Rediscovery and morpho-molecular characterization of three astome ciliates, with new insights into eco-evolutionary associations of astomes with their annelid hosts

Abstract

Astome ciliates live in the digestive tract of a broad spectrum of marine, freshwater, and terricolous annelids. In aquatic lumbriculid and criodrilid oligochaetes collected in Central Europe, we rediscovered three insufficiently known astomes: Hoplitophrya secans, Mesnilella clavata, and Buchneriella criodrili. Their morphology was studied using in vivo observation, protargol, and dry silver nitrate impregnation. Multiple nuclear and mitochondrial molecular markers were used to determine their phylogenetic positions and reconstruct their evolutionary history. According to our phylogenetic analyses: (1) mouthless ciliates isolated from annelids form a robustly supported monophylum within the class Oligohymenophorea, (2) the progenitor of astomes invaded the digestive tract of marine polychaetes during the Paleozoic era, (3) lumbricid earthworms likely served as a source of astomes for criodrilid, almid, and megascolecid earthworms, (4) the ancestral host of the earthworm-dwelling astome clade led an endogeic lifestyle, and (5) there were multiple independent transfers of astomes from endogeic to epigeic and anecic earthworms. These findings support previous views of the annelid phylogeny, suggesting that astomes reside and evolve in tandem with annelids for several hundred million years.

Keywords

Comparative network analyses / Endosymbionts / Integrative taxonomy / Oligochaeta / Oligohymenophorea / Phylogeny

References

[1] AlbaretJ-L, NjinéT. Description de cinq espèces nouvelles de ciliés heterotriches des genres Metanyctotherus Albaret, Pronyctotherus n. gen. et Plagiotoma Dujardin, endocommensaux d’oligochètes du Cameroun. Protistologica, 1975, 11: 305-311

[2] AndersonFE, WilliamsBW, HornKM, ErséusC, HalanychKM, SantosSR, JamesSW. Phylogenomic analyses of Crassiclitellata support major Northern and Southern Hemisphere clades and a Pangaean origin for earthworms. BMC Evol Biol, 2017, 17. e123

[3] AntipaGA, Strüder-KypkeMC, LynnDH. Molecular phylogeny, taxonomic relationships and North American distribution of Conchophthirus (Conchophthiridae, Scuticociliatia). Aquat Ecosys Health Manag, 2020, 23: 58-68.

[4] BollbackJP. SIMMAP: stochastic character mapping of discrete traits on phylogenies. BMC Bioinform, 2006, 7. e88

[5] BouckaertR, HeledJ, KühnertD, VaughanT, WuC-H, XieD, SuchardMA, RambautA, DrummondAJ. BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput Biol, 2014, 10. e1003537

[6] Buchholz R. Beiträge zur Anatomie der Gattung Enchytraeus, nebst Angabe der um Königsberg vorkommenden Formen derselben. Schr K Phys-Ökon Ges Königsberg, 1862, 1863: 93-132

[7] BütschliOBronnHG. Erster Band. Protozoa. III. Abtheilung: Infusoria und System der Radiolaria. Klassen und Ordnungen des Thier-Reichs, wissenschaftlich dargestellt in Wort und Bild. Erster Band, 1889HeidelbergCF Winter’sche Verlagshandlung1585-2035

[8] CépèdeC. Recherches sur les infusoires astomes. Anatomie, biologie, ethologie parasitaire, systématique. Arch Zool Exp Gén, 1910, 5: 341-609

[9] CheissinE. Morphologische und systematische Studien über Astomata aus dem Baikalsee. Arch Protistenkd, 1930, 70: 531-618

[10] Conway MorrisS, PeelJS. The earliest annelids: lower Cambrian polychaetes from the Sirius Passet Lagerstätte, Peary Land, North Greenland. Acta Palaeontol Pol, 2008, 53: 137-148.

