Magnetoreception in a freshwater ciliate arises from endosymbiosis

Domain: biology
Published: 2026-03-10
Editorial outcome: accept

Abstract
Magnetoreception is a remarkable ability found across a diverse range of organisms, including bacteria, birds, fish, insects, and mammals, enabling them to detect and harness the Earth's geomagnetic field. Recently, the recruitment of biomineralizing ectosymbionts by euglenozoans was evidenced as an ecological strategy for microeukaryotes to acquire this sense. Here, we report a case of magnetosymbiosis involving a ciliate and four populations of endosymbiotic bacteria experiencing genome reduction. Among these bacteria, one group of sulphate-reducing Desulfovibrionales was found to biomineralize bundles of bullet-shaped magnetite crystals. The ciliate’s magnetotaxis mirrors that of free-living magnetotactic bacteria and euglenozoans, enabling efficient navigation in chemically stratified aquatic environments. However, in this case, magnetotaxis arises from an endosymbiotic interaction. Using a combination of optical-, confocal-, electron- and X-ray-based microscopy techniques, together with genomic analyses, these findings demonstrate that magnetosymbiosis can emerge in unicellular eukaryotic lineages through endosymbiotic integration, expanding our understanding of such interactions in aquatic ecosystems. More broadly, this work contributes to the ongoing debate on the origins of magnetoreception in eukaryotes.

Introduction
Species biology has long been studied considering organisms as independent competing functional units. Today, however, it is clear that the evolution and functioning of living organisms can only be fully understood by integrating the role of additional levels of biological complexity and symbiotic interactions. Symbiosis is now recognized as one of the major forces that has shaped biodiversity1,2. It refers to the long-term coexistence of at least two species, where at least one depends on the other, regardless of whether the interaction is beneficial or detrimental to their fitness3. This interaction may therefore be based on conflict or cooperation, which can often be mediated by the same molecular mechanisms4,5. Symbioses are multi-scale systems; partners and associations may have different degrees of biological complexity, and a network of intimate interactions may be established between several partners on a permanent or transient basis, either as obligatory or facultative relationships. Symbionts may be simply attached to host cells (ectosymbiosis) or become integrated within the host cell (endosymbiosis). In their entirety, each host-symbiont consortium forms a supra-organism, i.e., an ecologically cohesive and functional unit also defined as a holobiont6. The level of physical integration and dependence of symbionts can even challenge the definition of the term “organism”, as some of them have progressively evolved into organelles over time2.

Today, the role of symbiosis and cooperation in the functioning and evolution of the microbial world is widely acknowledged, reaffirming the pioneering ideas of Lynn Margulis6,7. These mechanisms have gradually become central to evolutionary models of eukaryogenesis and organelle acquisition3,8. According to these scenarios, multiple independent and/or successive endosymbiosis events involving Archaea, Pseudomonadota (previously referred as Proteobacteria), Cyanobacteria and/or ancestral eukaryotic lineages led to the diversification of modern eukaryotic lineages9. These interactions led to the emergence of plastids, mitochondria and potentially also the nucleus. Many organelles as we know them today, have thus derived from the invagination, slavery or synergistic fusion of endosymbionts, granting several hosts novel functions, such as photosynthesis, nitrogen fixation, aerobic or anaerobic respiration.

Research in this field currently suffers from three weaknesses. First, much more attention has been paid to microbial symbioses involving at least one eukaryotic model macroorganism host10. As a result, most of our knowledge is based on microbiota associated with model macroorganisms (e.g. humans, corals, insects and plants)11–14. Second, the role of conflict in host-pathogen and host-parasite systems has been much better characterized than that of cooperation in mutualistic or commensal systems15. Most of the current knowledge is therefore based on the mechanisms and evolution associated with a single type of interaction. Finally, several technological hurdles have long limited the observation of microbial holobionts, the identification of partners and the visualization of their interaction. By overlooking holobionts involving eukaryotic-prokaryotic and prokaryotic-prokaryotic microorganisms, especially those based on cooperation and mutualism, we have neglected an entire dimension of molecular, biochemical, physiological and evolutionary mechanisms that govern species interactions across countless biological systems.

The establishment of symbiosis has often enabled hosts to “jump” into new ecological niches by conferring new capabilities. Interactions and benefits are often based on nutrition, detoxification and energy production through the exchange of metabolites, carbon or nitrogen sources, and electron acceptors/donors16. Such interactions gave rise to trophic dependency, also known as syntrophy. Photosynthesis is the most emblematic case17,18: a freshwater endosymbiotic cyanobacterium led to the emergence of the first photosynthetic eukaryote and its descendant lineages19. Additionally, some photosynthetic algae granted the ability to sense light and phototaxis20 to some warnowiid dinoflagellates. Another well-documented case is that of the anaerobic flagellates in the digestive tract of termites, themselves in symbiosis with their insect host21, some of which supply nitrogenous elements to endosymbiotic bacteria of the order Bacteroidales22. Several taxa also evolved hydrogen-based syntrophy to adapt to anaerobic conditions23,24. For example, some ciliates and amoebae-like protists produce molecular hydrogen from mitochondria-related organelles, that their symbionts use to reduce sulphate and produce energy25,26. Some predatory flagellated or ciliated protists can also use symbionts as a primary source of nutrients. For example, ciliates of the genus Kentrophoros, can cultivate Gammaproteobacteria and phagocyte them as a source of food27. Beyond syntrophic interactions, symbiosis confers additional advantages such as protection and motility. Certain ectosymbioses provide protection against competitors or predators, as seen in Euplotidium spp., which partner with Verrucomicrobia, expulsing harpoon-like structures when triggered28. Other microeukaryotes are propelled by the movement of ectosymbiotic spirochaetes in Mixotricha paradoxa29.

New associations continue to be discovered. In 2019, we described a novel ectosymbiosis between an euglenozoa and a bacterium affiliated with Symbiontida and Desulfobacterales, respectively, isolated from anoxic marine sediments of Carry-le-Rouet, France30. This relationship relies on classical hydrogen-based syntrophy involving sulphate-reduction, acetogenesis and hydrogen transfer from mitochondria-related organelles in the host. While similar syntrophic cases had been described in other protists inhabiting the same niche before3,26,31, this holobiont introduced a new collective behaviour based on magnetotaxis. Collective magnetotaxis involves the production and alignment of prokaryotic organelles known as magnetosomes, into chains along the symbiont and host motility axis, coupled with host-driven chemo-aerotaxis30,32. Together, the eukaryotic host and bacterial symbionts form a magnetosymbiosis, reproducing the same magnetoreception-driven motility as for free-living magnetotactic bacteria32. This behaviour is an adaptation to chemically stratified environments, whereby organisms move efficiently towards their optimal redox niche combining guidance by the Earth’s magnetic field lines and chemotaxis. Prior to this discovery, other magnetotactic protists had been observed33,34, but their magnetotaxis was transient only, relying on the grazing of magnetotactic bacteria accumulated in acidic food vacuoles. Meanwhile, some freshwater protists were hypothesized to exhibit magnetotaxis, biomineralizing magnetosomes within their cytoplasm35. However, the rarity of their observations in sediments from the Uruai and Ubatiba rivers (Brazil) has hampered comprehensive genetic and ultrastructural analyses necessary to confirm these conclusions. Altogether, these recent observations have prompted the search for additional magnetosymbioses in aquatic habitats that could be more widespread than currently known.

In this work, through multiple exploratory sampling campaigns, we identified a specific site along the Dordogne River (France) where magnetotactic ciliates thrive, morphologically resembling those documented by Leao et al 35. A comprehensive characterization was carried out using culture-independent molecular approaches, advanced microscopy and synchrotron analyses to elucidate the origin of their magnetotactic behaviour. We demonstrated that these unicellular eukaryotes are, in fact, multi-partner holobionts composed of a ciliate (Ciliophorea, Prostomatea) and four populations of endosymbiotic bacteria, one of which, belonging to the Desulfovibrionales, biomineralizes a bundle of bullet-shaped magnetosomes. We concluded that magnetosymbiosis has independently emerged in another protist phylum by endosymbiosis.

Results
Observation of anaerobic magnetotactic protists in freshwater sediments
Standard magnetic enrichment was performed on sediments from various sampling sites in France to target magnetically responsive microeukaryotes. Since the first identified magnetosymbiosis was south-seeking in the Northern Hemisphere, which is uncommon30, both south-seeking and north-seeking microorganisms were sorted and observed. Magnetotactic bacteria (MTB) were found in nearly all sampled locations. In addition to diverse MTB, a singular north-seeking, magnetically responsive organism was initially observed at a few sites in the Dordogne River near Beaulieu-sur-Dordogne (44°58’55.5”N, 1°50’14.5”E) (Fig. 1a and b and Supplementary Videos 1 and 2). The cells were rod-shaped, measuring 26.7 ± 2.3 µm in length and 10.9 ± 1.3 µm in width (n=19), suggesting they were likely protists. Their strong alignment and responsiveness to the reversion of the magnetic field, indicated active magnetotaxis rather than passive magnetism due to the grazing on MTB34. The cells had a helical motion facilitated by numerous short flagella surrounding them, visible under the light microscope (Fig. 1a-c; Supplementary Video 2). When reaching the edge of the droplet, the cells moved back and forth while the magnetic field remained unchanged (Supplementary Videos 1 and 2). This so-called “ping-pong motion” was previously described in multicellular magnetotactic prokaryotes36. Magnetic sorting at different sediment depths along the oxygen gradient showed that these microorganisms lived below the oxic-anoxic transition zone. The cells appeared sensitive to mechanical force as 1) the number of magnetotactic protists harvested using the magnet after magnetic concentration was higher when the magnetic enrichment was performed with a weak stirring bar magnet (~ 0.4 mT) for a maximum of 2 hours, and 2), the cells survived for up to 10 min under a hanging drop, while they rounded and exploded after less than one minute when transferred between a slide and a coverslip (Supplementary Video 3). These protists were particularly abundant in river areas rich in humic acids and leaf litter, with minimal water current exposure. Similar magnetotactic protists exhibiting a comparable magnetotactic behaviour were found in other freshwater habitats in France, including an unnamed spring (47°45’13.7”N, 3°28’54.1”W) (Supplementary Video 4), lake Lannénec (47°44,29.9”, 3°28’58.7”W) in Britany, Lake Aydat in Auvergne (45°39’54.1”N, 2°58’53.1”E) and the Cère River (44°55’1.3”N, 1°50’18.8”E), a few kilometers upstream from its confluence with the Dordogne River. For practical reasons, this study focuses on the description of populations observed in the Dordogne River, as it was the most easily accessible for sampling.

The magnetotactic protist belongs to an undescribed group of ciliates within Prostomatea (Alveolata)
The morphology of the magnetotactic protist initially observed under light microscopy, was further defined using scanning electron microscopy (SEM). This revealed a ciliate with a simple ovoid to cylindrical body, featuring a wide oral bulge and opening in apical position at the anterior pole and surrounded by cilia with a membranelle-like arrangement (Fig. 1c and Supplementary Fig. 1a-e). This organization consisted of twelve sets of 15-µm-long microtubules, arranged in a twisted pattern resembling a cyrtos (Supplementary Fig. 1). The extern part of this cyrtos-like structure is covered by the cilia of the membranelle and presents similarities with tooth-like capitulum (Supplementary Fig. 1). The cytostome opening measures about 0.5 µm, likely restricting predation to small bacterial prey.

Each of the apical cilia extended to the cell body as microtubules reaching the extremity of the posterior pole (Fig 1c, Supplementary Fig. 1b and 2a). Unlike previously described marine magnetotactic Euglenozoa30, no epibionts were observed; instead, the entire ciliate body was covered by a mucus-like envelope. Beneath this envelope, the cell membrane was homogeneously perforated with pores (Supplementary Fig. 2a and b). Cilia were aligned with the cell’s anterior-posterior axis along each longitudinal microtubule (Fig. 1c, Supplementary Fig. 1a and b). Transverse thin sections of several cilia revealed the canonical microtubule arrangement with 9 doublet microtubules in circle and two central microtubules (Supplementary Fig. 2c) as usually described for flagella and cilia in eukaryotes. Some rare observations indicated that the magnetotactic protists reproduce by binary fission with the cleavage furrow forming at the equator (Supplementary Fig. 3a), while other observations suggested that they also perform sexual reproduction (Supplementary Fig. 3b).

Molecular typing of several single magnetotactic ciliates sorted from four different sites where they were observed, yielded 17 nearly identical sequences of 18S rRNA gene (> 99.9% similarity). They form a monophyletic clade together with Prostomatea and Plagiopylea within the CONThreeP superphylum (Fig. 1d and Supplementary Data 1). Pairwise BLASTN alignments were performed with sequences of these two genetically closest groups retrieved from the PR2 database (https://app.pr2-database.org). Although average pairwise identities values between members of Prostomatea and Plagiopylea were approximately 90% and 85% respectively, they were on average 86% and 82% identical with those of the magnetotactic protists, respectively. These genetic distances along with their basal phylogenetic position relative to known families, placed these organisms incertae sedis in the paraphyletic class Prostomatea. Alternatively, the data could support their classification as a new undescribed class within CONThreeP. Pending a more formal taxonomic description in the future, we named the ciliate HBD-1 (HoloBiont Dordogne-1).

The ciliate harbours intracellular magnetite-bearing vesicles, accessory photosynthetic organisms and quartz
Three distinct types of inclusions were observed within magnetotactic protists under the light microscope: (i) one to three greenish to brownish oblong structures aligned along the antero-posterior axis and filling about 1/4 of the cell body (Fig. 1a), (ii) contrasted granules surrounding the cytostome at the anterior pole (Fig. 1a and b; Supplementary Videos 2 and 5) and (iii), five to six thin, dark and contrasted lines concentrated in the anterior two-thirds of the cells (Fig. 1b and Supplementary Video 5). Several minutes of exposure to air led to the disintegration of the magnetotactic protists, releasing their intracellular contents. The first type of structure was identified as diatom-like skeletons that, based on the shape of the frustules, could represent different species within a single cell (Fig. 1a; Fig 2; Supplementary Video 6). Elemental mapping conducted on a single protist using synchrotron-based nanobeam-scanning X-ray fluorescence (nano-XRF) confirmed the silica-based nature of these skeletons (Fig. 3a). Some chloroplasts and thylakoids appeared preserved, contributing to the greenish-brownish colour inside the magnetotactic protists (Fig. 1a, Fig. 2a and b). Although diatoms were the predominant intracellular photosynthetic microorganisms, TEM images of thin-sectioned cells showed the presence of other microalgae, with ultrastructural features resembling Chlorophyta species (Supplementary Fig. 4). We did not observe any protist with dismantled diatoms, but some were observed without intracellular diatoms. Nano-XRF also detected silica in the second structure, i.e., within the contrasted polar inclusions localized at the anterior pole surrounding the oral opening. Diffraction analyses indicated that these inclusions are crystalline, corresponding to quartz (Fig. 3b and c). Their morphology, size and number vary across cells but they are consistently located at the anterior pole. They are easily fractured by the diamond knife during the thin section preparation (Supplementary Fig. 1f and g), similar to diatom skeleton.

TEM and Nano-XRF analyses of the third structure visible under optical microscopy as thin, dark and contrasted lines, revealed that they consist of particles similar in size and shape to bullet-shaped magnetosomes found in magnetotactic bacteria37 (Fig. 3a, d and e and Supplementary Video 5). They form bundles of three to five chains filling almost entirely 5-6 rod-shaped vesicles that resemble cells, enclosed by a lipid-like bilayer (Fig. 3d and e). Unlike magnetosomes in grazers of magnetotactic bacteria34, all these particles are intact, and none of the cell-like inclusions are located within digestive vacuoles. High resolution TEM (HRTEM) coupled with chemical analysis confirmed these particles are rich in iron and made of magnetite (Figure 3a and Supplementary Fig. 5). Each vesicle contains about 131 ± 26 particles (n=13), which measure 143.6 ± 40.9 nm in length and 55.2 ± 8.8 nm in width (n=629), and are surrounded by a thin membrane similar to the membrane generally observed surrounding bullet-shaped magnetites38 (Supplementary Fig. 5d). Some particles are as large as 282.8 x 62.1 nm (Supplementary Fig. 5a). TEM images of thin-sectioned cells showed these vesicles surrounded by many other similar structures lacking magnetite particles, distributed throughout the protist (Fig. 4a and b).

