Interspecific hybrid lethality and sterility are a consequence of divergent evolution between species and serve to maintain the discrete identities of species. The evolution of hybrid incompatibilities has been described in widely accepted models by Dobzhansky and Muller where lineage-specific functional divergence is the essential characteristic of hybrid incompatibility genes. Experimentally tractable models are required to identify and test candidate hybrid incompatibility genes. Several  Drosophila melanogaster  genes involved in hybrid incompatibility have been identified but none has yet been shown to have functionally diverged in accordance with the Dobzhansky-Muller model. By introducing transgenic copies of the X-linked  Hybrid male rescue (Hmr)  gene into  D. melanogaster  from its sibling species  D. simulans  and  D. mauritiana,  we demonstrate that  Hmr  has functionally diverged to cause F1 hybrid incompatibility between these species. Consistent with the Dobzhansky-Muller model, we find that  Hmr  has diverged extensively in the  D. melanogaster  lineage, but we also find extensive divergence in the sibling-species lineage. Together, these findings implicate over 13% of the amino acids encoded by  Hmr  as candidates for causing hybrid incompatibility. The exceptional level of divergence at  Hmr  cannot be explained by neutral processes because we use phylogenetic methods and population genetic analyses to show that the elevated amino-acid divergence in both lineages is due to positive selection in the distant past—at least one million generations ago. Our findings suggest that multiple substitutions driven by natural selection may be a general phenomenon required to generate hybrid incompatibility alleles.Hybrid male rescue (Hmr)D. mauritiana,HmrHmrHmrHmr

Introduction Reproductive isolation is the most commonly used criterion to define species. Hybrid incompatibilities (HIs) such as hybrid sterility and lethality are widely observed examples of reproductive isolation. The Dobzhansky-Muller (D-M) model explains how the genes causing deleterious phenotypes in hybrids can evolve ( Dobzhansky 1937 ;  Muller 1942 ;  Turelli and Orr 2000 ) ( Figure 1 A). The model holds that HIs arise from the interaction between two or more genes that have evolved independently in two isolated populations; the deleterious phenotypes caused by these genes are a by-product of intraspecific divergence and will occur only when the genes interact in the interspecific hybrid. The essential criterion for defining HI genes, therefore, is that the alleles from the two species have distinct phenotypic properties in hybrids: for example, in  Figure 1 A the derived allele  A  from one species causes the incompatibility while the ancestral allele  a  from the other species does not. Figure 1 Models of Hybrid Incompatibility (A) D-M model of HI evolution. We diagram here an X–autosome incompatibility; for simplicity only haploid genotypes are shown. This model can be easily extended to include more complex multilocus interactions. (1) The ancestral species is fixed for the X-linked allele  x  and the autosomal allele  a . (2) As the two species independently diverge, one becomes fixed for allele  X  at the first locus and the other for allele  A  at the second locus. (3) HI is caused by the interaction between these derived alleles,  X  and  A.  (4) This interaction may cause misregulation of downstream effector genes  (d1, d2,  and  d3),  which in turn causes the HI phenotype. (B)  Hmr  is an HI gene.  Hmr mel  has evolved in the  D. melanogaster  lineage and interacts to cause HI with  A sib ,  an allele of a hypothesized autosomal gene that has evolved in the sibling-species lineage. Mutations in  Hmr mel  allow hybrid viability by eliminating the activity of this incompatibility allele. (C)  Hmr  is a downstream effector gene. Here, two unknown genes cause HI by misregulating  Hmr.  Mutations in  Hmr  allow hybrid viability by acting as downstream suppressors of the HI alleles. (D) Model with  Hmr  and gene  A  extensively diverging (see  Discussion ). Both  Hmr  and gene  A  coevolve with many changes along both lineages. HI could be caused by interactions between derived alleles or between a derived and an ancestral allele. All models to identify the codons in  Hmr  responsible for functional divergence have two constraints: first, that  Hmr  and gene  A  must be fully compatible with each other in each lineage, and second, that candidate codons must differ between  Hmr sib  from all three sibling species and  Hmr mel .
 This model makes clear and testable genetic predictions, namely, that experimental manipulation of allele  A  (or  X ) but not allele  a  (or  x ) will affect the HI phenotype. For example, increasing the dosage or activity of allele  A  should decrease hybrid fitness, while identical manipulations of allele  a  should not. In contrast, downstream effector genes such as  d1  in  Figure 1 A contribute to the phenotype of HI but are not expected to have functionally diverged alleles in the two hybridizing species; in other words, experimental manipulation of downstream effector alleles from either species will have equivalent effects on the HI phenotype. These alternative possibilities can only be addressed by genetically manipulating each species allele in a controlled hybrid background, but this has yet to be achieved in model organisms such as  Drosophila melanogaster.
 If the genetic changes that cause HI are rare, then genes that have undergone extensive divergence may be more likely to cause HI simply because there is more chance that they have experienced rare HI-causing mutations. But the D-M model itself offers no suggestions about the mode of evolution that leads to this divergence. One possibility is that HI genes accumulate genetic changes over time as they evolve neutrally. Alternatively, many models have suggested that speciation may be driven by molecules that are undergoing natural selection, for example, under the pressure of ecological divergence ( Schluter 2001 ) or sexual selection ( Lande 1981 ;  Parker and Partridge 1998 ). If genes associated with HI evolve under positive selection, one consequence is that they may be disproportionately X-linked, as positively selected, recessive X-chromosome alleles may go to fixation more quickly and therefore cause X-linked loci to evolve more rapidly relative to autosomal loci ( Charlesworth et al. 1987 ). This preferential X-linkage may contribute to Haldane's rule ( Haldane 1922 ), the observation that heterogametic (e.g., XY) hybrids suffer more incompatibilities than homogametic (XX) hybrids. A second consequence of selection is that HI genes may have unique phylogenetic and population genetic signatures ( Wang et al. 1997 ;  Ting et al. 2000 ). Molecular evolutionary analyses of speciation genes are essential to address the role of selection in reproductive isolation and speciation.
 D. melanogaster  can hybridize with three closely related species that we refer to collectively as its sibling species:  D. simulans, D. mauritiana,  and  D. sechellia.  Crosses between  D. melanogaster  females and sibling-species males result in invariantly lethal hybrid sons and temperature-dependent lethal hybrid daughters ( Sturtevant 1920 ;  Lachaise et al. 1986 ;  Barbash et al. 2000 ). The  Hybrid male rescue (Hmr)  gene has a major effect on the fitness of hybrids from this cross. This point is most strikingly demonstrated by the fact that  D. melanogaster Hmr  loss-of-function mutations such as  Hmr 1  suppress the lethality of both hybrid males and females ( Hutter and Ashburner 1987 ;  Barbash et al. 2000 ), as does the X-linked  In(1)AB  rescue mutation ( Hutter et al. 1990 ). Increasing the dosage of the wild-type gene  Hmr +  has the reciprocal property of decreasing hybrid viability ( Barbash et al. 2000 ;  Orr and Irving 2000 ). These studies led to the proposal that HI is caused by an interaction between the X-linked  D. melanogaster Hmr +  gene and an unknown autosomal gene(s) from the sibling species.  Barbash and colleagues (2003)  recently cloned the  Hmr  gene, which encodes a predicted DNA-binding protein similar to the ADF and MYB family of transcriptional regulators, and proposed that  D. melanogaster  HI may be caused by transcriptional misregulation. A limitation of previous genetic analyses of  Hmr  is that all genetic manipulations were done only with the  D. melanogaster  allele. These studies therefore cannot determine whether  Hmr  is an HI gene as modeled by Dobzhansky and Muller ( Figure 1 B) or is instead a downstream effector gene that suppresses hybrid lethality because it interacts with or is regulated by the actual HI genes ( Figure 1 C). Similar uncertainties apply to the  D. melanogaster Zhr  and  Nup96  genes ( Sawamura et al. 1993 ;  Presgraves et al. 2003 ), which also affect F1 hybrid viability. A preliminary analysis suggested that  Hmr  is highly diverged between  D. melanogaster  and the sibling species, with almost 8% divergence at nonsynonymous (amino-acid replacement) sites, a remarkable finding considering that  Hmr  encodes a predicted protein over 1,400 amino acids in length. Two other  Drosophila  genes that are involved in HI show elevated divergence that appears to have evolved under positive selection, but the divergence is confined to a small region of each gene ( Ting et al. 1998 ;  Presgraves et al. 2003 ) and  Hmr  is two times more diverged than either locus. These differences raise the question of whether the extensive divergence of  Hmr  may instead have evolved neutrally, or whether there are regional differences in selection and divergence at  Hmr.
 In this study we use transgenic assays to test whether  Hmr  has functionally diverged between  D. melanogaster  and its sibling species and thus fits the D-M model of HI evolution. We examine the functional consequences of this divergence and discuss models to determine which lineages and which codons have undergone functional divergence. We also test whether  Hmr  has evolved by positive selection and determine the number and locations of regions and codons that show particularly strong signals of adaptive evolution, asking whether the patterns of polymorphism at  Hmr  are consistent with ancient selection events.
Introduction
Reproductive isolation is the most commonly used criterion to define species. Hybrid incompatibilities (HIs) such as hybrid sterility and lethality are widely observed examples of reproductive isolation. The Dobzhansky-Muller (D-M) model explains how the genes causing deleterious phenotypes in hybrids can evolve ( Dobzhansky 1937 ;  Muller 1942 ;  Turelli and Orr 2000 ) ( Figure 1 A). The model holds that HIs arise from the interaction between two or more genes that have evolved independently in two isolated populations; the deleterious phenotypes caused by these genes are a by-product of intraspecific divergence and will occur only when the genes interact in the interspecific hybrid. The essential criterion for defining HI genes, therefore, is that the alleles from the two species have distinct phenotypic properties in hybrids: for example, in  Figure 1 A the derived allele  A  from one species causes the incompatibility while the ancestral allele  a  from the other species does not.AaThis model makes clear and testable genetic predictions, namely, that experimental manipulation of allele  A  (or  X ) but not allele  a  (or  x ) will affect the HI phenotype. For example, increasing the dosage or activity of allele  A  should decrease hybrid fitness, while identical manipulations of allele  a  should not. In contrast, downstream effector genes such as  d1  in  Figure 1 A contribute to the phenotype of HI but are not expected to have functionally diverged alleles in the two hybridizing species; in other words, experimental manipulation of downstream effector alleles from either species will have equivalent effects on the HI phenotype. These alternative possibilities can only be addressed by genetically manipulating each species allele in a controlled hybrid background, but this has yet to be achieved in model organisms such as  Drosophila melanogaster.AXaxAad1Drosophila melanogaster.If the genetic changes that cause HI are rare, then genes that have undergone extensive divergence may be more likely to cause HI simply because there is more chance that they have experienced rare HI-causing mutations. But the D-M model itself offers no suggestions about the mode of evolution that leads to this divergence. One possibility is that HI genes accumulate genetic changes over time as they evolve neutrally. Alternatively, many models have suggested that speciation may be driven by molecules that are undergoing natural selection, for example, under the pressure of ecological divergence ( Schluter 2001 ) or sexual selection ( Lande 1981 ;  Parker and Partridge 1998 ). If genes associated with HI evolve under positive selection, one consequence is that they may be disproportionately X-linked, as positively selected, recessive X-chromosome alleles may go to fixation more quickly and therefore cause X-linked loci to evolve more rapidly relative to autosomal loci ( Charlesworth et al. 1987 ). This preferential X-linkage may contribute to Haldane's rule ( Haldane 1922 ), the observation that heterogametic (e.g., XY) hybrids suffer more incompatibilities than homogametic (XX) hybrids. A second consequence of selection is that HI genes may have unique phylogenetic and population genetic signatures ( Wang et al. 1997 ;  Ting et al. 2000 ). Molecular evolutionary analyses of speciation genes are essential to address the role of selection in reproductive isolation and speciation.D. melanogaster  can hybridize with three closely related species that we refer to collectively as its sibling species:  D. simulans, D. mauritiana,  and  D. sechellia.  Crosses between  D. melanogaster  females and sibling-species males result in invariantly lethal hybrid sons and temperature-dependent lethal hybrid daughters ( Sturtevant 1920 ;  Lachaise et al. 1986 ;  Barbash et al. 2000 ). The  Hybrid male rescue (Hmr)  gene has a major effect on the fitness of hybrids from this cross. This point is most strikingly demonstrated by the fact that  D. melanogaster Hmr  loss-of-function mutations such as  Hmr 1  suppress the lethality of both hybrid males and females ( Hutter and Ashburner 1987 ;  Barbash et al. 2000 ), as does the X-linked  In(1)AB  rescue mutation ( Hutter et al. 1990 ). Increasing the dosage of the wild-type gene  Hmr +  has the reciprocal property of decreasing hybrid viability ( Barbash et al. 2000 ;  Orr and Irving 2000 ). These studies led to the proposal that HI is caused by an interaction between the X-linked  D. melanogaster Hmr +  gene and an unknown autosomal gene(s) from the sibling species.  Barbash and colleagues (2003)  recently cloned the  Hmr  gene, which encodes a predicted DNA-binding protein similar to the ADF and MYB family of transcriptional regulators, and proposed that  D. melanogaster  HI may be caused by transcriptional misregulation.D. simulans, D. mauritiana,D. sechellia.Hybrid male rescue (Hmr)D. melanogaster HmrHmr 1In(1)ABHmr +D. melanogaster Hmr +HmrA limitation of previous genetic analyses of  Hmr  is that all genetic manipulations were done only with the  D. melanogaster  allele. These studies therefore cannot determine whether  Hmr  is an HI gene as modeled by Dobzhansky and Muller ( Figure 1 B) or is instead a downstream effector gene that suppresses hybrid lethality because it interacts with or is regulated by the actual HI genes ( Figure 1 C). Similar uncertainties apply to the  D. melanogaster Zhr  and  Nup96  genes ( Sawamura et al. 1993 ;  Presgraves et al. 2003 ), which also affect F1 hybrid viability.HmrHmrD. melanogaster ZhrNup96A preliminary analysis suggested that  Hmr  is highly diverged between  D. melanogaster  and the sibling species, with almost 8% divergence at nonsynonymous (amino-acid replacement) sites, a remarkable finding considering that  Hmr  encodes a predicted protein over 1,400 amino acids in length. Two other  Drosophila  genes that are involved in HI show elevated divergence that appears to have evolved under positive selection, but the divergence is confined to a small region of each gene ( Ting et al. 1998 ;  Presgraves et al. 2003 ) and  Hmr  is two times more diverged than either locus. These differences raise the question of whether the extensive divergence of  Hmr  may instead have evolved neutrally, or whether there are regional differences in selection and divergence at  Hmr.HmrHmrDrosophilaHmrHmrHmr.In this study we use transgenic assays to test whether  Hmr  has functionally diverged between  D. melanogaster  and its sibling species and thus fits the D-M model of HI evolution. We examine the functional consequences of this divergence and discuss models to determine which lineages and which codons have undergone functional divergence. We also test whether  Hmr  has evolved by positive selection and determine the number and locations of regions and codons that show particularly strong signals of adaptive evolution, asking whether the patterns of polymorphism at  Hmr  are consistent with ancient selection events.HmrHmrHmrResults Testing  Hmr  for Functional Divergence In the absence of available  Hmr  mutations in the sibling species, we chose an alternative approach to test whether  Hmr  has functionally diverged by introducing cloned copies of  D. simulans  and  D. mauritiana Hmr +  into  D. melanogaster  ( Figure 2 ). It has been shown previously that increasing  Hmr +  activity using  P -element transgenes containing  D. melanogaster Hmr +  suppresses the hybrid-rescuing activity of  Hmr 1  and  In(1)AB  mutations ( Barbash et al. 2003 ). Those experiments can be interpreted as showing that the wild-type  D. melanogaster Hmr +  kills hybrids, with the  Hmr 1  and  In(1)AB  mutations rescuing hybrids by reducing or eliminating  Hmr +  activity. The transgenic copies of  D. melanogaster Hmr +  thus suppress rescue by increasing  Hmr +  activity in hybrids. We therefore asked whether or not sibling-species  Hmr +  has the same activity. We reasoned that if  Hmr  is functionally diverged between the species, as in  Figure 1 B, then transgenes containing sibling-species  Hmr +  would not have the property of suppressing hybrid rescue. On the other hand, if  Hmr  is not functionally diverged, as in  Figure 1 C, then these sibling-species constructs would have phenotypic properties similar to the  D. melanogaster Hmr +  transgenes and suppress hybrid rescue. Figure 2 Structure and Expression of Sibling-Species  Hmr +  Transgenes (A) Diagram of  Hmr +  transgenic constructs. The  Hmr  gene structure is shown, with the rightward arrow indicating the predicted translation start site. Sibling-species constructs used in this study are shown, together with  D. melanogaster  constructs previously shown ( Barbash et al. 2003 ) to be  Hmr + .
