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The order of species arrival can influence how species interact with one another and, consequently, which species may coexist in local communities. This phenomenon, called priority effects, has been observed in various types of communities, but it remains unclear whether priority effects persist over the long term spanning multiple generations of local communities in metacommunities. Focusing on bacteria and yeasts that colonize floral nectar of the sticky monkey flower, Mimulus aurantiacus, via hummingbirds and other flower-visiting animals, we experimentally manipulated initial microbial dominance on plants (regarded as metacommunities) to examine whether its effects persisted across multiple generations of flowers (regarded as local microbial habitats). The experimental introduction of Neokomagataea (= Gluconobacter) bacteria and Metschnikowia yeasts into wild flowers showed that the effects of initial dominance were observable across multiple floral generations. Three weeks after introduction, corresponding approximately to three floral generations, Neokomagataea introduction led to exclusion of yeasts, whereas Metschnikowia introduction did not result in the exclusion of Neokomagataea. Our results suggest that, even when local habitats are ephemeral, priority effects may influence multiple generations of local communities within metacommunities.

Abstract Background One of the major recent advances in evolutionary biology is the recognition that evolutionary interactions between species are substantially differentiated among geographic populations. To date, several authors have revealed natural selection pressures mediating the geographically-divergent processes of coevolution. How local, then, is the geographic structuring of natural selection in coevolutionary systems? Results I examined the spatial scale of a "geographic selection mosaic," focusing on a system involving a seed-predatory insect, the camellia weevil (Curculio camelliae), and its host plant, the Japanese camellia (Camellia japonica). In this system, female weevils excavate camellia fruits with their extremely-long mouthparts to lay eggs into seeds, while camellia seeds are protected by thick pericarps. Quantitative evaluation of natural selection demonstrated that thicker camellia pericarps are significantly favored in some, but not all, populations within a small island (Yakushima Island, Japan; diameter ca. 30 km). At the extreme, camellia populations separated by only several kilometers were subject to different selection pressures. Interestingly, in a population with the thickest pericarps, camellia individuals with intermediate pericarp thickness had relatively high fitness when the potential costs of producing thick pericarps were considered. Also importantly, some parameters of the weevil - camellia interaction such as the severity of seed infestation showed clines along temperature, suggesting the effects of climate on the fine-scale geographic differentiation of the coevolutionary processes. Conclusion These results show that natural selection can drive the geographic differentiation of interspecific interactions at surprisingly small spatial scales. Future studies should reveal the evolutionary/ecological outcomes of the "fine scale geographic mosaics" in biological communities.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions 1,2 . To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores 3 . The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms 4,5 ., (© 2024. The Author(s).)

Novel methods for sampling and characterizing biodiversity hold great promise for re-evaluating patterns of life across the planet. The sampling of airborne spores with a cyclone sampler, and the sequencing of their DNA, have been suggested as an efficient and well-calibrated tool for surveying fungal diversity across various environments. Here we present data originating from the Global Spore Sampling Project, comprising 2,768 samples collected during two years at 47 outdoor locations across the world. Each sample represents fungal DNA extracted from 24 m 3 of air. We applied a conservative bioinformatics pipeline that filtered out sequences that did not show strong evidence of representing a fungal species. The pipeline yielded 27,954 species-level operational taxonomic units (OTUs). Each OTU is accompanied by a probabilistic taxonomic classification, validated through comparison with expert evaluations. To examine the potential of the data for ecological analyses, we partitioned the variation in species distributions into spatial and seasonal components, showing a strong effect of the annual mean temperature on community composition., (© 2024. The Author(s).)

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., Competing Interests: H.T. is the founder and director of Sunlit Seedlings Ltd. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

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., Competing Interests: H.T. is the founder and director of Sunlit Seedlings Ltd. This had no impact on the design and implementation of the study. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Nakamura, Toju and Kitajima.)

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)., Competing Interests: HT is the founder and director of Sunlit Seedlings Ltd. This had no impact on the design and implementation of the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Fujita, Ushio, Suzuki, Abe, Yamamichi, Okazaki, Canarini, Hayashi, Fukushima, Fukuda, Kiers and Toju.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fujita, Ushio, Suzuki, Abe, Yamamichi, Okazaki, Canarini, Hayashi, Fukushima, Fukuda, Kiers and Toju.)

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., (© 2023. The Author(s).)

