*2.3. Litterbag Experiment*

Foliar litters of the four dominant trees species in this alpine forest were studied using an in situ litterbag method [22]. In the fall of 2012, senesced needles or leaves of each tree species were collected on tarpaulins from 20 or more trees (15–20 cm in diameter) by shaking their limbs. Green or partly decomposed needles (leaves), twigs and bark were removed, and only newly shed foliar litter was air-dried at room temperature for two weeks. Litter samples (10 ± 0.05 g) were placed into nylon litterbags (20 × 25 cm in size with mesh sizes of 1.0 mm on the top and 0.5 mm on the bottom). A total of 1172 litterbags (2 litterbags × 6 replicates × 4 litter species × 2 snow plots × 12 sampling times + 5 litterbags × 4 litter species) were incubated in the deep and shallow snow plots on November 15 and 16, 2012. Litterbags in the same subplots were strung together and kept 2–5 cm apart. Five litterbags of each litter species were randomly collected to determine the losses during

sample establishment and the gravimetric water contents of these air-dried litter samples before field incubation.

**Figure 1.** Snow depth, temperature and freeze–thaw cycle. (**a**) Snow depths (±SE, *n* = 18) in deep and shallow snow plots on each sampling date. The samplings were scheduled at the ends of the snow formation, snow coverage and snowmelt stages during winter from 2012 to 2014 (only the snowmelt stages from 2014 to 2016) and at the end of the growing season (beginning of the snow formation stage). The differences between deep and shallow snow plots are significant (*p* < 0.05) on all sampling dates. (**b**) Daily temperatures (*n* = 6) at the litter surface during the four years of decomposition. Winter times including the snow formation, snow coverage and snowmelt stages are shaded. The dashed line is drawn at the zero value. (**c**) Mean temperatures (±SE, *n* = 6) at individual stages. The times between two adjacent sampling dates were defined as separate stages. (**d**) Freeze–thaw cycles (±SE, *n* = 6) at individual stages. The values were calculated by the quotients of the total number of freeze–thaw cycles and the number of days of certain stages. For panel c and d, the winters in 2015 and 2016 were not divided into snow formation, snow coverage and snowmelt stages. SF: snow formation stage, SC: snow coverage stage, SM: snowmelt stage, W: winter, and GS: growing season. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

#### *2.4. Sampling and Analysis*

On each sampling date, snow depths were manually measured in triplicate in each deep and shallow snow plot (*n* = 18). Temperatures at the litter surface were recorded using data loggers (iButton DS1923-F5, Sunnyvale, CA, USA) placed in marked litterbags in deep and shallow snow plots (both *n* = 6). Based on hourly temperature data, a freeze–thaw cycle was defined as a transition above or below 0 ◦C for at least 3 h and then a transition back [25].

Two litterbags per subplot were randomly collected, carefully placed in separate plastic bags and returned to the laboratory. Roots, mosses and soils were carefully removed from the litter samples. One cleaned subsample was oven-dried at 105 ◦C for 48 h to measure the dry mass remaining and gravimetric water content in the decomposing litter. The remaining mass of decomposing litter was evaluated on a dry matter basis. Another subsample was air-dried, milled and used to extract the acid hydrolysable components using an acid hydrolysis method [26]. In our long-term study in this alpine forest, we also measured proximate fractions, elements, humic substances and microbial activities during

the decomposition of these foliar litters. In this study, we focused on the release pattern of acid hydrolysable components.

Here, the acid hydrolysable components were defined as the fraction that can be hydrolyzed by 2.5 mol/L H2SO4 [26]. These components were different with acid-soluble substances hydrolyzed by 72% H2SO4 as well as with dissolved organic carbon or hot-water extractable carbon, which are considered to be more labile with higher decomposability than acid hydrolysable components. In this study, the acid hydrolysable components include some polysaccharide, hemicellulose, cellulose and soluble lignin or phenols [6].

Specifically, a 0.10 g subsample was hydrolyzed by 2.5 mol/L H2SO4 at 105 ◦C for 30 min. The extracted acid hydrolysable components were diluted with deionized water, filtered through a 0.45 μm mesh and measured using a TOC analyzer (multi N/C 2100, Analytic Jena, Thüringen, Germany). Litter C and N contents were determined by elemental analyzer (Vario Max CN, Elementar, Germany) and were used to calculate litter C/N stoichiometry. Microbial biomass C content was measured using the chloroform fumigationincubation method [27]. At the same time, a 0.5 g subsample was oven-dried at 105 ◦C for 48 h to determine the gravimetric water content in the air-dried decomposing litter. The content of acid hydrolysable components was calculated based on the dry mass of decomposing litter.
