Dynamics of biomass and carbon sequestration across a chronosequence of Caragana intermedia plantations on alpine sandy land

Considering the variations in carbon concentrations among different plant components can significantly improve carbon storage estimates. The aim of this study was to estimate the biomass and carbon storage and sequestration in Caragana intermedia Kuang et H. C. Fu plantations for six different ages. The biomass and carbon sequestration in shrub biomass were quantified using a destructive method that involved analysing the carbon concentrations in the leaves, branches, stem bark, stem wood, roots > 5 mm, 2 mm < roots ≤ 5 mm, and roots ≤ 2 mm. The biomass and biomass carbon density of the C. intermedia plantations increased with the age of the stands. The biomass carbon density of the woody components was at its maximum in the 30-year-old plantation (14.27 ± 2.71 t·ha−1), indicating that C. intermedia plantations in alpine sandy land are an important carbon pool. The carbon accumulation rate of the woody components was higher during the early stages of plantation development. The carbon concentrations differed significantly among the components but changed only slightly with the stand age. The weighted mean carbon concentration of the woody components, which was found to be 44%, must be considered when estimating the long-term carbon pools in C. intermedia plantations.

forests and grasslands 21 . Tao and Zhang 22 reported that the vegetation types of temperate semi-shrubs and dwarf semi-shrubs exhibited the highest carbon storage capacities, and the average biomass carbon density of arid regions in Central Asia is 1.837 t·ha −1 . China has been severely affected by desertification, and the desertified land area accounts for 45% of the total territory 23 . Revegetation is an effective way to combat and control desertification 24 , and shrub species have been widely favoured in vegetation restoration efforts due to their tolerance of harsh environments 25,26 . In the context of global climate warming, studies on the carbon fixation function of vegetation in desert areas are very important 27 , especially in terms of carbon sequestration by shrubs after vegetation restoration 23 .
Most leguminous shrubs in the genus Caragana have strong drought resistance capabilities and fast growth, and they are important xerophytic shrub species for windbreaks, sand fixation, and soil and water conservation. For example, Caragana (Lam.) was intentionally introduced to the northern Great Plains of North America for erosion control 28 . Caragana microphylla Lam. is a pioneer leguminous shrub species for vegetation re-establishment that is widely distributed throughout the semi-fixed and fixed sandy lands of the Horqin region 29 . Caragana jubata (Pall.) thrives well under the extreme cold of the high altitudes of the Himalayas, and hence, this plant is expected to be a source of genes that might play an important role in tolerating low temperatures 30 . Caragana intermedia Kuang et H. C. Fu is a native desert shrub that is widespread in the desert land of western and north-western China 31 . The typical distribution range of C. intermedia is in the desert steppe area of the Mongolia Plateau, the typical steppe area and desert steppe area of the Erdos Plateau, and the typical steppe area and steppe area of the North Loess Plateau, forming a common C. intermedia sandy land shrub community 32 .
The Gonghe Basin of the Tibetan Plateau is one of the regions that has been severely affected by desertification, and the desertified area accounts for 91% of the area of the whole basin 33 . The establishment of sand barrier enclosures and the planting of shrubs on shifting sand dunes are effective methods for combating and controlling desertification in this region 14,34 . Large shrub plantations have been established with the help of straw checkerboards since 1958 33 . C. intermedia was widely planted on shifting sand dunes after the erection of the straw checkerboards due to its tolerance to drought, cold, and poor habitats and for its rapid growth. For example, some studies of C. intermedia plantations in this region have showed that C. intermedia development influences the chemical and physical properties of soil 26,35,36 ; it can increase water use efficiency to resist drought stress 37 , and the primary water sources are affected by its root distribution 38 . In addition, the root system distributions of the C. intermedia plantations showed that the absorptive roots (diameter ≤ 1 mm) of 5-, 9-and 25-year-old plantations are primarily distributed in the 10-30 cm, 10-50 cm and 10-60 cm soil layers, respectively, whereas the conductive roots (diameter >1 mm) primarily appear in the 10-50 cm, 10-60 cm and 10-90 cm soil layers, respectively 39 . However, there is little information on the biomass accumulation and carbon sequestration capacity of C. intermedia plantations, and the effects of these functions on the carbon sequestration capacity of shrubland should not be neglected. Therefore, a better understanding of the biomass carbon density and the carbon sequestration capacity of C. intermedia plantations could be helpful for improving the precision of shrubland carbon sequestration evaluations, and this information is also important for the sustainable management of sand-fixing plantations.
Therefore, we performed a sampling campaign of different shrub ages on sand dunes in C. intermedia plantations to test three general hypotheses as follows: (1) the carbon sequestration of C. intermedia plantations would increase as the stand aged, and the carbon accumulation rate would decrease as the stand aged; (2) there is no obvious change in carbon concentrations in the shrub components with the stand age; and (3) using the carbon fraction of 50% for converting C. intermedia biomass into carbon will overestimate the carbon stocks.

