Functional trait measurements
We quantified 13 physiological, morphological, and chemical traits for
each individual leaf. To do so, we executed 14-point photosynthetic
light-response curves on each leaf using a LI-6800 Portable
Photosynthesis System (Licor Bioscience, Lincoln, Nebraska, USA), to
measure area-based photosynthetic rates (A area;μ mol CO2 m-2s-1) across a range of photosynthetic photo flux
density (I ) levels (i.e., 2000, 1500, 1200, 1000, 800, 600, 400,
200, 100, 80, 60, 40, 20, and 0 μ mol photosynthetically active
radiation (PAR) m-2 s-1). All
photosynthetic rates were allowed to stabilize at each level of Ifor at least 120 seconds prior to data acquisition (Berry & Goldsmith,
2020; Salter, Merchant, Richards, Trethowan, & Buckley, 2019), with
each light response curve therefore taking a minimum of 30 minutes to
complete. All A area measurements were made
between 8:00-12:00 am to avoid mid-day stomatal closure, with leaf
chamber conditions maintained at CO2 concentrations of
400 ppm, relative humidity at 53.1-73.5%, leaf vapour pressure deficits
of 1.2-1.7 KPa, and leaf temperatures between 24.3-31.6 °C.
Physiological traits were calculated for each leaf by fitting
non-rectangular hyperbola to each of the 45 light response curves as:
\(A_{\text{area}}=R_{\text{area}}+\frac{\phi I+A_{\max}-\sqrt{\left(\phi I+A_{\max}\right)^{2}-4\theta\phi IA_{\max}}}{2\theta}\)Equation 1
where R area represents area-based leaf Rrates (μ mol CO2 m-2s-1), Φ is the apparent quantum yield of
photosynthesis (mol CO2 mol PPFD-1),A max is the light-saturated maximum area-based
photosynthetic rate, and θ represents a curvature parameter. From
these models, we also derived leaf-level light compensation points
(LLCP, μ mol PAR m-2 s-1),
calculated as I , where A area=0. Light
response curves were fitted using the ‘nls’ function in R v.3.3.3
statistical software (R Foundations for Statistical Computing, Vienna,
Austria).
After light response curves were completed in the field, each leaf was
immediately collected and transported to the University of Toronto
Scarborough for morphological and chemical trait determinations. First,
leaf area (cm2) was measured using a LI-3100C leaf
area meter (Licor Bioscience, Lincoln, Nebraska, USA), and all leaves
were then dried at 65 °C to constant mass and weighed (g). We then
calculated LMA (g m-2) as leaf mass/leaf area, and
used these LMA values to calculate maximum mass-based photosynthetic
(A mass) and mass-based dark respiration rates
(R mass), as A max orR area / LMA, respectively. Lastly, dried leaf
tissue was ground into a fine powder using a MM400 Retsch ball mill
(Retsch Ltd., Hann, Germany), and ~0.1 grams of tissue
was weighed and analyzed for C and N concentrations (both on a % mass
basis) on a LECO CN 628 elemental analyzer (LECO Instruments, Ontario,
Canada).