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 3 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 3 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 cwm ), Rubisco N allocation fraction (P R ), and mesophyll conductance (g m ) together explained 83% of PNUE variations, with P R and g m accounting for 65% of those variations. However, the P R effects were species-dependent on g m , meaning the contribution of P R on PNUE was substantially significant in high-g m species compared to low-g m species. Standard major axis (SMA) and path analyses revealed a weak correlation between PNUE and LMA (r 2  = 0.1), while the SMA correlation for PNUE-T cwm was robust (r 2  = 0.61). P R was inversely related to T cwm , paralleling the relationship between g m and T cwm , resulting in the internal CO 2 drawdown being only weakly proportional to T cwm . The coordination of P R and g m in relation to T cwm constrains PNUE during the course of evolution.