THE Y CHROMOSOMES OF DROSOPHILA SIMULANS ARE HIGHLY POLYMORPHIC FOR THEIR ABILITY TO SUPPRESS SEX-RATIO DRIVE

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1 Evolution, 55(4), 2001, pp THE Y CHROMOSOMES OF DROSOPHILA SIMULANS ARE HIGHLY POLYMORPHIC FOR THEIR ABILITY TO SUPPRESS SEX-RATIO DRIVE CATHERINE MONTCHAMP-MOREAU, 1,2 VALÉRIE GINHOUX, 2 AND ANNE ATLAN 2,3 1 Populations, Génétique et Evolution, CNRS, Gif-sur-Yvette cedex, France montchamp@pge.cnrs-gif.fr 2 Laboratoire Dynamique du Génome et Evolution, Institut Jacques Monod, CNRS-Université Paris 7, Paris cedex 05, France Abstract. The sex-ratio trait, known in several species of Drosophila including D. simulans, results from meiotic drive of the X chromosome against the Y. Males that carry a sex-ratio X chromosome produce strongly female-biased progeny. In D. simulans, drive suppressors have evolved on the Y chromosome and on the autosomes. Both the frequency of sex-ratio X and the strength of the total drive suppression (Y-linked and autosomal) vary widely among geographic populations of this worldwide species. We have investigated the pattern of Y-linked drive suppression in six natural populations representative of this variability. Y-linked suppressors were found to be a regular component of the suppression, with large differences between populations in the mean level of suppression. These variations did not correspond to differences in frequency of discrete types of Y chromosomes, but to a more or less wide continuum of phenotypes, from nonsuppressor to partial or total suppressor. We concluded that a large diversity of Y-linked suppressor alleles exists in D. simulans and that some populations are highly polymorphic. Our results support the hypothesis that a Y-chromosome polymorphism can be easily maintained by a balance between meiotic drive and the cost of drive suppression. Key words. Drosophila simulans, meiotic drive, sex ratio, Y chromosome. The sex-ratio trait is a case of meiotic drive expressed in males that naturally occurs in several species of Drosophila (see Cazemajor et al. 1997). Driving factors located on the X chromosome cause a deficiency of Y-bearing sperm, resulting in the production of nearly all female progeny. Because they are overrepresented among the functional gametes, the sex-ratio X chromosomes have a selective advantage and would increase to fixation in populations, unless they strongly reduce the fitness of their carriers (Edwards 1961; Hamilton 1967). If sex-ratio X chromosomes spread throughout populations, suppressors of their expression are expected to be selected on the Y and on the autosomes. Y chromosomes bearing suppressors are selected at the individual level, because they are transmitted better than sensitive Y chromosomes by males that carry a sex-ratio X (Thomson and Feldman 1975). Autosomal suppressors are selected at the population level when the sex ratio becomes biased toward females because of the advantage of any allele that increases the frequency of the rarer sex (Fisher 1930; Hamilton 1967; Wu 1983b). In agreement with the theory, autosomal and/or Y-linked suppressors have been found, or are strongly suspected to exist, in all the Drosophila species where they have been searched for, except in D. pseudoobscura (reviewed in Carvalho and Vaz 1999; Jaenike 1999). The sex-ratio X chromosomes are usually at moderate frequencies (10 20%) in natural populations of all the species known to harbor them. This implies that the selective advantage brought by the drive is balanced by deleterious effects on viability or fertility. Very few population studies have been made on drive suppressors, but the three species studied with respect to Y-linked suppressors, D. paramelan- 3 Present address: Laboratoire Evolution des Populations et des espèces, Université Rennes 1, Campus de Beaulieu Bâtiment 14, Rennes cedex, France; anne.atlan@univ-rennes1.fr 2001 The Society for the Study of Evolution. All rights reserved. Received May 24, Accepted November 13, ica (Stalker 1961), D. mediopunctata (Carvalho et al. 1997), and D. quinaria (Jaenike 1999), were found to be polymorphic. These observations support the hypothesis that drive suppression may also generally have a cost, and that sex-ratio meiotic drive can result in the maintenance of a balanced polymorphism of both sex chromosomes. Several models have been analyzed to determine the conditions required for a stable equilibrium. Under constant fitness models, with regard to the X chromosome alone, the conditions for the maintenance of a stable polymorphism of sex-ratio X are similar to those required for nondriving X-linked alleles: fitness differences at least among female genotypes are required (Edwards 1961). The proportion of the fitness parameter space that allows a stable polymorphism is weakly affected by the degree of drive (Curstinger and Feldman 1980). Alternatively, sex-ratio X polymorphism can be stabilized through frequency-dependent selection on male fertility (Jaenike 1996). This last model relies on the assumption that the males mate more frequently when the frequency of sex-ratio X increases in populations, because the sex ratio bias toward females increases accordingly. Such