5300-Year-old soil carbon is less primed than young soil organic matter.

Soils harbor more than three times as much carbon (C) as the atmosphere, a large fraction of which (stable organic matter) serves as the most important global C reservoir due to its long residence time. Litter and root inputs bring fresh organic matter (FOM) into the soil and accelerate the turnover of stable C pools, and this phenomenon is termed the "priming effect" (PE). Compared with knowledge about labile soil C pools, very little is known about the vulnerability of stable C to priming. Using two soils that substantially differed in age (500 and 5300 years before present) and in the degree of chemical recalcitrance and physical protection of soil organic matter (SOM), we showed that leaf litter amendment primed 264% more organic C from the young SOM than from the old soil with very stable C. Hierarchical partitioning analysis confirmed that SOM stability, reflected mainly by available C and aggregate protection of SOM, is the most important predictor of leaf litter-induced PE. The addition of complex FOM (i.e., leaf litter) caused a higher bacterial oligotroph/copiotroph (K-/r-strategists) ratio, leading to a PE that was 583% and 126% greater than when simple FOM (i.e., glucose) was added to the young and old soils, respectively. This implies that the PE intensity depends on the chemical similarity between the primer (here FOM) and SOM. Nitrogen (N) mining existed when N and simple FOM were added (i.e., Glucose+N), and N addition raised the leaf litter-induced PE in the old soil that had low N availability, which was well explained by the microbial stoichiometry. In conclusion, the PE induced by FOM inputs strongly decreases with increasing SOM stability. However, the contribution of stable SOM to CO2 efflux cannot be disregarded due to its huge pool size.

Applied phosphorus is maintained in labile and moderately occluded fractions in a typical meadow steppe with the addition of multiple nutrients.

Phosphorus (P) is a limiting nutrient second only to nitrogen (N) in the drylands of the world. Most previous studies have focused on N transformation processes in grassland ecosystems, particularly under artificial fertilization with N and atmospheric N deposition. However, P cycling processes under natural conditions and when P is applied as an inorganic P fertilizer have been understudied. Therefore, it is essential to examine the fate of applied P in grassland ecosystems that have experienced long-term grazing and, under certain circumstances, continuous hay harvest. We conducted a 3-year field experiment with the addition of multiple nutrient elements in a typical meadow steppe to investigate the fate of the applied P in various fractions of P pools in the top soil. We found that the addition of multiple nutrients significantly increased P concentrations in the labile inorganic P (Lab-Pi) and moderately occluded inorganic P (Mod-Pi) fractions but not in the recalcitrant inorganic P (Rec-Pi) fraction. An increase in the concentration of total inorganic P was found only when P and N were applied together. However, the addition of other nutrients did not change P concentrations in any fraction of the mineral soil. The addition of P and N significantly increased the total amount of P taken up by the aboveground plants but had no effect on the levels of organic and microbial P in the soil. Together, our results indicate that the P applied in this grassland ecosystem is taken up by plants, leaving most of the unutilized P as Lab-Pi and Mod-Pi rather than being immobilized in Rec-Pi or by microbial biomass. This implies that the grassland ecosystem that we studied has a relatively low P adsorption capacity, and the application of inorganic P to replenish soil P deficiency in degraded grasslands due to long-term grazing of livestock or continuous harvest of forage in the region could be a practical management strategy to maintain soil P fertility.

Plant genome size modulates grassland community responses to multi-nutrient additions.

Grassland ecosystems cover c. 40% of global land area and contain c. 40% of soil organic carbon. Understanding the effects of adding nutrients to grasslands is essential because they provide much of our food, support diverse ecosystem services and harbor rich biodiversity. Using the meadow steppe (grassland) study site of Inner Mongolia, we manipulated seven key nutrients and a cocktail of micronutrients to examine their effects on grassland biomass productivity and diversity. The results, explained in structural equation models, link two previously disparate hypotheses in grassland ecology: (1) the light asymmetry competition hypothesis and (2) the genome size-nutrient interaction hypothesis. We show that aboveground net primary productivity increases predominantly from species with large genome sizes with the addition of nitrogen, and nitrogen plus phosphorus. This drives an asymmetric competition for light, causing a decline in species richness mainly in species with small genome sizes. This dynamic is likely to be caused by the nutrient demands of the nucleus and/or the scaling effects of nuclear size on cell size which impact water use efficiency. The model will help inform the best management approaches to reverse the rapid and unprecedented degradation of grasslands globally.

Low carbon availability in paleosols nonlinearly attenuates temperature sensitivity of soil organic matter decomposition.

