Grazing effects on CH4 uptake
We found two contrasting patterns of CH4 flux in response to increasing stocking rate. In the first two years of grazing, CH4 uptake rate increased with stocking rate (Fig. 2A) with no significant differences detected among the treatments, suggesting that grazing effects on CH4 flux are not immediate. Indeed, the meadow steppe is more resistant to grazing than other steppe types in Inner Mongolia, due to its rich soil (fine textured, well-drained) and vegetation (height, diversity). The dominant plant species (i.e., Leymus chinensis) is more resistant to feeding and trampling than the Stipa spp. that dominate other steppes. Previous studies show that soil moisture and aeration conditions of the L. chinensis steppe are much less prone to grazing trampling, and its regrowth of biomass is also less affected by short-term herbivory, mainly due to the rhizomatous growth form of L. chinensis per se, in striking contrast to the bunch growth form of Stipa species (Li et al., 2020). As a result, the activity of MOB and CH4 uptake were also less affected by grazing in the initial few years in this steppe ecosystem.
An obviously decreasing trend in CH4 uptake with increasing grazing intensity appeared in the following years, with the highest stocking rate showing the greatest negative effects on CH4 uptake (Fig. 2A, 2B). Overall, grazing reduced CH4 uptake of the soil regardless of grazing intensity, which is consistent with most previous studies in typical steppes (Du et al. 1997; Dong et al. 2000; Wang et al. 2000; Wang et al. 2005a, 2005b; Liu et al. 2007; Holst et al. 2008), desert steppes (Tang et al. 2013), and alpine steppes (Wei et al. 2012). Wang et al. (2009) synthesized a number of relevant studies in the typical steppe and found that, on average, the CH4 uptake in grazed grasslands was approximately 68% of that in un-grazed grasslands. Treating three steppe types in China (i.e., desert, typical and meadow steppes) as a whole, Tang et al. (2013) showed that light grazing did not significantly change CH4 uptake compared with un-grazed steppe, but moderate and heavy grazing reduced CH4 uptake by 6.8% and 37.9%, respectively. In this study, reductions in CH4 uptake varied between 7.6% in the light grazing treatment (G0.23) and 25.4% in the heavy grazing treatment (G0.92), which fall within the range of Tang et al. (2013).
Three mechanisms may be responsible for the changes and differences in CH4 uptake under grazing in our study grassland. First, they may have resulted from changes in vegetation traits due to variable grazing. The CH4 uptake rate significantly positively correlated with the vegetation traits examined in our study (Fig. 5), indicating that the effects of grazing on methane uptake was partially mediated by vegetation. CH4 oxidation in the rhizosphere is the most important process for CH4 cycle, and methanotrophs are developed mainly in the oxidized soil layer and in the aerobic rhizosphere of plants. Here methanotrophs are associated with roots and rhizomes of plants and their activities should correlate with the oxidizing environment of the rhizosphere for MOB (Le Mer and Roger 2001). Changes in litter input and carbohydrate allocation into the soil may play a role in affecting CH4 uptake in this study, although SOM is not the principal energy source for MOB, mainly because SOM may influence the soil food-chain, indirectly affecting the abundance of MOB. By contrast, the effects of vegetation traits on soil moisture and thermal conditions may assume more importance in this regard.
