Delimitation of five astome ciliate species isolated from the digestive tube of three ecologically different groups of lumbricid earthworms, using the internal transcribed spacer region and the hypervariable D1/D2 region of the 28S rRNA gene

BACKGROUND: Various ecological groups of earthworms very likely constitute sharply isolated niches that might permit speciation of their symbiotic ciliates, even though no distinct morphological features appear to be recognizable among ciliates originating from different host groups. The nuclear highly variable ITS1–5.8S-ITS2 region and the hypervariable D1/D2 region of the 28S rRNA gene have proven to be useful tools for the delimitation of species boundaries in closely related free-living ciliate taxa. In the present study, the power of these molecular markers as well as of the secondary structure of the ITS2 molecule were tested for the first time in order to discriminate the species of endosymbiotic ciliates that were isolated from the gastrointestinal tract of three ecologically different groups of lumbricid earthworms. RESULTS: Nineteen new ITS1–5.8S-ITS2 region and D1/D2-28S rRNA gene sequences were obtained from five astome species (Anoplophrya lumbrici, A. vulgaris, Metaradiophrya lumbrici, M. varians, and Subanoplophrya nodulata comb. n.), which were living in the digestive tube of three ecological groups of earthworms. Phylogenetic analyses of the rRNA locus and secondary structure analyses of the ITS2 molecule robustly resolved their phylogenetic relationships and supported the distinctness of all five species, although previous multivariate morphometric analyses were not able to separate congeners in the genera Anoplophrya and Metaradiophrya. The occurrence of all five taxa, as delimited by molecular analyses, was perfectly correlated with the ecological groups of their host earthworms. CONCLUSIONS: The present study indicates that morphology-based taxonomy of astome ciliates needs to be tested in the light of molecular and ecological data as well. The use of morphological identification alone is likely to miss species that are well delimited based on molecular markers and ecological traits and can lead to the underestimation of diversity and overestimation of host range. An integrative approach along with distinctly increased taxon sampling would be helpful to assess the consistency of the eco-evolutionary trend in astome ciliates.