[11] CorlissJORavenPH. Biodiversity, classification, and numbers of species of protists. Nature and human society. The quest for a sustainable world, 2000Washington, DCNational Academies Press130-155

[12] de PuytoracP. Maupasella glossoscolecis sp. nov. et Maupasella lubeti sp. nov., Ciliés Astomes commensaux du tube digestif de Glossoscolex grandis (Michaelsen). Remarques sur les Maupasellinae. Bull Soc Zool Fr, 1952, 76: 323-333

[13] de PuytoracP. Contribution à l’étude cytologique et taxonomique les infusoires astomes. Ann Sci Nat Zool Biol Anim, 1954, 11: 85-270

[14] de PuytoracP. Nouvelles données sur les Ciliés Hoplitophryidae. Arch Zool Exp Gén Notes Rev, 1957, 94: 89-120

[15] de PuytoracP. Observations sur quelques Ciliés astomes des Oligochètes du lac d’Ohrid (Yougoslavie). II. Familles des Hoplitophryidae (Hoplitophryinae et Maupasellinae), des Intoshellinidae et des Anoplophryidae. J Protozool, 1960, 7: 278-289.

[16] de PuytoracP. Les ciliés astomes Hoplitophryidae. I Description De Nouvelles Espèces Protistologica, 1969, 5: 255-268

[17] de PuytoracP. Les ciliés astomes Hoplitophryidae. II. Révision de la systématique de ce groupe. Protistologica, 1972, 8: 5-42

[18] de PuytoracP, RakotoariveloJ. Sur quelques Ciliés Astomes des Oligochètes Megascolecidae. Arch Zool Exp Gén, 1965, 105: 229-243

[19] DomínguezJ, AiraM, BreinholtJW, StojanovicM, JamesSW, Pérez-LosadaM. Underground evolution: new roots for the old tree of lumbricid earthworms. Mol Phylogenet Evol, 2015, 83: 7-19.

[20] ErséusC, WilliamsBW, HornKM, HalanychKM, SantosSR, JamesSW, des ChâtelliersMC, AndersonFE. Phylogenomic analyses reveal a Palaeozoic radiation and support a freshwater origin for clitellate annelids. Zool Scr, 2020, 49: 614-640.

[21] FoissnerW. Comments on the neotypification of protists, especially ciliates (Protozoa, Ciliophora). Bull Zool Nomencl, 2002, 59: 165-169

[22] FoissnerW. An update of ‘basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa’. Int J Syst Evol Microbiol, 2014, 64: 271-292.

[23] FokamZ, NgassamP, Strüder-KypkeMC, LynnDH. Genetic diversity and phylogenetic position of the subclass Astomatia (Ciliophora) based on a sampling of six genera from West African oligochaetes (Glossoscolecidae, Megascolecidae), including description of the new genus Paraclausilocola n. gen. Eur J Protistol, 2011, 47: 161-171.

[24] FokamZ, NanaPA, NgassamP, BricheuxG, BouchardP, ViguesB, Sime-NgandoT. Morphological description of four new species of Nyctotherus (Ciliophora: Nyctotheridae: Heterotrichida), commensal ciliates of the digestive tract of a terrestrial Oligochaete (Megascolecidae) from the northwest region of Cameroon. Protistology, 2014, 8: 62-70

[25] Fokam Z (2012) Etude morphologique et phylogénie des Ciliés Astomes endocommensaux d’Oligochètes terricoles de la région de Yaoundé et ses environs. PhD Thesis, University of Yaoundé I

[26] HallTA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser, 1999, 41: 95-98

[27] HeidenreichE. Untersuchungen an parasitischen Ciliaten aus Anneliden. Teil I: Systematik. Arch Protistenkd, 1935, 84: 315-392

[28] HoangDT, ChernomorO, von HaeselerA, MinhBQ, VihnLS. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol, 2018, 35: 518-522.

