Phyllosphere fungal diversity generates pervasive nonadditive effects on plant performance.

Plants naturally harbor diverse microbiomes that can dramatically impact their health and productivity. However, it remains unclear how fungal microbiome diversity, especially in the phyllosphere, impacts intermicrobial interactions and consequent nonadditive effects on plant productivity. Combining manipulative experiments, field collections, culturing, microbiome sequencing, and synthetic consortia, we experimentally tested for the first time how foliar fungal community diversity impacts plant productivity. We inoculated morning glories (Ipomoea hederifolia L.) with 32 phyllosphere consortia of either low or high diversity or with single fungal taxa, and measured effects on plant productivity and allocation. We found the following: (1) nonadditive effects were pervasive with 56% of fungal consortia interacting synergistically or antagonistically to impact plant productivity, including some consortia capable of generating acute synergism (e.g. > 1000% increase in productivity above the additive expectation), (2) interactions among 'commensal' fungi were responsible for this nonadditivity in diverse consortia, (3) synergistic interactions were approximately four times stronger than antagonistic effects, (4) fungal diversity affected the magnitude but not frequency or direction of nonadditivity, and (5) diversity affected plant performance nonlinearly with the highest performance in low-diversity treatments. These findings highlight the importance of interpreting plant-microbiome interactions under a framework that incorporates intermicrobial interactions and nonadditive outcomes to understand natural complexity.

Heterogeneous landscape promotes distinct microbial communities in an imperiled scrub ecosystem.

Habitat heterogeneity is a key driver of biodiversity of macroorganisms, yet how heterogeneity structures belowground microbial communities is not well understood. Importantly, belowground microbial communities may respond to any number of abiotic, biotic, and spatial drivers found in heterogeneous environments. Here, we examine potential drivers of prokaryotic and fungal communities in soils across the heterogenous landscape of the imperiled Florida scrub, a pyrogenic ecosystem where slight differences in elevation lead to large changes in water and nutrient availability and vegetation composition. We employ a comprehensive, large-scale sampling design to characterize the communities of prokaryotes and fungi associated with three habitat types and two soil depths (crust and subterranean) to evaluate (i) differences in microbial communities across these heterogeneous habitats, (ii) the relative roles of abiotic, biotic, and spatial drivers in shaping community structure, and (iii) the distribution of fungal guilds across these habitats. We sequenced soils from 40 complete replicates of habitat × soil depth combinations and sequenced the prokaryotic 16S and fungal internal transcribed spacer (ITS) regions using Illumina MiSeq. Habitat heterogeneity generated distinct communities of soil prokaryotes and fungi. Spatial distance played a role in structuring crust communities, whereas subterranean microbial communities were primarily structured by the shrub community, whose roots they presumably interacted with. This result helps to explain the unexpected transition we observed between arbuscular mycorrhiza-dominated soils at low-elevation habitats to ectomycorrhiza-dominated soils at high-elevation habitats. Our results challenge previous notions of environmental determinism of microbial communities and generate new hypotheses regarding symbiotic relationships across heterogeneous environments.

Allelopathy-selected microbiomes mitigate chemical inhibition of plant performance.

Allelopathy is a common and important stressor that shapes plant communities and can alter soil microbiomes, yet little is known about the direct effects of allelochemical addition on bacterial and fungal communities or the potential for allelochemical-selected microbiomes to mediate plant performance responses, especially in habitats naturally structured by allelopathy. Here, we present the first community-wide investigation of microbial mediation of allelochemical effects on plant performance by testing how allelopathy affects soil microbiome structure and how these microbial changes impact germination and productivity across 13 plant species. The soil microbiome exhibited significant changes to 'core' bacterial and fungal taxa, bacterial composition, abundance of functionally important bacterial and fungal taxa, and predicted bacterial functional genes after the addition of the dominant allelochemical native to this habitat. Furthermore, plant performance was mediated by the allelochemical-selected microbiome, with allelopathic inhibition of plant productivity moderately mitigated by the microbiome. Through our findings, we present a potential framework to understand the strength of plant-microbial interactions in the presence of environmental stressors, in which frequency of the ecological stress may be a key predictor of microbiome-mediation strength.

