Conventional management has a greater negative impact on Phaseolus vulgaris L. rhizobia diversity and abundance than water scarcity.

Introduction: Drought is one of the biggest problems for crop production and also affects the survival and persistence of soil rhizobia, which limits the establishment of efficient symbiosis and endangers the productivity of legumes, the main source of plant protein worldwide. Aim: Since the biodiversity can be altered by several factors including abiotic stresses or cultural practices, the objective of this research was to evaluate the effect of water availability, plant genotype and agricultural management on the presence, nodulation capacity and genotypic diversity of rhizobia. Method: A field experiment was conducted with twelve common bean genotypes under irrigation and rain-fed conditions, both in conventional and organic management. Estimation of the number of viable rhizobia present in soils was performed before the crop establishment, whereas the crop yield, nodule number and the strain diversity of bacteria present in nodules were determined at postharvest. Results: Rainfed conditions reduced the number of nodules and of isolated bacteria and their genetic diversity, although to a lesser extent than the agrochemical inputs related to conventional management. In addition, the effect of water scarcity on the conventional management soil was greater than observed under organic conditions. Conclusions: The preservation of diversity will be a key factor to maintain crop production in the future, as problems caused by drought will be exacerbated by climate change and organic management can help to maintain the biodiversity of soil microbiota, a fundamental aspect for soil health and quality.

Co-inoculation with novel nodule-inhabiting bacteria reduces the benefits of legume-rhizobium symbiosis.

The ecologically and economically vital symbiosis between nitrogen-fixing rhizobia and leguminous plants is often thought of as a bi-partite interaction, yet studies increasingly show the prevalence of non-rhizobial endophytes (NREs) that occupy nodules alongside rhizobia. Yet, what impact these NREs have on plant or rhizobium fitness remains unclear. Here, we investigated four NRE strains found to naturally co-occupy nodules of the legume Medicago truncatula alongside Sinorhizobium meliloti in native soils. Our objectives were to (1) examine the direct and indirect effects of NREs on M. truncatula and S. meliloti fitness, and (2) determine whether NREs can re-colonize root and nodule tissues upon reinoculation. We identified one NRE strain (522) as a novel Paenibacillus species, another strain (717A) as a novel Bacillus species, and the other two (702A and 733B) as novel Pseudomonas species. Additionally, we found that two NREs (Bacillus 717A and Pseudomonas 733B) reduced the fitness benefits obtained from symbiosis for both partners, while the other two (522, 702A) had little effect. Lastly, we found that NREs were able to co-infect host tissues alongside S. meliloti. This study demonstrates that variation of NREs present in natural populations must be considered to better understand legume-rhizobium dynamics in soil communities.

Combining GWAS and population genomic analyses to characterize coevolution in a legume-rhizobia symbiosis.

The mutualism between legumes and rhizobia is clearly the product of past coevolution. However, the nature of ongoing evolution between these partners is less clear. To characterize the nature of recent coevolution between legumes and rhizobia, we used population genomic analysis to characterize selection on functionally annotated symbiosis genes as well as on symbiosis gene candidates identified through a two-species association analysis. For the association analysis, we inoculated each of 202 accessions of the legume host Medicago truncatula with a community of 88 Sinorhizobia (Ensifer) meliloti strains. Multistrain inoculation, which better reflects the ecological reality of rhizobial selection in nature than single-strain inoculation, allows strains to compete for nodulation opportunities and host resources and for hosts to preferentially form nodules and provide resources to some strains. We found extensive host by symbiont, that is, genotype-by-genotype, effects on rhizobial fitness and some annotated rhizobial genes bear signatures of recent positive selection. However, neither genes responsible for this variation nor annotated host symbiosis genes are enriched for signatures of either positive or balancing selection. This result suggests that stabilizing selection dominates selection acting on symbiotic traits and that variation in these traits is under mutation-selection balance. Consistent with the lack of positive selection acting on host genes, we found that among-host variation in growth was similar whether plants were grown with rhizobia or N-fertilizer, suggesting that the symbiosis may not be a major driver of variation in plant growth in multistrain contexts.

