Re-examining the evidence for the mother tree hypothesis - resource sharing among trees via ectomycorrhizal networks.

Seminal scientific papers positing that mycorrhizal fungal networks can distribute carbon (C) among plants have stimulated a popular narrative that overstory trees, or 'mother trees', support the growth of seedlings in this way. This narrative has far-reaching implications for our understanding of forest ecology and has been controversial in the scientific community. We review the current understanding of ectomycorrhizal C metabolism and observations on forest regeneration that make the mother tree narrative debatable. We then re-examine data and conclusions from publications that underlie the mother tree hypothesis. Isotopic labeling methods are uniquely suited for studying element fluxes through ecosystems, but the complexity of mycorrhizal symbiosis, low detection limits, and small carbon discrimination in biological processes can cause researchers to make important inferences based on miniscule shifts in isotopic abundance, which can be misleading. We conclude that evidence of a significant net C transfer via common mycorrhizal networks that benefits the recipients is still lacking. Furthermore, a role for fungi as a C pipeline between trees is difficult to reconcile with any adaptive advantages for the fungi. Finally, the hypothesis is neither supported by boreal forest regeneration patterns nor consistent with the understanding of physiological mechanisms controlling mycorrhizal symbiosis.

Carbon-nitrogen relations of ectomycorrhizal mycelium across a natural nitrogen supply gradient in boreal forest.

The supply of carbon (C) from tree photosynthesis to ectomycorrhizal (ECM) fungi is known to decrease with increasing plant nitrogen (N) supply, but how this affects fungal nutrition and growth remains to be clarified. We placed mesh-bags with quartz sand, with or without an organic N (15 N-, 13 C-labeled) source, in the soil along a natural N supply gradient in boreal forest, to measure growth and use of N and C by ECM extramatrical mycelia. Mycelial C : N declined with increasing N supply. Addition of N increased mycelial growth at the low-N end of the gradient. We found an inverse relationship between uptake of added N and C; the use of added N was high when ambient N was low, whereas use of added C was high when C from photosynthesis was low. We propose that growth of ECM fungi is N-limited when soil N is scarce and tree belowground C allocation to ECM fungi is high, but is C-limited when N supply is high and tree belowground C allocation is low. This suggests that ECM fungi have a major role in soil N retention in nutrient-poor, but less so in nutrient-rich boreal forests.

Carbon benefits from Forest Transitions promoting biomass expansions and thickening.

The growth of the global terrestrial sink of carbon dioxide has puzzled scientists for decades. We propose that the role of land management practices-from intensive forestry to allowing passive afforestation of abandoned lands-have played a major role in the growth of the terrestrial carbon sink in the decades since the mid twentieth century. The Forest Transition, a historic transition from shrinking to expanding forests, and from sparser to denser forests, has seen an increase of biomass and carbon across large regions of the globe. We propose that the contribution of Forest Transitions to the terrestrial carbon sink has been underestimated. Because forest growth is slow and incremental, changes in the carbon density in forest biomass and soils often elude detection. Measurement technologies that rely on changes in two-dimensional ground cover can miss changes in forest density. In contrast, changes from abrupt and total losses of biomass in land clearing, forest fires and clear cuts are easy to measure. Land management improves over time providing important present contributions and future potential to climate change mitigation. Appreciating the contributions of Forest Transitions to the sequestering of atmospheric carbon will enable its potential to aid in climate change mitigation.

Greater carbon allocation to mycorrhizal fungi reduces tree nitrogen uptake in a boreal forest.

The central role that ectomycorrhizal (EM) symbioses play in the structure and function of boreal forests pivots around the common assumption that carbon (C) and nitrogen (N) are exchanged at rates favorable for plant growth. However, this may not always be the case. It has been hypothesized that the benefits mycorrhizal fungi convey to their host plants strongly depends upon the availability of C and N, both of which are rapidly changing as a result of intensified human land use and climate change. Using large-scale shading and N addition treatments, we assessed the independent and interactive effects of changes in C and N supply on the transfer of N in intact EM associations with -15 yr. old Scots pine trees. To assess the dynamics of N transfer in EM symbioses, we added trace amounts of highly enriched 5NO3(-) label to the EM-dominated mor-layer and followed the fate of the 15N label in tree foliage, fungal chitin on EM root tips, and EM sporocarps. Despite no change in leaf biomass, shading resulted in reduced tree C uptake, ca. 40% lower fungal biomass on EM root tips, and greater 15N label in tree foliage compared to unshaded control plots, where more 15N label was found in fungal biomass on EM colonized root tips. Short-term addition of N shifted the incorporation of 15N label from EM fungi to tree foliage, despite no significant changes in below-ground tree C allocation to EM fungi. Contrary to the common assumption that C and N are exchanged at rates favorable for plant growth, our results show for the first time that under N-limited conditions greater C allocation to EM fungi in the field results in reduced, not increased, N transfer to host trees. Moreover, given the ubiquitous nature of mycorrhizal symbioses, our results stress the need to incorporate mycorrhizal dynamics into process-based ecosystem models to better predict forest C and N cycles in light of global climate change.

