The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered.

UNITE (https://unite.ut.ee) is a web-based database and sequence management environment for molecular identification of eukaryotes. It targets the nuclear ribosomal internal transcribed spacer (ITS) region and offers nearly 10 million such sequences for reference. These are clustered into ∼2.4M species hypotheses (SHs), each assigned a unique digital object identifier (DOI) to promote unambiguous referencing across studies. UNITE users have contributed over 600 000 third-party sequence annotations, which are shared with a range of databases and other community resources. Recent improvements facilitate the detection of cross-kingdom biological associations and the integration of undescribed groups of organisms into everyday biological pursuits. Serving as a digital twin for eukaryotic biodiversity and communities worldwide, the latest release of UNITE offers improved avenues for biodiversity discovery, precise taxonomic communication and integration of biological knowledge across platforms.

Modelling optimal ligninolytic activity during plant litter decomposition.

A large fraction of plant litter comprises recalcitrant aromatic compounds (lignin and other phenolics). Quantifying the fate of aromatic compounds is difficult, because oxidative degradation of aromatic carbon (C) is a costly but necessary endeavor for microorganisms, and we do not know when gains from the decomposition of aromatic C outweigh energetic costs. To evaluate these tradeoffs, we developed a litter decomposition model in which the aromatic C decomposition rate is optimized dynamically to maximize microbial growth for the given costs of maintaining ligninolytic activity. We tested model performance against > 200 litter decomposition datasets collected from published literature and assessed the effects of climate and litter chemistry on litter decomposition. The model predicted a time-varying ligninolytic oxidation rate, which was used to calculate the lag time before the decomposition of aromatic C is initiated. Warmer conditions increased decomposition rates, shortened the lag time of aromatic C oxidation, and improved microbial C-use efficiency by decreasing the costs of oxidation. Moreover, a higher initial content of aromatic C promoted an earlier start of aromatic C decomposition under any climate. With this contribution, we highlight the application of eco-evolutionary approaches based on optimized microbial life strategies as an alternative parametrization scheme for litter decomposition models.

Ectomycorrhizal fungi are more sensitive to high soil nitrogen levels in forests exposed to nitrogen deposition.

Ectomycorrhizal fungi are essential for nitrogen (N) cycling in many temperate forests and responsive to anthropogenic N addition, which generally decreases host carbon (C) allocation to the fungi. In the boreal region, however, ectomycorrhizal fungal biomass has been found to correlate positively with soil N availability. Still, responses to anthropogenic N input, for instance through atmospheric deposition, are commonly negative. To elucidate whether variation in N supply affects ectomycorrhizal fungi differently depending on geographical context, we investigated ectomycorrhizal fungal communities along fertility gradients located in two nemo-boreal forest regions with similar ranges in soil N : C ratios and inorganic N availability but contrasting rates of N deposition. Ectomycorrhizal biomass and community composition remained relatively stable across the N gradient with low atmospheric N deposition, but biomass decreased and the community changed more drastically with increasing N availability in the gradient subjected to higher rates of N deposition. Moreover, potential activities of enzymes involved in ectomycorrhizal mobilisation of organic N decreased as N availability increased. In forests with low external input, we propose that stabilising feedbacks in tree-fungal interactions maintain ectomycorrhizal fungal biomass and communities even in N-rich soils. By contrast, anthropogenic N input seems to impair ectomycorrhizal functions.

Metatranscriptomics sheds light on the links between the functional traits of fungal guilds and ecological processes in forest soil ecosystems.

Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.

The biological controls of soil carbon accumulation following wildfire and harvest in boreal forests: A review.

