Brendan Epstein

Rhizobia live as free-living microorganisms in the soil and in association with legume hosts. Both environments exert selective pressures on rhizobia, influencing the reproductive success of individual strains (e.g., fitness). The soil, a heterogeneous and fluctuating environment, is often overlooked, and little is known about whether selection in the soil influences the outcomes of the rhizobium-legume mutualism. We exposed a mixture of 68 Sinorhizobium meliloti strains to soil-mediated selection using eight different treatments (temperature, osmotic, and texture perturbations) and host-mediated selection with two Medicago species as hosts. We found that cold (4°C) and warm (32°C) temperatures, as well as salt addition, had the strongest effects on diversity, population composition, or population size. Strain relative fitness was strongly positively correlated among soil treatments, except cold, warm, and salinity, suggesting strains undergo similar selection in the soil. Genome-wide association analysis revealed a complex genetic architecture for soil fitness, characterized by numerous loci of small effect that did not show significant associations. In contrast, when comparing rhizobial fitness between soil and host environments, we found minimal strain fitness correlations, suggesting an independent genetic basis and habitat-specific adaptations. Finally, by examining the relationship between rhizobial fitness in soil and their benefits to the host plant, we found that soil selection influenced the relative abundance of high- and low-quality strains; however, whether these effects were positive or negative for the plant was host-dependent. Our results suggest that rhizobial evolution in soil and host are largely independent, but soil selection can alter mutualism benefits. IMPORTANCE: Rhizobium-legume mutualism is crucial for introducing nitrogen into agricultural and natural ecosystems, and rhizobial persistence in the soil is an important component of agroecosystems. However, we know little about how populations of rhizobia persist and adapt to this environment, especially in the context of the soil's spatial and temporal variations (temperature, moisture, and soil texture). We found that rhizobia are similarly selected across abiotic soil conditions, but their reproductive success in the soil is independent from their reproductive success in the host. Intriguingly, we found that certain soil conditions increase (or decrease) the relative abundance of more beneficial strains. Understanding how rhizobia adapt to diverse environments is crucial for developing effective bioinoculants that persist in the soil while remaining highly competitive for host colonization and beneficial to the plant.

Mutualism breakdown can be prevented if partner species preferentially select and reward partners that provide greater benefit. We examined these two components using the legume Medicago truncatula and its nitrogen-fixing symbiont Sinorhizobium meliloti. First, we re-analysed data from 202 accessions to show significant genetic variation in the capacity of Medicago to restrict strain diversity, finding that hosts with shorter nodules were more selective. A genome-wide association study on host selectivity identified genes including the hormone leginsulin, pectin degradation, multidrug and toxic compound efflux, zinc transport and DNA methylation. Second, we used two well-studied Medicago genotypes with contrasting nodule morphologies to assess the effectiveness of adaptive enrichment mechanisms by sampling the relative frequencies of rhizobial strains in pools of small nodules (indicating a lack of host investment) compared to large nodules (indicating increased host investment) and pairing these results with previous single-strain assessments of strain benefits to hosts. While both hosts enriched beneficial strains in large nodules, the host that formed larger and more variably sized nodules and thus had greater 'potential' to increase rhizobial populations was less effective. Our findings reveal that host genetic variation affects strain selectivity and suggest that nodule morphology traits warrant attention when exploring mutualism evolution.

In the mutualism between leguminous plants and rhizobial bacteria, rhizobia live inside root nodules, creating potential for host genes to shape the rhizobial selective environment. Many host genes that affect symbiosis have been identified; however, the extent to which these genes affect selection acting on rhizobia is unknown. In this study, we inoculated 18 Medicago truncatula symbiotic mutants (including mutants that alter Nodule Cysteine-Rich (NCR) peptide production, plant defence, and nodule number regulation) with a mixture of 86 Sinorhizobium meliloti strains. Most mutations resulted in reduced host benefits, but the effects on rhizobial benefit (i.e. relative strain fitness) varied widely, revealing widespread host-by-strain fitness interactions. Genome-wide association analyses identified variants on rhizobial replicons pSymA and pSymB as important in mediating strain fitness responses to host mutations. Whereas most top variants affected rhizobial fitness with one host mutation (limited effect variants), nine affected fitness across six or more host mutations. These pervasive variants occurred primarily on pSymA, the symbiotic replicon, and include fixL and some metabolic genes. In contrast to the limited effect variants, variants with pervasive positive effects on strain fitness when host genes were mutated tended to adversely affect fitness in wild-type hosts. Competition assays across Medicago genotypes confirmed a pervasive role for one candidate (malonyl-CoA synthase), and AlphaFold multimer modelling suggests that many rhizobial top candidates could interact with host NCR peptides. Our results reveal how host genetic mutations alter strain fitness, setting the stage for improving rhizobial inoculants and breeding legume hosts better adapted to multi-strain environments.

