Does ectomycorrhiza have a universal key role in the formation of soil organic matter in boreal forests?

Journal Pre-proof Does ectomycorrhiza have a universal key role in the formation of soil organic matter in boreal forests? Mona N. Högberg, Ulf Skyllberg, Peter Högberg, Heike Knicker PII:

S0038-0717(19)30299-8

DOI:

https://doi.org/10.1016/j.soilbio.2019.107635

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SBB 107635

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Soil Biology and Biochemistry

Received Date: 12 August 2019 Revised Date:

18 October 2019

Accepted Date: 20 October 2019

Please cite this article as: Högberg, M.N., Skyllberg, U., Högberg, P., Knicker, H., Does ectomycorrhiza have a universal key role in the formation of soil organic matter in boreal forests?, Soil Biology and Biochemistry (2019), doi: https://doi.org/10.1016/j.soilbio.2019.107635. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Does ectomycorrhiza have a universal key role in the formation of soil organic matter in

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boreal forests?

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Written for Soil Biology and Biochemistry (virtual issue: “Microbial necromass on the rise in

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SOM: advances, challenges, and perspectives”).

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Mona N. Högberg1*, Ulf Skyllberg1, Peter Högberg1, Heike Knicker2

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1

Department of Forest Ecology and Management, SLU, SE-901 83 Umeå, Sweden

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Instituto de Recursos Naturales y Agrobiologia de Sevilla, Consejo Superior Investigaciones

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Cientificas (IRNAS-CSIC, ES-41012, Seville, Spain

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*Corresponding author

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E-mail addresses: [email protected] [email protected] [email protected]

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[email protected]

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Key words: Boreal forest; Ectomycorrhizal fungi;

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N;

C.

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C NMR;

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N NMR; SOM formation;

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Abstract

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Forest soil organic matter (SOM) is an important dynamic store of C and N, which releases plant

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available N and the greenhouse gases CO2 and N2O. Early stages of decomposition of recent

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plant litters are better known than the formation of older and more stable soil pools of N and C,

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in which case classic theory stated that selective preservation of more resistant plant compounds

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was important. Recent insights heighten that all plant matter becomes degraded and that older

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SOM consists of compounds proximally of microbial origin. It has been proposed that in boreal

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forests, ectomycorrhizal fungi (ECMF), symbionts of trees, are actively involved in the

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formation of slowly-degrading SOM.

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We characterized SOM in the mor-layer along a local soil N supply gradient in a boreal forest, a

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gradient with large variations in chemical and biological characteristics, notably a decline in the

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biomass of ECMF in response to increasing soil N supply.

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We found contrasting and regular patterns in carbohydrates, lignin, aromatic carbon, and in N-

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containing compounds estimated by solid-state 13C and 15N nuclear magnetic resonance (NMR)

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spectroscopy. These occurred along with parallel changes in the natural abundances of the stable

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isotopes

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“bomb-14C” age of the lower layers studied ranged between 15 years at the N-poor end, to 70

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years at the N-rich end of the gradient. On average half the increase in δ13C with soil depth (and

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hence age) of the mor-layer can be attributed to soil processes and the other half to changes in

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the isotopic composition of the plant C inputs. There was a decrease in carbohydrates (O-alkyl

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C) with increasing depth. This supports the classical hypothesis of declining availability of easily

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decomposable substrates to microorganisms with increasing soil depth and age. The observed

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increase in δ13C with depth, however, speaks against the idea of selective preservation of more

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resistant plant compounds like lignin. Furthermore, from the N-poor to the N-rich end the

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C and

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N in both bulk SOM and extracted fractions of the SOM. The modelled

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difference between

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decreased in parallel with a decline in ECMF.

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The latter provides evidence that the role of ECMF as major sink for N diminishes, and hence

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their potential role in SOM stabilization, when the soil N supply increases. At the N-rich end,

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where bacteria dominate over fungi, other agents than ECMF must be involved in the large

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build-up of the H-layer with the slowest turnover rate found along the gradient.

N in plant litter N and N in the deeper part of the mor-layer, the H-layer,

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1. Introduction

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The decomposition of soil organic material (SOM) was commonly described (Stevenson, 1994;

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Berg and McClaugherty, 2003, 2014) as a continuum of processes starting with inputs of plant

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litter, which loses CO2, releases nutrients, and partially ends up as old humic compounds deeper

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down in the soil profile. Compounds like lignin and other phenols were thought to resist

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microbial degradation and ultimately form humus, a process to which various depolymerisation

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and recondensation reactions can contribute (Stevenson, 1994). Now, the ideas prevail that

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virtually all plant compounds are degraded in the end and that well defined humic molecules do

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not exist (e.g., Burdon, 2001; von Lützow et al., 2006; Schmidt et al., 2011; Lehman et al.,

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2015). It has also been heightened that the slowly-turning over SOM is relatively rich in N, and

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that C-N bonds may be a reason for low degradability rather than the other way around (e.g.,

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Knicker, 2011).

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Moreover, detailed characterisation of old SOM suggests that it is proximally of microbial rather

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than of plant origin (e.g., Gleixner, 2013; Paul, 2016). An increase in the natural abundance of

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proposed to indicate that ectomycorrhizal fungi (ECMF), root symbionts of many woody

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species, are producing

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(Högberg et al., 1996; Lindahl et al., 2007; Clemmensen et al., 2013).

N with increasing depth (and hence age) of the organic mor-layer in coniferous forests has been

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N-rich materials involved in the build-up of slowly-turning over SOM

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The interplays among the supplies of C and N and the organisms involved appears to be

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complex. Initially, the supply of N stimulates decomposition of plant litter, but after a few years,

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nitrogen has a negative effect on decomposition (Berg and Matzner, 1997; Berg and

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McClaugherty, 2014). Furthermore, saprotrophic fungi or saprobes, dominate in the litter, but

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ECMF become dominant with increasing depth of the mor-layer (Lindahl et al., 2007;

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Clemmensen et al., 2013). There, the ECMF receive fresh tree-derived C in form of exudates

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supplying sugars and can actively retain and even sequester N not transferred to the trees

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(Näsholm et al., 2013). This mechanism of N immobilisation is especially important in N-poor

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ecosystems, because of the particularly high below-ground C allocation by trees (TBCA) in such

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settings (Högberg et al., 2010), but less significant at a high plant N supply (Näsholm et al.,

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2013; Hasselquist et al., 2016). Moreover, ECMF can produce phenol-oxidases, and thus

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promote the active degradation of lignin and similar compounds (e.g., Sterkenburg et al., 2018),

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which should be important especially at a high TBCA (i.e., in response to a high N

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immobilisation and low plant N supply, see Högberg et al., 2017).

