Genes Genet. Syst. (2012) 87, p. 99–106 Genetic dissection of 160 (Nup160), a involved in multiple phenotypes of reproductive isolation in Drosophila

Kazunori Maehara1, Takayuki Murata1, Naoki Aoyama2, Kenji Matsuno2,3 and Kyoichi Sawamura4* 1Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan 2Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan 3Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan 4Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

(Received 11 December 2011, accepted 14 February 2012)

Previous reports have suggested that the Nucleoporin 160 (Nup160) gene of Drosophila simulans (Nup160sim) 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}Nup160EP372 insertion mutation of D. melanogaster. Null mutations of the Nup160 gene resulted in the three phenotypes caused by Nup160sim, but rever- tants 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 under- standing of the developmental mechanisms of reproductive isolation. Based on our analyses with the Nup160sim introgression line, the lethal phase of hybrid invi- ability was determined to be during the early pupal stage. Our analysis also sug- gested that homozygous Nup160sim in D. melanogaster leads to slow development. Thus, Nup160sim is involved in multiple aspects of reproductive isolation between these two species.

Key words: Drosophila, hybrid inviability, hybrid sterility, reproductive isola- tion, speciation

onic development. Sturtevant (1920) intercrossed the INTRODUCTION species using anomalies and was able to A century ago, Quackenbush (1910) claimed to have deduce the genetic causes of hybrid inviability, but “the observed unisexual broods in Drosophila melanogaster. complete sterility of surviving F1 hybrids frustrated It turned out later that his D. melanogaster flies actually Sturtevant and his vision of comprehensively exploring included two species, D. melanogaster and D. simulans the genetics of interspecific differences” (Barbash, 2010). (Sturtevant, 1919). The beauty of the latter new species Genetic tricks and the serendipitous discovery of rescue was that it could be crossed with D. melanogaster, the mutations were needed before further studies could shed most-studied and best-understood species of that genus light on his questions (Provine, 1991; Sawamura, 2000; (Provine, 1991). In crosses between D. melanogaster Barbash, 2010). females and D. simulans males, only sterile female Thanks to recent advances in molecular biology tech- hybrids are obtained, as male hybrids die during larval niques and genomic sequencing (Adams et al., 2000; Dros- development. In the reciprocal cross, sterile male ophila 12 Genomes Consortium, 2007), detailed study of hybrids appear, as most female hybrids die during embry- speciation has become feasible. As a result, several for hybrid inviability and sterility have recently Edited by Etsuko Matsuura been isolated in this pair of species and characterized at * Corresponding author. E-mail: [email protected] the molecular level (reviewed in Sawamura, 2012). The 100 K. MAEHARA et al.

D. melanogaster gene Hybrid male rescue (Hmr) encodes (Nup160) causes inviability and female sterility when a DNA-binding that is involved in hybrid inviabil- introgressed into D. melanogaster; the hybrid males with ity and female sterility (Hutter and Ashburner, 1987; the introgression (or deficiency) cannot be rescued by Lhr Barbash and Ashburner, 2003; Barbash et al., 2003). D. and introgression homozygous (or hemizygous) females simulans Lethal hybrid rescue (Lhr) encodes a heterochro- are sterile (Tang and Presgraves, 2009; Sawamura et al., matin protein that causes hybrid inviability (Watanabe, 2010). 1979; Brideau et al., 2006; Prigent et al., 2009). D. mel- Nup160 , like the other genes, has been mapped by anogaster zygotic hybrid rescue (zhr) consists of hetero- recombination and deficiencies, identified by complemen- chromatic 359-bp repetitive sequences and causes hybrid tation tests against mutations, and confirmed by gene inviability in crosses involving D. simulans females transformation (Sawamura, 2000; Presgraves, 2003; (Sawamura et al., 1993; Ferree and Barbash, 2009). Sawamura et al., 2004, 2010; Tang and Presgraves, 2009). JYalpha, a gene located on different in D. Three recessive lethal insertion mutations of Nup160 melanogaster and D. simulans, causes male sterility of have been reported in D. melanogaster (Fig. 1A; Tweedie introgression homozygotes (Muller and Pontecorvo, 1940; et al., 2009; http://flybase.org/). PBac{RB}RfC38e00704 Masly et al., 2006). D. simulans Nucleoporin 96 (Nup96) uncovers both hybrid inviability and female sterility when causes inviability when it is hemizygous in the hybrid; heterozygous with the wild-type allele of D. simulans, the hybrid males cannot be rescued by the Lhr mutation Nup160sim (Tang and Presgraves, 2009; Sawamura et al., (Presgraves et al., 2003). D. simulans Nucleoporin 160 2010). P{EP}Nup160EP372, which is synonymous with

