First molecular evidence of hybridization in endosymbiotic ciliates (Protista, Ciliophora).

Hybridization is an important evolutionary process that can fuel diversification via formation of hybrid species or can lead to fusion of previously separated lineages by forming highly diverse species complexes. We provide here the first molecular evidence of hybridization in wild populations of ciliates, a highly diverse group of free-living and symbiotic eukaryotic microbes. The impact of hybridization was studied on the model of Plagiotoma, an obligate endosymbiont of the digestive tube of earthworms, using split decomposition analyses and species networks, 2D modeling of the nuclear rRNA molecules and compensatory base change analyses as well as multidimensional morphometrics. Gene flow slowed down and eventually hampered the diversification of Lumbricus-dwelling plagiotomids, which collapsed into a single highly variable biological entity, the P. lumbrici complex. Disruption of the species boundaries was suggested also by the continuum of morphological variability in the phenotypic space. On the other hand, hybridization conspicuously increased diversity in the nuclear rDNA cistron and somewhat weakened the host structural specificity of the P. lumbrici complex, whose members colonize a variety of phylogenetically closely related anecic and epigeic earthworms. By contrast, another recorded species, P. aporrectodeae sp. n., showed no signs of introgression, no variability in the rDNA cistron, and very high host specificity. These contrasting eco-evolutionary patterns indicate that hybridization might decrease the alpha-diversity by dissolving species boundaries, weaken the structural host specificity by broadening ecological amplitudes, and increase the nuclear rDNA variability by overcoming concerted evolution within the P. lumbrici species complex.

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

Coevolution of endosymbionts with their hosts plays an important role in the processes of speciation and is among the most fascinating topics in evolutionary biology. Astome ciliates represent an interesting model for coevolutionary studies because they are so tightly associated with their host organisms that they completely lost the cell oral apparatus. In the present study, we used five nuclear markers (18S rRNA gene, ITS1-5.8S-ITS2 region, and 28S rRNA gene) and two mitochondrial genes (16S rRNA gene and cytochrome c oxidase subunit I) to explore the diversity of astomes inhabiting the digestive tract of lumbricid earthworms at temperate latitudes in Central Europe and to cast more light on their host specificity and coevolution events that shaped their diversification. The present coevolutionary and phylogenetic interaction-adjusted similarity analyses suggested that almost every host switch leads to speciation and firm association with the new host. Nevertheless, the suggested high structural host specificity of astomes needs to be tested with increased earthworm sampling, as only 52 out of 735 lumbricid earthworms (7.07%) were inhabited by ciliates. On the other hand, the diversification of astomes associated with megascolecid and glossoscolecid earthworms might have been driven by duplication events without host switching.

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.

Evolutionary Origin and Host Range of Plagiotoma lumbrici (Ciliophora, Hypotrichia), an Obligate Gut Symbiont of Lumbricid Earthworms.

Four common earthworm species, the anecic Lumbricus terrestris, the endogeic Octolasion tyrteum as well as the epigeic Eisenia fetida and Dendrobaena veneta, were examined for the presence of the microbial gut symbiont Plagiotoma lumbrici. The evolutionary origin of this endobiotic microbe was reconstructed, using the 18S rRNA gene, the ITS1-5.8S-ITS2 region, and the first two domains of the 28S rRNA gene. Plagiotoma lumbrici was exclusively detected in the anecic Lumbricus terrestris. Multigene analyses and the ITS2 secondary structure robustly determined the phylogenetic home of Plagiotoma lumbrici populations within the oxytrichid Dorsomarginalia (Spirotrichea: Hypotrichia) as a sister taxon of the free-living Hemiurosomoida longa. This indicates that earthworms obtained their gut endosymbiont by ingesting soil/leaf litter containing oxytrichine ciliates that became adapted to the intestinal tract of earthworms. Interestingly, according to the literature data, Plagiotoma lumbrici was detected in multiple anecic and some epigeic but never in endogeic earthworms. These observations suggest that Plagiotoma lumbrici might be adapted to certain gut conditions and the lifestyle of anecic Lumbricidae, such as Lumbricus, Aporrectodea, and Scherotheca, as well as of some co-occurring epigeic Lumbricus species.