長久以來，基因重複被認為是產生新基因的一個重要機制，並能夠增加生物體的複雜性和生物間的歧異度。然而，雖然基因重複也被認為可能參與在種化的機制中，但截至目前為止，並沒有任何實際的例子，能讓我們研究有關基因重複和種化之間的關連。關於Odysseus (OdsH)的研究或許能為基因重複和種化的關聯性提供一個可能的模式。OdsH是第一個被發現與種化有關的基因，它會造成果蠅的二個姊妹種(D. mauritiana 和 D. simulans)的雜交雄性子代不孕。先前的研究發現，OdsH和另一個基因unc-4是由原本單一個unc-4發生基因重複而來。黃果蠅的unc-4表現在胚胎發育中的神經細胞、雄性成蟲的的精巢和雌性成蟲的卵巢，而OdsH則主要表現在雄性成蟲的精巢內，顯示這二個重複基因可能已有功能分歧的現象。我們進一步研究了unc-4的長期演化，以及在基因重複後，OdsH和unc-4在幾個不同種中，序列和表現上的分歧。我們的結果顯示，在擬灰果蠅中同時存在著unc-4和OdsH的同源基因，因此unc-4和OdsH的基因重複應發生於擬灰果蠅和黃果蠅種果亞群分化之前。我們並且依據序列置換估算出unc-4和OdsH的基因重複應發生在四千七百萬年前左右。unc-4基因在序列及表現上，都具有高度的保守性，相對的，OdsH不論在序列或者表現上，在各個種內的歧異度都相當高。在擬灰果蠅中，OdsH在胚胎亦表現在與unc-4相似的位置，而在黃果蠅種亞群中，OdsH喪失了在胚胎的表現，但在雄蟲的精巢內有大量的表現。從序列的分析上，我們也發現OdsH在黃果蠅種亞群中，演化的速率變快，這個現象可能與功能上的改變有關，並且暗示OdsH在黃果蠅種亞群中可能獲得了新的功能。因此，由OdsH序列的變異造成的雄性子代不孕的現象，可能是由於OdsH和unc-4在基因重複發生後，unc-4保留原來的功能，而OdsH因得到新的功能所造成的快速演化而來。

Gene duplication has been regarded as the most important mechanism for genetic novelties, biological divergence and complexity. Gene duplication is also suggested involving in speciation, however, there is no empirical studies showing the correlation between known speciation genes and duplication. Duplication of Odysseus (OdsH) and unc-4 in Drosophila possibly provide a bridge between the two. OdsH is the first defined hybrid male sterility gene between D. mauritiana and D. simulans, and it was recently duplicated from unc-4. Previous studies showed that in D. melanogaster, unc-4 is highly expressed in developing neurons, testes, and ovaries, and OdsH is mainly expressed in adult male testes, suggesting the two genes have undergone functional divergence. In this study, we further investigated the long-term evolution of unc-4 homologs and compared sequences and expression of OdsH and unc-4 in diverged species of Drosophila. Our results showed that there are two unc-4 homologs in D. pseudoobscura but only one in Anopheles gambiae, indicating the duplication was occurred before the divergence of D. pseudoobscura and D. melanogaster subgroup. Based on the nucleotide substitution, we showed that OdsH and unc-4 were duplicated about 47 Myr ago. unc-4 is conserved both in sequences and expression in Drosophila, while OdsH is very diverged. In D. pseudoobscura, OdsH is also expressed in embryo, similar to the pattern of unc-4, where as OdsH lost the expression in embryo, and switched to high expression in adult male testes in D. melanogaster. Evolutionary analysis also showed that OdsH is merely rapidly evolved within the D. melanogaster subgroup, possibly correlating to the expression switch and involving in new functions. The rapidly evolved OdsH gene duplicated from the conserved unc-4 is the first evidence for the relationship between gene duplication and speciation.

ALLENDORF, F. W., F. M. UTTER and B. P. MAY, 1975 Gene duplication within the family Salmonidae: II. Detection and determination of the genetic control of duplicate loci through inheritance studies and the examination of populations in Isozymes, pp. 415-32, edited by C. L. MARKERT. Academic Press, New York.
