Mutualisms drive plant trait evolution beyond interaction-related traits.

Mutualisms have driven the evolution of extraordinary structures and behavioural traits, but their impact on traits beyond those directly involved in the interaction remains unclear. We addressed this gap using a highly evolutionarily replicated system - epiphytes in the Rubiaceae forming symbioses with ants. We employed models that allow us to test the influence of discrete mutualistic traits on continuous non-mutualistic traits. Our findings are consistent with mutualism shaping the pace of morphological evolution, strength of selection and long-term mean of non-mutualistic traits in function of mutualistic dependency. While specialised and obligate mutualisms are associated with slower trait change, less intimate, facultative and generalist mutualistic interactions - which are the most common - have a greater impact on non-mutualistic trait evolution. These results challenge the prevailing notion that mutualisms solely affect the evolution of interaction-related traits via stabilizing selection and instead demonstrate a broader role for mutualisms in shaping trait evolution.

Metagenomic analysis of ecological niche overlap and community collapse in microbiome dynamics.

Species utilizing the same resources often fail to coexist for extended periods of time. Such competitive exclusion mechanisms potentially underly microbiome dynamics, causing breakdowns of communities composed of species with similar genetic backgrounds of resource utilization. Although genes responsible for competitive exclusion among a small number of species have been investigated in pioneering studies, it remains a major challenge to integrate genomics and ecology for understanding stable coexistence in species-rich communities. Here, we examine whether community-scale analyses of functional gene redundancy can provide a useful platform for interpreting and predicting collapse of bacterial communities. Through 110-day time-series of experimental microbiome dynamics, we analyzed the metagenome-assembled genomes of co-occurring bacterial species. We then inferred ecological niche space based on the multivariate analysis of the genome compositions. The analysis allowed us to evaluate potential shifts in the level of niche overlap between species through time. We hypothesized that community-scale pressure of competitive exclusion could be evaluated by quantifying overlap of genetically determined resource-use profiles (metabolic pathway profiles) among coexisting species. We found that the degree of community compositional changes observed in the experimental microbiome was correlated with the magnitude of gene-repertoire overlaps among bacterial species, although the causation between the two variables deserves future extensive research. The metagenome-based analysis of genetic potential for competitive exclusion will help us forecast major events in microbiome dynamics such as sudden community collapse (i.e., dysbiosis).

Mycorrhizal mycelium as a global carbon pool.

For more than 400 million years, mycorrhizal fungi and plants have formed partnerships that are crucial to the emergence and functioning of global ecosystems. The importance of these symbiotic fungi for plant nutrition is well established. However, the role of mycorrhizal fungi in transporting carbon into soil systems on a global scale remains under-explored. This is surprising given that ∼75% of terrestrial carbon is stored belowground and mycorrhizal fungi are stationed at a key entry point of carbon into soil food webs. Here, we analyze nearly 200 datasets to provide the first global quantitative estimates of carbon allocation from plants to the mycelium of mycorrhizal fungi. We estimate that global plant communities allocate 3.93 Gt CO2e per year to arbuscular mycorrhizal fungi, 9.07 Gt CO2e per year to ectomycorrhizal fungi, and 0.12 Gt CO2e per year to ericoid mycorrhizal fungi. Based on this estimate, 13.12 Gt of CO2e fixed by terrestrial plants is, at least temporarily, allocated to the underground mycelium of mycorrhizal fungi per year, equating to ∼36% of current annual CO2 emissions from fossil fuels. We explore the mechanisms by which mycorrhizal fungi affect soil carbon pools and identify approaches to increase our understanding of global carbon fluxes via plant-fungal pathways. Our estimates, although based on the best available evidence, are imperfect and should be interpreted with caution. Nonetheless, our estimations are conservative, and we argue that this work confirms the significant contribution made by mycorrhizal associations to global carbon dynamics. Our findings should motivate their inclusion both within global climate and carbon cycling models, and within conservation policy and practice.

Exploring the dynamics of vascular adaptation.

A combination of in toto imaging and theory suggests a new mechanism for the remodeling of veins in vascular networks.

Symbioses shape feeding niches and diversification across insects.

