Interactive comment on “ Carbon and nitrogen dynamics in native Leymus chinensis grasslands along a 1000 km longitudinal transect in northeast China ”

General Comments The paper by Ma et al. advances a relevant topic by presenting observations of several carbon and nitrogen cycling diagnostics across a rainfall gradient. The primary interest of this data set is the synthesis of plant, soil, litter, and microbial community information, which are not typically included together in such studies. While the data set is new, complete, and potentially useful, there are significant issues with data analysis, the conclusions drawn, and integration with the vast literature on this topic. Most importantly, I am concerned about the use of field observations on a single day (or week) with climatology averaged over several years to address climate control on C-N cycling. Either the climate and ecosystem parameters should be collected at the same temporal scale (e.g., several years of field data) or an argument


Introduction
It is well known that climatic factors, especially precipitation, have important influences on ecosystem C and N processes in grasslands which covering ca. 25 % of the global terrestrial area and 40 % of the land area in China (He et al., 2008;Kang et al., Figures Back Close Full 2007; Zhou et al., 2009).Recently, the IPCC (2013) has anticipated that global climatic change will make drought and extreme precipitation events more frequent, which could have greater profound influence on ecosystem dynamics than the effects of warming and elevated CO 2 concentration (Weltzin et al., 2003;Zhou et al., 2009).Previous studies have demonstrated that ecosystem primary production (Barrett et al., 2002;Gao and Yu, 1998), litter mass (Zhou et al., 2009), soil organic matter reserve (Baumann et al., 2009;Wang et al., 2008) and trace gas flux (Niu et al., 2009) varied with precipitation gradients and their direct impacts on soil water availability at local, regional and global scales.However, ecosystem C and N processes responses to precipitation dynamic are complex, and usually interact with vegetation type, soil properties and microbial community commposition (Austin and Sala, 2002;Ma et al., 2012;Šantrucková et al., 2003;Zhou et al., 2009).Complexity in effects of changing precipitation regime on ecosystem C and N processes at a regional scale remains unclear.
Plant derived biomass, resources allocation in plant growth, litter accumulation, and storage and decomposition of soil organic matter are important processes contributing to ecosystem C and N cycling (Xiao et al., 2007).Many large-scale transect studies evaluated the spatial variability of climatic factors and annual aboveground net primary production (ANPP) in forest and grassland ecosystems, and indicated that ANPP is strongly and linearly correlated with water availability (Austin and Sala, 2002;Burke et al., 1997;Wang et al., 2003;Zhou et al., 2009).However, root production of grassland is also an important resource of organic matter, which represents 50-80 % of total plant biomass (Tomaskin, 2007).Therefore, a further understanding of total plant production to a large-scale precipitation gradient is crucial for predicting grassland ecosystem C and N dynamics.
Litter mass are closely associated with plant growth (Deutsch et al., 2010), soil carbon and nutrient pools (Rinnan et al., 2008), element fluxes (Michelsen et al., 2007) and hydrological cycle (Willms and Chanasyk, 2006).Therefore, litter accumulation plays important role in ecosystem structure and function.Available results show that litter mass is positively correlated with precipitation in water-limited regions, and the Introduction

