Copyright 0 1994 by the Genetics Society of America

Evidence for Complex Genic Interactions Between Conspecific Chromosomes Underlying Hybrid Female Sterility in the Drosophila simulans Clade

Andrew W. Davis, Erik G. Noonburg and Chung-I Wu

Department of Ecology and Evolution, University of Chicago, Chicago, Illinois 60637 Manuscript received September 3, 1993

Accepted for publication January 28, 1994

ABSTRACT F, hybrid females between the sibling species Drosophila simulans, Drosophila mauritiana and

Drosophila sechellia are completely fertile. However, we have found that female sterility can be observed in F, backcross females who are homozygous for D. simulans X chromosomes and homozygous for autosomal regions from either D. mauritiana or D. sechellia. Our results indicate that neither D. mau- ritiana autosome ( 2 or 3) can cause complete female sterility in a D. simulans background. The simul- taneous presence of homozygous regions from both the second and third chromosomes of D. mauritiana, however, causes nearly complete female sterility which cannot be accounted for by their individual effects. The two autosomes of D. sechellia may show a similar pattern. From the same crosses, we also obtained evidence against a role for cytoplasmic or maternal effects in causing hybrid male sterility between these species. Taken with the results presented elsewhere, these observations suggest that epistatic interactions between conspecific genes in a hybrid background may be the prevalent mode of hybrid sterility between recently diverged species.

E VOLUTIONARY biologists have long been in- trigued by the evolution of postzygotic reproduc-

tive isolation because sterility and inviability are traits which are expected to be strongly selected against in- traspecifically. In an attempt to explain the evolution of reproductive isolation, hybrid sterility and hybrid invi- ability have been the subject of intense genetic analysis, notably, in the genus Drosophila [see COYNE (1992) and Wu and DAVIS (1993) for recent reviews]. Studies con- tained in these reviews focused mostly on male inviability and male sterility because in crosses between species with heterogametic sex determination, the heteroga- metic sex tends to be more severely affected than the homogametic sex (HALDANE 1922). Recent analyses have suggested that HALDANE’S rule is most likely a composite phenomenon in which male sterility evolves much more rapidly than male inviability (Wu 1992; Wu and DAVIS 1993).

Interestingly, there appears to be an association be- tween male sterility and reduced female fertility but not between male sterility and male inviability (JOHNSON and WU 1993). Most studies, however, focused primarily on the effect of hybridization on male fertility and would have missed subtle affects of hybridization on female fertility. Only a handful of studies have been done which address the genetic basis of female sterility or inviability (LANCEFIELD 1929; ORR 1987, 1989; SAWAMURA et al. 1993). Thus sterility in hybrid females deserves more attention.

In this study we analyze the effect of hybridization on female fertility between the sibling species Drosophila simulans, Drosophila maurit iana and Drosophila sech-

Genetics 137: 191-199 (May, 1994)

ellia. D. simulans is a cosmopolitan human commensal while D. mauri t iana and D. sechellia are restricted to islands in the Indian Ocean (LACHAISE et al. 1986). F, female hybrids between these three species are com- pletely fertile (LACHAISE et al. 1986) as are F, female hybrids which inherit both X chromosomes from D. s imulans (COYNE 1985). On the basis of these results it has been assumed that hybridization between these three species has relatively little effect on female fertility.

We reexamined the genetic basis of hybrid female ste- rility between these three species. Our goal was to de- termine: (i) if genetic interactions between chromo- some segments of these species exist which cause female sterility and, if they do (ii) how do these genes interact to cause sterility? WRIGHT summarized three possible re- lationships of genotype to phenotype which we can use to predict how genes from these species might interact to cause female sterility (see Figure 1 of WRIGHT 1982): (1) replacement of a single gene from one species with its homologue from a sibling species, (2) replacement of many genes which cause sterility because of their addi- tive effects and (3) sterility results due to complex non- additive genetic interactions between many genes ( i . e., epistasis).

Previous studies have only measured the effect on fe- male fertility of interactions between heterozygous loci from one species and homozygous or heterozygous loci from another species (COYNE 1985; ORR 1987, 1989; JOHNSON and WU 1993). In this study, we tested whether or not interactions between homozygous X chromo- somal loci from one species and homozygous autosomal loci from another species can cause female sterility. A

192 A. W. Davis, E. G. Noonburg and C.-I Wu

phenotypic analysis was performed to determine the oo- genic phenotypes of the sterile hybrids.

The results show that interactions which cause female sterility do exist between these three species, Our analy- sis rules out the existence of a single gene on either autosome that by itself cause female sterility in a foreign background. Instead, several conspecific loci ( i . e., loci from the same species) need to be cointrogressed to cause sterility. The observations parallel those reported in the accompanying paper on hybrid male sterility (CABOT et al. 1994).

