2.6 Phylogenetic analysis and divergent time estimation
To define the phylogenetic tree, we identified the orthologous gene families by comparing the protein and cDNA sequences among the P. leopardus and nine teleosts, including Takifugu rubripes, Gasterosteus aculeatus, Oreochromis niloticus, Oryzias latipes, Danio rerio, Gadus morhua, Lepisosteus oculatus, Epinephelus lancelatus andEpinephelus akaara. TreeFam (v4.0) (Li et al., 2006) was used to define the orthologous and paralogous relationships among all the organisms. Using the single-copy orthologous genes, a phylogenetic tree was generated with Bayes model using PhyML (v3.0) (Guindon et al., 2010) with 500 bootstrap replications. The MCMCtree program implemented in the PAML package (Yang, 2007) was used to predict the divergence times. The divergent time of D. rerio and T. rubripes , G. aculeatus and T. rubripes , and O. latipes andG.morhua were used as calibration time, which was downloaded from the TimeTree database (http://www.timetree.org/).
Comparative genomic analyses
We compared the genome assembly of P. leopardus with the published genomes of grouper species, including E. lanceolatusand E. akaara . Firstly, to reveal their collinearity relationships, we aligned the chromosomes of the P. leopardus to that of the other Epinephelus species using the LASTZ tool (v1.02.00) with default options (Harris, 2007). The chromosomal collinearity were constructed with the mapped regions with lengths >2 Kb for visualizations using Circos (v0.69) (Krzywinski et al., 2009). Secondly, we identified the orthologous groups among these three species, using all-to-all Blast (E-value ≤1e-5, identity ≥ 80%) and identified the expanded and contracted gene families using CAFÉ (De Bie et al., 2006). The enrichment analyses based on GO and KEGG annotations were performed to identify functional implications of the expanded and contracted genes (Fisher’s exact test, adjusted p -value < 0.05).