Metabolic complexity drives divergence in microbial communities.

Microbial communities are shaped by environmental metabolites, but the principles that govern whether different communities will converge or diverge in any given condition remain unknown, posing fundamental questions about the feasibility of microbiome engineering. Here we studied the longitudinal assembly dynamics of a set of natural microbial communities grown in laboratory conditions of increasing metabolic complexity. We found that different microbial communities tend to become similar to each other when grown in metabolically simple conditions, but they diverge in composition as the metabolic complexity of the environment increases, a phenomenon we refer to as the divergence-complexity effect. A comparative analysis of these communities revealed that this divergence is driven by community diversity and by the assortment of specialist taxa capable of degrading complex metabolites. An ecological model of community dynamics indicates that the hierarchical structure of metabolism itself, where complex molecules are enzymatically degraded into progressively simpler ones that then participate in cross-feeding between community members, is necessary and sufficient to recapitulate our experimental observations. In addition to helping understand the role of the environment in community assembly, the divergence-complexity effect can provide insight into which environments support multiple community states, enabling the search for desired ecosystem functions towards microbiome engineering applications.

Restoring ecological complexity in a changing environment.

As land use leaves massive tracts of land vacant for recovery, restoration must undergo a substantial shift to incorporate a complexity perspective beyond the traditional community, biodiversity or functional views. With an interaction-function perspective, we may be able to achieve ecosystems with better chances to adapt to current environmental changes and, especially, to climate change. We explore combined approaches that include still unused and underexplored techniques that will soon go mainstream and produce massive amounts of information to address the complexity gap. As we understand how complexity reassembles after the end of agriculture, we will be able to design actions to restore or enhance it at unprecedented spatial scales while increasing its adaptability to environmental changes.

Ectomycorrhizal fungi enhance pine growth by stimulating iron-dependent mechanisms with trade-offs in symbiotic performance.

Iron (Fe) is crucial for metabolic functions of living organisms. Plants access occluded Fe through interactions with rhizosphere microorganisms and symbionts. Yet, the interplay between Fe addition and plant-mycorrhizal interactions, especially the molecular mechanisms underlying mycorrhiza-assisted Fe processing in plants, remains largely unexplored. We conducted mesocosms in Pinus plants inoculated with different ectomycorrhizal fungi (EMF) Suillus species under conditions with and without Fe coatings. Meta-transcriptomic, biogeochemical, and X-ray fluorescence imaging analyses were applied to investigate early-stage mycorrhizal roots. While Fe addition promoted Pinus growth, it concurrently reduced mycorrhiza formation rate, symbiosis-related metabolites in plant roots, and aboveground plant carbon and macronutrient content. This suggested potential trade-offs between Fe-enhanced plant growth and symbiotic performance. However, the extent of this trade-off may depend on interactions between host plants and EMF species. Interestingly, dual EMF species were more effective at facilitating plant Fe uptake by inducing diverse Fe-related functions than single-EMF species. This subsequently triggered various Fe-dependent physiological and biochemical processes in Pinus roots, significantly contributing to Pinus growth. However, this resulted in a greater carbon allocation to roots, relatively reducing the aboveground plant carbon content. Our study offers critical insights into how EMF communities rebalance benefits of Fe-induced effects on symbiotic partners.

Urbanization and edge effects interact to drive mutualism breakdown and the rise of unstable pathogenic communities in forest soil.

Temperate forests are threatened by urbanization and fragmentation, with over 20% (118,300 km2) of U.S. forest land projected to be subsumed by urban land development. We leveraged a unique, well-characterized urban-to-rural and forest edge-to-interior gradient to identify the combined impact of these two land use changes-urbanization and forest edge creation-on the soil microbial community in native remnant forests. We found evidence of mutualism breakdown between trees and their fungal root mutualists [ectomycorrhizal (ECM) fungi] with urbanization, where ECM fungi colonized fewer tree roots and had less connectivity in soil microbiome networks in urban forests compared to rural forests. However, urbanization did not reduce the relative abundance of ECM fungi in forest soils; instead, forest edges alone led to strong reductions in ECM fungal abundance. At forest edges, ECM fungi were replaced by plant and animal pathogens, as well as copiotrophic, xenobiotic-degrading, and nitrogen-cycling bacteria, including nitrifiers and denitrifiers. Urbanization and forest edges interacted to generate new "suites" of microbes, with urban interior forests harboring highly homogenized microbiomes, while edge forest microbiomes were more heterogeneous and less stable, showing increased vulnerability to low soil moisture. When scaled to the regional level, we found that forest soils are projected to harbor high abundances of fungal pathogens and denitrifying bacteria, even in rural areas, due to the widespread existence of forest edges. Our results highlight the potential for soil microbiome dysfunction-including increased greenhouse gas production-in temperate forest regions that are subsumed by urban expansion, both now and in the future.

