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 and has increased yield and vigor through improved genetics in conjunction with increased fertilizer and pesticide use, higher planting density, and agricultural mechanization. Stomata are expected to change in response to rising atmospheric CO₂ 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 Pioneer maize hybrids released from 1920 to 2022, grown under controlled conditions. Modern hybrids (2013-2022) had a smaller total stomatal pore area (9.17 × 10⁸ µm² leaf^-¹ vs. 9.94 × 10⁸ µm² leaf^-¹; p < 0.05), higher stomatal density (47.2 vs. 44.5 stomata mm^-²), and smaller leaf area (17.9 vs. 20.5 cm²) than historical hybrids (1920-2011). Stomatal size (μm²), length (μm), and width (μm) did not differ significantly between the two groups. Across all hybrids, stomatal density was negatively correlated with stomatal size, length, width, and leaf area (r = -0.54 to -0.62). Total stomatal pore area declined in the hybrid's year of release (r = -0.56, p < 0.05), whereas stomatal density increased over time (r = 0.51, p < 0.05). Stomatal traits were associated with release year and with release-year climate proxy variables (CO₂ and °C) under a shared contemporary growth environment. We found that stomatal density was positively correlated with atmospheric CO₂ concentration and temperature anomaly °C, whereas total stomatal pore area was negatively correlated with both variables. Our results indicate that modern maize hybrids differ from historical hybrids in stomatal density (increased), and total stomatal pore area (decreased), consistent with long-term breeding progress of inadvertent selection under changing production environments.