Boreal forests are frequently subjected to disturbances, including wildfire and clear-cutting. While these disturbances can cause soil carbon (C) losses, the long-term accumulation dynamics of soil C stocks during subsequent stand development is controlled by biological processes related to the balance of net primary production (NPP) and outputs via heterotrophic respiration and leaching, many of which remain poorly understood. We review the biological processes suggested to influence soil C accumulation in boreal forests. Our review indicates that median C accumulation rates following wildfire and clear-cutting are similar (0.15 and 0.20 Mg ha-1 year-1, respectively), however, variation between studies is extremely high. Further, while many individual studies show linear increases in soil C stocks through time after disturbance, there are indications that C stock recovery is fastest early to mid-succession (e.g. 15-80 years) and then slows as forests mature (e.g. >100 years). We indicate that the rapid build-up of soil C in younger stands appears not only driven by higher plant production, but also by a high rate of mycorrhizal hyphal production, and mycorrhizal suppression of saprotrophs. As stands mature, the balance between reductions in plant and mycorrhizal production, increasing plant litter recalcitrance, and ectomycorrhizal decomposers and saprotrophs have been highlighted as key controls on soil C accumulation rates. While some of these controls appear well understood (e.g. temporal patterns in NPP, changes in aboveground litter quality), many others remain research frontiers. Notably, very little data exists describing and comparing successional patterns of root production, mycorrhizal functional traits, mycorrhizal-saprotroph interactions, or C outputs via heterotrophic respiration and dissolved organic C following different disturbances. We argue that these less frequently described controls require attention, as they will be key not only for understanding ecosystem C balances, but also for representing these dynamics more accurately in soil organic C and Earth system models.

Do ectomycorrhizal exploration types reflect mycelial foraging strategies?

Ectomycorrhizal exploration types are commonly assumed to denote spatial foraging patterns and resource-related niches of extraradical mycelia. However, empirical evidence of the consistency of foraging strategies within exploration types is lacking. Here, we analysed ectomycorrhizal foraging patterns by incubating root-excluding ingrowth mesh bags filled with six different substrates in mature Picea abies forests. High-throughput sequencing was used to characterise ectomycorrhizal fungal communities in the mesh bags and on adjacent fine roots after one growing season. Contrary to expectations, many ectomycorrhizal genera of exploration types that are thought to produce little extraradical mycelium colonised ingrowth bags extensively, whereas genera commonly associated with ample mycelial production occurred sparsely in ingrowth bags relative to their abundance on roots. Previous assumptions about soil foraging patterns of exploration types do not seem to hold. Instead, we propose that variation in the proliferation of extraradical mycelium is related to intergeneric differences in mycelial longevity and the mobility of targeted resources.

Contrasting plant-soil-microbial feedbacks stabilize vegetation types and uncouple topsoil C and N stocks across a subarctic-alpine landscape.

Global vegetation regimes vary in belowground carbon (C) and nitrogen (N) dynamics. However, disentangling large-scale climatic controls from the effects of intrinsic plant-soil-microbial feedbacks on belowground processes is challenging. In local gradients with similar pedo-climatic conditions, effects of plant-microbial feedbacks may be isolated from large-scale drivers. Across a subarctic-alpine mosaic of historic grazing fields and surrounding heath and birch forest, we evaluated whether vegetation-specific plant-microbial feedbacks involved contrasting N cycling characteristics and C and N stocks in the organic topsoil. We sequenced soil fungi, quantified functional genes within the inorganic N cycle, and measured 15 N natural abundance. In grassland soils, large N stocks and low C : N ratios associated with fungal saprotrophs, archaeal ammonia oxidizers, and bacteria capable of respiratory ammonification, indicating maintained inorganic N cycling a century after abandoned reindeer grazing. Toward forest and heath, increasing abundance of mycorrhizal fungi co-occurred with transition to organic N cycling. However, ectomycorrhizal fungal decomposers correlated with small soil N and C stocks in forest, while root-associated ascomycetes associated with small N but large C stocks in heath, uncoupling C and N storage across vegetation types. We propose that contrasting, positive plant-microbial feedbacks stabilize vegetation trajectories, resulting in diverging soil C : N ratios at the landscape scale.

Ericoid shrubs shape fungal communities and suppress organic matter decomposition in boreal forests.

Mycorrhizal fungi associated with boreal trees and ericaceous shrubs are central actors in organic matter (OM) accumulation through their belowground carbon allocation, their potential capacity to mine organic matter for nitrogen (N) and their ability to suppress saprotrophs. Yet, interactions between co-occurring ectomycorrhizal fungi (EMF), ericoid mycorrhizal fungi (ERI), and saprotrophs are poorly understood. We used a long-term (19 yr) plant functional group manipulation experiment with removals of tree roots, ericaceous shrubs and mosses and analysed the responses of different fungal guilds (assessed by metabarcoding) and their interactions in relation to OM quality (assessed by mid-infrared spectroscopy and nuclear magnetic resonance) and decomposition (litter mesh-bags) across a 5000-yr post-fire boreal forest chronosequence. We found that the removal of ericaceous shrubs and associated ERI changed the composition of EMF communities, with larger effects occurring at earlier stages of the chronosequence. Removal of shrubs was associated with enhanced N availability, litter decomposition and enrichment of the recalcitrant OM fraction. We conclude that increasing abundance of slow-growing ericaceous shrubs and the associated fungi contributes to increasing nutrient limitation, impaired decomposition and progressive OM accumulation in boreal forests, particularly towards later successional stages. These results are indicative of the contrasting roles of EMF and ERI in regulating belowground OM storage.

