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Received Date : 15-Dec-2014 Accepted Date : 04-Feb-2015 Article type

: Primary Research Articles

Anthropogenic nitrogen deposition enhances carbon sequestration in boreal soils

NADIA I. MAAROUFI*1, ANNIKA NORDIN‡, NILES J. HASSELQUIST*, LISBET H.

BACH†, KRISTIN PALMQVIST†, and MICHAEL J. GUNDALE* *Department of Forest Ecology and Management, Swedish University of Agricultural Sciences (SLU), SE-901, Umeå, Sweden, ‡ Umeå Plant Science Center (UPSC), Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden, † Department of Ecology and Environmental Science (EMG), Umeå University, SE-901 87 Umeå, Sweden

1

Corresponding Author

Email: [email protected] Phone: +46 90 786 86 25 Fax: +46 90 786 81 63

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/gcb.12904 This article is protected by copyright. All rights reserved.

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Keywords: carbon sequestration, nitrogen deposition, boreal soil, soil respiration, soil C pool, boreal forest, carbon sink.

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Abstract It is proposed that carbon (C) sequestration in response to reactive nitrogen (Nr) deposition in

boreal forests accounts for a large portion of the terrestrial sink for anthropogenic CO2 emissions. While studies have helped clarify the magnitude by which Nr deposition enhances C sequestration by forest vegetation, there remains a paucity of long-term experimental studies evaluating how soil C pools respond. We conducted a long-term experiment, maintained since 1996, consisting of three N addition levels (0, 12.5, and 50 kg N ha-1 yr-1) in the boreal zone of northern Sweden in order to understand how atmospheric Nr deposition affects soil C accumulation, soil microbial communities, and soil respiration. We hypothesized that soil C sequestration will increase, and soil microbial biomass and soil respiration will decrease, with disproportionately large changes expected compared to low levels of N addition. Our data showed that the low N addition treatment caused a non-significant increase in the organic horizon C pool of ~15%, and a significant increase of ~30% in response to the high N treatment relative to the control. The relationship between C sequestration and N addition in the organic

horizon was linear, with a slope of 10 kg C kg-1 N. We also found a concomitant decrease in total microbial and fungal biomasses, and a ~11% reduction in soil respiration in response to the high N treatment. Our data complement previous data from the same study system describing aboveground C sequestration, indicating a total ecosystem sequestration rate of 26 kg C kg-1 N.

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These estimates are far lower than suggested by some previous modelling studies, and thus will help improve and validate current modelling efforts aimed at separating the effect of multiple global change factors on the C balance of the boreal region.

Introduction During the past century, anthropogenic activities such as the production and use of nitrogen (N) fertilizers and the combustion of fossil-fuels have greatly increased the quantity of reactive nitrogen (Nr) released into the atmosphere, which subsequently enters the biosphere via

deposition (IPCC, 2013). Many ecosystems in cold climate regions, such as boreal forests, are strongly N limited due to low rates of biological N2 fixation and slow soil mineralization rates

(Tamm, 1991; Vitousek & Howarth, 1991; Lindo et al., 2013). Thus increased inputs of anthropogenically derived Nr (NHx and NOy) have the potential to enhance productivity and carbon (C) sequestration in these systems (De Vries et al., 2006; Gruber & Galloway, 2008; Schlesinger, 2009; De Vries, 2014; Fernández-Martínez et al., 2014). In the last decade several studies have focused on quantifying the ratio of C sequestered per unit N deposition in the northern latitudes in order to understand the impacts of Nr deposition on the global C cycle.

Some modeling and experimental studies have suggested that as much as 500 parts of C are sequestered in plant biomass per unit of N deposition, which would account for a large portion of annual anthropogenic CO2 emissions (Holland et al., 1997; Magnani et al., 2007). In contrast,

long term forest N fertilization experiments have shown that N deposition causes a much lower quantity of C to be sequestered in boreal, temperate and tropical vegetation, ranging from 5-30 parts C per unit of N deposition (Högberg et al., 2006; Hyvönen et al., 2008; Pregitzer et al.,

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2008; Sutton et al., 2008; de Vries et al., 2009; de Vries et al., 2014; Gundale et al., 2014). While these studies help to clarify the magnitude by which Nr deposition enhances C sequestration by forest vegetation, there remains a paucity of studies evaluating whether corresponding changes in soil C pools occur in these ecosystems (Högberg et al., 2006; Pregitzer et al., 2008; de Vries et al., 2014), which is important when predicting how net ecosystem C

balances will respond to Nr deposition. Boreal forest ecosystems covers approximately 15% of terrestrial land surface area, and

store a substantial quantity of C in its aboveground biomass (≈88 Pg of C); however, to an even greater extent they store C belowground (≈471 Pg of C) (Malhi et al., 1999; Lal, 2005).