Fig. 1. Transposon insertions in the Nup160 gene and mating schemes to examine the effects of Nup160EP372 derivatives. A, Posi- tions 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, Nup160sim; *, Nup160EP372 derivative. Reproductive isolation in Drosophila 101

P{EP}CG4738EP372, does not lead to hybrid inviability or neighboring gene RfC38, they were made heterozygous female sterility (Tang and Presgraves, 2009; Sawamura with Nup160EP372, Nup160e00704, and RfC38k13807. At the et al., 2010). Interestingly, P{lacW}l(2)SH2055SH2055 opposite end of Nup160, the region containing Csl4s has does not lead to hybrid inviability but does partially lead not been examined genetically, because no appropriate to hybrid female sterility (Sawamura et al., 2010). These mutations or deletions are known. Flies heterozygous for three mutations raise questions about why they behave Nup160sim and each derivative were produced by crossing differently and about the possibility of distinct mecha- introgression carrier females (Int(2L)D+S, Nup160sim/CyO) nisms underlying hybrid inviability and female sterility. to male derivative heterozygotes (Fig. 1B), and morphologi- In the present study, we excised the P transposable ele- cal anomalies (abdomen, wing, and bristle defects) and ment from the P{EP}Nup160EP372 insertion and examined female fertility were examined as described (Sawamura et whether the new mutations cause hybrid inviability and al., 2010). To test hybrid inviability, derivative carrier female sterility. Because Nup160 has been implicated as females were crossed to D. simulans Lhr males (Fig. 1C). the cause of morphological anomalies by deficiency map- All experiments were conducted at 25°C. ping (Sawamura et al., 2010), we also examined the abdo- men, wings, and bristles of flies heterozygous for Molecular characterization of mutations In the Nup160sim and excision derivatives. Finally, we deter- original homozygous lethal Nup160EP372 chromosome, an mined the lethal phase of hybrid males and measured the 8-kb P{EP} element is inserted in the reverse orientation total duration of development of homozygous carriers. 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 MATERIALS AND METHODS (Berkeley Drosophila Genome Project coordinates, http:// Strain nomenclature Although PBac{RB}RfC38e00704/ genome.ucsc.edu/). GCCGGTGCC is the target site Df(2L)BSC242 and P{lacW}RfC38k13807/Df(2L)BSC242 duplication of the P element. are lethal because of the absence of Nup160 and RfC38 on DNA was extracted from derivative homozygotes (if via- Df(2L)BSC242 (Tweedie et al., 2009; http://flybase.org/), ble) or heterozygotes with CyO, and DNA fragments PBac{RB}RfC38e00704/P{lacW}RfC38k13807 flies are viable (our around the Nup160EP372 insertion site were amplified with unpublished observations). Thus, PBac{RB}RfC38e00704 the polymerase chain reaction (PCR). PCR primers and seems to carry a Nup160 mutation but not a loss-of-func- conditions are available upon request. When PCR prod- tion RfC38 mutation. In this report, therefore, we refer ucts were separated on an agarose gel, a single band (in to this insertion mutation as Nup160e00704. We also omit homozygotes) or double bands (in heterozygotes, one from the transposon symbols by designating insertion mutants the mutation allele and the other from the wild-type as Nup160EP372, Nup160SH2055, and RfC38k13807. Newly Nup160 allele on CyO) were expected. In four homozy- established excision mutants derived from Nup160EP372 gotes and ten heterozygotes, the target DNA band from were named Nup160EP372M# or Df(2L)Nup160EP372M# if each gel was purified and sequenced. In nine heterozy- the mutant has a deletion (# stands for digit numbers); gous derivatives, double bands were not obtained despite named Df(2L)Nup160M# if the derived mutation does not the use of several primer pairs, presumably because of a retain any sequences derived from the Nup160EP372 inser- large deletion or a large P element remnant. In these tion. cases, DNA was extracted from derivative carrier flies heterozygous for Int(2L)D+S, Nup160sim, and the PCR Generation of excision mutations The P{EP} element, products of regions of interest (outside the large deletion which includes the mini-white gene (w+), was excised from or the large P element remnant) were directly sequenced. Nup160EP372 in the germline of males in the w background Heterozygosity (derived from D. melanogaster and D. sim- by conventional methods using the defective P element ulans alleles) suggests that the derivative retains the cor- Δ2–3 as the transposase source (Robertson et al., 1988). responding region. If DNA from the adjacent positions A total of 219 white-eyed males were screened. The orig- to the insertion (~2L: 11,123,793 or 2L: 11,123,885~) was inal Nup160EP372 and 24 homozygous lethal derivatives present in the derivative chromosome, a partial P rem- were maintained using the CyO balancer chromosome. nant was suspected to remain at either side of the Homozygous viable derivatives seemed to be revertants Nup160EP372 insertion site. and were discarded except for 10 lines kept as controls. Derivatives having exactly the same sequences were The genetic symbol w is omitted hereafter unless such treated as being from the same excision event if they were indication is necessary, because all experiments were con- descendants of a single start vial containing target males ducted in the w background. that carried both Nup160EP372 and Δ2–3. If they were descendants of independent start vials, they were treated Genetic characterization of mutations To check as independent mutations, because the same excision whether derivatives have mutations in Nup160 and the event may have occurred more than once. 102 K. MAEHARA et al.