Discordant population structure among rhizobium divided genomes and their legume hosts.

Symbiosis often occurs between partners with distinct life history characteristics and dispersal mechanisms. Many bacterial symbionts have genomes comprising multiple replicons with distinct rates of evolution and horizontal transmission. Such differences might drive differences in population structure between hosts and symbionts and among the elements of the divided genomes of bacterial symbionts. These differences might, in turn, shape the evolution of symbiotic interactions and bacterial evolution. Here we use whole genome resequencing of a hierarchically structured sample of 191 strains of Sinorhizobium meliloti collected from 21 locations in southern Europe to characterize population structures of this bacterial symbiont, which forms a root nodule symbiosis with the host plant Medicago truncatula. S. meliloti genomes showed high local (within-site) variation and little isolation by distance. This was particularly true for the two symbiosis elements, pSymA and pSymB, which have population structures that are similar to each other, but distinct from both the bacterial chromosome and the host plant. Given limited recombination on the chromosome, compared to the symbiosis elements, distinct population structures may result from differences in effective gene flow. Alternatively, positive or purifying selection, with little recombination, may explain distinct geographical patterns at the chromosome. Discordant population structure between hosts and symbionts indicates that geographically and genetically distinct host populations in different parts of the range might interact with genetically similar symbionts, potentially minimizing local specialization.

Genome-Wide Association Studies across Environmental and Genetic Contexts Reveal Complex Genetic Architecture of Symbiotic Extended Phenotypes.

A goal of modern biology is to develop the genotype-phenotype (G→P) map, a predictive understanding of how genomic information generates trait variation that forms the basis of both natural and managed communities. As microbiome research advances, however, it has become clear that many of these traits are symbiotic extended phenotypes, being governed by genetic variation encoded not only by the host's own genome, but also by the genomes of myriad cryptic symbionts. Building a reliable G→P map therefore requires accounting for the multitude of interacting genes and even genomes involved in symbiosis. Here, we use naturally occurring genetic variation in 191 strains of the model microbial symbiont Sinorhizobium meliloti paired with two genotypes of the host Medicago truncatula in four genome-wide association studies (GWAS) to determine the genomic architecture of a key symbiotic extended phenotype-partner quality, or the fitness benefit conferred to a host by a particular symbiont genotype, within and across environmental contexts and host genotypes. We define three novel categories of loci in rhizobium genomes that must be accounted for if we want to build a reliable G→P map of partner quality; namely, (i) loci whose identities depend on the environment, (ii) those that depend on the host genotype with which rhizobia interact, and (iii) universal loci that are likely important in all or most environments. IMPORTANCE Given the rapid rise of research on how microbiomes can be harnessed to improve host health, understanding the contribution of microbial genetic variation to host phenotypic variation is pressing, and will better enable us to predict the evolution of (and select more precisely for) symbiotic extended phenotypes that impact host health. We uncover extensive context-dependency in both the identity and functions of symbiont loci that control host growth, which makes predicting the genes and pathways important for determining symbiotic outcomes under different conditions more challenging. Despite this context-dependency, we also resolve a core set of universal loci that are likely important in all or most environments, and thus, serve as excellent targets both for genetic engineering and future coevolutionary studies of symbiosis.

Contemporary evolution rivals the effects of rhizobium presence on community and ecosystem properties in experimental mesocosms.