a frequency-dependent mechanism is possibly widespread because sex-ratio X chromosomes are known in several species to reduce male fertility when mating occurs repeatedly (Wu 1983a; Jaenike 1996). Density-dependent deleterious effects, related to sexual competition between males, were also invoked to explain the elimination of sex-ratio X from experimental populations of D. simulans (Capillon and Atlan 1999). When X and Y chromosomes are considered jointly, fitness differences among the genotypes are still necessary to maintain the polymorphism of both chromosomes. Under a very general model, where both X and Y chromosomes can induce meiotic drive and have fitness effects in both sexes, the conditions for the maintenance of Y polymorphism appear restricted to a narrow range of the parameter space (1%), stay-

2 Y-LINKED SUPPRESSORS OF SEX-RATIO 729 ing within the range that allows the maintenance of X polymorphism (Clark 1987). However, when the parameter space is reduced to be in accordance with the known properties of sex-ratio, stable polymorphism or cycles of both sex chromosomes are frequently obtained (Carvalho et al. 1997). The analysis of Jaenike (1999) suggests that a polymorphic equilibrium of both sex chromosomes could be also easily reached when a Y-linked suppressor of drive arises in a population where a polymorphism for X-linked meiotic drive preexists. The worldwide species D. simulans originated in the East African region about 3 million years ago. It has recently colonized Europe, the New World, and Pacific islands (Lachaise et al. 1988; Irvin et al. 1998; Hamblin and Veuille 1999). This species is of peculiar interest for an evolutionary study of drive and drive suppression because both of them exhibit a strong geographical heterogeneity (Atlan et al. 1997), which possibly reflects a large variety of balanced polymorphisms. Sex-ratio X chromosomes have been detected in many locations, their observed frequencies vary widely (from 1% to 60%), with the highest values obtained in islands and in East Africa. They can induce more than 90% of females in the progeny of males carrying a sensitive genetic background. However, in the natural populations where driving X chromosomes reach a notable frequency, their expression is halted by the presence of drive suppressors, thus the average percentage of females always remains below 58%. Although the populations always have enough suppression factors to ensure an equal or nearly equal sex ratio, there is no significant correlation among populations between the frequency of sex-ratio X and the suppression ability. The highest levels of suppression were observed in central and East Africa, even in populations where sex-ratio X chromosomes were not detected. The whole set of data suggests that drive and drive suppression first evolved in the East African region, and that interactions between selective pressures and stochastic founder effects are responsible for the present differentiation on the world scale. In that geographical survey (Atlan et al. 1997), drive suppression was measured at the phenotypic level, and the chromosomes involved had not been identified. A more detailed study on two laboratory strains, derived from populations of Seychelles and Kenya, showed that Y-linked and autosomal suppressors were both present (Cazemajor et al. 1997; M. Cazemajor and C. Montchamp-Moreau, unpubl. data). Here, we analyze the pattern of Y-linked suppression in six natural populations of D. simulans covering a broad geographical area. The principle of the experiment is to test each Y chromosome of our samples for its ability to suppress the expression of a reference driving X (X SR6 ). Our work addresses three related questions with the objective to better understand the evolution of drive suppression: (1) Are Y- linked suppressors a systematical component of drive suppression? (2) Is there one or several genetic classes of suppressor Y chromosomes? (3) Do sensitive and suppressor Y chromosomes coexist within populations? MATERIALS AND METHODS Drosophila simulans Stocks ST is our reference standard stock, it harbors neither drive factors nor drive suppressors. The sex ratio within this strain is about 51% females (Cazemajor et al. 1997). SR is our reference sex-ratio stock. The X chromosomes of this strain are sex-ratio, but their expression is suppressed by Y and autosomal factors (Cazemajor et al. 1997). The sex-ratio trait is expressed in F 1 males from ST father and SR mother; F 1 males typically produce on average about 90% females. ST8 is an highly inbred line (20 generations of sib-pair mating) derived from the ST stock. All the ST8 males carry a single Y chromosome (Y ST8 ). ST8/C(1)RM, y w is a derivative of the ST8 line in which the females bear the compound X chromosomes from the lz[sp]/ C(1)RM, y w stock of the Bloomington Stock Center at Indiana University. The other chromosomes are from the ST8 line, therefore this line is free of drive suppressors. The stock CyUbxDl bears the dominant mutation Curly (Cy) on the second chromosome and two balanced markers Ultrabithorax (Ubx) and Delta (Dl) on the third chromosome. This stock was obtained by crossing between the stocks Cy[NC] and