 (B) Restriction map of RT-PCR fragments spanning part of exons 3 and 4, showing diagnostic restriction site polymorphisms found in the transgenic alleles and the stocks used to assay them. The  D. melanogaster  map corresponds to both the  Hmr 1  and  In(1)AB  rescue alleles, as well as all  D. melanogaster  alleles from our population sample. (C) RT-PCR products from interspecific hybrids. Hybrids were from the crosses described in  Table 1 . RNA was collected from 48- to 72-h-old larvae and 2- to 4-d-old adult males. Note that larval samples contain RNA from males and females, half of whom carry the sibling-species  Hmr +  transgene. The portion of the PCR product derived from the transgenes is that digested by XbaI or HpaI for the  D. simulans  and  D. mauritiana  transgenes, respectively. M, 100-bp ladder marker; G, undigested PCR from an  Hmr  genomic clone (this product contains a 59-bp intron); cD, undigested PCR from an  Hmr  cDNA clone. The following are all RT-PCR products: U, undigested; C, ClaI-digested; H, HindIII-digested; X, XbaI-digested; C/X, ClaI- and XbaI-digested; Hp, HpaI-digested; C/Hp, ClaI- and HpaI-digested; –, control containing no reverse transcriptase. Note that undigested lanes (U) contain half the amount of DNA as digested samples. Table 1 Viability of Hybrid Males Carrying  D. simulans  or  D. mauritiana Hmr +  Transgenes
 D. melanogaster / D. simulans  hybrids were from crosses at 25 °C of  In(1)AB,w/FM6,w; P{Hmr
 + }/+ females to  D. simulans
 w / Y  males, except for  In(1)AB,w/FM6,w; P{Dsim\Hmr +8.6 }2-4 /+ females, which were crossed to  D. simulans
 w m/Y  males.  D. melanogaster / D. mauritiana  hybrids were from crosses at 22 to 23 °C of  w Hmr 1  v/FM6,w; P{Hmr + } /+ females to  D. mauritiana  iso-207-males. The transgenes carried a copy of the w +  gene so that  P[Hmr + ] /+ hybrids were distinguished from +/+ hybrids by their w +  eye color To maximize the chances that the sibling-species transgenic lines would function normally, we made constructs that are similar to the largest  D. melanogaster Hmr +  transgenic construct and that exceed the minimal  Hmr +  region previously defined (see  Figure 2 A). We assayed two independent transformants each of  D. simulans Hmr +  and  D. mauritiana Hmr +  for suppression of hybrid male rescue by  Hmr 1  in  D. melanogaster / D. mauritiana  hybrids and by  In(1)AB  in  D. melanogaster / D. simulans  hybrids ( Table 1 ). In all cases hybrid males heterozygous for a sibling-species transgene were at least as viable as their brothers without the transgene. Some crosses showed an excess of transgene-carrying hybrids, which might suggest that the transgenes actually increase the effectiveness of hybrid rescue, perhaps by interfering with the pathway of  Hmr + -dependent lethality. This possibility requires further investigation, but we note that such a hypothetical effect must be minor because the sibling-species  Hmr +  transgenes by themselves do not rescue hybrid males. We also assayed the  D. simulans Hmr +  transformants for suppression of  In(1)AB -dependent rescue of hybrid female sterility ( Barbash and Ashburner 2003 ). In contrast to the complete suppression associated with  D. melanogaster Hmr +  transgenes ( Barbash et al. 2003 ), we found that our  D. simulans Hmr +  transgenes had little or no effect on egg counts in  D. melanogaster / D. simulans  hybrids.  In(1)AB,w/X sim ,w m  females heterozygous for the insertion  P{Dsim\Hmr +t8.6 }2-4  averaged 10.0 ± 8.9 eggs ( n  = 41) while their non-transgene-carrying sisters averaged 8.0 ± 12.4 eggs ( n  = 28). Using a second, independent transgenic line, we found that  In(1)AB,w/X sim ,w  females heterozygous for the insertion  P{Dsim\Hmr +t8.6 }4-1  had 12.2 ± 13.7 eggs ( n  = 24) while their non-transgene-carrying sisters had 17.7 ± 17.3 eggs ( n  = 31). RT-PCR analysis demonstrated that the  Hmr +  transgenes are expressed ( Figure 2 C). These results show that the sibling-species alleles of  Hmr +  have no phenotypic effect in species hybrids and strongly support the conclusion that  Hmr  has functionally diverged between the  D. melanogaster  and sibling-species lineages, demonstrating that  Hmr  meets the criteria for being a D-M HI gene as diagrammed in  Figure 1 B.
 Hmr  Divergence among  Drosophila  Lineages It has been shown previously that  Hmr  has a high level of average divergence per nonsynonymous (amino-acid replacement) site  (D N )  between  D. melanogaster  and its three sibling species ( Barbash et al. 2003 ). To understand this divergence in the context of genome-wide evolution, we calculated divergence of  Hmr  between  D. melanogaster  and  D. simulans  and compared it to compiled datasets containing over 250 genes from these two species ( Begun 2002 ;  Betancourt and Presgraves 2002 ). Pairwise comparisons revealed that  Hmr  has one of the highest levels of nonsynonymous divergence (0.089); only four other non– Accessory gland protein (Acp)  genes have a higher nonsynonymous divergence, and two of these include expressed sequence tag comparisons less than 350 bases in length. The remaining two loci, both X-linked, are  mei-218  and  Odysseus . In contrast, the average divergence per synonymous site  (Ds)  of 0.110 for  Hmr  is lower than the mean value of 0.125 for this dataset (again excluding  Accessory gland protein  genes). The exceptional divergence of  Hmr  raises a number of important questions: (1) Is divergence high on the lineage of either  D. melanogaster  or its sibling species, or both? (2) Was the divergence on either or both lineages caused by positive selection consistent with the time scale of speciation? (3) Can we identify specific regions and codons subject to positive selection on these lineages, and how does this compare to the few other known candidate speciation genes? (4) Is divergence on one or both lineages potentially responsible for causing the HI phenotype of  Hmr ? We first addressed whether  Hmr  began to diverge rapidly after the  D. melanogaster –sibling-species divergence by isolating and assembling an outgroup  Hmr  orthologous sequence from the  D. melanogaster  subgroup species  D. erecta , which is estimated to have diverged from  D. melanogaster  between 6 and 15 million years ago ( Powell 1997 ). The maximum-likelihood estimate of  D N  between  D. melanogaster  and  D. erecta Hmr  was 0.166, higher than the mean value of 0.057 in a survey of 53  D. erecta  genes ( Bergman et al. 2002 ). However, the  D N / D S  ratio was 0.556, consistent with a more selective constraint on nonsynonymous sites than on synonymous sites. In contrast, we found that the  D. melanogaster  lineage and the lineage leading to the sibling species both exhibited elevated levels of nonsynonymous divergence relative to synonymous divergence ( Figure 3 ). This observation suggests that the rate of amino-acid evolution at  Hmr  accelerated after the divergence of  D. melanogaster  from its sibling species. We also detected accelerated divergence along the sibling-species lineages, but because of the trichotomy for  D. simulans, D. sechellia,  and  D. mauritiana , these branch lengths have little confidence. Furthermore, this divergence appears to be irrelevant with respect to the HI phenotype because we showed above that transgenic copies of  Hmr +  from both  D. simulans  and  D. mauritiana  have no effect on hybrid viability ( Table 1 ). Figure 3 Maximum-Likelihood Estimates of  Hmr  Divergence among  Drosophila  Lineages Estimates of the number of changes per nonsynonymous site  (D N )  are shown above each lineage, and the number of changes per synonymous site  (D S )  are shown below each lineage, calculated separately for each branch. The coding region of  Hmr  is 1,390 to 1,427 amino acids long in the five species.  D N / D S  ratios differ significantly among branches as tested by the methods of  Nielsen and Yang (1998) . A model where all  D N / D S  ratios were free to vary along all branches (Model 2 [M2]) fit the data better than a model with a fixed  D N / D S  ratio for all branches (M1) (2Δ| = 308,  p  < 0.0001, chi-square distribution), as did a model where  D N /D S  ratios for the  D. melanogaster  lineage and the lineage leading to the sibling species differed from the rest of the tree (local clock, 2Δ| = 292,  p  < 0.0001). This suggests that most of the heterogeneity in the  D N / D S  ratio among branches of the phylogeny is due to an elevated ratio for the lineages leading from the ancestor of  D. melanogaster  and the sibling species. The tree is unrooted and we assume a trifurcation among the sibling species. Positive Selection at  Hmr
 We next asked whether  Hmr  nonsynonymous substitution rates are elevated along the  D. melanogaster  and sibling-species lineages because of relaxed selective constraints (e.g.,  McAllister and McVean 2000 ) or positive selection. Polymorphism data from populations in concert with interspecific divergence data allow one to detect departures from neutrality and to estimate the time in the past when selection events occurred. If the divergence of  Hmr  was due solely to a relaxed selective constraint (neutral processes), then one would expect that the ratio of nonsynonymous to synonymous divergence between species would be similar to the ratio of nonsynonymous to synonymous polymorphisms found within these species ( McDonald and Kreitman 1991 ). To test this we collected polymorphism data from 14  D. melanogaster  and seven  D. simulans Hmr  alleles. The average nonsynonymous  (π N )  and synonymous  (π S )  polymorphism per site within species was moderately low in both  D. melanogaster  ( π N  = 0.0017;  π S  = 0.0052) and  D. simulans  ( π N  = 0.0060;  π S  = 0.0123) relative to other loci ( Andolfatto 2001 ). However, in contrast to the neutral expectation, the ratio of nonsynonymous to synonymous substitutions was found to be in significant excess of the ratio of nonsynonymous to synonymous polymorphisms ( Table 2 ). When mutations were polarized along both lineages with respect to  D. erecta,  excess nonsynonymous changes were observed along both the  D. melanogaster  lineage and the  D. simulans  lineage. These results are highly indicative of positive selection acting in both the  D. melanogaster  and  D. simulans  lineages at coding positions in  Hmr . The same tests performed between  D. melanogaster  or  D. simulans  and  D. erecta  were not significant, reinforcing our inference that selection occurred along the  D. melanogaster  and sibling-species lineages and not along the  D. erecta  lineage. Table 2 MK Tests for Deviations from Neutrality at  Hmr
 Only the coding region of  Hmr  was used here. Results for the  D. melanogaster  and  D. simulans  lineages remain significant if both coding and noncoding regions are included in counting synonymous polymorphisms and divergence. Row 1 contains polymorphism data from both species. Lineage analyses (rows 2 and 3) include polymorphism data for the indicated species and polarized mutations for that species lineage. Comparisons with  D. erecta  contain polymorphism data from  D. melanogaster  only (row 4) or  D. simulans  only (row 5). All  p- values are exact and two-tailed. Comparisons between  D. melanogaster  and each sibling species were also significant but are not phylogenetically independent and are not shown Significant Regional Variation in Polymorphism and Divergence across  Hmr
 Polymorphism and divergence vary substantially for the separate exons and the DNA binding domains. In contrast to the neutral expectation, we found that there is highly significant heterogeneity in the ratio of polymorphism to divergence across the five exons of  Hmr  (Hudson-Kreitman-Aguade test [HKA test;  Hudson et al. 1987 ];  p  = 0.025; 10,000 simulations performed). This significant variation across the locus can be further refined by examining the ratio of nonsynonymous to synonymous variation across the locus and is visually displayed for  D. melanogaster  and  D. simulans  using sliding window analyses in  Figure 4 . These plots reveal multiple regions that have very high  D N / D S  ratios and low  π N / π S  ratios, in other words, much more amino-acid divergence than polymorphism. These contrasting ratios do not suggest that regional variation in substitution rates among the two classes of sites is due solely to mutation (and drift) but rather suggest that selection is contributing to the divergence pattern across the gene. Although this sliding window plot is highly suggestive of selection, we wished to obtain independent and statistically supported evidence for regional selection. We therefore tested rates of divergence relative to polymorphism for each exon in a manner similar to the McDonald-Kreitman test (MK test) described in the previous section. The data were partitioned a priori using each exon as a unit (rather than picking an arbitrary window size). Given the size of the sampled region, there is considerable power to address regional variation. These MK tests for each exon revealed that the fourth and fifth exons of  Hmr  appear to contribute the most to the overall MK test (Fisher's Exact Test [FET] and Bonferroni correction; exon 4,  p  = 0.0004; exon 5,  p  = 0.013). It is interesting to note that these regions are not homologous to any other proteins known in  D. melanogaster  (or in any other species), including other MADF domain–containing proteins. We conclude that multiple regions of  Hmr  show strong evidence for positive selection. Figure 4 Sliding Window Analysis of  Hmr  Divergence and Polymorphism Calculations were made with a window size of 150 nucleotides and a step size of 50 nucleotides. Nucleotide position 1 on the  x -axis is the start of the coding sequence, and the last position is the stop codon. The dashed line indicates where the ratio is one. Arrows at the top indicate the positions of codons identified as being under positive selection in  Figure 5 . Exon boundaries are indicated below the  x -axis with horizontal bars. A repeatability analysis ( Smith and Hurst 1998 ) revealed that polymorphism ratios for each window were not correlated ( p  = 0.43) with divergence ratios between  D. melanogaster  and  D. simulans . We also observed that the flanking regions of the  Hmr  locus appear to have evolved rapidly since  D. melanogaster  and the sibling species diverged, relative to both synonymous coding sites and introns. The ratios of divergence to polymorphism between  D. melanogaster  and  D. simulans  for the 5′ and 3′ flanking regions combined (including the UTRs) are significantly different from those of the coding and intron regions (3 × 2 FET;  p  < 0.0001) ( Table 3 ), but this significant difference is not observed between  D. simulans  and  D. mauritiana  or  D. sechellia  (FET;  p  = 0.543 and  p =  0.783, respectively). These observations suggest that adaptive fixations have occurred at a number of noncoding sites. Table 3 Observed Number of Replacement Substitutions Relative to Polymorphisms in  Hmr  for the Three Categories of Silent Sites Polymorphims and substitutions are from  D. melanogaster  and  D. simulans.  