The crosses used in our experiments also allowed us to investigate the contribution to hybrid sterility by cy- toplasmic and maternal effects (DOBZHANSKY 1937). Cy- toplasmic effects are reductions in fertility due to com- ponents inherited from the maternal cytoplasm (e.g., mitochondria or microorganisms) which are to a large extent unrelated to the maternal nuclear genotype. There also exist systems where the cytoplasmic effects are ultimately regulated by the nuclear genome, for ex- ample, the cytotype in the P-M dysgenic system (ENGELS 1989). Maternal effects are reductions in male fertility due to components contributed to the egg cytoplasm by the maternal genome during oogenesis ( e . g . , mRNA or protein). Our analysis indicates that the fertility of D. simulans-D. mauritiana male hybrids is unaffected by maternal contributions of either cytoplasmic or nuclear origin and extends the conclusion of ZENC and SINCH (1993).

MATERIALS AND METHODS

Flystocks: A D . simulans C (I) y w strain (i.e., compound X chromosomes bearing yellow and white mutations) was used in all experiments. For backcrosses to D. sechellia, two addi- tional C (1) y w strains were constructed, one homozygous for Curly (Cy, 2-6.1) and one heterozygous for Hairless (H, 3-69.5). A D. simulans y vfstrain (1089 from the University of Indiana Stock Center) was used in backcrosses designed to control for maternal effects. Males from wild-type strains of D. mauritiana and D. sechellia and a D. mauritiana strain car- rying singed ( s n 7D, 1-21.0), jaunty ( j 34E, 248.7), and ir- regular ( ir , 3- location unknown) were used for backcrosses. The map positions given above are all from D. melanogaster. The D. simulans C(1) y w, H , Cy, D. mauritiana sn; j ; ir, and the wild-type strains used in this analysis were kindly provided by J. COYNE. All crosses were done at 23-25" on standard corn- meal media.

Mating scheme: Virgin D. simulans C ( 1 ) y w females were mass mated to D. mauritiana or D. sechellia males. Virgin F, females were collected and backcrossed to males of one of the parental species and the resulting F, backcross females collected. (An example of the mating scheme is given in Figure 1.)

Female fertility experiments: The fertility of pure species and F, backcross females was tested by mating single females to 2 males from the backcross species for 5-7 days. Females were scored as fertile if the matings produced any larvae. For matings which did not produce larvae, the females were dis- sected and checked for insemination by examination of their spermathecae. Only inseminated females from matings which failed to produce larvae were scored as sterile. This is the same

criterion used in other studies on hybrid female sterility (Om 1987, 1989). An analysis of the ovarian phenotypes of sterile females was performed by squashing dissected ovaries in Dro- sophila Ringer's solution (182 mM KC1, 46 mM NaCl, 3 mM CaCI,, and 10 mM Tris, pH 8.0) and examining their mor- phology using phase contrast microscopy. The phenotypes were broken into three classes based on the most mature stage of oogenesis observed ( MAHOWLO and KAMBBELLIS 1980) : ( 1) agametic-no germ cell activity, (2) germinal-only previtello- genic egg chambers present (oogenic stages 1-7) and (3) vitellogenic-vitellogenic egg chambers present (stages 8-14) and usually defective.

Effect of cytoplasm and maternal effects on male fertility: In separate but identical crosses, the fertility of F2 males from backcrosses ofF, females (shown in Figure 1) to D. rnauritiana males was measured. For backcrosses to wild-type D. maul-i- tiana males, male fertility was determined by mass mating vir- gin males to virgin y vffemales for 5-7 days. Males which failed to produce progeny were considered sterile. For backcrosses to D. mauritiana sn; j ; ir males, male fertility was determined by checking for the presence of motile sperm in the seminal vesicle. Males which failed to produce any motile sperm after being held for three days were considered sterile.

RESULTS

Hybrid female sterility: To study the genetics of hy- brid female sterility, we constructed females which al- lowed completely homozygous X chromosomes from D. simulans to interact with homozygous autosomal re- gions from D. mauritania or D. sechellia. Homozygous and heterozygous, in this context, refer only to the spe- cies origin of a gene. Hybrid females with these geno- types were constructed by backcrossing D. simulans C(1) y w females to D. mauritiana or D. sechellia males for two generations. An example of such a cross and the genotypes obtained are shown in Figure 1. As a control for maternal contributions from F, hybrid females, the fertility of F, females produced from backcrossing F, hy- brid females to D. simulansy v f maleswas also analyzed. The D. simulans compound Xchromosomes of these F, females can only interact with some autosomal regions heterozygous for D. mauritiana-D. simulans or D. sechellia-D. simulans and are expected to be as fertile as their F, mothers unless they are affected by cytoplas- mic or maternal effects.

