Article https://doi.org/10.1038/s41467-024-54102-7 Aunique symbiosome in an anaerobic single- celled eukaryote Jon Jerlström-Hultqvist 1,2 , Lucie Gallot-Lavallée 2, Dayana E. Salas-Leiva 2,3, Bruce A. Curtis2, Kristína Záhonová 4,5,6,7, Ivan Čepička 8, Courtney W. Stairs 9, Shweta Pipaliya4, Joel B. Dacks 4,5,10, John M. Archibald 2 & Andrew J. Roger 2 Symbiotic relationships between eukaryotes and prokaryotes played pivotal roles in the evolution of life and drove the emergence of specialized symbiotic structures in animals, plants and fungi. The host-evolved symbiotic structures of microbial eukaryotes – the vast majority of such hosts in nature – remain largely unstudied. Here we describe highly structured symbiosomes within three free-living anaerobic protists (Anaeramoeba spp.). We dissect this sym- biosis using complete genome sequencing and transcriptomics of host and symbiont cells coupled with fluorescence in situ hybridization, and 3D reconstruction using focused-ion-beam scanning electron microscopy. The emergence of the symbiosome is underpinned by expansion of gene families encoding regulators ofmembrane trafficking and phagosomalmaturation and extensive bacteria-to-eukaryote lateral transfer. The symbionts reside deep within a symbiosomal membrane network that enables metabolic syntrophy by precisely positioning sulfate-reducing bacteria alongside host hydrogeno- somes. Importantly, the symbionts maintain connections to the Anaeramoeba plasma membrane, blurring traditional boundaries between ecto- and endosymbiosis. Symbioses between eukaryotes and prokaryotes are important sour- ces of novelty and drivers of evolutionary change. In a variety of multicellular eukaryote lineages, host body plans and physiologies have been adapted to support such interactions including the evolu- tion of specialized symbiotic “organs”1. If the symbionts are intracel- lular, such structures are often referred to as “symbiosomes” that have been defined as “membrane-bound compartment[s] housing one or more symbionts … located in the cytoplasm of eukaryotic cells”2. Symbiosomes have been described in a variety of symbiotic contexts such as in the root-nodules of plants1, aphid bacteriocytes1 and in protists2. Although many diverse unicellular eukaryote (protist) linea- ges are also known to host prokaryotic partners, the host-cell adap- tations to support such symbiotic interactions are generally poorly understood. In oxygen-poor environments, protists are sometimes found in syntrophic partnerships with ecto- or endosymbiotic prokaryotes3. Received: 7 November 2023 Accepted: 31 October 2024 Check for updates 1Department of Cell and Molecular Biology, Uppsala Universitet, Uppsala, Sweden. 2Institute for Comparative Genomics, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada. 3Department of Biochemistry, University of Cambridge, Cambridge, UK. 4Division of Infectious Diseases, Department of Medicine, Faculty of Medicine and Dentistry, and Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada. 5Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice (Budweis), Czechia. 6Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Vestec, Czechia. 7Life Science Research Centre, Department of Biology and Ecology, Faculty of Science, University of Ostrava, Ostrava, Czechia. 8Department of Zoology, Faculty of Science, Charles University, Prague, Czechia. 9Department of Biology, Lund University, Lund, Sweden. 10Centre for Life’s Origin and Evolution, Department of Genetics, Evolution, & Environment, University College, London, UK. e-mail: jon.jerlstrom.hultqvist@icm.uu.se; andrew.roger@dal.ca Nature Communications | (2024) 15:9726 1 12 34 56 78 9 0 () :,; 12 34 56 78 9 0 () :,; http://orcid.org/0000-0002-7992-7970 http://orcid.org/0000-0002-7992-7970 http://orcid.org/0000-0002-7992-7970 http://orcid.org/0000-0002-7992-7970 http://orcid.org/0000-0002-7992-7970 http://orcid.org/0000-0001-6763-3388 http://orcid.org/0000-0001-6763-3388 http://orcid.org/0000-0001-6763-3388 http://orcid.org/0000-0001-6763-3388 http://orcid.org/0000-0001-6763-3388 http://orcid.org/0000-0003-2356-3351 http://orcid.org/0000-0003-2356-3351 http://orcid.org/0000-0003-2356-3351 http://orcid.org/0000-0003-2356-3351 http://orcid.org/0000-0003-2356-3351 http://orcid.org/0000-0002-5766-0267 http://orcid.org/0000-0002-5766-0267 http://orcid.org/0000-0002-5766-0267 http://orcid.org/0000-0002-5766-0267 http://orcid.org/0000-0002-5766-0267 http://orcid.org/0000-0002-4322-0754 http://orcid.org/0000-0002-4322-0754 http://orcid.org/0000-0002-4322-0754 http://orcid.org/0000-0002-4322-0754 http://orcid.org/0000-0002-4322-0754 http://orcid.org/0000-0001-6650-0970 http://orcid.org/0000-0001-6650-0970 http://orcid.org/0000-0001-6650-0970 http://orcid.org/0000-0001-6650-0970 http://orcid.org/0000-0001-6650-0970 http://orcid.org/0000-0003-4520-5694 http://orcid.org/0000-0003-4520-5694 http://orcid.org/0000-0003-4520-5694 http://orcid.org/0000-0003-4520-5694 http://orcid.org/0000-0003-4520-5694 http://orcid.org/0000-0001-7255-780X http://orcid.org/0000-0001-7255-780X http://orcid.org/0000-0001-7255-780X http://orcid.org/0000-0001-7255-780X http://orcid.org/0000-0001-7255-780X http://orcid.org/0000-0003-1370-9820 http://orcid.org/0000-0003-1370-9820 http://orcid.org/0000-0003-1370-9820 http://orcid.org/0000-0003-1370-9820 http://orcid.org/0000-0003-1370-9820 http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-024-54102-7&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-024-54102-7&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-024-54102-7&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-024-54102-7&domain=pdf mailto:jon.jerlstrom.hultqvist@icm.uu.se mailto:andrew.roger@dal.ca www.nature.com/naturecommunications These interactions are usually centered on the exchange of metabo- lites producedby the host’smitochondrion-related organelles (MROs), that perform metabolism adapted to anaerobic conditions. H2 pro- duced by these “hydrogenosome”-typeMROs is an important currency in these interactions and is the end-product of an anaerobic ATP- producing metabolic pathway. Removal of H2 by prokaryotes likely increases themetabolic flux of the host by avoiding product inhibition of anaerobic metabolism. As a result, H2-consuming symbionts are sometimes found associated with host MROs, an arrangement that increases their access to substrate4. Although symbiont-associated subcellular structures have occasionally been observed in such anae- robic protists (e.g., ref. 5), little is known about these systems other than the metabolites likely being exchanged between host and symbionts. In this study, we comprehensively investigate a highly organized symbiosis involving Anaeramoebae, a recently described phylum of anaerobic protists within the Metamonada supergroup6,7. Anaera- moebid cells are predominantly ameboid, containing a densely packed mass of symbionts intricately interwoven with double-membraned electron-denseMROswehave recently identifiedashydrogenosomes6. These symbionts, although not in direct contact with the hydrogeno- somes, are enveloped by membranes of host origin (Fig. 1)7. By employing a combination of comparative genomic and tran- scriptomic analyses for both host and symbionts, phylogenomic investigations, focused-ion-beam scanning electron microscopy (FIB- SEM) tomography, and in situ localization techniques, we system- atically dissect the intricate metabolic interactions between Anaera- moebaeand their symbionts. Furthermore,wedelve into the structural aspects of the host-cell-derived symbiosome and explore the evolu- tionary roots of this distinctive symbiotic framework. Specifically, we: (i) reconstruct the symbiosome membrane network, (ii) identify the bacterial symbionts (iii) characterize the complete genomes of three host Anaeramoebae and each of their associated symbionts, (iv) reconstruct the host-symbiontmetabolic and cellular interactions, and (v) delineate the evolutionary trajectories of host and symbiont genomes. Our findings reveal that the symbionts are situated within a symbiosome, which comprises an interconnected host membrane network that positions the symbionts alongside host hydrogeno- somes. This network also features tubular connections that connect to the plasma membrane allowing access to the extracellular environ- ment. Notably, lateral gene transfer (LGT) and extensive gene dupli- cation emerge as pivotal factors that established and stabilized the incipient symbiosome within the ancestral lineage of Anaeramoebae. a f H H H H s ss s s s s s s N H H H H H H H N b c d e NN N fv s Fig. 1 |Anaeramoeba and symbionts. a Scanning electronmicrograph (SEM) of an Anaeramoeba flamelloides BUSSELTON2 amoebae showing a hyaline front and posterior trailing projections. Scale bar 5 µm. b Immunolocalization of alpha- tubulin (TAT1 antibody, 1:200) in A. flamelloides BUSSELTON2 cells using laser scanning confocal microscopy. Host nuclei and symbiont DNA were stained using DAPI (top panel) and the acentriolar centrosome and radiating microtubules by alpha-tubulin TAT1 antibody (middle panel) with overlay (bottom panel). Scale bar 5 µm. c TEM of chemically fixed A. flamelloidesBUSSELTON2. Scale bar 5 µm.d TEM of chemically fixedA.flamelloidesBUSSELTON2. Scale bar 2 µm. eTEMof cryo-fixed A. flamelloides BUSSELTON2.White arrow shows acentriolar centrosome. Scale bar 2 µm. f Transmission electron micrograph (TEM) of A. ignava BMAN showing vesicle-bound symbionts (S) and host hydrogenosomes (H) in close proximity. The boxed inlay shows narrow openings (red arrow) connecting outside media to the vesicles housing the symbionts. Scale bar 1 µm. Nucleus (N), Food-vacuole (fv). The experiment in (b) shows representative cells from three independent replicate immunostainings. Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 2 www.nature.com/naturecommunications Results The Anaeramoeba symbionts are directly connected to the extracellular milieu Actively feeding Anaeramoeba cells have a fan-shaped hyaline zone and trailing projections (Fig. 1a). Near the nucleus in the bulbous cell body lies a large mass of symbionts housed in compartments with single bounding membranes tightly positioned close to, but not in direct contact with, hydrogenosomes (Fig. 1b–e)6,7. Anaeramoeba cells have an acentriolar centrosome positioned below the hydrogenosome-symbiont mass and the nucleus from which micro- tubules radiate throughout the ventral side of the cell (Fig. 1b, e). The symbionts are stably maintained throughout the cell cycle and segre- gated in an organized fashion during cell division (Fig. S1). These observations indicate that the symbionts are housed in stable struc- tures but do not reveal if the Anaeramoeba symbionts are endo- symbionts completely enclosed within the host or are directly connected to the extracellular media. To distinguish between these two possibilities, we first used live- cell pulse-labeling experiments with fluorescent-labeled Wheat Germ Agglutinin (WGA), a lectin that stains sialic acid and N-acetyl glucosa- mine in bacterial cell walls. The Anaeramoeba flamelloides BUSSELTON2 symbiont showed clear WGA labeling after only 10min of incubation,with staining intensity comparable to free-livingbacteria (Fig. S2). This suggests that the symbionts have relatively quick “access” to the extracellular environment. Furthermore, transmission electron microscopy images of Anaeramoeba ignava BMAN show symbiont cells in pockets at the host cell membrane with connections to the surface (Fig. 1f). To better resolve the symbiosome structure, we conducted FIB- SEM tomography on A. flamelloides BUSSELTON2 holobionts. After 1780 serial section SEM images were collected in 7 nm deep incre- ments through a fixed cell, a computational 3D reconstruction was performedwith specific distinctions made between symbionts, other prokaryotes, hydrogenosomes, symbiosome-membrane, micro- tubules and the acentriolar centrosome, plasma membrane, the nucleus, and dense granules (Figs. 2 and S3). The 3D reconstructions show the mass of symbionts and the symbiosome structure housing them (Fig. 2d) is highly elaborate, tightly associated with the hydro- genosomes (Fig. 2e–g) (Movie S1), and located proximal to the nucleus. Reconstruction of the plasma membrane alongside the symbiosome revealed membrane connections between symbiosome compartments and the plasma membrane (arrowheads in Fig. 2h, i). a b c d e f g h i symbbioosoommee mmembbrannee pplassmmaa mmembbrannee othherr prokaaryryyoottess hyydrogeenosoommee syymmbbioontt ggrannuulless nnuccleuuss ssymbbiosoommee meemmmbraanee pplassmmaa memmbraanee symbbioontt nnnuucccleeuss pplassmmaa mmembraannee hyydroggeenosoommee nnuccleeuuss syymmbbiiossomee meeemmmbbrannee pllasmaa meemmbbrannee hhyydddrroooggeenossoommee syymmbiionntt nnuucllleeuss MTsTTs syymmbiiossomee meemmmbbbranee hhyddroogeennossomee symmbiionntt nuuclleuusss plasma membraneplasmpla a ee b anrmmembrane symbiosomesymbiosome pplassmmaa mmemmbraanee ssymbiosomee Fig. 2 | Anaeramoeba symbionts are housed in a membrane network with connections to the plasma membrane. a–c FIB-SEM slice of A. flamelloides BUS- SELTON2, showing (b) segmented regions of interest (symbiont–blue, hydrogenosome–red, symbiosome membrane–gold, nucleus–purple, granules–aubergine, other prokaryotes– blue green, plasma membrane–yellow). c overlay of (a, b). d–f Rendered volume of FIB-SEM slices showing segmented regions of interest. d Symbiont, symbiosome membrane, nucleus and plasma membrane. e Hydrogenosomes, nucleus and plasma membrane. f Overlay of all regions of interest in (d, e), and microtubules (MTs) (symbiont–blue, hydrogenosome–red, symbiosome membrane–gold, nucleus–purple, plasma membrane– yellow, MTs–pink). g A clipped 3D rendering of A. flamelloides BUSSELTON2 symbionts (blue), hydrogenosomes (red), symbiosome membrane (gold) and nucleus (purple) showing the internal structure of the symbiosome. h–i Two different views of 3D surface rendering of the components of the sym- biosome connected to the plasma membrane (light blue). Plasma membrane (yellow) and nucleus (purple). Symbiosome connections to the plasma membrane are indicated by black arrows. Scale bar (a–c), 1 µm. Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 3 www.nature.com/naturecommunications Analyses of the internal structure of the symbiosome revealed that the various compartments housing distinct symbionts (indicated in gold in Fig. 2g) are connected by tubular channels (Fig. S4). We observed 171 individual membrane connections between symbio- some subcompartments yielding fifteen connected componentswith two or more symbionts (Fig. S5A–H andMovie S2). In total, we found that in the specific Anaeramoeba cell analyzed, 86% of symbionts (158/183) have contact with at least one other symbiont while 25 symbionts are housed in individual compartments (Fig. S5 and Movie S2). The largest symbiosome component harbors 105 sym- bionts (Fig. S5i–l). Collectively, 108 symbionts are directly in contact with the outside media (Fig. 2h, i and S5). Even though the 25 indi- vidual compartments tend to be peripheral, it should be noted that they make very close approaches to the multi-symbiont compart- ments (Fig. S5 and Movie S2). Anaeramoeba symbionts belong to Desulfobacteraceae and were acquired independently in different host species We sequenced the nuclear genomes of three Anaerameoba isolates (A. ignava BMAN, A. flamelloides BUSSELTON2, and SCHOONER1) and the genomes of the associated prokaryotes using Nanopore long-read and Illumina short-read technologies (Table S1). The most abundant prokaryotes in the A. flamelloides and A. ignava genomic datasets were Desulfobacteraceae, hereafter referred to as Sym_BUSS2, Sym_SCH1, and Sym_BMAN. In A. flamelloides, where ameba separation from sus- pended bacteria was found to be the most efficient (Fig. S6), the symbiont lineages were detected almost exclusively in the amoeba enriched fraction and were virtually non-detectable in the culture supernatant (Fig. S6). Fluorescence in situ hybridization (FISH) using unique probes designed against the 16S rRNA genes in the genomes of Sym_BMAN (Fig. 3a–d) and Sym_BUSS2/Sym_SCH1 (Figs. 3f–l and S7) (as well as less specific probes for broader encompassing taxonomic groups (Fig. S8)) confirmed the identity of the symbionts. The average number of symbionts per host based on symbiont genome coverage relative to nuclear genome coverage was estimated to be 35.3–36.5 for A. flamelloides (Sym_BUSS2 & Sym_SCH1) and 12.6 for A. ignava (Sym_BMAN) (the ploidy of the host nuclear genome is unknown but for these analyses posited to be haploid). All three symbiont genomes are similar in size to free-living relatives (4.97–6.06Mbp) and relatively gene-rich (3823–5286 intact genes) (Table S2). The Sym_BUSS2 and Sym_SCH1 genomes are 99.7% identical at the nucleotide level but show large differences in synteny (Fig. S9), whereas the Sym_BMAN genome is highly divergent relative to the two A. flamelloides sym- bionts (average nucleotide identity of 74.1–74.2%). A second less abundant Desulfobacteraceae genome, Sym_BMAN2, was also detec- ted in the A. ignava dataset but its status as a symbiont could not be established using FISH. Thus, for A. ignava we focused on Sym_BMAN in subsequent analyses. 0.6 Desulfobacter sp UBA12168 Desulfobacterales bacterium RIFOXYA12_F Desulfatirhabdium butyrativorans DSM_18 Desulfospira joergensenii DSM_10085 Sym_BMAN Desulfobacter vibrioformis DSM_8776 Desulfobacter postgatei 2ac9 Desulfobotulus alkaliphilus ASO4_4 Desulfobacter curvatus DSM_3379 Desulfonema ishimotonii Tokyo_01 Desulfobacteraceae bacterium Glo_9 Desulfobacteraceae bacterium UBA2245 Desulfobacter sp UBA2225 Desulfobacteraceae bacterium UBA5852 Desulfobacter postgatei DOLJORAL78_47_2 Desulforegula conservatrix Mb1Pa Desulfobacteraceae bacterium UBA2174 Sym_SCH1 Desulfoluna spongiiphila AA1 Desulfococcus multivorans DSM_2059 Desulfatitalea tepidiphila S28bF Desulfobacter hydrogenophilus DSM_3380 Desulfobacteraceae bacterium UBA8212 Desulfobacteraceae bacterium UBA2771 Desulfamplus magnetovallimortis PRJEB14 Sym_BMAN2 Desulfosarcina ovata oXyS1 Desulfobacula sp S2_67 Desulfobacter latus AcRS2 Desulfotignum balticum DSM_7044 Desulfobacterium autotrophicum HRM2 Desulfobacteraceae bacterium 4572_89 Sym_BUSS2 Desulfobacula toluolica Tol2 Desulfobacteraceae bacterium Glo_11 100 100 84 100 100 100 100 100 100 100 100 100 95 100 99 92 100 100 99 100 99 100 100 100 100 100 91 100 100 100 100 100 82 D ESU LFO B A C TER A C EA E Desulfobacter Anaeramoeba ignava symbiont Anaeramoeba flamelloides symbionts m A. flamelloides SCH1A. flamelloides BUSS2A. ignava BMAN b BMAN d EUB338a DAPI a c DELTA495a f BUSS/SCH h EUB338a DAPI e g DELTA495a j BUSS/SCH l EUB338a DAPI i k DELTA495a Fig. 3 | Anaeramoeba symbionts are closely related to Desulfobacter and acquired in separate events. A. ignava BMAN stained with (a), DAPI and hybri- dized with (b), probe DSBA355-BMN-488, (c), probe Delta495a-Atto 550 and (d), probe EUB338a-Atto 633. A. flamelloides BUSSELTON2 stained with (e), DAPI and hybridized with (f), probe BUSS/SCH-BMN-488, (g), probe Delta495a-Atto 550 and (h), probe EUB338a-Atto 633. A. flamelloides SCHOONER1 stained with (i), DAPI and hybridized with (j), probe BUSS/SCH-BMN-488, (k), probe Delta495a-Atto 550 and (l), probe EUB338a-Atto 633. m The phylogenomic analysis was based on 36 taxa, 108 proteins, and 25,918 sites with IQTree v2.2.0.3 (LG +C60 + F +Gmodel of evolution). Bipartition support values are derived from 100 non-parametric boot- straps under the PMSFmodel. Scale bar indicates inferred number of substitutions per site. Tree files and alignments are available at FigShare: https://doi.org/10. 6084/m9.figshare.20375619. Scale bar (a–l), 1 µm. The experiments in (a–d), and (e–l), show representative images from duplicate and triplicate hybridizations respectively. Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 4 https://doi.org/10.6084/m9.figshare.20375619 https://doi.org/10.6084/m9.figshare.20375619 www.nature.com/naturecommunications Phylogenetic analysis of 15 ribosomal proteins from 165 Desulfo- bacterales genomes placed the symbionts as members of the family Desulfobacteraceae (Fig. S10), closely related to the genus Desulfo- bacter. To improve resolution, we performed phylogenetic analysis on a data set of 108 proteins from a phylogenetically restricted set of Desulfobacteraceae. Sym_BMAN and Sym_BMAN2 belong to a clade that branches sister to a large Desulfobacter group (Fig. 3m) whereas Sym_BUSS2/SCH1 branches separately, forming a well-supported group (BS 95%) with an environmental isolate (Glo_9) recovered from a metagenome of the benthic foraminiferan Globobulimina spp. The latter clade forms a sister group to theDesulfobacter – Sym_BMAN clade. TheDesulfobacter-like symbionts ofA. ignava andA. flamelloides thus appear to have been acquired independently by their respec- tive hosts. The Anaeramoeba-Desulfobacter symbiosis was recently established The Sym_BUSS2 and Sym_SCH1 genomes were found to have 922 and 1000 pseudogenes, respectively (Table S2 and Supplementary Data 1), and >600 Insertion Sequences (IS) per genome (Supplementary Data 2). In contrast, pseudogene and IS element abundance in Sym_BMAN is an order ofmagnitude lower and falls within the range of free-living Desulfobacteraceae species (Table S2). The IS elements of Sym_BUSS2 and Sym_SCH1, many of which have an intact transposase and are likely active, showed no strong evidence of clustering (Fig. S11A, B). However, >200 IS elements were found to be close (<1500 bp) to the edges of syntenic blocks, suggesting that the apparent high frequencyof rearrangements is connected to IS element activity (Fig. S11C, D). The Sym_BUSS2 and Sym_SCH1 genomes are enriched in pseudogenes with predicted gene ontologies related to signal transduction (T) and amino acid metabolism and transport (E) functional categories, whereas translation (J) and transcription (K) classes showed the opposite trend (Fig. S12). The genomes of these two symbionts have flagellar operons (Fig. S13), type IV pili, and CRISPR systems that are all extensively pseudogenized; both sym- bionts also appear to be impaired in their ability to decorate lipid A with O-antigen. In contrast, the Sym_BMAN genome encodes an intact type I-F CRISPR system with an array of 25 spacers as well as two convergent, 25 gene operons for a type VI secretion system (T6SS) (Fig. S14). The greater degree of genome degeneration seen in the Sym_BUSS2 and Sym_SCH1 genomes relative to the largely intact Sym_BMAN genome strongly suggests that the former are evolutio- narily “older” symbionts and that they are unable to survive without the host. However, the relatively large genomes of all Anaeramoeba sym- bionts and other genomic features are reminiscent of early-stage, recently-acquired symbionts8. Constitutive overexpression of the chaperonin GroEL/S is regarded as a critical factor in stabilizing endosymbionts in a wide range of insect symbioses9. Notably, meta- transcriptomic analysis of the A. ignava BMAN and A. flamelloides acetyl-CoA HysAB 2H+ H+ Na/H+ ATP NAD+ NADH/H+ ATP Fdox Fdox Fdred Fdred X[H ] X[H ] NADPH NADPH H+ acetate e- e- e- e- Qmo Tmc Nqr Hdr Hdr Hme Sulfate transporter Acetate transporter SO4 2- HS- SO3 2- APS SO4 2- CO2 CO2 CO2 CoA CoA CoA malate pyruvate propionyl-CoA propionate propionate acetyl-CoA succinyl-CoA succinate succinate 2H+ 2H 2H 2H O acetate ATP ATP CO2 CO2 F+ F- 2 2 AtpRnf HynAB M et hy lm al on yl -C oA pa th w ay W oo d- Lj un gd ah l pa th w ay Dissimilatory sulfate reduction pathway Desulfobacter symbionts Anaeramoeba hydrogenosome M et hy lm al on yl -C MM et hy lm al on yl -C a oAoA C o Fig. 4 | Anaeramoeba symbionts are metabolically poised for syntrophic interaction with hydrogenosomes. Suggested syntrophic interactions between Anaeramoeba hydrogenosomes (blue) and symbionts (pink/salmon) based on metabolic reconstruction from transcriptomic and genomic evidence. The ATP- producing hydrogenosomes generate H2, acetate, and propionate as end-products (in bold). Based on metatranscriptomic data, the symbionts use the hydrogeno- some products by prominently expressing the dissimilatory sulfate reduction (DSR), methylmalonyl-CoA, and the Wood-Ljungdahl pathways. The symbionts are in deep membrane-pits with a connection to the cell surrounding that gives ready access to sulfate (gold). HynAB periplasmic [NiFe] hydrogenase, HysAB [NiFeSe] hydrogenase, Qmo QmoABC complex, Tmc TmcABCD complex, Hdr Hetero- disulfide reductase, Hme DsrMKJOP complex, Nqr NADH:ubiquinone oxidor- eductase, Rnf Rnf complex, Atp ATP synthase, APS adenosine 5'-phosphosulfate, CoA Coenzyme A, NAD Nicotinamide adenine dinucleotide, NADPH Nicotinamide adenine dinucleotide phosphate, Fdred/ox Ferredoxin reduced/oxidized, ATP adenosine triphosphate. Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 5 www.nature.com/naturecommunications BUSSELTON2 cultures show that the groEL, groES, and HSP70- encoding dnaK genes are highly expressed in Sym_BUSS2 but moder- ately to lowly expressed in Sym_BMAN, in line with their respective degrees of genome erosion (Supplementary Data 3). Anaeramoeba symbionts are metabolically poised to use hydrogenosomal metabolites The hydrogenosomes of both Anaeramoeba species are predicted to produce H2, acetate, and propionate as end-products6. To investigate whether the symbionts express genes predicted to be important for H2-uptake, we performed metatranscriptomics of the A. ignava BMAN and A. flamelloides BUSSELTON2 cultures. Metatranscriptomics of Sym_BUSS2 showed that the most prominently symbiont-expressed genes include those involved in dissimilatory sulfate reduction (DSR), the group 1b uptake [Ni/Fe] hydrogenase (hynAB), the Wood–Ljungdahl (W–L) pathway, and ATP synthase (Fig. 4 and Sup- plementary Data 3). Similarly, in Sym_BMAN, the dsr genes, aprAB pathway and hynAB were highly expressed, while W–L pathway and ATP synthase genes are less expressed than in Sym_BUSS2 (Supple- mentary Data 3). Sym_BMAN shows high expression of an acetate permease (actP) that might act to bring in host-derived acetate to feed the W–L pathway. Although we could not identify acetate transporter genes (actP or satP) in the A. flamelloides symbiont genomes, we pre- dict these symbionts might be able to acquire acetate by diffusion across the membrane as reported in other systems10. The symbionts appear to be able to activate propionate via propionyl-CoA synthase (prpE), which is highly expressed in Sym_BUSS2 and Sym_BMAN (Supplementary Data 3). Propionyl-CoA likely feeds into the methylmalonyl-CoA (MMA) pathway to produce pyruvate that, via oxidative decarboyxlation by pyruvate:ferredoxin oxidoreductase, generates acetyl-CoA that could enter the W–L pathway (Fig. 4). Most enzymes of the MMApathway are highly andmoderately expressed in Sym_BUSS2 and Sym_BMAN, respectively. Because Sym_BUSS2 is clo- sely related to the Desulfobacter Glo_9 denitrifying symbiont of Glo- bobulimina spp. (discussed in refs. 11,12), we investigated whether the Anaeramoeba:Sym_BUSS2 symbiotic system could be based on deni- trification. However, we failed to find denitrification-related genes in the host (nrt, nirK, nor) or symbionts (napA, nozA). Given that many of the gene products described above are sen- sitive to oxygen, we examined the genomes of the symbionts for oxygen or reactive oxygen species (ROS) defense systems. Genes encoding the superoxide detoxification (superoxide reductase and rubredoxin) andhydrogen peroxide detoxification (rubrerythrin) were highly expressed by Sym_BUSS2 and Sym_BMAN (Supplemen- tary Data 3). Anaeramoeba nuclear genomes are A+T rich and have greatly expanded gene families The nuclear genomes of Anaeramoebae are A +T-rich, with the A. ignava BMAN nuclear genome (80.65% A+T) and intron sequences (91.86% A +T) being among the highest ever reported in eukaryotes (e.g., the Plasmodium falciparum nuclear genome and intron sequen- ces are 80.6% and 86.5% A +T, respectively13) (Tables S1 and S3A). Whereas the assembled genomes of A. flamelloides are almost six- times larger than for A. ignava, the number of protein coding genes is only two-fold higher (Table S1). The sequence divergence between the Anaeramoeba species is substantialwith only37.3%average aminoacid identity between orthologous proteins (Fig. S15). In contrast, the A. flamelloides BUSSELTON2 and SCHOONER1 strains are much more similar (94.8% identity between orthologs). To investigate the evolutionof gene content inAnaeramoebae,we compared the gene families of the three Anaeramoeba genomes to nine other microbial eukaryotes including species from Parabasalia, Oxymonadida, Fornicata, Heterolobosea and Amoebozoa (Supple- mentary Data 4). Overall 565 and 4704 families were deemed core and accessory, respectively (Supplementary Data 4A–C). Of the accessory families 67, 74 and 107 are specific to BUSSELTON2, SCHOONER1 and BMAN, respectively (Supplementary Data 4D) with 92 families uniquely found in all three of them (Supplementary Data 4E). When comparing only A. flamelloides to A. ignava, expansions were detected in 330 shared (core to Anaeramoebae) families while contractions were found in 13 such families (Supplementary Data 4H). The con- tractions seen in A. flamelloides likely correspond to expansions in A. ignava rather than contractions in A. flamelloides (Supplementary Data 4F–H). Together with the presence of BUSSELTON2, SCHOONER1 and BMAN-specific families already mentioned, these differences highlight the striking differences among these relatively closely related taxa. Many gene family expansions occur specifically in A. flamelloides and are enriched in genes involved in RNA and DNA metabolism and membrane-trafficking systems. One striking feature is the massive expansion of ribosomal proteins with more than 1100 genes each in A. flamelloides compared to the 82 genes in A. ignava, representing a more typical number for a eukaryote (Supplementary Note 1, Supple- mentaryData 4I–K and Fig. S16). However, expansions of gene families connected to nutrient exchange were also noted. For example, A. fla- melloides and A. ignava have sizable expansions of an Amt/MEP ammonium transporter gene, whose products likely function in the uptake or secretion of ammonia, a primary nitrogen source for cells14. Lateral gene transfer is ongoing in Anaeramoeba LGT is an important mechanism impacting genome evolution in many eukaryotes15,16. We performed a phylogenomic analyses to detect LGTs fromprokaryotes and viruses toAnaeramoeba.We identified 612, 1414, and 1359 putative foreign genes in the BMAN, BUSSELTON2, and SCHOONER1 genomes, respectively (4.1%, 4.7%, 4.5% of the total number of genes per genome). Accounting for gene duplications after acquisition, this corresponds to 388, 781, and 777 gene families (Table 1). These are someof the highest LGTproportions ever reported for protists17. Many of the LGTs appear to have happened after the divergence of A. ignava and A. flamelloides (Supplementary Data 5 and Fig. 5). 169 putative LGTs occurred in a common ancestor of the two species, whereas 577 LGTs map to the base of the divergence between the A. flamelloides BUSSELTON2 and SCHOONER1 strains, and 220 were acquired along the lineage leading to A. ignava (this pattern may also be explained in part by differential loss of LGT genes). Over 30 Table 1 | Predicted LGTs in the genomes of Anaeramoeba species Predicted proteins Ortho groups (OGs) OGs including at least one prokaryotic or viral sequence Treesa Inferred LGT events Total LGT- derived genes A. ignava BMAN 15,022 6168 2224 1743 388 612 A. flamelloides BUSSELTON2 29,927 14,259 3701 3156 781 1414 A. flamelloides SCHOONER1 30,041 14,246 3679 3115 777 1359 aTrees were estimated for only a subset of OGs that contained at least one prokaryotic or viral taxa and with >80 sites (see “Methods”) Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 6 www.nature.com/naturecommunications genes were inferred to have been acquired in the two A. flamelloides strains after their divergence. Most of the LGTs for which a taxonomic origin could be inferred are bacterial, although archaeal and viral contributions to Anaera- moeba are also apparent (Fig. 5a). Proteobacteria, Firmicutes and Bacteroidetes (Fig. 5a) are the most represented donor phyla con- tributing genes to the ancestor ofA. ignava andA. flamelloides, and the two species appear to have acquiredmore proteobacterial genes since they diverged from one another. Interestingly, Deltaproteobacteria is not the most represented class, despite the taxonomic affinity of Anaeramoeba’s symbionts (Fig. 3). That said, several dozen genes with amino acid identities >60% (Supplementary Data 5D) are inferred to have been transferred from Deltaproteobacteria to Anaeramoeba, such as an aspartate ammonia ligase (in A. flamelloides) and an FAD- dependent oxidoreductase (in both species) (Fig. 5d). A few Anaera- moeba genes have discernable homologs only in the symbionts and thus might represent symbiont-to-host LGTs. Curiously, these LGTs encode long repetitive proteins (up to 4100 amino acids). The relative contributions of Alpha- and Gammaproteobacteria are similar for A. ignava and A. flamelloides (Fig. 5b). 169 220 577 35 33 a c b Proteobacteria Firmicutes Cyanobacteria Bacteroidetes Chloroflexi Actinobacteria Planctomycetes Acidobacteria Spirochaetes Chlamydia Fibrobacteres Nitrospirae Thermotogae Other bacteria Archaea Unclassified Viruses A. ignava BMAN A. flamelloides BUSSELTON2 A. flamelloides SCHOONER1 0.18 0.1 0.2 0.3 0.4 0.5 Alpha Gamma BetaDelta / epsilon Unclassified 0 0.6 Pr op .p ro te ob ac te ria lL G Ts Functional Category Acquired in commonancestor 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0 Unique to A. flamelloides Unique to A. ignava Uncla- ssifiedZXWVUTSRQPONMLKJIHGFEDC A.ignava BMAN M0811_10053 A. flamelloides BUSSELTON2 M0812_12476 A. flamelloides SCHOONER1 M0813_22551 A. flamelloides BUSSELTON2 M0812_30147 A. flamelloides SCHOONER1 M0813_29272 A. flamelloides BUSSELTON2 M0812_29719 A. flamelloides SCHOONER1 M0813_20507 Bacteroidetes OFX25478.1 Bacteroidetes bacterium GWA2 31 9 PIE86397.1 Bacteroidia bacterium Firmicutes Bacteroidetes 95 59 100 66 97 43 43 51 79 71 97 10091 93 70 55 84 100 100 100 100 100 100 0.20 WP_051204005.1 Hugenholtzia roseola Bacteria Bacteria Bacteria Bacteria Eukaryotes A. ignava BMAN M0811_13764 A. ignava BMAN M0811_14384 A. ignava BMAN M0811_14385 A. flamelloides BUSSELTON2 M0812_10929 A. flamelloides SCHOONER1 M0813_17619 A. flamelloides BUSSELTON2 M0812_03420 A. flamelloides SCHOONER1 M0813_16958 A. flamelloides BUSSELTON2 M0812_09941 A. flamelloides SCHOONER1 M0813_25513 OGP64635.1 Deltaproteobacteria bacterium RBG 13 53 10 PKN22488.1 Deltaproteobacteria bacterium HGW Deltaproteobacteria 21 OPX38424.1 Desulfobacteraceae bacterium 4484 190.3 WP 012662852.1 Desulfobacterium autotrophicum OQY08337.1 Desulfobacteraceae bacterium 4572 123 OYT46111.1 Thermoplasmatales archaeon ex4484 6 Other prokaryotes 99 63 55 60 99 100 100 100 99 100 100 100 100 0.50 Eukaryotic Oxidosqualene cyclase Bacteria Proteobacteria Deltaproteobacteria Cyanobacteria Adiantum capillus Dryopteris crassirhizoma Polypodiodes niponica Ferns Proteobacteria Proteobacteria Rhodospirillum rubrum Gluconobacter oxydans Schizosaccharomyces japonicus Deltaproteobacteria A. flamelloides BUSSELTON M0812_08183 A. flamelloides SCHOONER M0813_23558 Bacteria Anaeromyxobacter sp. Fw109-5 Penicillium chrysogenum Aspergillus fumigatus Neosartorya fisheri Fungi (pezizomycotina) Actinomycetes Sq ua le ne ho pe ne cy cl as e Paratrimastix pyriformis Sawyeria marylandensis Stygiella incarcerata Brevimastigomonas motovehiculus 1 Paramesium tetraurelia Tetrahymena thermophila Brachionus plicatilis Breviata anathema Alvinella pompejana A. flamelloides BUSSELTON M0812_12004 A. flamelloides SCHOONER M0813_10392 Piromyces sp. E2 Trepomonas sp. PC1 Sq ua le ne te tra hy m en ol cy cl as e 97 100 100 94 100 100 100 89 89 75 93 100 96 84 100 74 100 99 100 100 99 100 100 100 95 92 99 95 44 88 65 49 100 94 65 42 100 97 79 79 100 100 100 0.20 d e f Brevimastigomonas motovehiculus 2 Fig. 5 | Taxonomic range and functional categories of LGT donors in Anaera- moeba. a Taxonomy of LGT donors for genes inferred to have been acquired in the common ancestor of Anaeramoeba and separately in A. flamelloides and A. ignava. The numbers on the branches are the inferred number of LGTs on each position of the tree. b Breakdown of LGTs from proteobacterial donors. c Functional cate- gories of genes inferred to have been acquired in the Anaeramoeba common ancestor, in A. flamelloides and A. ignava. Color coding is the same as for part (b). d Maximum-likelihood phylogeny of FAD-dependent oxidoreductases. e Maximum-likelihood phylogeny of vitamin B12-dependent methionine synthase MetH. The trees shown in (d) and (e) were produced by our LGT-detection pipeline (see text). f Maximum-likelihood phylogeny of oxidosqualene cyclase (OSC), squalene-hopene cyclase (SHC), and squalene-tetrahymanol cyclase (STC). The tree shown stems from analyses based on previously published datasets84,85. Sequences were aligned with MAFFT, sites were selected using BMGE, and the phylogeny inferred using IQTree model C20+G4 with 1000 ultrafast bootstraps. Anaera- moeba sequences are in red, eukaryotic sequences in blue, and prokaryotic sequences in bright green. Scale bars indicate the inferred number of amino acid substitutions per site. Abbreviations: INFORMATION STORAGE AND PROCESSING: [J] Translation, ribosomal structure and biogenesis; [K] Transcription; [L] Replica- tion, recombination and repair; CELLULAR PROCESSES AND SIGNALING: [D] Cell cycle control, cell division, chromosome partitioning; [V] Defensemechanisms; [T] Signal transductionmechanisms; [M]Cell wall/membrane/envelope biogenesis; [N] Cell motility; [Z] Cytoskeleton; [W] Extracellular structures; [U] Intracellular traf- ficking, secretion, and vesicular transport; [O] Posttranslational modification, protein turnover, chaperones. METABOLISM: [C] Energy production and conver- sion; [G] Carbohydrate transport and metabolism; [E] Amino acid transport and metabolism; [F] Nucleotide transport and metabolism; [H] Coenzyme transport andmetabolism; [I] Lipid transport andmetabolism; [P] Inorganic ion transport and metabolism; [Q] Secondary metabolites biosynthesis, transport and catabolism. POORLY CHARACTERIZED: [R] General function prediction only; [X] Mobilome: prophages, transposons; [S] Function unknown. Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 7 www.nature.com/naturecommunications Laterally transferred genes shape Anaeramoeba biology The predicted functions of LGTs in Anaeramoeba differ between those acquired in the common ancestor of A. ignava and A. flamelloides and those acquired after the divergence of the two species (Fig. 5c and Supplementary Note 2). Several genes acquired in the Anaeramoeba common ancestor may be related to accommodating sulfate-reducing symbionts. For example, genes encoding a cysteine synthase K and D-3-phosphoglycerate dehydrogenase are LGT-derived (Fig. S17D, E); together with SerC phosphoserine aminotransferase (which was not detected in our LGT screen but was found in the genomes), these proteins link glycolysis to the recycling of SH- produced by the sym- bionts and the generation of acetate that might be used by the sym- biont (Fig. S18). A prokaryotic acetate transporter gene (Fig. S17F) also appears to have been acquired in the Anaeramoeba ancestor and might have played a role in the establishment of the symbiosis (the symbionts are predicted to use acetate produced by Anaeramoeba). The foreign acetate transporter gene is present in >10 copies in each Anaeramoeba genome. We scanned the Anaeramoeba LGTs for putative host-symbiont recognition factors, which often have repetitive domains18. Amongst the LGT candidates, we identified 38 gene families that previously have been associated with host-symbiont interactions (Supplementary Data 5). Some of these gene families are highly amplified, have pre- dicted signal peptide-encoding regions, and show differential pre- sence/abundance between A. ignava and A. flamelloides, indicating that they might traffic in the secretory pathway and could potentially mediate symbiont interactions. Several foreign genes in A. ignava BMAN and A. flamelloides BUSSELTON2 are clearly involved in anae- robic life, including (i) MROmetabolism6, (see Supplementary Data 5), (ii) oxygen detoxification, (iii) ATP production in the absence of oxi- dative phosphorylation (e.g., pyruvate phosphate dikinase; Fig. S17G), and (iv) cell membrane composition modification (acquisition of a bacterial squalene hopene cyclase (SHC) involved in oxygen-free bio- synthesis of hopanoids (Supplementary Note 2 and Fig. 5f)). Several of these genes appear to have been acquired onmultiple occasions from various donors (Supplementary Note 2, e.g., Fig. S17M–O). Of the LGT-derived genes that are amenable to functional/meta- bolic prediction, very few make up a complete “module”19, suggesting that most LGT-derived proteins function in concert with host-origin enzymes in mosaic pathways. The sole exception is a complete set of genes encoding the enzymes of the Leloir pathway for galactose metabolism in A. flamelloides (see Supplementary Note 2). Anaeramoeba might acquire vitamin B12 from its symbionts Vitamin (vit)B12 is one of the most complex coenzymes known. Its biosynthesis involves ~30 enzymatic steps and is confined to certain bacterial and archaeal species20. The symbionts associated with both Anaeramoeba species encode a complete anaerobic pathway for vitB12 synthesis (except for alpha-ribazol phosphatase (CobC); below). Interestingly, our genome screens detected up to six vitB12-dependent enzymes encoded in the Anaeramoeba host genomes, a surprising number and a record among eukaryotes examined thus far. This includes two enzymes in A. flamelloides (Supplementary Note 3) that have not been found in a eukaryote before. Additionally, both Anae- ramoeba species have acquired a class II ribonucleotide reductase (RNR) and a methionine synthase (metH) (Fig. 5E) from bacteria and have a eukaryotic methylmalonyl-CoA mutase and its associated GTPase MeaB. These observations suggest that vitB12—or cobalamin— is produced by the symbionts and consumed by the host (both Anaeramoeba species encode a cobalamin adenosyl transferase that catalyzes the conversion of cobalamin into adenosylcobalamin, the form used as a cofactor by vitB12-dependent methylmalonyl-CoA mutase). The exchange of vitB12 between prokaryotic producers and B12-auxotrophic algae was hypothesized elsewhere21,22. Curiously, we founda laterally acquiredgene encodingCobC (Fig. S17W) inA. ignava. This enzyme catalyzes the last step of cobalamin synthesis, which is not predicted to occur in the symbionts. The cobC gene has also been acquired in some vitB12 auxotrophic diatoms23. Our analyses strongly suggest that vitB12 exchange is part of the host-symbiont metabolic interaction in Anaeramoeba, a hypothesis that can be tested in the future. Expansions of endosome/phagosome modulating membrane- trafficking proteins in Anaeramoeba The vesicle-bound symbiont mass in these Anaeramoebae, which is connected to the extracellular environment, is reminiscent of phago- somes that have been delayed or frozen in their maturation process and then elaborated. The general expansions in genes involved in membrane-trafficking systems in both Anaeramoeba spp. identified above prompted us to specifically investigate several sets of proteins, which in other eukaryotes have been implicated in regulation of pha- gosomalmaturation. Certain Rab GTPases are essential in this process, mediating early to late endosomal conversion, as well as endosomal function and are involved in other processes such as signal transduc- tion and acting as molecular switches that regulate formation and transportation of vesicles24,25. Consequently, we investigated not only Rabs but also their GTPase Activating Proteins (GAPs) and the Vps-C complexes HOPS and CORVET that mediate endosomal Rab conver- sion, with the former having recently been implicated in regulating phagosomal-lysosomal fusion26. The Anaeramoeba genomes encode many Rab GTPases, almost 400 in A. flamelloides. Rabs are small proteins, which made resolved classification by phylogenetic analysis intractable. Orthofinder27 was thus used for initial classification followed by targeted phylogenies of related Rab sub-families (when necessary). While 75–80% of Rab pro- teins could not be confidently classified, clear trends were never- theless apparent (Supplementary Data 6). We observed relatively low numbers of Rabs 6, 7, and 18, and no clear candidate orthologs for eight Rab sub-families were found, including some endosomal Rabs suchasRab4or 22. By contrast, RabGTPase families 1, 2, 5, 8, 11, 14, 20, 21, and 24 were found to be highly expanded (Fig. S19A–C and Sup- plementary Data 6).Wewere also able to confidently classifymost Tre- 2/Bub2/Cdc16 (TBC) proteins that serve as GAPs for Rabs. Although there are only one or several members of each of the TBC-I, M, and N subfamilies, the TBC-E, F, K, and Q subfamilies are notably expanded (Fig. S19D). Finally, we observed expansions in the complement of both HOPS and CORVET subunits, most obviously with increased numbers of the HOPS-specific subunits (Supplementary Data 6). We searched for evidence of gene duplications within the Rab and TBC subfamilies that might predate the divergence of the Anaera- moeba spp., and which could indicate Anaeramoeba-specific machin- ery involved in capture and regulated retention/digestion of symbionts. Rab1, or the metazoan-specific paralog Rab3528, has been directly implicated in phagosomematuration dynamics inmammalian cells29 and in the amoebozoan parasite Entamoeba30. Although the phylogenetic resolution between the subfamilies is poor, in the case of theRab1 sub-family (Fig. S19C), and its regulator TBC-E (Fig. S19D), tree topologies are consistent with gene duplication and diversification prior to the split of the Anaeramoeba spp. We also observed three strongly supported clades within TBC-K (Fig. S19D) that predate the Anaeramoeba split. TBC-K regulates another GTPase Arf631, which was recently shown in metazoan systems to regulate phagosomal maturation. Notably, Rab35 and Arf6 operate in a mutually antag- onistic mechanism in multiple mammalian systems32,33. Collectively, these analyses show expansions of the complement of membrane-trafficking components in Anaeramoeba spp. that, in other organisms, regulate phagosomal maturation. But this family complement expansion is not true of all Anaeramoeba membrane- trafficking machinery. A recent study34 of the vesicle coat forming machinery (including coats that act within the endosomal system) Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 8 www.nature.com/naturecommunications found these proteins to be largely encoded by a single copy in A. ignava, resembling the canonical eukaryotic complement. Overall, our findings are consistent with the existence of a specialized mod- ulation and control system for the symbiont-containing vacuoles that allows Anaeramoeba spp. to regulate the acquisition andmaintenance of its symbiont mass. Discussion Symbiosomes have evolved on numerous occasions across the tree of life and their origins are associated with massive changes to host cell biology and physiology1. Our analyses suggest that the ancestor of Anaeramoeba spp. evolved a subcellular symbiosome to house Desul- fobacter-related symbionts in a way that has drastically reshaped their cell biology and metabolic capacities. The notable expansion of some membrane-trafficking genes in the Anaeramoeba common ancestor suggests that the membrane compartment housing the symbionts is a highly evolved structure that might allow them to selectively manage their captured symbionts and position them in tight association with their hydrogenosomes. To our knowledge, the only superficially similar subcellular arrangements of hydrogenosomes and symbionts have been observed in certain anaerobic ciliates (Scuticociliatia and Karyorelictea)4,5, the heterolobosean Psalteriomonas lanterna35, and obligately symbiotic parabasalids (Trichonympha and Spiro- trichonympha), which are found in the termite digestive tract36,37. These organisms appear to have metabolic interdependencies with endosymbiotic and ectosymbiotic prokaryotes—often methanogens and/or sulfate-reducing bacteria—with some of the latter being housed in shallow host-derived cell membrane invaginations with hydro- genosomes in close proximity36,37. While the symbionts in Anaera- moeba are almost completely compartmentalized, they nevertheless retain connections to the external environment (Fig. 2h, i), presumably due tometabolic constraints such as the need to access sulfate, as seen in theDesulfovibrio symbionts of Trichonympha37,38. Brown-pigmented karyorelictid ciliates in anoxic sediments house several symbiont consortia including a member of Desulfobacteraceae that are not closely related to theAnaeramoebae symbionts (Fig. S10). These ciliate symbionts heavily transcribe genes needed for DSR even though they appear to reside in double-membraned host-derived vacuoles with no obvious mechanism for sulfate provisioning5. In the