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-B 12 -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., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hori, Fujita, Hiruma, Narisawa and Toju.)

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.

Constructing biological communities is a major challenge in both basic and applied sciences. Although model synthetic communities with a few species have been constructed, designing systems consisting of tens or hundreds of species remains one of the most difficult goals in ecology and microbiology. By utilizing high-throughput sequencing data of interspecific association networks, we here propose a framework for exploring "functional core" species that have great impacts on whole community processes and functions. The framework allows us to score each species within a large community based on three criteria: namely, topological positions, functional portfolios, and functional balance within a target network. The criteria are measures of each species' roles in maximizing functional benefits at the community or ecosystem level. When species with potentially large contributions to ecosystem-level functions are screened, the framework also helps us design "functional core microbiomes" by focusing on properties of species groups (modules) within a network. When embedded into agroecosystems or human gut, such functional core microbiomes are expected to organize whole microbiome processes and functions. An application to a plant-associated microbiome dataset actually highlighted potential functional core microbes that were known to control rhizosphere microbiomes by suppressing pathogens. Meanwhile, an example of application in mouse gut microbiomes called attention to poorly investigated bacterial species, whose potential roles within gut microbiomes deserve future experimental studies. The framework for gaining "bird's-eye" views of functional cores within networks is applicable not only to agricultural and medical data but also to datasets produced in food processing, brewing, waste water purification, and biofuel production., (Copyright © 2020 Toju, Abe, Ishii, Hori, Fujita and Fukuda.)

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., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Cyst and root-knot nematodes are major risk factors of agroecosystem management, often causing devastating impacts on crop production. The use of microbes that parasitize or prey on nematodes has been considered as a promising approach for suppressing phytopathogenic nematode populations. However, effects and persistence of those biological control agents often vary substantially depending on regions, soil characteristics and agricultural practices: more insights into microbial community processes are required to develop reproducible control of nematode populations. By performing high-throughput sequencing profiling of bacteria and fungi, we examined how root and soil microbiomes differ between benign and nematode-infected plant individuals in a soybean field in Japan. Results indicated that various taxonomic groups of bacteria and fungi occurred preferentially on the soybean individuals infected by root-knot nematodes or those uninfected by nematodes. Based on a network analysis of potential microbe-microbe associations, we further found that several fungal taxa potentially preying on nematodes ( Dactylellina (Orbiliales), Rhizophydium (Rhizophydiales), Clonostachys (Hypocreales), Pochonia (Hypocreales) and Purpureocillium (Hypocreales)) co-occurred in the soybean rhizosphere at a small spatial scale. This study suggests how 'consortia' of anti-nematode microbes can derive from indigenous (resident) microbiomes, providing basic information for managing anti-nematode microbial communities in agroecosystems., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as conflict of interest.

In terrestrial ecosystems, plants interact with diverse taxonomic groups of bacteria and fungi in the phyllosphere and rhizosphere. Although recent studies based on high-throughput DNA sequencing have drastically increased our understanding of plant-associated microbiomes, we still have limited knowledge of how plant species in a species-rich community differ in their leaf and root microbiome compositions. In a cool-temperate semi-natural grassland in Japan, we compared leaf- and root-associated microbiomes across 137 plant species belonging to 33 plant orders. Based on the whole-microbiome inventory data, we analyzed how sampling season as well as the taxonomy, nativeness (native or alien), lifeform (herbaceous or woody), and mycorrhizal type of host plants could contribute to variation in microbiome compositions among co-occurring plant species. The data also allowed us to explore prokaryote and fungal lineages showing preferences for specific host characteristics. The list of microbial taxa showing significant host preferences involved those potentially having some impacts on survival, growth, or environmental resistance of host plants. Overall, this study provides a platform for understanding how plant and microbial communities are linked with each other at the ecosystem level.

