Effects of mowing on N2O emission from a temperate grassland in Inner Mongolia, Northern China

L. Zhang, Q. Wang, H. J. Laanbroek, C. Wang, D. Guo, and L. Li State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, the Netherlands Institute of Environmental Biology, Utrecht University, Utrecht, the Netherlands Shandong Normal University, Jinan, 250014, China


Introduction
The temperate steppe in northern China is a typical vegetation type on the Eurasian continent and is sensitive to anthropogenic disturbances and climate changes (Christensen et al., 2004;Niu et al., 2009).Both mowing and grazing are prevailing management practices in these areas (Tix, 2005).Recently, mowing was highly recommended for sustainable grassland management in a national project called Grain for Green, which aims to restore the degraded ecosystems in western China (Liu et al., 2009).Mowing, the removal of a part of plant shoot tissue, has negative effects on overall plant growth and carbon allocation (Ferraro, 2002), which can influence the root carbon exudation and the rhizosphere organisms that rely on the nitrogen released from the plant roots (Hamilton et al., 2008).The removal of some plants by mowing inevitably leads to the adjustment of the size of the root system and thus causes the death and decay of the roots and nodules, followed by decomposition, mineralization of nitrogen, nitrification and denitrification.Mowing also reduces the input of above-ground litter into the soil (Valko et al., 2012), and consequently decreases the amount of coarse organic matter in the soil (Mikola et al., 2009) and related gas emissions from the soil, including nitrous oxide (N 2 O).
N 2 O is a vital greenhouse gas, and it contributes approximately 6 % to the anticipated global warming (IPCC, 2007).The global atmospheric N 2 O concentration increased from a preindustrial value of 270 to 322 ppb in 2008 (IPCC, 2007).N 2 O emitted from soils is considered as one of the major contributors to this rise (Wrage et al., 2001).Denitrification is a key ecological process that determines N 2 O production in an ecosystem.Related studies suggest that N 2 O emissions would be determined by soil properties and processes including soil temperature, soil moisture, substrate availability (Wrage et al., 2001), soil diffusive characteristics, air-fillled porosity (Neftel et al., 2000), the activity of nitrifying/denitrifying microbial communities (Steenwerth, 2008) and concurrent N 2 O consumption processes in the soil (Cavigelli, 2001).The complexity of these factors, which regulates N 2 O production, consumption and emission, Both grazing and mowing alter soil properties in grasslands, subsequently affecting the grassland N cycle, including soil N 2 O emission.Wolf et al. (2010) found that grazing decreased grassland N 2 O release via changing soil and environmental conditions.Sørensen et al. (2008) reported that mowing altered N cycling by decreasing soil N mineralization.Other aspects of grazing, such as random urine and faeces deposit of livestock, increased N 2 O emissions (Yanulki et al., 1998).Therefore, it is necessary to quantify the changes in N 2 O emission caused by mowing to fully understand the regional budget of trace gases.
However, there is no information available on how mowing affects N 2 O emission (Calanca et al., 2001), and findings from grazing management cannot be extrapolated to those from mowing, as defoliation by grazing is more frequent and not comparable to mowing.To quantify N

N 2 O flux measurements
N 2 O fluxes were measured by a static chamber technique following Zhang et al. (2007).Briefly, the chamber consists of a stainless steel permanent base (50 cm × 50 cm × 12 cm) and a stainless steel top (50 cm × 50 cm × 50 cm).The base, with a 3 cm-deep groove on the upper side for water sealing, was driven into soil down to 12 cm each year approximately a month before the measurement started in each plot (Song et al., 2009).The top is covered by heat-isolating and light-impenetrable materials outside and equipped with one rubber septa for gas sampling and two electric fans inside for mixing the air in the chamber headspace continuously and thoroughly (Wang and Wang, 2003).During the measurements, the top was installed over the base, with downsides into the groove.The grooves were filled with water to seal the chamber.Gas samples were collected at 10 min intervals for 30 min (i.e.0, 10, 20 and 30 min) through the septa using 60 l syringes with airtight stopcocks.All gas samples were brought to a laboratory at the research station for N 2 O analyses within 12 h after sampling.N 2 O concentration was analysed using a gas chromatograph (HP 5860, Agilent Technologies).N 2 O flux was calculated from the linear slope of the mixing ratio changes in the four samples taken at 0, 10, 20 and 30 min after the chamber was closed.The N 2 O flux was measured weekly from June to September in 2008 and biweekly from May to September in 2009.Concurrent with the N 2 O flux sampling, air temperature in the chamber, soil temperature and soil moisture next to the chambers were measured using a portable digital thermometer.Air temperature was measured at 40 cm height above the soil surface, and soil temperature was measured at 5 cm depth.

