Genetic Complexity Underlying Hybrid Male Sterility in Drosophila

Corresponding author: Kyoichi Sawamura, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan., sawamura{at}biol.tsukuba.ac.jp (E-mail)

Communicating editor: M. AGUADÉ

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Recent genetic analyses of closely related species of Drosophila have indicated that hybrid male sterility is the consequence of highly complex synergistic effects among multiple genes, both conspecific and heterospecific. On the contrary, much evidence suggests the presence of major genes causing hybrid female sterility and inviability in the less-related species, D. melanogaster and D. simulans. Does this contrast reflect the genetic distance between species? Or, generally, is the genetic basis of hybrid male sterility more complex than that of hybrid female sterility and inviability? To clarify this point, the D. simulans introgression of the cytological region 34D–36A to the D. melanogaster genome, which causes recessive male sterility, was dissected by recombination, deficiency, and complementation mapping. The 450-kb region between two genes, Suppressor of Hairless and snail, exhibited a strong effect on the sterility. Males are (semi-)sterile if this region of the introgression is made homozygous or hemizygous. But no genes in the region singly cause the sterility; this region has at least two genes, which in combination result in male sterility. Further, the males are less fertile when heterozygous with a larger introgression, which suggests that dominant modifiers enhance the effects of recessive genes of male sterility. Such an epistatic view, even in the less-related species, suggests that the genetic complexity is special to hybrid male sterility.

GENETIC bases of hybrid inviability and sterility have been analyzed in Drosophila species since the pioneering works of STURTEVANT 1920 and DOBZHANSKY 1936 . Conceptually, single-gene-based isolating mechanisms are difficult to achieve because populations have to cross adaptation valleys; A₁A₁ genotype can evolve to A₂A₂ genotype only through transitional individuals, A₁A₂, who are supposedly inviable or sterile. Thus, DOBZHANSKY 1937 and MULLER 1940 advocated a two-locus model of genetic incompatibility (the two-locus Dobzhansky-Muller model). In this classical model, A₁A₁B₁B₁ and A₂A₂B₂B₂ genotypes (species 1 and species 2, respectively) can be derived independently from A₁A₁B₂B₂, bypassing individuals equivalent to F₁ or backcross hybrid; A₂ is incompatible with B₁ here (note that inviable or sterile genotypes depend on the dominance of the alleles). Empirical data have been explained on the basis of this simple model of complementary genes, although more than two loci may be involved in some cases. This is still widely accepted as the standard model of reproductive isolation (GAVRILETS 1997 ; HUTTER 1997 ; ORR 1997 ; NEI and ZHANG 1998 ; TURELLI 1998 ; SAWAMURA 1999 ; JOHNSON 2000 ; ORR and PRESGRAVES 2000 ; HAYASHI and KAWATA 2001 ).

While the two-locus Dobzhansky-Muller model can be extrapolated to arbitrarily complex interactions involving both heterospecific and conspecific interactions as theoretical works have rigorously modeled (ORR 1995 ; ORR and TURELLI 1996 ; GAVRILETS 1997 ; TURELLI and ORR 2000 ), its popularity has much to do with its perceived simplicity (see WU and PALOPOLI 1994 ). According to this model, the genetics of hybrid incompatibility can be due to a very small number of loci, plausibly to one locus per species, whereas conspecific genic interactions are entirely missing. It is this most common interpretation of the Dobzhansky-Muller model that we wish to address in this study.

The two-locus Dobzhansky-Muller hypothesis of reproductive isolation implicitly predicts that interspecific introgressions of one of the complementary genes, e.g., homozygous B₁ introgression to species 2, would result in inviability or sterility. Thus, evolutionary biologists have tried to identify such major genes of reproductive isolation. But recent analyses of introgressions between Drosophila simulans and its sibling species, D. mauritiana or D. sechellia, have actually indicated that a large number of genes affect male fertility and that the sterility is a consequence of synergistic effects of multiple genes (WU and PALOPOLI 1994 ; WU et al. 1996 ; NAVEIRA and MASIDE 1998 ). For example, the D. mauritiana gene Odysseus (Ods) alone does not reveal its male sterility effect in the D. simulans genetic background, but the co-introgression with a second-site gene or genes linked to it does (PEREZ and WU 1995 ; TING et al. 1998 ). Thus, the genetic nature of hybrid male sterility is not as simple as what the two-locus Dobzhansky-Muller model assumes.

