Is there a latitudinal diversity gradient for symbiotic microbes? A case study with sensitive partridge peas.

Mutualism is thought to be more prevalent in the tropics than temperate zones and may therefore play an important role in generating and maintaining high species richness found at lower latitudes. However, results on the impact of mutualism on latitudinal diversity gradients are mixed, and few empirical studies sample both temperate and tropical regions. We investigated whether a latitudinal diversity gradient exists in the symbiotic microbial community associated with the legume Chamaecrista nictitans. We sampled bacteria DNA from nodules and the surrounding soil of plant roots across a latitudinal gradient (38.64-8.68 °N). Using 16S rRNA sequence data, we identified many non-rhizobial species within C. nictitans nodules that cannot form nodules or fix nitrogen. Species richness increased towards lower latitudes in the non-rhizobial portion of the nodule community but not in the rhizobial community. The microbe community in the soil did not effectively predict the non-rhizobia community inside nodules, indicating that host selection is important for structuring non-rhizobia communities in nodules. We next factorially manipulated the presence of three non-rhizobia strains in greenhouse experiments and found that co-inoculations of non-rhizobia strains with rhizobia had a marginal effect on nodule number and no effect on plant growth. Our results suggest that these non-rhizobia bacteria are likely commensals-species that benefit from associating with a host but are neutral for host fitness. Overall, our study suggests that temperate C. nictitans plants are more selective in their associations with the non-rhizobia community, potentially due to differences in soil nitrogen across latitude.

Generalized mutualisms promote range expansion in both plant and ant partners.

Mutualism improves organismal fitness, but strong dependence on another species can also limit a species' ability to thrive in a new range if its partner is absent. We assembled a large, global dataset on mutualistic traits and species ranges to investigate how multiple plant-animal and plant-microbe mutualisms affect the spread of legumes and ants to novel ranges. We found that generalized mutualisms increase the likelihood that a species establishes and thrives beyond its native range, whereas specialized mutualisms either do not affect or reduce non-native spread. This pattern held in both legumes and ants, indicating that specificity between mutualistic partners is a key determinant of ecological success in a new habitat. Our global analysis shows that mutualism plays an important, if often overlooked, role in plant and insect invasions.

Nonsymbiotic legumes are more invasive, but only if polyploid.

Both mutualism and polyploidy are thought to influence invasion success in plants, but few studies have tested their joint effects. Mutualism can limit range expansion when plants cannot find a compatible partner in a novel habitat, or facilitate range expansion when mutualism increases a plant's niche breadth. Polyploids are also expected to have greater niche breadth because of greater self-compatibility and phenotypic plasticity, increasing invasion success. For 847 legume species, we compiled data from published sources to estimate ploidy, symbiotic status with rhizobia, specificity on rhizobia, and the number of introduced ranges. We found that diploid species have had limited spread around the globe regardless of whether they are symbiotic or how many rhizobia partners they can host. Polyploids, by contrast, have been successfully introduced to many new ranges, but interactions with rhizobia constrain their range expansion. In a hidden state model of trait evolution, we also found evidence of a high rate of re-diploidization in symbiotic legume lineages, suggesting that symbiosis and ploidy may interact at macroevolutionary scales. Overall, our results suggest that symbiosis with rhizobia limits range expansion when legumes are polyploid but not diploid.

An emerging view of coevolution in the legume-rhizobium mutualism.