AMORES, A., A. FORCE, Y. L. YAN, L. JOLY, C. AMEMIYA et al., 1998 Zebrafish hox clusters and vertebrate genome evolution. Science 282: 1711-1714.
BLACK, J. A., and G. H. DIXON, 1968 Amino-acid sequence of alpha chains of human haptoglobins. Nature 218: 736-741.
BEVERLEY, S. M., and A. C. WILSON, 1984 Molecular evolution in Drosophila and the higher Diptera II. A time scale for fly evolution. J. Mol. Evol. 21: 1-13.
BRAUNITZER, G., R. GEHRING-MULLER, N. HILSCHMANN, K. HILSE, G. HOBORN et al., 1961 Die konstitution des normalen adulten human hamoglobinsr. A. Physiol. Chem. Hoppe Seyle 325: 283-286.
DOBZHANSKY, T., 1936 Studies on hybrid sterility. II. Localization of hybrid sterility factors in Drosophila pseudoobscura hybrids. Genetics 21: 113-135.
DUDA, T. F., JR., and S. R. PALUMBI, 1999 Molecular genetics of ecological diversification: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. Proc. Natl. Acad. Sci. USA 96: 6820-6823.
EDELMAN, G. M., R. MEECH, G. C. OWENS and F. S. JONES, 2000 Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. Proc. Natl. Acad. Sci. USA 97: 3038-3043.
FERRIS, S. D., and G. S. WHITT, 1979 Evolution of the differential regulation of duplicate genes after polyploidization. J. Mol. Evol.12: 267-317.
FISHER, R. A., 1935 The sheltering of lethals. Am. Nat. 69: 446-455.
FITCH, W. M., and E. MARGOLIASH, 1967 Construction of phylogenetic trees. A method based on mutation distances as estimated from cytochrome c sequences is of general applicability. Science 155: 279-284.
FORCE, A., M. LYNCH, F. B. PICKETT, A. AMORES, Y. L. YAN et al., 1999 Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151: 1531-1545.
GILBERT, W., 1978 Why gene in pieces? Nature 271: 50.
HALDANE, J. B. S., 1932 The Causes of Evolution. Longmans and Green, London.
HOLT, R. A., G. M. SUBRAMANIAN, A. HALPERN, G. G. SUTTON, R. CHARLAB et al., 2002 The genome sequence of the malaria mosquito Anopheles gambiae. Science 298: 129-149.
HUGHES, A. L., 1994 The evolution of functionally novel proteins after gene duplication. Proc. R. Soc. Lond. B. Biol. Sci. 256: 119-124.
HUGHES, A. L., 1999 Adaptive evolution of genes and genomes. Oxford University Press, New York.
HUGHES, A. L., J. A. GREEN, J. M. GARBAYO and R. M. ROBERTS, 2000 Adaptive diversification within a large family of recently duplicated, placentally expressed genes. Proc. Natl. Acad. Sci. USA 97: 3319-3323.
JEFFS, P. S., E. C. HOLMES and M. ASHBURNER, 1994 The molecular evolution of the alcohol dehydrogenase and alcohol dehydrogenase-related genes in the Drosophila melanogaster species subgroup. Mol. Biol. Evol. 11: 287-304.
KAPPEN, C., SCHUGHART, K., AND RUDDLE, F. H., 1989 Two steps in the evolution of Antennapedia-class vertebrate homeobox genes. Proc. Natl. Acad. Sci. USA 86: 5459-5463.
KIMURA, M., 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol.16: 111-120.
KMITA-CUNISSE, M., F. LOOSLI, J. BIERNE and W. J. GEHRING, 1998 Homeobox genes in the ribbonworm Lineus sanguineus: evolutionary implications. Proc. Natl. Acad. Sci. USA 95: 3030-3035.
KONIGSBERG, W., G. GUIDOTTI and R. J. HILL, 1961 The amino acid sequence of the α chain of human hemoglobin. J. Biol. Chem. 236: PC 55-56.
