"Candidatus Euplotechlamydia quinta," a novel chlamydia-like bacterium hosted by the ciliate Euplotes octocarinatus (Ciliophora, Spirotrichea).

Our knowledge of ciliate endosymbiont diversity greatly expanded over the past decades due to the development of characterization methods for uncultivable bacteria. Chlamydia-like bacteria have been described as symbionts of free-living amoebae and other phylogenetically diverse eukaryotic hosts. In the present work, a systematic survey of the bacterial diversity associated with the ciliate Euplotes octocarinatus strain Zam5b-1 was performed, using metagenomic screening as well as classical full-cycle rRNA approach, and a novel chlamydial symbiont was characterized. The metagenomic screening revealed 16S rRNA gene sequences from Polynucleobacter necessarius, three previously reported accessory symbionts, and a novel chlamydia-like bacterium. Following the full-cycle rRNA approach, we obtained the full-length 16S rRNA gene sequence of this chlamydia-like bacterium and developed probes for diagnostic fluorescence in situ hybridizations. The phylogenetic analysis of the 16S rRNA gene sequences unambiguously places the new bacterium in the family Rhabdochlamydiaceae. This is the first report of chlamydia-like bacterium being found in Euplotes. Based on the obtained data, the bacterium is proposed as a new candidate genus and species: "Candidatus Euplotechlamydia quinta."

Single-cell Microbiomics Unveils Distribution and Patterns of Microbial Symbioses in the Natural Environment.

Protist-bacteria associations are extremely common. Among them, those involving ciliates of the genus Euplotes are emerging as models for symbioses between prokaryotes and eukaryotes, and a great deal of information is available from cultured representatives of this system. Even so, as for most known microbial symbioses, data on natural populations is lacking, and their ecology remains largely unexplored; how well lab cultures represent actual diversity is untested. Here, we describe a survey on natural populations of Euplotes based on a single-cell microbiomic approach, focusing on taxa that include known endosymbionts of this ciliate. The results reveal an unexpected variability in symbiotic communities, with individual hosts of the same population harboring different sets of bacterial endosymbionts. Co-occurring Euplotes individuals of the same population can even have different essential symbionts, Polynucleobacter and "Candidatus Protistobacter," which might suggest that replacement events could be more frequent in nature than previously hypothesized. Accessory symbionts are even more variable: some showed a strong affinity for one host species, some for a sampling site, and two ("Candidatus Cyrtobacter" and "Candidatus Anadelfobacter") displayed an unusual pattern of competitive exclusion. These data represent the first insight into the prevalence and patterns of bacterial symbionts in natural populations of free-living protists.

The "Other" Rickettsiales: an Overview of the Family "Candidatus Midichloriaceae".

The family "Candidatus Midichloriaceae" constitutes the most diverse but least studied lineage within the important order of intracellular bacteria Rickettsiales. "Candidatus Midichloriaceae" endosymbionts are found in many hosts, including terrestrial arthropods, aquatic invertebrates, and protists. Representatives of the family are not documented to be pathogenic, but some are associated with diseased fish or corals. Different genera display a range of unusual features, such as full sets of flagellar genes without visible flagella or the ability to invade host mitochondria. Since studies on "Ca. Midichloriaceae" tend to focus on the host, the family is rarely addressed as a unit, and we therefore lack a coherent picture of its diversity. Here, we provide four new midichloriaceae genomes, and we survey molecular and ecological data from the entire family. Features like genome size, ecological context, and host transitions vary considerably even among closely related midichloriaceae, suggesting a high frequency of such shifts, incomplete sampling, or both. Important functional traits involved in energy metabolism, flagella, and secretion systems were independently reduced multiple times with no obvious correspondence to host or habitat, corroborating the idea that many features of these "professional symbionts" are largely independent of host identity. Finally, despite "Ca. Midichloriaceae" being predominantly studied in ticks, our analyses show that the clade is mainly aquatic, with a few terrestrial offshoots. This highlights the importance of considering aquatic hosts, and protists in particular, when reconstructing the evolution of these endosymbionts and by extension all Rickettsiales. IMPORTANCE Among endosymbiotic bacterial lineages, few are as intensely studied as Rickettsiales, which include the causative agents of spotted fever, typhus, and anaplasmosis. However, an important subgroup called "Candidatus Midichloriaceae" receives little attention despite accounting for a third of the diversity of Rickettsiales and harboring a wide range of bacteria with unique features, like the ability to infect mitochondria. Midichloriaceae are found in many hosts, from ticks to corals to unicellular protozoa, and studies on them tend to focus on the host groups. Here, for the first time since the establishment of this clade, we address the genomics, evolution, and ecology of "Ca. Midichloriaceae" as a whole, highlighting trends and patterns, the remaining gaps in our knowledge, and its importance for the understanding of symbiotic processes in intracellular bacteria.