Flanking regions include both 5′ and 3′ UTRs and nontranscribed regions. Heterogeneity among the three classes of sites in the relative numbers of polymorphic sites and substitutions was tested by FET (3 × 2;  p  < 0.0001) We next asked whether we could refine the targets of selection to the level of individual codons and correlate such data with different models of HI evolution ( Figure 5 ). Phylogenetic approaches similar to those used in  Figure 3  have been designed to detect recurrent positive selection at individual codons among species ( Yang and Nielsen 2002 ). We reasoned that the large amount of divergence at  Hmr  might offer a unique opportunity to apply these approaches to identify codons that exhibit positive selection and that could be contributing to HI. We identified 25 amino-acid positions that may have diverged due to selection, 20 of which fit possible models for the evolution of the  D. melanogaster –sibling-species incompatibility ( Figure 5 ). Many of these codons map to regions with peak  D N / D S  values (see  Figure 4 ). We conclude that multiple regions show evidence of positive selection and may have contributed to the functional divergence of  Hmr  between  D. melanogaster  and its sibling species. Figure 5 Phylogenetic Analysis of Positive Selection at Individual Codons Site-specific codon Model 8 (M8) in PAML was used to identify codons under selection. This model, which considers a discrete distribution of  D N / Ds  values plus a “selection category”— D N / D S  values greater than one—fit the data better than a neutral model (M1) (2Δ| = 25.43,  p  < 0.001). Codons listed are those with  p -values from posterior distributions greater than 0.5. The positions of these codons are also shown in  Figure 4 . Because  D. melanogaster  is incompatible with all three of its sibling species, we expect that  Hmr  codons involved in HI must be different between  D. melanogaster Hmr  and all three sibling-species alleles. Codons that fit a model of incompatibility between  Hmr mel  and  A sib  are shaded blue, and those that fit a model of incompatibility between  Hmr anc  and  A sib  are shaded yellow (see  Discussion ). The five remaining codons (unshaded) are identical between  D. melanogaster  and at least one of the sibling alleles and are thus excluded from both models. Selection Events Are Ancient Loci directly involved in reproductive isolation may reflect the true species history of divergence more accurately than a “random” locus sampled from the genome, because these loci cease exchanging alleles among species earlier than other loci ( Wang et al. 1997 ;  Ting et al. 2000 ). Both reduced gene flow and adaptive fixations can remove shared ancient polymorphisms.  D. melanogaster  and  D. simulans  separated approximately 2 to 3 million years ago ( Powell 1997 ), and many shared polymorphisms have been lost due to drift ( Clark 1997 ). However, shared polymorphisms are still observed in these genomes: among 15 loci ( Andolfatto and Przeworski 2000 ), we found that 5.2% and 3.5% of segregating sites in  D. melanogaster  and  D. simulans , respectively, are segregating at the same positions (among all classes of sites). Based on these values we expected to find approximately three to four shared polymorphic sites for  Hmr  among the 54 segregating sites in  D. melanogaster  and 94 segregating sites in  D. simulans , but instead found zero. Although the expectation of three to four shared polymorphisms is not a strong test, these findings are consistent with  Hmr  alleles not having been exchanged between these taxa since at or near the time of species divergence and/or with selection events having occurred throughout the  Hmr  gene and having removed recurrent mutations. We have shown by several methods that the elevated rate of amino-acid divergence at  Hmr  is due to positive selection. These findings raise the critical question of whether selection events occurred recently or at the time of speciation. The number of years separating  D. melanogaster  and the sibling-species lineage makes this a challenging task. We therefore concentrated on the more tractable problem of looking for evidence to exclude the possibility that  Hmr  divergence reflects the action of contemporary or recent selection. In other words, while it may not be possible to prove that selection occurred at  Hmr  at the time of speciation, it is nevertheless important to demonstrate that available data are, at the least, not inconsistent with more ancient selective events. Examination of the frequency spectrum of polymorphism within both  D. melanogaster  and  D. simulans  showed no evidence of recent sweeps, as the distribution of per-site heterozygosities did not differ from expectations of neutrality (Tajima's D is −0.61 and 0.75, respectively, which is not significant). However, the power for detecting a selective sweep with this method is not high (75%) even if the event occurred in the very recent past, and diminishes to 25% as early as 0.8 N  generations ago (where  N = population size) ( Przeworski 2002 ). Because we have prior knowledge from the analyses above that selection events occurred at  Hmr,  we reasoned that we could apply more powerful Bayesian approaches that use not only frequency spectrum information but other summary statistics of polymorphism and recombination to estimate the time at which these events happened ( Przeworski 2003 ). To estimate simultaneously the time and strength of selection events we conditioned on three summary statistics of the data: the polymorphism frequency spectrum (Tajima's D), the number of segregating sites, and the number of haplotypes. Prior distributions were chosen based on estimates of recombination, mutation rates, and effective population size. The most recent common ancestor for an  X -linked locus is on average 3 N  generations ago. We determined that in  D. melanogaster,  a recent selective sweep (within the last 0.25 N  generations) is clearly not consistent with the polymorphism data, whereas adaptive mutations occurring more than  N  generations ago are consistent with the polymorphism data, and selective sweeps more than 1.5 N  generations ago are possible ( Figure 6 ). Similarly, the marginal distribution of selection coefficients suggests that the strength of selection is inconsistent with small selection coefficients and recent selective sweeps (data not shown). Together, these results show that given our knowledge that selection has occurred at a large number of codons at  Hmr,  the last selection event occurred in the distant past. These procedures have been applied to infer selection events associated with human evolution ( Przeworski 2003 ) but have not previously been applied to HI loci. Estimating the time of fixation events is integral to determining whether selection events that are found at HI loci are consistent with the known time frame of speciation or might merely reflect ongoing contemporary selection. Figure 6 Posterior Distribution of Time (in Generations) Since the Most Recent Selective Sweep at  Hmr  for  D. melanogaster
 Population size  (N)  is assumed to be 1 × 10 6  for  D. melanogaster.  Samples were generated from the joint posterior distribution of five parameters of a selective sweep model assuming a selection event occurred sometime in the past at the  Hmr  locus, and from three summaries of polymorphism, including the number of segregating sites (54), Tajima's D (−0.61), the population recombination rate (4 Nr  = 43, where  N  = population size and  r  = per gene recombination rate;  McVean et al. 2002 ) and the number of haplotypes (11). The data is least consistent with a selective sweep in the recent past and is most consistent with selective sweeps occurring more than  N  generations ago. If there are ten generations per year, this suggests that the last selective sweep occurred at least 100,000 years ago. Data for  D. simulans  are not shown, as the population structure for the  D. simulans  Wolfskill populations we sampled would inflate estimates through increasing marginal frequencies of segregating sites ( Wall et al. 2002 ).
Results
Testing  Hmr  for Functional Divergence In the absence of available  Hmr  mutations in the sibling species, we chose an alternative approach to test whether  Hmr  has functionally diverged by introducing cloned copies of  D. simulans  and  D. mauritiana Hmr +  into  D. melanogaster  ( Figure 2 ). It has been shown previously that increasing  Hmr +  activity using  P -element transgenes containing  D. melanogaster Hmr +  suppresses the hybrid-rescuing activity of  Hmr 1  and  In(1)AB  mutations ( Barbash et al. 2003 ). Those experiments can be interpreted as showing that the wild-type  D. melanogaster Hmr +  kills hybrids, with the  Hmr 1  and  In(1)AB  mutations rescuing hybrids by reducing or eliminating  Hmr +  activity. The transgenic copies of  D. melanogaster Hmr +  thus suppress rescue by increasing  Hmr +  activity in hybrids. We therefore asked whether or not sibling-species  Hmr +  has the same activity. We reasoned that if  Hmr  is functionally diverged between the species, as in  Figure 1 B, then transgenes containing sibling-species  Hmr +  would not have the property of suppressing hybrid rescue. On the other hand, if  Hmr  is not functionally diverged, as in  Figure 1 C, then these sibling-species constructs would have phenotypic properties similar to the  D. melanogaster Hmr +  transgenes and suppress hybrid rescue. Figure 2 Structure and Expression of Sibling-Species  Hmr +  Transgenes (A) Diagram of  Hmr +  transgenic constructs. The  Hmr  gene structure is shown, with the rightward arrow indicating the predicted translation start site. Sibling-species constructs used in this study are shown, together with  D. melanogaster  constructs previously shown ( Barbash et al. 2003 ) to be  Hmr + .
 (B) Restriction map of RT-PCR fragments spanning part of exons 3 and 4, showing diagnostic restriction site polymorphisms found in the transgenic alleles and the stocks used to assay them. The  D. melanogaster  map corresponds to both the  Hmr 1  and  In(1)AB  rescue alleles, as well as all  D. melanogaster  alleles from our population sample. (C) RT-PCR products from interspecific hybrids. Hybrids were from the crosses described in  Table 1 . RNA was collected from 48- to 72-h-old larvae and 2- to 4-d-old adult males. Note that larval samples contain RNA from males and females, half of whom carry the sibling-species  Hmr +  transgene. The portion of the PCR product derived from the transgenes is that digested by XbaI or HpaI for the  D. simulans  and  D. mauritiana  transgenes, respectively. M, 100-bp ladder marker; G, undigested PCR from an  Hmr  genomic clone (this product contains a 59-bp intron); cD, undigested PCR from an  Hmr  cDNA clone. The following are all RT-PCR products: U, undigested; C, ClaI-digested; H, HindIII-digested; X, XbaI-digested; C/X, ClaI- and XbaI-digested; Hp, HpaI-digested; C/Hp, ClaI- and HpaI-digested; –, control containing no reverse transcriptase. Note that undigested lanes (U) contain half the amount of DNA as digested samples. Table 1 Viability of Hybrid Males Carrying  D. simulans  or  D. mauritiana Hmr +  Transgenes
 D. melanogaster / D. simulans  hybrids were from crosses at 25 °C of  In(1)AB,w/FM6,w; P{Hmr
 + }/+ females to  D. simulans
 w / Y  males, except for  In(1)AB,w/FM6,w; P{Dsim\Hmr +8.6 }2-4 /+ females, which were crossed to  D. simulans
 w m/Y  males.  D. melanogaster / D. mauritiana  hybrids were from crosses at 22 to 23 °C of  w Hmr 1  v/FM6,w; P{Hmr + } /+ females to  D. mauritiana  iso-207-males. The transgenes carried a copy of the w +  gene so that  P[Hmr + ] /+ hybrids were distinguished from +/+ hybrids by their w +  eye color To maximize the chances that the sibling-species transgenic lines would function normally, we made constructs that are similar to the largest  D. melanogaster Hmr +  transgenic construct and that exceed the minimal  Hmr +  region previously defined (see  Figure 2 A). We assayed two independent transformants each of  D. simulans Hmr +  and  D. mauritiana Hmr +  for suppression of hybrid male rescue by  Hmr 1  in  D. melanogaster / D. mauritiana  hybrids and by  In(1)AB  in  D. melanogaster / D. simulans  hybrids ( Table 1 ). In all cases hybrid males heterozygous for a sibling-species transgene were at least as viable as their brothers without the transgene. Some crosses showed an excess of transgene-carrying hybrids, which might suggest that the transgenes actually increase the effectiveness of hybrid rescue, perhaps by interfering with the pathway of  Hmr + -dependent lethality. This possibility requires further investigation, but we note that such a hypothetical effect must be minor because the sibling-species  Hmr +  transgenes by themselves do not rescue hybrid males. We also assayed the  D. simulans Hmr +  transformants for suppression of  In(1)AB -dependent rescue of hybrid female sterility ( Barbash and Ashburner 2003 ). In contrast to the complete suppression associated with  D. melanogaster Hmr +  transgenes ( Barbash et al. 2003 ), we found that our  D. simulans Hmr +  transgenes had little or no effect on egg counts in  D. melanogaster / D. simulans  hybrids.  In(1)AB,w/X sim ,w m  females heterozygous for the insertion  P{Dsim\Hmr +t8.6 }2-4  averaged 10.0 ± 8.9 eggs ( n  = 41) while their non-transgene-carrying sisters averaged 8.0 ± 12.4 eggs ( n  = 28). Using a second, independent transgenic line, we found that  In(1)AB,w/X sim ,w  females heterozygous for the insertion  P{Dsim\Hmr +t8.6 }4-1  had 12.2 ± 13.7 eggs ( n  = 24) while their non-transgene-carrying sisters had 17.7 ± 17.3 eggs ( n  = 31). RT-PCR analysis demonstrated that the  Hmr +  transgenes are expressed ( Figure 2 C). These results show that the sibling-species alleles of  Hmr +  have no phenotypic effect in species hybrids and strongly support the conclusion that  Hmr  has functionally diverged between the  D. melanogaster  and sibling-species lineages, demonstrating that  Hmr  meets the criteria for being a D-M HI gene as diagrammed in  Figure 1 B.