Multidimensional specialization and generalization are pervasive in soil prokaryotes.

Habitat specialization underpins biological processes from species distributions to speciation. However, organisms are often described as specialists or generalists based on a single niche axis, despite facing complex, multidimensional environments. Here, we analysed 236 environmental soil microbiomes across the United States and demonstrate that 90% of >1,200 prokaryotes followed one of two trajectories: specialization on all niche axes (multidimensional specialization) or generalization on all axes (multidimensional generalization). We then documented that this pervasive multidimensional specialization/generalization had many ecological and evolutionary consequences. First, multidimensional specialization and generalization are highly conserved with very few transitions between these two trajectories. Second, multidimensional generalists dominated communities because they were 73 times more abundant than specialists. Lastly, multidimensional specialists played important roles in community structure with ~220% more connections in microbiome networks. These results indicate that multidimensional generalization and specialization are evolutionarily stable with multidimensional generalists supporting larger populations and multidimensional specialists playing important roles within communities, probably stemming from their overrepresentation among pollutant detoxifiers and nutrient cyclers. Taken together, we demonstrate that the vast majority of soil prokaryotes are restricted to one of two multidimensional niche trajectories, multidimensional specialization or multidimensional generalization, which then has far-reaching consequences for evolutionary transitions, microbial dominance and community roles.

Past microbial stress benefits tree resilience.

Soil microbiota from stressful environments provide an avenue for climate resilience.

Salinity legacy: Foliar microbiome's history affects mutualist-conferred salinity tolerance.

The rapid human-driven changes in the environment during the Anthropocene have placed extreme stress on many plants and animals. Beneficial interactions with microorganisms may be crucial for ameliorating these stressors and facilitating the ecosystem services host organisms provide. Foliar endophytes, microorganisms that reside within leaves, are found in essentially all plants and can provide important benefits (e.g., enhanced drought tolerance or resistance to herbivory). However, it remains unclear how important the legacy effects of the abiotic stressors that select on these microbiomes are for affecting the degree of stress amelioration provided to their hosts. To elucidate foliar endophytes' role in host-plant salt tolerance, especially if salinity experienced in the field selects for endophytes that are better suited to improve the salt tolerance of their hosts, we combined field collections of 90 endophyte communities from 30 sites across the coastal Everglades with a manipulative growth experiment assessing endophyte inoculation effects on host-plant performance. Specifically, we grew >350 red mangrove (Rhizophora mangle) seedlings in a factorial design that manipulated the salinity environment the seedlings experienced (freshwater vs. saltwater), the introduction of field-collected endophytes (live vs. sterilized inoculum), and the legacy of salinity stress experienced by these introduced endophytes, ranging from no salt stress (0 parts per thousand [ppt] salinity) to high salt stress (40 ppt) environments. We found that inoculation with field-collected endophytes significantly increased mangrove performance across almost all metrics examined (15%-20% increase on average), and these beneficial effects typically occurred when the endophytes were grown in saltwater. Importantly, our study revealed the novel result that endophyte-conferred salinity tolerance depended on microbiome salinity legacy in a key coastal foundation species. Salt-stressed mangroves inoculated with endophyte microbiomes from high-salinity environments performed, on average, as well as plants grown in low-stress freshwater, while endophytes from freshwater environments did not relieve host salinity stress. Given the increasing salinity stress imposed by sea level rise and the importance of foundation species like mangroves for ecosystem services, our results indicate that consideration of endophytic associations and their salinity legacy may be critical for the successful restoration and management of coastal habitats.

Microbiome-mediated response to pulse fire disturbance outweighs the effects of fire legacy on plant performance.

