Alnus sibirica encroachment promotes dissolved organic carbon biodegradation in a boreal peatland

Science of the Total Environment 695 (2019) 133882

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Alnus sibirica encroachment promotes dissolved organic carbon biodegradation in a boreal peatland Yang Zhang a, Fu-Xi Shi a, Rong Mao a,b,⁎ a b

2011 Collaborative Innovation Center of Jiangxi Typical Trees Cultivation and Utilization, College of Forestry, Jiangxi Agricultural University, Nanchang 330045, China Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• A. sibirica expansion increased DIN and DTN concentrations in the extracts. • SUVA254 and humification index of DOC remained unchanged following A. sibirica expansion. • A. sibirica expansion enhanced the biodegradation of peat-derived DOC. • DOC biodegradation correlated negatively with C:N stoichiometry.

a r t i c l e

i n f o

Article history: Received 13 May 2019 Received in revised form 10 August 2019 Accepted 10 August 2019 Available online 11 August 2019 Editor: Paulo Pereira Keywords: Carbon quality Decomposition Nitrogen enrichment Permafrost Stoichiometry Woody plant expansion

a b s t r a c t Symbiotic dinitrogen (N2)-fixing trees have been expanding to boreal peatlands, yet its influence on dissolved organic carbon (DOC) biodegradation is unclear. Here, we measured DOC, ammonium nitrogen (NH+ 4 -N), nitrate nitrogen (NO− 3 -N), dissolved inorganic nitrogen (DIN), and dissolved total nitrogen (DTN) concentrations, specific ultraviolet absorbance at 254 nm (SUVA254), and humification index in the extracts obtained from peats in the 0–10 cm, 10–20 cm, and 20–40 cm depths in the open peatlands and Alnus sibirica islands in a boreal peatland, Northeast China. Afterwards, the peat extracts were used to assess the effect of N2-fixing woody plant expansion on DOC biodegradation with a 42-day incubation experiment. The expansion of − A. sibirica significantly increased NH+ 4 -N, NO3 -N, DIN, and DTN concentrations, but did not produce a significant effect on SUVA254 and humification index in the extracts in each depth. Following A. sibirica expansion, DOC biodegradation was enhanced by 24.5%, 15.4%, and 38.3% at 0–10 cm, 10–20 cm, and 20–40 cm depths, respectively. Furthermore, DOC biodegradation was significantly and negatively correlated with DOC:DIN and DOC:DTN ratios, but exhibited no significant relationship with SUVA254 and humification index. This implied that improved N availability and associated shifts in C:N stoichiometry determined the increase in DOC biodegradation following A. sibirica expansion. Our findings suggest that N2-fixing tree encroachment promotes microbial decomposition of DOC through improved N availability in boreal peatlands, which may cause organic C loss from soils in these C-enriched ecosystems. © 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: College of Forestry, Jiangxi Agricultural University, No. 1101 Zhimin Road, Nanchang 330045, China. E-mail address: [email protected] (R. Mao).

https://doi.org/10.1016/j.scitotenv.2019.133882 0048-9697/© 2019 Elsevier B.V. All rights reserved.

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Y. Zhang et al. / Science of the Total Environment 695 (2019) 133882

