Experimental design
In December 2020, when a growing stage of the current year was
completed, we selected tree A. nepalensis forest stands across a
wide range of tree age ranges. Our field experiments were conducted in
0.1 ha permanent plots in three age groups, i.e., the age group I: young
stage (5-8 years), age group II: mature stage (35- 40 years), and group
III: old stage (130-145 years) forest sites. All of the study sites were
separated by at least 500-800 m, and their topography (slope gradient,
slope aspect), micro-environments, and soil conditions were similar
(Table 1). The detailed information for the A. nepalensisindividuals in the different tree age classes is given in Table 1.
The leaf lifespan of A. nepalensis was between 9-10 months,
having leaflessness during the winter leaf age. Leaf budding starts
during spring (March to April), and leaf production completes in two to
four months. Leaf get fully expanded during the summer. We sampled five
healthy individuals of A. nepalensis of each age stage for a
total of 15 individuals. To measure the tree and leaf age-specific
photosynthetic physiology, we conducted gas exchange and potential water
measurements for all 15 individuals across the three-leaf age periods of
varying ambient moisture and temperature conditions. The investigation
dates for each sampling leaf age were 20-30 March 2021 (spring; leaf bud
burst), 20-30 June 2021 (early monsoon summer; fully expended leaf) and
20-30 October (fall; leaf senescence).
Area-based physiological traits e.g., photosynthetic rate
(Aarea ; µmol CO2 m− 2s − 1 ), stomatal conductance
(gswarea; mol H2O m− 2s − 1), transpiration rate (Earea; mol
H2O m− 2 s − 1) were
measured using an open-flow, portable measurement infrared gas analyzer
(IRGA) (Li-6800, Li-Cor, Lincoln, NE, USA) (Evans & Santiago 2014)
under ambient conditions and air temperature (T air,
°C), the leaf temperature (T leaf, °C) and
photosynthetic photon flux density (PPFD, µmol m− 2 s− 1) were recorded at each measurement by the IRGA
using a 6-cm2 chamber with red–blue light-emitting
diodes on normal cloudless days. To avoid the influence of fluctuating
environmental condition, Photosynthetically Active Radiation (PAR) was
set to 1200 µmol m− 2 s – 1, while
concentration CO2, temperature, and humidity was set as
per the ambient condition of the study site. Specific leaf area (SLA;
cm2 g-1) represents the inverse of
leaf mass area (LMA), and was calculated as the ratio of dry leaf area
and leaf mass (Poorter et al., 2009). Leaf area was measured by leaf
area meter (LI 3000C, LI-COR, Inc). Mass-based assimilation rate
(Amass; µmol CO2 m− 2s – 1); mass-based stomatal conductance
(gswmass; mol H2O m− 2s − 1), transpiration rate (Emass; mol
H2O m− 2 s − 1) were
calculated as Amass = A area × SLA; gswmass = gsw area × SLA and
Emass = Earea × SLA, respectively. Leaf
functional traits measured included specific leaf area (SLA;
cm2 g−1), total nitrogen (leaf N; g
kg-1), total phosphorus (leaf P; g
kg-1) concentrations, and total chlorophyll (Chl; mg
g-1) concentrations. For chemical analysis
(Nm and Pm mg nutrient
g-1), eight to ten leaf discs of definite area (1.60
cm2) were excised from the leaf (leaf without
petiole), dried at 64 °C to constant weight, and weighted for each
species. During the analysis, all samples were triplicated and averaged.
Leaf N, and leaf P concentrations were calculated by
K2Cr2O7-H2SO4oxidation, Kjeldahl method, and modifed
H2O2-H2SO4method (Rapp et al. 1999) respectively. The concentration of P were
determined at 725 nm using a spectrophotometer (UV-1800; Shimadzu Corp.,
Kyoto, Japan). Leaf chlorophyll concentration (mg g-1)
was measured on fresh leaf discs which were extracted by using 5 ml of
dimethylsulfoxide (DMSO), with three replicates for each tree and leaf
age. After the sample test tube was preheated to 64 °C in the water bath
for 4 hrs and sample tissues were decolorized, and cooled at room
temperature, the absorbance of the supernatant was measured using the
spectrophotometer (Shimadzu UV-1201, Kyoto, Japan). Chlorophyll a, and b
concentrations (mg g-1) were calculated using the
reading from 665 nm and 645 nm. Area-bases N and P concentrations and
total chlorophyll (Na, Pa mg
m-2, and µ g cm-2), were calculated
based on N, and P concentrations and multiplied by the specific leaf
area (i.e., Na and Pa =
Nm and Pm / SLA). Leaf N and P
concentrations were measured because they are important nutrient
elements for photosynthesis, namely RUBISCO and ATP. The photosynthetic
N – and P use efficiency were measured by calculating
nitrogen-phosphorus use efficiency (PNUE or PPUE=
Aarea/Narea or
Aarea/Parea µ mol CO2 N
and P s-1 g-1). Intrinsic water use
efficiency (WUEi; µ mol CO2 µ
mol-1 H2O) was measured as the ratio
of Aarea/gsw area and water use
efficiency (WUE; µ mol CO2 µ mol-1H2O) was derived as the ratio of Aarea/Earea (Farquhar & Sharkey 1982). We coupled leaf
physiological traits with the midday water potential on the same branch
on which leaf gas exchange experiments were conducted using a pressure
chamber (Model 1000, PMS Instrument, Corvallis, OR). We also measured
predawn water potential on each individual tree before leaf
physiological traits measurement.