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Full mass reaches a threshold after which it declines with increasing precipitation in mesic regions (Wang et al., 2011;Zhou et al., 2009).In the temperate grasslands of northern China, soil fertility and productivity are maintained primarily by recycling of nutrients through litter decomposition, with little fertilization or N deposition (Liu et al., 2006), whereas comparatively less observations of litter dynamic along a large-scale precipitation gradient in grassland ecosystem.About three-fourth of organic C contains in terrestrial ecosystems and the majority of N stores in plant litter and soil organic matter (Lal, 2008).Both C and N are mineralized to inorganic forms by microorganisms (Paul, 2007), and result in releasing CO 2 to the atmosphere and providing inorganic nutrient for microbial and plant growth (Manzoni and Porporato, 2009;Ma et al., 2013).Therefore, C and N mineralization play critical roles in the functioning of ecosystem C and nutrient cycling, structural formation and plant interactions (Harris et al., 2009).In water-limited region, soil C and N mineralization rates are greatly affected by soil water availability (Ma et al., 2013;Paul et al., 2003;Sierra, 1997;Wang et al., 2006), whereas little is known about pattern of soil mineralization processes across a precipitation gradient.
The use of natural precipitation gradient transects is an invaluable approach for understanding mechanisms of abiotic and biotic controls on ecosystem C and N processes (Austin and Sala, 2002).Northeast China Transect (NECT) is identified as a mid-latitude semiarid terrestrial transect and the vegetation shifts from desert steppes to typical steppes and moist meadows along the western half of the transect at a westeast precipitation gradient.Owing to relatively uniform soil origin and dominate specie of the NECT, it provides an ideal setting to investigate grassland ecosystem C and N dynamics along a precipitation gradient (Wang et al., 2003).The specific objectives addressed here were: (1) identify how precipitation regime and the indirect effects of precipitation on changes in soil property and microbial community interact to regulate regional C and N dynamics; (2) assess C and N sequestration and potential along a large-scale precipitation gradient.We hypothesize that regional precipitation regime is the primary driver to affect C and N dynamics in temperate grasslands, and the soil Introduction

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Full properties and microbial communities will modulate the responses of C and N dynamics to variation in precipitation.In addition, we also expect the greater precipitation will contribute to the higher C and N sequestration at this region.
2 Materials and methods

Study sites
The field study was conducted with 18 sites on native Leymus chinensis grasslands, located in northeastern plain and Inner Mongolia plateau of China (114-124  1).We selected sample sites have not been disturbed for at least 10 years (Wang et al., 2011).

Climate
This area has a continental monsoon climate, with large seasonal temperature and precipitation gradients.Long-term (1950Long-term ( -2000) ) mean annual temperature, mean annual precipitation and aridity index along the transect range from approximately 1.3-6.8Full

Plant biomass and litter mass
Aboveground living shoot, standing litter and surface litter were harvested from 10-12 randomly located, 0.5 × 0.5 m plot of each plot, respectively.Root biomass was measured by soil coring method sampling to a depth of 30 cm using a cylindrical root sampler (8 cm inner diameter).All samples of aboveground living tissue, litter and root were oven-dried at 65 • C to constant weight.C and N content of shoot, root and litter were determined by elemental analyzer (Elemetaranalysator vario Max CN, Germany).

Soil property and microbial community analyses
Soil samples from 18 sites were collected along the systematically located transect lines in 12-18 July 2012.8-16 soil core samples were collected randomly per site (100 × 100 m) for determination of soil properties.The samples were taken to a depth of 15 cm soil layer with a cylindrical soil sampler (5 cm inner diameter), and then immediately preserved at 4 • C in a cooler for transport to the laboratory within one week of collection.The fresh samples were processed using a 2 mm sieve and cleared of visible plant roots and organic debris.
Soil pH was measured with a pH electrode (PHS 29, China).Soil inorganic N content (NH + 4 -N and NO − 3 -N) was determined using a flow injection autoanalyzer (FIAstar 5000, Denmark).Soil total C and N content were measured by elemental analyzer (Elemetaranalysator vario Max CN, Germany).Soil light and heavy fractions of C and N content were measured by density fractionation method (Sollins et al., 1984).Soil texture was determined by the optical size analyzer (Mastersizer 2000, England).Gravimetric soil water content was measured by oven-drying samples at 105 • C for 24 h.
Phospholipid fatty acids (PLFAs) analysis was used to quantify microbial community composition.PLFAs were extracted from soil samples following a procedure described by Bossio et al. (1998).The separation and identification of extracted PLFAs were carried out using a Microbial Identification System (Newark, DE, USA).Methyl nonadecanoate fatty acid (19 : 0) was used as internal standard.Thirty-one fatty acids Introduction