The three species used in the previous analyses (the D. simulans clade) are relatively young and produce fertile female hybrids, allowing potential gene flow in nature. Molecular phylogenetic analyses have indeed detected such an ancient event (BALLARD 2000 ; KLIMAN et al. 2000 ). Having diverged beyond the point of fusion, the three species thus are "good species," but could still share a portion of their genomes, i.e., those in stage III of WU 2001 . This might be why no major genes of reproductive isolation have evolved. Then, what is the situation in highly diverged species that are completely isolated, i.e., in stage IV of WU 2001 ? Are there genes whose sole introgression, not co-introgression of multiple genes, causes inviability or sterility between D. melanogaster and D. simulans? [D. melanogaster is the outgroup of the D. simulans clade (LACHAISE et al. 1988 )]. In fact, the presence of such major genes causing inviability and female sterility has been suggested (PRESGRAVES 2003 ; SAWAMURA et al. 2004 ), and one of the hybrid inviability genes was molecularly identified in this particular species pair (PRESGRAVES et al. 2003 ). However, it is not clear whether the contrast is closely related species vs. less-related species or hybrid male sterility vs. hybrid inviability (and female sterility). Investigation of genes causing hybrid male sterility between D. melanogaster and D. simulans will clarify this point.

The cross of D. melanogaster and D. simulans produces only sterile unisexual hybrids (STURTEVANT 1920 ). Because backcross hybrids, which are necessary for conventional genetic analyses, are not obtainable, genetic tricks were introduced to map genes of hybrid sterility and inviability (MULLER and PONTECORVO 1940 ; SAWAMURA 2000 ; PRESGRAVES 2003 ). The discovery of "rescue mutations" that prevent hybrid inviability has facilitated the analyses of mechanisms of hybrid inviability (WATANABE 1979 ; HUTTER and ASHBURNER 1987 ; SAWAMURA et al. 1993A , SAWAMURA et al. 1993C ). The fact that single mutations have such dramatic effects on hybrid viability seems consistent with the two-locus Dobzhansky-Muller model of reproductive isolation (HUTTER et al. 1990 ; SAWAMURA et al. 1993B ). But it cannot be guaranteed that the rescue mutations are equivalent to the ancestral alleles. In fact, two of the rescue mutations have been shown to be loss of function (SAWAMURA and YAMAMOTO 1993 ; BARBASH et al. 2000 , BARBASH et al. 2003 ) and difficult to believe as ancestral. Interspecific introgressions would directly tell whether the two-locus Dobzhansky-Muller model is valid in both hybrid inviability and sterility.

In this analysis, a recently obtained D. simulans introgression to D. melanogaster, Int(2L)S (SAWAMURA et al. 2000 ), is utilized. This introgression (cytologically 30F3-31E1–36A2-36A7; see MATERIALS AND METHODS for the details) causes male sterility when homozygous. Previously the recessive genes responsible for this sterility have been mapped by examining fertility of males heterozygous for the introgression and a series of deficiencies. If the responsible genes of D. simulans are made hemizygous, males must be sterile. Thus, five genes of male sterility were mapped in this introgression (SAWAMURA et al. 2000 ; see Fig 1). In the present analysis, the genes were further mapped by molecular-marker-assisted recombination and genetically mapped by complementation with an additional series of deficiencies and male sterility mutations of D. melanogaster. The introgression tested here includes the Alcohol dehydrogenase (Adh) gene region, one of the best-characterized regions in D. melanogaster (ASHBURNER et al. 1999 ). This analysis of hybrid male sterility genes is therefore at a higher resolution than that of previous studies.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Mapping male sterility genes on Int(2L)S by recombination between the introgression-carrying chromosome and a multiply marked chromosome, wg b pr. The Int(2L)S carries D. simulans genes on the mid-2L region (indicated by a thick bar) in otherwise D. melanogaster genes (thin bars). Asterisks (*) represent putative male sterility genes detected in the previous analysis of deficiency mapping (SAWAMURA et al. 2000 ). Male fertility was tested in flies heterozygous for each recombinant and the original introgression, Int(2L)D+S. F, fertile; S, sterile; (S), semisterile.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Description of the original introgression:
As described previously (SAWAMURA et al. 2000 ), the introgression chromosome, Int(2L)D+S, carries two discrete regions of the left arm of chromosome 2 (2L) of D. simulans in an otherwise D. melanogaster genome. One of the introgressed segments, Int(2L)D, carries the 2L tip region and Int(2L)S the mid-2L region. The proximal boundary of Int(2L)D is between anterior open (aop, 22C3–D1) and Phosphoglycerate kinase (Pgk, 23A6–B1), and the distal boundary of Int(2L)S has been mapped between big brain (bib, 30F3–5) and daughterless (da, 31D11–E1; SAWAMURA et al. 2000 ; cytology according to FLYBASE 2003 ; http://flybase.org). The proximal boundary of Int(2L)S has been precisely determined in this study between dachshund (dac, 36A2) and grapes (grp, 36A6–7). Flies homozygous for Int(2L)D+S or Int(2L)S are sterile in both females and males, although Int(2L)D alone does not cause sterility (SAWAMURA 2000 ). The introgression is kept balanced with the CyO chromosome. For detailed information on marker genes, deficiencies, and other chromosomal aberrations used in the present analysis, see FLYBASE 2003 .