In(3R)Ubx, Ubx[m]/Dl[1] from the Stock Center at Bowling Green State University. SR6 is a line in which females bear the C(1)RM, y w compound X chromosomes and males a sex-ratio X chromosome (X SR6 ) extracted from the SR strain described in Cazemajor et al. (1997). The autosomes and the Y chromosome come from the ST8 line. SR6 males, bearing the sex-ratio X SR6 chromosome in the ST8 standard background, produce about 95% females when mated with normal females. When mated with females with compound X chromosomes, they transmit the X SR6 chromosome to their sons, which results in nearly all male progeny. Consequently, the SR6 line is maintained by repeated backcrosses of males with ST8/ C(1)RM, y w females; this also prevents the selection of drive suppressors. Samples from natural populations were collected with banana baits at six different locations: Saint Martin in West Indies (1995), Trouville in the western France (1997), Sticiano in Italy (1996), Kilimanjaro in Tanzania (1996), Harare in Zimbabwe (1997), and Bellepierre in Réunion Island (1996). The stocks were kept as isofemale lines, each founded with a wild-caught female. General Procedure for the Measure of Sex Ratios Each male under test (1 3 days old) was placed with a single virgin ST female into a vial containing fresh medium. Mating and egg-laying were allowed for three days, and the pair was transferred to a new vial every three days. The percentage of females in the progeny was measured by counting all the adults emerging from one or more vials. The point was to obtain at least 50 offspring per individual progeny, this was generally achieve with the first two broods. Crosses producing less than 50 flies were discarded. All experiments were carried out at 25 C. Characterization of the Natural Populations Individual tests of males were performed one generation after the foundation of the isofemale lines, following the procedure detailed in Atlan et al. (1997). For each population, three measures have been performed with regard to drive and drive suppression. To estimate the within-strain sex ratio, the

3 730 CATHERINE MONTCHAMP-MOREAU ET AL. FIG. 1. Crossing scheme for the extraction of Y chromosomes from isofemale lines. The parental cross was individual, whereas F 1 and F 2 crosses were made in mass. The Y line was founded with one F 3 male mated with five or six ST8 females. percentage of female was measured in the progeny of one or two males per isofemale line, and the mean percentage of females was calculated over the progenies. To estimate the frequency of sex-ratio X chromosomes, one or two females from each line were individually mated with males from the ST stock (devoid of suppressors). One F 1 male per parental cross was tested for its offspring sex ratio. Males that had produced more than 70% of females were assumed to have received a sex-ratio X from their mother. This is a rough procedure that may lead to underestimate the frequency of sex-ratio X. However, it was found to be reliable (see our remark in the Discussion section; Atlan et al. 1997). To test the drive resistance, one or two males per line were individually crossed with females from the SR stock (carrying sexratio X chromosomes). One F 1 male per parental cross was investigated for its offspring sex ratio. The mean percentage of females was calculated over those of the individual progenies and compared with the value obtained in a control test in which the parental cross was performed with males of the ST stock, devoid of suppressors (Wilcoxon test). Extraction of Y Chromosomes from the Natural Populations The Y chromosomes used in our study came from a random sample of the isofemale lines described above: 13 were from Saint Martin, 14 from Trouville, 22 from Sticiano, 24 from Kilimanjaro, 20 from Harare, and 14 from Bellepierre. A single Y chromosome was extracted from each isofemale line and put into the ST8 standard background according to the crossing scheme presented in Figure 1. From an individual cross between one male of the isofemale line and one Cy Ubx Dl female, we collected F 1 males with the Cy Ubx phenotype. These F 1 males were mass crossed with ST8 females. Similarly F 2 males with the Cy Ubx phenotype were crossed with ST8 females. We collected wild-type F 3 males, carrying the Y chromosome from the isofemale line, an X chromosome, and autosomes II and III from the standard ST8 stock (the dot fourth chromosomes were not controlled). Each of these males was crossed with six ST8 females. The lack of mutant phenotype was checked among the F 4 flies, and one of the F 3 crosses was retained as founder of the Y line. Drive Ability of X SR6 against Y ST8 (Standard Background) Fifteen replicates of the SR6 line were made, each started with a single male and five females. Five F 1 males of each replicate were individually tested for their offspring sex ratio, according to the general procedure described above. The drive ability of X SR6 was estimated for each replicate as the percentage of females averaged over the five progeny sets. The stability of X SR6 drive expression between replicates was analyzed with the Kruskal-Wallis test. Ability of the Y Chromosomes from the Populations to Suppress