A tipping point in carbon storage when forest expands into tundra is related to mycorrhizal recycling of nitrogen.

Tundra ecosystems are global belowground sinks for atmospheric CO2 . Ongoing warming-induced encroachment by shrubs and trees risks turning this sink into a CO2 source, resulting in a positive feedback on climate warming. To advance mechanistic understanding of how shifts in mycorrhizal types affect long-term carbon (C) and nitrogen (N) stocks, we studied small-scale soil depth profiles of fungal communities and C-N dynamics across a subarctic-alpine forest-heath vegetation gradient. Belowground organic stocks decreased abruptly at the transition from heath to forest, linked to the presence of certain tree-associated ectomycorrhizal fungi that contribute to decomposition when mining N from organic matter. In contrast, ericoid mycorrhizal plants and fungi were associated with organic matter accumulation and slow decomposition. If climatic controls on arctic-alpine forest lines are relaxed, increased decomposition will likely outbalance increased plant productivity, decreasing the overall C sink capacity of displaced tundra.

A group of ectomycorrhizal fungi restricts organic matter accumulation in boreal forest.

Boreal forest soils are important global carbon sinks, with significant storage in the organic topsoil. Decomposition of these stocks requires oxidative enzymes, uniquely produced by fungi. Across Swedish boreal forests, we found that local carbon storage in the organic topsoil was 33% lower in the presence of a group of closely related species of ectomycorrhizal fungi - Cortinarius acutus s.l.. This observation challenges the prevailing view that ectomycorrhizal fungi generally act to increase carbon storage in soils but supports the idea that certain ectomycorrhizal fungi can complement free-living decomposers, maintaining organic matter turnover, nutrient cycling and tree productivity under nutrient-poor conditions. The indication that a narrow group of fungi may exert a major influence on carbon cycling questions the prevailing dogma of functional redundancy among microbial decomposers. Cortinarius acutus s.l. responds negatively to stand-replacing disturbance, and associated population declines are likely to increase soil carbon sequestration while impeding long-term nutrient cycling.

Climate warming dominates over plant genotype in shaping the seasonal trajectory of foliar fungal communities on oak.

Leaves interact with a wealth of microorganisms. Among these, fungi are highly diverse and are known to contribute to plant health, leaf senescence and early decomposition. However, patterns and drivers of the seasonal dynamics of foliar fungal communities are poorly understood. We used a multifactorial experiment to investigate the influence of warming and tree genotype on the foliar fungal community on the pedunculate oak Quercus robur across one growing season. Fungal species richness increased, evenness tended to decrease, and community composition strongly shifted during the growing season. Yeasts increased in relative abundance as the season progressed, while putative fungal pathogens decreased. Warming decreased species richness, reduced evenness and changed community composition, especially at the end of the growing season. Warming also negatively affected putative fungal pathogens. We only detected a minor imprint of tree genotype and warming × genotype interactions on species richness and community composition. Overall, our findings demonstrate that warming plays a larger role than plant genotype in shaping the seasonal dynamics of the foliar fungal community on oak. These warming-induced shifts in the foliar fungal community may have a pronounced impact on plant health, plant-fungal interactions and ecosystem functions.

Root trait-microbial relationships across tundra plant species.

Fine roots, and their functional traits, influence associated rhizosphere microorganisms via root exudation and root litter quality. However, little information is known about their relationship with rhizosphere microbial taxa and functional guilds. We investigated the relationships of 11 fine root traits of 20 sub-arctic tundra meadow plant species and soil microbial community composition, using phospholipid fatty acids (PLFAs) and high-throughput sequencing. We primarily focused on the root economics spectrum, as it provides a useful framework to examine plant strategies by integrating the co-ordination of belowground root traits along a resource acquisition-conservation trade-off axis. We found that the chemical axis of the fine root economics spectrum was positively related to fungal to bacterial ratios, but negatively to Gram-positive to Gram-negative bacterial ratios. However, this spectrum was unrelated to the relative abundance of functional guilds of soil fungi. Nevertheless, the relative abundance of arbuscular mycorrhizal fungi was positively correlated to root carbon content, but negatively to the numbers of root forks per root length. Our results suggest that the fine root economics spectrum is important for predicting broader groups of soil microorganisms (i.e. fungi and bacteria), while individual root traits may be more important for predicting soil microbial taxa and functional guilds.