1. Introduction Dissolved organic carbon (DOC), despite a small proportion of organic carbon (C) pool, plays a critical role in regulating soil organic C sequestration, transporting C and other elements within terrestrial ecosystems, and linking hydrological and biogeochemical processes between terrestrial and aquatic ecosystems (Neff and Asner, 2001; Kalbitz et al., 2003). Generally, DOC acts as the main energy source for heterotrophic microbial communities and fuels C cycle in most ecosystems (Marschner and Kalbitz, 2003; Wickland et al., 2007). Therefore, DOC biodegradation is a key factor determining ecosystem C balance between C input and loss through greenhouse gas emissions, leaching, and surface runoff, and thus strongly influences C budget from regional to global scales (Dinsmore et al., 2010). Dissolved organic carbon is a heterogeneous mixture of soluble organic compounds ranging from labile fractions easily utilized by microorganisms to recalcitrant fractions that are resistant to microbial degradation (Marschner and Kalbitz, 2003). Thus, DOC biodegradation is influenced by the intrinsic chemical composition such as aromaticity and humification degree of DOC in most ecosystems (Kalbitz et al., 2003; Wickland et al., 2007; Pinsonneault et al., 2016; Mao et al., 2017). In addition to the initial C quality, DOC biodegradation also depends strongly on the nutrient availability, pH, temperature, moisture, and microbial community (Marschner and Kalbitz, 2003; Räsänen et al., 2014; Mao et al., 2017; Mao and Li, 2018). In general, DOC is primarily derived from the decomposition of soil organic matter and plant litters, root exudates, and microbial activity (Freeman et al., 2004; Kane et al., 2014), all of which are influenced by the vegetation community structure (Robroek et al., 2016). Therefore, the shifts in species composition would influence DOC biodegradation through altered DOC chemical composition, nutrient availability, and other biotic and abiotic factors, which is likely critical for accurately assessing and predicting C balance in terrestrial ecosystems (Neff and Asner, 2001). In recent decades, the coverage of dinitrogen (N2)-fixing trees (mainly Alnus spp.) have dramatically increased in the northern midand high latitudes as a consequence of climate warming and anthropogenic activities such as increased frequency of fire and disturbances (Lantz et al., 2010; Frost and Epstein, 2014; Hiltbrunner et al., 2014). For example, Lantz et al. (2013) have observed that alder (Alnus viridis) stem density increased by 68% in the upland tundra of Mackenzie Delta (Canada) between 1972 and 2004. The spreading of N2-fixing trees to these ecosystems can not only introduce large amounts of reactive N into soils (Rhoades et al., 2001; Mitchell and Ruess, 2009), but also produce woody plant litters containing high lignin concentration (Kaštoviká et al., 2018). Furthermore, like atmospheric N deposition, N2-fixing tree encroachment can lead to biodiversity loss, soil acidification, and altered C and nutrient cycles (Mitchell and Ruess, 2009; Hiltbrunner et al., 2014; Chen et al., 2017). Accordingly, this shift in species composition may cause substantial influences on DOC biodegradation in the northern mid- and high latitudes. Unfortunately, the direction and magnitude of the changes in DOC biodegradation following N2-fixing woody plant encroachment is still unclear due to the lack of empirical studies. Alnus sibirica, a common N2-fixing tree in many parts of Northeast China, has widely expanded to boreal peatlands and formed tree islands in this region (Chen et al., 2017). To assess the effect of N2-fixing tree encroachment on DOC biodegradation, we extracted DOC from peats at 0–10 cm, 10–20 cm, and 20–40 cm depths in the A. sibirica islands and adjacent open peatlands in a boreal peatland in the north of Da'xingan Mountain, Northeast China, and then assessed peat-derived DOC biodegradation under aerobic condition with a 42-day laboratory incubation experiment. Because of the importance of N availability and C quality to DOC biodegradation in boreal peatlands (Pinsonneault et al., 2016), we measured DOC, ammonium nitrogen (NH+ 4 -N), nitrate nitrogen (NO− 3 -N), dissolved inorganic nitrogen (DIN), and dissolved total nitrogen (DTN) concentrations, specific ultraviolet

