Dark respiration measurements

After the photosynthetic measurements, we collected seven individual plants for which the top canopies were not directly covered by the other plants, in each season or each species. Their stem heights were 113.5 ± 9.0 and 54.3 ± 16.9 cm (mean ± 1 S.D.) for S. flexicaulis and S. tashiroi, respectively. Their stem diameters near the ground were 9.0 ± 2.0 and 4.8 ± 1.1 mm for the monocarpic S. flexicaulis and the polycarpic S. tashiroi, respectively.

We separated the individual plants into three parts (leaves, stems, and roots) with scissors, and immediately measured their dark respiration rates under the field conditions. The plant parts were put into a closed plastic box with a small fan (1.3 L, 5 L or 14.5 L in volume; the size of the plastic box was selected according to the volume or length of the samples), and then the box was covered with black cloth. To measure the air temperature, copper-constantan thermocouples were placed in the box together with the plant parts. Here, we assumed that the plant temperature was in equilibrium with the air temperature in the box. We measured the increasing rates in air CO₂ concentration in the box for approximately 3–5 min, using a nondispersive infrared CO₂ gas analyzer (GMP343, Vaisala Inc., Helsinki, Finland) connected with a data logger (GL240, Graphtec Co. Ltd., Yokohama, Japan). From the CO₂ increasing rates and the box volume, we calculated the dark respiration rates. After the field measurements, the plant samples were dried at 60 °C for at least 3 days and then the dry mass was weighed. Dry mass-based respiration rates were calculated. Furthermore, to evaluate metabolic activity in cells at a given temperature, the respiration rates were standardized at 19 °C and 28 °C in both seasons by using Q₁₀ = 2^6,19. The chosen values of 19 °C and 28 °C were the mean box temperatures in winter and summer, respectively.