For over 300 million years, insects have relied on symbiotic microbes for nutrition and defence. However, it is unclear whether specific ecological conditions have repeatedly favoured the evolution of symbioses, and how this has influenced insect diversification. Here, using data on 1,850 microbe-insect symbioses across 402 insect families, we found that symbionts have allowed insects to specialize on a range of nutrient-imbalanced diets, including phloem, blood and wood. Across diets, the only limiting nutrient consistently associated with the evolution of obligate symbiosis was B vitamins. The shift to new diets, facilitated by symbionts, had mixed consequences for insect diversification. In some cases, such as herbivory, it resulted in spectacular species proliferation. In other niches, such as strict blood feeding, diversification has been severely constrained. Symbioses therefore appear to solve widespread nutrient deficiencies for insects, but the consequences for insect diversification depend on the feeding niche that is invaded.

Facilitative interaction networks in experimental microbial community dynamics.

Facilitative interactions between microbial species are ubiquitous in various types of ecosystems on the Earth. Therefore, inferring how entangled webs of interspecific interactions shift through time in microbial ecosystems is an essential step for understanding ecological processes driving microbiome dynamics. By compiling shotgun metagenomic sequencing data of an experimental microbial community, we examined how the architectural features of facilitative interaction networks could change through time. A metabolic modeling approach for estimating dependence between microbial genomes (species) allowed us to infer the network structure of potential facilitative interactions at 13 time points through the 110-day monitoring of experimental microbiomes. We then found that positive feedback loops, which were theoretically predicted to promote cascade breakdown of ecological communities, existed within the inferred networks of metabolic interactions prior to the drastic community-compositional shift observed in the microbiome time-series. We further applied "directed-graph" analyses to pinpoint potential keystone species located at the "upper stream" positions of such feedback loops. These analyses on facilitative interactions will help us understand key mechanisms causing catastrophic shifts in microbial community structure.

Alternative stable states, nonlinear behavior, and predictability of microbiome dynamics.

BACKGROUND: Microbiome dynamics are both crucial indicators and potential drivers of human health, agricultural output, and industrial bio-applications. However, predicting microbiome dynamics is notoriously difficult because communities often show abrupt structural changes, such as "dysbiosis" in human microbiomes. METHODS: We integrated theoretical frameworks and empirical analyses with the aim of anticipating drastic shifts of microbial communities. We monitored 48 experimental microbiomes for 110 days and observed that various community-level events, including collapse and gradual compositional changes, occurred according to a defined set of environmental conditions. We analyzed the time-series data based on statistical physics and non-linear mechanics to describe the characteristics of the microbiome dynamics and to examine the predictability of major shifts in microbial community structure. RESULTS: We confirmed that the abrupt community changes observed through the time-series could be described as shifts between "alternative stable states" or dynamics around complex attractors. Furthermore, collapses of microbiome structure were successfully anticipated by means of the diagnostic threshold defined with the "energy landscape" analysis of statistical physics or that of a stability index of nonlinear mechanics. CONCLUSIONS: The results indicate that abrupt microbiome events in complex microbial communities can be forecasted by extending classic ecological concepts to the scale of species-rich microbial systems. Video Abstract.

The evolution of plant cultivation by ants.

Outside humans, true agriculture was previously thought to be restricted to social insects farming fungus. However, obligate farming of plants by ants was recently discovered in Fiji, prompting a re-examination of plant cultivation by ants. Here, we generate a database of plant cultivation by ants, identify three main types, and show that these interactions evolved primarily for shelter rather than food. We find that plant cultivation evolved at least 65 times independently for crops (~200 plant species), and 15 times in farmer lineages (~37 ant taxa) in the Neotropics and Asia/Australasia. Because of their high evolutionary replication, and variation in partner dependence, these systems are powerful models to unveil the steps in the evolution and ecology of insect agriculture.

Evolution of manipulative microbial behaviors in the rhizosphere.

