Long-term stomatal and leaf trait dynamics in invasive knotweeds in Europe - insights from 160 years of herbarium records

1. Stomata and leaf traits are key regulators of plant water use efficiency and are expected to have changed in response to rising atmospheric CO2 concentrations and climate warming over the past centuries. However, long-term data documenting such changes are rare. 2. We leveraged herbarium collections to track changes in stomatal characteristics and leaf traits in 656 individuals of invasive Japanese knotweed and its hybrid Bohemian knotweed collected across their European range and spanning 160 years of invasive spread. 3. We found that several functional traits including stomatal density and maximum anatomical stomatal conductance did not show significant changes over time but that plants adjusted their stomatal size and shape over time, and that these changes were associated with increased atmospheric CO2 levels. Interestingly, Reynoutria japonica showed increases in stomatal size and stomatal elongation, while the hybrid R. x bohemica showed a reduction in stomatal size. Traits also varied systematically with climates of origin. Plants from warmer origins with higher evaporative demands during the growing season had thicker leaves, lower SLA, smaller stomata and higher stomatal density, indicating more conservative water-use strategies. Stomatal density and gas exchange capacity co-varied with leaf structural traits, and there was a trade-off between stomatal size and number. Overall, fast leaf economic traits were associated with slow physiological traits. 4. Our results suggest that stomatal anatomical plasticity may enhance climate resilience by maintaining a stable maximum gas exchange capacity across environmental gradients. Herbarium collections provide a unique resource for reconstructing plant responses to historical environmental changes and understanding intraspecific trait variation.