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

Abstract

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. 596 | MÜLLER Et aL.

A new species ofRhabdias(Nematoda: Rhabdiasidae), a lung parasite ofPseudopaludicola pocoto(Anura: Leptodactylidae) from north-eastern Brazil: description and phylogenetic analyses

“…Using molecular tools, in a recent study of Rhabdias populations of amphibians and lizards in Brazil, including those also from the Caatinga biome, Müller et al . (2018) reported that all studied species (five species, 26 specimens) exhibited an oral arrangement with four submedian lips and two lateral pseudolabia (pattern 1). However, this pattern was not observed in the morphology of species with no molecular data; although pattern 1 includes most known species.…”

“…Based on the set of morphological and molecular data produced (Tkach et al ., 2014; Müller et al ., 2018; Willkens et al , 2019) so far, we can observe three patterns of oral arrangements in species in the Neotropical region parasitizing amphibians and for which information about the oral arrangement is available: (1) four submedian lips and two lateral pseudolabia (five spp. R. manantlanensis , R. savage , R. kuzmini , R. pseudosphaerocephala and R. elegans ]); (2) six equal lips (seven spp.…”

“…Moreover, these relationships can be observed at another taxonomic level, such as the R. pseudosphaerocephala species complex, which is commonly found associated with bufonids (Müller et al ., 2018). Müller et al .…”

“…A Polymerase chain reaction was performed to amplify the partial mitochondrial coi gene using primers and cycling conditions, as well as purification and sequencing procedures in accordance with Müller et al . (2018). The sequence identity was verified using the Basic Local Alignment Search Tool (BLAST), and contiguous sequences were assembled using Sequencer v. 5.2.4 (Gene Codes, Ann Arbor, MI) and submitted to GenBank (accession numbers presented in table 1).…”

“…According to Poulin et al . (2019), the use of genetic information is fundamental in systematic parasite taxonomy, especially for descriptions of new species, allowing taxonomic problems among some groups to be solved using molecular phylogenies, description of patterns of gene flow among species and populations, and especially for understanding diversity in complex parasite groups such as Rhabdiasidae (Tkach et al ., 2014; Müller et al ., 2018).…”