[29] ICZNInternational Code of Zoological Nomenclature, 19994LondonInternational Trust for Zoological Nomenclature

[30] JefferyS, GardiC, JonesA, MontanarellaL, MarmoL, MikoL, RitzK, PeresG, RömbkeJ, van der PuttenWHEuropean Atlas of Soil Biodiversity, 2010LuxembourgPublications Office of the European Union

[31] JiangCQ, WangGY, XiongJ, YangWT, SunZY, FengJM, WarrenA, MiaoW. Insights into the origin and evolution of Peritrichia (Oligohymenophorea, Ciliophora) based on analyses of morphology and phylogenomics. Mol Phylogenet Evol, 2019, 132: 25-35.

[32] JiangCQ, GuSY, PanTT, WangXY, QinWW, WangGY, GaoXX, ZhangJ, ChenK, WarrenA, XiongJ, MiaoW. Dynamics and timing of diversification events of ciliated eukaryotes from a large phylogenomic perspective. Mol Phylogenet Evol, 2024, 197. e108110

[33] KaczanowskiA. About a presumable phylogenetic link between Mesnilella (Astomata) and Conchostoma longissimum Fauré-Fremiet (Trichostomata). Acta Protozool, 1965, 3: 225-232

[34] KalyaanamoorthyS, MinhBQ, WongTKF, von HaeselerA, JermiinLS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods, 2017, 14: 587-589.

[35] KarimiN, GroverCE, GallagherJP, WendelJF, AnéC, BaumDA. Reticulate evolution helps explain apparent homoplasy in floral biology and pollination in baobabs (Adansonia; Bombacoideae; Malvaceae). Syst Biol, 2020, 69: 462-478.

[36] KatohK, RozewickiJ, YamadaKD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform, 2019, 20: 1160-1166.

[37] KentWSA manual of the infusoria: including a description of all known flagellate, ciliate, and tentaculiferous protozoa British and foreign, and an account of the organization and affinities of the sponges, 1881LondonDavid Bogue433-7202

[38] KijenskijG. Nálevníci zažívací roury některých Oligochaetů pražského okolí (Morfologie. Nepohlavní množení. Systematika) [Ciliates in the intestinal cavity of Oligochaeta from the surroundings of Prague]. Věstník Královské České Společnosti Nauk, Třída Matematicko-Přírodovědecká Year, 1926, 1925: 1-32

[39] KishinoH, HasegawaM. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J Mol Evol, 1989, 29: 170-179.

[40] KishinoH, MiyataT, HasegawaM. Maximum likelihood inference of protein phylogeny and the origin of chloroplasts. J Mol Evol, 1990, 30: 151-160.

[41] KleinBM. The “dry” silver method and its proper use. J Protozool, 1958, 5: 99-103.

[42] LankesterER. Remarks on Opalina and its contractile vesicules, on Pachydermon and annelidan spermatophors. Quart J Microsc Sci, 1870, 10: 143-150

[43] LeidyI. Contributions towards a knowledge of the marine invertebrate fauna of the coasts of Rhode Island and New Jersey. J Acad Nat Sci Philad, 1855, 3: 132-152

[44] LiT, ZhangT, LiuM, ZhangZ, ZhangJ, NiuJ, ChenX, Al-FarrajSA, SongW. Findings on three endocommensal scuticociliates (Protista, Ciliophora) from freshwater mollusks, including their morphology and molecular phylogeny with descriptions of two new species. Mar Life Sci Technol, 2024, 6: 212-235.

[45] LynnDHThe ciliated protozoa. Characterization, classification, and guide to the literature, 20083DordrechtSpringer

[46] MackinnonDL, AdamDI. Notes on four astomatous ciliates from oligochaete worms. Quart J Microsc Sci, 1924, 68: 211-228

[47] MeierM. Parasitische Ciliaten bei Oligochäten. Arch Protistenkd, 1954, 100: 212-245

[48] Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop. Piscataway, NJ, New Orleans, LA, pp 1–8

[49] NgassamP. Description de quatre espèces nouvelles de ciliés hétérotriches des genres Nyctotherus Leidy et Paranyctotherus nov. gen. endocommensaux d’un oligochète terricole du Cameroun. Protistologica, 1983, 19: 167-175

[50] NgassamP. Trois espèces nouvelles de ciliés Astomes des genres: Almophrya de Puytorac et Dragesco, 1968, Maupasella Cépède, 1910, Njinella nov. g. endocommensaux d’Annélides oligochètes de la région de Yaoundé. Potistologica, 1983, 19: 131-135

[51] NguyenLT, SchmidtHA, von HaeselerA, MinhBQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol, 2015, 32: 268-274.