Anaeramoebae, it remains unclear the degree to which the cell surface connections to the symbiosomes are relatively static or continuously forming and closing. Regardless, given the many fine connections between indivi- dual symbiont-housing subcompartments there appears to be con- tinuity between the lumen of a large majority of these subcompartments (Fig. 2h, i and Fig. S4, S5) allowing metabolite exchange between symbionts and with the external environment. The WGA staining experiment indicates that this exchange is rapid and extensive. The specificity of symbiosomes for colonizing prokaryotes can be multifactorial, influenced by host and symbiont metabolic com- plementarity, receptor recognition, and host secondary metabolite production, which inhibits disruptive colonizers or pathogens39. Intriguingly, the Anaeramoeba genomes sequenced herein have many LGT-derived genes previously implicated in protein-protein interac- tions and cell-cell adhesion in host-symbiont systems18, as well as several non-ribosomal peptide synthases (Supplementary Data 7) whose products might be influencing which bacteria can successfully colonize the symbiosome. Hydrogen depletion is likely to be an important reason for host dependence on the Anaeramoeba symbionts. However, the large repertoire of host-encoded vitB12-dependent enzymes includes essential proteins involved in hydrogenosome metabolism and nucleotide scavenging, suggesting that a degree of metabolic com- plementarity exists and serves as a stabilizing factor. The lateral acquisition in A. ignava BMAN of cobC, encoding the final enzyme in vitB12 biosynthesis, in A. ignava BMAN may help to optimize the synthesis of vitB12. Diatoms have been shown to strongly induce vitB12-binding proteins under limiting growth conditions40 and mod- eling predicts active vitB12 release from bacteria in co-cultures22. How vitB12 is harvested from bacteria by microbial eukaryotes is not clear but directed release and lysis have both been suggested22,41. Whereas evidence for the obligate nature of the A. flamelloides symbionts is strong (i.e., the highdegreeof genomedegeneration), the degree of autonomy of the A. ignava symbiont is less clear. Although all threeAnaeramoeba strains examined herehaveDesulfobacteraceae symbionts, we do not know whether all Anaeramoebae exclusively establish partnerships with this group of bacteria or whether they can harbor other compatible strains or consortia that would similarly deplete hydrogen and provide vitB12. Hydrogen consumption may be carried out by other taxa such as Arcobacter spp42., some of which are predicted to also be vitB12 prototrophs, or additional Deltaproteo- bacteria that are present in our Anaeramoeba cultures (Fig. S7). Our LGT analyses indicate that no single bacterial lineage has dis- proportionately contributed to the gene repertoire of Anaeramoeba, which is reminiscent of other symbiotic systems in protists43 and insects44. Our data and these other examples are consistent with the shopping bagmodel of gene acquisitions in relation to the evolution of symbiont-derived organelles45. Although we detected several cases of transfers from Desulfobacteraceae, they are not obviously from the resident symbiont lineages. Some of the LGTs we observed might also be derived from past symbionts that have since been replaced by the current lineages. Thenumbers of predicted foreign genes inAnaeramoeba is higher than for most microbial eukaryote genomes investigated17. Many of these LGTs appear to be related to adaptation to life in low-oxygen environments, and only a few show obvious links to the establishment and maintenance of the symbiosis with their Desulfobacteraceae symbionts. The fact that the Desulfobacteraceae are not found to be major LGT donors suggests that the phagocytic capacity of Anaera- moeba may give rise to more foreign genes than does the symbiosis itself; symbiosome-associated bacteria may only rarely be fully inter- nalized and digested, thus providing few opportunities for gene transfer. We have provided deep insight into the evolution of a unique, elaborate symbiosome in an anaerobic protist lineage. We have revealed the detailed structure of this organelle as well as some of the metabolic interactions and selective pressures that led to its estab- lishment, and the evolutionary consequences on the genomes of the partner organisms. Future studies will focus on dissecting the fine- scale structure and function of the Anaeramoeba symbiosomes and host-symbiont interactions. Methods Culturing and harvesting of Anaeramoeba A. flamelloides BUSSELTON2 and SCHOONER1 and A. ignava BMAN cells were propagated as described in ref. 7. Large-scale cultures of Anaeramoeba were grown in 0.25× ATCC medium: 1525 Seawater 802 (SW802)mediummedia in fully filled 550mLCellstar® T175 low-profile flasks (Grenier Bio-OneGmbH).Cells weredislodgedwith a cell scraper and split 1:1 with fresh media every 2–4 days for A. flamelloides strains or 7–10 days for A. ignava BMAN. Large-scale cultures were harvested by decanting the culture supernatant and rinsing the ameba monolayer in each flask with 250mL 1× Artificial Seawater (ASW) (per liter medium: 24.72 g NaCl, 0.67 g KCl, 1.364g CaCl2× 2H2O, 4.66g MgCl2× 6H2O, 6.29g MgSO4× 7H2O, 0.18 g NaHCO3-). Cells were detached by cold-shock by adding 35mL of ice-cold 1× ASW and immersing the flasks in ice-slush for 15min. Percussive force was used to ensure efficient cell detach- ment. The cell suspensions were centrifuged at 500 × g for 8min at 4 °C and the combined cell pellets were resuspended in 10mL ice-cold Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 9 www.nature.com/naturecommunications 1× ASW. Centrifugation was repeated as above, and the cell pellet was processed further for RNA or DNA extraction. Cultures of Anaera- moeba strains are available upon request. RNA extraction and sequencing A. flamelloides BUSSELTON2 and A. ignava BMAN cells were harvested as above and total RNAwas extracted using TRIzol™ Reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Ten µg of total RNAwas treated by the TURBODNA-free™ Kit (Thermo Fisher Scientific) then treated with the DNase inactivation reagent. Total RNAwas submitted to GénomeQuébec for sequencing. Libraries were made using the Illumina TruSeq RNA strand-specific sequencing kit and were sequenced on an Illumina HiSeq 4000 using 100 bp paired reads. Illumina readswerequality checked using FastQC v.0.11.5 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and trimmed using Trimmomatic v0.3646. Enrichment of prokaryotic mRNA and sequencing Total RNA from enriched A. ignava BMAN and A. flamelloides BUSSELTON2 samples were treated by Terminator Exonuclease (Epi- centre) according to the manufacturer’s instructions. The total RNA samples were submitted to Génome Québec where polyA+ selection was performed to remove eukaryote mRNAs. One µl of supernatant from these RNA selections was used as input into the fragmentation step ahead of first-strand cDNA synthesis in the TruSeq protocol. The final library was sequenced on the Illumina NovaSeq 6000 S2 using 150 bp paired reads. Illumina reads were quality-checked and trimmed as above. DNA extraction and short-read sequencing DNAwas purified from large-scale cultures using theMagAttract HMW gDNA kit (Qiagen) using the tissue lysis protocol, then further purified on the GenomicTip G/20 column (Qiagen) by the manufacturer’s protocol. Sample quality and quantity were assessed by agarose gel electrophoresis, UV specrophotometry and the Qubit™ dsDNA BR Assay Kit (Thermo Fisher Scientific). DNA samples for Illumina short-read sequencing were submitted to Génome Québec for the construction of shotgun and PCR-free shotgun libraries (see Supplementary Data 8) using the Illumina Tru- Seq LT kit. The libraries were sequenced on an Illumina HiSeq X using 150 bp paired reads. Reads were quality-checked and trimmed as above. Long-read sequencing and basecalling Genomic DNAs (1–5 µg) prepared as above were processed using Oxford Nanopore LSK108, LSK109, or LSK308 kits to construct sequencing libraries. The libraries were loaded on FLO-MIN106 (R9.4 or R9.4.1 pore) or FLO-MIN107 (R9.5 pore) flowcells and sequenced on the MinIONMk2 nanopore sequencer (Oxford Nanopore) running the MinKNOW control software. The fast5 files were basecalled to fastq format using Guppy v2.3.5 (Oxford Nanopore). The fastq reads were trimmedusing Porechop v0.2.3_seqan2.1.1 (https://github.com/rrwick/ Porechop) with the --discard_middle flag. Assembly and correction The A. ignava BMAN read set was assembled using ABruijn (v1.0)47 using default parameters. A. flamelloides BUSSELTON2 and SCHOONER1 read sets were assembled in metagenomics mode (--meta) using Flye (v2.4) with 3000bpmin overlap and Flye (v2.4.2)48 with 1500bpmin overlap respectively. Read mapping steps for Illumina short-reads were performed by bowtie2 (v2.3.1)49 and Nanopore reads were mapped by minimap2 (v2.10-r761)50 with parameters (-ax map-ont). The BMAN assembly was corrected by three rounds of Racon (v0.5)51 followed by Nanopolish v0.8.4 (nanopolish-git-dec-18-2017)52. The final BMAN assembly was obtained by two rounds of Pilon (v1.22) polishing employing (--mindepth 5 –fix bases) parameters53. The BUSSELTON2 and SCHOONER1 assemblies were corrected by four rounds of Racon (v1.4.13) with settings: -u -m 8 -x -6 -g -8 -w 500. Draft genomes were polished using Medaka v0.6.2 (https:// github.com/nanoporetech/medaka) using the (-m r941_trans) model. The final assemblies were generated by five rounds of Pilon (v1.23)53 polishing employing (--mindepth 1 --fix bases,amb) parameters. Genome classification and binning Genome binning was performed manually utilizing the combined evidence of mapped polyA+ selected RNA sequencing data, sequence similarity searches (blastn and blastx) against the NCBI nr database, long-read coverage information, and GC-content of contigs. Chimeric and/or misassembles were identified by consistency of long-read mapping and split manually at the read-mapping border to retain the eukaryotic part of the contig. The presence of spliceosomal introns with GT-AG boundaries in genes was the main criterion for assigning a contig as being eukaryotic. RNAseq data forA. ignavaBMANandA. flamelloidesBUSSELTON2 were mapped using Hisat2 v2.1.054 using (--rna-strandness RF -- phred33 --max-intronlen 10000 -k 2) flags. For A. flamelloides SCHOONER1, the BUSSELTON2 dataset was mapped with Hisat2 v2.1.054 using relaxed parameters (--phred33 --max-intronlen 10000 -k 2 --mp 1,1 --sp 20). The GC% for each contig was calculated by countgc.sh in the BBMap package v38.20 (sourceforge.net/projects/bbmap/). Hybrid assembly of symbiont genomes Long- and short sequence reads mapping to each Desulfobacteraceae genome were extracted and re-assembled using the hybrid-assembler Unicycler. For A. flamelloides SCHOONER1, the long-read data was assembled using Flye (v2.4.2 with --meta flag)48. A detailed description of symbiont genome assemblies can be found in Supplementary Note 4. ANI values were calculated using the OAT software55. Prokaryotic annotation The reassembled symbiont genomes were annotated by Rapid Anno- tation using Subsystem Technology v2.0 (RAST56:) (Sym_BMAN; 2294.7, Sym_BUSS2, 2294.12; Sym_SCH1 2294.13). Insertion sequences were predicted using the ISsaga v2.0 webserver (http://issaga.biotoul. fr/ISsaga2/). Pseudogene candidates were identified using Pseudo- finder v0.1157 and additionally refined bymanual curation. Synteny was calculated using Sibelia v3.0.758. Circos plots were created with Circa (http://omgenomics.com/circa). 