Therefore gains or losses of soil C in response to Nr deposition could substantially influence the

magnitude to which forest ecosystems in the northern latitudes sequester C (Mack et al., 2004; De Vries et al., 2006; Janssens et al., 2010). The limited number of studies evaluating the long term impacts of chronic N enrichment on soil C sequestration have shown that Nr enrichment can enhance C storage in both temperate and boreal soils (Högberg et al., 2006; Hyvönen et al., 2008; Pregitzer et al., 2008). Pregitzer et al., (2008) showed more C sequestrated in soil than vegetation in northern temperate forest in response to long term chronic N enrichment , while Högberg et al. (2006) and Hyvönen et al. (2008) found the opposite trend with more C sequestrated in vegetation than soil in boreal forests. However, uncertainty remains regarding the magnitude to which Nr deposition enhances C sequestration in soils because previous studies

estimating this relationship have applied N at rates several times greater than maximum N deposition rates in each region of study. Furthermore, N addition experiments are often located in areas with high background Nr deposition rates, making it experimentally difficult to isolate the impact of actual Nr deposition (de Vries et al., 2009).

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While there remains uncertainty regarding the magnitude to which soil C pools change in response to Nr deposition, there also remains uncertainty regarding key mechanisms underlying

these changes (Janssens et al., 2010). One of several mechanisms by which Nr deposition is

thought to enhance soil C accumulation is by negatively impacting soil microbial biomass, composition, and activity (Treseder, 2008; Janssens et al., 2010). Several studies have shown reductions in microbial biomass and activity to high doses of N fertilizers (50-100 kg N ha-1 yr1

), with greater decreases in fungal than bacterial biomass (Högberg et al., 2007; Demoling et al.,

2008) or a concomitant decrease in both bacterial and fungal biomass (Treseder, 2008; Blaško et al., 2013). Likewise, several studies in temperate and boreal ecosystems have shown that the applications of high doses of N fertilizers (> 50kg N ha-1 yr-1) can have negative effects on total soil respiration (i.e. autotrophic and heterotrophic combined) (Olsson et al., 2005; Janssens et al., 2010), or a decline in microbial abundance and biomass (Treseder, 2008). Interestingly, recent studies have shown that relatively low rates of N addition can have contrasting effects relative to higher N doses. For example, several studies have shown that the addition of relatively low quantities of N (20 kg N ha-1 yr-1) over short time scales can have positive effect on autotrophic soil respiration (including tree root and mycorrhizal fungi components) (Hasselquist et al.,

2012), as well as decomposition rates (Allison et al., 2009). These studies highlight that low

chronic N addition rates (i.e. 2 mm) in the mineral soil across the study area. To estimate the stone volume from each pit, we first sieved (2 mm) the soil removed from each pit, and then placed the coarse material removed by the sieve in a large plastic box containing water where we quantified the volume of water displaced by this material. We then estimated the soil volume by subtracting the stone and boulder volumes from the total pit volume. We estimated the quantity of soil C and N present in each plot 16 years after the start of

the experiment by multiplying the mass of each soil core by the C or N concentration. These values were then scaled up to a surface area basis (Mg C ha-1) using the core surface area, and

then corrected using the stoniness constant (32.6% stones), as described above. Estimates from each plot were derived from the average of 5 subsamples within each plot.

Microbial and fungal composition Soil microbial communities were analyzed using the microbial phospholipid fatty acids method (PLFA). In summer 2011 (15 years after the start of the experiment), three organic horizon samples (i.e. the humus layer) were taken from each 0.1 ha plot (n=5), and sieved as described above. The samples were frozen immediately after sieving, until they were freeze-dried. The PLFAs were extracted from 1g (wet mass) subsamples of each soil sample using a modified method of Bligh and Dyer (Bligh & Dyer, 1959; White et al., 1979; Mcintosh et al., 2012). The abundance of PLFAs was quantified using a Perkin Elmer Clarus 500 gas chromatogragh (Waltham, Massachusetts, USA), and was converted to micromoles PLFA per gram of organic

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matter using conventional nomenclature (Tunlid et al., 1989). Different types of PLFAs represent diverse functional groups. PLFAs 18:2ω6 were used to estimate the contribution of fungi, while the branched fatty acids 10me16:0, 10me17:0 and 10me18:0 were used to estimate actinomycetes contribution. Bacterial PLFAs including i-15:0, α-15:0, 15:0, i-16:0, 16:1ω9, 16:1ω7, 16:0, i-17:0, cy-17:0, α-17:0, 18:1ω7, cy-19:0 were used to represent total bacteria. Gram positive bacteria were represented by branched fatty acids i-15:0, α-15:0, i-16:0, i-17:0 and α-17:0, while cy-17:0, cy-19:0 and 18:1ω7 were used as a measure of gram negative bacteria.