Bacteria and fungi form complex communities (microbiomes) in above- and below-ground organs of plants, contributing to hosts' growth and survival in various ways. Recent studies have suggested that host plant genotypes control, at least partly, plant-associated microbiome compositions. However, we still have limited knowledge of how microbiome structures are determined in/on grafted crop plants, whose above-ground (scion) and below-ground (rootstock) genotypes are different with each other. By using eight varieties of grafted tomato plants, we examined how rootstock genotypes could determine the assembly of leaf endophytic microbes in field conditions. An Illumina sequencing analysis showed that both bacterial and fungal community structures did not significantly differ among tomato plants with different rootstock genotypes: rather, sampling positions in the farmland contributed to microbiome variation in a major way. Nonetheless, a further analysis targeting respective microbial taxa suggested that some bacteria and fungi could be preferentially associated with particular rootstock treatments. Specifically, a bacterium in the genus Deinococcus was found disproportionately from ungrafted tomato individuals. In addition, yeasts in the genus Hannaella occurred frequently on the tomato individuals whose rootstock genotype was "Ganbarune". Overall, this study suggests to what extent leaf microbiome structures can be affected/unaffected by rootstock genotypes in grafted crop plants.

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.

Plants influence their soil environment, which affects the next generation of seedlings that can be established. While research has shown that such plant-soil feedbacks occur in the presence of mycorrhizal fungi, it remains unclear when and how mycorrhizal fungi mediate the direction and strength of feedbacks in tree communities. Here we show that arbuscular mycorrhizal and ectomycorrhizal fungal guilds mediate plant-soil feedbacks differently to influence large-scale patterns such as tree species coexistence and succession. When seedlings are grown under the same mycorrhizal type forest, arbuscular mycorrhizal plant species exhibit negative or neutral feedbacks and ectomycorrhizal plant species do neutral or positive feedbacks. In contrast, positive and neutral feedbacks dominate when seedlings are grown in associations within the same versus different mycorrhizal types. Thus, ectomycorrhizal communities show more positive feedbacks than arbuscular mycorrhizal communities, potentially explaining why most temperate forests are ectomycorrhizal., Competing Interests: The authors declare no competing interests.

Brassicaceae plants have lost symbiotic interactions with mutualistic mycorrhizal fungi, but, nonmycorrhizal Brassicaceae associate with diverse taxonomic groups of mutualistic root-endophytic fungi. Distantly related fungal endophytes of Brassicaceae plants transfer phosphorus to the hosts and promote plant growth, thereby suggesting that the beneficial function was independently acquired via convergent evolution. These beneficial interactions appear tightly regulated by the tryptophan-derived secondary metabolite pathway, which specifically developed in Brassicaceae. Importantly, phosphate availability and types of colonizing microbes appear to influence the metabolite pathway. Thus, endophytes of Brassicaceae may have evolved to adapt to the Brassicaceae-specific traits. Future comparative functional analyses among well-defined endophytic fungi and their relatives with distinct life strategies and host plants will help understand the mechanisms that establish and maintain beneficial interactions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Although fungi play essential roles in nutrient cycles and plant growth in forest ecosystems, limited information is currently available on the community compositions of soil fungi in tropical forests. Few studies have examined fungal community structures in seasonal tropical forests, in which forest fires potentially have a large impact on above- and belowground community processes. Based on high-throughput sequencing technologies, we herein examined the diversity and community structures of soil fungi in dry seasonal tropical forests in Sakaerat, northeast Thailand. We found that fungal community compositions diverged among dry evergreen, dry deciduous, and fire-protected dry deciduous forests within the region. Although tree species diversity did not positively correlate with soil fungal diversity, the coverage of an understory bamboo species (Vietnamosasa pusilla) showed a strong relationship with fungal community structures. Our community ecological analysis also yielded a list of fungi showing habitat preferences for either of the neighboring evergreen and deciduous forests in Sakaerat. The present results provide a basis for managing soil fungal communities and aboveground plant communities in seasonal tropical forests in Southeast Asia.

Metabolites of the intestinal microbiota are thought to be generated through metabolic pathways spanning multiple taxa of intestinal bacteria. We have previously shown that the level of putrescine, a polyamine found abundantly in the human intestinal lumen, is increased in the colonic lumen following administration of arginine and the probiotic Bifidobacterium sp.; however, the underlying mechanism remained poorly understood. We report a novel pathway for putrescine production from arginine through agmatine involving the collaboration of two bacterial groups, and triggered by environmental acidification (drop in pH to below 6.5 from neutral). This pathway comprises the acid tolerance system of Escherichia coli , representing bacteria that have an arginine-dependent acid resistance system; the energy production system of Enterococcus faecalis , representing bacteria that have an agmatine deiminase system; and the acid production system of the acid-producing bacteria, represented by Bifidobacterium spp. This pathway is unique in that it represents a relationship between the independent survival strategies of multiple bacteria.