Fire plays a major role in structuring plant communities across the globe. Interactions with soil microbes impact plant fitness, scaling up to influence plant populations and distributions. Here we present the first factorial manipulation of both fire and soil microbiome presence to investigate their interactive effects on plant performance across a suite of plant species with varying life history traits. We conducted fully factorial experiments on 11 species from the Florida scrub ecosystem to test plant performance responses to soils with varying fire histories (36 soil sources), the presence/absence of a microbiome, and exposure to an experimental burn. Results revealed interactive 'pulse' effects between fire and the soil microbiome on plant performance. On average, post-fire soil microbiomes strongly reduced plant productivity compared to unburned or sterilized soils. Interestingly, longer-term fire 'legacy' effects had minor impacts on plant performance and were unrelated to soil microbiomes. While pulse fire effects on plant-microbiome interactions are short-term, they could have long-term consequences for plant communities by establishing differential microbiome-mediated priority effects during post-disturbance succession. The prominence of pulse fire effects on plant-microbe interactions has even greater import due to expected increases in fire disturbances resulting from anthropogenic climate change.

Microbiome-mediated effects of habitat fragmentation on native plant performance.

Habitat fragmentation is a leading cause of biodiversity and ecosystem function loss in the Anthropocene. Despite the importance of plant-microbiome interactions to ecosystem productivity, we have limited knowledge of how fragmentation affects microbiomes and even less knowledge of its consequences for microbial interactions with plants. Combining field surveys, microbiome sequencing, manipulative experiments, and random forest models, we investigated fragmentation legacy effects on soil microbiomes in imperiled pine rocklands, tested how compositional shifts across 14 fragmentation-altered soil microbiomes affected performance and resource allocation of three native plant species, and identified fragmentation-responding microbial families underpinning plant performance. Legacies of habitat fragmentation were associated with significant changes in microbial diversity and composition (across three of four community axes). Experiments showed plants often strongly benefited from the microbiome's presence, but fragmentation-associated changes in microbiome composition also significantly affected plant performance and resource allocation across all seven metrics examined. Finally, random forest models identified ten fungal and six bacterial families important for plant performance that changed significantly with fragmentation. Our findings not only support the existence of significant fragmentation effects on natural microbiomes, but also demonstrate for the first time that fragmentation-associated changes in microbiomes can have meaningful consequences for native plant performance and investment.

Multiple Mutualism Effects generate synergistic selection and strengthen fitness alignment in the interaction between legumes, rhizobia and mycorrhizal fungi.

Nearly all organisms participate in multiple mutualisms, and complementarity within these complex interactions can result in synergistic fitness effects. However, it remains largely untested how multiple mutualisms impact eco-evolutionary dynamics in interacting species. We tested how multiple microbial mutualists-N-fixing bacteria and mycorrrhizal fungi-affected selection and heritability of traits in their shared host plant (Medicago truncatula), as well as fitness alignment between partners. Our results demonstrate for the first time that multiple mutualisms synergistically affect the selection and heritability of host traits and enhance fitness alignment between mutualists. Specifically, we found interaction with multiple microbial symbionts doubled the strength of natural selection on a plant architectural trait, resulted in 2- to 3-fold higher heritability of plant reproductive success, and more than doubled fitness alignment between N-fixing bacteria and plants. These findings show synergism generated by multiple mutualisms extends to key components of microevolutionary change, emphasising the importance of multiple mutualism effects on evolutionary trajectories.

Environmental stress destabilizes microbial networks.

Environmental stress is increasing worldwide, yet we lack a clear picture of how stress disrupts the stability of microbial communities and the ecosystem services they provide. Here, we present the first evidence that naturally-occurring microbiomes display network properties characteristic of unstable communities when under persistent stress. By assessing changes in diversity and structure of soil microbiomes along 40 replicate stress gradients (elevation/water availability gradients) in the Florida scrub ecosystem, we show that: (1) prokaryotic and fungal diversity decline in high stress, and (2) two network properties of stable microbial communities-modularity and negative:positive cohesion-have a clear negative relationship with environmental stress, explaining 51-78% of their variation. Interestingly, pathogenic taxa/functional guilds decreased in relative abundance along the stress gradient, while oligotrophs and mutualists increased, suggesting that the shift in negative:positive cohesion could result from decreasing negative:positive biotic interactions consistent with the predictions of the Stress Gradient Hypothesis. Given the crucial role microbiomes play in ecosystem functions, our results suggest that, by limiting the compartmentalization of microbial associations and creating communities dominated by positive associations, increasing stress in the Anthropocene could destabilize microbiomes and undermine their ecosystem services.