Measurements of vegetation and soil
Prior to experiments in 2008 and 2009, vegetation variables were measured once at peak biomass (i.e.August).A 1 m × 1 m frame with 100 equally distributed grids of 10 cm × 10 cm was put above the canopy adjacent to the flux chambers in each 10 m × 20 m plot.The coverage of each species was visually estimated following Yang et al. (2011) in all the grids, and then summed as the total coverage for the quadrat.After the measurement of the coverage, all plants were clipped at ground level within the quadrat of 1 m 2 and separated into living vegetation as total biomass (i.e.above-ground biomass, ANPP; see also Zhang et al., 2012) and dead plant materials as standing litter.The total litter was the sum of the standing litter and litter collected on the surface within the quadrat.All plant materials sampled were oven dried at 70 • C for 48 h and weighed.
Soil samples (0-10 cm layer) were collected monthly during the 2009 growing season using a soil corer (5 cm diameter) at the time of the fourth gas sampling.At each soil sampling, three soil cores were taken randomly at each plot and mixed evenly.These samples were separated into two set of sub-samples: one set was stored at 4 • C for microbial analysis, and the other was air-dried for soil organic carbon (SOC), total nitrogen (TN) and total phosphorus (TP) analyses as well as soil temperature (ST), soil moisture (SM), soil microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) for identifying the controls of N 2 O fluxes (for details see Zhang et al., 2012).

Statistical analysis
A repeated measure analysis of variance (ANOVA) was performed to examine interannual variability in SOC, TN and TP with the pooled data of all treatments during the growing season.Between-subject effects were evaluated as mowing effects and within-subject effects were the time-of-season.Regression analyses were made be- different times, we used the means of the whole growing season.Therefore, every variable had five replicates and five treatments.Differences in seasonal cumulative N 2 O flux, total litter and ANPP among treatments were determined by analysis of simple one-way ANOVA.To examine which variable had the most important effect on N 2 O fluxes, a stepwise multiple regression analysis was applied between the whole growing season mean N 2 O fluxes (as an independent variable) and the measured variables (as dependent variables).All statistical analyses were conducted with the SAS 8.0 software package (SAS Institute Inc., Cary, NC, USA).

Climate
The study area received 370 mm and 185 mm of rain for 2008 and 2009, respectively, and showed great variations in temporal distribution (Fig. 1).Most of the rain fell over the summer months (from June to August), which accounts for 90 % of the annual total precipitation in the two years.Soil temperature and moisture varied seasonally at this site (see Zhang et al., 2012).Briefly, soil temperature at 5 cm depth ranged from 13.1 to 31.1 • C with an average of 22.3 • C in 2008, and 8.5 to 27.9 • C with an average of 18.1 • C in 2009.The temperature also generally correlated negatively with mowing height.Soil moisture peaked in July and had similar temporal fluctuation patterns over the two growing seasons, although the rainfall that occurred in this area was substantially different during the same period.However, no consistent relationship was observed between soil moisture and mowing height.

