Genetic dissection of Nucleoporin 160 (Nup160), a gene involved in multiple phenotypes of reproductive isolation in Drosophila

ABSTRACT

Previous reports have suggested that the Nucleoporin 160 (Nup160) gene of Drosophila simulans (Nup160^sim) causes the hybrid inviability, female sterility, and morphological anomalies that are observed in crosses with D. melanogaster. Here we have confirmed this observation by transposon excision from the P{EP}Nup160^EP372 insertion mutation of D. melanogaster. Null mutations of the Nup160 gene resulted in the three phenotypes caused by Nup160^sim, but revertants of the gene did not. Interestingly, several mutations produced by excision partially complemented hybrid inviability, female sterility, or morphological anomalies. In the future, these mutations will be useful to further our understanding of the developmental mechanisms of reproductive isolation. Based on our analyses with the Nup160^sim introgression line, the lethal phase of hybrid inviability was determined to be during the early pupal stage. Our analysis also suggested that homozygous Nup160^sim in D. melanogaster leads to slow development. Thus, Nup160^sim is involved in multiple aspects of reproductive isolation between these two species.

INTRODUCTION

A century ago, Quackenbush (1910) claimed to have observed unisexual broods in Drosophila melanogaster. It turned out later that his D. melanogaster flies actually included two species, D. melanogaster and D. simulans (Sturtevant, 1919). The beauty of the latter new species was that it could be crossed with D. melanogaster, the most-studied and best-understood species of that genus (Provine, 1991). In crosses between D. melanogaster females and D. simulans males, only sterile female hybrids are obtained, as male hybrids die during larval development. In the reciprocal cross, sterile male hybrids appear, as most female hybrids die during embryonic development. Sturtevant (1920) intercrossed the species using chromosome anomalies and was able to deduce the genetic causes of hybrid inviability, but “the complete sterility of surviving F₁ hybrids frustrated Sturtevant and his vision of comprehensively exploring the genetics of interspecific differences” (Barbash, 2010). Genetic tricks and the serendipitous discovery of rescue mutations were needed before further studies could shed light on his questions (Provine, 1991; Sawamura, 2000; Barbash, 2010).

Thanks to recent advances in molecular biology techniques and genomic sequencing (Adams et al., 2000; Drosophila 12 Genomes Consortium, 2007), detailed study of speciation has become feasible. As a result, several genes for hybrid inviability and sterility have recently been isolated in this pair of species and characterized at the molecular level (reviewed in Sawamura, 2012). The D. melanogaster gene Hybrid male rescue (Hmr) encodes a DNA-binding protein that is involved in hybrid inviability and female sterility (Hutter and Ashburner, 1987; Barbash and Ashburner, 2003; Barbash et al., 2003). D. simulans Lethal hybrid rescue (Lhr) encodes a heterochromatin protein that causes hybrid inviability (Watanabe, 1979; Brideau et al., 2006; Prigent et al., 2009). D. melanogaster zygotic hybrid rescue (zhr) consists of heterochromatic 359-bp repetitive sequences and causes hybrid inviability in crosses involving D. simulans females (Sawamura et al., 1993; Ferree and Barbash, 2009). JYalpha, a gene located on different chromosomes in D. melanogaster and D. simulans, causes male sterility of introgression homozygotes (Muller and Pontecorvo, 1940; Masly et al., 2006). D. simulans Nucleoporin 96 (Nup96) causes inviability when it is hemizygous in the hybrid; the hybrid males cannot be rescued by the Lhr mutation (Presgraves et al., 2003). D. simulans Nucleoporin 160 (Nup160) causes inviability and female sterility when introgressed into D. melanogaster; the hybrid males with the introgression (or deficiency) cannot be rescued by Lhr and introgression homozygous (or hemizygous) females are sterile (Tang and Presgraves, 2009; Sawamura et al., 2010).

