Reconstructions of atmospheric CO₂ concentrations from 15-65 Ma have been made using leaf fossil records and modern experimental relationships between stomatal index (SI) and growth conditions of CO2 (for example, Ginkgo and Metasequoia by Beerling, Fox and Anderson 2009). For these two tree species, 25-50% reductions in SI were experimentally observed for trees grown under elevated CO2 conditions respectively (~500 ppm versus ambient CO2). Other experimental evidence suggests that not all plant species exhibit reduced SI in response to increasing CO2 developmental conditions. Within-canopy variability in stomatal characteristics have been repeatedly observed under ambient CO2 conditions, presumably in response to light and other microclimate gradients. Could it be possible that the variability of SI within a mature canopy could be greater than a species’ SI response to increasing CO2 conditions? This study evaluated the variability in SI and in other epidermal cell morphological characters within the tree crowns of young trees grown in two separate Free Air CO2 Enrichment (FACE) experiments.
Results/Conclusions

In September 2001, Acer saccharum (sugar maple), Betula papyrifera (paper birch) samples were collected from trees at the Wisconsin FACE site, and Liquidambar styraciflua (sweetgum) samples were collected from the Oak Ridge TN FACE site. Leaves were sampled at the top, bottom, and at various heights within the crowns of three trees per CO2 treatment (ambient ~360 pm, and elevated CO2, ~550 ppm) in each FACE ring at each site. Epidermal peels were made on leaf abaxial and adaxial surfaces, and slides were evaluated in the laboratory using light microscopy and NIH Image-J image analysis software. Data were recorded for 3 fields of view per leaf. The following were measured: number of stomata and epidermal cells in a given area, cell areas of epidermal cells and stomatal complexes, and stomatal pore length. Mean responses of SI were found to be greater ranging within canopies (e.g., 31, 20 and 17% for the top, middle, and bottom of the paper birch canopy) than across CO2 treatment (19 vs 18% and 25 vs 31% for ambient versus elevated CO2 at the bottom and top of the paper birch canopies, respectively).   SI, stomatal density, and leaf porosity results were similar across species and across sites, as treatments did not uniformly decrease with increasing CO2. The findings suggest species-specific patterns in leaf developmental morphological responses to elevated CO2, and that paleo reconstructions of atmospheric CO2 will continue to need experimental careful calibration.