Soil respiration measurements In order to estimate total soil respiration (i.e. autotrophic and heterotrophic respiration combined), we measured soil respiration every three weeks between May 30th and October 10th

2013, resulting in a total of seven sampling events. Soil respiration was measured between 9:00 am and 4:30 pm and the order in which plots were measured was changed at each sampling time with the intention of evenly dispersing any diurnal variation in these measurements evenly across treatments (Betson et al., 2007). The measurements were made by establishing five cylindrical collars in each 0.25 ha plot

(n=5). The collars (25 cm diameter, 10 cm high) were permanently inserted ca. 1 cm into the soil Oe horizon, and aboveground vegetation inside and 5 cm around the collar were removed in order to eliminate plant respiration. The collars were allowed to equilibrate for three days after they were set up before the first measurement. Additionally, before the first measurements, the height from soil surface to rim was measured in four cardinal dimensions within each collar in order to calculate the headspace volume. Soil respiration (CO2 efflux) was measured from the

linear rate of CO2 accumulation within sealed cylindrical headspaces. For each measurement,

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soil collars were covered by a removable plastic lid that contained an opening for the placement of a solid-state CO2 sensor (CARBOCAP model GMP 343, Vaisala, Finland). Directly after the closure of the lid, the buildup of CO2 within the headspace was monitored for 3 min, where after

the lid was removed. To prevent the development of a vertical CO2 gradient within the headspace, the lid was equipped with a fan. Individual respiration measurements were corrected to account for differences in air temperature and headspace volume among the different collars. Immediately following measurements of soil respiration, measurements of soil temperature (Model E514, Mingle Instrument, Willich, Germany) were taken inside each collar.

Statistical analyses All response variables were tested for normality and homoscedasticity prior to statistical analyses. Data were transformed using [Log (x+1)] when these assumptions were not met. Soil C and N contents, soil bulk density, O-horizon depth (see appendix), mean C-efflux, and PLFA data were tested to compare differences among N addition levels (0, 12.5, 50 kg N ha-1 yr-1)

using one-way analysis of variances (ANOVAs) with N addition levels used as a fixed factor and block as a random factor whenever significant. When significant differences between N addition levels were detected (α=0.05), post hoc pairwise comparisons between treatments were

conducted using the Student-Newman-Keuls test (Zar, 1999). In order to model the quantitative impact of N addition on C sequestration in the organic

horizon of our study system, we first subtracted all plot data by the mean organic horizon C pool per ha of the control plots. We then divided these values by the number of years where the treatments were applied, providing kg C sequestered per ha per year relative to the control. We then performed a linear regression using N addition rate as the independent variable, and relative

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C sequestration rate per year as the dependent variable. The slope of this regression indicated the quantity of C sequestered in the organic horizon per unit N added. For PLFA data, we first performed a detrended correspondence analysis (DCA) to be able

to choose between linear and unimodal methods. The gradient length was 0.32 SD-units and we thus used linear methods. We first performed a redundancy analysis (RDA) using a Monte Carlo

Permutation test (n=999, α=0.05) in order to determine whether multivariate differences in the overall PLFA signatures occurred in response to the N addition treatments. We then described multivariate PLFA signatures using principal component analysis (PCA). The first and second PCA of this ordination, as well as total PLFA, fungal, bacterial, fungi:bacteria, gram positive, gram negative, actinomycetes PLFAs were then compared using one-way ANOVA, as described above. When significant differences between N addition levels were detected (α=0.05), post hoc

pairwise comparisons between treatments were conducted using the Student-Newman-Keuls test. For soil respiration data, we performed a blocked repeated-measures ANOVA to test

whether the treatments had significant effects on respiration and soil temperature averaged across all sampling times. A pairwise Bonferonni post hoc test was subsequently used to compare whether significant differences between treatments occurred on average across all sampling times (α=0.05). All univariate analysis described above were performed using SPSS (Chicago, Illinois,

USA; version 20.0), while the multivariate analysis were done using CANOCO (Biometris, Wageningen, NL; version 5.0).