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Statistical analyses
Regression analyses were conducted on relationships of total plant biomass, shoot and root biomass, shoot biomass C and N, root biomass C and N, ratio of root to shoot biomass, litter mass, litter biomass C and N, soil C and N mineralization rates, soil total C and N content, soil light and heavy fractions of C and N content, soil water content, inorganic N, pH, microbial biomass (PLFAs), ratio of fungal to bacterial PLFAs and AMF biomass with MAP.Redundancy analysis (RDA) was used to represent the relationships among environmental factors and ecosystem C and N processes along the gradient (Lepš and Smilauer, 2003) using the Canoco for Windows 4.5 package (Ithaca, NY, USA).
Stepwise linear regression analyses were used to determine the relationship between ecosystem C or N processes with environmental factors.If the several independent variables are existed higher correlations, which may contribute to overfitting correlation, stepwise regression can delete one or more correlated independent variables to lest including complexity such as interactions.Significant differences among the sites in measured variables were determined using One-way ANOVA.Data management and statistical analyses were performed using SPSS 17.0 software (SPSS, Chicago, IL, USA).Introduction

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Full

Plant and litter properties
Total plant biomass (shoot plus root biomass), shoot biomass and ratio of root to shoot biomass (R : S) varied significantly along the large-scale precipitation gradient.In general, average total plant biomass and shoot biomass increased from 131 to 440 g m −2 (R 2 = 0.43, P < 0.0001; Fig. 2a) and 62 to 332 g m −2 (R 2 = 0.54, P < 0.0001; Fig. 2b), but R : S dropped from 1.38 to 0.32 with increasing mean annual precipitation (MAP) (R 2 = 0.46, P < 0.0001; Fig. 2d).Surprisingly, root biomass showed no remarkable differences among all sites (Fig. 2c).Similarity, shoot biomass C and N changed significantly with variation in precipitation, whereas root biomass C and N varied slightly.

Soil properties
In general, soil water content, total C and N content, inorganic N, C : N, light and heavy fractions of C and N content differed significantly along the large-scale precipitation gradient (Fig. 3).Soil water content linearly increased with MAP (R 2 = 0.72, P < 0.0001; j).In contrast, soil inorganic N content had a quadratic relationship with precipitation (R 2 = 0.40, P < 0.0001; Fig. 3c), and varied 4-fold at this region.

Soil microbial community, C and N mineralization
The viable microbial biomass (i.e. total PLFAs) presented to an increase trend (P < 0.0001, R 2 = 0.54; Fig. 4a), but the ratio of fungal to bacterial biomass displayed a decrease style (data not shown) from the dry west end to the moist east end along the gradient.The meadows had the highest microbial biomass (ca.33.8 nmol g −1 ), having 1.8-fold greater than the desert steppes, which had the lowest value (ca.19.2 nmol g −1 ).Inconsistent with microbial biomass, the desert steppes and meadows had higher AMF biomass than the typical steppes, which varied 1.5-fold across all sites.
The patterns of soil C and N mineralization rates at the depth of 0-15 cm both followed quadratic relationships with MAP, which increase at first, reached the highest values, and then decreased along the transect (R 2 = 0.35, P < 0.0001; R 2 = 0.37, P < 0.0001; Fig. 4c, d).The highest C mineralization rate from the typical steppe (site No. 10) was 28 % and 13 % higher than the values from the driest and wettest sites, respectively (P < 0.05; One-way ANOVA).Similarly, the highest soil net N mineralization rate was 26 % and 9 % higher than that from the driest and the wettest sites (P < 0.05; One-way ANOVA).Overall, ecosystem C and N sequestration enhanced with increasing precipitation in terms of C and N storage in shoot, root, litter and soil along the gradient (Fig. 4e, f).