The rhizosphere has been called "one of the most complex ecosystems on earth" because it is a hotspot for interactions among millions of microbial cells. Many of these are microbes are also participating in a dynamic interplay with host plant tissues, signaling pathways, and metabolites. Historically, breeders have employed a plant-centric perspective when trying to harness the potential of microbiome-derived benefits to improve productivity and resilience of economically important plants. This is potentially problematic because: (i) the evolution of the microbes themselves is often ignored, and (ii) it assumes that the fitness of interacting plants and microbes is strictly aligned. In contrast, a microbe-centric perspective recognizes that putatively beneficial microbes are still under selection to increase their own fitness, even if there are costs to the host. This can lead to the evolution of sophisticated, potentially subtle, ways for microbes to manipulate the phenotype of their hosts, as well as other microbes in the rhizosphere. We illustrate this idea with a review of cases where rhizosphere microbes have been demonstrated to directly manipulate host root growth, architecture and exudation, host nutrient uptake systems, and host immunity and defense. We also discuss indirect effects, whereby fitness outcomes for the plant are a consequence of ecological interactions between rhizosphere microbes. If these consequences are positive for the plant, they can potentially be misconstrued as traits that have evolved to promote host growth, even if they are a result of selection for unrelated functions. The ubiquity of both direct microbial manipulation of hosts and context-dependent, variable indirect effects leads us to argue that an evolutionary perspective on rhizosphere microbial ecology will become increasingly important as we continue to engineer microbial communities for crop production.

Rewilding plant microbiomes.

Microbiota of crop ancestors may offer a way to enhance sustainable food production.

Decreasing relatedness among mycorrhizal fungi in a shared plant network increases fungal network size but not plant benefit.

Theory suggests that relatives will cooperate more, and compete less, because of an increased benefit for shared genes. In symbiotic partnerships, hosts may benefit from interacting with highly related symbionts because there is less conflict among the symbionts. This has been difficult to test empirically. We used the arbuscular mycorrhizal symbiosis to study the effects of fungal relatedness on host and fungal benefits, creating fungal networks varying in relatedness between two hosts, both in soil and in-vitro. To determine how fungal relatedness affected overall transfer of nutrients, we fluorescently tagged phosphorus and quantified resource distribution between two root systems. We found that colonization by less-related fungi was associated with increased fungal growth, lower transport of nutrients across the network, and lower plant benefit - likely an outcome of increased fungal competition. More generally, we demonstrate how symbiont relatedness can mediate benefits of symbioses.

Fluorescent nanoparticles as tools in ecology and physiology.

Fluorescent nanoparticles (FNPs) have been widely used in chemistry and medicine for decades, but their employment in biology is relatively recent. Past reviews on FNPs have focused on chemical, physical or medical uses, making the extrapolation to biological applications difficult. In biology, FNPs have largely been used for biosensing and molecular tracking. However, concerns over toxicity in early types of FNPs, such as cadmium-containing quantum dots (QDs), may have prevented wide adoption. Recent developments, especially in non-Cd-containing FNPs, have alleviated toxicity problems, facilitating the use of FNPs for addressing ecological, physiological and molecule-level processes in biological research. Standardised protocols from synthesis to application and interdisciplinary approaches are critical for establishing FNPs in the biologists' tool kit. Here, we present an introduction to FNPs, summarise their use in biological applications, and discuss technical issues such as data reliability and biocompatibility. We assess whether biological research can benefit from FNPs and suggest ways in which FNPs can be applied to answer questions in biology. We conclude that FNPs have a great potential for studying various biological processes, especially tracking, sensing and imaging in physiology and ecology.

Temporal tracking of quantum-dot apatite across in vitro mycorrhizal networks shows how host demand can influence fungal nutrient transfer strategies.

Arbuscular mycorrhizal fungi function as conduits for underground nutrient transport. While the fungal partner is dependent on the plant host for its carbon (C) needs, the amount of nutrients that the fungus allocates to hosts can vary with context. Because fungal allocation patterns to hosts can change over time, they have historically been difficult to quantify accurately. We developed a technique to tag rock phosphorus (P) apatite with fluorescent quantum-dot (QD) nanoparticles of three different colors, allowing us to study nutrient transfer in an in vitro fungal network formed between two host roots of different ages and different P demands over a 3-week period. Using confocal microscopy and raster image correlation spectroscopy, we could distinguish between P transfer from the hyphae to the roots and P retention in the hyphae. By tracking QD-apatite from its point of origin, we found that the P demands of the younger root influenced both: (1) how the fungus distributed nutrients among different root hosts and (2) the storage patterns in the fungus itself. Our work highlights that fungal trade strategies are highly dynamic over time to local conditions, and stresses the need for precise measurements of symbiotic nutrient transfer across both space and time.

