Relationship between leaf traits
In the present study, there was a positive correlation between Emass, gswmass, PNUE, and PPUE with Amass; Earea, gswareawith Aarea; and Earea with gswarea. There was a negative correlation between WUEi and WUE with Aarea; gwsarea with WUEi and Earea with WUE in all three tree age stages (Fig.4). Intrinsic water use efficiency (WUEi) and water use efficiency were significantly negatively correlated with PNUE in all three tree age stages. There was a positive correlation between midday water potential (Ψmd) with Aarea, Earea and gswarea (Fig.5). The specific leaf area (SLA) was positively correlated with Amass, Emass, PNUE, and PPUE in all three tree age stages but negatively related to N concentration per unit leaf mass across all three tree age stages. In all three tree stages, the PNUE and PPUE were negatively correlated with N and P concentration per unit of leaf mass. The total chlorophyll concentration was positively correlated with Aarea (Fig.6).
DiscussionVariation in leaf ecophysiological traits along with the leaf age and tree age
Our first objective was to understand how key ecophysiological traits changing from resourse acquisition to resource conservation with plant age and leaf age. As expected, we noticed a shift toword resource-conservatite traits with tree age. Thus, our findings reinforce the idea that older tree have more conservative traits (Manson et al. 2013; Damián et al. 2018; Dayrell et al. 2018; Funk et al. 2021). Older tree had higher SLA, lower leaf Nmass and reduced photosynthetic traits compared with young trees. The modifications in photosynthetic rate were strongly dependent on E, gsw, PNUE, WUE, and SLA (Fig.4). Our results demonstrated that leaf age and tree age had a significant effect on gsw, which may be due the tree age-related changes in hydraulic conductance and differences in Ψleaf. The lower gsw of old tree than young and mature trees would be constant with the hydraulic limitation hypothesis and the influence of the gravitational hydrostatic gradient (Ryan and Yoder 1997). It is quite well recognised that stomatal closure is linked to decreased soil to leaf hydraulic conductance and variations in Ψleaf(Hubbard et al. 1999; Kolb and Stone 2000). As a result, plant strategies gradually shifted from more resource-acquisitive to more resource-conservative (Guariguata and Ostertag, 2001). This was also reflected in higher leaf chlorophyll concentrations and greater PNUE and PPUE in the young tree, which triggered a shift of leaf traits towards faster growth strategies involving greater photosynthesis and light capture to enhance the growth rate.
At the leaf scale, leaf age-driven differences in chemical and morphological traits have contributed significantly to the change in physiological traits along with the leaf age. The total chlorophyll, Nmass, and Pmass was higher in young tree which supports the higher growth rate, and productivity. This is because the combination of optimum morphological and chemical traits with more favorable environmental factors (light and temperature) of fully expanded leaf posed a positive impact on nutrient and water cycling. This supported optimum productivity during the summer season when the leaf was fully expanded than the leaf bud burst (spring) and leaf senescence stage (autumn). Previous studies also suggested the similar leaf age trends of the physiological traits related to leaf age; they showed peak values during fully expanded leaf (Wilson et al. 2000, 2001; Bauerle et al. 2004; Grassi et al. 2005). In the summer, day length was longer, highest photosynthetically active radiation, optimum air temperature and higher leaf Nmass, and Pmass combined to highest physiological traits (Grassi et al. 2005, Hikosaka et al. 2007, Wright et al. 2006). Leaf nitrogen and chlorophyll dropped to their lowest level in the leaf senescence stage due to the nutrient remobilization process after leaf senescence, associated with lowest physiological traits. Such leaf age and tree age-dependent decline in leaf morphological and chemical traits are significantly associated with changes in leaf physiological traits over time (i.e., photosynthetic rate) (Reich et al. 1991). In addition, the nutrient resorption process at the time of leaf senescence negatively affected the overall productivity during the leaf senescence stage (Crous et al. 2019). Photosynthetic rate in fully expanded leaf was significantly associated with other physiological traits including transpiration rate, stomatal conductance, water potential, and photosynthetic nutrient-use efficiency (Reich et al. 1998; Wright et al. 2005b). This indicated that, at a given photosynthesis rate, transpiration rate, stomatal conductance, and photosynthesis nutrient use efficiency were higher in the young stage than mature and old stages. The tight connection between the levels of photosynthesis, chlorophyll, and Nmass reflected contribution of nitrogen to the Calvin-Benson cycle enzyme (in particular, ribulose-1,5-biphosphate carboxylase/oxygenase—Rubisco) and chlorophyll for better light harvesting. The high chlorophyll concentrations in the young stage indicated supported the high light-harvesting and higher carbon assimilation per unit leaf area.
