Comparative molecular and morphological characterisations in the nematode genus Rotylenchus: Rotylenchus paravitis n. sp., an example of cryptic speciation

Abstract

The nematode Rotylenchus paravitis n. sp. infesting roots of commercial sunflowers in southern Spain is described. The new species is characterised by a truncate lip region with 7–9 annuli and continuous with the body contour, lateral fields areolated at pharyngeal region only, body without longitudinal striations, stylet length of 44–50μm, vulva position at 43–54%, tail rounded to hemispherical with 12–18 annuli. A comparative phenetic study based on a multivariate principal component analysis was developed to determine potential species discrimination. The degree of variation for most characters among specimens of Rotylenchus paravitis n. sp. and R. vitis was comparable to that observed among specimens belonging to each of the two studied populations of R. robustus from Spain and USA. Molecular comparison of the partial 18S, D2-D3 expansion segments of 28S-rRNA, ITS1-rRNA, partial COI and hsp90 from R. paravitis n. sp. and R. vitis, and other species in the genus, clearly supports the proposal of R. paravitis n. sp. as a new species. Consequently, R. paravitis n. sp. should be considered as an example of cryptic speciation within the genus Rotylenchus. PCR-ITS-RFLP was provided for diagnostics of R. paravitis n. sp. and PCR with specific primers were also developed for diagnostics of this new species, R. vitis and R. robustus. The results of the phylogenetic analysis based on the sequences of the D2-D3 expansion regions of the 28S, ITS1-rRNA genes, and the partial COI, have proven to be a powerful tool for providing accurate species identification and assessing phylogenetic relationships within the genus Rotylenchus. Phylogenetic testing of D2-D3 expansion segments of 28S-rRNA gene sequences did not refute the monophyly of the genera Rotylenchus, Helicotylenchus, Hoplolaimus, based on tree topologies and the Shimodaira–Hasegawa test even with the split in several clades for some of the genera.