Nup160, like the other genes, has been mapped by recombination and deficiencies, identified by complementation tests against mutations, and confirmed by gene transformation (Sawamura, 2000; Presgraves, 2003; Sawamura et al., 2004, 2010; Tang and Presgraves, 2009). Three recessive lethal insertion mutations of Nup160 have been reported in D. melanogaster (Fig. 1A; Tweedie et al., 2009; http://flybase.org/). PBac{RB}RfC38^e00704 uncovers both hybrid inviability and female sterility when heterozygous with the wild-type allele of D. simulans, Nup160^sim (Tang and Presgraves, 2009; Sawamura et al., 2010). P{EP}Nup160^EP372, which is synonymous with P{EP}CG4738^EP372, does not lead to hybrid inviability or female sterility (Tang and Presgraves, 2009; Sawamura et al., 2010). Interestingly, P{lacW}l(2)SH2055^SH2055 does not lead to hybrid inviability but does partially lead to hybrid female sterility (Sawamura et al., 2010). These three mutations raise questions about why they behave differently and about the possibility of distinct mechanisms underlying hybrid inviability and female sterility.

Fig. 1.

Transposon insertions in the Nup160 gene and mating schemes to examine the effects of Nup160^EP372 derivatives. A, Positions and directions of three transposon insertions (triangles and arrows, not to scale). Open reading frames (full or partial) of Csl4, Nup160, and RfC38 are indicated (exons are numbered). UTR, untranslated region. B, Cross used to test the effects of mutations on morphology and female fertility. Open chromosome regions are from D. melanogaster; shaded regions are from D. simulans. C, Cross used to test the effects of mutations on hybrid viability. Int, Int(2L)D+S, Nup160^sim; *, Nup160^EP372 derivative.

In the present study, we excised the P transposable element from the P{EP}Nup160^EP372 insertion and examined whether the new mutations cause hybrid inviability and female sterility. Because Nup160 has been implicated as the cause of morphological anomalies by deficiency mapping (Sawamura et al., 2010), we also examined the abdomen, wings, and bristles of flies heterozygous for Nup160^sim and excision derivatives. Finally, we determined the lethal phase of hybrid males and measured the total duration of development of homozygous carriers.

MATERIALS AND METHODS

Strain nomenclature

Although PBac{RB}RfC38^e00704/Df(2L)BSC242 and P{lacW}RfC38^k13807/Df(2L)BSC242 are lethal because of the absence of Nup160 and RfC38 on Df(2L)BSC242 (Tweedie et al., 2009; http://flybase.org/), PBac{RB}RfC38^e00704/P{lacW}RfC38^k13807 flies are viable (our unpublished observations). Thus, PBac{RB}RfC38^e00704 seems to carry a Nup160 mutation but not a loss-of-function RfC38 mutation. In this report, therefore, we refer to this insertion mutation as Nup160^e00704. We also omit the transposon symbols by designating insertion mutants as Nup160^EP372, Nup160^SH2055, and RfC38^k13807. Newly established excision mutants derived from Nup160^EP372 were named Nup160^EP372M# or Df(2L)Nup160EP372M# if the mutant has a deletion (# stands for digit numbers); named Df(2L)Nup160M# if the derived mutation does not retain any sequences derived from the Nup160^EP372 insertion.

Generation of excision mutations

The P{EP} element, which includes the mini-white gene (w⁺), was excised from Nup160^EP372 in the germline of males in the w background by conventional methods using the defective P element Δ2–3 as the transposase source (Robertson et al., 1988). A total of 219 white-eyed males were screened. The original Nup160^EP372 and 24 homozygous lethal derivatives were maintained using the CyO balancer chromosome. Homozygous viable derivatives seemed to be revertants and were discarded except for 10 lines kept as controls. The genetic symbol w is omitted hereafter unless such indication is necessary, because all experiments were conducted in the w background.

Genetic characterization of mutations

To check whether derivatives have mutations in Nup160 and the neighboring gene RfC38, they were made heterozygous with Nup160^EP372, Nup160^e00704, and RfC38^k13807. At the opposite end of Nup160, the region containing Csl4s has not been examined genetically, because no appropriate mutations or deletions are known. Flies heterozygous for Nup160^sim and each derivative were produced by crossing introgression carrier females (Int(2L)D+S, Nup160^sim/CyO) to male derivative heterozygotes (Fig. 1B), and morphological anomalies (abdomen, wing, and bristle defects) and female fertility were examined as described (Sawamura et al., 2010). To test hybrid inviability, derivative carrier females were crossed to D. simulans Lhr males (Fig. 1C). All experiments were conducted at 25°C.

