Molecular basis of symbiotic promiscuity.

Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the "keys" to a succession of legume "doors". Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys.     

Standing genetic variation in host preference for mutualist microbial symbionts

Many models of mutualisms show that mutualisms are unstable if hosts lack mechanisms enabling preferential associations with mutualistic symbiotic partners over exploitative partners. Despite the theoretical importance of mutualism-stabilizing mechanisms, we have little empirical evidence to infer their evolutionary dynamics in response to exploitation by non-beneficial partners. Using a model mutualism—the interaction between legumes and nitrogen-fixing soil symbionts—we tested for quantitative genetic variation in plant responses to mutualistic and exploitative symbiotic rhizobia in controlled greenhouse conditions. We found significant broad-sense heritability in a legume host''s preferential association with mutualistic over exploitative symbionts and selection to reduce frequency of associations with exploitative partners. We failed to detect evidence that selection will favour the loss of mutualism-stabilizing mechanisms in the absence of exploitation, as we found no evidence for a fitness cost to the host trait or indirect selection on genetically correlated traits. Our results show that genetic variation in the ability to preferentially reduce associations with an exploitative partner exists within mutualisms and is under selection, indicating that micro-evolutionary responses in mutualism-stabilizing traits in the face of rapidly evolving mutualistic and exploitative symbiotic bacteria can occur in natural host populations.     

Evolutionary Dynamics of Nitrogen Fixation in the Legume–Rhizobia Symbiosis

The stabilization of host–symbiont mutualism against the emergence of parasitic individuals is pivotal to the evolution of cooperation. One of the most famous symbioses occurs between legumes and their colonizing rhizobia, in which rhizobia extract nutrients (or benefits) from legume plants while supplying them with nitrogen resources produced by nitrogen fixation (or costs). Natural environments, however, are widely populated by ineffective rhizobia that extract benefits without paying costs and thus proliferate more efficiently than nitrogen-fixing cooperators. How and why this mutualism becomes stabilized and evolutionarily persists has been extensively discussed. To better understand the evolutionary dynamics of this symbiosis system, we construct a simple model based on the continuous snowdrift game with multiple interacting players. We investigate the model using adaptive dynamics and numerical simulations. We find that symbiotic evolution depends on the cost–benefit balance, and that cheaters widely emerge when the cost and benefit are similar in strength. In this scenario, the persistence of the symbiotic system is compatible with the presence of cheaters. This result suggests that the symbiotic relationship is robust to the emergence of cheaters, and may explain the prevalence of cheating rhizobia in nature. In addition, various stabilizing mechanisms, such as partner fidelity feedback, partner choice, and host sanction, can reinforce the symbiotic relationship by affecting the fitness of symbionts in various ways. This result suggests that the symbiotic relationship is cooperatively stabilized by various mechanisms. In addition, mixed nodule populations are thought to encourage cheater emergence, but our model predicts that, in certain situations, cheaters can disappear from such populations. These findings provide a theoretical basis of the evolutionary dynamics of legume–rhizobia symbioses, which is extendable to other single-host, multiple-colonizer systems.     

Evolutionary changes in symbiont community structure in ticks

Ecological specialization to restricted diet niches is driven by obligate, and often maternally inherited, symbionts in many arthropod lineages. These heritable symbionts typically form evolutionarily stable associations with arthropods that can last for millions of years. Ticks were recently found to harbour such an obligate symbiont, Coxiella‐LE, that synthesizes B vitamins and cofactors not obtained in sufficient quantities from blood diet. In this study, the examination of 81 tick species shows that some Coxiella‐LE symbioses are evolutionarily stable with an ancient acquisition followed by codiversification as observed in ticks belonging to the Rhipicephalus genus. However, many other Coxiella‐LE symbioses are characterized by low evolutionary stability with frequent host shifts and extinction events. Further examination revealed the presence of nine other genera of maternally inherited bacteria in ticks. Although these nine symbionts were primarily thought to be facultative, their distribution among tick species rather suggests that at least four may have independently replaced Coxiella‐LE and likely represent alternative obligate symbionts. Phylogenetic evidence otherwise indicates that cocladogenesis is globally rare in these symbioses as most originate via horizontal transfer of an existing symbiont between unrelated tick species. As a result, the structure of these symbiont communities is not fixed and stable across the tick phylogeny. Most importantly, the symbiont communities commonly reach high levels of diversity with up to six unrelated maternally inherited bacteria coexisting within host species. We further conjecture that interactions among coexisting symbionts are pivotal drivers of community structure both among and within tick species.     

