsis zone, which were likely to generate the spermatozoa present in the oviducts, whereas these two elements were absent from R. chamaeleonis. The parasitic females of R. jarki thus appeared to be hermaphroditic, whereas those of R. chamaeleonis appeared to be parthenogenetic. The free living phase of these Rhabdias species was heterogonic. The infective larva of R. chamaeleonis was 360-590 microns long, unmolile and at third stage inside the maternal cuticle. The free-living male of R. jarki was described. The numerous infective larvae recovered from cultures of unidentified Rhabdias were all in maternal cuticle (one larva/female instead of two as in R. gemellipara from Calumma parsonii, from Madagascar) and two kinds of larvae were identified, R. chamaeleonis and larvae 700-900 microns long provisionally identified to R. jarki. The behaviour of infective larvae and a few successful infections of insects suggest that, in the field, insect transport hosts are involved in transmission and in preventing infective larvae from drying out.     

Phylogenetic affinities and systematic position of Entomelas sylvestris Baker, 1982 (Nematoda: Rhabdiasidae), a parasite of Breviceps sylvestris FitzSimons (Amphibia: Brevicipitidae) in South Africa

The genus Entomelas Travassos, 1930 currently includes nine species of rhabdiasid nematodes, eight of them parasitic in lizards and only one, Entomelas sylvestris Baker, 1982, parasitic in amphibians. Entomelas sylvestris was originally described from the Forest Rain Frog Breviceps sylvestris FitzSimons in South Africa and was not reported since. It was placed in the genus Entomelas without any specific arguments for this taxonomic decision, presumably mainly based on details of the buccal capsule morphology. We have found this species in the same host in Limpopo province, South Africa. Molecular phylogenetic analysis based on the newly-obtained sequence of complete ITS region and partial nuclear large ribosomal subunit (28S) gene of E. sylvestris and previously published sequences of a variety of other rhabdiasid taxa, has convincingly demonstrated that this species does not belong in Entomelas. Instead, it clustered together with the members of Rhabdias Stiles & Hassall, 1905 from amphibian hosts. Therefore, we transfer E. sylvestris into Rhabdias as Rhabdias sylvestris (Baker, 1982) n. comb. In our analysis E. sylvestris appears, albeit with weak support, as a basal/sister taxon to the rest of Rhabdias spp. which explains to some extent the differences in the buccal capsule morphology between this species and other Rhabdias spp.     

<Emphasis Type="Italic">Rhabdias lacertae</Emphasis> n. sp. (Nematoda: Rhabdiasidae), the first rhabdiasid species parasitising lizards in Europe

A new nematode species, Rhabdias lacertae n. sp. (Rhabdiasidae), is described from the body-cavity of the common lizard Lacerta vivipara Jacquin (Lacertidae) from the Ridge of Malá Fatra (Sokolie Hill), north-western Slovakia. The new species differs from its congeners mainly in possessing 3 min cuticular spikes at the tail tip and some other features. This is the first species of Rhabdias Stiles & Hassall, 1905 described from lizards in Europe and the first species of this genus parasitising hosts belonging to the Lacertidae.     

Rhabdias esculentarum n. sp. (Nematoda: Rhabdiasidae) from green frogs of the Rana esculenta species complex in Italy: molecular evidence, morphological description and genetic differentiation from its congeners in frogs and toads

