Investigations on the type species of two anaerobic ciliate genera, Metopus Claparède & Lachmann, 1858 and Brachonella Jankowski, 1964 (Alveolata, Ciliophora, Armophorea), focusing on their morphogenesis and molecular phylogeny

Abstract

Ciliates in the order Metopida exhibit a global distribution and play essential roles as consumers of and common hosts to prokaryotes in both hypoxic and anoxic environments. Based on detailed morphological and morphometric data of Metopus es and Brachonella contorta, type species of two common and relatively species-rich metopid genera, we investigate the morphogenesis, and molecular phylogeny of each, and analyze the secondary structure of the V9 region of their 18S rRNA gene sequences. The new findings include: (1) These two species present two different stomatogenetic modes, which are stable within their respective genera and each can be regarded as a reliable generic feature for differentiation. (2) Both species share the same outcome regarding the parental oral apparatus, i.e., the old paroral membrane and adoral zone of membranelles are entirely reorganized in situ in the proter. (3) The paroral membrane is diplostichomonadal in the examined isolates, a feature which has long been overlooked and may be enigmatically present in other previously described populations. As concerns their phylogeny, the clear delineation of each species is supported by the high conservation of SSU rRNA gene sequences, and the close phylogenetic clustering, of different populations of each species from geographically distant localities. In agreement with previous phylogenetic studies, the MeBr clade (Metopus, including M. es and marine/brackish Metopus members, and all Brachonella species) is recovered repeatedly with moderate to high support.

Keywords

Anaerobe / Metopida / Morphology / Ontogenesis / Protozoa / Systematics

References

[1] Alekperov IK. New species of freshwater ciliates (Heterotrichida) from artificial water bodies of Azerbaijan. Zool Zh, 1984, 63: 1731-1734

[2] Bourland WA, Wendell L. Redescription of Atopospira galeata (Kahl, 1927) nov. comb. and A. violacea (Kahl, 1926) nov. comb. with redefinition of Atopospira Jankowski, 1964 nov. stat. and Brachonella Jankowski, 1964 (Ciliophora, Armophorida). Eur J Protistol, 2014, 50: 356-372.

[3] Bourland WA, Rotterová J, Čepička I. Morphologic and molecular characterization of seven species of the remarkably diverse and widely distributed metopid genus Urostomides Jankowski, 1964 (Armophorea, Ciliophora). Eur J Protistol, 2017, 61: 194-232.

[4] Bourland WA, Rotterová J, Cepicka I. Redescription and molecular phylogeny of the type species for two main metopid genera, Metopus es (Müller, 1776) Lauterborn, 1916 and Brachonella contorta (Levander, 1894) Jankowski, 1964 (Metopida, Ciliophora), based on broad geographic sampling. Eur J Protistol, 2017, 59: 133-154.

[5] Bourland WA, Rotterová J, Čepička I. Morphologic and molecular characterization of Brachonella pulchra (Kahl, 1927) comb. nov. (Armophorea, Ciliophora) with comments on cyst structure and formation. Int J Syst Evol Microbiol, 2018, 68: 3052-3065.

[6] Bourland WA, Rotterová J, Luo X, Čepička I. The little-known freshwater metopid ciliate, Idiometopus turbo (Dragesco and Dragesco-Kernéis, 1986) nov. gen., nov. comb., originally discovered in Africa, found on the Micronesian Island of Guam. Protist, 2018, 169: 494-506.

[7] Bourland WA, Rotterová J, Čepička I. Description of three new genera of Metopidae (Metopida, Ciliophora): Pileometopus gen. nov., Castula gen. nov., and Longitaenia gen. nov., with notes on the phylogeny and cryptic diversity of metopid ciliates. Protist, 2020, 171: 125740.

