Isoprime 100 isotope ratio mass spectrometer

According to the method of Sack and Scoffoni³⁹ , recent fully expanded leaves from each provenance were sampled at pre-drought, and then, minimum leaf conductance (g_min; the rate of water loss through the leaf when stomata are closed) was estimated. Leaf area was measured using a Licor-3100A (Li-Cor Inc., Lincoln, NE, USA). Samples were then dried in a growth chamber, with the air temperature of 25 °C, and a light intensity of < 5 μmol m⁻² s⁻¹. Afterwards, samples were weighed every 20 min at 6 to 15 intervals using a high precision balance. The g_min (mmol m⁻² s⁻¹) was calculated from the slope of the linear part of leaf mass vs. time regression in conjunction with chamber VPD and leaf area^{40 (link)}.
Other leaf samples were placed in the oven at 110 ℃ for 1 h and then oven-dried at 70 ℃ for at least 72 h. Leaf carbon isotopic composition (δ¹³C, ‰) was measured on these dried samples, using a PE2400 elemental analyzer (PerkinElmer, USA) connected to an IsoPrime100 isotope ratio mass spectrometer (Elementar, Germany).

All tin capsules were combusted in an Elementar Pyrocube and the resulting CO₂ gas was analyzed with an Elementar Isoprime100 isotope ratio mass spectrometer at The Academy of Natural Sciences at Drexel University. δ¹³C values are reported relative to Vienna Peedee Belemnite Limestone Standard (vPDB) (δ¹³C = per mil deviation of the ratio of stable carbon ¹³C:¹²C relative to vPDB). Samples were analyzed in duplicate. Standards, B2150 (EA Consumables, LLC, Marlton, NJ), internal elk tissue, DORM (fish muscle) and bird feather standards had a precision of ± 0.14‰ for δ¹³C.

Leaf material was oven-dried at 60°C for 48 h and used to determine δ¹³C and the C/N ratio. The material was analyzed using the Isoprime 100 isotope ratio mass spectrometer coupled to an isotope cube elemental analyzer (Elementar, Hanau, Germany), as described by Gowik and Bra (2011) (link).

Plant height (H, cm) and basal diameter (D, mm) were measured regularly over the experimental period. Basal diameter was measured at 5 cm above the soil. The plants used for hydraulic measurements were harvested and we separated the leaves, stems and roots, which were washed free of soil. All harvested fresh organs were placed into an oven at 110°C for 1 h to eliminate biological activity and then oven-dried at 70°C for 72 h for dry mass determination. Leaf area (cm²) was determined using a portable leaf area meter (LI-3100A, Li-Cor, Lincoln, NE, United States). Specific leaf area (SLA, cm² g⁻¹) was calculated as the ratio of leaf area to leaf dry mass. Leaf carbon isotopic composition (δ¹³C, ‰) was measured on dried samples, using a PE2400 elemental analyzer (PerkinElmer, United States) connected to an IsoPrime100 isotope ratio mass spectrometer (Elementar, Germany). Leaf δ¹³C was used to estimate the integrated, long-term leaf water-use efficiency. One stem segment per harvested seedling was used to determine stem wood density (g cm⁻³). The volume of the fresh segment (without bark) was determined gravimetrically by the water displacement method. The dry mass was measured after 72 h by oven-drying at 70°C. Wood density was calculated as the stem dry mass divided by the stem volume.