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Results Our data did not reveal any significant differences in soil C and N concentrations at any depth, except for in the organic horizon where the high N treatment resulted in a higher N concentration relative to the control (Tab. S1). A difference in soil bulk density was found only in the deepest layer (10-20 cm), where the low N treatment showed a higher bulk density relative to the control. The mean thickness of the organic horizon increased with increasing N addition rates; however, they did not significantly differ among the treatments (Tab. S1). When C pools were scaled to a per unit area basis (see methods), the data showed that

organic horizon C pools significantly differed among the N treatments, with significantly more C present in the high N plots (50 kg ha-1 yr-1) relative to the low N (12.5 kg ha-1 yr-1) or control

plots (Tab. 1; Fig. 1a). A similar pattern was found for the O-horizon N pool, with significantly more N in the high N treatment compared to the low N and control treatments (Tab. 1, Fig. 1b). The data showed no significant difference among the treatments in mineral soil C and N pools at both depths (Table 1, Fig. 1a, b). The amount of C stored in soil in the O-horizon ranged between 19.4-27.8 Mg ha-1 C, which was considerably greater than the amount stored in living root biomass, 6.1-6.3 Mg ha-1 C (Fig. 1). No significant differences in living root biomass C or N were found at any of the soil depths (Fig. 1c, d). When soil N, root N, and the combination of soil and root N were summed across all soil

layers, we found that the high N treatment caused a significant increase in these values relative to the low N or control (Tab. 2). For soil C, there was no significant difference among the treatments when these pools were summed across all soil layers. Our regression analysis showed the presence of a significant linear relationship between

the relative annual C accrual in the O-horizon and the annual N addition rate (R2=0.54, P=0.002).

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The slope of this relationship was 10 kg C sequestrated in the O-horizon per kg of N added after 16 years of simulated N addition (Fig. 2). Comparisons of the PLFAs using ANOVA showed that significant differences occurred

for total PLFAs, fungal markers, and the first axis of the principle component analysis (Tab. 3, Fig. 3). In each of these cases, the high N addition treatment significantly differed from the low N treatment and the control. For total PLFA and fungal PLFA, concentrations were significantly lower in the high N plots relative to the low N or control plots. We found that the total PLFA concentration was non-significantly reduced by ~12% in the low N treatment and significantly reduced by ~28% in the high N treatment relative to the control plots. Fungal PLFA concentrations showed a non-significant reduction of ~12.5% in the low N treatment and a significant reduction of ~42% in the high N treatment relative to the control. The data showed no significant changes among the N treatments for the bacteria, fungi:bacteria, gram positive, gram negative, actinomycetes PLFA markers, or the second principle component axis. For axis 1 values of the first principle component (i.e. PC1), values were significantly higher in response to the high N treatment compared to the low N and control plots (Tab. 3, Fig. 3). The RDA analysis using Monte Carlo permutations tests confirmed that significant differences in PLFA signatures were present among the N treatments (P 1st axis=0.013, P 2nd axis=0.012). The first axis

of the PCA explained 83.1% of the variation among PLFA markers, with positive loading score values most influenced by the i-16:0 gram positive PLFA marker (loading score =0.03), the10me16:0 (loading score =0.01,) and 10me17:0 actinomycete markers (loading score =0.02) which were relatively more abundant in the high N level treatment. The negative loading score values were influenced by 16:0 (loading score =-0.29), 16:1ω7 total bacteria PLFA markers (loading score =-0.19), i-15:0 gram positive PLFA marker (loading score =-0.17), 18:1ω7

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(loading score =-0.18), cy-19:0 gram negative PLFA markers (loading score =-0.17), and 18:2ω6 (loading score =-0.80), which were relatively more abundant in the control plots (Fig. 3).

Soil respiration ranged between 19.3 and 51.7 g C ha-1 h-1 and showed temporal variation

over the growing season (Fig 4a). The data showed a significant effect of time, a significant effect of N treatment, while there was no significant interaction between time and N treatment. The significant effect of time was due to higher soil respiration fluxes between late July through September compared to the other sampling times. There were no significant differences between the treatments at any individual sampling time; however, there was a significant effect of the N treatments when averaged across all sampling times (Fig. 4). Post-hoc analyses showed that soil respiration was significantly reduced by ~4 g C ha-1 h-1 on average across all sampling events in the high N treatment compared to the low N treatment and the control (Fig. 4b). There were no significant differences in soil temperature among N treatments (Fig. S1). Discussion Our aim was to investigate how soil C pools respond to long term N addition as well as to study the response of soil microbial composition and total soil respiration, which have been proposed as key mechanisms by which Nr deposition affects soil C sequestration in boreal forests. No

other long term experiments has simulated realistic levels of Nr deposition in the boreal region