Symbionts of the ciliate Euplotes: diversity, patterns and potential as models for bacteria-eukaryote endosymbioses.

Endosymbioses between bacteria and eukaryotes are enormously important in ecology and evolution, and as such are intensely studied. Despite this, the range of investigated hosts is narrow in the context of the whole eukaryotic tree of life: most of the information pertains to animal hosts, while most of the diversity is found in unicellular protists. A prominent case study is the ciliate Euplotes, which has repeatedly taken up the bacterium Polynucleobacter from the environment, triggering its transformation into obligate endosymbiont. This multiple origin makes the relationship an excellent model to understand recent symbioses, but Euplotes may host bacteria other than Polynucleobacter, and a more detailed knowledge of these additional interactions is needed in order to correctly interpret the system. Here, we present the first systematic survey of Euplotes endosymbionts, adopting a classical as well as a metagenomic approach, and review the state of knowledge. The emerging picture is indeed quite complex, with some Euplotes harbouring rich, stable prokaryotic communities not unlike those of multicellular animals. We provide insights into the distribution, evolution and diversity of these symbionts (including the establishment of six novel bacterial taxa), and outline differences and similarities with the most well-understood group of eukaryotic hosts: insects.

Detecting Associations Between Ciliated Protists and Prokaryotes with Culture-Independent Single-Cell Microbiomics: a Proof-of-Concept Study.

Symbioses between prokaryotes and microbial eukaryotes, particularly ciliated protists, have been studied for a long time. Nevertheless, researchers have focused only on a few host genera and species, mainly due to difficulties in cultivating the hosts, and usually have considered a single symbiont at a time. Here, we present a pilot study using a single-cell microbiomic approach to circumvent these issues. Unicellular ciliate isolation followed by simultaneous amplification of eukaryotic and prokaryotic markers was used. Our preliminary test gave reliable and satisfactory results both on samples collected from different habitats (marine and freshwater) and on ciliates belonging to different taxonomic groups. Results suggest that, as already assessed for many macro-organisms like plants and metazoans, ciliated protists harbor distinct microbiomes. The applied approach detected new potential symbionts as well as new hosts for previously described ones, with relatively low time and cost effort and without culturing. When further developed, single-cell microbiomics for ciliates could be applied to a large number of studies aiming to unravel the evolutionary and ecological meaning of these symbiotic systems.

Use of bio-containers from seagrass wrack with nursery planting to improve the eco-sustainability of coastal habitat restoration.

Traditional revegetation techniques employed to restore seagrass meadows and coastal dunes have recently been criticized for their impact on donor populations as well as for the installation of plant anchoring structures made of non-biodegradable or not natural materials in recipient habitats. To improve the ecological sustainability of restoration practices, a novel plantable biodegradable container made of beach-cast seagrass wrack and a bio-based polymer was produced. The long-term performance of two seagrasses, Cymodocea nodosa and Zostera noltei, and two dune plants, Euphorbia paralias and Thinopyrum junceum, grown in nurseries from seeds using the bio-container or a non-biodegradable container of equal size/form made of a conventional plastic (control) was also examined. In addition, the development of bio-container-raised C. nodosa plants in the field was compared to that of plants removed from control containers at the installation and anchored with a traditional system. The bio-container degraded slowly in seawater and in sand and lost its functionality after about three years. In nurseries, all the tested species grown in bio-containers performed as well as, or better than, those raised in non-biodegradable ones. Six months after transplanting into the field, 80% of the C. nodosa nursery-raised plants installed with their bio-container have colonized the surrounding substrate while most of those planted with the traditional system was lost. These results indicate that the new bio-container may support plant growth, and it may also provide protection and anchorage to plants in the field. The use of this bio-container in combination with nursery techniques could improve the environmental sustainability of coastal restoration interventions by providing large plant stocks from seed, thus reducing the impact of collection on donor populations. This approach would also limit the introduction of extraneous materials in recipient habitats and offer an opportunity for valorizing seagrass beach-cast material.