Mycorrhizal fungi control phosphorus value in trade symbiosis with host roots when exposed to abrupt 'crashes' and 'booms' of resource availability.

Biological market theory provides a conceptual framework to analyse trade strategies in symbiotic partnerships. A key prediction of biological market theory is that individuals can influence resource value - meaning the amount a partner is willing to pay for it - by mediating where and when it is traded. The arbuscular mycorrhizal symbiosis, characterised by roots and fungi trading phosphorus and carbon, shows many features of a biological market. However, it is unknown if or how fungi can control phosphorus value when exposed to abrupt changes in their trade environment. We mimicked an economic 'crash', manually severing part of the fungal network (Rhizophagus irregularis) to restrict resource access, and an economic 'boom' through phosphorus additions. We quantified trading strategies over a 3-wk period using a recently developed technique that allowed us to tag rock phosphate with fluorescing quantum dots of three different colours. We found that the fungus: compensated for resource loss in the 'crash' treatment by transferring phosphorus from alternative pools closer to the host root (Daucus carota); and stored the surplus nutrients in the 'boom' treatment until root demand increased. By mediating from where, when and how much phosphorus was transferred to the host, the fungus successfully controlled resource value.

Compartmentalization drives the evolution of symbiotic cooperation.

Across the tree of life, hosts have evolved mechanisms to control and mediate interactions with symbiotic partners. We suggest that the evolution of physical structures that allow hosts to spatially separate symbionts, termed compartmentalization, is a common mechanism used by hosts. Such compartmentalization allows hosts to: (i) isolate symbionts and control their reproduction; (ii) reward cooperative symbionts and punish or stop interactions with non-cooperative symbionts; and (iii) reduce direct conflict among different symbionts strains in a single host. Compartmentalization has allowed hosts to increase the benefits that they obtain from symbiotic partners across a diversity of interactions, including legumes and rhizobia, plants and fungi, squid and Vibrio, insects and nutrient provisioning bacteria, plants and insects, and the human microbiome. In cases where compartmentalization has not evolved, we ask why not. We argue that when partners interact in a competitive hierarchy, or when hosts engage in partnerships which are less costly, compartmentalization is less likely to evolve. We conclude that compartmentalization is key to understanding the evolution of symbiotic cooperation. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Tetranychus evansi spider mite populations suppress tomato defenses to varying degrees.

Plant defense suppression is an offensive strategy of herbivores, in which they manipulate plant physiological processes to increase their performance. Paradoxically, defense suppression does not always benefit the defense-suppressing herbivores, because lowered plant defenses can also enhance the performance of competing herbivores and can expose herbivores to increased predation. Suppression of plant defense may therefore entail considerable ecological costs depending on the presence of competitors and natural enemies in a community. Hence, we hypothesize that the optimal magnitude of suppression differs among locations. To investigate this, we studied defense suppression across populations of Tetranychus evansi spider mites, a herbivore from South America that is an invasive pest of solanaceous plants including cultivated tomato, Solanum lycopersicum, in other parts of the world. We measured the level of expression of defense marker genes in tomato plants after infestation with mites from eleven different T. evansi populations. These populations were chosen across a range of native (South American) and non-native (other continents) environments and from different host plant species. We found significant variation at three out of four defense marker genes, demonstrating that T. evansi populations suppress jasmonic acid- and salicylic acid-dependent plant signaling pathways to varying degrees. While we found no indication that this variation in defense suppression was explained by differences in host plant species, invasive populations tended to suppress plant defense to a smaller extent than native populations. This may reflect either the genetic lineage of T. evansi-as all invasive populations we studied belong to one linage and both native populations to another-or the absence of specialized natural enemies in invasive T. evansi populations.

Parallel Evolution in the Integration of a Co-obligate Aphid Symbiosis.