(≤12.5 kg N ha-1 yr-1) (Dentener et al., 2006; de Vries et al., 2009), and thus our study provides a

unique opportunity to reveal both the magnitude and response relationship of soil properties and processes to a gradient of N addition. In support of our first hypothesis, our data showed that long term N addition increased

soil C sequestration. After 16 years of N addition, we estimated an increase of C sequestered in

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the organic horizon by ~30% (8.5 Mg ha-1) on average in the high N treatment relative to the control and a non-significant increase for the low N treatment of ~15% (3.4 Mg ha-1) on average (Fig. 1a). Our data also showed a long-term accumulation of N in the O-horizon. We estimated that ~75% (0.15 Mg ha-1) and ~50% (0.40 Mg ha-1) of the total N added over the course of the

experiment (16 years) were sequestered in the soil O-horizon for the low and high N treatments, respectively. This amount of sequestered N represents approximately half of the total amount of N added over the course of the experiment in each treatment, indicating that the soil serves as long-term sink for anthropogenic N (Templer et al., 2012; Gundale et al., 2014a). De Vries et al.,

(2014) proposed that the decrease of soil N retention in long-term N addition studies may be due to N losses by leaching and denitrification. The estimated N losses in the low and high N treatments were in line with previous studies which estimate N losses < 30% on average between 10-25 kg N ha-1 yr-1 and >30% on average above 25 kg N ha-1 yr-1 (MacDonald et al., 2002; de Vries et al., 2007, 2014; van der Salm et al., 2007; Dise et al., 2009). The increase in C in the

organic horizon was due to the combined effect of a non-significant increase in both the Ohorizon thickness, bulk density, and C concentration (Fig. S1). They are also consistent with a study by Pregitzer et al. (2008) who showed that 10 years of N fertilization (30 kg N ha-1 yr-1) increased O-horizon C by 6.9 Mg ha-1 in temperate hardwood forests. They are also consistent with a study by Franklin et al. (2003) who used the 14C bomb signature within a 20 year

simulated N addition experiment (30 kg N ha-1 yr-1) to estimate an increase of approximately 13 Mg soil C ha-1 in a boreal forest. Despite the significant increase in O-horizon C in response to N enrichment, we did not detect any response in living root biomass at any depth, or soil C in either of the mineral soil layers. The absence of change in mineral soil C could be the result of several factors, including inherently lower rates of biological activity with potential to be

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responsive to N compared to the O-horizon (Högberg et al., 2001; Lindahl et al., 2007), greater spatial heterogeneity that may disguise subtle changes in C pool sizes, the predominance of older C that accumulates more slowly and is inherently less responsive (Gleixner, 2013), or the absence of soil fauna capable of redistributing newly produced surface C into the mineral horizons (e.g. anecic earthworms) (Jégou et al., 1998). Contrary to our first hypothesis, we did not find any evidence that the amount of C added

per unit N changed with increasing N addition rates. Instead, our data show that the relationship between N enrichment and C sequestration in the O-horizon followed a linear relationship with a slope of 10 kg C sequestered in the soil per kg of N added (Fig. 2). This relationship suggests that even low levels of Nr deposition should promote soil C sequestration, albeit in quantities too small to appear significantly different using ANOVA analysis in our experiment. The estimate of C sequestration derived from our regression is consistent with Hyvönen et al. (2008), who

estimated on average 11 kg C kg N-1 in boreal forest in response to a much higher range of N addition rates (30-200 kg N ha-1 yr-1) than applied in our study; however, they showed that N addition rates ≤50 kg N ha-1 yr-1 were more effective in C accumulation than N addition rates between 50 and 200 kg N ha-1 yr-1. Our finding is also in the same order of magnitude as

evaluated by de Vries et al. (2009), who estimated on average 23 kg C kg N-1 after a decade of N

fertilization in a northern deciduous forest (Pregitzer et al., 2008). Consistent with our second hypothesis, we found a significant decrease in the total

microbial and fungal PLFAs in response to chronic N addition, but only in response to the high N treatment (i.e. 50 kg N ha-1 yr-1) (Tab. 3). Although, inconsistent with our second hypothesis, we did not observe a significant reduction in the fungal:bacterial ratio due to a simultaneous nonsignificant reduction also in bacterial biomass. The decrease in total and fungal PLFA markers

