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.

Vegetation, topography, and soil depth drive microbial community structure in two Swedish grasslands.

Soil microbial diversity and community composition are shaped by various factors linked to land management, topographic position, and vegetation. To study the effects of these drivers, we characterized fungal and bacterial communities from bulk soil at four soil depths ranging from the surface to below the rooting zone of two Swedish grasslands with differing land-use histories, each including both an upper and a lower catenary position. We hypothesized that differences in plant species richness and plant functional group composition between the four study sites would drive the variation in soil microbial community composition and correlate with microbial diversity, and that microbial biomass and diversity would decrease with soil depth following a decline in resource availability. While vegetation was identified as the main driver of microbial community composition, the explained variation was significantly higher for bacteria than for fungi, and the communities differed more between grasslands than between catenary positions. Microbial biomass derived from DNA abundance decreased with depth, but diversity remained relatively stable, indicating diverse microbial communities even below the rooting zone. Finally, plant-microbial diversity correlations were significant only for specific plant and fungal functional groups, emphasizing the importance of functional interactions over general species richness.

Fire severity as a key determinant of aboveground and belowground biological community recovery in managed even-aged boreal forests.

Changes in fire regime of boreal forests in response to climate warming are expected to impact postfire recovery. However, quantitative data on how managed forests sustain and recover from recent fire disturbance are limited.Two years after a large wildfire in managed even-aged boreal forests in Sweden, we investigated how recovery of aboveground and belowground communities, that is, understory vegetation and soil microbial and faunal communities, responded to variation in the severity of soil (i.e., consumption of soil organic matter) and canopy fires (i.e., tree mortality).While fire overall enhanced diversity of understory vegetation through colonization of fire adapted plant species, it reduced the abundance and diversity of soil biota. We observed contrasting effects of tree- and soil-related fire severity on survival and recovery of understory vegetation and soil biological communities. Severe fires that killed overstory Pinus sylvestris promoted a successional stage dominated by the mosses Ceratodon purpureus and Polytrichum juniperinum, but reduced regeneration of tree seedlings and disfavored the ericaceous dwarf-shrub Vaccinium vitis-idaea and the grass Deschampsia flexuosa. Moreover, high tree mortality from fire reduced fungal biomass and changed fungal community composition, in particular that of ectomycorrhizal fungi, and reduced the fungivorous soil Oribatida. In contrast, soil-related fire severity had little impact on vegetation composition, fungal communities, and soil animals. Bacterial communities responded to both tree- and soil-related fire severity. Synthesis: Our results 2 years postfire suggest that a change in fire regime from a historically low-severity ground fire regime, with fires that mainly burns into the soil organic layer, to a stand-replacing fire regime with a high degree of tree mortality, as may be expected with climate change, is likely to impact the short-term recovery of stand structure and above- and belowground species composition of even-aged P. sylvestris boreal forests.

Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons.

Fungal species participate in vast numbers of processes in the landscape around us. However, their cryptic mycelial growth, inside various substrates and in highly diverse species assemblages, has been a major obstacle to thorough analysis of fungal communities, hampering exhaustive description of the fungal kingdom. Technological developments allowing rapid, high-throughput sequencing of mixed communities from many samples at once are currently having a tremendous impact in fungal community ecology. Universal DNA extraction followed by amplification and sequencing of fungal species-level barcodes such as the nuclear internal transcribed spacer (ITS) region now enables identification and relative quantification of fungal community members across well-replicated experimental settings.Here, we present the sample preparation procedure presently used in our laboratory for fungal community analysis by high-throughput sequencing of amplified ITS2 markers. We focus on the procedure optimized for studies of total fungal communities in humus-rich soils, wood, and litter. However, this procedure can be applied to other sample types and markers. We focus on the laboratory-based part of sample preparation, i.e., the procedure from the point where samples enter the laboratory until amplicons are submitted for sequencing. Our procedure comprises four main parts: (1) universal DNA extraction, (2) optimization of PCR conditions, (3) production of tagged ITS amplicons, and (4) preparation of the multiplexed amplicon pool to be sequenced. The presented procedure is independent of the specific high-throughput sequencing technology used, which makes it highly versatile.

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.

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.

Reindeer control over subarctic treeline alters soil fungal communities with potential consequences for soil carbon storage.

