Results and discussion
Mean stomatal density estimates for both species are reported in Table 1. The results from this study are similar to those reported for P. ponderosa (Cregg, 1994), and for P. taeda (Teskey, 1997). Stomatal density is one common measure of plant response to rising atmospheric CO2 concentrations (Woodward and Bazzaz, 1988; Van de Water et al., 1994; Teskey, 1997; Bettarini et al., 1998), climate change (Beerling and Chaloner, 1993) and water availability (Beerling et al., 1996). As information continues to increase, and new experimental methods for acquiring stomatal densities continue to expand, it is becoming increasingly difficult to compare and interpret stomatal densities reported in the literature. Accordingly, it is useful to consider causes of variation among methods, including changes in leaf area and geometry.
Stomatal density estimates from SEM were similar to dissecting scope estimates (t=0.509, P=0.62, n=21, Table 2; Fig. 2). Because stomatal densities are measured per unit area, variations in needle width were considered between SEM and the dissecting scope due to differences in drying techniques. Needles were air-dried prior to measurement under the dissecting scope and were vacuum-dried prior to using scanning electron microscopy. The results of this study indicate that the widths did not change after the air-dried needles were vacuum-dried. Nevertheless, future observations should consider potential differences in shrinkage among other drying methods, including critical point drying (dehydration in ethanol and liquid CO2), and freeze drying (see Porter et al., 1972, for a comparison of drying methods and their influences on cell integrity).
Stomatal densities obtained by light microscopy varied from those obtained from the dissecting scope (t=-2.307, P=0.04, n=13, Table 2; Fig. 3A). Because preparation for light microscopy required the construction of temporary slides in distilled H2O, the widths for the degree of expansion of each macerated needle were adjusted. After adjusting for geometry and width expansion, stomatal densities were not significantly different (t=-1.205, P=0.25, n=13, Table 2; Fig. 3B). Hydration-induced changes in needle length could also produce proportional changes in stomatal densities. The results indicate that needle length increased 1.3% after saturation (t=6.908, P=n=12). However, unlike width expansion, the increase in length was not large enough to account for the difference in stomatal density estimates between methods.
It was found that stomatal densities increased 14% when fresh P. taeda needles are oven-dried (t=-3.503, P=0.0086, n=6), but no variation occurred in fresh P. ponderosa needles after oven-drying (Table 3). After re-wetting, both species retained similar stomatal densities as when the needles are fresh (Table 3). The results suggest that stomatal density estimates made from dried leaves, such as herbarium samples, potentially overestimate stomatal densities of fresh leaves. Therefore, stomatal densities measured from air-dried herbarium sheets may closer represent fresh leaves if the samples are first re-wetted before counting.
Is leaf expansion inherent in other plants when dry leaves become saturated? The relative increase in leaf area and needle width of seven broad-leaved genera and three conifer genera, respectively, was measured after the dried leaves were saturated in distilled H2O for 48 h. The relative increase in broad-leaved areas ranged from 3.9% in Magnolia to 15.3% in Aesculus with a mean of 10.8% (Table 4). For conifers, needle width increased from 5.3% in Abies to 42.5% in Pseudotsuga, mean increase was 21.1% (Table 4). These data show that changes in leaf area occur in other genera and will produce proportional changes in stomatal densities when comparing estimates between dry and saturated leaves. The tests with Pinus leaves also indicate that the correction for shrinkage may be sufficient to adjust the stomatal densities to a common base.
It is concluded that stomatal densities measured under a dissecting scope are comparable with those obtained from SEM and light microscopy. Agreement between dissecting scope and SEM indicates that air-dried needle widths do not significantly change as needles are vacuum- dried after being air-dried. Stomatal densities are also comparable between dissecting scope and light microscopy after needle widths are adjusted for saturation and changes in geometry. These results indicate that changes in leaf structure should be considered when comparing stomatal densities obtained from more than one method, particularly if maceration techniques are used. Future observations should consider methodological differences in analyses of large data sets where several methods are used. Such interpretations will improve palaeo-atmospheric reconstructions and assessments of plant response to environmental change.
We thank P Van de Water, J King, and an anonymous reviewer for comments on an earlier version of this manuscript. Thanks to D Jennings, C Davitt, and V Lynch-Holm for technical assistants at the Washington State University, Electron Microscope Center. This research was supported by a grant from the National Science Foundation.
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