Asking the pikas politely…
Measure leaf morphological traits
Measure height of leaf above ground using a metre stick (cm). Calculate leaf mass per area (dry mass divided by fresh area; g/m²) and leaf dry matter content (dry mass divided by fresh mass; dimensionless). Measure fresh leaf area (cm²) using a flatbed scanner (Canon, LIDE 220) at 300 pixels per inch resolution, then dry mass (g) using a digital balance after drying the leaf sample at 60°C for at least 72 hr. Remove petioles before analysis. For compound leaves, include all leaflets and rachis in the measurement.
Quantities: 267 leaves measured, scanning at 300 pixels per inch, drying at 60°C for minimum 72 hoursDuration: Fresh measurements immediately after collection, drying for minimum 72 hoursConditions: Laboratory conditions for scanning and weighing
Equipment: metre stick, flatbed scanner (Canon, LIDE 220), digital balance, drying oven
Measure leaf chemistry
Measure leaf nitrogen content (%) as a proxy of photosynthetic capacity, and δ¹³C as a proxy of water use efficiency, from dried leaf tissue. Grind dried leaf samples in liquid nitrogen and ground them to fine powder with a mortar and pestle. Send samples for elemental analysis at the University of California at Davis' Stable Isotope Facility using an elemental analyzer (Sercon, PDZ Europa ANCA-GSL) interfaced to an isotope ratio mass spectrometer (Sercon, PDZ Europa 20-20). Express δ¹³C values relative to international standard VPDB (Vienna PeeDee Belemnite).
Quantities: 267 leaf samples analyzed for nitrogen content and δ¹³CDuration: Sample preparation followed by laboratory analysisConditions: Laboratory conditions, samples ground in liquid nitrogen
Equipment: liquid nitrogen, mortar and pestle, elemental analyzer (Sercon, PDZ Europa ANCA-GSL), isotope ratio mass spectrometer (Sercon, PDZ Europa 20-20)
Measure leaf spectral properties
Measure leaf absorptance (dimensionless) using a field spectroradiometer (ASD, FieldSpec HandHeld 2) calibrated against white and black references before usage. Measure spectral reflectance (R) from 325 to 1,075 nm over three replicates per leaf. Estimate transmittance (T) at each wavelength from R = 2,500 nm via inversion of the PROSPECT-5B radiative transfer model after optimization to correspond with reflectance data from 400 to 1,075 nm. Calculate absorptance as the mean value of 1 - T - R across the spectral band 400-2,500 nm.
Quantities: Spectral measurements 325-1,075 nm, 3 replicates per leaf, 715/757 spectra retained after quality controlDuration: Field measurements with immediate calibrationConditions: Field conditions with white and black reference calibration
Equipment: field spectroradiometer (ASD, FieldSpec HandHeld 2), white and black references
Assess stomatal distribution
Measure stomatal distribution (dimensionless) via a stomatal peel (clear nail polish) of the abaxial and adaxial side of the leaf. Image each peel using a brightfield microscope (Leica, DM750) at 40x magnification. Score a leaf as hypostomatous if only the abaxial side showed guard cells, and amphistomatous if the abaxial and adaxial side showed guard cells.
Quantities: Stomatal peels on both leaf surfaces, imaging at 40x magnificationDuration: Laboratory analysis following field collectionConditions: Laboratory microscopy conditions
Equipment: clear nail polish, brightfield microscope (Leica, DM750)
Estimate canopy exposure
Estimate canopy exposure of the leaf by scoring each leaf as having a low, medium or high value (<2, <10 or >10 cm protrusion beyond the approximate upper canopy layer of the site). Calculate leaf width (cm) as the maximum breadth of the leaf. For compound or highly lobed leaves, measure the width of a leaflet or leaflet lobe when leaflets were highly dissected.
Quantities: Categorical scoring: <2 cm, <10 cm, or >10 cm protrusionDuration: Field assessment during leaf collectionConditions: Visual assessment in field conditions
Equipment: measuring tools for leaf width assessment