A Fine-Scale Genetic Analysis of Hybrid Incompatibilities in Drosophila
a Department of Biology, University of Rochester, Rochester, New York 14627^4?*[a:, 百拇医药
ABSTRACT^4?*[a:, 百拇医药
The sterility and inviability of species hybrids is thought to evolve by the accumulation of genes that cause generally recessive, incompatible epistatic interactions between species. Most analyses of the loci involved in such hybrid incompatibilities have suffered from low genetic resolution. Here I present a fine-resolution genetic screen that allows systematic counting, mapping, and characterizing of a large number of hybrid incompatibility loci in a model genetic system. Using small autosomal deletions from D. melanogaster and a hybrid rescue mutation from D. simulans, I measured the viability of hybrid males that are simultaneously hemizygous for a small region of the D. simulans autosomal genome and hemizygous for the D. melanogaster X chromosome. These hybrid males are exposed to the full effects of any recessive-recessive epistatic incompatibilities present in these regions. A screen of ~ 70% of the D. simulans autosomal genome reveals 20 hybrid-lethal and 20 hybrid-semilethal regions that are incompatible with the D. melanogaster X. In further crosses, I confirm the epistatic nature of hybrid lethality by showing that all of the incompatibilities are rescued when the D. melanogaster X is replaced with a D. simulans X. Combined with information from previous studies, these results show that the number of recessive incompatibilities is approximately eightfold larger than the number of dominant ones. Finally, I estimate that a total of ~ 191 hybrid-lethal incompatibilities separate D. melanogaster and D. simulans, indicating extensive functional divergence between these species' genomes.
THE last decade has seen important progress in the genetics of speciation. In particular, there is now broad agreement on three aspects of intrinsic postzygotic isolation, the sterility and inviability of species hybrids::hw, 百拇医药
Hybrid fitness problems evolve gradually as incompatible epistatic interactions accumulate between species (COYNE and ORR 1989 , COYNE and ORR 1997 ; SASA et al. 1998 ; PRESGRAVES 2002 ; PRICE and BOUVIER 2002 ). The so-called Dobzhansky-Muller model posits that incompatibilities evolve by the substitution of advantageous or neutral mutations in one species, which, having never been tested in combination with those in other species, have harmful effects when brought together in hybrids (DOBZHANSKY 1937 ; MULLER 1940 , MULLER 1942 ; ORR 1995 , ORR 1996 ).:hw, 百拇医药
The alleles involved in these hybrid incompatibilities are thought to be, on average, partially recessive. This "dominance theory" neatly accounts for several phenomena, including Haldane's rule (the observation that XY hybrids typically suffer more severe hybrid problems than do XX hybrids; HALDANE 1922 ) and F2 hybrid breakdown (MULLER 1942 ; ORR 1993 ; TURELLI and ORR 1995 , TURELLI and ORR 2000 ).
Incompatibilities causing hybrid male sterility accumulate faster than those causing other types of hybrid fitness problems (WU and DAVIS 1993 ; HOLLOCHER and WU 1996 ; TRUE et al. 1996 ; WU et al. 1996 ; NAVEIRA and MASIDE 1998 ; PRESGRAVES and ORR 1998 ; WU and HOLLOCHER 1998 ; SINGH 1999 ). The "faster-male" theory posits that the rapid evolution of hybrid male sterility is caused by the faster divergence of male-specific fertility genes, perhaps driven by sexual selection, or by the inherent sensitivity of spermatogenesis to the genetic perturbations experienced by hybrids (WU and DAVIS 1993 ).vz5:|^, http://www.100md.com
Nevertheless, progress in the fine-genetic and molecular characterization of hybrid incompatibility loci has been slow. Despite much effort, few studies have succeeded in precisely and systematically counting, fine mapping, and, ultimately, identifying a large number of "speciation genes." The reason is that the traits of interest, hybrid sterility and inviability, are by their very nature barriers to crossing and thus are refractory to standard genetic approaches. This problem is especially severe in one of our best model organisms, the fruit fly Drosophila melanogaster: All hybrids between D. melanogaster and its closest known relatives (D. simulans, D. mauritiana, and D. sechellia) are completely dead or sterile (STURTEVANT 1920 , STURTEVANT 1929 ; LACHAISE et al. 1986 ). Consequently, nearly a century's worth of accumulated genetic tools has not been brought to bear on the genetics of speciation, and genetic analyses of the D. melanogaster-D. simulans hybridization, despite an 80-year history, have suffered poor genetic resolution. The rough portrait that has emerged from this work is that a large number of hybrid male steriles but a modest number of hybrid lethals have accumulated between D. melanogaster and D. simulans (MULLER and PONTECORVO 1940 , MULLER and PONTECORVO 1942 ; PONTECORVO 1943A , PONTECORVO 1943B ; PROVINE 1991 ; SANCHEZ et al. 1994 ; COYNE et al. 1998 ; SAWAMURA et al. 2000 ; SAWAMURA 2000 ).