Effect of mowing on total coverage, above-ground biomass and litter
Total coverage, above-ground biomass and litter measure in 2009 (prior to the N 2 O experiment) are presented in Table 1.The mowing treatments did not significantly affect plant coverage, regardless of mowing heights (P > 0.05).The above-ground biomass and total litter decreased progressively as cutting height decreased from 15 cm to 2 cm, except for the above-ground biomass of M 5 in 2008, and significantly lower litter mass was found for the M 2 and M 5 treatments (P < 0.01).This was also observed for ANPP, indicating that a cutting height > 10 cm has little effect on net primary productivity.Above-ground biomass and total litter were markedly lower in 2008 compared with 2009, except for the M 2 treatment in ANPP.These results indicated that the effects of higher cutting heights (M 15 and M 10 ) on net primary production were limited in this grassland ecosystem.We also observed that the above-ground biomass and total litter were significant differences in the two years, except for the above-ground biomass of treatment M 2 , suggesting that a significant year effect existed in the grassland (Table 1).
In addition, the biomass of Artemisia frigida, a species with low height, contributed most of the total biomass of M 2 and M 5 plots relative to that of the control, M 15 and M 10 plots (data not shown).M ck , M 15 , M 10 , M 5 , and M 2 , respectively, in 2009 (Fig. 2).The variation of N 2 O fluxes was relatively narrow in the dry year ( 2009) compared with the wet year (2008) (Fig. 2).

Temporal dynamics in
The higher variation in N 2 O emission rate resulted in high variation in the monthly cumulative N 2 O flux, with a range of 0.48 to −0.35 kg N 2 O ha −1 over the two growing seasons (Fig. 3). Figure 3 1).Over the two-year period, the M 15 , M 10 , M 5 and M 2 mowing treatments decreased the total N 2 O emission by 159, 73, 36 and 77 %, respectively, relative to the control (Table 1).

Controls of N 2 O fluxes
The seasonal N 2 O flux increased with increasing soil moisture content, indicating a strong influence of soil moisture on seasonal N 2 O flux (Fig. 4a).The results of regression analyses showed that the closest relationship existed between the N 2 O fluxes and soil moisture (r 2 = 0.8, P < 0.0001), followed by above-ground plant biomass (r 2 = 0.30, P = 0.02), and microbial biomass nitrogen (r 2 = 0.16, P = 0.14) in this experiment (Fig. 4a (Fig. 4f).Our stepwise regression analyses showed that a combination of soil moisture (partial R 2 = 0.42, P = 0.0005), soil temperature (partial R 2 = 0.13, P = 0.02) and total phosphorus (partial R 2 = 0.08, P = 0.047) explained 62.5 % of the variation in the N 2 O fluxes (P < 0.005) in May.Microbial biomass nitrogen explained 21.2 % of the variation in the N 2 O fluxes (P = 0.02) in June, and soil moisture explained 21.4 % of the variation (P = 0.02) in July.Other factors did not contribute significantly to the explanation of the variation in N 2 O fluxes in these summer months.In addition, we did not find a significant correlation between N 2 O fluxes and any other variables measured at P < 0.05 in August and September.
Using the pooled data of the whole growing season in the different treatments, stepwise regression analyses show that microbial biomass nitrogen was a major controlling factor in N 2 O fluxes in the control and the M 2 treatment, explaining 25 % and 27 % of the variation in the N 2 O fluxes, respectively (P = 0.01, P = 0.008).Microbial biomass carbon was a major controlling factor in the M 10 treatment, explaining 23 % of the variation in the N 2 O fluxes (P = 0.014), and soil moisture was the most important controlling factor in the M 5 treatment, explaining 21 % of the variation in the N 2 O fluxes (P = 0.02).