Molecular characterization of mutations

In the original homozygous lethal Nup160^EP372 chromosome, an 8-kb P{EP} element is inserted in the reverse orientation into the 5’UTR of the Nup160 gene, which is also in the forward orientation adjacent to the 5’UTR of the Csl4 gene, at the site designated 2L: 11,123,814–11,123,822 (Berkeley Drosophila Genome Project coordinates, http://genome.ucsc.edu/). GCCGGTGCC is the target site duplication of the P element.

DNA was extracted from derivative homozygotes (if viable) or heterozygotes with CyO, and DNA fragments around the Nup160^EP372 insertion site were amplified with the polymerase chain reaction (PCR). PCR primers and conditions are available upon request. When PCR products were separated on an agarose gel, a single band (in homozygotes) or double bands (in heterozygotes, one from the mutation allele and the other from the wild-type Nup160 allele on CyO) were expected. In four homozygotes and ten heterozygotes, the target DNA band from each gel was purified and sequenced. In nine heterozygous derivatives, double bands were not obtained despite the use of several primer pairs, presumably because of a large deletion or a large P element remnant. In these cases, DNA was extracted from derivative carrier flies heterozygous for Int(2L)D+S, Nup160^sim, and the PCR products of regions of interest (outside the large deletion or the large P element remnant) were directly sequenced. Heterozygosity (derived from D. melanogaster and D. simulans alleles) suggests that the derivative retains the corresponding region. If DNA from the adjacent positions to the insertion (~2L: 11,123,793 or 2L: 11,123,885~) was present in the derivative chromosome, a partial P remnant was suspected to remain at either side of the Nup160^EP372 insertion site.

Derivatives having exactly the same sequences were treated as being from the same excision event if they were descendants of a single start vial containing target males that carried both Nup160^EP372 and Δ2–3. If they were descendants of independent start vials, they were treated as independent mutations, because the same excision event may have occurred more than once.

Determination of hybrid lethal phase

We made a y w; Int(2L)D+S/CyO, y⁺ strain by conventional crosses. To determine the lethal phase of Nup160^sim carrier hybrid males, heterozygous females were crossed to D. simulans Lhr males (Fig. 2), and the viability of yellow (y) offspring (i.e., Int(2L)D+S carrier males) was examined at different developmental stages. All other offspring must have the y⁺ phenotype, which is distinguishable from y by mouth hook and denticle bands color during early development. Because sexing larvae by the size of gonadal imaginal discs is difficult in sterile interspecific hybrids (Shen, 1932), larvae were sexed based on Malpighian tubule color, which was white (w) in males.

Measurement of total development time

There is a possibility that homozygous Nup160^sim introgression affects not only female reproduction but also non-reproductive characteristics (e.g., development) in both sexes. To examine if Nup160^sim homozygotes develop normally, eggs were collected at 2-hr intervals from Int(2L)D+S/CyO females crossed with Int(2L)D+S/CyO males, and emerging flies of introgression homozygotes and heterozygotes were counted every 2 hr.

Fig. 2.

Cross used to examine the phase of hybrid lethality caused by Nup160^sim. Int, Int(2L)D+S, Nup160^sim. Open chromosome regions are from D. melanogaster; shaded regions are from D. simulans. Introgression carrier hybrid males (circled) are phenotypically distinguishable from the others.

RESULTS

Nup160^EP372 excisions

Thirty-four derivative strains were established by transposon excision from the recessive semi-lethal Nup160^EP372. Among the ten homozygous viable derivatives examined, seven were complete revertants (Nup160^EP372rev) that retained no transposon sequences (strains M164, M177, M187, M188, M195, M209, and M249). Nup160^EP372M206 retained a 31-bp insertion remnant (transposon footprint), and Nup160^EP372M230 (and M241, potentially of the same origin) had a 276-bp footprint.

