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

Abstract

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.

2D approach to reconstruct the evolutionary history of clevelandellids (Ciliophora, Armophorea) inhabiting the hindgut of the Panesthiinae cockroaches

“…More specifically, the V9 region has gradually shortened from N. nakhoni (166 nt) and N. galerus (132 nt), the sister group of all other species included in the present analyses, through N. angustipennis (115 nt) and N. ovalis (118 nt), the sister group of the family Clevelandellidae, to the family Clevelandellidae, whose members share the shortest helix 44 (103–105 nt) (Figure 8). A relatively long hypervariable region V9 was also found in the ciliate genera Haptophrya (145–148 nt), Hysterocineta (124 nt), Tetrahymena (166–169 nt), and Trichodina (122–123 nt) (Obert et al, 2023; Rataj et al, 2022; Zhang et al, 2023; Zhang & Vďačný, 2023). Remarkable structural features of the 16S rRNA molecule in ciliates include the absence/reduction of helices 26 and 26a in Trichodina (Zhang et al, 2023) as well as an extraordinarily long and branched helix 40 in Haptophrya (Rataj et al, 2022).…”

“…As expected, mutations are situated mainly in the hypervariable regions of the 18S rRNA molecule, especially in the V2, V4, and V7–V9 regions (Li et al, 2020; present study). Similarly to clevelandellids, the majority of mutations was detected within the hypervariable regions of the 18S rRNA molecule in the ciliate genera Haptophrya Stein, 1867, Plagiotoma Dujardin, 1841, and Trichodina Ehrenberg, 1830 (Obert et al, 2022; Rataj et al, 2022; Zhang et al, 2023). A common trait shared by clevelandellids isolated from the digestive tract of cockroaches is the absence of helix 9es3b in the V2 region.…”

“…Interestingly, the lack of this helix was also reported for the spirotrich genus Plagiotoma (Obert et al, 2022). On the other hand, helix 9es3b was recognized in Haptophrya , Hysterocineta Diesing, 1866, Protoptychostomum Raabe, 1949, Tetrahymena Furgason, 1940, Trichodina , and Urceolaria Stein, 1867 (Lee & Gutell, 2012; Obert et al, 2023; Rataj et al, 2022; Zhang et al, 2023), whereas the stem of this helix is shortened to only two base pairs in Trichodina (Zhang et al, 2023). It is not clear whether the absence of helix 9es3b is a molecular synapomorphy of the SAL supercluster (Spirotrichea + Armophorea + Litostomatea) in which Plagiotoma and clevelandellids are classified, or whether its loss may have occurred independently in the Spirotrichea and Armophorea.…”

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

“…It was, therefore, assumed that hybridization in nature may occur only seldom in Euplotes ( Valbonesi et al, 1988 ) and possibly also in other ciliate genera. Indeed, obvious discordances between nuclear and mitochondrial data, which are predominantly caused by hybridization, have been up to now not detected in wild populations of free-living and symbiotic ciliates (e.g., Shazib et al, 2019 ; Rataj and Vďačný, 2020 , 2021 , 2022 ; Obert et al, 2021 ; Zhang and Vďačný, 2021a , b , 2022 ; Rataj et al, 2022 ). It was, therefore, a great surprise when we noticed a strong conflicting signal between the nuclear rDNA cistron and the mitochondrial COI phylogenies in the genus Plagiotoma Dujardin, 1841 (family Plagiotomidae Bütschli, 1887, class Spirotrichea Bütschli, 1889), which lives in the digestive tube of earthworms.…”

“…Such uneven distribution of diversity provokes a question of what are the reasons for this pronounced asymmetry among these four phylogenetically distant ciliate groups. Diversification and distribution of endobiotic ciliates are very likely governed by the geographic range and biology of their hosts (e.g., Irwin et al, 2017 ; Vďačný, 2018 ; Rataj and Vďačný, 2020 , 2021 ; Obert et al, 2021 ; Zhang and Vďačný, 2021a , b , 2022 ; Rataj et al, 2022 ). However, host switching and hybridization also have a significant impact on diversification and speciation processes.…”