To examine the Q10 difference between soil depths and the associated mechanisms, we established two manipulative experiments based on 330-day incubations at 10° and 20°C: One was conducted to compare Q10 in the topsoil and subsoil, and also to test the effects of aggregate protection through the uncrushed (control) and crushed (remove aggregate protection) soils (termed “aggregate disruption experiment”). The other was performed to examine the depth difference in microbial controls by inoculating soils with active microorganism from away (collected in the other depth) and own (the same depth) (termed “microorganism reciprocal transplant experiment”).

In the aggregate disruption experiment, we compared Q10 between the topsoil and the subsoil and then examined the effects of aggregate protection on Q10. The treatments included uncrushed (control) and crushed soil samples. For the crushed treatment, air-dried soil samples were crushed for 1 min by a ball mill to disrupt aggregates and remove aggregate protection (19). Together, a 3 × 2 × 2 × 2 × 3 factorial experiment was conducted corresponding to three study sites, two soil depths, two levels of aggregate disruption, two incubation temperatures, and three replicates. For each of these treatments, 10 g of air-dried topsoil and 20 g of air-dried subsoil from each replicate were evenly distributed into 250 ml of airtight amber jars and incubated at both 10° and 20°C for 330 days (fig. S1D). Soil moisture was adjusted to 60% of water holding capacity (46). The rate of CO2 release was determined on the basis of the changes in headspace CO2 concentration during the incubation interval (17) using an infrared gas analyzer (EGM-5; PP Systems, Haverhill, MA, USA). The measurements were taken every 1 to 4 days for the first 3 weeks and then 1 to 2 weeks for 3 months, followed by 1 to 2 months for the rest of the incubation. It should be noted that the CO2 production from the uncrushed (control) soils was also used to explore the difference in Q10 between soil depths.

In the microorganism reciprocal transplant experiment, we explored whether microorganisms were functionally different between soil depths (20). This experiment was conducted by applying two microbial inoculums (derived from topsoil and subsoil) to both sterilized topsoil and subsoil. Specifically, soil samples were sterilized using γ-irradiation (60Co) at a dose of 30 kGy (37). The sterilized soil was then divided into two subsamples, receiving the inoculum derived from itself (own) and from the other corresponding depth (away), respectively; the former treatment (own) was treated as control. In total, 72 microcosms (3 sites × 2 soil depths × 2 inoculums × 2 temperatures × 3 replicates) were constructed. Inoculums from each soil depth were made by adding 1 g of dry weight fresh soil to 100 ml of sterilized deionized water. Then, the inoculum was shaken at 150 rpm for 0.5 hour and filtered through a Whatman GF/C filter. Inoculum (0.4 ml) was applied to per gram soil. The procedure of subsequent CO2 measurement was the same as the first experiment.

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