Aboveground herbivores drive stronger plant species-specific feedback than belowground fungi to regulate tree community assembly.

Ectomycorrhizal (EcM) tree species often become more dominant than arbuscular mycorrhizal (AM) tree species in temperate forests, but they generally coexist. Theory predicts that ecological feedback mediated by aboveground herbivory and/or belowground microbes could explain these dominance/coexistence patterns. An experimental test of how aboveground/belowground organisms associated with AM/EcM trees mediate ecological feedbacks has been lacking at the community-level. By establishing AM and EcM tree sapling assemblages in mesocosms and then introducing seedlings of each type in a reciprocal planting experiment, we compared seedling performance under varying sapling species (conspecifics, heterospecifics within the same and different mycorrhizal types), using traits that reflect either aboveground herbivory-mediated feedback or belowground fungal-mediated feedback or both. When examining seedling traits that reflect aboveground herbivory-mediated feedbacks (i.e., foliar damage), AM plants tended to experience less foliar damage and EcM plants more damage under conspecific versus heterospecific saplings within the same mycorrhizal types, and aboveground herbivory-mediated feedback was species-specific rather than mycorrhizal type-specific. Conversely, when examining traits that reflect belowground fungal-mediated feedbacks, both AM and EcM plant species often exhibited mycorrhizal type-specific feedbacks (e.g., greater aboveground biomass under the same versus different mycorrhizal-type saplings) rather than species-specific feedbacks. Furthermore, tree species affected by herbivory-mediated feedback were less affected by belowground feedback, indicating that the relative importance of the feedbacks varied among plant species. Analysis of plant-associated organisms verified that the feedback outcomes corresponded with species accumulation of belowground fungi (but not of aboveground herbivores). Thus, aboveground herbivores drive stronger plant species-specific feedback than belowground fungi to regulate temperate tree diversity.

Timing of evolutionary innovation: scenarios of evolutionary diversification in a species-rich fungal clade, Boletales.

Acquisition of mutualistic symbiosis could provide hosts and/or symbionts with novel ecological opportunities for evolutionary diversification. Such a mechanism is one of the major components of coevolutionary diversification. However, whether the origin of mycorrhizal symbiosis promotes diversification in fungi still requires clarification. Here, we aimed to reveal evolutionary diversification in a clade comprising ectomycorrhizal (ECM) fungi. Based on a phylogenic tree inferred from the sequences of 87 single-copy genes, we reconstructed the origins of ECM symbiosis in a species-rich basidiomycetous order, Boletales. High-resolution phylogeny of Boletales revealed that ECM symbiosis independently evolved from non-ECM states at least four times in the group. Among them, only the second most recent event, occurring in the clade of Boletaceae, was inferred to involve an almost synchronous rapid diversification and rapid transition from non-ECM to ECM symbiosis. Our results contradict the hypothesis of evolutionary priority effect, which postulates the greatest ecological opportunities in the oldest lineages. Therefore, the novel resources that had not been pre-empted by the old ECM fungal lineages - supposedly the coevolving angiosperm hosts - could be available for the young ECM fungal lineages, which resulted in evolutionary diversification occurring only in the young ECM fungal lineages.

Structural diversity across arbuscular mycorrhizal, ectomycorrhizal, and endophytic plant-fungus networks.

BACKGROUND: Below-ground linkage between plant and fungal communities is one of the major drivers of terrestrial ecosystem dynamics. However, we still have limited knowledge of how such plant-fungus associations vary in their community-scale properties depending on fungal functional groups and geographic locations. METHODS: By compiling a high-throughput sequencing dataset of root-associated fungi in eight forests along the Japanese Archipelago, we performed a comparative analysis of arbuscular mycorrhizal, ectomycorrhizal, and saprotrophic/endophytic associations across a latitudinal gradient from cool-temperate to subtropical regions. RESULTS: In most of the plant-fungus networks analyzed, host-symbiont associations were significantly specialized but lacked "nested" architecture, which has been commonly reported in plant-pollinator and plant-seed disperser networks. In particular, the entire networks involving all functional groups of plants and fungi and partial networks consisting of ectomycorrhizal plant and fungal species/taxa displayed "anti-nested" architecture (i.e., negative nestedness scores) in many of the forests examined. Our data also suggested that geographic factors affected the organization of plant-fungus network structure. For example, the southernmost subtropical site analyzed in this study displayed lower network-level specificity of host-symbiont associations and higher (but still low) nestedness than northern localities. CONCLUSIONS: Our comparative analyses suggest that arbuscular mycorrhizal, ectomycorrhizal, and saprotrophic/endophytic plant-fungus associations often lack nested network architecture, while those associations can vary, to some extent, in their community-scale properties along a latitudinal gradient. Overall, this study provides a basis for future studies that will examine how different types of plant-fungus associations collectively structure terrestrial ecosystems.