X SR6 Drive The crossing scheme for this test is presented in Figure 2. For each of the 107 Y lines, males were mass-mated with ST8/C(1)RM, y w females. Crosses between F 1 females (bearing compound X chromosomes) and SR6 males produced F 2 males with the Y chromosome from the corresponding Y line, the X SR6 sex-ratio chromosome, and a standard ST8 autosomal background (the dot fourth chromosomes were not controlled). The drive ability of X SR6 against each Y was given by the average percentage of females, among the individual progenies of three to five F 2 males. The Kruskal-Wallis test was used to compare the strengths of suppression between the Y chromosomes from the same location. When these were found statistically different, we attempted to identify homogeneous blocks of Y chromosomes by a Duncan test on the transformed data arcsin p, where p is the frequency of females. Checking of Within-Population Polymorphism of Y Chromosomes This test was performed to determine whether microenvironmental or autosomal differences may be responsible for

4 Y-LINKED SUPPRESSORS OF SEX-RATIO 731 FIG. 2. Crossing scheme for the measure of Y-linked suppression of drive. The parental and F 1 crosses were made in mass. The sex ratio was measured in individual progenies of F 2 males. a part of the heterogeneity observed between the Y chromosomes in the previous experiment. For each location, one or two paired comparisons were performed between Y chromosomes that had produced extreme values of sex ratios, including, when possible, those that had shown the lowest and the highest values. For each paired comparison, the autosomes of the two Y lines were first made homogeneous by reciprocal crosses to suppress any differences that could result from a residual polymorphism in the inbred ST8 stock. Six males of the line corresponding to a low drive suppression were mated with five females of the line corresponding to a high drive suppression and the reciprocal cross was performed similarly. Then, from each reciprocal cross, we collected 10 male offspring that were used in a crossing scheme similar to that of the previous experiment (Fig. 2) to test again the Y chromosomes against X SR6 drive. The 10 males were crossed to eight females with compound X chromosomes (ST8 C(1)RM, y w stock), F 1 females were mated with SR6 males in three replicate crosses, each with one male and three females. Three F 2 males of each replicate were individually tested for their offspring sex ratio. The proportion p of females in each progeny was transformed to arcsin p before statistical analysis by nested ANOVA (nesting replicates within Y), which was performed with the software SAS (SAS Institute, Inc., Cary, NC). RESULTS Characterization of Natural Populations The results of the tests are summarized in Table 1. The within-strain sex ratio was close to 50% in all locations, the higher frequency of females being observed in Saint Martin (57%). Sex-ratio X chromosomes (i.e., that produce more than 70% female in our standard conditions) have not been detected in Trouville; because 36 X chromosomes were sampled, the frequency of sex-ratio X in this location must be 0.08 ( 0.05). Otherwise, the observed frequencies ranged from 0.04 in Harare to 0.46 in Bellepierre. The resistance to drive, that is, the joint effects of Y-linked and autosomal suppressors, varies widely between populations. It was not detected in Trouville, whereas Kilimanjaro and Bellepierre exhibit nearly complete drive suppression. The three other samples showed intermediate levels of suppression. X SR6 Drive Ability Against Y ST8 The drive ability of X SR6 against Y ST8 was measured in males of the SR6 line, that is, in the standard autosomal background ST8. The results are presented on Figure 3A. Among the 15 replicate sublines, with five males from each TABLE 1. Characterization of the natural populations. N is the number of males individually tested for their offspring sex ratio (see Materials and Methods for details). Location control ST 1 Saint Martin 1 Trouville Stitciano Killimanjaro Harare Bellepierre N Within-strain sex ratio Mean female % N Distortion against ST Mean female % Frequency of sex-ratio X N Resistance against SR Mean female % 2 1 Data from Atlan et al. (1997). 2 Mean percentage of females among individual progenies. 3 Males that produce more than 70% of females are assumed to carry a sex-ratio X. * Value significantly lower than that obtained in the control test of the ST stock (Wilcoxon test), indicating that drive suppressors are present in the population * * 53.0* 63.9* 53.7*

5 732 CATHERINE MONTCHAMP-MOREAU ET AL. FIG. 3. Variability of drive suppression expressed by the Y chromosomes against X SR6. On each diagram, Y chromosomes (replicates for the Y ST8 control) are ordered along the x-axis according to a decreasing suppression strength. N is the total number of Y chromosomes tested (number of replicates for the Y ST8 control), and m is the average percentage of females over the Y chromosomes (replicates for Y ST8 ). For each chromosome (replicates for Y ST8 ), crosses represent the percentage of females in individual progenies and the black circle the mean percentage over these progenies.

