Trends in stomatal density and size in maize hybrids representing 100 years of long-term breeding for yield

Maize hybrid breeding started over 100 years ago, has increased yield and vigor through improved genetics in conjunction with increased fertilizer and pesticide use, planting density, and agricultural mechanization. Stomata are expected to change in response to rising atmospheric CO 2 concentration and average temperature anomalies (°C). Yet, the impact of long-term maize breeding over the past century on stomatal traits and their responses to climate factors remains poorly understood. We evaluated stomatal traits at the seedling stage in 27 maize hybrids released from 1920 to 2022, grown under controlled conditions. Modern hybrids (2013 ‒ 2022) had a smaller total stomatal pore area (9.17 x 10 8 μm 2 ) than (1920 ‒ 2012) historical ERA hybrids (9.94 x 10 8 μm 2 ; p ≤ 0.001), a higher stomatal density (47.2 per mm -2 ) vs. historical ERA hybrids (44.5), and a smaller leaf area (17.9 cm 2 vs. 20.5 cm 2 ). No significant differences were found in the size (μm 2 ), length (μm), or width (μm) of stomata between the two groups. Stomatal density increased, while all other traits decreased in modern hybrids. Stomatal density was negatively correlated with stomatal size (r = -0.62), length (r = -0.57), width (r = - 0.54), and leaf area (r = -0.54). Stomatal size had a negative correlation with atmospheric CO 2 concentration (r = -0.22) and average temperature anomalies (°C) (r = -0.35) in the hybrid’s year of release and climate proxies. In contrast, stomatal density had a positive correlation with both atmospheric CO 2 concentration and average temperature anomalies (°C) (r = 0.44) in year of release. Total stomatal pore area negatively correlated with atmospheric CO 2 concentration (r = -0.45) and average temperature anomalies (°C) (r = -0.36). Our study indicates that maize stomatal traits suggest inadvertent selection for key stomatal traits (density and size), total stomatal pore area (per year decline of about 0.02%) associated with yield stability and environmental adaptation. Highlights Maize stomatal traits changed through environmental (CO 2 and °C) adaptation but total stomatal pore area, effects indirectly by decreased leaf area in maize hybrids representing 100 years of long-term breeding for yield. A negative correlation was observed across 27 ERA hybrids between stomatal density and stomatal size, length, width and leaf area. Stomatal density increases while stomata size, length width and leaf area decrease per se. Over the past 100 years, the total stomatal pore area on leaves decreased, while stomatal density increased as leaf area declined, revealing a connection between these two patterns. A negative correlation was found between total stomatal pore area and atmospheric CO 2 concentrations, and temperature over the past century.