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However, so far no method has enabled detailed tracing of plant litter compounds and their

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transformations by microbes to the several decades old SOM in the mor of boreal forests. Here,

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we describe the natural variations of the concentration of the stable isotopes 13C and 15N in bulk

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soil and SOM fractions with soil depth in the mor-layers of three common forest types in

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Fennoscandian boreal forests occurring along a single strong gradient of plant N supply. SOM

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fractions were used as they are operationally defined as fractions of SOM with different

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solubility in aqueous solution at different pH. In addition, we characterized the chemical

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structure and degree of decomposition of the SOM of the bulk soils and their fractions by

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and

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resonance (NMR) spectroscopy. To address the hypothesis whether ectomycorrhiza have a

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universal key role in the formation of soil organic matter in boreal forest, we discuss the data

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C

N solid-state cross polarization magic angle spinning (CPMAS) nuclear magnetic

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obtained in the light of changes in soil chemistry (notably variations in soil pH and N supply)

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and microbial community composition (especially mycorrhizal associations).

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2. Materials and methods

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2.1 Sampling site

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Soil samples were taken along a 90 m long transect (slope 2%), northwest of Betsele, in northern

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Sweden (64°39’ N, 18°30’ E, 235 m above sea level). This and other parallel transects through

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this boreal coniferous forest (Table 1) are described in detail by Högberg et al. (1990), Giesler et

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al. (1998) and Högberg et al. (2003, 2006b, 2007). The soil chemistry and the microbiological

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variables in Table 1 are stable according to various methodologies applied and repeated

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measurements. For example, the use of DNA sequencing (Högberg et al., 2014), signature lipid

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biomarker analysis (Högberg et al., 2003, 2007), growth of ECMF using ingrowth mesh bags

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(Nilsson et al., 2005), ergosterol analysis (Högberg, 2006), and counts of ECM root-tips

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(Hoffland et al., 2003; Toljander et al., 2006) all shows the same trend in ECMF abundance. The

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soils are uniform sandy till soils with many boulders and classified as Haplic Podzols (FAO,

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1988). The superficial organic mor-layer (O-horizon) was approximately 0.05 m thick

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throughout the transect, but varied in density, with a higher bulk density at the N-rich end of the

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transect. The S-layer, which is the uppermost layer, is usually not considered a part of the mor-

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layer, but can be considered as the starting point for plant litter decomposition, just as the L-

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(litter) layer is in other forests. Unlike an L-, the S-layer also includes forest floor vegetation like

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mosses and lichens, which are typical of nutrient-poor, open boreal forests. Under the S-layer,

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there is a layer traditionally designated the F-layer (Högberg et al., 2017), in which structural

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remains of plants can still be identified, although most is fragmentized. The transition from S- to

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the F-layer also marks the transition from a dominance by saprotrophic fungi to dominance by

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ECMF (Lindahl et al., 2007). In the lower part of the mor-layer, the H-layer, the plant material

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no longer can be identified and the SOM is amorphous. The colour often changes from mostly

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brown to blackish in the transition from F- to H-layers. The age of the SOM was estimated after

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“bomb-14C-analysis” (Harrison et al., 2000) to vary between 4 and 8 years in the S-layer and

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between 15 and 70 years in the H-layer (Fig. 1), with the highest age at the N-rich end of the N-

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supply gradient.

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In the N-poor, low plant productivity end of the transect with a plant field-layer dominated by

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ericaceous dwarf shrubs (forest type DS), the H-layer was very thin. It increased in thickness

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along the gradient, i.e. through a field-layer of short herbs (forest type SH) to the high-N end,

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where tall herbs dominate (forest type TH). The F-layer showed the reverse pattern, dominating

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in the N-poor end and decreasing in thickness towards the N-rich end.

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Hydrologically, the first 70 m of the transect is in a groundwater recharge area in an

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approximately 145 year old (in 2000) Pinus sylvestris forest with a site height index (tree height

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at 100 years) H100, of 17 m at the low N supply end (at 0 m). At 90 m, there is a discharge area

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and a dense Picea abies forest with a H100 of 28 m. The groundwater level may rise and fall

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several decimetres in a day. However, most of the summer the water table is > 0.7 m below the

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surface (Högberg et al., 2006b). Sampling was not conducted during or shortly after discharge

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events. The transect was divided into three parts according to the field-layer vegetation; DS from

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0 to 40 m, SH from 50 to 80 m and TH at 90 m. Hence, there was also a shift in the field-layer

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from ericoid mycorrhizal plants, to plants with arbuscular mycorrhizal in the direction from low

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to high soil N supply, along with a large decrease in the ratio fungi-to-bacteria in the F- and H-

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layers (Högberg et al., 2003, 2007, see Table 1). Picea abies is becoming the dominant trees

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species at about 40 m distance (Fig. 1). The canopy trees are ECM throughout the gradient.

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The total N pool in the mor-layer, on a kilogram per hectare basis, increased approximately 4.7

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times along the transect, whereas the increase in C was more modest (2.1 times). The NH4+ in

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soil solution increased from 0 to 80 m, but from 70 to 90 m NO3- became the most abundant

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inorganic form in the soil solution (Giesler et al., 1998). Based on traditional salt extraction, the

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quantity of amino acid-N exceeded that of inorganic-N in the DS type, was equal to inorganic-N

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in the SH type and lower than inorganic-N in the TH type (Nordin et al., 2001, recent studies in

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boreal forests suggest amino acids are likely more important in general, see Näsholm et al.,

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2009). We used a new transect for this study, to avoid contamination from previous tracer

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experiments with

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described by Giesler et al. (1998) and Högberg (2004). Thus, this study was conducted along a

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third transect established in 2000.