6 Y-LINKED SUPPRESSORS OF SEX-RATIO 733 TABLE 2. Expression of X SR6 in males carrying Y chromosomes extracted from natural populations. N is the number of Y tested from each location. For each Y, the percentage of females was measured in three to five individual progenies. Y minimum and Y maximum are the Y chromosomes that have produced the lower and the higher percentage of females (averaged over the individual progenies), respectively. Mean is the mean percentage of females over all the Y from each location. The within-population homogeneity of the Y chromosomes is tested by a Kruskal-Wallis test. Y being tested Location Saint Martin Trouville Stitciano Kilimanjaro Harare Bellepierre N Percentage of females in progeny Y min Y max Mean * * 55.7* 79.9* 67.4* Kruskal-Wallis test P * Mean percentage of females significantly lower than against the standard Y ST8 (Wilcoxon test). replicate being individually tested, the mean percentage of females in progeny was 95.5 (SE 3.08). As expected, the ST8 background behaves as though it is devoid of drive suppressors. Given the multigenic nature of the autosomal drive suppression, a residual polymorphism at some loci controlling X-drive ability might remain in the inbred ST8 stock, and result in variable expression of drive between replicates. In addition, each replicate could differ for uncontrolled culture conditions, possibly acting on drive expression. We looked for possible variation in drive expression among replicates, and the comparison of offspring sex ratios between replicates did not show any significant difference (Kruskal- Wallis test, , df 14, P 0.74). We therefore concluded that our experimental conditions allowed repeatable expression of X SR6 drive against Y ST8 between replicate sublines. Ability of the Y Chromosomes from the Natural Populations to Suppress X SR6 Drive We studied the suppression ability of the 107 Y chromosomes from the six populations in the reference standard autosomal background ST8. For each Y line, according to the crossing scheme presented in Figure 2, three to five F 2 males were individually tested for their offspring sex ratio. Figures 3B G present for each population and each Y the individual measures of sex ratios and their mean. For each location, except Trouville, the mean percentage of females observed over all the Y chromosomes studied was significantly lower than that obtained against Y ST8 (Table 2, Wilcoxon test). This is in accordance with the fact that, in our search of drive resistance (Table 1), a negative result was obtained in Trouville only. The five other populations presented significant Y-linked suppression of drive, when compared to our reference standard Y ST8 chromosome, but the level of suppression differed widely between them, the slightest mean suppression was observed in Saint Martin (92.4% females, Fig. 3C), whereas the suppression appeared nearly complete in Kilimanjaro (55.7% females, Fig. 3G). The three other populations (Sticiano, Harare, and Bellepierre) exhibited intermediate levels. A nested ANOVA, nesting Y within populations, confirmed major differences between populations (F , df 5,101, P 10 4 ). A two-by-two comparison of means by a Scheffe test showed no difference between the samples from Trouville and Saint Martin and no difference between Harare and Sticiano. The differences were significant at the 1% level for all the other comparisons. The Kruskal-Wallis tests for within-population polymorphism of the Y chromosomes are presented on Table 2. In both Trouville and Saint Martin, the Y chromosomes sampled were found homogenous for their sensitivity to X SR6 drive. In contrast, the samples from Sticiano, Kilimanjaro, Bellepierre, and Harare clearly appeared heterogeneous. For each of these four samples, a Duncan test generally identified several overlapping blocks of means (data not shown). Therefore, it was not possible to classify the Y chromosomes in distinct homogeneous groups. This confirms the continuous variation observed on the graphs. Checking of Within-Population Polymorphism for Y-Linked Suppressors This test was performed for the four populations where the Y chromosomes had been found heterogeneous with regard to their sensitivity to X SR6 drive in the previous experiment and for the Saint Martin sample. For each population, paired comparisons were performed between the Y that had produced the extreme sex ratio values presented on Figure 3, following the procedure described in the Material and Methods section. A nested ANOVA, nesting replicates within Y chromosome (Table 3), showed no replicate effect, excepted in the Zimbabwe sample (P 0.04). Thus, microenvironmental differences in our experimental conditions have little or no effect on the expression of Y-linked suppressors. In contrast, a highly significant Y effect was found, at least when comparing the more extreme Y chromosomes, in the samples from Sticiano, Kilimanjaro, and Harare (P 10 4 ). The Y chromosomes tested from Bellepierre, however, were found homogeneous. In this case, the two Y chromosomes that had produced the highest percentage of females in the previous experiment (89.7% and 75.5%, respectively) had been lost, thus the comparison was made between the third one (R150, 74.5% of females) and the less sensitive Y of this sample (R149, 56.7% of females). It must be noted that R150 and R149 had