The uptake and excretion of partially oxidized sulfur expands the repertoire of energy resources metabolized by hydrothermal vent symbioses

Symbiotic associations between animals and chemoautotrophic bacteria crowd around hydrothermal vents. In these associations, symbiotic bacteria use chemical reductants from venting fluid for the energy to support autotrophy, providing primary nutrition for the host. At vents along the Eastern Lau Spreading Center, the partially oxidized sulfur compounds (POSCs) thiosulfate and polysulfide have been detected in and around animal communities but away from venting fluid. The use of POSCs for autotrophy, as an alternative to the chemical substrates in venting fluid, could mitigate competition in these communities. To determine whether ESLC symbioses could use thiosulfate to support carbon fixation or produce POSCs during sulfide oxidation, we used high-pressure, flow-through incubations to assess the productivity of three symbiotic mollusc genera—the snails Alviniconcha spp. and Ifremeria nautilei, and the mussel Bathymodiolus brevior—when oxidizing sulfide and thiosulfate. Via the incorporation of isotopically labelled inorganic carbon, we found that the symbionts of all three genera supported autotrophy while oxidizing both sulfide and thiosulfate, though at different rates. Additionally, by concurrently measuring their effect on sulfur compounds in the aquaria with voltammetric microelectrodes, we showed that these symbioses excreted POSCs under highly sulfidic conditions, illustrating that these symbioses could represent a source for POSCs in their habitat. Furthermore, we revealed spatial disparity in the rates of carbon fixation among the animals in our incubations, which might have implications for the variability of productivity in situ. Together, these results re-shape our thinking about sulfur cycling and productivity by vent symbioses, demonstrating that thiosulfate may be an ecologically important energy source for vent symbioses and that they also likely impact the local geochemical regime through the excretion of POSCs.     

Stability of Sponge-Associated Bacteria over Large Seasonal Shifts in Temperature and Irradiance

Complex microbiomes reside in marine sponges and consist of diverse microbial taxa, including functional guilds that may contribute to host metabolism and coastal marine nutrient cycles. Our understanding of these symbiotic systems is based primarily on static accounts of sponge microbiota, while their temporal dynamics across seasonal cycles remain largely unknown. Here, we investigated temporal variation in bacterial symbionts of three sympatric sponges (Ircinia spp.) over 1.5 years in the northwestern (NW) Mediterranean Sea, using replicated terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of bacterial 16S rRNA gene sequences. Bacterial symbionts in Ircinia spp. exhibited host species-specific structure and remarkable stability throughout the monitoring period, despite large fluctuations in temperature and irradiance. In contrast, seawater bacteria exhibited clear seasonal shifts in community structure, indicating that different ecological constraints act on free-living and on symbiotic marine bacteria. Symbiont profiles were dominated by persistent, sponge-specific bacterial taxa, notably affiliated with phylogenetic lineages capable of photosynthesis, nitrite oxidation, and sulfate reduction. Variability in the sponge microbiota was restricted to rare symbionts and occurred most prominently in warmer seasons, coincident with elevated thermal regimes. Seasonal stability of the sponge microbiota supports the hypothesis of host-specific, stable associations between bacteria and sponges. Further, the core symbiont profiles revealed in this study provide an empirical baseline for diagnosing abnormal shifts in symbiont communities. Considering that these sponges have suffered recent, episodic mass mortalities related to thermal stresses, this study contributes to the development of model sponge-microbe symbioses for assessing the link between symbiont fluctuations and host health.     

A roadmap of plant membrane transporters in arbuscular mycorrhizal and legume–rhizobium symbioses

Most land plants live in close contact with beneficial soil microbes: the majority of land plant species establish symbiosis with arbuscular mycorrhizal fungi, while most legumes, the third largest plant family, can form a symbiosis with nitrogen-fixing rhizobia. These microbes contribute to plant nutrition via endosymbiotic processes that require modulating the expression and function of plant transporter systems. The efficient contribution of these symbionts involves precisely controlled integration of transport, which is enabled by the adaptability and plasticity of their transporters. Advances in our understanding of these systems, driven by functional genomics research, are rapidly filling the gap in knowledge about plant membrane transport involved in these plant–microbe interactions. In this review, we synthesize recent findings associated with different stages of these symbioses, from the pre-symbiotic stage to nutrient exchange, and describe the role of host transport systems in both mycorrhizal and legume–rhizobia symbioses.

