The Montgomery-Koyama-Smith (MKS) equation predicts that total leaf area per shoot is proportional to the product of the sum of individual leaf widths and maximum individual leaf length, which has been validated for some herbaceous and woody plants. The equation is also predicted to be valid in describing the relationship between the total stomatal area per micrograph (A_T) and the product of the sum of individual stomatal widths (denoted as L_KS) and maximum individual stomatal length (denoted by W_KS) in any particular micrograph. To test the validity of the MKS equation, 69,931 stomata (from 720 stomatal micrographs from 12 Magnoliaceae species) were examined. The area of each stoma was calculated using empirical measurements of stomatal length and width multiplied by a constant. Six equations describing the relationships among A_T, L_KS, and W_KS were compared. The root-mean-square (RMSE) and the Akaike information criterion (AIC) were used to measure the goodness of fit, and the trade-off between the goodness of fit and the structural complexity of each model, respectively. Analyses supported the validity of the MKS equation and the power-law equation A_T ∝ (L_KS∙W_KS)^α, where a is a scaling exponent. The estimated values of α at the species level and for the pooled data were all statistically smaller than unity, which did not support the hypothesis that A_T ∝ L_TS∙W_TS. The power-law equation had smaller RMSE and AIC values than the MKS equation for the data from the 12 individual species and the pooled data. These results indicate that A_T tends to allometrically scale with L_KS∙W_KS, and that increases in A_T do not keep pace with increases in L_TS∙W_TS. In addition, using the product of L_KS and W_KS is better than using only one of the two variables.

The intra-plant plasticity of leaves plays a vital role in enabling plants to adapt to changing climatic conditions. However, limited research has investigated the extent of intra-plant leaf trait variation and leaf biomass allocation strategies in herbaceous plants. To address this gap, we collected a total of 1746 leaves from 217 Lamium barbatum Siebold and Zucc. plants and measured their leaf dry mass (M) and leaf area (A). Leaves were categorized by vertical position (upper vs. lower canopy layer) and leaf–shoot orientation (east, south, west, north). ANOVA with Tukey’s HSD test was used to compare differences in M, A, and leaf dry mass per unit area (LMA). Reduced major axis regression was employed to evaluate the scaling relationship between M and A, and the bootstrap percentile method was used to determine differences in scaling exponents. The data indicated that: (i) M, A, LMA, and the scaling exponents of M versus A did not differ significantly among leaf–shoot orientations, and (ii) lower layer leaves exhibited significantly greater M, A, and LMA than upper layer leaves, but their scaling exponents were significantly smaller. These findings highlight that plant vertical growth brings significant intra-plant plasticity in leaf traits and their scaling relationships in herbaceous plants. This plasticity differs from that observed in trees, but is also critical for balancing weight load and optimizing light-use efficiency, potentially enhancing stress resilience in herbaceous plants.

The scaling relationship of leaf dry mass (M) versus leaf area (A) is crucial for understanding the tradeoff between leaf construction cost and the photosynthetic area return across diverse environmental stresses. Because leaves at different spatial positions on a plant receive varying intensities of light, this can affect the overall assessment of leaf scaling; however, the impact has not been tested. We collected a total of 1,746 leaves from 217 Lamium barbatum plants and measured their M and A. These leaves were grouped based on vertical (upper layer and lower layer) and horizontal (east, south, west, and north) positions. Reduced major axis regression protocols were used to evaluate the scaling relationship between M and A, and the bootstrap percentile method was employed to determine the differences in scaling exponents across different positions. ANOVA with Tukey’s HSD test was utilized to compare the M, A, and leaf dry mass per unit area (LMA) across these spatial positions. The results showed that: (1) M, A, and LMA were not significantly different among horizontal positions; however, these traits in the lower layer leaves were significantly greater compared to the upper layer leaves; (2) The scaling exponents of M versus A were also not significantly different across horizontal positions, but the scaling exponents of the lower layer leaves were significantly smaller than those of the upper layer leaves. Although the leaf scaling of M versus A tends to be consistent horizontally, it is necessary to consider the impact of their longitudinal positions on biomass investment for the photosynthetic area when conducting experiments on leaves.

Leaf vein density (LVD) is a critical trait linked to leaf hydraulic efficiency, commonly quantified using an automated tool—LEAF GUI, which analyzes vein structures via pixel-based algorithms. However, concerns persist about its accuracy for species with complex hierarchical venation networks. To evaluate the reliability of LEAF GUI, we compared its LVD measurements against those from ImageJ—a manual tracing platform renowned for its precision—using leaf specimens from nine Magnoliaceae species. Paired t-tests revealed no statistically significant differences between the two methods (P = 0.534), demonstrating comparable accuracy under standardized conditions. However, LEAF GUI’s reliability is constrained by its reliance on laborious threshold calibration and stringent image quality standards. For studies prioritizing precision, especially in taxa with heterogeneous or low-resolution samples, we recommend ImageJ as the standard approach. Its manual tracing protocol achieves consistency in resolving complex vein networks, balancing analytical rigor with adaptability to diverse sample conditions