Strengths and Biases of High-Throughput Sequencing Data in the Characterization of Freshwater Ciliate Microbiomes.

Molecular surveys of eukaryotic microbial communities employing high-throughput sequencing (HTS) techniques are rapidly supplanting traditional morphological approaches due to their larger data output and reduced bench work time. Here, we directly compare morphological and Illumina data obtained from the same samples, in an effort to characterize ciliate faunas from sediments in freshwater environments. We show how in silico processing affects the final outcome of our HTS analysis, providing evidence that quality filtering protocols strongly impact the number of predicted taxa, but not downstream conclusions such as biogeography patterns. We determine the abundance distribution of ciliates, showing that a small fraction of abundant taxa dominates read counts. At the same time, we advance reasons to believe that biases affecting HTS abundances may be significant enough to blur part of the underlying biological picture. We confirmed that the HTS approach detects many more taxa than morphological inspections, and highlight how the difference varies among taxonomic groups. Finally, we hypothesize that the two datasets actually correspond to different conceptions of "diversity," and consequently that neither is entirely superior to the other when investigating environmental protists.

Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria.

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss.

"Candidatus Midichloriaceae" fam. nov. (Rickettsiales), an ecologically widespread clade of intracellular alphaproteobacteria.

"Candidatus Midichloria mitochondrii" is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus. Bacteria phylogenetically related to "Ca. Midichloria mitochondrii" (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales. This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family "Candidatus Midichloriaceae." In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

Morphological, ultrastructural, and molecular characterization of Euplotidium rosati n. sp. (Ciliophora, Euplotida) from Guam.

We combined morphological (i.e. live, stained, scanning, and transmission electron microscopy) with morphometric and molecular analysis to describe a ciliate species collected from shallow reefs in Guam, grown, and maintained in our laboratory. The species was recognized as a member of Euplotidium, and compared with established species of the genus: Euplotidium itoi Ito 1958; Euplotidium psammophilus (Vacelet 1961) Borror 1972; Euplotidium arenarium Magagnini and Nobili 1964; Euplotidium helgae Hartwig 1980; Euplotidium prosaltans Tuffrau 1985, and Euplotidium smalli Lei, Choi and Xu, 2002. To obtain more elements to compare the species, new morphometric data and additional SSU rRNA gene sequences of E. itoi and of E. arenarium are reported. On the basis of this comparison, we established the new species Euplotidium rosati that has a cirral pattern composed of 12 frontoventral and six transverse cirri, and lacks the left marginal cirrus. Euplotidium rosati harbors on its dorsal surface epixenosomes, the peculiar extrusive symbionts described in other Euplotidium species. The whole body of our observations together with the analysis of the data available in the literature leads us to propose a redefinition of the genus. The results may also be useful to clarify the tangled relationship between Euplotidium and Gastrocirrhus.

"Candidatus Defluviella procrastinata" and "Candidatus Cyrtobacter zanobii", two novel ciliate endosymbionts belonging to the "Midichloria clade".