Host shifts enhance diversification of ectomycorrhizal fungi: diversification rate analysis of the ectomycorrhizal fungal genera Strobilomyces and Afroboletus with an 80-gene phylogeny.

Mutualisms with new host lineages can provide symbionts with novel ecological opportunities to expand their geographical distribution, thereby leading to evolutionary diversification. Because ectomycorrhizal (ECM) fungi provide ideal opportunities to test the relationship between host shifts and diversification, we tested whether mutualism with new host lineages could increase the diversification rates of ECM fungi. Using a Bayesian tree inferred from 23 027-base nucleotide sequences of 80 single-copy genes, we tested whether the diversification rate had changed through host-shift events in the monophyletic clade containing the ECM fungal genera Strobilomyces and Afroboletus. The results indicated that these fungi were initially associated with Caesalpinioideae/Monotoideae in Africa, acquired associations with Dipterocarpoideae in tropical Asia, and then switched to Fagaceae/Pinaceae and Nothofagaceae/Eucalyptus. Fungal lineages associated with Fagaceae/Pinaceae were inferred to have approximately four-fold and two-fold greater diversification rates than those associated with Caesalpinioideae/Monotoideae and Dipterocarpoideae or Nothofagaceae/Eucalyptus, respectively. Moreover, the diversification rate shift was inferred to follow the host shift to Fagaceae/Pinaceae. Our study suggests that host-shift events, particularly those occurring with respect to Fagaceae/Pinaceae, can provide ecological opportunities for the rapid diversification of Strobilomyces-Afroboletus. Although further studies are needed for generalization, we propose a possible diversification scenario of ECM fungi.

Diet disparity among sympatric herbivorous cichlids in the same ecomorphs in Lake Tanganyika: amplicon pyrosequences on algal farms and stomach contents.

BACKGROUND: Lake Tanganyika, an ancient lake in the Great Rift Valley, is famous for the adaptive radiation of cichlids. Five tribes of the Cichlidae family have acquired herbivory, with five ecomorphs: grazers, browsers, scrapers, biters and scoopers. Sixteen species of the herbivorous cichlids coexist on a rocky littoral slope in the lake. Seven of them individually defend feeding territories against intruding herbivores to establish algal farms. We collected epiphyton from these territories at various depths and also gathered fish specimens. Algal and cyanobacteria community structures were analysed using the amplicon-metagenomic method. RESULTS: Based on 454-pyrosequencing of SSU rRNA gene sequences, we identified 300 phototrophic taxa, including 197 cyanobacteria, 57 bacillariophytes, and 31 chlorophytes. Algal farms differed significantly in their composition among cichlid species, even in the same ecomorph, due in part to their habitat-depth segregation. The algal species composition of the stomach contents and algal farms of each species differed, suggesting that cichlids selectively harvest their farms. The stomach contents were highly diverse, even between species in the same tribe, in the same feeding ecomorph. CONCLUSIONS: In this study, the amplicon-metagenomic approach revealed food niche separation based on habitat-depth segregation among coexisting herbivorous cichlids in the same ecomorphs in Lake Tanganyika.

Deterministic and stochastic processes generating alternative states of microbiomes.