Among 24 homozygous lethal derivatives (Table 1), 18 contained insertion remnants. Nup160^EP372M18 (and M25, M54, M55, M64, M75) and Nup160^EP372M121 (and M123) each had a 53-bp footprint; Nup160^EP372M85 had a 249-bp footprint; and Nup160^EP372M161 had a 926-bp footprint. The others, Nup160^EP372M26, Nup160^EP372M39, Nup160^EP372M94, Nup160^EP372M133, Nup160^EP372M142, Nup160^EP372M185 (and M215), and Nup160^EP372M227, presumably retained long partial transposon insertions not amplified by PCR. Among them, Nup160^EP372M133 and Nup160^EP372M142 must be functional revertants, because heterozygotes with Nup160^EP372 or Nup160^e00704 were viable, indicating that these strains carry recessive lethal mutations elsewhere in the second chromosome.

Table 1. Molecular, genetic, and phenotypic characteristics of homozygous lethal Nup160^EP372 derivatives

Nup160EP372 derivativea	Insertion/deletionb	Csl4c	Nup160		RfC38f	Hybrid viabilityg	Female fertilityh	Morphological anomalyi	Mutation typej
			EP372d	e00704e
EP372 originalk	+8 kbp	+	(–)	(–)	+	V	F	No	Class iii
M180	+13 bp; –451 bp	+	(–)	(–)	+	L	S	Yes	Null
M190 (M201, M203)	–881 bp	–	–	–	+	L	S	Yes	Null
M69	+2,083 bp; –2,890 bp	+	–	–	+	L	S	Yes	Null
M219	–nd; +?	–	–	–	+	L	F	No	Class v
M18 (M25, M54, M55, M64, M75)	+53 bp	+	(+)	–	+	L	S	No	Class ii
M121 (M123)	+53 bp	+	(+)	–	+	L	S	No	Class ii
M85	+249 bp	+	–	–	+	L	S	Yes	Class i
M161	+926 bp	+	(+)	(–)	+	V	F	No	Class iii
M26	+nd	+	(–)	–	+	V	F	No	Class iii
M39	+nd	+	(–)	–	+	V	F	No	Class iii
M94	+nd	+	–	(–)	+	V	F	No	Class iii
M185 (M215)	+nd	+	–	(–) or –	+	V	F	No	Class iii
M227	+nd	+	(–)	–	+	V	S	No	Class iv
M133	+nd	+	+	+	+	V	F	No	Revertant
M142	+nd	+	+	+	+	V	F	No	Revertant

^a Derivatives potentially of same origin noted in parentheses.

^b +, insertion; –, deletion; nd, size not determined; +?, presence unknown.

^c Based on sequence data: +, complete; –, absent or disrupted.

^d Complementation test versus Nup160^EP372: –, lethal (relative viability 0); (–), leaky (0.01–0.16); (+), not completely viable (0.23–0.62); +, viable (0.81–1.07).

^e Complementation test versus Nup160^e00704: –, lethal (relative viability 0); (–), leaky (0.004–0.05); +, viable (1.24–1.27).

^f Complementation test versus RfC38^k13807: +, viable.

^g Male viability in derivative/CyO females × D. simulans Lhr males: L, lethal; V, viable.

^h Fertility of In(2L)D+S/derivative heterozygous females: S, sterile; F, fertile.

ⁱ Abdomen, wing, and bristle defects in In(2L)D+S/derivative heterozygotes (for detailed descriptions see Sawamura et al., 2010).

^j Classified by the genotypes and phenotypes (hybrid viability, female sterility, morphological anomaly): Class i, L, S, Yes; Class ii, L, S, No; Class iii, V, F, No; Class iv, V, S, No; Class v, L, F, No.

^k Data after Sawamura et al. (2010).

The other six homozygous inviable derivatives had deletions (Table 1, Fig. 3). Df(2L)Nup160EP372M180 was a 451-bp deletion (2L: 11,123,823–11,124,273; 5’UTR to exon 3 of Nup160) that retained a 13-bp fragment of the P{EP} element. Df(2L)Nup160M190 (and M201, M203) was an 881-bp deletion (2L: 11,123,413–11,124,293; 5’UTR to exon 3 of Nup160 and 5′UTR to exon 2 of Csl4). Df(2L)Nup160EP372M69 was a 2,890-bp deletion (2L: 11,123,823–11,126,712; 5’UTR to exon 9 of Nup160) that retained a 2,083-bp fragment of the P{EP} element. Df(2L)Nup160EP372M219 was a large deletion (left breakpoint not determined); Csl4, CG14921, and CG6230 are absent (at least partially for the latter locus). And it is unknown whether this deficiency retains a partial sequence of the P{EP} element. Df(2L)Nup160EP372M180, Df(2L)Nup160M190, and Df(2L)Nup160EP372M69 must be null mutations of the Nup160 gene, because several exons from the beginning are absent. As the molecular data suggested, none of the derivatives included mutations in the RfC38 gene; heterozygotes with RfC38^k13807 were viable.