16S rDNA amplicon typing The Anaeramoeba-enriched cell material was prepared by cold-shock and monolayer rinsing as described above. The supernatant samples were obtained by pelleting 10mL of culture supernatant poured off prior to monolayer rinsing. The samples were extracted using the DNeasy PowerSoil Pro Kit (Qiagen) according to the manufacturer’s instructions. The V4V5 (Bacteria) or V6V8 (Archaea) regions of the 16S rRNA genes were amplified and sequenced at the Integrated Microbiome Resource (IMR) at Dalhousie University, as described in ref. 59. Bioin- formatic processing of raw reads was carried out by IMR (see ref. 59), updated in the Standard Operating Procedures (SOPs) for creation of ASV in QIIME2 (v2019.7) as outlined on the current MicrobiomeHelper website (https://github.com/LangilleLab/microbiome_helper/wiki). Phylogenomics Phylogenomic analysis was performed on the symbiont genomes and genomes from Desulfobacterales (NCBI:txid213118) supplemented by Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 10 http://www.bioinformatics.babraham.ac.uk/projects/fastqc https://github.com/rrwick/Porechop https://github.com/rrwick/Porechop https://github.com/nanoporetech/medaka https://github.com/nanoporetech/medaka http://issaga.biotoul.fr/ISsaga2/ http://issaga.biotoul.fr/ISsaga2/ http://omgenomics.com/circa https://github.com/LangilleLab/microbiome_helper/wiki www.nature.com/naturecommunications data from outgroup taxa. The initial selection of representative gen- omes and marker genes/proteins was done using phyloSkeleton v1.160 from 353 Desulfobacterales genomes from NCBI. One representative genome per genus was selected in Desulfobacteraceae except for Desulfobacter where all available genomes were selected. The Bac109 protein markers were identified with phyloSkeleton v1.160 and only genomes with >80% of the marker genes were selected for further analyses. The UPF0081 marker was removed since it was only present in the out-group genomebut not in any of the in-group genomes. Each of the 108 remaining marker proteins was aligned by MAFFT-linsi v7.45861 and trimmed by BMGE v1.1262, using the BLOSUM30 matrix and stationary-based character trimming. Alignments were con- catenated and a phylogenetic tree was inferred by IQTree v2.0.363 using the LG4X64 substitution matrix with 1000 ultrafast bootstraps65. The resulting tree was inspected, closely related taxa were removed, and the procedure was repeated. The final dataset consisted of 35 genome-derived sequences: Desulfovibrio desulfuricans ND132 (1 gen- ome), Desulfobacteraceae (23 genomes), Desulfobacter (8 genomes) and the Anaeramoeba symbionts (3 genomes). A maximum-likelihood tree was inferred using IQ-TREE v2.0.363 using the LG +C60+ F+ Gammamixturemodel66. The PMSFmodel67 generated from the above guide-tree and fitted model was used to perform 100 nonparametric bootstrap replicates. Protein-coding gene family expansions and contractions We employed the PANTHER family database (v15)68 to examine which protein families may have undergone expansions or contractions. Assignment of proteins to families was done with direct hidden Mar- kov models (HMM) searches and the Panther Score tool69 (set for hmmsearchwithHMMERv3.1b270) using the PANTHER familydatabase with e-value cut-off <1e-5. Once classified, proteins assigned to each PANTHER familywere counted to investigate relative family expansion or contraction in any Anaeramoeba species relative to other groups. (Protein families that were taxon-specific, exclusive to each group being compared, apparently heavily expanded in diplomonads, Tri- chomonas, DictyosteliumorNaegleria, and thosewithmediancountsof zero for any compared group were not considered in the expansion/ contraction analysis.) To detect signatures of expansion/contraction, outlier values within each group were replaced by the median value of their respective group when the value had a 1.5 > z-score < -1.5. Data were normalized by the median value of their respective families and log2FC among groups was calculated. To identify what metabolic pathways were relevant for further analyses, we investigated families with signature of expansions or contractions ≥ 5-fold (2.35 > log2FC or log2FC< −2.35). Relative contractions and expansions are reported for Anaeramoeba taxa and protein families were considered “core’ if they were present in all studied proteomes and “accessory’ if they were missing in at least one. Possible expansions/contractions were visua- lizedwith Circos71. A large proportion of the protein families expanded beyond ≥ 5-fold belong to RNA synthesis, DNA synthesis and repair, and membrane trafficking metabolisms. Hence, we carried out path- way reconstruction for RNA synthesis and membrane trafficking (see below) by validating protein orthology and recording absence/pre- sence patterns. In the case of RNA systems, we first classified query proteins of interest (from yeast or human) with PANTHER Score (as above) to identify their PANTHER family. Query proteins and proteins identified for each PANTHER family of interest were then aligned together with a random taxonomic sample from the same family of interest from the PANTHER database. Alignments were trimmed to carry out phylogenetic reconstructions as described in ref. 72. For membrane-trafficking proteins, Anaeramoeba datasets were searched by BLAST using queries from previous studies73–75. Identified Rab and TBC proteins were subjected to orthologous clustering by OrthoFinder v2.0.027 including proteins from human, cnidarian Nematostella vectensis, and the heterolobosean Naegleria gruberi. Phylogenetic analyses for selected Rab sub-families and TBC proteins were conducted. Sequences were aligned by MAFFT v7.45861 under L-INS-i strategy, and poorly aligned positions were removed by trimAl v1.4.rev1576 using -gt 0.8. Maximum-likelihood trees were inferred by IQ-TREE v1.6.877 using the PMSF method67 and the LG +C20 + F +G model, with the guide tree inferred under the LG + F +G model. Ultrafast bootstrap (UFBOOT2) branch supports were obtained with 1000 replicates. Lateral gene transfer in Anaeramoeba In order to assess LGT from prokaryotic and viral donors, we con- ducted a large-scale screen of the predicted proteomes of the three Anaeramoeba genomes. Initial clustering was performed using Orthofinder v2.5.427 with the following outgroup proteomes: Arabi- dopsis thaliana, Dictyostelium discoideum, Acanthamoeba castellanii str. Neff, Giardia intestinalis, Homo sapiens, Kipferlia bialata, Mono- cercomonoides sp., Naegleria gruberi, Trepomonas PC1, Trypanosoma brucei, Trichomonas vaginalis, Saccharomyces cerevisiae. In some instances, Orthofinder appeared to cluster proteins that were assigned different PANTHER annotations obtained in the protein-coding gene family expansion/contraction analysis, indicating they were unlikely to be true orthologs. In such cases, clusters were further split according to the PANTHER annotations. Proteins for phylogenetic trees were inferred by gathering homologous sequences in the nr database using blastp using an E-value cutoff of 1 × 10−5. All Anaeramoeba orthologs/paralogs con- stituting a cluster together with the 500 best database hits to each were grouped. Clusters containing at least one prokaryotic or viral protein together with Anaeramoeba orthologs/paralogs were aligned using MAFFT v7.310 61 with the default setting and sites were selected using BMGE v1.062. Initial phylogenetic reconstructions were done using FastTree v1.0.178. These phylogenies were used to reduce the taxonomic redundancy of the initial sequence files using in-house scripts. The reduced files were realigned using MAFFT v7.310 61 with the accurate option (L-INS-i), and sites were selected using BMGE v1.062. Phylogenetic reconstructions were done using IQ-TREE multi- core v1.5.577 with the LG4X model and 1000 UFBOOT for alignments ≥ 80 sites (shorter alignments were treated separately, see below). Phylogenetic trees were then parsed to find putative LGTs, using the following criteria: i) When Anaeramoeba sequence(s) and prokaryotic or viral sequences constituted a clade supported by an UFBOOT value of ≥ 70%, the Anaeramoeba homologs were considered as possible LGTs. To allow formis-annotation, one other sequence in that clade could be eukaryotic. ii) When no clades were well-supported, if ≥ 95% of the sequences in the tree were prokaryotic and/or viral, then this was counted as an LGT. iii) The taxonomic group of the donor could be inferred when >50% of the taxa in a clade containing the Anaeramoeba protein(s) were from a particular taxonomic group. iv) Treeswere estimated for only a subset ofOGs that contained at least one prokaryotic or viral taxa and >80 sites. However, when all sequences constituting the cluster, besides Anaeramoeba, were pro- karyotic or viral, it was counted as LGT. FISH probe design and testing The symbiont 16S rDNA sequences were extracted from the RAST annotations and aligned by MAFFT v7 (https://mafft.cbrc.jp/ alignment/software/) to selected full-length 16S rDNA sequences selected from the Ribosomal Database Project (RDP). Probes targeting Sym_BUSS2 and Sym_SCH1 to the exclusion of outgroups in the 16S rDNA alignment were designed using Decipher79. The probes were evaluated by matching against the SILVA and RDP databases and did not match any other sequences in the database at 1 Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 11 https://mafft.cbrc.jp/alignment/software/ https://mafft.cbrc.jp/alignment/software/ www.nature.com/naturecommunications mismatch. The probes were ordered from biomers.net GmBH. Probe sequences,fluorophores, andhybridization conditions canbe found in Supplementary Data 9. Control probing for probe BUSS/SCH is shown in Fig. S7. Fluorescence in situ hybridization (FISH) Anaeramoeba cell cultures in 10mL slanted culture tubes were dec- anted and the adhered cells were resuspended in the remaining volume of culture media (typically 200 µl) by percussive shock. The suspended cells were applied to Teflon multi-well microscope slides (Electron Microscopy Sciences (ER-264)) for 5min at room tempera- ture. The cells were fixed for 10min at room temperature by adding a suitable volume of 16% methanol-free formaldehyde (w/v) (Pierce- ThermoFisher Scientific,CatNo28908) to give afinal concentrationof 4% paraformaldehyde in ASW. The cells were rinsed in 2 × 50mL dis- tilled water for a total of 3min and then air-dried. The slide was immersed in 100% ethanol for 5min and air-dried again. Hybridization buffer (20mM Tris-HCl, pH 7.6, 0.01% SDS, 900mM NaCl) with an appropriate concentration of formamide and probe (5 ng/µl) (Sup- plementary Data 9) was added to dried cells and incubated for 2–3 h at 47 °C in amoisture chamber. Post-hybridization, the slides were rinsed with buffer (20mM Tris-HCl, pH 7.6, 0.01% SDS, 5mM EDTA and an appropriate NaCl concentration depending on the %FA in the hybri- dization buffer; see Supplementary Data 9) and incubated with 50mL for 45min at 48 °C, finally rinsed in water for 40 s and air-dried. The slides were mounted in ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific, Cat No P36971) using #1.5H cover slips (Ibidi, Cat No 10811). The slides were incubated at room tempera- ture ≥ 12 hours before imaging. Images were acquired using either wide-field microscopy on a Zeiss Axio Imager Z2 or by confocal microscopy on a Zeiss LSM 710 or Leica SP8. Pseudo color, merging of channels and projections were made in Zeiss ZEN v3.1 or BioImageXD80. Scanning electron microscopy A. flamelloides BUSSELTON2 cells were cultured and harvested as described above with cells finally being resuspended in 500 µL of ice- cold filtered growthmedia. 