2.2 Common garden experiments
We performed two common garden experiments to test separately the effect of Cd and Cu on DNA methylation and gene expression in S. cataractae . First, we clonally propagated each population in growth chambers at 22 ºC and 16h light/8h dark for several months (conditions maintained for all experiments); for this, we cleaned individual gametophores with deionized (DI) water and a brush under the dissection microscope, cut them into small pieces with a razor blade, and spread them into 4x4 cm pots containing a 2:1 mixture of clay (Turface) and commercial soil. Second, we selected 50-70 gametophores from each lab grown culture, cleaned them with DI water as explained above, cut them into small pieces, and spread them in 4x4 cm pots containing a previously autoclaved 2:1 mixture of clay and potting soil. We grew five replicates per population (n = 4) and treatment (n = 3) combination (n = 60 pots in total) for 3 months in a growth chamber watering the pots with DI water every two days. After this period, we applied the treatments by watering the plants every two days with 20 mL of water (controls), or water containing 1 mM Cu (Cu), or 0.1 mM Cd (Cd) (n=4-5 replicates per population and treatment). After 30 days, we harvested the plants, blotted them with filter paper and made several aliquots that were immediately frozen in liquid N and stored at -80ºC for DNA methylation and gene expression analyses. Other aliquots of these same samples were used for the phenotypic characterization (see68).