Background: Although a number of recent studies have uncovered remarkable diversity of microbes associated with plants, understanding and managing dynamics of plant microbiomes remain major scientific challenges. In this respect, network analytical methods have provided a basis for exploring "hub" microbial species, which potentially organize community-scale processes of plant-microbe interactions., Methods: By compiling Illumina sequencing data of root-associated fungi in eight forest ecosystems across the Japanese Archipelago, we explored hubs within "metacommunity-scale" networks of plant-fungus associations. In total, the metadata included 8080 fungal operational taxonomic units (OTUs) detected from 227 local populations of 150 plant species/taxa., Results: Few fungal OTUs were common across all the eight forests. However, in each of the metacommunity-scale networks representing northern four localities or southern four localities, diverse mycorrhizal, endophytic, and pathogenic fungi were classified as "metacommunity hubs," which were detected from diverse host plant taxa throughout a climatic region. Specifically, Mortierella (Mortierellales), Cladophialophora (Chaetothyriales), Ilyonectria (Hypocreales), Pezicula (Helotiales), and Cadophora (incertae sedis) had broad geographic and host ranges across the northern (cool-temperate) region, while Saitozyma/Cryptococcus (Tremellales/Trichosporonales) and Mortierella as well as some arbuscular mycorrhizal fungi were placed at the central positions of the metacommunity-scale network representing warm-temperate and subtropical forests in southern Japan., Conclusions: The network theoretical framework presented in this study will help us explore prospective fungi and bacteria, which have high potentials for agricultural application to diverse plant species within each climatic region. As some of those fungal taxa with broad geographic and host ranges have been known to promote the survival and growth of host plants, further studies elucidating their functional roles are awaited.

In an era of ecosystem degradation and climate change, maximizing microbial functions in agroecosystems has become a prerequisite for the future of global agriculture. However, managing species-rich communities of plant-associated microbiomes remains a major challenge. Here, we propose interdisciplinary research strategies to optimize microbiome functions in agroecosystems. Informatics now allows us to identify members and characteristics of 'core microbiomes', which may be deployed to organize otherwise uncontrollable dynamics of resident microbiomes. Integration of microfluidics, robotics and machine learning provides novel ways to capitalize on core microbiomes for increasing resource-efficiency and stress-resistance of agroecosystems.

Arbuscular mycorrhizal and ectomycorrhizal symbioses are among the most important drivers of terrestrial ecosystem dynamics. Historically, the two types of symbioses have been investigated separately because arbuscular mycorrhizal and ectomycorrhizal plant species are considered to host discrete sets of fungal symbionts (i.e., arbuscular mycorrhizal and ectomycorrhizal fungi, respectively). Nonetheless, recent studies based on high-throughput DNA sequencing technologies have suggested that diverse non-mycorrhizal fungi (e.g., endophytic fungi) with broad host ranges play roles in relationships between arbuscular mycorrhizal and ectomycorrhizal plant species in forest ecosystems. By analyzing an Illumina sequencing dataset of root-associated fungi in a temperate forest in Japan, we statistically examined whether co-occurring arbuscular mycorrhizal ( Chamaecyparis obtusa ) and ectomycorrhizal ( Pinus densiflora ) plant species could share non-mycorrhizal fungal communities. Among the 919 fungal operational taxonomic units (OTUs) detected, OTUs in various taxonomic lineages were statistically designated as "generalists," which associated commonly with both coniferous species. The list of the generalists included fungi in the genera Meliniomyces, Oidiodendron, Cladophialophora, Rhizodermea, Penicillium , and Mortierella . Meanwhile, our statistical analysis also detected fungi preferentially associated with Chamaecyparis (e.g., Pezicula ) or Pinus (e.g., Neolecta ). Overall, this study provides a basis for future studies on how arbuscular mycorrhizal and ectomycorrhizal plant species interactively drive community- or ecosystem-scale processes. The physiological functions of the fungi highlighted in our host-preference analysis deserve intensive investigations for understanding their roles in plant endosphere and rhizosphere.