[52] ObertT, VďačnýP. Integrative taxonomy of five astome ciliates (Ciliophora, Astomatia) isolated from earthworms in Central Europe. Eur J Taxon, 2019, 559: 1-37

[53] ObertT, VďačnýP. Evolutionary origin and host range of Plagiotoma lumbrici (Ciliophora, Hypotrichia), an obligate gut symbiont of lumbricid earthworms. J Eukaryot Microbiol, 2020, 67: 176-189.

[54] ObertT, VďačnýP. 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. BMC Evol Biol, 2020, 20. e37

[55] ObertT, VďačnýP. Multi-gene phylogeny of the subclass Astomatia (Protista: Ciliophora) refreshed with two rare astome ciliates from the digestive tube of endogeic earthworms. Org Divers Evol, 2021, 21: 59-77.

[56] ObertT, RurikI, VďačnýP. Diversity and eco-evolutionary associations of endosymbiotic astome ciliates with their lumbricid earthworm hosts. Front Microbiol, 2021, 12. e689987

[57] ObertT, ZhangT, RurikI, VďačnýP. First molecular evidence of hybridization in endosymbiotic ciliates (Protista, Ciliophora). Front Microbiol, 2022, 13. e1067315

[58] ObertT, ZhangT, VďačnýP. The search finds an end: the morphologically chimeric hysterocinetids belong to the subclass Hymenostomatia (Ciliophora: Oligohymenophorea). Zool J Linn Soc, 2023, 199: 97-123.

[59] ParfreyLW, LahrDJ, KnollAH, KatzLA. Estimating the timing of early eukaryotic diversification with multigene molecular clocks. Proc Natl Acad Sci USA, 2011, 108: 13624-13629.

[60] ParkE, LeanderB. Coinfection of slime feather duster worms (Annelida, Myxicola) by different gregarine apicomplexans (Selenidium) and astome ciliates reflects spatial niche partitioning and host specificity. Parasitology, 2024, 151: 400-411.

[61] PecinaL, RurikI, VďačnýP. Macroevolutionary trends and diversification dynamics in the hindgut order Clevelandellida (Ciliophora Armophorea). Zool Scr, 2024.

[62] PenardEÉtudes sur les infusoires d’eau douce, 1922GenèveGeorg & Cie.

[63] RaabeZ. Protoanoplophrya bithyniae sp. n., eine neue parasitische Ciliaten-Art aus dem Subordo Astomata. Ann Mus Zool Pol, 1932, 9: 354-358

[64] RaabeZ. Desmophrya contorta gen. nov., sp. nov., ein im Darm von Pisidium parasitierender Ciliate aus der Familie Hoplitophryidae Cheissin (Infusoria, Astomata). Ann Mus Zool Pol, 1933, 10: 51-56

[65] RaabeZ. Ordo Thigmotricha (Ciliata − Holotricha). V. Familiae Hysterocinetidae et Protoanoplophryidae. Acta Protozool, 1972, 10: 115-184

[66] RatajM, VďačnýP. Dawn of astome ciliates in light of morphology and time-calibrated phylogeny of Haptophrya planariarum, an obligate endosymbiont of freshwater turbellarians. Eur J Protistol, 2018, 64: 54-71.

[67] RatajM, VďačnýP. Living morphology and molecular phylogeny of oligohymenophorean ciliates associated with freshwater turbellarians. Dis Aquat Org, 2019, 134: 147-166.

