A novel mitochondrion-related organelle and complex endosymbiosis in a rare anaerobic single-celled predator

A novel mitochondrion-related organelle and complex endosymbiosis in a rare anaerobic single-celled predator | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A novel mitochondrion-related organelle and complex endosymbiosis in a rare anaerobic single-celled predator Ivan Čepička, Ondřej Pomahač, Daniel Méndez-Sánchez, Yong Heng Phua, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8743768/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Anaerobic single-celled eukaryotes illustrate the remarkable diversity of adaptations to life without oxygen, including multiple mitochondrial reductions and various symbiotic interactions with prokaryotes. However, rare lineages remain virtually unexplored. From 11 collected cells, we conducted an in-depth investigation of the ciliate Legendrea loyezae , a rare predator of microscopic animals. Using single-cell transcriptomics and metagenomics, we generated a high-completeness host transcriptome and macronuclear genome and characterized a novel type of mitochondrion-related organelle. By integrating omics data with microscopy and 3D imaging, we uncover an exceptionally complex endosymbiotic consortium with two methanogenic archaea and two bacteria, including the first strictly anaerobic member of Legionellales , and the most reduced member of Thermodesulfobacteriota , with a 0.5-Mb genome. We infer the metabolic potential and extensive complementarity within this pentapartite system and align it with spatial relationships. Our results expand the known complexity of anaerobic eukaryotes and demonstrate the feasibility of comprehensive analyses of rare protists. Biological sciences/Microbiology/Microbial communities/Metagenomics Biological sciences/Cell biology/Cellular imaging Biological sciences/Microbiology/Microbial communities/Symbiosis Biological sciences/Cell biology/Organelles/Mitochondria Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterials.pdf Figures S1-S4, and Table S5 TableS1.Symbiontsspatialstatisticsanalysis.xlsx Related Manuscript File TableS2Contaminantsinmetagenomicandtranscriptomicdata.xlsx Related Manuscript File TableS3.AnnotatedputativeMethanobacteriumsp.proteinsinvolvedinmethanogenesis.xlsx Related Manuscript File TableS4.LegendreaLoyezeaeMROtargetingpredictiontables.xlsx Related Manuscript File MovieS1.3DmodelofLegendrealoyezaeandtheirprokaryoticsymbionts.avi Movie S1. 3D model of Legendrea loyezae and their prokaryotic symbionts FigureS5MitochondrialrelatedorganelleMROsingleproteintreescollection.pdf Fig. S5. Mitochondrial-related organelle (MRO) single protein trees collection Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8743768","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":610189238,"identity":"7e8f1606-1a7e-413f-8740-027dd5647a3c","order_by":0,"name":"Ivan 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