50 µL of cells were transferred onto poly-L- lysine-coated 12mm round coverslips, left to adhere for 5min at room temperature and immediately fixed with a drop of 25% glutaraldehyde andOsO4 vapor for 1 h. After fixation, the coverslipswerewashed three times in filtered ASW, and then subjected to a dehydration series of ethanol-water mixtures, as follows: 30%, 50%, 70%, 80%, 90%, 95%, 100% (three times). This was followed by critical-point drying with CO2 on a Leica EM CPD300, then an ∼15 nm Au-Pd coat was added with a Leica EM ACE200 sputter-coater. Samples were imaged on a Hitachi S4700 scanning electron microscope. Transmission electron microscopy Fixation (both chemical and cryofixation), embedding, sectioning, and TEM were conducted as described in detail by ref. 7. Focused-ion-beam scanning electron microscopy (FIB-SEM) Cells were adhered to the surface of a gridded MatTek dish and fixed with 2.5% glutaraldehyde (TAAB) in 0.1M PHEM-buffer. All samples were processed using a Pelco Biowave Pro+ microwave tissue pro- cessor (Ted Pella, Redding, CA) according to81 with minor modifica- tions: no calcium was used during fixation and the contrasting steps with lead aspartate was omitted to reduce the risk of overstaining. Samples were detached from the glass using liquid nitrogen and glued to an SEM-stub with epoxy and silver glue. Samples were further coatedwith 5 nmplatinum to reduce charging. Volumeswere acquired using a Scios dual-beam (Thermo Fischer Scientific) with the electron beam operating at 2 kV/0.2 nA detected with a T1 In-lens detector. To automate volume acquisition, we used the Auto Slice and View 4 softwareprovidedwith themicroscope. A 700nmprotective layer of platinumwas deposited on the selected area beforemilling. A FIB-SEM volume of 1780 slices of A. flamelloides BUSSELTON2 was acquired close to isotropic resolution (6.7 × 6.7 × 7 nm). Volumes were further registered andprocessed using the ImageJ plugins Linear alignment by SIFT andMultistackreg. After registration the volumes were converted tomrc-files and headerwasmodified to recover the pixel-sizes that got lost during conversion. Segmentation and visualization of cell structures We segmented eight cell structures (nucleus, symbionts, hydrogeno- somes, symbiosome-membrane, dense granules, other prokaryotes, plasma membrane, and the acentriolar centrosome with individual microtubules) usingMicroscopy Image Browser v2.8482,83. The nucleus was segmented using the Graphcut semi-automatic segmentation function in MIB. Symbionts, symbiosome-membrane, and hydro- genosomes were segmented by the deep-learning segmentation tool in MIB (DeepMIB). Briefly, symbionts and hydrogenosomes were manually annotated in a 50-slice segment of the FIB-SEM volume abundant in symbionts and hydrogenosomes. Image segments (patch size 256× 256) were extracted and used to train DeepLabV3 ResNet50 model using default settings. The trained model was then used to predict segmentations for symbionts and hydrogenosomes. The resulting symbiont model was manually refined using the MIB seg- mentation tools. The hydrogenosomes and dense granules were pre- dicted together by the classifier and weremanually separated by hand using the MIB segmentation tools. Other prokaryotes were partially annotated by the symbiont+hydrogenosome model and were sepa- rated manually for additional curation by hand using the MIB seg- mentation tools. The symbiosome-membranes were predicted by training amodel that segments whole symbionts (symbiont+symbiont subcompartment and membrane) as described as for symbiont and hydrogenosomes above. The outer membranes were obtained by eroding the whole symbiont predictions by 3 pixels (approximated thickness of the symbiosome-membrane) and using this selection as a mask to cut out the outermost 3 pixels of the whole symbiont model. The plasma membrane was manually annotated using black-white thresholding of brush-traced selections every 5–10 slices and shape interpolation was applied between those slices. In segments where the membrane was sharply shifting between slices individual slices were segmented by hand using black-white thresholding of brush selec- tions. The acentriolar centrosomewas segmented using Graphcut and individualmicrotubulesweremanually annotatedusing thebrush tool. Symbiosome subcompartment connectivity was manually traced, annotated and visualized inMIB v2.8482,83. The volumes were rendered using ORS Dragonfly v2022.2.0.1399. Further information about FIB- SEM is reported in Supplementary Note 5. Tubulin staining For tubulin staining cells were harvested and fixed for 10min in 4% paraformaldehyde according to the procedure described in the “Fluorescence in situ hybridization (FISH)’ section. Fixative was removed, and the cells were washed twice with ASW and once with PBS. Cells were then incubated 15min in PBS with 50mM NH4Cl to quench remaining aldehyde fixative. The cells were washed twice with PBS and permeabilized for 15min in PBS with 0.1% Triton X100. Cells were blocked in antibody dilution buffer (ADB - 1% BSA-c (Aurion) in PBS with 0.1%Triton X100) for 1 h at room temperature. The cells were then incubated with primary antibodies diluted in ADB (TAT1; 1:200 dilution or KMX-1; 1:200 dilution) overnight at 4 °C in a moisture chamber. The slides were droplet-washed six times using excess ADB and incubated 1 hour at room temperature with secondary antibody (goat anti-mouse Alexa Fluor 594, 1:250 dilution, Thermo Fisher Sci- entific, Cat No A-11032). The cells were washed six times using ADB, twice with PBS, and then mounted in ProLong™ Diamond Antifade Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 12 www.nature.com/naturecommunications Mountant with DAPI (Thermo Fisher Scientific, Cat No P36971) using #1.5H cover slips (Ibidi, Cat No 10811). Images were acquired using wide-field microscopy on the Zeiss Axio Imager Z2 or by confocal microscopy on Zeiss LSM 710 or Leica SP8. Pseudo color, merging of channels and projections were made in Zeiss ZEN or BioImageXD80. Wheat Germ Agglutinin (WGA) staining For WGA staining two different protocols were used, the first was a live stain protocol, and the second relied on staining after aldehyde fixation. In both protocols, cells were harvested and attached to multi-well slides according to the FISH procedure (above). Live stain cells were washed twice using 100 µl ASW and then incubated for 10min in 50 µg/ml WGA-CF633 conjugate (Biotium, Cat No #29024-1) in ASW. The stain was removed, and cells were washed twice in 100 µl ASW and then post-fixed in 4% formaldehyde in ASW for 10min at room temperature. For the aldehyde fixation protocol, cells were fixed in 4% for- maldehyde in ASW immediately after attachment and the first two washes. The fixative was removed, and the cells were washed twice in 100 µl ASW. Each slide was rinsed twice in a large volume of distilled water for 1min 30 s each and air-dried. The slides were incubated 5min in 100% ethanol, air-dried and the slide was mounted and imaged according to the procedure described in the FISH section. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability Sequencing reads and the annotated genomes of A. flamelloides BUS- SELTON2, A. flamelloides SCHOONER1, A. ignava BMAN, Sym_BUSS2, Sym_SCH1 and Sym_BMAN were deposited to NCBI under the BioPro- ject number PRJNA634776 [https://ncbi.nlm.nih.gov/bioproject/ 634776]. FIB-SEM raw data, aligned mrc-files and segmentation mod- els inORSObject format are available fromFigshare: https://doi.org/10. 6084/m9.figshare.24033777. Tree-files and alignments are available from Figshare: Fig. 3 at https://doi.org/10.6084/m9.figshare.20375619, Figure S10 at https://doi.org/10.6084/m9.figshare.20375601, Fig- ure S19 at https://doi.org/10.6084/m9.figshare.22193497. Animations are available at Figshare: Movie S1 at https://doi.org/10.6084/m9. figshare.27108724, Movie S2 at https://doi.org/10.6084/m9.figshare. 27108751. References 1. Fronk, D. C. & Sachs, J. L. Symbiotic organs: the nexus of host–microbe evolution. Trends Ecol. Evol. 37, 599–610 (2022). 2. Roth, L. E., Jeon, K. & Stacey, G. Homology in endosymbiotic sys- tems: the term “symbiosome’. In Molecular Genetics of Plant- Microbe Interactions 220–225 (APS Press, St Paul, Minnesota). 3. Husnik, F. et al. Bacterial and archaeal symbioseswithprotists.Curr. Biol. 31, R862–R877 (2021). 4. Rotterová, J., Edgcomb, V. P., Čepička, I. & Beinart, R. Anaerobic ciliates as a model group for studying symbioses in oxygen‐ depleted environments. J. Eukaryot. Microbiol. 69, e12912 (2022). 5. Beinart, R. A., Beaudoin, D. J., Bernhard, J. M. & Edgcomb, V. P. Insights into the metabolic functioning of a multipartner ciliate symbiosis from oxygen‐depleted sediments. Mol. Ecol. 27, 1794–1807 (2018). 6. Stairs, C. W. et al. Anaeramoebae are a divergent lineage of eukaryotes that shed light on the transition from anaerobic mitochondria to hydrogenosomes. Curr. Biol. 31, 5605–5612.e5 (2021). 7. Táborský, P., Pánek, T. & Čepička, I. Anaeramoebidae fam. nov., a novel lineage of anaerobic amoebae and amoeboflagellates of uncertain phylogenetic position. Protist 168, 495–526 (2017). 8. Moran, N. A. & Plague, G. R. Genomic changes following host restriction in bacteria. Curr. Opin. Genet. Dev. 14, 627–633 (2004). 9. Kupper, M., Gupta, S. K., Feldhaar, H. & Gross, R. Versatile roles of the chaperonin GroEL in microorganism-insect interactions. FEMS Microbiol. Lett. 353, 1–10 (2014). 10. Grime, J. M. A., Edwards, M. A., Rudd, N. C. & Unwin, P. R. Quanti- tative visualization of passive transport across bilayer lipid mem- branes. Proc. Natl. Acad. Sci. USA 105, 14277–14282 (2008). 11. Woehle, C. et al. A novel eukaryotic denitrification pathway in for- aminifera. Curr. Biol. 28, 2536–2543.e5 (2018). 12. Woehle, C. et al. Denitrification in foraminifera has an ancient origin and is complemented by associated bacteria. Proc. Natl. Acad. Sci. USA 119, e2200198119 (2022). 13. Gardner, M. J. et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511 (2002). 14. Li, X.-D., Lupo, D., Zheng, L. & Winkler, F. Structural and functional insights into the AmtB/Mep/Rh protein family. Transfus. Clin. Biol. 13, 65–69 (2006). 15. Sibbald, S. J., Eme, L., Archibald, J. M. & Roger, A. J. Lateral gene transfer mechanisms and pan-genomes in eukaryotes. Trends Parasitol. 36, 927–941 (2020). 16. Cote-L’Heureux, A., Maurer-Alcalá, X. X. & Katz, L. A. Old genes in new places: a taxon-rich analysis of interdomain lateral gene transfer events. PLoS Genet. 18, e1010239 (2022). 17. Van Etten, J. & Bhattacharya, D. Horizontal gene transfer in eukar- yotes: not if, but how much? Trends Genet. 36, 915–925 (2020). 18. Hinzke, T. et al. Host-microbe interactions in the chemosynthetic Riftia pachyptila symbiosis. mBio 10, e02243–19 (2019). 19. Aramaki, T. et al. KofamKOALA: KEGG ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics 36, 2251–2252 (2020). 20. Raux, E., Schubert, H. L. & Warren, M. J. Biosynthesis of cobalamin (vitamin B12): a bacterial conundrum. Cell. Mol. Life Sci. 57, 1880–1893 (2000). 21. Croft, M. T., Lawrence, A. D., Raux-Deery, E., Warren, M. J. & Smith, A. G. Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature 438, 90–93 (2005). 22. Grant, M. A. A., Kazamia, E., Cicuta, P. & Smith, A. G. Direct exchange of vitamin B12 is demonstrated by modelling the growth dynamics of algal-bacterial cocultures. ISME J. 8, 1418–1427 (2014). 23. Vancaester, E., Depuydt, T., Osuna-Cruz, C. M. & Vandepoele, K. Comprehensive and functional analysis of horizontal gene transfer events in diatoms. Mol. Biol. Evol. 37, 3243–3257 (2020). 24. Homma, Y., Hiragi, S. & Fukuda, M. Rab family of small GTPases: an updated view on their regulation and functions. FEBS J. 288, 36–55 (2021). 25. Hutagalung, A. H. & Novick, P. J. Role of rab GTPases in membrane traffic and cell physiology. Physiol. Rev. 91, 119–149 (2011). 26. Jeschke, A. & Haas, A. Sequential actions of phosphatidylinositol phosphates regulate phagosome-lysosome fusion. Mol. Biol. Cell 29, 452–465 (2018). 27. Emms, D. M. & Kelly, S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 20, 238 (2019). 28. Diekmann, Y. et al. Thousands of rab GTPases for the cell biologist. PLoS Comput. Biol. 7, e1002217 (2011). 29. Haley, R. & Zhou, Z. The small GTPase RAB-35 facilitates the initia- tion of phagosome maturation and acts as a robustness factor for apoptotic cell clearance. Small GTPases 12, 188–201 (2021). 30. Verma, K. & Datta, S. The monomeric GTPase Rab35 regulates phagocytic cup formation and phagosomal maturation in Enta- moeba histolytica. J. Biol. Chem. 292, 4960–4975 (2017). Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 13 https://ncbi.nlm.nih.gov/bioproject/634776 https://ncbi.nlm.nih.gov/bioproject/634776 https://doi.org/10.6084/m9.figshare.24033777 https://doi.org/10.6084/m9.figshare.24033777 https://doi.org/10.6084/m9.figshare.20375619 https://doi.org/10.6084/m9.figshare.20375601 https://doi.org/10.6084/m9.figshare.22193497 https://doi.org/10.6084/m9.figshare.27108724 https://doi.org/10.6084/m9.figshare.27108724 https://doi.org/10.6084/m9.figshare.27108751 https://doi.org/10.6084/m9.figshare.27108751 www.nature.com/naturecommunications 31. Falace, A. et al. TBC1D24, an ARF6-interacting protein, is mutated in familial infantile myoclonic epilepsy. Am. J. Hum. Genet. 87, 365–370 (2010). 32. Chesneau, L. et al. An ARF6/Rab35 GTPase cascade for endocytic recycling and successful cytokinesis. Curr. Biol. 22, 147–153 (2012). 33. Kobayashi, H. & Fukuda, M. Rab35 regulates Arf6 activity through centaurin β2/ACAP2 during neurite outgrowth. J. Cell Sci. jcs.098657 https://doi.org/10.1242/jcs.098657 (2012). 34. Maciejowski, W. J., Gile, G. H., Jerlström-Hultqvist, J. & Dacks, J. B. Ancient and pervasive expansion of adaptin-related vesicle coat machinery across Parabasalia. Int. J. Parasitol. 