[68] RatajM, ZhangT, VďačnýP. Nuclear and mitochondrial SSU rRNA genes reveal hidden diversity of Haptophrya endosymbionts in freshwater planarians and challenge their traditional classification in Astomatia. Front Microbiol, 2022, 13. e830951

[69] RevellLJ. Phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol Evol, 2012, 3: 217-223.

[70] RonquistF, TeslenkoM, van der MarkP, AyresDL, DarlingA, HöhnaS, LargetB, LiuL, SuchardMA, HuelsenbeckJP. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol, 2012, 61: 539-542.

[71] RossolimoLL, PerzewaTA. Zur Kenntnis einiger astomen Infusorien: Studien an Skelettbildung. Arch Protistenkd, 1929, 67: 237-252

[72] SauvadetA-L, LynnDH, RousselEG, Le PanseS, BigeardE, SchrévelJ, GuillouL. Redescription and phylogenetic analyses of Durchoniella spp. (Ciliophora, Astomatida) associated with the polychaete Cirriformia tentaculata (Montagu, 1808). Eur J Protistol, 2017, 61: 265-277.

[73] SchewiakoffW. Organizacia i sistematika infusoria Aspirotricha (Holotricha auctorum) [The organization and systematics of the infusoria Aspirotricha (Holotricha auctorum)]. Zap Imp Akad Nauk (8e Sér), 1896, 4: 1-395

[74] SchmelzRM, ErséusC, MartinP, van HaarenT, TimmT. A proposed order-level classification in Oligochaeta (Annelida, Clitellata). Zootaxa, 2021, 5040: 589-597.

[75] SchusterF. Vývoj opalin. Spisů Poctěných Jubilejní Cenou Královské České Společnosti Náuk v Praze, 1912, 21: 1-62

[76] ShimodairaH. An approximately unbiased test of phylogenetic tree selection. Syst Biol, 2002, 51: 492-508.

[77] ShimodairaH, HasegawaM. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol, 1999, 16: 1114-1116.

[78] SteinF. Über die mir bisher bekannt gewordenen und von mir genauer erforschten Infusionsthiere, welche im Inneren anderer Thiere eine parasitische Lebensweise führen. Abh K Böhm Ges Wiss, 1859, 1857–1859: 35-38

[79] SteinF. Über die Eintheilung der holotrichen Infusionsthiere und einige neue Gattungen und Arten aus dieser Ordnung. Sber K Böhm Ges Wiss Prag, 1860, 1860: 56-62

[80] SteinF. Über ein neues parasitisches Infusionsthier aus dem Darmkanal von Paludinen und über die mit demselben zunächst verwandten Infusorienformen. Sber K Böhm Ges Wiss Prag, 1861, 1861: 85-90

[81] TimmT. A guide to the freshwater Oligochaeta and Polychaeta of Northern and Central Europe. Lauterbornia, 2009, 66: 1-235

[82] TrifinopoulosJ, NguyenLT, von HaeselerA, MinhBQ. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res, 2016, 44: W232-W235.

[83] WarpachowskyN. Eine neue Form von Opalina. Bull Acad Imp Sci St Pétersbourg, 1886, 30: 512-514

[84] WilbertN. Eine verbesserte Technik der Protargolimprägnation für Ciliaten. Mikrokosmos, 1975, 64: 171-179

[85] ZhangT, VďačnýP. Multiple independent losses of cell mouth in phylogenetically distant endosymbiotic lineages of oligohymenophorean ciliates: a lesson from Clausilocola. Mol Phylogenet Evol, 2022, 166. e107310

[86] ZhangT, VďačnýP. Deciphering phylogenetic relationships of and delimiting species boundaries within the controversial ciliate genus Conchophthirus using an integrative morpho-evo approach. Mol Phylogenet Evol, 2024, 190. e107931

Funding

Comenius University in Bratislava

RIGHTS & PERMISSIONS

The Author(s)