Background: Understanding feedback between above- and below-ground processes of biological communities is a key to the effective management of natural and agricultural ecosystems. However, as above- and below-ground food webs are often studied separately, our knowledge of material flow and community dynamics in terrestrial ecosystems remains limited., Results: We developed a high-throughput sequencing method for examining how spiders link above- and below-ground food webs as generalist predators. To overcome problems related to DNA-barcoding-based analyses of arthropod-arthropod interactions, we designed spider-specific blocking primers and Hexapoda-specific primers for the selective PCR amplification of Hexapoda prey sequences from spider samples. By applying the new DNA metabarcoding framework to spider samples collected in a temperate secondary forest in Japan, we explored the structure of a food web involving 15 spider species and various taxonomic groups of Hexapoda prey. These results support the hypothesis that multiple spider species in a community can prey on both above- and below-ground prey species, potentially coupling above- and below-ground food-web dynamics., Conclusions: The PCR primers and metabarcoding pipeline described in this study are expected to accelerate nuclear marker-based analyses of food webs, illuminating poorly understood trophic interactions in ecosystems., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Changes in species richness along climatological gradients have been instrumental in developing theories about the general drivers of biodiversity. Previous studies on microbial communities along climate gradients on mountainsides have revealed positive, negative and neutral richness trends. We examined changes in richness and composition of Fungi, Bacteria and Archaea in soil along a 50-1000 m elevation, 280-3280 mm/yr precipitation gradient in Hawai'i. Soil properties and their drivers are exceptionally well understood along this gradient. All three microbial groups responded strongly to the gradient, with community ordinations being similar along axes of environmental conditions (pH, rainfall) and resource availability (nitrogen, phosphorus). However, the form of the richness-climate relationship varied between Fungi (positive linear), Bacteria (unimodal) and Archaea (negative linear). These differences were related to resource-ecology and limiting conditions for each group, with fungal richness increasing most strongly with soil carbon, ammonia-oxidizing Archaea increasing with nitrogen mineralization rate, and Bacteria increasing with both carbon and pH. Reponses to the gradient became increasingly variable at finer taxonomic scales and within any taxonomic group most individual OTUs occurred in narrow climate-elevation ranges. These results show that microbial responses to climate gradients are heterogeneous due to complexity of underlying environmental changes and the diverse ecologies of microbial taxa., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Understanding how ecological and evolutionary processes interdependently structure biosphere dynamics is a major challenge in the era of worldwide ecosystem degradation. However, our knowledge of 'eco-evolutionary feedbacks' depends largely on findings from simple systems representing limited spatial scales and involving few species. Here we review recent conceptual developments for the understanding of multispecies coevolutionary processes and then discuss how new lines of concepts and methods will accelerate the integration of ecology and evolutionary biology. To build a research workflow for integrating insights into spatiotemporal dynamics of species-rich systems, we focus on the roles of 'metacommunity hub' species, whose population size and/or genetic dynamics potentially control landscape- or regional-scale phenomena. As large amounts of network data are becoming available with high-throughput sequencing of various host-symbiont, prey-predator, and symbiont-symbiont interactions, we suggest it is now possible to develop bases for the integrated understanding and management of species-rich ecosystems.

Fungi in soil play pivotal roles in nutrient cycling, pest controls, and plant community succession in terrestrial ecosystems. Despite the ecosystem functions provided by soil fungi, our knowledge of the assembly processes of belowground fungi has been limited. In particular, we still have limited knowledge of how diverse functional groups of fungi interact with each other in facilitative and competitive ways in soil. Based on the high-throughput sequencing data of fungi in a cool-temperate forest in northern Japan, we analyzed how taxonomically and functionally diverse fungi showed correlated fine-scale distributions in soil. By uncovering pairs of fungi that frequently co-occurred in the same soil samples, networks depicting fine-scale co-occurrences of fungi were inferred at the O (organic matter) and A (surface soil) horizons. The results then led to the working hypothesis that mycorrhizal, endophytic, saprotrophic, and pathogenic fungi could form compartmentalized (modular) networks of facilitative, antagonistic, and/or competitive interactions in belowground ecosystems. Overall, this study provides a research basis for further understanding how interspecific interactions, along with sharing of niches among fungi, drive the dynamics of poorly explored biospheres in soil., Competing Interests: We declare that we have no competing interests.