Making recombinants:
Females heterozygous for Int(2L)S and a multiply marked chromosome, wg b pr pk cn tuf ltd, were crossed to wg b pr pk cn tuf ltd/CyO males. From the progeny, strains carrying a recombinant or nonrecombinant second chromosome were established by using CyO as a balancer chromosome. Each strain was checked for the marker mutations, wingless (wg, 27F1–3), black (b, 34D1–3), and purple (pr, 38B4–6). These mutations are located distally, within and proximal to the region of the introgression, respectively (Fig 1). Therefore, recombinant introgressions are recovered effectively.

To obtain smaller introgressions, a second recombination cross was set up with females heterozygous for chromosome 37 marked with wg and b (see RESULTS) and a chromosome marked with reduced ocelli (rdo, 36E1-3; Fig 2). Two markers, b and rdo, are useful to recover recombinant introgressions, because the locations are close to the distal and proximal boundaries of the introgression, respectively. Recombinant lines were again established from single flies using CyO as a balancer chromosome.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Molecular demarcation of recombinant introgressions. The megabase scale at the top [the 2.9-Mb Adh region (ASHBURNER et al. 1999 )] indicates the physical distance between the known molecular markers used in this study (the start point, 0, is in the B4 gene). Thick bars denote D. simulans introgressions on each recombinant (the species boundaries should be located in the shaded regions). The origin of the R and L series of recombinants is also shown at the bottom.

Molecular determination of recombinant introgressions:
Genomic DNA was extracted from five flies of recombinant/CyO heterozygotes with phenol/chloroform, precipitated by ethanol, dissolved in 30 µl of Tris-EDTA buffer, and diluted 100 times in water. The wg b pr pk cn tuf ltd/CyO strain of D. melanogaster and the C167.4 strain of D. simulans [the latter was the progenitor of the introgression (DAVIS et al. 1996 )] were used as controls. Polymerase chain reaction (PCR) mixture was prepared according to the manufacturer's instructions. PCR conditions were 30 cycles of denaturation (described in Table 1). Digested DNA was separated in 1% or 2% agarose gel. The loci examined (Fig 2) were Angiotensin converting enzyme (Ance, 34D6–8), spellchecker 1 (spel1, 35A1), Adh (35B3), Suppressor of Hairless [Su(H), 35B10–C1], guftagu (gft, 35C4), snail (sna, 35D2–3), Cyclin E (CycE, 35D4–6), twine (twe, 35F6), and dac (36A2).

