Differential Responses of Community Nonstructural Carbohydrate to Drought Manipulation Along a Natural Aridity Gradient

Plant nonstructural carbohydrates (NSC) can reect community and ecosystem responses to environmental changes such as water availability. Climate change is predicted to increase aridity and the frequency of extreme drought events in grasslands, but it is unclear how community-scale NSC will respond to drought or how such responses may vary along aridity gradients. We experimentally imposed a 4-year drought in six grasslands along a natural aridity gradient and measured the community-weighted mean of leaf soluble sugar (SS CWM ) and total leaf NSC (NSC CWM ) concentrations. We observed a bell-shape relationship across this gradient, where SS CWM and total NSC CWM concentrations were lowest at intermediate aridity, with this pattern driven primarily by species turnover. Drought manipulation increased both SS CWM and total NSC CWM concentrations at intermediately arid grassland but decreased total NSC CWM concentrations at one site. These differential responses to experimental drought depended on the relative role of species turnover and intraspecic variation in driving SS CWM and total NSC CWM . Specically, the synergistic effects of species turnover and intraspecic variation driven the responses of leaf NSC concentrations to drought, while their antagonistic effects diminished the effect of drought on plant SS CWM and total NSC CWM concentrations. Plant resource strategies were more acquisitive, via increasing chlorophyll CWM content, to offset reduced NSC CWM concentrations with increasing aridity at drier sites, but more conservative (i.e., decreased plant height CWM ) to reduce NSC consumption at more mesic sites. The relationship between water availability and NSC CWM concentrations may contribute to community drought resistance and improve plant viability and adaptation strategies to a changing climate.


Introduction
Grasslands cover over 40% of the earth's terrestrial surface and provide many critical ecosystem services, such as soil stability, water conservation, biodiversity, and forage for livestock (Gao et al. 2016). Climate change projections for the 21st century suggest that summer droughts will become more frequent (IPCC 2013) and grasslands are particularly sensitive to drought (Knapp et  dynamics have been used in vegetation models to represent metabolism, species habitat range shifts, plant vulnerability to climate extremes, and even species extinction risks (Rosas et al. 2013). Thus, an indepth understanding of long-term impacts of drought on community scale NSC is potentially important for forecasting shifts in ecosystem processes and functioning during drought.
To date, much research has focused on assessing NSC responses to drought at the individual species level, whereas such studies are less common at the community scale, especially in grasslands. However, changes in NSC dynamics in response to drought are more relevant to community structure and ecosystem functions when assessed at the community level (Violle et al. 2012). Shifts in plant community NSC concentrations in response to drought can be due to species turnover (i.e., species with different NSC concentrations replacing others and a shift in relative abundance of each species) and/or intraspeci c variation (i.e., plasticity and genetic differentiation) in NSC concentrations (Albert et al. 2010, Violle et al. 2012. The synergistic effects of species turnover and intraspeci c variation in NSC concentrations can magnify community NSC responses to drought, whereas antagonistic effects can weaken responses. Shorter-term shifts in water availability may impact communities through shifts in both species turnover and intraspeci c variation, while longer-term droughts are likely to in uence community functional responses primarily through species turnover (Volf et al. 2016). Therefore, quantifying the relative contributions of species turnover and intraspeci c variation is important for understanding the responses of community NSC concentrations to water limitation.
We established a coordinated 4-yr drought experiment at six grasslands sites spanning an aridity gradient in northern China. At each site, we measured plant NSC concentrations of all species cumulatively representing ~ 90% of total plant biomass to estimate community-weighted mean NSC (NSC CWM ) concentrations. We tested the hypothesis that experimental drought would have no effect on NSC CWM concentrations due to the opposing effects of intraspeci c trait responses (i.e., stomatal closure and reductions in NSC reserves) and species turnover (i.e., increased abundance of drought tolerant species with high NSC concentrations). Additionally, we predicted that NSC CWM would be maximized at the extremes of the aridity gradient as xeric-adapted species accumulate NSC for osmotic regulation and more productive mesic-adapted species produce more photosynthate in general. Thus, this trend would largely be driven by species turnover.

