Tree stems are a net source of CH₄ and N₂O in a hemiboreal drained peatland forest during the winter period

The study was conducted in a drained peatland forest site (58°17'N, 27°17'E; 38 m.a.s.l.) in the eastern part of Estonia, which belongs to the hemiboreal vegetation zone [28]. The stand was drained in the early 1970s using an open-ditch network drainage system [29] and belongs to the Oxalis site type. The soil was classified as Drainic Eutric Histosol [30] with low dry bulk density, high organic C content, and low pH [6, 29]. The area is covered mainly by Downy birch (Betula pubescens Ehrh.) and Norway spruce (Picea abies (L.) H. Karst.) trees. The long-term average precipitation in the area is 650 mm, the average temperature is 17 °C in July and −6.7 °C in January, and the growing season lasts 175–180 days [31]. The tree stand characteristics are presented in Supplementary table 1.

Twelve representative monitoring points were selected in the study area. Each of the first six points consists of one Downy Birch and one Norway Spruce tree with installed stem chambers and one automatic dynamic soil chamber. The remaining six monitoring points are set pairs of one birch tree and one soil chamber.

2.2.1. Tree stem gas fluxes

2.2.2. Soil gas fluxes

Stem and soil CH₄ and N₂O flux rates were calculated based on the linear regression approach of chamber headspace gas concentration change over time using the following equation (equation (1)):

$$\begin{eqnarray}&&\begin{array}{c}F[\mu {\mathrm{gm}}^{-2}{{\rm{h}}}^{-1}]\\ \,=\,\frac{{\rm{M}}[{\rm{g}}\,{\mathrm{mol}}^{-1}]\times {\rm{P}}[\mathrm{Pa}]\times {\rm{V}}[{{\rm{m}}}^{3}\times \sigma {\rm{v}}[\mathrm{ppm}({\rm{v}})]}{R[{{\rm{m}}}^{3}{\mathrm{PaK}}^{-1}{\mathrm{mol}}^{-1}]\times {\rm{T}}[{\circ }^{}K]\times t[h]\times A[{{\rm{m}}}^{2}]\times \mathrm{ppm}}\end{array}\end{eqnarray} \tag{ 1 }$$

Where M = molecular mass of the gas, P = air pressure, V = chamber volume, $\sigma {\rm{v}}$ = the slope of linear regression gas concentration change in chamber headspace during the sampling time, R = gas constant (8.314), T = temperature, t = sampling time, and A = soil surface area covered by the chamber.

The quality of the chamber measurement session was validated using the the adjusted R² value of the linear regression for the CO₂ measurements. Flux values were accepted only if the R² value exceeded 0.9. To compare the contribution of soil and stem CH₄ and N₂O fluxes, stem fluxes averaged across the three heights were up-scaled to a hectare of ground area, calculated based on tree stand characteristics brought out in Supplementary table 1, assuming a cylindric shape of the tree stems [17, 33].

Soil and air temperature, SWC and WTD were continuously measured during the study period. Soil temperature (107, CAMPBELL SCIENTIFIC. INC, Logan, Utah, USA) and SWC sensors (ML3 ThetaProbe, Delta-T Devices, Cambridge, United Kingdom) were placed vertically at 0.1 m soil depth inside the soil chambers. In groundwater wells, the WTD was observed using automatic data loggers (Hobo U20L-04, Onset Computer Corporation, Bourne, Massachusetts, USA).

Statistical analysis was performed using R version 4.0.3 [34]. The normality of data distribution was examined using the Kolmogorov-Smirnov, Lilliefors and Shapiro-Wilks tests. As flux data were not normally distributed, non-parametric tests were used. Kruskal–Wallis one-way analysis of variance was used to determine the significance of temporal variability of gas fluxes, and differences between stem fluxes at different heights. Dunn's multiple comparison, corrected with the Bonferroni method, was conducted as a post hoc test to determine which groups differed. Spearman's rank correlation was used to analyse relationships between soil and stem CH₄ and N₂O fluxes and meteorological parameters. The significance level was defined at p < 0.05 for all tests.

Stems were a net source of CH₄ and N₂O during the winter period. On average, birch stems emitted more CH₄ (0.18 ± 0.03 μg C m⁻² h⁻¹, mean ± standard error (SE)) and N₂O (1.44 ± 0.22 μg N m⁻² h⁻¹) than spruce stems (0.10 ± 0.03 μg C m⁻² h⁻¹, 0.003 ± 0.01 μg N m⁻² h⁻¹). Fluxes were statistically different in time and between tree species (p < 0.05).