KUMAR, S., K. TAMURA, I. B. JAKOBSEN and M. NEI, 2001 MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17: 1244-1245.
LI, W. H., and D. GRAUR, 1997 Fundamentals of Molecular Evolution. 1st ed. Sinauer Associates, Sunderland, MA.
LONG, M., and C. H. LANGLEY, 1993 Natural selection and the origin of jingwei, a chimeric processed functional gene in Drosophila. Science 260: 91-95.
LONG, M., and K. THORNTON, 2001 Gene duplication and evolution. Science 293: 1551.
LYNCH, M., and J. S. CONERY, 2000 The evolutionary fate and consequences of duplicate genes. Science 290: 1151-1155.
LYNCH, M., and A. FORCE, 2000 The probability of duplicate gene preservation by subfunctionalization. Genetics 154: 459-473.
LYNCH, M., M. O'HELY, B. WALSH and A. FORCE, 2001 The probability of preservation of a newly arisen gene duplicate. Genetics 159: 1789-1804.
MANSOURI, A., Y. YOKOTA, R. WEHR, N. G. COPELAND, N. A. JENKINS et al., 1997 Paired-related murine homeobox gene expressed in the developing sclerotome, kidney, and nervous system. Dev. Dyn. 210: 53-65.
MAYR, E., 1942 Systematics and the Origin of Species. Columbia University Press, New York.
MILLER, D. M., M. M. SHEN, C. E. SHAMU, T. R. BURGLIN, G. RUVKUN et al., 1992 C. elegans unc-4 gene encodes a homeodomain protein that determines the pattern of synaptic input to specific motor neurons. Nature 355: 841-845.
MULLER, H. J., 1935 The origination of chromatin deficiencies as minute deletions subject to insertion elsewhere. Genetics 17: 237-252.
MULLER, H. J., 1940 Bearing of the Drosophila work on systematics in The New Systematics, edited by J. HUXLEY. Clarendon Press, Oxford.
MULLER, H. J., 1942 Isolating mechanisms, evolution and temperature. Biol. Symp. 6: 71-125.
MUNOZ-MARMOL, A. M., J. R. BAYASCAS-RAMIREZ, E. CASTILLO, A. CASALI and E. SALO, 1997 Planarian homeobox gene Dtprd-1 is expressed in specific gland cells, and belongs to a new family within the paired-like class. Dev. Genes Evol. 306: 316-1997.
NEI, M., and S. KUMAR, 2000 Molecular evolution and phylogenetics. Oxford University Press, New York.
NEI, M., and J. ZHANG, 1998 Molecular origin of species. Science 282: 1428-1429.
NURMINSKY, D. I., M. V. NURMINSKAYA, D. DE AGUIAR and D. L. HARTL, 1998 Selective sweep of a newly evolved sperm-specific gene in Drosophila. Nature 396: 572-575.
OHNO, S., 1970 Evolution by gene duplication. Springer, Berlin.
OHNO, S., 1973 Ancient linkage groups and frozen accidents. Nature 244: 259-262.
PALOPOLI, M. F., and C.-I WU, 1994 Genetics of hybrid male sterility between drosophila sibling species: a complex web of epistasis is revealed in interspecific studies. Genetics 138: 329-341.
PALOPOLI, M. F., and C.-I WU, 1996 Rapid evolution of a coadapted gene complex: evidence from the Segregation Distorter (SD) system of meiotic drive in Drosophila melanogaster. Genetics 143: 1675-1688.
PEREZ, D. E., and C.-I WU, 1995 Further characterization of the Odysseus locus of hybrid sterility in Drosophila: one gene is not enough. Genetics 140: 201-206.
PEREZ, D. E., C.-I WU, N. A. JOHNSON and M. L. WU, 1993 Genetics of reproductive isolation in the Drosophila simulans clade: DNA marker-assisted mapping and characterization of a hybrid-male sterility gene, Odysseus (Ods). Genetics 134: 261-275.
RHINESMITH, H. S., W. A. SCHROEDER and N. MARTIN, 1958 The N-terminal sequence of the α chains of normal adult human hemoglobin. J. Am. Chem. Soc. 80: 3358-3361.