The membrane transport system functions in establishing and maintaining arbuscular mycorrhiza and legume–rhizobium symbioses.     

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host–symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host–symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host–symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed.     

Symbiotic diversity in marine animals: the art of harnessing chemosynthesis

Chemosynthetic symbioses between bacteria and marine invertebrates were discovered 30 years ago at hydrothermal vents on the Galapagos Rift. Remarkably, it took the discovery of these symbioses in the deep sea for scientists to realize that chemosynthetic symbioses occur worldwide in a wide range of habitats, including cold seeps, whale and wood falls, shallow-water coastal sediments and continental margins. The evolutionary success of these symbioses is evident from the wide range of animal groups that have established associations with chemosynthetic bacteria; at least seven animal phyla are known to host these symbionts. The diversity of the bacterial symbionts is equally high, and phylogenetic analyses have shown that these associations have evolved on multiple occasions by convergent evolution. This Review focuses on the diversity of chemosynthetic symbionts and their hosts, and examines the traits that have resulted in their evolutionary success.     

Several deep-sea mussels and their associated symbionts are able to live both on wood and on whale falls

Bathymodiolin mussels occur at hydrothermal vents and cold seeps, where they thrive thanks to symbiotic associations with chemotrophic bacteria. Closely related genera Idas and Adipicola are associated with organic falls, ecosystems that have been suggested as potential evolutionary 'stepping stones' in the colonization of deeper and more sulphide-rich environments. Such a scenario should result from specializations to given environments from species with larger ecological niches. This study provides molecular-based evidence for the existence of two mussel species found both on sunken wood and bones. Each species specifically harbours one bacterial phylotype corresponding to thioautotrophic bacteria related to other bathymodiolin symbionts. Phylogenetic patterns between hosts and symbionts are partially congruent. However, active endocytosis and occurrences of minor symbiont lineages within species which are not their usual host suggest an environmental or horizontal rather than strictly vertical transmission of symbionts. Although the bacteria are close relatives, their localization is intracellular in one mussel species and extracellular in the other, suggesting that habitat choice is independent of the symbiont localization. The variation of bacterial densities in host tissues is related to the substrate on which specimens were sampled and could explain the abilities of host species to adapt to various substrates.     

The influence of competing root symbionts on below‐ground plant resource allocation

1. Plants typically interact with multiple above‐ and below‐ground organisms simultaneously, with their symbiotic relationships spanning a continuum ranging from mutualism, such as with arbuscular mycorrhizal fungi (AMF), to parasitism, including symbioses with plant‐parasitic nematodes (PPN).
2. Although research is revealing the patterns of plant resource allocation to mutualistic AMF partners under different host and environmental constraints, the root ecosystem, with multiple competing symbionts, is often ignored. Such competition is likely to heavily influence resource allocation to symbionts.
3. Here, we outline and discuss the competition between AMF and PPN for the finite supply of host plant resources, highlighting the need for a more holistic understanding of the influence of below‐ground interactions on plant resource allocation. Based on recent developments in our understanding of other symbiotic systems such as legume–rhizobia and AMF‐aphid‐plant, we propose hypotheses for the distribution of plant resources between contrasting below‐ground symbionts and how such competition may affect the host.
4. We identify relevant knowledge gaps at the physiological and molecular scales which, if resolved, will improve our understanding of the true ecological significance and potential future exploitation of AMF‐PPN‐plant interactions in order to optimize plant growth. To resolve these outstanding knowledge gaps, we propose the application of well‐established methods in isotope tracing and nutrient budgeting to monitor the movement of nutrients between symbionts. By combining these approaches with novel time of arrival experiments and experimental systems involving multiple plant hosts interlinked by common mycelial networks, it may be possible to reveal the impact of multiple, simultaneous colonizations by competing symbionts on carbon and nutrient flows across ecologically important scales.

     

Independent phylogenetic origins of methanotrophic and chemoautotrophic bacterial endosymbioses in marine bivalves.

