2.2 Sex ratio investigation
To reveal the geographical distribution of parthenogenetic (female-dominant) and sexual populations of S. lomentaria , sex ratios of the 38 populations (localities; Table 1) were investigated. Sex of S. lomentaria gametophytes, which is morphologically indistinguishable, was identified by sex-check PCR and/or crossing experiments. In the sex-check PCR, partial sequences of the sex-specific non-recombining region on the sex chromosome (female U chromosome and male V chromosome) of S. lomentaria were used as sex markers and were amplified as in Hoshino et al. (2019). The sex of each samples was confirmed by the presence/absence of PCR products in 1% agarose-gel electrophoresis. In the crossing experiments, gametes of each sample were crossed with male and female gametes from sexual populations using culture strains following the methods outlined in Hoshino et al. (2018).