Sub‐Functionalization of Duplicated Genes in the Evolution of Nine‐Spined Stickleback Hatching Enzyme MARI KAWAGUCHI 1 *, HIROSHI TAKAHASHI 2 , YUSUKE TAKEHANA 3 , KIYOSHI NARUSE 3 , MUTSUMI NISHIDA 4 , AND SHIGEKI YASUMASU 1 1 Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Chiyoda‐kuTokyo, Japan 2 Department of Applied Aquabiology, National Fisheries University, Yamaguchi, Japan 3 Laboratory of Bioresources, National Institute for Basic Biology, Okazaki, Aichi, Japan 4 Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Chiba, Japan Gene and genome duplication have been proposed as primary driving forces in molecular evolution (Ohno, '70). It has also been suggested that one duplicated gene maintains the original function, while the other evolves a new function or is lost (Ohno, '70). This is known as the classical hypothesis of gene duplication and diversification. The modern hypothesis suggests additional evolutionary fates of the duplicated genes as follows (the first two correspond to the classical hypothesis): (1) non‐ functionalization through silencing mutation, (2) neo‐function- alization through gain of novel function, and (3) sub‐function- alization through the partitioning of ancestral function by ABSTRACT 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, a‐type and b‐type, suggesting that a duplication of the LCE gene occurred during the evolution of sticklebacks. The a‐type and b‐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, a‐type and b‐ type. Herein, we provide a molecular mechanism for an evolutionary adaptation driven by gene duplication and sub‐functionalization. J. Exp. Zool. (Mol. Dev. Evol.) 320B:140–150, 2013. © 2013 Wiley Periodicals, Inc. How to cite this article: Kawaguchi M, Takahashi H, Takehana Y, Naruse K, Nishida M, Yasumasu S. 2013. Sub‐functionalization of duplicated genes in the evolution of nine‐spined stickleback hatching enzyme. J. Exp. Zool. (Mol. Dev. Evol.) 320:140–150. J. Exp. Zool. (Mol. Dev. Evol.) 320B:140–150, 2013 Grant sponsor: JSPS KAKENHI; Grant number: 23870025, 24770231. Additional supporting information may be found in the online version of this article. The nucleotide data reported in the present paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers AB745718–745731. *Correspondence to: Mari Kawaguchi, Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7‐1 Kioi‐ cho, Chiyoda‐ku, Tokyo 102‐8554, Japan. E‐mail: k‐mari@sophia.ac.jp Received 10 October 2012; Revised 16 January 2013; Accepted 22 January 2013 Published online 1 April 2013 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/jez.b.22490 RESEARCH ARTICLE © 2013 WILEY PERIODICALS, INC.