Several studies have found shifts in stomatal density (number of pores per leaf surface area) attributed to rising atmospheric CO2 concentrations (Van de Water et al., 1994, McElwain et al., 1995), climate change (Beerling and Chaloner, 1993), or plant water status (Ridolfi et al., 1996). Likewise, stomatal density records have been applied to palaeo-atmospheric reconstructions for periods considerably longer than ice core data can provide (Beerling et al., 1993; McElwain and Chaloner, 1995). However, because of the potential for evolutionary changes (Masterson, 1994), such reconstructions generally gain rigour if they include data from several species.
Comparing the stomatal density among separate species is often complicated by differences in leaf structure. Variation in stomatal size and distribution, presence of trichomes (small hairs on the leaf surface), and leaf venation can make one method valuable for analysing some species but not others. These problems are worsened when comparing fossil leaves against modern leaves because alternative methods must often be used for the fossils. Thus, several methods have been developed to measure stomatal densities. Common methods include the use of acetate peels (Beerling and Chaloner, 1992), silicone impressions (Weyers and Johnson, 1985), cuticular maceration (McElwain et al., 1995), scanning electron microscopy (Alvin, 1970), and light microscopy.
Here a comparison of three methods used to estimate the stomatal density of two species of three-needle pines, Pinus taeda and Pinus ponderosa is reported. Methods include: (1) direct stomatal counts of intact leaves under a binocular dissecting scope, (2) scanning electron microscopy (SEM) of intact leaves, and (3) epidermal peels photographed under a compound light microscope. Sample preparation varies among methods, which may lead to changes in leaf surface area. Dissecting scope measurements are frequently made on fresh tissues or air-dried herbarium samples, SEM requires vacuum-drying or critical-point drying of the specimen, and light microscopy generally uses needles saturated in water or alcohol. Hence, the epidermal surface may expand when saturated and shrink when dried. Because stomatal densities are reported per unit area, changes in leaf area will cause a proportional change in stomatal densities. It is hypothesized that variation in stomatal densities among methods can be attributed entirely to changes in needle width which results from differences in hydration and geometry.