Background
Atmospheric CO2 is an effective antitranspirant; as its concentration is experimentally increased, the apertures of the stomatal openings of the leaves or needles of most plants gradually become smaller, reducing the rate of evaporative water loss from the plants.  Over much longer periods of time, increases in the air's CO2 content also lead to the creation of fewer stomates per unit leaf or needle area.  This latter phenomenon has been used in recent years to infer past atmospheric CO2 concentrations from stomatal frequency counts of old herbarium specimens and fossilized leaves.

Most studies of stomatal frequency response to atmospheric CO2 enrichment have been conducted on the leaves of woody angiosperm taxa (Woodward, 1987; Peņuelas and Matamala, 1990; Paoletti and Gellini, 1993, Kurschner et al., 1996; Wagner et al., 1996; Wagner, 1998); and they have revealed, in the words of the authors, that "the maximum effect of the current CO2 increase on stomatal frequency has already been reached."  Kouwenberg et al. thus concentrated their efforts on conifers, restricting their study to the range of atmospheric CO2 concentrations experienced over the past century.

What was done
Plots of needle stomatal frequency as a function of atmospheric CO2 concentration were constructed for four native North American conifer species - Tsuga heterophylla, Picea mariana, Picea glauca and Larix laricina - based on stomatal frequency measurements made on needles collected from living trees, herbarium samples and well-dated peat cores that could be assigned atmospheric CO2 concentrations corresponding to the times of the needles' creation on the basis of historical atmospheric CO2 measurements and CO2 measurements of air bubbles trapped in shallow Antarctic ice cores.

What was learned
The authors report that "the four conifer species analyzed in this study show a significant reduction in stomatal frequency as a response to a CO2 rise of 80 ppm over the last century."  Specifically, they identify a 4.49% decrease in stomatal frequency or density per unit needle length for Tsuga heterophylla and decreases of 7.34% and 8.09% for Picea mariana/glauco and Larix laricina, respectively, which responses are of the same order as those observed for angiosperm tree taxa.  In addition, they note that "Tsuga heterophylla and Picea glauca/mariana have not reached their response limit yet at the current CO2 level of 370 ppm," whereas most woody angiosperms have reached their limits.

What it means
In the words of the authors, "because of their sensitive response to CO2, combined with a high preservation capacity, fossil needles of Tsuga heterophylla, Picea glauca, P. mariana, and Larix laricina have great potential for detecting and quantifying past atmospheric CO2 fluctuations," even "well above present levels (Royer et al., 2001)."

References
Kurschner, W.M., van der Burgh, J., Visscher, H. and Dilcher, D.L.  1996.  Oak leaves as biosensors of late Neogene and early Pleistocene paleoatmospheric CO2 concentrations.  Marine Micropaleontology 27: 299-312.

Paoletti, E. and Gellini, R.  1993.  Stomatal density in beech and holm oak leaves collected over the last 200 years.  Acta Ecologica 14: 173-178.

Peņuelas, J. and Matamala, R.  1990.  Changes in N and S leaf content, stomatal density and specific leaf area of 14 plant species during the last three centuries of CO2 increase.  Journal of Experimental Botany 41: 1119-1124.

Royer, D.L.  2001.  Stomatal density and stomatal index as indicators of paleoatmospheric CO2 concentration.  Review of Palaeobotamy and Palynology 114: 1-28.

Wagner, F.  1998.  The Influence of Environment on the Stomatal Frequency in Betula.  Ph.D. Thesis. Laboratory of Palaeobotany and Palynology, Utrecht University, Utrecht, The Netherlands.

Wagner, F., Below, R., de Klerk, P., Dilcher, D.L., Joosten, H., Kurschner, W.M. and Visscher, H.  1996.  A natural experiment on plant acclimation: lifetime stomatal frequency response of an individual tree to annual atmospheric CO2 increase.  Proceedings of the National Academy of Science USA 93: 11,705-11,708.

Woodward, F.I.  1987.  Stomatal numbers are sensitive to increases in CO2 concentration from pre-industrial levels.  Nature 327: 617-618.