Results
Biomass and its distribution into components. The component, aboveground, belowground and total biomass for the C. intermedia plantation stands significantly differ with the plant age (P < 0.05) ( Table 1). The branch, stem wood, roots > 5 mm, 2 mm < roots ≤ 5 mm, belowground and total biomass showed the same trend with an increase in the stand age following the natural order of increases of 3-year-old stand < 5-year-old stand < 8-, 10-, and 16-year-old stands < 30-year-old stand, but there was no consistent difference among the 8-, 10-and 16-year-old stands. The aboveground biomass for the 16-year-old stand was only slightly higher than that recorded for the 3-year-old stand and lower than that for stands of other ages; however, the belowground biomass for the 16-year-old stand was slightly lower than that recorded for the 30-year-old stand and higher than that for stands of other ages. The woody component biomass (leafless) of the C. intermedia plantations increased as the stand aged, peaking in the 30-year-old plantation at 32.26 ± 6.15 t·ha −1 .
The proportion of leaf biomass decreased and the proportion of the biomass of woody components increased as the stand age increased (Fig. 1). The proportions of branch, stem bark and stem wood biomass showed the same increasing trend with the stand age in the 3-, 5-, 8-, and 10-year-old stands, declining in the 16-year-old stand, and increasing again in the 30-year-old stand. The root biomass proportion showed the opposite trend, declining with the stand age in the 3-, 5-, 8-, and 10-year-old stands, increasing in the 16-year-old stand, and declining again in the 30-year-old stand. The percentage of biomass in woody components that accounted for the total shrub biomass of all the stand age plantations ranged from 83-94%, and the average percentage was 88 ± 2%. The percentage of belowground biomass that accounted for the biomass of woody components of all ages of stand plantations ranged from 57-77%, and the average percentage was 66 ± 3%.
Carbon concentrations. The carbon concentrations of the C. intermedia components in the stands of the same age were significantly different (P < 0.05) ( Table 2). The carbon concentrations in the components did not change significantly with the stand age. The carbon concentrations of the C. intermedia components ranged from 41% to 46%, and the weighted average carbon concentration of the wood components for the six stand ages was Biomass carbon density and its distribution in different components. The component, aboveground, belowground and total biomass carbon densities for the C. intermedia plantation stands of different ages were significantly different (P < 0.05) ( Table 3). The branch, stem wood, roots > 5 mm, 2 mm < roots ≤ 5 mm, belowground and total biomass carbon densities showed the same trend as the stand age increased, with 3-year-old stand < 5-year-old stand < 8-, 10-, and 16-year-old stands < 30-year-old stand, but there was no consistent difference among the 8-, 10-and 16-year-old stands. The aboveground biomass carbon density for the 16-year-old   stand was only slightly higher than that found for the 3-year stand and lower than that found for the stands of other ages. The belowground biomass carbon density for the 16-year-old stand was slightly lower than that recorded for the 30-year-old stand and higher than that for the stands of other ages. The biomass carbon density for the woody components of C. intermedia plantations increased as the stand aged, peaking in the 30-year-old plantation at 14.27 ± 2.71 t·ha −1 .
The proportion of biomass carbon density in the leaves decreased and it increased in the woody components as the stand age increased (Fig. 2). The proportions of branch, stem bark and stem wood biomass carbon density showed the same trend as the stand age increased, increasing in the 3-, 5-, 8-, and 10-year-old stands, declining in the 16-year-old stand, and increasing again in the 30-year-old stand. The proportion of the root biomass carbon density showed the opposite pattern, declining in the 3-, 5-, 8-, and 10-year-old stands, increasing in the 16-year-old stand, and declining again in the 30-year-old stand. The percentage of biomass carbon density in woody components that accounted for the total shrub biomass carbon density of all ages of stand plantations ranged from 82-94%, and the average percentage was 88 ± 2%. The percentage of belowground biomass carbon density that accounted for the biomass carbon density of woody components of all the stand age plantations ranged from 57-78%, and the average percentage was 67 ± 3%.

