Distinct evolutionary origins and mixed-mode transmissions of methanogenic endosymbionts are revealed in anaerobic ciliated protists

Abstract

Methanogenic endosymbionts are the only known intracellular archaeans and are especially common in anaerobic ciliated protists. Studies on the evolution of associations between anaerobic ciliates and their methanogenic endosymbionts offer an excellent opportunity to broaden our knowledge about symbiosis theory and adaptation of eukaryotes to anoxic environments. Here, the diversity of methanogenic endosymbionts was analyzed with the addition of nine anaerobic ciliate populations that were newly studied by various methods. Results showed that diverse anaerobic ciliates host methanogenic endosymbionts that are limited to a few genera in orders Methanomicrobiales, Methanobacteriales, and Methanosarcinales. For the first time, anaerobic ciliates of the classes Muranotrichea and Prostomatea were found to host methanogenic endosymbionts. Distinct origins of endosymbiosis were revealed for classes Armophorea and Plagiopylea. We posit that armophoreans and plagiopyleans might have harbored Methanoregula (order Methanomicrobiales) and Methanocorpusculum (order Methanomicrobiales), respectively, as methanogenic endosymbionts at the beginning of their evolution. Subsequently, independent endosymbiont replacement events occurred in methanogen-ciliate associations, probably due to ecological transitions, species radiation of ciliate hosts, and vertical transmission bottlenecks of endosymbionts. Our results shed light on the evolution of associations between anaerobic ciliates and methanogens, and identifies the necessary preconditions for illustrating mechanisms by which endosymbioses between these partners were established.

Keywords

Anaerobic ciliates / Endosymbiont replacement / Evolution of associations / Methanogenic endosymbionts

References

[1] Akinyemi TS, Shao N, Whitman WB. Trujillo ME, Dedysh S, DeVos P, Hedlund B, Kämpfer P, Rainey FA, Whitman WB. Methanotrichales ord. nov.. Bergey’s manual of systematics of archaea and bacteria. 2025, New York, John Wiley & Sons: 12

[2] Beinart RA, Rotterová J, Čepička I, Gast RJ, Edgcomb VP. The genome of an endosymbiotic methanogen is very similar to those of its free-living relatives. Environ Microbiol. 2018, 20: 2538-2551

[3] Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019, 37: 852-857

[4] Boscaro V, Kolisko M, Felletti M, Vannini C, Lynn D, Keeling PJ. Parallel genome reduction in symbionts descended from closely related free-living bacteria. Nat Ecol Evol. 2017, 1: 1160-1167

[5] Boscaro V, Fokin S, Petroni G, Verni F, Keeling PJ, Vannini C. Symbiont replacement between bacteria of different classes reveals additional layers of complexity in the evolution of symbiosis in the ciliate Euplotes. Protist. 2018, 169: 43-52

[6] Boscaro V, Husník F, Vannini C, Keeling PJ. Symbionts of the ciliate Euplotes: diversity, patterns and potential as models for bacteria–eukaryote endosymbioses. Proc Roy Soc B-Biol Sci. 2019, 286: 20190693

[7] Bright M, Bulgheresi S. A complex journey: transmission of microbial symbionts. Nat Rev Microbiol. 2010, 8: 218-230

[8] Callahan BJ, McMurdie PJ, Rosen MJ. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016, 13: 581-583

[9] Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, et al. . QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010, 7: 335-336

[10] Chen Z, Li J, Salas-Leiva DE, Chen M, Chen S, Li S, Wu Y, Yi Z. Group-specific functional patterns of mitochondrion-related organelles shed light on their multiple transitions from mitochondria in ciliated protists. Mar Life Sci Technol. 2022, 4: 609-623

[11] Clarke KJ, Finlay BJ, Esteban G, Guhl BE, Embley TM. Cyclidium porcatum n. sp.: a free-living anaerobic scuticociliate containing a stable complex of hydrogenosomes, eubacteria and archaeobacteria. Eur J Protistol. 1993, 29: 262-270

[12] Coolen MJ, Hopmans EC, Rijpstra WI, Muyzer G, Schouten S, Volkman JK, Damste JSS. Evolution of the methane cycle in Ace Lake (Antarctica) during the Holocene: response of methanogens and methanotrophs to environmental change. Org Geochem. 2004, 35: 1151-1167