been classified in different groups by the Duncan test in the previous experiment. As for the control sample, Saint Martin, of which the 13 Y chromosomes had been previously found homogenous, no significant difference was found between the extreme Y A19 and A8. DISCUSSION The 107 Y chromosomes studied here have been sampled from six populations whose properties in the sex-ratio system of drive and drive suppression are representative of the different situations found in the wild. The Trouville population represents a standard or nearly standard state: Neither sexratio X nor drive suppression have been detected. The five other populations showed various frequencies of sex-ratio X and various levels of suppression. As previously observed (Atlan et al. 1997), the suppression is strong enough to ensure

7 734 CATHERINE MONTCHAMP-MOREAU ET AL. TABLE 3. Comparisons of drive suppression ability between pairs of Y chromosomes that had produced extreme sex ratio values in the first experiment (see Fig. 3B G). For each Y, the percentages of females within parentheses correspond to the mean obtained in the first experiment. Y effect and replicate effect were tested by an ANOVA with replicates nested within Y chromosomes. ns, not significant. Location Y being tested Saint Martin A19 A8 Sticiano I30 I48 I30 I45 Kilimanjaro T41 T39 T5 T39 Harare Z6.2 Z6.1 Z6.2 Z6.24 Bellepierre R149 R150 Mean female percent 89.4 (91.6) 89.6 (95.0) 76.7 (65.6) 94.0 (96.4) 76.7 (65.6) 83.2 (85.9) 49.8 (39.2) 72.7 (73.0) 52.3 (49.1) 71.6 (73.0) 47.8 (59.7) 78.2 (86.7) 62.1 (59.7) 80.4 (71.4) 60.2 (56.7) 67.3 (74.5) F 1,12 Y effect P F 4,12 Replicate effect P 0.0 ns 0.9 ns ns ns ns ns ns ns 0.6 ns a roughly equal sex ratio when sex-ratio X are at notable frequencies (Saint Martin, Sticiano, Kilimanjaro, and Bellepierre). Also in accordance with the previous results, there is no significant correlation between the observed frequencies of sex-ratio X and the strength of the total drive suppression among populations: Strong suppression can exists in populations where sex-ratio X are rare or absent, such as Harare in the present study. Y-Linked Suppressors Are a Regular Component of Drive Suppression Whenever we looked for Y-linked suppressors in populations showing drive suppression, we found some: Sticiano, Saint Martin, Kilimanjaro, and Bellepierre (this article), in Nairobi, Kenya and Mahe, Seychelles (Cazemajor et al. 1997; C. Montchamp-Moreau, unpubl. data). Again, there is a discrepancy between the frequency of sex-ratio X and the strength of suppression when we look specifically at its Y- linked part (cf. Tables 1 and 2). Strong Y suppression occurs in Harare, where sex-ratio X chromosomes are rare. Moreover, the respective parts of Y-linked and autosomal suppressions can differ according to the population. The total suppression is similar in Bellepierre and Kilimanjaro (53.7% and 53.0% females in the tests, respectively), but the mean level of Y sensitivity is significantly lower in the former than in the later. In Saint Martin, the Y chromosomes are very sensitive to drive in spite of a notable frequency of sex-ratio X (0.23), whereas the total suppression is rather strong. The autosomes thus appear to be responsible for most of the suppression in this population. It seems probable that drive suppression systematically includes a strong Y-linked component in the populations from the African region, around the putative center of origin of the sex-ratio system. However, the sample from Saint Martin shows that this is not the case over the whole range of the species, and we cannot exclude the possibility of suppression being sometimes purely autosomal. Several Types of Suppressor Y Chromosomes Exist in Drosophila simulans The Y chromosomes can differ substantially from each other, with respect to their ability to suppress the drive induced by X SR6, one of the strongest drivers we have found in our survey of X chromosomes (unpubl. data). The populations appear monomorphic or polymorphic for the Y chromosome, depending on their geographic origin: Those from France and the West Indies are monomorphic and sensitive, whereas those from Italy, Zimbabwe, Tanzania, and Réunion Island are polymorphic and differ in their mean level of Y- linked suppression. None of the statistical tests we used could separate discrete classes of Y chromosomes, either within populations or when pooling the six samples of Y chromosomes. The whole set of Y chromosomes exhibits a continuous range of variation, from totally resistant to totally sensitive. Such a continuum may have several origins. It may result from environmental variation, creating so much noise around a given number of discrete types that the phenotypes overlap. The same phenomenon would be expected if there is uncontrolled polymorphism for autosomal genes that interact pleiotropically on the expression of the Y-linked suppressors. We have compared Y-lines with the same autosomal background from a highly inbred standard stock (ST8) to minimize the latter source of variation. However, because of the multigenic and probably multiallelic nature of drive suppression (Cazemajor et al. 1997; M. Cazemajor and C. Montchamp-Moreau, unpubl. data), we did not exclude the possibility of a residual polymorphism of ST8 autosomes. Environmental and autosomal sources of variation were controlled in the additional experiment performed to check the within-population polymorphism of Y chromosomes (Table 3). Environmental variation was found to have little or no effect in our experimental conditions, and we still observed large differences between pairs of Y chromosomes in the level of suppression despite the previous homogenization of autosomes. Clearly, environmental and autosomal variability