The "Midichloria clade" is a recently discovered but well-established evolutionary lineage clustering inside the order Rickettsiales (Alphaproteobacteria). Not much is known about the biology of these organisms. The best characterized ones are endocellular symbionts of very different eukaryotic hosts, ranging from arthropods to protists. "Candidatus Midichloria mitochondrii", the most studied organism of the group, is an interesting object of study because of its unique capability to infect metazoans' mitochondria and the presence of flagellar genes in its genome. With this work, we aim at increasing the knowledge on the biodiversity and phylogeny of the "Midichloria group". We characterized according to the "full cycle rRNA approach" two novel endosymbionts of ciliated protozoa, i.e. Paramecium nephridiatum and Euplotes aediculatus. According to the nomenclatural rules for uncultivated prokaryotes, we established the novel taxa "Candidatus Defluviella procrastinata" and "Candidatus Cyrtobacter zanobii" for the two bacterial symbionts. Our phylogenetic analysis based on 16S rRNA gene sequences confirms that the evolutionary histories of "Midichloria clade" representatives and of their hosts are very different. This suggests that the symbiotic processes arose many times independently, perhaps through ways of transmission still not described in Rickettsiales.

Characterization and comparison of bacterial communities selected in conventional activated sludge and membrane bioreactor pilot plants: a focus on Nitrospira and Planctomycetes bacterial phyla.

A pilot-scale membrane bioreactor (MBR) and a conventional activated sludge system (CAS) were in parallel operated to investigate the impact of the separation technology on the structure and functionality of the selected microbial community. Microbial communities as well as nitrogen removal efficiency of the biomass were characterized. Kinetics and microbial community structure turned out to be duly correlated. The impact of the separation technology on selective conditions and, in particular, the higher variability of solid separation efficiency in CAS with respect to MBR pilot plant possibly represented the main factor influencing the selection of bacterial communities. Concerning nitrifiers, bacteria of the genus Nitrospira were predominant in the MBR. This was in accordance with kinetics of nitrite-oxidizing bacteria that suggested the presence of k-strategists, while r-strategists were selected in the CAS plant, possibly because of the presence of transient higher concentrations of nitrite (in the range of 0.05-0.18 and of 0.05-4.4 mg [Formula: see text]-N L(-1) in the MBR and CAS effluents, respectively). An unexpectedly high presence of bacteria belonging to two specific phylogenetic clades of Planctomycetes was found in both reactors.

Protists' microbiome: A fine-scale, snap-shot field study on the ciliate Euplotes.

Host-microbiome relationships play a fundamental role in the evolution and ecology of any living being. As unicellular organisms, protists represent a unique eukaryotic model to investigate selection mechanisms of the prokaryotic microbiome at the cellular level. Field investigations are central to disentangle relative importance of selective drivers in nature. Here we performed an analysis on data from a snap-shot field study reported previously on bacterial microbiomes associated to natural populations of protist ciliates of the genus Euplotes to detect at a fine scale any influence of habitat and/or host identity in microbiome selection. Comparative analyses revealed environment at a relatively large scale (sampling area) as the main driving factor in shaping prokaryotic communities' structures. No evidence of habitat as key-factor emerged when a smaller spatial scale was considered (pond/channel or site). When only microbiomes of ciliates from the same site were compared, a clear assessment on the influence of host identity at the species level was not achieved, probably due to the small and unbalanced number of individuals for the two considered host species. Starting from this point, wider sampling campaigns will contribute in the future to depict a general view of the drivers influencing the prokaryotic microbiomes of natural protist populations.

Wild Avian Gut Microbiome at a Small Spatial Scale: A Study from a Mediterranean Island Population of Alectoris rufa.

This research is one of the few comparative descriptions at an intraspecific level of wild non-passerine microbiomes. We investigated for the first time the gut microbiome of red-legged partridges (Alectoris rufa) using fecal pellets in order to provide a more informed management. We focused on a small Italian population consisting of two demes (WEST, EAST) separated by about 20 km on the opposite sides of Elba Island. Given the small spatial scale, we set up a sampling protocol to minimize contamination from environmental bacteria, as well as differences due to variations in-among others-habitat, season, and age of feces, that could possibly affect the investigation of the three Elban sites. We found a significant divergence between the WEST and EAST Elban subpopulations in terms of microbial composition and alpha diversity. Although most represented bacterial phyla were the same in all the sites (Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes), microbiomes displayed a much higher diversity in western than in eastern partridges. This result might be related to locally diverging individual physiological needs and/or to different intensities in past releases of captive-bred birds between the two sides of Elba. We suggest that the two subpopulations should be treated as distinct management units.

