2.3 Heavy metal treatments
The concentrations of Cd (as CdCl2) and Cu (as CuCl2) used in this study were selected to induce significant effects in moss performance without causing its death, according to the available literature (e.g. Ares, Itouga, Kato, & Sakakibara, 2018; Carginale et al., 2004; Konno, Nakashima, & Katoh, 2010), and culturing trials carried out in our laboratory.
For S. cataractae, we cut approximately 50-70 clean gametophores from each population into small pieces with a razor blade, mixed each with 2 ml of DI water, and spread them in 4x4 cm pots containing a previously autoclaved 2:1 mixture of clay (Turface) and potting soil. We cultured a total of 60 pots (4 populations x 3 treatments x 5 replicates per population and treatment) for 3 months in the growth chamber, and watered the pots every two days with DI water. We applied the following treatments by watering the plants every two days for exactly 30 days with 20 ml of: water (control), 1 mM Cu (Cu), and 0.1 mM Cd (Cd) (n=4-5 replicates per population in each of 3 treatments).
For C. purpureus, we transplanted 7-day old protonema into new petri dishes overlaid with sterilized cellophane discs. Each plate contained BCD medium enriched with metals under the following treatments: control (C), 0.02 mM Cu (Cu), 0.01 mM Cd (Cd) (n=7 replicates per population and treatment). The levels of Cd and Cu in the treatments differed between species due to their obvious differences in tolerance. The treatment lasted 21 days and we took pictures of each replicate at the beginning and at the end of the experiment to measure the individual growth of each protonemal mat (n=5 mats per replicate).
In both experiments, we changed the position of the replicates within the chamber every week to minimize local microenvironmental effects. At the end of each common garden experiment, we harvested the plants, blotted them with filter paper, and separated several aliquots from each population and treatment in order to perform different analyses: several aliquots were immediately frozen in liquid N and stored at -80ºC for lipid peroxidation and microscopy analyses; one aliquot was kept in the oven at 50 ºC in plastic tubes for total Cd and Cu determination; one last aliquot was stored at 4ºC in the fridge to phenotype the plants (only in S. cataractae ).