In the legume-rhizobia mutualism, symbiotic success changes with time, complicating early strain quality evaluations. We measured host biomass at three and six months after inoculation of bearded clover ( Trifolium barbigerum ) with 77 strains of Rhizobium leguminosarum . Across timepoints, strain performance rankings based on host biomass varied: some top strains at three months later declined, whereas initially low-ranking strains ultimately surpassed them. This suggests biological tradeoffs in the timing of nitrogen fixation and the allocation of the host resources. Our results highlight that symbiont function can vary over time and that single timepoint data collection risks inaccurately identifying long-term beneficial strains.

Theories explaining the evolution of plant defensive strategies are difficult to experimentally test. Biological invasion scenarios can serve as helpful natural experiments for examining the evolutionary dynamics of plant defenses when plants become established as potential hosts in new environments. This study uses a historical invasion by Medicago polymorpha (Burr Clover) to test the predictive power of the Shifting Defense Hypothesis (SDH) by investigating variation in plant defenses to herbivorous insects. We compared the feeding preferences of a generalist and a specialist herbivore on native and invasive populations of M. polymorpha. We document a shift in herbivore preference patterns for constitutive versus herbivore-induced tissues when comparing plants from native and invaded ranges. However, specific biochemical defenses showed a conserved negative correlation between constitutive and inducible defenses across both ranges, indicating a fundamental trade-off in defense strategy that persists despite allocation differences, suggesting defense evolution that was not revealed by tests in this study. These results provide evidence of evolutionary shifts in plant palatability that are consistent with predictions of the SDH, which predicts evolutionary shifts in defense allocation. Our findings reveal complex evolutionary dynamics that underlie invasion success and demonstrate that invasive M. polymorpha have undergone evolutionary adaptation in defense strategy beyond any immediate ecological advantages of enemy release, providing insight into how invasive plants successfully adapt to novel herbivore communities over time.

Saint John’s wort, Hypericum perforatum, is a medicinally and ecologically important perennial plant species that has a broad global distribution. Despite the species’ importance, little is known about the factors that structure its microbial communities and the identity of microbes that enhance plant growth and fitness. Here we aim to describe the microbial communities associated with Hypericum perforatum and elucidate factors that structure these communities. We collected H. perforatum root samples in three adjacent habitat types: wet and dry alvars (two types of limestone barren) and fallow agricultural fields (i.e. old-fields), in Jefferson County, New York. We used high-throughput amplicon sequencing to characterize the bacterial and fungal root microbiome. We also quantified aspects of the plant phenotype and soil characteristics to evaluate habitat variables that correlate with the root microbiome. Habitat and plant height were correlated with shifts in microbial community composition. We identified two bacterial taxa positively associated with plant height, both belonging to the bacterial phylum Actinobacteria. This work contributes to our understanding of the environmental determinants of microbial community composition and identifies microbial taxa that may be important in promoting plant growth.

Less than 1% of native prairie lands remain in the United States. Located in eastern Washington, the rare habitat called Palouse prairie was largely converted to wheat monocropping. With this conversion came numerous physical, chemical, and biological changes to the soil that may ultimately contribute to reduced wheat yields. Here, we explored how wheat (Tritcum aestivum L.) seedling establishment, plant size, and heading, signifying the developmental transition to flowering, were affected by being planted in prairie soil versus agricultural soils. We then sought to understand whether the observed effects were the result of changes to the soil microbiota due to agricultural intensification. We found that prairie soil enhanced both the probability of wheat seedling survival and heading compared to agricultural soil; however, wheat growth was largely unaffected by soil source. We did not detect effects on wheat developmental transitions or phenotype when inoculated with prairie microbes compared with agricultural microbes, but we did observe general antagonistic effects of microbes on plant size, regardless of soil source. This work indicates that agricultural intensification has affected soils in a way that changes early seedling establishment and the timing of heading for wheat, but these effects may not be caused by microbes, and instead may be caused by soil nutrient conditions.

