10.1071/BT11009_AC CSIRO 2011 Australian Journal of Botany, 2011, 59(7), 630–639 Supplementary Data Table 1: Stomatal density values of cycad species grown under elevated [CO2] of 1500ppm and sub-ambient [O2] of 13% in combination and isolation relative to control of 380ppm [CO2] and 20.9% [O2]. Atmospheric Treatment Species Control st dev Low O2 st dev High CO2 st dev Low O2 / High CO2 st dev Cycas revoluta Stomatal Density (mm2) 51.9 12.9 53.7 12.1 54.2 2.4 40.7 2.9 Cv 24.9 22.6 4.4 7.1 Change Relative to Control (%) - 103.6 104.5 78.6 Dioon merolae Stomatal Density (mm2) 66.2 9.5 69.0 5.8 62.0 7.1 80.1 17.3 Cv 14.3 8.4 11.5 21.5 Change Relative to Control (%) - 104.2 93.7 121.0 Lepidozamia hopei Stomatal Density (mm2) 31.0 2.9 32.4 5.8 31.5 0.8 36.1 7.7 Cv 9.3 17.8 2.5 21.4 Change Relative to Control (%) - 104.5 101.5 116.4 Lepidozamia peroffskyana Stomatal Density (mm2) 33.3 4.2 39.4 5.6 35.6 3.5 36.6 7.6 Cv 12.5 14.3 9.8 20.9 Change Relative to Control (%) - 118.1 106.9 109.7 Macrozamia miquelii Stomatal Density (mm2) 62.5 2.4 50.9 7.0 53.2 10.4 55.6 4.2 Cv 3.8 13.7 19.6 7.5 Change Relative to Control (%) - 81.5 85.2 88.9 Zamia floridiana Stomatal Density (mm2) 72.7 8.5 74.5 18.7 61.1 1.4 70.4 14.7 Cv 11.7 25.0 2.3 20.9 Change Relative to Control (%) - 102.5 84.1 96.8 1 Supplementary Data Table 2: Stomatal index values of cycad species grown under elevated [CO2] of 1500ppm and sub-ambient [O2] of 13% in combination and isolation relative to control of 380ppm [CO2] and 20.9% [O2]. Atmospheric Treatment Species Control st dev Low O2 st dev High CO2 st dev Low O2 / High CO2 st dev Cycas revoluta Stomatal Index (%) 4.7 0.5 4.8 0.4 4.4 0.3 4.2 0.1 Cv 11.2 9.1 7.1 2.7 Change Relative to Control (%) - 102.4 94.8 90.2 Dioon merolae Stomatal Index (%) 6.8 0.7 6.5 0.4 5.8 0.6 6.6 0.9 Cv 9.6 6.3 10.5 13.4 Change Relative to Control (%) - 96.2 85.1 97.6 Lepidozamia hopei Stomatal Index (%) 4.6 0.1 4.3 0.5 4.3 0.5 4.3 0.5 Cv 1.6 11.0 10.8 10.9 Change Relative to Control (%) - 92.4 92.0 92.2 Lepidozamia peroffskyana Stomatal Index (%) 4.6 0.7 4.8 0.3 4.2 0.4 4.2 0.4 Cv 15.1 7.2 8.6 8.3 Change Relative to Control (%) - 104.0 92.3 92.4 Macrozamia miquelii Stomatal Index (%) 8.1 0.1 6.8 0.5 7.3 1.3 7.4 0.7 Cv 1.6 7.2 18.2 10.0 Change Relative to Control (%) - 84.6 90.1 91.3 Zamia floridiana Stomatal Index (%) 6.7 0.4 6.4 0.7 6.3 0.2 6.9 0.3 Cv 5.9 10.4 3.9 4.0 Change Relative to Control (%) - 96.0 94.4 103.1 2 Supplementary Data Table 3: Stomatal Pore Lengths of cycad species grown under elevated [CO2] of 1500ppm and sub-ambient [O2] of 13% in combination and isolation relative to control of 380ppm [CO2] and 20.9% [O2]. Atmospheric Treatment Species Control st dev Low O2 st dev High CO2 st dev Low O2 / High CO2 st dev Cycas revoluta Stomatal Pore Length ( m) 31.8 9.4 23.9 5.2 25.9 5.9 26.3 4.5 Cv 29.7 21.8 22.6 17.3 Change Relative to Control (%) - 75.1 81.5 82.7 Dioon merolae Stomatal Pore Length ( m) 17.5 4.7 22.3 4.6 22.9 4.5 24.4 5.1 Cv 26.7 20.5 19.8 20.7 Change Relative to Control (%) - 127.9 131.4 139.8 Lepidozamia hopei Stomatal Pore Length ( m) 41.2 8.4 38.0 8.3 46.7 5.2 43.6 5.5 Cv 20.5 21.8 11.2 12.7 Change Relative to Control (%) - 92.1 113.4 105.8 Lepidozamia peroffskyana Stomatal Pore Length ( m) 35.6 5.5 39.8 4.0 42.9 5.7 39.3 5.9 Cv 15.5 10.1 13.3 15.1 Change Relative to Control (%) - 111.7 120.4 110.3 Macrozamia miquelii Stomatal Pore Length ( m) 28.6 3.3 26.4 3.8 31.5 6.7 29.8 5.0 Cv 11.7 14.5 21.2 16.8 Change Relative to Control (%) - 92.1 110.2 104.3 Zamia floridiana Stomatal Pore Length ( m) 21.5 5.4 19.4 3.7 17.2 3.1 18.0 2.7 Cv 25.2 18.8 17.8 15.1 Change Relative to Control (%) - 90.3 79.8 