Long-term declines in stream and river inorganic nitrogen (N) export correspond to forest change.

Human activities have exerted a powerful influence on the biogeochemical cycles of nitrogen (N) and carbon (C) and drive changes that can be a challenge to predict given the influence of multiple environmental stressors. This study focused on understanding how land management and climate change have together influenced terrestrial N storage and watershed inorganic N export across boreal and sub-arctic landscapes in northern Sweden. Using long-term discharge and nutrient concentration data that have been collected continuously for over three decades, we calculated the hydrologic inorganic N export from nine watersheds in this region. We found a consistent decline in inorganic N export from 1985 to 2011 over the entire region from both small and large watersheds, despite the absence of any long-term trend in river discharge during this period. The steepest declines in inorganic N export were observed during the growing season, consistent with the hypothesis that observed changes are biologically mediated and are not the result of changes in long-term hydrology. Concurrent with the decrease in inorganic N export, we report sustained increases in terrestrial N accumulation in forest biomass and soils across northern Sweden. Given the close communication of nutrient and energy stores between plants, soils, and waters, our results indicate a regional tightening of the N cycle in an already N-limited environment as a result of changes in forest management and climate-mediated growth increases. Our results are consistent with declining inorganic N efflux previously reported from small headwater streams in other ecosystems and shed new light on the mechanisms controlling these patterns by identifying corresponding shifts in the terrestrial N balance, which have been altered by a combination of management activities and climate change.

Forests trapped in nitrogen limitation--an ecological market perspective on ectomycorrhizal symbiosis.

Ectomycorrhizal symbiosis is omnipresent in boreal forests, where it is assumed to benefit plant growth. However, experiments show inconsistent benefits for plants and volatility of individual partnerships, which calls for a re-evaluation of the presumed role of this symbiosis. We reconcile these inconsistencies by developing a model that demonstrates how mycorrhizal networking and market mechanisms shape the strategies of individual plants and fungi to promote symbiotic stability at the ecosystem level. The model predicts that plants switch abruptly from a mixed strategy with both mycorrhizal and nonmycorrhizal roots to a purely mycorrhizal strategy as soil nitrogen availability declines, in agreement with the frequency distribution of ectomycorrhizal colonization intensity across a wide-ranging data set. In line with observations in field-scale isotope labeling experiments, the model explains why ectomycorrhizal symbiosis does not alleviate plant nitrogen limitation. Instead, market mechanisms may generate self-stabilization of the mycorrhizal strategy via nitrogen depletion feedback, even if plant growth is ultimately reduced. We suggest that this feedback mechanism maintains the strong nitrogen limitation ubiquitous in boreal forests. The mechanism may also have the capacity to eliminate or even reverse the expected positive effect of rising CO2 on tree growth in strongly nitrogen-limited boreal forests.

Are ectomycorrhizal fungi alleviating or aggravating nitrogen limitation of tree growth in boreal forests?

Symbioses between plant roots and mycorrhizal fungi are thought to enhance plant uptake of nutrients through a favourable exchange for photosynthates. Ectomycorrhizal fungi are considered to play this vital role for trees in nitrogen (N)-limited boreal forests. We followed symbiotic carbon (C)-N exchange in a large-scale boreal pine forest experiment by tracing (13) CO(2) absorbed through tree photosynthesis and (15) N injected into a soil layer in which ectomycorrhizal fungi dominate the microbial community. We detected little (15) N in tree canopies, but high levels in soil microbes and in mycorrhizal root tips, illustrating effective soil N immobilization, especially in late summer, when tree belowground C allocation was high. Additions of N fertilizer to the soil before labelling shifted the incorporation of (15) N from soil microbes and root tips to tree foliage. These results were tested in a model for C-N exchange between trees and mycorrhizal fungi, suggesting that ectomycorrhizal fungi transfer small fractions of absorbed N to trees under N-limited conditions, but larger fractions if more N is available. We suggest that greater allocation of C from trees to ectomycorrhizal fungi increases N retention in soil mycelium, driving boreal forests towards more severe N limitation at low N supply.