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N and

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C along the second transect laid out through the N supply gradient

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2.2 Sampling

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We sampled soils for SOM analysis on June 13, 2000, and for studies of microbial community

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structure on September 21, 2000. Five to seven samples of the mor-layer were taken every 10th

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meter along the gradient using a corer (inner diameter = 0.15 m). The thickness of the mor-layer

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was measured and its division into S-, F- and H-layers were made in situ. Five to 7 samples (per

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10 m location) were bulked together into three composite samples representing the S-, F- and H-

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layers, respectively. For the analysis of microbial community structure the F + H layer was

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sampled. The soil was sieved (5 mm mesh), remaining roots were picked out by hand and the dry

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mass of soil was determined after oven-drying (105°C, 24 h). Organic content was determined as

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loss-on-ignition (600 °C, 4 h). Dried soils were ground to a fine powder using a ball mill.

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2.3 Extraction of SOM fractions

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Conventional fractions of SOM, or humus (Paul, 2016), have been operationally defined as

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fulvic acid, humic acid and humin, lately including a more advanced classification based on their

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solubility in water at different pH (MacCarthy et al., 1990). Here, a method of Wolf et al. (1994)

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was used for the extraction of SOM fractions. Approximately 3.5 g of air dried soil was pre-

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treated in a centrifugation tube with 30 ml of 0.05 M HCl for 30 minutes and thereafter

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centrifuged. The supernatant was transferred to a flask. To wash the soil, thirty ml of distilled

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water was added and the soil-water mixture was centrifuged again as above. The supernatant was

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transferred into the same flask as before. Thirty ml of 0.5 M NaOH was added to the soil pellet

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and air was evacuated. The soil slurries were shaken overnight (18 hours) and centrifuged as

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above. Two more 30 ml portions of 0.5 M NaOH were added and the mixture was centrifuged as

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before after each addition. Then, 30 ml of water was added and after 10 minutes of shaking, the

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mixture was centrifuged. The total volume of supernatant fractions in the flask was 180 ml. The

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pellet consisted of the fraction insoluble at high pH (IH-fraction), conventionally named humin

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fraction. The alkaline supernatant fraction was then adjusted to pH = 1 using HCl and allowed to

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settle overnight. The mixture was centrifuged, after which the supernatant was transferred to a

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250 ml flask. Additional 30 ml of water was added to the pellet and the mixture was centrifuged

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again as above. The supernatant was decanted to the former flask with in total 220 ml of what

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was then classified as the soluble fraction not sensitive to pH (soluble-fraction), conventionally

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named fulvic acid. The fraction was not desalted, but the amounts of soluble-fraction reported

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are corrected for the weight contributed by NaCl. The remaining pellet consisted of the fraction

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insoluble at low pH (IL-fraction), c.f. humic acid. The soluble-fraction was reduced to about 30

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ml using a rotary evaporator.

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Thus, the IH-fraction is insoluble at high pH and the IL-fraction insoluble at low pH, while the

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solubility of the soluble fraction is insensitive to the pH changes. All three fractions were freeze-

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dried and kept in a desiccator until analysis. Standard peat soil BS103P from IHSS (International

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Humic Substance Society) was treated in the same way as the other samples. The recovery of C

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was calculated as the amount extracted C out of the total C in bulk soil. The yields of, or specific

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distribution of C into soluble-, IL-, and IH-fractions were expressed as the amount of C in each

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fraction multiplied by the weight yield of the fraction and normalized to the sum of the C yields

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of all three fractions (Kögel-Knabner et al., 1988).

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2.4 Stable isotope analysis

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Percentage N, δ15N (‰), percentage C and δ13C (‰) were analysed using an online C and N

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analyser coupled to an isotope ratio spectrometer (an ANCA-NT solid/liquid preparation module

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coupled to a model 20-20 isotope ratio mass spectrometer (IRMS), Europe Scientific Ltd. Crewe,

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U.K.) run in dual isotope mode (Ohlsson and Wallmark, 1999). Wheat flour was used as internal

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standard. The standard deviation based on samples was ± 0.3 ‰ for δ15N, ± 0.04 for % N, ± 0.5

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‰ for δ13C, and ± 1.1 for % C.

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The differences in isotopic compositions considered here are small. Thus, the δ value is used

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instead of the absolute isotope ratio,

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δ 13 C (‰) = 

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where R = 13C/12C for the sample or standard. The standard used for C, a limestone fossil from

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South Carolina, PDB, has thus been assigned a δ13C value of 0 ‰, while the absolute ratio

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(Rstandard) has been reported to be 0.01118 (Boutton, 1991). The δ15N (‰) is calculated using the

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same equation as for C, but the isotopic ratio of N2-gas in the atmosphere 0.0036765 (Mariotti,

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1983) is used as the standard ratio.

 (R sample − R standard )  × 1000 , R standard  

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2.5 NMR Spectroscopy

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The

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Bruker DSX 200 spectrometer located at the Technical University of Munich, Germany using, at

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a resonance frequency of 50.3 MHz using zirconium rotors (outside diameter = 7 mm) with caps

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C NMR spectra of bulk soils and SOM fractions at 0, 60, and 90 m were recorded on a

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made of polychlorotrifluoroethylene (PCTFE; trade name Kel-F). The cross-polarization magic

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angle spinning (CPMAS) technique was applied during magic-angle spinning of the rotor at 6.8

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kHz (Schaefer and Stejskal, 1976). A ramped 1H-pulse was used during the contact time in order

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to circumvent Hartman-Hahn mismatches. A contact time of 1 ms and a 90° degree 1H-pulse

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width of 5.5 µs were used for all spectra. The

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tetramethylsilane (at 0 ppm) and were adjusted with glycine (176.04 ppm). A pulse delay of 200

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or 250 ms between the single scans was used. Between 50 and 15 000 scans were accumulated.

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A line-broadening of 50 and 100 Hz was applied prior to Fourier transformation. For

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determination of the relative contribution of different carbon species, the NMR spectra of soils

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were divided into four major chemical shift regions: the alkyl C (region 1: 0 to 45 ppm); O/N-

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alkyl C (region 2: 45 to 110 ppm); aromatic/olefinic C (region 3: 110 to 160 ppm); carboxyl-,

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carbonyl-, and amide-C (region 4: 160 to 220 ppm). These regions were further assigned to

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different chemical shift ranges (Table 2) representing various possible carbon species (Knicker

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and Lüdemann, 1995; Dignac et al., 2002).