Fertility test:
The male fertility of flies (1) heterozygous for each recombinant and the original intact introgression, Int(2L)D+S; (2) homozygous for each recombinant; and (3) trans-heterozygous for recombinant introgressions of different sizes was tested. Introgression-carrying flies were crossed to flies carrying a series of deficiencies or male-sterile mutations, and the fertility of the trans-heterozygotes was tested. For initial fertility measurement, the five males to be examined were crossed with five females from the Oregon-R strain of D. melanogaster (at least four replicates were made) as described previously (SAWAMURA et al. 2000 ). Male fertility was also measured by pair matings of 3-day-old virgin flies transferred every 3–5 days until day 21 (when the flies were discarded).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Making recombinant introgressions:
The Int(2L)S chromosome, which carries the mid-2L region introgression from D. simulans, causes recessive male sterility. First, to make smaller introgressions, 120 lines carrying chromosomes descended from females heterozygous for Int(2L)S and wg b pr were established. When the chromosomes were made heterozygous with the original Int(2L)D+S chromosome, nonintrogression controls (wg b pr) produced fertile males (chromosome 3) whereas intact introgression controls (+++) produced sterile males. Thirty single crossovers between wg and pr were isolated (Fig 1). Among them, 2 ++ pr and 4 wg ++ chromosomes apparently inherited male sterility genes detected in the previous analysis (SAWAMURA et al. 2000 ). On the other hand, 7 + b pr chromosomes produced fertile males (but there is a possibility that none of these seven carried a D. simulans introgression). The results of wg b + chromosomes are of interest. Among the 17 chromosomes tested, 3 (chromosomes 5, 37, and 101) resulted in male sterility and 1 (chromosome 91) resulted in male semisterility (0.6 progeny from a pair). These 4 chromosomes were used to map the male sterility genes more precisely.

Chromosomes 91 and 101 have recessive lethals, which presumably were floating in the original multiply marked strain or introgression strain and were crossed into our experimental lines. Because the two lethals did not complement each other, the chromosomes must carry the same mutation. The lethal gene was eliminated from chromosome 91 by recombination with Oregon-R, and the newly established chromosome utilized when necessary.

Among the three male-sterile introgressions, chromosome 37 is the smallest in size (see below). Smaller introgressions were made by further recombination between this chromosome (marked with b) and rdo ("R" and "L" recombinants; see Fig 2). Among 23 b rdo and 24 + + recombinant chromosomes established, molecular mapping (see below) indicated that 5 b rdo chromosomes (L3, L7, L11, L12, and L22) and 3+ + chromosomes (R2, R10, and R16) carried smaller introgressions than did chromosome 37 (R and L recombinants carry the right- and left-hand tip of introgression 37, respectively).

Molecular-marker-assisted mapping of male sterility genes:
Molecular demarcation of the recombinant introgressions is depicted in Fig 2 and fertility of males heterozygous for Int(2L)D+S and each recombinant introgression is shown in Table 2. The data are explicable if we predict a gene or genes having strong effects on male sterility in the region between gft and sna [or maybe between Su(H) and sna]. An exception is chromosome 91, which resulted in low fertility (or semisterility in the preliminary test mentioned above). This suggests that there might be more genes responsible for low male fertility between sna and twe, although the effect is detectable in a certain genetic background (chromosome 91 has a different genetic background than chromosomes R2, R10, and R16). The proximal boundary of introgression L12 is between gft and sna and this introgression may not include the whole region having strong effects on male sterility. This might be why the introgression L12 exhibited low male fertility.

Fertility was also examined in males homozygous for the recombinant introgressions (Table 2). (We have no data for introgression 101 because homozygotes are lethal.) A similar conclusion above is met; a gene or genes having strong effects on male sterility exist in the region between gft and sna. In this case the region between Su(H) and gft is excluded because L12 homozygotes were fertile. Exceptionally the fertility of chromosome 91 homozygotes was lower than that of R10 homozygotes, which is parallel to the data of heterozygotes with Int(2L)D+S. Another exception is R16 homozygotes, which were semisterile. But this might be caused by a recessive male-sterile mutation linked to the chromosome not by the introgression itself because Int(2L)D+S/R16 heterozygous males exhibited normal fertility. The fact that L12 homozygotes were fertile but Int(2L)D+S/L12 heterozygotes exhibited low fertility suggests that heterozygous D. simulans genes on Int(2L)D+S may be involved in this low male fertility. In other words, dominant modifiers of recessive male sterility exist on the introgression (but not necessarily in the region now studied).

The fertility of males trans-heterozygous for introgressions of different sizes was examined (Table 3). Males trans-heterozygous for any combination of the introgressions 101, 5, and 37 are almost completely sterile. Interestingly, males trans-heterozygous for those and 91 were fertile. This contrasts with Int(2L)D+S/91 heterozygous males, which were semisterile. Male sterility genes apparently exist in introgression 91, but the effect can be detected only when heterozygous with a very large introgression. This again suggests the presence of dominant modifier(s) of recessive male sterility genes on Int(2L)D+S.