Because genotypes within a species commonly differ in traits that influence other species, whole communities, or even ecosystem functions, evolutionary change within one key species may affect the community and ecosystem processes. Here we use experimental mesocosms to test how the evolution of reduced cooperation in rhizobium mutualists in response to 20 years of nitrogen fertilization compares to the effects of rhizobium presence on soil nitrogen availability and plant community composition and diversity. The evolution of reduced rhizobium cooperation caused reductions in soil nitrogen, biological nitrogen fixation, and leaf nitrogen concentrations that were as strong as, or even stronger than, experimental rhizobium inoculation (presence/absence) treatments. Effects of both rhizobium evolution and rhizobium inoculation on legume dominance, plant community composition, and plant species diversity were often smaller in magnitude, but suggest that rhizobium evolution can alter the relative abundance of plant functional groups. Our findings indicate that the consequences of rapid microbial evolution for ecosystems and communities can rival the effects resulting from the presence or abundance of keystone mutualists.

Phenotypic and genomic signatures of interspecies cooperation and conflict in naturally occurring isolates of a model plant symbiont.

Given the need to predict the outcomes of (co)evolution in host-associated microbiomes, whether microbial and host fitnesses tend to trade-off, generating conflict, remains a pressing question. Examining the relationships between host and microbe fitness proxies at both the phenotypic and genomic levels can illuminate the mechanisms underlying interspecies cooperation and conflict. We examined naturally occurring genetic variation in 191 strains of the model microbial symbiont Sinorhizobium meliloti, paired with each of two host Medicago truncatula genotypes in single- or multi-strain experiments to determine how multiple proxies of microbial and host fitness were related to one another and test key predictions about mutualism evolution at the genomic scale, while also addressing the challenge of measuring microbial fitness. We found little evidence for interspecies fitness conflict; loci tended to have concordant effects on both microbe and host fitnesses, even in environments with multiple co-occurring strains. Our results emphasize the importance of quantifying microbial relative fitness for understanding microbiome evolution and thus harnessing microbiomes to improve host fitness. Additionally, we find that mutualistic coevolution between hosts and microbes acts to maintain, rather than erode, genetic diversity, potentially explaining why variation in mutualism traits persists in nature.

MGEs as the MVPs of Partner Quality Variation in Legume-Rhizobium Symbiosis.

Despite decades of research, we are only just beginning to understand the forces maintaining variation in the nitrogen-fixing symbiosis between rhizobial bacteria and leguminous plants. In their recent work, Alexandra Weisberg and colleagues use genomics to document the breadth of mobile element diversity that carries the symbiosis genes of Bradyrhizobium in natural populations. Studying rhizobia from the perspective of their mobile genetic elements, which have their own transmission modes and fitness interests, reveals novel mechanisms for the generation and maintenance of diversity in natural populations of these ecologically and economically important mutualisms.

When does mutualism offer a competitive advantage? A game-theoretic analysis of host-host competition in mutualism.

Due to their non-motile nature, plants rely heavily on mutualistic interactions to obtain resources and carry out services. One key mutualism is the plant-microbial mutualism in which a plant trades away carbon to a microbial partner for nutrients like nitrogen and phosphorous. Plants show much variation in the use of this partnership from the individual level to entire lineages depending upon ecological, evolutionary and environmental context. We sought to determine how this context dependency could result in the promotion, exclusion or coexistence of the microbial mutualism by asking if and when the partnership provided a competitive advantage to the plant. To that end, we created a 2 × 2 evolutionary game in which plants could either be a mutualist and pair with a microbe or be a non-mutualist and forgo the partnership. Our model includes both frequency dependence and density dependence, which gives us the eco-evolutionary dynamics of mutualism evolution. As in all models, mutualism only evolved if it could offer a competitive advantage and its net benefit was positive. However, surprisingly the model reveals the possibility of coexistence between mutualist and non-mutualist genotypes due to competition between mutualists over the microbially obtained nutrient. Specifically, frequency dependence of host strategies can make the microbial symbiont less beneficial if the microbially derived resources are shared, a phenomenon that increasingly reduces the frequency of mutualism as the density of competitors increases. In essence, ecological competition can act as a hindrance to mutualism evolution. We go on to discuss basic experiments that can be done to test and falsify our hypotheses.