Metabolic complexity drives divergence in microbial communities.

Microbial communities are shaped by the metabolites available in their environment, but the principles that govern whether different communities will converge or diverge in any given condition remain unknown, posing fundamental questions about the feasibility of microbiome engineering. To this end, we studied the longitudinal assembly dynamics of a set of natural microbial communities grown in laboratory conditions of increasing metabolic complexity. We found that different microbial communities tend to become similar to each other when grown in metabolically simple conditions, but diverge in composition as the metabolic complexity of the environment increases, a phenomenon we refer to as the divergence-complexity effect. A comparative analysis of these communities revealed that this divergence is driven by community diversity and by the diverse assortment of specialist taxa capable of degrading complex metabolites. An ecological model of community dynamics indicates that the hierarchical structure of metabolism itself, where complex molecules are enzymatically degraded into progressively smaller ones, is necessary and sufficient to recapitulate all of our experimental observations. In addition to pointing to a fundamental principle of community assembly, the divergence-complexity effect has important implications for microbiome engineering applications, as it can provide insight into which environments support multiple community states, enabling the search for desired ecosystem functions.

Soils at the temperate forest edge: An investigation of soil characteristics and carbon dynamics.

Global proliferation of forest edges through anthropogenic land-use change and forest fragmentation is well documented, and while forest fragmentation has clear consequences for soil carbon (C) cycling, underlying drivers of belowground activity at the forest edge remain poorly understood. Increasing soil C losses via respiration have been observed at rural forest edges, but this process was suppressed at urban forest edges. We offer a comprehensive, coupled investigation of abiotic soil conditions and biotic soil activity from forest edge to interior at eight sites along an urbanization gradient to elucidate how environmental stressors are linked to soil C cycling at the forest edge. Despite significant diverging trends in edge soil C losses between urban and rural sites, we did not find comparable differences in soil % C or microbial enzyme activity, suggesting an unexpected decoupling of soil C fluxes and pools at forest edges. We demonstrate that across site types, soils at forest edges were less acidic than the forest interior (p < 0.0001), and soil pH was positively correlated with soil calcium, magnesium and sodium content (adj R2 = 0.37), which were also elevated at the edge. Compared to forest interior, forest edge soils exhibited a 17.8 % increase in sand content and elevated freeze-thaw frequency with probable downstream effects on root turnover and decomposition. Using these and other novel forest edge data, we demonstrate that significant variation in edge soil respiration (adj R2 = 0.46; p = 0.0002) and C content (adj R2 = 0.86; p < 0.0001) can be explained using soil parameters often mediated by human activity (e.g., soil pH, trace metal and cation concentrations, soil temperature), and we emphasize the complex influence of multiple, simultaneous global change drivers at forest edges. Forest edge soils reflect legacies of anthropogenic land-use and modern human management, and this must be accounted for to understand soil activity and C cycling across fragmented landscapes.

Urbanization and fragmentation have opposing effects on soil nitrogen availability in temperate forest ecosystems.

Nitrogen (N) availability relative to plant demand has been declining in recent years in terrestrial ecosystems throughout the world, a phenomenon known as N oligotrophication. The temperate forests of the northeastern U.S. have experienced a particularly steep decline in bioavailable N, which is expected to be exacerbated by climate change. This region has also experienced rapid urban expansion in recent decades that leads to forest fragmentation, and it is unknown whether and how these changes affect N availability and uptake by forest trees. Many studies have examined the impact of either urbanization or forest fragmentation on nitrogen (N) cycling, but none to our knowledge have focused on the combined effects of these co-occurring environmental changes. We examined the effects of urbanization and fragmentation on oak-dominated (Quercus spp.) forests along an urban to rural gradient from Boston to central Massachusetts (MA). At eight study sites along the urbanization gradient, plant and soil measurements were made along a 90 m transect from a developed edge to an intact forest interior. Rates of net ammonification, net mineralization, and foliar N concentrations were significantly higher in urban than rural sites, while net nitrification and foliar C:N were not different between urban and rural forests. At urban sites, foliar N and net ammonification and mineralization were higher at forest interiors compared to edges, while net nitrification and foliar C:N were higher at rural forest edges than interiors. These results indicate that urban forests in the northeastern U.S. have greater soil N availability and N uptake by trees compared to rural forests, counteracting the trend for widespread N oligotrophication in temperate forests around the globe. Such increases in available N are diminished at forest edges, however, demonstrating that forest fragmentation has the opposite effect of urbanization on coupled N availability and demand by trees.