The structure of microbiomes is often classified into discrete or semi-discrete types potentially differing in community-scale functional profiles. Elucidating the mechanisms that generate such "alternative states" of microbiome compositions has been one of the major challenges in ecology and microbiology. In a time-series analysis of experimental microbiomes, we here show that both deterministic and stochastic ecological processes drive divergence of alternative microbiome states. We introduced species-rich soil-derived microbiomes into eight types of culture media with 48 replicates, monitoring shifts in community compositions at six time points (8 media × 48 replicates × 6 time points = 2304 community samples). We then confirmed that microbial community structure diverged into a few state types in each of the eight medium conditions as predicted in the presence of both deterministic and stochastic community processes. In other words, microbiome structure was differentiated into a small number of reproducible compositions under the same environment. This fact indicates not only the presence of selective forces leading to specific equilibria of community-scale resource use but also the influence of demographic drift (fluctuations) on the microbiome assembly. A reference-genome-based analysis further suggested that the observed alternative states differed in ecosystem-level functions. These findings will help us examine how microbiome structure and functions can be controlled by changing the "stability landscapes" of ecological community compositions.

Soil prokaryotic and fungal biome structures associated with crop disease status across the Japan Archipelago.

Archaea, bacteria, and fungi in the soil are increasingly recognized as determinants of agricultural productivity and sustainability. A crucial step for exploring soil microbiomes with important ecosystem functions is to perform statistical analyses on the potential relationship between microbiome structure and functions based on comparisons of hundreds or thousands of environmental samples collected across broad geographic ranges. In this study, we integrated agricultural field metadata with microbial community analyses by targeting 2,903 bulk soil samples collected along a latitudinal gradient from cool-temperate to subtropical regions in Japan (26.1-42.8 °N). The data involving 632 archaeal, 26,868 bacterial, and 4,889 fungal operational taxonomic units detected across the fields of 19 crop plant species allowed us to conduct statistical analyses (permutational analyses of variance, generalized linear mixed models, randomization analyses, and network analyses) on the relationship among edaphic factors, microbiome compositions, and crop disease prevalence. We then examined whether the diverse microbes form species sets varying in potential ecological impacts on crop plants. A network analysis suggested that the observed prokaryotes and fungi were classified into several species sets (network modules), which differed substantially in association with crop disease prevalence. Within the network of microbe-to-microbe coexistence, ecologically diverse microbes, such as an ammonium-oxidizing archaeon, an antibiotics-producing bacterium, and a potentially mycoparasitic fungus, were inferred to play key roles in shifts between crop-disease-promotive and crop-disease-suppressive states of soil microbiomes. The bird's-eye view of soil microbiome structure will provide a basis for designing and managing agroecosystems with high disease-suppressive functions.IMPORTANCEUnderstanding how microbiome structure and functions are organized in soil ecosystems is one of the major challenges in both basic ecology and applied microbiology. Given the ongoing worldwide degradation of agroecosystems, building frameworks for exploring structural diversity and functional profiles of soil microbiomes is an essential task. Our study provides an overview of cropland microbiome states in light of potential crop-disease-suppressive functions. The large data set allowed us to explore highly functional species sets that may be stably managed in agroecosystems. Furthermore, an analysis of network architecture highlighted species that are potentially used to cause shifts from disease-prevalent states of agroecosystems to disease-suppressive states. By extending the approach of comparative analyses toward broader geographic ranges and diverse agricultural practices, agroecosystem with maximized biological functions will be further explored.

Interaction network rewiring and species' contributions to community-scale flexibility.

The architecture of species interaction networks is a key factor determining the stability of ecological communities. However, the fact that ecological network architecture can change through time is often overlooked in discussions on community-level processes, despite its theoretical importance. By compiling a time-series community dataset involving 50 spider species and 974 Hexapoda prey species/strains, we quantified the extent to which the architecture of predator-prey interaction networks could shift across time points. We then developed a framework for finding species that could increase the flexibility of the interaction network architecture. Those "network coordinator" species are expected to promote the persistence of species-rich ecological communities by buffering perturbations in communities. Although spiders are often considered as generalist predators, their contributions to network flexibility vary greatly among species. We also found that detritivorous prey species can be cores of interaction rewiring, dynamically interlinking below-ground and above-ground community dynamics. We further found that the predator-prey interactions between those network coordinators differed from those highlighted in the standard network-analytical framework assuming static topology. Analyses of network coordinators will add a new dimension to our understanding of species coexistence mechanisms and provide platforms for systematically prioritizing species in terms of their potential contributions in ecosystem conservation and restoration.