Assembly of wood-inhabiting archaeal, bacterial and fungal communities along a salinity gradient: common taxa are broadly distributed but locally abundant in preferred habitats.

Wood decomposition in water is a key ecosystem process driven by diverse microbial taxa that likely differ in their affinities for freshwater, estuarine and marine habitats. How these decomposer communities assemble in situ or potentially colonize from other habitats remains poorly understood. At three watersheds on Coiba Island, Panama, we placed replicate sections of branch wood of a single tree species on land, and in freshwater, estuarine and marine habitats that constitute a downstream salinity gradient. We sequenced archaea, bacteria and fungi from wood samples collected after 3, 9 and 15 months to examine microbial community composition, and to examine habitat specificity and abundance patterns. We found that these microbial communities were broadly structured by similar factors, with a strong effect of salinity, but little effect of watershed identity on compositional variation. Moreover, common aquatic taxa were also present in wood incubated on land. Our results suggest that either taxa dispersed to both terrestrial and aquatic habitats, or microbes with broad habitat ranges were initially present in the wood as endophytes. Nonetheless, these habitat generalists varied greatly in abundance across habitats suggesting an important role for habitat filtering in maintaining distinct aquatic communities in freshwater, estuarine and marine habitats.

Elevated carbon dioxide reduces a common soybean leaf endophyte.

Free-air CO2 enrichment (FACE) experiments have elucidated how climate change affects plant physiology and production. However, we lack a predictive understanding of how climate change alters interactions between plants and endophytes, critical microbial mediators of plant physiology and ecology. We leveraged the SoyFACE facility to examine how elevated [CO2 ] affected soybean (Glycine max) leaf endophyte communities in the field. Endophyte community composition changed under elevated [CO2 ], including a decrease in the abundance of a common endophyte, Methylobacterium sp. Moreover, Methylobacterium abundance was negatively correlated with co-occurring fungal endophytes. We then assessed how Methylobacterium affected the growth of co-occurring endophytic fungi in vitro. Methylobacterium antagonized most co-occurring fungal endophytes in vitro, particularly when it was more established in culture before fungal introduction. Variation in fungal response to Methylobacterium within a single fungal operational taxonomic unit (OTU) was comparable to inter-OTU variation. Finally, fungi isolated from elevated vs. ambient [CO2 ] plots differed in colony growth and response to Methylobacterium, suggesting that increasing [CO2 ] may affect fungal traits and interactions within the microbiome. By combining in situ and in vitro studies, we show that elevated [CO2 ] decreases the abundance of a common bacterial endophyte that interacts strongly with co-occurring fungal endophytes. We suggest that endophyte responses to global climate change will have important but largely unexplored implications for both agricultural and natural systems.

Soil origin and plant genotype structure distinct microbiome compartments in the model legume Medicago truncatula.

BACKGROUND: Understanding the genetic and environmental factors that structure plant microbiomes is necessary for leveraging these interactions to address critical needs in agriculture, conservation, and sustainability. Legumes, which form root nodule symbioses with nitrogen-fixing rhizobia, have served as model plants for understanding the genetics and evolution of beneficial plant-microbe interactions for decades, and thus have added value as models of plant-microbiome interactions. Here we use a common garden experiment with 16S rRNA gene amplicon and shotgun metagenomic sequencing to study the drivers of microbiome diversity and composition in three genotypes of the model legume Medicago truncatula grown in two native soil communities. RESULTS: Bacterial diversity decreased between external (rhizosphere) and internal plant compartments (root endosphere, nodule endosphere, and leaf endosphere). Community composition was shaped by strong compartment × soil origin and compartment × plant genotype interactions, driven by significant soil origin effects in the rhizosphere and significant plant genotype effects in the root endosphere. Nevertheless, all compartments were dominated by Ensifer, the genus of rhizobia that forms root nodule symbiosis with M. truncatula, and additional shotgun metagenomic sequencing suggests that the nodulating Ensifer were not genetically distinguishable from those elsewhere in the plant. We also identify a handful of OTUs that are common in nodule tissues, which are likely colonized from the root endosphere. CONCLUSIONS: Our results demonstrate strong host filtering effects, with rhizospheres driven by soil origin and internal plant compartments driven by host genetics, and identify several key nodule-inhabiting taxa that coexist with rhizobia in the native range. Our results set the stage for future functional genetic experiments aimed at expanding our pairwise understanding of legume-rhizobium symbiosis toward a more mechanistic understanding of plant microbiomes. Video Abstract.