A new taxon, Rhabdias esculentarum n. sp., is described based on DNA sequence analysis at multiple loci (i.e. mtDNA cox-1, 12S rRNA, ITS-1 and partial ITS-2 regions of the nuclear rDNA) and morphometric analysis carried out on specimens collected from the green frogs of the Rana esculenta species complex in Italy (i.e. R. lessonae Camerano and R. esculenta Linnaeus, identified genetically by diagnostic allozyme loci). Rhabdias esculentarum n. sp. was differentiated genetically, at both mitochondrial and nuclear levels, from Rh. bufonis (Schrank, 1788) (sensu Hartwich, 1972) and Rh. sphaerocephala Goodey, 1924 recovered from the toad Bufo bufo Linnaeus collected sympatrically with the specimens of Rana lessonae and R. esculenta examined in the present study. Moreover, the new taxon proved to be different from the other species of Rhabdias from anurans, which had previously been sequenced using the same genes and deposited in GeneBank. Phylogenetic analyses (MP and ML) inferred from mitochondrial (mtDNA cox-1 and 12S ribosomal RNA) and nuclear (ITS-1 and ITS-2 of the rDNA regions) sequences datasets were congruent in depicting Rh. esculentarum n. sp. as forming a highly supported clade distinct from the sympatric species Rh. bufonis, as well as from Rh. sphaerocephala, characterised on the basis of the same loci. Morphometric analysis and the differential diagnosis of genetically characterised specimens of the new species have revealed differences in several features in comparison with the type-species, Rh. bufonis. Material of the latter species included voucher specimens from Germany deposited by Hartwich (1972) and other specimens collected from B. bufo in Italy. Among the diagnostic characters, the particular cup-shaped buccal capsule characterising Rh. esculentarum is clearly different from the tear-shaped buccal capsule observed in material of R. bufonis obtained from Berlin Museum and collected in the same geographical area as the green frogs under study. Rh. esculentarum was also found to differ in some measurements and allometric characters from Rh. bufonis (sensu Moravec et al., 1997). The data so far collected appear to indicate a host-preference of Rh. esculentarum for Rana lessonae and R. esculenta, which belong to the R. esculenta hybridogenetic species complex in Italy.     

A new species of Rhabdias (Nematoda: Rhabdiasidae) in Calotes versicolor (Squamata: Agamidae) from Singapore

Rhabdias singaporensis n. sp. (Rhabditida: Rhabdiasidae) from the lungs of Calotes versicolor (Squamata: Agamidae) from Singapore is described and illustrated. Rhabdias singaporensis n. sp. represents the 77th species assigned to the genus, the eighth of the Asian region, and the second from Singapore. The distinguishing characteristic of the new species is the location of the excretory pore. In all species of Rhabdias for which excretory pore location data are available, the excretory pore is situated just posterior to the level of the nerve ring; in R. singaporensis , it lies near the esophageointestinal junction.     

New species of Rhabdias (Nematoda: Rhabdiasidae) and other helminths from Norops capito (Sauria: Polychrotidae) from Nicaragua

Rhabdias nicaraguensis n. sp. (Rhabditida: Rhabditidae) from the lungs of Norops capito (Sauria: Polychrotidae) is described and illustrated. Rhabdias nicaraguensis n. sp. represents the 54th species assigned to the genus and the 12th from the Neotropical realm. Of the 12 Neotropical Rhabdias species, nicaraguensis is most similar to tobagoensis and vellardi. These 3 species have equatorial placement of the vulva, inflated cuticle, and 6 small circumoral lips. Rhabdias nicaraguensis is easily separated from R. tobagoensis by the shape of the buccal cavity and from R. vellardi by body size and shape of the tail. Rhabdias nicaraguensis differs from both species by host preference, the amount of inflated cuticle covering the body, and the phasmids situated posterior to the midpoint of the tail.     

Rhabdias collaris n. sp. (Nematoda: Rhabdiasidae) from frogs of Tanzania

Rhabdias collaris n. sp. (Rhabditoidea: Rhabdiasidae) is described from Leptopelis vermiculatus (Boulenger, 1909) (Hyperoliidae: Leptopelinae) (type host) and Hyperolius sp. (Hyperoliinae) of Amani, Tanzania. This species differs from all others in the genus in possessing a cephalic end which is markedly thickened by four peripheral muscle bundles. The muscular mass extends anteriorly forming a concave depression at the base of which the mouth is located.     

Two new species of Rhabdias (Nematoda: Rhabdiasidae) from the marine toad, Bufo marinus (L.) (Lissamphibia: Anura: Bufonidae), in Central America

Two new Rhabdias species are described from the lungs of the cane toad Bufo marinus (L.) from Costa Rica and Nicaragua. Rhabdias alabialis n. sp. differs from other known species of the genus by the remarkable morphology of its head end, i.e., the absence of lips or pseudolabia, the slitlike oral opening, and the triangular shape of the buccal capsule in apical view. Rhabdias pseudosphaerocephala n. sp. is identified as a form previously known in Central and South America as Rhabdias sphaerocephala Goodey, 1924, a species initially described from toads in Europe. The new species is differentiated from R. sphaerocephala based on head-end morphology and sequences of nuclear rDNA.     