Fig. 3.

Deficiencies produced by excision of Nup160^EP372. Open reading frames (full or partial) of Csl4, Nup160, and RfC38 are shown at the top (exons are numbered). Brackets, deficiency breakpoints; triangle, original Nup160^EP372 insertion; partial triangles, transposon remnants.

Phenotypic effects of derivatives

Hybrid viability, female fertility, and several aspects of adult morphology were examined using appropriate genotypes of the Nup160^EP372 derivatives as shown in Tables 2 and 3. The results are summarized in Table 1. The original Nup160^EP372 does not lead to hybrid inviability, female sterility, or morphological anomalies (Tang and Presgraves, 2009; Sawamura et al., 2010). The three null mutations of Nup160 (i.e., Df(2L)Nup160EP372M180, Df(2L)Nup160M190, and Df(2L)Nup160EP372M69) exhibited hybrid inviability, female sterility, and morphological anomalies. Thus, the previous conclusion that Nup160^sim, not the introgression of Csl4 or RfC38, is responsible for hybrid inviability and female sterility (Tang and Presgraves, 2009; Sawamura et al., 2010) was confirmed. Furthermore, it is now apparent that morphological anomalies were caused by the same gene, which was not conclusive in the previous analysis (Sawamura et al., 2010). The results for Nup160^EP372M85 were the same as for the three nulls, suggesting that this might also be a null mutation (class i). Nup160^EP372M18 and Nup160^EP372M121 exhibited hybrid inviability and female sterility, but not morphological anomaly. These might be partial loss-of-function mutations (class ii).

Table 2. Fertility of females heterozygous for In(2L)D+S, Nup160^sim and Nup160^EP372 derivatives

Nup160EP372 derivative	Fertilitya	Eggs collected	Eggs hatched	% Hatched
M18	S	–	–	–
M25	S	–	–	–
M26	F	100	81	81.0
M39	F	200	144	72.0
M54	S	–	–	–
M55	S	–	–	–
M64	S	–	–	–
M69	S	–	–	–
M75	S	109	0	0
M85	S	–	–	–
M94	F	200	126	63.0
M121	S	–	–	–
M123	S	–	–	–
M133	F	140	95	67.9
M142	F	250	205	82.0
M161	F	210	149	71.1
M180	S	–	–	–
M185	F	100	90	90.0
M190	S	–	–	–
M201	S	–	–	–
M203	S	–	–	–
M215	F	100	85	85.0
M219	F	225	147	65.3
M227	S	100	0	0

^a Determined by emergence of adults when mated to Oregon-R males: F, fertile; S, sterile.

Table 3. Hybrid viability of crosses between females heterozygous for Nup160^EP372 derivatives and CyO and D. simulans Lhr males

Nup160EP372 derivative	Females			Males
	Cy	Cy+	Viabilitya	Cy	Cy+	Viabilitya
M18	53	54	1.02	45	0	0
M25	498	487	0.98	67	0	0
M26	76	55	0.72	52	63	1.21
M39	135	185	1.37	42	52	1.24
M54	128	106	0.82	79	0	0
M55	269	249	0.93	81	0	0
M64	52	62	1.19	30	0	0
M69	65	78	1.20	37	0	0
M75	184	212	1.15	34	0	0
M85	67	65	0.97	32	0	0
M94	188	186	0.99	77	65	0.84
M121	418	424	1.01	32	0	0
M123	74	61	0.82	45	0	0
M133	109	105	0.96	18	70	3.89
M142	221	214	0.97	69	140	2.03
M161	101	111	1.10	48	25	0.52
M180	88	104	1.18	51	0	0
M185	134	150	1.12	71	83	1.17
M190	69	72	1.04	30	0	0
M201	77	67	0.87	52	0	0
M203	41	55	1.34	29	0	0
M215	62	73	1.18	36	36	1.00
M219	127	113	0.89	41	0	0
M227	201	204	1.01	58	19	0.32

^a Viability of Cy⁺ flies relative to Cy flies was calculated as the number of Cy⁺ flies divided by the number of Cy flies.