Controls of changes in N 2 O fluxes
Our observations, showing a decrease in N 2 O with mowing height, generally supported the hypothesis that mowing would decrease the grassland N 2 O emissions into the atmosphere.This was in line with others, though the mowing effects were not statistically significant in most cases.In this experiment, these observations are in line with the results of another similar investigation also conducted in a grazed grassland in Inner Mongolia (Wolf et al., 2010), but opposite to the findings in a heavily grazed alpine grassland by Gao et al. (2008).Mowing not only led to lower N 2 O emission rates, but also led to lower above-ground plant and litter biomass as well as total plant cov- erage (Table 1), and most likely a removal of nitrogen from the system.Nitrification is commonly repressed by nitrogen limitation, which explains the decline in N 2 O with mowing height.Indeed, we observed a linear relationship between N 2 O fluxes and total soil nitrogen (Fig. 4f).However, this relationship was only found with soil temperatures ranging from 16 to 18 • C.This was in line with findings that N mineralization and nitrification were repressed during the growing season, but increased after that period (Both et al., 1992).
In July of 2008 and 2009, a number of weak N 2 O uptake peaks occurred in some of the mowing treatments (Fig. 3a and b), leading to significant uptake of N 2 O on a monthly basis (Fig. 3b).Though the temperature was favourable in July, microbial activity may decrease due to the low precipitation (Fig. 1a).Additionally, plants strongly compete for the mineralized N, leaving less N for nitrifying and denitrifying bacteria (Verhagen et al., 1994;Mikola et al., 2009).This competition may be the reason for the N 2 O uptake observed in grasslands during July in our study.This result confirms that grassland soils may act as net sinks of atmospheric N 2 O under certain conditions due to high N 2 O reduction activity by denitrifying microorganisms (Wrage et al., 2004;Flechard et al., 2005).N 2 O reduction by denitrifying microorganisms is bound to reduced levels of oxygen, which is usually related to increased moisture content (Schlesinger, 2013).Despite the different patterns of N 2 O fluxes in July 2008 and 2009, soil moisture content was not very different between these months (Zhang et al., 2012).
Hence, unknown soil factors might have been responsible for the observed differences in the fluxes in July.
In August, however, there was a net N 2 O emission (Fig. 3b).At this time of the year, the temperature is still high enough for soil microbial activity (data not shown).Therefore soil microbes will decompose plant material and likely result in mineralization of N.However, most of the plants get senescent with more substrate released into the soil, and require fewer nutrients from the soil for growth at that time; subsequently the competition between plants and microorganisms for mineralized N will be reduced.The Using the pooled data of the whole growing season and all treatments, stepwise regression analyses show that soil moisture was the most important controlling factor in N 2 O fluxes, which explains 80 % of the observed variations in seasonal average N 2 O fluxes (P < 0.0001) at our study site (Fig. 4a).Similar results were reported for grasslands in Ireland (Leahy et al., 2004) and New Zealand (Müller et al., 2004).Therefore, soil moisture could be considered as a major control of seasonal N 2 O fluxes, and determines microbial N 2 O emissions (Merino et al., 2001).In some agricultural regions, soil moisture was also found to be the controlling factor in N 2 O fluxes (Izaurrade et al., 2004).Because soil moisture is the key determinant of the microbial processes that consume or produce N 2 O, soil moisture shifts in arid and semiarid regions will likely affect N 2 O fluxes.