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we observed corresponded with a significant increase in soil C that accumulated in the high N plots, suggesting that reductions in soil microbial activity likely contributed to this accumulation of C. Several studies have suggested that reduction in fungi in response to N addition may result in a competitive release of r-strategist microbes that are relatively more N-demanding (e.g. bacteria) at the expense of N-conservative microbes less efficient in C assimilation (Andrews & Harris, 1986; Fog, 1988; Ågren et al., 2001). However, our data do not support these suggestions, as bacteria showed a non-significant concomitant decline with fungi. This pattern is instead more consistent with suggestions that fungi, and ectomycorrhizal fungi specifically, can have priming affects that can enhance soil saprotrophs, including bacteria, through the production of hyphal exudates and the breakdown of complex carbon substrates (Högberg & Högberg, 2002; Janssens et al., 2010). A variety of mechanisms have been proposed to explain the fungal declines in response

to N addition. For instance, several studies have suggested that alleviation of N limitation by trees results in reduced belowground C allocation to support mycorrhizae (Haynes & Gower, 1995; Högberg et al., 2010; Kaiser et al., 2010). Soil fungal biomass in boreal forests is often

dominated by ectomycorrhizal species (Wallander et al., 2001, 2003), thus reduced belowground

tree C allocation in response to N additions may help explain the greater sensitivity of fungi relative to bacteria that we observed in this study. An additional mechanism that may explain the greater sensitivity of fungi relative to bacteria is that nitrate (NO3-) has been shown to slow down several fungal lignolytic enzymes, including fungal phenol oxidases and peroxidases of white rot Basidiomycetes (Waldrop & Zak, 2006; Ekberg et al., 2007; Kaiser et al., 2010). This

impairment of enzyme activity can reduce the ability of fungi to decompose compounds rich in lignin (e.g. spruce needles), resulting in a reduction in fungal biomass and an increase in soil C

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(Waldrop et al., 2004), as we observed. Additionally, abiotic stabilization mechanism caused by N addition may also be responsible for the decline of fungal biomass. It has been proposed that added N and high soil organic matter density can interact and produce recalcitrant compounds that are protected from microbial decay (Neff et al., 2002; Swanston et al., 2004), thereby

potentially impacting fungal metabolism. The disproportionately large reduction in fungal biomass relative to microbial biomass we observed emphasizes the need of future research to understand the mechanisms controlling their decline (Litton et al., 2007; Fernández-Martínez et al., 2014). In support of our third hypothesis, the high N addition treatment caused an approximately

11% decrease in total soil respiration (i.e. autotrophic and heterotrophic combined), which corresponded with the decline in total microbial and fungal biomass we observed. Consistent with our third hypothesis, the impact of the high N treatment was greater than the low N treatment, whereas no significant change occurred for the low N treatment relative to the control (Fig. 3b). The decrease in respiration caused by our high N treatment is substantially lower (~30% lower) than what has previously been reported in other long term N fertilization studies in boreal forest (Franklin et al., 2003; Olsson et al., 2005), yet these previous studies had much

higher N addition rates (60-180 kg N ha-1 yr-1). This result also contrasts Hasselquist et al. (2012), where relatively low N addition rates (20 kg N ha-1 yr-1) caused an increase of soil respiration. These discrepancies may be explained by differences in forest age, forest productivity, tree species and soil characteristics (Pregitzer & Euskirchen, 2004; Hyvönen et al., 2008; Thomas et al., 2010).The study by Hasselquist et al. (2012) was carried out in a relatively young Pinus sylvestris forest where the application of N fertilizer was much shorter (5 years) compared to the current study. Similarly, Bowden et al. (2004) measured soil respiration in

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temperate forests in response to chronic N additions and found an initial increase of soil respiration, followed by an eventual decrease after more than a decade of chronic N addition. They proposed that the initial increase could be due to an increase in tree productivity that resulted in increased C allocation to roots and mycorrhizae, whereas belowground allocation would eventually decline as tree N limitation gradually decreases. These studies emphasize the value of long term response data derived from realistic experiments for clarifying the effects of chronic environmental change factors, such as Nr deposition. As such, our long term data clearly

show that N input levels approximating upper level atmospheric N deposition rates in the boreal region are likely to have relatively subtle effects on soil respiration. Our estimates of soil C accumulation in response to simulated chronic Nr deposition have

