2.3 Phylogenetic analyses of mitochondrial and nuclear markers
To roughly understand the genetic structure of parthenogenetic populations, we conducted phylogenetic analyses using the 5’ end of cytochrome oxidase I (cox 1) gene as a mitochondrial marker and the second intron of centrin gene (cetn -int2) which is a single copy nuclear gene in S. lomentaria (Nagasato et al., 2004). Gene amplification (PCR) and sequencing were performed as in Kogame et al. (2015a). Samples from p24 and p27contained double peaks in the PCR amplified region of their cetn -int2 gene, suggesting that these samples had multiple cetn -int2 haplotypes. Therefore, fusion cloning (Fu et al., 2014a) was performed to separate each sequence. Newly generated sequences (Table S1; cox 1: 207 sequences from 26 populations; cetn -int2: 98 sequences from 34 populations) were aligned with the previously generated sequences including those of related Scytosiphon species (Table S1; Kogame et al., 2015a; McDevit & Saunders, 2017; Hoshino et al., 2018, 2020c) using CLUSTAL W (Thompson et al., 1994) in MEGA v. 7 (Kumar et al., 2016) with default parameters. The cox 1 dataset was 600 bp in length, and sequences less than 600 bp were excluded. For the cetn -int2 dataset, gaps and highly variable regions were omitted using Gblocks web server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html; Castresana, 2000) and the final dataset consisted of 604 bp.
For the cox 1 dataset, phylogenetic relationships among the haplotypes were reconstructed using median-joining methods (Bandelt et al., 1999) in Network v. 5.0.1.0. For the cetn -int2 dataset, a maximum likelihood tree was constructed using RAxML version 8.2.10 (Stamatakis, 2014) with 1000 bootstrap pseudoreplicates under the GTR-GAMMA, through Cipres Science Gateway version 3.3 (Miller et al., 2010).