Deficiency and complementation mapping of male sterility genes:
The male sterility genes in the introgression generally behave as recessives; if D. simulans alleles of such genes are made homozygous or hemizygous by deficiencies, then males are sterile. In fact, five overlapping deficiencies uncovered male (semi-)sterility genes in this region of the introgression (proximal to the b gene) when tested against the original introgression, Int(2L)D+S (SAWAMURA et al. 2000 ; Fig 3, deficiencies 1–5; Table 4). Three male sterility genes were tentatively located in this region by assuming that such genes are within the overlap of deficiencies uncovering male sterility. But the actual number of male sterility genes can be much larger. When 16 other deficiencies covering a similar region (Fig 3, deficiencies 6–21) were examined against Int(2L)D+S, most of the deficiencies uncovered male sterility genes: sterile, semisterile, or weakly fertile in trans-heterozygous males (Table 4). It is difficult to map major genes of male sterility from these data; some blocks not including male sterility genes were necessary to localize the genes.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Deficiencies and male-sterile mutations used in these analyses. To make this figure easily comparable with Fig 2, the same scale is presented at the top. Male sterility loci of D. melanogaster are indicated by arrows, and the deleted region of each deficiency chromosome is shown by hatched bars (uncertainty boundaries indicated by shaded bars). The deficiency breakpoints are from FLYBASE 2003 and M. ASHBURNER and J. ROOTE (unpublished results). Deficiencies 1–5 are the deficiencies used in the previous analysis (SAWAMURA et al. 2000 ). See Table 4 for the name of each deficiency.

Then what is the situation when the same series of deficiencies are made heterozygous with smaller introgressions like 101, 5, or 37? Because dominant modifiers of recessive male sterility genes exist on the original introgression (mentioned above), male fertility must be higher when the smaller introgressions are utilized. This was the case in most of the deficiencies. The extreme cases are Df(2L)H20 (deficiency 1) and Df(2L)TE35B-9 (deficiency 9), which resulted in constantly high male fertility. Such regions may not have male sterility genes with a strong effect, although the effect can be detected when heterozygous with the larger introgression. Contrastingly, some of the deficiencies exhibited semisterility when heterozygous with an introgression, such as 101, 5, or 37. Those are Df(2L)osp29 (deficiency 4), Df(2L)b88c75 (deficiency 7), Df(2L)Sco7 (deficiency 8), Df(2L)TE35BC-8 (deficiency 11), Tp(2;2)A446 (deficiency 12), Df(2L)TE35D-5 (deficiency 16), Df(2L)TE35BC-24 (deficiency 17), and Df(2L)TE35BC-3 (18). The deletions generally make the Su(H)-sna region hemizygous. Thus, male sterility genes with a strong effect exist in this region of introgression, which is consistent with the conclusion from the recombination mapping above. But it should be stressed here that the semisterility is not caused by a single gene. This region can be subdivided to two overlapping deficiencies exhibiting high male fertility: e.g., In(2L)C158^LSco^rv11R (deficiency 15) and Df(2L)TE35D-4 (deficiency 19). Thus, the Su(H)-sna region has at least two genes that, in combination, are sufficient to cause male semisterility.

The same series of deficiencies were made heterozygous with the smaller introgression 91. Almost all resulted in male fertility comparative to the controls. [The deleted regions of Df(2L)b87e25 (deficiency 5), Df(2L)b80k (deficiency 6), Df(2L)TE35B-9 (deficiency 9), T(Y;2)A80^DR15^P (deficiency 10), Df(2L)A267 (deficiency 13), and Df(2L)A72 (14) do not overlap with the introgression region of 91 and thus can be controls.] This is presumably because this small introgression does not have enough dominant modifiers of recessive male sterility to be detected.

