Competitive interference among rhizobia reduces benefits to hosts.

The capacity of beneficial microbes to compete for host infection-and the ability of hosts to discriminate among them-introduces evolutionary conflict that is predicted to destabilize mutualism. We investigated fitness outcomes in associations between legumes and their symbiotic rhizobia to characterize fitness impacts of microbial competition. Diverse Bradyrhizobium strains varying in their capacity to fix nitrogen symbiotically with a common host plant, Acmispon strigosus, were tested in full-factorial coinoculation experiments involving 28 pairwise strain combinations. We analyzed the effects of interstrain competition and host discrimination on symbiotic-interaction outcomes by relativizing fitness proxies to clonally infected and uninfected controls. More than one thousand root nodules of coinoculated plants were genotyped to quantify strain occupancy, and the Bradyrhizobium strain genome sequences were analyzed to uncover the genetic bases of interstrain competition outcomes. Strikingly, interstrain competition favored a fast-growing, minimally beneficial rhizobia strain. Host benefits were significantly diminished in coinoculation treatments relative to expectations from clonally inoculated controls, consistent with competitive interference among rhizobia that reduced both nodulation and plant growth. Competition traits appear polygenic, linked with inter-strain allelopathic interactions in the rhizosphere. This study confirms that competition among strains can destabilize mutualism by favoring microbes that are superior in colonizing host tissues but provide minimal benefits to host plants. Moreover, our findings help resolve the paradox that despite efficient host control post infection, legumes nonetheless encounter rhizobia that vary in their nitrogen fixation.

Dynamic Interactions Between Mega Symbiosis ICEs and Bacterial Chromosomes Maintain Genome Architecture.

Acquisition of mobile genetic elements can confer novel traits to bacteria. Some integrative and conjugative elements confer upon members of Bradyrhizobium the capacity to fix nitrogen in symbiosis with legumes. These so-called symbiosis integrative conjugative elements (symICEs) can be extremely large and vary as monopartite and polypartite configurations within chromosomes of related strains. These features are predicted to impose fitness costs and have defied explanation. Here, we show that chromosome architecture is largely conserved despite diversity in genome composition, variations in locations of attachment sites recognized by integrases of symICEs, and differences in large-scale chromosomal changes that occur upon integration. Conversely, many simulated nonnative chromosome-symICE combinations are predicted to result in lethal deletions or disruptions to architecture. Findings suggest that there is compatibility between chromosomes and symICEs. We hypothesize that the size and structural flexibility of symICEs are important for generating combinations that maintain chromosome architecture across a genus of nitrogen-fixing bacteria with diverse and dynamic genomes.

Symbiotic organs: the nexus of host-microbe evolution.

Diverse plants and animals have evolved specialized structures to filter and house beneficial microbes. These symbiotic organs form crucial points of exchange between host and symbiont, are often shaped by both partners, and exhibit features that facilitate a suite of microbial services. While symbiotic organs exhibit varied function, morphology, and developmental plasticity, they share core features linked to the evolutionary maintenance of beneficial symbiosis. Moreover, these organs can have a significant role in altering the demographic forces that shape microbial genomes, driving population bottlenecks and horizontal gene transfer (HGT). To advance our understanding of these 'joint phenotypes' across varied systems, future research must consider the emergent forces that can shape symbiotic organs, including fitness feedbacks and conflicts between interacting genomes.

Pangenome Evolution Reconciles Robustness and Instability of Rhizobial Symbiosis.