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 Nup160sim 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 Nup160sim introgression affects not only female reproduction but also non-repro- Fig. 2. Cross used to examine the phase of hybrid lethality caused by Nup160sim. Int, Int(2L)D+S, Nup160sim. Open chro- ductive characteristics (e.g., development) in both sexes. mosome regions are from D. melanogaster; shaded regions are from D. simulans. Introgression carrier hybrid males (circled) are phenotypically distinguishable from the others.

Table 1. Molecular, genetic, and phenotypic characteristics of homozygous lethal Nup160EP372 derivatives

Nup160 EP372 a b c f Hybrid Female Morphological Mutation Nup160 derivative Insertion/deletion Csl4 RfC38 g h i j EP372d e00704e viability fertility anomaly type

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 Nup160EP372: –, 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 Nup160e00704: –, lethal (relative viability 0); (–), leaky (0.004–0.05); +, viable (1.24–1.27). f Complementation test versus RfC38k13807: +, 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). Reproductive isolation in Drosophila 103

To examine if Nup160sim homozygotes develop normally, Nup160EP372 or Nup160e00704 were viable, indicating that eggs were collected at 2-hr intervals from Int(2L)D+S/ these strains carry recessive lethal mutations elsewhere CyO females crossed with Int(2L)D+S/CyO males, and in the second chromosome. emerging flies of introgression homozygotes and heterozy- The other six homozygous inviable derivatives had gotes were counted every 2 hr. 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 RESULTS the P{EP} element. Df(2L)Nup160M190 (and M201, Nup160EP372 excisions Thirty-four derivative strains M203) was an 881-bp deletion (2L: 11,123,413– were established by transposon excision from the reces- 11,124,293; 5’UTR to exon 3 of Nup160 and 5′UTR to exon sive semi-lethal Nup160EP372. Among the ten homozy- 2 of Csl4). Df(2L)Nup160EP372M69 was a 2,890-bp gous viable derivatives examined, seven were complete deletion (2L: 11,123,823–11,126,712; 5’UTR to exon 9 of revertants (Nup160EP372rev) that retained no transposon Nup160) that retained a 2,083-bp fragment of the P{EP} sequences (strains M164, M177, M187, M188, M195, element. Df(2L)Nup160EP372M219 was a large dele- M209, and M249). Nup160EP372M206 retained a 31-bp inser- tion (left breakpoint not determined); Csl4, CG14921, and tion remnant (transposon footprint), and Nup160EP372M230 CG6230 are absent (at least partially for the latter (and M241, potentially of the same origin) had a 276-bp ). And it is unknown whether this deficiency footprint. retains a partial sequence of the P{EP} element. Among 24 homozygous lethal derivatives (Table 1), 18 Df(2L)Nup160EP372M180, Df(2L)Nup160M190, and contained insertion remnants. Nup160EP372M18 (and M25, Df(2L)Nup160EP372M69 must be null mutations of the M54, M55, M64, M75) and Nup160EP372M121 (and M123) Nup160 gene, because several exons from the beginning each had a 53-bp footprint; Nup160EP372M85 had a 249-bp are absent. As the molecular data suggested, none of the footprint; and Nup160EP372M161 had a 926-bp footprint. derivatives included mutations in the RfC38 gene; The others, Nup160EP372M26, Nup160EP372M39, Nup160EP372M94, heterozygotes with RfC38k13807 were viable. Nup160EP372M133, Nup160EP372M142, Nup160EP372M185 (and M215), and Nup160EP372M227, presumably retained long Phenotypic effects of derivatives Hybrid viability, partial transposon insertions not amplified by PCR. female fertility, and several aspects of adult morphology Among them, Nup160EP372M133 and Nup160EP372M142 must were examined using appropriate genotypes of the be functional revertants, because heterozygotes with Nup160EP372 derivatives as shown in Tables 2 and 3. The

Fig. 3. Deficiencies produced by excision of Nup160EP372. Open reading frames (full or partial) of Csl4, Nup160, and RfC38 are shown at the top (exons are numbered). Brackets, deficiency breakpoints; triangle, original Nup160EP372 insertion; partial triangles, transpo- son remnants. 104 K. MAEHARA et al.