The 2.9-Mb chromosome region from kuzbanian (kuz, 34C1–4) to dac, including Adh, has been extensively characterized in D. melanogaster at the molecular and genetic level (ASHBURNER et al. 1999 ). The D. simulans introgression studied in the present analysis is almost all included in that region. Five loci whose mutation or deficiency causes recessive male sterility are known in the region between b and dac as shown in Fig 3. Complementation tests were performed with mutations of the loci against the original introgression, Int(2L)D+S. Mutations tested are ms(2)34Fe¹, ms(2)35Bi^z0090, ms(2)35Ci⁰²³¹⁶, ms(2)35Eb^z3860, and twe¹ [(ms(2)35Bi^z0090 and ms(2)35Eb^z3860 are newly discovered mutant alleles of genes previously predicted from the sterility of overlapping deletions (ASHBURNER et al. 1999 ; D. LINDSLEY and J. ROOTE, unpublished results)]. None resulted in male sterility in heterozygotes (results not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The D. simulans introgression of the mid-2L region, Int(2L)S, causes male and female sterility when homozygous in an otherwise D. melanogaster genetic background (SAWAMURA et al. 2000 ). To identify recessive genes involved in male sterility, this introgression was genetically dissected, especially for the region around the Adh locus. First, molecular-marker-assisted recombination mapping indicated that the gft-sna region [or maybe the Su(H)-sna region, 450 kb] has at least one gene with strong effects, although other regions (e.g., the introgression region of 91) may also have male sterility genes whose effect is detectable in a certain genetic background. Interestingly, dominant enhancers of the effects of the recessive genes have been detected on the introgression. Small recombinant introgressions (e.g., 91 and L12) resulted in low male fertility only when heterozygous with a larger introgression, Int(2L)D+S. This fits the type II incompatibility of JOHNSON 2000 or the H₁ incompatibility of TURELLI and ORR 2000 (between a homozygote at one locus and a heterozygote at the other).

Second, deficiency mapping was performed. Most of the deficiencies resulted in male semisterility when tested against Int(2L)D+S and the responsible genes could not be localized. Because dominant enhancers exist on the introgression, a more sensitive test is possible if such elements are decreased by utilizing smaller introgressions. In fact, trans-heterozygous males were more fertile when introgression 101, 5, 37, or 91 was used. The results from 101, 5, and 37 were consistent with the conclusion from recombination mapping: trans-heterozygous males were semisterile when the Su(H)-sna region was deleted. (Introgression 91 exhibited normal fertility when heterozygous with any deficiencies, presumably because this introgression does not have enough dominant enhancers to be detected in this analysis.)

It is important to note that male sterility is not caused by a single gene. Deficiencies including the whole Su(H)-sna region result in male semisterility when heterozygous with introgressions 101, 5, or 37, but those partially including the region (which can overlap each other) do not. Thus, this region has at least two genes that, in combination, cause male semisterility. The complementation tests against male sterility mutations in this region also support that any single genes are not responsible for male sterility. In summary, no single gene substitutions between species in this region of the genome result in male sterility, which contrasts with what the two-locus Dobzhansky-Muller model of reproductive isolation assumes. The epistatic aspect of postzygotic reproductive isolation is not specific to the early stage of speciation (cf. WU and PALOPOLI 1994 ; WU et al. 1996 ; NAVEIRA and MASIDE 1998 ), but is general for hybrid male sterility. This contrasts with the genetic system of hybrid inviability and female sterility, where major genes that individually cause inviability and sterility have been suggested or identified (PRESGRAVES 2003 ; PRESGRAVES et al. 2003 ; SAWAMURA et al. 2004 ). It is possible that functional divergence of genes involved in male reproduction is accelerated by sexual selection (COULTHART and SINGH 1988 ; TSAUR and WU 1997 ; NURMINSKY et al. 1998 ; SINGH and KULATHINAL 2000 ; SWANSON et al. 2001 ).

A concern remains that the male sterility genes detected in this analysis might be simple null mutations. In fact, a recessive lethal detected on the introgression was shown to be caused by a new transposable element (TE) insertion; TEs might have been mobilized as a result of the initial species cross (SAWAMURA et al. 2000 ). However, in this analysis we detected dominant enhancers of male sterility genes and showed that not a single gene but instead a combination of at least two genes causes male sterility. We believe that not all the genes detected in this analysis are new mutations but rather true hybrid male sterility genes.

	ACKNOWLEDGMENTS

We are grateful to A. Garcia-Bellido and the Bloomington and Umeå Drosophila stock centers for providing fly strains. M. Ashburner and D. Lindsley shared unpublished data. Thanks are due to K. Hirai, M. Itoh, T. L. Karr, and K. Ogura for discussion and/or comments. Comments from M. Aguadé and two anonymous reviewers improved this report. This work was supported by grants from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology to K.S. and M.T.Y.

Manuscript received January 5, 2003; Accepted for publication November 4, 2003.

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