Temporal dynamics of both birch and spruce stem CH₄ emissions were characterised by a notable peak in November (daily mean reached 1.78 ± 0.47 μg C m⁻² h⁻¹ for birch and 1.96 ± 0.49 μg C m⁻² h⁻¹ for spruce), fluctuations between small emissions and uptake in the winter, and a slight increase in emissions in spring (figure 1(B)). Birch CH₄ fluxes had the highest correlation with SWC, while spruce CH₄ fluxes did not have statistically significant correlations with any meteorological parameters (Supplementary table 2).

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Birch trees emitted N₂O throughout the study period, with the highest emissions in October (up to 11.43 ± 3.89 μg N m⁻² h⁻¹), after which emissions dropped to near-zero in the winter months. Fluxes increased from the end of February to the end of March but dropped again in April and May. Spruce stem fluxes fluctuated around zero between small emissions and uptake throughout the study period (figure 1(C)). Both birch and spruce N₂O fluxes were mostly driven by changes in WTD (Supplementary table 2).

CH₄ stem fluxes did not follow a statistically significant vertical tree stem flux trend. Average birch fluxes were lowest at 0.1 m (0.16 ± 0.04 μg C m⁻² h⁻¹; averaged across plots during the whole study period) and increase to 0.20 ± 0.05 μg C m⁻² h⁻¹ at both 0.8 and 1.7 m. Spruce CH₄ stem fluxes stayed nearly equal at all heights (figure 2(A)). On the other hand, a clear trend was observed for birch N₂O fluxes. Birch fluxes decreased from 2.56 ± 0.42 μg N m⁻² h⁻¹ at 0.1 m stem height to 0.18 ± 0.05 μg N m⁻² h⁻¹ at 1.7 m. Spruce N₂O fluxes were highest at 0.1 m (0.07 ± 0.03 μg N m⁻² h⁻¹) and emissions turned to N₂O uptake at 0.8 m (−0.02 ± 0.02 μg N m⁻² h⁻¹) and 1.7 m (−0.04 ± 0.02 μg N m⁻² h⁻¹) (figure 2(B)).

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Soil was a net sink for CH₄ (−2.00 ± 0.12 μg C m⁻² h⁻¹) and a source of N₂O (50.46 ± 2.77 μg N m⁻² h⁻¹) across the study period. Fluxes were statistically different in time (p < 0.05). Methane uptake was higher during winter onset, with the largest consumption at the beginning of January (up to −8.13 μg C m⁻² h⁻¹). From late January to early April, fluxes hovered around zero, with occasional CH₄ release. From April through May, soils again turned into a CH₄ sink (figure 1(B)). Soil CH₄ fluxes had the most significant relationship with soil temperature, but also SWC and WTD. No correlation was found between soil and stem CH₄ fluxes (Supplementary table 2).

Hot moments—short periods of notably higher daily average emissions compared to the period average—contributed 64.2% of the whole study period N₂O emissions, while accounting for 31.8% of the time in days (figure 1(C); Supplementary table 3). We identified three hot moments during the study period—(I) 20/01/2021–27/01/2021, (II) 22/02/2021–09/03/2021, and (III) 27/03/2021–20/04/2021. Daily average N₂O emissions during hot moments were (I) 76.60 ± 9.65 (8.4% of study period total), (II) 72.33 ± 7.83 (15.1%), and (III) 118.73 ± 10.4 (40.7%) μg N m⁻² h⁻¹ (Supplementary table 3).

During the whole study period, soil N₂O fluxes correlated positively with air temperature, SWC, soil temperature, and WTD. Soil N₂O fluxes correlated with stem N₂O fluxes from birch, but not from spruce (Supplementary table 2). Relationships between soil fluxes and SWC had higher statistical significance during hot moments (II) (p < 0.00001, r = 0.88) and (III) (p < 0.00001, r = −0.88). In addition, N₂O flux correlated positively with air temperature during hot moment (I) (p < 0.01, r = 0.83) and with WTD during hot moment (II) (p < 0.00001, r = 0.86).

Stem and soil fluxes, when upscaled to hectare of forest ground, showed that birch and spruce stem CH₄ emissions offset the soil CH₄ sink by 10.4% and 3.5%, respectively (figure 3(A)). Stem emissions accounted for 1.9% of the total soil and stem N₂O emissions, almost entirely related to birch fluxes (figure 3(B)).