Rhizobia live as free-living microorganisms in the soil and in association with legume hosts. Both environments exert selective pressures on rhizobia, influencing the reproductive success of individual strains (e.g., fitness). The soil, a heterogeneous and fluctuating environment, is often overlooked, and little is known about whether selection in the soil influences the outcomes of the rhizobia-legume mutualism. We exposed a mixture of 68 Sinorhizobium meliloti strains to eight soil treatments (temperature, osmotic, and texture perturbations) and selection by two Medicago plant hosts. We found that cold (4°C) and warm (32°C) temperatures, as well as salt addition, had the strongest effects on diversity, community composition, or population size. Strain relative fitness was strongly positively correlated among soil treatments, except for cold. Genome-wide association analysis revealed a complex genetic architecture for soil fitness. In contrast, when comparing rhizobial fitness between soil and host environments, we found minimal strain fitness correlations, suggesting independent genetic bases and habitat-specific adaptations. Lastly, by examining the relationship between rhizobial fitness in the soil and their nitrogen-fixing plant benefits, we found that soil selection influenced the relative abundance of high- and low-quality strains; However, whether these effects were positive or negative for the plant was host dependent. Our results suggest that rhizobial evolution in soil and host are largely independent, but soil selection can alter mutualism benefits. IMPORTANCE: Rhizobia-legume mutualism is crucial for introducing nitrogen into agricultural and natural ecosystems, and rhizobia persistence in the soil is an important component of agroecosystems. However, we know little about how individual strains of rhizobia persist and adapt to this environment, especially in the context of the soil’s spatial and temporal variations (temperature, moisture, and soil texture). We found that rhizobia similarly adapt to abiotic soil conditions but their reproductive success in the soil is independent from their reproductive success in the host. Intriguingly, we found that certain soil conditions increase (or decrease) the relative abundance of more effective nitrogen-fixing strains. Understanding how rhizobia adapt to diverse environments is crucial for developing effective bioinoculants that maintain high persistence in the soil while are also highly competitive to colonize the host and are beneficial to the plant.

Mutualism breakdown can be prevented if partner species preferentially select and reward partners that provide greater benefit. We examined these two components using the legume Medicago truncatula and its nitrogen-fixing symbiont Sinorhizobium meliloti . First, we reanalyzed data from 202 accessions to show significant genetic variation in the capacity of Medicago to restrict strain diversity, finding that hosts with shorter nodules were more selective. A genome-wide association study on host selectivity identified genes including the hormone leginsulin, pectin degradation, multidrug and toxic compound efflux, Zinc transport, and DNA methylation. Second, we used two well-studied Medicago genotypes with contrasting nodule morphologies to assess the effectiveness of adaptive enrichment mechanisms by sampling the relative frequencies of rhizobial strains in pools of small nodules (indicating a lack of host investment) compared to large nodules (indicating increased host investment) and pairing these results with previous single-strain assessments of strain benefits to hosts. While both hosts enriched beneficial strains in large nodules, the host that formed larger and more variably sized nodules and thus had greater ’potential’ to increase rhizobial populations was less effective. Our findings reveal that host genetic variation affects strain selectivity and suggest that nodule traits warrant attention when exploring mutualism evolution.

