Sub-functionalization of duplicated genes in the evolution of nine-spined stickleback hatching enzyme

Gene duplication is the primary source of novel genes, and is followed by non-, sub-, or neo-functionalization. In this study, we compared the egg envelope digestion mechanism of hatching enzymes between three-spined stickleback and nine-spined stickleback species, and found that the function of the hatching enzymes of nine-spined sticklebacks was uniquely derived by gene duplication, followed by sub-functionalization. The hatching enzyme of euteleosts consists of two metalloproteases, high choriolytic enzyme (HCE), and low choriolytic enzyme (LCE). LCE, especially, has an important role in solubilizing egg envelope protein by cleaving two specific sites. Three-spined stickleback had a single copy of the LCE gene, like other euteleosts. However, nine-spined stickleback had two types of LCE genes, α-type and β-type, suggesting that a duplication of the LCE gene occurred during the evolution of sticklebacks. The α-type and β-type each cleaved one of the two sites. Therefore, in the nine-spined stickleback, the function of the ancestral LCE was driven by a single copy gene, which was partitioned into two functions separately driven by two duplicated genes, and egg envelope was solubilized by the cooperative action of the two LCEs, α-type and β-type. Herein, we provide a molecular mechanism for an evolutionary adaptation driven by gene duplication and sub-functionalization.