Leaf, root, and soil microbiomes of an invasive plant, Ardisia crenata, differ between its native and exotic ranges.

Introduction: Ecological underpinnings of the invasion success of exotic plants may be found in their interactions with microbes, either through the enemy release hypothesis and the enhanced mutualism hypothesis. Whereas recent high-throughput sequencing techniques have significantly expanded our understanding of plant-associated microbiomes and their functional guilds, few studies to date have used these techniques to compare the microbiome associated with invasive plants between their native and exotic ranges. Methods: We extracted fungal and bacterial DNA within leaf endosphere, root endosphere and soil of an invasive plant, Ardisia crenata, sampled from their native range Japan and exotic range Florida, USA. Using Illumina sequencing data, we compared microbial community compositions and diversity between the native and exotic ranges, and tested whether abundance of pathogenic or mutualistic microbes differ between the native or exotic ranges in accordance to the enemy release hypothesis or the enhanced mutualism hypothesis. Results: Fungal and bacterial community compositions differed among leaves, roots and soil, and between the native and exotic ranges. Despite a higher microbial diversity in the soil in the exotic range than in the native range, the microbial diversity within leaf and root was lower in the exotic range compared to the native range. In addition, leaves in the native range harbored a greater number of plant pathogenic fungi compared to those in the exotic range. Discussion: These patterns suggest plant controls over what microbes become associated with leaves and roots. The higher abundance of leaf pathogenic fungi, including the pathogen which is known to cause specific disease in A. crenata in the exotic range than in the native range, support the enemy release hypothesis and highlighted potential importance of examining microbial communities both above- and below-ground.

Dynamics of species-rich predator-prey networks and seasonal alternations of core species.

In nature, entangled webs of predator-prey interactions constitute the backbones of ecosystems. Uncovering the network architecture of such trophic interactions has been recognized as the essential step for exploring species with great impacts on ecosystem-level phenomena and functions. However, it has remained a major challenge to reveal how species-rich networks of predator-prey interactions are continually reshaped through time in the wild. Here, we show that dynamics of species-rich predator-prey interactions can be characterized by remarkable network structural changes and alternations of species with greatest impacts on community processes. On the basis of high-throughput detection of prey DNA from 1,556 spider individuals collected in a grassland ecosystem, we reconstructed dynamics of interaction networks involving, in total, 50 spider species and 974 prey species and strains through 8 months. The networks were compartmentalized into modules (groups) of closely interacting predators and prey in each month. Those modules differed in detritus/grazing food chain properties, forming complex fission-fusion dynamics of belowground and aboveground energy channels across the seasons. The substantial shifts of network structure entailed alternations of spider species located at the core positions within the entangled webs of interactions. These results indicate that knowledge of dynamically shifting food webs is crucial for understanding temporally varying roles of 'core species' in ecosystem processes.

Core species and interactions prominent in fish-associated microbiome dynamics.

BACKGROUND: In aquatic ecosystems, the health and performance of fish depend greatly on the dynamics of microbial community structure in the background environment. Nonetheless, finding microbes with profound impacts on fish's performance out of thousands of candidate species remains a major challenge. METHODS: We examined whether time-series analyses of microbial population dynamics could illuminate core components and structure of fish-associated microbiomes in the background (environmental) water. By targeting eel-aquaculture-tank microbiomes as model systems, we reconstructed the population dynamics of the 9605 bacterial and 303 archaeal species/strains across 128 days. RESULTS: Due to the remarkable increase/decrease of constituent microbial population densities, the taxonomic compositions of the microbiome changed drastically through time. We then found that some specific microbial taxa showed a positive relationship with eels' activity levels even after excluding confounding effects of environmental parameters (pH and dissolved oxygen level) on population dynamics. In particular, a vitamin-B12-producing bacteria, Cetobacterium somerae, consistently showed strong positive associations with eels' activity levels across the replicate time series of the five aquaculture tanks analyzed. Network theoretical and metabolic modeling analyses further suggested that the highlighted bacterium and some other closely-associated bacteria formed "core microbiomes" with potentially positive impacts on eels. CONCLUSIONS: Overall, these results suggest that the integration of microbiology, ecological theory, and network science allows us to explore core species and interactions embedded within complex dynamics of fish-associated microbiomes.  Video Abstract.