Environmental microbiome engineering for the mitigation of climate change.

Environmental microbiome engineering is emerging as a potential avenue for climate change mitigation. In this process, microbial inocula are introduced to natural microbial communities to tune activities that regulate the long-term stabilization of carbon in ecosystems. In this review, we outline the process of environmental engineering and synthesize key considerations about ecosystem functions to target, means of sourcing microorganisms, strategies for designing microbial inocula, methods to deliver inocula, and the factors that enable inocula to establish within a resident community and modify an ecosystem function target. Recent work, enabled by high-throughput technologies and modeling approaches, indicate that microbial inocula designed from the top-down, particularly through directed evolution, may generally have a higher chance of establishing within existing microbial communities than other historical approaches to microbiome engineering. We address outstanding questions about the determinants of inocula establishment and provide suggestions for further research about the possibilities and challenges of environmental microbiome engineering as a tool to combat climate change.

Alternative stable states of the forest mycobiome are maintained through positive feedbacks.

Most trees on Earth form a symbiosis with either arbuscular mycorrhizal or ectomycorrhizal fungi. By forming common mycorrhizal networks, actively modifying the soil environment and other ecological mechanisms, these contrasting symbioses may generate positive feedbacks that favour their own mycorrhizal strategy (that is, the con-mycorrhizal strategy) at the expense of the alternative strategy. Positive con-mycorrhizal feedbacks set the stage for alternative stable states of forests and their fungi, where the presence of different forest mycorrhizal strategies is determined not only by external environmental conditions but also mycorrhiza-mediated feedbacks embedded within the forest ecosystem. Here, we test this hypothesis using thousands of US forest inventory sites to show that arbuscular and ectomycorrhizal tree recruitment and survival exhibit positive con-mycorrhizal density dependence. Data-driven simulations show that these positive feedbacks are sufficient in magnitude to generate and maintain alternative stable states of the forest mycobiome. Given the links between forest mycorrhizal strategy and carbon sequestration potential, the presence of mycorrhizal-mediated alternative stable states affects how we forecast forest composition, carbon sequestration and terrestrial climate feedbacks.

Ectomycorrhizal fungi are associated with reduced nitrogen cycling rates in temperate forest soils without corresponding trends in bacterial functional groups.

Microbial processes play a central role in controlling the availability of N in temperate forests. While bacteria, archaea, and fungi account for major inputs, transformations, and exports of N in soil, relationships between microbial community structure and N cycle fluxes have been difficult to detect and characterize. Several studies have reported differences in N cycling based on mycorrhizal type in temperate forests, but associated differences in N cycling genes underlying these fluxes are not well-understood. We explored how rates of soil N cycle fluxes vary across gradients of mycorrhizal abundance (hereafter "mycorrhizal gradients") at four temperate forest sites in Massachusetts and Indiana, USA. We paired measurements of N-fixation, net nitrification, and denitrification rates with gene abundance data for specific bacterial functional groups associated with each process. We find that the availability of NO3 and rates of N-fixation, net nitrification, and denitrification are reduced in stands dominated by trees associated with ECM fungi. On average, rates of N-fixation and denitrification in stands dominated by trees associated with arbuscular mycorrhizal fungi were more than double the corresponding rates in stands dominated by trees associated with ectomycorrhizal fungi. Despite the structuring of flux rates across the mycorrhizal gradients, we did not find concomitant shifts in the abundances of N-cycling bacterial genes, and gene abundances were not correlated with process rates. Given that AM-associating trees are replacing ECM-associating trees throughout much of the eastern US, our results suggest that shifts in mycorrhizal dominance may accelerate N cycling independent of changes in the relative abundance of N cycling bacteria, with consequences for forest productivity and N retention.

Omics analyses and biochemical study of Phlebiopsis gigantea elucidate its degradation strategy of wood extractives.

Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi.

Soil microbiome predictability increases with spatial and taxonomic scale.