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We also report on a few solid-state 15N NMR spectra (two S-layer bulk samples from 0 and 90 m

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and the corresponding humus fractions). They were obtained on a Bruker DMX 400 located at

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the Chair of soil science at the Technical University of Munich and operating at 40.56 MHz. The

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cross-polarization technique with a ramped 1H pulse was used. The contact time was 1 ms, and a

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90° pulse width of 6.5 µs, a pulse delay of 200 ms. a line broadening between 100 and 150 Hz

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were applied. Around 1 million scans were accumulated at a magic-angle spinning speed of 5.0

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kHz. The chemical shift was standardized to the nitromethane scale (0 ppm) and adjusted with

239

15

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et al. (1993) and Knicker (2000). Due to the low signal-to-noise ratio of the solid-state 15N NMR

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spectra, they were not quantified.

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C-chemical shifts were calibrated to

N-labeled glycine (-347.6 ppm). The different integrals were assigned according to Witanowski

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2.6. Signature lipid fatty acid analysis

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Total lipids were extracted according to the method of Bligh and Dyer (1959), as modified by

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Frostegård et al. (1991), from fresh soil corresponding to 0.3 g organic matter (F + H layer).

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Lipids were dried in 1 mL portions using a cooled vacuum evaporation system at Umeå Plant

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Science Centre at Umeå University, Sweden, and then stored at -80 ºC before transportation on

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dry-ice to the Max Planck Institute for Biogeochemistry in Jena, Germany, for fractionation and

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subsequent steps and analysis, following the methods in Kramer and Gleixner (2006).

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3. Results

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3.1. SOM fractions and their distribution

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Using the IHSS standard, the recovery of C after the extraction procedure was 91 ± 14% and for

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N, 88 ± 14%. The ratio of C in soluble-fraction, IL-fraction, IH-fraction was 9 : 29 : 62 (c.f. ratio

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FA : HA : humin; Table 5 in Kögel-Knabner et al. (1988)). In all three layers, the IH-fraction

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dominated followed by IL- and soluble-fraction (Fig. 2). Throughout the gradient the amounts of

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C (in 3.5 g of soil) extracted were similar in the S- and F-layers, but then declined down to the

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H-layer by 33 – 58 %. The IL- and soluble-fraction C declined significantly from the F- to the H-

259

layer throughout the gradient (One-way ANOVA, P < 0.01, N = 9 – 10), whereas the IH-fraction

260

C did not. In the S-layer, the percentage contribution (means ± 1.0 SE, N = 3) by the three

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fractions remained stable throughout the three forest types (Fig. 2), with on average 9.6 (0.3),

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22.4 (0.4), and 68.0 (0.6) % for the soluble-, IL- and IH-fractions, respectively. The F- and H-

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layers showed some variability in IL-fraction distribution, but there was no significant difference

264

between the forest types or soil layers along the gradient (Fig. 2).

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3.2 Concentrations of N and C and isotopic analysis of bulk soil and SOM fractions

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The concentration of N of the bulk soil increased along the gradient from the DS type to the TH

268

type, whereas the organic C concentration remained rather constant, leading to a successive

269

decrease of the C/N ratios from slightly above 40 down to 16 (Figs. 3a-c). At 0 m, we found

270

some H-layer samples with very high C/N ratios (Fig. 3c), and which contained a lot of charcoal.

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These also differed in many respects from the other samples, and from previous samples of the

272

H-layer in similar positions at Betsele. Among the SOM fractions, the decline in C/N was most

273

evident in the IH-fraction, which showed the most drastic decline across the gradient and in all

274

layers. In the IH-fraction of the H-layer, the C/N ratio decreased from 60 at 10 m down to ca. 20

275

at 90 m, the N rich end of the gradient, whereas the C/N ratios of the IL- and soluble-fractions

276

decreased from c. 30 to 10 from 0 to 90 m. For IL-fraction in the F- and H-layers, the

277

concentrations of C and N were negatively correlated (R = -0.91, N = 7, P <0.01, and R = -0.97,

278

N = 7, P < 0.000, respectively).

279

In general, the δ13C in the bulk soil and SOM fractions increased by around 2 to 3 ‰ with soil

280

depth (Figs. 4a-d). The δ13C increased in the order IH-, IL-, and soluble-fraction for all layers;

281

the δ13C of soluble-fraction was enriched by 0.5 to 2 ‰ compared to IL- and IH-fractions in the

282

S-, F-, and H-layers.

283

The 15N abundance of the S-layer, in particular, increased towards the N-rich end of the gradient

284

and with soil depth (Figs. 5a-d). Simultaneously, the F- and the H-layer δ15N was rather constant

285

whereas the H-layer was enriched by approx. 2 ‰ compared to the F-layer. The difference in

286

δ15N between the S- and H-layers was at most 6‰, which occurred in the DS type at the N-poor

287

end, but declined to 2‰ in the TH type in the N-rich end. At most positions the IL was the

288

fraction most enriched in δ15N and IH the least enriched fraction (difference 1 to 4‰), except at

289

90 m (i.e., in the TH type), where differences were small.

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C NMR and

15

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3.3 Solid state

N NMR spectroscopic analysis of bulk soil and SOM

292

fractions

293

The mor-layer soils are dominated by the O-alkyl (-C-O-C-) carbon signal at 72 ppm (Fig. 6,

294

Table 2). The ratio alkyl carbon (-C-C-) to carboxyl carbon (-COOH) is mostly < 3, that means

295

less than three times higher abundance of alkyl C compared with carboxyl C (C ratio 3 : 1)

296

(Tables 2, S1), indicating an average chain length of a maximum of 4 carbon atoms (3 alkyl C +

297

1 carboxyl C). This indicates a considerable contribution of peptide structures or short-chain

298

acids to the total organic C fraction. The aromatic region has signals from tannins, lignins and

299

other substances containing phenol groups contributing to the signal at 141 ppm. This signal

300

becomes clearer with depth, indicating a relative enrichment of phenolic structures.

301

Carboxyl C also increased with increasing soil depth and in the direction of the N-rich (TH) end

302

of the gradient in bulk soil, IL- and IH-fractions (Fig. 6, Table 2). This peak, “carboxyl C”,

303

contains some N in amides (Knicker and Lüdemann, 1995). As indicated by the solid-state 15N

304

NMR spectra, the amide N (signal at -260 ppm), most likely originate from peptide structures of

305

biogenic origin (Knicker, 2011), and is the major organic N form both in the bulk soil but also in

306

the IL fractions (Fig. S1). O-alkyl C, denoting sugars, on the other hand, decreased with depth

307

from S-, through F- to the H-layer in the bulk soil in DS (note that H-layer sample at 0 m

308

contains charcoal), SH, and TH (Table 2). A similar trend with depth was seen among the SOM

309

fractions. O-alkyl C was more abundant in the IH-fraction from DS and SH, than in IH-fraction

310

from TH. As regards aromatic carbon (O- and N-aryl C), higher levels were commonly found

311

across layers in TH as compared to in DS and SH, with the exception of the high value found for

312

the charcoal containing sample in DS. In all forest types, carbonyl-C increased with soil depth in

313

both bulk soil and IH-fraction, but not clearly so in the IL-fraction. The IL-fraction was

314

relatively rich in carboxyl C and aromatic C, whereas the IH-fraction contained relatively more

315

O-alkyl C (Fig. 6).