Testing  Hmr  for Functional Divergence
HmrIn the absence of available  Hmr  mutations in the sibling species, we chose an alternative approach to test whether  Hmr  has functionally diverged by introducing cloned copies of  D. simulans  and  D. mauritiana Hmr +  into  D. melanogaster  ( Figure 2 ). It has been shown previously that increasing  Hmr +  activity using  P -element transgenes containing  D. melanogaster Hmr +  suppresses the hybrid-rescuing activity of  Hmr 1  and  In(1)AB  mutations ( Barbash et al. 2003 ). Those experiments can be interpreted as showing that the wild-type  D. melanogaster Hmr +  kills hybrids, with the  Hmr 1  and  In(1)AB  mutations rescuing hybrids by reducing or eliminating  Hmr +  activity. The transgenic copies of  D. melanogaster Hmr +  thus suppress rescue by increasing  Hmr +  activity in hybrids. We therefore asked whether or not sibling-species  Hmr +  has the same activity. We reasoned that if  Hmr  is functionally diverged between the species, as in  Figure 1 B, then transgenes containing sibling-species  Hmr +  would not have the property of suppressing hybrid rescue. On the other hand, if  Hmr  is not functionally diverged, as in  Figure 1 C, then these sibling-species constructs would have phenotypic properties similar to the  D. melanogaster Hmr +  transgenes and suppress hybrid rescue.HmrHmrD. mauritiana Hmr +Hmr +PD. melanogaster Hmr +Hmr 1In(1)ABD. melanogaster Hmr +Hmr 1In(1)ABHmr +D. melanogaster Hmr +Hmr +Hmr +HmrHmr +HmrD. melanogaster Hmr +Viability of Hybrid Males Carrying  D. simulans  or  D. mauritiana Hmr +  Transgenes
D. mauritiana Hmr +D. melanogaster / D. simulans  hybrids were from crosses at 25 °C of  In(1)AB,w/FM6,w; P{Hmr
 + }/+ females to  D. simulans
 w / Y  males, except for  In(1)AB,w/FM6,w; P{Dsim\Hmr +8.6 }2-4 /+ females, which were crossed to  D. simulans
 w m/Y  males.  D. melanogaster / D. mauritiana  hybrids were from crosses at 22 to 23 °C of  w Hmr 1  v/FM6,w; P{Hmr + } /+ females to  D. mauritiana  iso-207-males. The transgenes carried a copy of the w +  gene so that  P[Hmr + ] /+ hybrids were distinguished from +/+ hybrids by their w +  eye colorIn(1)AB,w/FM6,w; P{HmrwYIn(1)AB,w/FM6,w; P{Dsim\Hmr +8.6 }2-4w m/Yw Hmr 1  v/FM6,w; P{Hmr + }P[Hmr + ]To maximize the chances that the sibling-species transgenic lines would function normally, we made constructs that are similar to the largest  D. melanogaster Hmr +  transgenic construct and that exceed the minimal  Hmr +  region previously defined (see  Figure 2 A). We assayed two independent transformants each of  D. simulans Hmr +  and  D. mauritiana Hmr +  for suppression of hybrid male rescue by  Hmr 1  in  D. melanogaster / D. mauritiana  hybrids and by  In(1)AB  in  D. melanogaster / D. simulans  hybrids ( Table 1 ). In all cases hybrid males heterozygous for a sibling-species transgene were at least as viable as their brothers without the transgene. Some crosses showed an excess of transgene-carrying hybrids, which might suggest that the transgenes actually increase the effectiveness of hybrid rescue, perhaps by interfering with the pathway of  Hmr + -dependent lethality. This possibility requires further investigation, but we note that such a hypothetical effect must be minor because the sibling-species  Hmr +  transgenes by themselves do not rescue hybrid males. We also assayed the  D. simulans Hmr +  transformants for suppression of  In(1)AB -dependent rescue of hybrid female sterility ( Barbash and Ashburner 2003 ). In contrast to the complete suppression associated with  D. melanogaster Hmr +  transgenes ( Barbash et al. 2003 ), we found that our  D. simulans Hmr +  transgenes had little or no effect on egg counts in  D. melanogaster / D. simulans  hybrids.  In(1)AB,w/X sim ,w m  females heterozygous for the insertion  P{Dsim\Hmr +t8.6 }2-4  averaged 10.0 ± 8.9 eggs ( n  = 41) while their non-transgene-carrying sisters averaged 8.0 ± 12.4 eggs ( n  = 28). Using a second, independent transgenic line, we found that  In(1)AB,w/X sim ,w  females heterozygous for the insertion  P{Dsim\Hmr +t8.6 }4-1  had 12.2 ± 13.7 eggs ( n  = 24) while their non-transgene-carrying sisters had 17.7 ± 17.3 eggs ( n  = 31).D. melanogaster Hmr +Hmr +D. simulans Hmr +D. mauritiana Hmr +Hmr 1In(1)ABHmr +Hmr +D. simulans Hmr +In(1)ABD. melanogaster Hmr +D. simulans Hmr +In(1)AB,w/X sim ,w mP{Dsim\Hmr +t8.6 }2-4nnIn(1)AB,w/X sim ,wP{Dsim\Hmr +t8.6 }4-1nnRT-PCR analysis demonstrated that the  Hmr +  transgenes are expressed ( Figure 2 C). These results show that the sibling-species alleles of  Hmr +  have no phenotypic effect in species hybrids and strongly support the conclusion that  Hmr  has functionally diverged between the  D. melanogaster  and sibling-species lineages, demonstrating that  Hmr  meets the criteria for being a D-M HI gene as diagrammed in  Figure 1 B.Hmr +Hmr +HmrHmrHmr  Divergence among  Drosophila  Lineages It has been shown previously that  Hmr  has a high level of average divergence per nonsynonymous (amino-acid replacement) site  (D N )  between  D. melanogaster  and its three sibling species ( Barbash et al. 2003 ). To understand this divergence in the context of genome-wide evolution, we calculated divergence of  Hmr  between  D. melanogaster  and  D. simulans  and compared it to compiled datasets containing over 250 genes from these two species ( Begun 2002 ;  Betancourt and Presgraves 2002 ). Pairwise comparisons revealed that  Hmr  has one of the highest levels of nonsynonymous divergence (0.089); only four other non– Accessory gland protein (Acp)  genes have a higher nonsynonymous divergence, and two of these include expressed sequence tag comparisons less than 350 bases in length. The remaining two loci, both X-linked, are  mei-218  and  Odysseus . In contrast, the average divergence per synonymous site  (Ds)  of 0.110 for  Hmr  is lower than the mean value of 0.125 for this dataset (again excluding  Accessory gland protein  genes). The exceptional divergence of  Hmr  raises a number of important questions: (1) Is divergence high on the lineage of either  D. melanogaster  or its sibling species, or both? (2) Was the divergence on either or both lineages caused by positive selection consistent with the time scale of speciation? (3) Can we identify specific regions and codons subject to positive selection on these lineages, and how does this compare to the few other known candidate speciation genes? (4) Is divergence on one or both lineages potentially responsible for causing the HI phenotype of  Hmr ? We first addressed whether  Hmr  began to diverge rapidly after the  D. melanogaster –sibling-species divergence by isolating and assembling an outgroup  Hmr  orthologous sequence from the  D. melanogaster  subgroup species  D. erecta , which is estimated to have diverged from  D. melanogaster  between 6 and 15 million years ago ( Powell 1997 ). The maximum-likelihood estimate of  D N  between  D. melanogaster  and  D. erecta Hmr  was 0.166, higher than the mean value of 0.057 in a survey of 53  D. erecta  genes ( Bergman et al. 2002 ). However, the  D N / D S  ratio was 0.556, consistent with a more selective constraint on nonsynonymous sites than on synonymous sites. In contrast, we found that the  D. melanogaster  lineage and the lineage leading to the sibling species both exhibited elevated levels of nonsynonymous divergence relative to synonymous divergence ( Figure 3 ). This observation suggests that the rate of amino-acid evolution at  Hmr  accelerated after the divergence of  D. melanogaster  from its sibling species. We also detected accelerated divergence along the sibling-species lineages, but because of the trichotomy for  D. simulans, D. sechellia,  and  D. mauritiana , these branch lengths have little confidence. Furthermore, this divergence appears to be irrelevant with respect to the HI phenotype because we showed above that transgenic copies of  Hmr +  from both  D. simulans  and  D. mauritiana  have no effect on hybrid viability ( Table 1 ). Figure 3 Maximum-Likelihood Estimates of  Hmr  Divergence among  Drosophila  Lineages Estimates of the number of changes per nonsynonymous site  (D N )  are shown above each lineage, and the number of changes per synonymous site  (D S )  are shown below each lineage, calculated separately for each branch. The coding region of  Hmr  is 1,390 to 1,427 amino acids long in the five species.  D N / D S  ratios differ significantly among branches as tested by the methods of  Nielsen and Yang (1998) . A model where all  D N / D S  ratios were free to vary along all branches (Model 2 [M2]) fit the data better than a model with a fixed  D N / D S  ratio for all branches (M1) (2Δ| = 308,  p  < 0.0001, chi-square distribution), as did a model where  D N /D S  ratios for the  D. melanogaster  lineage and the lineage leading to the sibling species differed from the rest of the tree (local clock, 2Δ| = 292,  p  < 0.0001). This suggests that most of the heterogeneity in the  D N / D S  ratio among branches of the phylogeny is due to an elevated ratio for the lineages leading from the ancestor of  D. melanogaster  and the sibling species. The tree is unrooted and we assume a trifurcation among the sibling species.