Insects evolve dependence-often extreme-on microbes for nutrition. This includes cases in which insects harbor multiple endosymbionts that function collectively as a metabolic unit [1-5]. How do these dependences originate [6], and is there a predictable sequence of events leading to the integration of new symbionts? While co-obligate symbioses, in which hosts rely on multiple nutrient-provisioning symbionts, have evolved numerous times across sap-feeding insects, there is only one known case in aphids, involving Buchnera aphidicola and Serratia symbiotica in the Lachninae subfamily [7-9]. Here, we identify three additional independent transitions to the same co-obligate symbiosis in different aphids. Comparing recent and ancient associations allow us to investigate intermediate stages of metabolic and anatomical integration of Serratia. We find that these uniquely replicated evolutionary events support the idea that co-obligate associations initiate in a predictable manner-through parallel evolutionary processes. Specifically, we show how the repeated losses of the riboflavin and peptidoglycan pathways in Buchnera lead to dependence on Serratia. We then provide evidence of a stepwise process of symbiont integration, whereby dependence evolves first. Then, essential amino acid pathways are lost (at ∼30-60 mya), which coincides with the increased anatomical integration of the companion symbiont. Finally, we demonstrate that dependence can evolve ahead of specialized structures (e.g., bacteriocytes), and in one case with no direct nutritional basis. More generally, our results suggest the energetic costs of synthesizing nutrients may provide a unified explanation for the sequence of gene losses that occur during the evolution of co-obligate symbiosis.

Tradeoffs in the evolution of plant farming by ants.

Diverse forms of cultivation have evolved across the tree of life. Efficient farming requires that the farmer deciphers and actively promotes conditions that increase crop yield. For plant cultivation, this can include evaluating tradeoffs among light, nutrients, and protection against herbivores. It is not understood if, or how, nonhuman farmers evaluate local conditions to increase payoffs. Here, we address this question using an obligate farming mutualism between the ant Philidris nagasau and epiphytic plants in the genus Squamellaria that are cultivated for their nesting sites and floral rewards. We focused on the ants' active fertilization of their crops and their protection against herbivory. We found that ants benefited from cultivating plants in full sun, receiving 7.5-fold more floral food rewards compared to shade-cultivated plants. The higher reward levels correlated with higher levels of crop protection provided by the ants. However, while high-light planting yielded the greatest immediate food rewards, sun-grown crops contained less nitrogen compared to shade-grown crops. This was due to lower nitrogen input from ants feeding on floral rewards instead of insect protein gained from predation. Despite this tradeoff, farming ants optimize crop yield by selectively planting their crops in full sun. Ancestral state reconstructions across this ant-plant clade show that a full-sun farming strategy has existed for millions of years, suggesting that nonhuman farmers have evolved the means to evaluate and balance conflicting crop needs to their own benefit.

Mycorrhizal Fungi Respond to Resource Inequality by Moving Phosphorus from Rich to Poor Patches across Networks.

The world's ecosystems are characterized by an unequal distribution of resources [1]. Trade partnerships between organisms of different species-mutualisms-can help individuals cope with such resource inequality [2-4]. Trade allows individuals to exchange commodities they can provide at low cost for resources that are otherwise impossible or more difficult to access [5, 6]. However, as resources become increasingly patchy in time or space, it is unknown how organisms alter their trading strategies [7, 8]. Here, we show how a symbiotic fungus mediates trade with a host root in response to different levels of resource inequality across its network. We developed a quantum-dot-tracking technique to quantify phosphorus-trading strategies of arbuscular mycorrhizal fungi simultaneously exposed to rich and poor resource patches. By following fluorescent nanoparticles of different colors across fungal networks, we determined where phosphorus was hoarded, relocated, and transferred to plant hosts. We found that increasing exposure to inequality stimulated trade. Fungi responded to high resource variation by (1) increasing the total amount of phosphorus distributed to host roots, (2) decreasing allocation to storage, and (3) differentially moving resources within the network from rich to poor patches. Using single-particle tracking and high-resolution video, we show how dynamic resource movement may help the fungus capitalize on value differences across the trade network, physically moving resources to areas of high demand to gain better returns. Such translocation strategies can help symbiotic organisms cope with exposure to resource inequality.