absorbance at 254 nm (SUVA254), and humification index in the extracts. We hypothesized that: (1) A. sibirica encroachment would in− crease NH+ 4 -N, NO3 -N, DIN, and DTN concentrations in the extracts due to the symbiotic N2 fixation, and (2) A. sibirica encroachment would elevate SUVA254 and humification index of DOC because of increased lignified woody litter inputs to soils. Considering the contrasting roles of N availability and DOC aromaticity and humification degree in regulating microbial decomposition (Wickland et al., 2007; Pinsonneault et al., 2016; Mao et al., 2017), we raised the third hypothesis: (3) A. sibirica encroachment would not affect DOC biodegradation because the positive effect of improved N availability would be mitigated by the negative effect of increased DOC aromaticity and humification degree. 2. Materials and methods 2.1. Study site This study was conducted in a permafrost peatland (52.94°N, 122.86°E) located in the north of Da'xingan Mountain, Heilongjiang Province, China (Fig. 1). The area of permafrost peatland is about 13,300 km2 in the Da'xingan Mountain (Jin et al., 2007). In this region, − annual NH+ 4 -N, NO3 -N, and DIN deposition is about 2.9, 1.6, and 4.5 kg N per hectare (Zhan et al., 2014). The climate of the study site is cool continental monsoon. Mean (1980–2010) annual temperature is −3.9 °C ranging from −29.7 °C in January to 18.4 °C in July. Mean annual precipitation is about 450 mm, 45% of which falls in July and August. This peatland is an ombrotrophic bog that began forming approximately 3000 years ago (Xing et al., 2015) over alluvium. Peat depth ranged from approximately 30 cm at the margins to 150 cm near the center, and active layer depth is nearly 50 cm. The soil is classified as Histosol according to the World Reference Base for Soil Resources (IUSS Working Group WRB, 2006). The surface microtopography of this peatland includes hummock, lawn, and hollow. The dominant species of this peatland are dwarf shrubs, sedges, and Sphagnum mosses. Since the 1980s, tree species, such as A. sibirica, Betula platyphylla, and Larix gmelinii, have expanded to the peatland as a result of climate warming and associated soil drying (Fig. S1). In this study, we chose A. sibirica as a focal species because it is a common N2-fixing tree and generally formed dense tree islands in this peatland. In the study area, A. sibirica is widely distributed in the forests, especially in the riparian forests. In this peatland, deciduous shrub biomass increased, while moss species disappeared following about 28 years of A. sibirica expansion (Chen et al., 2017). 2.2. Experimental design and sample collection In August 2015, six pairs of A. sibirica islands (tree age ranging from 27 to 29 years) and open peatlands were selected. Tree canopy cover was about 55% in the A. sibirica islands (Fig. S1). For both tree islands and corresponding open peatlands, the microtopography of the sampling plots was hummock. The mean distance between tree islands and the adjacent open peatlands was N30 m, and the mean distance between pairs was about 150 m. In the tree islands, A. sibirica stem density was 2.32 per m2. In addition, aboveground plant biomass in the open peatlands and A. sibirica islands were 5.70 and 50.51 Mg ha−1, respectively (Chen et al., 2017). In each tree island, three subplots under about half of tree canopy were selected to collect peat samples, and then three subplots along the same directions were chosen in the open peatlands (Fig. 1). In each subplot, one peat profile to a depth of 40 cm was sampled with a peat auger (12 cm in diameter), divided into 0–10 cm, 10–20 cm, and 20–40 cm using a knife, and then transported back to the laboratory on ice at 0–4 °C instantly. Peat samples collected from the three subplots were combined according to their depth gradient, resulting in 36 peat samples. Soil microbial biomass was measured by the fumigation-

Y. Zhang et al. / Science of the Total Environment 695 (2019) 133882

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Fig. 1. Location of the study site and the spatial arrangement of sampling subplots.

extraction method (Wu et al., 1990), and pH was measured in a 1:5 soil: water suspension with a pH meter. The peat characteristics are listed in Table 1. Dissolved organic carbon and N fractions were assessed by a water extraction method proposed by Pinsonneault et al. (2016). Each combined peat sample was extracted in triplicate, and in total, there were 108 water samples. Field-moist peat equivalent to 2 g dry-weighted (oven-dried at 105 °C for 48 h) peat was placed in 100 mL deionized water in a 200 mL centrifuge vial, gently mixed by hand, and soaked overnight for 12 h at about 20 °C. Subsequently, the extracts were filtered first with ashless filter paper to remove the suspended particulate organic matter, re-filtered through pre-combusted (450 °C for 4 h) and pre-washed (with both Milli-Q and peat extracts) 0.7 μm Whatman GF/ F glass microfiber filters, and then subdivided into two subgroups. To observe the shaking and filtering processes, the extracts will be compared to process blanks (n = 3). For the blank treatment, 100 mL deionized water were added to centrifuge vials and filtered by the procedure as described above. One subgroup of re-filtered extracts was used to determine the chemical and optical properties, and the second subgroup was instantly used to assess DOC biodegradation.

