Rapidly Evolving Genes of Drosophila: Differing Levels of Selective Pressure in Testis, Ovary, and Head Tissues Between Sibling Species Santosh Jagadeeshan and Rama S. Singh Department of Biology, McMaster University, Hamilton, Ontario, Canada Investigations of rapidly evolving sex- and reproduction-related genes are expected to reveal important information about the process of speciation and species divergence. We screened testis, ovary, and head tissues to identify and characterize rapidly evolving genes (REGs) between closely related species. The results show differential patterns of evolution of genes expressed in reproductive and nonreproductive tissues. (1) There is a differential distribution of REGs in the Drosophila genome, with most REGs localized in the testis, followed by ovary, and then head. (2) Sequence analysis indicates that differential selective pressures are driving the rapid evolution of genes expressed in sex and nonsex tissues. Testis REGs from our data, on average, yielded higher rates of nonsynonymous substitutions relative to transcripts in ovary and head, indicating stronger selective pressures on the male reproductive system. (3) We identified REGs in the testis, ovary, as well as in head tissue that show evidence of evolving under positive selection. Identification of rapidly evolving sex genes is important for detailed investigations of cryptic female choice, sexual conflict, and faster male evolution and is pertinent to our understanding of the process of species divergence and speciation. Introduction About 30% of the Drosophila genome has been shown to be rapidly evolving (Werman, Davidson, and Britten 1990; Schmid and Tautz 1997). This fraction of the Dro- sophila genome still remains to be characterized systemat- ically, although some interesting trends have been revealed. Two-dimensional electrophoresis studies have suggested that a large proportion of this rapidly evolving fraction may be localized in the reproductive tissues (Coulthart and Singh 1988). In addition, many proteins in testis and ovary showed higher divergence over nonreproductive tissue proteins (Civetta and Singh 1995). Numerous recent comparative DNA studies have shown that several sex- and reproduction-related genes (SRR) evolve rapidly and often adaptively between closely related species (see Singh and Kulathinal 2000; Swanson et al. 2001a, 2004; Swanson and Vacquier 2002). This trend has been extended to transcrip- tional patterns of sex-biased genes between closely related species (Ranz et al. 2003). Male-biased genes show higher variation in expression patterns (Meiklejohn et al. 2003; Ranz et al. 2003). Whole-genome comparisons of Anoph- eles gambiae and Drosophila melanogaster (Parisi et al. 2003) and more recently of two Drosophila species di- verged about 35 MYA (Powell 1997) also support this trend. Together, these data reveal, at the molecular level, an important trend that had previously been reported at the morphological level (Eberhard 1985)––differential di- vergence of sex traits over nonsex traits. This differential divergence of sex versus nonsex genes conforms to evolu- tion by sexual selection (Darwin 1871; Eberhard 1985). The challenge remains, however, to understand how rapidly evolving genes (REGs) relate to important phenom- ena such as species divergence and speciation (Singh and Kulathinal 2000; Wu and Ting 2004). How do REGs affect reproductive isolation and hybrid sterility or inviability? The higher divergence of male and female genes could result from coadaptation (Miller and Pitnick 2002) or con- flict (Gavrilets 2000) driven by interacting male and female reproductive molecules. On the other hand, the higher divergence of male genes over female genes could imply male-driven evolution. The first step to resolve this issue is to identify genes that are rapidly evolving in both male and female reproductive systems and then to characterize their functions. It is also important to identify REGs in non- reproductive tissues such as head or brain, which may con- tribute to sexual behavior or affect other sensory stimuli. We have conducted a survey to systematically iden- tify, isolate, and investigate REGs in Drosophila species that have diverged for different lengths of time. We used complete cDNAs from testis, ovary, and head cDNA librar- ies to identify REGs and then sequenced candidate REGs from each tissue for further analysis. This allows us not only to dissect the rapidly evolving fraction of the Drosophila transcriptome but also to comprehensively study patterns of sequence evolution of REGs expressed in different tissues. We focused on genes that are evolving rapidly between closely related species of Drosophila, with an operational definition of an REG as a gene that shows relatively high divergence between two species separated for the least amount of time. In this contribution, we particularly focused on the sibling species pair D. melano- gaster and Drosophila simulans, diverged about 2.5–5 MYA (Powell 1997). We asked the following questions: (1) Do reproductive tissues have larger proportions of REGs? (2) If so, are there differences in the proportion of REGs be- tween the male and female reproductive systems? (3) Are they evolving neutrally or in response to selection? And most importantly, (4) What are these REGs? How do they affect the reproductive biology of Drosophila? Materials and Methods Screening for REGs We used a modification of the original screen of Schmid and Tautz (1997) to isolate fast-evolving genes from Drosophila. We procured D. melanogaster adult testis cDNA library from Justin Andrews (National Institutes of Diabetes and Digestive and Kidney Diseases of the Na- tional Institute of Health, Bethesda, Maryland, NIH), adult ovary cDNA library from Tulle Hazellrigg (Columbia University), and adult head cDNA library from Richard Key words: speciation, sexual selection, sex- and reproduction- related genes, rapidly evolving genes. E-mail: singh@mcmaster.ca. Mol. Biol. Evol. 22(9):1793–1801. 2005 doi:10.1093/molbev/msi175 Advance Access publication May 25, 2005 Ó The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org by guest on March 7, 2016 http://mbe.oxfordjournals.org/ Downloaded from