Page 13 of 34

316 317

3.4. Correlations among the measured soil parameters and their relations to microbial

318

community structure

319

In order to identify general trends and relations among parameters, we made a series of

320

regression analyses using the bulk soil data for the S-, F- and H-layers across the gradient.

321

Strong relationships were found among percentage of C contributed by specific compound

322

classes, some ratios thereof, δ13C, and δ15N.

323

The O-alkyl C and aromatic C were negatively related (R2adj = 0.680, P = 0.003, N = 9; Fig. 7a).

324

The δ13C increased as O-alkyl carbon decreased (R2adj = 0.864, P < 0.001, N = 9; Fig. 7b). In

325

contrast, there was, as expected from the above, a positive relationship between δ13C and

326

aromatic C (R2adj= 0.541, P < 0.014, N = 9; Fig. 7 d). Similarly, the δ15N increased along with a

327

decrease in O-alkyl C (R2adj = 0.838, P < 0.001, N = 9; Fig. 7c) and an increase in aromatic C

328

and (R2adj = 0.467, P = 0.025, N = 9; Fig. 7e).

329

The δ15N of IL-fraction in the H-layer was negatively related to soil N % (R2adj = 0.879, P <

330

0.000, N = 8) and decreased with decreasing fungi-to-bacteria ratio and fungal abundance (Table

331

1, Fig. 8). Meanwhile, in the S-layer the δ15N of the IL-fractions were positively related to N %

332

(IL-fraction, R2adj = 0.735, N = 9, P = 0.002, IH-fraction, R2adj = 0.568, N = 9, P = 0.011, and

333

soluble-fraction, R2adj = 0.789, N = 9, P < 0.000). From the above it is clear that there should also

334

be a strong positive relationship between δ13C and δ15N and this was the case for all SOM

335

fractions and bulk soil (R2adj = 0.507 to 0.752, N = 26, P = 0.001 to 0.019, Table S2).

336 337

4. Discussion

338

The gradient studied encompasses most of the variation in soil pH and N % found in mor-layers

339

of Fennoscandian boreal forests (Högberg et al., 2017). Thus, this study has bearings on a much

340

larger area, but has the advantage that the relevant variations in soils occur at one location. Page 14 of 34

341

Moreover, we have conventional soil chemistry data from several proximal, parallel transects at

342

this location (Högberg et al. 1990; Giesler et al. 1998; Högberg et al., 2003, 2006b), and also

343

detailed data on the microbial community composition (Högberg et al., 2003, 2006ab, 2014).

344

Bulk soil data on pH, C/N, δ13C, δ15N and signature lipid biomarkers of functional groups of

345

microorganisms, were similar to data from previous studies. The only exceptions worth

346

mentioning are the sample from the H-layer at 0 m, which was rich in charcoal, and the relatively

347

low δ13C of SOM at 90 m, which was not seen previously (Högberg et al., 2005). These

348

deviations from what we have seen earlier have no major importance for our interpretations in

349

general, as follows.

350

We observed 1) changes in the SOM (humus) fractions with soil depth and along the gradient, 2)

351

changes in compounds classified by NMR spectroscopy, such as aromatic C, carboxyl and O-

352

alkyl C, 3) changes in the natural abundances of stable isotopes of C and N with soil depth and

353

along the gradient, and 4) large changes in the microbial community composition along the

354

gradient. In the following, we will discuss how these changes support or refute the idea that

355

ECMF are actively involved in the formation of slowly turning over SOM.

356

This hypothesis is primarily based on the observation that ECMF are highly enriched in

357

(Gebauer and Dietrich, 1993; Handley et al., 1996; Taylor et al., 1997, 2003), and that the

358

increasing enrichment of

359

1989, Högberg et al., 1996) is associated with the occurrence and function of ECM symbiosis

360

(Högberg et al., 1996, 2011; Billings and Richter, 2006; Lindahl et al., 2007; Wallander et al.,

361

2009, Clemmensen et al., 2013; Blasko et al., 2015). More specifically, Lindahl et al., (2007)

362

showed that during decomposition of needle litter, the δ15N did not change during the first years,

363

a phase dominated by saprotrophic fungi, but the δ15N increased rapidly when ECMF took over

364

the dominance. This is consistent with the observation that ECMF become enriched by 15N, and

365

pass

15

15

15

N

N with soil depth (e.g., Nadelhoffer and Fry, 1988; Melillo et al.,

N-depleted N to their plant host (Högberg et al., 1999; Hobbie et al., 2009; Hobbie and

Page 15 of 34

366

Högberg, 2012). Importantly, our characterization of the microbial community structure (which

367

is in line with previous analyses, Table 1) by the analysis of PLFA in the F + H-layers,

368

demonstrates a phase when ECMF have totally taken over the dominance from the saprotrophic

369

fungi (Lindahl et al., 2007).

370

Hence, the very clear decrease in the difference in δ15N between the S-layer and the H-layer

371

along the gradient from the N-poor to the N-rich end (Figs. 5d, 8), suggests that the role of ECM

372

symbiosis in redistributing the stable N isotopes diminishes as the contribution from these fungi

373

to the microbial community declines (Table 1). This provides indirect evidence that ECMF may

374

be involved in the formation of slowly-turning SOM (e.g., Högberg et al., 1996; Lindahl et al.,

375

2007; Clemmensen et al., 2013), especially in direction towards the N-poor end of the gradient,

376

where ECMF dominates the soil microbial community. However, we can also observe i) that the

377

soil with the oldest SOM, the H-layer at 90 m (Fig. 1), has a very low fungi-to-bacteria ratio and

378

low ECMF abundance, and ii) that the general increase in δ15N by 2‰ from F- to the H-layer

379

cannot be explained by the mechanism of ECM-plant redistribution of

380

questions the idea of a universal key role for ECMF in the formation of SOM in boreal forests. In

381

fact, in the bacteria-dominated soil at 90 m, we find not only the oldest SOM (Fig. 1), but also

382

the largest accumulation of soil C (Table 1). This is the N-richest community, with the most

383

productive forest, and hence the largest litter C input (Högberg et al., 2003), which is important,

384

but of significance is also the low biomass of ECMF. We would like to stress that the microbial

385

biomass C changes very little along the gradient, which means that the decline in the ratio fungi-

386

to-bacteria is correlated with a real decline in fungal biomass.