Nitrogen isotopes link mycorrhizal fungi and plants to nitrogen dynamics.

In this review, we synthesize field and culture studies of the 15N/14N (expressed as δ15N) of autotrophic plants, mycoheterotrophic plants, parasitic plants, soil, and mycorrhizal fungi to assess the major controls of isotopic patterns. One major control for plants and fungi is the partitioning of nitrogen (N) into either 15N-depleted chitin, ammonia, or transfer compounds or 15N-enriched proteinaceous N. For example, parasitic plants and autotrophic hosts are similar in δ15N (with no partitioning between chitin and protein), mycoheterotrophic plants are higher in δ15 N than their fungal hosts, presumably with preferential assimilation of fungal protein, and autotrophic, mycorrhizal plants are lower in 15N than their fungal symbionts, with saprotrophic fungi intermediate, because mycorrhizal fungi transfer 15N-depleted ammonia or amino acids to plants. Similarly, nodules of N2-fixing bacteria transferring ammonia are often higher in δ15N than their plant hosts. N losses via denitrification greatly influence bulk soil δ15N, whereas δ15N patterns within soil profiles are influenced both by vertical patterns of N losses and by N transfers within the soil-plant system. Climate correlates poorly with soil δ15N; climate may primarily influence δ15N patterns in soils and plants by determining the primary loss mechanisms and which types of mycorrhizal fungi and associated vegetation dominate across climatic gradients.

Allocation of carbon to fine root compounds and their residence times in a boreal forest depend on root size class and season.

Fine roots play a key role in the forest carbon balance, but their carbon dynamics remain largely unknown. We pulse labelled 50 m(2) patches of young boreal forest by exposure to (13)CO(2) in early and late summer. Labelled photosynthates were traced into carbon compounds of < 1 and 1-3 mm diameter roots (fine roots), and into bulk tissue of these and first-order roots (root tips). Root tips were the most strongly labelled size class. Carbon allocation to all size classes was higher in late than in early summer; mean residence times (MRTs) in starch increased from 4 to 11 months. In structural compounds, MRTs were 0.8 yr in tips and 1.8 yr in fine roots. The MRT of carbon in sugars was in the range of days. Functional differences within the fine root population were indicated by carbon allocation patterns and residence times. Pronounced allocation of recent carbon and higher turnover rates in tips are associated with their role in nutrient and water acquisition. In fine roots, longer MRTs but high allocation to sugars and starch reflect their role in structural support and storage. Accounting for heterogeneity in carbon residence times will improve and most probably reduce the estimates of fine root production.

Recovery of ectomycorrhiza after 'nitrogen saturation' of a conifer forest.

Trees reduce their carbon (C) allocation to roots and mycorrhizal fungi in response to high nitrogen (N) additions, which should reduce the N retention capacity of forests. The time needed for recovery of mycorrhizas after termination of N loading remains unknown. Here, we report the long-term impact of N loading and the recovery of ectomycorrhiza after high N loading on a Pinus sylvestris forest. We analysed the N% and abundance of the stable isotope (15) N in tree needles and soil, soil microbial fatty acid biomarkers and fungal DNA. Needles in N-loaded plots became enriched in (15) N, reflecting decreased N retention by mycorrhizal fungi and isotopic discrimination against (15) N during loss of N. Meanwhile, needles in N-limited (control) plots became depleted in (15) N, reflecting high retention of (15) N by mycorrhizal fungi. N loading was terminated after 20yr. The δ(15) N and N% of the needles decreased 6yr after N loading had been terminated, and approached values in control plots after 15yr. This decrease, and the larger contributions compared with N-loaded plots of a fungal fatty acid biomarker and ectomycorrhizal sequences, suggest recovery of ectomycorrhiza. High N loading rapidly decreased the functional role of ectomycorrhiza in the forest N cycle, but significant recovery occurred within 6-15yr after termination of N loading.

Quantification of effects of season and nitrogen supply on tree below-ground carbon transfer to ectomycorrhizal fungi and other soil organisms in a boreal pine forest.