Dissolved organic carbon concentration (mg L−1) was determined with a total organic carbon analyzer (TOC-5000, Shimadzu, Japan), −1 − NH+ ) were measured with a 4 -N and NO3 -N concentrations (μg L continuous-flow autoanalyzer (AA3, Seal Analytical, Germany), and dissolved total N concentration (mg L−1) was assessed with a continuousflow autoanalyzer following alkaline peroxodisulfate oxidation (Ebina et al., 1983). Dissolved inorganic nitrogen concentration (μg L−1) was − the sum of NH+ 4 -N and NO3 -N concentrations. For all peat extracts, SUVA254 (L mg C−1 m−1) was measured by a 1 cm quartz cell with a UV–Visible spectrophotometer (UV-1750, Shimadzu Corporation, Japan), and was calculated by dividing the ultraviolet absorbance at 254 nm by the DOC concentration (Weishaar et al., 2003). Fluorescence excitation-emission matrices were determined at excitation wavelengths between 220 and 400 nm and emission wavelengths between 250 and 500 nm with 2-nm intervals with a Hitachi F-7000 fluorescence spectrometer (Hitachi High Technologies, Japan) (Fellman et al., 2008). Humification index was obtained by dividing the area under the emission spectra 435–480 nm by 300–445 nm at excitation wavelength 254 nm (Zsolnay et al., 1999). In the previous studies, SUVA254 and humification index have been widely used to indicate aromaticity and

Table 1 Peat characteristics of three different soil depths (0–10 cm, 10–20 cm, and 20–40 cm) in the open peatlands and adjacent Alnus islands. Soil properties

Soil moisture (g g−1) Organic carbon (mg g−1) Total nitrogen (mg g−1) Ammonium-nitrogen (μg g−1) Nitrate-nitrogen (μg g−1) Microbial biomass C (mg g−1) pH

0–10 cm soil depth

10–20 cm soil depth

20–40 cm soil depth

Open peatlands

Alnus islands

p-Value

Open peatlands

Alnus islands

p-Value

Open peatlands

Alnus islands

p-Value

5.12(0.11) 399(7) 15.6(1.1) 38.2(5.5) 3.69(0.50) 16.48(1.05) 4.84(0.05)

4.14(0.14) 404(7) 18.9(1.4) 166.3(18.9) 6.47(0.48) 22.62(1.20) 4.49(0.05)

b0.001 0.515 0.001 b0.001 0.008 0.003 0.001

5.67(0.33) 351(14) 16.3(1.3) 23.1(2.9) 4.50(0.77) 10.16(0.68) 5.04(0.04)

4.72(0.42) 385(10) 19.6(1.1) 37.8(2.4) 10.81(0.83) 13.52(0.51) 4.80(0.04)

0.031 0.104 0.002 0.001 0.007 0.001 0.002

5.15(0.23) 359(26) 18.6(1.4) 23.1(2.9) 6.35(0.66) 5.62(0.25) 5.05(0.09)

3.59(0.29) 324 (30) 20.9(1.5) 37.8(2.4) 10.17(0.47) 6.78(0.21) 4.83(0.07)

0.002 0.004 0.001 b0.001 0.005 0.023 0.001

Values are means and data in the parentheses are standard errors (n = 6). At each peat depth, p b 0.05 indicates significant differences between open peatlands and Alnus islands.

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humification degree of DOC, respectively (Weishaar et al., 2003; Fellman et al., 2008). Furthermore, DOC with a higher humification degree has a greater proportion of condensed aromatic components, leading to a lower DOC biodegradation (Fellman et al., 2008; Chen et al., 2011). Dissolved organic carbon biodegradation was measured by the standard laboratory incubation method (McDowell et al., 2006; Vonk et al., 2015). To prevent the excessive growth of microorganisms, the extracts were diluted to about 10 mg DOC L−1 prior to the start of incubation experiment. For each water sample, 20 mL of diluted extract was added to 100-mL glass jars, and three additional jars with 20 mL filtered deionized water were used as blanks. In a recent study, Vonk et al. (2015) have proposed that inoculation of soil extracts is not needed because filtration through 0.7-μm filters allows for a sufficient amount of microbes to pass the filter in the northern high latitudes, so we did not add inoculum to the extracts. All jars were sealed using Teflon-lined stoppers, and then incubated at 20 °C in the dark for 42 days. Approximately 20 mL headspace gas was taken on days 2, 6, 12, 18, 24, 30, 36, and 42, and CO2 concentration (mg L−1) was measured using an Agilent 7890A gas chromatograph (Agilent Technologies, USA). The jars were purged with CO2-free air after gas sampling and then sealed. Before and after incubation, dissolved inorganic C concentration (mg L−1) in the extracts was determined using a total organic carbon analyzer. For the diluted extracts, cumulative CO2 production and DIC concentration were calculated by multiplying the dilution factor. Considering that the changes in dissolved inorganic C concentration in the extracts were negligible over the incubation periods (Table S1), DOC biodegradation was obtained by the difference in the cumulative CO2 production between soil extracts and blanks, and expressed as the percentage of initial DOC concentration in the extracts. 2.3. Statistical analyses Before analysis, all data were test for normality and homogeneity of variances by the Levene's test, and the data were normally distributed − when p N 0.05. The data including humification index, NH+ 4 -N, NO3 -N, DIN, DTN, and DOC:DIN ratio were log-transformed to meet the assumption of normality. We used General Linear Models to test the effects of vegetation type and peat depth on DOC parameters. In the models, soil depth was initially included as a fixed effect, and vegetation type was a random effect. When there was a significant interaction, a paired t-test was used to examine the significant differences in DOC parameters at each depth. A Pearson's correlation analysis was performed to examine relationships between DOC biodegradation and initial properties of peat extracts. The statistical analyses were executed using the SPSS 13.0 for Windows, and p b 0.05 indicates significant differences. 3. Results Compared to open peatlands, Alnus islands had lower soil moisture − and pH value, and greater concentrations of NH+ 4 -N, NO3 -N, and total N at each peat depth (Table 1). However, Alnus islands had lower soil

