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

Abstract

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.

Helminth Parasites of thePelophylax esculentusComplex (Anura: Ranidae) in Hortobágy National Park (Hungary)

“…Recent research suggests that specimens identified as R. bufonis represent a species complex (Tkach et al, 2014). Cipriani et al (2012) recently described R. esculentarum from both P. lessonae and P. esculentus in Italy, and our specimens in water frogs from HNP correspond to that species. We have not collected parasites from Bufo bufo (Linnaeus, 1758) or B. viridis, so we do not know if both R. bufonis and R. esculentarum do, or did, co-occur in the HNP.…”

“…The absence of C. retusus, C. ornata, G. cygnoides, H. asper, H. cylindracea, and P. loossi were confirmed by sampling in 2013. Murai et al (1983) did not report members of 2 common amphibian helminth taxa that we detected, including nematodes belonging to the genus Rhabdias, which includes species living in the lungs of both amphibians and some lizards (Cipriani et al, 2012;Kuzmin, 2013). Most studies of Rhabdias in European amphibians have reported R. bufonis, and Edelényi (1960) reported this worm from P. esculentus, P. ridibundus, and Bufo viridis near the HNP.…”

“…Type host and type locality: Pelophylax lessonae, Lake Vico, Latium, central Italy (Cipriani et al, 2012).…”

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

“…Molecular data have been successfully applied to the study of nematodes in general and, in particular, the genus Rhabdias (Cipriani, Mattiucci, Paoletti, Santoro, & Nascetti, ; Dare, Nadler, & Forbes, ; Dubey & Shine, ; Langford & Janovy, ; Lhermitte‐Vallarino et al, ; Morais et al, ; Nascimento et al, ; Tkach, Kuzmin, et al, ). Several molecular markers have been used for Rhabdiasidae, including ribosomal and mitochondrial genes (Cipriani et al, ; Dare et al, ; Dubey & Shine, ; Junker et al, ; Langford & Janovy, ; Lhermitte‐Vallarino et al, ; Nascimento et al, ; Tkach, Halajian, & Kuzmin, ; Tkach, Kuzmin, et al, ).…”

“…Regarding the molecular studies conducted for this genus, some used DNA sequences only to distinguish closely related species (Cipriani et al, ; Junker et al, ; Lhermitte‐Vallarino et al, ) although others reported on a broader taxon sampling, improving the phylogenetic reconstruction (Dare et al, ; Dubey & Shine, ; Langford & Janovy, ; Tkach, Halajian, et al, ; Tkach, Kuzmin, et al, ). Using morphological and molecular data, Tkach, Kuzmin, et al, added considerable information on the taxonomy of Rhabdiasidae studying a broad phylogeny with species from several continents, in which a new genus ( Serpentirhabdias ) was proposed, together with notes on its biology, morphology, geographical distribution and host–parasite relationships.…”