[13] Doddema HJ, Vogels GD. Improved identification of methanogenic bacteria by fluorescence microscopy. Appl Environ Microbiol. 1978, 36: 752-754

[14] Dziallas C, Allgaier M, Monaghan MT, Grossart HP. Act together-implications of symbioses in aquatic ciliates. Front Microbiol. 2012, 3: 288

[15] Edgcomb VP, Leadbetter ER, Bourland W, Beaudoin D, Bernhard JM. Structured multiple endosymbiosis of bacteria and archaea in a ciliate from marine sulfidic sediments: a survival mechanism in low oxygen, sulfidic sediments. Front Microbiol. 2011, 2: 55

[16] Embley TM, Finlay BJ. The use of small subunit rRNA sequences to unravel the relationships between anaerobic ciliates and their methanogen endosymbionts. Microbiology. 1994, 140: 225-235

[17] Embley TM, Finlay BJ, Brown S. RNA sequence analysis shows that the symbionts in the ciliate Metopus contortus are polymorphs of a single methanogen species. FEMS Microbiol Lett. 1992, 97: 57-61

[18] Embley TM, Finlay BJ, Thomas RH, Dyal PL. The use of rRNA sequences and fluorescent probes to investigate the phylogenetic positions of the anaerobic ciliate Metopus palaeformis and its archaeobacterial endosymbiont. Microbiology. 1992, 138: 1479-1487

[19] Fenchel T, Finlay BJ. Synchronous division of an endosymbiotic methanogenic bacterium in the anaerobic ciliate Plagiopyla frontata Kahl. J Protozool. 1991, 38: 22-28

[20] Fenchel T, Finlay BJ. Production of methane and hydrogen by anaerobic ciliates containing symbiotic methanogens. Arch Microbiol. 1992, 157: 475-480

[21] Fenchel T, Finlay BJ. Free-living protozoa with endosymbiotic methanogens. (Endo)symbiotic methanogens. 2010, New York, Springer: 111

[22] Finlay BJ, Fenchel T. An anaerobic ciliate as a natural chemostat for the growth of endosymbiotic methanogens. Eur J Protistol. 1992, 28: 127-137

[23] Finlay BJ, Embley TM, Fenchel T. A new polymorphic methanogen, closely related to Methanocorpusculum parvum, living in stable symbiosis within the anaerobic ciliate Trimyema sp. Microbiology. 1993, 139: 371-378

[24] Gijzen HJ, Broers CA, Barughare M, Stumm CK. Methanogenic bacteria as endosymbionts of the ciliate Nyctotherus ovalis in the cockroach hindgut. Appl Environ Microbiol. 1991, 57: 1630-1634

[25] Goosen NK, Wagener S, Stumm CK. A comparison of two strains of the anaerobic ciliate Trimyema compressum. Arch Microbiol. 1990, 153: 187-192

[26] Gouy M, Guindon S, Gascuel O. SeaView Version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol. 2010, 27: 221-224

[27] Guo H, Yu Z, Liu R, Zhang H, Zhong Q, Xiong Z. Methylotrophic methanogenesis governs the biogenic coal bed methane formation in Eastern Ordos Basin, China. Appl Microbiol Biotechnol. 2012, 96: 1587-1597

[28] Hackstein JH. (Endo)symbiotic methanogenic archaea. 2018, New York, Springer19

[29] Hackstein JH, Vogels GD. Endosymbiotic interactions in anaerobic protozoa. Anton Leeuw Int J G. 1997, 71: 151-158

[30] Hirakata Y, Oshiki M, Kuroda K, Hatamoto M, Kubota K, Yamaguchi T, Harada H, Araki N. Identification and detection of prokaryotic symbionts in the ciliate Metopus from anaerobic granular sludge. Microbes Environ. 2015, 30: 335-338

[31] Husnik F, Tashyreva D, Boscaro V, George EE, Lukeš J, Keeling PJ. Bacterial and archaeal symbioses with protists. Curr Biol. 2021, 31: 862-877

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

[33] Jin D, Li C, Chen X, Byerly A, Stover NA, Zhang T, Shao C, Wang Y. Comparative genome analysis of three euplotid protists provides insights into the evolution of nanochromosomes in unicellular eukaryotic organisms. Mar Life Sci Technol. 2023, 5: 300-315