The climate-driven encroachment of shrubs into the Arctic is accompanied by shifts in soil fungal communities that could contribute to a net release of carbon from tundra soils. At the same time, arctic grazers are known to prevent the establishment of deciduous shrubs and, under certain conditions, promote the dominance of evergreen shrubs. As these different vegetation types associate with contrasting fungal communities, the belowground consequences of climate change could vary among grazing regimes. Yet, at present, the impact of grazing on soil fungal communities and their links to soil carbon have remained speculative. Here we tested how soil fungal community composition, diversity and function depend on tree vicinity and long-term reindeer grazing regime and assessed how the fungal communities relate to organic soil carbon stocks in an alpine treeline ecotone in Northern Scandinavia. We determined soil carbon stocks and characterized soil fungal communities directly underneath and >3 m away from mountain birches (Betula pubescens ssp. czerepanovii) in two adjacent 55-year-old grazing regimes with or without summer grazing by reindeer (Rangifer tarandus). We show that the area exposed to year-round grazing dominated by evergreen dwarf shrubs had higher soil C:N ratio, higher fungal abundance and lower fungal diversity compared with the area with only winter grazing and higher abundance of mountain birch. Although soil carbon stocks did not differ between the grazing regimes, stocks were positively associated with root-associated ascomycetes, typical to the year-round grazing regime, and negatively associated with free-living saprotrophs, typical to the winter grazing regime. These findings suggest that when grazers promote dominance of evergreen dwarf shrubs, they induce shifts in soil fungal communities that increase soil carbon sequestration in the long term. Thus, to predict climate-driven changes in soil carbon, grazer-induced shifts in vegetation and soil fungal communities need to be accounted for.

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.

Root associated fungi respond more strongly than rhizosphere soil fungi to N fertilization in a boreal forest.

Nitrogen (N) fertilization is a routine practice in boreal forests but its effects on fungal functional guilds in Pinus sylvestris forests are still incompletely understood. Sampling is often restricted to the upper organic horizons and based on DNA extracted from mixtures of soil and roots without explicitly analysing different spatial niches. Fungal community structure in soil and roots of an 85-y-old Pinus sylvestris forest was investigated using high throughput sequencing. Fertilized plots had been treated with a single dose of N fertilizer, 15 months prior to sampling. Species richness of fungi colonizing roots was reduced in all horizons by N fertilization. In contrast, species richness of soil fungi in the organic horizon was increased by N fertilization, but unaffected in the mineral horizons. Community composition of fungi colonizing roots differed from that of soil fungi, and both communities were significantly influenced by soil horizon and N. The ectomycorrhizal community composition in both roots and soil was significantly affected by N fertilization but no significant effect was found on saprotrophic fungi. The results highlight the importance of analysing the rhizosphere soil and root compartments separately since the fungal communities in these two niches appear to respond differently to environmental perturbations involving the addition of nitrogen.

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.

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.

Contrasting effects of ectomycorrhizal fungi on early and late stage decomposition in a boreal forest.

Symbiotic ectomycorrhizal fungi have received increasing attention as regulators of below-ground organic matter storage. They are proposed to promote organic matter accumulation by suppressing saprotrophs, but have also been suggested to play an active role in decomposition themselves. Here we show that exclusion of tree roots and associated ectomycorrhizal fungi in a boreal forest increased decomposition of surface litter by 11% by alleviating nitrogen limitation of saprotrophs-a "Gadgil effect". At the same time, root exclusion decreased Mn-peroxidase activity in the deeper mor layer by 91%. Our results show that ectomycorrhizal fungi may hamper short-term litter decomposition, but also support a crucial role of ectomycorrhizal fungi in driving long-term organic matter oxidation. These observations stress the importance of ectomycorrhizal fungi in regulation of below-ground organic matter accumulation. By different mechanisms they may either hamper or stimulate decomposition, depending upon stage of decomposition and location in the soil profile.

Below-ground organic matter accumulation along a boreal forest fertility gradient relates to guild interaction within fungal communities.

Plant-soil interactions link ecosystem fertility and organic matter accumulation below ground. Soil microorganisms play a central role as mediators of these interactions, but mechanistic understanding is still largely lacking. Correlative data from a coniferous forest ecosystem support the hypothesis that interactions between fungal guilds play a central role in regulating organic matter accumulation in relation to fertility. With increasing ecosystem fertility, the proportion of saprotrophic basidiomycetes increased in deeper organic layers, at the expense of ectomycorrhizal fungal species. Saprotrophs correlated positively with the activity of oxidative enzymes, which in turn favoured organic matter turnover and nitrogen recycling to plants. Combined, our findings are consistent with a fungus-mediated feedback loop, which results in a negative correlation between ecosystem fertility and below-ground carbon storage. These findings call for a shift in focus from plant litter traits to fungal traits in explaining organic matter dynamics and ecosystem fertility in boreal forests.

Boreal Forests Sequester Large Amounts of Mercury over Millennial Time Scales in the Absence of Wildfire.

Alterations in fire activity due to climate change and fire suppression may have profound effects on the balance between storage and release of carbon (C) and associated volatile elements. Stored soil mercury (Hg) is known to volatilize due to wildfires and this could substantially affect the land-air exchange of Hg; conversely the absence of fires and human disturbance may increase the time period over which Hg is sequestered. Here we show for a wildfire chronosequence spanning over more than 5000 years in boreal forest in northern Sweden that belowground inventories of total Hg are strongly related to soil humus C accumulation (R2 = 0.94, p < 0.001). Our data clearly show that northern boreal forest soils have a strong sink capacity for Hg, and indicate that the sequestered Hg is bound in soil organic matter pools accumulating over millennia. Our results also suggest that more than half of the Hg stock in the sites with the longest time since fire originates from deposition predating the onset of large-scale anthropogenic emissions. This study emphasizes the importance of boreal forest humus soils for Hg storage and reveals that this pool is likely to persist over millennial time scales in the prolonged absence of fire.