Terrestrial plants host phylogenetically and functionally diverse groups of below-ground microbes, whose community structure controls plant growth/survival in both natural and agricultural ecosystems. Therefore, understanding the processes by which whole root-associated microbiomes are organized is one of the major challenges in ecology and plant science. We here report that diverse root-associated fungi can form highly compartmentalized networks of coexistence within host roots and that the structure of the fungal symbiont communities can be partitioned into semi-discrete types even within a single host plant population. Illumina sequencing of root-associated fungi in a monodominant south beech forest revealed that the network representing symbiont-symbiont co-occurrence patterns was compartmentalized into clear modules, which consisted of diverse functional groups of mycorrhizal and endophytic fungi. Consequently, terminal roots of the plant were colonized by either of the two largest fungal species sets (represented by Oidiodendron or Cenococcum). Thus, species-rich root microbiomes can have alternative community structures, as recently shown in the relationships between human gut microbiome type (i.e., 'enterotype') and host individual health. This study also shows an analytical framework for pinpointing network hubs in symbiont-symbiont networks, leading to the working hypothesis that a small number of microbial species organize the overall root-microbiome dynamics., (© 2016 The Author(s).)

In nature, plants and their pollinating and/or seed-dispersing animals form complex interaction networks. The commonly observed pattern of links between specialists and generalists in these networks has been predicted to promote species coexistence. Plants also build highly species-rich mutualistic networks below ground with root-associated fungi, and the structure of these plant-fungus networks may also affect terrestrial community processes. By compiling high-throughput DNA sequencing data sets of the symbiosis of plants and their root-associated fungi from three localities along a latitudinal gradient, we uncovered the entire network architecture of these interactions under contrasting environmental conditions. Each network included more than 30 plant species and hundreds of mycorrhizal and endophytic fungi belonging to diverse phylogenetic groups. The results were consistent with the notion that processes shaping host-plant specialization of fungal species generate a unique linkage pattern that strongly contrasts with the pattern of above-ground plant-partner networks. Specifically, plant-fungus networks lacked a "nested" architecture, which has been considered to promote species coexistence in plant-partner networks. Rather, the below-ground networks had a conspicuous "antinested" topology. Our findings lead to the working hypothesis that terrestrial plant community dynamics are likely determined by the balance between above-ground and below-ground webs of interspecific interactions.

Root-associated fungi, including ectomycorrhizal and root-endophytic fungi, are among the most diverse and important belowground plant symbionts in dipterocarp rainforests. Our study aimed to reveal the biodiversity, host association, and community structure of ectomycorrhizal Basidiomycota and root-associated Ascomycota (including root-endophytic Ascomycota) in a lowland dipterocarp rainforest in Southeast Asia. The host plant chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) region and fungal internal transcribed spacer 2 (ITS2) region were sequenced using tag-encoded, massively parallel 454 pyrosequencing to identify host plant and root-associated fungal taxa in root samples. In total, 1245 ascomycetous and 127 putative ectomycorrhizal basidiomycetous taxa were detected from 442 root samples. The putative ectomycorrhizal Basidiomycota were likely to be associated with closely related dipterocarp taxa to greater or lesser extents, whereas host association patterns of the root-associated Ascomycota were much less distinct. The community structure of the putative ectomycorrhizal Basidiomycota was possibly more influenced by host genetic distances than was that of the root-associated Ascomycota. This study also indicated that in dipterocarp rainforests, root-associated Ascomycota were characterized by high biodiversity and indistinct host association patterns, whereas ectomycorrhizal Basidiomycota showed less biodiversity and a strong host phylogenetic preference for dipterocarp trees. Our findings lead to the working hypothesis that root-associated Ascomycota, which might be mainly represented by root-endophytic fungi, have biodiversity hotspots in the tropics, whereas biodiversity of ectomycorrhizal Basidiomycota increases with host genetic diversity.

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.

Species in ecological communities build complex webs of interaction. Although revealing the architecture of these networks is fundamental to understanding ecological and evolutionary dynamics in nature, it has been difficult to characterize the structure of most species-rich ecological systems. By overcoming this limitation through next-generation sequencing technology, we herein uncover the network architecture of below-ground plant-fungus symbioses, which are ubiquitous to terrestrial ecosystems. The examined symbiotic network of a temperate forest in Japan includes 33 plant species and 387 functionally and phylogenetically diverse fungal taxa, and the overall network architecture differs fundamentally from that of other ecological networks. In contrast to results for other ecological networks and theoretical predictions for symbiotic networks, the plant-fungus network shows moderate or relatively low levels of interaction specialization and modularity and an unusual pattern of 'nested' network architecture. These results suggest that species-rich ecological networks are more architecturally diverse than previously recognized.