The ciliate cytoplasm is filled with intracellular bacteria
We tested the hypothesis that rod-shaped vesicles filling the protist cytoplasm, including those with magnetic particles were in fact bacteria. For this purpose, we sorted single magnetotactic protists and applied fluorescent in situ hybridization (FISH) using a fluorescent probe targeting Bacteria. Confocal laser scanning microscopy (CLSM), combined with previous TEM analyses of thin-sectioned cells, validated our hypothesis and revealed the presence of 50-100 bacteria within each protist, along with diatoms (Fig. 4). All bacteria appeared to be fully enclosed within their host, with no detectable connection to the plasma membrane or the extracellular medium, and were therefore considered endosymbionts. The bacteria were rod-shaped and, when observed outside the disaggregated protist, flagella were never observed. The intracellular organization of the protist conserved in near-native form was imaged with cryo-soft X-ray tomography (cryo-SXT) (Supplementary Video 7, diatom and four magnetosome-bearing bacteria are labelled). Bacteria appear to be embedded in an organic compound surrounded by a membrane-like envelope, but none were located within digestive vacuoles of the host. The bacterial cells measured approximately 5 µm in length and 0.5 µm in width. Most of them exhibited a uniform electron contrast, except for 5-10 cells that contained electron dense particles (Fig. 4). These observations confirm that the magnetotactic protist forms a microbial holobiont with endosymbiotic bacterial species. By validating the cellular nature of these structures, we concluded that particles were, in fact, magnetosomes sensus lato formed by some of the endosymbionts. Additionally, structures resembling mitochondria-related organelles, i.e., hydrogenosomes39, were observed enveloping some endosymbiotic bacteria, including those with magnetosomes (Fig. 4b). Similar observations were made in protists isolated from Lake Aydat in Auvergne (Supplementary Fig. 6a), the unnamed spring in Britany (Supplementary Fig. 6b-d) and Lake Lannénec, Britany (Supplementary Fig. 6e and f).

Intracellular bacteria belong to four taxonomic groups, some of which are known for their symbiotic lifestyle
The identity of the endosymbionts was investigated through pyrosequencing cloned 16S rRNA gene sequences of sorted magnetotactic holobionts (MHB) collected from the Dordogne River and three other sites where these magnetotactic ciliates were found (Supplementary Fig. 7a-d). Whole genome sequencing of one representative hologenome HBD1-SC9 was also carried out after genome amplification. Three high-quality bacterial genomes (i.e., > 90% complete and < 5 % redundancy according to checkM values on GTDB release 220) and one medium-quality genome (i.e., 86.17% complete and 0.77% redundant) were obtained using both long- and short-read technologies (Supplementary Data 4). However, only few eukaryotic reads were obtained after the hologenome amplification— approximately 31 million, representing 15.3 % of the total reads. They mapped to 2 209 contigs representing a total assembly length of 19.95 Mbp. According to the Protista_83 single-copy core gene (SCG) collection in anvi’o40, the assembly corresponds to a highly incomplete genome (< 6.0% completeness) with substantial redundancy (> 9.6%). Only a single 18S rRNA gene sequence associated with the ciliate was detected. A 16S rRNA gene sequence was assembled with each of the four bacterial bins of the single amplified hologenome. These four 16S rRNA gene sequences were identical to those obtained by multiple amplicon sequencing of various MHB samples.

Molecular phylogenetics revealed the identities of the four endosymbiotic populations detected in the MHB (Fig. 5a-d). The first two endosymbiotic populations belong to two groups of sulphate-reducing bacteria within the Desulfobacterota phylum. One population, represented by the DsD-1 genome, belongs to a diverse family well described in the literature: the Desulfovibrionaceae (Fig. 5a), that includes several freshwater MTB, such as the reference magnetotactic bacterium Solidesulfovibrio magneticus RS-141. GTDB-Tk v2.1.142 assigned DsD-1 to an undescribed genus forming a monophyletic group with both Oceanidesulfovibrio and Megalodesulfovibrio genera composed of free-living bacteria. The second population, represented by the DcD-1 genome, is affiliated with a poorly known family: the Desulfarculaceae (Fig. 5b), for which few genomes and 16S rRNA gene sequences are available. This family has long been a separate and distant lineage within the former Deltaproteobacteria class and is not known for being a group of intracellular symbionts43–45. GTDB-Tk v2.1.142 assigned DcD-1 to an undescribed genus g_JACRDC01 with no representative cultivated strain.

The third symbiotic species belongs to the Ca. Midichloriaceae family: a diverse but understudied lineage within the important order of intracellular bacteria Rickettsiales of the Alphaproteobacteria class46. GTDB-Tk v2.1.142 assigned the HBD1-SC9 endosymbionts (represented by the MD-1 genome), to an undescribed genus clustering with endosymbiotic species of another family of ciliates (Oligohymenophorea) isolated from a wastewater treatment plant in Rio de Janeiro (Brazil)47 (Fig. 5c). Although the phylogenetic tree based on the 16S rRNA gene sequences is weakly supported, these endosymbionts appear to also cluster with the genus Ca. Euplotella, endosymbionts of ciliates from the same family48.

The fourth population affiliates with the family Coxiellaceae (Gammaproteobacteria class; Legionellales order) (Fig. 5d). This group is best known for being represented solely by intracellular bacteria infecting microeukaryotes and opportunistic pathogens in humans49. A growing number of environmental surveys highlight the underestimated diversity of this group and their eukaryotic hosts, including arthropods and microeukaryotes such as amoebae49. This classification within Legionellales and this family specifically, might be revised in the future, as GTDB currently splits them into different orders50. Indeed, GTDB-Tk v2.1.142 assigned the fourth population of HBD1-SC9 endosymbionts (represented by the genome GD-1) to an undescribed order o_UBA9339 clustering with the order Coxiellales51 represented by the obligate intracellular pathogen Coxiella burnetii49. The few small sized genomes (<1.5 Mbp) within the same cluster were assembled from metagenomes of diverse aquatic sediments across the world and none have been linked to cultivated organisms (Fig. 5d). Few close 16S rRNA gene sequences of uncultured bacteria associated to various freshwater and saline habitats, could be retrieved from public databases (Supplementary Fig. 7d).

Endosymbionts, including magnetosome-forming Desulfovibrionales, exhibit a specific pattern of cellular organization
FISH assays, using specific probes designed from the 16S rRNA gene sequence of each of the four bacterial populations, confirmed the taxonomic affiliation of the different endosymbiotic bacteria, and further identified their spatial distribution and relative abundance (Fig. 6a and Supplementary Videos 8 and 9). Multiple FISH and TEM analyses carried out with different MHB from the Dordogne River revealed a unique organizational pattern, where each symbiont is systematically observed in the same position within the host cytoplasm. Bacteria producing magnetosomes bundles were stretched across the whole length of the host as shown in Supplementary Videos 5, 8 and 9, and Fig. 3a. CLSM images revealed that DsD-1 of the Desulfovibrionales is the biomineralizing endosymbiont (Fig. 6a and Supplementary Videos 8 and 9). This is further supported by genomics, as the DsD-1 genome is the only one containing magnetosome genes, assembled into a partial cluster composed of mad genes and some of the mam genes involved in magnetite formation in the Desulfobacterota52 (Supplementary Fig. 7e and Supplementary Data 5). Mam proteins of DsD-1 share the highest sequence identity with their corresponding homologs in the reference strains Solidesulfovibrio magneticus RS-153, and Fundidesulfovibrio magnetotacticus FSS-154, with average identities of 54.4% and 51.8%, respectively. The 5 to 6 biomineralizing cells cluster with the Desulfarculaceae cells, which occupy most of the host’s cytoplasm and extend over two-thirds of its length at the anterior pole. In contrast, the gammaproteobacterium GD-1 and alphaproteobacterium MD-1 lack specific spatial localization.

All endosymbionts possess reduced genomes and have lost the ability to form flagella
Genome sizes of the endosymbionts are 1.3 Mbp, 2.0 Mbp, 2.3 Mbp, and 2.8 Mbp for MD-1, GD-1, DsD-1, and DcD-1, respectively (Supplementary Data 4). Despite its reduced size, the genome of DcD-1 has a relatively high G+C content (60.5%) compared to the other three genomes, whose G+C contents are significantly lower (from 37.2% to 45.7%, Supplementary Data 4). The average protein-coding density of GD-1 genome is within the typical range of 85–90% for free-living bacteria55, whereas this density is lower for other endosymbionts, down to 57.6% for DsD-1, that contains the highest number of pseudogenes (> 6%) compared to the three others (< 1%).

Protein-coding genes were classified into Clusters of Orthologous Groups (COGs) using eggNOG-mapper v2.1.1256 (Fig. 5e and Supplementary Data 6). The gene repertoires of the ciliate symbionts are typical of obligate endosymbionts. Six out of 22 COG categories detected once in all genomes were prominently represented in all endosymbionts aside “unknown functions”—ranging from 8 to 16 % (Fig. 5e). Three of them are essential for cellular metabolism of primary endosymbionts57, representing on average 16% of the genome: “Translation, ribosomal structure and biogenesis” , “Amino acid transport and metabolism”, and “Energy production and conversion”. Two others—“Intracellular trafficking, secretion, and vesicular transport" and “Cell wall/membrane/envelope biogenesis”—accounted for 4-6% of the proteins. The COG category “Replication, recombination, and repair” represented 14%, 16%, and 30% of the predicted proteins in the endosymbionts GD-1, MD-1, and DcD-1, respectively. Of these, 46%, 63%, and 94% corresponded to mobile genetic element–related genes. In contrast, such genes were rare in DsD-1, where this COG category accounted for less than 0.02% of all annotated proteins. All symbionts have a minimal machinery involved in DNA replication, homologous recombination, base excision repair or mismatch repair. The set of conserved genes includes polymerases genes together with dna, rec, ruv, uvr, and mut genes (Fig. 5e and Supplementary Data 7).

Functional annotation predicted genes encoding proteins with leucine-rich repeats (LRRs) and ankyrin repeats—domains involved in interactions with eukaryotic hosts58— in three out of four symbiont genomes (Supplementary Data 8). While the DsD-1, DcD-1, GD-1, and MD-1 genomes encode 0, 3, 8, and 7 ankyrin repeat-containing proteins, respectively, only GD-1 harbours four proteins with leucine-rich repeats, alongside several Type IV secretion systems (Supplementary Data 9), which are often repurposed by endosymbionts to deliver host-targeted effectors58.

Conversely, COGs related to “Cell motility” and “Signal transduction mechanisms” are highly reduced in all symbionts compared to free-living counterparts (Fig. 5e). MacSyFinder v 2.1.259 did not predict the ability to form a flagellum in any of the four endosymbionts. Few flagella genes are present in MD-1 and DsD-1 (Supplementary Data 10). No additional macromolecular system is encoded, except in the GD-1 genome, in which several secretion systems were predicted, in addition to the three type IV secretion system proteins (T4SS_TypeFL_6, pT4SS, pT4SS )60. Other secretion systems include three type V secretion systems—including Can autotransporter T5aSS and a two-partners system (T5bSS)—and a mannose-sensitive hemagglutinin (MSHA) pilus which is a type IV pilus61 (Supplementary Data 9).

We found 21 Dot/Icm genes (colocalized with the pT4SSi) involved in the virulence of Legionella pneumophila and Coxiella burnetii62 (Supplementary Data 11). These genes are colocalized in the genome with the pT4SS a secretion system that was suggested to be implied in host association in these lineages63. A total of 118 genes in GD-1 showing remote homology to known virulence factors were also predicted, including a phtA-like gene, a phagosomal transporter involved in the establishment and persistence of L. pneumophila within its replication vacuole64, as well as enhanced entry (Enh) proteins that facilitate host cell invasion and infection65. Many transporters involved in iron uptake, adherence and toxins (e.g., Enterobactin, Hemolysin, LPS, Microcin H47) in pathogens such as Neisseria meningitidis MC58, Escherichia coli O157:H7, Haemophilus influenzae Rd KW20, were also predicted. However, some sequence identities being low (i.e., 30-40%), these homologs may be functionally different. No such genes were detected in MD-1, except antophagocytosis genes, like the two other symbionts exhibiting similar but less numerous defensive systems (Supplementary Data 11).

Metabolic network reconstruction of endosymbionts reveals hydrogen and sulphur-based syntrophies
Metabolic pathways were inferred using the MetaCyc database66 and Pathways Tools software67. Besides revealing auxotrophy for several amino-acids, cofactors and vitamins, a common feature in many symbionts, the analysis indicates possible syntrophies based on the degradation of organic compounds such as acetate or glucose, the oxidation of hydrogen and sulphate reduction (Supplementary Fig. 8 and Supplementary Data 12). Together with previous analyses, these predictions helped us to develop a model describing the holobiont functioning (Fig. 6b).

Both Desulfobacterota symbionts, DcD-1 and DsD-1, have the potential to perform glycolysis and produce acetate and ATP from acetyl-CoA. They may also be able to reduce sulphate (SO 2-) to hydrogen sulphide (H S) using H or organic substrates as electron donors. The Desulfarculales endosymbiont DcD-1, which accounts for most of the endosymbionts, may perform autotrophy. Indeed, we identified genes involved in the carbonyl branch (acs genes; EC 1.2.7.4), and the methyl branch (folD, metF and acsE genes) (Supplementary Data 13) of the Wood–Ljungdahl pathway, which enables acetyl-CoA synthesis from CO reduction in sulphate-reducing prokaryotes68. This partial prediction may result from incomplete genome assembly and requires further validation. No other canonical CO fixation pathways were found in the four endosymbionts. The biomineralizing symbiont seems to play a central role in respiration and electron flux within the whole consortium, as only the Dsd-1 genome harbours genes encoding the synthesis of a low-potential menaquinone that is central to anaerobic respiration69.

The GD-1 and MD-1 genomes show very different metabolic profiles. None of the two genomes encodes for cytochrome c-type of cbb -type oxidases or other enzymes associated to inorganic terminal electron acceptors (Supplementary Data 12). MD-1 has a severely reduced energy metabolism and lacks the ability to perform glycolysis as well as the tricarboxylic acid (TCA) cycle—similar to many Rickettsiales, which import metabolites from their host70. However, MD-1 has the potential to generate reducing power (NADH), acetyl-CoA, and CO through the decarboxylation of exogenous pyruvate. It may also produce the (2S)-ethylmalonyl-CoA intermediate required for the ethylmalonyl-CoA pathway, an alternative to the TCA cycle. Notably, this endosymbiont is the only one in HBD-1 to encode three ADP/ATP translocases (tlc; InterPro entry IPR004667), which enable the direct import of ATP from the host cytosol58. In contrast, the genome of the Gammaproteobacteria GD-1 appears less reduced, and is likely able to perform glycolysis through a variant pathway based on sucrose and glucose. Additionally, GD-1 could utilize the non-oxidative branch of the pentose phosphate pathway, which is absent in MD-1. Symbionts harbour a repertoire of functions that sustain these relationships including detoxification pathways for superoxide radicals, 8-oxo-(d)GTP (EC 3.6.1.55) or acidic resistance (EC 4.1.1.19) (Supplementary Data 13). MD-1 genome also encodes ahpD and ahpC genes (EC 1.11.1.28/1.11.1.26) involved in antioxidant defence in bacteria, specifically in protecting cells from oxidative stress caused by reactive oxygen species (ROS)71.

Methods
Site description and sample collection
Sediment samples were collected from the shore of the river Dordogne at Beaulieu-sur-Dordogne, Nouvelle-Aquitaine, France (44.9783°N, 1.8378°E) at different seasons between August 2021 and January 2023. Samples were collected in 1 L bottles filled with one third of sandy sediments and completed to full capacity with water from the site to exclude air bubbles. Samples stored at 10°C with the cap slightly opened allowed the conservation of magnetotactic protists for more than 3 months.

Magnetic enrichment and light microscope observation
Prior the concentration, chemical gradients of the microcosm were broken, and the sediments were resuspended by mixing the bottle. Magnetotactic organisms were concentrated by placing the south pole of a magnetic stirring bar next to the sample bottle above the water-sediment interface for 1h. Magnetically concentrated cell were collected and observed using the hanging drop assay91 with a ZEISS Primo Star light microscope equipped with phase-contrast optic.

Mesocosms measurements of chemical and cell count profiles
Dissolved oxygen concentration profiles were measured in the water column and sediments of the microcosms using a fiber-optic oxygen sensor (50-µm tip diameter, REF OXR50) and a FireStingO 2 meter, both from Pyroscience (Germany). High-resolution profiles (100 µm steps, from 10 mm above the sediment to -30 mm below the sediment) were achieved with a Pyroscience MU1 motorized micromanipulator. Sensor calibration was made with a 50 mL milliQ water solution flushed with air for 30 min (O saturation = 100%) and a milliQ water solution flushed with N for 30 min (O saturation = 0%). Magnetotactic protists were not sufficiently concentrated to carry a cell count profile. Therefore, sediment was harvested every 1 cm after oxygen profile measurements and transfer into a 50 mL FalconTM tube containing filtered water from the Dordogne River. A magnetic concentration was then performed for 1 h and magnetotactic organisms were observed using the hanging drop assay. Magnetotactic protists were always observed in regions depleted with oxygen, i.e., below 1 cm of sediment. Measurements were systematically carried out on three replicates of three different samples.