387

Do our more detailed chemical analyses give any insights about the role of ECMF in the

388

development of SOM? A strong negative correlation was found between the biomass of ECMF

389

biomarker across the gradient and the δ15N signature in the uppermost S-layer (Fig. 8) i.e., the

390

layer reflecting the plant signature, which is affected by the transfer of N from the soil through

Page 16 of 34

15

N alone (Fig. 8). This

391

ECMF (e.g., Hobbie and Högberg, 2012). Across the gradient and soil depths we also found a

392

negative correlation between O-alkyl C and δ15N in bulk soil (Fig. 7c), suggesting a greater

393

contribution by ECMF as the SOM is losing O-alkyl C i.e., SOM is decomposed and lose the

394

more easily available C. At 0 m, O-alkyl C decreased from 52 to 22 % of organic matter C when

395

δ15N increased with depth by 6.0 ‰, whereas at 90 m, O-alkyl C decreased from 43 to 32 % and

396

δ15N increased only 1.8 ‰. Despite the large differences in change in the two parameters, all

397

data from the three different forest types and soil depths fell on the same regression line in Fig.

398

7c (R2adj > 0.8). Notably, this does not prove that ECMF are the agents of the change in O-alkyl

399

C abundance. The relation may simply be there, because ECMF take over when saprotrophs,

400

which have broken down carbohydrates or O-alkyl C, lose the competition for nutrients with the

401

ECMF (Lindahl et al., 2002).

402

As regards variations in δ13C, the picture is much less clear, although we observe, as others, an

403

increase in δ13C with increasing soil depth (Nadelhoffer and Fry, 1988; Gebauer and Schulze,

404

1991; Boström et al., 2007). However, there are some couplings between the abundance of 13C

405

and some components of SOM, but the changes in δ13C are smaller than the changes in δ15N and

406

there is a lack of an alleged agent of change like the ECMF in the case of δ15N. Nadelhoffer and

407

Fry (1988) proposed four mechanisms that could explain the enrichment of 13C with increasing

408

soil depth. First, an overall discrimination against

409

during organic matter decomposition. Second, differential preservation of components enriched

410

in 13C. Third, changes through time from litter inputs with high δ13C values to litter inputs with

411

lower values because of increasing concentrations of depleted fossil-fuel 13CO2 over time (e. g.,

412

Rubinho et al., 2013), and lastly, illuviation of 13C enriched dissolved organic matter into lower

413

soil layers. Ehleringer et al. (2000) and others expanded these earlier hypotheses by suggesting

414

that the frequently observed progressive δ13C enrichment of SOM with soil depth may be related

415

to a gradual shift in the relative contributions of microbial vs. plant components in the residual

13

C (thus respiration of

Page 17 of 34

13

C-depleted CO2)

416

SOM and not to differential SOM degradation or to microbial isotope fractionation during

417

decomposition. Subsequent research has shown that the relation between the signatures of soil-

418

respired CO2 and SOM are not always in one direction, and that there may be considerable

419

variations among compounds in their δ13C (Hobbie and Werner, 2004).

420

We observe that δ13C increases 1.6 - 4.5 ‰ with increasing soil depth (Fig. 4d). The highest

421

value was found at 0 m, the sample with charcoal in the H-layer, and which seems to be an

422

anomaly also with regard to the high C/N ratio (see above) and the low age of the H-layer (Fig.

423

1). If this sample is discarded, the increase in δ13C is 1.6 - 2.8 ‰. A significant portion of this

424

increase should not be attributed to soil processes, but rather to changes in the isotopic signature

425

of the C fixed through photosynthesis. First, there is the decline in δ13C of around 0.015 ‰ year-1

426

of the atmospheric CO2 due to anthropogenic emissions of isotopically light CO2 (the so called

427

Suess-effect, Ehleringer et al., 2000; Rubino et al., 2013). Second, there is also an additional

428

effect of increasing fractionation against 13CO2 during photosynthesis in C3 plants as the [CO2]

429

increases, with an effect of around -0.014 ‰ ppm-1 (Keeling et al., 2017). Given the average

430

increase in atmospheric [CO2] during the period most relevant here (1960 to 1990; the S-layer

431

contains C which was fixed from the atmosphere on average 7 years earlier as compared to 33

432

years for the H-layer) this would mean an average decrease of 0.02 ‰ year-1. Third, there may

433

actually be an added effect related to tree age-related processes; Betson et al. (2007) found a

434

decline of 0.03 ‰ year-1 in current needles of trees during a period of thirty years (this decline

435

would include the process described by Keeling et al., 2017). Thus, altogether we would expect a

436

total decline of 0.045 ‰ year-1 of the δ13C of the plant C inputs. With an estimated difference in

437

age between the S- and H-layers of between 17 and 64 years, this would cause changes in the

438

δ13C with depth of 0.8 - 2.9 ‰, which account for 56 ± 4 % of the variations with depth. Hence,

439

such changes in the isotopic signal of the plant C inputs explain the whole change with soil depth

440

at the N-rich end of the gradient, but less at the N-poor end (Table 1 and Fig. 4).

Page 18 of 34

441

In most cases, the soluble-fraction is the humus fraction with the highest δ13C. As regards the

442

bulk soil, δ13C is negatively correlated with O-alkyl C, but positively correlated with aromatic C.

443

In the laboratory, bacterial cultures respire C depleted in

444

source or the total cell (Blair et al., 1985; Macko et al., 1987). Gleixner et al. (1993) analysed 13C

445

of primary and secondary products from different cell compartments and that of decomposing

446

basidiomycete sporocarps and concluded that saprophytic fungi preferentially metabolise

447

isotopically “light” glucose molecules with respect to

448

preferentially used to make cell wall polymers. Ågren et al. (1996) presented field data (from an

449

arable soil without crops), where the δ13C of SOM increased as soil C content decreased with

450

time. This also suggests that microbes respire C depleted in

451

which leads to 13C-enrichment of the remaining SOM.