Variations in epilithic microbial biofilm composition and recruitment of a canopy-forming alga between pristine and urban rocky shores.

Brown algae of the genus Ericaria are habitat formers on Mediterranean rocky shores supporting marine biodiversity and ecosystem functioning. Their population decline has prompted attempts for restoration of threatened populations. Although epilithic microbial biofilms (EMBs) are determinant for macroalgal settlement, their role in regulating the recovery of populations through the recruitment of new thalli is yet to be explored. In this study, we assessed variations in microbial biofilms composition on the settlement of Ericaria amentacea at sites exposed to different human pressures. Artificial fouling surfaces were deployed in two areas at each of three study sites in the Ligurian Sea (Capraia Island, Secche della Meloria and the mainland coast of Livorno), to allow bacterial biofilm colonization. In the laboratory, zygotes of E. amentacea were released on these surfaces to evaluate the survival of germlings. The EMB's composition was assessed through DNA metabarcoding analysis, which revealed a difference between the EMB of Capraia Island and that of Livorno. Fouling surfaces from Capraia Island had higher rates of zygote settlement than the other two sites. This suggests that different environmental conditions can influence the EMB composition on substrata, possibly influencing algal settlement rate. Assessing the suitability of rocky substrata for E. amentacea settlement is crucial for successful restoration.

Plastic litter changes the rhizosphere bacterial community of coastal dune plants.

The presence of plastic litter in coastal environments like beach-dune systems has been well documented, and recent studies have shown that this pollutant can influence sand properties as well as dune vegetation. However, the effects of plastics on rhizosphere bacterial communities of dune plants have largely been neglected. This is an ecologically relevant issue since these communities may play an important role in improving plant growth and resilience of dune systems. Here, we explored the impact of plastic litter made of either non-biodegradable polymers (NBP) or biodegradable/compostable polymers (BP) on the structure and composition of rhizosphere bacterial communities associated with two widespread species along coastal European dunes, Thinopyrum junceum and Sporobolus pumilus, by using a one-year field experiment combined with metabarcoding techniques. Both plastics did not affect neither the survival nor the biomass of T. junceum plants, but they significantly increased alpha-diversity of rhizosphere bacterial communities. They also changed rhizosphere composition by increasing the abundance of the phyla Acidobacteria, Chlamydiae, and Nitrospirae, and of the family Pirellulaceae, and reducing the abundance of the family Rhizobiaceae. NBP reduced drastically the survival of S. pumilus while BP increased its root biomass compared to controls. BP also increased the abundance of the phylum Patescibacteria of the rhizosphere bacterial communities. Our findings provide the first evidence that NBP and BP can change rhizosphere bacterial communities associated with dune plants and highlight the importance of investigating how these changes can affect the resilience of coastal dunes to climate change.

Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association.

The Polynucleobacter-Euplotes association is an obligatory symbiotic system between a monophyletic group of ciliate species belonging to the genus Euplotes and bacteria of the species Polynucleobacter necessarius (Betaproteobacteria). Both organisms are unable to survive independently. Several studies revealed the existence of free-living populations of Polynucleobacter bacteria which are phylogenetically closely related to the endosymbiotic ones, but never share associations with Euplotes in the natural environment. Hence, following the most parsimonious explanation on the origin of the association, this symbiosis should represent a synapomorphic character for the hosts' clade. Nevertheless, phylogenetic analyses performed on an increased number of strains here presented suggest that Euplotes species, during their evolution, recruited Polynucleobacter bacteria as symbionts more than once. Moreover, in three cases, we observed different bacteria as obligate symbionts. These symbionts are the first characterized representatives of a phylogenetic lineage branching in a basal position with respect to the genus Polynucleobacter. The hypothesis that the original obligate symbionts belonged to this newly discovered clade and that, only subsequently, in most cases they have been replaced by Polynucleobacter bacteria recruited from the environment is proposed and discussed. The evolutionary path of this association seems anyway to have been more complex than so far supposed.

Unexpected genetic integrity boosts hope for the conservation of the red-legged partridge (Alectoris rufa, Galliformes) in Italy.