The second mechanism may be that grazing changes soil NH4+-N content and thus affects methane uptake. A significant negative relationship between the CH4 uptake rate and soil NH4+-N content was found in this study (Fig. 6). Our long-term data showed that the multi-year mean soil NH4+-N content had increased by all grazing treatments, particularly at the highest stocking rate (Zhang et al. 2021). For some years, one- to two-fold increases in soil NH4+-N content were observed under the highest stocking rate compared to the reference unit (G0.00). Increases in available nitrogen content, soil NH4+-N in particular, resulted mainly from livestock excretion of dung and urine. Quite a few studies have showed that methane oxidation potential of upland soils may be reduced by ammonium N-fertilizer application (Mosier et al. 1991; Le Mer and Roger 2001; Täumer et al. 2020). When soil CH4 concentration (< 12 ppm) is low, high soil NH4+-N concentration can significantly inhibit the methanotroph process (i.e., CH4 oxidation) (Topp and Pattey 1997). It has been reported in an alpine meadow that animal dung was the primary CH4 source, while urine-soaked soil consumed much less CH4 (Lin et al. 2009). In a desert steppe in Inner Mongolia, Jiang et al. (2012) showed that CH4 uptake from urine and dung units decreased by 25.7% and 33.3%, respectively, compared with a control unit. Wang et al. (2013) also measured CH4 emission from urine and dung patches in a typical steppe. This nitrogen fertilization that led directly or indirectly to an increased soil NH4+-N content produced an inhibitory effect on CH4 oxidation through competition of methane monooxygenase towards nitrification and nitrite (Le Mer and Roger 2001). In addition, methanotrophs significantly contributed to nitrification in the rhizosphere, while the contribution of nitrifiers to CH4 oxidation was insignificant (Han et al. 1999).
Thirdly, altered soil moisture by grazing may be another mechanism that regulates CH4 uptake. Our long-term data showed that soil moisture was higher in most cases under various stocking rates of grazing than the no grazing (G0.00) treatment, although no linearly increasing trend of soil moisture with increasing stocking rate was found (Zhang et al. 2021). Stocking-resultant increases in soil moisture were likely related in part to the corresponding decreases in ANPP which consumed less soil water. In addition, we observed that dicotyledonous forbs with deep taproots were significantly more abundant in the no-grazing treatment than in all grazing treatments except the intermediate-grazing treatment, which could also be a cause in this regard. Given the significant negative relationship between the CH4 uptake and soil water content (Fig. 6), the decrease in CH4 uptake with stocking rate can be partially explained through the mediating role of soil moisture and increases soil bulk density. Soil submersion allows the development of the methanogenic activity and reduces methanotrophic activity by reducing the size of the oxidized zone. In a typical steppe, CH4 uptake was primarily determined by soil temperature and soil moisture of the topsoil (7 cm) (Wang et al. 2005a). Negative correlation between CH4 uptake and soil moisture was reported in an alpine steppe (Wei et al. 2012) and temperate steppes (Tang et al. 2013). At Swiss grassland sites, the soil methanotrophic activity is related to its water content. With the increase of water content, it gradually decreases and is close to the field capacity (Imer et al. 2013). Negative correlations between CH4 consumption and soil moisture were also reported in Canadian forests and in a Massachusetts forest (Le Mer and Roger 2001). Upland soils, when temporarily submerged, may become CH4 sources. This was also observed in a Canadian grassland with well drained soils (Wang and Bettany 1995).
A positive correlation is often observed between the methanogenic potential and the SOM content in rice field soils, and almost all in situ studies have shown that organic matter incorporation markedly increased CH4 emission. However, a positive relationship between CH4 uptake and soil SOC content was detected in this study (Fig. 6), which may reflect the fact that grassland soils per se have rather low methanogenic potentials. This can be further corroborated by the fact that CH4 production is often positively correlated with SOM content in soils exhibiting a high methanogenic activity (Le Mer and Roger 2001).
Complexity of grazing regulation on CH4 uptake
Our multivariate linear regression analysis indicated that livestock grazing had substantially changed the interannual variation of both CH4 uptake and its biophysical regulators in this steppe ecosystem (Fig. 3A, 3B; Table 1). The increasing trends of CH4 uptake with year in the no-grazing (G0.00) and light grazing treatments (G0.23) may reflect a progressive successional process of the ecosystem under these treatments. In fact, this steppe had been under heavy grazing for ages prior to our experiment. As such, the vegetation and soil nutrient conditions should have improved compared with the start of the experiment under no grazing or light grazing conditions. Nevertheless, the control stand was far from the pristine stand in terms of vegetation and soil regimes. This may partially explain the non-significant differences in soil conditions, making it difficult to explain variation in CH4 uptake along the grazing gradient. The positive correlations between CH4 uptake and vegetation characteristics, SOC and total nitrogen contents (Figs. 5, 6) provide empirical evidences of an ecosystem recovery process (i.e., reduced grazing impacts). The positive relationships between CH4 uptake rate and vegetation coverage and BGB (Table 1) also support this assumption. The negative relationships between CH4 uptake and the soil NH4+-N content (Tables 1, 2), however, suggest that other factors may be responsible for the interannual variations of CH4 uptake under these light grazing treatments.
