Grasslands have been subject to considerable alteration due to human activities globally, including widespread changes in populations and composition of large mammalian herbivores and elevated supply of nutrients. Grassland soils remain important reservoirs of carbon (C) and nitrogen (N). Herbivores may affect both C and N pools and these changes likely interact with increases in soil nutrient availability. Given the scale of grassland soil fluxes, such changes can have striking consequences for atmospheric C concentrations and the climate. Here, we use the Nutrient Network experiment to examine the responses of soil C and N pools to mammalian herbivore exclusion across 22 grasslands, under ambient and elevated nutrient availabilities (fertilized with NPK + micronutrients). We show that the impact of herbivore exclusion on soil C and N pools depends on fertilization. Under ambient nutrient conditions, we observed no effect of herbivore exclusion, but under elevated nutrient supply, pools are smaller upon herbivore exclusion. The highest mean soil C and N pools were found in grazed and fertilized plots. The decrease in soil C and N upon herbivore exclusion in combination with fertilization correlated with a decrease in aboveground plant biomass and microbial activity, indicating a reduced storage of organic matter and microbial residues as soil C and N. The response of soil C and N pools to herbivore exclusion was contingent on temperature – herbivores likely cause losses of C and N in colder sites and increases in warmer sites. Additionally, grasslands that contain mammalian herbivores have the potential to sequester more N under increased temperature variability and nutrient enrichment than ungrazed grasslands. Our study highlights the importance of conserving mammalian herbivore populations in grasslands worldwide. We need to incorporate local-scale herbivory, and its interaction with nutrient enrichment and climate, within global-scale models to better predict land-atmosphere interactions under future climate change.

Sitters.et.al.GCB.CNpool.RR.script

R script required to: (1) quantify the impact of herbivore exclusion and fertilization on soil C and N pools by linear mixed models with block nested within site as random effect. And (2) examine the impact of the exclusion of herbivores on soil C and N pools and C:N ratio under unfertilized and fertilized conditions by performing one sample t-tests on the log response ratios (RR = ln(fenced/unfenced)). If the 95% confidence interval values of the RRs did not overlap with zero, there was a significant decrease or increase with herbivore exclusion.

Sitters.et.al.GCB.MuMIn.script

R script required to examine: (1) which local controls over soil C and N were responsible for changes in soil C and N pools due to herbivore exclusion, and (2) which across-site environmental drivers affected the impact of herbivore exclusion on soil C and N pools. Multi-model inference was used by modelling the effects of the predictor variables (either the local controls or the environmental variables) on the C and N response ratios with a full LMM with site ID as a random effect. The models also included fertilization as a fixed factor to observe any significant interactions between fertilization and local controls/environmental drivers. Multi-model inference uses model averaging based on Akaike’s information criterion (AIC) to arrive at consistent parameter estimates of the most important explanatory variables in the full LMM, by averaging a set of top models which share similarly high levels of parsimony. Top models were defined as those that fell within 4 AIC units of the model with the lowest AIC value. 

soil.CNpool_4trt

Data file required to run the LMMs on soil C and N pools (analysis 1 in Sitters.et.al.GCB.CNpool.RR.script). Data included in the file are the site ID (site_code), the blocks at each site, the treatment (Control à in Fig. 2: +H-F, NPK à +H+F, Fence à -H-F, NPK+Fence à -H+F), the year soil samples were taken, the number of years treatments were in place before the soil samples were taken (after_n_treatment_yrs), soil C and N concentrations in % (pct_C and pct_N), soil bulk density in g/cm³ (bulk_density) and soil C and N pools in kg m^-2 (kgm2_C and kgm2_N).

soil.RR.CNpool_local_env

Data file required to: (1) run the one sample t-tests on the RRs of the exclusion of herbivores on soil C and N pools (RR = ln(fenced/unfenced); analysis 2 in Sitters.et.al.GCB.CNpool.RR.script). And (2) use multi-model inference to examine which local controls and environmental drivers affect the impact of herbivore exclusion on soil C and N pools (analyses in Sitters.et.al.GCB.MuMIn.script). Data included in the file are the site ID (site_code), the blocks at each site, the fertilization treatment (Unfertilized or Fertilized) for which the RRs were calculated, the year soil samples were taken (year), the number of years treatments were in place before the soil samples were taken (after_n_treatment_yrs), log response ratios of soil C and N concentrations, pools and C:N ratio (RR_Ccon, RR_Ncon, RR_Cpool, RR_Npool, RR_CN), log response ratio of soil bulk density (RR_bulk_density). Then follow the candidate local controls used in the multi-model inference analysis: log response ratios of aboveground live plant biomass (RR_live.biom), aboveground dead biomass (RR_dead.biom), belowground plant biomass (RR_root.biom), microbial activity (RR_mic.act) and microbial biomass (RR_mic.biom). After that the candidate environmental controls used in the multi-model inference analysis: mean annual temperature (°C; MAT), temperature seasonality (SD of temperature among months; TEMP_VAR), mean temperature of wettest quarter (°C; TEMP_WET_Q), mean annual precipitation (mm; MAP), precipitation seasonality (coefficient of variation in precipitation among months; MAP_VAR), annual atmospheric N deposition (kg N ha^-1 year^-1; N_Dep), pre-treatment aboveground plant biomass (plant.biom_yr0), and pre-treatment soil N concentrations (pct_N_y0).