Diverse clades of mycorrhizal and endophytic fungi are potentially involved in competitive or facilitative interactions within host-plant roots. We investigated the potential consequences of these ecological interactions on the assembly process of root-associated fungi by examining the co-occurrence of pairs of fungi in host-plant individuals. Based on massively-parallel pyrosequencing, we analyzed the root-associated fungal community composition for each of the 249 Quercus serrata and 188 Quercus glauca seedlings sampled in a warm-temperate secondary forest in Japan. Pairs of fungi that co-occurred more or less often than expected by chance were identified based on randomization tests. The pyrosequencing analysis revealed that not only ectomycorrhizal fungi but also endophytic fungi were common in the root-associated fungal community. Intriguingly, specific pairs of these ectomycorrhizal and endophytic fungi showed spatially aggregated patterns, suggesting the existence of facilitative interactions between fungi in different functional groups. Due to the large number of fungal pairs examined, many of the observed aggregated/segregated patterns with very low P values (e.g., < 0.005) turned non-significant after the application of a multiple comparison method. However, our overall results imply that the community structures of ectomycorrhizal and endophytic fungi could influence each other through interspecific competitive/facilitative interactions in root. To test the potential of host-plants' control of fungus-fungus ecological interactions in roots, we further examined whether the aggregated/segregated patterns could vary depending on the identity of host plant species. Potentially due to the physiological properties shared between the congeneric host plant species, the sign of hosts' control was not detected in the present study. The pyrosequencing-based randomization analyses shown in this study provide a platform of the high-throughput investigation of fungus-fungus interactions in plant root systems.

Plant-mycorrhizal fungal interactions are ubiquitous in forest ecosystems. While ectomycorrhizal plants and their fungi generally dominate temperate forests, arbuscular mycorrhizal symbiosis is common in the tropics. In subtropical regions, however, ectomycorrhizal and arbuscular mycorrhizal plants co-occur at comparable abundances in single forests, presumably generating complex community structures of root-associated fungi. To reveal root-associated fungal community structure in a mixed forest of ectomycorrhizal and arbuscular mycorrhizal plants, we conducted a massively-parallel pyrosequencing analysis, targeting fungi in the roots of 36 plant species that co-occur in a subtropical forest. In total, 580 fungal operational taxonomic units were detected, of which 132 and 58 were probably ectomycorrhizal and arbuscular mycorrhizal, respectively. As expected, the composition of fungal symbionts differed between fagaceous (ectomycorrhizal) and non-fagaceous (possibly arbuscular mycorrhizal) plants. However, non-fagaceous plants were associated with not only arbuscular mycorrhizal fungi but also several clades of ectomycorrhizal (e.g., Russula) and root-endophytic ascomycete fungi. Many of the ectomycorrhizal and root-endophytic fungi were detected from both fagaceous and non-fagaceous plants in the community. Interestingly, ectomycorrhizal and arbuscular mycorrhizal fungi were concurrently detected from tiny root fragments of non-fagaceous plants. The plant-fungal associations in the forest were spatially structured, and non-fagaceous plant roots hosted ectomycorrhizal fungi more often in the proximity of ectomycorrhizal plant roots. Overall, this study suggests that belowground plant-fungal symbiosis in subtropical forests is complex in that it includes "non-typical" plant-fungal combinations (e.g., ectomycorrhizal fungi on possibly arbuscular mycorrhizal plants) that do not fall within the conventional classification of mycorrhizal symbioses, and in that associations with multiple functional (or phylogenetic) groups of fungi are ubiquitous among plants. Moreover, ectomycorrhizal fungal symbionts of fagaceous plants may "invade" the roots of neighboring non-fagaceous plants, potentially influencing the interactions between non-fagaceous plants and their arbuscular-mycorrhizal fungal symbionts at a fine spatial scale.

