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.