Two methods have been used to circumvent the problematic sterility and inviability of D. melanogaster species hybrids. First, the discovery of hybrid rescue mutations—alleles that restore the viability and fertility of normally unfit hybrids—raised the possibility of introgressing foreign genes into a D. melanogaster background, where they might be further analyzed (DAVIS et al. 1996 ). Unfortunately, hybrid female fertility rescue has proved extremely weak, and so far only two small introgressions of the D. simulans second chromosome have been studied (SAWAMURA et al. 2000 ). Second, COYNE et al. 1998 used a battery of deficiencies (small chromosomal deletions) from D. melanogaster to uncover recessive D. simulans factors causing hybrid lethality in otherwise heterozygous F1 females. Their screen of ~ 50% of the D. simulans genome revealed only a handful of hybrid lethals, none of which were unconditionally lethal. One reason why so few were detected may be that the screen was limited to incompatibilities involving a hemizygous (recessive) factor from D. simulans and a heterozygous (dominant) one from D. melanogaster (elsewhere in the genome). If most incompatibility alleles are recessive, as the dominance theory posits, then the most abundant class of hybrid incompatibility should be that in which the interacting loci are both homozygous or both hemizygous, i.e., genotypes normally produced only in F2 (or backcross) hybrids.
Here I present a new screen that modifies and combines these two approaches, taking advantage of both a hybrid rescue mutation from D. simulans and the genetic tools of D. melanogaster to test for the presence of these extreme recessive-recessive hybrid incompatibilities. In particular, I test the viability of F1 hybrid males hemizygous for a small region of the D. simulans autosomal genome and simultaneously hemizygous for the D. melanogaster X. These males are exposed to the full effects of any recessive-recessive X-autosome hybrid incompatibilities in the regions tested. Using the many deficiencies available in D. melanogaster, I systematically screened most of the D. simulans autosomal genome for such incompatibilities.'lmx(, 百拇医药
Deficiency mapping in hybrids offers several advantages over recombination mapping. First, large numbers of the relevant genotypes can be assayed in the F1 generation. Second, the existence and precise location of hybrid lethals in most cases can be easily confirmed using overlapping but independently derived deficiencies with defined cytological breakpoints on the polytene chromosome map (BRIDGES 1935 ). Third, the fitness effects of hybrid incompatibilities can be assessed without being confounded with recombination distance between the relevant locus and a genetic marker. Finally, and perhaps most important, the locations of hybrid lethals can be quickly narrowed to such small chromosome regions that the next step—identifying the particular genes causing hybrid lethality—becomes routine.
The present large-scale, fine-resolution screen reveals the existence of many new hybrid lethals between D. melanogaster and D. simulans. In later work, these hybrid lethals will be the subject of molecular study. Here, I use these data to address the following questions:;f@v, http://www.100md.com
How many hybrid lethals separate D. melanogaster and D. simulans? This number is a proxy for functional divergence between the viability-essential components of the two species' genomes.;f@v, http://www.100md.com
Where are the hybrid lethals? Are they randomly distributed throughout the genome or clustered in particular regions?;f@v, http://www.100md.com
Is hybrid lethality caused by epistasis between incompatible loci, as predicted by the Dobzhansky-Muller model?;f@v, http://www.100md.com
Are most hybrid incompatibilities recessive, as predicted by the dominance theory? There are few direct tests of the dominance of incompatible alleles. Furthermore, comparing the number of recessive hybrid lethals discovered here with the number of dominant ones from other studies provides a quantitative test of the dominance theory.
