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Gene Transposition as a Cause of Hybrid Sterility in Drosophila

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Science  08 Sep 2006:
Vol. 313, Issue 5792, pp. 1448-1450
DOI: 10.1126/science.1128721

Abstract

We describe reproductive isolation caused by a gene transposition. In certain Drosophila melanogasterD. simulans hybrids, hybrid male sterility is caused by the lack of a single-copy gene essential for male fertility, JYAlpha. This gene is located on the fourth chromosome of D. melanogaster but on the third chromosome of D. simulans. Genomic and molecular analyses show that JYAlpha transposed to the third chromosome during the evolutionary history of the D. simulans lineage. Because of this transposition, a fraction of hybrids completely lack JYAlpha and are sterile, representing reproductive isolation without sequence evolution.

Reproductive isolation can be a by-product of divergent evolution between populations and is a necessary step in speciation. Dobzhansky and Muller described a model for the evolution of reproductive isolation wherein functional divergence between interacting loci in different lineages yields incompatible interactions in their hybrids (1, 2). Although evidence for Dobzhansky-Muller interactions is well established, few genes involved in these incompatibilities have been identified and characterized (3). Furthermore, it remains unclear whether molecular evolutionary processes other than functional divergence cause postzygotic reproductive isolation (2).

The genetic basis of postzygotic isolation between Drosophila melanogaster and D. simulans has been studied previously by crossing triploid D. melanogaster females to heavily x-irradiated D. simulans males (4). This approach avoided the normal sterility and inviability of D. melanogasterD. simulans F1 hybrids, producing hybrids with backcross-like genotypes. One of these individuals, a fertile female, was used to establish a stock that carried the tiny “dot” fourth chromosome of D. simulans in an otherwise D. melanogaster genetic background. Hybrid males homozygous for the D. simulans fourth (4-sim) chromosome were completely sterile because mature sperm were immotile (5). Because the fourth chromosome does not recombine during meiosis (6), the location of the hybrid sterility gene(s) was mapped by using deletions and translocations to cytological regions 101E to 101F and/or 102A5 to 102B5 (5, 7).

It remained unclear, however, whether 4-sim hybrid male sterility was genuine or an artifact of the x-irradiation used to construct the hybrid stock. By using recently characterized mutations that rescue the viability (8) and fertility (9) of D. melanogasterD. simulans F1 hybrid females, we introgressed a new D. simulans fourth chromosome into an otherwise D. melanogaster background without use of radiation (Materials and Methods).

Hybrid male fertility in the new 4-sim introgression line was scored by both sperm motility and number of offspring sired by individual males (Materials and Methods). Heterozygous 4-sim males produce abundant motile sperm, whereas homozygous 4-sim males typically produce immotile sperm (Table 1); sterile hybrid males thus show the same spermatogenic phenotype as those described previously (5). Although pure species D. melanogaster and heterozygous 4-sim males (eyD/4-sim) produce many off-spring [232.1 ± 21.5 (SEM), N = 13, and 157.7 ± 13.9, N = 15, respectively], homozygous 4-sim males produce none (0.0 ± 0.0, N = 39). In females, the 4-sim chromosome has no effect on fertility. The D. simulans fourth chromosome thus causes true hybrid male sterility.

Table 1.

Hybrid male sterility and deficiency mapping. eyD indicates eyeless-Dominant mutation.

GenotypeSperm motilityχ2
ManyFewNone
eyD/4-sim 43 50 18 96.31View inline
4-sim/4-sim 0 32 92
Df(4)M101-62f/eyD 110 175 36 289.2View inline
Df(4)M101-62f/4-sim 0 59 211
Df(4)Gley D 87 71 11 3.22
Df(4)G/4-sim 146 118 8
Df(4)ED6369/ey D 29 8 0 1.10
Df(4)ED6369/4-sim 82 15 1
Df(4)ED6366/ey D 105 36 1 2.47
Df(4)ED6366/4-sim 132 66 2
Df(4)ED6364/ey D 205 34 1 9.09View inline
Df(4)ED6364/4-sim 125 43 2
Df(4)Δ3M/ey D 112 13 0 4.63
Df(4)Δ3M/4-sim 94 22 1
Df(4)Δ9M/ey D 131 11 0 1.98
Df(4)Δ9M/4-sim 99 12 1
  • View inline*** P « 0.0001.