Soil microorganisms shape ecosystem function, yet it remains an open question whether we can predict the composition of the soil microbiome in places before observing it. Furthermore, it is unclear whether the predictability of microbial life exhibits taxonomic- and spatial-scale dependence, as it does for macrobiological communities. Here, we leverage multiple large-scale soil microbiome surveys to develop predictive models of bacterial and fungal community composition in soil, then test these models against independent soil microbial community surveys from across the continental United States. We find remarkable scale dependence in community predictability. The predictability of bacterial and fungal communities increases with the spatial scale of observation, and fungal predictability increases with taxonomic scale. These patterns suggest that there is an increasing importance of deterministic versus stochastic processes with scale, consistent with findings in plant and animal communities, suggesting a general scaling relationship across biology. Biogeochemical functional groups and high-level taxonomic groups of microorganisms were equally predictable, indicating that traits and taxonomy are both powerful lenses for understanding soil communities. By focusing on out-of-sample prediction, these findings suggest an emerging generality in our understanding of the soil microbiome, and that this understanding is fundamentally scale dependent.

The National Ecological Observatory Network's soil metagenomes: assembly and basic analysis.

The largest dataset of soil metagenomes has recently been released by the National Ecological Observatory Network (NEON), which performs annual shotgun sequencing of soils at 47 sites across the United States. NEON serves as a valuable educational resource, thanks to its open data and programming tutorials, but there is currently no introductory tutorial for accessing and analyzing the soil shotgun metagenomic dataset. Here, we describe methods for processing raw soil metagenome sequencing reads using a bioinformatics pipeline tailored to the high complexity and diversity of the soil microbiome. We describe the rationale, necessary resources, and implementation of steps such as cleaning raw reads, taxonomic classification, assembly into contigs or genomes, annotation of predicted genes using custom protein databases, and exporting data for downstream analysis. The workflow presented here aims to increase the accessibility of NEON's shotgun metagenome data, which can provide important clues about soil microbial communities and their ecological roles.

Soil Microbes Trade-Off Biogeochemical Cycling for Stress Tolerance Traits in Response to Year-Round Climate Change.

Winter air temperatures are rising faster than summer air temperatures in high-latitude forests, increasing the frequency of soil freeze/thaw events in winter. To determine how climate warming and soil freeze/thaw cycles affect soil microbial communities and the ecosystem processes they drive, we leveraged the Climate Change across Seasons Experiment (CCASE) at the Hubbard Brook Experimental Forest in the northeastern United States, where replicate field plots receive one of three climate treatments: warming (+5°C above ambient in the growing season), warming in the growing season + winter freeze/thaw cycles (+5°C above ambient +4 freeze/thaw cycles during winter), and no treatment. Soil samples were taken from plots at six time points throughout the growing season and subjected to amplicon (rDNA) and metagenome sequencing. We found that soil fungal and bacterial community composition were affected by changes in soil temperature, where the taxonomic composition of microbial communities shifted more with the combination of growing-season warming and increased frequency of soil freeze/thaw cycles in winter than with warming alone. Warming increased the relative abundance of brown rot fungi and plant pathogens but decreased that of arbuscular mycorrhizal fungi, all of which recovered under combined growing-season warming and soil freeze/thaw cycles in winter. The abundance of animal parasites increased significantly under combined warming and freeze/thaw cycles. We also found that warming and soil freeze/thaw cycles suppressed bacterial taxa with the genetic potential for carbon (i.e., cellulose) decomposition and soil nitrogen cycling, such as N fixation and the final steps of denitrification. These new soil communities had higher genetic capacity for stress tolerance and lower genetic capacity to grow or reproduce, relative to the communities exposed to warming in the growing season alone. Our observations suggest that initial suppression of biogeochemical cycling with year-round climate change may be linked to the emergence of taxa that trade-off growth for stress tolerance traits.

Warming and disturbance alter soil microbiome diversity and function in a northern forest ecotone.