[34] Jones J, Nagle DPJr, Whitman WB. Methanogens and the diversity of archaebacteria. FEMS Microbiol Rev. 1987, 51: 135-177

[35] Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013, 30: 772-780

[36] Kim KH, Kim JM, Baek JH, Jeong SE, Kim H, Yoon HS, Jeon CO. Metabolic relationships between marine red algae and algae-associated bacteria. Mar Life Sci Technol. 2024, 6: 298-314

[37] Letunic I, Bork P. Interactive tree of life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019, 47: 256-259

[38] Lewis WH, Sendra KM, Embley TM, Esteban GF. Morphology and phylogeny of a new species of anaerobic ciliate, Trimyema finlayi n. sp., with endosymbiotic methanogens. Front Microbiol. 2018, 9: 140

[39] Lewis WH, Lind AE, Sendra KM, Onsbring H, Williams TA, Esteban GF, Hirt RP, Ettema TJG, Embley TM. Convergent evolution of hydrogenosomes from mitochondria by gene transfer and loss. Mol Biol Evol. 2019, 37: 524-539

[40] 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

[41] 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

[42] Lind AE, Lewis WH, Spang A, Guy L, Embley TM, Ettema TJ. Genomes of two archaeal endosymbionts show convergent adaptations to an intracellular lifestyle. ISME J. 2018, 12: 2655-2667

[43] López-García P, Philippe H, Gail F, Moreira D. Autochthonous eukaryotic diversity in hydrothermal sediment and experimental microcolonizers at the Mid-Atlantic Ridge. Proc Natl Acad Sci. 2003, 100: 697-702

[44] Lu B, Hu X, Warren A, Song W, Yan Y. From oral structure to molecular evidence: new insights into the evolutionary phylogeny of the ciliate order Sessilida (Protista, Ciliophora), with the establishment of two new families and new contributions to the poorly studied family Vaginicolidae. Sci China Life Sci. 2023, 66: 1535-1553

[45] Lyu Z, Shao N, Akinyemi T, Whitman WB. Methanogenesis. Curr Biol. 2018, 28: 727-732

[46] Mayumi D, Mochimaru H, Tamaki H, Yamamoto K, Yoshioka H, Suzuki Y, Kamagata Y, Sakata S. Methane production from coal by a single methanogen. Science. 2016, 354: 222-225

[47] Medlin L, Elwood HJ, Stickel S, Sogin ML. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene. 1988, 71: 491-499

[48] Méndez-Sánchez D, Schrecengost A, Rotterová J, Koštířová K, Beinart RA, Čepička I. Methanogenic symbionts of anaerobic ciliates are host and habitat specific. ISME J. 2024, 18: wrae164

[49] Narayanan N, Priya M, Haridas A, Manilal VB. Isolation and culturing of a most common anaerobic ciliate, Metopus sp. Anaerobe. 2007, 13: 14-20

[50] Narayanan N, Krishnakumar B, Anupama VN, Manilal VB. Methanosaeta sp., the major archaeal endosymbiont of Metopus es. Res Microbiol. 2009, 160: 600-607

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

[52] Nitla V, Serra V, Fokin SI, Modeo L, Verni F, Sandeep BV, Kalavati C, Petroni G. Critical revision of the family Plagiopylidae (Ciliophora: Plagiopylea), including the description of two novel species, Plagiopyla ramani and Plagiopyla narasimhamurtii, and redescription of Plagiopyla nasuta Stein, 1860 from India. Zool J Linn Soc. 2019, 186: 1-45

[53] Nowack EC, Melkonian M. Endosymbiotic associations within protists. Phil Trans R Soc B. 2010, 365: 699-712

[54] Nylander J (2004) MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden

[55] Omar A, Zhang Q, Zou S, Gong J. Morphology and phylogeny of the soil ciliate Metopus yantaiensis n. sp. (Ciliophora, Metopida), with identification of the intracellular bacteria. J Eukaryot Microbiol. 2017, 64: 792-805

[56] Øvreås L, Forney L, Daae FL, Torsvik V. Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl Environ Microbiol. 1997, 63: 3367-3373