  • View inline* Although marginally significant (P < 0.05), this deficiency does not uncover the severe “none” sperm motility phenotype seen in 4-sim hybrids.

  • By using chromosomal deficiencies, we confirmed previous results (5, 7) showing that the gene(s) causing hybrid male sterility resides within Df(4)M101-62f, which includes the proximal-most 21 genes on chromosome 4 (10). We dissected this region further by using deficiencies and genomic sequence data unavailable to earlier workers (Fig. 1). None of the new deficiencies uncovers hybrid sterility (Table 1 and Fig. 1). Assuming that 4-sim hybrid sterility is caused by a single gene, we excluded all loci distal to cubitus interruptus as the cause of sterility. We also excluded plexinB on the basis of complementation tests. Our results thus show that one of the requisite loci for 4-sim hybrid male sterility lies proximal to plexinB even if multiple genes within Df(4)M101-62f are involved (Fig. 1).

    Fig. 1.

    Chromosome 4 region uncovered by Df(4)M101-62f. Horizontal bars represent deficiencies. White bars show deficiencies that uncover hybrid sterility when heterozygous with 4-sim; black bars show deficiencies that fail to uncover sterility when heterozygous with 4-sim. Df(4)G (not pictured) lies distal to Df(4)M101-62f and uncovers region 102E2 to 102F2. The distal breakpoint of Df(4)ED6369 extends beyond Df(4)M101-62f. Map adapted from Entrez Genomes Build 4.3 (28).

    JYAlpha (CG17923), a 4.1-kb gene that encodes the alpha subunit of a Na+ and K+ adenosine triphosphatase (Na+/K+ ATPase), a transmembrane protein involved in ion exchange (11), was identified as a strong candidate from the four remaining loci in the region. Four mammalian isoforms of the Na+/K+ ATPase alpha subunit exist (12). One of these, α4, is expressed exclusively in testes (13) and is essential for sperm motility (14). JYAlpha from D. melanogaster (JYAlphamel) shows ∼60% amino acid identity to mouse α4.

    To test whether JYAlpha is the cause of 4-sim hybrid male sterility, we performed complementation tests with the 4-sim chromosome by using a P-element insertion in JYAlphamel (P{y+,w+}JYAlpha). Because P{y+,w+}JYAlpha does not appear to be a null mutation (Table 2), we remobilized the element. Two of the resulting excisions were chosen for analysis. JYAlphamel.8c truncated JYAlphamel after the first 268 amino acids, excluding the presumed active site. JYAlphamel.12a restored wild-type sequence at JYAlphamel. JYAlphamel.8c/4-sim males were sterile, and their sperm motility resembled that of Df(4)M101-62f/4-sim (Table 2). As expected, JYAlphamel.12a/4-sim males were fully fertile (Table 2). JYAlpha is thus both necessary and sufficient for hybrid male sterility. Further analysis shows that JYAlpha is essential for sperm motility within D. melanogaster (Table 2).

    Table 2.

    JYAlpha complementation tests. ciD, cubitus interruptus-Dominant mutation; eyD, eyeless-Dominant mutation.

    GenotypeSperm motilityχ2
    ManyFewNone
    ciD/4-sim 91 45 10 173.1View inline
    P{y+,w+}JYAlpha/4-sim 18 110 148
    P{y+,w+}JYAlpha/ey D 71 61 27 115.7View inline
    P{y+,w+}JYAlpha/4-sim 6 114 116
    P{y+,w+}JYAlpha/ci D 186 20 2 313.1View inline
    P{y+,w+}JYAlpha/P{y+,w+}JYAlpha 17 174 57
    ciD/4-sim 94 25 3 173.4View inline
    JYAlphamel.8c/4-sim 0 53 78
    JYAlphamel.12a/ciD 67 13 0 2.91
    JYAlphamel.12a/4-sim 90 9 1
    JYAlphamel.8c/ciD 112 33 3 151.7View inline
    JYAlphamel.8c/JYAlphamel.8c 0 37 53
    JYAlphamel.12a/ciD 67 18 5 8.35View inline
    JYAlphamel.12a/JYAlphamel.12a 92 8 2
  • View inline*** P << 0.0001.