The discovery of bacterium-bivalve symbioses capable of utilizing methane as a carbon and energy source indicates that the endosymbionts of hydrothermal vent and cold seep bivalves are not restricted to sulfur-oxidizing chemoautotrophic bacteria but also include methanotrophic bacteria. The phylogenetic origin of methanotrophic endosymbionts and their relationship to known symbiotic and free-living bacteria, however, have remained unexplored. In situ localization and phylogenetic analysis of a symbiont 16S rRNA gene cloned from the gills of a recently described deep-sea mussel species demonstrate that this symbiont represents a new taxon which is closely related to free-living, cultivable Type I methanotrophic bacteria. This symbiont is distinct from known chemoautotrophic symbionts. Thus, despite compelling similarities between the symbioses, chemoautotrophic and methanotrophic symbionts of marine bivalves have independent phylogenetic origins.     

An out-of-body experience: the extracellular dimension for the transmission of mutualistic bacteria in insects

Across animals and plants, numerous metabolic and defensive adaptations are a direct consequence of symbiotic associations with beneficial microbes. Explaining how these partnerships are maintained through evolutionary time remains one of the central challenges within the field of symbiosis research. While genome erosion and co-cladogenesis with the host are well-established features of symbionts exhibiting intracellular localization and transmission, the ecological and evolutionary consequences of an extracellular lifestyle have received little attention, despite a demonstrated prevalence and functional importance across many host taxa. Using insect–bacteria symbioses as a model, we highlight the diverse routes of extracellular symbiont transfer. Extracellular transmission routes are unified by the common ability of the bacterial partners to survive outside their hosts, thereby imposing different genomic, metabolic and morphological constraints than would be expected from a strictly intracellular lifestyle. We emphasize that the evolutionary implications of symbiont transmission routes (intracellular versus extracellular) do not necessarily correspond to those of the transmission mode (vertical versus horizontal), a distinction of vital significance when addressing the genomic and physiological consequences for both host and symbiont.     

Phylogenetic analysis of host–symbiont specificity and codivergence in bioluminescent symbioses

Several groups of marine fishes and squids form mutualistic bioluminescent symbioses with luminous bacteria. The dependence of the animal on its symbiont for light production, the animal's specialized anatomical adaptations for harboring bacteria and controlling light emission, and the host family bacterial species specificity characteristic of these associations suggest that bioluminescent symbioses are tightly coupled associations that might involve coevolutionary interactions. Consistent with this possibility, evidence of parallel cladogenesis has been reported for squid–bacterial associations. However, genetic adaptations in the bacteria necessary for and specific to symbiosis have not been identified, and unlike obligate endosymbiotic associations in which the bacteria are transferred vertically, bacterially bioluminescent hosts acquire their light‐organ symbionts from the environment with each new host generation. These contrasting observations led us to test the hypotheses of species specificity and codivergence in bioluminescent symbioses, using an extensive sampling of naturally formed associations. Thirty‐five species of fish in seven teleost families (Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae, Monocentridae, Acropomatidae, Leiognathidae) and their light‐organ bacteria were examined. Phylogenetic analysis of a taxonomically broad sampling of associations was based on mitochondrial 16S rRNA and cytochrome oxidase I gene sequences for the fish and on recA, gyrB and luxA sequences for bacteria isolated from the light organs of these specimens. In a fine‐scale test focused on Leiognathidae, phylogenetic analysis was based also on histone H3 subunit and 28S rRNA gene sequences for the fish and on gyrB, luxA, luxB, luxF and luxE sequences for the bacteria. Deep divergences were revealed among the fishes, and clear resolution was obtained between clades of the bacteria. In several associations, bacterial species identities contradicted strict host family bacterial species specificity. Furthermore, the fish and bacterial phylogenies exhibited no meaningful topological congruence; evolutionary divergence of host fishes was not matched by a similar pattern of diversification in the symbiotic bacteria. Re‐analysis of data reported for squids and their luminous bacteria also revealed no convincing evidence of codivergence. These results refute the hypothesis of strict host family bacterial species specificity and the hypothesis of codivergence in bioluminescent symbioses. © The Willi Hennig Society 2007.     

Aphid-Symbiotic Bacteria Cultured in Insect Cell Lines

The cells and tissues of many aphids contain bacteria known as “secondary symbionts,” which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names “Candidatus Consessoris aphidicola” for T type and “Candidatus Adiaceo aphidicola” for U type.     