Mutualisms are often framed as 'delicately balanced antagonisms' (Bronstein, 1994), with the net fitness benefits to both partners potentially masking underlying conflicts of interest. How commonly symbionts evolve to 'cheat' their hosts and hosts evolve to 'sanction' or 'control' uncooperative symbionts is the subject of debate, especially in legume-rhizobium interactions (Frederickson, 2013; Kiers et al., 2003). This kind of antagonistic coevolution should result in either arms-race dynamics characterized by repeated selective sweeps or fluctuating selection dynamics that leave signatures of balancing selection in host and symbiont genomes (Frederickson, 2013; Kortright et al., 2022; O'Brien et al., 2021). In a From the Cover article in this issue of Molecular Ecology, Epstein et al. (2022) combine GWAS and population genomic analyses to assess the evidence for positive or balancing selection consistent with ongoing, antagonistic coevolution between legumes and rhizobia. They found few genomic signatures of fitness conflicts between mutualistic partners, suggesting that legume and rhizobium fitness interests are largely aligned and symbiotic traits are mostly under stabilizing selection. In combination with other recent work (e.g. Batstone et al., 2020), the results of Epstein et al. (2022) indicate that there is little ongoing fitness conflict between legumes and rhizobia that shapes host and symbiont genomes in this system. It may be time to move beyond symbiont 'cheating' and host 'control' as the dominant paradigm for understanding how partners in mutualism coevolve.

Genetic architecture of multiple mutualisms and mating system in Turnera ulmifolia.

Plants often associate with multiple arthropod mutualists. These partners provide important services to their hosts, but multiple interactions can constrain a plant's ability to respond to complex, multivariate selection. Here, we quantified patterns of genetic variance and covariance among rewards for pollination, biotic defence and seed dispersal mutualisms in multiple populations of Turnera ulmifolia to better understand how the genetic architecture of multiple mutualisms might influence their evolution. We phenotyped plants cultivated from 17 Jamaican populations for several mutualism and mating system-related traits. We then fit genetic variance-covariance (G) matrices for the island metapopulation and the five largest individual populations. At the metapopulation level, we observed significant positive genetic correlations among stigma-anther separation, floral nectar production and extrafloral nectar production. These correlations have the potential to significantly constrain or facilitate the evolution of multiple mutualisms in T. ulmifolia and suggest that pollination, seed dispersal and defence mutualisms do not evolve independently. In particular, we found that positive genetic correlations between floral and extrafloral nectar production may help explain their stable coexistence in the face of physiological trade-offs and negative interactions between pollinators and ant bodyguards. Locally, we found only small differences in G among our T. ulmifolia populations, suggesting that geographic variation in G may not shape the evolution of multiple mutualisms.

Microplastics shift impacts of climate change on a plant-microbe mutualism: Temperature, CO2, and tire wear particles.

Anthropogenic stressors can affect individual species and alter species interactions. Moreover, species interactions or the presence of multiple stressors can modify the stressor effects, yet most work focuses on single stressors and single species. Plant-microbe interactions are a class of species interactions on which ecosystems and agricultural systems depend, yet may be affected by multiple global change stressors. Here, we use duckweed and microbes from its microbiome to model responses of interacting plants and microbes to multiple stressors: climate change and tire wear particles. Climate change is occurring globally, and microplastic tire wear particles from roads now reach many ecosystems. We paired perpendicular gradients of temperature and carbon dioxide (CO2) treatments with factorial manipulation of leachate from tire wear particles and duckweed microbiomes. We found that tire leachate and warmer temperatures enhanced duckweed and microbial growth, but caused effects of microbes on duckweed to become negative. However, induced negative effects of microbes were less than additive with warming and leachate. Without tire leachate, we observed that higher CO2 and temperature induced positive correlations between duckweed and microbial growth, which can strengthen mutualisms. In contrast, with tire leachate, growth correlations were never positive, and shifted negative at lower CO2, again suggesting leachate disrupts this plant-microbiome mutualism. In summary, our results demonstrate that multiple interacting stressors can affect multiple interacting species, and that leachate from tire wear particles could potentially disrupt plant-microbe mutualisms.

Priority effects alter interaction outcomes in a legume-rhizobium mutualism.

