Cell wall thickness has phylogenetically consistent effects on the photosynthetic nitrogen-use efficiency of terrestrial plants.

Leaf photosynthetic nitrogen-use efficiency (PNUE) diversified significantly among C species. To date, the morpho-physiological mechanisms and interrelationships shaping PNUE on an evolutionary time scale remain unclear. In this study, we assembled a comprehensive matrix of leaf morpho-anatomical and physiological traits for 679 C species, ranging from bryophytes to angiosperms, to comprehend the complexity of interrelationships underpinning PNUE variations. We discovered that leaf mass per area (LMA), mesophyll cell wall thickness (T ), Rubisco N allocation fraction (P ), and mesophyll conductance (g ) together explained 83% of PNUE variations, with P and g accounting for 65% of those variations. However, the P effects were species-dependent on g , meaning the contribution of P on PNUE was substantially significant in high-g species compared to low-g species. Standard major axis (SMA) and path analyses revealed a weak correlation between PNUE and LMA (r  = 0.1), while the SMA correlation for PNUE-T was robust (r  = 0.61). P was inversely related to T , paralleling the relationship between g and T , resulting in the internal CO drawdown being only weakly proportional to T . The coordination of P and g in relation to T constrains PNUE during the course of evolution.