Annals of Botany

• Background and Aims The inverse relationship between the number of stomata and atmospheric CO₂ levels observed in different plant species is increasingly used for reconstructions of past CO₂ concentrations. To validate this relationship, the potential influence of other environmental conditions and ontogenetical development stage on stomatal densities must be investigated as well. Quantitative data on the changes in stomatal density of conifers in relation to leaf development is reported.

• Methods Stomatal frequency and epidermal cells of Tsuga heterophylla needles during different stages of budburst were measured using computerized image analysis systems on light microscope slides.

• Key Results Stomata first appear in the apical region and subsequently spread basipetally towards the needle base during development. The number of stomatal rows on a needle does not change during ontogeny, but stomatal density decreases nonlinearly with increasing needle area, until about 50% of the final needle area. The total number of stomata on the needle increases during the entire developmental period, indicating that stomatal and epidermal cell formation continues until the needle has matured completely.

• Conclusions Epidermal characteristics in developing conifer needles appear to be fundamentally different from angiosperm dicot leaves, where in general leaf expansion in the final stages is due to cell expansion rather than cell formation. The lack of further change in either stomatal density or stomatal density per millimetre needle length (the stomatal characteristic most sensitive to CO₂ in conifers) in the final stages of leaf growth indicates that in conifers the stage of leaf maturation would not influence CO₂ reconstructions based on stomatal density.