Molecular identification of a new species of Rhigonema (Nematoda: Rhigonematidae) and phylogenetic relationships within the infraorder Rhigonematomorpha

Abstract

Background The infraorder Rhigonematomorpha comprises a group of obligate parasitic nematodes of millipedes (Arthropoda: Diplopoda). The current species identification of Rhigonematomorpha nematodes remains mainly based on morphological features, with molecular-based identification still in its infancy. Also, current knowledge of the phylogeny of Rhigonematomorpha is far from comprehensive. Methods The morphology of Rhigonematomorpha nematodes belonging to the genus Rhigonema, collected from the millipede Spirobolus bungii Brandt (Diplopoda: Spirobolida) in China, was studied in detail using light and scanning electron microscopy. Five different genetic markers, including the nuclear small ribosomal subunit (18S), internal transcribed spacer (ITS) and large ribosomal subunit (28S) regions and the mitochondrial cox1 and cox2 genes of these Rhigonematomorpha nematodes collected from China and Rhigonema naylae collected from Japan were sequenced and analyzed using Bayesian inference (BI) and Assemble Species by Automatic Partitioning (ASAP) methods. Phylogenetic analyses that included the most comprehensive taxa sampling of Rhigonematomorpha to date were also performed based on the 18S + 28S genes using maximum likelihood (ML) and BI methods. Results The specimens of Rhigonema collected from S. bungii in China were identified as a new species, Rhigonema sinense n. sp. Striking variability in tail morphology was observed among individuals of R. sinense n. sp. ASAP analyses based on the 28S, ITS, cox1 and cox2 sequences supported the species partition of R. sinense n. sp. and R. naylae, but showed no evidence that the different morphotypes of R. sinense n. sp. represent distinct genetic lineages. BI analyses also indicated that R. sinense n. sp. represents a separated species from R. naylae based on the cox1 and cox2 genes, but showed that R. naylae nested in samples of R. sinense n. sp. based on the ITS and 28S data. Phylogenetic results showed that the representatives of Rhigonematomorpha formed two large clades. The monophyly of the families Carnoyidae and Ichthyocephalidae and the genus Rhigonema was rejected. The representatives of the family Ransomnematidae clustered together with the family Hethidae with strong support. Conclusions A new species of Rhigonematomorpha, R. sinense n. sp. is described based on morphological and molecular evidence. ASAP analyses using 28S, ITS, cox1 and cox2 data indicate the striking variability in tail morphology of R. sinense n. sp. as intraspecific variation, and also suggest that partial 28S, ITS, cox1 and cox2 markers are effective for molecular identification of Rhigonematomorpha nematodes. The phylogenetic results support the traditional classification of Rhigonematomorpha into the two superfamilies Rhigonematoidea and Ransomnematoidea, and indicate that the families Carnoyidae and Ichthyocephalidae and the genus Rhigonema are non-monophyletic. The present phylogeny strongly supports resurrection of the family Brumptaemiliidae, and also indicates that the family Ransomnematidae is sister to the family Hethidae. Graphical Abstract

Anatomical and molecular characterization of some rhigonematid parasites of millipedes in Nigeria, with new insights into their phylogeny

“…2015; Mejia-Madrid 2018; Zhang et al . 2022). However, the low availability of ITS rRNA sequences makes it difficult to infer phylogenetic relationships with this marker.…”

“…2015; Mejia-Madrid 2018; Zhang et al . 2022). Our results on 28S and 18S rRNA genes suggest that genera within Ransomnematoidea ( Ransomnema , Heth , Carnoya , Brumptaemilius , Cattiena , Insulanema , Gilsonema ) and Rhigonematoidea ( Rhigonema , Obainia , Xystrognathus , Trachyglossoides , Ichthyocephaloides ) clustered closer than could be expected in view of their morphological differences, these groups having been lumped together mainly on host association, although it would appear from the molecular data that they are actually closely linked (Figures 11, 13).…”

“…2015; Mejia-Madrid 2018; Zhang et al . 2022). Multiple sequence alignments of the different genes were made using the FFT-NS-2 algorithm of MAFFT V.7.450 (Katoh et al 2019).…”

“…As a result, the present information on the molecular phylogeny of rhigonematids is far from complete (Mejia-Madrid 2018; Zhang et al . 2022).…”

“…(Hunt 2001a(Hunt , 2001b. Traditionally, species identification of rhigonematid nematodes was mainly based on morphological features, whereas integrative taxonomical approaches, including molecular-based identification, are still scarce (Zhang et al 2022). However, some recent studies have provided some ribosomal and mitochondrial (mt) DNA sequence data (i.e., the small subunit ribosomal DNA (18S), the large subunit ribosomal DNA (28S), and the mitochondrial cytochrome c oxidase subunit I (COI) gene, or the mitochondrial genome), which can be used for species identification or phylogeny (Kim et al 2014;Malysheva et al 2015).…”