2.8 Phylogenetic construction and divergence time estimation
The phylogenetic relationships between M. japonicus and other
species were determined using 174 single-copy orthologous families.
Protein sequences in each single-copy family were aligned using MUSCLE
(version 3.8.31) with default parameters (Edgar et al., 2004) and a
super alignment matrix was constructed by concatenating the alignments.
Then, RAxML (version 8.0.19) (Stamatakis, 2014) was used to construct
the phylogenetic tree using the maximum-likelihood (ML) method with the
PROTGAMMAAUTO model for amino acid substitution. The MCMCTree program
implemented in the PAML package was used to estimate the divergence
times between the 21 species. Several calibration points applied in the
present study were retrieved from the TimeTree website
(http://timetree.org/ ), including D. rerio and C.
semilaevis (206-252 Mya), A. purpuratus and C. gigas(395-551 Mya), C. elegans and P. trituberculatus (623–877
Mya), D. melanogaster and P. trituberculatus (452-557
Mya), D. melanogaster and L. migratoria , P.
trituberculatus and C. quadricarinatus (281-410 Mya), P.
trituberculatus and P. monodon (271-430 Mya), F.
chinensis , and L. vannamei (55-108 Mya).