Root nodulating rhizobia are nearly ubiquitous in soils and provide the critical service of nitrogen fixation to thousands of legume species, including staple crops. However, the magnitude of fixed nitrogen provided to hosts varies markedly among rhizobia strains, despite host legumes having mechanisms to selectively reward beneficial strains and to punish ones that do not fix sufficient nitrogen. Variation in the services of microbial mutualists is considered paradoxical given host mechanisms to select beneficial genotypes. Moreover, the recurrent evolution of non-fixing symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. Here, we deconstructed hundreds of genome sequences from genotypically and phenotypically diverse Bradyrhizobium strains and revealed mechanisms that generate variation in symbiotic nitrogen fixation. We show that this trait is conferred by a modular system consisting of many extremely large integrative conjugative elements and few conjugative plasmids. Their transmissibility and propensity to reshuffle genes generate new combinations that lead to uncooperative genotypes and make individual partnerships unstable. We also demonstrate that these same properties extend beneficial associations to diverse host species and transfer symbiotic capacity among diverse strains. Hence, symbiotic nitrogen fixation is underpinned by modularity, which engenders flexibility, a feature that reconciles evolutionary robustness and instability. These results provide new insights into mechanisms driving the evolution of mobile genetic elements. Moreover, they yield a new predictive model on the evolution of rhizobial symbioses, one that informs on the health of organisms and ecosystems that are hosts to symbionts and that helps resolve the long-standing paradox. IMPORTANCE Genetic variation is fundamental to evolution yet is paradoxical in symbiosis. Symbionts exhibit extensive variation in the magnitude of services they provide despite hosts having mechanisms to select and increase the abundance of beneficial genotypes. Additionally, evolution of uncooperative symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. We analyzed genome sequences of Bradyrhizobium, bacteria that in symbioses with legume hosts, fix nitrogen, a nutrient essential for ecosystems. We show that genes for symbiotic nitrogen fixation are within elements that can move between bacteria and reshuffle gene combinations that change host range and quality of symbiosis services. Consequently, nitrogen fixation is evolutionarily unstable for individual partnerships, but is evolutionarily stable for legume-Bradyrhizobium symbioses in general. We developed a holistic model of symbiosis evolution that reconciles robustness and instability of symbiosis and informs on applications of rhizobia in agricultural settings.

No disruption of rhizobial symbiosis during early stages of cowpea domestication.

Modern agriculture intensely selects aboveground plant structures, while often neglecting belowground features, and evolutionary tradeoffs between these traits are predicted to disrupt host control over microbiota. Moreover, drift, inbreeding, and relaxed selection for symbiosis in crops might degrade plant mechanisms that support beneficial microbes. We studied the impact of domestication on the nitrogen-fixing symbiosis between cowpea and root-nodulating Bradyrhizobium. We combined genome-wide analyses with a greenhouse inoculation study to investigate genomic diversity, heritability, and symbiosis trait variation among wild and early-domesticated cowpea genotypes. Cowpeas experienced modest decreases in genome-wide diversity during early domestication. Nonetheless, domesticated cowpeas responded efficiently to variation in symbiotic effectiveness, by forming more root nodules with nitrogen-fixing rhizobia and sanctioning nonfixing strains. Domesticated populations invested a larger proportion of host tissues into root nodules than wild cowpeas. Unlike soybean and wheat, cowpea showed no compelling evidence for degradation of symbiosis during domestication. Domesticated cowpeas experienced a less severe bottleneck than these crops and the low nutrient conditions in Africa where cowpea landraces were developed likely favored plant genotypes that gain substantial benefits from symbiosis. Breeders have largely neglected symbiosis traits, but artificial selection for improved plant responses to microbiota could increase plant performance and sustainability.

Wild legumes maintain beneficial soil rhizobia populations despite decades of nitrogen deposition.

Natural landscapes are increasingly impacted by nitrogen enrichment from aquatic and airborne pollution sources. Nitrogen enrichment in the environment can eliminate the net benefits that plants gain from nitrogen-fixing microbes such as rhizobia, potentially altering host-mediated selection on nitrogen fixation. However, we know little about the long-term effects of nitrogen enrichment on this critical microbial service. Here, we sampled populations of the legume Acmispon strigosus and its associated soil microbial communities from sites spanning an anthropogenic nitrogen deposition gradient. We measured the net growth benefits plants obtained from their local soil microbial communities and quantified plant investment into nodules that house nitrogen-fixing rhizobia. We found that plant growth benefits from sympatric soil microbes did not vary in response to local soil nitrogen levels, and instead varied mainly among plant lines. Soil nitrogen levels positively predicted the number of nodules formed on sympatric plant hosts, although this was likely due to plant genotypic variation in nodule formation, rather than variation among soil microbial communities. The capacity of all the tested soil microbial communities to improve plant growth is consistent with plant populations imposing strong selection on rhizobial nitrogen fixation despite elevated soil nitrogen levels, suggesting that host control traits in A. strigosus are stable under long-term nutrient enrichment.

Experimental evolution can enhance benefits of rhizobia to novel legume hosts.