The red-legged partridge (Alectoris rufa) is a medium-sized galliform endemic to southwestern Europe. In the easternmost part of the species' range, the population inhabiting Elba Island (Tuscan Archipelago National Park, Italy) is of undisputed conservation value. While we found nuclear and maternal DNA introgression with the exotic chukar partridge (A. chukar) in previous studies based on microsatellite DNA (n = 25) and two mitochondrial markers (n = 103), respectively, we disclosed a limited or null admixture in a few Elban partridges (n = 4) in a recent genomic investigation relying on 168,675 Single Nucleotide Polymorphisms (SNPs). We herein carried out an extended microsatellite DNA survey including additional 65 samples (total, 90) and six loci (total, 11) to determine both spatial structure and genetic integrity of local A. rufa. A sharp divergence between the subpopulations inhabiting the two sides of the island was disclosed, and the microsatellites indicated that all Elban partridges were not admixed with the chukar, thus fully reflecting the picture inferred using SNPs. We hypothesized that the spreading of chukar genes was constrained by negative selection, with the persistence of only the maternal lineage being indicative of thermal adaptation. The two subpopulations should be treated as distinct Management Units, and an envisaged plan to secure a stock onto nearby Pianosa Island could not only warrant endurance of the Elban population but also establish a source of valuable founders for the ex-situ management of the species in Italy. Our study exemplifies how a suitable samples/loci combination is the key to solve wildlife issues dealing with introgression.

At the threshold of symbiosis: the genome of obligately endosymbiotic 'Candidatus Nebulobacter yamunensis' is almost indistinguishable from that of a cultivable strain.

Comparing obligate endosymbionts with their free-living relatives is a powerful approach to investigate the evolution of symbioses, and it has led to the identification of several genomic traits consistently associated with the establishment of symbiosis. 'Candidatus Nebulobacter yamunensis' is an obligate bacterial endosymbiont of the ciliate Euplotes that seemingly depends on its host for survival. A subsequently characterized bacterial strain with an identical 16S rRNA gene sequence, named Fastidiosibacter lacustris, can instead be maintained in pure culture. We analysed the genomes of 'Candidatus Nebulobacter' and Fastidiosibacter seeking to identify key differences between their functional traits and genomic structure that might shed light on a recent transition to obligate endosymbiosis. Surprisingly, we found almost no such differences: the two genomes share a high level of sequence identity, the same overall structure, and largely overlapping sets of genes. The similarities between the genomes of the two strains are at odds with their different ecological niches, confirmed here with a parallel growth experiment. Although other pairs of closely related symbiotic/free-living bacteria have been compared in the past, 'Candidatus Nebulobacter' and Fastidiosibacter represent an extreme example proving that a small number of (unknown) factors might play a pivotal role in the earliest stages of obligate endosymbiosis establishment.

All essential endosymbionts of the ciliate Euplotes are cyclically replaced.

Symbiotic systems vary in the degree to which the partners are bound to each other1. At one extreme, there are intracellular endosymbionts in mutually obligate relationships with their host, often interpreted as mutualistic. The symbiosis between the betaproteobacterium Polynucleobacter and the ciliate Euplotes (clade B) challenges this view2: although freshwater Euplotes species long ago became dependent on endosymbionts, the many extant Polynucleobacter lineages they harbour arose recently and in parallel from different free-living ancestors2. The host requires the endosymbionts for reproduction and survival3, but each newly established symbiont is ultimately driven to extinction in a cycle of establishment, degeneration, and replacement. Similar replacement events have been observed in sap-feeding insects4-6, a model for bacteria-eukaryote symbioses7, but usually only affect a small subset of the host populations. Most insects retain an ancient coevolving symbiont, suggesting that long-term mutualism and permanent integration remain the rule and symbiont turnovers are mere evolutionary side-stories. Here we show that this is not the case for Euplotes. We examined all known essential Euplotes symbionts and found that none are ancient or coevolving; rather, all are recently established and continuously replaced over relatively short evolutionary time spans, making the symbiosis ancient for the host but not for any bacterial lineage.