8 Y-LINKED SUPPRESSORS OF SEX-RATIO 735 cannot explain the wide range of sex-ratio suppression and we are left to conclude that there must be extensive Y-linked variation for suppression. This continuous variation contrasts with the situation described in D. mediopunctata, where two discrete classes of sensitive and suppressor Y chromosomes have been distinguished (Carvalho et al. 1997). Unfortunately, there are no data on the distribution of Y suppression levels in the other species. We therefore cannot know to what extent the pattern observed in D. simulans populations is uncommon. A continuous phenotypic variation may result from the molecular nature of the Y-linked suppression factors. For instance, the suppression could be induced by a multiple copy region, with the level of suppression depending on the number of copies. Such a situation is observed in D. melanogaster for Rsp, the target locus of Segregation Distorter (Wu et al. 1988) and Su(Ste), suspected to be the target locus of a relict sex-ratio driver (Lyckegaard and Clark 1989; Hurst 1996). A system of tandemly repeated sequences, with a high rate of mutational change in the number of copies, would generate a continuous variation resulting in a continuum of phenotypes. Another hypothesis is that several sequences of different natures are involved, acting as enhancers or modifiers, so that the Y suppression has a quantitative determinism. Such a quantitative sex ratio determination may be a general result of genetic conflicts, as discussed in Gigord et al. (1999). Within-Population Polymorphism of Y Chromosomes Both monomorphic and polymorphic populations were observed in our survey. It is noteworthy that the monomorphic populations are those where the Y chromosomes are highly sensitive to drive (producing 92 94% females against X SR6 ), whereas the polymorphic ones are those where the Y chromosomes are at least partly resistant (producing from 56% to 81% females on average, depending on the population). This suggests that the maintenance of Y-linked suppression in D. simulans involves a nonneutral mechanism that also maintains its polymorphism. In other words, the selective advantage due to the suppression must be balanced by deleterious effects. Providing that both drive and drive suppression are deleterious, sex-ratio is a case where interactions in the population dynamics of X (driving vs. nondriving) and Y (suppressor vs. nonsuppressor) alleles can stabilize Y polymorphism for many combinations of fitness values, within the range that allows X polymorphism (Carvalho et al. 1997). Such a mechanism could underlie the situation that we report in D. simulans. First, the necessary condition of a polymorphic X is satisfied, because driving and nondriving X coexist in the populations that have been found polymorphic for Y- linked suppression. Second, driving X were found to cause fitness loss in D. simulans (Capillon and Atlan 1999). Third, drive expression is not totally suppressed in natural populations: The frequency of females can rise up to 58% in populations where sex-ratio X have been detected, whereas it stays below 54% in the other populations (Atlan et al. 1997; Table 1; unpubl. data). Because this residual expression of the drive maintains a selective pressure in favor of suppressors, the apparent inability of the most powerful suppressor Y chromosomes to get fixed in populations argues strongly against the hypothesis of a neutral Y polymorphism. The coexistence of Y chromosomes with various levels of suppression ability within the same population suggests a positive correlation between the strength of the suppression and its cost. In the absence of a model including a multiallelic Y-linked suppression, we do not know whether the conditions for the maintenance of a multiallelic polymorphism are more or less restrictive than those found for the pair suppressor/ nonsuppressor by Carvalho et al. (1997). On the one hand, we believe that the more numerous the alleles are, the narrower the range of fitness values authorized for each Y, because of the correlation noted above. On the other hand, the drive/suppression system is highly complex in D. simulans. In addition to the diversity of Y-linked suppressors (this paper), we have found differences among the sex-ratio X chromosomes for their drive ability and their sensitivity to autosomal suppressors (unpubl. data). The assumption that multiple and specific interactions between drivers, between suppressors, and between drivers and suppressors as well could help to stabilize Y polymorphism appears quite reasonable. A deleterious effect could explain the low level of Y-linked suppression in Saint Martin. This population results from a recent expansion of D. simulans from its East African center of origin, where the sex-ratio system is supposed to have arisen. The frequency of sex-ratio X may have been too low to allow the maintenance of Y-linked suppressors through the colonization process, so