Priority effects occur when the order of species arrival affects the final community structure. Mutualists often interact with multiple partners in different orders, but if or how priority effects alter interaction outcomes is an open question. In the field, we paired the legume Medicago lupulina with two nodulating strains of Ensifer bacteria that vary in nitrogen-fixing ability. We inoculated plants with strains in different orders and measured interaction outcomes. The first strain to arrive primarily determined plant performance and final relative abundances of rhizobia on roots. Plants that received effective microbes first and ineffective microbes second grew larger than plants inoculated with the same microbes in the opposite order. Our results show that mutualism outcomes can be influenced not just by partner identity, but by the interaction order. Furthermore, hosts receiving high-quality mutualists early can better tolerate low-quality symbionts later, indicating that priority effects may help explain the persistence of ineffective symbionts.

Whose trait is it anyways? Coevolution of joint phenotypes and genetic architecture in mutualisms.

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.

No selection for cheating in a natural meta-population of rhizobia.

Whether natural selection favours 'cheating' in mutualisms is hotly debated. Gano-Cohen et al. (2019a) report a negative correlation between fitness and mutualist quality in rhizobia, suggesting that rhizobia evolve to cheat. However, reanalysis of their data shows that the correlation is an artefact of unequal sampling across populations.

Using text-mined trait data to test for cooperate-and-radiate co-evolution between ants and plants.

Mutualisms may be "key innovations" that spur lineage diversification by augmenting niche breadth, geographic range, or population size, thereby increasing speciation rates or decreasing extinction rates. Whether mutualism accelerates diversification in both interacting lineages is an open question. Research suggests that plants that attract ant mutualists have higher diversification rates than non-ant associated lineages. We ask whether the reciprocal is true: does the interaction between ants and plants also accelerate diversification in ants, i.e. do ants and plants cooperate-and-radiate? We used a novel text-mining approach to determine which ant species associate with plants in defensive or seed dispersal mutualisms. We investigated patterns of lineage diversification across a recent ant phylogeny using BiSSE, BAMM, and HiSSE models. Ants that associate mutualistically with plants had elevated diversification rates compared to non-mutualistic ants in the BiSSE model, with a similar trend in BAMM, suggesting ants and plants cooperate-and-radiate. However, the best-fitting model was a HiSSE model with a hidden state, meaning that diversification models that do not account for unmeasured traits are inappropriate to assess the relationship between mutualism and ant diversification. Against a backdrop of diversification rate heterogeneity, the best-fitting HiSSE model found that mutualism actually decreases diversification: mutualism evolved much more frequently in rapidly diversifying ant lineages, but then subsequently slowed diversification. Thus, it appears that ant lineages first radiated, then cooperated with plants.

Interactions between seed-dispersing ant species affect plant community composition in field mesocosms.

In generalized mutualisms, species vary in the quality of services they provide to their partners directly via traits that affect partner fitness and indirectly via traits that influence interactions among mutualist species that play similar functional roles. Myrmecochory, or seed dispersal by ants, is a generalized mutualism with ant species varying in the quality of dispersal services they provide to their plant partners. Variation in ant species identity can directly impact seed dispersal patterns and plant community composition; however, we know less about how interactions among seed-dispersing ant species indirectly influence plant partners. The invasive ant Myrmica rubra, is a high-quality seed-disperser in its native range that interacts with myrmecochores (ant-dispersed plants) and the high-quality seed disperser Aphaenogaster sp. in its invaded range. We use this system to examine how interactions between two functionally similar mutualist ant species influence the recruitment and community composition of ant-dispersed plants. We performed a field mesocosm experiment and a laboratory behavioural experiment to compare discovery and dominance behaviours between ant species, and seed dispersal and seedling recruitment of four myrmecochore species among intraspecific interaction treatments of each ant species and an interspecific interaction treatment. We found that M. rubra was better at discovering and dispersing seeds, but Aphaenogaster sp. was dominantly aggressive over M. rubra. Interspecific interactions dampened seed dispersal relative to dispersal by the better disperser. Despite this dampening, we found no effect of interspecific interactions on seedling recruitment. However, community composition of seedlings in the interspecific interaction treatment was more similar to composition in the aggressively dominant ant (Aphaenogaster sp.) treatment than in the better discoverer ant M. rubra treatment. We show that interspecific interactions between mutualist species in the same functional guild affect the outcome of mutualistic interactions with partner species. Despite the native ant dispersing fewer seeds, its dominance over the subordinate (invasive) ant has the potential to allow for some level of biotic resistance against the effects of M. rubra on plant communities when these species coexist.