Nup160 revertants (i.e., Nup160^EP372M133 and Nup160^EP372M142) did not exhibit hybrid inviability, female sterility, or morphological anomaly. This, too, is consistent with the conclusion that Nup160^sim is responsible for the three phenotypes. The results for Nup160^EP372M161, Nup160^EP372M26, Nup160^EP372M39, Nup160^EP372M94, and Nup160^EP372M185 were the same as for the revertants. They are apparently not Nup160 revertants because heterozygotes with Nup160^EP372 or Nup160^e00704 were lethal, but they behave like revertants in terms of hybrid phenotypes (class iii). This is similar to the original Nup160^EP372.

Interestingly, the remaining two derivatives exhibited different combinations of hybrid phenotypes, although none affected morphology. Nup160^EP372M227 resulted in complete female sterility but not hybrid inviability (class iv). This is similar to Nup160^SH2055, which leads to incomplete female sterility but has no effect on hybrid inviability (Sawamura et al., 2010). Df(2L)Nup160EP372M219 exhibited the opposite trend, resulting in hybrid inviability but not in female sterility (class v).

Developmental analyses

In crosses between y w; Int(2L)D+S/CyO, y⁺ females and D. simulans Lhr males (Fig. 2), Int(2L)D+S, Nup160^sim carrier males (phenotypically yellow white) were observed in the third instar larval and early pupal stages but not as late pupae and adults (Table 4). Thus, the lethal phase of the Nup160^sim carrier males seems to be during the early pupal stage.

Table 4. Cross of y w; Int(2L)D+S/CyO, y⁺ females and D. simulans Lhr males

Larvae collecteda					Adults eclosed
Phenotype	1st instar	2nd instar	3rd instar	Total	Phenotype	Number	% Eclosed
y+ w+	1	5	105	111	y+ w+ Cy females	57	99.1b
					y+ w+ Cy+ females	53
y+ w	31	30	49	110	y+ w Cy males	72	65.5
y w	20	23	105	148	y w Cy+ males	0c	0

^a Simultaneously collected; developmental speed difference among phenotypes might be reflected.

^b Total of In(2L)D+S carrier and CyO carrier females.

^c Lethal at the early pupal stage.

The total duration of development was 10.7 hr longer in female introgression homozygotes than in female heterozygotes (t = 6.104, df = 153, P = 8.15 × 10^–9), and 13.9 hr longer in male introgression homozygotes than in male heterozygotes (t = 6.430, df = 130, P = 2.23 × 10^–9; Table 5). Thus, a recessive gene (or genes) on the introgression chromosome makes development slower in both females and males.

Table 5. Total duration of development

Genotype	Sex	Mean ± SE (hr)	n
Int(2L)D+S/CyO	Female	227.7 ± 1.0	110
Int(2L)D+S/Int(2L)D+S	Female	238.4 ± 1.2	45
Int(2L)D+S/CyO	Male	231.1 ± 1.2	94
Int(2L)D+S/Int(2L)D+S	Male	245.0 ± 1.8	38

DISCUSSION

We obtained 13 imprecise recessive lethal excisions from Nup160^EP372 and examined their effects on hybrid viability, female fertility, and morphology in the appropriate genotypes. Our results confirm previous observations that the D. simulans allele of the Nup160 gene (Nup160^sim), not introgression of Csl4 or RfC38, is responsible for hybrid inviability and female sterility in crosses between D. simulans and D. melanogaster (Tang and Presgraves, 2009; Sawamura et al., 2010). In addition, we discovered that morphological anomalies are also caused by Nup160^sim. The Nup160^EP372 derivatives resulted in variable hybrid phenotypes, ranged from class i to class v. For example, the class v derivative affected hybrid viability more severely than female fertility, whereas the class iv derivative had the opposite effect. All the exons of Nup160 were intact in class i–v derivatives; the derivatives differed in the partial transposon remnants found in the 5’UTR or in a deletion of adjacent sequences. Such exogenous sequences might negatively regulate Nup160 expression both temporally and spatially. There is also a possibility that Df(2L)Nup160EP372M219 lacks an upstream regulatory region of Nup160. These mutations partially complement the hybrid phenotypes and will be useful in future analyses to examine the developmental mechanisms of hybrid inviability and female sterility.