Legumes preferentially associate with and reward beneficial rhizobia in root nodules, but the processes by which rhizobia evolve to provide benefits to novel hosts remain poorly understood. Using cycles of in planta and in vitro evolution, we experimentally simulated lifestyles where rhizobia repeatedly interact with novel plant genotypes with which they initially provide negligible benefits. Using a full-factorial replicated design, we independently evolved two rhizobia strains in associations with each of two Lotus japonicus genotypes that vary in regulation of nodule formation. We evaluated phenotypic evolution of rhizobia by quantifying fitness, growth effects and histological features on hosts, and molecular evolution via genome resequencing. Rhizobia evolved enhanced host benefits and caused changes in nodule development in one of the four host-symbiont combinations, that appeared to be driven by reduced costs during symbiosis, rather than increased nitrogen fixation. Descendant populations included genetic changes that could alter rhizobial infection or proliferation in host tissues, but lack of evidence for fixation of these mutations weakens the results. Evolution of enhanced rhizobial benefits occurred only in a subset of experiments, suggesting a role for host-symbiont genotype interactions in mediating the evolution of enhanced benefits from symbionts.

Evolution of specialization in a plant-microbial mutualism is explained by the oscillation theory of speciation.

Specialization in mutualisms is thought to be a major driver of diversification, but few studies have explored how novel specialization evolves, or its relation to the evolution of other niche axes. A fundamental question is whether generalist interactions evolve to become more specialized (i.e., oscillation hypothesis) or if partner switches evolve without any change in niche breadth (i.e., musical chairs hypothesis). We examined alternative models for the evolution of specialization by estimating the mutualistic, climatic, and edaphic niche breadths of sister plant species, combining phylogenetic, environmental, and experimental data on Acmispon strigosus and Acmispon wrangelianus genotypes across their overlapping ranges in California. We found that specialization along all three niche axes was asymmetric across species, such that the species with broader climatic and edaphic niches, Acmispon strigosus, was also able to gain benefit from and invest in associating with a broader set of microbial mutualists. Our data are consistent with the oscillation model of specialization, and a parallel narrowing of the edaphic, climatic, and mutualistic dimensions of the host species niche. Our findings provide novel evidence that the evolution of specialization in mutualism is accompanied by specialization in other niche dimensions.

Dysregulation of host-control causes interspecific conflict over host investment into symbiotic organs.

Microbial mutualists provide substantial benefits to hosts that feed back to enhance the fitness of the associated microbes. In many systems, beneficial microbes colonize symbiotic organs, specialized host structures that house symbionts and mediate resources exchanged between parties. Mutualisms are characterized by net benefits exchanged among members of different species, however, inequalities in the magnitude of these exchanges could result in evolutionary conflict, destabilizing the mutualism. We investigated joint fitness effects of root nodule formation, the symbiotic organ of legumes that house nitrogen-fixing rhizobia in planta. We quantified host and symbiont fitness parameters dependent on the number of nodules formed using near-isogenic Lotus japonicus and Mesorhizobium loti mutants, respectively. Empirically estimated fitness functions suggest that legume and rhizobia fitness is aligned as the number of nodules formed increases from zero until the host optimum is reached, a point where aligned fitness interests shift to diverging fitness interests between host and symbiont. However, fitness conflict was only inferred when analyzing wild-type hosts along with their mutants dysregulated for control over nodule formation. These data demonstrate that to avoid conflict, hosts must tightly regulate investment into symbiotic organs maximizing their benefit to cost ratio of associating with microbes.

Agriculture and the Disruption of Plant-Microbial Symbiosis.

Domestication has transformed hundreds of wild plant species into productive cultivars for human utility. However, cultivation practices and intense artificial selection for yield may entail a hidden cost: the disruption of interactions between plants and beneficial microbiota. Here, we synthesize theory predicting that evolutionary trade-offs, genetic costs, and relaxed selection disrupt plant-microbial symbiosis under domestication, and review the wealth of new data interrogating these predictions in crops. We describe the agronomic practices, ecological scenarios, and genomic attributes that can result in the disruption of symbiosis, and highlight new work probing its molecular basis. To improve agricultural output and sustainability, research should develop breeding methods to optimize symbiotic outcomes in crop species.

Polyploid plants obtain greater fitness benefits from a nutrient acquisition mutualism.