Effect of mowing on N 2 O fluxes
The relationship between N availability and N 2 O flux is commonly examined for predicting N 2 O fluxes on large scales, and Millar et al. (2010) had even reported that N availability is the only factor affecting N 2 O fluxes at metre scales.However, in our study, we did not find any significant correlation between total nitrogen and N 2 O fluxes; combing with another result, the mowing treatments did not alter the concentrations of soil TN (including NH + 4 and NO − 3 ) (Table 2), and we may explain why there was no significant effect of mowing on the cumulative N 2 O fluxes in 2008 in this study (Table 1).
Our other hypothesis is that both soil biotic and abiotic factors play important roles underlying mowing effects of decreasing plant biomass on N 2 O fluxes.Since the cumulative seasonal N 2 O fluxes were only slightly different among different mowing treatments, and no consistent changes were found among any of the mowing treatments (Table 1) biomass (Table 1), since the decreased plant litter and biomass might result in decreases in the substrates supplied to microbes, which are in charge of nitrification and denitrification processes.
The above finding agrees with that of some previous studies.Zou et al. (2005) established a positive linear relationship between above-ground plant biomass and N 2 O emissions.Kammann et al. (1998) found that increasing numbers of cuts reduced N 2 O emissions.However, in other cases, for instance, Beck and Christensen (Beck, 1987) found that N 2 O emissions increased when all above-ground grass was removed, and Klumpp et al. (2011) only observed that some small peaks of N 2 O emission occurred in response to cutting events.Further analysis indicates that there was a significant effect of the differences in soil moisture on the N 2 O fluxes after mowing (Fig. 5), which explains 76 % of the variation in the N 2 O emissions induced by mowing (P < 0.001).Similar results were found in a study performed in grazed grasslands by Wolf et al. (2010).
Overall, our results showed that the N 2 O emissions from the grassland mowed at plant heights of 5 cm were smaller than those from the grasslands not mowed during the growing season in Inner Mongolia.We may extrapolate from this finding that by changing grassland management, such as introducing a proper mowing intensity, the greenhouse effects of N 2 O emission might be mitigated in the grassland.Thus, emission estimates from grasslands may need to take into account more specific management practices, for instance, mowing intensity.The most significant finding from this study is that soil moisture played a key role in the seasonal cumulative N 2 O fluxes.and F, panels G and H, panels I and J represent the treatments of plant mowed at heights of 15 cm 534 (M 15 ), 10 cm (M 10 ), 5 cm (M 5 ) and 2 cm (M 2 ) above soil surface, respectively.Data are mean ±SE 535 (n=5).536  (mg kg -1 , B), total soil phosphorus (g kg -1 , C), above-ground plant biomass (g m -2 , D), total soil 555 carbon (g kg -1 , E), total soil nitrogen (g kg -1 , F). Solid circles in F only plotted from the data 556

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Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | results in considerable uncertainties in estimating actual N 2 O exchange rates for given management scenarios.
2 O fluxes in response to different mowing intensities, we conducted an experiment setup in 2003 with different mowing heights in a steppe ecosystem in Inner Mongolia of northern China.Here, we present results of N 2 O fluxes over the two growing seasons (from May to September) of 2008 and 2009, analysing the relation between N 2 O emission and abiotic and biotic factors to identify the controls of the emission.We hypothesize that (1) mowing will decrease N 2 O emission due to the removal of a part of plants above the soil surface, which can result in continuous decreases in the availability of substrate and nutrient for N 2 O production (Berliner, 1999) and the related soil microbes; (2) both soil biotic and abiotic factors play important roles in underlying mowing effects on N 2 O flux.Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | tween N 2 O fluxes and the measured variables (e.g.ST, SM, SOC, TN, TP, MBC, MBN and ANPP, total litter, total coverage).Because some parameters were measured at 19225 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | N 2 O fluxes Mean N 2 O emission rates from the different mowing treatments varied in a range of −31.7 to 67.2 µg N 2 O m −2 h −1 , with means of 12.7, −4.2, 4.8, −5.2 and 4.1 µg N 2 O m −2 h −1 for treatments of M ck , M 15 , M 10 , M 5 , and M 2 , respectively, over the growing season of 2008, and means of 13.6, −9.0, 3.7, −2.9 and 5.5 µg N 2 O m −2 h −1 for also indicates that the grassland could possibly function as a N 2 O source (positive value), but that it has also frequently acted as a N 2 O sink (negative flux values).Regardless of the mowing heights, surprisingly, the monthly cumulative N 2 O flux was negative in July for most of the treatments, except for the controls both in 2008 and 2009 and the M 10 and M 2 in 2008 (Fig. 3).19227 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , b, and d).A significant linear relationship between N 2 O fluxes and soil total nitrogen was only observed within soil temperatures ranging from 16 to 18 • C Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

