Introduction
Tree age may result in variation in leaf morphological, chemical, physiological traits and the ability to absorb nutrients. Incresing tree age can cause alterations in leaf morphological traits, vegetative/reproductive allocation, nutritional absorption, hormonal control, and environmental adaptability, all of which can lead to variation in tree structure and function (Thomas et al., 2002; Thomas 2011; Damián et al. 2018). All of these traits may induce the associated diffrances in the structure and function of the forest (Thomas 2010; Thomas 2011; Martin and Thomas 2013; Damián et al. 2018).
The leaf ecophysiological traits vary significantly with plant life history, light and nutrients availabilities (Reich et al., 1992; Ackerly et al. 2000; Han et al. 2020). Many studies  investigated the physiological traits (i.e. photosynthesis rate) appear to decline in tall trees, because of the limitation of hydraulic transponrt (Mencuccini and Grace 1996a). It has been objerved that hydraulic transport may reduced with tree age as a results of the longer path lenth from soil to stomata, which derive the reductions in stomatal conductance and photosynthetic rate that directly influence tree productivity (Ryan and Yoder 1997). The high values of leaf physiological traits represent acquisitive strategies (high productivity) for plants, and low values represent conservative strategies (low productivity) (Wright et al. 2005a; Gorne et al. 2020). Several investigations evaluated that younger tree have greater trait values related to resource acquisition, but mature and older have more conservative traits (Damián et al. 2018; Dayrell et al. 2018). During the forest chronosequence, plants shift from acquisitive physiological strategy to conservative physiological strategy (Han et al. 2020) to maintain the plant overall productivity at its optimum as per the metabolic need.
The leaf phenological cycle offers an intriguing system for investigating the interconnection between leaf gas exchange, water relations, and leaf functional traits with respect to leaf phenology (Fajardo, and Siefert 2016). Leaf physiological traits significantly change with phenological phases. During the growing leaf age, leaf physiological traits change as a result of the in seasonal environmental conditions and leaf age (Escudero et al. 2003; Chvaana et al. 2017; Chvaana et al. 2019). Leaf phenological cycle and leaf economic spectrum (i.e. leaf initiation, expansion, and senescence) varied among species, among individual plants, and also among leaves on a plant (Chabot and Hicks 1982; Reich et al. 1991; Mediavilla et al. 2014; Bai et al. 2015). Previous research has revealed that with leaf age, leaf mass area (LMA) rises while leaf nitrogen and phosphorus (N and P) are decline (Niinemets and Lukjanova 2003; Niinemets et al. 2006; Athokpam and Garkoti 2015). Previous workers also revealed the leaf and tree age-related decline in photosynthesis traits which could be associated with the change in leaf morphology and nutrient over time (Reich et al. 1991). Leaf N is positively correlated with the activity of Rubisco (ribulose-1,5-biphosphate carboxylase/oxygenase), and its concentration often decreased with tree and leaf age (Kitajima et al. 2002, Wright et al. 2006, Fajardo and Siefert 2016, Chavana-Bryant et al. 2019). Leaf P is involved in various metabolic activities during the photosynthesis processes and also decreases with leaf and tree age (Wright et al. 2006, Mediavilla et al. 2011, Chavana-Bryant et al. 2017, 2019). Likewise stomatal conductance and stomatal control (i.e, stomatal opening and closing) also decrease with leaf and tree age (Reich and Borchet 1988) and affect photosynthesis.
Water (H2O), N, and P are essential resources for plant survival, growth, and photosynthesis. Leaf physiological traits such as net photosynthetic capacity, leaf diffusive conductance, and transpiration rate are indicators of CO2 assimilation, resource use strategies, and water exchange. The water use efficiency (WUE) and photosynthetic N and P use efficiency (PNUE and PPUE) is an essential characteristic of different species and determines the leaf physiology, leaf economics, strategy, and competition expected to change with plant age and leaf age (Robinson et al. 2001; Wright et al. 2004; Nabeshima and Hiura 2004; Bai et al. 2015). The WUE, PNUE and PPUE are excellent ecological indicator of species performance in different environmental conditions expected to change with palnt age, and leaf age (Funk and Vitousek 2007). The PNUE and WUE describe the N concentration per unit leaf area and the amount of water transpired, respectively, for a given rate of photosynthesis. At the leaf level, WUE is the ratio between net CO2 assimilation and water loss via transpiration, and intrinsic water-use efficiency (WUEi) is the quick ratio between net CO2 assimilation and stomatal conductance. The PNUE, PPUE, and WUE predict how the photosynthetic assimilation is optimized per unit of N, P, and water in leaves (e.g., Poorter and Evans 1998; Castellanos et al. 2005; Sheng et al. 2011). Thus, leaf phenological cycle and resource allocation strategies are physiologically related (Ackerly and Bazzaz 1995; Hikosaka 2005), making leaf lifespan a crucial characteristic controlling plant carbon and nutrient economies, which eventually result in adaptive modification in response to ecological heterogeneity.
Nitrogen-fixing Alnus nepalensis (D. Don) is one of the fast-growing early successional tree species that often forms pure stands in areas affected by landslide/ slip stages over 1400 m.a.s.l. but also occurred in a forest chronosequence in the central Himalaya (Joshi and Garkoti 2021b). A. nepalensis is an important economic and reforestation tree species in central Himalaya. Previous research has demonstrated that A. nepalensis is an extremely important for soil restoration and degraded forest management (Joshi and Garkoti 2021b). Most studies of A. nepalensis have focused on ecosystem carbon dynamics (Joshi and Garkoti 2021b), soil physicochemical properties, below ground biomass and litter dynamics (Joshi and Garkoti 2020; Joshi and Garkoti 2021a). However, it is still not understood how the leaves of A. nepalensis response (photosynthetically, chemically and morphologically) along with the leaf age, and tree age. Therefore, we investigated the effect of leaf age, and tree age on leaf ecophysiology traits in A. nepalensis . In this study, we proposed two questions 1) how do the leaf ecophysiological and morphological traits change with leaf and plant age? We expect a shift from a more acquisitive physiological strategy in the young stage to a conservative physiological strategy in the older stage, and 2) A resource conservation strategy adopted by the older stage may have high WUE, tough leaf construction, and low leaf N and P concentration, low photosynthetic N-, and P-use efficiency and low rates of carbon assimilation when compared to the young stage.
Material and Methods