Transmission Electronic Microscopy (TEM)
TEM was used to analyse the ultrastructure of the magnetic protists that were directly deposited onto TEM copper grids coated with a carbon film and with Poly-L-Lysine. Electron micrographs were acquired with a Tecnai G2 BioTWIN (FEI Company) equipped with a CCD camera (Megaview III, Olympus Soft imaging Solutions GmbH) operating at an accelerating voltage of 100 kV. High-resolution TEM (HRTEM) was performed using a JEOL 2100F microscope. This machine, run at 200 kV, was equipped with a Schottky emission gun, an ultra-high-resolution pole piece and an ultrathin window JEOL XEDS detector. HR-TEM images were recording using a Gatan US 4000 CCD camera.

Thin-sectioned samples were obtained from magnetically concentrated protists fixed in 0.25% (w/v) glutaraldehyde in sodium cacodylate buffer (10 mM, pH 7.4) and stored at 4°C for at least 24 h. Cells were subsequently washed with the same buffer solution and post-fixed for 1 h with 1% (w/v) of osmium tetroxide in cacodylate buffer. Magnetic cells were then dehydrated through a graded series of ethanol solutions with increasing concentrations and finally embedded in Epon 812 monomeric resin. All the chemicals used for histological preparation were purchased from Electron Microscopy Sciences. Sections (80 nm thick and 3 mm long) were cut using an ultramicrotome UC7RT (Leica Microsystems GmbH), mounted onto TEM copper grids, and stained with uranyless and 3% lead citrate (Reynolds Lead Citrate, Uranyless) for 3 min.

Magnetosome and endosymbiotic cell sizes were determined from TEM images using ImageJ software (v 1.48). Several cells were used to calculate the mean value and standard deviation for each feature.

Scanning Electronic Microscopy (SEM)
SEM was employed to examine the preserved morphology of the magnetotactic protist. Magnetically concentrated magnetic protists were (i) micromanipulated and chemically fixed with 0.25% glutaraldehyde on a glass coverslip coated with Poly-L-Lysine, (ii) dehydrated through a graded ethanol series (50%; 70%; 96%; 100%), (iii) dried through critical point drying (CPD) (Leica EM CPD300), and finally (iv) coated with carbon using a Leica EM SCD500 carbon coater. Observations were carried out using a Zeiss ultra 55 field emission gun SLEM. Secondary electron images were collected with an InLens detector and an accelerating voltage of 1-3 kV with a working distance of 3.5 mm. Backscattered electron imaging (AsB detector) and chemical mapping by EDXS were performed at accelerating voltages between 10 and 15 kV with a working distance of 7.5 mm.

Synchrotron-based nanobeam-scanning X-ray fluorescence (nano-XRF)
Sorted and 0.25% glutaraldehyde fixed magnetotactic protists were deposited on Si N membrane for measurement at the ID16B hard X-ray nanoprobe beamline (ESRF, Grenoble)92. Nanobeam-scanning X-ray fluorescence (nano-XRF) data was collected under ambient conditions using an incident photon energy of 17.5 keV with 11 Si-drift detectors to measure X-ray fluorescence (XRF) ~ 15 degrees to the sample in forward scattering direction. The focused X-ray beam was measured to be 55 X 56 nm (FWHM) before XRF mapping. A raster scanning step size of 50 nm was used with a dwell time of 50 ms to collect high-resolution XRF maps. XRF spectra were energy calibrated and fitted with Kα emission lines of second/third row periodic elements (e.g. Si, P, S, Ca, Fe, Cu, Zn) using pyMCA.

Cryo Soft X-ray Tomography (cryo-SXT)
Imaging was conducted at ALBA synchrotron using cryo transmission X-ray microscopy at Mistral beamline (Barcelona, Spain)93 under the awarded proposal number 2022025566. Using a similar approach to that described above, magnetotactic cells were magnetically concentrated on the wall of an environmental sample bottle and then extracted by micropipette. Micromanipulated magnetotactic protists were then fixed with 0.25% of glutaraldehyde for 10 sec and deposited on a poly-L-lysine coated transmission electron microscopy grids (Quantafoil R2/2 holey carbon, gold) along with 1 µL of 100 nm Au nanoparticles (BBI Solutions concentrated 5X). Gold nanoparticles deposited on the grid were used as fiducial references for projection alignment prior to tomographic reconstruction. The grid was incubated horizontally for 1-2 min to facilitate the settling of protists onto its surface. The grid was subsequently mounted vertically into a Leica EM GP plunge freezer at 95% humidity, blotted from the back of the grid with a filter paper for 3 s and then rapidly plunged into a liquid ethane maintained at -180°C, cooled by liquid nitrogen. Ice-vitrified cells were maintained under cryogenic conditions until transfer to the Mistral beamline cryo chamber for analysis.

A tilt series of projections from – 65° to + 65° was collected every 1° with an incident X-ray energy of 520 eV. Exposure time varied from 1-2 s for each projection (2 s at higher angles). The sample was imaged at 0° before and after collecting the tilt series to ensure there was no significant beam damage at the achievable resolution. A 40 nm Fresnel zone plate was used with an effective pixel size of 12 nm. The projections were normalized with the incoming flux and deconvolved with the measured point spread function (PSF) of the optical system94. Alignment of projections was done with Etomo using Au fiducials of 100 nm. Tomographic reconstruction and SIRT deconvolution were performed using IMOD. Although this approach conserves the organization of the magnetotactic protists in their near native-state configuration, they slightly flattened due to their thickness. Nevertheless, the overall ultrastructure of the magnetotactic protists was maintained.

Cell sorting and whole genome amplification (WGA)
Cell sorting was performed on sediment samples collected in Dordogne River with an InjectMan® 4 micromanipulator and a CellTram® 4r, hydraulic, manual microinjector from Eppendorf mounted on a ZEISS Axio Vert.A1 KMAT microscope. The microscope and micromanipulator were installed inside a clean chamber, previously sterilized by 1 h germicidal ultraviolet irradiation (UV lamp 2  15 W, 254 nm). A 10-µL drop containing magnetically concentrated cells was gently added to a 30-µL drop of filtered Dordogne River water on a hydrophobic coverslip to enable magnetic migration of magnetotactic cells toward the filtered water. One magnetotactic protist was transferred with a sterile microcapillary (Drummond Scientific 3-000-203-g/X) into a 4-µL droplet of phosphate buffer saline (PBS). This droplet was stored at –20°C prior to WGA. To generate sufficient gDNA for 16S rRNA gene and shotgun metagenomic sequencing, WGA was conducted using multiple displacement amplification (MDA) with the REPLI-g Single Cell Kit (QIAGEN), according to the manufacturer’s protocol. The concentration of double-stranded gDNA was quantified using a QuBitTM 4 fluorimeter (ThermoFisher Scientific).

Cloning and sequencing of the 18S rRNA / 16S rRNA gene sequences of magnetically concentrated and sorted cells
The 18S and 16S rRNA genes were used to identify magnetically-concentrated eukaryotic and prokaryotic cells, respectively, from samples collected in the Dordogne River, an unnamed spring and the lake Lannénec in Britany, Lake Aydat in Auvergne and the Cere River few kilometres before it flows in the Dordogne River. DNA was amplified using the Phusion® Hot Start Flex DNA Polymerase following the manufacturer’s instructions. Two set of primers were used: for the 18S rRNA gene, EukA 5′-AACCTGGTTGATCCTGCCAGT-3′ and EukB 5′-TGATCCTTCTGCAGGTTCACCTAC-3′ 95, and 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-TACGGHTACCTTGTTACGACTT-3′ for the 16S rRNA gene96. Blunt-end fragments of these gene sequences were inserted into vectors using the Zero Blunt® TOPO®PCR Cloning Kit with One Shot® TOP10 chemically competent E. coli cells. The resulting cloned inserts were submitted for sequencing (Eurofins Genomics Germany GmbH). Obtained sequences were aligned against the NCBI nucleotide database with Basic Local Alignment Search Tool97. The rRNA gene sequences were screened for chimeric structures using the UCHIME2 algorithm98 and subsequently deposited in GenBank under accession numbers (see Data availability).

Confocal laser scanning microscopy and fluorescence in situ hybridization (FISH)
FISH was performed according to Pernthaler et al.99. Specific probes for the four different endosymbiotic bacteria were designed based on the alignment of the most similar 16S rRNA gene sequences and labelled with different fluorochrome. Probe specificity was assessed using the PROBE_MATCH tool available in the RDP-II100. The nearest non-target sequences displayed at least one mismatch with the designed specific probes. The oligonucleotide probes used in this study were ordered from Eurofins Genomics (Supplementary Table 1).

Micromanipulated magnetotactic protists were deposited on a SuperFrost Plus® Gold, Menzel-Gläser (Thermo Scientific) microscope slide. Cells were fixed during 2 h with 30 µL of a solution of 1% paraformaldehyde. Fixed cells were then dehydrated by serial incubation for 5 min each in 50%, 80% and 100% ethanol. The hybridization solution contained 10 ng/ml of the probe, 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM Na EDTA, and 0.01% sodium dodecyl sulphate (SDS), using the hybridization and washing stringencies of 30% for all probes (i.e., 0.6 mL of formamide per mL of hybridization buffer). Hybridization was performed at 46°C for 2 h. Slides were mounted with a ProLong™ Diamond Antifade Mountant (Invitrogen™) with or without 4,6-diamidino-2-phenyl- indole (DAPI) and then were immediately observed with the Zeiss LSM980 confocal laser scanning microscope. The four specific probes HBD1-ALPHAp, HBD1-GAMMA1-2p, HBD1-DARCU1-2p, and HBD1-DARCU1-2p were designed with four different fluorochrome Alexa555, ATTO633, ATTO425 and ATTO488, respectively, to be able to distinguish the different cells and localize them. The specificity of each of the four probes was tested using usual controls with DAPI and the EUBp. The specific probe HBD1-GAMMA1p with the fluorochrome ATTO488 was used for these tests to distinguish between the fluorescence of all bacteria at 633 nm (EUBp-ATTO633) and the endosymbiotic Gammaproteobacteria.

The multiplex labelling with ATTO425, ATTO488, Alexa555 and ATTO633 dyes were excited simultaneously with 4 lasers: 405 nm (0.3% power), 488 nm (0.7% power), 561 nm (1% power), and 639 nm (0.3% power). Images were acquired using the spectral mode (with MBS filters 405/488/561/639) and the signal was collected from 411 to 694 nm using a GaSEP spectral detector. Reference emission spectra were recorded for each dye (using their corresponding laser excitation and MBS filters 405/488/561/633). In addition, a reference spectrum corresponding to autofluorescence in cell was also recorded on unstained samples simultaneously with all laser excitation (405, 488, 561, and 639) and using the MBS filters 405/488/561/633. A complete set of these reference spectra was recorded. Spectral separation using these reference spectra was applied on multiplex images with online-fingerprinting method, resulting in four separate dye channels and one autofluorescence channel. Images acquired in spectral mode represent the maximum intensity projection of a subset of 16 axial planes.

The image (Fig. 4c) acquired in Airyscan SR (Super-Resolution) mode using the Airyscan 2 detector were processed using the Airyscan joint deconvolution (jDCV) plugin of the ZEN blue software to enhance image resolution and clarity. The image was acquired in 16 bits mode, and processed using Zen bleu software (Zen 3.6) and imageJ (ImageJ2, version 2.16.0/1.54).

Shotgun metagenomic sequencing, assembly and functional annotation
The genomic DNA of the holobiont was sequenced at Genoscope with a combination of Illumina and Oxford Nanopore Technologies. First, 3 μg of amplified DNA was treated with T7 Endonuclease I (New England Biolabs) to resolve branches due to the multiple displacement mechanism. For Illumina sequencing, 250 ng DNA was sonicated to a 100–1,000 bp size range using the E220 Covaris instrument (Covaris, Inc.). The fragments were end repaired and 3′ adenylated, and NEXTflex HT barcodes were added (BioScientific Inc.). The ligated product was amplified using 12 PCR cycles with the Hifi HotStart ReadyMix kit (Kapa Biosystems) and purified with 0.8× AMPure XP reagent. After library profile analysis using the Agilent 2100 Bioanalyzer and quantitative PCR (qPCR) (MxPro; Agilent Technologies), the library was sequenced on an Illumina NovaSeq 6000 instrument. A total of 1.20 × 108 paired-end reads were obtained. The Illumina reads were trimmed by removing low-quality (Q < 20) nucleotides, sequencing adaptors, and sequences of < 30 nucleotides (nt), using the FastX-Toolkit package.

For Nanopore sequencing, 1 μg DNA was used for the library preparation following the 1D Native barcoding genomic DNA protocol with the EXP-NBD104 and SQK-LSK109 ligation kit (Oxford Nanopore). The library was sequenced using a Nanopore R9.4.1 revD flow cell and the PromethION device with MinKNOW v.4.5.4 and Guppy v.5.1.13 + b292f4d13 software. A total of 7,444,240 reads were obtained with an N50 value of 4.97 kb.

A hybrid assembly was launched using SPAdes v.3.13.0 with –sc option101. This assembly resulted in 280,856 contigs for a total length of 157 Mb. First, EukRep102 was used to identify eukaryotic contigs within the assembly. Then all reads were mapped to these contigs with Bowtie 2103 to estimate the number of eukaryotic reads. Contigs longer than 1000 bp were used as input for the Anvi’o metagenomic workflow40 to perform the metagenomic binning based on tetra-nucleotide frequency. The binning resulted in four Bins corresponding to four bacterial SAGs (Supplementary Data 4). The automatic annotation of the different SAGs was performed with the MicroScope platform (https://mage.genoscope.cns.fr/microscope)104. Pseudogenes were detected by aligning each symbiont CDS to its ortholog in E. coli K-12 substr. MG1655 and identifying orthologs with disrupted structure. A gene was classified as fragmented when a single E. coli CDS matched two colinear ORFs in the symbiont genomes, indicative of frameshift mutations or premature stop codons. Assembly completeness, contamination and redundancy of the different SAGs assembly were estimated using CheckM2 v.1.0.2105 with the “allmodel” option leading to the selection of the general model, and with anvi’o using anvi-estimate-genome-completeness tool with the defaults HMM collections of single-copy core genes named Bacteria_71106. The rRNA genes were predicted using RNAmmer107 in all the global assembly before binning, and we looked if the predicted 16S / 18S rRNA belonged to a binned SAG. Then we performed a BLASTN108 comparison to the rRNA / ITS NCBI database, selecting the 16S rRNA from Bacteria and Archaea type strains.

Genome-based taxonomic classification and molecular phylogeny
We used the GTDB-Tk v2.1.142 software to assign a taxonomic classification to each bacterial SAGs. We could notice that the taxonomic classification was mostly defined by topology, suggesting that all our SAGs correspond to new bacterial genera and species.

The taxonomic position of each SAG was confirmed building phylogenetic trees and the 120 (bac120) phylogenetically informative markers42 used by GTDB. First, all genomes of good quality (i.e., > 90% complete with < 5% redundancy according to checkM values on GTDB) were downloaded from the Genbank database109 in April 2024, and rapidly re-annotated with PROKKA v1.14.6110 to homogenize coding sequence predictions. The number of high-quality genomes being scarce for some taxa, we lowered our stringency for genome completion to 80% for some of them. The conserved markers were extracted using PyHMMER 0.8.0111 and the Hidden Markov Models (HMMs) of each of the 120 markers using the bit score gathering threshold (GA) for each profile to define a corresponding homologue. We proceeded to an additional quality checking as previously112. We removed genomes in which more than 90% of markers we detected, and eliminated markers detected in less than 90% of genomes. Multiple sequence alignments (MSAs) were then performed with MAFFT v7.490113, trimmed with BMGE v1.12114 for each marker and concatenated into a single alignment. Trees were inferred using the maximum likelihood method implemented in the IQ-TREE v2.2 software115,116 and a substitution model selected from the global alignment with ModelFinder117 with the –MFP option. Branch robustness was estimated by the SH-like approximate likelihood ratio test118 (1,000 replicates) and the ultrafast bootstrap approximation119 (1,000 replicates). Trees were drawn and edited using FigTree v1.4.4120 and Inkscape software (Inkscape Project, 2020, https://inkscape.org).

Identification of the protist relied on a maximum-likelihood phylogenetic tree of the Ciliophora constructed from SSU 18S rRNA gene sequences alone, as no eukaryotic draft genome and other conserved marker genes could be obtained. A sample of 294 sequences at least 1634 pb long representing 205 Ciliophora families were retrieved from the PR2 database (https://app.pr2-database.org). The tree was rooted with the Colponemidia based on Tikhonenkov et al.121, who demonstrated their monophyly and position as the sister lineage to all other known Alveolata. The alignment of the 319 sequences was performed using MAFFT and trimmed using a relaxed parameterized Gblocks v 0.91 program122. The tree was then inferred as described above for whole genome trees.