Contribution of fungal and invertebrate communities to wood decay in tropical terrestrial and aquatic habitats.

Wood is a major carbon input into aquatic ecosystems and is thought to decay slowly, yet surprisingly little terrestrial carbon accumulates in marine sediments. A better mechanistic understanding of how habitat conditions and decomposer communities influence wood decay processes along the river-estuary-ocean continuum can address this seeming paradox. We measured mass loss, wood element, and polymer concentrations, quantified invertebrate-induced decay, and sequenced fungal communities associated with replicate sections of Guazuma branch wood submerged in freshwater, estuarine, and near-shore marine habitats and placed on the soil surface in nearby terrestrial habitats in three watersheds in the tropical eastern Pacific. Over 15 months, we found that wood decayed at similar rates in estuarine, marine, and terrestrial sites, reflecting the combined activity of invertebrate and microbial decomposers. In contrast, in the absence of shipworms (Teredinidae), which accounted for ~40% of wood mass loss in the estuarine habitats, decay proceeded more slowly in freshwater. Over the experiment, wood element chemistry diverged among freshwater, estuarine, and marine habitats, due to differences in both nutrient losses (e.g., potassium and phosphorus) and gains (e.g., calcium and aluminum) through decay. Similarly, we observed changes in wood polymer content, with the highest losses of cellulose, hemicellulose, and lignin moieties in the marine habitat. Aquatic fungal communities were strongly dominated by ascomycetes (88-99% of taxa), compared to terrestrial communities (55% ascomycetes). Large differences in fungal diversity were also observed across habitats with threefold higher richness in terrestrial than freshwater habitats and twofold higher diversity in freshwater than estuarine/marine habitats. Divergent decay trajectories across habitats were associated with widespread order-level differences in fungal composition, with distinct communities found in freshwater, estuarine and marine habitats. However, few individual taxa that were significantly associated with mass loss were broadly distributed, suggesting a high level of functional redundancy. The rapid processing of wood entering tropical rivers by microbes and invertebrates, comparable to that on land, indicates that estuaries and coastal oceans are hotspots not just for the processing of particulate and dissolved organic carbon, but also for woody debris and for the breakdown of lignin, the most recalcitrant polymer in plant tissue.

Light availability and rhizobium variation interactively mediate the outcomes of legume-rhizobium symbiosis.

PREMISE: Nutrients, light, water, and temperature are key factors limiting the growth of individual plants in nature. Mutualistic interactions between plants and microbes often mediate resource limitation for both partners. In the mutualism between legumes and rhizobia, plants provide rhizobia with carbon in exchange for fixed nitrogen. Because partner quality in mutualisms is genotype-dependent, within-species genetic variation is expected to alter the responses of mutualists to changes in the resource environment. Here we ask whether partner quality variation in rhizobia mediates the response of host plants to changing light availability, and conversely, whether light alters the expression of partner quality variation. METHODS: We inoculated clover hosts with 11 strains of Rhizobium leguminosarum that differed in partner quality, grew plants under either ambient or low light conditions in the greenhouse, and measured plant growth, nodule traits, and foliar nutrient composition. RESULTS: Light availability and rhizobium inoculum interactively determined plant growth, and variation in rhizobium partner quality was more apparent in ambient light. CONCLUSIONS: Our results suggest that variation in the costs and benefits of rhizobium symbionts mediate host responses to light availability and that rhizobium strain variation might more important in higher-light environments. Our work adds to a growing appreciation for the role of microbial intraspecific and interspecific diversity in mediating extended phenotypes in their hosts and suggests an important role for light availability in the ecology and evolution of legume-rhizobium symbiosis.