[8] Bourland WA, Pomahač O, Čepička I. Morphology and phylogeny of two anaerobic freshwater ciliates: Brachonella comma sp. nov. and the widely distributed but little-known caenomorphid, Ludio parvulus Penard, 1922. J Eukaryot Microbiol, 2022, 69. e12892

[9] Bourland WA, Pomahač O, Čepička I. Redescription and molecular phylogeny of the freshwater metopid, Castula strelkowi (Jankowski, 1964) from the Czech Republic and synonymization of Pileometopus with Castula. Protist, 2024, 175. 126034

[10] Calkins GN. The protozoan life cycle. Biol Bull, Woods Hole, 1906, 11: 229-244.

[11] Corliss JO. The value of ontogenetic data in reconstructing protozoan phylogenies. Trans Am Microsc Soc, 1968, 87: 1-20.

[12] Darty K, Denise A, Ponty Y. VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics, 2009, 25: 1974-1975.

[13] Dragesco J. Ciliés mésopsammiques littoraux: Systématique, morphologie, écologie. Trav Stn Biol Roscoff, 1960, 12: 1-356

[14] Esteban GF, Fenchel T, Finlay BJ. Diversity of free-living morphospecies in the ciliate genus Metopus. Arch Protistenkd, 1995, 146: 137-164.

[15] Esteban GF, Fenchel T (2020) The biology of free-living phagotrophic protists. In: Cham (Ed) Ecology of protozoa, 2nd edn. Springer, Nature, Switzerland, p. 186

[16] Feng X, Méndez-Sánchez D, Zhuang W, Li R, Pomahač O, Čepička I, Rotterová J, Hu X. Morphology, morphogenesis, and molecular characterization of Castula specialis sp. nov. (Ciliophora, Armophorea, Metopida). J Eukaryot Microbiol, 2024, 71. e13014

[17] Feng X, Zhuang W, Li R, Hu X. Ontogeny and phylogeny of the anaerobic genus Urostomides with supplementing morphological characterization of the type and one little-known species (Alveolata: Ciliophora: Armophorea). Zool J Linn Soc, 2025, 203: zlae055.

[18] Fernandes NM, Vizzoni VF, Borges BDN, Soares CAG, da Silva-Neto ID, Paiva TDS. Molecular phylogeny and comparative morphology indicate that odontostomatids (Alveolata, Ciliophora) form a distinct class-level taxon related to Armophorea. Mol Phylogenet Evol, 2018, 126: 382-389.

[19] Foissner W. Taxonomische Studien über die Ciliaten des Grossglocknergebietes IX. Ordnungen Heterotrichida Und Hypotrichida. Ber Nat-Med Ver Salzbg, 1980, 5: 71-117

[20] Foissner W, Agatha S. Morphology and morphogenesis of Metopus hasei Sondheim, 1929 and M. inversus (Jankowski, 1964) nov. comb. (Ciliophora, Metopida). J Eukaryot Microbiol, 1999, 46: 174-193.

[21] Foissner W, Agatha S, Berger H. Soil ciliates (Protozoa, Ciliophora) from Namibia (Southwest Africa), with emphasis on two contrasting environments, the Etosha region and the Namib Desert. Denisia, 2002, 5: 1-1459

[22] Foissner W (1996) Ontogenesis in ciliated protozoa, with emphasis on stomatogenesis. In: Hausmann K, Bradbury BC (Eds) Ciliates: cells as organisms, 1st edn. Fischer Verlag, Stuttgart, Jena, New York, pp 95–177

[23] Frankel J (1989) Pattern formation. Ciliate studies and models. Oxford University Press, New York, Oxford

[24] Gao F, Katz LA, Song W. Insights into the phylogenetic and taxonomy of philasterid ciliates (Protozoa, Ciliophora, Scuticociliatia) based on analyses of multiple molecular markers. Mol Phylogenet Evol, 2012, 64: 308-317.

[25] Gao F, Warren A, Zhang Q, Gong J, Miao M, Sun P, Xu D, Huang J, Yi Z, Song W. The all-data-based evolutionary hypothesis of ciliated protists with a revised classification of the phylum Ciliophora (Eukaryota, Alveolata). Sci Rep, 2016, 6: 24874.