In the present analysis, the lethal phase of the Nup160^sim carrier hybrid males was determined to be during the early pupal stage, which is later than that for regular hybrid males from crossing D. melanogaster females with D. simulans males (i.e., those not rescued by Lhr or Hmr; Sturtevant, 1920; Hadorn, 1961; Bolkan et al., 2007). And it has been suggested that Nup160^sim does not directly interact with the rescuing genes Lhr and Hmr, but rather that Nup160^sim results in hybrid inviability through an independent genetic system (Tang and Presgraves, 2009; Sawamura et al., 2010). Also in the present analysis, a recessive gene (or genes) on the Int(2L)D+S introgression was found to slow development of the homozygous carriers. Nup160^sim is a candidate, although we cannot rule out the possibility that other linked genes are responsible. Because homozygous (or hemizygous) Nup160^sim in the D. melanogaster genetic background results in not only female sterility but also morphological anomalies in both sexes, it is not a surprise that the same gene perturbs development in a pleiotropic manner. The Nup160^sim gene is apparently involved in multiple reproductive isolation phenotypes in the cross between D. melanogaster and D. simulans.

ACKNOWLEDGMENT

We are grateful to the Bloomington, Exelixis, Kyoto, and Szeged Drosophila stock centers for providing fly strains. This work was supported by a Grant-in-Aid for Scientific Research (21570001) from the Japan Society for the Promotion of Science to K. S.