Environmental variation impacts trait expression and selection in the legume-rhizobium symbiosis.

PREMISE: The ecological outcomes of mutualism are well known to shift across abiotic or biotic environments, but few studies have addressed how different environments impact evolutionary responses, including the intensity of selection on and the expression of genetic variance in key mutualism-related traits. METHODS: We planted 30 maternal lines of the legume Medicago lupulina in four field common gardens and compared our measures of selection on and genetic variance in nodulation, a key trait reflecting legume investment in the symbiosis, with those from a previous greenhouse experiment using the same 30 M. lupulina lines. RESULTS: We found that both the mean and genetic variance for nodulation were much greater in the greenhouse than in the field and that the form of selection on nodulation significantly differed across environments. We also found significant genotype-by-environment (G × E) effects for fitness-related traits that were generated by differences in the rank order of plant lines among environments. CONCLUSIONS: Overall, our results suggest that the expression of genotypic variation and selection on nodulation differ across environments. In the field, significant rank-order changes for plant fitness potentially help maintain genetic variation in natural populations, even in the face of directional or stabilizing selection.

Resilience to multiple stressors in an aquatic plant and its microbiome.

PREMISE: Outcomes of species interactions, especially mutualisms, are notoriously dependent on environmental context, and environments are changing rapidly. Studies have investigated how mutualisms respond to or ameliorate anthropogenic environmental changes, but most have focused on nutrient pollution or climate change and tested stressors one at a time. Relatively little is known about how mutualisms may be altered by or buffer the effects of multiple chemical contaminants, which differ fundamentally from nutrient or climate stressors and are especially widespread in aquatic habitats. METHODS: We investigated the impacts of two contaminants on interactions between the duckweed Lemna minor and its microbiome. Sodium chloride (salt) and benzotriazole (a corrosion inhibitor) often co-occur in runoff to water bodies where duckweeds reside. We tested three L. minor genotypes with and without the culturable portion of their microbiome across field-realistic gradients of salt (3 levels) and benzotriazole (4 levels) in a fully factorial experiment (24 treatments, tested on each genotype) and measured plant and microbial growth. RESULTS: Stressors had conditional effects. Salt decreased both plant and microbial growth and decreased plant survival more as benzotriazole concentrations increased. In contrast, benzotriazole did not affect microbial abundance and even benefited plants when salt and microbes were absent, perhaps due to biotransformation into growth-promoting compounds. Microbes did not ameliorate duckweed stressors; microbial inoculation increased plant growth, but not at high salt concentrations. CONCLUSIONS: Our results suggest that multiple stressors matter when predicting responses of mutualisms to global change and that beneficial microbes may not always buffer hosts against stress.

Mutualistic Outcomes Across Plant Populations, Microbes, and Environments in the Duckweed Lemna minor.