Temperature sensitivity (Q10 ) of soil organic matter (SOM) decomposition is an important parameter in models of the global carbon (C) cycle. Previous studies have suggested that substrate quality controls the intrinsic Q10 , whereas environmental factors can impose large constraints. For example, physical protection of SOM and its association with minerals attenuate the apparent Q10 through reducing substrate availability and accessibility ([S]). The magnitude of this dampening effect, however, has never been quantified. We simulated theoretical Q10 changes across a wide range of [S] and found that the relationship between Q10 and the log10 -transformed [S] followed a logistic rather than a linear function. Based on the unique Holocene paleosol chronosequence (7 soils from ca. 500 to 6900 years old), we demonstrated that the Q10 decreased nonlinearly with soil age up to 1150 years, beyond which Q10 remained stable. Hierarchical partitioning analysis indicated that an integrated C availability index, derived from principal component analysis of DOC content and parameters reflecting physical protection and mineral association, was the main explanatory variable for the nonlinear decrease of Q10 with soil age. Microbial inoculation and 13 C-labelled glucose addition showed that low C availability induced by physical protection and minerals association attenuated Q10 along the chronosequence. A separate soil incubation experiment indicated that Q10 increased exponentially with activation energy (Ea ) in the modern soil, suggesting that SOM chemical complexity regulates Q10 only when C availability is high. In conclusion, organic matter availability strongly decreased with soil age, whereas Michelis-Menten kinetics defines the Q10 response depending on C availability, but Arrhenius equation describes the effects of increasing substrate complexity.

Carbon limitation overrides acidification in mediating soil microbial activity to nitrogen enrichment in a temperate grassland.

Higher ecosystem nitrogen (N) inputs resulting from human activities often suppress soil microbial biomass and respiration, thereby altering biogeochemical cycling. Soil acidification and carbon (C) limitation may drive these microbial responses, yet their relative importance remains elusive, which limits our understanding of the longer term effects of increasing N inputs. In a field experiment with continuous N addition at seven different rates from 0 to 50 g N m-2  year-1 over 6 years in a temperate grassland of Inner Mongolia, China, we examined the responses of soil microbial biomass and respiration to changes in soil acidity and C availability by adding lime and/or glucose to soil samples. Soil microbial biomass and respiration did only weakly respond to increasing soil pH, but increased strongly in response to higher C availability with increasing N addition rates. Soil net N immobilization increased in response to glucose addition, and soil microbial biomass increased at higher rates than microbial respiration along the gradient of previous N addition rates, both suggesting increasingly reinforced microbial C limitation with increasing N addition. Our results provide clear evidence for strong N-induced microbial C limitation, but only little support for soil acidity effects within the initial pH range of 4.73-7.86 covered by our study. Field data support this conclusion by showing reduced plant C allocation belowground in response to N addition, resulting in soil microbial C starvation over the long term. In conclusion, soil microbial biomass and respiration under N addition were strongly dependent on C availability, most likely originating from plant belowground C inputs, and was much less affected by changes in soil pH. Our data help clarify a long-standing debate about how increasing N input rates affect soil microbial biomass and respiration, and improve the mechanistic understanding of the linkages between ecosystem N enrichment and C cycling.

Spatial patterns and ecological drivers of soil nematode ß-diversity in natural grasslands vary among vegetation types and trophic position.

Understanding biogeographic patterns of community assemblages is a core objective in ecology, but for soil communities these patterns are poorly understood. To understand the spatial patterns and underlying mechanisms of ß-diversity in soil communities, we investigated the ß-diversity of soil nematode communities along a 3,200-km transect across semi-arid and arid grasslands. Spatial turnover and nested-resultant are the two fundamental components of ß-diversity, which have been attributed to various processes of community assembly. We calculated the spatial turnover and nested-resultant components of soil nematode ß-diversity based on the ß-partitioning framework. Distance matrices for the dissimilarity of soil nematode communities were computed using the 'Sørensen' method. We fitted negative exponential models to compare the distance decay patterns in nematode community similarity with geographic distance and plant community distance in three vegetation types (desert, desert steppe and typical steppe) and along the whole transect. Variation partitioning was used to distinguish the contribution of geographic distance and environmental variables to ß-diversity and the partitioned components. Geographic distance and environmental filtering jointly drove the ß-diversity patterns of nematode community, but environmental filtering explained more of the variation in ß-diversity in the desert and typical steppe, whereas geographic distance was important in the desert steppe. Nematode community assembly was explained more by the spatial turnover component than by the nested-resultant component. For nematode feeding groups, the ß-diversity in different vegetation types increased with geographic distance and plant community distance, but the nested-resultant component of bacterial feeders in the desert ecosystem decreased with geographic distance and plant community distance. Our findings show that spatial variation in soil nematode communities is regulated by environmental processes at the vegetation type scale, while spatial processes mainly work on the regional scale, and emphasize that the spatial patterns and drivers of nematode ß-diversity differ among trophic levels. Our study provides insight into the ecological processes that maintain soil biodiversity and biogeographic patterns of soil community assemblage at large spatial scales.