[26] Hall TA. 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] Hu X. Ciliates in extreme environments. J Eukaryot Microbiol, 2014, 61: 410-418.

[28] Hu X, Lin X, Song WCiliate atlas: species found in the South China Sea, 2019BeijingScience Press

[29] Jankowski AW. Morphology and evolution of Ciliophora I. The new system of sapropelobiotic Heterotrichida. Zool Zh, 1964, 43: 503-517

[30] Jankowski AW. Morphology and evolution of Ciliophora. III. Diagnoses and phylogenesis of 53 sapropelebionts, mainly of the oder Heterotrichida. Arch Protistenkd, 1964, 107: 185-330

[31] Jiang L, Wang C, Al-Farraj SA, Hines HN, Hu X. Morphological and molecular examination of the ciliate family Lagynusidae (Protista, Ciliophora, Prostomatea) and descriptions of two new genera and two new species from China. Mar Life Sci Technol, 2023, 5: 178-195.

[32] Jiang L, Wang C, Liao L, Al-Farraj SA, Warren A, Hu X. Re-evaluation of the systematic position of the order Prostomatida (Protista: Ciliophora), with the establishment of two new genera and two new species. Zool J Linn Soc, 2024, 200: 849-864.

[33] Kahl A. Neue und ergänzende Beobachtungen heterotricher Ciliaten. Arch Protistenkd, 1927, 57: 121-203

[34] Kahl A. Urtiere oder Protozoa. I: Wimpertiere oder Ciliata (Infusoria). 3. Spirotricha Tierwelt Dtl, 1932, 25: 399-650

[35] Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform, 2019, 20: 1160-1166.

[36] Li M, Hu G, Li C, Zhao W, Zou H, Li W, Wu S, Wang G, Ponce-Gordo F. Morphological and molecular characterization of a new ciliate Nyctotheroides grimi n. sp. (Armophorea, Clevelandellida) from Chinese frogs. Acta Trop, 2020, 208. e105531

[37] Li S, Zhuang W, Pérez-Uz B, Zhang Q, Hu X. Two anaerobic ciliates (Ciliophora, Armophorea) from China: morphology and SSU rDNA sequence, with report of a new species, Metopus paravestitus nov. spec. J Eukaryot Microbiol, 2021, 68. e12822

[38] Li R, Zhuang W, Feng X, Al-Farraj SA, Warren A, Hu X. Phylogeny of the anaerobic ciliate genus Sonderia (Protista: Ciliophora: Plagiopylea), including the description of three novel species and a brief revision of the genus. Mar Life Sci Technol, 2022, 4: 493-512.

[39] Li J, Li S, Su H, Yu M, Xu J, Yi Z. Comprehensive phylogenomic analyses reveal that order Armophorida is most closely related to class Armophorea (Protista, Ciliophora). Mol Phylogenet Evol, 2023, 182. 107737

[40] Li R, Zhuang W, Feng X, Al-Farraj SA, Schrecengost A, Rotterová J, Beinart RA, Hu X. Molecular phylogeny and taxonomy of three anaerobic plagiopyleans (Alveolata: Ciliophora), retrieved from two geographically distant localities in Asia and North America. Zool J Linn Soc, 2023, 199: 493-510.

[41] Li R, Zhuang W, Feng X, Hines H, Hu X. First redescription and molecular phylogeny of Trimyema claviforme Kahl, 1933 with the description of a Chinese population of Plagiopyla nasuta Stein, 1860 (Ciliophora, Plagiopylea). Eur J Protistol, 2023, 90. 126003

[42] Li R, Zhuang W, Feng X, Zhu X, Hu X. Morphology and molecular phylogeny of three Parasonderia species including a new species (Ciliophora, Plagiopylea). Eur J Protistol, 2024, 94. 126087

[43] Liao L, Jiang L, Hu X. Integrative data of a novel ciliate (Alveolata, Ciliophora) propose the establishment of Heterodeviata nantongensis nov. sp. BMC Microbiol, 2024, 24: 27.