substantial implications for understanding the impacts of Nr deposition on the global C cycle. Boreal forests cover approximately 15% of the terrestrial land surface area and they serve as a major global C pool and sink, and approximately 2/3 of the C in these systems exists in the soil (Malhi et al., 1999; Lal, 2005; DeLuca & Boisvenue, 2012). Some studies have proposed that C sequestration in response to Nr deposition in boreal and temperate forests accounts for a large

portion of the yet unidentified terrestrial sink for anthropogenic CO2 emissions, but in doing so

assume C sequestration rates in the range of 200-500 parts C per unit of N deposition (Holland et

al., 1997; Magnani et al., 2007; Reay et al., 2008). Modelling the quantitative impacts of Nr deposition on C sequestration in the boreal region has been controversial, and remains unresolved due to uncertainty in the magnitude and linearity of the relationship between Nr deposition and C sequestration at the stand level. Our data complements a recent study from the same experimental system describing aboveground C sequestration (Gundale et al., 2014a) and thus provides a complete accounting of C at our experimental system, which is the longest

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running experiment in the boreal region simulating realistic levels of Nr deposition. The accumulation of 10 kg C kg-1 N sequestered in soils reported in this study, and 16 kg C kg-1 N

sequestered in aboveground biomass (reported in Gundale et al., 2014), result in a total C sequestration rate of 26 kg C kg-1 N at our study site. While this rate is likely to vary somewhat among tree species, stand ages, or soil type (Pregitzer & Euskirchen, 2004; Thomas et al., 2010), they are consistent with more recent model estimates (De Vries et al., 2006; Mol Dijkstra et al.,

2009; de Vries et al., 2014), as well as previous experimental data using much higher N addition rates (Hyvönen et al., 2008; de Vries et al., 2009). As such, our data provide a substantial contribution to the growing consensus that Nr deposition in the boreal region has a relatively minor impact on the global C cycle (Hyvönen et al., 2008; Sutton et al., 2008; de Vries et al., 2009; Gundale et al., 2014) than what has previously been proposed (Holland et al., 1997;

Magnani et al., 2007).

Acknowledgements We thank Ann Sehlstedt, Agnes Väppling and Maja Sandström for assistance with field and lab work. We thank Jonatan Klaminder and Cédric L. Meunier for helpful comments on a previous draft of this manuscript. The project was supported by the center for Environmental Research in Umeå (CMF), the Swedish Research Council (FORMAS) for the project Sustainable Management of Carbon and Nitrogen in Future Forests (NiCaf), the Mistra Future Forests program, and the SLU strong research environment Trees and Crops for the Future (TC4F).

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Table 1 The F-values, degrees of freedom (DF) and P-values from one-way ANOVAs comparing whether C and N pool sizes at different soil depths (organic horizon, mineral soil from 0 to10 cm depth, and mineral

soil from 10 to 20 cm depth) stored in soil or roots differed across three simulated chronic N deposition treatments (0,12.5, and 50 kg N ha-1 yr-1; n=5). F-value

DF

P-value

Organic horizon Soil C pool

7.47

2,12

0.008

N pool

15.58

2,12

0.000

C pool

0.18

2,12

0.838

N pool

7.81

2,12

0.013*

C pool

0.48

2,12

0.631

N pool

0.88

2,12

0.441

C pool

0.39

2,12

0.684

N pool

0.06

2,12

0.940

0.20

2,9

0.825

Roots

Mineral soil (0-10 cm depth) Soil

Roots

Mineral soil (10-20 cm depth) Soil C pool

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N pool

0.09

2,9

0.915

C pool

1.72

2,9

0.234

N pool

1.44

2,9

0.287

Roots

*Block was used as a significant factor Ɨ the data were transformed using the function Log(X+1) to meet the assumption of normality

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Table 2

Means (±SE) of C and N total pools, in organic horizon and mineral soil combined, roots from organic horizon and mineral soil combined, and soil and roots combined in response to three simulated chronic N deposition treatments (0, 12.5, and 50 kg N ha-1 yr-1). Each pool were estimated in replicated (n=5)

0.1 ha plots 16 years after treatments were initiated. Samples were collected using soil cores. Root C and N pools were estimated using the root biomass collected by soil coring the same year in each plot and %C and %N of each plot from a previous study. The F- and P-values were derived from one-factor ANOVAs and the different letters a or b next to means indicate significant differences determined using

Nitrogen deposition (Kg N ha-1yr-1) 0

12.5

50

Fvalue

DF

Pvalue

Total pools

Organic horizon and mineral soil C pool (Mg ha-1)

34.70

±2.94

38.09

±1.09

41.50

±1.84

2.63

2,12

0.113

N pool (Mg ha-1)