organic C concentration than open peatlands only in the 20–40 cm peat depth (Table 1). In addition, soil microbial biomass C was observed to be higher in the Alnus islands than in the open peatlands across all peat depths (Table 1). There were significant differences between the two vegetation types − and peat depth regarding concentrations of DOC, NH+ 4 -N, NO3 -N, DIN, and DTN, and DOC:DTN ratio (Tables 2 and 3). Alnus sibirica islands had greater DOC concentration in the extracts than open peatlands at 0–10 cm and 10–20 cm depths (Table 2). Compared with open − peatlands, A. sibirica islands generally had higher NH+ 4 -N, NO3 -N, DIN, and DTN concentrations in the extracts at all depths, and thus had lower DOC:DIN and DOC:DTN ratios (Table 2). However, vegetation types had no significant effect on SUVA254 and humification index in each peat depth (Table 3 and Fig. 2). Dissolved organic carbon biodegradation varied significantly with vegetation types (Table 3). By the end of 42-day incubations, DOC biodegradation in the A. sibirica islands was 24.5%, 15.4%, and 38.3% greater than that in the open peatlands at 0–10 cm, 10–20 cm, and 20–40 cm depths, respectively (Fig. 3). When all data were pooled together, DOC biodegradation in the extracts was significantly and negatively related to DOC:DIN and DOC:DTN ratios (r = −0.617 and −0.655, respectively), but did not exhibit significant relationships with SUVA254 and humification index (Fig. 4). 4. Discussion As hypothesized, A. sibirica expansion to this peatland increased the − concentrations of NH+ 4 -N, NO3 -N, DIN, and DTN, leading to the declined DOC:DIN and DOC:DTN ratios in the soil extracts. It is well-documented that Alnus species could enhance N inputs to soils and increased N availability primarily because of the association with the N2-fixing actinomycete Frankia in northern mid- and high latitudes (Rhoades et al., 2001; Titus, 2009). For instance, Mitchell and Ruess (2009) have observed that mean annual N inputs by A. viridis ranged from 2.5 to 6.6 kg N per hectare via N2 fixation in interior Alaska. Moreover, the expansion of A. sibirica to this peatland could accelerate organic matter decomposition by producing plant litters with high nutrient concentrations (Handa et al., 2014), enhancing microbial growth (Bragazza et al., 2006), and increasing the dominance of vascular plants over mosses (Chen et al., 2017), and thus enhance N returns to soils. In this study, − A. sibirica islands had greater soil NH+ 4 -N, NO3 -N, and total N concentrations than the open peatlands (Table 1), resulting in higher DIN and DTN concentrations in soil extracts across all peat depths. Increased N inputs following A. sibirica expansion can alleviate the N limitation of plant growth and microbial activity in boreal peatlands (Bragazza et al., 2006; Song et al., 2017). Furthermore, Alnus-fixed N might exceed the demands of plants and microbes, leading to N leaching from soils, especially NO− 3 -N in the peatland. Generally, N leaching can not only cause eutrophication in boreal aquatic systems, but also result in soil acidification, cation losses, and nutrient imbalances (Cairns and Lajtha, 2005). Indeed, we observed a decline in pH values across all peat depths following A. sibirica expansion (Table 1). Therefore, increased N