Nitrogen deposition weakens plant-microbe interactions in grassland ecosystems.

Soil carbon (C) and nitrogen (N) stoichiometry is a main driver of ecosystem functioning. Global N enrichment has greatly changed soil C : N ratios, but how altered resource stoichiometry influences the complexity of direct and indirect interactions among plants, soils, and microbial communities has rarely been explored. Here, we investigated the responses of the plant-soil-microbe system to multi-level N additions and the role of dissolved organic carbon (DOC) and inorganic N stoichiometry in regulating microbial biomass in semiarid grassland in northern China. We documented a significant positive correlation between DOC and inorganic N across the N addition gradient, which contradicts the negative nonlinear correlation between nitrate accrual and DOC availability commonly observed in natural ecosystems. Using hierarchical structural equation modeling, we found that soil acidification resulting from N addition, rather than changes in the plant community, was most closely related to shifts in soil microbial community composition and decline of microbial respiration. These findings indicate a down-regulating effect of high N availability on plant-microbe interactions. That is, with the limiting factor for microbial biomass shifting from resource stoichiometry to soil acidity, N enrichment weakens the bottom-up control of soil microorganisms by plant-derived C sources. These results highlight the importance of integratively studying the plant-soil-microbe system in improving our understanding of ecosystem functioning under conditions of global N enrichment.

Effects of multi-resource addition on grassland plant productivity and biodiversity along a resource gradient.

The change of plant biodiversity caused by resource-enhancing global changes has greatly affected grassland productivity. However, it remains unclear how multi-resource enrichment induces the effects of multifaceted biodiversity on grassland productivity under different site resource constraints. We conducted a multiple resource addition (MRA) experiment of water and nutrients at three sites located along a resource gradient in northern China. This allowed us to assess the response of aboveground net primary productivity (ANPP), species (species richness and plant density), functional (functional richness and community-weighted mean of traits) and phylogenetic (phylogenetic richness) diversity to increasing number of MRA. We used structural equation model (SEM) to examine the direct and indirect effects of MRA and multifaceted biodiversity on ANPP. The combined addition of the four resources increased ANPP at all three sites. But with increasing number of MRA, biodiversity varied at the three sites. At the high resource constraint site, species richness, plant density and leaf nitrogen concentration (LNC) increased. At the medium resource constraint site, plant height and LNC increased, leaf dry matter content (LDMC) decreased. At the low resource constraint site, species, functional and phylogenetic richness decreased, and height increased. The SEM showed that MRA increased ANPP directly at all three sites, and indirectly by increasing plant density at the high constraint site and height at the medium constraint site. Independent of MRA, ANPP was affected by height at the high resource constraint site and LNC at the low resource constraint site. Our results illustrate that multi-resource addition positively affects productivity, while affects biodiversity depending on site resource constraint. The study highlights that site resource constraint conditions need to be taken into consideration to better predict grassland structure and function, particularly under the future multifaceted global change scenarios.

ß-diversity in temperate grasslands is driven by stronger environmental filtering of plant species with large genomes.

Elucidating mechanisms underlying community assembly and biodiversity patterns is central to ecology and evolution. Genome size (GS) has long been hypothesized to potentially affect species' capacity to tolerate environmental stress and might therefore help drive community assembly. However, its role in driving ß-diversity (i.e., spatial variability in species composition) remains unclear. We measured GS for 161 plant species and community composition across 52 sites spanning a 3200-km transect in the temperate grasslands of China. By correlating the turnover of species composition with environmental dissimilarity, we found that resource filtering (i.e., environmental dissimilarity that includes precipitation, and soil nitrogen and phosphorus concentrations) affected ß-diversity patterns of large-GS species more than small-GS species. By contrast, geographical distance explained more variation of ß-diversity for small-GS than for large-GS species. In a 10-year experiment manipulating levels of water, nitrogen, and phosphorus, adding resources increased plant biomass in species with large GS, suggesting that large-GS species are more sensitive to the changes in resource availability. These findings highlight the role of GS in driving community assembly and predicting species responses to global change.

