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.