2.1 Sampling, DNA extraction, and taxonomic identity
Scytosiphon lomentaria gametophytes examined in this study were collected from Asia (33 localities in Japan; Figure 1; population code p1–p33), Europe [two localities in Ireland (Slea Head and Portrush) and one locality in Norway (Ona)], and South America [two localities in Argentina (Puerto Madryn and San Antonio Oeste); Table 1]. These samples include specimens collected for previous studies (Kogame et al., 2005; Kogame et al., 2015a; Kogame et al., 2015b; Hoshino et al., 2019, 2020c). For newly collected samples, a fragment of each specimen was preserved in silica-gel for molecular analyses and unialgal culture isolates were also established for some samples as previously described (Kogame et al., 2015a). Pressed specimens were made for all samples and deposited as vouchers in the Herbarium of Faculty of Science, Hokkaido University, Sapporo, Japan (SAP) or in the Herbarium of Universidad Nacional der Sur, Bahia Blanca, Argentina (BBB); herbarium acronyms follow Index Herbariorum (http://sweetgum.nybg.org/science/ih/). For molecular analyses, total genomic DNA was extracted from the cultured or silica gel-dried thalli using GenCheck® DNA Extraction Reagent (FASMAC, Atsugi, Japan) following Hoshino et al. (2020b) and used as template DNA for PCR.
Since six cryptic species of S. lomentaria have been reported in Japan (Hoshino et al., 2020c), all samples were screened by species-check PCR. In this PCR, the partial mitochondrial cox 1 gene is amplified only in S. lomentaria . Primers used and PCR conditions are described in Hoshino et al. (2019). The presence/absence of PCR products was determined by 1% agarose-gel electrophoresis.