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

Abstract

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.

Nuclear and Mitochondrial SSU rRNA Genes Reveal Hidden Diversity of Haptophrya Endosymbionts in Freshwater Planarians and Challenge Their Traditional Classification in Astomatia

“…With the application of multiple molecular markers (especially the mitochondrial 16S rRNA and COI genes), cryptic or near-cryptic speciation (subtle, easily overlooked differences, or only statistically supported differences) has been detected in a variety of symbiotic ciliates. For instance, in clevelandellids associated with wood-feeding cockroaches ( Pecina and Vďačný, 2020 , 2021 ), in histophagous tetrahymenids parasitizing mollusks ( Zhang and Vďačný, 2021 ), in astomes occupying the digestive tube of lumbricid earthworms ( Obert and Vďačný, 2019 , 2020 ; Obert et al, 2021 ), and in mobilids living on the surface of freshwater planarians ( Rataj and Vďačný, 2021 ). Cryptic or near-cryptic speciation has apparently occurred also in haptophryans associated with freshwater planarians, as documented by the huge differences in the primary structure (interspecies p -distances range from 11.03 to 17.03%, while maximum intraspecies distances are only 0.26%), the secondary structure of the V4, V7, and V9 regions ( Figure 4 ; Supplementary Figures S6, S7 ) as well as by 9–12 CBCs in the 16S rRNA molecule.…”

Putative ITS2 secondary structure model and multi‐gene phylogenies of tetrahymenids (Ciliophora, Hymenostomatia) parasitizing planarians and crayfish worms

“…Several studies tested the discriminatory power of this marker in different groups of ciliates with various results. For instance, ITS2 helped to reliably distinguish 16 astome species belonging to nine genera isolated from the digestive tube of earthworms, and multiple CBCs and hemi-CBCs were detected among congeners Obert & Vďačný, 2020. On the other hand, no CBCs were detected among distinct Spirostomum (Shazib et al, 2016(Shazib et al, , 2019, Trichodina (Rataj & Vďačný, 2021) and clevelandellid (Pecina & Vďačný, 2021) species, which very likely corresponds to the shortness and high GC content of their ITS2 molecules.…”

Diversity and Eco-Evolutionary Associations of Endosymbiotic Astome Ciliates With Their Lumbricid Earthworm Hosts

“…The putative secondary structures of the astome ITS2 molecules were modeled in mfold ver. 3.0 (Zuker, 2003), following the pipeline described in our previous study (Obert and Vd'ačný, 2020a). The single difference was that the hybridized 5.8S-28S rRNA imperfect helix was much longer; that is, it contained 16 instead of five nucleotide pairs (Coleman, 2005;Keller et al, 2009).…”

“…In our previous study, we proposed for the first time a secondary structure model for the astome ITS2 molecules (Obert and Vd'ačný, 2020a). Unfortunately, we used only five pairs to constrain the 5.8S-28S rRNA imperfect helix, which led to incorrect determination of the 3'-end of the ITS2 molecules.…”

“…To promote stability and universality, we state the following: The type species of Subanoplophrya Obert and Vd'ačný, 2020a, is now fixed under Article 70.3 of the Code, as S. nodulata nov. spec., misidentified as Leucophrys nodulata Dujardin, 1841 (basionym Leucophra nodulata Müller, 1779) in the original designation by Obert and Vd'ačný (2020a). A new species needs to be established for the misidentified and dubious Leucophrys nodulata, which is done in the Taxonomic Summary section.…”