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Birch and spruce stem CH₄ fluxes in drained peatlands have not been previously studied and cold-period measurements from other ecosystems are limited. Thus, comparison with other studies is challenging. Compared to our results of average birch stem emissions of 0.18 μg C m⁻² h⁻¹, previous studies on cold-period emissions from various deciduous tree species showed much higher emissions. For example, emissions around 10 μg C m⁻² h⁻¹ have been reported from alder stems in a hemiboreal riparian forest [19], whereas average stem emissions reached around 50 μg C m⁻² h⁻¹ from birch stems and 110 μg C m⁻² h⁻¹ from alder stems during winter months in a temperate forested peatland [21]. In addition, we found spruce stem CH₄ emissions to be lower than birch emissions, similarly to boreal trees measured by Vainio et al [35]. Spatial and species-level variability in stem fluxes can been linked to hydrologic conditions in the soil [14, 17], aspects of stem morphology and tree physiology, such as wood density, lenticel abundance, transpiration, sap flow rates, and wood vessel structure [22, 26, 27, 36, 37], as well as differences in microbiology in stems and soils [17, 21].

Stem CH₄ emissions peaked in November, with fluctuations between small emissions and uptake in the winter and a slight increase in emissions in spring (figure 1(B)). Similar seasonal patterns were observed in a hemiboreal riparian forest [19] and in a temperate forested peatland [21]. Several previous studies with smaller sampling frequencies [20] and only growing season measurements [22, 37] have not found clear seasonal trends in CH₄ stem fluxes. Moreover, Barba et al [15] found seasonality only with automated high-frequency measurements, but not with manual measurements, emphasising the need for increased high-frequency stem CH₄ and N₂O measurements to better understand the temporal and spatial variations.

Birch and spruce stem CH₄ emissions combined offset the soil sink by 14%. A wide range of estimates have been proposed previously, from stem flux contributions of 3.5% [37] and 1%–6% [26] to 63% [38] in temperate forests. In addition, in ecosystems where soil is a net CH₄ source, stem emissions can add up to 27% to the total soil source in a temperate wetland [21] and up to 83% in a riparian forest [19]. Therefore, CH₄ budgets that do not incorporate stem emissions can significantly overestimate the sink strength or underestimate the total emissions of the ecosystem [38].

To the best of our knowledge, our study is among the first to focus on winter-time N₂O stem fluxes. Stems were a net source of N₂O with birch stems emitting significantly more than spruce. Conversely, spruce stems in a boreal forest emitted significantly more N₂O anually, as well as during the dormant season, than birch stems [23]. Inter-species variability of N₂O fluxes could be explained by spatial variability of N₂O concentration in the soil or differences in trees' physiological properties, such as projected leaf area per tree [18, 23]. Further examination of these variables in different ecosystems is needed to explain the discrepancy in species-level fluxes in different ecosystems.

Seasonality of stem N₂O fluxes is generally driven by ecosystem activity and physiological activity of trees [23]. We observed birch N₂O emissions throughout the study period, but spruce fluxes were negligible. Birch emissions were highest in late autumn and early spring, and fluctuated near-zero in the winter. Early autumn and early spring also contribute most to the cumulative stem N₂O emissions from Alnus incana in a riparian forest [12]. Boreal trees also emit N₂O during dormancy, agreeing with our results of prevailing emissions during winter months [23].

Stem fluxes added about 2% to forest floor N₂O emissions, almost entirely related to birch fluxes. Previous studies also report low contributions on annual scales, such as Machacova et al [23] (birch 0.75% and spruce 2.5%), and Mander et al [12] (alder 0.8%). However, there are no studies to compare stem fluxes' contributions solely during the winter months. Although the contribution of stem N₂O fluxes to total emissions is relatively insignificant, even minor emissions from trees can accumulate to a substantial addition to the annual balance of forest N₂O emissions.

