The functional explanation for increased plant leaf stomatal density (SD) with elevation remains a topic of much debate among plant eco-physiologists (Friend and Woodward, 1990; Gale, 1972, 2004; Smith and Donahue, 1991; Terashima et al., 1995; Woodward and Bazzaz, 1988). Johnson et al. raise an interesting aspect of this ongoing debate. Based on the observation that gaseous diffusion increases with decreasing atmospheric pressure these authors argue that any negative impact of decreasing carbon dioxide partial pressure (pCO2) with elevation on plant photosynthesis would likely be compensated for by greater diffusion rates of CO2 into the leaf. Using these theoretical considerations Johnson et al. argue that decreasing CO2 partial pressure is unlikely to select for increased SD in plant species evolving at high elevation, and thus, the highly significant relationship demonstrated between Californian black oak SD and elevation is unlikely to be generally applicable. In their theoretical calculations, however, Johnson et al. fail to take into account the observation that transpiration rates are often extremely elevated at high elevation due to (1) higher light intensity, (2) increased diffusion of H2O in air at reduced atmospheric pressure, and (3) an increased density gradient of H2O vapor out of the leaf into the ambient air (Gale, 2004). As H2O vapor efflux through stomata has been shown to hamper CO2 diffusion into the leaf (von Caemmerer and Farquhar, 1981), it is highly likely that increased transpirational water vapor loss at higher elevations would act to cancel out the higher diffusion coefficient of CO2 at reduced barometric pressure (Gale, 2004), thereby selecting strongly for increased SD at high elevation to maintain adequate conductance to CO2 diffusion.
Johnson et al. argue that many abiotic factors in addition to pCO2 change with altitude and suggest that these other factors rather than pCO2 may be responsible for the observed increase in SD in Quercus kelloggii. The potential role of other biotic and abiotic factors on SD, including temperature, light intensity, water availability, sex of the plant, etc., is well known (McElwain and Chaloner, 1996; Royer, 2001). Carbon dioxide and to a much lesser extent light intensity are the only factors which are known to control the actual development of stomata from epidermal initials. The effect of CO2 on leaf development can be tracked by measuring stomatal index (SI); a ratio of the number of stomata to the total number of cells (stomata plus epidermal) on the leaf surface. In contrast all other biotic/abiotic variables which influence SD do so indirectly (Beerling and Kelly, 1997). In other words, although factors like temperature, water availability, and humidity do not directly influence the absolute number of stomata developing from initials, these variables can influence the size and/or spacing of epidermal cells, resulting in stomata being packed more closely together or further apart resulting in higher or lower densities but importantly the same SI.
The significant increase in both SI and SD with elevation in both sun and shade leaves of Californian black oak which I document in my paper can only therefore be explained by the effect of increasing CO2 partial pressure on stomatal development from epidermal initials. Increasing SI rules out the effect of all other biotic and abiotic variables. In addition, to reiterate the results in the paper, a historical investigation into the effect of four climatic variables showed no significant correlation with black oak SD. However, the same historical black oak SD data set showed a highly significant inverse correlation with CO2 concentration. Contrary to the assertions of Johnson et al. therefore, these two lines of evidence suggest that decreasing CO2 partial pressure is indeed a primary altitudinal factor driving SD changes in Californian black oak.
This does not simply imply however, and I do not state as such in my paper, that the stomatal pCO2 paleoaltimeter will be universally applicable. Certain taxa are not CO2 sensitive—they do not show the classic inverse relationship between pCO2 and SD or SI. These include many grasses and forbs (Reid et al., 2003) and plants with C4 photosynthesis (Raven and Ramden, 1989). These taxa have evolved alternative strategies for optimizing CO2 uptake for photosynthesis against transpirational water loss, which negates the need to adjust SD. These include fine-tuned stomatal control which enable much more rapid stomatal opening and closing times within minutes rather than hours (as in grasses) and bio-chemical means of concentrating CO2 internally thereby ensuring a high CO2 gradient from the air to the leaf (as in C4 plants). It is imperative therefore that the stomatal pCO2 paleoaltimeter is only applied to extant taxa, which are known to be CO2 sensitive. Meta analysis of woody plant SD and SI responses to historical increases in pCO2 over the past 200 yr and at naturally elevated CO2 springs has shown that >70% (n = 44) of the taxa analyzed demonstrate the expected inverse relationship (Royer, 2001). This high CO2 sensitivity among the majority of woody C3 taxa indicates that increased SD with elevation is likely to be the general rule for this group of plants. Coupled with the fact that woody C3 plants are the most well represented group in the plant fossil record, they are an ideal group for application of the stomatal pCO2 paleoaltimeter.