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.