SUMMARY: *The flux of carbon from tree photosynthesis through roots to ectomycorrhizal (ECM) fungi and other soil organisms is assumed to vary with season and with edaphic factors such as nitrogen availability, but these effects have not been quantified directly in the field. *To address this deficiency, we conducted high temporal-resolution tracing of (13)C from canopy photosynthesis to different groups of soil organisms in a young boreal Pinus sylvestris forest. *There was a 500% higher below-ground allocation of plant C in the late (August) season compared with the early season (June). Labelled C was primarily found in fungal fatty acid biomarkers (and rarely in bacterial biomarkers), and in Collembola, but not in Acari and Enchytraeidae. The production of sporocarps of ECM fungi was totally dependent on allocation of recent photosynthate in the late season. There was no short-term (2 wk) effect of additions of N to the soil, but after 1 yr, there was a 60% reduction of below-ground C allocation to soil biota. *Thus, organisms in forest soils, and their roles in ecosystem functions, appear highly sensitive to plant physiological responses to two major aspects of global change: changes in seasonal weather patterns and N eutrophication.

Short-term dynamics of abiotic and biotic soil 13CO2 effluxes after in situ 13CO2 pulse labelling of a boreal pine forest.

Physical diffusion of isotopic tracers into and out of soil pores causes considerable uncertainty for the timing and magnitude of plant belowground allocation in pulse-labelling experiments. Here, we partitioned soil CO(2) isotopic fluxes into abiotic tracer flux (physical return), heterotrophic flux, and autotrophic flux contributions following (13)CO(2) labelling of a Swedish Pinus sylvestris forest. Soil CO(2) efflux and its isotopic composition from a combination of deep and surface soil collars was monitored using a field-deployed mass spectrometer. Additionally, (13)CO(2) within the soil profile was monitored. Physical (abiotic) efflux of (13)CO(2) from soil pore spaces was found to be significant for up to 48 h after pulse labelling, and equalled the amount of biotic label flux over 6 d. Measured and modelled changes in (13)CO(2) concentration throughout the soil profile corroborated these results. Tracer return via soil CO(2) efflux correlated significantly with the proximity of collars to trees, while daily amplitudes of total flux (including heterotrophic and autotrophic sources) showed surprising time shifts compared with heterotrophic fluxes. The results show for the first time the significance of the confounding influence of physical isotopic CO(2)-tracer return from the soil matrix, calling for the inclusion of meaningful control treatments in future pulse-chase experiments.

No diurnal variation in rate or carbon isotope composition of soil respiration in a boreal forest.

Characterization of soil respiration rates and delta(13)C values of soil-respired CO(2) are often based on measurements at a particular time of day. A study by Gower et al. (2001) in a boreal forest demonstrated diurnal patterns of soil CO(2) flux using transparent measurement chambers that included the understory vegetation. It is unclear whether these diurnal patterns were solely the result of photosynthetic CO(2) uptake during the day by the understory or whether there were underlying trends in soil respiration, perhaps driven by plant root allocation, as recently demonstrated in Mediterranean oak savannah. We undertook intensive sampling campaigns in a boreal Picea abies L. Karst. forest to investigate whether diurnal variations in soil respiration rate and stable carbon isotope ratio (delta(13)C) exist in this ecosystem when no understory vegetation is present in the measurement chamber. Soil respiration rates and delta(13)C were measured on plots in which trees were either girdled (to terminate the fraction of soil respiration directly dependent on recent photosynthate from the trees), or not girdled, every 4 h over two 48-hour cycles during the growth season of 2004. Shoot photosynthesis and environmental parameters were measured concurrently. No diurnal patterns in soil respiration rates and delta(13)C were observed in either treatment, despite substantial variations in climatic conditions and shoot photosynthetic rates in non-girdled trees. Consequently, assessment of daily soil respiration rates and delta(13)C in boreal forest systems by single, instantaneous daily measurements does not appear to be confounded by substantial diurnal variation.

15 N natural abundance as a possible marker of the ectomycorrhizal habit of trees in mixed African woodlands.

The partial contribution by fixed nitrogen in N2 -fixing plants can be estimated if the 15 N natural abundance of non-N2 -fixing reference species, which derive their N from the soil, deviates from that of atmospheric N2 (the 15 N natural abundance method). Data from Tanzanian miombo woodland showed a significant difference (= 1.0-2.5 δ15 N%0 ) in 15 N abundance between ectomycorrhizal and vesicular-arbuscular (VA) mycorrhizal reference species. This finding casts doubt upon arbitrary selection of reference species, and also raises the possibility of using 15 N abundance as a marker of the ectomycorrhizal habit. It is suggested that the higher 15 N abundance of ectomycorrhizal tree species could result from differences in discrimination during uptake or from greater utilization of organic N.

Tansley Review No. 95 15 N natural abundance in soil-plant systems.