Study sites and experimental design
In 2014, we selected six sites arrayed across the east-west extent of the grassland biome in northern China (Table S1). These six sites encompass the three major grassland types in China (i.e. meadow steppe, typical steppe and desert steppe) and vary in mean annual precipitation (MAP), mean annual temperature (MAT), plant species composition and edaphic properties. We extracted climatic variables (e.g., MAT, MAP, and potential evapotranspiration (PET, mm)) for each site from the global Worldclim data set with a resolution of 0.0083° × 0.0083° (http://www.worldclim.org). We de ned the aridity index (AI) of each site as 1 -MAP/PET (Delgado-Baquerizo et al. 2013). Among the six sites, aridity increases from east (SMR) to west (UDR) ( Table S1). Soil texture also varies across the six sites from sandy to clay loams, as well as other soil characteristics (Table S1). All six sites had not been grazed by domestic herbivores for the 3 years prior to the 2015 drought. The dominant species are Stipa baicalensis and Leymus chinensis in the meadow steppe, S. baicalensis and L. chinensis in the typical steppe, and S. brevi ora and Peganum harmala in the desert steppe.
We established experimental drought infrastructure at each site which remained in place from 2015 to 2018. Using a randomized complete block design, we established twelve 6×6 m plots per site (6 drought; 6 control) in a topographically uniform area. Plots were located at least 2 m from each other and were hydrologically isolated by trenching the perimeter to a depth of 1 m and installing 6-mm-thick plastic barriers to prevent lateral water ow. We constructed large rainout shelters to block 66% of ambient growing season precipitation from May to August in each year ( Figure S1). These rainout shelter roofs were built with transparent polyethylene panels covering 66% of the surface and were supported by a light scaffolding structure. To allow for air ow below the panels and minimize any potential greenhouse effect, we installed shelters 2 m above the ground (Delgado-Baquerizo et al. 2017). Similar experimental infrastructure has been used in previous experiments with minimal effects on light environment (permitting nearly 90% transmission) (Yahdjian and Sala 2002; Wilcox et al. 2015). For control plots, we established similar scaffolding structures but did not install polyethylene panels.

Sampling and measurements
Each plot contained a 4×4 m sampling plot, which we divided into four 1×1 quadrats. In August 2017, we randomly selected one of these quadrats and further divided it into four 50×50 cm sub-quadrats. We designated two diagonal sub-quadrats for destructive measurements of plant biomass, and the other two for surveys of plant traits (Luo et al. 2019).
We estimated aboveground net primary productivity (ANPP) in the two designated sub-quadrats by clipping plant material at the ground level during peak growth. We sorted biomass by species before oven-drying (48 hr at 65 °C) and weighing to estimate species-speci c ANPP. We calculated species relative abundance as the species' percent contribution towards total ANPP.
In the other two diagonal sub-quadrats, we measured plant traits of the most abundant species (i.e., cumulatively representing at least 90% of the total ANPP). First, we estimated plant height for three individuals of each species per plot. We then collected the youngest, fully expanded leaf from the same individuals (Perez-Harguindeguy et al. 2016) and oven-dried these leaves at 105°C for 30 min to stop enzymatic activity before drying at 65°C until constant weight. We estimated foliar soluble sugar (SS) and starch content spectrophotometrically (ultraviolet-visible spectrophotometer 723 S, Yoke Precision Instruments Co., Ltd, Shanghai, China) at 620 nm using the Sulfuric acid-Anthrone method (Li et al. 2008b). We used the sum of starch and SS concentrations as an estimate of total NSC concentration for each species. The concentrations of SS and total NSC have been widely used as indicators of NSC In August 2018, we sampled leaves of the same species to determine leaf chlorophyll content. We stored samples at -20°C until foliar chlorophyll was extracted using spectra-analyzed grade N, N dimethylformamide. We measured absorbance at 663, 646 and 480 nm using a Shimadzu UV-1700 spectrophotometer (Wellburn 1994;Chen et al. 2013) and total chlorophyll content was estimated according to Porra et al. (1989).