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).

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.

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.

Climatic gradients of arms race coevolution.

In nature, spatiotemporally dynamic coevolutionary processes play major roles in the foundation and maintenance of biodiversity. Here, we examined the arms race coevolution involving a seed-eating weevil with a long snout and its camellia plant host with a thick fruit coat (pericarp) throughout the marked climatic gradient observed across the Japanese islands. Results demonstrated that female weevils, which bored holes through camellia pericarps to lay eggs into seeds, had evolved much longer snouts than males, especially in areas in which Japanese camellia pericarps were very thick. The thickness of the plant pericarp was heritable, and the camellia plant evolved a significantly thicker pericarp on islands with the weevil than on islands without it. Across populations with weevils, resource allocation to plant defense increased with increasing annual mean temperature or annual precipitation, thereby geographically differentiating the evolutionary and ecological interactions between the two species. Given that the coevolutionary relationship exhibited appreciable variation across a relatively small range of annual mean temperatures, ongoing global climatic change can dramatically alter the coevolutionary process, thereby changing the ecological interaction between these species.

Diversity and infection prevalence of endosymbionts in natural populations of the chestnut weevil: relevance of local climate and host plants.

Many insects are ubiquitously associated with multiple endosymbionts, whose infection patterns often exhibit spatial and temporal variations. How such endosymbiont variations are relevant to local adaptation of the host organisms is of ecological interest. Here, we report a comprehensive survey of endosymbionts in natural populations of the chestnut weevil Curculio sikkimensis, whose larvae are notorious pests of cultivated chestnuts and also infest acorns of various wild oaks. From 968 insects representing 55 localities across the Japanese Archipelago and originating from 10 host plant species, we identified six distinct endosymbiont lineages, namely Curculioniphilus, Sodalis, Serratia, Wolbachia, Rickettsia and Spiroplasma, at different infection frequencies (96.7%, 12.8%, 82.3%, 82.5%, 28.2% and 6.8%, respectively) and with different geographical distribution patterns. Multiple endosymbiont infections were very common; 3.18±0.61 (ranging from 1.74 to 5.50) endosymbionts per insect on average in each of the local populations. Five pairs of endosymbionts (Curculioniphilus-Serratia, Curculioniphilus-Wolbachia, Sodalis-Rickettsia, Wolbachia-Rickettsia and Rickettsia-Spiroplasma) co-infected the same host individuals more frequently than expected, while infections with Serratia and Wolbachia were negatively correlated to each other. Infection frequencies of the endosymbionts were significantly correlated with climatic and ecological factors: for example, higher Sodalis, Wolbachia and Rickettsia infections at localities of higher temperature; lower Wolbachia and Rickettsia infections at localities of greater snowfall; and higher Curculioniphilus, Sodalis, Serratia, Wolbachia and Rickettsia infections on acorns than on chestnuts. These patterns are discussed in relation to potential host-endosymbiont co-evolution via local adaptation across geographical populations.

Arms race between weevil rostrum length and camellia pericarp thickness: Geographical cline and theory.

