2.2 Experimental design and sampling
After a reconnaissance survey, a series of A. nepalensis stands with different ages were selected. Selected forest stands were categorized into six age gradients namely alder-early regeneration (AER, 2-5-year-old) forest, alder-late regeneration (ALR, 7–9-year-old) forest, alder young-mixed (AYM, 20-25-year-old) forest, alder mixed mature oak (AMOM, 80-110-year-old) forest, alder mixed mature-rhododendron (AMR, 85-120-year-old forest) forest, and alder mixed old-oak (AOOM, 250-270-year-old) forest (Joshi & Garkoti 2021b). As the exact details of forest age were not present, we used A. nepalensis basal area (a proxy for tree age) and validated it by interviewing elderly local people who had information about the year of landslide and establishment of A. nepalensis . We further cross-checked it from the Forest Department.
We evaluated the ecosystem C:N:P stoichiometry and N-P stocks for the following ecosystem components: (i) aboveground components (bole, branch, twigs, and foliage) of trees, (ii) belowground components (fine root, stamp root, and lateral root) of trees species, (iii) aboveground and belowground components of shrubs (stem, leaf, root) (iv) above and belowground parts of herbaceous plants, (v) forest litter and (vi) soil including soil microbial biomass at different soil depths.
In each forest stand, three plots of 0.1 ha were established in August-October 2018. In each plot, ten stratified random quadrats of 10 m × 10 m sizes were laid to evaluate dendrometric attributes (basal area, diameter, density, and important value index (IVI)) and also vegetation biomass and N and P stocks. The biomass of different tree components was estimated using the allometric equations developed by previous workers for the species in the region (Supplementary Table 1). Allometric equations for A. nepalensis belowground tree components were not available. Therefore, we used interspecies allometric equations developed by Rawat & Singh (1988).
Understory (shrubs and herbs) biomass was estimated by using the harvest method (Singh & Yadava, 1974). Shrub biomass was estimated by laying three 5m × 5m quadrats, and the same number of 1m × 1m sized quadrats were established to evaluate the litter and herbaceous biomass in each plot (Garkoti & Singh, 1995; Joshi & Garkoti, 2021b). Shrubs were harvested and differentiated into the root, branches, and leaves; herbs were harvested and differentiated into belowground parts and aboveground.
Soil profile varied among the chronosequence of A. nepalensisforest stands. Five replicates of soil samples were collected with a stainless-steel corer (diameter, 5 cm) at the four corners and the center of the sampling plots up to the maximum soil depths (0–10 cm in AER, 0–30 cm in AYM, 0–50 cm in ALR, AMOM, AMR, and AOOM plots) and divided in 0–10, 10–20, 20–30, 30–50 cm and then mixed to form one homogenous sample per depth per plot.Each replicate of the soil sample was passed through a 2-mm mesh size sieve and divided into two parts (1) one part was stored at 4 °C for soil microbial biomass (i.e., soil microbial biomass carbon (MBC), soil microbial biomass nitrogen (MBN), and soil microbial biomass phosphorus (MBP) analysis and (2) second part was air-dried and used for chemical analysis. Soil bulk density of the different soil layers was measured by collecting five replicates of soil in each plot using the same stainless-steel corer as above.