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).