Abstract Aims Saint John’s wort, Hypericum perforatum, is a medicinally and ecologically important perennial plant species that has a broad global distribution. Despite the species’ importance, little is known about the factors that structure its microbial communities and the identity of microbes that enhance plant growth and fitness. Here we aim to describe the microbial communities associated with Hypericum perforatum and elucidate factors that structure these communities.Methods We collected H. perforatum root samples in three adjacent habitat types: wet and dry alvars (two types of limestone barren) and fallow agricultural fields (i.e. old-fields), in Jefferson County, New York. We used high-throughput amplicon sequencing of the SSU-rRNA gene (16S, bacteria) and the internal transcribed spacer region 1 (ITS1, fungi) to characterize the root microbiome of H. perforatum. At each root sampling location, we quantified aspects of the plant phenotype and soil characteristics to evaluate habitat variables that correlate with root microbial communities.Results Alvars had ~ 13% higher bacterial richness compared to old-fields. In contrast, old-fields had 28% higher fungal richness than dry alvars, but similar fungal richness to wet alvars. Habitat and plant height were important predictors of microbial community composition. We identified two bacterial taxa positively associated with plant height, both belonging to the bacterial phylum Actinobacteria.Conclusions This work contributes to our understanding of the environmental determinants of microbial community composition. Additionally, we were able to identify bacterial taxa that are correlated with plant health and should be investigated further as indicators of soil health or plant-growth promoting rhizobacteria.

Evolutionary biologists typically envision a trait’s genetic basis and fitness effects occurring within a single species. However, traits can be determined by and have fitness consequences for interacting species, thus evolving in multiple genomes. This is especially likely in mutualisms, where species exchange fitness benefits and can associate over long periods of time. Partners may experience evolutionary conflict over the value of a multi-genomic trait, but such conflicts may be ameliorated by mutualism’s positive fitness feedbacks. Here, we develop a simulation model of a host–microbe mutualism to explore the evolution of a multi-genomic trait. Coevolutionary outcomes depend on whether hosts and microbes have similar or different optimal trait values, strengths of selection and fitness feedbacks. We show that genome-wide association studies can map joint traits to loci in multiple genomes and describe how fitness conflict and fitness feedback generate different multi-genomic architectures with distinct signals around segregating loci. Partner fitnesses can be positively correlated even when partners are in conflict over the value of a multi-genomic trait, and conflict can generate strong mutualistic dependency. While fitness alignment facilitates rapid adaptation to a new optimum, conflict maintains genetic variation and evolvability, with implications for applied microbiome science.

The appeal of using microbial inoculants to mediate plant traits and productivity in managed ecosystems has increased over the past decade, because microbes represent an alternative to fertilizers, pesticides, and direct genetic modification of plants. Using microbes bypasses many societal and environmental concerns because microbial products are considered a more sustainable and benign technology. In our desire to harness the power of plant?microbial symbioses, are we ignoring the possibility of precipitating microbial invasions, potentially setting ourselves up for a microbial Jurassic Park? Here, we outline potential negative consequences of microbial invasions and describe a set of practices (Testing, Regulation, Engineering, and Eradication, TREE) based on the four stages of invasion to prevent microbial inoculants from becoming invasive. We aim to stimulate discussion about best practices to proactively prevent microbial invasions.