  • View inline* Although marginally significant (P < 0.05), the effect is in the wrong direction and probably reflects a mild ciD marker effect.

  • Genes causing hybrid incompatibilities often evolve rapidly and show population genetic signs of divergence under positive natural selection (3, 1517). However, preliminary analysis of JYAlpha revealed an apparent difference in its location between D. simulans and D. melanogaster. A BLAST search of the D. simulans whole genome assembly (18) suggests that JYAlpha is flanked proximally by CG9766 and distally by complexin (cpx), two loci that reside on the right arm of the third chromosome (3R) in D. melanogaster, D. simulans, and their sister species (Fig. 2A). This 3R-linked locus represents the single best BLAST hit for JYAlpha (reciprocal e values = 0.0) and appears to be JYAlpha's only location in D. simulans. We confirmed these results in several ways.

    Fig. 2.

    JYAlpha chromosomal locations. (A)(Top) D. melanogaster chromosome 4. JYAlphamel (exons in red) appears to be the proximal-most gene on 4 (10). Shared sequences between both D. melanogaster chromosome 4 and D. simulans chromosome arm 3R are coded yellow. Upstream of JYAlphamel, chromosome 4 becomes highly repetitive (striped) and then “centromeric” (black). Arrows indicate the direction of transcription. (Bottom) D. simulans chromosome arm 3R. About 1.6 kb of sequence showing weak homology to sequence found on all major chromosomes (white) is present upstream and downstream of JYAlphasim (red). This sequence shows no significant homology to known DNA-mediated transposable elements (29). 3R material including CG9766 and cpx is shown in gray. Half arrows give approximate primer locations for PCR across the 3R-4 breakpoints. Coding sequence between JYAlphamel and JYAlphasim shows no signature of divergence by positive selection, at least as crudely measured by the ratio of amino-acid changing to non-amino-acid changing substitutions: Ka/Ks = 0.05, consistent with purifying selection. (B) PCR-amplified region across the 3R-4 CG9766-JYAlphasim breakpoint. Analogous results were obtained from the JYAlphasim-cpx breakpoint. Divergence timesareshownatspeciationeventsonthe phylogeny. The branch onto which the JYAlpha transposition event maps is bolded. yak indicates D. yakuba; mel, D. melanogaster; sim, D. simulans; sech, D. sechellia; maur, D. mauritiana; and 4-sim, 4-sim homozygotes.

    To determine JYAlpha's chromosomal location in D. simulans, we performed crosses to track genetically the chromosome with which JYAlpha segregates. Strain-specific molecular markers in JYAlphasim confirmed that the locus segregates with chromosome 3 (Materials and Methods).

    To confirm JYAlpha's precise location within the third chromosome of D. simulans, we attempted to amplify polymerase chain reaction (PCR) product across the putative 3R-4 break-points from pure D. simulans C167.4, control pure D. melanogaster, and homozygous 4-sim hybrids. Amplification across both the proximal (CG9766-JYAlphasim) and the distal (JYAlphasim-cpx) breakpoints succeeded in pure D. simulans but failed in pure D. melanogaster and in homozygous 4-sim hybrids, as expected if JYAlpha resides on 3R in D. simulans but on 4 in D. melanogaster (Fig. 2B).

    Next, we sequenced a large region of 3R from pure D. simulans. In particular, we sequenced ∼9.8 kb from D. simulans C167.4; this region extends from CG9766 proximally to cpx distally. In D. melanogaster, CG9766 and cpx are adjacent genes on 3R (19). In D. simulans, however, the region between CG9766 and cpx is interrupted by JYAlpha (Fig. 2A). JYAlpha is the only gene found on chromosome 4 of D. melanogaster that resides in this region of 3R in D. simulans (fig. S2).

    We also attempted to PCR-amplify JYAlpha from homozygous 4-sim hybrids. We were unable to PCR-amplify JYAlpha product from homozygous 4-sim hybrids with use of any primer pairs, despite routine amplification from purespecies D. melanogaster and D. simulans individuals. This confirms that an intact JYAlpha locus does not exist on the D. simulans fourth chromosome.