Wood decomposition in aquatic and terrestrial ecosystems in the tropics: contrasting biotic and abiotic processes.

Wood decomposition, a critical process in carbon and nutrient cycles, is influenced by environmental conditions, decomposer communities and substrate composition. While these factors differ between land and stream habitats, across-habitat comparisons of wood decay processes are rare, limiting our ability to evaluate the context- dependency of the drivers of decay. Here we tracked wood decomposition of three tree species placed in stream and terrestrial habitats in a lowland tropical forest in Panama. At 3 and 11 months we measured mass loss, wood nitrogen and wood polymer concentrations, and sampled wood-associated fungal and bacterial communities. After 11 months of decay we found that mass loss occurred 9% faster in streams than on land, but loss of cellulose, hemicellulose and lignin did not differ between habitats. We also observed large differences in microbial decomposer communities between habitats. Overall, we found faster mass loss of wood in water, but no differences in biotic decay processes between habitats despite distinct microbial communities in streams and on land. Our research challenges the assumption that wood decays relatively slowly in water reflecting unfavorable environmental conditions and a limited capacity of aquatic microbial communities to effectively degrade wood polymers.

Geographically structured genetic variation in the Medicago lupulina-Ensifer mutualism.

Gene flow between genetically differentiated populations can maintain variation in species interactions, especially when population structure is congruent between interacting species. However, large-scale empirical comparisons of the population structure of interacting species are rare, particularly in positive interspecific interactions (mutualisms). One agriculturally and ecologically important mutualism is the partnership between legume plants and rhizobia. Through characterizing and comparing the population genomic structure of the legume Medicago lupulina and two rhizobial species (Ensifer medicae and E. meliloti), we explored the spatial scale of population differentiation between interacting partners in their introduced range in North America. We found high proportions of E. meliloti in southeastern populations and high proportions of E. medicae in northwestern populations. Medicago lupulina and the Ensifer genus showed similar patterns of spatial genetic structure (isolation by distance). However, we detected no evidence of isolation by distance or population structure within either species of bacteria. Genome-wide nucleotide diversity within each of the two Ensifer species was low, suggesting limited introduction of strains, founder events, or severe bottlenecks. Our results suggest that there is potential for geographically structured coevolution between M. lupulina and the Ensifer genus, but not between M. lupulina and either Ensifer species.

Intrapopulation genomics in a model mutualist: Population structure and candidate symbiosis genes under selection in Medicago truncatula.

Bottom-up evolutionary approaches, including geographically explicit population genomic analyses, have the power to reveal the mechanistic basis of adaptation. Here, we conduct a population genomic analysis in the model legume, Medicago truncatula, to characterize population genetic structure and identify symbiosis-related genes showing evidence of spatially variable selection. Using RAD-seq, we generated over 26,000 SNPs from 191 accessions from within three regions of the native range in Europe. Results from STRUCTURE analysis identify five distinct genetic clusters with divisions that separate east and west regions in the Mediterranean basin. Much of the genetic variation is maintained within sampling sites, and there is evidence for isolation by distance. Extensive linkage disequilibrium was identified, particularly within populations. We conducted genetic outlier analysis with FST -based genome scans and a Bayesian modeling approach (PCAdapt). There were 70 core outlier loci shared between these distinct methods with one clear candidate symbiosis related gene, DMI1. This work sets that stage for functional experiments to determine the important phenotypes that selection has acted upon and complementary efforts in rhizobium populations.