Relationship among ecosystem C and N dynamics and environmental factors
The ordination biplot diagram from redundancy analysis (RDA) exhibited the effects of environmental factors on C and N dynamics along the gradient (Fig. 5).Environmental factors and response variables with more similar locations had higher correlations than those with less similar locations.The first and second axes of RDA ordination explained Introduction

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Full 55.5 % and 16.7 % of the total variation, respectively.Along the transect, MAP was positively correlated with MAT, mean July temperature, soil water content and inorganic N content, and negatively correlated with aridity index and elevation, suggesting that these environmental factors likely affect responses of ecosystem C and N dynamics to precipitation (P < 0.05; Fig. 5).Total C and N sequestration, shoot biomass C and N, soil total C and N and light fractions of C and N content showed positively dependence on soil water content and MAP (P < 0.05).In contrast, litter biomass C and N were only correlated with soil water content.In addition, soil C and N mineralization rates and heavy fractions of C and N content were positively correlated not only with MAP, MAT and July temperature, but also with inorganic N content and AMF biomass (P < 0.05).
Environmental factors such as soil microbial biomass (total PLFAs) and pH were not important here.
Stepwise multiple regression analyses demonstrated that 46.2 % and 48 % of the spatial variation in shoot biomass C and N could be only explained by soil water content.Similarity, soil water content alone contributed to 32.7 %, 35 %, 53 % and 49.2 % of the variation in litter biomass C and N and soil C and N content, respectively.Along the gradient, soil water content (CMR: partial R 2 = 0.11; NMR: partial R 2 = 0.11), mean July temperature (CMR: partial R 2 = 0.32; NMR: partial R 2 = 0.35), inorganic N (CMR: partial R 2 = 0.10; NMR: partial R 2 = 0.11) and AMF biomass (CMR: partial R 2 = 0.12; NMR: partial R 2 = 0.13) together accounted for 65 % and 70 % of the spatial variation in CMR and NMR.

Discussion
Climatic factors, especially precipitation and their direct influence on water availability are dominant drives to impact ecosystem C and nutrient cycles directly through C sequestration, mineralization, and uptake as well as controlling inputs and outputs of nutrients at global and regional extent (Austin and Sala, 2002).In this study, total plant and shoot biomass increased linearly with MAP along the gradient (Fig. 2a, b), sug-Introduction

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Full gesting that primary production in this precipitation gradient is limited, at least in part, by water availability.Across the gradient, although shoot biomass showed positively dependence upon mean annual temperature (MAT) (P < 0.05; Table 2), stepwise multiple regression analyses demonstrated that MAT didn't contribute to the variation in shoot biomass (partial R 2 = 0.02; P > 0.05).These results are similar with that reported in grasslands (Barrett et al., 2002;Wang et al., 2003;Zhou et al., 2009), forests (Austin et al., 2002;Schuur and Matson, 2001), woodlands (Zerihun et al., 2006) along the large-scale precipitation gradients, and a former developed regional vegetation models in this area (Gao and Yu, 1998).Consistent with the variation in shoot biomass, we also found that litter mass (standing plus surface litter) was positively related to increasing MAP along the gradient (Fig. 2).These findings indicate that the increasing precipitation facilitates aboveground C and N accumulation in this water-limited region.Contrary to our expectation, root biomass changed only slightly along the gradient (Fig. 2c), resulting the decreases in R : S from the dry to the mesic sites (Fig. 2d).Our results are consistent with those of studies on effects of increased precipitation on root biomass at local and regional scales (Ma et al., 2012;Zhou et al., 2009), in which plants respond to increasing precipitation by decreasing the photosynthates allocation to root system relative to the aboveground parts (to enhance light capture).Such lack of response of the root biomass to changes in precipitation regime was likely because nutrients limit root growth along the gradient.Although we cannot prove this point, manipulating nutrient and water experiments demonstrated that nutrient addition increased plant growth, especially root growth, whereas water addition showed modest effect in this study region (Ma et al., 2012(Ma et al., , 2013;;Xiao et al., 2007).
Previous transect and manipulative studies have showed that soil C mineralization (CMR) and N mineralization rate (NMR) were positively related to increasing precipitation at local and regional scales (McCully et al., 2005;Xiao et al., 2007;Kim et al., 2012).However, surprisingly, both soil CMR and NMR followed quadratic relationships with MAP, which increase at first, reached the highest value, and then decreased along the gradient (R 2 = 0.35, R 2 = 0.37; Fig. 4c, d).The trends could have been attributed to