1.19a

±0.08

1.32a

±0.14

1.65b

±0.08

5.24

2,12

0.023

C pool (Mg ha-1)

8.32

±0.78

8.83

±1.66

8.13

±1.34

0.08

2,12

0.928

N pool (Mg ha-1)

0.08a

±0.01

0.09a

±0.02

0.12b

±0.02

5.091

2,12

0.037*

C pool (Mg ha-1)

43.01

±3.14

46.92

±0.70

49.63

±2.67

1.90

2,12

0.192

N pool (Mg ha-1)

1.28a

±0.08

1.41a

±0.12

1.78b

±0.07

7.69

2,12

0.007

Total roots

Total roots and soils

Student-Newman-Keuls post-hoc analyses.

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*Block was used as a significant factor Ɨ the data were transformed using the function Log(X+1) to meet the assumption of normality

Table 3

Mean (±SE) response of Phospholipid Fatty Acid Analysis (PLFA) expressed in µmol PLFA g-1 organic

Nitrogen deposition (kg N ha-1 yr-1) 0

12.5

50

Fvalue

Pvalue

Total PLFA

2.24b

±0.15

1.97ab

±0.10

1.61a

±0.09

7.33

0.008

Bacteria

0.66

±0.05

0.56

±0.06

0.50

±0.04

2.66

0.111

Fungi

0.48b

±0.05

0.42b

±0.01

0.28a

±0.02

9.84

0.003

Fungi:Bacteria

0.74

±0.05

0.80

±0.11

0.57

±0.03

2.55

0.120

Gram Positive

0.24

±0.02

0.21

±0.02

0.19

±0.01

1.79

0.209

Gram Negative

0.35

±0.26

0.29

±0.04

0.25

±0.02

2.88

0.095

Actinomycetes

0.13

±0.01

0.11

±0.01

0.13

±0.01

0.98

0.402

PCA 1 (83.06%)

-0.11a

±0.06

-0.03a

±0.02

0.14b

±0.03

10.72

0.002

PCA 2 (11.98%)

0.01

±0.02

-0.01

±0.03

0.01

±0.01

0.26

0.778

matter of the soil organic horizon in 0.1 ha plots treated with 0, 12.5, 50 kg N ha-1 yr-1 annually for 15

years.

The F- and P-values were derived from one-factor ANOVA (df=2, 12) for each variable, and

different letters (a or b) next to means which each row indicate differences determined using StudentNewman-Keuls post-hoc analyses. The percentage in brackets next to the variables PCA 1 and PCA 2 indicate the percentage of total variation explained by axis 1 and axis 2, respectively.

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Fig. 1 Mean (+SE) carbon (left panels) or nitrogen (right panels) pool size at different soil depths that is stored in soil (a and b) or roots (c and d) in response to long-term N additions (0, 12.5 or

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50 kg N ha-1 yr-1; n=5). Different letters (a or b) next to each group of bars indicate significant differences between treatments determined using Student-Newman-Keuls post-hoc tests. Nonsignificant differences are indicated by n.s. Fig. 2 Regression relationship between carbon accumulation (kg C ha-1 yr-1) in the organic

horizon and simulated N deposition rate (kg N ha-1 yr-1). Data are derived from plots fertilized since 1996 with three different simulated Nr deposition treatments (0, 12.5, or 50 kg N ha-1 yr-1,

n=5). Carbon accumulation was calculated by first estimating the C pool size in the organic horizon of each plot, then subtracting all plot data by the mean organic C pool per ha of the control plots, and dividing by the number of years the treatments had been applied at the time of measurement. Fig. 3 Total PLFA samples scores from PCA for each N addition levels. Treatments consisted of 0 kg N ha-1 yr-1;(white circle), 12.5 kg N ha-1 yr-1; (grey circle), and 50 kg N ha-1 yr-1; (black

square). Small symbols correspond to mean PLFA for each plot, large symbols correspond to mean PLFA per treatment. Bars indicate 95% confidence intervals. Fig. 4 Soil respiration measured during summer 2013. (a) Data points represent mean (± SE) of all plots (n=5) at each sampling time. Treatments consisted of 0 kg N ha-1 yr-1; (white circle),

12.5 kg N ha-1 yr-1; (grey circle), and 50 kg N ha-1 yr-1; (black square). (b) Mean (±SE) soil respiration averaged across all sampling times. Data were analyzed using a repeated -measures ANOVA, and a post-hoc Bonferoni test was used to compare mean effect of treatments averaged across all times. Different letters (a or b) in sub-panel b indicate significant differences between treatments.

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