The response to global change by soil microbes is set to affect important ecosystem processes. These impacts could be most immediate in transitional zones, such as the temperate-boreal forest ecotone, yet previous work in these forests has primarily focused on specific subsets of microbial taxa. Here, we examined how bacterial and fungal communities respond to simulated above- and below-ground warming under realistic field conditions in closed and open canopy treatments in Minnesota, USA. Our results show that warming and canopy disturbance shifted bacterial and fungal community structure as dominant bacterial and fungal groups differed in the direction and intensity of their responses. Ectomycorrhizal and saprotrophic fungal communities with greater connectivity (higher prevalence of strongly interconnected taxa based on pairwise co-occurrence relationships) were more resistant to compositional change. Warming effects on soil enzymes involved in the hydrolytic and oxidative liberation of carbon from plant cell walls and nutrients from organic matter were most strongly linked to fungal community responses, although community structure-function relationships differed between fungal guilds. Collectively, these findings indicate that warming and disturbance will influence the composition and function of microbial communities in the temperate-boreal ecotone, and fungal responses are particularly important to understand for predicting future ecosystem functioning.

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

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

Global imprint of mycorrhizal fungi on whole-plant nutrient economics.

Mycorrhizal fungi are critical members of the plant microbiome, forming a symbiosis with the roots of most plants on Earth. Most plant species partner with either arbuscular or ectomycorrhizal fungi, and these symbioses are thought to represent plant adaptations to fast and slow soil nutrient cycling rates. This generates a second hypothesis, that arbuscular and ectomycorrhizal plant species traits complement and reinforce these fungal strategies, resulting in nutrient acquisitive vs. conservative plant trait profiles. Here we analyzed 17,764 species level trait observations from 2,940 woody plant species to show that mycorrhizal plants differ systematically in nitrogen and phosphorus economic traits. Differences were clearest in temperate latitudes, where ectomycorrhizal plant species are more nitrogen use- and phosphorus use-conservative than arbuscular mycorrhizal species. This difference is reflected in both aboveground and belowground plant traits and is robust to controlling for evolutionary history, nitrogen fixation ability, deciduousness, latitude, and species climate niche. Furthermore, mycorrhizal effects are large and frequently similar to or greater in magnitude than the influence of plant nitrogen fixation ability or deciduous vs. evergreen leaf habit. Ectomycorrhizal plants are also more nitrogen conservative than arbuscular plants in boreal and tropical ecosystems, although differences in phosphorus use are less apparent outside temperate latitudes. Our findings bolster current theories of ecosystems rooted in mycorrhizal ecology and support the hypothesis that plant mycorrhizal association is linked to the evolution of plant nutrient economic strategies.

An evolutionary signal to fungal succession during plant litter decay.

Ecologists have frequently observed a pattern of fungal succession during litter decomposition, wherein different fungal taxa dominate different stages of decay in individual ecosystems. However, it is unclear which biological features of fungi give rise to this pattern. We tested a longstanding hypothesis that fungal succession depends on the evolutionary history of species, such that different fungal phyla prefer different decay stages. To test this hypothesis, we performed a meta-analysis across studies in 22 different ecosystem types to synthesize fungal decomposer abundances at early, middle and late stages of plant litter decay. Fungal phyla varied in relative abundance throughout decay, with fungi in the Ascomycota reaching highest relative abundance during early stages of decay (P < 0.001) and fungi in the Zygomycota reaching highest relative abundance during late stages of decay (P < 0.001). The best multiple regression model to explain variation in abundance of these fungal phyla during decay included decay stage, as well as plant litter type and climate factors. Most variation in decay-stage preference of fungal taxa was observed at basal taxonomic levels (phylum and class) rather than finer taxonomic levels (e.g. genus). For many finer-scale taxonomic groups and functional groups of fungi, plant litter type and climate factors were better correlates with relative abundance than decay stage per se, suggesting that the patchiness of fungal community composition in space is related to both resource and climate niches of different fungal taxa. Our study indicates that decomposer fungal succession is partially rooted in fungal decomposers' deep evolutionary history, traceable to the divergence among phyla.

Microbial carbon use efficiency predicted from genome-scale metabolic models.

Respiration by soil bacteria and fungi is one of the largest fluxes of carbon (C) from the land surface. Although this flux is a direct product of microbial metabolism, controls over metabolism and their responses to global change are a major uncertainty in the global C cycle. Here, we explore an in silico approach to predict bacterial C-use efficiency (CUE) for over 200 species using genome-specific constraint-based metabolic modeling. We find that potential CUE averages 0.62 ± 0.17 with a range of 0.22 to 0.98 across taxa and phylogenetic structuring at the subphylum levels. Potential CUE is negatively correlated with genome size, while taxa with larger genomes are able to access a wider variety of C substrates. Incorporating the range of CUE values reported here into a next-generation model of soil biogeochemistry suggests that these differences in physiology across microbial taxa can feed back on soil-C cycling.