Tight coupling between leaf δ¹³ C and N content along leaf ageing in the N₂ -fixing legume tree black locust (Robinia...

N₂ -fixing legumes can strongly affect ecosystem functions by supplying nitrogen (N) and improving the carbon-fixing capacity of vegetation. Still, the question of how their leaf-level N status and carbon metabolism are coordinated along leaf ageing remains unexplored. Leaf tissue carbon isotopic composition (δ¹³ C) provides a useful indicator of time-integrated intrinsic water use efficiency (WUEi). Here, we quantified the seasonal changes of leaf δ¹³ C, N content on a mass and area basis (N_mass , N_area , respectively), Δ¹⁸ O (leaf ¹⁸ O enrichment above source water, a proxy of time-integrated stomatal conductance) and morphological traits in an emblematic N₂ -fixing legume tree, the black locust (Robinia pseudoacacia L.), at a subtropical site in Southwest China. We also measured xylem, soil and rainwater isotopes (δ¹⁸ O, δ² H) to characterize tree water uptake patterns. Xylem water isotopic data reveal that black locust primarily used shallow soil water in this humid habitat. Black locust exhibited a decreasing δ¹³ C along leaf ageing, which was largely driven by decreasing leaf N_mass , despite roughly constant N_area . In contrast, the decreasing δ¹³ C along leaf ageing was largely uncoupled from parallel increases in Δ¹⁸ O and leaf thickness. Leaf N content is used as a proxy of leaf photosynthetic capacity; thus, it plays a key role in determining the seasonality in δ¹³ C, whereas the roles of stomatal conductance and leaf morphology are minor. Black locust leaves can effectively adjust to changing environmental conditions along leaf ageing through LMA increases and moderate stomatal conductance reduction while maintaining constant N_area to optimize photosynthesis and carbon assimilation, despite declining leaf N_mass and δ¹³ C.