Fungal ecological strategies reflected in gene transcription - a case study of two litter decomposers.

Microbial communities interplay with their environment through their functional traits that can be a response or an effect on the environment. Here, we explore how a functional trait-the decomposition of organic matter, can be addressed based on genetic markers and how the expression of these markers reflect ecological strategies of two fungal litter decomposer Gymnopus androsaceus and Chalara longipes. We sequenced the genomes of these two fungi, as well as their transcriptomes at different steps of Pinus sylvestris needles decomposition in microcosms. Our results highlighted that if the gene content of the two species could indicate similar potential decomposition abilities, the expression levels of specific gene families belonging to the glycoside hydrolase category reflected contrasting ecological strategies. Actually, C. longipes, the weaker decomposer in this experiment, turned out to have a high content of genes involved in cell wall polysaccharides decomposition but low expression levels, reflecting a versatile ecology compare to the more competitive G. androsaceus with high expression levels of keystone functional genes. Thus, we established that sequential expression of genes coding for different components of the decomposer machinery indicated adaptation to chemical changes in the substrate as decomposition progressed.

Optimized metabarcoding with Pacific biosciences enables semi-quantitative analysis of fungal communities.

Recent studies have questioned the use of high-throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi-quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking. We used artificially assembled communities consisting of 10 ITS-like fragments of varying lengths and guanine-cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies. Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under-represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (> 447 bases) were lost when sequenced with Illumina MiSeq. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases related to GC content were observed. Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.

Rhizosphere allocation by canopy-forming species dominates soil CO2 efflux in a subarctic landscape.

In arctic ecosystems, climate change has increased plant productivity. As arctic carbon (C) stocks predominantly are located belowground, the effects of greater plant productivity on soil C storage will significantly determine the net sink/source potential of these ecosystems, but vegetation controls on soil CO2 efflux remain poorly resolved. In order to identify the role of canopy-forming species in belowground C dynamics, we conducted a girdling experiment with plots distributed across 1 km2 of treeline birch (Betula pubescens) forest and willow (Salix lapponum) patches in northern Sweden and quantified the contribution of canopy vegetation to soil CO2 fluxes and belowground productivity. Girdling birches reduced total soil CO2 efflux in the peak growing season by 53%, which is double the expected amount, given that trees contribute only half of the total leaf area in the forest. Root and mycorrhizal mycelial production also decreased substantially. At peak season, willow shrubs contributed 38% to soil CO2 efflux in their patches. Our findings indicate that C, recently fixed by trees and tall shrubs, makes a substantial contribution to soil respiration. It is critically important that these processes are taken into consideration in the context of a greening arctic because productivity and ecosystem C sequestration are not synonymous.

Accounting for environmental variation in co-occurrence modelling reveals the importance of positive interactions in root-associated fungal communities.

Understanding the role of interspecific interactions in shaping ecological communities is one of the central goals in community ecology. In fungal communities, measuring interspecific interactions directly is challenging because these communities are composed of large numbers of species, many of which are unculturable. An indirect way of assessing the role of interspecific interactions in determining community structure is to identify the species co-occurrences that are not constrained by environmental conditions. In this study, we investigated co-occurrences among root-associated fungi, asking whether fungi co-occur more or less strongly than expected based on the environmental conditions and the host plant species examined. We generated molecular data on root-associated fungi of five plant species evenly sampled along an elevational gradient at a high arctic site. We analysed the data using a joint species distribution modelling approach that allowed us to identify those co-occurrences that could be explained by the environmental conditions and the host plant species, as well as those co-occurrences that remained unexplained and thus more probably reflect interactive associations. Our results indicate that not only negative but also positive interactions play an important role in shaping microbial communities in arctic plant roots. In particular, we found that mycorrhizal fungi are especially prone to positively co-occur with other fungal species. Our results bring new understanding to the structure of arctic interaction networks by suggesting that interactions among root-associated fungi are predominantly positive.

Soil fertility in boreal forest relates to root-driven nitrogen retention and carbon sequestration in the mor layer.