Polyploidy is a key driver of ecological and evolutionary processes in plants, yet little is known about its effects on biotic interactions. This gap in knowledge is especially profound for nutrient acquisition mutualisms, despite the fact that they regulate global nutrient cycles and structure ecosystems. Generalism in mutualistic interactions depends on the range of potential partners (niche breadth), the benefits obtained and ability to maintain benefits across a variety of partners (fitness plasticity). Here, we determine how each of these is influenced by polyploidy in the legume-rhizobium mutualism. We inoculated a broad geographic sample of natural diploid and autotetraploid alfalfa (Medicago sativa) lineages with a diverse panel of Sinorhizobium bacterial symbionts. To analyze the extent and mechanism of generalism, we measured host growth benefits and functional traits. Autotetraploid plants obtained greater fitness enhancement from mutualistic interactions and were better able to maintain this across diverse rhizobial partners (i.e. low plasticity in fitness) relative to diploids. These benefits were not attributed to increases in niche breadth, but instead reflect increased rewards from investment in the mutualism. Polyploid plants displayed greater generalization in bacterial mutualisms relative to diploids, illustrating another axis of advantage for polyploids over diploids.

Recurrent mutualism breakdown events in a legume rhizobia metapopulation.

Bacterial mutualists generate major fitness benefits for eukaryotes, reshaping the host phenotype and its interactions with the environment. Yet, microbial mutualist populations are predicted to generate mutants that defect from providing costly services to hosts while maintaining the capacity to exploit host resources. Here, we examined the mutualist service of symbiotic nitrogen fixation in a metapopulation of root-nodulating Bradyrhizobium spp. that associate with the native legume Acmispon strigosus. We quantified mutualism traits of 85 Bradyrhizobium isolates gathered from a 700 km transect in California spanning 10 sampled A. strigosus populations. We clonally inoculated each Bradyrhizobium isolate onto A. strigosus hosts and quantified nodulation capacity and net effects of infection, including host growth and isotopic nitrogen concentration. Six Bradyrhizobium isolates from five populations were categorized as ineffective because they formed nodules but did not enhance host growth via nitrogen fixation. Six additional isolates from three populations failed to form root nodules. Phylogenetic reconstruction inferred two types of mutualism breakdown, including three to four independent losses of effectiveness and five losses of nodulation capacity on A. strigosus. The evolutionary and genomic drivers of these mutualism breakdown events remain poorly understood.

Interspecific conflict and the evolution of ineffective rhizobia.

Microbial symbionts exhibit broad genotypic variation in their fitness effects on hosts, leaving hosts vulnerable to costly partnerships. Interspecific conflict and partner-maladaptation are frameworks to explain this variation, with different implications for mutualism stability. We investigated the mutualist service of nitrogen fixation in a metapopulation of root-nodule forming Bradyrhizobium symbionts in Acmispon hosts. We uncovered Bradyrhizobium genotypes that provide negligible mutualist services to hosts and had superior in planta fitness during clonal infections, consistent with cheater strains that destabilise mutualisms. Interspecific conflict was also confirmed at the metapopulation level - by a significant negative association between the fitness benefits provided by Bradyrhizobium genotypes and their local genotype frequencies - indicating that selection favours cheating rhizobia. Legumes have mechanisms to defend against rhizobia that fail to fix sufficient nitrogen, but these data support predictions that rhizobia can subvert plant defenses and evolve to exploit hosts.

Host investment into symbiosis varies among genotypes of the legume Acmispon strigosus, but host sanctions are uniform.

Efficient host control predicts the extirpation of ineffective symbionts, but they are nonetheless widespread in nature. We tested three hypotheses for the maintenance of symbiotic variation in rhizobia that associate with a native legume: partner mismatch between host and symbiont, such that symbiont effectiveness varies with host genotype; resource satiation, whereby extrinsic sources of nutrients relax host control; and variation in host control among host genotypes. We inoculated Acmispon strigosus from six populations with three Bradyrhizobium strains that vary in symbiotic effectiveness on sympatric hosts. We measured proxies of host and symbiont fitness in single- and co-inoculations under fertilization treatments of zero added nitrogen (N) and near-growth-saturating N. We examined two components of host control: 'host investment' into nodule size during single- and co-inoculations, and 'host sanctions' against less effective strains during co-inoculations. The Bradyrhizobium strains displayed conserved growth effects on hosts, and host control did not decline under experimental fertilization. Host sanctions were robust in all hosts, but host lines from different populations varied significantly in measures of host investment in both single- and co-inoculation experiments. Variation in host investment could promote variation in symbiotic effectiveness and prevent the extinction of ineffective Bradyrhizobium from natural populations.

Legumes versus rhizobia: a model for ongoing conflict in symbiosis.