Equilibrium and kinetic isotope fractionations during incomplete reactions result in minute differences in the ratio between the two stable X isotopes, 15 N and 14 N, in various N pools. In ecosystems such variations (usually expressed in per mil [δ15 N] deviations from the standard atmospheric N2 ) depend on isotopic signatures of inputs and outputs, the input-output balance, N transformations and their specific isotope effects, and compartmentation of N within the system. Products along a sequence of reactions, e.g. the N mineralization-N uptake pathway, should, if fractionation factors were equal for the different reactions, become progressively depleted. However, fractionation factors van. For example, because nitrification discriminates against 15 N in the substrate more than does N mineralization, NH4 + can become isotopically heavier than the organic N from which it is derived. Levels of isotopic enrichment depend dynamically on the stoichiometry of reactions, as well as on specific abiotic and biotic conditions. Thus, the δ15 N of a specific N pool is not a constant, and 15 N of a N compound added to the system is not a conservative, unchanging tracer. This fact, together with analytical problems of measuring 15 N in small and dynamic pools of N in the soil-plant system, and the complexity of the X cycle itself (for instance the abundance of reversible reactions), limit the possibilities of making inferences based on observations of 15 N abundance in one or a few pools of N in a system. Nevertheless, measurements of δ15 N might offer the advantage of giving insights into the N cycle without disturbing the system by adding 15 N tracer. Such attempts require, however, that the complex factors affecting 15 N in plants be taken into account, viz. (i) the source(s) of N (soil, precipitation, NOX , NH3 , N2 -fixation), (ii) the depth(s) in soil from which N is taken up, (iii) the form(s) of soil-N used (organic N, NH4 + , NO3 - ), (iv) influences of mycorrhizal symbioses and fractionations during and after N uptake by plants, and (v) interactions between these factors and plant phenology. Because of this complexity, data on δ15 N can only be used alone when certain requirements are met, e.g. when a clearly discrete N source in terms of amount and isotopic signature is studied. For example, it is recommended that N in non-N2 -fixing species should differ more than 5% from N derived by N2 -fixation, and that several non-N2 -fixing references are used, when data on δ15 N are used to estimate Na -fixation in poorly described ecosystems. As well as giving information on N source effects, δ15 N can give insights into N cycle rates. For example, high levels of N deposition onto previously N-limited systems leads to increased nitrification, which produces 15 N-enriched NH4 and N-depleted NO3 . As many forest plants prefer NH4 - they become enriched in 15 N in such circumstances. This change in plant 15 N will subsequently also occur in the soil surface horizon after litter-fall, and might be a useful indicator of N saturation, especially since there is usually an increase in 15 N with depth in soils of N-limited forests. Generally, interpretation of 15 N measurements requires additional independent data and modelling, and benefits from a controlled experimental setting. Modelling will be greatly assisted by the development of methods to measure the 15 N of small dynamic pools of N in soils. Direct comparisons with parallel low tracer level 15 N studies will be necessary to further develop the interpretation of variations in 15 N in soil-plant systems. Another promising approach is to study ratios of 15 N: 14 N together with other pairs of stable isotopes, e.g. 13 C: 12 C or 18 O:16 O, in the same ion or molecules. This approach can help to tackle the challenge of distinguishing isotopic source effects from fractionations within the system studied. CONTENTS Summary 179 I. Introduction 180 II. Units, causes of isotope effects, stoichiometry, modelling 181 III. N dynamics and variations in 15 N abundance in soil-plant systems 183 IV. Applications 189 V. Conclusions and suggestions for future research 197 Acknowledgements 198 References 198.

Extramatrical ectomycorrhizal mycelium contributes one-third of microbial biomass and produces, together with associated roots, half the dissolved organic carbon in a forest soil.

• A large-scale tree-girdling experiment enabled estimates in the field of the contribution of extramatrical mycelium of ectomycorrhizal (ECM) fungi to soil microbial biomass and by ECM roots and fungi to production of dissolved organic carbon (DOC). • Tree-girdling was made early (EG) or late (LG) during the summer to terminate the flow of photosynthate to roots and ECM fungi. Determination of microbial C (Cmicr ) and microbial N in root-free organic soil was performed by using the fumigation-extraction technique; extractable DOC was determined on unfumigated soil. • Soil Cmicr was 41% lower on LG than on control plots 1 month after LG, whereas at the same time (that is, 3 months after EG), the Cmicr was 23% lower on EG than on control plots. Extractable DOC was 45% lower on girdled plots than control plots. • Our results, which are of particular interest as they were obtained directly in the field, clearly demonstrate the important contribution by extramatrical ECM mycelium to soil microbial biomass and by ECM roots to the production of DOC, a carbon source for other microbes.