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Genetics, Vol. 154, 1747-1771, April 2000, Copyright © 2000

The Drosophila melanogaster Hybrid male rescue Gene Causes Inviability in Male and Female Species Hybrids

Daniel A. Barbasha, John Rootea, and Michael Ashburnera
a Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom

Corresponding author: Daniel A. Barbash, Department of Genetics, University of Cambridge, Downing St., Cambridge CB2 3EH, United Kingdom., d.barbash{at}gen.cam.ac.uk (E-mail)

Communicating editor: C.-I WU


*  ABSTRACT
*TOP
*ABSTRACT
*MATERIALS AND METHODS
*RESULTS
*DISCUSSION
*LITERATURE CITED

The Drosophila melanogaster mutation Hmr rescues inviable hybrid sons from the cross of D. melanogaster females to males of its sibling species D. mauritiana, D. simulans, and D. sechellia. We have extended previous observations that hybrid daughters from this cross are poorly viable at high temperatures and have shown that this female lethality is suppressed by Hmr and the rescue mutations In(1)AB and D. simulans Lhr. Deficiencies defined here as Hmr- also suppressed lethality, demonstrating that reducing Hmr+ activity can rescue otherwise inviable hybrids. An Hmr+ duplication had the opposite effect of reducing the viability of female and sibling X-male hybrid progeny. Similar dose-dependent viability effects of Hmr were observed in the reciprocal cross of D. simulans females to D. melanogaster males. Finally, Lhr and Hmr+ were shown to have mutually antagonistic effects on hybrid viability. These data suggest a model where the interaction of sibling species Lhr+ and D. melanogaster Hmr+ causes lethality in both sexes of species hybrids and in both directions of crossing. Our results further suggest that a twofold difference in Hmr+ dosage accounts in part for the differential viability of male and female hybrid progeny, but also that additional, unidentified genes must be invoked to account for the invariant lethality of hybrid sons of D. melanogaster mothers. Implications of our findings for understanding Haldane's rule—the observation that hybrid breakdown is often specific to the heterogametic sex—are also discussed.


THE sterility and lethality of species hybrids is a defining characteristic of species (MAYR 1942 Down), but little is known about why hybrids are unfit or what allelic changes are responsible (WU and PALOPOLI 1994 Down; COYNE and ORR 1998 Down). Without such information, it is not possible to determine whether there are general patterns among the genes and alleles that cause hybrid breakdown or to understand the evolutionary forces that lead to allelic divergence between species.

Lack of progress cannot be attributed to the lack of a model for explaining hybrid breakdown. DOBZHANSKY 1937 Down and MULLER 1940 Down proposed that hybrid breakdown results from interactions between alleles that have evolved independently in the parental species. This theory remains compelling because of its simplicity and generality, but the supporting evidence is largely indirect (COYNE and ORR 1998 Down). The lack of more direct evidence is due to the great difficulties in finding species groups that both display hybrid breakdown and are amenable to the identification and experimental manipulation of incompatibility alleles.

When STURTEVANT 1919 Down discovered Drosophila simulans and its close relationship to D. melanogaster, he quickly realized its potential for investigating questions of species divergence (PROVINE 1991 Down). D. simulans is now known to form a three-member clade with D. mauritiana and D. sechellia; we refer collectively to these three species as the "siblings" of D. melanogaster. Hybrids between D. melanogaster and its sibling species generally show the same pattern of viability as described for D. melanogaster/D. simulans hybrids (STURTEVANT 1920 Down; reviewed in ASHBURNER 1989 Down; SAWAMURA et al. 1993B Down; HUTTER 1997 Down; SAWAMURA 2000 Down). D. melanogaster females crossed to sibling species males produce viable but sterile hybrid daughters and lethal sons, while hybrid progeny of sibling species mothers include viable but sterile sons and poorly viable daughters. These sibling species are more closely related to one another than to D. melanogaster because they produce viable hybrids of both sexes, with the daughters being fertile; their greater evolutionary distance from D. melanogaster is also supported by cytological and molecular data (LEMEUNIER et al. 1986 Down; CACCONE et al. 1996 Down). The genetics of hybrid breakdown among the sibling species has been characterized extensively (HOLLOCHER and WU 1996 Down; TRUE et al. 1996 Down; JOLY et al. 1997 Down; MASIDE et al. 1998 Down; TING et al. 1998 Down).

The complete sterility of D. melanogaster hybrids has been the primary obstacle to identifying the genes that distinguish D. melanogaster from its siblings. The recent discovery of D. simulans strains that produce fertile F1 female hybrids with D. melanogaster provides reason for optimism (DAVIS et al. 1996 Down), but it remains to be determined whether hybrid incompatibility genes can be identified in D. melanogaster by backcross analysis or introgression (WU and PALOPOLI 1994 Down), let alone by direct selection through mutagenesis. Researchers have therefore searched natural populations or laboratory stocks for alleles that suppress the inviability of F1 hybrids. Three mutations that rescue lethal hybrid sons of D. melanogaster mothers have been discovered: D. simulans Lethal hybrid rescue (Lhr; WATANABE 1979 Down) and D. melanogaster Hybrid male rescue (Hmr) and In(1)AB (HUTTER and ASHBURNER 1987 Down; HUTTER et al. 1990 Down). Two mutations that rescue subviable daughters from the reciprocal cross to sibling species females have also been discovered: D. simulans maternal hybrid rescue (mhr; SAWAMURA et al. 1993A Down) and D. melanogaster Zygotic hybrid rescue (Zhr; SAWAMURA et al. 1993C Down). The existence of these distinct sets of rescuing mutations suggests that two independent mechanisms of lethality exist in D. melanogaster hybrids (SAWAMURA et al. 1993B Down).

It is not unreasonable to suppose that these rescue mutations are alleles of genes that actually cause hybrid lethality, but it is also possible that they are mutations that suppress lethal interactions between other unknown genes (COYNE 1992 Down; WU and PALOPOLI 1994 Down). Distinguishing between these possibilities requires the ability to manipulate the wild-type alleles of the rescue mutations in hybrids. This has been convincingly achieved only for the Zhr locus. Zhr+ appears to cause hybrid lethality, because deletions of the locus mimic Zhr rescue activity, while Zhr+ duplications reduce hybrid viability (SAWAMURA and YAMAMOTO 1993 Down). Less is known about Hmr. HUTTER et al. 1990 Down showed that an Hmr+ duplication suppresses Hmr-dependent male rescue, but it is unclear whether Hmr+ itself is deleterious to hybrids. Because Hmr is X linked, deletions of the Hmr region are lethal to hemizygous males and therefore cannot be assayed in hybrid males. This limitation, together with the absence of an Hmr phenotype within D. melanogaster, has impeded the characterization and isolation of Hmr.

We have looked, therefore, for possible phenotypes of Hmr in hybrid females. It has long been known that D. melanogaster/D. simulans hybrid females are fully viable only at low temperatures (STURTEVANT 1929 Down; KERKIS 1933B Down). We have found that high-temperature lethality is even stronger in D. sechellia hybrids and, more importantly, that it is suppressed by Lhr, In(1)AB, Hmr, and by deletions that we define here as being Hmr-. We have used the suppression of high-temperature female lethality, as well as other assays, to investigate in greater detail the relationship between Hmr and hybrid viability. Our results suggest that Hmr+ gene dosage is a major factor in determining the viability of D. melanogaster interspecific hybrids.


*  MATERIALS AND METHODS
*TOP
*ABSTRACT
*MATERIALS AND METHODS
*RESULTS
*DISCUSSION
*LITERATURE CITED

Culture conditions:
All crosses were done at 25°. Progeny were collected for 1 or 2 days; after removing the parents, cultures were immediately shifted to the temperatures indicated in each table, with the following two exceptions. Crosses with D. simulans mhr mothers (see Table 9 and Table 10) were kept at 25° for ~24 hr after removing the parents and then shifted to the appropriate temperature. Progeny for the temperature-shift experiments shown in Fig 4 were collected at 25° for 6–9 hr (29° to 18° shifts) or 12–14 hr (18° to 29° shifts); shorter collections were used for the 29 to 18° shifts because we found the viability of these cultures to be particularly sensitive to overcrowding. After removing the parents, cultures were placed immediately at the appropriate starting temperature and then shifted at the times indicated in Fig 4.




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Figure 1. Schematic of viability comparisons performed in this study. Genotypes within each section are hybrid siblings (with the exception of section F, see below). D. melanogaster chromosomes are represented as thin lines, and sibling species chromosomes as thick lines. Only those chromosomes relevant to each experiment are shown; the X, Y, and autosomes are indicated as rod-shaped acrocentric, J-shaped submetacentric, and metacentric chromosomes, respectively. (A) Comparison of female hybrids heterozygous for deletions in the 9D region [or for Hmr1 or In(1)AB; left] with wild-type females (right) to assay aberrations and rescue mutations for dominant suppression of female lethality (Table 3 and Table 4). (B) Comparison of female hybrids heterozygous for Hmr1 (left) with females heterozygous for In(1)AB or for Hmr- deletions (right) to determine whether Hmr1 is a null mutation (Table 5). (C) Comparison of matroclinous exceptional female hybrids (right) with sibling females of the same genotypes as in A. See Table 6 and Fig 6 for details of the method used to generate these hybrids. (D) Comparison of females carrying a duplication of Hmr+ (left) with wild-type female siblings (right) to determine whether additional doses of Hmr+ reduce hybrid viability (Table 7B and Table 7C). (E) Comparison of Xsib male progeny of C(1)mel mothers to determine whether a duplication of Hmr+ reduces hybrid male viability and whether the deleterious effect of Hmr+ occurs even in the absence of Xmel (Table 8). (F and G) Crosses to determine whether Hmr-dependent lethality occurs in progeny of D. simulans mothers. (F) Comparison of females heterozygous for Hmr1 (top left) with wild-type females (bottom left). Viabilities of these females were determined in separate crosses, relative to their Xsim brothers (right; see Table 9). (G) Comparison of females heterozygous for Hmr- deletions (top right) relative to siblings with a wild-type dosage of Hmr+ (top left). This cross (see Table 10) also allows the comparison of Xsim males carrying a duplication of Hmr+ (bottom left), relative to nonduplication brothers (bottom right).



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Figure 2. Hybrid daughters from the cross of D. melanogaster Df(1)N110/FM6 females to D. simulans ryi83 males at 25°. (A) A Df(1)N110, Hmr-/Xsim animal. (B) An FM6, Hmr+/Xsim sibling. Note the rough eyes, necrotic tissue patches, and malformed wings. At least one such necrotic patch was observed in 93% (n = 115) of FM6/Xsim females that eclosed, with none observed in Df(1) N110/Xsim siblings (n = 182).



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Figure 3. Genetic map of the Hmr region. Duplicated regions are indicated by solid, thick lines and deleted regions are represented by dashed lines. Presence or absence of Hmr+ was determined from Table 3 for deficiencies, and from Table 7 and HUTTER et al. 1990 Down for duplications. Breakpoints relative to sesB/Ant2 and other genes shown are from ZHANG et al. 1999 Down and references therein.



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Figure 4. Viability of female D. melanogaster/D. simulans hybrids shifted between 18° and 29° during development. Percentage viability was calculated as the number of FM6/Xsim hybrids relative to Df(1)N110/Xsim siblings, from the cross of D. melanogaster Df(1)N110/FM6 females to D. simulans ryi83 males. Cultures were shifted from 18° to 29° (open circles) or 29° to 18° (solid circles) at the times indicated; the curves were drawn by hand. Developmental times correspond to Df(1)N110/Xsim hybrids; FM6/Xsim hybrids grown at 29° were delayed in development by ~1–2 days. A minimum of 183 Df(1)N110/Xsim animals were scored for each data point (mean 372). See MATERIALS AND METHODS for further experimental details.


 
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Table 1. Temperature-dependent viability of hybrids from wild-type and marker stocks


 
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Table 2. Mapping suppression by Hmr1 of male and female hybrid lethality, relative to a 9D marker


 
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Table 3. Assaying Hmr1 and 9D/9E aberrations for suppression of hybrid female lethality


 
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Table 4. Quantification of postpupal lethality in hybrid females


 
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Table 5. Direct comparison of Hmr1, In(1)AB, and Hmr- deletions for suppression of female lethality in D. sechellia hybrids


 
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Table 6. Hmr- deletions partially suppress lethality of exceptional female hybrids


 
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Table 7. An Hmr+ duplication reduces hybrid female viability and interacts with Lhr


 
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Table 8. An Hmr+ duplication reduces the viability of Xsib/Ymel hybrid sons of compound-Xmel mothers


 
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Table 9. Hmr1 suppresses postembryonic lethality in mhr-rescued hybrid females


 
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Table 10. Hmr- deficiencies suppress postembryonic lethality in mhr-rescued hybrid females

For some experiments it was informative to count and score dead pharate and eclosed adults. We defined pharate adults as those stages where sex and eye color (w or wa vs. w+) could be scored easily; this corresponds approximately to stage P10 of BAINBRIDGE and BOWNES 1981 Down(cited in ASHBURNER 1989 Down, pp. 181–187) until eclosion.

Stocks:
Sibling species marker stocks were from the Drosophila Species Center (DSC, Bowling Green, OH, or Cambridge, United Kingdom) stock collections, with the exception of D. sechellia f, which was obtained from J. Coyne. Wild-type stocks were as follows:

D. melanogaster deficiency and duplication stocks were obtained from the Bloomington or Umeå stock centers. Their breakpoints are shown in Fig 3; we verified the published cytologies (with the exception of Df(1)ras-v17) by analyzing orcein-stained squashes of polytene chromosomes. All D. melanogaster marker mutations and aberrations are described in LINDSLEY and ZIMM 1992 Down and in FLYBASE 1999 Down.

Nomenclature:
Chromosomes from the melanogaster complex species D. melanogaster, D. mauritiana, D. sechellia, and D. simulans are indicated by the subscripts mel, mau, sec, or sim, respectively. The latter three species are referred to collectively as siblings, abbreviated as sib. For clarity we use the designation Hmr1 to refer explicitly to the rescue allele described in HUTTER and ASHBURNER 1987 Down; this remains the only known allele of Hmr.

Experimental design:
Most of the experimental crosses involved comparisons of sibling hybrids of different genotypes with respect to Hmr. A summary diagram is shown in Fig 1.


*  RESULTS
*TOP
*ABSTRACT
*MATERIALS AND METHODS
*RESULTS
*DISCUSSION
*LITERATURE CITED

High-temperature lethality in female hybrids:
D. simulans hybrid daughters from D. melanogaster mothers vary in their viability at 25°, depending on the stocks used (WATANABE et al. 1977 Down; LEE 1978 Down). To investigate whether female hybrids with D. mauritiana and D. sechellia show similar properties, we measured hybrid viability at three temperatures with a small number of stocks from each species (Table 1). We used two D. melanogaster stocks, Oregon-R and Nguruman-4, and found in most cases that hybrids with Oregon-R had viability lower than that of Nguruman-4 hybrids. Quite unintentionally, we used the same Oregon-R stock used previously by LEE 1978 Down to measure viability in D. simulans female hybrids, who also found it to be strongly biased against hybrid viability. We placed hybrids into three classes—viable, dead eclosed, and dead pharate (see MATERIALS AND METHODS). The calculated viability will therefore be an overestimate of the true viability, if there is significant prepharate lethality. This may be the case for D. sechellia hybrids, because a cursory examination of the hybrid cultures often revealed a large number of dead embryos and young larvae; we did not, however, determine the sex of these dead early-stage animals.

Our results with D. simulans hybrids were consistent with previous studies: all female hybrids were at least 78% viable at 18° but varied from fully viable to fully lethal at 25°. Viable escapers at 25° often displayed morphological defects including crinkled wings, rough eyes, and multiple necrotic patches similar to those shown in Fig 2B.

To investigate the genetic basis of the variation among D. simulans stocks, we made reciprocal crosses between a stock that produced fully viable female hybrids with Oregon-R at 25° (v f2) and a second stock (ryi83) that produced lethal female hybrids at 25° and crossed the resulting F1 males to Oregon-R (Table 1B). Female hybrids from both crosses had ~50% viability, suggesting that the difference in hybrid viability between the D. simulans ryi83 and v f2 stocks is caused by an autosomal gene (or genes). This result contrasts with the report of LEE 1978 Down, which implicated the X chromosome as being largely responsible for differences in hybrid viability between D. simulans stocks (based on less direct measurements with different D. simulans stocks). Determining whether or not this discrepancy reflects the existence of distinct systems of hybrid viability modifiers within D. simulans will require more extensive mapping efforts.

All D. simulans hybrids were essentially lethal at 29°, with rare escapers being quite sickly (Table 1B). Intriguingly, the one exception occurred in hybrids between Oregon-R and a stock homozygous for the mutation Lhr (WATANABE 1979 Down). Female hybrids were 85% viable at 29° and showed none of the morphological abnormalities characteristic of hybrid escapers. Male hybrids were poorly rescued at 29° (3 live males vs. 104 females) but fully rescued at 18° and 25°. Several attempts at mating Nguruman-4 females to Lhr males failed.

D. mauritiana hybrid females had significantly higher viabilities than D. simulans hybrids. Of four stocks tested, three produced female hybrids with Oregon-R at both 25° and 29° that were >=89% viable (Table 1A). Hybrids from the fourth stock, Iso 197, however, were only 23 and 20% viable at 29° with Oregon-R and Nguruman-4, respectively. Escapers from these crosses had morphological defects similar to those seen in D. simulans hybrid escapers, albeit at a reduced frequency and intensity. At 18° a small number of pharate males were found in some crosses (see Table 1, footnote a). These animals typically had extreme morphological defects including split and malformed nota and greatly reduced eyes. Because patroclinous males (Xmau) are viable (HUTTER et al. 1990 Down; see also Table 6 below), these malformed pharate males may be nonexceptional hybrid "escapers" (i.e., Xmel). The observation of these rare pharate escapers does not contradict the accepted fact that Xmel/Ymau hybrid males are invariably lethal; they do serve to emphasize a point revealed by the results presented below, namely that hybrid males and females exist on a single Hmr-dependent continuum of viability.

Female hybrids with D. sechellia had much lower viability than D. mauritiana or D. simulans hybrids (Table 1C). Female hybrids at 25° were essentially lethal with all five D. sechellia stocks tested; rare escapers were severely necrotic. Even at 18° female hybrids between two different stocks (v and Iso 24) and Oregon-R were only 10% viable, and many of the surviving adults had rough eyes.

The data presented in Table 1 are derived from a small number of strains and therefore may not be representative of the range of variability within each species. However, the relative designations of D. mauritiana, D. simulans, and D. sechellia female hybrids as having high, intermediate, and low viabilities, respectively, seems to be a justified generalization. We note the striking observation that these qualitative descriptions for each species are the same for the strength of suppression of hybrid male lethality by Hmr1 (HUTTER and ASHBURNER 1987 Down).

Suppression of female lethality by Hmr1:
We began our investigation of temperature-sensitive hybrid female lethality after obtaining the unexpected mapping results shown in Table 2. On the basis of its rescue of male hybrids, Hmr was mapped distally to ras (1-32.41) and estimated to be in cytological region 9D1-9E4 (HUTTER et al. 1990 Down). Here we have mapped Hmr1 relative to a ry+-marked homozygous, viable P element inserted at 9D; this insertion had no effect on hybrid female viability (see Table 2). Hybrids were generated using the D. simulans stock ryi83, which showed strong female lethality effects (Table 1). At 18°, all hybrid males were ry, demonstrating the expected close linkage to 9D, while both ry and ry+ females were obtained in roughly equal proportions. At 27°, no hybrid males survived, a result consistent with the known temperature sensitivity of male rescue (HUTTER and ASHBURNER 1987 Down). Surprisingly, however, only 2 ry+ females survived compared with >1000 ry siblings (which appeared generally wild type in morphology). One of these ry+ females was necrotic and had rough eyes, suggesting that it was an escaper, while the other was wild type in appearance and, thus, may have been either an escaper or a recombinant between Hmr and the marker. At 29° the cultures contained a large number of dead pharate and eclosed females, and the relatively small number of viable females were all ry (data not shown). We propose that the closely linked suppressors of male and female hybrid lethality are both in fact Hmr1.

Suppression by In(1)AB and deficiencies:
In Table 3 we show that In(1)AB also suppressed the lethality of female hybrids with the D. simulans ryi83 stock, by comparing the viability of In(1)AB/Xsim females relative to their FM6/Xsim sisters (Fig 1A). As in Table 1, we have also estimated the viability of In(1)AB/Xsim females by scoring the total number of females that reached pharate adulthood and beyond. At 18° there was little difference in viability between the sibling classes, but at 25° and 29° only In(1)AB/Xsim hybrids survived; they were of normal morphology. We note that a similar cross using Hmr1/FM6 mothers also produced many viable female hybrids at 29°, and these were probably of genotype Hmr1/Xsim, but many had misshapen eyes and, thus, we could not unambiguously distinguish them from their FM6/Xsim (B/B+) siblings (data not shown). Hmr1/Xsim daughters of this cross did appear to be fully viable at 18° and 25° (Table 3).

Because this rescue of hybrid female lethality is dominant, we could determine whether homozygous lethal deletions in the Hmr region have similar rescuing properties. In crosses with 12 different deletions, +/Xsim females generally had similar viabilities relative to Df/Xsim siblings at 18° (Table 3). At 25° and particularly at 29°, however, the crosses fell into two discrete classes, those that included viable Df/Xsim females of normal morphology and lethal +/Xsim siblings and those that produced only occasional highly necrotic escapers of both genotypes (see Fig 2). We define the first class as being Hmr-; these deficiencies all delete cytological region 9D and place Hmr between the distal 9D1 breakpoint of Df(1)ras203, Df(1)B13, and Df(1)ras-v17 and the proximal 9D3-4 breakpoint of Df(1)N110 (Fig 3). The second class of nonrescuing deficiencies does not delete 9D. It is worth mentioning that most of the rescuing deficiencies were generated independently in unrelated screens, including Df(1)ras-v17, which was induced on the balancer chromosome Binsc (LINDSLEY and ZIMM 1992 Down).

Our results are consistent with previous mapping of Hmr to region 9D1-9E4 (HUTTER et al. 1990 Down), as well as mapping based on duplications (see Effects of an Hmr+ duplication in female hybrids below). We also note that there was no correlation between rescue of hybrid female lethality and complementation of sesB (Fig 3), which agrees with the conclusion that Hmr and sesB/Ant2 are distinct loci (ZHANG et al. 1999 Down).

Quantification of pharate and posteclosion lethality:
The viabilities calculated in Table 3 assumed that Hmr-/Xsim and +/Xsim hybrid females are equally viable up to the pharate adult stage, with +/Xsim hybrids dying at high temperature as adults. To test this assumption, we performed crosses where dead hybrid females could be genotyped readily on the basis of whether they had wild-type (red) or white eyes (Table 4). This allowed us to measure both viability within each sibling class as well as relative viability between classes. In crosses to a D. simulans w strain, very few +/Xsim females survived at 29°, in contrast to their In(1)AB/Xsim or Df(1)N110/Xsim siblings. The majority (83 and 78%, respectively) of the absent +/Xsim hybrids, though, could be found among the dead adults.

Similar results were obtained with D. sechellia hybrids at 25°, where >=75% of the relative viability difference was due to pharate adult and posteclosion lethality. At 29°, however, unrescued D. sechellia hybrids suffered from extensive prepharate lethality, as only 23–58% of +/Xsec hybrids reached the pharate adult stage relative to their rescued siblings. In some cases there was also significant lethality within the rescued class. For example, only 27% of the Df(1)N110/Xsec hybrids that reached the pharate adult stage were viable (Table 4C). These data suggest that D. sechellia female hybrids suffer from both Hmr-dependent and Hmr-independent lethality at high temperatures.

All five genotypes shown in Table 4 were crossed to D. mauritiana w males at 18°, 25°, and 29° (data not shown). No significant viability effects were found within or between sibling classes. At 29° the +/Xmau hybrids were >=82% viable relative to reference siblings, and the maximum pharate/posteclosion lethality was 24%.

The lethal phase of hybrid females:
Hybrid females die predominantly as pharate adults or after eclosion at high temperatures, but the time of death does not reveal at what stage(s) development is disrupted. We therefore performed reciprocal temperature shifts of unrescued FM6/Xsim D. melanogaster/D. simulans hybrid females and compared their viability relative to Df(1)-N110/Xsim siblings (Fig 4). FM6/Xsim hybrids grown at 18° until approximately the mid-third instar larval stage (L3) and then shifted to 29° had high viability, while cultures shifted before L3 were poorly viable or lethal. There was little apparent difference between siblings in time of development, even in crosses where FM6/Xsim females were poorly viable. Escaper females often had rough eyes and necrotic leg patches, as seen in nontemperature shifted escapers (Fig 2B).

In the reciprocal shift from 29° to 18°, however, FM6/Xsim females were delayed in development by ~1–2 days relative to their Df(1)N110/Xsim siblings. We did not determine the precise phase of this developmental delay, but it was apparent in cultures shifted from 29° at 76 hr after egg laying (AEL), where FM6/Xsim hybrids were fully viable (but often had rough eyes). In cultures shifted from 29° at 96 hr AEL or later, the FM6/Xsim hybrids that eclosed first often had extreme morphological defects, including severely misshapen eyes, missing ocelli, and disarrayed notal microchaetes. Their wings were typically normal in length but reduced in width, with absent or incomplete crossveins (Fig 5B); STURTEVANT 1920 Down also noted crossvein defects in hybrid females (that were not temperature shifted). Later eclosing FM6/Xsim females from the same cultures generally were more normal in morphology.



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Figure 5. Wings from D. melanogaster/D. simulans hybrid females grown at 29° for 96–104 hr and then shifted to 18° as described in Fig 4. (A) Wing from a Df(1)N110/Xsim animal. (B) Wing from an FM6/Xsim animal. Note the missing posterior crossvein, incomplete longitudinal veins, and substantial reduction in width. Wings from less severely affected animals ranged from having incomplete posterior crossveins but normal shape to being comparable to Df(1)N110/Xsim siblings as in A.

These results suggest that culture at high temperature causes a general delay and disruption of development beginning in L2 or early L3 larvae that can be alleviated by transfer to low temperature, with the severity of lethality and morphological defects depending on how far development proceeds before the temperature shift. Both the general time course of viability and the developmental delay at high temperature are comparable to that observed for Hmr1-dependent rescue of D. melanogaster/D. mauritiana hybrid males (HUTTER and ASHBURNER 1987 Down).

Comparison of Hmr1, In(1)AB, and deficiencies:
In crosses to the D. simulans ryi83 stock described in Table 2 and Table 3, Hmr1 only weakly suppressed the lethality of female hybrids at 29° (data not shown), but Hmr- deficiencies fully suppressed lethality at 29° (Table 3). In contrast, the crosses with the D. sechellia w stock in Table 4 showed little difference between Hmr1 and deficiencies for high-temperature rescue. These discrepancies could reflect a difference between hybrid rescue in D. simulans and D. sechellia or could merely be a consequence of comparing results from different genetic backgrounds. To determine more directly whether or not rescue by Hmr1 and deletions are equivalent, we compared the viabilities of hybrids heterozygous for Hmr1, In(1)AB, and Hmr- deficiencies as sibling progeny of the same mothers (Table 5; Fig 1B). Hybrids were made with the D. sechellia v stock, which produced poorly viable hybrids with Oregon-R (Table 1). At 25° all pairwise comparisons had similar viabilities. At 29°, Hmr1/Xsec hybrids were ~50% viable compared to Df/Xsec siblings, and many had wing defects (Table 5A and Table 5B). Hmr1/Xsec hybrids were only 9% viable compared to In(1)AB/Xsec siblings; this cross used a different Hmr1 stock than the deficiency-containing crosses (Table 5C). In(1)AB/Xsec and Df(1)v-L11/Xsec hybrids were equally viable at 29°.

We conclude that Hmr1 is a somewhat weaker dominant suppressor of hybrid female lethality than In(1)AB or deficiencies. It is important to note, however, that the partial rescue by Hmr1 of D. melanogaster/D. sechellia female hybrids at 29° stands in marked contrast to its rescue of D. sechellia hybrid males, which is low at 18° and absent at higher temperatures (HUTTER and ASHBURNER 1987 Down; our unpublished data).

Dominant rescue of exceptional female hybrids:
D. mauritiana and D. simulans hybrid females carrying two Xmel chromosomes are lethal (STURTEVANT 1920 Down; BIDDLE 1932 Down; KERKIS 1933A Down; HUTTER et al. 1990 Down), but can be rescued by Lhr (TAKAMURA and WATANABE 1980 Down) or by Hmr1 and In(1)AB (HUTTER et al. 1990 Down). In several crosses with Hmr- deficiencies, we observed occasional matroclinous exceptional hybrids (Xmel, Hmr-/Xmel, Hmr+; see footnotes d–f in Table 3). To generate exceptional hybrid females at high frequency, we took advantage of the fact that females carrying a normal sequence X chromosome, an inverted X chromosome, and a Y chromosome produce X-X nondisjunctional progeny at much greater frequencies compared to the wild type (STURTEVANT and BEADLE 1936 Down). Besides being less laborious than constructing compound chromosomes, this technique allowed us to compare the viabilities of exceptional and nonexceptional sibling progeny from a single cross (Fig 1C); the expected progeny of such crosses are shown in Fig 6.



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Figure 6. Expected progeny from a hybrid cross of Df(1)Hmr-/Xmel/Ymel females and Xsib/Ysib males. To reflect their similarity to the products of nondisjunction typically observed in XX females, we refer to the products of maternal X-Y nondisjunction (a and b) as regular or nonexceptional progeny and the products of maternal X-X nondisjunction (c) as exceptional progeny. Note that the frequency of X-X nondisjunctional maternal gametes varies depending on the specific stocks used, as shown by control crosses to D. melanogaster males in Table 6. Rare progeny of XXY eggs or XY sperm are not shown. Viability designations are as follows: ++, viable; +, semiviable due to Hmr+-dependent hybrid lethality; – –, lethal due to aneuploidy; –, lethal due to Hmr+-dependent hybrid lethality; ?, the experimental class. The +, –, and ? classes are viable in control crosses to D. melanogaster males. Crosses were also performed with In(1)AB instead of Df(1)Hmr-; hybrid sons carrying In(1)AB are semiviable. Df(1)Hmr-/Xmel/Xmau metafemale (3X; 2A) hybrids with D. mauritiana also appear to be semiviable (see Table 6, footnote b).

We constructed marked-Y stocks with In(1)AB/FM7, Df(1)ras203/FM7, and Df(1)N110/FM4 (see Table 6 for complete genotypes). Because Df(1)ras-v17 is present on the balancer chromosome Binsc, we constructed a marked-Y stock with Df(1)ras-v17 and the normal sequence X chromosome y v f. Control crosses with D. melanogaster males demonstrated the success of this technique in generating exceptional progeny. The In(1)AB, Df(1)ras203, and Df(1)ras-v17 marked-Y stocks produced exceptional females at between 57 and 72% the rate of nonexceptional In(1)AB/+ or Df/+ siblings (Table 6A, Table 6C, and Table 6D), while exceptional females from the Df(1)N110 stock were twice as frequent as nonexceptional siblings (Table 6B). Because these frequencies varied among the different stocks, the viabilities of exceptional female hybrids relative to regular siblings must be evaluated in comparison to the intraspecific control for each cross.

One caveat in the interpretation of these crosses is that Ymel or its markers might have effects on hybrid viability unrelated to Hmr. The Dp(1;Y)Bs chromosome present in crosses with Df(1)N110/FM4 could be scored in all progeny (Table 6B). Hybrid females with this Ymel were >=65% viable relative to siblings without it (e.g., 106 Df(1)N110/FM4/Dp(1;Y)Bs vs. 162 Df(1)N110/FM4, in the cross to D. simulans v f2 at 18°); the high viability of exceptional Xsib/Ymel sons also suggests that this marked Y chromosome did not have significant viability effects. Dp(1;Y), y+ and especially Dp(1;Y), y+ v+ present in the Df(1)ras-v17/y v f stock (Table 6D) did appear to reduce Xsib/Ymel viability compared to the intraspecific controls, presumably due to the duplicated material. Such effects should be less severe in females (because of the absence of dosage compensation), and only one-half of the nonexceptional females will carry Ymel; nonetheless, any Ymel-induced viability reduction of these females would increase inappropriately the relative viabilities calculated for exceptional females.

The partial rescue of In(1)AB/FM7 D. mauritiana female hybrids at 18° confirms the discovery of HUTTER et al. 1990 Down, who used a compound-X chromosome. In the cross to D. mauritiana Iso 197 males at 25°, these hybrids were fully rescued (Table 6A). In(1)AB/FM7 hybrids were 71.0% viable relative to their In(1)AB/Xmau sisters, which is essentially identical to the 71.6% viability of In(1)AB/FM7 females relative to their In(1)AB/Xmel sisters in the intraspecific control. At 29° exceptional female hybrids with Iso 197 were partially rescued; most (10 of 11 scored) had rough eyes but were not necrotic, while 11 of 38 of their FM7/Xmau sisters had necrotic thoracic patches (but were fully viable relative to In(1)AB/Xmau siblings). Surprisingly, the In(1)AB/FM7 exceptional females appeared to have equivalent or greater viability at 25° and 29° than In(1)AB/Ymau sibling males.

All three deficiencies also produced exceptional female hybrids with D. mauritiana; in some cases relative viability again approached that seen in intraspecific controls. The relative viability of exceptional females could not be precisely measured in crosses with Df(1)N110/FM4/Dp(1;Y)Bs (see Table 6, footnote a), but we believe the higher estimated limits shown in Table 6B for D. mauritiana hybrids are more accurate for two reasons: first, the nonexceptional hybrids are likely to be half Df(1)N110/Xmau and half FM4/Xmau on the basis of results with other deficiencies in Table 6 and other results described above, and second, the higher estimates are more consistent with the number of exceptional males observed.

Several unexpected features concerning rescue of exceptional D. simulans female hybrids deserve comment. First, viability at 25° was always similar to or even higher than viability at 18°, which is the opposite temperature profile consistently observed for nonexceptional Xmel/Xsib hybrids throughout this study. This phenomenon is most clearly demonstrated in the cross of Df(1)ras203/FM7/Dp(1;Y), y+ to D. simulans ryi83 males (Table 6C). The exceptional Df(1)ras203/FM7 females were not only significantly more viable at 25° than at 18°, but they were also more viable than their nonexceptional FM7/Xsim sisters. The same appeared to be true for hybrids between Df(1)N110/FM4 and D. simulans ryi83 (Table 6B). For this cross, we suspect that the higher relative viability estimate of exceptional females is more accurate at 18° (29%) and the lower is more accurate at 25° (55%) and 29° (4%), for reasons analogous to those noted above for the D. mauritiana crosses. An independent estimate of viability at 25° was provided by using the ectopic mesopleural hair phenotype associated with the sc8 marker of FM4. This phenotype was 50% penetrant in Df(1)N110/FM4 exceptional females and was observed in 8% of the nonexceptional females, allowing us to estimate that 16% of the Xmel/Xsim females in this cross were FM4/Xsim. We therefore estimate that the 238 total nonexceptional females included 38 (16%) FM4/Xsim and 200 (84%) Df(1)N110/Xsim; the viability of Df(1)N110/FM4 exceptions relative to Df(1)N110/Xsim would then be 65.5% (131/200).

A second intriguing result involves the genetic background difference between the ryi83 and v f2 D. simulans stocks. In these crosses and others (Table 1), the ryi83 stock caused much greater lethality to Xmel/Xsim hybrids at 25° and 29° than the v f2 stock; recall that the apparent variation appeared to be entirely autosomal (Table 1B). Yet both Df(1)N110/FM4 and Df(1)ras203/FM7 exceptional hybrids were more viable at 25° with D. simulans ryi83 than with the v f2 stock. These dissimilar genetic background and temperature effects on Xmel/Xmel vs. Xmel/Xsim hybrids suggest that X-linked factors other than Hmr may influence hybrid viability (see DISCUSSION).

No exceptional female D. sechellia hybrids were recovered at either 18° or 25° with Df(1)N110 (crossed to D. sechellia v and f stocks) or with Df(1)ras203 and Df(1)ras-v17 crossed to the D. sechellia w stock (data not shown). We also generated exceptional females heterozygous for Hmr1 with a stock of y Hmr1 v/FM7/Dp(1;Y), y+. A high frequency of exceptional females was produced in control crosses to D. melanogaster males, but none were observed in crosses to D. mauritiana w or Iso 197 males at either 18° or 25° (data not shown). This negative result is consistent with the data of HUTTER et al. 1990 Down, who found that Hmr1 only rescues compound-Xmel hybrid females with D. mauritiana when homozygous.

Effects of an Hmr+ duplication in female hybrids:
The above results show that Hmr1 and deficiencies in the 9D region are qualitatively equivalent in suppressing hybrid lethality and thus imply that the wild-type Hmr+ product is deleterious to hybrids. We therefore tested whether increasing the dosage of Hmr+ would decrease the viability of hybrids, using the Hmr+ duplication Dp(1;2)v+75d (Table 7).

HUTTER et al. 1990 Down showed that Dp(1;2)v+75d causes a modest decrease in viability of D. mauritiana female hybrids at 18° (as well as a developmental delay). In Table 7 we have extended this analysis to all three sibling species at a range of temperatures. We first did a series of control crosses to generate Df(1)HC133/Xsib; Dp(1;2)v+75d/2sib and Df(1)HC133/Xsib; 2mel/2sib sibling female hybrids (Table 7A). If Hmr is the only gene affecting hybrid viability within these aneuploid segments, then the hybrids should be equivalent in viability to +/Xsib and Hmr-/Xsib hybrids, respectively, and display the same temperature-sensitive viability profile described above (Table 1, Table 3, Table 4, and Table 6). The control crosses suggest that this assumption is correct: Df(1)HC133/Xsib; Dp(1;2)v+75d/2sib females had high viability at all temperatures in D. mauritiana hybrids, but reduced viability at 29° in D. simulans hybrids and at 25° in D. sechellia hybrids. Lhr suppressed female lethality but failed to rescue males at 29°, as was also observed earlier (Table 1B).

We used two different stocks to assay the effect of Dp(1;2)v+75d in the presence of an Hmr+ Xmel (diagrammed in Fig 1D). The second stock used (Table 7C) generally produced stronger lethal effects than the first (Table 7B), but qualitatively, the results were similar. At 25°, Dp(1;2)v+75d reduced the viability of D. mauritiana hybrids and, more strongly, that of D. simulans hybrids. D. sechellia female hybrids carrying Dp(1;2)v+75d were lethal at both 18° and 25° (Table 7B).

The one notable exception involved the D. simulans Lhr stock (Table 7B and Table 7C). Dp(1;2)v+75d had no significant effect on female viability in crosses to Lhr males, suggesting that Lhr suppressed the deleterious effect of duplicating Hmr+. The opposite effect was observed in male progeny, where Dp(1;2)v+75d appeared to strongly suppress the rescue activity of Lhr. Duplication-containing sons were <=7% viable relative to nonduplication brothers at all temperatures. These reciprocal effects support a model where the Hmr+ and Lhr+ loci form a pair of interacting genes that causes hybrid lethality (see DISCUSSION).

The distal breakpoint of Dp(1;2)v+63i is at 9E, which is very close to the proximal limit of the Hmr region defined by deficiencies (Fig 3). HUTTER et al. 1990 Down suggested that this duplication does not carry Hmr+, but the sole evidence was the absence of effects in D. mauritiana hybrid females at low temperature. In contrast to the suppression by Dp(1;2)v+75d described above, we found that Dp(1;2)v+63i did not suppress rescue of hybrid males by Lhr (Table 7D). It also had little effect on male rescue by Hmr1, even at 25°, where nonduplication males were only 12% viable (relative to their nonduplication sisters; Table 7E). We also conclude that Dp(1;2)v+63i does not carry Hmr+.

Effects of an Hmr+ duplication in male hybrids:
The results described above show that an extra copy of Hmr+ reduces the viability of Xmel/Xsib D. simulans and D. mauritiana hybrid females, but does not cause unconditional lethality. Yet Xmel/Ysib hybrid males, which have an equivalent Hmr+ dosage, are invariably lethal. Does this discrepancy reflect a sex-specific effect of Hmr+, or the fact that hybrid males are hemizygous for all Xmel genes, including Hmr+? One way to address this question is to measure the effect of the Hmr+ duplication on Xsib/Ymel hybrids derived from compound-X D. melanogaster mothers. Assuming that the duplication is fully dosage compensated, these males will have the same dosage of Hmr+ as Xmel hybrid males. In Table 8 we have compared the viability of Xsib/Ymel; Dp(1;2)v+75d, Hmr+/2sib hybrids with their nonduplication-carrying brothers (diagrammed in Fig 1E).

Control crosses with D. melanogaster showed that nonhybrid males heterozygous for Dp(1;2)v+75d have reduced viability; this result was not unexpected considering the large size of the duplication. Xsim/Ymel; Dp(1;2)v+75d/2sim hybrid males showed little reduction in viability at 18° and 25° but were essentially lethal at 29°. Xsec/Ymel; Dp(1;2)v+75d/2sec males were completely lethal at both 18° and 25°; scoring of dead animals showed that most of the lethality must have occurred before the pharate adult stage (Table 8, footnotes d and e). Attempts to make hybrids with a D. mauritiana v stock failed to produce any progeny. The results with D. simulans hybrids in Table 7 and Table 8 show that Hmr+ reduces the viability of both male and female hybrids, but also that unconditional lethality requires hemizygosity of Xmel. While a male-like dosage of Hmr+ appears to be sufficient to kill both male and female D. sechellia hybrids, other results suggest that additional Xmel genes also influence viability (see Dose dependence of Hmr+ in DISCUSSION).

Dominant effects of Hmr+ in hybrids from D. simulans mothers:
The reciprocal cross of D. simulans females to D. melanogaster males produces viable sons but poorly viable daughters. The lethality of these female hybrids is embryonic and can be rescued by the D. melanogaster mutation Zhr and the D. simulans mutation mhr, but not by Hmr1. These rescued hybrids are also sensitive to pupal and posteclosion lethality at 23°, but not at 18° (SAWAMURA et al. 1993A Down, SAWAMURA et al. 1993C Down). Because this late lethality seemed similar to the Hmr-dependent lethality of female hybrids from D. melanogaster mothers that we have described above, we decided to investigate further the potential role of Hmr in hybrids derived from D. simulans mothers.

The viability of female hybrids from D. simulans mothers is highly dependent on genetic background variation (SAWAMURA and YAMAMOTO 1993 Down; SAWAMURA et al. 1993A Down; DAVIS et al. 1996 Down; ORR 1996 Down), which complicates the effort to determine whether Hmr might influence pupal but not embryonic lethality in this cross. We therefore performed two different schemes of parallel crosses to mhr females using Hmr1 and Hmr+ sibling brothers (Table 9; diagrammed in Fig 1F). The first scheme utilized the close linkage of Hmr and v (<2 cM; HUTTER et al. 1990 Down) to distinguish Hmr1 and Hmr+ males (Table 9A). When postembryonic cultures were grown at 18°, females derived from Hmr+ and Hmr1 fathers were 44 and 32% viable, respectively, relative to their Xsim/Ymel brothers. At 25°, hybrid daughters from Hmr1 fathers had similar viability (29%), but those from Hmr+ fathers were essentially lethal (<2% viability). When dead pharate and eclosed adults (many of which were necrotic) are included, the proportion of females in the latter cross rose to 18%, indicating that much of the lethality was postpupal stage (Table 9A, footnotes c and d).

A second crossing scheme (Table 9B) used a homozygous viable w+ P element inserted in 9E to distinguish Hmr+ from Hmr1 males. Female viability was lower than in scheme A and there was also substantial variation among different cultures of identical genotypes. For example, female viability in crosses from Hmr1 fathers ranged from 3 to 10% at 25°. As in scheme A, however, temperature-dependent late lethality of female hybrids was observed and only in female progeny of Hmr+ sons. At 25° these hybrids were essentially lethal with ~12% of the females dying as pharate adults or posteclosion (Table 9B, footnotes e and f).

Effects of Hmr- deletions:
We also assayed two Hmr- deficiencies for their ability to suppress late lethality in daughters of mhr mothers. Xsim/Df(1)Hmr-; 2sim/2mel females were compared with their Xsim/Df(1)Hmr-; 2sim/Dp(1;2)v+75d, Hmr+ siblings (Table 10, diagrammed in Fig 1G). At 25°, Xsim/Df(1)N110; 2sim/Dp(1;2)v+75d females were lethal, while Xsim/Df(1)HC133; 2sim/Dp(1;2)v+75d females were nearly lethal at 29° and had reduced viability at 25° compared to their nonduplication siblings. Although dead animals were not genotyped for whether or not they carried the duplication, the number of dead females in each cross was in approximate correspondence to the expected number of missing Xsim/Df(1)Hmr-; 2sim/Dp(1;2)v+75d females. In combination with the data in Table 9, the results of Table 10 suggest that Hmr1 and Hmr- deficiencies are dominant suppressors of late lethality in female hybrids of D. simulans mothers and D. melanogaster fathers.

Xsim/Ymel; 2sim/Dp(1;2)v+75d sons of mhr mothers also were less viable than their nonduplication brothers at 25°, with the number of dead animals again suggesting that the missing duplication-carrying males were dying after the pupal stage (Table 10). Together with the results of Table 8, these data suggest that Xsib males are sensitive to Hmr+ dosage, regardless of the direction of crossing. Deleterious effects of Dp(1;2)v+75d on hybrid male viability have also been observed independently by H. A. ORR and S. IRVING (personal communication).


*  DISCUSSION
*TOP
*ABSTRACT
*MATERIALS AND METHODS
*RESULTS
*DISCUSSION
*LITERATURE CITED

Temperature-sensitive pupal lethality in hybrids:
Hybrid Xmel sons and Xmel/Xmel daughters of D. melanogaster mothers die as larvae or pseudopupae (STURTEVANT 1920 Down, STURTEVANT 1929 Down; HUTTER et al. 1990 Down), and much effort has been made to understand the genetic and developmental basis of this lethality. Less is known about the temperature-sensitive lethality of Xmel/Xsib females, first noted by STURTEVANT 1929 Down in D. melanogaster/D. simulans hybrids. After SAWAMURA et al. 1993B Down, we refer to the lethality of Xmel male and Xmel/Xmel female hybrids as larval lethality and that of Xmel/Xsib females as pupal lethality (although many females in fact survive until eclosion). Investigating this female lethality can potentially overcome the limitations associated with assaying X-linked alleles in hemizygous males.

We quantified female viability in hybrids between D. melanogaster and its three sibling species at three temperatures (Table 1). D. mauritiana hybrids had the highest viability, followed by D. simulans and finally by D. sechellia hybrids, which were not fully viable even at 18°. The genetic basis of these species-specific differences in hybrid viability is unknown. Although F1 hybrid males of the sibling species are sterile, interspecific heterozygous introgressions created by repeated backcrossing of hybrid females are often male fertile (HOLLOCHER and WU 1996 Down; TRUE et al. 1996 Down). Such introgressions could be used to map the genetic differences responsible for this variation in hybrid viability.

Even with the small number of strains sampled, there was substantial variation in hybrid lethality among different stocks of each species, as observed in previous studies of D. melanogaster/D. simulans hybrids (WATANABE et al. 1977 Down; LEE 1978 Down). While it appears from Table 1 that variability within D. mauritiana and D. sechellia is somewhat less than that observed among D. simulans stocks, this result may merely reflect the fact that D. melanogaster/D. simulans viability falls in the middle of the phenotypic range detectable by our assay. Alternatively, the insular species D. mauritiana and D. sechellia may in fact harbor less variation for hybrid lethality than the cosmopolitan D. simulans.

The substantial genetic background variation observed can complicate the analysis of hybrid viability to the point where meaningful conclusions about particular genotypes cannot be easily reached from any single cross. On a more positive note, however, understanding the evolutionary forces responsible for the origin and maintenance of this type of variation is relevant to understanding the process of speciation. Several other studies have identified intraspecific variation for traits that cause hybrid breakdown and reproductive isolation. WADE et al. 1997 Down discovered substantial population-level variation in beetles for inviability and morphological defects of interspecific hybrids, while TAKANO 1998 Down has found that loss of macrochaetes in D. melanogaster/D. simulans hybrids is highly dependent on variation within D. simulans stocks.

The wild-type Hmr+ causes hybrid lethality:
One important question raised by the discovery of rescue alleles such as Lhr and Hmr1 is whether the wild-type allele of the rescue gene causes hybrid lethality. Addressing this requires the ability to manipulate the wild-type gene in hybrids. We have done so for Hmr and found that Hmr- deficiencies and an Hmr+ duplication have reciprocal effects on hybrid viability. The qualitatively similar activities of Hmr1 and Hmr- deficiencies further suggest that Hmr1 rescues hybrids by reducing the level of Hmr+, as proposed by HUTTER et al. 1990 Down. In other words, hybrid rescue does not require a mutation that switches Hmr+mel to an Hmr+sib-like allele.

Two previous studies failed to detect any effect on hybrid female viability of deletions that we have defined here as being Hmr-. This discrepancy probably reflects the fact that viability was assayed under conditions less stringent than used in this study: HUTTER et al. 1990 Down looked only in D. mauritiana hybrids while COYNE et al. 1998 Down examined D. simulans hybrids at 24°.

On the basis of his pioneering analysis of D. melanogaster/D. simulans hybrids, STURTEVANT 1929 Down proposed that hybrid lethality is caused by either the presence of the D. simulans Y chromosome or the absence of the D. simulans X chromosome. The first hypothesis was ruled out by YAMAMOTO 1992 Down, who used a D. simulans C(1;Y) chromosome to generate Xmel/O hybrid males and found that they remain inviable. So is Xsim (and more generally Xsib) required for hybrid viability? Although not explicitly stated by Sturtevant, his second hypothesis, that hybrids require Xsib, implies that hybrid lethality results from a gene (or genes) on Xmel that fails to function in hybrids or, alternatively, that Xsib provides a function that counteracts a deleterious effect of Xmel (SAWAMURA et al. 1993A Down).

These predictions are in contrast to our suggestion that the activity of Hmr+ causes hybrid lethality and that reducing its function is sufficient to rescue hybrids. The most direct demonstration of this point is that simply removing one copy of Hmr+ partially rescued exceptional Xmel/Xmel female hybrids (Table 6)—Xsib is clearly not absolutely required for hybrid viability. The rescue of hybrid males by Lhr, Hmr1, and In(1)AB first suggested that Xsib is not required for hybrid viability, but was subject to the reservation that the precise nature of these alleles is unknown. Although it remains possible that Xsib may have some positive effect on hybrid viability (see Dose dependence of Hmr below), in accordance with Sturtevant's second hypothesis, our results strongly support a third, alternative hypothesis that hybrid lethality results from the presence of Xmel and, more specifically, Hmr+.

Are larval and pupal lethality caused by the same mechanism?
If we wish to use the temperature-sensitive pupal lethality of hybrid females as a new assay for investigating Hmr, it is important to consider whether this lethality is caused by the same mechanism that causes larval lethality. ORR et al. 1997 Down have proposed that hybrid larval lethality may be due to a mitotic defect. The rough eyes, malformed wings, and necrotic tissue found in hybrid female escapers are consistent with this hypothesis, as similar phenotypes also occur in certain hypomorphic cell cycle alleles (WHITE-COOPER et al. 1996 Down; SECOMBE et al. 1998 Down). However, this syndrome of defects is also reminiscent of phenotypes associated with mutations in pleiotropic signaling molecules such as Notch (ARTAVANIS-TSAKONAS et al. 1999 Down) and epidermal growth factor (FREEMAN 1998 Down). These possibilities are not mutually exclusive and can be addressed by detailed examination of rescued and unrescued female hybrids. The temperature dependence of unrescued female hybrids and the use of temperature shifts at different developmental stages will be particularly useful for identifying the most direct consequences of Hmr+ activity in hybrids.

The strongest available evidence that larval and pupal lethality are caused by the same mechanism is that both are suppressed by the rescue mutations Hmr1, In(1)AB, and Lhr. Using similar logic, SAWAMURA et al. 1993A Down, SAWAMURA et al. 1993C Down have convincingly argued that the embryonic lethality of hybrid daughters of sibling species mothers and D. melanogaster fathers is mechanistically unrelated to larval lethality because it is rescued by a distinct set of mutations. The relative degree of lethality with the different sibling species is a second common character. As noted above for pupal lethality, larval lethality appears to be strongest with D. sechellia, intermediate with D. simulans, and weakest with D. mauritiana, with strength of lethality measured by its inverse correlation to strength of rescue of exceptional females by Hmr- deletions (Table 6) and of hybrid males by Hmr1 (HUTTER and ASHBURNER 1987 Down). This ranking of the sibling species also holds for the effects of the Hmr+ duplication on both female (Table 7) and Xsib male (Table 8) hybrids.

However, the patterns of conditional variability for larval and pupal lethality are not entirely equivalent. First, larval lethality was more severe with the D. simulans v f2 stock than with the ryi83 stock, while the opposite was true for pupal lethality (Table 1 and Table 6; note that larval lethality here refers to that found in Xmel/Xmel exceptional females). Second, pupal lethality is clearly temperature sensitive, with little or no lethality detected at 18° and increasing lethality at higher temperatures. Larval lethality, however, appears to be temperature insensitive (below 29°) or even somewhat cold sensitive. Rescue was generally equivalent or lower at 18° than at 25° for In(1)AB males (Table 3; see also HUTTER et al. 1990 Down) and for Xmel, Hmr-/Xmel females (Table 6). [The temperature profile of Hmr1 is more complicated. Rescue of male lethality is most effective at 18° (HUTTER and ASHBURNER 1987 Down; our unpublished data). If larval lethality is not itself a temperature-sensitive trait, as suggested by our results with deficiencies in females, then the preferential rescue at cold temperatures by Hmr1 may mean that it is a cold-sensitive loss-of-function allele.]

Unknown gene(s) on the X chromosome are pro