Diversity and characterization of bacterial communities of five co‐occurring species at a hydrothermal vent on the Tonga Arc

Host–symbiont relationships in hydrothermal vent ecosystems, supported by chemoautotrophic bacteria as primary producers, have been extensively studied. However, the process by which densely populated co‐occurring invertebrate hosts form symbiotic relationships with bacterial symbionts remains unclear. Here, we analyzed gill‐associated symbiotic bacteria (gill symbionts) of five co‐occurring hosts, three mollusks (“Bathymodiolus” manusensis, B. brevior, and Alviniconcha strummeri) and two crustaceans (Rimicaris variabilis and Austinograea alayseae), collected together at a single vent site in the Tonga Arc. We observed both different compositions of gill symbionts and the presence of unshared operational taxonomic units (OTUs). In addition, the total number of OTUs was greater for crustacean hosts than for mollusks. The phylogenetic relationship trees of gill symbionts suggest that γ‐proteobacterial gill symbionts have coevolved with their hosts toward reinforcement of host specificity, while campylobacterial Sulfurovum species found across various hosts and habitats are opportunistic associates. Our results confirm that gill symbiont communities differ among co‐occurring vent invertebrates and indicate that hosts are closely related with their gill symbiont communities. Considering the given resources available at a single site, differentiation of gill symbionts seems to be a useful strategy for obtaining nutrition and energy while avoiding competition among both hosts and gill symbionts.     

Efficiency of partner choice and sanctions in Lotus is not altered by nitrogen fertilization

Eukaryotic hosts must exhibit control mechanisms to select against ineffective bacterial symbionts. Hosts can minimize infection by less-effective symbionts (partner choice) and can divest of uncooperative bacteria after infection (sanctions). Yet, such host-control traits are predicted to be context dependent, especially if they are costly for hosts to express or maintain. Legumes form symbiosis with rhizobia that vary in symbiotic effectiveness (nitrogen fixation) and can enforce partner choice as well as sanctions. In nature, legumes acquire fixed nitrogen from both rhizobia and soils, and nitrogen deposition is rapidly enriching soils globally. If soil nitrogen is abundant, we predict host control to be downregulated, potentially allowing invasion of ineffective symbionts. We experimentally manipulated soil nitrogen to examine context dependence in host control. We co-inoculated Lotus strigosus from nitrogen depauperate soils with pairs of Bradyrhizobium strains that vary in symbiotic effectiveness and fertilized plants with either zero nitrogen or growth maximizing nitrogen. We found efficient partner choice and sanctions regardless of nitrogen fertilization, symbiotic partner combination or growth season. Strikingly, host control was efficient even when L. strigosus gained no significant benefit from rhizobial infection, suggesting that these traits are resilient to short-term changes in extrinsic nitrogen, whether natural or anthropogenic.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

Nitrogen fixation and transfer in open ocean diatom–cyanobacterial symbioses

Many diatoms that inhabit low-nutrient waters of the open ocean live in close association with cyanobacteria. Some of these associations are believed to be mutualistic, where N₂-fixing cyanobacterial symbionts provide N for the diatoms. Rates of N₂ fixation by symbiotic cyanobacteria and the N transfer to their diatom partners were measured using a high-resolution nanometer scale secondary ion mass spectrometry approach in natural populations. Cell-specific rates of N₂ fixation (1.15–71.5 fmol N per cell h⁻¹) were similar amongst the symbioses and rapid transfer (within 30 min) of fixed N was also measured. Similar growth rates for the diatoms and their symbionts were determined and the symbiotic growth rates were higher than those estimated for free-living cells. The N₂ fixation rates estimated for Richelia and Calothrix symbionts were 171–420 times higher when the cells were symbiotic compared with the rates estimated for the cells living freely. When combined, the latter two results suggest that the diatom partners influence the growth and metabolism of their cyanobacterial symbionts. We estimated that Richelia fix 81–744% more N than needed for their own growth and up to 97.3% of the fixed N is transferred to the diatom partners. This study provides new information on the mechanisms controlling N input into the open ocean by symbiotic microorganisms, which are widespread and important for oceanic primary production. Further, this is the first demonstration of N transfer from an N₂ fixer to a unicellular partner. These symbioses are important models for molecular regulation and nutrient exchange in symbiotic systems.