Plant material and growth conditions
Eighteen C4 grasses (Table 1) representing the three classical biochemical subtypes and 8 evolutionary lineages were selected for this study. However, the aim of our study was not to look at subtype or lineage effect but to maximize leaf physiological, structural and anatomical trait diversity. Each species was given a unique identification number (Table S1) for presentation in Fig. 1-5, 7 and S2-S5.
As previously described by Pathare et al ., (2020) plants were grown in 3-L free drainage pots in a controlled environment growth chamber (model GC-16; Enconair Ecological Chambers Inc., Winnipeg, MB, Canada). The photoperiod was 14 h including a 2 h ramp at the beginning and end of the light period. Light and dark temperatures were maintained at 26 and 22 °C, respectively. Light was provided by 400-W metal halide and high-pressure sodium lamps with maximum photosynthetic photon flux density (PPFD) of ca. 1000 µmol photons m-2s-1 at plant height. One individual per species was grown per pot in a Sunshine mix LC-1 soil (Sun Gro Horticulture, Agawam, MA, USA) with 6-7 replicate pots per species. The plants were irrigated daily to pot saturation and fertilized twice a week with Peters 20-20-20 (2.5 g L-1). Plants were supplemented with Spring 330 iron chelate (BASF, Ludwigshafen, Germany) and Scott-Peters Soluble Trace Element Mix (The Scotts Co., Marysville, OH, USA) once a week at concentrations of 10 mg L-1. Pot locations were randomized daily within the growth chamber.