Acquisitive vs. conservative resource-use strategy
In the present study, we found a significant PNUE-WUE trade-off for the young, mature, and old stage. Young stage achieved higher PNUE and lower WUE, whereas mature and old trees followed the reverse trend. The trade-off between WUE and PNUE may explain the greater rates of physiological traits in young stage. The PNUE for the young stage was 12 % higher than the mature stage and 18 % higher than the old stage. Young stage exhibited higher water potential suggesting a trade-off between leaves with greater photosynthetic rates in young stage and leave are water-stressed (low water potential) in the old stage. The PNUE and PPUE were higher in the young stage, favoring low investment and quick return resource strategy, than mature and old stages, which favored slow resource return strategy (Wright et al. 2004; Reich and Flores- Moreno 2017). In the present study, the young tree showed greater N assimilation efficiency and more focused allocation of N to chlorophyll tissues than other non-photosynthetic tissue to ensure optimum growth. This was supported by the morphological traits, as SLA showed comparatively lower values in the young stage than the older one (Abdul and Mencuccini 2009), to reduce the recourse investment in non-photosynthetic tissues in leaves of young stage. The trade-off N partitioning between photosynthetic and non-photosynthetic tissues in varying tree age has also been reported by previous studies (Hikosaka and Hirose, 2000; Hikosaka, 2004). In the present study, the majority of mass based-physiological traits (i.e Amass, Emass, and gswmass) and photosynthesis nutrient-use efficiency, showed a positive correlation with SLA, which is similar to many previous studies (Wright et al. 2005b; Bahar et al. 2017; Crous et al. 2017). Onoda et al. (2017) also revealed that leaves with greater SLA tend to enhance Amass, Emass, and PNUE to support fast growth. Our results indicated that young trees allocate more nutrients to photosynthetic tissues to support their rapid growth than mature and old trees. This reveals the differences between leaf size and weight (Shipley et al. 2006). The old trees had higher WUE than young and mature trees, leading to significantly lower physiological traits. In general, the high WUE indicates a more conservative resource use pattern (Lambers et al. 1998). The midday water potential (Ψmd) was more negative (indicating higher potential stress) during the summer in old tree, because of the enhanced water supply pressure and higher hydraulic pressure. Consequently, to adapt to the pressure and balance of demand and supply, the old tree acquires the more conservative approach to shift toward higher WUE.
Ecosystem implication
Leaf age and tree age leads to adaptive changes in ecophysiological process, such as variation in water relation, gas exchange, and growth rate among varying tree age stages (Sala et al., 2010). Physiological responses, including evolution and adaptation to environmental shifts, characterize phenotypic plasticity, which is believed to be the dominant underlying process with implications for ecological processes (Hovenden and Vander Schoor, 2003; Thomas, 2011). A better comprehension of the physiological traits of major native trees is required for introducing resilient forest management strategies to minimize the expected effects of climatic change on plant development and water stress. The stage-specific physiological traits of A. nepalensis appear to have important for the ecosystem process. The WUE, PNUE, and PPUE apparently play an important regulatory role in the functioning ofA. nepalensis -dominated ecosystem. The PNUE and PPUE was significantly greater in the young stage, providing additional evidence that A. nepalensis has developed a mechanism for efficient use of N and P nutrient and the modifications in physiological traits among theA. nepalensis dominated forest stages accordingly. Ishida et al. (2005) have also explained the similar ontogenetic morphological, anatomical, and chemical modifications leading to evolution and adaptation in leaf physiology along the age gradient for the pioneer tree species, Macaranga gigantean . All the stage-specific changes of physiological traits of A. nepalensis had important implications with respect to nutrient cycling, carbon assimilation, and feedback interaction between leaf, stand, and ecosystem-level process of degraded forests in central Himalaya.
Conclusion
In conclusion, our findings indicate that most of the physiological traits of A. nepalensis decreased with a natural age gradient, indicating that the ecological strategy of A. nepalensis changed from a resource acquisitive approach to conservative resource approach with a change in age gradient with time. The relationship between the leaf traits and the structural and chemical traits changed during the age gradients, indicating different trade-off strategies across the age gradient. High photosynthetic nitrogen- and phosphorus use efficiency in the young stage could support the rapid growth of A. nepalensis . Specific leaf area and total chlorophyll concentration strongly influenced most of the physiological traits, being one of the vital regulators. Our results contributed to a more dynamic understanding of the relationship between leaf physiological traits and their interaction with leaf morphological and chemical traits. We also anticipated ecophysiological response to varying age gradients through the A. nepalensis forest stands. Additional studies are needed to understand the interaction with soil physicochemical properties and soil moisture concentration along the age gradient in the A. nepalensis forest stand in central Himalaya.