What is the probability that any two divergent substitutions, one from D. melanogaster and one from D. simulans, are incompatible, causing hybrid lethality?6, http://www.100md.com
What is the distribution of fitness effects of hybrid-lethal incompatibilities? Do most have weak or strong effects on viability? How does this distribution compare with that for hybrid male sterility, which often appears to have a polygenic basis (NAVEIRA and MASIDE 1998 ; WU and HOLLOCHER 1998 )?6, http://www.100md.com
At what stage of development do hybrid lethals act? If genes acting early in development are more evolutionarily constrained than later-acting ones, and thus less diverged, then embryonic hybrid lethals should be rarer than postembryonic ones.6, http://www.100md.com
MATERIALS AND METHODS6, http://www.100md.com
A screen for X-autosome incompatibilities:6, http://www.100md.com
The screening method is diagrammed in and represents a modification of the crossing design of COYNE et al. 1998 . Using many D. melanogaster (mel) stocks, each heterozygous for a deficiency chromosome with known cytological breakpoints and a dominantly marked balancer chromosome (LINDSLEY and ZIMM 1992 ), I crossed mel Deficiency/Balancer (Df/Bal) females to D. simulans (sim) males carrying the hybrid rescue mutation, Lethal hybrid rescue (Lhr). Lhr rescues normally dead F1 males from lethality that typically occurs at the larval-pupal transition (WATANABE 1979 ; TAKAMURA and WATANABE 1980 ; SAWAMURA et al. 1993 ). Half of the F1 hybrid progeny inherit the deficiency and half inherit the balancer. If hybrids of both sexes appear with both Df and Bal genotypes in roughly equal proportions, then no recessive hybrid lethal resides in the sim region uncovered by the deficiency. If, on the other hand, only Df hybrid males die, then a hybrid lethal resides in the sim autosomal region exposed by the deficiency. The fact that only Df hybrid males die, while their Df hybrid sisters do not, suggests either that the sim hybrid lethal is incompatible with a mostly recessive factor on the mel X (females are heterozygous for the X) or that the sim hybrid lethal is male specific. These possibilities are distinguished using follow-up crosses described below.
fig.ommitted(?s*&04, http://www.100md.com
Figure 1. Crosses used in F1 deficiency screen for lethal hybrid incompatibilities. Sex chromosomes and one set of representative autosomes are shown. Gray, D. melanogaster; white, D. simulans. (A) Deficiency/Balancer females crossed to Lhr males produce (in the absence of hybrid lethality) four types of offspring in Mendelian ratios within each sex. (B) Attached-X Deficiency/Balancer female crossed to Lhr male. See text for further details.(?s*&04, http://www.100md.com
shows the 193 deficiencies used. Together these deficiencies uncover ~ 69–77% of 2L (chromosome 2, left arm), 49–59% of 2R, 72–77% of 3L, and 65–79% of 3R. I did not test the small-dot fourth chromosome of D. simulans as it is known to carry no hybrid lethals (MULLER and PONTECORVO 1940 , MULLER and PONTECORVO 1942 ; PONTECORVO 1943A ; ORR 1992 ). All stocks are available through the Bloomington or Umeå Stock Centers (Bloomington Stock Center, ). Soichi Tanda kindly provided a stock with a newly extracted Df(2L)al. The most common dominant markers on chromosome 2 balancers were CurlyO and Glazed, and those on chromosome 3 balancers were Stubble, Serrate, Tubby, or a combination. When possible, deficiency stocks with a chromosome 3 balancer marked only with Tubby or Ultrabithorax were rebalanced over a different chromosome with a more easily scored marker (e.g., Stubble). Complete descriptions of all stocks, including the particular balancers used, are available upon request.