Hmr  Divergence among  Drosophila  Lineages
HmrDrosophilaIt has been shown previously that  Hmr  has a high level of average divergence per nonsynonymous (amino-acid replacement) site  (D N )  between  D. melanogaster  and its three sibling species ( Barbash et al. 2003 ). To understand this divergence in the context of genome-wide evolution, we calculated divergence of  Hmr  between  D. melanogaster  and  D. simulans  and compared it to compiled datasets containing over 250 genes from these two species ( Begun 2002 ;  Betancourt and Presgraves 2002 ). Pairwise comparisons revealed that  Hmr  has one of the highest levels of nonsynonymous divergence (0.089); only four other non– Accessory gland protein (Acp)  genes have a higher nonsynonymous divergence, and two of these include expressed sequence tag comparisons less than 350 bases in length. The remaining two loci, both X-linked, are  mei-218  and  Odysseus . In contrast, the average divergence per synonymous site  (Ds)  of 0.110 for  Hmr  is lower than the mean value of 0.125 for this dataset (again excluding  Accessory gland protein  genes).Hmr(D N )HmrHmrAccessory gland protein (Acp)mei-218Odysseus(Ds)HmrAccessory gland proteinThe exceptional divergence of  Hmr  raises a number of important questions: (1) Is divergence high on the lineage of either  D. melanogaster  or its sibling species, or both? (2) Was the divergence on either or both lineages caused by positive selection consistent with the time scale of speciation? (3) Can we identify specific regions and codons subject to positive selection on these lineages, and how does this compare to the few other known candidate speciation genes? (4) Is divergence on one or both lineages potentially responsible for causing the HI phenotype of  Hmr ?HmrHmrWe first addressed whether  Hmr  began to diverge rapidly after the  D. melanogaster –sibling-species divergence by isolating and assembling an outgroup  Hmr  orthologous sequence from the  D. melanogaster  subgroup species  D. erecta , which is estimated to have diverged from  D. melanogaster  between 6 and 15 million years ago ( Powell 1997 ). The maximum-likelihood estimate of  D N  between  D. melanogaster  and  D. erecta Hmr  was 0.166, higher than the mean value of 0.057 in a survey of 53  D. erecta  genes ( Bergman et al. 2002 ). However, the  D N / D S  ratio was 0.556, consistent with a more selective constraint on nonsynonymous sites than on synonymous sites. In contrast, we found that the  D. melanogaster  lineage and the lineage leading to the sibling species both exhibited elevated levels of nonsynonymous divergence relative to synonymous divergence ( Figure 3 ). This observation suggests that the rate of amino-acid evolution at  Hmr  accelerated after the divergence of  D. melanogaster  from its sibling species. We also detected accelerated divergence along the sibling-species lineages, but because of the trichotomy for  D. simulans, D. sechellia,  and  D. mauritiana , these branch lengths have little confidence. Furthermore, this divergence appears to be irrelevant with respect to the HI phenotype because we showed above that transgenic copies of  Hmr +  from both  D. simulans  and  D. mauritiana  have no effect on hybrid viability ( Table 1 ).HmrHmrD ND. erecta HmrD ND SHmrD. simulans, D. sechellia,Hmr +Positive Selection at  Hmr
 We next asked whether  Hmr  nonsynonymous substitution rates are elevated along the  D. melanogaster  and sibling-species lineages because of relaxed selective constraints (e.g.,  McAllister and McVean 2000 ) or positive selection. Polymorphism data from populations in concert with interspecific divergence data allow one to detect departures from neutrality and to estimate the time in the past when selection events occurred. If the divergence of  Hmr  was due solely to a relaxed selective constraint (neutral processes), then one would expect that the ratio of nonsynonymous to synonymous divergence between species would be similar to the ratio of nonsynonymous to synonymous polymorphisms found within these species ( McDonald and Kreitman 1991 ). To test this we collected polymorphism data from 14  D. melanogaster  and seven  D. simulans Hmr  alleles. The average nonsynonymous  (π N )  and synonymous  (π S )  polymorphism per site within species was moderately low in both  D. melanogaster  ( π N  = 0.0017;  π S  = 0.0052) and  D. simulans  ( π N  = 0.0060;  π S  = 0.0123) relative to other loci ( Andolfatto 2001 ). However, in contrast to the neutral expectation, the ratio of nonsynonymous to synonymous substitutions was found to be in significant excess of the ratio of nonsynonymous to synonymous polymorphisms ( Table 2 ). When mutations were polarized along both lineages with respect to  D. erecta,  excess nonsynonymous changes were observed along both the  D. melanogaster  lineage and the  D. simulans  lineage. These results are highly indicative of positive selection acting in both the  D. melanogaster  and  D. simulans  lineages at coding positions in  Hmr . The same tests performed between  D. melanogaster  or  D. simulans  and  D. erecta  were not significant, reinforcing our inference that selection occurred along the  D. melanogaster  and sibling-species lineages and not along the  D. erecta  lineage. Table 2 MK Tests for Deviations from Neutrality at  Hmr
 Only the coding region of  Hmr  was used here. Results for the  D. melanogaster  and  D. simulans  lineages remain significant if both coding and noncoding regions are included in counting synonymous polymorphisms and divergence. Row 1 contains polymorphism data from both species. Lineage analyses (rows 2 and 3) include polymorphism data for the indicated species and polarized mutations for that species lineage. Comparisons with  D. erecta  contain polymorphism data from  D. melanogaster  only (row 4) or  D. simulans  only (row 5). All  p- values are exact and two-tailed. Comparisons between  D. melanogaster  and each sibling species were also significant but are not phylogenetically independent and are not shown
Positive Selection at  Hmr
HmrWe next asked whether  Hmr  nonsynonymous substitution rates are elevated along the  D. melanogaster  and sibling-species lineages because of relaxed selective constraints (e.g.,  McAllister and McVean 2000 ) or positive selection. Polymorphism data from populations in concert with interspecific divergence data allow one to detect departures from neutrality and to estimate the time in the past when selection events occurred. If the divergence of  Hmr  was due solely to a relaxed selective constraint (neutral processes), then one would expect that the ratio of nonsynonymous to synonymous divergence between species would be similar to the ratio of nonsynonymous to synonymous polymorphisms found within these species ( McDonald and Kreitman 1991 ).HmrHmrTo test this we collected polymorphism data from 14  D. melanogaster  and seven  D. simulans Hmr  alleles. The average nonsynonymous  (π N )  and synonymous  (π S )  polymorphism per site within species was moderately low in both  D. melanogaster  ( π N  = 0.0017;  π S  = 0.0052) and  D. simulans  ( π N  = 0.0060;  π S  = 0.0123) relative to other loci ( Andolfatto 2001 ). However, in contrast to the neutral expectation, the ratio of nonsynonymous to synonymous substitutions was found to be in significant excess of the ratio of nonsynonymous to synonymous polymorphisms ( Table 2 ). When mutations were polarized along both lineages with respect to  D. erecta,  excess nonsynonymous changes were observed along both the  D. melanogaster  lineage and the  D. simulans  lineage. These results are highly indicative of positive selection acting in both the  D. melanogaster  and  D. simulans  lineages at coding positions in  Hmr . The same tests performed between  D. melanogaster  or  D. simulans  and  D. erecta  were not significant, reinforcing our inference that selection occurred along the  D. melanogaster  and sibling-species lineages and not along the  D. erecta  lineage.D. simulans Hmr(π N )(π S )π Nπ Sπ Nπ SD. erecta,HmrMK Tests for Deviations from Neutrality at  Hmr
HmrOnly the coding region of  Hmr  was used here. Results for the  D. melanogaster  and  D. simulans  lineages remain significant if both coding and noncoding regions are included in counting synonymous polymorphisms and divergence. Row 1 contains polymorphism data from both species. Lineage analyses (rows 2 and 3) include polymorphism data for the indicated species and polarized mutations for that species lineage. Comparisons with  D. erecta  contain polymorphism data from  D. melanogaster  only (row 4) or  D. simulans  only (row 5). All  p- values are exact and two-tailed. Comparisons between  D. melanogaster  and each sibling species were also significant but are not phylogenetically independent and are not shownHmrp-Significant Regional Variation in Polymorphism and Divergence across  Hmr
 Polymorphism and divergence vary substantially for the separate exons and the DNA binding domains. In contrast to the neutral expectation, we found that there is highly significant heterogeneity in the ratio of polymorphism to divergence across the five exons of  Hmr  (Hudson-Kreitman-Aguade test [HKA test;  Hudson et al. 1987 ];  p  = 0.025; 10,000 simulations performed). This significant variation across the locus can be further refined by examining the ratio of nonsynonymous to synonymous variation across the locus and is visually displayed for  D. melanogaster  and  D. simulans  using sliding window analyses in  Figure 4 . These plots reveal multiple regions that have very high  D N / D S  ratios and low  π N / π S  ratios, in other words, much more amino-acid divergence than polymorphism. These contrasting ratios do not suggest that regional variation in substitution rates among the two classes of sites is due solely to mutation (and drift) but rather suggest that selection is contributing to the divergence pattern across the gene. Although this sliding window plot is highly suggestive of selection, we wished to obtain independent and statistically supported evidence for regional selection. We therefore tested rates of divergence relative to polymorphism for each exon in a manner similar to the McDonald-Kreitman test (MK test) described in the previous section. The data were partitioned a priori using each exon as a unit (rather than picking an arbitrary window size). Given the size of the sampled region, there is considerable power to address regional variation. These MK tests for each exon revealed that the fourth and fifth exons of  Hmr  appear to contribute the most to the overall MK test (Fisher's Exact Test [FET] and Bonferroni correction; exon 4,  p  = 0.0004; exon 5,  p  = 0.013). It is interesting to note that these regions are not homologous to any other proteins known in  D. melanogaster  (or in any other species), including other MADF domain–containing proteins. We conclude that multiple regions of  Hmr  show strong evidence for positive selection. Figure 4 Sliding Window Analysis of  Hmr  Divergence and Polymorphism Calculations were made with a window size of 150 nucleotides and a step size of 50 nucleotides. Nucleotide position 1 on the  x -axis is the start of the coding sequence, and the last position is the stop codon. The dashed line indicates where the ratio is one. Arrows at the top indicate the positions of codons identified as being under positive selection in  Figure 5 . Exon boundaries are indicated below the  x -axis with horizontal bars. A repeatability analysis ( Smith and Hurst 1998 ) revealed that polymorphism ratios for each window were not correlated ( p  = 0.43) with divergence ratios between  D. melanogaster  and  D. simulans . We also observed that the flanking regions of the  Hmr  locus appear to have evolved rapidly since  D. melanogaster  and the sibling species diverged, relative to both synonymous coding sites and introns. The ratios of divergence to polymorphism between  D. melanogaster  and  D. simulans  for the 5′ and 3′ flanking regions combined (including the UTRs) are significantly different from those of the coding and intron regions (3 × 2 FET;  p  < 0.0001) ( Table 3 ), but this significant difference is not observed between  D. simulans  and  D. mauritiana  or  D. sechellia  (FET;  p  = 0.543 and  p =  0.783, respectively). These observations suggest that adaptive fixations have occurred at a number of noncoding sites. Table 3 Observed Number of Replacement Substitutions Relative to Polymorphisms in  Hmr  for the Three Categories of Silent Sites Polymorphims and substitutions are from  D. melanogaster  and  D. simulans.  Flanking regions include both 5′ and 3′ UTRs and nontranscribed regions. Heterogeneity among the three classes of sites in the relative numbers of polymorphic sites and substitutions was tested by FET (3 × 2;  p  < 0.0001) We next asked whether we could refine the targets of selection to the level of individual codons and correlate such data with different models of HI evolution ( Figure 5 ). Phylogenetic approaches similar to those used in  Figure 3  have been designed to detect recurrent positive selection at individual codons among species ( Yang and Nielsen 2002 ). We reasoned that the large amount of divergence at  Hmr  might offer a unique opportunity to apply these approaches to identify codons that exhibit positive selection and that could be contributing to HI. We identified 25 amino-acid positions that may have diverged due to selection, 20 of which fit possible models for the evolution of the  D. melanogaster –sibling-species incompatibility ( Figure 5 ). Many of these codons map to regions with peak  D N / D S  values (see  Figure 4 ). We conclude that multiple regions show evidence of positive selection and may have contributed to the functional divergence of  Hmr  between  D. melanogaster  and its sibling species. Figure 5 Phylogenetic Analysis of Positive Selection at Individual Codons Site-specific codon Model 8 (M8) in PAML was used to identify codons under selection. This model, which considers a discrete distribution of  D N / Ds  values plus a “selection category”— D N / D S  values greater than one—fit the data better than a neutral model (M1) (2Δ| = 25.43,  p  < 0.001). Codons listed are those with  p -values from posterior distributions greater than 0.5. The positions of these codons are also shown in  Figure 4 . Because  D. melanogaster  is incompatible with all three of its sibling species, we expect that  Hmr  codons involved in HI must be different between  D. melanogaster Hmr  and all three sibling-species alleles. Codons that fit a model of incompatibility between  Hmr mel  and  A sib  are shaded blue, and those that fit a model of incompatibility between  Hmr anc  and  A sib  are shaded yellow (see  Discussion ). The five remaining codons (unshaded) are identical between  D. melanogaster  and at least one of the sibling alleles and are thus excluded from both models.