The geographical cline of the coevolving traits of weevil rostrum (mouthpart) length and camellia pericarp (fruit coat) thickness provides an opportunity to test the arms race theory of defense (pericarp thickness) and countermeasure (rostrum length) between antagonistically interacting species. By extending the previous model for the coevolution of quantitative traits to introduce nonlinear costs for exaggerated traits, the generation overlap, and density-dependent regulation in the host, we studied the evolutionarily stable (ES) pericarp thickness in the Japanese camellia (Camellia japonica) and the ES rostrum length in the camellia-weevil (Curculio camelliae). The joint monomorphic ES system has a robust outcome with nonlinear costs, and we analyzed how the traits of both species at evolutionary equilibrium depend on demographic parameters. If camellia demographic parameters vary latitudinally, data collected over the geographical scale of rostrum length and pericarp thickness should lie on an approximately linear curve with the slope less than that of the equiprobability line A/B of boring success, where A and B are coefficients for the logistic regression of boring success to pericarp thickness and rostrum length, respectively. This is a robust prediction as long as the cost of rostrum length is nonlinear (accelerating). As a result, boring success should be lower in populations with longer rostrum length, as reported in the weevil-camellia system (Toju, H., and Sota, T., 2006a. Imbalance of predator and prey armament: Geographic clines in phenotypic interface and natural selection. American Naturalist 167, 105-117). The nonlinearity (exponent) for the cost of rostrum length estimated from the geographical cline data for the weevil-camellia system was 2.2, suggesting nonlinearity between quadratic and cubic forms.

Synergistic and Offset Effects of Fungal Species Combinations on Plant Performance.

In natural and agricultural ecosystems, survival and growth of plants depend substantially on residing microbes in the endosphere and rhizosphere. Although numerous studies have reported the presence of plant-growth promoting bacteria and fungi in below-ground biomes, it remains a major challenge to understand how sets of microbial species positively or negatively affect plants' performance. By conducting a series of single- and dual-inoculation experiments of 13 plant-associated fungi targeting a Brassicaceae plant species (Brassica rapa var. perviridis), we here systematically evaluated how microbial effects on plants depend on presence/absence of co-occurring microbes. The comparison of single- and dual-inoculation experiments showed that combinations of the fungal isolates with the highest plant-growth promoting effects in single inoculations did not have highly positive impacts on plant performance traits (e.g., shoot dry weight). In contrast, pairs of fungi with small/moderate contributions to plant growth in single-inoculation contexts showed the greatest effects on plants among the 78 fungal pairs examined. These results on the offset and synergistic effects of pairs of microbes suggest that inoculation experiments of single microbial species/isolates can result in the overestimation or underestimation of microbial functions in multi-species contexts. Because keeping single-microbe systems under outdoor conditions is impractical, designing sets of microbes that can maximize performance of crop plants is an important step for the use of microbial functions in sustainable agriculture.

"Candidatus Curculioniphilus buchneri," a novel clade of bacterial endocellular symbionts from weevils of the genus Curculio.

Here we investigated the bacterial endosymbionts of weevils of the genus Curculio. From all four species of Curculio weevils examined, a novel group of bacterial gene sequences were consistently identified. Molecular phylogenetic analyses demonstrated that the sequences formed a distinct clade in the Gammaproteobacteria, which was not related to previously known groups of weevil endosymbionts such as Nardonella spp. and Sodalis-allied symbionts. In situ hybridization revealed that the bacterium was intracellularly harbored in a bacteriome associated with larval midgut. In adult females, the bacterium was localized in the germalia at the tip of each overiole, suggesting vertical transmission via ovarial passage. Diagnostic PCR surveys detected high prevalence of the bacterial infection in natural host populations. Electron microscopy identified the reduced cell wall of the bacterial cells, and the bacterial genes exhibited AT-biased nucleotide composition and accelerated molecular evolution, which are suggestive of a long-lasting endosymbiotic association. On the basis of these results, we conclude that the novel endocellular bacteria represent the primary symbiont of Curculio weevils and proposed the designation "Candidatus Curculioniphilus buchneri." In addition to "Ca. Curculioniphilus," we identified Sodalis-allied gammaproteobacterial endosymbionts from the chestnut weevil, Curculio sikkimensis, which exhibited partial infection frequencies in host insect populations and neither AT-biased nucleotide composition nor accelerated molecular evolution. We suggest that such Sodalis-allied secondary symbionts in weevils might provide a potential source for symbiont replacements, as has occurred in an ancestor of Sitophilus grain weevils.