Functional annotation and metabolic network modelling
Metabolic pathways were inferred from the MetaCyc database66 as described previously112, which offers an extensive catalogue of metabolic pathways, enzymatic reactions, enzymes, chemical compounds, genes and review-level information for organisms in the three domains of life. This database includes 3,105 pathways and 18,566 reactions in the version 27.0 that was used in this study. Metabolic pathway reconstruction was performed using the PathoLogic algorithm of Pathway Tools . Beforehand, Prokka v 1.14.5110 was used to perform gene calling for each genome. Then, functional annotation was performed with KofamScan v.1.3.0123, that assigns KEGG Orthologs (KO) family numbers to protein sequences using HMMs database of KO and the pre-computed adaptive score thresholds. Next, we created Pathway Tools input files with associated enzymatic activities for protein-coding genes described with either Enzyme Commission (EC) numbers or MetaCyc reaction identifiers when cross-references between KO, KEGG and MetaCyc identifiers were available. Finally, the obtained Pathway/Genome Databases (PGDB) were queried using the PythonCyc API v2.0.2 (https://github.com/networkbiolab/PythonCyc) to compute pathway completeness rates for each genome and a pathway/genome matrix. Scripts that were used to generate Pathway Tools input files and the matrix are available at: https://github.com/labgem/metacoco124.

