Positive Darwinian selection drives the evolution of several female reproductive proteins in mammals Willie J. Swanson*†, Ziheng Yang‡, Mariana F. Wolfner*, and Charles F. Aquadro* *Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703; and ‡Department of Biology, University College London, 4 Stephenson Way, London NW1 2HE, United Kingdom Communicated by M. T. Clegg, University of California, Riverside, CA, December 20, 2000 (received for review May 15, 2000) Rapid evolution driven by positive Darwinian selection is a recur- neutral evolution, purifying selection, and positive diversifying rent theme in male reproductive protein evolution. In contrast, selection, respectively. This criterion has been used to demon- positive selection has never been demonstrated for female repro- strate rapid evolution of male reproductive proteins in a variety ductive proteins. Here, we perform phylogeny-based tests on three of invertebrate and vertebrate species (1–17). Recently, Wyckoff female mammalian fertilization proteins and demonstrate positive et al. (1) used the ␻ ratio and other criteria to demonstrate the selection promoting their divergence. Two of these female fertil- rapid evolution of male reproductive proteins in primates. In ization proteins, the zona pellucida glycoproteins ZP2 and ZP3, are their study, female reproductive proteins, including OGP and part of the mammalian egg coat. Several sites identified in ZP3 as ZP3, were placed within a control group of nonrapidly evolving likely to be under positive selection are located in a region genes expressed in a variety of tissues (1). To elucidate the previously demonstrated to be involved in species-specific sperm- selective forces underlying the rapid divergence of reproductive egg interaction, suggesting the selective pressure is related to proteins, it is important to analyze female, as well as male, male-female interaction. The results provide long-sought evidence reproductive proteins. for two evolutionary hypotheses: sperm competition and sexual The diversity of female reproductive proteins was previously conflict. analyzed by two-dimensional denaturing PAGE (24). Both fe- male and male reproductive proteins were more diverse than zona pellucida ͉ ZP3 ͉ fertilization ͉ sperm competition ͉ sexual conflict proteins from other tissues. However, because DNA sequences were unavailable for these proteins, it was not possible to test for ne of the striking features that has emerged from the study positive selection and therefore impossible to rule out that the of reproduction is diversity (1–17). Diversity has been divergence was simply caused by lack of constraint and neutral O drift. In the one case of a female reproductive protein for which observed in several morphological traits that are involved in ␻ ␻ Ͻ reproduction, including sperm (14) and reproductive organ (15) was estimated, an egg coat gene in abalone, was 1, morphology. Recently, male-derived molecules involved in re- indicating no signs of positive selection (25). Additional tests of production have been shown to be extraordinarily divergent neutrality for that gene, including methods presented herein and between closely related species (1–13). These molecules include analyses of a polymorphism survey, are consistent with that proteins involved in signaling between males and females (3–5), conclusion (52). However, the repetitive nature of the abalone fertilization (6–11), sperm chromosome condensation (1, 12), female protein suggested a hypothesis for the rapid evolution of and sex-specific transcription (16, 17). However, only the diver- its male ligand that did not require positive selection acting on gence of molecules from males has been shown to be driven by the female receptor (25) and was consistent with a previous positive selection. The divergence of female reproductive pro- model for the evolution of species-specific interaction of gametes teins has never been demonstrated to be promoted by selection. (6, 26). This finding is surprising because many of the models that Here we present an analysis suggesting that positive Darwin- account for rapid evolution of reproductive proteins are based ian selection promotes the divergence of three mammalian on male-female interaction and therefore would predict that female reproductive proteins (ZP3, ZP2, and OGP) from a female proteins also would be subjected to positive selection. variety of mammalian species. ZP3 is the primary species- These models include sperm competition, a process in which it specific binding site for sperm, and species-specifically induces has been demonstrated that females play a significant role (19, sperm to undergo the acrosome reaction (21). ZP2 binds acro- 20), and sexual