Premise of research. Plants in natural and agricultural systems are influenced in myriad ways by their microbial communities, particularly by providing goods and services that change plant functional traits. Microbes are considered an influential part of the environmental context that change plant trait expression, but often, microbe-mediated effects are contingent on local resources, such as nitrogen. Here, we ask how microbes and nitrogen affect belowground functional traits and patterns of phenotypic selection.Methodology. We performed a fully factorial greenhouse experiment with switchgrass (Panicum virgatum), manipulating microbial community composition and nitrogen availability. We measured plant performance and belowground functional traits and performed 16S amplicon sequencing of the root-associated microbial communities. We looked for correlations between microbial taxa and root functional traits, and we performed phenotypic selection analysis on five belowground functional traits to determine how traits affect plant relative performance across biotic and abiotic contexts.Pivotal results. All belowground plant functional traits except root tissue density were affected by adding nitrogen. We found that a microbial taxon (amplicon sequence variant [ASV]) in the genus Micromonospora correlated with shorter root lengths. We also found strong positive selection for longer roots regardless of the abiotic or biotic environment. In contrast, selection favored lower root-to-shoot ratios in high-nitrogen conditions, and selection on root tissue density was highest in treatments that had high nitrogen levels and perturbed microbial communities.Conclusions. We did not detect microbial effects on the expression of plant traits (ecological effects); however, patterns of phenotypic selection (evolutionary effects) on root tissue density differed depending on the biotic and abiotic environment. Additionally, we detected strong selection for increased root length across treatments; we also found that one ASV correlated with decreased root length, indicating potential conflict between root microbiome components and plant fitness. Future work would be to include microbial taxa in phenotypic selection analysis and to conduct manipulations of the microbes correlated with functional traits to determine causality.

Premise of the StudyCurrent methods for quantifying herbivore‐induced alterations in plant biochemistry are often unusable by researchers due to practical constraints. We present a cost‐effective, high‐throughput protocol to quantify multiple biochemical responses from small plant tissue samples using spectrophotometric techniques.Methods and ResultsUsing Solanum lycopersicum and Medicago polymorpha leaves pre‐ and post‐herbivory, we demonstrate that our protocol quantifies common plant defense responses: peroxidase production, polyphenol oxidase production, reactive oxygen species production, total protein production, and trypsin‐like protease inhibition activity.ConclusionsCurrent protocols can require 500 mg of tissue, but our assays detect activity in less than 10 mg. Our protocol takes two people 6 h to run any of the assays on 300 samples in triplicate, or all of the assays on 20 samples. Our protocol enables researchers to plan complex experiments that compare local versus systemic plant responses, quantify environmental and genetic variation, and measure population‐level variation.

Abstract The Enemy Release Hypothesis posits that invasion of novel habitats can be facilitated by the absence of coevolved herbivores. However, a new environment and interactions with unfamiliar herbivores may impose selection on invading plants for traits that reduce their attractiveness to herbivores or for enhanced defenses compared to native host plants, leading to a pattern similar to enemy release but driven by evolutionary change rather than ecological differences. The Shifting Defense Hypothesis posits that plants in novel habitats will shift from specialized defense mechanisms to defense mechanisms effective against generalist herbivores in the new range. We tested these ideas by comparing herbivore preference and performance of native (Eurasia)‐ and invasive (New World)‐range Medicago polymorpha, using a generalist herbivore, the soybean looper, that co‐occurs with M. polymorpha in its New World invaded range. We found that soybean loopers varied in preference and performance depending on host genotype and that overall the herbivore preferred to consume plant genotypes from naïve populations from Eurasia. This potentially suggests that range expansion of M. polymorpha into the New World has led to rapid evolution of a variety of traits that have helped multiple populations become established, including those that may allow invasive populations to resist herbivory. Thus, enemy release in a novel range can occur through rapid evolution by the plant during invasion, as predicted by the Shifting Defense Hypothesis, rather than via historical divergence.

Microbial inhabitants of the rhizosphere can have substantial impacts on the fitness of their associated host plants, in both beneficial and detrimental ways. Soil-borne pathogens can impose severe fitness costs that can be mitigated or eliminated in many cases by co-occurring beneficial bacteria that directly or indirectly temper pathogens’ antagonistic effects. Rhizobial bacteria are best known for their role as nitrogen fixing symbionts in the rhizobia-legume mutualism but there is growing evidence that they can also act as protective agents against microbial pathogens. This study examined the role of rhizobial bacterium Ensifer medicae in protecting the burclover, Medicago polymorpha, against antagonistic soil microbes in complex soil communities. Exposing plants to concentrated slurries of soil inoculum had an adverse effect on all aspects of plant fitness in M. polymorpha. However, inoculating plants with rhizobia increased plant survival in the presence of live soil inoculum from 18% to over 80%. In addition, the soil microbiome changed the symbiotic relationship between plants and rhizobia; plants that were co-inoculated with dilute soil inoculum and a beneficial rhizobium produced more nodules with a higher nodule biomass than plants inoculated with rhizobium alone. Finally, we found that the effects of soil microbes and rhizobia on root biomass, root:shoot ratio, and nodule number differed between host genotypes, indicating there is potential for complex plant-bacterial interactions to respond to selection and potentially contribute to the maintenance of both plant genetic variation and bacterial diversity.