The genomes were also analyzed using the MicroScope platform104 for further comparative analysis and expert functional annotation. Protein-coding genes were classified by eggNOG-mapper version 2.1.1256 and the public database eggnog v 5.0.2125. Putative virulence-associated genes were identified using VirulenceFinder126 and BLASTP searches against hits of the Virulence Factor Database (VFDB)127 applying a minimal identity of 30%.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I am satisfied with the revisions the authors made to the manuscript and I appreciate the time and effort they took to address all reviewers' comments. In my opinion, the revision has improved the manuscript and I am looking forward to seeing this wonderful discovery published.\nI have only a few last small suggestions for text edits:\nLine 56: to me ‚diversification‘ is redundant here and could be deleted\nLine 75-76: It is my impression that while the endosymbiotic origin of plastids and mitochondria is largely undisputed, discussions regarding the origin of nucleus persist (i.e. the autogenic models). Hence, I’d suggest to rephrase it here as such (e.g. : ‘… emergence of plastids, mitochondria, and potentially also the nucleus’).\nLine 76: Consider replacing ‘most’ with ‘many’.\nLine 78: mention also aerobic respiration, as the primary role of mitochondria\nLine 140: ‘multi-partner’ instead of ‘multi-partners’\nLine 250: replace ‘particles’ with ‘which’\nLine 254: consider adding ‘magnetite’ before ‘particles’ for clarity.\nLine 262 – 264: consider rephrasing: ‘All bacteria were considered endosymbionts as they appeared to be completely enclosed … and the extracellular space.’\nLine 272: replace ‘support’ with ‘confirm’\nLine 280: replace ‘genetic’ with ‘taxonomic’\nLine 299: replace ‘population belongs’ with ‘populations belong’\nLine 356: As it is only 4 genomes, please add also the sizes of the two other genomes (DsD and GD-1).\nLine 363: replace ‘proteins-coding’ with ‘protein-coding’\nLine 378: please italicize gene names.\nLine 382: what about DsD? Please add.\nLine 421: replace ‘account’ with ‘accounts’\nLine 428 – 431: sentence is unclear. Does ‘exclusively’ mean that only menaquinone and no other quinone was detected in DsD or that menaquinone was exclusively detected in DsD and in no other symbiont? Please clarify. Also, it is not obvious to me how can the quinones of one endosymbiont have such broad implications for the whole consortium?\nLine 433: Do you mean ‘or’ instead of ‘of’?\nLine 453: I suggest to slightly rephrase, e.g.: ‘A recent discovery of marine magnetic holobionts revealed a unique ability of certain microeukaroytes …’\nLine 472: replace ‘with’ with ‘from’\nLine 473 – 474: replace with ‘…not possible as the genome of the host could not be assembled …’\nLine 562-565: ‘On the other hand, DcD-1 and DsD-1 could enable their host to breathe sulphate instead of oxygen.’\nPlease rephrase. Indeed, DcD-1 and DsD-1 can breathe sulphate, and thus so can the holobiont but not the host (= ciliate) as there is no evidence that the ciliate can access the energy obtained from sulfate reduction, unlike in ref. 81. The authors themselves proposed that the ciliate obtains energy from fermentation in the hydrogenosomes. This should be clarified.\nLine 653: replace ‘highlights’ with ‘reports the discovery of’ and replace ‘capable to sense’ with ‘capable of sensing’\nFigure Legend:\nFig. 1b: In the text white and grey arrows are mentioned but in the image there are only black arrows. Can you check and correct if necessary?\nFig. 4a: I can only see 2 arrows (=2 cells with magnetosomes), whereas 4 are mentioned in the figure legend.\nFig. 5a,b,c,d: the symbionts from this study should be in bold in the respective trees but they are not (in my version of the figures). Please check and make sure to highlight them (in bold or colour).\nLine 1356: Replace 'under study' with 'from this study'.\nFig. 6b: replace 'preys' with 'prey' in sketch\nLine 1377: replace 'cell host' with 'host cell'\nLine 1390 - 1392: These two sentences are essentially identical, please delete one.", "reviewer_2": "The authors have carefully addressed our comments, as well as those of the other reviewers, and have made substantial improvements to the manuscript. All suggested changes were thoroughly considered and implemented where possible, either through additional analyses or by removing debatable statements. The main story is now much more clearly articulated and supported by additional data, while more speculative aspects have been either removed or appropriately rephrased.\nSome of our comments proposing additional analyses, such as phylogenetic analysis of mam genes or more in-depth comparative analyses, were intended as general suggestions. While these were not fully pursued, we understand and agree with the authors’ decision, as incorporating them would have substantially expanded the scope of the manuscript and blurred the main focus.\nWe have no further comments, and we would like to congratulate the Authors on this exciting and well-supported discovery in protist symbiosis and look forward to seeing the paper published.", "reviewer_3": "The authors have answered all of my concerns. Congratulations on a fantastic manuscript.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We would like to express our gratitude to the editor for giving us the opportunity to\naddress the concerns raised by the reviewers in this new submission. We have carefully\nconsidered their feedback and found the overall comments and suggestions constructive. We\nbelieve we have addressed all the requested changes.\nIn this revised manuscript, which we believe has been significantly improved, we have provided\na point-by-point response to each concern. We have included line by line where changes were\nmade in the revised version.\nReviewers’ comments/questions are written in black while our answers are written in blue.\nSee below.\nReviewer’s Comments:\nReviewer #1 (Remarks to the Author)\nReview of Bolzoni, Monteil et al. (NCOMMS-25-28667-T)\nBolzoni, Monteil et al. describe the discovery of a novel magnetosymbiosis between a\nfreshwater ciliate and its prokaryotic endosymbionts. The ciliate, belonging to a novel clade\nwithin Prostomatea, is magnetotactic (north-seeking) thanks to magnetite minerals produced\nby its symbiont (belonging to the Desulfovibrionaceae). This symbiosis is reminiscent of the\nmagnetosymbiosis between a distantly related euglenozoan and a magnetotactic\nDesulfobacterales ectobacterium, with the key difference that here the symbiont appears to\nhave been adapted to intracellular lifestyle. The ciliate additionally hosts 3 other non-\nmagnetotactic endosymbionts (Desulfoarculaceae, Midichloriaceae and Coxiellaceae) as well\nas diverse diatoms, whose role for the host is not clear.\nThe authors’ discovery not only describes a wholly novel intriguing symbiosis but it further\nreveals the profound effect symbiotic interactions have on eukaryotic evolution.\nI very much enjoyed reading this manuscript; I find the topic of protist symbioses fascinating,\nthe manuscript is very well written, and the images and videos are spectacular. The study\nencompasses a truly impressive collection of data to support the authors’ conclusions,\nincluding advanced microscopy (SEM, TEM, nano-XRF) techniques and metagenomics. My\ncomments largely pertain to minor clarifications in text (see below) but I have one general\nquestion:\nResponse: We thank the reviewer for their kind comments.\n1. The authors don’t explicitly state whether the ciliate host is anaerobic or not. As it lives below\nthe oxic-anoxic transition zone, is ‘sensitive to oxygen’ and contains putative hydrogenosomes\nit might likely be an anaerobe. On the other hand, phylogenetically, it belongs to a sister clade\nof the mainly aerobic Prostomatea. Do the authors lean one way or another? Did they maybe\nfind indications in their molecular data (e.g. potential mitochondrial proteins etc.) that would\nallow them to address this point?\nregard to the metabolic interplay with its many endosymbionts. Two of these are obligate\nanaerobes, the Coxiellaceae is presumably an aerobe (is it?), and then there is the\nphotosynthetic diatom and maybe even microalgae. How can these all sensibly work together?\nI’d very much like to see this addressed with a few sentences in the manuscript.\nResponse: Indeed, it is difficult to make strong conclusions on whether the ciliate is strictly\nanaerobic or not. With the genome of the ciliate it would have been possible to make stronger\nconclusions. In this study, we unfortunately did not obtain any usable bins for the analysis of\nthe ciliate genome, our single-cell sequencing only allowed the assembly of the bacterial\ngenomes.\nWe agree with this reviewer that it is confusing to see such interactions below the oxic-\nanoxic transition zone. Our observations do not allow us to determine whether the\nmicroalgae within the ciliate are alive, nor whether they were phagocytosed by the ciliate as\na result of their sedimentation in the sediment. It is also possible that the ciliate is a\nfacultative anaerobe, although our observations indicate that they are consistently found in\noxygen-depleted sediments.\nIn the discussion we addressed the comment of this reviewer with the addition of the\nfollowing sentences line 544, page 12: “The emergence of these symbioses is likely\nfacilitated in environments where magnetotactic prokaryotes are prevalent and common.\nThis suggests that environmental pressures in oxygen-deprived aquatic habitats might\ncommonly favour the development of such symbiotic relationships among microeukaryotes.\nHowever, the strict anaerobic lifestyle of the ciliate host remains under question. Indeed,\nalthough it lives below the oxic-anoxic interface, is sensitive to air, contains putative\nhydrogenomes and has established symbiosis with anaerobic bacteria, we are missing\ngenomic information that would confirm this point. Moreover, the presence of a microalgae\nquestions on how these can all sensibly work together”.\nImportantly, no cytochrome c oxidases were detected in the gammaproteobacterium and the\nalphaprotobacterium genomes (see KOfamScan outputs in Supplementary Table 9 in the\nrevised manuscript). If such absence is not related to the draft assembly status, it means\nthat they are not aerobic, but could, as the protist, rely on the sulphate reducing symbionts\nto offload reduced products (as discussed line 432, page 10).\nBased on this comment, and after re-reading the article and preparing a revised version, we\nbelieve that it would be more appropriate to remove the term “anaerobic” from the title of the\narticle. Accordingly, we have modified the title of the manuscript to: “Magnetoreception in a\nfreshwater ciliate arises from endosymbiosis.”\nLine-specific comments:\nLine 55: It is not my impression that the importance of symbiotic interactions has been ‘long-\ndenigrated’. Is this with regard to a specific reference? I’d suggest removing this.\nResponse: It is in fact due to an old assumption before Margulis’ work, we removed this\nnotion that was making the sentence more difficult to read.\nLine 65: replace ‘within the host cytoplasm’ with ‘within the host cell’ to encompass also\nintranuclear bacteria.\nResponse: The correction has been made (line 64, page 3).\nLine 73-79: Please include corresponding references. Also, given the recent re-organization in\n2\ntaxonomy, it might be worthwhile mentioning that Pseudomonatota were previously referred to\nas proteobacteria.\nResponse: The following reference was added at the end of this sentence: López-García,\nP. & Moreira, D. The symbiotic origin of the eukaryotic cell. C R Biol 346, 55–73 (2023).\nLine 73, page 3, in parentheses we mentioned that Pseudomonatota was “previously\nreferred as Proteobacteria.\nLine 105+118+275: replace ‘degenerated’ with ‘reduced’\nResponse: The correction has been made.\nLine 126-127: it is not clear to me how magnetotaxis alone would allow organisms to ‘move\nefficiently towards their optimal redox niche’. To find redox conditions (e.g. oxygen) the\norganisms would need to be additionally aerotactic or chemotactic.\nResponse: Indeed, magnetic orientation alone does not allow magnetotactic\nmicroorganisms to find their optimal redox niche, magnetotaxis is defined as a combination\nof the magnetic alignment of the cell while they swim and chemotaxis. We understand the\nconfusion while reading the sentence lines 126-127, we have modified this sentence, line\n125, page 4, by: “whereby organisms move efficiently towards their optimal redox niche\ncombining guidance by the Earth’s magnetic field lines and chemotaxis”.\nLine 165ff: the link between the arguments 1 and 2 and the conclusions that the host is ‘fragile\nto osmotic changes and sensitive to oxygen’ is not clear to me. Both argument 1 – that less\nciliates were enriched using a stirring bar and argument 2- that the cells survived longer in a\nhanging drop than on a glass slide would suggest that they are sensitive to mechanical\nforce (and maybe heat). I don’t see how the conclusion about sensitivity to osmotic change and\nparticularly oxygen was made. The oxygen tolerance is an important issue and evidence\nregarding this should be clearly outlined.\nResponse: We agree with this comment, line 165, page 5, we modified the sentence by:\n“The cells appeared sensitive to mechanical force”.\nLine 191: this statement is in contrast to the one made later (Line 622) where it is suggested\nthat the ciliate can take up whole intact diatoms. Please remove or revise.\nResponse: Line 622, now line 626, page 14, of the revised version, we modified the\nsentence to avoid this confusion: “Because diatoms and protists are similar in size and given\nthe small diameter of the cytostome opening (0.5 μm), it is unlikely that the cytostome can\nexpand enough to engulf the entire rigid silica frustule of diatoms, as also observed in other\nciliates such as Didinium nasutum88. Moreover, the silica composition of the diatom's frustule\nmay hinder flexibility. Alternatively, diatoms and other algae may be phagocytosed through\nthe plasma membrane of the ciliate and subsequently internalized within phagosomes89.”\nLine 209-211: It is not clear to which organisms/clades the different identity values refer to.\nResponse: We clarified the sentence in the revised version of the manuscript, line 208,\npage 6, as follows: “Although average pairwise identities values between members of\nProstomatea and Plagiopylea were approximately 90% and 85% respectively, they were on\naverage 86% and 82% identical with those of the magnetotactic protists, respectively.”\nLine 212-216: I might have missed the corresponding method section that describes how the\n3\n18S phylogeny was calculated and which sequences were included in the tree. If indeed\nmissing, this information needs to be added to the manuscript. Additionally, the 18S tree in\nSuppl. Fig. 4 is hard to read, as bootstrap values and names overlap. This should be checked\nand amended.\nResponse: The method for the phylogenetic reconstruction based on the 18S rRNA gene\nsequences was included in the Figure 1d caption in the previous version but was indeed\nmissing in the materials and methods section. This information is now included in the section\n“Genome-based taxonomic classification and molecular phylogeny” line 898, page 19:\n“Identification of the protist relied on a maximum-likelihood phylogenetic tree of the\nCiliophora constructed from SSU 18S rRNA gene sequences alone, as no eukaryotic draft\ngenome and other conserved marker genes could be obtained. A sample of 294 sequences\nat least 1634 pb long representing 205 Ciliophora families were retrieved from the PR2\ndatabase (https://app.pr2-database.org). The tree was rooted with the Colponemidia based\non Tikhonenkov et al.120, who demonstrated their monophyly and position as the sister\nlineage to all other known Alveolata. A total of. The alignment of the 319 sequences was\nperformed using MAFFT and trimmed using a relaxed parameterized Gblocks v 0.91\nprogram121. The tree was then inferred as described above for whole genome trees”. The\nFigure 1d caption was also simplified in the revised version.\nSupplementary Fig. S4 has been improved in the revised version of the manuscript. We\nalso included a new supplementary data (now Supplementary Data S1) that corresponds to\nthe newick format of the tree in which all the boostraps value are visible.\nLine 224: ‘exposure to oxygen’ – do you mean air?\nResponse: We replaced “oxygen” by “air”.\nLine 237: delete ‘as well’ as it is repetitive with ‘also’.\nResponse: The correction has been made.\nLine 264-265: What it meant with ‘…the (bacteria) showed no connection to the extracellular\nenvironment…’ ? Please clarify and revise.\nResponse: To be defined as endosymbiont, a bacterium has to be completely enclosed\nwithin the host in the cytoplasm. If a symbiont is physically encapsulated within its host but\nis in contact with to the extracellular media, it will be considered as an ectosymbiotic\nbacterium. For instance, in the model host Anaeramoeba, some symbionts present within\nthe host are considered as ectosymbionts as they are housed in a membrane network with\nconnections to the plasma membrane (Jerlström-Hultqvist et al. 2024, Nat Commun,\n10.1038/s41467-024-54102-7). In this study, no comparable observations were noted.\nInstead, only endosymbiotic bacteria completely residing within the cytoplasm were\nobserved. To make the sentence clearer, we split this sentence in two, to read line 262,\npage 7: “All bacteria appeared to be completely enclosed within their host without\nconnection to the plasma membrane and the extracellular media, they were then considered\nas endosymbionts. The bacteria were rod-shaped and, when observed outside the\ndisaggregated protist, flagella were never observed.”\nLine 323: should read ‘burnetii’\nResponse: The correction has been made.\nLine 326: unclear what is meant with ‘the same ecological niche’, please clarify.\nisolated from various aquatic habitats. We modified the sentence line 331, page 8, by: “Few\nclose 16S rRNA gene sequences of uncultured bacteria associated to various freshwater\nand saline habitats, could be retrieved from public databases (Supplementary Fig. 8d).”\nLine 336-337: It is not obvious to me that the Desulfovibrionales in the CLSM picture is at the\nanterior part of the cell, as mentioned in text. Rather it seems to stretch across the whole length\nof the host. As the FISH image in Fig. 6a and in Video 8 are the same cell, the authors should\ninclude a few more representative FISH images to support the claims about symbionts’\nlocation and abundance.\nResponse: Indeed, the Desulfovibrionales appear to stretch across the whole length of the\nhost. We wrote in the text that these biomineralizing cell “were generally located at the\nanterior pole” which is not confirmed by the CLSM picture. This pattern of cell distribution\nwas observed in several holobionts. However, to avoid the discrepancy with some of the\nimages we decided to show in the manuscript, we modified the sentence line 341, page 8,\nby: “Bacteria producing magnetosomes bundles were stretched across the whole length of\nthe host as shown in Supplementary Videos 5, 8 and 9, and Fig. 3a.”\nIn the new Supplementary Video 9, we now present three different holobionts that\nconfirmed symbionts’ location and abundance.\nLine 343-345: From this one FISH picture it seems to me that the gammas are in fact more\nabundant than the Desulfovibrionales, in contrast to what is stated in text.\nResponse: We agree with this reviewer. Similarly to the positioning of the\nDesulfovibrionales, the abundance of the Gammaproteobacteria but also of the\nAlphaproteobacteria symbionts was not consistent between holobionts. This sentence, line\n353, page 8, in the revised version, has been modified by: “In contrast, the\ngammaproteobacterium GD-1 and alphaproteobacterium MD-1 lack specific spatial\nlocalization.”\nLine 351: what is meant with ‘symbiotic counterparts’, who is this referring to?\nResponse: ‘symbiotic counterparts’ was used for the Desulfovirbionales,\nAlphaproteobacteria and Gammaproteobacteria symbionts. We understand the confusion\nhere. To make the sentence clearer it has been modified line 357, page 9, by: “Despite its\nreduced size, the genome of DcD-1 has a relatively high G+C content (60.5%) compared to\nthe other three genomes, whose G+C contents are significantly lower (from 37.2% to 45.7%,\nSupplementary Table 1).”\nLine 354: I find a protein-coding density of 57% very low and rather atypical for an\nendosymbiont – the authors could touch on this and the high pseudogene presence in their\ndiscussion.\nResponse: We agree, this is an interesting result that should be discussed. The following\nsentence has been added line 597, page 13, in the revised manuscript: “The association of\nGD-1 and MD-1 with their ciliate host appears ancient as their genome structures reflect an\nadvanced stage of reductive evolution. However, further investigation is required to date the\nrelative emergence of this symbiosis in their respective taxonomic groups. Indeed, although\nfamilies of the Rickettsiales order are considered to be relatively old groups of intracellular\nbacteria58,83,84, associations with particular hosts may have arisen more recently. On the\nother hand, UBA9339 symbiont resides in a poorly explored group of orders related to\nLegionellales and Coxiellales. While there are suggestions that host association in general\n5\nmay be ancestral in these lineages, even its obligatory nature may be a more recent trait63..\nDespite its relatively large genome size (>2 Mbp), the magnetic symbiont DsD-1 shows clear\nsignatures of a long-term obligate symbiotic lifestyle as well. DsD-1 contains very few mobile\ngenetic element–related genes and almost no pseudogenes, and it has a minimal set of\nconserved genes for DNA replication recombination an repair54. However, in such advanced\nreductive evolutionary processes, one would not expect the unusually low protein-coding\ndensity observed in DsD-1 (57.43%), which indicates that nonfunctional sequences have\nnot yet been fully purged. This genomic architecture suggests that DsD-1 may be\nundergoing an advanced, deletion-driven stage of genome reduction that followed the earlier\nloss of MGEs and pseudogenes. In contrast, the symbiosis establishment could have been\nmore recent for the Desulfarculales endosymbiont whose genome is highly pseudogenized\n(6 %) and contains more than 1.5 k MBEs. Such a genomic structure could be the result of,\nor a remnant of ongoing reductive evolutionary processes following endosymbiosis\nestablishment, as observed in other symbiotic bacteria54,74,84,85.