Most terrestrial plants interact with diverse clades of mycorrhizal and root-endophytic fungi in their roots. Through belowground plant-fungal interactions, dominant plants can benefit by interacting with host-specific mutualistic fungi and proliferate in a community based on positive plant-mutualistic fungal feedback. On the other hand, subordinate plant species may persist in the community by sharing other sets (functional groups) of fungal symbionts with each other. Therefore, revealing how diverse clades of root-associated fungi are differentially hosted by dominant and subordinate plant species is essential for understanding plant community structure and dynamics. Based on 454-pyrosequencing, we determined the community composition of root-associated fungi on 36 co-occurring plant species in an oak-dominated forest in northern Japan and statistically evaluated the host preference phenotypes of diverse mycorrhizal and root-endophytic fungi. An analysis of 278 fungal taxa indicated that an ectomycorrhizal basidiomycete fungus in the genus Lactarius and a possibly endophytic ascomycete fungus in the order Helotiales significantly favored the dominant oak (Quercus) species. In contrast, arbuscular mycorrhizal fungi were generally shared among subordinate plant species. Although fungi with host preferences contributed to the compartmentalization of belowground plant-fungal associations, diverse clades of ectomycorrhizal fungi and possible root endophytes were associated not only with the dominant Quercus but also with the remaining plant species. Our findings suggest that dominant-ectomycorrhizal and subordinate plant species can host different subsets of root-associated fungi, and diverse clades of generalist fungi can counterbalance the compartmentalization of plant-fungal associations. Such insights into the overall structure of belowground plant-fungal associations will help us understand the mechanisms that facilitate the coexistence of plant species in natural communities.

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used "1-nearest-neighbor" (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research.

In natural forests, hundreds of fungal species colonize plant roots. The preference or specificity for partners in these symbiotic relationships is a key to understanding how the community structures of root-associated fungi and their host plants influence each other. In an oak-dominated forest in Japan, we investigated the root-associated fungal community based on a pyrosequencing analysis of the roots of 33 plant species. Of the 387 fungal taxa observed, 153 (39.5%) were identified on at least two plant species. Although many mycorrhizal and root-endophytic fungi are shared between the plant species, the five most common plant species in the community had specificity in their association with fungal taxa. Likewise, fungi displayed remarkable variation in their association specificity for plants even within the same phylogenetic or ecological groups. For example, some fungi in the ectomycorrhizal family Russulaceae were detected almost exclusively on specific oak (Quercus) species, whereas other Russulaceae fungi were found even on "non-ectomycorrhizal" plants (e.g., Lyonia and Ilex). Putatively endophytic ascomycetes in the orders Helotiales and Chaetothyriales also displayed variation in their association specificity and many of them were shared among plant species as major symbionts. These results suggest that the entire structure of belowground plant-fungal associations is described neither by the random sharing of hosts/symbionts nor by complete compartmentalization by mycorrhizal type. Rather, the colonization of multiple types of mycorrhizal fungi on the same plant species and the prevalence of diverse root-endophytic fungi may be important features of belowground linkage between plant and fungal communities.

The processes and mechanisms underlying the diversification of host-microbe endosymbiotic associations are of evolutionary interest. Here we investigated the bacteriocyte-associated primary symbionts of weevils wherein the ancient symbiont Nardonella has experienced two independent replacement events: once by Curculioniphilus symbiont in the lineage of Curculio and allied weevils of the tribe Curculionini, and once by Sodalis-allied symbiont in the lineage of grain weevils of the genus Sitophilus. The Curculioniphilus symbiont was detected from 27 of 36 Curculionini species examined, the symbiont phylogeny was congruent with the host weevil phylogeny, and the symbiont gene sequences exhibited AT-biased nucleotide compositions and accelerated molecular evolution. These results suggest that the Curculioniphilus symbiont was acquired by an ancestor of the tribe Curculionini, replaced the original symbiont Nardonella, and has co-speciated with the host weevils over evolutionary time, but has been occasionally lost in several host lineages. By contrast, the Sodalis-allied symbiont of Sitophilus weevils exhibited no host-symbiont co-speciation, no AT-biased nucleotide compositions and only moderately accelerated molecular evolution. These results suggest that the Sodalis-allied symbiont was certainly acquired by an ancestor of the Sitophilus weevils and replaced the original Nardonella symbiont, but the symbiotic association must have experienced occasional re-associations such as new acquisitions, horizontal transfers, replacements and/or losses. We detected Sodalis-allied facultative symbionts in populations of the Curculionini weevils, which might represent potential evolutionary sources of the Sodalis-allied primary symbionts. Comparison of these newcomer bacteriocyte-associated symbiont lineages highlights potential evolutionary trajectories and consequences of novel symbionts after independent replacements of the same ancient symbiont.