Table 2 Chemical properties of soil extracts in the open peatlands and adjacent Alnus islands. Initial chemical properties

DOC (mg L−1) DTN (mg L−1) −1 NH+ ) 4 -N (μg L −1 NO− ) 3 -N (μg L DIN (μg L−1) DOC:DTN ratio DOC:DIN ratio

0–10 cm soil depth

10–20 cm soil depth

20–40 cm soil depth

Open peatlands

Alnus islands

p-Value

Open peatlands

Alnus islands

p-Value

Open peatlands

Alnus islands

p-Value

23.9(1.2) 0.78(0.02) 69.8(9.2) 71.7(7.6) 141.5(12.8) 30.6(1.1) 175.4(17.0)

33.0(1.0) 2.03(0.05) 153.7(5.5) 218.2(4.0) 372.0(38.9) 16.3(0.6) 93.6(9.6)

b0.001 b0.001 0.001 0.021 0.003 b0.001 0.007

18.7(0.8) 0.78(0.01) 50.9(4.1) 73.0(6.2) 123.9(8.2) 24.2(1.4) 153.6(9.7)

22.9(0.9) 1.54(0.10) 106.7(3.4) 129.1(25.7) 235.8(25.2) 15.3(1.4) 102.8(10.8)

0.001 b0.001 b0.001 0.110 0.009 b0.001 0.015

16.1(0.6) 0.84(0.02) 46.2(3.3) 69.3(4.0) 115.4(2.8) 19.2(1.0) 140.2(6.0)

16.3(1.1) 1.10(0.04) 66.1(4.4) 106.5(8.7) 172.6(11.5) 14.8(0.5) 95.8(7.3)

0.943 0.001 0.012 0.004 0.003 0.018 0.009

Values are means and data in the parentheses are standard errors (n = 6). At each peat depth, p b 0.05 indicates significant differences between open peatlands and Alnus islands. − DOC, dissolved organic carbon; DTN, dissolved total nitrogen; NH+ 4 -N, ammonium-nitrogen; NO3 -N, nitrate-nitrogen; DIN, dissolved inorganic nitrogen.

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Table 3 Results (F-values) of two-way ANOVAs on the effects of vegetation type (V), soil depth (D), and their interaction on chemical properties and DOC biodegradation in the extracts.

V D V×D

DOC

DTN

NH+ 4 -N

NO− 3 -N

DIN

mg L−1

mg L−1

μg L−1

μg L−1

μg L−1

2.9 7.5 11.2⁎⁎⁎

6.9 0.8 53.0⁎⁎⁎

8.2 3.0 17.9⁎⁎⁎

5.6 1.0 4.1⁎

6.8 1.7 9.4⁎⁎

DOC:DIN ratio

DOC:DTN ratio

SUVA254

Humification index

L mg C−1 m−1 26.1⁎ 0.7 1.8

10.6⁎ 1.7 10.6⁎⁎⁎

0.6 19.1⁎ 0.3

DOC biodegradation %

0.5 35.5⁎ 0.4

16.1⁎ 1.0 3.6

− DOC, dissolved organic carbon; DTN, dissolved total nitrogen; NH+ 4 -N, ammonium nitrogen; NO3 -N, nitrate nitrogen; DIN, dissolved inorganic nitrogen; SUVA254, specific ultraviolet absorbance at 254 nm. ⁎ p b 0.05. ⁎⁎ p b 0.01. ⁎⁎⁎ p b 0.001.