    Lastly, we asked whether JYAlpha is singlecopy. We already possess strong genomic and genetic evidence that D. melanogaster carries a single functional copy of JYAlpha (10) (Table 2). We also performed a Southern blot analysis of pure D. melanogaster, pure D. simulans, and homozygous 4-sim flies. As expected from genome sequence data, JYAlpha appears to be single-copy in both species (fig. S3). Also as expected, no hybridization was observed in our Southern blot for homozygous 4-sim hybrids (fig. S3). This again demonstrates that no intact JYAlpha resides on chromosome 4 of D. simulans.

    The cause of 4-sim hybrid male sterility appears, therefore, to be surprisingly simple. A copy of JYAlpha exists on the fourth chromosome of D. melanogaster but not of D. simulans. Thus, a heterozygous 4-sim hybrid male carries one D. melanogaster chromosome 4 and remains fertile, whereas a homozygous 4-sim hybrid male lacks JYAlpha and is sterile. The sterility of this hybrid genotype reflects the complete absence of a locus essential for male fertility.

    To determine the evolutionary direction of JYAlpha's transposition, we performed PCR assays across the 3R-4 breakpoints in several species closely related to D. melanogaster. PCR amplification across both the proximal and distal break points succeeded in D. sechellia and D. mauritiana, showing that these species also carry JYAlpha on 3R (Fig. 2B). The D. sechellia genome also confirmed that JYAlpha resides on 3R in this species [Supporting Online Material (SOM) Text and fig. S2]. Amplification across the 3R-4 breakpoints did not succeed, however, in D. yakuba. Consistent with this, genome sequence data show that JYAlpha resides on the fourth chromosome in this species (SOM Text). Thus, JYAlpha appears to have resided ancestrally on chromosome 4 and was transposed to chromosome arm 3R after the split of D. melanogaster from the simulans clade species but before the split of D. simulans from its sister species. This dates JYAlpha's transposition to roughly 0.3 to 5.5 million years ago (20, 21).

    The transposition of JYAlpha raises several evolutionary questions. Because the transposition is evolutionarily old (at least ∼3 × 106 generations), it is unlikely that population genetic data would allow detection of a selective sweep associated with this event (22). Although the coding region of JYAlpha shows no obvious signs of divergence by positive natural selection between D. melanogaster and D. simulans (Fig. 2A legend), we cannot exclude a history of selection at this locus. Similarly, we cannot infer the exact mechanism of JYAlpha's transposition. We can, however, rule out retroposition, because JYAlphasim possesses introns (Fig. 2A).

    It has been hypothesized that movement of gene function between chromosomes might cause postzygotic isolation, either by simple transposition or translocation (1) or by gene duplication-transposition followed by divergent evolution (23). Although gene duplication-transposition events are fairly common in Drosophila (2426), our results suggest that JYAlpha is currently single-copy in both D. melanogaster and D. simulans. But it seems likely that a JYAlpha duplication existed sometime during the evolutionary history of the simulans clade. In any case, JYAlpha represents a clear example of a gene transposition causing reproductive isolation.

    These findings raise the possibility that gene transposition could be important in the evolution of reproductive isolation. Although the present example sterilizes only a fraction of F2 hybrids and has no effect on the fertility of F1 hybrids, an analogous gene transposition event between the sex chromosomes could sterilize or kill F1 hybrids between allopatric populations. If, for example, a Y-linked gene essential for male fertility transposed to the X chromosome, crosses between transposed and nontransposed populations would yield sterile F1 hybrid males in one direction of the hybridization, consistent with Haldane's rule (27). Similarly, transpositions between sex chromosomes and autosomes, or between autosomes, could affect a fraction of hybrid backcross or F2 genotypes (23). Gene transposition events between chromosomes need not, therefore, be common to have a large effect on hybrid fitness, because any hybrid that lacks a single essential gene would be inviable or sterile. The transposition of essential genes could represent a largely overlooked cause of reproductive isolation.

    Supporting Online Material

    www.sciencemag.org/cgi/content/full/313/5792/1448/DC1

    Materials and Methods

    SOM Text

    Figs. S1 to S3

    Tables S1 to S2

    References

    References and Notes

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