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).