In the mutualism between leguminous plants and rhizobia bacteria, rhizobia live inside root nodules, creating potential for host genes to shape the rhizobial selective environment. Many host genes that affect symbiosis have been identified; however, the extent to which these genes affect selection acting on rhizobia is unknown. In this study, we inoculated 18 Medicago truncatula symbiotic mutants (including mutants that alter Nodule Cysteine-Rich (NCR) peptide production, plant defense, and nodule number regulation) with a mixture of 86 Sinorhizobium meliloti strains. Most mutations resulted in reduced host benefits, but the effects on rhizobial benefit (i.e., relative strain fitness) varied widely, revealing widespread host-by-strain fitness interactions. Genome-wide association analyses identified variants on rhizobial replicons pSymA and pSymB as important in mediating strain fitness responses to host mutations. Whereas most top variants affected rhizobial fitness with one host mutant (limited effect variants), nine affected fitness across six or more host mutations. These pervasive variants occurred primarily on pSymA, the symbiotic replicon, and include fixL and some metabolic genes. In contrast to the limited effect variants, variants with pervasive positive effects on strain fitness when host genes were mutated tended to adversely affect fitness in wild-type hosts. Competition assays across Medicago genotypes confirmed a pervasive role for one candidate (malonyl-CoA synthase), and AlphaFold multimer modelling suggests that many rhizobial top candidates could interact with host NCR peptides. Our results reveal how host genetic variation affects rhizobial fitness, setting the stage for improving rhizobial inoculants and breeding legume hosts better adapted to multi-strain environments.

Adaptive evolution can facilitate species' range expansions across environmentally heterogeneous landscapes. However, serial founder effects can limit the efficacy of selection, and the evolution of increased dispersal during range expansions may result in gene flow swamping local adaptation. Here, we study how genetic drift, gene flow and selection interact during the cane toad's (Rhinella marina) invasion across the heterogeneous landscape of Australia. Following its introduction in 1935, the cane toad colonised eastern Australia and established several stable range edges. The ongoing, more rapid range expansion in north‐central Australia has occurred concomitant with an evolved increase in dispersal capacity. Using reduced representation genomic data of Australian cane toads from the expansion front and from two areas of their established range, we test the hypothesis that high gene flow constrains local adaptation at the expansion front relative to established areas. Genetic analyses indicate the three study areas are genetically distinct but show similar levels of allelic richness, heterozygosity and inbreeding. Markedly higher gene flow or recency of colonisation at the expansion front have likely hindered local adaptation at the time of sampling, as indicated by reduced slopes of genetic‐environment associations (GEAs) estimated using a novel application of geographically weighted regression that accounts for allele surfing; GEA slopes are significantly steeper in established parts of the range. Our work bolsters evidence supporting adaptation of invasive species post‐introduction and adds novel evidence for differing strengths of evolutionary forces among geographic areas with different invasion histories.

Mercury (Hg) is highly toxic and has the potential to cause severe health problems for foraging animals and humans when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Using long-read sequencing, we assembled the genomes of Sinorhizobium medicae and Rhizobium leguminosarum from the Almadén mercury mine in Spain with high variation in Hg-tolerance to identify structural and transcriptomic differences between low and high Hg-tolerant strains. While independent mercury reductase A (merA) genes are prevalent in α-proteobacteria, Mer operons are rare and often vary in their gene organization. Our analyses identified multiple structurally conserved merA homologs in the genomes of S. medicae , including a dihydrolipoamide 2-oxoglutarate dehydrogenase (D2OD), but only the strains that possessed a Mer operon exhibited hypertolerance to Hg. Using RNAseq reads mapped to the unique genome assemblies, we found the Hg-tolerant strains which possessed a Mer operon, that nearly all genes within the operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of DEGs in the genome, indicating a rapid and efficient detoxification of Hg ²⁺ from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while inside of nodules showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant, indicating a genotype-by-genotype interaction that influences nitrogen-fixation under stress conditions. Transfer of the Mer operon to non-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the operon is solely necessary to confer hypertolerance to Hg, despite paralogous merA genes present elsewhere in the genome. This study demonstrated that the Mer operon can be exchanged via horizontal gene transfer into non-tolerant rhizobia strains naturally and experimentally.

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.

Legume crops establish beneficial associations with rhizobial bacteria that perform biological nitrogen fixation, providing nitrogen to plants without the economic and greenhouse gas emission costs of chemical nitrogen inputs. Here, we examine the influence of three environmental factors that vary in agricultural fields on strain relative fitness in nodules.