The origin and drivers of CH₄ and N₂O stem fluxes are contested. Stems have been suggested to act as conduits for CH₄ [16–18, 26, 36, 39] and N₂O [23, 24] produced in the soil. However, CH₄ can also be produced on the stem bark [40] or inside the tree stem by methanogenic microbes [15, 38, 41, 42]. The origin of tree stem CH₄ emissions is not clear based on our results. Stems constituted a CH₄ source, while the soil was a net sink, which could be evidence for methanogenic activity inside the woody tissue [27, 42]. Birch emissions were driven by changes in SWC (figures 1(A), (B); Supplementary table 2), but the vertical stem flux profile was irregular (figure 2(A)). Therefore, it is possible that the CH₄ that is taken up by the tree roots originates from deeper soil layers where roots are more abundant, whereas methanotrophic activity leading to net soil CH₄ uptake dominates over methanogenesis in the shallower layers of the soil. This could also explain higher emissions from birch stems than from spruce, as the fine root density of Norway spruce is generally higher closer to the soil surface [43]. Thus, only birch roots may reach the deeper soil layers to facilitate CH₄ transport [35]. To further examine the origin of tree stem CH₄ emissions, information about CH₄ concentrations in the soil vertical profile and identification of methanotrophic and methanogenic bacteria in different soil layers, as well as in the tree heartwood, is needed.

Our results suggest that N₂O fluxes may originate from the soil. Birch emissions decreased with increasing stem height (figure 2(B)) and were driven by soil N₂O emissions, changes in WTD and soil temperature (Supplementary table 2). In agreement with previous studies reporting declining N₂O stem fluxes with tree height [12, 20, 33], this supports that N₂O is predominantly produced in the soil.

Soil in the drained peatland forest was a net CH₄ sink during the winter with higher uptake during winter onset (figure 1(B)). Fluxes stayed nearly dormant in February, coinciding with constant low soil temperatures. Snow cover may be a barrier for gas exchange and the frozen topsoil layer could restrict gas diffusion due to ice in soil pores [44]. Spring onset presented fluxes fluctuating between emissions and uptake, agreeing with results from an adjacent drained peatland location [11]. Methane stored in frozen soils can be released to the atmosphere through changes in pressure during freeze-thaw periods [45]. Soils turned into a CH₄ sink in mid-April, together with increasing soil temperatures. Soil temperature plays a role in determining CH₄ dynamics by increasing the metabolic activities of microorganisms [46, 47]. In addition to soil temperature, soil CH₄ seasonality was also determined by SWC and WTD (Supplementary table 2). Previous studies consider SWC as the primary factor determining CH₄ dynamics in forest ecosystems [3, 19, 46, 48]. Methanogenesis is facilitated by anaerobic conditions induced by higher SWC [46, 49], whereas methanotrophic CH₄ consumption is increased under drier aerobic conditions [44, 46].

Soils were a net source of N₂O during the winter period. Hot moments and hot spots predominantly determine spatio-temporal dynamics of N₂O emissions from soils [12]. We identified three hot moments, during which daily average N₂O emissions were notably higher than the period average, accounting for 64.2% of the total release of N₂O (figure 1(C); Supplementary table 3). Hot moments are primarily controlled by changes in SWC and they can be induced by drying-rewetting or freeze-thaw cycles [12, 50, 51].

Hot moments (I) and (II) were likely the result of soil freeze-thaw events. The increases in daily average N₂O emissions coincided with the rising WTD and air temperature (figures 1(A), (C)). During hot moment (II), SWC also increased simultaneously with N₂O fluxes, reaching 0.88 m³m⁻³, indicating a high SWC optimum for N₂O release. Various explanations have been proposed to describe the effects of freeze-thaw on N₂O emissions. Disruption of soil aggregates can lead to rapid mineralisation of previously physically protected organic matter [52]. Dead cells of microorganisms, fine roots, and mycorrhiza in the soil, destroyed by freezing, can rapidly decompose during thawing [50, 52]. Death of fine roots can also leave more nitrate available for microorganisms for N₂O production [12, 51]. As snow cover keeps deeper soil layers unforozen, soil microorganisms can remain active, stimulating organic matter decomposition and N mineralisation, prompting N₂O production and release [52, 53]. In addition, rapid increase in N₂O fluxes at high SWC during the freeze-thaw hot moments can be explained by increased O₂ content in snow melt water which can inhibit the full denitrification pathway from N₂O to N₂ [54]. In low temperature zones (below 10 °C), nitrifier denitrification is likely to dominate total denitrification in fluctuating aerobic-anaerobic conditions [55].

Hot moment (III) showed the highest emissions of N₂O and is related to fluctuations in SWC. An increase in SWC happened directly prior to the hot moment, followed by a drop in SWC at the beginning of the hot moment (figures 1(A), (B)). Therefore, before the hot moment, SWC may have exceeded the optimum, and the water level has decreased to reach the optimum SWC for N₂O release to create the peak in emissions. Furthermore, the initial increase in N₂O emissions concurred with rising air and soil temperatures, indicating that temperature increase may be driving the N₂O emissions rise by stimulating microbial activity in the soil [12].