Table 2. Fertility of females heterozygous for In(2L)D+S, Table 3. Hybrid viability of crosses between females heterozy- Nup160sim and Nup160EP372 derivatives gous for Nup160EP372 derivatives and CyO and D. simulans Lhr males Nup160EP372 Eggs Eggs Fertilitya % Hatched derivative collected hatched Nup160EP372 Females Males M18 S – – – derivative Cy Cy+ Viabilitya Cy Cy+ Viabilitya M25 S – – – M18 53 54 1.02 45 0 0 M26 F 100 81 81.0 M25 498 487 0.98 67 0 0 M39 F 200 144 72.0 M26 76 55 0.72 52 63 1.21 M54 S – – – M39 135 185 1.37 42 52 1.24 M55 S – – – M54 128 106 0.82 79 0 0 M64 S – – – M55 269 249 0.93 81 0 0 M69 S – – – M64 52 62 1.19 30 0 0 M75 S 109 0 0 M69 65 78 1.20 37 0 0 M85 S – – – M75 184 212 1.15 34 0 0 M94 F 200 126 63.0 M85 67 65 0.97 32 0 0 M121 S – – – M94 188 186 0.99 77 65 0.84 M123 S – – – M121 418 424 1.01 32 0 0 M133 F 140 95 67.9 M123 74 61 0.82 45 0 0 M142 F 250 205 82.0 M133 109 105 0.96 18 70 3.89 M161 F 210 149 71.1 M142 221 214 0.97 69 140 2.03 M180 S – – – M161 101 111 1.10 48 25 0.52 M185 F 100 90 90.0 M180 88 104 1.18 51 0 0 M190 S – – – M185 134 150 1.12 71 83 1.17 M201 S – – – M190 69 72 1.04 30 0 0 M203 S – – – M201 77 67 0.87 52 0 0 M215 F 100 85 85.0 M203 41 55 1.34 29 0 0 M219 F 225 147 65.3 M215 62 73 1.18 36 36 1.00 M227 S 100 0 0 M219 127 113 0.89 41 0 0 a Determined by emergence of adults when mated to Oregon- M227 201 204 1.01 58 19 0.32 R males: F, fertile; S, sterile. a Viability of Cy+ flies relative to Cy flies was calculated as the number of Cy+ flies divided by the number of Cy flies. results are summarized in Table 1. The original Nup160EP372 does not lead to hybrid inviability, female Nup160 revertants (i.e., Nup160EP372M133 and sterility, or morphological anomalies (Tang and Nup160EP372M142) did not exhibit hybrid inviability, female Presgraves, 2009; Sawamura et al., 2010). The three sterility, or morphological anomaly. This, too, is consis- null mutations of Nup160 (i.e., Df(2L)Nup160EP372M180, tent with the conclusion that Nup160sim is responsible for Df(2L)Nup160M190, and Df(2L)Nup160EP372M69) exhib- the three phenotypes. The results for Nup160EP372M161, ited hybrid inviability, female sterility, and morphological Nup160EP372M26, Nup160EP372M39, Nup160EP372M94, and anomalies. Thus, the previous conclusion that Nup160EP372M185 were the same as for the revertants. Nup160sim, not the introgression of Csl4 or RfC38, is They are apparently not Nup160 revertants because responsible for hybrid inviability and female sterility heterozygotes with Nup160EP372 or Nup160e00704 were (Tang and Presgraves, 2009; Sawamura et al., 2010) was lethal, but they behave like revertants in terms of hybrid confirmed. Furthermore, it is now apparent that mor- phenotypes (class iii). This is similar to the original phological anomalies were caused by the same gene, Nup160EP372. which was not conclusive in the previous analysis Interestingly, the remaining two derivatives exhibited (Sawamura et al., 2010). The results for Nup160EP372M85 different combinations of hybrid phenotypes, although were the same as for the three nulls, suggesting that this none affected morphology. Nup160EP372M227 resulted in might also be a null mutation (class i). Nup160EP372M18 complete female sterility but not hybrid inviability (class and Nup160EP372M121 exhibited hybrid inviability and iv). This is similar to Nup160SH2055, which leads to incom- female sterility, but not morphological anomaly. These plete female sterility but has no effect on hybrid inviabil- might be partial loss-of-function mutations (class ii). ity (Sawamura et al., 2010). Df(2L)Nup160EP372M219 Reproductive isolation in Drosophila 105

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+ Cy females 57 + + b y w 1 5 105 111 + + + 99.1 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.