Plants engage in complex multipartite interactions with mutualists and antagonists, but these interactions are rarely included in studies that explore plant invasiveness. When considered in isolation, we know that beneficial microbes can enhance an exotic plant's invasive ability and that herbivorous insects often decrease an exotic plant's likeliness of success. However, the effect of these partners on plant fitness has not been well characterized when all three species coevolve. We use computational evolutionary modeling of a trait-based system to test how microbes and herbivores simultaneously coevolving with an invading plant affect the invaders' probability of becoming established. Specifically, we designed a model that explores how a beneficial microbe would influence the outcome of an interaction between a plant and herbivore. To model novel interactions, we included a phenotypic trait shared by each species. Making this trait continuous and selectable allows us to explore how trait similarities between coevolving plants, herbivores and microbes affect fitness. Using this model, we answer the following questions: (1) Can a beneficial plant-microbe interaction influence the evolutionary outcome of antagonistic interactions between plants and herbivores? (2) How does the initial trait similarity between interacting organisms affect the likelihood of plant survival in novel locations? (3) Does the effect of tripartite interactions on the invasion success of a plant depend on whether organisms interact through trait similarity [Enemy Release Hypothesis (ERH)] or dissimilarity (Biotic Resistance Hypothesis)? We found that it was much more difficult for plants to invade under the ERH but that beneficial microbes increase the probability of plant survival in a novel range under both hypotheses. To our knowledge, this model is the first to use tripartite interactions to explore novel species introductions. It represents a step towards gaining a better understanding of the factors influencing establishment of exotic species to prevent future invasions.

Interaction conditions can change the balance of cooperation and conflict in multicellular groups. After aggregating together, cells of the social amoeba Dictyostelium discoideum may migrate as a group (known as a slug) to a new location. We consider this migration stage as an arena for social competition and conflict because the cells in the slug may not be from a genetically homogeneous population. In this study, we examined the interplay of two seemingly diametric actions, the solitary action of kin recognition and the collective action of slug migration in D. discoideum, to more fully understand the effects of social competition on fitness over the entire lifecycle. We compare slugs composed of either genetically homogenous or heterogeneous cells that have migrated or remained stationary in the social stage of the social amoeba Dictyostelium discoideum. After migration of chimeric slugs, we found that facultative cheating is reduced, where facultative cheating is defined as greater contribution to spore relative to stalk than found for that clone in the clonal state. In addition our results support previous findings that competitive interactions in chimeras diminish slug migration distance. Furthermore, fruiting bodies have shorter stalks after migration, even accounting for cell numbers at that time. Taken together, these results show that migration can alleviate the conflict of interests in heterogeneous slugs. It aligns their interest in finding a more advantageous place for dispersal, where shorter stalks suffice, which leads to a decrease in cheating behavior.

One condition for the evolution of altruism is genetic relatedness between altruist and beneficiary, often achieved through active kin recognition. Here, we investigate the power of a passive process resulting from genetic drift during population growth in the social amoeba Dictyostelium discoideum . We put labelled and unlabelled cells of the same clone in the centre of a plate, and allowed them to proliferate outward. Zones formed by genetic drift owing to the small population of actively growing cells at the colony edge. We also found that single cells could form zones of high relatedness. Relatedness increased at a significantly higher rate when food was in short supply. This study shows that relatedness can be significantly elevated before the social stage without a small founding population size or recognition mechanism.