The picture emerging from the rapidly growing literature on host-associated microbiota is that host traits and fitness often depend on interactive effects of host genotype, microbiota, and abiotic environment. However, testing interactive effects typically requires large, multi-factorial experiments and thus remains challenging in many systems. Furthermore, most studies of plant microbiomes focus on terrestrial hosts and microbes. Aquatic habitats may confer unique properties to microbiomes. We grew different populations of duckweed (Lemna minor), a floating aquatic plant, in three microbial treatments (adding no, "home", or "away" microbes) at two levels of zinc, a common water contaminant in urban areas, and measured both plant and microbial performance. Thus, we simultaneously manipulated plant source population, microbial community, and abiotic environment. We found strong effects of plant source, microbial treatment, and zinc on duckweed and microbial growth, with significant variation among duckweed genotypes and microbial communities. However, we found little evidence of interactive effects: zinc did not alter effects of host genotype or microbial community, and host genotype did not alter effects of microbial communities. Despite strong positive correlations between duckweed and microbe growth, zinc consistently decreased plant growth, but increased microbial growth. Furthermore, as in recent studies of terrestrial plants, microbial interactions altered a duckweed phenotype (frond aggregation). Our results suggest that duckweed source population, associated microbiome, and contaminant environment should all be considered for duckweed applications, such as phytoremediation. Lastly, we propose that duckweed microbes offer a robust experimental system for study of host-microbiota interactions under a range of environmental stresses.

Invasive ants disperse seeds farther than native ants, affecting the spatial pattern of seedling recruitment and survival.

Mutualists can vary in the quantity and quality of service which they provide to their partners. Variation in seed disperser quality depends on seed-processing traits, dispersal distance, and deposition location, all of which ultimately affect plant fitness. Here, we compared these aspects of seed dispersal quality between a native and an invasive ant species, and examined how they affect competition and plant performance. Using experimental mesocosm communities, we examined how these two ant species affect the spatial pattern of recruitment and establishment for four myrmecochorous plant species, including one invasive species. We measured the locations of dispersed seedlings relative to ant nests, adult plants, and other dispersed seedlings, as well as measured the effects of location on plant performance. The invasive ant, Myrmica rubra, secondarily dispersed seeds farther from its nests, creating a less clumped pattern of seedling recruitment compared to the native ant, Aphaenogaster rudis. Plant species responded differently to dispersal. Invasive seedlings recruited farther from adult plants than native seedlings, and had higher survival the farther they were from conspecifics. In contrast, native plants had higher survival and grew taller when dispersed farther from invasive plants. We show that seed-dispersing ant partners differ in mutualist quality creating differences in dispersal distance and deposition location that affects a plant's competitive environment. Our results reveal the potential for long-term consequences on plant community structure with changing ant partner identity. We emphasize the need to examine dispersal quality in addition to quantity to uncover the importance of partner identity in structuring communities.

Asynchrony between ant seed dispersal activity and fruit dehiscence of myrmecochorous plants.

PREMISE OF THE STUDY: Phenological mismatch has received attention in plant-pollinator interactions, but less so in seed dispersal mutualisms. We investigated whether the seasonal availability of myrmecochorous seeds is well matched to the seasonal activity patterns of seed-dispersing ants. METHODS: We compared seasonal timing of seed removal by a keystone seed-dispersing ant, Aphaenogaster rudis, and fruit dehiscence of several species of plants whose seeds it disperses in a deciduous forest in southern Ontario, Canada. We examined the timing of elaiosome "robbing" by the nonnative slug Arion subfuscus and tested whether seed removal by ants declines in response to supplementation with additional elaiosome-bearing seeds (ant "satiation"). KEY RESULTS: Seed removal from experimental depots peaked early in the season for all plant species and correlated with temperature. In contrast, elaiosome robbing by slugs increased late in the season and thus may disproportionately affect plants with late-dehiscing fruits. Ant colonies removed seeds at similar rates regardless of seed supplementation, indicating that satiation likely does not impact seasonal patterns of seed dispersal in this system. Fruits of the five myrmecochorous plant species in our study dehisced at discrete intervals throughout the season, with minimal overlap among species. Peak dehiscence did not overlap with peak seed removal for any plant species. CONCLUSIONS: Fruit dehiscence of myrmecochorous plants and peak ant seed dispersal activity occur asynchronously. Whether future climate warming will shift ant and plant phenologies in ways that have consequences for seed dispersal remains an open question.

Using niche breadth theory to explain generalization in mutualisms.