fig.ommitted)!i}, 百拇医药
Table 1. Results from deficiency screen for hybrid-lethal X-autosome incompatibilities)!i}, 百拇医药
Each species cross was made by mass mating 15–20 mel Df/Bal females with 15–25 sim Lhr males. All crosses were done at 24° and flies were reared on standard cornmeal-yeast-agar medium. For each deficiency, I repeated the cross at least four times and report the pooled number of progeny. Contamination was detectable in several ways: consistency among replicate crosses, the segregation of dominant markers among progeny, the presence of diagnostic hybrid male genitalia, and the absolute sterility of all progeny. Although the cross shown in is in principle straightforward, some stocks showed strong sexual isolation, requiring many replicate crosses to obtain sufficient progeny.)!i}, 百拇医药
Most crosses deviated from the expected 1:1:1:1 segregation ratio of Df females, Bal females, Df males, and Bal males, because Lhr rescue of hybrid males is incomplete. Hybrid males were significantly rarer than females in most crosses, with an average of 45% rescue. In a few crosses, no hybrid males were rescued (see also GRANADINO et al. 1996 ); since these crosses are not informative, they are not reported here. For crosses that produced hybrid females and at least 10 Bal hybrid males, I estimated the relative viability of the deficiency by simply tabulating the ratio of Df:Bal progeny for each hybrid sex. I used 2 tests to detect deviations from the expected 1:1 ratio of Df:Bal progeny within each sex and Fisher's exact tests to detect differences in the ratio of Df:Bal progeny between sexes. I defined candidate X-autosome hybrid incompatibilities as those causing a significant deficit of Df hybrid males and a significantly smaller Df:Bal ratio in hybrid males than in hybrid females. I then classified hybrid incompatibilities by viability following the scheme of Crow and colleagues for deleterious mutations within species (reviewed in SIMMONS and CROW 1977 ; CROW and SIMMONS 1983 ): For regions causing significant reductions in Df hybrid male viability, those with viabilities "50% (i.e., a Df:Bal ratio <0.50 at least for one of two overlapping deficiencies) were considered hybrid "semilethals," and those with viabilities of "10% were considered hybrid "lethals."
Further investigation of hybrid-lethal deficiencies:1iu.9.$, http://www.100md.com
I performed three further tests for most of the regions that caused hybrid lethality. First, to ensure that the lethality of deficiencies is hybrid specific, I confirmed the viability of deficiencies in nonhybrids by crossing Df/Bal mel females to wild type, Wolbachia-free Canton-S mel males. (Note, however, that the very existence of the deficiency stocks shows that they are not lethal when heterozygous within species.)1iu.9.$, http://www.100md.com
Second, when possible, I confirmed the presence of each hybrid lethal by testing at least one other overlapping deficiency. Such confirmation with multiple, independently derived deficiencies rules out the possibility that a putative hybrid lethal is an artifact of the genetic background of any particular stock. Those regions shown to be lethal by a single deficiency, but which could not be confirmed because overlapping deficiencies were unavailable, should be viewed as candidate hybrid-lethal regions.
Third, I tested whether the exposed sim factors caused lethality by epistasis with the mel X. To do this, I constructed mel attached-X stocks, each carrying a hybrid-lethal deficiency over a balancer, and crossed females from these stocks to sim Lhr males. As shows, the attached-X chromosome forces mother-to-daughter inheritance of the mel attached-X and father-to-son inheritance of the sim free-X. Hybrid males from this cross thus inherit a sim X rather than a mel X, while the rest of their genotype, including their cytoplasm, remains identical in species origin to the hybrid males from the original cross (compare F1 males in and ). The important point is that if Df hybrid male lethality is caused by an incompatibility between a recessive sim autosomal factor and a recessive factor(s) on the mel X, then replacing the mel X with a sim X should rescue these males (as the sim X should be compatible with the sim autosomal factor). If, on the other hand, hybrid male lethality is caused by a male-specific incompatibility, replacing the X chromosome will not necessarily rescue hybrid males. The reason is that a male-specific incompatibility could involve an interaction between a recessive sim factor and a dominant mel factor anywhere in the D. melanogaster genome. Unfortunately, mel attached-X hybrid females from this cross, which would also have been informative, were not rescued in sufficient numbers (see RESULTS).
Lethal phase:0, 百拇医药
For each incompatibility, I determined if hybrid lethality occurred at embryonic or postembryonic stages. If Df hybrid males suffer embryonic lethality, 25% of all embryos should die (i.e., 50% of all male embryos). To test if embryonic lethality was sufficient to account for the absent Df hybrid males, I calculated the ratio of the number of dead (necrotized) embryos divided by half the number of hybrid females: dead embryos/[(Df females + Bal females)/2]. If this ratio was ">="0, 百拇医药
1, the deficiency was scored as embryonic lethal. If the ratio was <<1, the number of dead embryos could not plausibly explain the deficit of Df hybrid males, and the incompatibility was scored as postembryonic lethal. Establishing the exact phase of lethality for postembryonic hybrid lethals is, unfortunately, complicated by background mortality and the imperfect degree of overall hybrid male rescue by Lhr.0, 百拇医药
RESULTS
Fine-mapping hybrid lethals:9#tp#}, http://www.100md.com
gives the results for all tested deficiencies. The distributions of viability effects for the two sexes are shown in . The most striking result is that while very few deficiencies uncover lethality in hybrid females, many uncover lethality in hybrid males : The distribution of viability for Df hybrid females is unimodal, with most crosses showing normal viability, i.e., roughly equal representation of Df and Bal females. In contrast, the distribution of viability for Df hybrid males is bimodal, with one peak at normal viability and a second near complete lethality. Rare "escaper" males bearing hybrid-lethal deficiencies are typically weak and sometimes show developmental anomalies (e.g., malformed abdominal tergites, bristles, wings, and eyes). In all, 40 nonoverlapping autosomal regions in D. simulans cause significant lethality when uncovered in Df hybrid males. Recessive hybrid lethals are thus common in the sim autosomal genome.