Significant Regional Variation in Polymorphism and Divergence across  Hmr
HmrPolymorphism and divergence vary substantially for the separate exons and the DNA binding domains. In contrast to the neutral expectation, we found that there is highly significant heterogeneity in the ratio of polymorphism to divergence across the five exons of  Hmr  (Hudson-Kreitman-Aguade test [HKA test;  Hudson et al. 1987 ];  p  = 0.025; 10,000 simulations performed). This significant variation across the locus can be further refined by examining the ratio of nonsynonymous to synonymous variation across the locus and is visually displayed for  D. melanogaster  and  D. simulans  using sliding window analyses in  Figure 4 . These plots reveal multiple regions that have very high  D N / D S  ratios and low  π N / π S  ratios, in other words, much more amino-acid divergence than polymorphism. These contrasting ratios do not suggest that regional variation in substitution rates among the two classes of sites is due solely to mutation (and drift) but rather suggest that selection is contributing to the divergence pattern across the gene. Although this sliding window plot is highly suggestive of selection, we wished to obtain independent and statistically supported evidence for regional selection. We therefore tested rates of divergence relative to polymorphism for each exon in a manner similar to the McDonald-Kreitman test (MK test) described in the previous section. The data were partitioned a priori using each exon as a unit (rather than picking an arbitrary window size). Given the size of the sampled region, there is considerable power to address regional variation. These MK tests for each exon revealed that the fourth and fifth exons of  Hmr  appear to contribute the most to the overall MK test (Fisher's Exact Test [FET] and Bonferroni correction; exon 4,  p  = 0.0004; exon 5,  p  = 0.013). It is interesting to note that these regions are not homologous to any other proteins known in  D. melanogaster  (or in any other species), including other MADF domain–containing proteins. We conclude that multiple regions of  Hmr  show strong evidence for positive selection.HmrpD ND Sπ Nπ SHmrppHmrWe also observed that the flanking regions of the  Hmr  locus appear to have evolved rapidly since  D. melanogaster  and the sibling species diverged, relative to both synonymous coding sites and introns. The ratios of divergence to polymorphism between  D. melanogaster  and  D. simulans  for the 5′ and 3′ flanking regions combined (including the UTRs) are significantly different from those of the coding and intron regions (3 × 2 FET;  p  < 0.0001) ( Table 3 ), but this significant difference is not observed between  D. simulans  and  D. mauritiana  or  D. sechellia  (FET;  p  = 0.543 and  p =  0.783, respectively). These observations suggest that adaptive fixations have occurred at a number of noncoding sites.Hmrppp =Observed Number of Replacement Substitutions Relative to Polymorphisms in  Hmr  for the Three Categories of Silent Sites
HmrPolymorphims and substitutions are from  D. melanogaster  and  D. simulans.  Flanking regions include both 5′ and 3′ UTRs and nontranscribed regions. Heterogeneity among the three classes of sites in the relative numbers of polymorphic sites and substitutions was tested by FET (3 × 2;  p  < 0.0001)D. simulans.pWe next asked whether we could refine the targets of selection to the level of individual codons and correlate such data with different models of HI evolution ( Figure 5 ). Phylogenetic approaches similar to those used in  Figure 3  have been designed to detect recurrent positive selection at individual codons among species ( Yang and Nielsen 2002 ). We reasoned that the large amount of divergence at  Hmr  might offer a unique opportunity to apply these approaches to identify codons that exhibit positive selection and that could be contributing to HI. We identified 25 amino-acid positions that may have diverged due to selection, 20 of which fit possible models for the evolution of the  D. melanogaster –sibling-species incompatibility ( Figure 5 ). Many of these codons map to regions with peak  D N / D S  values (see  Figure 4 ). We conclude that multiple regions show evidence of positive selection and may have contributed to the functional divergence of  Hmr  between  D. melanogaster  and its sibling species.HmrD ND SHmrSelection Events Are Ancient Loci directly involved in reproductive isolation may reflect the true species history of divergence more accurately than a “random” locus sampled from the genome, because these loci cease exchanging alleles among species earlier than other loci ( Wang et al. 1997 ;  Ting et al. 2000 ). Both reduced gene flow and adaptive fixations can remove shared ancient polymorphisms.  D. melanogaster  and  D. simulans  separated approximately 2 to 3 million years ago ( Powell 1997 ), and many shared polymorphisms have been lost due to drift ( Clark 1997 ). However, shared polymorphisms are still observed in these genomes: among 15 loci ( Andolfatto and Przeworski 2000 ), we found that 5.2% and 3.5% of segregating sites in  D. melanogaster  and  D. simulans , respectively, are segregating at the same positions (among all classes of sites). Based on these values we expected to find approximately three to four shared polymorphic sites for  Hmr  among the 54 segregating sites in  D. melanogaster  and 94 segregating sites in  D. simulans , but instead found zero. Although the expectation of three to four shared polymorphisms is not a strong test, these findings are consistent with  Hmr  alleles not having been exchanged between these taxa since at or near the time of species divergence and/or with selection events having occurred throughout the  Hmr  gene and having removed recurrent mutations. We have shown by several methods that the elevated rate of amino-acid divergence at  Hmr  is due to positive selection. These findings raise the critical question of whether selection events occurred recently or at the time of speciation. The number of years separating  D. melanogaster  and the sibling-species lineage makes this a challenging task. We therefore concentrated on the more tractable problem of looking for evidence to exclude the possibility that  Hmr  divergence reflects the action of contemporary or recent selection. In other words, while it may not be possible to prove that selection occurred at  Hmr  at the time of speciation, it is nevertheless important to demonstrate that available data are, at the least, not inconsistent with more ancient selective events. Examination of the frequency spectrum of polymorphism within both  D. melanogaster  and  D. simulans  showed no evidence of recent sweeps, as the distribution of per-site heterozygosities did not differ from expectations of neutrality (Tajima's D is −0.61 and 0.75, respectively, which is not significant). However, the power for detecting a selective sweep with this method is not high (75%) even if the event occurred in the very recent past, and diminishes to 25% as early as 0.8 N  generations ago (where  N = population size) ( Przeworski 2002 ). Because we have prior knowledge from the analyses above that selection events occurred at  Hmr,  we reasoned that we could apply more powerful Bayesian approaches that use not only frequency spectrum information but other summary statistics of polymorphism and recombination to estimate the time at which these events happened ( Przeworski 2003 ). To estimate simultaneously the time and strength of selection events we conditioned on three summary statistics of the data: the polymorphism frequency spectrum (Tajima's D), the number of segregating sites, and the number of haplotypes. Prior distributions were chosen based on estimates of recombination, mutation rates, and effective population size. The most recent common ancestor for an  X -linked locus is on average 3 N  generations ago. We determined that in  D. melanogaster,  a recent selective sweep (within the last 0.25 N  generations) is clearly not consistent with the polymorphism data, whereas adaptive mutations occurring more than  N  generations ago are consistent with the polymorphism data, and selective sweeps more than 1.5 N  generations ago are possible ( Figure 6 ). Similarly, the marginal distribution of selection coefficients suggests that the strength of selection is inconsistent with small selection coefficients and recent selective sweeps (data not shown). Together, these results show that given our knowledge that selection has occurred at a large number of codons at  Hmr,  the last selection event occurred in the distant past. These procedures have been applied to infer selection events associated with human evolution ( Przeworski 2003 ) but have not previously been applied to HI loci. Estimating the time of fixation events is integral to determining whether selection events that are found at HI loci are consistent with the known time frame of speciation or might merely reflect ongoing contemporary selection. Figure 6 Posterior Distribution of Time (in Generations) Since the Most Recent Selective Sweep at  Hmr  for  D. melanogaster
 Population size  (N)  is assumed to be 1 × 10 6  for  D. melanogaster.  Samples were generated from the joint posterior distribution of five parameters of a selective sweep model assuming a selection event occurred sometime in the past at the  Hmr  locus, and from three summaries of polymorphism, including the number of segregating sites (54), Tajima's D (−0.61), the population recombination rate (4 Nr  = 43, where  N  = population size and  r  = per gene recombination rate;  McVean et al. 2002 ) and the number of haplotypes (11). The data is least consistent with a selective sweep in the recent past and is most consistent with selective sweeps occurring more than  N  generations ago. If there are ten generations per year, this suggests that the last selective sweep occurred at least 100,000 years ago. Data for  D. simulans  are not shown, as the population structure for the  D. simulans  Wolfskill populations we sampled would inflate estimates through increasing marginal frequencies of segregating sites ( Wall et al. 2002 ).
Selection Events Are Ancient
Loci directly involved in reproductive isolation may reflect the true species history of divergence more accurately than a “random” locus sampled from the genome, because these loci cease exchanging alleles among species earlier than other loci ( Wang et al. 1997 ;  Ting et al. 2000 ). Both reduced gene flow and adaptive fixations can remove shared ancient polymorphisms.  D. melanogaster  and  D. simulans  separated approximately 2 to 3 million years ago ( Powell 1997 ), and many shared polymorphisms have been lost due to drift ( Clark 1997 ). However, shared polymorphisms are still observed in these genomes: among 15 loci ( Andolfatto and Przeworski 2000 ), we found that 5.2% and 3.5% of segregating sites in  D. melanogaster  and  D. simulans , respectively, are segregating at the same positions (among all classes of sites). Based on these values we expected to find approximately three to four shared polymorphic sites for  Hmr  among the 54 segregating sites in  D. melanogaster  and 94 segregating sites in  D. simulans , but instead found zero. Although the expectation of three to four shared polymorphisms is not a strong test, these findings are consistent with  Hmr  alleles not having been exchanged between these taxa since at or near the time of species divergence and/or with selection events having occurred throughout the  Hmr  gene and having removed recurrent mutations.HmrHmrHmrWe have shown by several methods that the elevated rate of amino-acid divergence at  Hmr  is due to positive selection. These findings raise the critical question of whether selection events occurred recently or at the time of speciation. The number of years separating  D. melanogaster  and the sibling-species lineage makes this a challenging task. We therefore concentrated on the more tractable problem of looking for evidence to exclude the possibility that  Hmr  divergence reflects the action of contemporary or recent selection. In other words, while it may not be possible to prove that selection occurred at  Hmr  at the time of speciation, it is nevertheless important to demonstrate that available data are, at the least, not inconsistent with more ancient selective events. Examination of the frequency spectrum of polymorphism within both  D. melanogaster  and  D. simulans  showed no evidence of recent sweeps, as the distribution of per-site heterozygosities did not differ from expectations of neutrality (Tajima's D is −0.61 and 0.75, respectively, which is not significant). However, the power for detecting a selective sweep with this method is not high (75%) even if the event occurred in the very recent past, and diminishes to 25% as early as 0.8 N  generations ago (where  N = population size) ( Przeworski 2002 ). Because we have prior knowledge from the analyses above that selection events occurred at  Hmr,  we reasoned that we could apply more powerful Bayesian approaches that use not only frequency spectrum information but other summary statistics of polymorphism and recombination to estimate the time at which these events happened ( Przeworski 2003 ). To estimate simultaneously the time and strength of selection events we conditioned on three summary statistics of the data: the polymorphism frequency spectrum (Tajima's D), the number of segregating sites, and the number of haplotypes. Prior distributions were chosen based on estimates of recombination, mutation rates, and effective population size. The most recent common ancestor for an  X -linked locus is on average 3 N  generations ago. We determined that in  D. melanogaster,  a recent selective sweep (within the last 0.25 N  generations) is clearly not consistent with the polymorphism data, whereas adaptive mutations occurring more than  N  generations ago are consistent with the polymorphism data, and selective sweeps more than 1.5 N  generations ago are possible ( Figure 6 ). Similarly, the marginal distribution of selection coefficients suggests that the strength of selection is inconsistent with small selection coefficients and recent selective sweeps (data not shown). Together, these results show that given our knowledge that selection has occurred at a large number of codons at  Hmr,  the last selection event occurred in the distant past. These procedures have been applied to infer selection events associated with human evolution ( Przeworski 2003 ) but have not previously been applied to HI loci. Estimating the time of fixation events is integral to determining whether selection events that are found at HI loci are consistent with the known time frame of speciation or might merely reflect ongoing contemporary selection.HmrHmrHmrNNHmr,XND. melanogaster,NNNHmr,Discussion We have shown here that transgenes carrying  D. simulans  or  D. mauritiana Hmr +  have no effect on hybrid fitness, in contrast to the strong deleterious effects previously observed with  D. melanogaster Hmr + .  These transgenic experiments demonstrate that  Hmr  has functionally diverged between  D. melanogaster  and its sibling species and thus meets the experimental criteria defined by the D-M model. To our knowledge this is the first demonstration of functional divergence for a  D. melanogaster  HI gene. Although transgenic technologies have limitations—for example, genes with large and complex regulatory regions may not function correctly when transformed—they have the clear advantage of providing unambiguous analyses of single genes. Transgenics was previously used ( Winkler et al. 1994 ) to show that the  Xmrk-2  gene from the platyfish  Xiphophorus maculatus  causes HI (although for technical reasons it was assayed in the related fish  Oryzias latipes  rather than the actual hybridizing species  X. helleri  [Weis and  Schartl 1998 ]). Functional divergence is inferred in this case because  Xmrk-2  (also called ONC-X mrk ) is a gene duplication that is present in  X. maculatus  but appears to be absent from  X. helleri ). Functional divergence can also be inferred in other HI systems where multigene regions are transferred between species by repeated backcrossing. This approach has been used to show that  Odysseus (Ods)  has functionally diverged: X-chromosome introgressions from  D. mauritiana  into  D. simulans  that contain the  D. mauritiana Ods  region are sterile, while related introgressions that lack  D. mauritiana Ods  remain fertile ( Ting et al. 1998 ). Both  Hmr  and  Ods,  as well as  Nup96,  which has recently been implicated in causing lethality in  D. melanogaster / D. simulans  male hybrids ( Presgraves et al. 2003 ), show high rates of divergence for part  (Ods  and  Nup96)  or most  (Hmr)  of their coding regions. If generally true, this finding may reflect the fact that a high rate of substitution is required in order to generate a rare HI-causing mutation. Alternatively, it may be an indication that the combined effects of multiple substitutions are required to generate an HI allele. Our analysis of  Hmr  suggests two additional characteristics of HI genes. One is that while the D-M model requires divergence along only one lineage in order to generate HI, we have strong statistical support for accelerated divergence of  Hmr  along both lineages since  D. melanogaster  and the sibling species split. One consequence of this extensive divergence is that it becomes unexpectedly complicated to identify the divergent codons in  Hmr  that are candidates for causing the incompatibility. The simple model shown in  Figure 1 B, where HI is caused by the interaction of  Hmr mel  and  A sib ,  implies that any codons in  Hmr mel  that have diverged from the ancestral allele could potentially be causing the incompatibility; these codons could also diverge in the sibling-species alleles as long as they remain different from the  D. melanogaster  allele. There are 137 amino acids in  Hmr  (plus two sites of  D. melanogaster –specific insertions) that fit these criteria. However, considering the extensive divergence we have detected at  Hmr  along both lineages, a more realistic model is shown in  Figure 1 D, where both  Hmr  and gene  A  go through multiple changes in both  D. melanogaster  and the sibling species. This model raises a possibility described by  Muller (1942) , namely, that a derived allele may become incompatible with an ancestral allele. For example,  A sib  may become incompatible with  Hmr anc ,  which means that  Hmr mel  will also be incompatible if it retains the interacting residues present in  Hmr anc .  Since  Hmr sib  must be compatible with  A sib ,  the candidate codons are those where  Hmr sib  differs from  Hmr anc  and  Hmr mel ,  and  Hmr mel  remains identical to  Hmr anc .  There are 49 amino acids that fit this model, all of which are different from those identified in the previous model. Distinguishing between these models may be possible by using our transgene assays on  Hmr +  constructs containing site-directed changes at candidate amino acids. Divergence due to positive selection is a second striking characteristic of  Hmr  as well as  Ods  and  Nup96.  Adaptive fixations and reproductive isolation at  Hmr  have clearly shaped the pattern of polymorphism relative to divergence and swept away any shared polymorphisms that may have been present. This characteristic raises the general question of what forms of selection are responsible and whether genes involved in certain traits or phenotypes that are under strong directional selection may preferentially contribute to HI. In allopatric models, where speciation occurs between two populations in geographical isolation, HI between species is strictly a secondary consequence of divergence of HI genes that has occurred within each species. The target of selection must therefore be sought by looking at gene function within species. We do not yet know the function of  Hmr; Hmr 1  mutants are viable and fertile, but this allele is clearly hypomorphic ( Barbash et al. 2003 ) and the null phenotype remains unknown. Because  Hmr  is expressed and causes HI in both sexes, however, it appears unlikely that it diverged under sexual selection. We do know that the pattern of variation in  Hmr  is not consistent with only weak selection events that have occurred recently. Rather,  Hmr  polymorphism is consistent with selective events occurring more than 0.5 N  generations ago and with a potentially large range of selection coefficients. Also unique to  Hmr  is that we have shown that it is likely to have been subject to multiple selective sweeps in both the  D. melanogaster  and sibling-species lineages and that the signal of positive selection comes from multiple sites and regions. We have argued here that experimental demonstrations of functional divergence are required to prove that a gene is a bona fide HI locus. While man has undoubtedly been aware of the phenomenon of plant and animal HI for thousands of years, and hundreds of examples have been described in the scientific literature, identifying the genes involved has progressed rather slowly, with candidates generally being discovered by either genetic mapping or suppressor screens. As genomic sequences become available for species closely related to model organisms such as  D. melanogaster,  we suggest that the characteristics of  Hmr,  including high levels of divergence due to positive selection, may provide an alternative means of identification, with comparative genomics being used to identify candidate HI genes.
