Study site and sampling
Soil samples were collected from Tatajia, the saddle of Yushan, in central Taiwan (23°28′N, 120°54′E). The altitude of the study location was about 2700 m, with mean annual precipitation 4100 mm and temperature 9.5 °C. The study area geologically consisted of metamorphosed sedimentary rock (Miocene Epoch) comprising sandstone and shale.
Two distinct vegetation zones, coniferous forest and grassland, were visually identified in the study locations. Chinese hemlock (Tsuga chinensis) was the dominant species in the coniferous forest, and Taiwan red cypress (Chamaecyparis formosensis), Morrison spruce (Picea morrisonicola) and Armand’s pine (Pinus armandi) were found in the area. A grassland formed from a wildfire event approximately 50 years ago adjacent to the forest was used as a control, with dwarfed bamboo (Yushania niitakayamensis) and alpine silver grass (Miscanthus transmorrisonensis) the dominant plants. Between the grassland and forest was a transition zone inhabited by both bamboo and hemlock.
Soil was poorly developed due to the steep erosive terrain and classified as Typic Haplorthod in the forest, Typic Haplumbtept in the transition zone, and Umbric Dystrochrept in the grassland (Imberger and Chiu 2001).
After carefully removing the litter layer, one surface (0–10 cm) soil sample was collected from the center of each vegetation zone including forest, transition, and grassland by using a soil auger (8 cm diameter and 10 cm deep). Visible materials such as litter and roots were manually removed before sieving through a 2-mm sieve. The fresh soil samples were kept at 4 °C in the dark before analysis.
Sample preparation and analyses
Part of the sieved soil samples (<2 mm) were air-dried and used for physical and chemical analysis. Soil texture (Gee and Bauder 1986), pH (McLean 1982), cation exchange capacity (Thomas 1982), and percentage of base saturation (Thomas 1982) were determined.
Other parts of sieved soil samples were treated with low-energy sonication and then separated to different particle-size fractions by a combination of wet sieving and continuous flow centrifugation (Oades et al. 1987). Six fractions, coarse (>250 μm), large (53–250 μm), medium (20–53 μm), small (2–20 μm), fine (2–0.4 μm), and very fine (<0.4 μm), were separated by sieving and centrifugation (Chiu et al. 2006). All fractionated samples were freeze-dried and stored for further analyses. Subsamples of each soil fractions were used to analyze total organic carbon (TOC) and total nitrogen (TN) using an elemental analyzer (NA1500 Series 2, Fisons, Italy).
Because of the complexity of separating soil samples to different particle-size fractions, only one set of soil particle-size fractions for each site was prepared for analyzing its 13C NMR.
13C NMR spectroscopy
The C functional groups of whole soils and particle-size fractions were examined by solid-state CP-MAS 13C NMR spectroscopy (Bruker DSX 400 MHz solid-state NMR, Germany). About 500 mg of the whole soils and particle-size fraction samples from each vegetation zone was used for each analysis. Acquisition conditions were spectrometer frequency, 100.46 MHz; spectra width, 20 kHz; spinning speed, 7 kHz; contact time, 6 ms; pulse delay time, 1 s; number of scans, 5000; spectra plotted region, 0–200 ppm.
Experimental procedures basically followed Jien et al. (2011). Briefly, the C functional groups were divided into 4 categories based on their chemical-shift areas: 0–50 ppm (alkyl-C), 50–110 ppm (O-alkyl-C), 110–165 ppm (aromatic-C), and 165–190 ppm (carboxyl-C). Each C functional group was determined by integrating the signal intensity of the designated spectrum ranges from 13C NMR. Aromaticity was calculated as the ratio of aromatic-C to the sum of alkyl-C, O-alkyl-C and aromatic-C (Hatcher et al. 1981).
Statistical analysis
Because only one whole soil sample was obtained from each vegetation type, the degree of freedom of the data was not enough to process statistical analysis.
While for the particle size fractions, effects of difference of TOC, TN, C/N ratio, alkyl-C, O-alkyl-C, aromatic-C, carboxyl-C, aromaticity and humification degree on vegetation types and soil particle sizes were each analyzed by crossed two-way factorial analysis (3 vegetations × 6 particle sizes). Particle size was treated as a numerical variable and the average particle size was used to represent each fraction. When testing different variables on vegetations, different particle sizes were treated as random effect and vice versa. Tukey’s honestly significant difference (HSD) test and simple linear regression were respectively applied to further test the difference of each variable with different vegetations and with different particle sizes. Percentages of different C functional groups were compared with paired t test.
Because of the limited amounts of samples, P < 0.1 was considered statistically significant for all analyses.