conflict, a model that explicitly assumes adaptive some-reacted sperm and has been termed the secondary sperm receptor (22). The role of OGP in fertilization remains unclear, evolution of female proteins (2, 20). To examine whether female but estrous oviductal fluid, which would contain OGP, plays a reproductive molecules undergo adaptive evolution, we per- role in species-specific gamete interaction (28). For comparison, formed phylogeny-based statistical tests of positive selection on we reanalyzed the three male reproductive proteins (protamine three mammalian female reproductive proteins. Our goal was to 1, protamine 2, and transition protein 2) identified as being determine whether any female reproductive proteins were sub- under positive selection by Wyckoff et al. (ref. 1; see also refs. 12 ject to positive selection. Because such selection is most likely to and 29). Furthermore, to demonstrate the reliability of these new act on extracellular or surface proteins involved in male-female analyses, we perform calculations on two control proteins: (i) the (sperm-egg or oviduct) recognition (rather than on all repro- class I MHC glycoprotein, which is known to be subjected to ductive proteins) we concentrated on those reproductive pro- positive selection (thus serving as a positive control) and whose EVOLUTION teins that are known to play important roles in sperm-egg structure and variable regions are known (30) and (ii) a house- interaction [ZP2 and ZP3, vertebrate egg coat (zona pellucida) keeping gene, carbonic anhydrase I, for which there is no proteins; ref. 21] or that have been suggested to be involved in evidence of positive selection (thus serves as a negative control). fertilization [oviductal glycoprotein (OGP); ref. 22]. A stringent and unequivocal signal of positive Darwinian selection in molecular evolution is a significantly higher non- Abbreviations: ZP, zona pellucida; OGP, oviductal glycoprotein. synonymous (dN; amino acid replacing) than synonymous (dS; † ͞ To whom reprint requests should be addressed. Email: [email protected]. silent) substitution rate (23). The ratio of the two rates, dN dS, ␻ The publication costs of this article were defrayed in part by page charge payment. This denoted herein, measures the magnitude and direction of article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. selective pressure on a protein, with ␻ ϭ 1, Ͻ1, and Ͼ1 indicating §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.051605998 PNAS ͉ February 27, 2001 ͉ vol. 98 ͉ no. 5 ͉ 2509–2514 With the control proteins confirming the robustness of the with ␻ limited in the interval (0, 1). This is a flexible distribution, methodology used, we demonstrate that ZP3, ZP2, and OGP are but ␻ is limited to the interval (0, 1), where 0 indicates complete subjected to positive Darwinian selection. Furthermore, we constraint and 1 is the expectation under no selective constraint. identify specific residues of these proteins that may be the targets The alternative model M8 (beta and ␻) adds an extra class of ␻ of selection and are perhaps involved in species-specific gamete sites with estimated, so that a proportion p0 of sites come from ϭ Ϫ interaction. the beta distribution B(p, q) and the remaining sites (p1 1 ␻ p0) have a ratio estimated from the data that can be greater Materials and Methods than 1. M8 has two more parameters than M7, so d.f. ϭ 2. Sequences Analyzed. GenBank accession numbers (organisms) for Likelihood analyses using other models of variable ␻ ratios sequences used are as follows: among sites also were performed and produced consistent ZP2: M90366 (Homo sapiens), Y10690 (Macaca radiata), results with those presented here (data not shown). Y10767 (Callithrix jacchus), U05776 (Felis catus), D45064 (Sus The second major step of the analysis is to identify residues scrofa), D45069 (Canis familiaris), M34148 (Mus musculus), under positive selection when the likelihood ratio test suggests AB000929 (Rattus norvegicus). their presence. This is achieved by using the Bayes theorem to ZP3: M20026 (M. musculus), Y10823 (Rattus rattus), X56777 calculate the (posterior) probabilities that each site, given the (H. sapiens), S71825 (marmosets), X82639 (M. radiata), D45070 data at that site, are from the different ␻ classes (32, 33). Sites (C. familiaris), D45068 (F. catus), D45065 (S. scrofa). with a high probability of coming from the class with ␻ Ͼ 1 are OGP: U09550 (H. sapiens), M59903 (Papio hamadryas likely to be under positive selection. Positions predicted to be anubis), U87259 (Macaca mulatta), D16639 (Bos taurus), under positive selection for the MHC were mapped onto the U16719 (Ovis aries), U15048 (Mesocricetus auratus), U43490 (S. crystal structure