Discussion
The biomass quantification of woody plants has been recognized as a crucial step in the calculation of forest biomass and carbon stocks 40 . In our results, the biomass and carbon density of the measured C. intermedia shrubs increased as the stand age increased, which indicates that shrubs continuously absorb CO 2 during the photosynthesis and subsequently store the resulting carbohydrates in their biomass. Therefore, the stand age has positive impacts on the forest ecosystem and nutrient cycling 10,41 . However, each species has its own lifespan, and the biomass and carbon sequestration capacity peak in the mature stage and decrease afterwards 42 . In this study, the   In the arid zone of India, the carbon density of Calligonum polygonoides L. ranged between 0.37 and 1.84 t·ha −1 44 , which was obviously lower than it was in our study. The 16-year-old stand showed a greater root biomass proportion (77%), which might be attributed to the accelerated development of root systems reaching its maximum by this age, and further studies are required to confirm the findings. Osone and Tateno reported that plants allocate more biomass to underground organs to absorb more soil moisture or nutrients when water or nutrients are the primary limiting factors 45 . In our study, the average percentages of the root biomass and the biomass carbon density of the total values for the woody components were 66 ± 3% and 67 ± 3%, respectively, demonstrating that the C. intermedia roots were the dominant organs. This result probably occurred because C. intermedia needs to produce more roots to absorb water and nutrients to survive in barren alpine sandy land. Deng et al. reported that the ratio of roots to the total biomass of C. korshinskii plantations on the Loess Plateau was higher during the mature and post-mature stages, at 58.7% 42  The carbon accumulation rate of woody components was higher during the early stages of plantation development, and the 3-year-old stand reached 1.17 t·ha −1 ·yr −1 , which is consistent with the results of Dixon et al., who reported that young forests take up carbon dioxide at relatively high rates 1 . The study about the biomass carbon sequestration of a C. korshinskii plantation on the Loess Plateau also found that the annual rate of biomass carbon accumulation decreased with the plantation development and that of young plantations reached 0.76 Mg·ha −1 ·yr −1 42 . Francis Justine et al. also found that changes in the biomass accumulation rate of Pinus massoniana decreased significantly with the forest age and that the highest accumulation rate occurred during the early stages of forest growth 10 .
The weighted average carbon concentration of the wood components was 44%, and this result is supported by those of Fonseca et al., who reported carbon fractions in the range of 0.40 to 0.45 11 . These results clearly show that the assumed carbon concentration of 50% 6,7 for the components was generally higher than the measured carbon concentrations in the C. intermedia plantations and may result in an approximate 13% overestimation of the carbon sequestration of C. intermedia plantations. The high variation in the carbon concentrations among components from a single species may be affected by a range of factors, such as the sample chemical composition and origin and the selected analytical methods 47 . However, the carbon concentrations did not differ significantly with the stand age, which is consistent with the assumption that the ability of wood to store carbon in mature stands is the same as that in young stands 10 . Our findings are consistent with those of previous studies conducted by Maiti et al., who found that the carbon concentration differed significantly among tree organs, while there was no significant difference among the ages 48 .
In summary, the biomass and biomass carbon density of C. intermedia plantations increased as the stand age increased, and the 30-year-old plantation showed the highest biomass carbon stock, although the carbon accumulation rate peaked in younger, 3-to 8-year-old stands. The biomass carbon density of woody components from C. intermedia plantations reached 14.27 ± 2.71 t·ha −1 , which shows that the C. intermedia plantations in alpine sandy land are an important carbon pool. The carbon accumulation rate of wood components was higher during the early stages of plantation development, and the 3-year-old stand reached 1.17 t·ha −1 ·yr −1 . The roots presented the primary biomass and biomass carbon stocks. The carbon concentrations of the C. intermedia components in stands of the same age were significantly different, whereas the carbon concentrations in the components hardly changed with the stand age. Therefore, measuring the carbon concentrations once in the components of C. intermedia would suffice for its entire lifetime when estimating the plantation carbon sequestration. Using a constant value of 50% for the carbon concentration in C. intermedia biomass may result in an approximate 13% overestimation of the carbon sequestration of C. intermedia plantations. Therefore, the weighted mean carbon concentration of the wood components of 44% rather than the generic value of 50% must be considered for long-term carbon pool estimations in C. intermedia plantations.