\nLine 358: ‘typical of obligate endosymbionts’ is vague and unclear what this refers to. Be more\nconcrete or remove such generic statement.\nResponse: We removed this generic statement.\nLine 360: please include how many % of all proteins these three categories amounted to.\nResponse: We added this information line 367, page 9, in the revised manuscript.\nLine 366: ‘apart from Dsd-1, a last category outcompeted all the others:…’ – what do you mean\nwith this? Please revise and split these two statements into two sentences for better readability.\nResponse: We modified this sentence for better readability, line 372, page 9, by: “The COG\ncategory “Replication, recombination, and repair” represented 14%, 16%, and 30% of the\npredicted proteins in the endosymbionts GD-1, MD-1, and DcD-1, respectively. Of these,\n46%, 63%, and 94% corresponded to mobile genetic element–related genes. In contrast,\nsuch genes were rare in DsD-1, where this COG category accounted for less than 0.02% of\nall annotated proteins.”\nLine 372: Who are the Pseudomonadota symbionts? In the diversity description\nPseudomonadota are not mentioned.\nResponse: Here the Pseudomonadota symbionts are the Alphaproteobacteria MD-1 and\nthe Gammaproteobacteria GD-1. Strains DcD-1 and DsD-1 belong to another phyla, the\nDesulfobacterota. This sentence has been totally modified based on a comment by reviewer\n#2. To avoid any ambiguity, we now specify the strain name whenever a taxon is mentioned\nthroughout the revised manuscript.\nLine 382: Please use the taxonomic names for each symbiont consistently throughout the text\nand do not switch between e.g. GD-1, Legionellales, Gammaproteobacteria when describing\nthe same organism as this complicates the reading.\nResponse: The modification has been made throughout the Results and Discussion.\nLine 406: do you mean oxidation of organic compounds (such as acetate)? Or really\ndegradation (of polymers, such as PHB or such)?\nResponse: We agree that this statement may not be detailed enough. We rephrased line\n413, page 10, as follows: “…the analysis indicates possible syntrophies based on the\n6\ndegradation of organic compounds such as acetate or glucose, the oxidation of hydrogen\nand sulphate reduction… ”\nLine 413: do you mean it was not complete (rather than not fully predicted) in the genome?\nResponse: Indeed not all genes involved in the Wood-Ljungdahl pathway were predicted,\nwe modified the sentence, now line 422, page 10, by: “although this pathway was not\ncomplete in the draft genome.”. We also included the KOFamScan outputs in a new\nSupplementary Table 10 listing genes with KEGG Orthology annotations.\nLine 441ff: I don’t understand this sentence. This study does not report the euglenozoan\nholobiont. Please revise.\nResponse: We start the discussion by a short reminding of the discovery in 2019 of the first\ncollective magnetotaxis between an euglenozoan host and tens of ectosymbionts that\nbiomineralize magnetite. We modified these sentences (now line 453, page 10, in the\nrevised manuscript) to improve clarity at the beginning of the Discussion.\nLine 446: I’d recommend to remove the ‘five-member’ as it is not clear if other symbionts other\nthan the Dsd-1 are necessary for functioning of the consortium and also 5 member reference\nomits the diatom.\nResponse: The modification has been made.\nLine 458: ‘Unfortunately, …’ What is meant with this sentence? It does not make sense here, I\nsuggest removing.\nResponse: We aimed to mention that we hoped to get a more resolved tree of the Ciliophora\nwith more markers, but that we could not because no eukaryotic DNA was amplified or host\ngenome assembled. To clarify, we rephrased as follows line 470 page 11: “The 18S rRNA\ngene sequence might not be resolutive enough to get a proper insight into the relationships\nwith other ciliates. The tree topology lacks support and differs with those reconstructed from\nnumerous orthologs72. A more resolved phylogenetic reconstruction was not possible as no\ngenome associated with the host could be assembled from the amplified DNA of the\nholobiont.”\nLine 484: Please include the number.\nResponse: This sentence has been modified, line 500, page 11, by: “This number of\nmagnetosome chains far exceeds the minimum number of few dozen magnetosomes\nrequired to generate sufficient torque in such MHB to align with Earth’s magnetic field\nlines32.”\nLine 496: ‘should be largely enough’. As you document magenotactic behavior of the ciliate, it\nis clearly enough. Please revise accordingly.\nResponse: This sentence, now line 503, page 11, has been modified by: “In total,\napproximately 800 magnetosomes are produced by E. magneticus DsD-1 cells from a single\nhost to induce a magnetotactic behaviour in the ciliate.”\nLine 503: reminding how? Please amend the statement to be more concrete.\n#2 considered it too speculative.\nLine 528: I am not sure I follow why the discovery in France and Brazil would raise question\nabout the presence of magnetite biomineralization in microeukaryotes. Do you maybe mean\nbiogeography or distribution or origin? Please be more specific.\nResponse: To date, the only microeukaryote proposed to biomineralize magnetite is the\nmagnetotactic protist described by Leao et al. (2019). However, our study shows that this\nprotist is actually in symbiosis with magnetite-biomineralizing bacteria, indicating that no\nmicroeukaryote is currently known to biomineralize magnetite. Nonetheless, it remains\npossible that undiscovered microeukaryotes with this capability exist. We rephrased this\nsentence, now line 541, page 12, by: “Magnetite biomineralization by microeukaryotes still\nneed to be confirmed.”\nLine 530: Revise this formulation – I guess you mean association of protist host from different\nlineages with symbionts from different phyla?\nResponse: We agree, it is clearer to formulate the sentence that way. It has been modified,\nline 542, page 12, in the revised manuscript, as follows: “The association of protist hosts\nfrom diverse phyla with symbionts from various classes suggests that magnetosymbiosis\nhas independently emerged multiple times across both the Bacteria and Eukaryota\ndomains.”\nLine 532-533: It should also be considered here that the emergence of these symbioses will\nbe likely facilitated in environments where magnetotactic prokaryotes are prevalent and\ncommon.\nResponse: We agree, this notion is relevant in this paragraph, now line 544, page 12. We\nadded the following sentence: “Moreover, the emergence of these symbioses will be likely\nfacilitated in environments where magnetotactic prokaryotes are prevalent and common.”\nLine 539: should be ‘consuming’ not ‘producing’\nResponse: Modification has been made, now line 557, page 12.\nLine 542 - 547: which other hydrogen-producing specialized organelles are meant here? The\nprotist can utilize ‘external inorganic electron acceptors’? Which would these be? ‘Offload’\nsounds a bit like slang. Also, hydrogen doesn’t need to be ‘offloaded’, it will diffuse out of a\nswimming ciliate without problems. What about the sulfide that is the end product of the\nDesulfobacterota metabolism – how does the host deal with that toxic substance being\nproduced intracelullarly?\nResponse: Concerning the “other hydrogen-producing specialized organelles” it was not\nmeant to point out a specific organelle but rather suggest the possibility that other organelles\ncould produce hydrogen. We simply removed these words.\nWe agree that this whole section was not really well written. We rephrased line 559, page\n12, in the revised manuscript as follows: “Predatory protists metabolize organic matter and\nproduce hydrogen (H ) as a byproduct via their hydrogenosomes and small molecules such\n2\nas CO , acetate or alcohols. In the magnetotactic ciliate, these intermediates are likely\n2\nfurther oxidized by their endosymbionts for biomass and ATP generation. On the other hand,\nDcD-1 and DsD-1 could enable their host to breathe sulphate instead of oxygen. Such\nutilization of symbionts as electron acceptors or sinks in metabolic coupling has been\n8\npreviously documented in other ciliates81. In addition, hydrogen sulphide produced by\nsulphate respiration of DcD-1 and DsD-1 could help maintain a low redox potential inside\nthe consortium or could be eliminated outside the protist by diffusion to avoid toxic effects.”\nLine 567: I would not say that Rickettsialles are better known for mutualism as many people\n(including me) might associate them with human pathogens. Or do you mean the specific group\nthat includes your symbiont?\nResponse: Indeed, in this sentence we are discussing about the specific family to which\nbelongs MD-1. In this sentence, now line 588, page 13, we specified that we are talking\nabout the Midichloriaceae family.\nLine 570: What is MHB? I missed this explanation.\nResponse: MHB referred to “magnetotactic holobionts and was defined line 283, page 7\n(in the revised manuscript), when the acronym appears for the first time. This acronym is\nused numerous times throughout the text. This acronym was also previously used\nthroughout the manuscript Chevrier et al 2023, PNAS.\nLine 571: Spell out whose genome.\nResponse: This paragraph line 589, page 13, was modified for clarity.\nLine 575: Which associations and lost from where, which genomes/organisms?\nResponse: We agree, it was unclear if we were talking of our model or of the example from\nthe previous sentence. In fact, we are discussing about our model, we specified this point in\nthis sentence to avoid the ambiguity. Line 597, page 13: “The association of GD-1 and MD-\n1 with their ciliate host appears ancient as their genome structures reflect an advanced stage\nof reductive evolution.”\nLine 582: I would not describe being prey as a facultative relationship – or what is meant here?\nResponse: This sentence, now line 621, page 14, was modified and the term “facultative\nrelationship” was removed: “The presence of different diatom species within the same\npopulation of magnetotactic ciliates from a single sample supports the idea that intracellular\ndiatoms are not essential for the functioning of the ciliate or the holobiont.”\nLine 590-591: Is this the case in this host? Could you include a reference to a Figure then?\nResponse: Yes it does. It was observed in some of our thin sections such as the\nSupplementary Figure 5e. Based on a comment of Reviewer #2, this paragraph has been\nshortened (from line 584 to line 619 in the former manuscript) and this sentence has been\nremoved.\nLine 598 – 599: Also, I assume that sediments will not be limited in any of these nutrients,\nunlike the water column.\nResponse: We agree that sediments should be less limited than the water column in CO ,\n2\ninorganic nutrients, such as phosphorus, nitrogen, or trace elements. However, we do not\nknow if the diatoms found in the ciliate were living in the water column or the sediments\nbefore their engulfment. Based on a comment of Reviewer #2, this paragraph has been\nshortened and this sentence has been removed.\nwhat happens with the produced oxygen? Can you speculate how the obligately anaerobic\nsulfate reducers can possibly deal with this?\nResponse: The relationship between the diatoms and the ciliate is only hypothetical here\nand we hope that our future research will allow to decipher what exactly linked both\nmicroeukaryotes. Here the ciliate appears to be living in anaerobic conditions but it might\nalso be able to respire oxygen as most of ciliates do. Thus, it is possible that the oxygen\nproduced by the diatom during photosynthesis is directly used by the ciliate and that\nprokaryotic endosymbionts are not in contact with this oxygen. It is also possible that the\noxygen diffuse outside the ciliate and its time inside the ciliate is limited to avoid potential\ntoxic effect. In this case the ciliate would benefit only from the energy produced during\nphotosynthesis. We could also hypothesize that the host is not the only one to benefit from\nthe oxygen released by the diatom, the bacterial symbionts could also use oxygen as a\nterminal electron acceptor, even the Desulfobacterota as some strains of the\nDesulfovibrionia have been shown to be able to respire oxygen, including Solidesulfovibrio\nmagneticus (doi: 10.1111/1758-2229.12479), a magnetotactic bacterium related to strain\nDsD-1. However, based on current assemblies, it is unlikely as no cytochrome c or cbb\n3\noxidases were identified (Supplementary Table 9).\nWe wanted to add the sentence: “The oxygen produced during photosynthesis could also\nbenefit to the bacterial symbionts even the Desulfobacterota as some have been shown to\ngrow with oxygen83. Alternatively, the oxygen could be toxic for the host and the symbionts\nonce released inside the ciliate, its rapid diffusion outside the ciliate could potentially avoid\ndamages.” However, the whole paragraph has been removed as the Reviewer #2 found it\ntoo speculative at this stage.\nLine 652: Care should be taken to clarify that in the euglenozoa symbiosis magnetotaxis of the\nectosymbiont is combined with the chemotaxis (aerotaxis) of the host, and thus allow\nnavigation in chemically-stratified aquatic environments. Is this ciliate host also capable of\naero- or chemo-taxis?\nResponse: We do not have evidence of host ability of aero- or chemo-taxis in both models\nof magnetotactic protists. It is only an hypothesis to explain the potential advantage\nconferred by magnetotaxis based on what has been demonstrated in free-living\nmagnetotactic bacteria. The sentence was modified to the conditional, line 656, page 14:\n“The magnetotaxis observed in the magnetotactic holobiont HBD-1 from the Dordogne river\ncould mirror that of free-living magnetotactic bacteria and euglenozoan, enabling efficient\nnavigation in chemically stratified aquatic environments”\nLine 657: This is to me an unclear statement and it is a pity to end up this really nice paper on\nthis kind of vague sentence. Maybe try to revise to be more specific.\nResponse: In this sentence we meant that the evolution of collective magnetotaxis based\non an endosymbiosis between a ciliate and biomineralizing bacteria could be at the origin of\nmagnetoreception in eukaryotes. We agree that we should have been more specific in this\nsentence. We modified this sentence, line 661, page 14, by: “However, although the\nintegration of magnetotactic bacteria into a modern lineage of protists is now proven, further\nresearch is needed to determine whether such integration and further organellogenesis or\nlateral gene transfers could have occurred in early holobionts that gave rise to the first\neukaryotes lineages able of magnetoreception.”\nParagraph Line 675ff: What is meant by microcosms? Please explain. It is unclear to me how\n10\nthe oxygen sensor was calibrated – what is ‘saturated humid air (O2 saturation 100%)’? Do\nyou mean pure oxygen? What is humid? Why was the calibration not done with air-saturated\nwater, as outlined in the instruction manual? What is ‘water solution flushed with N2’?\nIs it MilliQ? Was it flushed or bubbled? How long? Please include all relevant information here.\nResponse: Microcosms is commonly used to mean mesocosms or aquaria. Since\n“mesocosms” seems to be more often used, we replaced “microcosms” by “mesocosms”.\nThe calibration of the oxygen probe was made with 1) a 50 mL solution of milliQ water\nflushed (i.e. bubbled) for 30 min with air in order to get 100% of oxygen saturation = 21%\noxygen in the water. “humid” is generally employed to show that the calibration is made in a\nliquid, here milliQ water; 2) the second point of calibration was made in 50 mL milliQ water\nflushed (i.e. bubbled) for 30 min with 100% nitrogen. This information have now been\nincluded in the Methods section, line 687, page 15, in the revised manuscript: “Sensor\ncalibration was made with a 50 mL milliQ water solution flushed with air for 30 min (O\n2\nsaturation = 100%) and a milliQ water solution flushed with N for 30 min (O saturation =\n2 2\n0%).”\nParagraph Line 788ff: It is essential to include here the formamide concentrations of the\nhybridization buffers, which were used for FISH.\nResponse: The stringency used during FISH protocol was already indicated in the Methods:\n“using the hybridization and washing stringencies of 30% for all probes”. In general, we do\nnot need to indicate the formamide concentration since it is proportional to the stringency.\nHere a stringency of 30% mean that the formamide concentration in the hybridization buffer\nwas 0.6 mL/mL. We added this information in the revised Materials and Methods section\nline 807, page 17.\nLine 1260: replace ‘blows’ with ‘breaks apart’\nResponse: Modification has been made line 1310, page 30, and also in the caption of\nSupplementary Video 3.\nLine 1279: replace ‘presence of the extremity of a diatom’ with ‘part of a diatom’.\nResponse: Modification has been made line 1329, page 30.\nLine 1325: replace ‘authentication’ with ‘identification’ or ‘visualization’\nResponse: Modification has been made. We preferred to use “visualization” line 1375, page\n31.\nLine 1328: replace ‘authenticate’ with ‘target’\nResponse: Modification has been made line 1378, page 31.\nLine 1337ff: Please revise this sentence for clarity. Also, sulfate reducers typically do not use\nmore complex organic compounds.\nResponse: The sentence, line 1388, page 31, has been modified by: “Complex\ncarbohydrates such as glucose would benefit to the endosymbiotic Gammaproteobacteria\nand Alphaproteobacteria that could feed from the primary breakdown of organic material in\ndigestive vacuoles.”\nsymbionts for their growth’.\nResponse: The legend of Figure 6b has been modified, this sentence does not exist\nanymore, although the information is still present line 1393, page 31.\n__________________________________________________________________________\nReviewer #2 (Remarks to the Author)\nThe paper entitled \"Magnetoreception in a freshwater anaerobic ciliate arises from\nendosymbiosis\" represents the first documented case of magnetoreception in a protist\nresulting from an endosymbiotic association with bacteria. While magnetoreception in protists\nremains a rare and poorly understood phenomenon - previously observed only in protists with\nectosymbionts, this study reveals an astonishingly complex and novel system. The discovery\nnot only expands our understanding of magnetoreception in eukaryotes, but also contributes\nto the growing body of research highlighting the remarkable diversity and evolutionary\nsignificance of interactions between bacteria and protists.\nAs the first discovery of a system in which magnetoreception is acquired through\nendosymbiosis, this research is both novel and timely and fits well into the current wave of\nstudies investigating endosymbiotic interactions between bacteria and protists. The results are\nthoroughly documented and supported by multiple lines of evidence, including advanced\nmicroscopy techniques and comprehensive genome analyses.\nResponse: We thank the reviewer for their kind comments. Their comments not only\nstrengthened the manuscript but also broadened our personal understanding of protist\nsymbioses.\nHowever, there are concerns regarding the interpretation of the results. While the involvement\nof the endosymbiont DsD-1, a member of the Desulfovibrionaceae, in magnetoreception is well\nsupported, the specific contributions of other members of the holobiont, both bacterial and\neukaryotic, remain unclear. These additional partners may play an important role in supporting\nthe anaerobic lifestyle and overall fitness of the host, but their direct involvement in\nmagnetoreception has not been convincingly demonstrated. Therefore, characterising the\nphenomenon as “magnetotaxis of a multi-partner endosymbiotic interaction” appears\npremature based on current data and their interpretation.\nThe manuscript repeatedly emphasises that magnetoreception arises from the interaction\nbetween several partners; however, the evidence presented does not sufficiently support this\nclaim. Either more direct evidence is needed to demonstrate the involvement of all partners in\nmagnetoreception, or the current formulation should be weakened to more accurately reflect\nthe limitations of the data. Ideally, experiments on cultures would clarify the critical role of\nindividual partners, although this is often difficult to achieve. Therefore, a more detailed\nreconstruction of metabolism that emphasises the interdependence of all holobiont\ncomponents is required, together with more careful interpretation.\nResponse: We agree with the reviewer #2: we believe that magnetoreception in the protist\nis possible only thanks to one of the four endosymbionts, DsD-1. We apologize for the\nawkward expression “magnetotaxis arises from a multi-partner endosymbiotic interaction”\nthat led to this confusion in the abstract and suggests that several symbionts are involved in\nmagnetoreception. In the revised manuscript, we comprehensively specified the organisms\ninvolved in magnetotaxis when describing the holobiont.\nremoving the term “multi-partner” as follows: “However, in this case, magnetotaxis arises\nfrom an endosymbiotic interaction.” The origin of magnetoreception was also specified in\nthe introductory section line 139, page 4: “We demonstrated that these unicellular\neukaryotes are, in fact, multi-partners holobionts composed of a ciliate (Ciliophorea,\nProstomatea) and four populations of endosymbiotic bacteria, one of which, belonging to\nthe Desulfovibrionales, biomineralizes a bundle of bullet-shaped magnetosomes.\"\nDetailed comments on these and other issues are provided below. Overall, the study presents\na convincing result that is supported by solid data. However, our main concerns relate to the\nway in which the results are communicated. In some cases, the claims are presented too\nbroadly, and in others, the interpretations appear too speculative. We believe that a more\nfocused presentation on the magnetoreception aspect would strengthen the manuscript.\nResponse: Based on the corrections proposed below by this reviewer, we believe that the\npresentation of the results and the discussion is now more focused on the magnetoreception\naspect. In addition, the entire discussion section from line 584 to line 619 on the relationships\nof diatoms with the ciliate was removed as it was found too speculative.\nDetailed comments\nLine 42 – “However, in this case, magnetotaxis arises from a multi-partner endosymbiotic\ninteraction.” Is the magnetoreception in the case presented strictly dependent on multiple\nendosymbionts (instead of just single magnetotactic symbiont), in contrast to previously known\nmagnetoreception in euglenozoans (relying on extracellular bacteria)?\nResponse: Indeed, this sentence is confusing. As mentioned above, we removed the term\n“multi-partner” line 42, page 2, in the revised version.