ROVESCALLI, A. C., S. ASOH and M. NIRENBERG, 1996 Cloning and characterization of four murine homeobox genes. Proc. Natl. Acad. Sci. USA 93: 10691-10696.
ROZAS, J., and R. ROZAS, 1999 DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15: 174-175.
SAITO, T., L. LO, D. J. ANDERSON and K. MIKOSHIBA, 1996 Identification of novel paired homeodomain protein related to C. elegans unc-4 as a potential downstream target of MASH1. Dev Biol 180: 143-155.
SAITOU, N., and M. NEI, 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
SATOU, Y., N. TAKATORI, S. FUJIWARA, T. NISHIKATA, H. SAIGA et al., 2002 Ciona intestinalis cDNA projects: expressed sequence tag analyses and gene expression profiles during embryogenesis. Gene 287: 83-96.
SMITH, S. T., and J. B. JAYNES, 1996 A conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and msh-class homeoproteins, mediates active transcriptional repression in vivo. Development 122: 3141-3150.
SPRING, J., 1997 Vertebrate evolution by interspecific hybridisation - are we polyploid? FEBS Lett. 400: 2-8.
SWANSON, W. J., and V. D. VACQUIER, 1995 Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa. Proc. Natl. Acad. Sci. USA 92: 4957-4961.
TABUCHI, K., S. YOSHIKAWA, Y. YUASA, K. SAWAMOTO and H. OKANO, 1998 A novel Drosophila paired-like homeobox gene related to Caenorhabditis elegans unc-4 is expressed in subsets of postmitotic neurons and epidermal cells. Neurosci. Lett. 257: 49-52.
THROCKMORTON, L. H., 1975 The phylogeny, ecology and geography of Drosophila., pp. 421-469 in Handbook of genetics, edited by E. R. KING. Plenum, New York.
TING, C.-T., S. C. TSAUR and C.-I WU, 2000 The phylogeny of closely related species as revealed by the genealogy of a speciation gene, Odysseus. Proc. Natl. Acad. Sci. USA 97: 5313-5316.
TING, C.-T., S. C. TSAUR, M.-L. WU and C.-I WU, 1998 A rapidly evolving homeobox at the site of a hybrid sterility gene. Science 282: 1501-1504.
TING, C.-T., S. C. Tsaur, S. Sun, W. Browne, Y.-C. Chen, N. Patel, and C.-I Wu 2003 Gene duplication and speciation in Drosophila- evidence from the Odysseus locus. (submitted)
WAGNER, A., 2001 Birth and death of duplicated genes in completely sequenced eukaryotes. Trends. Genet. 17: 237-239.
WANG, W., F. G. BRUNET, E. NEVO and M. LONG, 2002 Origin of sphinx, a young chimeric RNA gene in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 99: 4448-4453.
WANG, W., J. ZHANG, C. ALVAREZ, A. LLOPART and M. LONG, 2000 The origin of the Jingwei gene and the complex modular structure of its parental gene, yellow emperor, in Drosophila melanogaster. Mol. Biol. Evol. 17: 1294-1301.
WHITE, J. G., E. SOUTHGATE and J. N. THOMSON, 1992 Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons. Nature 355: 838-841.
WHITFIELD, C. W., M. R. BAND, M. F. BONALDO, C. G. KUMAR, L. LIU et al., 2002 Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee. Genome Res. 12: 555-566.
WINNIER, A. R., J. Y. MEIR, J. M. ROSS, N. TAVERNARAKIS, M. DRISCOLL et al., 1999 UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans. Genes Dev. 13: 2774-2786.
WITTBRODT, J., A. MEYER and M. SCHARTL, 1998 More genes in fish? BioEssays 20: 511-512.
WU, C.-I, and M. F. PALOPOLI, 1994 Genetics of postmating reproductive isolation in animals. Annu. Rev. Genet. 28: 283-308.
ZHANG, J., H. F. ROSENBERG and M. NEI, 1998 Positive Darwinian selection after gene duplication in primate ribonuclease genes. Proc. Natl. Acad. Sci. USA 95: 3708-3713.