[44] Liu M, Liu Y, Zhang T, Lu B, Gao F, Gu J, Al-Farraj SA, Hu X, Song W. Integrative studies on the taxonomy and molecular phylogeny of four new Pleuronema species (Protozoa, Ciliophora, Scuticociliatia). Mar Life Sci Technol, 2022, 4: 179-200.

[45] Lynn DHThe ciliated protozoa: characterization, classification and guide to the literature, 20083DordrechtSpringer

[46] Méndez-Sánchez D, Pomahač O, Rotterová J, Bourland WA, Čepička I. Diversity and phylogenetic position of Bothrostoma Stokes, 1887 (Ciliophora: Metopida), with description of four new species. Protist, 2022, 173. 125887

[47] Méndez-Sánchez D, Pomahač O, Rotterová J, Bourland WA, Čepička I. Morphology and phylogenetic position of three anaerobic ciliates from the classes Odontostomatea and Muranotrichea (Ciliophora). J Eukaryot Microbiol, 2023, 70. e12965

[48] Müller OF (1776) Zoologicae Danicae, prodromus, seu animalium Daniae et Norvegiae indigenarum, characteres, nomina, et synonyma imprimis popularium. Havniae.

[49] Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol, 2015, 32: 268-274.

[50] Noland LE. Conjugation in the ciliate Metopus sigmoides C. and L. J Morphol, 1927, 44: 341-361.

[51] Orsi W, Edgcomb VP, Faria J, Foissner W, Fowle WH, Hohmann T, Suarez P, Taylor C, Taylor GT, Vd’ačný P, Epstein SS. Class Cariacotrichea, a novel ciliate taxon from the anoxic Cariaco Basin, Venezuela. Int J Syst Evol Microbiol, 2012, 62: 1425-1433.

[52] Pan X, Bourland WA, Song W. Protargol synthesis: an in-house protocol. J Eukaryot Microbiol, 2013, 60: 609-614.

[53] Pecina L, Vďačný P. 2D approach to reconstruct the evolutionary history of clevelandellids (Ciliophora, Armophorea) inhabiting the hindgut of the Panesthiinae cockroaches. Zool Scr, 2023, 52: 662-687.

[54] Pomahač O, Méndez-Sánchez D, Čepička I. Bit by bit toward the diversity of metopids: description of the genus Pidimetopus n. gen. (Ciliophora: Armophorea). J Eukaryot Microbiol, 2024, 71: e13034.

[55] Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol, 2012, 61: 539-542.

[56] Rotterová J, Bourland WA, Čepička I. Tropidoatractidae fam. nov., a deep branching lineage of Metopida (Armophorea, Ciliophora) found in diverse habitats and possessing prokaryotic symbionts. Protist, 2018, 169: 362-405.

[57] Rotterová J, Salomaki E, Pánek T, Bourland WA, Žihala D, Táborský P, Edgcomb VP, Beinart RA, Kolísko M, Čepička I. Genomics of new ciliate lineages provides insight into the evolution of obligate anaerobiosis. Curr Biol, 2020, 30: 2037-2050.e6.

[58] Rotterová J, Edgcomb VP, Čepička I, Beinart R. Anaerobic ciliates as a model group for studying symbioses in oxygen-depleted environments. J Eukaryot Microbiol, 2022, 69. e12912

[59] Song W. On cell development of ciliated protozoa: diversity and progress. J Ocean Univ China, 2004, 5: 72-82(in Chinese)

[60] Song W, Warren A, Hu XFree-living ciliates in the Bohai and Yellow Seas, 2009BeijingScience Press

[61] Song W, Zhang S, Li Y, Ma H, Li Q, Luo X, Al-Rasheid KAS, Hines HN, Lu X. Multi-gene-based investigation on the molecular phylogeny of the hypotrichous family Strongylidiidae (Protista, Ciliophora), with notes on the ontogeny of a new genus and new species. Mar Life Sci Technol, 2024, 6: 442-461.