For a mutualism to remain evolutionarily stable, theory predicts that mutualists should limit their associations to high-quality partners. However, most mutualists either simultaneously or sequentially associate with multiple partners that confer the same type of reward. By viewing mutualisms through the lens of niche breadth evolution, we outline how the environment shapes partner availability and relative quality, and ultimately a focal mutualist's partner breadth. We argue that mutualists that associate with multiple partners may have a selective advantage compared to specialists for many reasons, including sampling, complementarity, and portfolio effects, as well as the possibility that broad partner breadth increases breadth along other niche axes. Furthermore, selection for narrow partner breadth is unlikely to be strong when the environment erodes variation in partner quality, reduces the costs of interacting with low-quality partners, spatially structures partner communities, or decreases the strength of mutualism. Thus, we should not be surprised that most mutualists have broad partner breadth, even if it allows for ineffective partners to persist.

More partners, more ranges: generalist legumes spread more easily around the globe.

How does mutualism affect range expansion? On the one hand, mutualists might thrive in new habitats thanks to the resources, stress tolerance or defence provided by their partners. On the other, specialized mutualists might fail to find compatible partners beyond their range margins, limiting further spread. A recent global analysis of legume ranges found that non-symbiotic legumes have been successfully introduced to more ranges than legumes that form symbioses with rhizobia, but there is still abundant unexplained variation in introduction success within symbiotic legumes. We test the hypothesis that generalist legumes have spread to more ranges than specialist legumes. We used published data and rhizobial 16S rRNA sequences from GenBank to quantify the number of rhizobia partners that associate with 159 legume species, spanning the legume phylogeny and the globe. We found that generalist legumes occur in more introduced ranges than specialist legumes, suggesting that among mutualists, specialization hinders range expansions.

An ant-plant mutualism through the lens of cGMP-dependent kinase genes.

In plant-animal mutualisms, how an animal forages often determines how much benefit its plant partner receives. In many animals, foraging behaviour changes in response to foraging gene expression or activation of the cGMP-dependent protein kinase (PKG) that foraging encodes. Here, we show that this highly conserved molecular mechanism affects the outcome of a plant-animal mutualism. We studied the two PKG genes of Allomerus octoarticulatus, an Amazonian ant that defends the ant-plant Cordia nodosa against herbivores. Some ant colonies are better 'bodyguards' than others. Working in the field in Peru, we found that colonies fed with a PKG activator recruited more workers to attack herbivores than control colonies. This resulted in less herbivore damage. PKG gene expression in ant workers correlated with whether an ant colony discovered an herbivore and how much damage herbivores inflicted on leaves in a complex way; natural variation in expression levels of the two genes had significant interaction effects on ant behaviour and herbivory. Our results suggest a molecular basis for ant protection of plants in this mutualism.

Distinctive fungal communities in an obligate African ant-plant mutualism.

Three ant species nest obligately in the swollen-thorn domatia of the African ant-plant Vachellia (Acacia) drepanolobium, a model system for the study of ant-defence mutualisms and species coexistence. Here we report on the characteristic fungal communities generated by these ant species in their domatia. First, we describe behavioural differences between the ant species when presented with a cultured fungal isolate in the laboratory. Second, we use DNA metabarcoding to show that each ant species has a distinctive fungal community in its domatia, and that these communities remain characteristic of the ant species over two Kenyan sampling locations separated by 190 km. Third, we find that DNA extracted from female alates of Tetraponera penzigi and Crematogaster nigriceps contained matches for most of the fungal metabarcodes from those ant species' domatia, respectively. Fungal hyphae and other debris are also visible in sections of these alates' infrabuccal pockets. Collectively, our results indicate that domatium fungal communities are associated with the ant species occupying the tree. To the best of our knowledge, this is the first record of such ant-specific fungal community-level differences on the same myrmecophytic host species. These differences may be shaped by ant behaviour in the domatia, and by ants vectoring fungi when they disperse to establish new colonies. The roles of the fungi with respect to the ants and their host plant remain to be determined.