concentrations in the soil extracts following N2-fixing tree expansion would exert remarkable influences on ecological processes at the whole watershed level. Alnus sibirica expansion increased the amount of peat-extracted DOC in the 0–10 and 10–20 cm depths, but had no effect in the 20–40 cm depth (Table 2). Generally, DOC concentration is largely dependent on the amount of organic C stored in soils (Kane et al., 2014). In this study, organic C concentration kept unchanged at 0–10 and 10–20 cm depth, and declined at 20–40 cm depth (Table 1). Thus, the differential responses of peat-derived DOC concentration to A. sibirica expansion might be explained by the different changes in organic C concentration among peat layers. Although A. sibirica expansion did not increase organic C concentration in the peats, but may enhance DOC production via the following mechanisms. First, increased N availability following A. sibirica expansion stimulated plant growth and increased plant productivity (Chen et al., 2017), which probably increased the quantity of vegetation-derived DOC inputs to the peats such as leaf leachate and root exudate. Furthermore, A. sibirica expansion could cause increases in substrates and nutrients for microbial growth (Bragazza et al., 2006; Song et al., 2017), and thus elevate soil microbial biomass and increase microbial production of DOC in peats. In addition, A. sibirica expansion might stimulate organic matter decomposition through improved substrate quality (Giudice and Lindo, 2017; Bell et al., 2018; Mao et al., 2018) and increased microbial activity (Bragazza et al., 2006), resulting in enhanced DOC release from decomposing peats. Therefore, A. sibirica expansion increased DOC concentration in the extracts at both 0–10 and 10–20 cm depth, and did not decrease DOC

concentration at 20–40 cm depth. In boreal peatlands, peat-derived DOC concentration is also influenced by permafrost thawing and precipitation regime (Hodgkins et al., 2014; Bragazza et al., 2016). Considering the critical role of DOC in regulating C cycle, additional studies are needed to examine the temporal changes in peat-derived DOC following A. sibirica expansion in boreal peatlands. Inconsistent with the second hypothesis, SUVA254 and humification index of DOC in the extracts remained unchanged following A. sibirica expansion in this peatland. Although the effect of plant species on DOC quality has been examined, the results are still inconclusive. Some studies have found that woody plants can produce greater SUVA254 values of litter-derived DOC than mosses (Kaštoviká et al., 2018; Mastný et al., 2018). However, in a plant removal experiment, Robroek et al. (2016) have showed a decrease of low molecular weight organic compounds in the dissolved organic matter after vascular plant removal because of the declined microbial enzymatic activities in a peatland, Southern Sweden. Furthermore, several other studies have also observed that vascular plant-derived DOC, especially deciduous shrub, had lower SUVA254 values and recalcitrant, humic-like components of DOC than mosses (Pinsonneault et al., 2016; Giudice and Lindo, 2017). In this peatland, the increased biomass of deciduous shrubs as well as the disappearance of mosses following A. sibirica expansion might stimulate microbial decomposition of recalcitrant DOC (Robroek et al., 2016; Chen et al., 2017), which would offset A. sibirica expansion-induced increases in DOC aromaticity and humification degree. Moreover, because phenol oxidase activity is often inhibited by the anerobic and nutrient-poor conditions in the peatlands (Freeman

Fig. 2. Effect of Alnus expansion on optical properties of DOC in the soil extracts. The boxes represent the mean value, and the error bars are the standard error (n = 6). At each peat depth, ns indicates no significant differences (p N 0.05) between open peatlands and Alnus islands.

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Fig. 3. Effect of Alnus expansion on DOC biodegradation in the soil extracts. The boxes represent the mean value, and the error bars are the standard error (n = 6). At each peat depth, p b 0.05 indicates significant differences between open peatlands and Alnus islands.

et al., 2001; Bragazza et al., 2006), increased N availability and decreased soil moisture following A. sibirica expansion (Table 1) might stimulate the decomposition of phenolic compounds. The expansion of A. sibirica to the peatland enhanced the biodegradation of DOC in the peat extracts, and the magnitude was greatest in the relatively deep layer (20–40 cm depth). Thus, our result did not support the third hypothesis. In boreal and subarctic regions, DOC biodegradation is often controlled by C:N stoichiometry and/or initial C quality in wetland ecosystems (Wickland et al., 2007; Fellman et al., 2008; Roehm et al., 2009; Kane et al., 2014; Mao et al., 2017). For example, Pinsonneault et al. (2016) have pointed out that C:N ratio and SUVA254 can effectively indicate the biodegradability of vegetationderived DOC in a cool temperate ombrotrophic bog, Canada. In this study, DOC biodegradation was negatively correlated with DOC:DTN and DOC:DIN ratios, and did not show significant relationships with SUVA254 and humification index. Therefore, these results clearly indicated that enhanced DOC biodegradation following A. sibirica expansion was largely driven by the increase in N availability and associated shifts in C:N stoichiometry in this boreal peatland. In the northern mid- and high latitudes, DOC:DIN and DOC:DTN ratios are believed to be important factors influencing DOC biodegradation due to the importance of DIN to heterotrophic growth and metabolism and the overriding control of C:N stoichiometry on microbial processes (Wickland et al., 2012; Pinsonneault et al., 2016). In this peatland, increased DIN and DTN concentrations and associated declines in DOC:

DIN and DOC:DTN ratios following A. sibirica expansion could alleviate the N limitation of microbial growth and activity, resulting in enhanced DOC biodegradation. Moreover, because of the relatively lower DOC: DTN and DOC:DIN ratios, the magnitude of the increase in DOC biodegradation was greater at both 0–10 cm and 20–40 cm depths than at 10–20 cm depth. In this study, peat-derived DOC biodegradation ranged from 34% to 50% by the end of 42-day incubation, which were comparable to those found in the soil extracts from wetlands in Alaska, USA (23–42% in a 30-day incubation; Fellman et al., 2008) or in Sweden (21–56% in a 12-day incubation, Roehm et al., 2009). Compared with open peatlands, DOC biodegradation in the 0–10, 10–20, and 20–40 cm depths increased by 25%, 15%, and 38% in the A. sibirica islands. In a cool temperate bog, Pinsonneault et al. (2016) observed approximately three-fold changes in vegetation-derived DOC biodegradation among species. These findings highlight that vegetation exerts a substantial influence on DOC biodegradation in boreal peatlands. In the present study, we only investigated the effect of A. sibirica expansion on DOC biodegradation during the peak growing season in this peatland. Previous studies found seasonal variations of soil-derived DOC chemical composition and N availability due to the substantial differences in DOC sources, hydrological regimes, microclimate condition, microbial activity, and permafrost thawing (Yano et al., 2004; Lambert et al., 2013; Hodgkins et al., 2014). To fully understand C dynamics of boreal peatlands, further studies are required to evaluate the effect of

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Fig. 4. The correlations between DOC biodegradation and the initial properties of soil extracts.

A. sibirica expansion on DOC biodegradation throughout the permafrost-free season. In boreal peatlands, DOC is an appreciable fraction of organic C in soils, and plays an essential role in regulating ecosystem and regional C cycle (Limpens et al., 2008). Thus, our findings will provide insights into future C budget in these ecosystems. On one hand, the expansion of N2-fixing woody plants stimulated microbial decomposition of DOC, which would increase greenhouse gas emissions from soils and reduce the C sink of boreal peatlands. On the other hand, enhanced DOC biodegradation following N2-fixing woody plant expansion would result in a decline in the amounts of DOC delivery to the boreal river networks. Given that terrestrial-derived DOC is the primary energy source for aquatic heterotrophic microbes (Neff and Asner, 2001; Kalbitz et al., 2003), this may substantially alter ecosystem structure and function in boreal rivers. In addition, the broad occurrence of N2-fixing woody plants in boreal peatlands can introduce large amounts of N to soils, leading to N saturation in these formerly N-poor ecosystems (Bühlmann et al., 2016). Previous studies have observed that soil N inputs by Alnus species did not increase with increasing tree age (Bühlmann et al., 2016). Therefore, the effect of A. sibirica expansion on DOC biodegradation may be complex, and vary with tree size and coverage in boreal peatlands. Considering the key role of DOC in regulating ecosystem C cycle, an age sequence of A. sibirica islands is urgently needed to comprehensively assess the effect of N2-fixing tree expansion on DOC biodegradation in boreal peatlands.

5. Conclusions This study showed that A. sibirica expansion promoted the biodegradation of peat-derived DOC in the 0–10 cm, 10–20 cm, and 20–40 cm depths in a boreal peatland, Northeast China. Moreover, DOC biodegradation correlated significantly and negatively with DOC: DIN and DOC: DTN ratios, indicating that the shift in DOC biodegradation after A. sibirica expansion was largely caused by the improved N availability and subsequently altered C:N stoichiometry. These findings suggest that expansion of N2-fixing woody plants has the potential to result in DOC losses from soils as greenhouse gases and reduce C sink function in boreal peatlands.

Acknowledgments We wish to acknowledge Wen-Wen Tan and Yu Du for the field work, and Hui-Min Chen for the laboratory analyses. We also thank the editor and two anonymous reviewers for their constructive comments on a previous draft of this manuscript. This work was supported by the National Natural Science Foundation of China (No. 31570479). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.133882.

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