Species selection and sample collection
We collected samples from 147 tropical forest species from 54 families
from lowlands up to pre-montane forests in the Republic of Panama
between February 2019 and January 2020. We also included two species ofAgave cultivated at the Smithsonian Tropical Research Institute’s
Santa Cruz plant growth facilities in Gamboa, and an invasive grass
common in forest edges. In Panama there is no evidence for seasonal
changes in heat tolerance for the species for which this has been tested
(Krause et al . 2010) as temperature seasonality is minimal. Fig.
S2 shows measured heat tolerance parameters through the collection
period, showing no indication of seasonal patterns. Species included
gymnosperms (5) but mostly consisted of angiosperms (145)—primarily
trees (129), but also palms (2), lianas and vines (5), large woody
shrubs (5), large forbs (3), and a grass (1) (Table S2). ForAcacia mangium , we measured phyllodes instead of leaves. The vast
majority of the species are native to Panama (134), but several common
non-native species were included (16), most of them ornamentals.
Sun-exposed outer canopy leaves were collected using (pole) pruners,
where possible from multiple individuals (n=1–4, mean 1.5). For each
species vouchers were collected and deposited at the herbarium of the
University of Panama (PMA). At Parque Nacional San Lorenzo, canopy
leaves were accessed with the aid of a construction crane maintained by
the Smithsonian Tropical Research Institute. CAM plants were collected
in the afternoon of sunny days to make sure that leaf acid content was
low, as vacuolar release of these acids from fully acidified tissues
during sample preparation can exacerbate leaf damage and significantly
lower estimates of heat tolerance (Krause et al . 2016). All other
species were collected in the morning to reduce the risk of sampling
heat-stressed or photoinhibited leaves—this included the facultative
CAM species Clusia minor and C. pratensis that were
sampled during the wet season when expressing C3.
Branches were enclosed in large opaque plastic bags with moist tissue
paper until processed in the laboratory in Panama City on the same day.
Chlorophyll a fluorescence protocol
Following Krause et al . (2010) we measured
Fv/Fm on leaf disks 24 hours after they
were incubated for 15 minutes in a temperature-controlled water bath,
using 8–12 incubation temperatures between 44 and 54°C (where necessary
58°C) , with a minimum of five leaf disks at each temperature. When
multiple individuals were sampled for a species, they were pooled, and
at each temperature a random set of leaf discs was used. Leaf surfaces
were first cleaned with distilled water. Leaf disks (typically 2 cm
diameter, or smaller when leaves were narrow) were wrapped into strips
of miracloth (Calbiochem, La Jolla, CA)—to avoid hypoxic
conditions—and then put into individual small zip-lock bags with small
glass rods at the bottom. The bags were placed in preheated water baths
(Lauda RM6/RMS circulating water bath or Lauda Alpha immersion
thermostat; Analytical Instruments, LLC, Golden Valley, MN, USA). After
15 minutes the miracloth was removed and the disks transferred to moist
tissue paper in petri dishes. Samples were allowed to recover for 24
hours at low (<10 µmol m–2s–1) photosynthetically active radiation to ensure
that the reductions in fluorescence yield were irreversible and not
transient in nature. We then measured the initial Chl afluorescence emission (F0), maximum fluorescence
(Fm), and recorded the ratio of variable
(Fm–F0) to maximum fluorescence
(Fv/Fm) after dark adaptation for 15
min. Measurements were made with a PAM-2000 and a mini-PAM fluorometer
(Walz GmbH, Effeltrich, Germany).