Not that kind of tree: Assessing the potential for decision tree-based plant identification using trait databases.

PREMISE: Advancements in machine learning and the rise of accessible "big data" provide an important opportunity to improve trait-based plant identification. Here, we applied decision-tree induction to a subset of data from the TRY plant trait database to (1) assess the potential of decision trees for plant identification and (2) determine informative traits for distinguishing taxa. METHODS: Decision trees were induced using 16 vegetative and floral traits (689 species, 20 genera). We assessed how well the algorithm classified species from test data and pinpointed those traits that were important for identification across diverse taxa. RESULTS: The unpruned tree correctly placed 98% of the species in our data set into genera, indicating its promise for distinguishing among the species used to construct them. Furthermore, in the pruned tree, an average of 89% of the species from the test data sets were properly classified into their genera, demonstrating the flexibility of decision trees to also classify new species into genera within the tree. Closer inspection revealed that seven of the 16 traits were sufficient for the classification, and these traits yielded approximately two times more initial information gain than those not included. DISCUSSION: Our findings demonstrate the potential for tree-based machine learning and big data in distinguishing among taxa and determining which traits are important for plant identification.

Do plant-microbe interactions support the Stress Gradient Hypothesis?

The Stress Gradient Hypothesis (SGH), which predicts increasing ratios of facilitative:competitive interactions with increasing stress, has long been a guiding framework for conceptualizing plant-plant interactions. Recently, there has been a growing recognition of the roles of microbes in mitigating or exacerbating environmental stress for their plant hosts. As such, we might predict, based on the SGH, that beneficial microbial effects on plant performance should be positively associated with stress. Specifically, we hypothesized that support for the SGH would depend on the host plant's habitat specialization such that species that specialize in high stress habitats and thus likely coevolved with the resident microbes would exhibit stronger support for the SGH than non-specialist plant species. We further hypothesized that support for the SGH would vary with germination frequency, since boosting germination of low-frequency germinators is one effective means by which microbes can benefit plant species performance. Here, we explore whether plant-microbial interactions support the SGH using 12 plant species native to the Florida rosemary scrub. We conducted factorial experiments that manipulated the presence of microbes in nine soils collected along an elevational stress gradient, and recorded germination frequency and biomass. Microbes increased the germination frequency of four species, all of which had relatively low germination rates. Furthermore, we found support for the SGH in nearly one-half of the species examined, with soil microbes facilitating germination with increasing stress for 5 of the 12 species tested, and none of the species exhibiting the opposite trend. Support for the SGH was not predicted by either the plant hosts' habitat specialization or germination frequency. In contrast to germination, biomass results showed little support for the SGH, with four of 12 species refuting and one species supporting SGH predictions. Taken together, our study documents that interactions between the soil microbial community and plant species along a stress gradient can support the SGH, but emphasizes that these effects are life-history-stage dependent. This work also identifies a common mechanism (germination facilitation) by which microbes can benefit plant species in stressful habitats.

Tripartite mutualisms as models for understanding plant-microbial interactions.

All plants host diverse microbial assemblages that shape plant health, productivity, and function. While some microbial effects are attributable to particular symbionts, interactions among plant-associated microbes can nonadditively affect plant fitness and traits in ways that cannot be predicted from pairwise interactions. Recent research into tripartite plant-microbe mutualisms has provided crucial insight into this nonadditivity and the mechanisms underlying plant interactions with multiple microbes. Here, we discuss how interactions among microbial mutualists affect plant performance, highlight consequences of biotic and abiotic context-dependency for nonadditive outcomes, and summarize burgeoning efforts to determine the molecular bases of how plants regulate establishment, resource exchange, and maintenance of tripartite interactions. We conclude with four goals for future tripartite studies that will advance our overall understanding of complex plant-microbial interactions.

Fungal functional ecology: bringing a trait-based approach to plant-associated fungi.

