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However, due to large number of exon/intron structure types of the grouper β -hsd genes, only a few genes shared similar structures. The functional domains of grouper β-HSD were predicted by the CD-Search (Marchler-Bauer et al., 2017) based on their protein sequences (Figure 2C). As shown in Figure 2C, 18 of the 19 grouper β-HSDs possessed an SDR superfamily domain, except Hsdl2, indicating a high level of conservation of β -hsd genes belonging to the SDR superfamily. In addition to the SDR superfamily domain, Hsd17b4 also contained two other domains: PLN02864 superfamily domain and SCP2 superfamily domain. Notably, Hsdl2 was a member of the SDR superfamily, but no SDR superfamily domain existed in grouper Hsdl2, according to the CDD prediction. Therefore, we also predicted the functional domain of Hsdl2 protein sequences of other species by CD-Search (Supplementary Figure S1). As shown in Supplementary Figure S1, six Hsdl2 protein sequences contained the SDR superfamily domain and did not contain the NADB_Rossmann superfamily domain, while the other eight Hsdl2 protein sequences, including grouper Hsdl2, were the opposite. To clarify the evolutionary relationships among β -hsd genes, a phylogenetic tree was constructed with amino acid sequences of 96 β-HSDs collected from grouper, human, zebrafish, stickleback, and tilapia. Both maximum likelihood (ML, Figure 3) and Neighboring-Joining method (NJ, Supplementary Figure S2) strategies were devoted to construct phylogenetic trees based on the alignment of their amino acid sequences. The trees produced by these two methods differed only in a small number of branches, indicating that the evolutionary tree is credible.
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