452

However, typical of the N-poor boreal forest soils, is the large contribution by roots and their

453

associated microorganisms, chiefly ECMF (Högberg and Högberg, 2002), to both respiration

454

(Högberg et al., 2001), and SOM, which also needs consideration in this context. When the total

455

soil CO2 efflux (i.e., combining root respiration, including that of ECMF, and the respiration by

456

decomposers) was measured in the field along the N supply gradient, the δ13C of the CO2 was 5

457

‰ heavier than the H-layer SOM at the N-poor end, as compared to 2 ‰ at the N-rich end

458

(Högberg et al., 2004). This difference is explained by the greater tree below-ground allocation

459

of C in response to the low N supply at the N-poor end. It also raises the possibility that the C

460

contributed by roots and ECMF to SOM may have an isotopic signature different from that of

461

above-ground litter.

462

If more readily decomposable compounds, e.g., carbohydrates and proteins, are metabolised first,

463

there will be an accumulation of lignin, lipids and waxes, which are more depleted in δ13C (Stout

464

et al., 1981, Benner et al., 1987). As regards conifer needles, lignin and lipids were depleted by

465

1-2‰ relative to the carbohydrates (Gleixner et al., 1993). Moreover, Kögel-Knabner et al.

Page 19 of 34

13

C by 2 to 3.4 ‰ compared to the C

13

C, whereas “heavy” molecules are

13

C as decomposition proceeds,

466

(1988) and Kögel-Knabner (2002) expected the major portion of biodegraded lignin to be solved

467

in 0.5 M NaOH and depleted lignin C should thus be found in IL- and soluble-fraction. However,

468

these fractions showed higher δ13C enrichments than the IH-fraction. Furthermore, if selective

469

preservation of lignin prevails, as stated by classic theory, the δ13C would not increase with

470

depth, which it does. Thus, our data speak against the idea of selective preservation of lignin.

471

What about potential contributions of fungal material to the variations in δ13C in SOM? The

472

fungal wall constitutes up to 30 % of the cellular dry weight and is well characterized (De Nobel

473

et al., 2001). The classical cell wall fractionation procedures often include alkali extraction

474

followed by neutralization. Alkali-soluble polymers from a variety of fungi include proteins,

475

glycosylated proteins like mannoprotein, and some α-(1,3)-D-glucan. The dominant alkali-

476

insoluble carbohydrate polymers include β-(1,3)-D-glucan and β-(1,6)-D-glucan, chitin

477

(acetylated aminosugar), chitosan (non-acetylated aminosugar), polyglucoronic acid, and

478

cellulose. Of all these cell wall components, the alkali-insoluble polymers β-(1,3)-D-glucan, β-

479

(1,6)-D-glucan, chitin, chitosan, polyglucoronic acid and cellulose (Debono and Gordee, 1994)

480

are most likely found in the IH-fraction c.f. humin fraction (MacCarthy et al., 1990). The

481

separation into alkali-soluble and alkali-insoluble components, however, is not fully clear

482

because alkali-soluble glucans are often cross-linked to chitin and other alkali-insoluble

483

macromolecules (Wessels, 1993). However, chitin should be enriched in

484

compared to the carbohydrates (Gleixner et al., 1993), which means that the contribution by

485

chitin to the

486

from fungi using C isotope signatures is further complicated by the fact that saprotrophic fungi

487

are in general more enriched in

488

contribute greatly to the formation of SOM at the N-rich end, where their biomass is very low

489

and bacteria dominate. Thus, further detailed exploration of soil biology and biochemistry under

13

13

C by 1 to 2‰

C-depleted humin fraction cannot be large. The possibilities to trace any imprint

13

C than ECMF (Taylor et al., 2003). Finally, fungi cannot

Page 20 of 34

490

such conditions clearly deserve much further attention (e.g., Knicker et al., 1995ab; Dignac et al.,

491

2002; Knicker, 2011).

492 493

5. Conclusions

494

We found several strong correlations among the chemical composition and the stable isotope

495

signatures of both N and C in the SOM. These variations e.g., that of O-alkyl C, reflecting

496

decomposition of easily degradable carbohydrates, were associated with SOM age-dependent

497

processes as well as with differences in soil N supply. Apart from the very clear effect of ECMF,

498

direct recipients of recent plant sugars on the δ15N of SOM, we found no unambiguous indicator

499

enabling tracing the effect of a specific agent over the decades of ageing of the SOM studied.

500

The considerable enrichment in

501

ECMF are involved in forming slowly turning over SOM under conditions of low soil N supply.

502

At higher N supply, other agents must be involved.

15

N with soil depth and age provides indirect evidence that

503 504

Acknowledgements

505

We thank Christian Brun for help in the field and in the laboratory, Douglas D. Harkness for

506

performing the 14C analysis and the ‘bomb-14C’ age model calculations, Håkan Wallmark for

507

performing the 13C and 15N analyses, Karin Ljung for technical advice, and Christiane Kramer

508

and Gerd Gleixner for the PLFA analysis. This work was supported by The Swedish Research

509

Council Formas (2016-01658), The Swedish Research Council (621-2014-5356), and the

510

Swedish University of Agricultural Sciences (KoN grants).

511

Page 21 of 34

512

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Figure legends

756

Figure 1. Modelled C age (years) of mor-layers along the Betsele N supply gradient. S-layer (unfilled

757

circles) represents the uppermost layer composed of a mix of bottom-layer mosses or lichens and plant

758

litter. H-layer (filled circles) is the lowest, most highly decomposed part of the mor-layer. The insert

759

illustrates the forest types across the gradient: dwarf-shrub (DS) between 0 and 40 m, short-herb (SH)

760

between 50 and 80 m and tall-herb (TH) at 90 m distance.

761 762

Figure 2. Per cent distribution of humus layer C among SOM fractions across the N supply gradient at

763

Betsele. SOM fractions are operationally defined by their solubility/insolubility in aqueous solution.

764

Soluble fraction is insensitive to pH, whereas the IL- and IH-fractions are insoluble at low and high pH,

765

respectively.

766 767

Figure 3. Soil C/N ratio of bulk soil and the three SOM fractions along the Betsele N supply gradient.

768

Soluble-fraction (circles), IL-fraction (triangles), IH-fraction (squares), bulk soil (diamonds). a) S-layer,

769

b) F-layer, c) H-layer.