\nLine 65 – There are known cases of endosymbionts residing in the nucleus (which formally is\ndistinguished from the cytoplasm) (10.1016/j.tcb.2015.01.002), thus it is better to change for\nthe “integrated within the host protoplasm” or “integrated within the host cytoplasm and/or\nnucleus”.\nResponse: A similar comment was raised by Reviewer #1, we replaced “integrated within\nthe host cytoplasm” by “integrated within the host cell” line 64, page 3, in the revised version.\nLine 104 - please change protozoa to protist\nResponse: Modification has been made line 103, page 4, in the revised version.\nLine 105 - please use mitochondria-related organelles (MRO) as a well-established term for\nreduced mitochondria in anaerobic lineages of protists\nResponse: Modification has been made line 104, page 4, in the revised version.\nLine 110 – rather “protection against competitors or predators”, not “chemical camouflage” (the\nlatter not mentioned in the original publication).\nResponse: Modification has been made line 109, page 4, in the revised version.\nmake reliable taxonomic decisions, as evolutionary rates vary greatly between lineages. The\nguidelines of the Zoological Code should be followed when describing new ciliate species. The\ndata presented may not be sufficient to formally describe a new species or genus, as this\nrequires the identification of type material. Furthermore, a formal taxonomic description does\nnot appear to be necessary in this case, and its absence would not diminish the significance\nof the results. We therefore suggest reconsidering whether such a description is needed.\nResponse: We agree that further work would be required to give a formal taxonomic\ndescription to this protist. We replaced the statement line 214, page 6, by “Pending a more\nformal taxonomic description in the future, we named the ciliate HBD-1” in the revised\nversion. In addition, we removed the reference to Candidatus Magnetocilia dordonia\nthroughout the manuscript when referring to the host and replaced the proposed genus and\nspecies name by the term “magnetotactic protist”, “ciliate” or “HBD-1”.\nLine 287 - we suggest providing the information on the average genome coverage for the\nobtained bacterial genomes. This will better illustrate the quality of data presented and may\ninform about relative abundances of the symbionts in the host cell.\nResponse: We added this information as requested in the new Supplementary Table 1.\nLine 288 - how the estimation of the amount of eukaryotic reads was performed? As the authors\nattempted total rRNA extraction from the raw assembly, is it correct to state that no 18S\nfragments of either host or intracellular photosynthetic algae were recovered? If so, please\nmention this in an appropriate section of the results.\nResponse: The number of eukaryotic reads was estimated as follows: first, EukRep was\nused to identify eukaryotic contigs within the assembly. Then all reads were mapped to these\ncontigs with Bowtie 2 to estimate the number of eukaryotic reads. This information has been\nadded in the materials and methods section line 857, page 18, in the revised manuscript.\nThe rRNA genes were predicted using RNAmmer in all the global assembly before binning\nas stated line 871, page 18, in the revised manuscript and led to the identification of only\none 18S rRNA gene sequence matching with the 18S rRNA sequence of the ciliate obtained\nby pyrosequencing. No other sequence was recovered.\nThe corresponding result section was rewritten and corrected as follows: “However, only few\neukaryotic reads were obtained after the hologenome amplification— approximately 31\nmillion, representing 15.3 % of the total reads. They mapped to 2 209 contigs representing\na total assembly length of 19.95 Mbp. According to the Protista_83 single-copy core gene\n(SCG) collection in anvi’o, the assembly corresponds to a highly incomplete genome (< 6.0%\ncompleteness) with substantial redundancy (> 9.6%). Only a single 18S rRNA gene\nsequence associated with the ciliate was detected.” line 289, page 7, in the revised\nmanuscript.\nLines 298-299 - as Dsd-1 is currently the only endosymbiotic magnetotactic bacterium in its\nfamily, authors may consider comparing its genomic capabilities with that of the free-living\nrelatives (Oceanidesulfovibrio and Megalodesulfovibrio). Similarly, comparison with Ca.\nDesulfarcum epimagneticum CR-1 may inform about the differences in the integration between\neukaryotic hosts and their magnetotactic symbionts.\nResponse: We fully agree with Reviewer #2. We initially began conducting this broader\nanalysis for this symbiont as well as others, including some unpublished ones. However, we\nsoon realized that the volume of data and the number of insights were substantial enough\n14\nto write a dedicated manuscript of their own on the emergence of a symbiotic lifestyle in\nmagnetotactic Desulfobacterota. For the present manuscript, we believe it is more\nappropriate to focus specifically on magnetoreception.\nLines 339-341 - Presence of the mam genes is mentioned in support of the ability of Dsd-1 to\nform magnetite particles. While this proof seems sufficient, Extended data Fig. 8e shows that\nsome of the core mam genes (according to Lefevre et al. 2013 https://doi.org/10.1111/1462-\n2920.12128) are missing (mamA, mamE, mamQ, mamB). Was there an attempt to find those\ngenes in the assembly in order to confirm or rule out that their absence was caused by binning\ninaccuracy?\nResponse: This is correct. As stated in the original manuscript, the MGC is incomplete. The\nabsence of the mentioned mam genes is likely due to amplification, sequencing, or assembly\nissues, as the MGC lies at the end of a truncated contig. These genes were not detected\nelsewhere in the genome. The remaining 27 genes are conserved in deep-branching MTB\nand appear to be specific to magnetotaxis. Additional sequencing projects are planned to\nimprove the assembly of the MGC.\nAdditionally, while unnecessary here, the phylogenetic analysis of mam genes would be\ninstrumental in understanding the process of biomineralisation cluster acquisition.\nGood point. A phylogenetic analysis of mam genes or Mam proteins would be very\ninteresting indeed, and is complementing the comparative genomics analysis mentioned\nabove. As the reviewer noted, several mam genes that are among the most conserved in\nMTB (see doi:10.1038/nrmicro.2016.99) are missing, which currently limits the robustness\nof phylogenetic inference. However, we agree that we could have provided more information\nregarding the potential origins of these genes.\nBased on BLASTP alignments, Mam proteins of DsD-1 share the highest sequence identity\n(i.e., 50.6 % and 47.7 % on average) with their corresponding homologs in the reference\nstrains Solidesulfovibrio magneticus RS-1 (https://doi-org/10.1101/gr.088906.108), and\nFundidesulfovibrio magnetotacticus FSS-1 (https://doi-org/10.1099/ijsem.0.005516),\nrespectively. Together DsD-1, RS-1 and FSS-1 represent the only three genera containing\nmagnetotactic species (Figure 5a and the new Supplementary Table 2 in the revised\nmanuscript). A phylogenetic tree of the most conserved Mam protein among the all-shared\nMam proteins, MamK, supports that the MGC of RS-1 and FSS-1 have a more recent\ncommon ancestor than both RS-1 and FSS-1 MGC with that of DsD-1. Such topology can\nsupport several scenarios given the species tree in Figure 5A, involving and not, ancient\nhorizontal gene transfers—even if the first scenario (with HGT) is the more parsimonious. In\nthe revised manuscript, we simply specified the relatedness of Mam proteins with their\nclosest MTB, line 348, page 8: “Mam proteins of DsD-1 share the highest sequence identity\nwith their corresponding homologs in the reference strains Solidesulfovibrio magneticus RS-\n152, and Fundidesulfovibrio magnetotacticus FSS-153, with average identities of 50.6 % and\n47.7 %, respectively.”. In addition a new supplementary table was provided showing these\nidentities as proposed by the reviewer #3 (Supplementary Table 2)”.\nLine 354 - How were the pseudogenes identified? This should be included in the methods.\nResponse: Pseudogenes were identified using the Microscope platform\n(https://doi.org/10.1093/database/bap021), based primarily on ortholog length comparison.\nEach CDS of the symbionts was aligned to its ortholog in Escherichia coli K-12 substr.\nMG1655 (GCF_000005845.2). Cases in which a single E. coli CDS corresponded to two\ncolinear ORFs in the symbiont genomes were interpreted as gene fragmentation, typically\n15\nresulting from frameshift mutations or premature stop codons. Because no closely related\ncomplete genomes were available for comparison, the extent of pseudogenization reported\nhere is likely conservative. This clarification has been added to the Methods section (line\n864, page 18, in the revised manuscript): “Pseudogenes were detected by aligning each\nsymbiont CDS to its ortholog in E. coli K-12 substr. MG1655 and identifying orthologs with\ndisrupted structure. A gene was classified as fragmented when a single E. coli CDS matched\ntwo colinear ORFs in the symbiont genomes, indicative of frameshift mutations or premature\nstop codons.”.\nLines 364-366 – we find the information about the proportion of proteins related to\nglycosyltransferases a bit out of context, as it is not interpreted or mentioned further. Is this\nproportion different in other symbionts or somehow remarkable in this case?\nResponse: We are sorry, we can’t justify this statement in the original version. We agree\nwith the reviewer, and we removed the sentence in the revised manuscript.\nLines 366-368 - there is an enormous number of replication, recombination & repair genes\nidentified in genome Dcd-1 (as shown in Supplementary Table 1), an order of magnitude larger\nthan in other symbionts or free-living bacteria used for comparison. Is it possible to assess\nwhether this inflated number is not caused by the mobile genetic elements-related genes?\nThey often fall within the same COG category and may contribute to the assembly\nfragmentation. In addition, their identification informs about the proliferation of mobile genetic\nelements, observed in some symbiont genomes.\nResponse: Excellent point, we missed it in the original manuscript. Indeed, the number of\nreplication, recombination, and repair genes is highly inflated in DcD-1 due to mobile genetic\nelement–related genes, which we had initially overlooked. To quantify their contribution, we\nscreened the eggNOG-mapper output for COG category “L” and identified genes whose\nPFAM domains or annotation descriptions contained the terms “IS,” “transposase,” or\n“integrase” (see the new Supplementary Table 4). Mobile genetic elements accounted for\n94%, 63%, 46%, and 4% of category L predicted genes in DcD-1, MD-1, GD-1, and DsD-1,\nrespectively. This observation and interpretation have been added to the revised manuscript,\nline 374, page 9.\nLines 368-371 - the loss of the replication, recombination & repair machinery in Dsd-1 is very\ninteresting, as those processes are highly conserved and mostly retained even in extremely\nreduced endosymbionts with genomes below 1 Mbp (10.1146/annurev-micro-091213-112901\nand 10.1371/journal.pbio.3002577). Therefore, we suggest presenting more information on the\ncomponents that are preserved or lost, and whether this loss would impact the bacterial\ncapability to maintain its genome. It is important information for understanding the genome\nreduction processes in endosymbionts.\nResponse: We apologize, this statement was not correctly written, because indeed, it\nsuggests that ALL conserved genes were lost and this is not correct. We meant that DsD-1\nmachinery was reduced. We carefully checked the eggNOG-mapper output for COG\ncategory “L” (see the new Supplementary Table 4), and we confirm that a minimal\nreplication, recombination & repair machinery is present in all symbionts. We corrected this\nsentence and provided more information on the genes preserved in the results section line\n376, page 9, in the revised manuscript: “All symbionts have a minimal machinery involved\nin DNA replication, homologous recombination, base excision repair or mismatch repair. The\nset of conserved genes includes polymerases genes together with dna, rec, ruv, uvr, and\nmut genes (Fig. 5e and Supplementary Table 4)”.\nsection of the revised manuscript: “Despite its relatively large genome size (>2 Mbp), the\nmagnetic symbiont DsD-1 shows clear signatures of a long-term obligate symbiotic lifestyle.\nDsD-1 contains very few mobile genetic element–related genes and almost no\npseudogenes, and it has a minimal set of conserved genes for DNA replication\nrecombination an repair54.”.\nLine 371 - We suggest adding a reference to the Supplementary table 1 as well, because Fig.\n5e shows the relative frequencies of gene categories and not the absolute number of genes,\nwhich is important for interpreting the gene losses of conserved cellular systems.\nResponse: Reference to this Supplementary table has been added, in the new version of\nthe manuscript it is Supplementary Table 4.\nLine 372 - Proteins with typical eukaryotic-interacting domains (including leucine-rich repeats)\nare presumed to participate in symbiont manipulation of the host cell after delivery via secretion\nsystems (10.1016/j.cub.2021.05.049). While it is possible that they were acquired via HGT,\nthere is no direct evidence for that presented, and we would suggest focusing on their potential\nrole in interaction with the host. In particular, we suggest providing information regarding the\nnumber of such genes for a particular symbiont genome.\nResponse: We agree. First, we removed the sentence “Genomes of both Pseudomonadota\nendosymbionts specifically, show evidence for lateral gene transfers.”. Second, we built a\nlist of proteins containing leucine-rich repeats (LRRs) and ankyrin repeats—domains\ninvolved in interactions with eukaryotic hosts (new Supplementary Table 5). Homology\nsearches were performed using the The InterPro database (http://www.ebi.ac.uk/interpro/)\nintegrating together predictive models or ‘signatures’ representing protein domains, families\nand functional sites from multiple, diverse source databases: Gene3D, PANTHER, Pfam,\nPIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY and TIGRFAMs. Finally, we\nreported the number of such genes in symbiont genomes line 379, page 9: “Functional\nannotation predicted genes encoding proteins with leucine-rich repeats (LRRs) and ankyrin\nrepeats—domains involved in interactions with eukaryotic hosts58—in three out of four\nsymbiont genomes (Supplementary Table 5). While the DcD-1, GD-1, and MD-1 genomes\nencode 3, 8, and 7 ankyrin repeat-containing proteins, respectively, only GD-1 harbors four\nproteins with leucine-rich repeats, alongside several Type IV secretion systems\n(Supplementary Table 6), which are often repurposed by endosymbionts to deliver host-\ntargeted effectors58”.\nLine 387 - We suggest moving the details about the virulence factor search into the methods\nsection.\nResponse: Modification has been made. In the Methods section, line 926, page 20, we\nadded the following sentence: “Putative virulence-associated genes were identified using\nVirulenceFinder125 and BLASTP searches against hits of the Virulence Factor Database\n(VFDB)126 applying a minimal identity of 30%.”\nLine 395-397 - We suggest toning down this interpretation of function as many of the presented\nvirulence factor predictions are based on relatively low (30%) identity hits and may indicate\nonly distant similarity and not the same function.\nResponse: We fully agree. The following section was rephrased line 398, page 9, as\nfollows: “A total of 118 genes in GD-1 showing remote homology to known virulence factors\nwere also predicted, including a phtA-like gene, a phagosomal transporter involved in the\nestablishment and persistence of L. pneumophila within its replication vacuole65, as well as\n17\nenhanced entry (Enh) proteins that facilitate host cell invasion and infection66. Many\ntransporters involved in iron uptake, adherence and toxins (e.g. Enterobactin, Hemolysin,\nLPS, Microcin H47) in pathogens such as Neisseria meningitidis MC58, Escherichia coli\nO157:H7, Haemophilus influenzae Rd KW20, were also predicted. However, some\nsequence identities being low (i.e., 30-40%), these homologs may be functionally different.\nNo such genes were detected in MD-1, except antophagocytosis genes, like the two other\nsymbionts exhibiting similar but less numerous defensive systems (Supplementary Table\n8).”\nLine 439 - We suggest rephrasing this, as there is no evidence for reliance of\nmagnetoreception specifically on syntrophy, to the best of our knowledge. E.g. there is a\npossibility that those layers of interaction are uncoupled, or that magnetoreception was a\ndriving force for the evolution of this association, with established syntrophy being a later\nbyproduct.\nResponse: We clarified and simplified the sentence line 455, page 10, in the revised\nmanuscript: “This finding introduced the concept of collective magnetotaxis characterized by\na mutualistic and obligatory symbiosis. The labour in magnetotactic holobionts is divided\nbetween partners: the euglenozoan host performs chemo-aerotaxis, while the ectosymbiotic\nbacteria provide magnetoreception.”\nLines 474-476 - we strongly encourage authors to provide full, proper descriptions of the newly\ndescribed bacteria (see examples, 10.1038/s41396-023-01499-6 and 10.1038/s41467-024-\n54047-x). Authors may also consider using SeqCode (10.1038/s41564-022-01214-9) for new\ntaxa submission.\nResponse: We are convinced that providing proper descriptions of the newly identified taxa\nis highly valuable. The protologue has now been included in the Supplementary Results 1\nsection. All names have been deposited in the SeqCode Registry under accession number\nr:dgcae3s5. Because the genome quality of DcD-1 was not sufficient, the curators\nrecommended removing the name Desella intracellularis from the list and assigning\nit Candidatus status instead. The name will be formally published in SeqCode once higher-\nquality assemblies become available.\nLines 501-504 - presented statement suggests that the magnetite-forming endosymbiont has\nlost many of its metabolic capabilities, preserving only essential pathways for cell functioning.\nHowever, it still possesses a relatively large genome (2 Mbp) and in terms of the number of\nmetabolic pathways identified is only the second lowest after the Midichloriaceae\nendosymbiont. Additionally, it is suggested to participate in syntrophy. We suggest to tone this\ndown a bit or provide more details about the relevant pathway losses. For example, severe\ndeficiencies of systems important for cell maintenance (phospholipid/fatty acids synthesis,\npeptidoglycan/cell wall synthesis, division, nucleotide and amino acid synthesis) may\nsupport this claim.\nResponse: We agree that this whole section was confusing. We deleted it in the revised\nmanuscript. Instead, we focused on the magnetosome aspect line 502, page 11:\n“Conversely, HBD-1 is larger than the marine MHB but harbours 25 times fewer\nendosymbiotic E. magneticus cells a total of approximately 800 magnetosomes to induce a\nmagnetotactic behaviour in the holobiont. Despite their lower numbers, each cell produces\nalmost five times more magnetosomes aligned into six chains of bullet-shaped magnetite\ncrystals. These particles are nearly twice as large as those of free-living magnetotactic\nbacteria from the Desulfovibrionaceae family, such as Solidesulfovibrio magneticus strain\nRS-178 or Fundidesulfovibrio magnetotacticus FSS-154, which typically form about ten\nparticles of similar width but less than 100 nm in length. However, they are smaller than\n18\nthose in a magnetotactic eukaryote described by Leão et al.35, which had magnetosomes-\nlike particles averaging 276.6 ± 61.3 nm in length and 52.7 ± 5.1 nm in width. Another striking\ndifference between both magnetosome-producing symbionts is that the endosymbiont\napparently evolved to overproduce magnetosomes to the point that they fill the entire\ncytoplasm. Indeed, cells of DsD-1 produce about 131 particles for a length and width of\napproximately 5 μm and 0.5 μm, whereas cells of the cultivated strain FSS-1 produce about\n10 magnetosomes for a length and width of approximately 4 μm and 0.8 μm, respectively54.”.\nLine 569-570 - did authors consider performing a search for the ATP/ADP translocases in\nendosymbiont genomes? This information may be useful for assessing the potential impact of\nPseudomonadota symbionts on the energy metabolism of the holobiont.\nResponse: We agree that the presence of ATP/ADP translocases in endosymbiont\ngenomes is important to understand the nature of symbiont/host interactions. Homology\nsearches were performed using the InterPro model IPR004667 and only three significant\nmatches were identified in the alphaprotobacterium genome. This information is now\nincluded in a revised version of the Figure 6b and line 434, page 10, in the revised\nmanuscript: “MD-1 has a severely reduced energy metabolism and lacks the ability to\nperform glycolysis and to use the tricarboxylic acid (TCA) cycle—similar to many\nRickettsiales, which import metabolites from their host70. However, MD-1 has the potential\nto generate reducing power (NADH), acetyl-CoA and CO through the decarboxylation of\n2\nexogenous pyruvate. It may also produce the (2S)-ethylmalonyl-CoA intermediate required\nfor the ethylmalonyl-CoA pathway, an alternative to the TCA cycle. Notably, this\nendosymbiont is the only one in HBD-1 to encode three ADP/ATP translocases (tlc; InterPro\nentry IPR004667), which enable the direct import of ATP from the host cytosol58.”. We also\ninclude this result in the Discussion section line 591, page 13, in the revised version.\nLine 571-573 - we find this part slightly confusing, are those auxotrophies present in\nPseudomonadota symbionts? If yes, how do they contribute to the consortium? Are there any\ncontributions from the Desulfobacterota symbionts? Figure 6b uses the generalised term\n“amino acids” and only provides information about vitamins B1 (provided by all symbionts),\nB9 (provided by three symbionts) and B12 (provided by two symbionts), impairing interpretation.\nResponse: We agree that this part is confusing as we don’t mention which symbiont has\nthe inability to synthetize amino acid, vitamin, or nucleotides. We rephrased this section line\n591, page 13, in the revised version as follows : «They likely do not participate directly to\nthe global energy metabolism of the MHB and they are unable to synthetize a large number\nof compounds required for their growth. However, they may support the consortium by\nsynthetizing essential compounds for both the host and other symbiotic partners, such as\nfew vitamins and key cofactors». The Figure 6b was also revised and now includes the\ndetailed list of amino acids/vitamins & co-factors synthetized by each bacterial partner.