Discussion
We have shown here that transgenes carrying  D. simulans  or  D. mauritiana Hmr +  have no effect on hybrid fitness, in contrast to the strong deleterious effects previously observed with  D. melanogaster Hmr + .  These transgenic experiments demonstrate that  Hmr  has functionally diverged between  D. melanogaster  and its sibling species and thus meets the experimental criteria defined by the D-M model. To our knowledge this is the first demonstration of functional divergence for a  D. melanogaster  HI gene. Although transgenic technologies have limitations—for example, genes with large and complex regulatory regions may not function correctly when transformed—they have the clear advantage of providing unambiguous analyses of single genes. Transgenics was previously used ( Winkler et al. 1994 ) to show that the  Xmrk-2  gene from the platyfish  Xiphophorus maculatus  causes HI (although for technical reasons it was assayed in the related fish  Oryzias latipes  rather than the actual hybridizing species  X. helleri  [Weis and  Schartl 1998 ]). Functional divergence is inferred in this case because  Xmrk-2  (also called ONC-X mrk ) is a gene duplication that is present in  X. maculatus  but appears to be absent from  X. helleri ). Functional divergence can also be inferred in other HI systems where multigene regions are transferred between species by repeated backcrossing. This approach has been used to show that  Odysseus (Ods)  has functionally diverged: X-chromosome introgressions from  D. mauritiana  into  D. simulans  that contain the  D. mauritiana Ods  region are sterile, while related introgressions that lack  D. mauritiana Ods  remain fertile ( Ting et al. 1998 ).D. mauritiana Hmr +D. melanogaster Hmr + .HmrXmrk-2Xmrk-2mrkOdysseus (Ods)D. mauritiana OdsD. mauritiana OdsBoth  Hmr  and  Ods,  as well as  Nup96,  which has recently been implicated in causing lethality in  D. melanogaster / D. simulans  male hybrids ( Presgraves et al. 2003 ), show high rates of divergence for part  (Ods  and  Nup96)  or most  (Hmr)  of their coding regions. If generally true, this finding may reflect the fact that a high rate of substitution is required in order to generate a rare HI-causing mutation. Alternatively, it may be an indication that the combined effects of multiple substitutions are required to generate an HI allele. Our analysis of  Hmr  suggests two additional characteristics of HI genes. One is that while the D-M model requires divergence along only one lineage in order to generate HI, we have strong statistical support for accelerated divergence of  Hmr  along both lineages since  D. melanogaster  and the sibling species split.HmrOds,Nup96,(OdsNup96)(Hmr)HmrHmrOne consequence of this extensive divergence is that it becomes unexpectedly complicated to identify the divergent codons in  Hmr  that are candidates for causing the incompatibility. The simple model shown in  Figure 1 B, where HI is caused by the interaction of  Hmr mel  and  A sib ,  implies that any codons in  Hmr mel  that have diverged from the ancestral allele could potentially be causing the incompatibility; these codons could also diverge in the sibling-species alleles as long as they remain different from the  D. melanogaster  allele. There are 137 amino acids in  Hmr  (plus two sites of  D. melanogaster –specific insertions) that fit these criteria. However, considering the extensive divergence we have detected at  Hmr  along both lineages, a more realistic model is shown in  Figure 1 D, where both  Hmr  and gene  A  go through multiple changes in both  D. melanogaster  and the sibling species. This model raises a possibility described by  Muller (1942) , namely, that a derived allele may become incompatible with an ancestral allele. For example,  A sib  may become incompatible with  Hmr anc ,  which means that  Hmr mel  will also be incompatible if it retains the interacting residues present in  Hmr anc .  Since  Hmr sib  must be compatible with  A sib ,  the candidate codons are those where  Hmr sib  differs from  Hmr anc  and  Hmr mel ,  and  Hmr mel  remains identical to  Hmr anc .  There are 49 amino acids that fit this model, all of which are different from those identified in the previous model. Distinguishing between these models may be possible by using our transgene assays on  Hmr +  constructs containing site-directed changes at candidate amino acids.HmrHmr melA sib ,Hmr melHmrHmrHmrAA sibHmr anc ,Hmr melHmr anc .Hmr sibA sib ,Hmr sibHmr ancHmr mel ,Hmr melHmr anc .Hmr +Divergence due to positive selection is a second striking characteristic of  Hmr  as well as  Ods  and  Nup96.  Adaptive fixations and reproductive isolation at  Hmr  have clearly shaped the pattern of polymorphism relative to divergence and swept away any shared polymorphisms that may have been present. This characteristic raises the general question of what forms of selection are responsible and whether genes involved in certain traits or phenotypes that are under strong directional selection may preferentially contribute to HI. In allopatric models, where speciation occurs between two populations in geographical isolation, HI between species is strictly a secondary consequence of divergence of HI genes that has occurred within each species. The target of selection must therefore be sought by looking at gene function within species. We do not yet know the function of  Hmr; Hmr 1  mutants are viable and fertile, but this allele is clearly hypomorphic ( Barbash et al. 2003 ) and the null phenotype remains unknown. Because  Hmr  is expressed and causes HI in both sexes, however, it appears unlikely that it diverged under sexual selection. We do know that the pattern of variation in  Hmr  is not consistent with only weak selection events that have occurred recently. Rather,  Hmr  polymorphism is consistent with selective events occurring more than 0.5 N  generations ago and with a potentially large range of selection coefficients. Also unique to  Hmr  is that we have shown that it is likely to have been subject to multiple selective sweeps in both the  D. melanogaster  and sibling-species lineages and that the signal of positive selection comes from multiple sites and regions.HmrOdsNup96.HmrHmr; Hmr 1HmrHmrHmrNHmrWe have argued here that experimental demonstrations of functional divergence are required to prove that a gene is a bona fide HI locus. While man has undoubtedly been aware of the phenomenon of plant and animal HI for thousands of years, and hundreds of examples have been described in the scientific literature, identifying the genes involved has progressed rather slowly, with candidates generally being discovered by either genetic mapping or suppressor screens. As genomic sequences become available for species closely related to model organisms such as  D. melanogaster,  we suggest that the characteristics of  Hmr,  including high levels of divergence due to positive selection, may provide an alternative means of identification, with comparative genomics being used to identify candidate HI genes.D. melanogaster,Hmr,Materials and Methods Nomenclature The subscripts  mel, sib,  and  anc  are used to designate genes from  D. melanogaster,  its three sibling species, and their (hypothetical) ancestor, respectively. RT-PCR RT-PCR was performed from total RNA as described in  Barbash et al. (2003) . Thirty-five or 40 cycles of PCR were performed in a 50-μl volume with the oligos 5′-AAATCGAATCGCTTGTTTGG-3′ and 5′-CTCGAGCGGATGGTAGCGCAC-3′ at an annealing temperature of 61 °C. Two reactions per template were processed with QIAquick PCR Purification (Qiagen, Valencia, California, United States), eluted in 50 μl of 10mM Tris-HCl (pH 8.0), ethanol precipitated in the presence of 10 μg of glycogen, and resuspended in 10 μl of 10mM Tris-HCl (pH 8.0). Two microliters of DNA was digested with the appropriate restriction enzyme and run on a 2% agarose/TAE gel. Clones and constructs Sibling-species  Hmr  constructs were derived from  D. simulans  and  D. mauritiana  lambda genomic libraries ( Ting et al. 1998 ). A  D. simulans  phage clone was isolated using the  D. melanogaster Hmr  cDNA clone RE54143 ( Barbash et al. 2003 ) as a probe. An approximately 3.8-kb BamHI/NotI fragment of phage DNA was cloned into pBSII KS(+) (Stratagene, La Jolla, California, United States) to create p88. The adjacent approximately 5.8-kb BamHI phage DNA fragment was then cloned into the BamHI site of p88 to create p89; the correct orientation was determined by PCR across the BamHI junction. Using an XbaI site near the end of the p89 insert, p92 was made by cloning the approximately 8.6-kb NotI/XbaI fragment from p89 into pCasper4. End sequencing of this construct demonstrated that it extends from approximately 2.8 kb 5′ of  Hmr  to approximately 1.05 kb 3′ of  Hmr ; this construct also contains the complete predicted coding region of  CG2124.  The formal designation of this construct is  P{w +mc  Dsim\CG2124 +t8.6  Dsim\Hmr +t8.6 = Dsim\Hmr +t8.6 }.
 A  D. mauritiana  phage clone was isolated using a  D. mauritiana  PCR product from exon 2 of  Hmr  as a probe. The entire phage insert was cloned into the NotI site of pBSII KS(+) to make the p94 plasmid. An approximately 9.4-kb NotI/XbaI fragment of the p94 insert was cloned into the corresponding sites of pCasper4. End sequencing of the insert demonstrated that this construct extends from approximately 2.8 kb 5′ of  Hmr  to approximately 1.8 kb 3′ of  Hmr.  This construct also contains the complete predicted coding regions of  Rab9D  and  CG2124.  The formal designation of this construct is  P{w +mc  Dmau\Rab9D +t9.4  Dmau\Hmr +t9.4  Dmau\CG2124 +t9.4 =Dmau\Hmr +t9.4 }.

 D. erecta Hmr  was isolated by screening a gridded fosmid library (BACPAC Resource Center, Oakland, California, United States) with  D. melanogaster Hmr  cDNA clone RE54143 as a probe. An approximately 8-kb XbaI fragment was subcloned from fosmid 11D-19 into pBSII KS(+) and sequenced using a GPS-1 Genome Priming System (New England Biolabs, Beverly, Massachusetts, United States) and custom sequencing primers. We also identified an  Hmr  ortholog from the more distant  D. pseudoobscura,  but an unalignable repetitive region in the second exon made unambiguous calculations of amino-acid and silent divergence impossible. As is found in  D. melanogaster , the ortholog of  CG2124  is located 5′ of  Hmr  in opposite orientation in both  D. erecta  and  D. pseudoobscura,  demonstrating that we have correctly identified the ortholog of  Hmr  in both of these species. Population samples Ten  D. melanogaster Hmr  alleles were sequenced from a collection of iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe ( Begun and Aquadro 1993 ). Five  D. simulans Hmr  alleles were sequenced from a collection of highly inbred isofemale lines collected in Wolfskill, California, United States ( Begun and Whitley 2000 ).  Hmr  alleles were amplified by PCR in five overlapping segments of approximately 1 to 1.5 kb and sequenced directly using Big Dye chemistry (version 3; Applied Biosystems, Foster City, California, United States). We also included in our analyses four  D. melanogaster  alleles and one each from the three sibling species reported previously ( Barbash et al. 2003 ), as well as the  D. simulans  p92 clone described above. Our population data set contains the complete coding region of  Hmr  as well as 447 bases upstream and 326 bases downstream of the coding region. Sequence analysis Sequences were aligned using ClustalX. Phylogenetic analyses were performed with PAML ( Yang 1997 ). Population genetic analyses were performed using C++ libraries from K. Thornton. Estimates of the population recombination rate were calculated using Ldhat ( McVean et al. 2002 ). Exact  p- values for FETs were derived using the R statistical package. The HKA test was performed using the HKA program by Jody Hey. Estimates of the posterior distribution for time in generations since the most recent selective sweep were estimated using the method of  Przeworski (2003) .
Materials and Methods
Nomenclature The subscripts  mel, sib,  and  anc  are used to designate genes from  D. melanogaster,  its three sibling species, and their (hypothetical) ancestor, respectively. RT-PCR RT-PCR was performed from total RNA as described in  Barbash et al. (2003) . Thirty-five or 40 cycles of PCR were performed in a 50-μl volume with the oligos 5′-AAATCGAATCGCTTGTTTGG-3′ and 5′-CTCGAGCGGATGGTAGCGCAC-3′ at an annealing temperature of 61 °C. Two reactions per template were processed with QIAquick PCR Purification (Qiagen, Valencia, California, United States), eluted in 50 μl of 10mM Tris-HCl (pH 8.0), ethanol precipitated in the presence of 10 μg of glycogen, and resuspended in 10 μl of 10mM Tris-HCl (pH 8.0). Two microliters of DNA was digested with the appropriate restriction enzyme and run on a 2% agarose/TAE gel. Clones and constructs Sibling-species  Hmr  constructs were derived from  D. simulans  and  D. mauritiana  lambda genomic libraries ( Ting et al. 1998 ). A  D. simulans  phage clone was isolated using the  D. melanogaster Hmr  cDNA clone RE54143 ( Barbash et al. 2003 ) as a probe. An approximately 3.8-kb BamHI/NotI fragment of phage DNA was cloned into pBSII KS(+) (Stratagene, La Jolla, California, United States) to create p88. The adjacent approximately 5.8-kb BamHI phage DNA fragment was then cloned into the BamHI site of p88 to create p89; the correct orientation was determined by PCR across the BamHI junction. Using an XbaI site near the end of the p89 insert, p92 was made by cloning the approximately 8.6-kb NotI/XbaI fragment from p89 into pCasper4. End sequencing of this construct demonstrated that it extends from approximately 2.8 kb 5′ of  Hmr  to approximately 1.05 kb 3′ of  Hmr ; this construct also contains the complete predicted coding region of  CG2124.  The formal designation of this construct is  P{w +mc  Dsim\CG2124 +t8.6  Dsim\Hmr +t8.6 = Dsim\Hmr +t8.6 }.
 A  D. mauritiana  phage clone was isolated using a  D. mauritiana  PCR product from exon 2 of  Hmr  as a probe. The entire phage insert was cloned into the NotI site of pBSII KS(+) to make the p94 plasmid. An approximately 9.4-kb NotI/XbaI fragment of the p94 insert was cloned into the corresponding sites of pCasper4. End sequencing of the insert demonstrated that this construct extends from approximately 2.8 kb 5′ of  Hmr  to approximately 1.8 kb 3′ of  Hmr.  This construct also contains the complete predicted coding regions of  Rab9D  and  CG2124.  The formal designation of this construct is  P{w +mc  Dmau\Rab9D +t9.4  Dmau\Hmr +t9.4  Dmau\CG2124 +t9.4 =Dmau\Hmr +t9.4 }.