Contents Summary 1199 I. Introduction 1199 II. Selecting beneficial symbionts: one problem, many solutions 1200 III. Control and conflict over legume nodulation 1201 IV. Control and conflict over nodule growth and senescence 1204 V. Conclusion 1204 Acknowledgements 1205 References 1205 SUMMARY: The legume-rhizobia association is a powerful model of the limits of host control over microbes. Legumes regulate the formation of root nodules that house nitrogen-fixing rhizobia and adjust investment into nodule development and growth. However, the range of fitness outcomes in these traits reveals intense conflicts of interest between the partners. New work that we review and synthesize here shows that legumes have evolved varied mechanisms of control over symbionts, but that host control is often subverted by rhizobia. An outcome of this conflict is that both legumes and rhizobia have evolved numerous traits that can improve their own short-term fitness in this interaction, but little evidence exists for any net improvement in the joint trait of nitrogen fixation.

Symbiotic nitrogen fixation by rhizobia-the roots of a success story.

By evolving the dual capacity of intracellular survival and symbiotic nitrogen fixation in legumes, rhizobia have achieved an ecological and evolutionary success that has reshaped our biosphere. Despite complex challenges, including a dual lifestyle of intracellular infection separated by a free-living phase in soil, rhizobial symbiosis has spread horizontally to hundreds of bacterial species and geographically throughout the globe. This symbiosis has also persisted and been reshaped through millions of years of history. Here, we summarize recent advances in our understanding of the molecular mechanisms, ecological settings, and evolutionary pathways that are collectively responsible for this symbiotic success story. We offer predictions of how this symbiosis can evolve under new influences and for the benefit of a burgeoning human population.

Lotus japonicus alters in planta fitness of Mesorhizobium loti dependent on symbiotic nitrogen fixation.

Rhizobial bacteria are known for their capacity to fix nitrogen for legume hosts. However ineffective rhizobial genotypes exist and can trigger the formation of nodules but fix little if any nitrogen for hosts. Legumes must employ mechanisms to minimize exploitation by the ineffective rhizobial genotypes to limit fitness costs and stabilize the symbiosis. Here we address two key questions about these host mechanisms. What stages of the interaction are controlled by the host, and can hosts detect subtle differences in nitrogen fixation? We provide the first explicit evidence for adaptive host control in the interaction between Lotus japonicus and Mesorhizobium loti. In both single inoculation and co-inoculation experiments, less effective rhizobial strains exhibited reduced in planta fitness relative to the wildtype M. loti. We uncovered evidence of host control during nodule formation and during post-infection proliferation of symbionts within nodules. We found a linear relationship between rhizobial fitness and symbiotic effectiveness. Our results suggest that L. japonicus can adaptively modulate the fitness of symbionts as a continuous response to symbiotic nitrogen fixation.

Cell autonomous sanctions in legumes target ineffective rhizobia in nodules with mixed infections.

PREMISE OF THE STUDY: To maximize benefits from symbiosis, legumes must limit physiological inputs into ineffective rhizobia that nodulate hosts without fixing nitrogen. The capacity of legumes to decrease the relative fitness of ineffective rhizobia-known as sanctions-has been demonstrated in several legume species, but its mechanisms remain unclear. Sanctions are predicted to work at the whole-nodule level. However, whole-nodule sanctions would make the host vulnerable to mixed-nodule infections, which have been demonstrated in the laboratory and observed in natural settings. Here, we present and test a cell-autonomous model of legume sanctions that can resolve this dilemma. METHODS: We analyzed histological and ultrastructural evidence of sanctions in two legume species, Acmispon strigosus and Lotus japonicus. For the former, we inoculated seedlings with rhizobia that naturally vary in their abilities to fix nitrogen. In the latter, we inoculated seedlings with near-isogenic strains that differ only in the ability to fix nitrogen. KEY RESULTS: In both hosts, plants inoculated with ineffective rhizobia exhibited evidence for a cell autonomous and accelerated program of senescence within nodules. In plants that received mixed inoculations, only the plant cells harboring ineffective rhizobia exhibited features consistent with programmed cell death, including collapsed vacuoles, ruptured symbiosomes, and bacteroids that are released into the cytosol. These features were consistently linked with ultrastructural evidence of reduced survival of ineffective rhizobia in planta. CONCLUSIONS: Our data suggest an elegant cell autonomous mechanism by which legumes can detect and defend against ineffective rhizobia even when nodules harbor a mix of effective and ineffective rhizobial genotypes.