A new species of Rhabdias (Nematoda: Rhabdiasidae) from agamid lizards on Luzon Island, Philippines

Rhabdias odilebaini n. sp. is described on the basis of specimens found in the lungs of 2 species of agamid lizards: the Philippine flying lizard Draco spilopterus and the marbled bloodsucker Bronchocela marmorata . Specimens were collected in Aurora Province, Luzon Island, Philippines. The new species of Rhabdias is characterized by presence of 4 submedian lips, inconspicuous lateral lips, rounded cross-shaped oral opening, and tail end bent dorsally. This species is morphologically distinct from other Rhabdias spp. that parasitize reptilian and amphibian hosts, including 3 other species known to parasitize lizards of the Agamidae.     

Rhabdias anolis n. sp. (Nematoda: Rhabdiasidae) from the lizard,Anolis frenatus (Sauria: Polychrotidae), from Panama

Rhabdias anolis n. sp. from the lungs of Anolis frenatus collected in Panama is described and illustrated. Rhabdias anolis n. sp. represents the 45th species assigned to the genus and the ninth from the Notropical Realm. It is distinguished from all other Neotropical species by the presence of a pair of sessile lateral papillae on its tail.     

Jirovecia branchilis n. sp. (Microsporidia) from glands of Branchiura sowerbyi (Oligochaeta: Tubificidae) in China

Jirovecia species primarily infect oligochaetes and are typically characterized by large rod-shaped spores with a tail-like posterior prolongation. Presently, seven Jirovecia spp. are reported worldwide with only one described in China. Here, a new species, Jirovecia branchilis n. sp. was discovered in glands of oligochaetes Branchiura sowerybi Beddard, 1892 in China. Jirovecia branchilis n. sp. elicited the formation of numerous opaque xenomas of 0.12 to 0.20 mm (n = 30) in diameter. Electron microscopic observations demonstrated that the earliest developmental stages observed were uninucleate meronts residing directly with the host cytoplasm. Mature spores were rod-shaped with blunt ends and possessed a collar-like anchoring disk, a manubrium-type polar filament, a bipartite polarplast, and a three-layered spore wall. A tail-like prolongation was distinctly observed in the posterior of spores and measured 13.2–28.6 μm long (n = 30). Jirovecia branchilis n. sp. showed 98.54% sequence similarity with Janacekia tainunus isolated from the fat body of chironomidae larvae Kiefferulus tainanus based on obtained partial SSU rDNA gene sequence, but was significantly different in morphology, host, and infection sites. SSU rDNA-based phylogenetic analysis indicated Jirovecia branchilis n. sp. clustered with Janacekia tainanus within the Jirovecia-Bacillidium-Janacekia clade. In conclusion, a new species within Jirovecia, Jirovecia branchilis n. sp. is erected herein based mainly on its morphological, ecological, and to a lesser degree on its molecular characteristics. The whole relationship between Jirovecia spp., Janacekia spp., and Bacillidium spp. is in need of revision and could potentially be elucidated by using additional makers and sequencing a broader diversity of the already described species.     

Ortholinea nupchi n. sp. (Myxosporea: Ortholineidae) from the urinary bladder of the cultured olive flounder Paralichthys olivaceus,South Korea

A new myxosporean parasite, Ortholinea nupchi n. sp. (Myxozoa; Bivalvulida), was isolated from the urinary bladder of the olive flounder Paralichthys olivaceus cultured on Jeju Island, Korea. Mature spores were subspherical in the valvular and apical views and ellipsoidal in the sutural view. The spores measured 7.6 ± 0.5 μm in length, 6.7 ± 0.3 μm in thickness, and 7.3 ± 0.5 μm in width. Two pyriform polar capsules measured 3.2 ± 0.1 μm in length and 2.7 ± 0.1 μm in width and were located at the same level at the anterior half of the myxospores. The suture line was straight in the middle of the spores, and the surface ridges ranged between five and seven, forming an intricate pattern. The result of the 18S rDNA comparison showed ≤ 93.0% similarity with other Ortholinea species. The phylogenetic tree demonstrated that O. nupchi n. sp. was closest to O. auratae and clustered with oligochaete-infecting myxosporeans (OIM) having urinary system infection tropism. Based on the comparison of environmental and host factors in the phylogenetic groups of the OIM clade, we propose that the infection of O. nupchi n. sp. originated from marine oligochaetes.