REFERENCES

•  Adams,  M. D.,  Celniker,  S. E.,  Holt,  R. A.,  Evans,  C. A.,  Gocayne,  J. D.,  Amanatides,  P. G.,  Scherer,  S. E.,  Li,  P. W.,  Hoskins,  R. A.,  Galle,  R. F., et al. (2000) The genome sequence of Drosophila melanogaster. Science 287, 2185–2195.
•  Barbash,  D. A. (2010) Ninety years of Drosophila melanogaster hybrids. Genetics 186, 1–8.
•  Barbash,  D. A., and  Ashburner,  M. (2003) A novel system of fertility rescue in Drosophila hybrids reveals a link between hybrid lethality and female sterility. Genetics 163, 217–226.
•  Barbash,  D. A.,  Siino,  D. F.,  Tarone,  A. M., and  Roote,  J. (2003) A rapidly evolving MYB-related protein causes species isolation in Drosophila. Proc. Natl. Acad. Sci. USA 100, 5302–5307.
•  Bolkan,  B. J.,  Booker,  R.,  Goldberg,  M. L., and  Barbash,  D. A. (2007) Developmental and cell cycle progression defects in Drosophila hybrid males. Genetics 177, 2233–2241.
•  Brideau,  N. J.,  Flores,  H. A.,  Wang,  J.,  Maheshwari,  S.,  Wang,  X., and  Barbash,  D. A. (2006) Two Dobzhansky-Muller genes interact to cause hybrid lethality in Drosophila. Science 314, 1292–1295.
• Drosophila 12 Genomes Consortium (2007) Evolution of genes and genomes on the Drosophila phylogeny. Nature 450, 203–218.
•  Ferree,  P. M., and  Barbash,  D. A. (2009) Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila. PLoS Biol. 7, e1000234.
•  Hadorn,  E. (1961) Zur Autonomie und Phasenspezifität der Latalität von Bastarden zwischen Drosophila melanogaster und Drosophila simulans. Rev. Suisse Zool. 68, 197–207.
•  Hutter,  P., and  Ashburner,  M. (1987) Genetic rescue of inviable hybrids between Drosophila melanogaster and its sibling species. Nature 327, 331–333.
•  Masly,  J. P.,  Jones,  C. D.,  Noor,  M. A. F.,  Locke,  J., and  Orr,  H. A. (2006) Gene transposition as a cause of hybrid sterility in Drosophila. Science 313, 1448–1450.
•  Muller,  H. J., and  Pontecorvo,  G. (1940) Recombinants between Drosophila species the F₁ hybrids of which are sterile. Nature 146, 199–200.
•  Presgraves,  D. C. (2003) A fine-scale genetic analysis of hybrid incompatibilities in Drosophila. Genetics 163, 955–972.
•  Presgraves,  D. C.,  Balagopalan,  L.,  Abmayr,  S. M., and  Orr,  H. A. (2003) Adaptive evolution drives divergence of a hybrid inviability gene between two species of Drosophila. Nature 423, 715–719.
•  Prigent,  S.,  Matsubayashi,  H., and  Yamamoto,  M. T. (2009) Transgenic Drosophila simulans strains prove the identity of the speciation gene Lethal hybrid rescue. Genes Genet. Syst. 84, 353–360.
•  Provine,  W. B. (1991) Alfred Henry Sturtevant and crosses between Drosophila melanogaster and Drosophila simulans. Genetics 129, 1–5.
•  Quackenbush,  L. S. (1910) Unisexual broods of Drosophila. Science 32, 183–185.
•  Robertson,  H. M.,  Preston,  C. R.,  Phillis,  R. W.,  Johnson-Schlitz,  D. M.,  Benz,  W. K., and  Engels,  W. R. (1988) A stable genomic source of P-element transposase in Drosophila melanogaster. Genetics 118, 461–470.
•  Sawamura,  K. (2000) Genetics of hybrid inviability and sterility in Drosophila: The Drosophila melanogaster-Drosophila simulans case. Plant Species Biol. 15, 237–247.
•  Sawamura,  K. (2012) Chromatin evolution and molecular drive in speciation. Int. J. Evol. Biol. 2012, 301894.
•  Sawamura,  K.,  Yamamoto,  M. T., and  Watanabe,  T. K. (1993) Hybrid lethal systems in the Drosophila melanogaster species complex. II. The Zygotic hybrid rescue (Zhr) gene of D. melanogaster. Genetics 133, 307–313.
•  Sawamura,  K.,  Karr,  T. L., and  Yamamoto,  M. T. (2004) Genetics of hybrid inviability and sterility in Drosophila: dissection of introgression of D. simulans genes in D. melanogaster genome. Genetica 120, 253–260.
•  Sawamura,  K.,  Maehara,  K.,  Mashino,  S.,  Kagesawa,  T.,  Kajiwara,  M.,  Matsuno,  K.,  Takahashi,  A., and  Takano-Shimizu,  T. (2010) Introgression of Drosophila simulans Nuclear Pore Protein 160 in Drosophila melanogaster alone does not cause inviability but does cause female sterility. Genetics 186, 669–676.
•  Shen,  T. H. (1932) Zytologische Untersuchungen über Sterilität bei Männchen von Drosophila melanogaster und bei F₁-Männchen der Kreuzung zwischen D. simulans-Weibchen und D. melanogaster-Männchen. Z. Zellforsch. Mikrosk. Anat. 15, 547–580.
•  Sturtevant,  A. H. (1919) A new species closely resembling Drosophila melanogaster. Psyche 26, 153–155.
•  Sturtevant,  A. H. (1920) Genetic studies on Drosophila simulans. I. Introduction. Hybrids with Drosophila melanogaster. Genetics 5, 488–500.
•  Tang,  S., and  Presgraves,  D. C. (2009) Evolution of the Drosophila nuclear pore complex results in multiple hybrid incompatibilities. Science 323, 779–782.
•  Tweedie,  S.,  Ashburner,  M.,  Falls,  K.,  Leyland,  P.,  McQuilton,  P.,  Marygold,  S.,  Millburn,  G.,  Osumi-Sutherland,  D.,  Schroeder,  A.,  Seal,  R., et al. (2009) FlyBase: enhancing Drosophila Gene Ontology annotations. Nucleic Acids Res. 37, D555–D559.
•  Watanabe,  T. K. (1979) A gene that rescues the lethal hybrids between Drosophila melanogaster and D. simulans. Jpn. J. Genet. 54, 325–331.