[62] Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol, 2021, 38: 3022-3027.

[63] Vďačný P, Foissner W. A huge diversity of metopids (Ciliophora, Armophorea) in soil from the Murray River floodplain, Australia. II. Morphology and morphogenesis of Lepidometopus platycephalus nov. gen., nov. spec. Acta Protozool, 2017, 56: 39-57

[64] Vďačný P, Foissner W. A huge diversity of metopids (Ciliophora, Armophorea) in soil from the Murray River floodplain, Australia. III. Morphology, ontogenesis and conjugation of Metopus boletus nov. spec., with implications for the phylogeny of the SAL supercluster. Eur J Protistol, 2019, 69: 117-137.

[65] Wang Z, Chi Y, Li T, Song W, Wang Y, Wu T, Zhang G, Liu Y, Ma H, Song W, Al-Rasheid KAS, Warren A, Lu B. Biodiversity of freshwater ciliates (Protista, Ciliophora) in the Lake Weishan wetland, China: the state of the art. Mar Life Sci Technol, 2022, 4: 429-451.

[66] Wilbert N. Eine verbesserte Technik der Protargolimprägnation für Ciliaten. Mikrokosmos, 1975, 64: 171-179

[67] Wu T, Chen T, Jiang Y, Al-Rasheid Khaled AS, Warren A, Wang Z, Lu B. On four epibiotic peritrichous ciliates (Protozoa, Ciliophora) found in the Lake Weishan wetland: morphological and molecular data support the establishment of a new genus, Parapiosoma gen. nov., and two new species. Mar Life Sci Technol, 2023, 5: 337-358.

[68] Zhang G, Sheng Y, Liu Y, Cao X, Al-Farrajn SA, Vďačný P, Pan H. Integrative studies on three freshwater Amphileptus species (Ciliophora, Pleurostomatida) discovered in northern China. Mar Life Sci Technol, 2022, 4: 452-470.

[69] Zhang Y, Liang F, Fu Y, Chi Y, Song W, Warren A, Li L. Comparative genomics provides insights into the evolutionary history of the phylum Ciliophora (Eukaryota, Alveolata) and uncovers the adaptive evolution of anaerobic ciliate classes. Zool Scr, 2025, 54: 270-290.

[70] Zhuang W, Li S, Bai Y, Zhang T, Al-Rasheid KAS, Hu X. Morphology and molecular phylogeny of the anaerobic freshwater ciliate Urostomides spinosus nov. spec. (Ciliophora, Armophorea, Metopida) from China. Eur J Protistol, 2021, 81: 125823.

[71] Zhuang W, Li R, Feng X, Al-Farraj SA, Hu X. New contribution to the diversity of the anaerobic genus Metopus (Ciliophora, Armophorea), with descriptions of three new marine species. Front Mar Sci, 2022, 9. 884834

[72] Zhuang W, Feng X, Li R, Al-Farraj SA, Hu X. Morphogenesis of an anaerobic ciliate Heterometopus palaeformis (Kahl, 1927) Foissner, 2016 (Ciliophora, Armophorea) with notes on its morphological and molecular characterization. Protist, 2024, 175. 126007

[73] Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res, 2003, 31: 3406-3415.

RIGHTS & PERMISSIONS

Ocean University of China

Just Accepted

This article has successfully passed peer review and final editorial review, and will soon enter typesetting, proofreading and other publishing processes. The currently displayed version is the accepted final manuscript. The officially published version will be updated with format, DOI and citation information upon launch. We recommend that you pay attention to subsequent journal notifications and preferentially cite the officially published version. Thank you for your support and cooperation.