Boreal forest soils retain significant amounts of carbon (C) and nitrogen (N) in purely organic layers, but the regulation of organic matter turnover and the relative importance of leaf litter and root-derived inputs are not well understood. We combined bomb 14 C dating of organic matter with stable isotope profiling for Bayesian parameterization of an organic matter sequestration model. C and N dynamics were assessed across annual depth layers (cohorts), together representing 256 yr of organic matter accumulation. Results were related to ecosystem fertility (soil inorganic N, pH and litter C : N). Root-derived C was estimated to decompose two to 10 times more slowly than leaf litter, but more rapidly in fertile plots. The amounts of C and N per cohort declined during the initial 20 yr of decomposition, but, in older material, the amount of N per cohort increased, indicating N retention driven by root-derived C. The dynamics of root-derived inputs were more important than leaf litter dynamics in regulating the variation in organic matter accumulation along a forest fertility gradient. N retention in the rooting zone combined with impeded mining for N in less fertile ecosystems provides evidence for a positive feedback between ecosystem fertility and organic matter turnover.

Exploring the role of ectomycorrhizal fungi in soil carbon dynamics.

The extent to which ectomycorrhizal (ECM) fungi enable plants to access organic nitrogen (N) bound in soil organic matter (SOM) and transfer this growth-limiting nutrient to their plant host, has important implications for our understanding of plant-fungal interactions, and the cycling and storage of carbon (C) and N in terrestrial ecosystems. Empirical evidence currently supports a range of perspectives, suggesting that ECM vary in their ability to provide their host with N bound in SOM, and that this capacity can both positively and negatively influence soil C storage. To help resolve the multiplicity of observations, we gathered a group of researchers to explore the role of ECM fungi in soil C dynamics, and propose new directions that hold promise to resolve competing hypotheses and contrasting observations. In this Viewpoint, we summarize these deliberations and identify areas of inquiry that hold promise for increasing our understanding of these fundamental and widespread plant symbionts and their role in ecosystem-level biogeochemistry.

Multiscale patterns and drivers of arbuscular mycorrhizal fungal communities in the roots and root-associated soil of a wild perennial herb.

Arbuscular mycorrhizal (AM) fungi form diverse communities and are known to influence above-ground community dynamics and biodiversity. However, the multiscale patterns and drivers of AM fungal composition and diversity are still poorly understood. We sequenced DNA markers from roots and root-associated soil from Plantago lanceolata plants collected across multiple spatial scales to allow comparison of AM fungal communities among neighbouring plants, plant subpopulations, nearby plant populations, and regions. We also measured soil nutrients, temperature, humidity, and community composition of neighbouring plants and nonAM root-associated fungi. AM fungal communities were already highly dissimilar among neighbouring plants (c. 30 cm apart), albeit with a high variation in the degree of similarity at this small spatial scale. AM fungal communities were increasingly, and more consistently, dissimilar at larger spatial scales. Spatial structure and environmental drivers explained a similar percentage of the variation, from 7% to 25%. A large fraction of the variation remained unexplained, which may be a result of unmeasured environmental variables, species interactions and stochastic processes. We conclude that AM fungal communities are highly variable among nearby plants. AM fungi may therefore play a major role in maintaining small-scale variation in community dynamics and biodiversity.

Soil microclimate changes affect soil fungal communities in a Mediterranean pine forest.

Soil microclimate is a potentially important regulator of the composition of plant-associated fungal communities in climates with significant drought periods. Here, we investigated the spatio-temporal dynamics of soil fungal communities in a Mediterranean Pinus pinaster forest in relation to soil moisture and temperature. Fungal communities in 336 soil samples collected monthly over 1 year from 28 long-term experimental plots were assessed by PacBio sequencing of ITS2 amplicons. Total fungal biomass was estimated by analysing ergosterol. Community changes were analysed in the context of functional traits. Soil fungal biomass was lowest during summer and late winter and highest during autumn, concurrent with a greater relative abundance of mycorrhizal species. Intra-annual spatio-temporal changes in community composition correlated significantly with soil moisture and temperature. Mycorrhizal fungi were less affected by summer drought than free-living fungi. In particular, mycorrhizal species of the short-distance exploration type increased in relative abundance under dry conditions, whereas species of the long-distance exploration type were more abundant under wetter conditions. Our observations demonstrate a potential for compositional and functional shifts in fungal communities in response to changing climatic conditions. Free-living fungi and mycorrhizal species with extensive mycelia may be negatively affected by increasing drought periods in Mediterranean forest ecosystems.