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Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | higher substrate and N availabilities likely promote microbial nitrification, leading to high N 2 O production.
, we tested the effects of mowing on N 2 O fluxes by comparing the pooled data of all the mowing treatments, including those of the control.The result suggests that mowing might decrease N 2 O emissions by reducing plant litter and above-ground 19231 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

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Figure 2
Figure 2 Temporal variations in N 2 O fluxes under different mowing treatments in 2008 (left) and 532

Figure 4
Figure 4 Relationships between N 2 O fluxes and soil moisture (%, A), microbial biomass nitrogen 554

Fig. 3 .
Fig. 3. Monthly cumulative N 2 O fluxes in 2008 (A) and 2009 (B) under different mowing heights.Values are mean ± SE (n = 5).Different letters represent statistically significant differences between treatments in the same month at P < 0.05.

Figure 5 Fig. 5 .
Figure 5 The relationship between soil moisture and N 2 O fluxes at sites after mowing.562 This study was carried out at the Duolun Restoration Ecology Research Station, Institute of Botany, Chinese Academy of Sciences (IBCAS), which is located in Duolun County (116 • 17 E, 42 • 02 N) in Inner Mongolia, China.The area is situated in a semiarid, middle temperate zone and characterized by a continental monsoon climate.The mean annual air temperature is around 2.1 • C, with monthly mean temperatures ranging from −17.5 • C in January to 18.9 • C in July.The mean annual precipitation is approximately 385 mm, with 80 % of precipitation occurring from mid-June to late September.The topography is featured by low foothills at elevations of 1150-1800 m.The soil at the study site is classified as chestnut soil in the Chinese soil classification, containing 62.75 ± 0.04 % sand, 20.30 ± 0.01 % silt and 16.95 ± 0.01 % clay, with mean soil bulk density of 1.31 g cm −3 and a pH value of 7.12.The dominant plant species in the temperate grassland are Stipa krylovii Roshev., Cleistogenes squarrosa (Trin.)Keng., Artemisia frigida Willda, Potentilla acaulis L., Allium bidentatum Fisch.Ex Prokh.and Table 1 presents the seasonal cumulative N 2 O emissions based on a linear interpolation, spatially averaged, of daily or monthly mean fluxes.Over the two growing seasons, an estimated amount of 0.22 kg N 2 O ha −1 per season was emitted in the control treatment, of which more than 88 % occurred in May and June.Occa-Table 1).M ck and most of the mowing treatments acted as net sources of N 2 O, except for the M 5 treatment, which promoted the grassland to uptake N 2 O from atmosphere, with an estimated cumulative flux of −0.08 kg N 2 O ha −1 (Table 1) in both 2008 and 2009.For the total fluxes over the two growing seasons, the value for M 5 was the only mowing treatment significantly different from that of the control (p < 0.05).Compared with the control, the M 15 , M 10 , M 5 and M 2 mowing treatments decreased N 2 O emission by 96, 40, 180 and 0 %, respectively, in 2008 (Table 1), and by 182, 82, 124 and 118 %, respectively, in 2009 (Table

Table 1 .
Means of total plant litter (g m −2 ), above-ground plant biomass (g m −2 ), total plant coverage (%), and cumulative N 2 O emission/uptake (kg ha −1 ) under different mowing treatments in a steppe ecosystem in Inner Mongolia, northern China.Values represent the mean of five replicates.Different letters in a column indicate a significant difference between treatments at P < 0.05.M ck represent the no-mowing control.M 15 , M 10 , M 5 and M 2 represent the treatments of plants mowed at heights of 15 cm, 10 cm, 5 cm and 2 cm above the soil surface, respectively.

Table 2 .
P values of repeated measures ANOVAs on the effects of mowing (M), sampling date (D) and their interactions on C/N ratio, soil organic carbon (SOC), total nitrogen (TN) and total phosphorus (TP) under different mowing treatments.Data with * indicate a significant difference at the P < 0.05 level.