 D. erecta Hmr  was isolated by screening a gridded fosmid library (BACPAC Resource Center, Oakland, California, United States) with  D. melanogaster Hmr  cDNA clone RE54143 as a probe. An approximately 8-kb XbaI fragment was subcloned from fosmid 11D-19 into pBSII KS(+) and sequenced using a GPS-1 Genome Priming System (New England Biolabs, Beverly, Massachusetts, United States) and custom sequencing primers. We also identified an  Hmr  ortholog from the more distant  D. pseudoobscura,  but an unalignable repetitive region in the second exon made unambiguous calculations of amino-acid and silent divergence impossible. As is found in  D. melanogaster , the ortholog of  CG2124  is located 5′ of  Hmr  in opposite orientation in both  D. erecta  and  D. pseudoobscura,  demonstrating that we have correctly identified the ortholog of  Hmr  in both of these species. Population samples Ten  D. melanogaster Hmr  alleles were sequenced from a collection of iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe ( Begun and Aquadro 1993 ). Five  D. simulans Hmr  alleles were sequenced from a collection of highly inbred isofemale lines collected in Wolfskill, California, United States ( Begun and Whitley 2000 ).  Hmr  alleles were amplified by PCR in five overlapping segments of approximately 1 to 1.5 kb and sequenced directly using Big Dye chemistry (version 3; Applied Biosystems, Foster City, California, United States). We also included in our analyses four  D. melanogaster  alleles and one each from the three sibling species reported previously ( Barbash et al. 2003 ), as well as the  D. simulans  p92 clone described above. Our population data set contains the complete coding region of  Hmr  as well as 447 bases upstream and 326 bases downstream of the coding region. Sequence analysis Sequences were aligned using ClustalX. Phylogenetic analyses were performed with PAML ( Yang 1997 ). Population genetic analyses were performed using C++ libraries from K. Thornton. Estimates of the population recombination rate were calculated using Ldhat ( McVean et al. 2002 ). Exact  p- values for FETs were derived using the R statistical package. The HKA test was performed using the HKA program by Jody Hey. Estimates of the posterior distribution for time in generations since the most recent selective sweep were estimated using the method of  Przeworski (2003) .

Nomenclature The subscripts  mel, sib,  and  anc  are used to designate genes from  D. melanogaster,  its three sibling species, and their (hypothetical) ancestor, respectively.
Nomenclature
The subscripts  mel, sib,  and  anc  are used to designate genes from  D. melanogaster,  its three sibling species, and their (hypothetical) ancestor, respectively.mel, sib,ancD. melanogaster,RT-PCR RT-PCR was performed from total RNA as described in  Barbash et al. (2003) . Thirty-five or 40 cycles of PCR were performed in a 50-μl volume with the oligos 5′-AAATCGAATCGCTTGTTTGG-3′ and 5′-CTCGAGCGGATGGTAGCGCAC-3′ at an annealing temperature of 61 °C. Two reactions per template were processed with QIAquick PCR Purification (Qiagen, Valencia, California, United States), eluted in 50 μl of 10mM Tris-HCl (pH 8.0), ethanol precipitated in the presence of 10 μg of glycogen, and resuspended in 10 μl of 10mM Tris-HCl (pH 8.0). Two microliters of DNA was digested with the appropriate restriction enzyme and run on a 2% agarose/TAE gel.
RT-PCR
RT-PCR was performed from total RNA as described in  Barbash et al. (2003) . Thirty-five or 40 cycles of PCR were performed in a 50-μl volume with the oligos 5′-AAATCGAATCGCTTGTTTGG-3′ and 5′-CTCGAGCGGATGGTAGCGCAC-3′ at an annealing temperature of 61 °C. Two reactions per template were processed with QIAquick PCR Purification (Qiagen, Valencia, California, United States), eluted in 50 μl of 10mM Tris-HCl (pH 8.0), ethanol precipitated in the presence of 10 μg of glycogen, and resuspended in 10 μl of 10mM Tris-HCl (pH 8.0). Two microliters of DNA was digested with the appropriate restriction enzyme and run on a 2% agarose/TAE gel.Clones and constructs Sibling-species  Hmr  constructs were derived from  D. simulans  and  D. mauritiana  lambda genomic libraries ( Ting et al. 1998 ). A  D. simulans  phage clone was isolated using the  D. melanogaster Hmr  cDNA clone RE54143 ( Barbash et al. 2003 ) as a probe. An approximately 3.8-kb BamHI/NotI fragment of phage DNA was cloned into pBSII KS(+) (Stratagene, La Jolla, California, United States) to create p88. The adjacent approximately 5.8-kb BamHI phage DNA fragment was then cloned into the BamHI site of p88 to create p89; the correct orientation was determined by PCR across the BamHI junction. Using an XbaI site near the end of the p89 insert, p92 was made by cloning the approximately 8.6-kb NotI/XbaI fragment from p89 into pCasper4. End sequencing of this construct demonstrated that it extends from approximately 2.8 kb 5′ of  Hmr  to approximately 1.05 kb 3′ of  Hmr ; this construct also contains the complete predicted coding region of  CG2124.  The formal designation of this construct is  P{w +mc  Dsim\CG2124 +t8.6  Dsim\Hmr +t8.6 = Dsim\Hmr +t8.6 }.
 A  D. mauritiana  phage clone was isolated using a  D. mauritiana  PCR product from exon 2 of  Hmr  as a probe. The entire phage insert was cloned into the NotI site of pBSII KS(+) to make the p94 plasmid. An approximately 9.4-kb NotI/XbaI fragment of the p94 insert was cloned into the corresponding sites of pCasper4. End sequencing of the insert demonstrated that this construct extends from approximately 2.8 kb 5′ of  Hmr  to approximately 1.8 kb 3′ of  Hmr.  This construct also contains the complete predicted coding regions of  Rab9D  and  CG2124.  The formal designation of this construct is  P{w +mc  Dmau\Rab9D +t9.4  Dmau\Hmr +t9.4  Dmau\CG2124 +t9.4 =Dmau\Hmr +t9.4 }.

 D. erecta Hmr  was isolated by screening a gridded fosmid library (BACPAC Resource Center, Oakland, California, United States) with  D. melanogaster Hmr  cDNA clone RE54143 as a probe. An approximately 8-kb XbaI fragment was subcloned from fosmid 11D-19 into pBSII KS(+) and sequenced using a GPS-1 Genome Priming System (New England Biolabs, Beverly, Massachusetts, United States) and custom sequencing primers. We also identified an  Hmr  ortholog from the more distant  D. pseudoobscura,  but an unalignable repetitive region in the second exon made unambiguous calculations of amino-acid and silent divergence impossible. As is found in  D. melanogaster , the ortholog of  CG2124  is located 5′ of  Hmr  in opposite orientation in both  D. erecta  and  D. pseudoobscura,  demonstrating that we have correctly identified the ortholog of  Hmr  in both of these species.
Clones and constructs
Sibling-species  Hmr  constructs were derived from  D. simulans  and  D. mauritiana  lambda genomic libraries ( Ting et al. 1998 ). A  D. simulans  phage clone was isolated using the  D. melanogaster Hmr  cDNA clone RE54143 ( Barbash et al. 2003 ) as a probe. An approximately 3.8-kb BamHI/NotI fragment of phage DNA was cloned into pBSII KS(+) (Stratagene, La Jolla, California, United States) to create p88. The adjacent approximately 5.8-kb BamHI phage DNA fragment was then cloned into the BamHI site of p88 to create p89; the correct orientation was determined by PCR across the BamHI junction. Using an XbaI site near the end of the p89 insert, p92 was made by cloning the approximately 8.6-kb NotI/XbaI fragment from p89 into pCasper4. End sequencing of this construct demonstrated that it extends from approximately 2.8 kb 5′ of  Hmr  to approximately 1.05 kb 3′ of  Hmr ; this construct also contains the complete predicted coding region of  CG2124.  The formal designation of this construct is  P{w +mc  Dsim\CG2124 +t8.6  Dsim\Hmr +t8.6 = Dsim\Hmr +t8.6 }.HmrD. melanogaster HmrHmrHmrCG2124.P{w +mc  Dsim\CG2124 +t8.6  Dsim\Hmr +t8.6 = Dsim\Hmr +t8.6 }.A  D. mauritiana  phage clone was isolated using a  D. mauritiana  PCR product from exon 2 of  Hmr  as a probe. The entire phage insert was cloned into the NotI site of pBSII KS(+) to make the p94 plasmid. An approximately 9.4-kb NotI/XbaI fragment of the p94 insert was cloned into the corresponding sites of pCasper4. End sequencing of the insert demonstrated that this construct extends from approximately 2.8 kb 5′ of  Hmr  to approximately 1.8 kb 3′ of  Hmr.  This construct also contains the complete predicted coding regions of  Rab9D  and  CG2124.  The formal designation of this construct is  P{w +mc  Dmau\Rab9D +t9.4  Dmau\Hmr +t9.4  Dmau\CG2124 +t9.4 =Dmau\Hmr +t9.4 }.HmrHmrHmr.Rab9DCG2124.P{w +mc  Dmau\Rab9D +t9.4  Dmau\Hmr +t9.4  Dmau\CG2124 +t9.4 =Dmau\Hmr +t9.4 }.D. erecta Hmr  was isolated by screening a gridded fosmid library (BACPAC Resource Center, Oakland, California, United States) with  D. melanogaster Hmr  cDNA clone RE54143 as a probe. An approximately 8-kb XbaI fragment was subcloned from fosmid 11D-19 into pBSII KS(+) and sequenced using a GPS-1 Genome Priming System (New England Biolabs, Beverly, Massachusetts, United States) and custom sequencing primers.D. erecta HmrD. melanogaster HmrWe also identified an  Hmr  ortholog from the more distant  D. pseudoobscura,  but an unalignable repetitive region in the second exon made unambiguous calculations of amino-acid and silent divergence impossible.HmrD. pseudoobscura,As is found in  D. melanogaster , the ortholog of  CG2124  is located 5′ of  Hmr  in opposite orientation in both  D. erecta  and  D. pseudoobscura,  demonstrating that we have correctly identified the ortholog of  Hmr  in both of these species.CG2124HmrD. pseudoobscura,HmrPopulation samples Ten  D. melanogaster Hmr  alleles were sequenced from a collection of iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe ( Begun and Aquadro 1993 ). Five  D. simulans Hmr  alleles were sequenced from a collection of highly inbred isofemale lines collected in Wolfskill, California, United States ( Begun and Whitley 2000 ).  Hmr  alleles were amplified by PCR in five overlapping segments of approximately 1 to 1.5 kb and sequenced directly using Big Dye chemistry (version 3; Applied Biosystems, Foster City, California, United States). We also included in our analyses four  D. melanogaster  alleles and one each from the three sibling species reported previously ( Barbash et al. 2003 ), as well as the  D. simulans  p92 clone described above. Our population data set contains the complete coding region of  Hmr  as well as 447 bases upstream and 326 bases downstream of the coding region.
Population samples
Ten  D. melanogaster Hmr  alleles were sequenced from a collection of iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe ( Begun and Aquadro 1993 ). Five  D. simulans Hmr  alleles were sequenced from a collection of highly inbred isofemale lines collected in Wolfskill, California, United States ( Begun and Whitley 2000 ).  Hmr  alleles were amplified by PCR in five overlapping segments of approximately 1 to 1.5 kb and sequenced directly using Big Dye chemistry (version 3; Applied Biosystems, Foster City, California, United States). We also included in our analyses four  D. melanogaster  alleles and one each from the three sibling species reported previously ( Barbash et al. 2003 ), as well as the  D. simulans  p92 clone described above. Our population data set contains the complete coding region of  Hmr  as well as 447 bases upstream and 326 bases downstream of the coding region.D. melanogaster HmrD. simulans HmrHmrHmrSequence analysis Sequences were aligned using ClustalX. Phylogenetic analyses were performed with PAML ( Yang 1997 ). Population genetic analyses were performed using C++ libraries from K. Thornton. Estimates of the population recombination rate were calculated using Ldhat ( McVean et al. 2002 ). Exact  p- values for FETs were derived using the R statistical package. The HKA test was performed using the HKA program by Jody Hey. Estimates of the posterior distribution for time in generations since the most recent selective sweep were estimated using the method of  Przeworski (2003) .
Sequence analysis
Sequences were aligned using ClustalX. Phylogenetic analyses were performed with PAML ( Yang 1997 ). Population genetic analyses were performed using C++ libraries from K. Thornton. Estimates of the population recombination rate were calculated using Ldhat ( McVean et al. 2002 ). Exact  p- values for FETs were derived using the R statistical package. The HKA test was performed using the HKA program by Jody Hey. Estimates of the posterior distribution for time in generations since the most recent selective sweep were estimated using the method of  Przeworski (2003) .p-Supporting Information Accession Numbers The GenBank accession numbers of the genes discussed in this paper are  Hmr  coding region from  D. erecta  (AY568390),  Hmr  coding region from  D. mauritiana  contained in plasmid p94 (AY573924),  Hmr  coding region from  D. simulans  contained in plasmid p92 (AY568391),  D. melanogaster Hmr  alleles sequenced from iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe (AY568380–AY568389), and  D. simulans Hmr  alleles sequenced from highly inbred isofemale lines collected in Wolfskill, California, United States (AY568392–AY568396).
Supporting Information
Accession Numbers The GenBank accession numbers of the genes discussed in this paper are  Hmr  coding region from  D. erecta  (AY568390),  Hmr  coding region from  D. mauritiana  contained in plasmid p94 (AY573924),  Hmr  coding region from  D. simulans  contained in plasmid p92 (AY568391),  D. melanogaster Hmr  alleles sequenced from iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe (AY568380–AY568389), and  D. simulans Hmr  alleles sequenced from highly inbred isofemale lines collected in Wolfskill, California, United States (AY568392–AY568396).
Accession Numbers
The GenBank accession numbers of the genes discussed in this paper are  Hmr  coding region from  D. erecta  (AY568390),  Hmr  coding region from  D. mauritiana  contained in plasmid p94 (AY573924),  Hmr  coding region from  D. simulans  contained in plasmid p92 (AY568391),  D. melanogaster Hmr  alleles sequenced from iso-X-chromosome stocks derived from isofemale lines collected in Zimbabwe (AY568380–AY568389), and  D. simulans Hmr  alleles sequenced from highly inbred isofemale lines collected in Wolfskill, California, United States (AY568392–AY568396).HmrHmrHmrD. melanogaster HmrD. simulans Hmr