Data analysis
We used R statistical programming (R version i386 3.6.1) to run all data analyses described below. First, we used Shapiro-Wilk and Levene's tests to con rm the normality and heteroscedasticity of all trait data.
Based on this con rmation, we used the original non-transformed data in all statistical analyses. We To determine the spatial relationships between aridity and NSC concentrations, we regressed AI against CWM of SS and total NSC concentrations using linear or curvilinear (quadratic) equations. Based on both explained variation and Akaike's Information Criterion (AIC) values, we found that the relationships between AI and both SS CWM and total NSC CWM concentrations were best described by a second-order polynomial, with lowest levels at a site with intermediate aridity. To determine trait-trait relationships, we used mixed linear models to relate SS CWM and total NSC CWM concentrations to height CWM and chlorophyll CWM concentration along the sampled aridity gradient as well as on each side of the aridity gradient (i.e., moist vs. dry regions) with blocks nested within site as random effect. Here, the two sites with intermediate aridity were included as both moist and dry regions. To determine the effect of experimental drought on both SS CWM and total NSC CWM concentrations, we ran a mixed model analysis of variance with drought treatment and site as xed factors and block as a random factor. As interactive effects of drought treatment and site were all signi cant (P < 0.05), mixed-effect models were applied separately for each site with drought treatment as xed factor and block as a random factor. Additionally, the total effects of experimental drought on SS CWM and total NSC CWM concentrations were analyzed with mixed models with drought treatment as a xed factor and site and block as random factors.
Shifts in plant SS CWM and total NSC CWM can be attributed to both species turnover (C Turn ) and intraspeci c variation (C Intra ); thus, we isolated their relative contributions using the following equation and total NSC CWM concentrations in the control and drought plots, respectively, calculated from relative biomass and SS and total NSC concentrations of each species measured in their respective plot. N Dr* is SS CWM and total NSC CWM concentrations in the drought plots, recalculated using a species' relative biomass in the drought plots, but the SS and total NSC concentrations measured in the control plots.

Results
Along the aridity gradient, plant SS CWM and total NSC CWM concentrations were lowest at the IMG site with intermediate aridity, and highest at the driest and wettest sites (Fig. 1). In contrast, plant height CWM and chlorophyll CWM concentrations were highest at the intermediate aridity site (Figure S2). Accordingly, plant SS CWM concentrations were negatively related to plant height CWM (R 2 = 0.79, P = 0.004) and chlorophyll CWM concentrations (R 2 = 0.68, P = 0.096), and plant total NSC CWM concentrations were negatively related to plant height CWM (R 2 = 0.76, P = 0.003) and chlorophyll CWM concentration (R 2 = 0.64, P = 0.012) along the entire natural aridity gradient ( Figure S3).
These relationships showed a strong tradeoff between these two functional traits and plant NSC concentrations, but the strength of the tradeoff differed by region (Fig. 2). Speci cally, in the moist region, plant SS CWM concentrations and plant total NSC CWM concentrations were more strongly related to chlorophyll CWM concentration (R 2 = 0.69 and 0.68, respectively; P < 0.001) than plant height CWM (R 2 = 0.15 and 0.15, respectively; P < 0.01). In the dry region, however, plant SS CWM concentrations plant total NSC CWM concentrations were more strongly correlated with plant height CWM (R 2 = 0.52 and 0.67, respectively; P < 0.001) than chlorophyll CWM concentrations (R 2 = 0.09 and 0.24, respectively; P < 0.05) (Fig. 2).
The treatment×site interactions were signi cant for SS CWM and total NSC CWM concentrations, suggesting that drought effect on these variables depended on the site characteristics (Table 1). Experimental drought led to an increase in SS CWM at the IMG site but showed negligible effects at the other sites ( Fig. 1). Plant total NSC CWM concentrations signi cantly increased with experimental drought at the IMG site, decreased at the EFS site and remained unchanged at the remaining sites (Fig. 1). The overall effect of experimental drought on plant SS CWM and total NSC CWM concentrations was non-signi cant with site as a random effect ( Figure S4). Plant total starch CWM concentrations signi cantly decreased with experimental drought at the EFS site and remained unchanged at the remaining sites ( Figure S5). Shifts in plant SS CWM and total NSC CWM along the aridity gradient were primarily caused by species turnover, which explained more than 90% of the variance (data not shown). In contrast, changes in plant SS CWM and total NSC CWM concentrations caused by experimental drought were due to both intraspeci c variation and species turnover (Fig. 3). The effects of species turnover and intraspeci c variation were synergistic for plant SS CWM at the IMG site and for total NSC CWM at the IMG and EFS sites (Fig. 3). In contrast, the effects were antagonistic for plant SS CWM and total NSC CWM concentrations for the remaining grassland sites (Fig. 3).

