Belowground carbon estimation

To compare C content of the steppe and the forest, eight quantitative soil pits (50 ×

50 cm) at each ecosystem were excavated in 2009 and 2010, according to a method

described by Hamburg (1984). In the steppe, vegetation cover and the thin litter layer was

removed before excavation. Three to five mineral soil samples were taken at depths of 0-

10, 10-20, 20-30, 30-50, 50-70 and 70-100 cm during excavation of each pit in the steppe

but samples from same depth and pit were composited before any analysis. Likewise,

several mineral soil samples were taken at depths of 0-10, 10-20, 20-30, 30-50 and 50-70

cm of each forest pit and were composited before any analysis. In the forest, coarse

woody debris and vegetation cover was removed from the surface before excavation.

Organic horizons (Oi, Oe, Oa) of the forest were also weighed and subsamples were

taken. Care was taken to limit error in our bulk density estimations by maintaining

straight sides for each pit and by measuring profile depths as precisely as possible.

During the excavations, rocks in the pit walls were removed and weighed whenever

possible. Larger rocks that could not be removed during the excavations were removed

and weighed afterwards. In two cases, the rocks were too large to be removed so

equivalent volume of rocks was used to estimate mass. The amount of rock material

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mineral soil were weighed separately. Subsamples of soil were weighed, air-dried in the

field, and re-weighed to determine soil moisture content.

Soil laboratory analyses:

Whole-soil bulk density of each depth interval was estimated using the total air-

dried weight of soil excavated from each depth interval. The soil samples were further

separated using a 2-mm sieve into coarse (2-10 mm) and < 2 mm fractions, and the

relative weight percentage of < 2 mm fraction was calculated to estimate air dried weight

of the < 2 mm fraction of a depth interval. Approximately 2 g subsamples of < 2 mm soil

fraction were weighed and dried at 105° for 24 hr to determine weight conversion from

air-dry to oven-dry weight. Bulk density of the < 2 mm fraction of any depth interval was

based on the estimated oven-dry weight of the < 2 mm fraction of that depth interval. For

further chemical analyses, < 2 mm soil samples were used.

Soil texture was determined by the hydrometer method, modified from Gee and Or

(1996). No pretreatment was applied to the samples. Approximately 30 g of < 2mm soil

fractions were dispersed with 5% w/v Na-hexametaphosphate (HMP) by shaking for 12

hr. The soil suspensions were transferred to sedimentation columns and manually

inverted end-over-end for 30 seconds prior to initiation of sedimentation. Hydrometer

readings were carried out at 1.5 hr and 24 hr to determine the clay fraction. After the

hydrometer reading, each soil suspension was wet sieved using a 50 µm sieve and rinsed

until no visible particles passed through the sieve. The texture classification was carried

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hydrometer data, and silt by difference, and using the USDA soil texture classification

scheme (Gee and Or 1996).

Soil pH was measured using 1:1 deionized water-to-soil ratio and an OAKTON®

Waterproof pH Meter (Thomas 1996). Exchangeable cations were determined by

displacement of cations with 1N NH4Cl. The extraction was carried out with 25:1

solution-to-soil ratio on an extraction machine for eight hours (Sumner and Miller 1996).

The extracted solution was analyzed for concentrations of K+, Ca2+, Mg2+, Na+ and Al+

by inductively-coupled plasma emission spectroscopy (Spectro Genesis, Mahwah, NJ).

Plant available phosphorus was analyzed using the method of Kuo (1996) and Tiessen et

al. (1984). Soil samples were extracted by shaking for 16 hours with 0.5 M NaHCO3

(60:1 solution-to-soil ratio). Before adding sodium bicarbonate to soil, its pH was brought

to pH=8 by adding 4 M NaOH. After extraction, the solution was centrifuged at 10000

rpm and -1 °C for 12 min. Due to excess Na, the supernatant was diluted before analysis

and concentrations of plant available P were determined using inductively coupled

plasma spectroscopy (Spectro Genesis, Mahwah, NJ).

Concentrations of organic C and total nitrogen were analyzed by dry combustion

method using an elemental analyzer (Carlo Erba NA 1500 C/N Analyzer and Costech

ECS 4010 CHNSO Analyzer). Prior research suggests that calcium carbonate was

leached out (Batkhishig 2006), and field tests with dilute HCl suggested no presence of

calcium carbonate. Therefore, we assumed that total C measurements reflected the

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Statistical analysis:

The element concentrations (μg g-1

) as well as the C and nitrogen percentage

concentrations were converted to content per meter square area (kg m-2 or g m-2) using

bulk density of the <2 mm soil fraction. Visual examination of the data suggested that

assumptions of the ANOVA analysis had been violated. Therefore, normality of the data

and error terms were tested using the Shapiro-Wilk’s test. Homogeneity of the variance

of the data and error terms were tested using O’Brien, Brown-Forsythe, Levene and

Bartlett’s tests. The majority of the data and error terms were neither normally distributed

nor in accordance with the variance homogeneity assumption. Hence, we transformed

data using Log10 and tested again for ANOVA assumptions. In a few cases, ANOVA

assumptions had been violated. In those conditions, Welch’s and Kruskal-Wallis’s tests,

instead of ANOVA, were used to test whether ecosystems differ in C and nutrient

content. When ANOVA assumptions were met, Log10 transformed data were tested using

a one-way ANOVA with ecosystems treated as a fixed factor for each profile depth. All

these analyses were carried out with JMP v8 (SAS Institute, Cary, NC).

4.2.3. Aboveground carbon estimation