*4.2. RNA Extraction*

Total RNA from *G. imbricata* samples was isolated, as previously described [30]. In short, total RNA was extracted separately from the apical regions (100 mg) of fertilised, fertile, and infertile *G. mbricate* thalli using 1 mL of Tri-Reagent (Sigma, St. Louis, MO, USA) pursuant to the manufacturer's instructions. The isolated RNA samples were suspended individually in 20 μL of 1 M Tris-HCl, pH 8, 0.5 M EDTA and treated with DNase (1 U mg<sup>−</sup>1; Promega, Madison, WI, USA) to destroy contaminating DNA. Total RNA was quantified in a TrayCell cuvette using a Beckman Coulter DU 530 spectrophotometer.

RNA (~1 μg from each sample) was reverse transcribed to first strand cDNA using an iScript Select cDNA Synthesis Kit (Bio-Rad; Hercules, CA, USA). The reverse transcriptase reaction was performed at 25 ◦C for 5 min, 42 ◦C for 30 min, and 85 ◦C for 5 min. The integrity of the cDNA was validated using a Nanodrop spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). The products were kept at 4 ◦C until used.