One of the challenges of microbial life is that the best location for feeding and growth may not be the best location for dispersal. This is likely to be the case for the social amoebae Dictyostelium discoideum and Dictyostelium purpureum that feed on soil bacteria in the amoeba stage, but then group into a multicellular slug that moves towards light before forming a fruiting body. Here we examine this short-range social dispersal in the social amoebae, Dictyostelium discoideum and D. purpureum. We predicted D. purpureum would have higher migration costs and travel less far because it forms a dead stalk from living cells as it moves, while D. discoideum delays stalk formation until movement ceases. We found that D. purpureum migrated shorter distances than D. discoideum, in accord with our prediction. D. discoideum slugs moved an average of 2.46 +/- 0.19 cm while D. purpureum slugs moved an average of 1.04 +/- 0.06 cm. In both species, migration incurred a cost in reduced spore production, compared to experimental conditions where slugs did not migrate. D. discoideum under the no migration treatment produced 0.55 +/- 0.05 spores per cell and under the migration treatment produced 0.25 +/- 0.04 spores per cell. D. purpureum under the no migration treatment produced 1.01 +/- 0.06 spores per cell and under the migration treatment produced 0.85 +/- 0.06 spores per cell. We also found that D. discoideum produced fruiting bodies with fewer spores after migrating while D. purpureum did not. It appears that the evolutionary loss of stalked migration gives D. discoideum cells the advantage of delaying specialization and the ability to colonize more distant locations, but has significant costs due to migration distance, such as the fraction of cells that become fertile spores.

Background: A major challenge for evolutionary biology is explaining altruism, particularly when it involves death of one party and occurs across species. Chimeric fruiting bodies of Dictyostelium discoideum and Dictyostelium purpureum develop from formerly independent amoebae, and some die to help others. Here we examine co-aggregation between D. discoideum and D. purpureum, determine its frequency and which party benefits, and the extent of fair play in contribution to the altruistic caste.Results: We mixed cells from both species in equal proportions, and then we analyzed 198 individual fruiting bodies, which always had either a D. discoideum or D. purpureum phenotype (D. discoideum-98, D. purpureum-100). Fifty percent of the fruiting bodies that looked like D. discoideum and 22% of the fruiting bodies that looked like D. purpureum were chimeric, though the majority of spores in any given fruiting body belonged to one species (D. discoideum fruiting bodies-0.85 +/- 0.03, D. purpureum fruiting bodies-0.94 +/- 0.02). Clearly, there is species level recognition occurring that keeps the cells mostly separate. The number of fruiting bodies produced with the D. discoideum phenotype increased from 225 +/- 32 fruiting bodies when D. discoideum was alone to 486 +/- 61 in the mix treatments. However, the number of D. discoideum spores decreased, although not significantly, from 2.75e(7) +/- 1.29e(7) spores in the controls to 2.06e(7) +/- 8.33e(6) spores in the mix treatments. D. purpureum fruiting body and spore production decreased from 719 +/- 111 fruiting bodies and 5.81e(7) +/- 1.26e(7) spores in the controls to 394 +/- 111 fruiting bodies and 9.75e(6) +/- 2.25e(6) spores in the mix treatments.Conclusion: Both species appear to favor clonality but can cooperate with each other to produce fruiting bodies. Cooperating amoebae are able to make larger fruiting bodies, which are advantageous for migration and dispersal, but both species here suffer a cost in producing fewer spores per fruiting body.

Given the right circumstances, even an amoeba chooses to be altruistic towards its relatives.

Kin recognition helps cooperation to evolve in many animals, but it is uncertain whether microorganisms can also use it to focus altruistic behaviour on relatives. Here we show that the social amoeba Dictyostelium purpureum prefers to form groups with its own kin in situations where some individuals die to assist others. By directing altruism towards kin, D. purpureum should generally avoid the costs of chimaerism experienced by the related D. discoideum.