Table 5. Total duration of development affected hybrid viability more severely than female fer- tility, whereas the class iv derivative had the opposite Genotype Sex Mean ± SE (hr) n effect. All the exons of Nup160 were intact in class i–v Int(2L)D+S/CyO Female 227.70 ± 1. 110 derivatives; the derivatives differed in the partial trans- Int(2L)D+S/Int(2L)D+S Female 238.42 ± 1. 45 poson remnants found in the 5’UTR or in a deletion of ± Int(2L)D+S/CyO Male 2231.1 1. 94 adjacent sequences. Such exogenous sequences might Int(2L)D+S/Int(2L)D+S Male 8245.0 ± 1. 38 negatively regulate Nup160 expression both temporally and spatially. There is also a possibility that exhibited the opposite trend, resulting in hybrid inviabil- Df(2L)Nup160EP372M219 lacks an upstream regulatory ity but not in female sterility (class v). region of Nup160. These mutations partially comple- ment the hybrid phenotypes and will be useful in future Developmental analyses In crosses between y w; analyses to examine the developmental mechanisms of Int(2L)D+S/CyO, y+ females and D. simulans Lhr males hybrid inviability and female sterility. (Fig. 2), Int(2L)D+S, Nup160sim carrier males (phenotypi- In the present analysis, the lethal phase of the cally yellow white) were observed in the third instar lar- Nup160sim carrier hybrid males was determined to be dur- val and early pupal stages but not as late pupae and ing the early pupal stage, which is later than that for reg- adults (Table 4). Thus, the lethal phase of the Nup160sim ular hybrid males from crossing D. melanogaster females carrier males seems to be during the early pupal stage. with D. simulans males (i.e., those not rescued by Lhr or The total duration of development was 10.7 hr longer in Hmr; Sturtevant, 1920; Hadorn, 1961; Bolkan et al., female introgression homozygotes than in female 2007). And it has been suggested that Nup160sim does heterozygotes (t = 6.104, df = 153, P = 8.15 × 10–9), and not directly interact with the rescuing genes Lhr and 13.9 hr longer in male introgression homozygotes than in Hmr, but rather that Nup160sim results in hybrid invia- male heterozygotes (t = 6.430, df = 130, P = 2.23 × 10–9; bility through an independent genetic system (Tang and Table 5). Thus, a recessive gene (or genes) on the intro- Presgraves, 2009; Sawamura et al., 2010). Also in the gression chromosome makes development slower in both present analysis, a recessive gene (or genes) on the females and males. Int(2L)D+S introgression was found to slow development of the homozygous carriers. Nup160sim is a candidate, although we cannot rule out the possibility that other DISCUSSION linked genes are responsible. Because homozygous (or We obtained 13 imprecise recessive lethal excisions hemizygous) Nup160sim in the D. melanogaster genetic from Nup160EP372 and examined their effects on hybrid background results in not only female sterility but also viability, female fertility, and morphology in the appro- morphological anomalies in both sexes, it is not a surprise priate genotypes. Our results confirm previous obser- that the same gene perturbs development in a pleiotropic vations that the D. simulans allele of the Nup160 gene manner. The Nup160sim gene is apparently involved in (Nup160sim), not introgression of Csl4 or RfC38, is multiple reproductive isolation phenotypes in the cross responsible for hybrid inviability and female sterility in between D. melanogaster and D. simulans. crosses between D. simulans and D. melanogaster (Tang and Presgraves, 2009; Sawamura et al., 2010). In addi- We are grateful to the Bloomington, Exelixis, Kyoto, and tion, we discovered that morphological anomalies are Szeged Drosophila stock centers for providing fly strains. This also caused by Nup160sim. The Nup160EP372 derivatives work was supported by a Grant-in-Aid for Scientific Research (21570001) from the Japan Society for the Promotion of Science resulted in variable hybrid phenotypes, ranged from to K. S. class i to class v. For example, the class v derivative 106 K. MAEHARA et al.

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