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

Like many other aquatic animals, freshwater planarians have also become partners of symbiotic ciliates from the class Oligohymenophorea. In the present study, we explored the hidden diversity and addressed the questionable systematic position of mouthless obligatory gut endosymbionts of freshwater planarians, using the nuclear and mitochondrial SSU rRNA genes. Although all isolated ciliates morphologically corresponded to a single species, molecular analyses suggested the existence of three genetically distinct entities: Haptophrya planariarum, Haptophrya dugesiarum nov. spec., and Haptophrya schmidtearum nov. spec. The two former species share the same planarian host, which indicates a speciation model involving one duplication event without host switching. Such a diversification pattern was recognized also in astome ciliates inhabiting megascolecid and glossoscolecid earthworms. The present multi-gene phylogenies along with the secondary structure of the mitochondrial 16S rRNA molecule, however, challenge the traditional classification of Haptophrya within the subclass Astomatia. Haptophrya very likely evolved from an orphan scuticociliate lineage by the loss of oral apparatus and by the transformation of the thigmotactic field into an adhesive sucker. Since astomy evolved multiple times independently within the Oligohymenophorea, the loss of cell mouth cannot be used as a sole argument for the assignment of Haptophrya to the Astomatia anymore.

Cryptic host-driven speciation of mobilid ciliates epibiotic on freshwater planarians.

Mobilids are among the most taxonomically diverse but morphologically uniform groups of epibiotic ciliates. They attach to their hosts by means of an adhesive disc as harmless commensals such as Urceolaria, or as parasites causing significant economic loss such as some Trichodina species. We investigated the diversity, species boundaries, and phylogenetic relationships of mobilids associated with freshwater planarians, using 114 new sequences of two mitochondrial (16S rRNA gene and cytochrome c oxidase gene) and five nuclear (18S rRNA gene, ITS1-5.8S-ITS2 region, D1/D2 domains of 28S rRNA gene) markers. Although the morphological disparity of the isolated trichodinids and urceolariids was low, Bayesian coalescent analyses revealed the existence of five distinct evolutionary lineages/species given the seven molecular markers. The occurrence of mobilids perfectly correlated with their planarian hosts: Trichodina steinii and two Urceolaria mitra-like taxa were associated exclusively with the planarian Dugesia gonocephala, Trichodina polycelis sp. n. with the planarian Polycelis felina, and Trichodina schmidtea sp. n. with the planarian Schmidtea polychroa. Host organisms thus very likely constitute sharply isolated niches that might permit speciation of their epibiotic ciliates, even though no distinct morphological features appear to be recognizable among ciliates originating from different hosts.

Multi-gene phylogeny of Tetrahymena refreshed with three new histophagous species invading freshwater planarians.

Planarians represent an insufficiently explored group of aquatic invertebrates that might serve as hosts of histophagous ciliates belonging to the hymenostome genus Tetrahymena. During our extensive research on freshwater planarians, parasitic tetrahymenas were detected in two of the eight planarian species investigated, namely, in Dugesia gonocephala and Girardia tigrina. Using the 16S and 18S rRNA genes as well as the barcoding cytochrome oxidase subunit I, one ciliate species was identified as T. scolopax and three species were recognized as new forms: T. acanthophora, T. dugesiae, and T. nigricans. Thus, 25% of the examined planarian taxa are positive for Tetrahymena species and three of them represent new taxa, indicating a large undescribed ciliate diversity in freshwater planarians. According to phylogenetic analyses, histophagous tetrahymenas show a low phylogenetic host specificity. Although T. acanthophora, T. dugesiae, and T. scolopax clustered together within the "borealis" clade, the former species has been detected exclusively in G. tigrina, while the two latter species only in D. gonocephala. Tetrahymena nigricans, which has been isolated only from G. tigrina, was classified within the "paravorax" clade along with T. glochidiophila which feeds on glochidia. The present phylogenetic reconstruction of ancestral life strategies suggested that the last common ancestor of the family Tetrahymenidae was free-living, unlike the progenitor of the subclass Hymenostomatia which was very likely parasitic. Consequently, there were at least seven independent shifts back to parasitism/histophagy within Tetrahymena: one each in the "paravorax" and "australis" clades and at least five transfers back to parasitism in the "borealis" clade.

Dawn of astome ciliates in light of morphology and time-calibrated phylogeny of Haptophrya planariarum, an obligate endosymbiont of freshwater turbellarians.

Morphology, systematic position and time-calibrated phylogeny of Haptophrya planariarum were investigated. This endosymbiont of freshwater turbellarians is characterized by: (i) a length of about 200-900 µm; (ii) a campanulate to truncate claviform body carrying an anterior adhesive sucker; (iii) an ellipsoidal macronucleus localized in the rear body end; (iv) a contractile canal extending along the dorsal margin; and (v) usually more than 150 meridional ciliary rows, a horseshoe-shaped suture line along the sucker, and two inconspicuous secant systems at lateral ends of the suture line. In 18S rRNA gene phylogenies, astomes were depicted as a non-monophyletic group within the scuticociliate clade, whereby H. planariarum clustered with the loxocephalid genus Dexiotricha. After considering morphological evidence, statistical tree topology tests and evolutionary distances, we find astomes as a distinct group that evolved from a free-living scuticociliate ancestor in the early Paleozoic. Molecular clock analyses indicated that astomes living in annelids diverged from those inhabiting turbellarians within about 50 Ma during the Late Cambrian and the Upper Ordovician. This comparatively short time span might have not sufficed for fixation of molecular synapomorphies in the 18S rRNA gene and/or they might have been erased by substitutions during the almost 500 Ma-long evolutionary history of astomes.

Evaluation of Systematic Position of Helicoprorodontids and Chaeneids (Ciliophora, Litostomatea): An Attempt to Break Long Branches in 18S rRNA Gene Phylogenies.

Phylogenetic position of some free-living litostomatean taxa has not been correctly determined because of long-branch artifacts in 18S rRNA gene trees. The main aim of this study was to test the effectiveness of various masking algorithms, tree-building techniques, binarization of DNA data as well as combining morphological and molecular data to eliminate long-branch attraction of two problematic groups, helicoprorodontids and chaeneids. Guidance and SlowFaster masking in a combination with PhyloBayesian tree construction erased the artifactual positions of helicoprorodontids and chaeneids. On the other hand, binarization of DNA sequences and the strategy of combining morphological and molecular data eliminated only the artifactual position of chaeneids but not that of helicoprorodontids which were still being attracted by out-group taxa. According to statistical tree topology tests and comparative morphological studies, helicoprorodontids are classified as a distinct order while chaeneids are considered to be fast evolving members of the order Lacrymariida. The high body contractility, "cephalization" of the anterior body end, and helicalization of the anterior portion of some or all somatic ciliary rows indicate relatedness of helicoprorodontids, chaeneids, and lacrymariids. On the other hand, the dorsal brush separated from the circumoral kinety by dense ciliary files supports kinships of chaeneids, lacrymariids, and didiniids.