Discussion
In our study, SS CWM and total NSC CWM concentrations were lowest in the site with intermediate aridity and highest in sites at the opposing extremes of the gradient (Fig. 1). This U-shaped relationship is perhaps due to differences in resource-use tradeoffs exhibited by plants on opposing extremes of the gradient. We document a clear tradeoff between plant investment in acquisitive growth-related traits (e.g., height and chlorophyll) and conservative resource storage traits (e.g., NSC). It is well established that water limitation leads to stomatal closure at the expense of photosynthesis and NSC production (Casson and Interestingly, this intermediate aridity site experienced the greatest increase in NSC CWM concentrations following experimental drought (Fig. 1), which suggest the co-occurrence of species with divergent resource-use strategies manifests as a higher capacity for community NSC adjustment following drought. These results were consistent with the positive covariation observed between species turnover and intraspeci c variation at this site (Fig. 3), which implies that experimental drought acted as an environmental lter selecting both species with inherently higher NSC CWM concentrations and those with a greater potential to increase NSC CWM concentrations in response to drought (Jung et al. 2014).
Consistent with our expectation, plant total NSC CWM signi cantly reduced with drought treatment at the EFS site, which was caused by the reduced starch CWM  While plant NSC CWM concentrations changed along the aridity gradient ( Fig. 1), it did not respond strongly to experimental drought treatments ( Figs. 1 and S4). The mismatch between experimental drought and aridity trends is not surprising and can be explained by differences in the temporal scale of aridity. Manipulative experiments reveal the initial plastic responses of plant communities and ecosystem functioning following extreme drought (Smith 2011;Yuan et al. 2017). In contrast, natural aridity gradients allow for observations of the long-term responses of plant communities to water-limitation as well as shifts towards optimal function for a given aridity level (Smith 2011;Yuan et al. 2017). Thus, manipulative and gradient experiments measure the plastic and evolutionary responses to drought, respectively. Ecosystem models should thus incorporate both experimental and gradient data to properly assess the responses of plant community structure and functioning to climate change such as increased drought frequency and intensity.
From these observed trends, we propose a conceptual model to evaluate the effects of short-and longterm water limitation on patterns of SS CWM and total NSC CWM concentrations in grassland ecosystem (Fig. 4). We propose a three-phase hypothetical process by which plant NSC CWM concentrations respond to aridity. Initially (phase A), there is a short-term plastic response during which a community shows stochastic uctuation over time around some equilibrium NSC concentrations.   Plant community-weighted mean of soluble sugar and total nonstructural carbohydrate (NSC) concentrations in plots exposed to experimental drought and control plots for six grassland sites along a natural aridity gradient in northern China. Soluble sugar and total NSC concentrations are shown as mean ± 1SE (n = 6). Statistical signi cance is represented by asterisks: *P<0.05, **P<0.01.  Changes in plant community-weighted mean of soluble sugar and total nonstructural carbohydrate (NSC) concentrations in response to drought in grasslands of northern China due to species turnover and intraspeci c variation or species turnover only for each site. Black squares correspond to communityweighted mean of soluble sugar and total NSC concentrations in control and drought plots; Red circles correspond to community-weighted mean of soluble sugar and total NSC concentrations in the drought plots recalculated from soluble sugar and total NSC concentrations in the control plots. Arrows indicate the contributions of species turnover (red arrows) and intraspeci c variation (black arrows) to the change in community-weighted mean of soluble sugar and total NSC concentrations. Species turnover and intraspeci c variation are expressed as percentages of their cumulative magnitude. A parallel direction of shift in species turnover and intraspeci c variation effects indicates positive (synergistic) covariation, whereas an opposite direction of shift in species turnover and intraspeci c variation indicates negative (antagonistic) covariation. Community-weighted mean of soluble sugar and total NSC concentrations are shown as mean ± SE (n = 6).