Materials and Methods
Study site. The study was carried out at the desertification-combating experimental site of the Qinghai Gonghe Desert Ecosystem Research Station (99°45′-100°30′E, 36°03′-36°40′N and altitude 2871 m), and the total area of the experimental site is 1094 ha. The station is located in the Gonghe Basin of the north-eastern Tibetan Plateau, and it is a part of the Chinese Desert Ecosystem Research Network (CDERN) of the State Forestry Administration of P.R. China. The station was constructed by the Chinese Academy of Forestry and the Desertification Combating Station of Qinghai Province. The mean annual temperature is 2.4 °C, the mean annual precipitation is 246.3 mm, and the mean annual potential evaporation is 1716.7 mm. The mean annual number of windy days is 50.6 days, and the mean annual number of sandstorm days is 20.7 days. The mean annual wind speed is 2.7 m·s −1 , and the maximum wind speed reaches 40 m·s −1 . Chestnut soil and brown soil are the zonal soils in the study area, while aeolian, meadow and bog soils are the azonal soils. The primary vegetation type is sand-fixing plantation, which includes the tree species Populus simonii Carr. and Populus cathayana Rehd. and the shrub species C. intermedia, Caragana korshinskii Kom., Salix cheilophila Schneid., Hippophae rhamnoides L., and Salix psammophila C. Wang et Chang Y. Yang 27,31 . Zhang et al. reported that C. intermedia is a variant of C. korshinskii 44 . In the study area, the morphological characteristics of these two species are noticeably different, and C. intermedia plantations are distributed more widely; therefore, they were selected for this study.
Experimental design and sampling. Our study was conducted in mid-August 2016, when the biomass of the C. intermedia stands peaked. All the C. intermedia plantations in the study area were sown. Therefore, each shrub grows from multiple seeds. In addition, C. intermedia has strongly asexual germination, resulting in multiple stems, and all the stems from different seeds grow in a staggered fashion. Three representative plots (10 × 10 m) with slopes within 15° were established in 3-, 5-, 8-, 10-, 16-and 30-year-old mono-species plantations of C. intermedia as the sampling sites (Fig. 3). The plots were more than 50 m apart. The areas of the 3-, 5-, 8-, 10-, 16-and 30-year-old stands are 8.71, 5.72, 5.65, 4.82, 5.67 and 20.91 ha, respectively. In all the plantations, straw checkerboards (1 × 1 m) were established on shifting sand dunes for sand fixation and then sowed with C. intermedia. In each plot, the morphological characteristics of 10 plants were measured, including the maximum height (H), basal diameter, crown diameter (maximum diameter of the shrub canopy in the northeast-southwest (C NE-SW ) and northwest-southeast (C NW-SE ) directions), and ground diameter (the maximum diameter of the area shrub shoots emerged at ground level in the northeast-southwest (G NE-SW ) and northwest-southeast (G NW-SE ) directions). Caragana intermedia is a shrub with multiple stems; thus, the stems were categorized into three diameter classes (maximum, medium and minimum), and a representative stem basal diameter was measured first to calculate a mean value of each shrub's basal diameter (D). One standard shrub was selected from each plot according to the mean morphological characteristics for different C. intermedia plantation ages, for a total of 18 plants harvested for biomass measurements. The morphological characteristics of the standard shrubs are shown in Table 4.
For the 3-, 5-and 8-year-old C. intermedia plantations, C. intermedia was sown in lines; therefore, we regarded a C. intermedia line in one straw checkerboard (1 × 1 m) as one standard shrub when we conducted experiments. Each harvested shrub was separated into the leaves, branches, stem bark, stem wood, and roots with diameters > 5 mm (roots > 5 mm), roots with diameters between 2 mm and 5 mm (2 mm < roots ≤ 5 mm) and roots with diameters ≤ 2 mm (roots ≤ 2 mm). Root samples were obtained by the total excavation of the standard shrubs extending out from the shrub centre to the middle of the lines and plants (Fig. 4). Therefore, the sampling area for the 3-, 5-, 8-and 10-year-old plantations was 1 m 2 , and the area for the 16-and 30-year-old plantations was 2 m 2 . The soil samples were sieved through a 0.6-mm sieve to obtain the roots. The sampling depth was 80 cm, at which point no more roots were visible. The fresh weights of all the components were measured in situ, and samples of every component from each standard shrub were oven-dried at 65 °C for 48 h and weighed to determine the ratio of fresh weight to dry biomass.

Carbon concentration analysis.
Dried samples of the different shrub components in each plantation were ground and sieved through a 100-mesh sieve, and the carbon concentrations were quantified using the induction furnace method with a CHNOS elemental analyser.
Biomass carbon density and carbon accumulation rate calculation. The following equations were used to calculate the shrub biomass carbon density: where B is the shrub biomass density (t·ha −1 ), B it is the i component biomass of the whole standard shrub (g), A is the sampling area (m 2 ), C is the biomass carbon density (t·ha −1 ), B i is the i component biomass density (t·ha −1 ), and C i is the i component biomass carbon concentration (%).
The following equation was used to calculate the shrub carbon accumulation rate: where C r is the shrub carbon accumulation rate (t·ha −1 ·yr −1 ) and Y is the stand age (yr).

Data analysis.
All the results are reported as the means (three measurements) ± standard error (SE). A one-way ANOVA was used to compare the biomass, carbon concentration, biomass carbon density and annual carbon sequestration among stands of different ages, and Duncan's multiple range test was used to evaluate the differences among the means. The SPSS ver. 16.0 software was used for all the statistical analyses.
Data Availability. The datasets analysed during the current study are available from the corresponding author upon reasonable request.  Table 4. The morphological characteristics of C. intermedia plantation stands of different ages. C NE-SW , crown diameter in the northeast-southwest direction; C NW-SE, crown diameter in the northwest-southeast direction; G NE-SW , ground diameter in the northeast-southwest direction; G NW-SE , ground diameter in the northwestsoutheast direction. The values are the means ± SE (n = 3).