Fungi play many essential roles in ecosystems. They facilitate plant access to nutrients and water, serve as decay agents that cycle carbon and nutrients through the soil, water and atmosphere, and are major regulators of macro-organismal populations. Although technological advances are improving the detection and identification of fungi, there still exist key gaps in our ecological knowledge of this kingdom, especially related to function. Trait-based approaches have been instrumental in strengthening our understanding of plant functional ecology and, as such, provide excellent models for deepening our understanding of fungal functional ecology in ways that complement insights gained from traditional and -omics-based techniques. In this review, we synthesize current knowledge of fungal functional ecology, taxonomy and systematics and introduce a novel database of fungal functional traits (FunFun ). FunFun is built to interface with other databases to explore and predict how fungal functional diversity varies by taxonomy, guild, and other evolutionary or ecological grouping variables. To highlight how a quantitative trait-based approach can provide new insights, we describe multiple targeted examples and end by suggesting next steps in the rapidly growing field of fungal functional ecology.

Soil Microbiomes Underlie Population Persistence of an Endangered Plant Species.

Microbiomes can dramatically alter individual plant performance, yet how these effects influence higher-order processes is not well resolved. In particular, little is known about how microbiome effects on individual plants alter plant population dynamics, a question critical to imperiled species conservation. Here we integrate bioassays, multidecadal demographic data, and integral projection modeling to determine how the presence of the natural soil microbiome underlies plant population dynamics. Simulations indicated that the presence of soil microbiomes boosted population growth rates (λ) of the endangered Hypericum cumulicola by 13% on average, the difference between population growth versus decline in 76% of patches. The greatest benefit (47% increase in λ) occurred in low-nutrient, high-elevation habitats, suggesting that the soil microbiome may help expand H. cumulicola's distribution to include these stressful habitats. Our results demonstrate that soil microbiomes can significantly affect plant population growth and persistence and support the incorporation of soil microbiomes into conservation planning.

Cooperation and coexpression: How coexpression networks shift in response to multiple mutualists.

A mechanistic understanding of community ecology requires tackling the nonadditive effects of multispecies interactions, a challenge that necessitates integration of ecological and molecular complexity-namely moving beyond pairwise ecological interaction studies and the "gene at a time" approach to mechanism. Here, we investigate the consequences of multispecies mutualisms for the structure and function of genomewide differential coexpression networks for the first time, using the tractable and ecologically important interaction between legume Medicago truncatula, rhizobia and mycorrhizal fungi. First, we found that genes whose expression is affected nonadditively by multiple mutualists are more highly connected in gene networks than expected by chance and had 94% greater network centrality than genes showing additive effects, suggesting that nonadditive genes may be key players in the widespread transcriptomic responses to multispecies symbioses. Second, multispecies mutualisms substantially changed coexpression network structure of 18 modules of host plant genes and 22 modules of the fungal symbionts' genes, indicating that third-party mutualists can cause significant rewiring of plant and fungal molecular networks. Third, we found that 60% of the coexpressed gene sets that explained variation in plant performance had coexpression structures that were altered by interactive effects of rhizobia and fungi. Finally, an "across-symbiosis" approach identified sets of plant and mycorrhizal genes whose coexpression structure was unique to the multiple mutualist context and suggested coupled responses across the plant-mycorrhizal interaction to rhizobial mutualists. Taken together, these results show multispecies mutualisms have substantial effects on the molecular interactions in host plants, microbes and across symbiotic boundaries.

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.

Microbial mitigation-exacerbation continuum: a novel framework for microbiome effects on hosts in the face of stress.

A key challenge to understanding microbiomes and their role in ecological processes is contextualizing their effects on host organisms, particularly when faced with environmental stress. One influential theory, the Stress Gradient Hypothesis, might predict that the frequency of positive interactions increases with stressful conditions such that microbial taxa would mitigate harmful effects on host performance. Yet, equally plausible is that microbial taxa could exacerbate these effects. Here, we introduce the Mitigation-Exacerbation Continuum as a novel framework to conceptualize microbial mediation of stress. We (1) use this continuum to quantify microbial mediation of stress for six plant species and (2) test the association between these continuum values and natural species' abundance. We factorially manipulated a common stress (allelopathy) and the presence of soil microbes to quantify microbial effects in benign and stressed environments for two critical early life-history metrics, seed germination and seedling biomass. Although we found evidence of both mitigation and exacerbation among the six species, exacerbation was more common. Across species, the degree of microbial-mediated effects on germination explained >80% of the variation of natural field abundances. Our results suggest a critical role of soil microbes in mediating plant stress responses, and a potential microbial mechanism underlying species abundance.