Backcrosses to D . mauritiana: Initial experiments used wild-type strains of D. mauritiana for the backcross experiment outlined in Figure 1. The autosomal con- stitution of such F, females are designated f s im o r maul/ mau ( i e . , [maternal autosomal contribution]/paternal autosomal contribution) and represent the aggregate of genotypes I-IV in Figure 1. The results are summarized in Table 1. For backcrosses to wild-type D. mauritiana males, 25.2% of the F, C (1) y w ; [sim or maul/mau females were sterile. The sterility suggests that interac- tions between homozygous X chromosome loci from D. simulans and homozygous autosomal loci from D. mau- ritiana can cause female sterility in contrast with the observation that F, hybrid females of Figure 1 are nearly completely fertile (see Table 1 and COYNE 1985). If a

Genetics of Hybrid Female Sterility

XIY 2 3

193

P:

F1:

> 7

j ir " "I " x 7 / --- J Ir

J.

F2 Autosomal Genotypic Classes:

Genotype I Genotype I1 Genotype In Genotype IV

FIGURE I.-An example of the crossing scheme used to construct F2 hybrid females for sterility analysis. D. simulans C (I) y w females were backcrossed to D. mauritiana sn; j ; irmales for hvo generations and the fertility of females from the four autosomal genotypic classes deter- mined. Due to recombination in F, females, the autosomal markers only identify the species ori- gin of a small region of each chromosome. Varia- tions from this scheme were carried out using different strains for the backcrosses and are dis- cussed in the text.

= D. mauritiana

= either

TABLE 1

Fertility of the parental species, F, hybrid, and F2 hybrid females generated in backcross experiments using wild-type strains of D. mauritiana and D. sechellia

No. No. No. No. No. Female tested Autosomal genotype tested dead unmated fertile sterile

D. simulans C ( 1 ) y w D. simutans 119 18 0 100 1 (1.0)"

D. mauritiana wild type D. mauritiana 104 8 3 90 3 (3.2) F, D. simulans/D. mauritiana sim/mau 24 1 11 0 230 0 F2 D. simulans/D. mauritiana [sim or maulb/s im 237 24 3 207 3 (1.4) F2 D. simulans/D. mauritiana [sim or m a ~ ] ~ / m a u 626 110 5 382 129 (25.2) D. sechellia wild type D. sechellia 109 1 0 107 1 (0.9) F2 D. simulans/D. secheltia [sim or seclb/sim 242 8 14 183 37 (16.8) F2 D. simutans/D. sechetlia [sim or seclc/sec 525 90 N/A 164 271 (62.3)

All F, and F2 females have D. simulans C ( 1 ) y w X chromosomes. " Numbers in parentheses are percentage sterile/(sterile + fertile).

Maternally inherited autosomes are of mixed species origin due to recombination in the F,. All autosomal material is either heterozygous for

Because of the absence of autosomal markers, the genotypes of these females is the average of genotypes I-lV shown in Figure 1. the second species or homozygous for D. simulans.

single homozygous factor with complete from D. mau- ritiana was responsible for hybrid female sterility be- tween these species, 50% of the F2 females should be sterile not 25.2%. Therefore, hybrid female sterility between D. simulans and D. mauritiana is caused by genes which reduce female fertility less than expected for a single gene of complete effect. For the control backcrosses to D. simulans y v f males, 1.4% of the F, C (1 ) y w ; [sim or mau]/sim females tested were sterile. The degree of sterility observed is not significantly dif-

ferent from that observed for the D. simulans C(1) y w strain (Gadj = 0.094; P > 0.25) suggesting that F, hybrid maternal effects do not affect the fertility of D. simulans-D. mauritiana hybrid females.

To investigate further the effects each D. mauritiana autosome and their interactions have on female fertility, D. simulans C(1) y w females were backcrossed for two generations to a D. mauritiana strain homozygous for jaunty ( j ) on chromosome 2 and irregular ( i r ) on chromosome 3.This crossing scheme generated four

194 A. W. Davis, E. G. Noonburg and C.-I Wu

TABU 2

Fertility of hybrid females generated in backcrosses to D. mauritiana sn; j ; ir males as shown in Figure 1

No. No. No. No. No. Female tested Autosomal genotype tested dead' unmated fertile sterile

D. mauritiana j / j ; ir/ir D. mauritians 102 30 0 72 F, D. simulans/D. mauritiana I : ~ j : / j ; i r+ / i r 98 18 7 54 F, D. simulans/D. mauritiana 11: 14; i r + / i r 69 15 3 19 F, D. simulans/D. mauritiana 111: j+/j; i r / i r 77 19 2 33 F, D. simulans/D. mauritiana Tv: j / j ; ir/ ir 79 21 2 3 Total over all classes: genotypes I-IV 323 73 14 109 Marginal totals: j+/j-genotypes I + 111 87

ir+/ir-genotypes I + 11 73

All F, females have D. simulans C (1) y w X chromosomes. Alleles from D. mauritiana are shown in boldface. ' The difference in viability observed between the phenotypic classes was not significant (2 = 5.607; P > 0.10). The autosomal genotype designation refers to those shown in Figure 1. Numbers in parentheses are percentages.

