Sample collection:
All data in this package were collected at the Black Rock Forest (BRF) in Cornwall, NY (41.39, -74.027; https://www.blackrockforest.org). BRF receives an average of 1.2 m of precipitation per growing season, and has a mean growing season temperature of 23.4°C.
We collected branches predawn using a 12-gauge shotgun and steel bird shot. After branches were removed from the upper canopy, we immediately recut them (at least 20 cm) under water to prevent cavitation or embolism of the xylem. Subsequently, multiple smaller cuts (5-15 cm) were made to progressively ease xylem tension before the branches were transported to a nearby lab for analysis.
Gas exchange:
Gas exchange was performed using three LI-6800 and two LI-6400XT portable photosynthesis systems (LI-COR Inc., Lincoln, NE, USA). We conducted stomatal response curves by adjusting irradiance to propagate changes in photosynthesis, while maintaining a constant level of sample CO2 concentration and VPDleaf for the length of the curve. Leaf temperature for each curve ranged from 24-27ºC, and VPDleaf ranged between 0.8 and 1.4 kPa. Irradiance levels measured during each curve were 1500, 900, 700, 450, 250, 175, 90, 40, 20, 0 μmol m-2 s-1.
Parameters estimated from response curves included stomatal slope (g1), stomatal intercept (g0), minimum stomatal conductance (gsmin), the maximum carboxylation rate of rubisco (Vcmax), and the dark respiration rate (Rdark). Stomatal parameters were estimated using the Unified Stomatal Optimization model (USO, Medlyn et al., 2011, doi: 10.1111/j.1365-2486.2010.02375.x). Vcmax was estimated using the “one-point method” (De Kauwe et al., 2016, doi: 10.1111/nph.13815). Rdark was calculated from dark-adapted measurements of photosynthesis.
Both Vcmax and Rdark were normalized to 25°C. We calculated Vcmax.25 from Vcmax using a modified Arrhenius equation (Leuning, 2002 doi: 10.1046/j.1365-3040.2002.00898.x). We calculated Rdark.25 from Rdark using an inverse Arrhenius equation (Bernacchi et al., 2001 doi: 10.1111/j.1365-3040.2001.00668.x).
Functional Traits:
Multiple leaf discs of a known area were weighed then dried to a constant mass in a 60ºC oven. After drying they were again weighed to obtain dry mass. Using these data we estimated LMA (dry mass/leaf area) and leaf dry matter content (LDMC; dry mass/fresh mass). A subset of the dried leaf samples were ground, and elemental nitrogen and carbon was quantified using a 2400 Series II CHN analyzer (PerkinElmer, Waltham, MA, USA) to estimate nitrogen as expressed on an area and mass basis (Narea, Nmass).
Leaf hydraulics:
After branch collection, a single leaf per branch was removed at the base of the petiole, and sealed in an airtight, humid, cool, dark box for transport to the laboratory. We then quantified leaf water potential (Ψleaf) using a Scholander type pressure chamber (Scholander et al., 1964).
We also collected pressure-volume (PV) curves following the method of Bartlett et al., (2012, doi: 10.1111/j.1461-0248.2012.01751.x). The relationship between Ψleaf and relative water content was assessed using the R package ‘pvldcurve’ (v 1.2.6; Raesch, 2020, https://cran.r-project.org/web/packages/pvldcurve/index.html). This analysis enabled us to estimate the turgor loss point (ΨTLP), relative water deficit at the turgor loss point (RWDTLP, %), and the bulk elastic modulus (ε).
Stomatal anatomy:
We obtained impressions of the abaxial side of two leaves per branch sample using clear varnish. Then using a stage microscope with a digital camera attachment (Nikon ECLIPSE Ci/Ni, Tokyo, Japan) at 400x magnification we collected images of these impressions. These images allowed us to estimate stomatal density (number of stomata per mm2) and stomatal area, (length x width of the guard cells).
Reflectance spectroscopy:
We used a full-range spectroradiometer (HR-1024i; Spectra Vista Corporation, Spectra Vista Corp., Poughkeepsie, NY, USA) together with an LC-RPPro leaf clip foreoptic containing an internal, full-spectrum calibrated light source to measure leaf reflectance. Before each spectra was collected we measured leaf temperature using an MI-210 Infrared Radiometer (Apogee Instruments, Inc., Logan, UT, USA).