Shift in fungal communities and associated enzyme activities along an age gradient of managed Pinus sylvestris stands.

Forestry reshapes ecosystems with respect to tree age structure, soil properties and vegetation composition. These changes are likely to be paralleled by shifts in microbial community composition with potential feedbacks on ecosystem functioning. Here, we assessed fungal communities across a chronosequence of managed Pinus sylvestris stands and investigated correlations between taxonomic composition and extracellular enzyme activities. Not surprisingly, clear-cutting had a negative effect on ectomycorrhizal fungal abundance and diversity. In contrast, clear-cutting favoured proliferation of saprotrophic fungi correlated with enzymes involved in holocellulose decomposition. During stand development, the re-establishing ectomycorrhizal fungal community shifted in composition from dominance by Atheliaceae in younger stands to Cortinarius and Russula species in older stands. Late successional ectomycorrhizal taxa correlated with enzymes involved in mobilisation of nutrients from organic matter, indicating intensified nutrient limitation. Our results suggest that maintenance of functional diversity in the ectomycorrhizal fungal community may sustain long-term forest production by retaining a capacity for symbiosis-driven recycling of organic nutrient pools.

Changes in turnover rather than production regulate biomass of ectomycorrhizal fungal mycelium across a Pinus sylvestris chronosequence.

In boreal forest soils, ectomycorrhizal fungi are fundamentally important for carbon (C) dynamics and nutrient cycling. Although their extraradical mycelium (ERM) is pivotal for processes such as soil organic matter build-up and nitrogen cycling, very little is known about its dynamics and regulation. In this study, we quantified ERM production and turnover, and examined how these two processes together regulated standing ERM biomass in seven sites forming a chronosequence of 12- to 100-yr-old managed Pinus sylvestris forests. This was done by determining ERM biomass, using ergosterol as a proxy, in sequentially harvested in-growth mesh bags and by applying mathematical models. Although ERM production declined with increasing forest age from 1.2 to 0.5 kg ha-1  d-1 , the standing biomass increased from 50 to 112 kg ha-1 . This was explained by a drastic decline in mycelial turnover from seven times to one time per year with increasing forest age, corresponding to mean residence times from 25 d up to 1 yr. Our results demonstrate that ERM turnover is the main factor regulating biomass across differently aged forest stands. Explicit inclusion of ERM parameters in forest ecosystem C models may significantly improve their capacity to predict responses of mycorrhiza-mediated processes to management and environmental changes.

Modelling the influence of ectomycorrhizal decomposition on plant nutrition and soil carbon sequestration in boreal forest ecosystems.

Tree growth in boreal forests is limited by nitrogen (N) availability. Most boreal forest trees form symbiotic associations with ectomycorrhizal (ECM) fungi, which improve the uptake of inorganic N and also have the capacity to decompose soil organic matter (SOM) and to mobilize organic N ('ECM decomposition'). To study the effects of 'ECM decomposition' on ecosystem carbon (C) and N balances, we performed a sensitivity analysis on a model of C and N flows between plants, SOM, saprotrophs, ECM fungi, and inorganic N stores. The analysis indicates that C and N balances were sensitive to model parameters regulating ECM biomass and decomposition. Under low N availability, the optimal C allocation to ECM fungi, above which the symbiosis switches from mutualism to parasitism, increases with increasing relative involvement of ECM fungi in SOM decomposition. Under low N conditions, increased ECM organic N mining promotes tree growth but decreases soil C storage, leading to a negative correlation between C stores above- and below-ground. The interplay between plant production and soil C storage is sensitive to the partitioning of decomposition between ECM fungi and saprotrophs. Better understanding of interactions between functional guilds of soil fungi may significantly improve predictions of ecosystem responses to environmental change.

Changes in fungal communities along a boreal forest soil fertility gradient.

Boreal forests harbour diverse fungal communities with decisive roles in decomposition and plant nutrition. Although changes in boreal plant communities along gradients in soil acidity and nitrogen (N) availability are well described, less is known about how fungal taxonomic and functional groups respond to soil fertility factors. We analysed fungal communities in humus and litter from 25 Swedish old-growth forests, ranging from N-rich Picea abies stands to acidic and N-poor Pinus sylvestris stands. 454-pyrosequencing of ITS2 amplicons was used to analyse community composition, and biomass was estimated by ergosterol analysis. Fungal community composition was significantly related to soil fertility at the levels of species, genera/orders and functional groups. Ascomycetes dominated in less fertile forests, whereas basidiomycetes increased in abundance in more fertile forests, both in litter and humus. The relative abundance of mycorrhizal fungi in the humus layer remained high even in the most fertile soils. Tolerance to acidity and nitrogen deficiency seems to be of greater importance than plant carbon (C) allocation patterns in determining responses of fungal communities to soil fertility, in old-growth boreal forests.