0 19 (26.0)' 32 (62.7) 23 (41.1) 53 (94.6)

127 (53.8) 42 (32.6) 51 (41.1)

autosomal phenotypic classes of females as outlined in Figure 1. Type I females are j + / j ; ir+/ir, type I1 females are j / j ; ir'/ir, type I11 are j + / j ; ir/ir, and type IV fe- males are j / j ; ir/ir. It is important to note that the markers used only identify a relatively small portion of each D. mauritiana autosome. Therefore, the size of the region homozygous for the D. mauritiana markers var- ies among F, individuals [see Wu and DAVIS (1993) for further discussion]. The results of these experiments are summarized in Table 2.

A glance at Table 2 suggests that the X autosome in- compatibility between species plays a crucial role in hy- brid sterility. The most sterile class (genotype IV) has most of its autosomes from D . mauritiana while both X chromosomes are from D. simulans. Genotype IV has the least autosome-autosome incompatibility among all F, females implying that X autosome interactions are responsible for the sterility. It is also clear that both au- tosomes (genotypes I1 and 111) contribute to hybrid fe- male sterility. In analyzing the results, we attempt to ad- dress two questions. First, does either chromosome 2 or chromosome 3 contain a gene of major effect, which causes complete female sterility when homozygous for the D. mauritiana allele? Second, are the sterility effects of the two autosomes independent from each other? The answers to both questions appear to be negative as described below.

To address the first question, we note that the ob- served sterility of the j + / j genotype of the second chro- mosome, averaged over both genotypes of the third chromosome (i. e. , genotype I and I11 of Table 2), was only 0.33. If the unselected ir-bearing third chromo- some carried a gene with complete sterility effect, a p proximately 50% of the j ' / j should have been sterile. This is because half of the j ' / j genotype would have carried this putative major gene on chromosome III. The sterility should, in fact, have been greater than 0.5 since the second chromosomes with the j ' / j markers contributed some of the sterility effect on their own. A comparison of genotype I of Table 2 which was 26%

sterile, with F, females of Figure 1, which were com- pletely fertile (Table l ) , supports this claim. The pre- diction that 50% or more of thej'/jfemales should have been sterile is also based on the assumption that the putative sterility genes have no appreciable effect on vi- ability.JOHNSON and Wu (1993) have provided empirical data that corroborate such an assumption. Moreover, the relative frequencies of the four genotypic classes in Table 2 do not deviate significantly from the expected even distribution (x' = 5.607; P > 0.10). The slight varia- tion in the number recovered did not affect the con- clusion because the observed sterility of the j ' / j geno- type only changes from 0.326 to 0.355 if the calculation is not weighted by the apparent viability differences. Thus, the observed sterility of thej+/j (0.326) genotype was much less than the expected sterility of greater than 0.5 (Gadj = 15.971; P < 0.0005), if the unselected third chromosome carried a complete female sterility gene. If the third chromosome carried more than one complete sterility gene, the expected sterility should be even higher. Similarly, the observed sterility of the ir'/zr genotype (genotype I and 11), 0.411, was also less than the expected number of greater than 0.5 (Gadj = 3.908; P < 0.05). Our observations thus fail to corroborate the presence of genes of major sterility effect on either autosome.

To address the second question of between- chromosome interactions, we construct the following null model on the assumption of independent action: Let the probability that the second chromosomes with the j / j or j ' / j genotype have no sterility effect be a* or 1 - a, respectively. If the sterility were caused by a single factor of complete penetrance on the D. mauritiana second chromosome, then a* = a, which is the recom- bination distance between the markerj and this putative factor. Without assuming a* = a, we make no suppo- sition about the genetic architecture underlying sterility within each chromosome. Similarly, let the correspond- ing probabilities be b* and 1 - b for ir/ir and ir+/ir, respectively. The expected proportions of fertile females

Genetics of Hybrid Female Sterility 195

are therefore: genotype I, (1 - a ) (1 - b) [0.74, ob- served]; genotype 11, a* (1 - b) [0.37]; genotype 111, (1 - a ) b* [0.59]; and genotype IV, a*b* [0.053]. Under the null model of independent action, the ratio of fertile females, type 1:type I1 = (1 - a):a*, should equal that of type 1II:type IV = (1 - a ) :a*. The observed ratio for the former is 0.74/0.37 = 2.0, whereas the latter is 0.59/ 0.053 = 11.1. This suggests the sterility effect of j / j rela- tive to that of j + / j is 5.5-fold greater when the back- ground is i r / i r than when it is i r+ / i r . The comparison of the ratios type 1:type 111 [1.25 observed] and type 1I:type IV [6.98 observed] yields the same conclusion for the third chromosome: the sterility effect of ir / ir is also 5.5-fold greater when the background is j / j than when it is j+/ j . In other words, the joint effect of homozygosity for both autosomes is much greater than the product of the individual effects. To show that the interaction is statistically significant, we did a 2 test on the number of fertile males in each genotypic class with a conservative adjustment for the uneven sample size (We used the proportions of fertility of Table 2 but used the smallest sample size in the four classes, n = 51, in our calcula- tion). Even with the conservative test, the 2 value is 6.51 with one degree of freedom (P< 0.01). Thus, the answer to the second question is that there exist betweenchro- mosome epistatic interactions leading to female sterility.

Overall F, female sterility was higher in backcrosses to the D. mauritiana sn; j ; ir strain than in backcrosses to the D. mauritiana wild-type strain (e.g. , 53.8% us. 25.2%). The difference between the two experiments most likely represents strain specific differences. The two strains could differ in the total number of loci presentwhich can interactwith D. simulans loci to cause female sterility ( i . e . , more in sn; j ; i r ) . The between strain difference is compatible with the view that hybrid sterility is due to multigenic interactions.

Backcrosses to D . sechellia: Initial experiments used a wild-type strain of D . sechellia for backcrosses. The results of these experiments and the control backcrosses to D. simulans y v f males are listed in Table 1. Sur- prisingly, 16.8% of the control F, C ( l ) y w ; [sim or see]/ sim females were sterile. This degree of sterility is sig- nificantly different from that observed for the D . simulans C ( l ) y w strain (Gadj = 22.583; P< 0.0005). The sterility was unexpected because these backcross geno- types have fewer incompatible interactions than the F, females of Figure 1. It is possible that there are complex epistatic interactions such that the absence of some D . sechellia genes in the F,’s, relative to the Fl’s, causes sterility in a mostly D. simulans background.

Alternatively, maternal effects may have been a con- tributing factor to the sterility of F, hybrid females. F, females have a hybrid nuclear genome that could have produced maternal components antagonistic to the mostly D. simulans zygotic genome of their F, daugh- ters. It is unlikely that cytoplasmic effects were respon-

sible for the reduction in F, fertility because F, females are fully fertile (COYNE 1985) and because the genome of the F, females was mostly of D. simulans origin. Cy- tological observations on these sterile F, females are con- sistent with the maternal effect hypothesis. As shown in Table 4, all 10 sterile F, C(1) y w ; [sim or sec]/sim fe- males examined were agametic; a phenotype resembling those of the maternal effect grandchildless mutations found in D . melanogasterand D . subobscura (MAHOWALD

et al . 1979; MARIoL 1981; NIKI and OKADA 1981). In backcrosses to D . sechellia wild-type males, 62.3%

of the F, C ( I ) y w ; [sim or sec]/sec females were sterile. The autosomal genotype of these females represents the aggregate of genotypes I-IV shown in Figure 1. As with the D. mauritiana backcross, these results demonstrate that interactions between homozygous X chromosome loci from D. simulans and homozygous autosomal loci from D. sechellia can cause female sterility. Based on these results alone, the existence of genes capable of causing complete female sterility cannot be ruled out for the D. simulans-D.sechellia hybridization because the observed sterility is greater than 50%.

To determine the relative effect of each major auto- some on female fertility, two D. simulans C(1) y w strains were constructed, one homozygous for chromosome 2 dominant marker Curly (Cy) and one heterozygous for chromosome 3 dominant marker Hairless ( H ) . These strains were then used in backcross experiments and the fertility of the two phenotypic classes tested. The results are summarized in Table 3.

In the experiments using Cy as the dominant marker, 67.5% of the females homozygous for Cy’ from D . sech- ellia were sterile (average of genotypes I1 and IV in Fig- ure 1) and 46.3% of the females heterozygous for Cy were sterile (average of genotypes I and 111). For the backcrosses using H, 75.4% of the females homozygous for H+ were sterile (average of genotypes I11 and IV) and 59.7% of the females heterozygous for H were sterile (average of genotypes I and 11). The difference in fer- tility between the homozygous and heterozygous F, phe- notypic classes was significant for both backcross experi- ments. The difference in fertility of F, females homozygous for Cy’ or H’ between experiments was not significant (G!dj = 2.749; P > 0.05) suggesting that the two D. sechellza autosomes have a comparable effect on female fertility.

The genetic basis of female sterility in the D . simulans-D. sechellia hybrids is somewhat more difficult to interpret than the D . simulans-D. mauritiana hybrid- izations mainly because of the more limited resolution of the experimental design. Since the two autosomes from D. sechellia appear to have a comparable sterility effect, we can rule out the presence of a gene of major effect on both chromosomes. If this were the case, we would expect altogether 75% of the F, females to be sterile. The combined data of Tables 1 and 3 give 735

196 A. W. Davis, E. G. Noonburg and C.-I Wu

TABLE 3

Fertility of D. simulans C (1) y w; Cy/Cy and C (1) y w ; H/H' females and the F, hybrid females produced by backcrossing these females to D. sechellia wild-type males

No. No. No. No. No. Female tested Autosomal genotype tested dead unmated fertile sterile

D. simulans C (1) y w ; Cy/Cy D. simulans 74 2 0 71 1 (1.4)= F, D. simulans/D. sechettia Cy/Cy' (type I + 111) 199 21 2 94 81 (46.3) F, D. simulans/D. sechellia Cy+/Cy+ (type I1 + IV) 191 20 9 53 110 (67.5) Total over both classes: 390 41 11 147 191 (56.5) D. simulans C (1) y w ; H/H' D. simulans 50 1 0 46 3 (6.1)

F2 D. simulans/D. sechellia H /H+ (genotype I + 11) 239 25 8 83 123 (59.7) F2 D. simulans/D. sechellia H + / H + (genotype 111 + IV) 234 31 4 49 150 (75.4) Total over both classes: 473 56 12 132 273 (67.4)

All F, females have D. simulans C ( I ) Y w X chromosomes. Alleles from D. sechellia are shown in boldface. Numbers in parentheses are percentages.

. , ,

sterile females out of 1178 examined (62.3%). Further- more, the actual sterility due to the genotypic interac- tions in the F, could be even smaller-maybe as low as 55% (= 1 - [ l - 0.623]/[1 - 0.1681) if the maternal effect of F, females contributes 16.8% of the F, sterility (see Table 1). Whether there is a maternal effect on F, female sterility or not, the observed percentage is sig- nificantly lower than the expected 75% for a major effect gene on each autosome (Gadj = 91.196; P < 0.0005).

Although it is still possible that a single major effect gene exists on only one of the two autosomes that results in 50% sterility, the other chromosome should be much weaker in its sterility effect in order to explain the overall sterility in the F, (e.g., 62.3%). This interpretation is inconsistent with the results of Table 3 which show that the effect of homozygosity of the second chromo- some, Cy'/Cy+ over Cy +/Cy, on female fertility is 1.65 (= [ 1 - 0.4631 / [ 1 - 0.675]), is exactly the same as the third chromosome effect, which is 1.64 ( = [ 1 - 0.5971 / [ 1 - 0.7541). The basis of this calculation is similar to the null model forD. mauritiana described above. Thus, our observations suggest that single genes of major sterility effect may not be present in this hybridization either.

Ovarian phenotypes: Results from phenotypic analy- sis of a subset of the sterile F, females listed in Tables 1-3 are summarized in Table 4 and examples of the range of ovarian phenotypes observed are shown in Figure 2. Sterile females showed defects in oogenesis ranging from complete absence of germ cell activity to fused egg chambers to defective chorion and egg shape. For back- crosses to the D. mauritiana sn; j ; ir stock, type N fe- males showed the most severe disruption in oogenesis. Seventy percent of the sterile females in this class showed no ovarian development. The wide range of sterile phe- notypes observed provides further support for a multi- genic basis of female sterility.

Maternal and cytoplasmic effects on hybrid male fertility: F, males from the crosses depicted in Figure 1 can be used to analyze cytoplasmic and maternal effects on male fertility. The cytoplasmic effects are relatively easy to assess by repeated backcrossing that would re-

TABLE 4

Ovarian phenotypes of sterile hybrid C ( I ) y w females produced in backcross experiments

Phenotypic class

Autosomal genotype Agametic Germinal Vitellogenic

[sim or m a u ] / m a u 31 (33.7)a 13 (14.1) 48 (52.2)

I: j + / j ; i r+/ i r 3 (37.5) 1 (12.5) 4 (50.0) 11: j / j ; ir+/ir 4 (33.3) 1 (8.3) 7 (58.3)

111: j + / j ; ir/ir 1 (12.5) 0 7 (87.5) N j / j ; ir/ir 14 (70.0) 1 (5.0) 5 (25.0) Total over all classes: 22 (44.9) 3 (6.1) 24 (49.0)

[ s im or seclb"/sim 10 (100) 0 0 [sim or secIC/sec 34 (45.3) 9 (12.0) 32 (42.7) Cy/Cy' (genotype I + 111) 32 (39.0) 8 (10.0) 42 (51.2) Cy+/Cy+ (genotype 111 + IV) 27 (24.5) 3 (2.7) 80 (72.7) H/H+ (genotype I + 11) 19 (15.4) 0 104 (84.6) H'/H' (genotype 111 + IV) 50 (42.4) 0 68 (57.6)

The females listed are a subset of those listed in Tables 1-3. Alleles from D. mauritiana or D. sechellia are shown in boldface.

Numbers in parentheses are percentages. ' Ovarian phenotypes were determined for only 10 of the 37 sterile

females listed in Table 1. The [sim or m a u ] / s i m females were nearly all fertile (98.6%) and hence not included.

Because of the absence of autosomal markers, the genotypes of these females is the average of genotypes I-IV shown in Figure 1.

store the purity of the nuclear genome in the cytoplas- mic background of another species, as done previously (e .g . , ZENG and SINGH 1993). On the other hand, it is more difficult to analyze maternal effects cleanly be- cause maximal incompatibility between the genomes of the mother and offspring may be required to see an effect. The crosses of Figure 1 are as close to meeting that requirement as is technically feasible in these spe- cies: the maternal genome is either homozygous (for the X chromosome) or at least heterozygous for each D. simulans chromosome whereas the zygotic genome is mostly of D. mauritiana origin (Le. , Xmau/Ymau; fsim or mau]/mau) and may be entirely from that species. The results are summarized in Table 5.

In the wild-type backcross experiments where the D. mauritiana males bear no visible markers, 3.8% of F,

Genetics of Hybrid Female Sterility 197

FIGURE 2.-Examples of the ovarian phenotypes observed in sterile F2 hybrid females. (A) Nor- mal ovary from a fertile F2 D. simulans-D. mauritiana hybrid female ( X 100). (B) Agametic ova- ries from a sterile F2 D. simulans-D. mauritiana hybrid female (X400). (C) Germinal stage egg chamber from a sterile F2 D. simulans-D. mauritiana hy- brid female. Normally germinal chambers contain a maximum of 8 cells. The chamber indicated by the arrow has more than 60 (X1000). (D) Vitellogenic stages from a sterile F2 D. simulans-D. maun'tiana hybrid female. Note the irregular yolk distribution of the egg indicated by the arrow (X400). An identical range of phenotypes was observed for ster- ile D. simulans-D.sechellia hybrid females.

males were fertile. [The sterility of the rest is due to the incompatibilitywithin the nuclear genome (COYNE 1984; Wu et al. 1993) .] The presence of fertile males suggests that maternal components, mostly of D. simulans ori- gin, may not be incompatible with a zygotic genome that is mostly D. mauritiana in origin. We predicted that the fertile males must have had an essentially reconstituted D. mauritiana wild-type nuclear genome. The data of Table 5 from backcross experiments using j and ir- marked males agree with our prediction. Fertile males were only observed in the genotypic class which was ho- mozygous for both D. mauritiana autosomal markers. Thus, the maternal components of predominantly D. simulans origin are not incompatible with the D. mau-

ritiana nuclear genome. We can also conclude that D. simulans cytoplasm-D. mauritiana nuclear interactions do not effect male fertility. Because the cytoplasm was derived from D. simulans only two generations ago, such rapid replacement of the nuclear genome avoids the possible nuclear influence on the cytoplasmic effect like the cytotype phenomenon in hybrid dysgenesis (ENGELS 1989).

DISCUSSION

We have examined the genetic basis of hybrid female sterility between the sibling species D. simulans, D. mau- ritiana and D. sechellia. Our results demonstrate that

198 A. W. Davis, E. G. Noonburg and C.-I Wu

TABLE 5

Fertility of male hybrids produced from backcrosses of D. simulans C (1) y w females to D. mauritiana wild-type

or sn; j ; ir males

Sex Autosomal No. No. chromosomes background sterile fertile

X mau/Y mau [sim or m a u ] / m a u a 203 8 (3.8)b X mau/Y mau I:' j + / j ; i r+ / i r 28 0

11: j / j ; i r+ / i r 22 0 111: j + / j ; i r / i r 22 0 Iv: j / j ; i r / i r 33 10 (23.3)

Total over all classes: 105 10 (8.7) Total for both experiments: 308 18 (5.5)

Alleles from D. mauritiana are shown in boldface. 'The autosomal genotypes of these males are equivalent to the

average of genotypes I-IV shown in Figure 1. Numbers in parentheses are percentages.

'The autosomal genotype designation refers to those shown in Figure 1.

genetic interactions exist between these species which can cause female sterility. The reason hybrid female ste- rility was not observed in earlier studies of these species (COYNE 1985; LACHAISE et al. 1986) is because hybrid genotypes were not constructed which allowed homozy- gous loci from each species to interact. Such genotypes cannot be constructed in standard hybridization cross- ing schemes because hybrid males are sterile. We con- structed hybrids with these genotypes by backcrossing a D. simulans strain with compound X chromosomes to D. mauritiana or D. sechellia males for two generations (see Figure 1). The resulting F, hybrids are completely homozygous for X chromosomes from D. simulans and for various combinations of autosomal regions from D. mauritiana or D. sechellia. The results demonstrate that negative epistatic interactions capable of causing female sterility do exist between these three species.

That female sterility is only observed when homozy- gous loci from each species are allowed to interact points out the limitations of standard F, and F, hybrid back- cross analysis. For some species, evidence of reproduc- tive isolation may only occur when such genotypes can be constructed. An example is the sterility of F, male D. hydei and D. neohydei hybrids (HENNIG 1977).

For D. simulans-D. mauritiana hybrids, both the sec- ond and third chromosome of D. mauritiana cause par- tial female sterility when homozygous in a background with homozygous D. simulans X chromosomes (Table 2). Similarly, both the second and third chromosome of D. sechellia cause partial sterility when homozygous in D. simulans-D. sechellia female hybrids (Table 3 ) . We observed more sterility in D. simulans-D. sechellia hy- brids compared to D. simulans-D. mauritiana hybrids (62% us. 25-5076, see Table 1). This difference may be due to a maternal effect contributed by F, D. simulans-D. sechellia females. Examples of maternal ef- fects on hybrid female fertility have been suggested for

other Drosophila hybridizations (ORR 1987; ORR and COYNE 1989).

We were able to show that the second and third chro- mosomes of D. mauritiana do not exert their effects independently; that is they show strong epistatic inter- actions which cause almost complete female sterility in this background. Ninety-five percent of F, females si- multaneously homozygous for regions from chromo- somes 2 and 3 from D. mauritiana were sterile (Table 2). Another example of epistatic interactions which can lead to female sterility has been reported for hybrids between D. uirilis and D. novamexicana (ORR and COYNE 1989). In a companion study (COOT et al. 1994), such epistasis between conspecific loci has also been demonstrated for hybrid male sterility. The conclusion is contrary to the common assumption that hybrid ste- rility is caused by interaction between a single pair of genes (WU and BECKENBACH 1983; VIGNEAULT and ZOUROS 1986; COYNE and CHARLESWORTH 1989; PANTAZIDIS et al. 1993). The observation that hybrid sterility results from complex genetic interactions is significant because it bears directly on the genetic basis of species differences and speciation (DOBZHANSKY 1937; MAW 1963; WRIGHT 1977).

Phenotypic analysis of the ovarian defects observed in sterile F, females supports our conclusion that female sterility has a multigenic basis between these species. No consistent ovarian defect was observed in any of the backcrosses (Table 4 and Figure 2) as might be expected if single loci of large effect were responsible for the ste- rility. The distribution of phenotypes is similar to those observed for a collection of D. melanogaster zygotic le- thals which, when made homozygous as germ line clones, cause ovarian defects or have maternal effects resulting in female sterility (PERRIMON et al. 1989). In fact, most female sterile mutations are actually hypomor- phic alleles of zygotic genes (PERRIMON et al. 1986). The extreme developmental pleiotropy of oogenesis in D. melanogaster suggests that the loci responsible for hy- brid female sterility between D. simulans, D. mauriti- ana, and D. sechellia are not specific to oogenesis. We have not determined, however, if the F, females in our study have maternal effects on F, viability although cur- sory observations suggest that they may. Comparison of the viability of progeny produced by [sim or mau]/sim females us. [sim or mau]/mau females will resolve this issue.

Finally, we showed that hybrid male sterility is not likely to result from the incompatibility between either the maternal nuclear genome or cytoplasm and the zy- gotic genome of their sons. These results extend the conclusion of ZENC and SINCH (1993) by showing that the cytoplasmic effects, even under the influence of the maternal genome, do not play an important role in hybrid male sterility.

We thank MIKE PALOPOU, NORMAN JOHNSON, ERIC CABOT and HOPE HOLLOCHER for helpful comments and stimulating discussions. We

Genetics of Hybrid Female Sterility 199

thank JERRY COWE for fly stocks. ED STEPHENSON taught us Drosophila oogenesis. A.W.D. was supported by a William B. Graham Fellowship from the Baxter Foundation and C.1 W. by an National Institutes of Health (NIH) grant (GM 39902) and an NIH Research Career De- velopment Award (GM 00553).

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Communicating editor: C. C. LAURIE