Biotic factors dominantly determine soil inorganic carbon stock across 1 Tibetan alpine grasslands 2

. Soil inorganic carbon (SIC) pool is a major component of soil C pools, and 15 clarifying the predictors of SIC stock is urgent for decreasing soil C losses and 16 maintaining soil health and ecosystem functions. However, the drivers and their relative 17 effects on the SIC stock at different soil depths remain largely unexplored. Here, we 18 conducted a large-scale sampling to investigate the effects and relative contributions of 19 abiotic (climate and soil) and biotic (plant and microbe) drivers on the SIC stock 20 between topsoils (0–10 cm) and subsoils (20–30 cm) across Tibetan alpine grasslands. 21 Results showed that the SIC stock had no significant differences between the topsoil 22 and subsoil. The SIC stock was positively associated with altitude, pH, and sand 23 proportion, but negatively correlated with mean annual precipitation, plant 24 aboveground biomass, plant coverage, root biomass, soil available nitrogen, microbial

At each site, we selected four 1 m ×1 m plots for soil and plant samplings and the 114 distance between nearby sampling plots was 25 m. In each plot, a 7.5-cm diameter soil 115 drill was used to take five soil cores at fixed soil depths (0-10 cm, 10-20 cm, and 20-30 116 cm), and a 2-mm mesh was used to remove stones. We used soil samples from 0-10 cm 117 and 20-30 cm to represent the topsoil and subsoil, respectively, according to previous  The rest soil samples about 700 g were also sent back to the laboratory and air-dried for 122 measurements of other soil properties. A 40 cm ×40 cm ×40 cm (length × width × depth) 123 pit was dug for measuring soil bulk density (BD) by using a constant volume soil 124 sampling drill (100 cm 3 ), and the undisturbed soil was preserved in aluminum specimen 125 boxes returning to the laboratory and oven-dried for 48 hours at 105°C and weighed.

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The oven-dried soil (20 g) was screened into gravel by sifting through a 2-mm mesh 127 sieve and gravels larger than 2 mm were collected and weighed to determine the 128 percentage of gravels. Soil pH (1:25 soil: H2O) was measured using a soil pH meter,     Table S2. SIC density (g C m -2 ) = SIC (g C kg -1 ) × BD (g cm -3 ) ×d (cm) × (1-g) /100 (1) where SIC is soil inorganic C content, d is the depth of the soil layer (0.1 m), BD is 175 bulk density, and g is the percentage of gravel fraction (>2 mm). subsoil, but bulk density in the subsoil was much higher compared with the topsoil.

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Specifically, SIC density in the topsoil and subsoil ranged from 1.8 g C m -2 to 3271 g 207 C m -2 and 5.4 g C m -2 to 3214 g C m -2 across 25 sampling sites, with an average of 802 208 ± 220 g C m -2 and 814 ± 236 g C m -2 , respectively ( Fig. 1). No significant changes in 209 SIC density with soil depth were observed in both the alpine steppe and alpine desert 210 (p=0.113 and p=0.068, respectively; Fig. 1), but SIC density was higher in the subsoil 211 than that in the topsoil in the alpine meadow (p = 0.002, Fig. 1).

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Meanwhile, the majority of abiotic and biotic drivers had significant differences 213 between the topsoil and subsoil (Table 1). RB, AN, MBC, BA, and FA in the topsoil 214 were significantly larger than those in the subsoil (all p<0.001). In contrast, pH was 215 significantly lower in the topsoil than in the subsoil (p<0.001, Table 1). However, the 216 sand proportion between the two soil depths had no significant differences (Table 1).

Associations of SIC density with abiotic and biotic variables 218
The SIC density was closely related to multiple abiotic and biotic variables (Fig.s 2 and   219 3). For both the topsoil and subsoil, the SIC density was positively associated with  (Fig. 2), but not in the subsoil (Fig. 3). Meanwhile, the SIC density in both two 223 soil depths did not correlate with MAT (Figs. 2 and 3).  (Fig. 4).

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Among these variables, PC, BA, and FA exhibited larger effects on the SIC density 230 compared with other controlling factors (Fig. 4). Also, the VPA analysis illustrated that 231 biotic factors explained the majority variation of SIC density compared with abiotic 232 factors (Fig. 5). For the subsoil, the linear model showed that edaphic variables largely 233 explained the variation in SIC density, followed by microbial and plant variables, and 234 climate contributed the least (Fig. 4). Among these variables, the soil pH had larger 235 contributions to the variation of SIC density rather than others (Fig. 4). Meanwhile, the 236 VPA analysis confirmed that the effects of biotic factors on SIC density were larger 237 than those of abiotic factors in the subsoil (Fig. 5).  . 303 We also revealed that bacterial and fungal gene abundance contributed significantly 304 to the variation of SIC stock (Figs. 2 and 3), which was likely to account for decreasing

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Microbial factors also affected SIC stock more in the topsoil than in the subsoil.

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The large plant residues incorporated into the topsoil provided substantial amounts of among edaphic variables (Fig. 4). The buffering capacity in soil solutions determines 326 the equilibrium of ion inputs and outputs by soil pH (Huang et al, 2015). In this study, soil pH in the subsoil (7.85) was much higher than that (7.66) in the topsoil (Table 1).