Symbioses with nitrogen-fixing bacteria: nodulation and phylogenetic data across legume genera.

How species interactions shape global biodiversity and influence diversification is a central - but also data-hungry - question in evolutionary ecology. Microbially based mutualisms are widespread and could cause diversification by ameliorating stress and thus allowing organisms to colonize and adapt to otherwise unsuitable habitats. Yet the role of these interactions in generating species diversity has received limited attention, especially across large taxonomic groups. In the massive angiosperm family Leguminosae, plants often associate with root-nodulating bacteria that ameliorate nutrient stress by fixing atmospheric nitrogen. These symbioses are ecologically-important interactions, influencing community assembly, diversity, and succession, contributing ~100-290 million tons of N annually to natural ecosystems, and enhancing growth of agronomically-important forage and crop plants worldwide. In recent work attempting to determine whether mutualism with N-fixing bacteria led to increased diversification across legumes, we were unable to definitively resolve the relationship between diversification and nodulation. We did, however, succeed in compiling a very large searchable, analysis-ready database of nodulation data for 749 legume genera (98% of Leguminosae genera; LPWG 2017), which, along with associated phylogenetic information, will provide a valuable resource for future work addressing this question and others. For each legume genus, we provide information about the species richness, frequency of nodulation, subfamily association, and topological correspondence with an additional data set of 100 phylogenetic trees curated for database compatibility. We found 386 legume genera were confirmed nodulators (i.e., all species examined for nodulation nodulated), 116 were non-nodulating, four were variable (i.e., containing both confirmed nodulators and confirmed non-nodulators), and 243 had not been examined for nodulation in published studies. Interestingly, data exploration revealed that nodulating legume genera are ~3 × more species-rich than non-nodulating genera, but we did not find evidence that this difference in diversity was due to differences in net diversification rate. Our metadata file describes in more detail the structure of these data that provide a foundational resource for future work as more nodulation data become available, and as greater phylogenetic resolution of this ca. 19,500-species family comes into focus. We release this data set under the Creative Commons 4.0 Attribution-ShareAlike License (https://creativecommons.org/licenses/by-sa/4.0/). The data may be used, distributed, and reproduced with proper citation of this article.

Multiple mutualist effects on genomewide expression in the tripartite association between Medicago truncatula, nitrogen-fixing bacteria and mycorrhizal fungi.

While all species interact with multiple mutualists, the fitness consequences and molecular mechanisms underlying these interactions remain largely unknown. We combined factorial ecological experiments with genomewide expression analyses to examine the phenotypic and transcriptomic responses of model legume Medicago truncatula to rhizobia and mycorrhizal fungi. We found synergistic effects of these mutualists on plant performance and examined unique features of plant gene expression responses to multiple mutualists. There were genomewide signatures of mutualists and multiple mutualists on expression, with partners often affecting unique sets of genes. Mycorrhizal fungi had stronger effects on plant expression than rhizobia, with 70% of differentially expressed genes affected by fungi. Fungal and bacterial mutualists had joint effects on 10% of differentially expressed genes, including unexpected, nonadditive effects on some genes with important functions such as nutrient metabolism. For a subset of genes, interacting with multiple mutualists even led to reversals in the direction of expression (shifts from up to downregulation) compared to interacting with single mutualists. Rhizobia also affected the expression of several mycorrhizal genes, including those involved in nutrient transfer to host plants, indicating that partner species can also impact each other's molecular phenotypes. Collectively, these data illustrate the diverse molecular mechanisms and transcriptional responses associated with the synergistic benefits of multiple mutualists.