770 771

Figure 4. Natural abundance of 13C (‰) of the bulk soil and the three SOM fractions soluble-, IL-, and

772

IH-fraction along the N supply gradient. Soluble-fraction (circles), IL-fraction (triangles), IH-fraction

773

(squares), bulk soil (diamonds). a) S-layer, b) F-layer, c) H-layer, and d) bulk soil.

774 775

Figure 5. Natural abundance of 15N (‰) of bulk soil and the three SOM fractions soluble-, IL-, and IH-

776

fraction along the Betsele N supply gradient. Soluble-fraction (circles), IL-fraction (triangles), IH-fraction

777

(squares), bulk soil (diamonds). The 15N signature of atmospheric N2 (0 ‰) is indicated. a) S-layer, b) F-

778

layer, c) H-layer, and d) bulk soil.

779 780

Figure 6. Percent distribution (% of organic C) of C species in bulk soil and SOM fractions in the S-, F-,

781

and H-layer across the N supply gradient. Note that the figure shows the charcoal containing sample (H-

782

layer at 0 m) discussed in the text.

Page 33 of 34

783 13

C and

15

784

Figure 7. Relationship between natural abundance of

N in bulk soil at 0, 60 and 90 m and

785

specific compound classes (chemical shift range) in the 13C NMR spectra. O-alkyl C (60-110 ppm) and

786

aromatic C (110-160 ppm). Symbol followed by an asterisk (*) indicates the charcoal containing H-layer

787

sample at 0 m. Relationship between a) aromatic C and O-alkyl C, b) δ13C and O-alkyl C, c) δ15N and O-

788

alkyl C, d) δ13C and aromatic C, and e) δ15N and aromatic C.

789 790

Figure 8. Natural abundance of 15N in S-, F-, and H-layer soil and contribution by PLFA 18:2ω6,9 to the

791

soil microbial community, measured as mol % of the total amount of PLFAs. This signature lipid is a

792

good biomarker of ectomycorrhizal fungi across the N supply gradient (Näsholm et al. 2013, and

793

references therein, note also the declining number of ECM root-tips per soil volume in Table 1).

Page 34 of 34

Table 2. Organic carbon species in bulk soils and SOM fractions for the S-, F-, and H-layers in dwarf shrub (DS), short herb (SH), and tall herb (TH) forest type, sampled at 0, 60, and 90 m respectively, along the N supply gradient at Betsele in N. Sweden. Carbon species are determined by solid state 13C nuclear magnetic resonance (13C CPMAS NMR) spectroscopy analysis. For quantification, the spectra were divided into different chemical shift regions assigned to tentative C species (Knicker and Lüdemann, 1995; Dignac et al., 2002). Data are relative signal distributions (% of total spectrum area). The sample highlighted in italics contains charcoal.

Alkyl C Sample

Layer

Distance (m)

N-alkyl C O-alkyl C

Chemical shift range (ppm) 0-45 45-60 60-110

Aryl C

Carboxyl C

O/N-aryl

Amide-C

Carbonyl C

110-160

160-185

185-220

Bulk soil

S S S F F F H H H

0 60 90 0 60 90 0 60 90

20 18 17 20 19 21 13 19 22

7 8 9 7 8 10 4 8 9

52 47 44 44 39 40 21 34 32

15 18 20 20 21 19 48 24 22

5 6 7 6 8 8 9 10 11

1 2 3 2 4 3 5 5 5

IL

S S S F F F H H H

0 60 90 0 60 90 0 60 90

36 29 25 33 25 25 19 24 31

9 9 10 8 10 10 5 9 7

23 20 24 26 25 24 15 25 15

22 27 25 22 24 24 44 25 27

8 12 13 9 12 13 15 14 15

2 4 4 3 4 4 3 3 5

IH

S S S F F F H H H

0 60 90 0 60 90 0 60 90

16 18 15 19 20 18 14 22 19

7 9 9 6 9 10 3 8 8

57 50 47 46 43 41 16 32 28

13 17 20 19 18 21 52 23 28

5 6 7 7 8 9 9 8 13

2 1 2 4 3 3 6 7 4

Table 1. Characteristics of the mor-layer soil (F + H layer) along the 90-m-long N supply gradient from a dwarf shrub type, through a short herb type to a tall herb type, near Betsele, N. Sweden (pH and C/N data are from Högberg et al., 2007, Ctot, Ntot data and gross N mineralisation rate are from Högberg et al. (2006), and microbial data are from Högberg et al. (2003) and Hoffland et al. (2003). Data are means (± 1.0 SE). Seasonal means (N = 3 – 4) are given except for soil pH and C/N (n = 9), total C and N (n = 24), and number of ectomycorrhizal (ECM) root-tips at 0, 65, and 90 m (n = 8).

Parameter

Dwarf shrub (0 – 40 m)

Short herb (50 – 80 m)

Tall herb (90 m)

pHH20

4.0 (0.1)

4.6 (0.1)

5.3 (0.1)

C/N ratio

38.1 (2.4)

22.9 (1.1)

14.9 (0.3)

Ctot (kg/ha)

12,754 (435)

12,679 (510)

26,515 (1695)

312 (10)

529 (10)

1,472 (94)

Ntot (kg/ha) -1

-1

Gross N mineralisation rate (kg ha day )

0.3 (0.1)

1.1 (0.3)

4.1 (1.1)

-1

10.9 (2.2)

10.2 (3.5)

11.0 (3.5)

-1

Microbial biomass N (mg g OM)

0.9 (0.1)

1.4 (0.3)

2.3 (0.1)

Microbial C/N

11.7 (2.0)

6.9 (1.6)

4.8 (1.3)

0.44 (0.10)

0.18 (0.02)

0.02 (0.00)

ECM root-tips per soil surface area (cm )

170 (15)

77 (15)

20 (4)

Microbial C out of total soil C (%)

1.42 (0.06)

1.45 (0.29)

1.45 (0.18)

Microbial N out of total soil N (%)

7.6 (1.0)

6.9 (1.3)

7.3 (0.4)

Microbial biomass C (mg g OM)

Ratio fungi/bacteria -2

Highlights •

Ectomycorrhizal fungi (ECMF) have been proposed as key agents of SOM formation.

•

We show that this may occur in N-poor boreal soils.

•

The largest buildup of SOM occurred at low abundance of ECMF in N-rich soils.