Nitrogen enrichment buffers phosphorus limitation by mobilizing mineral-bound soil phosphorus in grasslands.

Phosphorus (P) limitation is expected to increase due to nitrogen (N)-induced terrestrial eutrophication, although most soils contain large P pools immobilized in minerals (Pi ) and organic matter (Po ). Here we assessed whether transformations of these P pools could increase plant available pools alleviating P limitation under enhanced N availability. The mechanisms underlying these possible transformations were explored by combining results from a 10-year field N addition experiment and a 3700-km transect covering wide ranges in soil pH, soil N, aridity, leaching, and weathering that could affect soil P status in grasslands. Nitrogen addition promoted the dissolution of immobile Pi (mainly Ca-bound recalcitrant P) to more available forms of Pi (including Al- and Fe-bound P fractions and Olsen P) by decreasing soil pH from 7.6 to 4.7, but did not affect Po . Soil total P declined by 10% from 385 ± 6.8 to 346 ± 9.5 mg kg-1 , whereas available P increased by 546% from 3.5 ± 0.3 to 22.6 ± 2.4 mg kg-1 after the 10-year N addition, associated with an increase in Pi mobilization, plant uptake, and leaching. Similar to the N addition experiment, the drop in soil pH from 7.5 to 5.6 and increase in soil N concentration along the grassland transect were associated with an increased ratio between relatively mobile Pi and immobile Pi . Our results provide a new mechanistic understanding of the important role of soil Pi mobilization in maintaining plant P supply and accelerating biogeochemical P cycles under anthropogenic N enrichment. This mobilization process temporarily buffers ecosystem P limitation or even causes P eutrophication, but will extensively deplete soil P pools in the long run.

Sampling date, leaf age and root size: implications for the study of plant C:N:p stoichiometry.

Plant carbon : nitrogen : phosphorus (C:N:P) ratios are powerful indicators of diverse ecological processes. During plant development and growth, plant C:N:P stoichiometry responds to environmental conditions and physiological constraints. However, variations caused by effects of sampling (i.e. sampling date, leaf age and root size) often have been neglected in previous studies. We investigated the relative contributions of sampling date, leaf age, root size and species identity to stoichiometric flexibility in a field mesocosm study and a natural grassland in Inner Mongolia. We found that sampling date, leaf age, root size and species identity all significantly affected C:N:P stoichiometry both in the pot study as well as in the field. Overall, C:N and C:P ratios increased significantly over time and with increasing leaf age and root size, while the dynamics of N:P ratios depended on species identity. Our results suggest that attempts to synthesize C:N:P stoichiometry data across studies that span regional to global scales and include many species need to better account for temporal variation.

Nitrogen addition regulates soil nematode community composition through ammonium suppression.

Nitrogen (N) enrichment resulting from anthropogenic activities has greatly changed the composition and functioning of soil communities. Nematodes are one of the most abundant and diverse groups of soil organisms, and they occupy key trophic positions in the soil detritus food web. Nematodes have therefore been proposed as useful indicators for shifts in soil ecosystem functioning under N enrichment. Here, we monitored temporal dynamics of the soil nematode community using a multi-level N addition experiment in an Inner Mongolia grassland. Measurements were made three years after the start of the experiment. We used structural equation modeling (SEM) to explore the mechanisms regulating nematode responses to N enrichment. Across the N enrichment gradient, significant reductions in total nematode abundance, diversity (H' and taxonomic richness), maturity index (MI), and the abundance of root herbivores, fungivores and omnivores-predators were found in August. Root herbivores recovered in September, contributing to the temporal variation of total nematode abundance across the N gradient. Bacterivores showed a hump-shaped relationship with N addition rate, both in August and September. Ammonium concentration was negatively correlated with the abundance of total and herbivorous nematodes in August, but not in September. Ammonium suppression explained 61% of the variation in nematode richness and 43% of the variation in nematode trophic group composition. Ammonium toxicity may occur when herbivorous nematodes feed on root fluid, providing a possible explanation for the negative relationship between herbivorous nematodes and ammonium concentration in August. We found a significantly positive relationship between fungivores and fungal phospholipid fatty acids (PLFA), suggesting bottom-up control of fungivores. No such relationship was found between bacterivorous nematodes and bacterial PLFA. Our findings contribute to the understanding of effects of N enrichment in semiarid grassland on soil nematode trophic groups, and the cascading effects in the detrital soil food web.