83.7 3 Supplementary Data Table 4: Calculated maximum stomatal conductance (Gsmax) of cycad species (see Supplementary Data Formulae 1) grown under elevated [CO2] of 1500ppm and sub-ambient [O2] of 13% in combination and isolation relative to control of 380ppm [CO2] and 20.9% [O2]. Atmospheric Treatment Species Control st dev Low O2 st dev High CO2 st dev Low O2 / High CO2 st dev -2 -1 Cycas revoluta Gsmax (mmol m s ) 112.1 27.9 74.2 16.8 85.2 3.8 65.6 4.7 Cv 24.9 22.6 4.4 7.1 Change Relative to Control (%) - 66.2 76.0 58.5 -2 -1 Dioon merolae Gsmax (mmol m s ) 59.6 8.5 92.4 7.7 86.8 10.0 123.5 26.6 Cv 14.3 8.4 11.5 21.5 Change Relative to Control (%) - 154.9 145.5 207.1 -2 -1 Lepidozamia hopei Gsmax (mmol m s ) 89.0 8.3 82.1 14.6 109.1 2.8 112.8 24.2 Cv 9.3 17.8 2.5 21.4 Change Relative to Control (%) - 92.2 122.6 126.7 -2 -1 Lepidozamia peroffskyana Gsmax (mmol m s ) 77.2 9.7 108.0 15.4 109.6 10.7 98.5 20.6 Cv 12.5 14.3 9.8 20.9 Change Relative to Control (%) - 139.9 142.0 127.6 -2 -1 Macrozamia miquelii Gsmax (mmol m s ) 108.8 4.2 77.7 10.7 107.8 21.1 103.2 7.7 Cv 3.8 13.7 19.6 7.5 Change Relative to Control (%) - 71.4 99.1 94.9 -2 -1 Zamia floridiana Gsmax (mmol m s ) 97.8 11.4 85.4 21.4 57.4 1.3 71.4 14.9 Cv 11.7 25.0 2.3 20.9 Change Relative to Control (%) - 87.3 58.7 73.0 4 Supplementary Data Graph 1: Stomatal density and size of cycad species relative to other plant groups grown under ambient atmospheric [CO2] and [O2]. Stomatal size calculated as stomatal length x width of closed guard cells. Data taken from Franks and Beerling (2009). 1000 900 800 ) Franks and Beerling 2009 2 700 Cycas revoluta 600 Dioon merolae 500 Lepidozamia hopei 400 Lepidozamia peroffskyana Macrozamia miquelii 300 Stomatal Density (mm Stomatal Zamia integrifolia 200 100 0 0 500 1000 1500 2000 2500 3000 3500 4000 Stomatal Size ( m2) Franks PJ, Beerling DJ. 2009. Maximum leaf conductance driven by CO2 effects on stomatal size and density over geologic time. Proceedings of the National Academy of Sciences of the United States of America 106: 10343-10347 5 Supplementary Data Graph 2: Calculated maximum stomatal conductance (Gsmax) (see supplementary data for additional information) of cycad species grown sub-ambient [O2] of 13.0 % and elevated [CO2] of 1500 ppm (see Table 2 for growth conditions): a) Cycas revoluta; b) Dioon merolae; c) Lepidozamia hopei; d) Lepidozamia peroffskyana; e) Macrozamia miquelii, and; f) Zamia integrifolia. Letters indicates significant difference in treatment SPL to control SPL value (a = P > 0.05; b = P < 0.05; c = P < 0.01). Error bars indicate one standard deviation either side of the mean. 6 Supplementary Data Graph 3: Maximum stomatal conductance (Gsmax) of cycad species grown under elevated [CO2] and sub-ambient [O2]. Cycas revoluta (closed squares); Dioon merolae (open squares); Lepidozamia hopei (closed triangles); Lepidozamia peroffskyana (open triangles); Macrozamia miquelii (closed diamonds); Zamia integrifolia (open diamonds). Error bars indicate stomatal deviation either side of mean. 7 Supplementary Data Formulae 1: Maximum stomatal conductance formulae of Cowan (1977) use in Figure 3. -2 -1 Gsmax = maximum stomatal conductance of water vapour (mol m s ) S = stomatal density (number of stomata per m2) D = diffusivity of water in air (m-2 s-1) A = area of the stomatal pore (m2) V = molar volume of air (m3 mol-1) L = depth of stomatal pore (m) Area of the stomatal pore was calculated as an ellipse as cycad species analysed possess large kidney shaped stomata. Following the methods of Beerling and Chaloner (1993) stomatal width at full opening was assumed to be half stomatal pore length.