Under the light (G0.34) and intermediate (G0.46) grazing treatments (Fig. 3A), both increase and decrease in CH4 uptake with year may reflect a standstill status over the successional process of the ecosystem since the start of the fencing. Here no significant changes in the vegetation characteristics and soil nutrient conditions had occurred. Climatic factors, especially rainfall, became the predominant driver for interannual dynamics of CH4 uptake (Table 1). The negative relationships between rainfall and CH4 uptake under these treatments may reflect the negative impacts of the soil NH4+-N content on CH4 uptake, whereas the positive relationships between CH4 uptake and soil available nitrogen (Table 1) are likely due to the impacts of soil NO3+-N on the CH4 dynamics, coupled with the methanotrophic and the nitrification process (Han et al. 1999; Le Mer and Roger 2001) where the excretion of dung and urine played an important role.
With the heavy (G0.69) and over-grazing (G0.92) treatments, the decreasing trends of CH4 uptake with year may reflect a retrogressive successional process by grazing. Here vegetation characteristics and soil conditions were significantly affected over the years (Li et al. 2021) and, consequently regulate CH4 dynamics (Table 1). The positive correlations between CH4 uptake and vegetation, SOC and total nitrogen contents (Figs. 5, 6) demonstrated their coupled influences on CH4 uptake (Table 1). The negative relationship between CH4 uptake and the soil NH4+-N content (Table 1) further indicate that NH4+-N content continued its role in regulating the interannual dynamics of CH4 uptake under the over-grazing treatment. However, the relative importance of soil NH4+-N was substantially underestimated in our multivariate linear regression analysis (Tables 1, 2). As previously discussed, dung was a significant CH4 source, while urine patches restricted CH4 consumption (Lin et al. 2009; Wang et al. 2013). The dung and urine patches under the over-grazing treatment in this study had covered about one-tenth of the entire steppe ground during each stocking season, which should have much more substantial impacts on the soil nitrogen budget. However, the nitrogen input via this way is difficult to be captured by measuring the soil contents of available components (NH4+-N, NO3−-N) due to their rapid volatile nature and their being prone to plant absorption. The negative relationship between the CH4 uptake rate and soil water content detected in the heavy grazing treatment (Table 1) is mechanistically clear, while we did find that the interannual CV of soil moisture under this treatment was the second largest among all the treatments.
We found some inconsistent and even contradictory results with previous reports on the impacts of livestock grazing on grassland soil CH4 uptake, partially due to the inconsistency in the number of study years and grazing intensity. Most previous studies were conducted in a single season or over 1–3 years. These studies did not have a broad range of stocking rates, either (i.e., limited to 1–3 grazing intensities). Clearly, long-term treatments with multiple stocking rates should be studied in other worldwide grasslands.
Our hypothesis that livestock grazing imposes impacts on methane dynamics in grassland soils through trampling, selective feeding, and excretion of dung and urine is generally accepted. These impacts are induced through alterations of soil moisture, aboveground biomass, and soil total nitrogen and NH4+-N contents. Our findings are partially in agreement with some previous studies (Du et al. 1997; Wang et al. 2009; Wei et al. 2012). However, we have not teased apart the contributions of three grazing actions in the present study: trampling, vegetation-feeding, and excretion of dung and urine. Continued efforts are needed to see if the findings may shift over even longer time scales (e.g., decades), along with added efforts in partitioning the three specific grazing actions in the future.