that when sex-ratio X locally increased in frequency, only autosomal suppressors were still present. These have a multigenic nature (Cazemajor et al. 1997) that could have preserved them from elimination, even in the absence of selection pressure induced by a high frequency of sex-ratio X. Assuming that the cost of Y-linked suppression is compensated by its advantage in the presence of driving X, it is difficult to understand the situation observed in the Harare population of Zimbabwe. Indeed, the frequency of sex-ratio X in this area is probably very low, as found in the samples collected in 1995 (Atlan et al. 1997) and 1997 (Table 1), but the Y chromosomes exhibit a high level of suppression. The procedure used to detect sex-ratio X could have led us to underestimate their frequency if dominant autosomal suppressors were present. However this hypothesis does not hold at least for the 1997 sample, because in this case X drive ability has been elsewhere tested in a completely standard context, and a similarly low frequency was observed (data not shown). A second hypothesis is that short episodes of high sex-ratio X frequency occur and are sufficient to maintain a high level of Y suppression. Seasonal variations in sexratio X frequency cannot be excluded and have been already described in D. testacea (James and Jaenike 1990), but such effects of temporal variation have not been theoretically explored. A third hypothesis is that Y suppression is maintained by migration. Indeed, the closest location studied (Kilimanjaro, Tanzania) exhibits a high frequency of sex-ratio X, together with highly suppressor Y chromosomes. Migration from that kind of population could explain the situation observed in Harare, providing that the local deleterious effects

9 736 CATHERINE MONTCHAMP-MOREAU ET AL. of sex-ratio X chromosomes is higher than those of suppressor Y chromosomes. Experimental population studies have shown that the deleterious effect of sex-ratio X chromosomes may be very strong and depends on the density (Capillon and Atlan 1999). The same experiments failed to detect strong deleterious effects of Y-linked suppressors, suggesting that ecological and demographic conditions may exist where migrant sex-ratio X would be lost more rapidly than a migrant suppressor Y. Under the hypothesis that the cost of drive and drive suppression are dependent on the demographic and ecological context, a fourth possibility is that Harare represents a case where the cost of suppression is particularly low. The numerical simulations of Jaenike (1999) shows that, in such a case, it is possible to reach a stable equilibrium where suppressor Y chromosomes are frequent and driving X chromosomes are rare. Finally, we cannot exclude the hypothesis that Y polymorphism is maintained by a side effect of a selective pressure on a character independent of drive suppression. Drosophila simulans is the fourth Drosophila species, after D. paramelanica (Stalker 1961), D. mediopunctata (Carvalho et al. 1997), and D. quinaria (Jaenike 1999), in which populations have been found to be polymorphic for Y-linked suppressors of sex-ratio drive. This supports the hypothesis that such a polymorphism can be stabilized easily. However, the existence of extensive variation among Y chromosomes, within and between populations, led us to ask if the geographic pattern observed in D. simulans corresponds to a variety of balanced polymorphisms, due to differences in local conditions or to transient states that reflect over the species range different stages of an endless arm race between X and Y. Drive Suppression and the Polymorphism of Y-Liked Genes The high level of within- and among-population polymorphism that we have found for Y-linked drive suppression may appear contradictory with data showing that genes of this chromosome have a reduced polymorphism. Such is the case in Silene latifolia (Filatov et al. 2000), humans (reviewed in Jaruzelska et al. 1999), and D. melanogaster and D. simulans (Zurovcova and Eanes 1999). Several phenomena such as reduced effective size, selective sweep, and background selection enhanced by the absence of recombination may lead to the reduced diversity of the coding sequences (McAllister and Charlesworth 1999). Zurovcova and Eanes (1999) propose that some of the Y-linked selective sweep events result from periodic emergence of sex-ratio chromosomes polymorphism if transmission modifiers arise on the Y. In D. simulans, it is clear that the presence of sex-ratio X chromosomes does not lead to the directional selection of a single type of suppressor Y chromosome at the species level, but, on the contrary, allows the maintenance of a wide range of Y suppression levels. The maintenance of such a diversity on a chromosome that otherwise exhibits a monomorphism of coding sequences reinforces the idea that the locus involved in suppression is a sequence with a particularly high mutation rate, such as repeated heterochomatic sequences. The amount of heterochromatin of the Y chromosome can be highly variable within species (e.g., example Miller and Roy 1964; McKay et al. 1978). We thus propose the following scenario: The first step would be the selection of a suppressor Y chromosome, inducing a selective sweep of the whole chromosome because of its clonal transmission. 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