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).