[57] Pomahač O, Méndez-Sánchez D, Poláková K, Müller M, Solito MM, Bourland WA, Čepička I. Rediscovery of remarkably rare anaerobic tentaculiferous ciliate genera Legendrea and Dactylochlamys (Ciliophora: Litostomatea). Biology. 2023, 12: 707

[58] Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013, 41: 590-596

[59] Rognes T, Flouri T, Nichols B, Quince C, Mahé F. VSEARCH: a versatile open source tool for metagenomics. PeerJ. 2016, 4: e2584

[60] Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, 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

[61] Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F. The prokaryotes: other major lineages of bacteria and the archaea. 2014, New York, Springer

[62] Rotterová J, Salomaki E, Pánek T, Bourland W, 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

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

[64] Schrecengost A, Rotterová J, Poláková K, Čepička I, Beinart RA. Divergent marine anaerobic ciliates harbor closely related Methanocorpusculum endosymbionts. ISME J. 2024, 18: wrae125

[65] Shinzato N, Watanabe I, Meng X, Sekiguchi Y, Tamaki H, Matsui T, Kamagata Y. Phylogenetic analysis and fluorescence in situ hybridization detection of archaeal and bacterial endosymbionts in the anaerobic ciliate Trimyema compressum. Microb Ecol. 2007, 54: 627-636

[66] Skillman LC, Evans PN, Naylor GE, Morvan B, Jarvis GN, Joblin KN. 16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs. Anaerobe. 2004, 10: 277-285

[67] Su H, Xu J, Li J, Yi Z. Four ciliate-specific expansion events occurred during actin gene family evolution of eukaryotes. Mol Phylogenet Evol. 2023, 184: 107789

[68] Su H, Hao T, Yu M, Zhou W, Wu L, Sheng Y, Yi Z. Complex evolutionary patterns within the tubulin gene family of ciliates, unicellular eukaryotes with diverse microtubular structures. BMC Biol. 2024, 22: 170

[69] Takeshita K, Yamada T, Kawahara Y, Narihiro T, Ito M, Kamagata Y, Shinzato N. Tripartite symbiosis of an anaerobic scuticociliate with two hydrogenosome-associated endosymbionts, a Holospora-related alphaproteobacterium and a methanogenic archaeon. Appl Environ Microbiol. 2019, 85: e00854-e919

[70] Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021, 38: 3022-3027

[71] Tang D, Liu Y, Wang C, Li L, Al-Farraj SA, Chen X, Yan Y. Invasion by exogenous RNA: cellular defense strategies and implications for RNA inference. Mar Life Sci Technol. 2023, 5: 573-584

[72] Van Bruggen JJA, Claudius KS, Vogels GD. Symbiosis of methanogenic bacteria and sapropelic protozoa. Arch Microbiol. 1983, 136: 89-95

[73] Van Bruggen JJA, Zwart KB, van Assema RM, Stumm CK, Vogels GG. Methanobacterium formicicum, an endosymbiont of the anaerobic ciliate Metopus striatus McMurrich. Arch Microbiol. 1984, 139: 1-7

[74] Van Bruggen JJA, Zwart KB, Hermans JGF, van Hove EM, Stumm CK, Vogels GD. Isolation and characterization of Methanoplanus endosymbiosus sp. nov., an endosymbiont of the marine sapropelic ciliate Metopus contortus Quennerstedt. Arch Microbiol. 1986, 144: 367-374

[75] Van Hoek AH, van Alen TA, Sprakel VS, Leunissen JA, Brigge T, Vogels GD, Hackstein JHP. Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates. Mol Biol Evol. 2000, 17: 251-258

[76] Xu J, Shen Z, Hao T, Su H, Chen M, Pan X, Yi Z. Exploring the evolution of anaerobes within ciliate class Prostomatea by phylogenomic analyses and metabolisms of mitochondrion-related organelles. Mol Phylogenet Evol. 2025, 207 108345

[77] Zhang C, Pan J, Liu Y, Duan C, Li M. Genomic and transcriptomic insights into methanogenesis potential of novel methanogens from mangrove sediments. Microbiome. 2020, 8: 1-12

[78] Zhou Z, Zhang C, Liu P, Fu L, Laso-Pérez R, Yang L, Bai L, Li J, Yang M, Lin J, Wang W. Non-syntrophic methanogenic hydrocarbon degradation by an archaeal species. Nature. 2022, 601: 257-262

[79] 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

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