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Full the relative changes in mean July temperature, soil arbuscular mycorrhizal fungi (AMF) biomass, and soil inorganic N content (IN) along the gradient (Figs. 3,4,5), because stepwise multiple regression analyses showed that both CMR and NMR were positively correlated with July temperature and IN, and were negatively correlated with soil AMF.In this study, strong positive correlation between CMR (and NMR) and July temperature indicating that temperature was the most primary determinant of the spatial variation in soil mineralization processes, whereas the precipitation effect was relative weaker but still significant.Another possible explanation is that the variation in soil AMF biomass probably controls the decomposition process of soil organic matter.AMF is seen as an important component of microbial biomass, which makes a distinct and significant contribution to soil organic matter accumulation (Rillig et al., 2001).Like other soil fungi, AMF hyphae can enmesh and entangle soil microaggregates into macroaggregates (Tisdall et al., 1997).In addition, AMF hyphae produce an extracellular glycoproteinaceous substance called glomalin, which favor stabilization and protection of soil organic matter (Rillig, 2004).In this study, soil AMF biomass were negatively correlated with C and N mineralization rates (Fig. 5), and thus the relative higher AMF biomass may contribute to the lower soil organic matter decomposition.Additionally, we also found soil IN content had a quadratic relationship with MAP (R 2 = 0.30; Fig. 3c).The relative reduction in soil N availability in mesic sites likely intensifies nutrient competition between plants and soil microorganisms and results in nutrient limitation of microbial activity.Soil C and N storage is the result of a balance between inputs from plant production and outflows from organic matter decomposition (Zhou et al., 2009).Related research involving field measurements and regional model predictions have demonstrated that the responses of soil C and N content to increased precipitation represent increases (Niu et al., 2009;Zhou et al., 2002) and no changes (Zhou et al., 2009) at local and regional scales in the temperate grasslands.However, in this study, although regional soil C and N storage rapidly increased with MAP, heavy fractions of C and N content followed by steady phases with MAP from 422-472 mm long the gradient (Fig. 3).The

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Full findings indicate that soil heavy fractions of C and N storage in the temperate grasslands subjected to site No. 12 (MAP: 422 mm; Table 1) may reach stable or saturation and have some important implications to ecosystem C and N sequestration.A large number of shoot or litter biomass to soils only increased soil light rather than heavy organic matter in mesic sites suggesting that mineral soils in the L. chinensis grasslands have a limited capacity to accumulate mineral component (Chung et al., 2008;Stewart et al., 2007).
In generally, ecosystem C and N sequestration enhanced with water availability in terms of C and N storage in shoot, root, litter, and soil along the precipitation gradient (Fig. 4e, f).Although we did not detected belowground C and N allocation in deeper soil layer (30-100 m) due to sampling difficulties, about 80 % of the root and soil C and N were focused in the top 20 cm soil layer (Zhou et al., 2007;He et al., 2008).Therefore, these results may have not altered the overall patterns of C and N sequestration along the precipitation gradient.
Based on our results, changing in global precipitation regime would strongly influence on ecosystem C and N dynamics.These findings indicate regional precipitation regime and the changes in temperature, nutrient availability and microbial communities may play important roles in determining ecosystem C and N dynamics, and suggest that the L. chinensis grasslands of northeast China exhibit tremendous potential for enhancing C and N sequestration at the regional scale.The temperate grasslands of northeastern China could be utilized as significant ecosystem C and N sinks in the context of mitigating climate change.Introduction

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Fig. 3a )
Fig.3a), and varied 7.4-fold along the gradient.However, soil pH fluctuated only slightly, with very little significant variation (Fig.3b).Soil total C and N content and C : N to a depth of 15 cm layer increased from 1316 to 4232 g m −2 (R 2 = 0.60, P < 0.0001; Fig.3d), 196 to 438 g m −2 (R 2 = 0.30, P < 0.0001; Fig.3e), and 5.8 to 12.8 (R 2 = 0.37, P < 0.0001; Fig.3f) with MAP along the gradient, respectively.However, although soil light fractions of C and N content rapidly increased with MAP, heavy fractions of C and Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |