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. 2007 May;176(1):543–552. doi: 10.1534/genetics.107.072827

Nup96-Dependent Hybrid Lethality Occurs in a Subset of Species From the simulans Clade of Drosophila

Daniel A Barbash 1,1
PMCID: PMC1893067  PMID: 17409061

Abstract

The cross of Drosophila melanogaster females to D. simulans males typically produces lethal F1 hybrid males. F1 male lethality is suppressed when the D. simulans Lhr1 hybrid rescue strain is used. Viability of these F1 males carrying Lhr1 is in turn substantially reduced when the hybrids are heterozygous for some mutant alleles of the D. melanogaster Nup96 gene. I show here that similar patterns of Nup96-dependent lethality occur when other hybrid rescue mutations are used to create F1 males, demonstrating that Nup96 does not reduce hybrid viability by suppressing the Lhr1 rescue effect. The penetrance of this Nup96-dependent lethality does not correlate with the penetrance of the F1 hybrid rescue, arguing that these two phenomena reflect genetically independent processes. D. simulans, together with two additional sister species, forms a clade that speciated after the divergence of their common ancestor from D. melanogaster. I report here that Nup96− reduces F1 viability in D. melanogaster hybrids with one of these sister species, D. sechellia, but not with the other, D. mauritiana. These results suggest that Nup96-dependent lethality evolved after the speciation of D. melanogaster from the common ancestor of the simulans clade and is caused by an interaction among Nup96, unknown gene(s) on the D. melanogaster X chromosome, and unknown autosomal gene(s), at least some of which have diverged in D. simulans and D. sechellia but not in D. mauritiana. The genetic properties of Nup96 are also discussed relative to other hybrid lethal genes.

THE use of chemical and transposon mutagens to systematically identify mutations has been a powerful technique for investigating the genetic basis of many biological processes. Speciation is not one of the many examples that could be listed. The study of interspecific hybrid incompatibilities in the model organism Drosophila melanogaster was begun by Sturtevant (1920) many decades before the application of high-efficiency mutagenesis techniques in Drosophila, but until recently has advanced solely by the characterization of variants segregating in natural or laboratory populations. Watanabe (1979) began this approach with his discovery of the D. simulans Lethal hybrid rescue (Lhr1) strain, which suppresses lethality of D. melanogaster/D. simulans F1 hybrid males, and three additional hybrid rescue strains were subsequently identified and characterized (Hutter and Ashburner 1987; Sawamura et al. 1993a,b). All four of these rescue strains increase the viability of F1 hybrids. The D. melanogaster mutations Hybrid male rescue (Hmr) and Zygotic hybrid rescue (Zhr) appear to be loss-of-function mutations (Sawamura and Yamamoto 1993; Barbash et al. 2000), while duplications of Hmr+ and Zhr+ reduce hybrid viability (Sawamura and Yamamoto 1993; Barbash et al. 2000; Orr and Irving 2000). These observations suggest that the wild-type Hmr+ and Zhr+ genes cause hybrid lethality, but only Hmr has been molecularly cloned and shown to correspond to a single gene (Barbash et al. 2003).

Two genetic screens have attempted to systematically find regions of the genome that affect the viability of D. melanogaster hybrids. F1 daughters of D. melanogaster females crossed to D. simulans males are viable at low temperatures. Coyne et al. (1998) used a collection of D. melanogaster strains deleted for different autosomal regions to create F1 female hybrids and then measured the viability of Deficiency/+ hybrids relative to +/+ control hybrid females. The Df/+ hybrids would have only D. simulans genes in the deleted region, while the +/+ hybrids would have a full set of genes from both species. Deletions that reduced hybrid viability could then be inferred to contain D. simulans genes that act as recessive lethals when uncovered in F1 hybrids or genes that are haplo-insufficient in hybrids. It is important to note that the genes detectable in this assay might not normally cause lethality in F1 hybrids, but rather could potentially cause lethality in F1 or backcross hybrids. Only a handful of lethal regions with variable penetrance were identified, and the authors concluded that relatively few genes cause hybrid lethality.

Presgraves (2003) subsequently made the insightful observation that even if the genes recoverable in the screen of Coyne et al. (1998) were recessive, they would have to cause lethality by interacting with other genes that are dominant. Presgraves (2003) therefore designed a similar deficiency screen that could detect lethal effects in males; such a screen can detect lethal interactions between the deficiencies and recessive genes on the hemizygous X chromosome. Because F1 hybrid males are normally lethal, the screen was conducted by crossing Df/+ D. melanogaster females to Lhr1 D. simulans males. Twenty autosomal regions that are essentially lethal were identified, meaning that few or no Df/+ F1 hybrid males were recovered. Comparing these results to those of Coyne et al. (1998), Presgraves concluded that recessive–recessive incompatibilities are much more frequent than dominant–recessive incompatibilities. Sawamura et al. (2004) subsequently observed that an introgression of part of the D. simulans chromosome II into D. melanogaster suppressed rescue by Lhr1 of F1 hybrid males but did not affect F1 female viability; this observation can also be interpreted as evidence for a recessive–recessive incompatibility.

The deficiency screen also suggested a straightforward approach for identifying the genes responsible for the effects observed with deficiencies. Presgraves et al. (2003) showed that the gene Nup96 is responsible for the lethality observed for one region, because some loss-of-function Nup96 alleles mimic the properties of the chromosomal deficiencies.

In this article I attempt to address several issues arising from this study of Nup96 as well as the genomewide deficiency screen. These issues stem largely from the fact that the hybrid lethal effects are detectable only in males, which are normally lethal and thus first must be rescued from lethality by Lhr1. The following issues are addressed in this article:

  1. Presgraves et al. (2003) interpret the lethal effect that they observed as reflecting an interaction between Nup96 and unknown gene(s) on the D. melanogaster X chromosome. Since the Nup96 effect was discovered as a suppressor of the hybrid-lethality-suppressing mutation Lhr1, an alternative possibility is that hemizygosity for Nup96 suppresses the hybrid rescue ability of Lhr1. In other words, it remains unclear whether Nup96 affects hybrid viability independently of Lhr. This possibility cannot be easily excluded since the mechanism of how Lhr1 rescues hybrid males remains unknown, despite the recent identification of the wild-type Lhr+ locus (Brideau et al. 2006). Lhr was also independently identified and named heterochromatin protein 3 (HP3) by Greil et al. (2007).

  2. A related question is whether the magnitude of the lethal effect depends on the strength of hybrid rescue. Lhr1 rescue of male lethality was incomplete in many of the crosses with deficiency stocks, so that lethal effects of deficiencies were often observed in genotypes that are subviable to begin with (Presgraves 2003).

  3. D. simulans is one of three sibling species of D. melanogaster, the other two being D. mauritiana and D. sechellia. These three sibling species are referred to as forming the simulans clade of species. Lethal interactions could be assayed only in D. melanogaster/D. simulans hybrids because Lhr1-like mutations are not known in these other two sibling species. If Nup96-dependent lethality occurs only in D. melanogaster/D. simulans hybrids, then it likely reflects an incompatibility that arose after D. simulans speciated from D. mauritiana and/or D. sechellia and thus well after the time of speciation of D. melanogaster and its sibling species.

  4. Many hybrid lethal effects have variable penetrance depending on genetic background. I have investigated here whether Nup96-dependent lethality is a fixed trait among sibling-species stocks.

A remaining question is whether Nup96 may cause lethality because it is haplo-insufficient in F1 hybrid males. This possibility cannot be addressed with the methods used here but is considered in the discussion.

MATERIALS AND METHODS

Stocks and nomenclature:

The Drosophila Nup98 locus produces two transcripts, the larger of which is predicted to encode a single polypeptide, which is proposed to be cleaved into two separate proteins, NUP96 and NUP98 (Presgraves et al. 2003), on the basis of structural similarities to orthologs from other species (Rosenblum and Blobel 1999; Teixeira et al. 1999). The hybrid lethal effect of the Nup98 locus has been mapped by Presgraves et al. (2003) to the region of the gene that encodes the NUP96 protein; following these authors, I therefore refer to the Nup98E53.1 allele as Nup96− and to the wild-type allele as Nup96+. The D. simulans Lhr rescue allele discovered by Watanabe (1979) and used in the Presgraves et al (2003) study is referred to here as Lhr1.

Df(1)Hmr− was generated by transposase-mediated imprecise excision of the P-element insertion upstream from the hypomorphic Hmr1 allele and is described in Barbash and Lorigan (2007). This derivative allele deletes the first 96 amino acids of the predicted HMR protein and has a stronger hybrid rescue phenotype than Hmr1.

Stocks of In(1)AB,w/FM6; Nup98E53.1/TM3, Sb e and Df(1)Hmr−, y w v/FM6; Nup98E53.1/TM3, Sb,e were made by crossing males from the Nup96− mutant stock to females from either In(1)AB,w/FM6; D/TM3 or Df(1)Hmr−, y w v/FM6; D/TM3 and backcrossing F1 males to the females of the parental genotype.

D. simulans SZ5 (Zimbabwe), Wolfskill 1, and MD199S were obtained from David Begun (University of California at Davis); the latter two stocks are inbred. The D. simulans v stock is described in Barbash and Ashburner (2003) and the Tsimbazaza stock in Lachaise et al. (1986). D. mauritiana “W” stocks were from the National Institute of Genetics (Mishima, Japan) and obtained from Shun-Chern Tsaur (Academia Sinica, Taipei, Taiwan). Remaining D. mauritiana and all D. sechellia stocks were obtained from the Tucson Drosophila Stock Center.

Crosses and scoring of progeny:

Crosses were designed so that the viability of Nup96−/+ vs. +/+ hybrids could be compared in four different genotypes of progeny (Figure 1). Sb was used to distinguish Nup96−/+ vs. TM3, Sb Nup96+/+ hybrids. Animals that did not have at least one macrochaete on both the head and notum were scored as being of uncertain genotype and eliminated from further analysis.

Figure 1.—

Figure 1.—

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Viability effects tested in this study. Only the sex and third chromosomal genotypes are shown. “Rescue” represents either the In(1)AB or the Df(1)Hmr− hybrid rescue mutation. FM6/Y hybrid sons are lethal since they do not carry a rescue mutation. Four surviving genotypes can be compared for viability with and without the Nup96− mutation. (A) Comparison of F1 hybrid females that have high viability due to the rescue mutation to confirm that Nup96− does not cause lethality in females. (B) Comparison of F1 hybrid females that have low viability due to the presence of D. melanogaster Hmr+ (Barbash et al. 2000). This genotype provides a more sensitive assay for possible effects of Nup96− in female hybrids than the genotype in A. (C) Comparison of F1 hybrid males carrying the D. melanogaster X chromosome to test whether Nup96-dependent lethality occurs with hybrid rescue mutations other than Lhr1. (D) Comparison of F1 hybrid males carrying the sibling-species X chromosome to confirm that lethality occurs only with the D. melanogaster X. This class of exceptional males is generated by nondisjunction in the female parents and was observed at high rates, presumably due to the presence of two balancer chromosomes. “Rescue”/FM6/Y hybrid females would also be produced by nondisjunction and were previously shown to be semiviable (Barbash et al. 2000). Such females would be distinguishable from FM6/X+ regular females by visible markers in crosses with Df(1)Hmr− in Table 2; however, no such females were observed in this study.

Two vials of each cross were established, each with ∼25 0- to 1-day-old females and ∼40 3- to 4-day-old males. Crosses were changed every 3-4 days for a total of six collections, all done at room temperature (∼22°–23°). Progeny were counted for at least 20 days after removing the parents, until no more progeny eclosed.

Dead pupae and pharate adults were then examined and scored in a subset of crosses. Pharate adults were classified as having well-developed thoracic, head, and genital cuticular structures and at least some abdominal bristles. Late pupae were classified as having well-developed thoracic, head, and genital cuticular structures but no abdominal bristles. This is a conservative classification of late pupae because eclosed F1 hybrid males often are missing a portion of their abdominal cuticle and bristles.

RESULTS

Tests using In(1)AB:

Nup96 was first assayed in hybrids containing In(1)AB. In(1)AB rescues F1 male lethality and high-temperature F1 female lethality although the genetic basis of rescue remains unknown (Hutter et al. 1990; Barbash et al. 2003). The effect of hemizygosity for Nup96 on viability of male hybrids rescued by In(1)AB was determined by crossing to four different lines of D. simulans (Table 1). In(1)AB/Y; Nup96−/+ males were only 13–15% viable relative to their In(1)AB /Y; +/+ siblings in crosses to D. simulans v and SZ5. No significant difference in viability was observed in crosses with D. simulans Wolfskill 1, although relatively few hybrid males survived, while the fourth cross to D. simulans Tsimbazaza failed to produce any In(1)AB male hybrids. I conclude that hemizygosity for Nup96 reduces viability of In(1)AB-rescued F1 males.

TABLE 1.

Hybrid progeny from In(1)AB/FM6; Nup96−/TM3 D. melanogaster females and D. simulans, D. sechellia, or D. mauritiana males

In(1)AB/+ females
FM6/+ females
In(1)AB malesa
Xsib males
Male parent No. of TM3/+ No. of Nup−/+ Relative viability No. of TM3/+ No. of Nup−/+ Relative viability No. of TM3/+ No. of Nup−/+ Relative viability No. of TM3/+ No. of Nup−/+ Relative viability
1. D. simulans Wolfskill 1
 Alive 126 167 1.33 122 143 1.17 12 7 0.58 (NS) 31 39 1.26
 Pharate 2 1 4 3 33 19 11 0
 Pupae 0 0 0 0 18 32 0 0
 Total 128 168 1.31 126 146 1.16 63 58 0.92 (NS) 42 39 0.93
2. D. simulans v
 Alive 296 342 1.16 173 193 1.12 75 10 0.13*** 63 74 1.17
 Pharate 1 1 57 27 91 26 23 0
 Pupae 0 0 3 3 26 115 0 0
 Total 297 343 1.15 233 223 0.96 192 151 0.79* 86 74 0.86
3. D. simulans SZ5
 Alive 59 71 1.20 44 37 0.84 27 4 0.15*** 13 16 1.23
 Pharate 1 1 9 4 11 8 1 0
 Pupae 1 0 29
 Total 60 72 1.20 54 41 0.76 38 41 1.08 (NS) 14 16 1.14
4. D. simulans Tsimbazaza
 Alive 58 92 1.59 22 36 1.64 0 0 5 12 2.40
 Pharate 2 5 4 6 7 0 0 1
 Pupae 0 0 0 0 0 3 0 0
 Total 60 97 1.62 26 42 1.62 7 3 0.43 (NS) 5 13 2.60
5. D. sechellia Robertson line 1
 Alive 195 237 1.22 13 42 3.23 44 13 0.30*** 15 45 3.00
 Pharate 10 9 40 48 23 15 9 3
 Pupae 0 0 0 0 2 19 1 0
 Total 205 246 1.20 53 90 1.70 69 47 0.68* 25 48 1.92
6. D. mauritiana Iso 105
 Alive 191 225 1.18 145 174 1.20 153 144 0.94 (NS) 41 54 1.32
7. D. mauritiana Iso 152
 Alive 141 185 1.31 174 185 1.06 145 173 1.19 (NS) 53 44 0.83
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In(1)AB,w/FM6; Nup96−/TM3,Sb D. melanogaster females were crossed to males indicated in the first column. Progeny of crosses are as diagrammed in Figure 1. Some crosses produced B Hw males, as described originally by Hutter (1990) and proposed to be of mosaic origin. In cross 2, these males carried the v marker. Among live progeny only, crosses produced the following: cross 1, none; cross 2, 15+, 3 Sb; cross 3, none; cross 4, 12+, 5 Sb; cross 5, none; cross 6, 9 +, 0 Sb; cross 7, 12+, 1 Sb. NS, not significant. *P < 0.01; ***P < 0.0001.

a

The significance of differences in the two classes of In(1)AB males compared to In(1)AB/+ females was tested using Fisher's exact test (one tailed) for the “Alive” and the “Total” classes.

These crosses confirmed two additional conclusions shown previously in crosses of Nup96− point mutations or deficiencies to D. simulans Lhr1 (Presgraves 2003; Presgraves et al. 2003). First, hemizygosity for Nup96 had no effect on hybrid female viability, with Nup96−/+ females generally having higher viability than their +/+ siblings. This was true even in FM6, Hmr+/+ females, which in some crosses had reduced viability relative to In(1)AB/+ females, due to the dominant lethal effect of Hmr+ previously observed at ≥25° (Barbash et al. 2000).

Second, all crosses produced exceptional hybrid males, which are viable since they contain an X chromosome from D. simulans (Sturtevant 1920). The relatively high rate observed likely reflects an increased rate of nondisjunction in the mothers caused by the presence of two multiply inverted balancer chromosomes. Nup96−/+ exceptional males had similar viability to +/+ exceptional males, confirming that Nup96 hemizygosity interacts with the D. melanogaster X but not with the D. simulans X to cause lethality.

I also examined viability effects in D. sechellia hybrids and found a similar pattern described above for D. simulans hybrids (Table 1). In(1)AB/Y; Nup96−/+ males were only 30% viable relative to their In(1)AB/Y; +/+ siblings.

In all of these crosses, the level of hybrid male rescue by In(1)AB was rather low. I therefore scored dead pharate and late-stage pupae to increase the number of hybrids analyzed. This analysis also allows one to determine whether the differences in viability observed between classes of eclosed animals are caused by late-stage lethality or by lethality occurring earlier in development. Note that, in these crosses, substantial pre-eclosion lethality was observed even in the In(1)AB; +/+ control males. In all cases, I found that the majority of “missing” Nup96−/+ males were found among the late-stage pupae and pharate adults. For example, in the cross to D. simulans SZ5 males, In(1)AB; Nup96−/+ hybrid sons were only 15% viable at the adult stage relative to In(1)AB;+/+ hybrid sons. However, 108% of In(1)AB; Nup96−/+ males reached the late pupal stage relative to In(1)AB;+/+. Among the four crosses, only that to D. simulans v had a significant reduction in viability of In(1)AB/Y; Nup96−/+ male hybrids when considering total progeny (late pupal, pharate, and eclosed adults). These data indicate that Nup96 hemizygosity causes lethality predominantly in late-stage pupae.

Crosses were also performed with three different strains of D. mauritiana, two of which were successful (Table 1). In contrast to the results with D. simulans and D. sechellia hybrids, no significant difference in viability was observed between In(1)AB; Nup96−/+ and In(1)AB; +/+ eclosed males. I conclude that Nup96 hemizygosity does not cause lethality in D. melanogaster/D. mauritiana hybrids.

Tests using Df(1)Hmr−:

One possible explanation for these different results with D. melanogaster/D. mauritiana hybrids is that Nup96-dependent lethality occurs only in F1 males that are semiviable. In(1)AB fully rescued hybrid males in crosses to D. mauritiana, as shown by the comparable viability of In(1)AB/Y; +/+ males and In(1)AB/+; +/+ females in Table 1. In contrast, similar comparisons show that male rescue varied from 0 to 46% in crosses to D. simulans and D. sechellia in Table 1 (considering only eclosed animals). I attempted to address whether the Nup96 effect correlates with the level of F1 hybrid male rescue by using a stronger rescue allele, Df(1)Hmr− (Table 2). When Df(1)Hmr− females were crossed to D. simulans v, Df(1)Hmr−/Y; +/+ hybrid males were fully viable (n = 111) compared to Df(1)Hmr−/+; +/+ hybrid females (n = 95). This result contrasts with the 25% rescue observed in crosses of this same D. simulans stock with In(1)AB (Table 1). These data demonstrate that Df(1)Hmr− is indeed a stronger rescue allele than In(1)AB. Df(1)Hmr−; Nup96−/+ males were completely lethal, however, compared to the 13% relative viability of In(1)AB; Nup96−/+ males. Similar results were obtained with hybrids made with the D. sechellia Robertson 1 stock, with In(1)AB; Nup96−/+ hybrid males having a higher relative viability than Df(1)Hmr−; Nup96−/+ males. I conclude that the penetrance of Nup96-dependent lethality varies depending on genetic background but does not correlate with the strength of the rescue allele used.

TABLE 2.

Hybrid progeny from Df(1)Hmr−/FM6; Nup96−/TM3 D. melanogaster females and D. simulans, D. sechellia, or D. mauritiana males

Df(1)Hmr−/+ females
FM6/+ females
Df(1)Hmr− malesa
Xsib males
Male parent No. of TM3/+ No. of Nup−/+ Relative viability No. of TM3/+ No. of Nup−/+ Relative viability No. of TM3/+ No. of Nup−/+ Relative viability No. of TM3/+ No. of Nup−/+ Relative viability
1. D. simulans v 95 166 1.75 51 32 0.63 111 0 0.00*** 18 29 1.61
2. D. simulans MD199S 294 290 0.99 116 146 1.26 153 0 0.00*** 50 41 0.82
3. D. sechellia Sy007 63 89 1.41 1 17 17.00 39 0 0.00*** 10 11 1.10
4. D. sechellia Robertson 83 103 1.24 2 4 2.00 46 1 0.02*** 4 13 3.25
5. D. sechellia Praslin Island 107 92 0.86 1 6 6.00 71 0 0.00*** 18 16 0.89
6. D. sechellia Iso 81 80 79 0.99 0 13 — 22 0 0.00*** 14 13 0.93
7. D. mauritiana w f 37 41 1.11 22 29 1.32 8 15 1.88 (NS) 4 8 2.00
8. D. mauritiana Iso 75 160 111 0.69 130 124 0.95 122 114 0.93 (NS) 18 21 1.17
9. D. mauritiana Iso 105 72 68 0.94 46 60 1.30 64 54 0.84 (NS) 12 12 1.00
10. D. mauritiana Iso 152 78 84 1.08 42 84 2.00 52 56 1.08 (NS) 16 20 1.25
11. D. mauritiana W138 69 94 1.36 56 79 1.41 65 64 0.98 (NS) 9 14 1.56
12. D. mauritiana W139 121 143 1.18 106 152 1.43 115 128 1.11 (NS) 31 29 0.94
13. D. mauritiana W140 169 153 0.91 116 125 1.08 131 131 1.00 (NS) 6 26 4.33
14. D. mauritiana W146 58 77 1.33 36 64 1.78 58 32 0.55* 8 9 1.13
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Df(1)Hmr−, y w v/FM6; Nup96−/TM3 D. melanogaster females were crossed to males indicated in the first column. Progeny of crosses are as diagrammed in Figure 1. Some crosses produced B Hw males, as described originally by Hutter (1990) and proposed to be of mosaic origin. Among live progeny only, crosses produced the following: cross 1, 20+, and 1 Sb; cross 2, 3+, and 1 Sb; cross 3, 7+, and 0 Sb; cross 4, 3+, and 0 Sb; cross 5, 4+, and 0 Sb; cross 6, none; cross 7, none; cross 8, 4+, and 0 Sb; cross 9, 6+, and 0 Sb; cross 10, 2+, and 0 Sb; cross 11, 1+, and 0 Sb; cross 12, none; cross 13, 8+, and 0 Sb; cross 14, 2+, and 0 Sb. NS, not significant. *P < 0.01; ***P < 0.0001.

a

The significance of differences in the two classes of Df(1)Hmr− males compared to Df(1)Hmr−/+ females was tested using Fisher's exact test (one tailed).

I used the stronger rescue allele Df(1)Hmr− to test three additional D. sechellia stocks and one additional D. simulans stock (Table 2). No Df(1)Hmr−; Nup96−/+ males survived in these crosses, strengthening the conclusion that Nup96-dependent lethality occurs in both D. melanogaster/D. simulans and D. melanogaster/D. sechellia hybrids.

No significant effect on Df(1)Hmr−; Nup96−/+ male viability was observed in crosses to the same two D. mauritiana lines tested above (Table 2). Six additional D. mauritiana stocks were then tested, with five producing a similar lack of effect on Df(1)Hmr−; Nup96−/+ male viability. Hybrids from the sixth line, W146, showed a modest reduction (55%) in viability compared to Df(1)Hmr−; +/+ male siblings that was statistically significant when compared to female controls. Considering the results from all crosses with D. mauritiana, Nup96 hemizygosity appears to have little effect in D. melanogaster/D. mauritiana hybrids.

DISCUSSION

Nup96 is not a suppressor of Lhr1:

I have shown here that hemizygosity for Nup96 reduces the viability of F1 hybrid males that have been rescued from lethality by either In(1)AB or Df(1)Hmr−. These findings demonstrate that Nup96 is not a specific suppressor of the Lhr1 rescue mutation. I have also found that the penetrance of the lethality induced by Nup96 does not correlate with the degree of rescue obtained with these different hybrid rescue alleles. These results suggest that Nup96 also is not a general suppressor of hybrid male rescue. Rather, these data support the hypothesis of Presgraves et al. (2003) that Nup96 interacts with unknown gene(s) on the D. melanogaster X chromosome to cause lethality.

The effect of Nup96 on hybrid male development, however, is rather mild. Lhr1-rescued males heterozygous for a Nup96 deletion were previously shown to die sometime postembryogenesis, but the precise lethal phase was not determined (Presgraves 2003). Using In(1)AB, I found that hybrid males that failed to eclose due to Nup96 hemizygosity died during the late pupal or pharate adult stages. Note that unrescued hybrid males die as larvae (Sturtevant 1920). Nup96 hemizygosity can therefore be described as causing late pupal lethality in F1 hybrid males.

Nup96-dependent lethality postdates the speciation of D. melanogaster and the simulans clade:

Reproductive isolation between species is the defining characteristic of the biological species concept. Interspecific hybrid incompatibilities are one cause of reproductive isolation, and understanding the genetic basis of hybrid incompatibility (HI) is therefore a key step toward understanding the process of speciation. As species continue to diverge postspeciation, many genes will continue to diverge and cause additional incompatibilities. Only the HI genes that evolved to cause incompatibilities at the time of speciation can truly be designated as speciation genes (Coyne 1992).

Such a distinction is clearly difficult to make when one is attempting to isolate speciation genes in organisms that may have completed speciation hundreds of thousands or millions of years ago. For HIs observed in D. melanogaster/D. simulans hybrids, one can separate relatively ancient from relatively recent HIs by determining whether or not the incompatibility predates the divergence of the three species in the simulans clade. Hmr- and Zhr-dependent lethalities appear to predate this divergence point, because loss-of-function alleles suppress lethality in all three hybridizations (Hutter and Ashburner 1987; Sawamura et al. 1993b). In contrast, F1 hybrid bristle loss likely postdates divergence of the simulans clade because it occurs in D. melanogaster/D. simulans hybrids, but is essentially absent in D. melanogaster/D. mauritiana and D. melanogaster/D. sechellia hybrids (Takano 1998). I found here that Nup96-dependent lethality occurs in D. melanogaster hybrids with D. simulans and D. sechellia, but not with D. mauritiana.

I suggest three possible explanations for this pattern. First, the incompatibility may be ancestral to the divergence of the simulans clade, with suppressors having evolved in the D. mauritiana lineage postspeciation. This possibility cannot be excluded but seems unlikely: if incompatibilities are caused by deleterious interactions among rare alleles, then specific suppressing alleles becoming fixed in populations and reverting the incompatibility phenotype are likely to be even more rare. Note that the hybrid rescue alleles Hmr1 and Lhr1 were identified as alleles segregating at low frequencies (Watanabe 1979; Hutter and Ashburner 1987), presumably because they are loss-of-function mutations that cause some reduction in fitness within their species. Second, the incompatibility alleles may have been present but not fixed in the common ancestor of the simulans clade. During speciation, lineage sorting could have led to segregation of the incompatibility alleles into D. simulans and D. sechellia but not D. mauritiana. Third, the incompatibility may have evolved after the divergence of the simulans clade. Under this hypothesis, the pattern of incompatibility suggests that D. mauritiana speciated prior to D. simulans and D. sechellia (Figure 2). The phylogenetic relationship of the simulans clade species has been difficult to resolve, with data from individual loci supporting different phylogenies (Kliman et al. 2000). Ting et al. (2000) have proposed that loci involved in reproductive isolation may more accurately reflect the phylogenetic relationships among species. They analyzed polymorphism and divergence of the Odysseus gene, which causes male sterility when introgressed from D. mauritiana into D. simulans, and obtained support for D. sechellia having speciated before D. mauritiana and D. simulans. When considering possible reasons for this apparent phylogenetic discrepancy, it is important to emphasize that Nup96 is known only to cause HI between simulans clade species and D. melanogaster. There is no evidence available addressing whether Nup96 and interacting genes cause reproductive isolation between or among the simulans clade species. These data do not therefore speak directly to the issue of whether or not different regions of the genome containing isolating factors will show different genealogical histories when nascent species go through a stage of incompletely penetrant isolation (Ting et al. 2000). However, if hypothesized Nup96-interacting genes have diverged under positive selection as recently suggested (Presgraves and Stephan 2007), then our genetic analysis here suggests that they have experienced a shared pattern of selection in D. simulans and D. sechellia. A recent study of the Iris gene also supports a phylogeny where D. mauritiana branches off before D. simulans and D. sechellia, with the authors suggesting that Iris and Odysseus have experienced different patterns of selection among the simulans clade species (Malik and Henikoff 2005).

Figure 2.—

Figure 2.—

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Three evolutionary events leading to Nup96-dependent hybrid lethality. (1) Lethality requires X-linked gene(s) that have diverged on the D. melanogaster lineage because lethality occurs in hybrids with the D. melanogaster but not with the D. simulans or D. sechellia X chromosome (Presgraves et al. 2003; this study). (2) The divergence of Nup96 is inferred to have occurred before the divergence of D. simulans and D. mauritiana because all replacement substitutions in D. simulans are also found in D. mauritiana (Presgraves et al. 2003). (3) Results presented in this study suggest that additional genetic changes must have evolved to cause lethality only in D. melanogaster/D. simulans and D. melanogaster/D. sechellia hybrids but not in D. melanogaster/D. mauritiana hybrids.

The genetic basis of Nup96-dependent lethality:

Interestingly, all amino acid changes that occurred on the lineage leading to D. simulans are also found in D. mauritiana (Presgraves et al. 2003). It is possible that Nup96 has functionally diverged between D. mauritiana and D. simulans due to unidentified changes in noncoding regions. However, since Nup96 is so similar between these two species but HI occurs only with D. simulans (and D. sechellia), a more likely possibility is that Nup96-dependent hybrid lethality depends on interactions with additional genes that have diverged in D. simulans and D. sechellia but not in D. mauritiana. These additional genes must be autosomal, since F1 male hybrids with all three species carry a D. melanogaster X chromosome. From the available data it is impossible to determine how many additional interacting genes may be involved in causing lethality. If Nup96-dependent lethality is complex, then many of the interacting alleles may be present in D. mauritiana as well as in D. simulans and D. sechellia. The results here do suggest that D. mauritiana must be missing at least one interacting gene found in the other two sibling species. In summary, one can infer the existence of two distinct times of divergence for genes that cause Nup96-dependent lethality (Figure 2).

Functional properties and dominance relations of hybrid lethality genes:

The following discussion assumes that Nup96 has functionally diverged between D. melanogaster and D. simulans, meaning that hybrid lethality is caused specifically by the D. simulans Nup96+ ortholog but not by D. melanogaster Nup96+. The rejection of the McDonald–Kreitman test due to an apparent excess of nonsynonymous substitutions between these species suggests that Nup96 has functionally diverged (Presgraves et al. 2003), but this suggestion remains untested. If Nup96 has not functionally diverged, then the observed lethal effect would instead suggest that Nup96 is haplo-insufficient in F1 male hybrids, meaning that removing either the D. melanogaster or the D. simulans ortholog would cause lethality. If Nup96 is haplo-insufficient, then much of the discussion below, such as considering dominance relations between the species, would be irrelevant.

Nup96 and Hmr are the best-characterized genes causing hybrid lethality in D. melanogaster, and it is therefore of interest to compare their genetic and inferred mechanistic properties. These properties appear to be quite different: for Hmr, removing the gene suppresses hybrid lethality, while, for Nup96, removing the non-HI-causing ortholog causes hybrid lethality. Comparisons can be further illustrated by considering two different meanings of dominance that are relevant to HI genes.

Dominance relations between orthologs:

One definition concerns the dominance relationship between the orthologs of the HI gene from each of the two hybridizing species. For Hmr, genetic assays suggested that the wild-type D. melanogaster ortholog causes hybrid lethality and that hybrid rescue results from loss-of-function mutations in D. melanogaster Hmr+ (Hutter et al. 1990; Barbash et al. 2000; Orr and Irving 2000). This model was confirmed by showing that transgenic copies of D. melanogaster Hmr+ reduce hybrid viability and suppress hybrid rescue (Barbash et al. 2003). Hmr+ from D. simulans and D. mauritiana was then tested in similar assays and found to not have such activity (Barbash et al. 2004). These studies suggest that Hmr+ from the sibling species is neutral with respect to hybrid fitness and thus demonstrate that D. melanogaster Hmr+ is fully dominant over sibling-species Hmr+. It is interesting to consider the alternative possibility. If D. melanogaster Hmr+ were recessive, then adding D. simulans or D. mauritiana Hmr+ transgenes would suppress the lethality of hybrid males by masking this deleterious effect. No such suppression was observed in FM6/Y, P{D. simulans-Hmr+}/+ or FM6/Y, P{D. mauritiana-Hmr+}/+ males (Barbash et al. 2004).

Nup96 appears to have the opposite pattern of dominance, because the deleterious effect of the D. simulans ortholog becomes apparent only when removing the D. melanogaster ortholog. A possible mechanistic explanation is that the D. simulans ortholog has diverged in sequence so that it cannot function in hybrids (that is, in male hybrids with a D. melanogaster X chromosome). D. simulans Nup96+ would then be analogous to a loss-of-function allele in hybrids, and F1 male hybrids rescued by Lhr1 or other rescue alleles would be viable because they contain a functional copy of Nup96+ from D. melanogaster. In this scenario, D. simulans Nup96+ is fully recessive to D. melanogaster Nup96+.

The unusual pattern of complementation for D. simulans Nup96+, however, is inconsistent with this interpretation. If D. simulans Nup96+ is a recessive loss-of-function allele in hybrids, then it should be complemented only by functional D. melanogaster copies. D. melanogaster mutant alleles predicted to truncate Nup96 at positions 1021 and 1534 failed to complement hybrid lethality, but a third allele predicted to encode a truncated Nup96 at position 1142 (Nup96F1.15) did complement (Presgraves et al. 2003). Note that all three of these alleles are homozygous lethal within D. melanogaster. These data suggest that D. simulans Nup96+ may be acting as a dominant negative in hybrids rather than (or in addition to) being a loss-of-function allele. The Nup96 protein is part of the large multi-protein nuclear pore complex (Suntharalingam and Wente 2003), so that a heterospecific ortholog with a moderate level of sequence divergence might bind to and inhibit other proteins in the complex. A speculative explanation is that different length forms of truncated D. melanogaster Nup96+ would have varying abilities to block the deleterious effect of D. simulans Nup96+, independently of their ability to function normally themselves. If D. simulans Nup96+ does have a dominant-negative activity, then the lethality in male hybrids hemizygous for this gene is caused by both the absence of the D. melanogaster ortholog and the presence of the D. simulans ortholog. An experimentally testable prediction is that adding additional copies of D. simulans Nup96+ to hybrids containing a wild-type D. melanogaster Nup96+ would reduce their viability. If true, then D. simulans Nup96+ would not be fully recessive to D. melanogaster Nup96+.

Dominance of fitness effects:

The second definition of dominance relevant to HI genes is in the population genetic sense of fitness. In this second context, one attempts to quantitate the fitness effects of the HI allele in a heterozygote relative to a homozygote.

When measuring this dominance property, it is necessary to consider the magnitude of fitness effects separately from the issue of incomplete penetrance of HI phenotypes. F1 hybrid daughters of D. melanogaster females are viable at low temperatures and lethal at high temperatures (Sturtevant 1929; Kerkis 1933; Barbash et al. 2000). Deletions of Hmr suppress this high-temperature lethality, demonstrating that D. melanogaster Hmr+ causes female lethality (Barbash et al. 2000). A similar variable penetrance occurs with lethality in F1 daughters of the reciprocal cross of D. simulans mothers and D. melanogaster fathers (Sawamura et al. 1993a; Orr 1996). When lethality is observed in this cross, it can be rescued by mutations in D. melanogaster Zhr (Sawamura et al. 1993b). Further genetic analyses suggested that Zhr mutations are deletions or loss-of-function mutations of D. melanogaster Zhr+ (Sawamura and Yamamoto 1993). Both Hmr+ and Zhr+ are therefore dominant F1 female hybrid lethal genes of variable penetrance.

This definition of Hmr+ and Zhr+ as dominant in their effects in F1 females is not surprising, because it must be true for any zygotically acting gene causing F1 hybrid female lethality. Nevertheless, Hmr+ does not conform to a strict definition of a dominant effect. We have argued previously that hybrid fitness is dependent not only on the presence or absence of D. melanogaster Hmr+, but also on its dosage (Barbash et al. 2000). This conclusion did not derive from the observation of variable penetrance of lethality in wild-type F1 females, but rather from experimental manipulation of Hmr+ gene dosage in hybrids.

Is Nup96 similar to Hmr in dominance with respect to fitness? A key question is whether D. simulans Nup96+ causes any deleterious phenotype when heterozygous in hybrids. If it acts like a loss-of-function allele in hybrids, then it should be completely recessive in its deleterious effect, because a heterozygote would always have a functional copy from D. melanogaster. If, as suggested above, it has some dominant-negative or neomorphic activity in hybrids, then it might cause a dominant fitness effect.

There are clearly conditions under which D. simulans Nup96+ is fully recessive, since both Lhr and Hmr mutations can sometimes rescue to full-viability F1 males, which are wild type for Nup96. But the magnitude of rescue varies widely in different backgrounds, and it would be of interest to test whether D. simulans Nup96+ contributes to this variation. The most straightforward experiment to test whether Nup96 has any dominant effect would require testing whether a D. simulans Nup96 loss-of-function mutation increases F1 viability under conditions of intermediate rescue by Hmr1 or Lhr1. A less direct experiment might be to test whether increased doses of D. simulans Nup96+ reduce fitness in hybrids.

To summarize, Hmr and Nup96 may share an important common feature, namely that in each case it is the presence of the wild-type form, D. melanogaster Hmr+ and D. simulans Nup96+, that is deleterious to hybrids. Differences in the magnitude of their deleterious effects may explain the differences in dominance properties discussed above. D. melanogaster Hmr+ is semidominant in its fitness effect and cannot be suppressed by the presence of its sibling species ortholog, while D. simulans Nup96+ is largely recessive in its fitness effect and this fitness effect can be suppressed by the D. melanogaster Nup96+ ortholog.

Dosage effects of HI genes:

If the severity of HI phenotypes depends on HI gene dosage, then genotypes homozygous for HI genes will always have reduced fitness compared to heterozygotes. The relationship between dosage and fitness is unlikely to be linear, and the precise degree of recessiveness of HI genes is one factor in determining conformity to Haldane's rule in species hybrids (Turelli and Orr 2000), namely the reduced fitness of the heterogametic sex (XY or ZW) relative to the homogametic sex (XX or ZZ). Several studies have identified numerous regions that, when introgressed between Drosophila species, cause sterility when homozygous but not when heterozygous (Hollocher and Wu 1996; True et al. 1996; Tao et al. 2003); similar results have also been reported in tomatoes (Moyle and Graham 2005). While these introgressions are thus recessive in effect, it is important to emphasize that the fitness effects are measured in a genetic background that is otherwise composed solely of the foreign-species genome. At least for Drosophila, hybrid sterility appears to be caused by complex interactions among genes (Cabot et al. 1994; Maside et al. 1998), suggesting that fitness effects of individual HI genes may be quite different in F1, F2, or early generation backcross hybrids compared to introgression lines.

I suggest that the comparison of the mechanisms of how Hmr and Nup96 cause hybrid lethality reveals why hybrid incompatibility alleles that are fully dominant in their fitness effect are likely to be rare. HI alleles like D. melanogaster Hmr+ encode products that are deleterious to hybrids due to their sequence divergence between the hybridizing species. While the amount of Hmr divergence is high relative to average genes between D. melanogaster and D. simulans, the respective proteins are still >80% identical and retain intact MADF domains (Barbash et al. 2003), suggesting that they may retain ancestral function in both species. Likewise, Nup96 is likely to be required for nuclear pore complex function within both D. melanogaster and D. simulans despite containing a region of substantial divergence (Presgraves et al. 2003). If HI is caused by relatively subtle changes in protein structure or function, one would expect that HI phenotypes will typically be proportional to gene dosage. These subtle-effect mutations would also make HI phenotypes highly susceptible to variation caused by environmental fluctuations and genetic background differences, as observed here and elsewhere for both hybrid rescue and hybrid lethality alleles.

Acknowledgments

I thank David Begun, Corbin Jones, Daven Presgraves, Shun-Chern Tsaur, and the Tucson Drosophila Stock Center for fly stocks. This work was supported in part by National Institutes of Health grant R01 GM074737-01.

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