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).