\nLine 575-576 - Families of Rickettsiales are considered to be relatively old groups of\nintracellular bacteria, but associations with particular hosts may be more recent\n(10.1038/s41467-021-23645-4, 10.1038/s41467-024-45351-7 and\n10.1016/j.cub.2021.05.049). On the other hand, UBA9339 symbiont resides in a poorly\nexplored group of orders related to Legionellales and Coxiellales. While there are suggestions\nthat host association in general may be ancestral in these lineages, even its obligatory nature\nmay be a more recent trait (10.1038/s41564-022-01174-0). We suggest explaining this\nnotion in more detail or citing a specific reference.\nResponse: We agree. We rephrased this section and included the following statement line\n597, page 13: “The association of GD-1 and MD-1 with their ciliate host appears ancient as\ntheir genome structures reflect an advanced stage of reductive evolution. However, further\n19\ninvestigation is required to date the relative emergence of this symbiosis in their respective\ntaxonomic groups. Indeed, although families of the Rickettsiales order are considered to be\nrelatively old groups of intracellular bacteria58,83,84, associations with particular hosts may\nhave arisen more recently. On the other hand, UBA9339 symbiont resides in a poorly\nexplored group of orders related to Legionellales and Coxiellales. While there are\nsuggestions that host association in general may be ancestral in these lineages, even its\nobligatory nature may be a more recent trait63.”\nLine 577 - The role of diatoms in this system remains highly speculative. Ciliates often form\nphotosymbioses with green algae, although they have never acquired permanent plastids.\nSuch interactions have not yet been observed between ciliates and diatoms, and although the\npossibility is intriguing, convincing evidence is lacking. We therefore recommend focussing on\nthe most important discoveries and not on the uncertain role of diatoms in this context.\nResponse: We agree with the reviewer that the role of the diatoms and other microalgae\ninside the ciliate remains speculative at this stage. We shortened this paragraph to keep\nonly the essential information while highlighting directions for future research to elucidate\nthe nature of this association line 618, page 14, in the revised manuscript.\nLines 589-590 and 621-623 - Previously (lines 190-191), authors suggest that examined\nmagnetotactic ciliates are unable to phagocyte prey larger than small bacteria due to the small\ncytostome size, contradicting themselves.\nResponse: This issue was also raised by Reviewer#1. Accordingly, we rephrased the section\nto avoid any ambiguity line 624, page 14, in the revised manuscript (former line 622):\n“Nevertheless, additional evidence is needed to clearly define the nature of this relationship.\nThe exact process by which diatoms and other microalgae are internalized remains unclear as\ndirect observations are lacking. Given that diatoms and protists are similar in size, and\nconsidering the small size of the cytostome opening (0.5 μm), it is unlikely that the cytostome\ncan expand sufficiently to engulf the entire rigid silica frustule of diatoms, as observed in other\nciliates such as Didinium nasutum88. ”.\nLines 623-624 - This sentence is confusing, as it seems to conflict with the prior one.\nResponse: The previous sentence was confusing, but after being revised according to the\ncomment mentioned just above, it is now clear and no longer confusing .\nLines 624-625 - citation needed.\nResponse: The following reference has been added: Wessenberg, H. & Antipa, G. Capture\nand Ingestion of Paramecium by Didinium nasutum. The Journal of Protozoology 17, 250–\n270 (1970).\nLines 602-603 - wouldn’t the immediate proximity of oxygen producers be highly detrimental\nfor all anaerobic members of the consortium?\nResponse: This point was also raised by the Reviewer #1, and we responded as follows:\nHere the ciliate appears to be living in anaerobic conditions but it might also be able to\nrespire oxygen as most of ciliates do. Thus, it is possible that the oxygen produced by the\ndiatom during photosynthesis is directly used by the ciliate and that prokaryotic\nendosymbionts are not in contact with this oxygen. It is also possible that the oxygen diffuse\noutside the ciliate and its time inside the ciliate is limited to avoid potential toxic effect. In this\ncase the ciliate would benefit only from the energy produced during photosynthesis.\nthe most important discoveries and not on the uncertain role of diatoms.\nLines 652-658 - the link between particular magnetotactic symbiosis and eukaryogenesis\nsuggested here is unclear.\nResponse: In this sentence we meant that the evolution of collective magnetotaxis based on\nan endosymbiosis between a ciliate and biomineralizing bacteria open questions on the origin\nof magnetoreception in eukaryotes. As mentioned by Reviewer #1, we should have been more\nspecific in this sentence. We rephrased it line 659, page 14, in the revised manuscript as\nfollows: “Considering the current evolutionary scenarios of eukaryogenesis and eukaryotes\ndiversification9, it is appealing to link these findings to the origin of magnetoreception in\neukaryotes more largely. However, although the integration of magnetotactic bacteria into a\nmodern lineage of protists is now proven, further research is needed to determine whether\nsuch integration and further organellogenesis or lateral gene transfers could have occurred in\nearly holobionts that gave rise to the first eukaryotes lineages able of magnetoreception.”\n__________________________________________________________________________\nReviewer #3 (Remarks to the Author):\nThe manuscript by Bolzoni et al. presents the identification a new class of magnetotactic\neukaryotes distinguished by an symbiotic relationship between a magnetotactic bacterium and\na protist. Previous work, including groundbreaking studies from this group, had shown that\nprotists can be magnetically sensitive in two different ways: passively through the ingestion of\nmagnetotactic bacteria and actively through an ectosymbiotic relationship with a magnetotactic\nbacterium. This work presents the incredible discovery of an endosymbiotic magnetotactic\nbacterium. The cell biology, multi-layered endosymbiotic relationships, minerology, phylogeny,\nand genomics of this new group of protists is detailed through a series of carefully performed\nexperiments. The findings are highly impactful and open up a host of new questions for\nmicrobiologists, cell biologists, and evolutionary biologists. I am thoroughly impressed by the\nwork and cannot wait to see it published. I have only a few minor comments which can be\naddressed through changes to the text and figures.\nResponse: We thank the reviewer for their kind comments. We deeply appreciate your\nfeedback, it’s very encouraging and motivating.\n1. Line 247: There is no reference to a figure but there is a closed parens.\nResponse: Correct. Reference to Fig. 3d and “e” has been added.\n2. Line 253: There is a reference to \"membrane similar to bacterial magnetosomes\" in ED\nFigure 6d. First, I had to zoom in quite a bit to see the relevant magnetosomes. To my eyes\nthese do not look as convincingly like the ones in the Gorby reference. Given that there is\nsome dispute over the presence of membranes around bullet-shaped magnetosomes, I would\nencourage the authors to provide a more convincing image or acknowledge the possible\nambiguity.\nResponse: It has initially been thought that bullet-shaped magnetosomes might lack a\nsurrounding membrane (doi: 10.1073/pnas.1001290107). However, more recent studies\nhave shown that bullet-shaped magnetites were indeed surrounded by a thin membrane\n21\n(e.g. doi:10.1111/1462-2920.13677). Although this membrane is thinner than the one\ndescribed for cubo-octahedral magnetite magnetosomes in Magnetospirillum\nmagnetotacticum MS-1 by Gorby et al 1988, it is now widely accepted that all types of\nmagnetosomes are surrounded by a membrane. In Supplementary Figure 6d, we pointed\nwith black arrows the bullet-shaped particles for which a membrane is visible. We believe\nthe resolution of this figure submitted at 600 dpi allows to see the membrane of some of the\nbullet-shaped particles produced by the endosymbiotic strain DsD-1. These membranes\nhave a similar contrast and thickness than those described for instance in doi:10.1111/1462-\n2920.13677. In the text, we modified the end of the sentence line 249, page 6, in the revised\nmanuscript: “Each vesicle contains about 131 ± 26 particles (n=13), particles measure 143.6\n± 40.9 nm in length and 55.2 ± 8.8 nm in width (n=629), and are surrounded by a thin\nmembrane similar to the membrane generally observed surrounding bullet-shaped\nmagnetites38 (Supplementary Fig. 6d)”. The reference from Gorby et al 1988 was replaced\nby doi:10.1111/1462-2920.13677.\n3. Lines 339-341: I was surprised at how little attention and space was given to analyzing the\nmagnetosome genes of the endosymbiont. This is incredibly important in thinking through the\nevolutionary diveristy of magnetotactic bacteria and should be given a more visibility. I would\nencourage the authors to have a dedicated figure for these genes (extended data is fine\nalthough it could also work as a portion of a main text figure). The current extended data figure\n(8E) has some ambiguous descriptions in its legend. For instance, line 1425 says that each\narrow represents a gene corresponding to MSR-1 operons. However, MSR-1 does not have\nmad genes. In the legend, \"mam\" should be italicized and CR-1 should be defined in the\nlegend. I would request a table comparing the magnetosome proteins of Dsd-1 with their\naccession numbers to those of other magnetotactic bacteria (for instance, including the %\nidentity to homolog in RS-1 or others). This would be a big contribution to the community.\nResponse: We agree with the reviewer #3 that the study of the evolution of magnetosome\ngenes of the symbiotic magnetite producing bacteria would be very interesting. However,\nour assembly of DsD-1 genome enabled us to obtain a partial magnetosome gene cluster\nonly and most of the mam genes usually used to study magnetotaxis evolution are missing.\nTo get a robust phylogeny to make assumptions about magnetotaxis evolution we would\nneed the sequence of all the magnetosome genes that are conserved in all magnetotactic\nbacteria including mamABEIKLMOPQ. We are currently attempting to improve the genome\nsequencing of DsD-1 to get the entire sequence of the magnetosome gene cluster and we\nare currently writing another manuscript specifically focusing on the evolutionary history of\nbiomineralizing bacteria including free-living magnetotactic bacteria and symbiotic\nmagnetosomes-producing bacteria. Please, see our response to the Reviewer #2 page 15,\nof this letter.\nIn the revised manuscript, we specified the relatedness of Mam proteins with their closest\nMTB line 348, page 8: “Mam proteins of DsD-1 share the highest sequence identity with\ntheir corresponding homologs in the reference strains Solidesulfovibrio magneticus RS-153,\nand Fundidesulfovibrio magnetotacticus FSS-154, with average identities of 50.6 % and 47.7\n%, respectively.”. In addition a new supplementary table was provided showing these\nidentities as proposed by the reviewer #3 (Supplementary Table 2)”.\nWe also agree that the legend of Supplementary Figure 8 contained ambiguous\ndescriptions. This is an error caused by copying and pasting a similar caption from a previous\narticle. We removed the sentence “Each arrow represents a gene of a color corresponding\nto a specific operon in MSR-1” and replaced it by the appropriate reference in the legend of\nSupplementary Figure 8e.\n22\n4. Finally, I feel that the extrapolation from genome sequences to metabolic capability of the\nendosymbionts was too strong. The authors used the words \"can\" and \"cannot\" based on the\npresence or absence of certain genes. However, one cannot say that a certain reaction will\nhappen because genes are there as some might be incative. Similarly, novel pathways using\ngenes annotated as hypothetical or as having different function may fulfill the missing pieces\nin a metabolic pathway. In other sentences, the authors used \"may\" to describe the potential\nactivities of these organisms. This is more appropriate and should be repeated throughout.\nResponse: We fully agree with this comment, we revised the entire manuscript to use the\nconditional form to show that our genomic analyses give predictions of metabolic capability\nsection in the revised manuscript.\n5. Finally, I could not open videos 1, 2, and 5. I am not sure if it is an issue with my current\nsetup or something is wrong with the files.\nResponse: These three movies are in AVI format the other movies were in MPG or MP4\nformats. We checked all the videos on both a Mac and a PC and found that, depending on\nthe software used, some videos could not be opened. We therefore converted these videos\nto ensure that they can be opened.\n__________________________________________________________________________\nReviewer #4 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank this reviewer for their help at improving this manuscript.\nTitle: Magnetoreception in a freshwater ciliate arises from endosymbiosis\nResponse: We would like to express our gratitude to the editor for giving us the opportunity to\naddress the concerns raised by the reviewers in this new submission. We have carefully\nconsidered their feedback and found the overall comments and suggestions constructive. We\nbelieve we have addressed all the requested changes.\nIn this revised manuscript, we have provided a point-by-point response to each concern. We\nhave included line by line where changes were made in the revised version.\nReviewers’ comments/questions are written in black while our answers are written in blue.\nSee below.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nI am satisfied with the revisions the authors made to the manuscript and I appreciate\nthe time and effort they took to address all reviewers' comments. In my opinion, the\nrevision has improved the manuscript and I am looking forward to seeing this\nwonderful discovery published.\nResponse: We thank the reviewer for their kind comments.\nI have only a few last small suggestions for text edits:\nLine 56: to me ‚diversification‘ is redundant here and could be deleted\nResponse: The correction has been made.\nLine 75-76: It is my impression that while the endosymbiotic origin of plastids and\nmitochondria is largely undisputed, discussions regarding the origin of nucleus persist\n(i.e. the autogenic models). Hence, I’d suggest to rephrase it here as such (e.g. : ‘…\nemergence of plastids, mitochondria, and potentially also the nucleus’).\nResponse: The correction has been made, page 3, line 77: “These interactions led to the\nemergence of plastids, mitochondria and potentially also the nucleus.”\nLine 76: Consider replacing ‘most’ with ‘many’.\nResponse: The correction has been made.\nLine 78: mention also aerobic respiration, as the primary role of mitochondria\nResponse: The correction has been made.\nLine 140: ‘multi-partner’ instead of ‘multi-partners’\nResponse: The correction has been made.\nLine 250: replace ‘particles’ with ‘which’\nResponse: The correction has been made.\nLine 254: consider adding ‘magnetite’ before ‘particles’ for clarity.\nResponse: The correction has been made.\nLine 262 – 264: consider rephrasing: ‘All bacteria were considered endosymbionts as\nthey appeared to be completely enclosed … and the extracellular space.’\nResponse: This sentence has been modified, page 7, line264, by: “All bacteria appeared to\nbe fully enclosed within their host, with no detectable connection to the plasma membrane\nor the extracellular medium, and were therefore considered endosymbionts.”\nLine 272: replace ‘support’ with ‘confirm’\nResponse: The correction has been made.\nLine 280: replace ‘genetic’ with ‘taxonomic’\nResponse: The correction has been made.\nLine 299: replace ‘population belongs’ with ‘populations belong’\nResponse: The correction has been made.\nLine 356: As it is only 4 genomes, please add also the sizes of the two other\ngenomes (DsD and GD-1).\nResponse: This sentence has been modified, page 9, line 358, by: “Genome sizes of the\nendosymbionts are 1.3 Mbp, 2.0 Mbp, 2.3 Mbp and 2.8 Mbp for MD-1, GD-1, DsD-1 and\nDcD-1, respectively (Supplementary Table 1).”\nLine 363: replace ‘proteins-coding’ with ‘protein-coding’\nResponse: The correction has been made.\nLine 378: please italicize gene names.\nResponse: The correction has been made.\nLine 382: what about DsD? Please add.\nResponse: This sentence has been modified, page 9, line 385, by: “While the DsD-1, DcD-\n1, GD-1, and MD-1 genomes encode 0, 3, 8, and 7 ankyrin repeat-containing proteins”.\nLine 421: replace ‘account’ with ‘accounts’\nResponse: The correction has been made.\nLine 428 – 431: sentence is unclear. Does ‘exclusively’ mean that only menaquinone\nand no other quinone was detected in DsD or that menaquinone was exclusively\ndetected in DsD and in no other symbiont? Please clarify. Also, it is not obvious to me\nhow can the quinones of one endosymbiont have such broad implications for the\nwhole consortium?\nResponse: Only menaquinone and no other quinone was detected in the genome of DsD-\n1. The implication of the quinones of DsD-1 on the whole consortium has been added,\npages 9, line 430: “The biomineralizing symbiont seems to play a central role in respiration and\nelectron flux within the whole consortium, as only the Dsd-1 genome harbours genes\nencoding the synthesis of a low-potential menaquinone that is central to anaerobic\nrespiration69”.\nLine 433: Do you mean ‘or’ instead of ‘of’?\nResponse: This sentence is correct, we mean “or” in the sentence page 10, line 435: “None\nof the two genomes encodes for cytochrome c-type of cbb3-type oxidases or other enzymes\nassociated to inorganic terminal electron acceptors (Supplementary Table 9).”\nLine 453: I suggest to slightly rephrase, e.g.: ‘A recent discovery of marine magnetic\nholobionts revealed a unique ability of certain microeukaroytes …’\nResponse: The correction has been made.\nLine 472: replace ‘with’ with ‘from’\nResponse: The correction has been made.\nLine 473 – 474: replace with ‘…not possible as the genome of the host could not be\nassembled …’\nResponse: The correction has been made.\nLine 562-565: ‘On the other hand, DcD-1 and DsD-1 could enable their host to\nbreathe sulphate instead of oxygen.’\nPlease rephrase. Indeed, DcD-1 and DsD-1 can breathe sulphate, and thus so can\nthe holobiont but not the host (= ciliate) as there is no evidence that the ciliate can\naccess the energy obtained from sulfate reduction, unlike in ref. 81. The authors\nthemselves proposed that the ciliate obtains energy from fermentation in the\nhydrogenosomes. This should be clarified.\nResponse: This sentence has been modified, page 12, line 564, by: “On the other hand, the\nhost could access energy produced by its sulphate-reducing endosymbionts DcD-1 and\nDsD-1.”\nLine 653: replace ‘highlights’ with ‘reports the discovery of’ and replace ‘capable to\nsense’ with ‘capable of sensing’\nResponse: The correction has been made.\nFigure Legend:\nFig. 1b: In the text white and grey arrows are mentioned but in the image there are\nonly black arrows. Can you check and correct if necessary?\nResponse: The legend of Fig. 1b has been modified by: “b, transmitted CLSM image of a\nmagnetotactic protist showing contrasted granules in the anterior part and dark lines aligned\nin the anteroposterior axis (black arrows).” “grey” has been replaced by “black” while the\nreference of “white arrow” was unnecessary since the contrasted granules are obvious on\nthe image and therefore do not need to be indicated on the image.\nFig. 4a: I can only see 2 arrows (=2 cells with magnetosomes), whereas 4 are\nmentioned in the figure legend.\nResponse: Panel a corresponds to the entire upper grey panel and contains three electron\nmicroscopy images: the central image shows the whole protist, while the left and right\nimages are higher-magnification views, each displaying two endosymbionts containing\nmagnetosomes, which are indicated by two white arrows. We have slightly modified the\nlegend of panel a for greater clarity “(white arrows indicated in the left and right images)”.\nFig. 5a,b,c,d: the symbionts from this study should be in bold in the respective trees\nbut they are not (in my version of the figures). Please check and make sure to\nhighlight them (in bold or colour).\nResponse: The correction has been made, a new Figure 5 has been submitted.\nLine 1356: Replace 'under study' with 'from this study'.\nResponse: The correction has been made.\nFig. 6b: replace 'preys' with 'prey' in sketch\nResponse: The correction has been made, a new Figure 6 has been submitted.\nLine 1377: replace 'cell host' with 'host cell'\nResponse: The correction has been made.\nLine 1390 - 1392: These two sentences are essentially identical, please delete one.\nResponse: The correction has been made.\nReviewer #2 (Remarks to the Author):\nThe authors have carefully addressed our comments, as well as those of the other\nreviewers, and have made substantial improvements to the manuscript. All suggested\nchanges were thoroughly considered and implemented where possible, either\nthrough additional analyses or by removing debatable statements. The main story is\nnow much more clearly articulated and supported by additional data, while more\nspeculative aspects have been either removed or appropriately rephrased.\nSome of our comments proposing additional analyses, such as phylogenetic analysis\nof mam genes or more in-depth comparative analyses, were intended as general\nsuggestions. While these were not fully pursued, we understand and agree with the\nauthors’ decision, as incorporating them would have substantially expanded the\nscope of the manuscript and blurred the main focus.\nWe have no further comments, and we would like to congratulate the Authors on this\nexciting and well-supported discovery in protist symbiosis and look forward to seeing\nthe paper published.\nResponse: We thank the reviewer for their kind comments.\nReviewer #3 (Remarks to the Author):\nThe authors have answered all of my concerns. Congratulations on a fantastic\nmanuscript.\nResponse: We thank the reviewer for their kind comments.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: We thank the reviewer for their kind comments."}]

Decision letter