53, 233–245 (2023). 35. Broers, C. A. M., Stumm, C. K., Vogels, G. D. & Brugerolle, G. Psal- teriomonas lanterna gen. nov., sp. nov., a free-living amoebo- flagellate isolated from freshwater anaerobic sediments. Eur. J. Protistol. 25, 369–380 (1990). 36. Sato, T. et al. Candidatus Desulfovibrio trichonymphae, a novel intracellular symbiont of the flagellate Trichonympha agilis in ter- mite gut. Environ. Microbiol. 11, 1007–1015 (2009). 37. Kuwahara, H., Yuki,M., Izawa, K., Ohkuma,M. &Hongoh, Y. Genome of “Ca. Desulfovibrio trichonymphae”, an H2-oxidizing bacterium in a tripartite symbiotic system within a protist cell in the termite gut. ISME J. 11, 766–776 (2017). 38. Takeuchi, M. et al. Parallel reductive genome evolution in Desulfo- vibrio ectosymbionts independently acquired by Trichonympha protists in the termite gut. ISME J. 14, 2288–2301 (2020). 39. Clay, K. Defensive symbiosis: a microbial perspective. Funct. Ecol. 28, 293–298 (2014). 40. Bertrand, E. M. et al. Influence of cobalamin scarcity on diatom molecular physiology and identification of a cobalamin acquisition protein. Proc. Natl. Acad. Sci. USA 109, E1762-71 (2012). 41. Nef, C. et al. Sharing vitamin B12 between bacteria and microalgae does not systematically occur: case study of the haptophyte Tiso- chrysis lutea. Microorganisms 10, 1337 (2022). 42. Hamann, E. et al. Environmental breviatea harbour mutualistic Arcobacter epibionts. Nature 534, 254–258 (2016). 43. Singer, A. et al. Massive protein import into the early-evolutionary- stage photosynthetic organelle of the amoeba Paulinella chroma- tophora. Curr. Biol. 27, 2763–2773.e5 (2017). 44. Husnik, F. et al. Horizontal gene transfer from diverse bacteria to an insect genomeenables a tripartite nestedmealybug symbiosis.Cell 153, 1567–1578 (2013). 45. Howe, C. J., Barbrook, A. C., Nisbet, R. E. R., Lockhart, P. J. & Larkum, A. W. D. The origin of plastids. Philos. Trans. R. Soc. B 363, 2675–2685 (2008). 46. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trim- mer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). 47. Lin, Y. et al. Assembly of long error-prone reads using de Bruijn graphs. Proc. Natl. Acad. Sci. USA 113, E8396–E8405 (2016). 48. Kolmogorov, M. et al. MetaFlye: scalable long-read metagenome assembly using repeat graphs. Nat. Methods 17, 1103–1110 (2020). 49. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with bowtie 2. Nat. Methods 9, 357–359 (2012). 50. Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018). 51. Vaser, R., Sović, I., Nagarajan, N. & Šikić, M. Fast and accurate de novogenomeassembly from longuncorrected reads.GenomeRes. 27, 737–746 (2017). 52. Loman,N. J.,Quick, J. &Simpson, J. T. Acompletebacterial genome assembled de novo using only nanopore sequencing data. Nat. Methods 12, 733–735 (2015). 53. Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014). 54. Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph- based genome alignment and genotyping with HISAT2 and HISAT- genotype. Nat. Biotechnol. 37, 907–915 (2019). 55. Lee, I., Ouk Kim, Y., Park, S.-C. & Chun, J. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol. 66, 1100–1103 (2016). 56. Aziz, R. K. et al. The RAST Server: rapid annotations using sub- systems technology. BMC Genom. 9, 75 (2008). 57. Syberg-Olsen, M. J., Garber, A. I., Keeling, P. J., McCutcheon, J. P. & Husnik, F. Pseudofinder: detection of pseudogenes in prokaryotic genomes. Mol. Biol. Evol. 39, msac153 (2022). 58. Minkin, I., Pham, H., Starostina, E., Vyahhi, N. & Pham, S. C-Sibelia: an easy-to-use and highly accurate tool for bacterial genome comparison. F1000Research2, 258 (2013). 59. Comeau, A. M., Douglas, G. M. & Langille, M. G. I. Microbiome helper: a custom and streamlined workflow for microbiome research. mSystems 2, e00127–16 (2017). 60. Guy, L. phyloSkeleton: taxon selection, data retrieval and marker identification for phylogenomics. Bioinformatics. btw824. https:// doi.org/10.1093/bioinformatics/btw824 (2017). 61. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). 62. Criscuolo, A. & Gribaldo, S. BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic infor- mative regions frommultiple sequence alignments. BMC Evol. Biol. 10, 210 (2010). 63. Minh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534 (2020). 64. Le, S. Q., Dang, C. C. & Gascuel, O. Modeling protein evolutionwith several amino acid replacement matrices depending on site rates. Mol. Biol. Evol. 29, 2921–2936 (2012). 65. Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2018). 66. Si Quang, L., Gascuel, O. & Lartillot, N. Empirical profile mixture models for phylogenetic reconstruction. Bioinformatics 24, 2317–2323 (2008). 67. Wang, H.-C., Minh, B. Q., Susko, E. & Roger, A. J. Modeling site heterogeneity with posterior mean site frequency profiles accel- erates accurate phylogenomic estimation. Syst. Biol. 67, 216–235 (2018). 68. Mi, H. et al. PANTHER version 11: expanded annotation data from gene ontology and reactome pathways, and data analysis tool enhancements. Nucleic Acids Res. 45, D183–D189 (2017). 69. Mi, H. et al. Protocol update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0). Nat. Protoc. 14, 703–721 (2019). 70. Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011). 71. Krzywinski, M. et al. Circos: an information aesthetic for compara- tive genomics. Genome Res. 19, 1639–1645 (2009). 72. Salas-Leiva, D. E. et al. Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist. Nat. Commun. 12, 6003 (2021). 73. Elias, M., Brighouse, A., Castello, C. G., Field, M. C. & Dacks, J. B. Sculpting the endomembrane system in deep time: high resolution phylogenetics of Rab GTPases. J. Cell Sci. jcs.101378 https://doi. org/10.1242/jcs.101378 (2012). 74. Gabernet-Castello, C., O’Reilly, A. J., Dacks, J. B. & Field, M. C. Evolution of Tre-2/Bub2/Cdc16 (TBC) Rab GTPase-activating pro- teins. Mol. Biol. Cell 24, 1574–1583 (2013). 75. Klinger, C. M., Klute, M. J. & Dacks, J. B. Comparative genomic analysis of multi-subunit tethering complexes demonstrates an Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 14 https://doi.org/10.1242/jcs.098657 https://doi.org/10.1093/bioinformatics/btw824 https://doi.org/10.1093/bioinformatics/btw824 https://doi.org/10.1242/jcs.101378 https://doi.org/10.1242/jcs.101378 www.nature.com/naturecommunications ancient pan-eukaryotic complement and sculpting in apicomplexa. PLoS ONE 8, e76278 (2013). 76. Capella-Gutierrez, S., Silla-Martinez, J. M. & Gabaldon, T. TrimAl: a tool for automated alignment trimming in large-scale phyloge- netic analyses. Bioinformatics 25, 1972–1973 (2009). 77. Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ- TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015). 78. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009). 79. Wright, E. S., Yilmaz, L. S., Corcoran, A. M., Ökten, H. E. & Noguera, D. R. Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification. Appl. Environ. Microbiol. 80, 5124–5133 (2014). 80. Kankaanpää, P. et al. BioImageXD: an open, general-purpose and high-throughput image-processing platform. Nat. Methods 9, 683–689 (2012). 81. Deerinck, T.J., Bushong, E.A., Thor, A. & Ellisman, M.H. NCMIR methods for 3D EM: A new protocol for preparation of biological specimens for serial block face scanning electron microscopy e SBEM Protocol v7_01_10. Retrieved from https://www.ncmir.ucsd. edu/sbem-protocol (2010). 82. Belevich, I., Joensuu, M., Kumar, D., Vihinen, H. & Jokitalo, E. Microscopy image browser: a platform for segmentation and ana- lysis of multidimensional datasets. PLoS Biol. 14, e1002340 (2016). 83. Belevich, I. & Jokitalo, E. DeepMIB: user-friendly and open-source software for training of deep learning network for biological image segmentation. PLoS Comput. Biol. 17, e1008374 (2021). 84. Takishita, K. et al. Microbial eukaryotes that lack sterols. J. Eukaryot. Microbiol. 64, 897–900 (2017). 85. Bouwknegt, J. et al. A squalene–hopene cyclase in Schizosacchar- omyces japonicus represents a eukaryotic adaptation to sterol- limited anaerobic environments. Proc. Natl. Acad. Sci. USA 118, e2105225118 (2021). Acknowledgements Gordon Lax and Yana Eglit are acknowledged for help during SEM sample preparation and imaging. The majority of the work and J.J.H. were supported by a Foundation grant (FRN-142349) from the Canadian Institutes of Health Research awarded to A.J.R. J.J.H. was additionally supported by a grant fromVetenskapsrådet, (VR-NT grant 2022-04490). The research was also supported by grant GMBF12188 from the Gordon and Betty Moore Foundation. Archibald Lab contributions to this study were supportedby theGordon andBettyMoore Foundation (GBMF5782) and a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2019-05058). Work from the Čepička lab was funded by Czech Science Foundation grant no. 21- 30563S. Computational resources were provided by the e-INFRA CZ project (ID:90254) supported by the Ministry of Education, Youth and Sports of the Czechia. Research in the Dacks Lab was supported by grants from the Natural Sciences and Engineering Research Council of Canada (RES0043758, and RES0046091). The authors acknowledge the facilities and technical assistance of the Umeå Core Facility Electron Microscopy (UCEM) at the Chemical Biological Centre (KBC), Umeå University, a part of the National Microscopy Infrastructure NMI (VR-RFI 2019-00217). Author contributions Conceptualization, J.J.-H., and A.J.R.; Investigation, J.J.-H., I.Č.; Formal analysis, J.J-H., L.G.L., D.E.S.L., B.A.C., K.Z., C.W.S., S.P.; Resources, I.Č., A.J.R.; Writing—Original Draft, J.J.-H., L.G.L., D.E.S.L., B.A.C., K.Z., J.B.D., J.M.A. and A.J.R.; Writing – Review & Editing, J.J.-H, L.G.L., D.E.S.L., B.A.C., K.Z., I.Č., C.W.S., S.P., J.B.D., J.M.A. and A.J.R.; Funding Acquisi- tion, J.J.-H., I.Č., J.B.D., J.M.A., and A.J.R. Funding Open access funding provided by Uppsala University. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-54102-7. Correspondence and requests for materials should be addressed to Jon Jerlström-Hultqvist or Andrew J. Roger. Peer review information Nature Communications thanks Virginia Edg- comb and the other, anonymous, reviewers for their contribution to the peer review of this work. A peer review file is available. Reprints and permissions information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2024 Article https://doi.org/10.1038/s41467-024-54102-7 Nature Communications | (2024) 15:9726 15 https://www.ncmir.ucsd.edu/sbem-protocol https://www.ncmir.ucsd.edu/sbem-protocol https://doi.org/10.1038/s41467-024-54102-7 http://www.nature.com/reprints http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ www.nature.com/naturecommunications A unique symbiosome in an anaerobic single-celled eukaryote Results The Anaeramoeba symbionts are directly connected to the extracellular milieu Anaeramoeba symbionts belong to Desulfobacteraceae and were acquired independently in different host species The Anaeramoeba-Desulfobacter symbiosis was recently established Anaeramoeba symbionts are metabolically poised to use hydrogenosomal metabolites Anaeramoeba nuclear genomes are A + T rich and have greatly expanded gene families Lateral gene transfer is ongoing in Anaeramoeba Laterally transferred genes shape Anaeramoeba biology Anaeramoeba might acquire vitamin B12 from its symbionts Expansions of endosome/phagosome modulating membrane-trafficking proteins in Anaeramoeba Discussion Methods Culturing and harvesting of Anaeramoeba RNA extraction and sequencing Enrichment of prokaryotic mRNA and sequencing DNA extraction and short-read sequencing Long-read sequencing and basecalling Assembly and correction Genome classification and binning Hybrid assembly of symbiont genomes Prokaryotic annotation 16S rDNA amplicon typing Phylogenomics Protein-coding gene family expansions and contractions Lateral gene transfer in Anaeramoeba FISH probe design and testing Fluorescence in situ hybridization (FISH) Scanning electron microscopy Transmission electron microscopy Focused-ion-beam scanning electron microscopy (FIB-SEM) Segmentation and visualization of cell structures Tubulin staining Wheat Germ Agglutinin (WGA) staining Reporting summary Data availability References Acknowledgements Author contributions Funding Competing interests Additional information