fig.ommitted!3}, 百拇医药
Figure 2. Distribution of relative viability of Df hybrids (deficiency bearing/total) for (A) hybrid females and (B) hybrid males.!3}, 百拇医药
Of these regions, 20 are hybrid-lethal and 20 are hybrid-semilethal incompatibilities . These regions appear to be distributed randomly among the two major autosomes (2 = 3.333, d.f. = 1, P = 0.068) and the four autosomal arms, with 14 on 2L, 14 on 2R, 6 on 3L, and 6 on 3R (2 = 3.195, d.f. = 3, P = 0.363; ). I performed further analyses on 20 hybrid lethals and 3 hybrid semilethals (these semilethals were nearly classifiable as lethal; i.e., the ratio of Df:Bal hybrid males was "0.12 for all 3). The lethal effects of these deficiencies are hybrid specific because none caused strong haploinsufficiency when heterozygous in pure species individuals . Of the 23 hybrid lethals and semilethals, I confirmed 18 (78%) with overlapping deficiencies that yielded consistent results . Each of these regions thus contains at least one hybrid incompatibility factor.
fig.ommittedy%, 百拇医药
Figure 3. Locations of 20 hybrid lethals (solid bars) and 3 hybrid semilethals (shaded bars) on cytological map of major autosomes.y%, 百拇医药
fig.ommittedy%, 百拇医药
Table 2. Deficiencies are not male lethal within D. melanogaster (Df/Bal females x Canton-S males)y%, 百拇医药
It is important to note, however, that more hybrid lethals probably exist than the above numbers suggest because the available deficiencies from D. melanogaster cumulatively uncover only 64–73% of the autosomal genome. Correcting for this incomplete coverage yields an estimate closer to 27 hybrid lethals and ~ 27 hybrid semilethals. These remain minimum estimates as each region could harbor more than one hybrid lethal. However, the fact that most deficiencies fail to uncover hybrid lethality suggests that hybrid lethals are sparsely distributed. Therefore, either most hybrid-lethal regions harbor a single hybrid-lethal locus or hybrid lethals are tightly clustered. If the former is true, as seems most plausible, it follows that many incompatibilities have major effects on hybrid viability.
The recessivity of the incompatible factors on the mel X can be illustrated in three ways. First, if factors on the mel X were completely dominant (and the incompatibilities are not sex specific), Df hybrid female viability would be correlated with Df hybrid male viability, with slope 1. (The slope of this relationship can be thought of as a proxy for the mean dominance coefficient of incompatible factors on the mel X.) Instead, while there is a weak positive correlation between Df hybrid female and male viability, the slope of the least-squares relationship is b = 0.103—a value <<1.0—consistent with the general recessivity of incompatible factors on the mel X (; Spearman's r = 0.326, d.f. = 163, P < 0.0001; statistics use only crosses producing ">="pp, 百拇医药
10 Bal hybrid males). Second, of 16 regions that cause significant lethality in Df hybrid females, 9 cause significantly stronger lethality in Df hybrid males (e.g., , line 4). Third, and most convincing, 30 of 40 deficiencies that caused significant lethality in Df hybrid males have no discernable effects in Df hybrid females. These findings strongly suggest that the incompatible factors on the mel X are also overwhelmingly close to completely recessive.
fig.ommitted3c+11ko, 百拇医药
Figure 4. Relative viability of Df hybrid females (Df-bearing females/total females) plotted against viability of Df hybrid males (Df-bearing males/total males).3c+11ko, 百拇医药
These results are consistent with those of YAGYU and YAMAMOTO 1996 , who independently performed similar crosses using 15 deficiencies on chromosome 2.3c+11ko, 百拇医药
Are hybrid lethals epistatic?3c+11ko, 百拇医药
The hybrid-lethal and semilethal sim autosomal factors detected above could be either incompatible with factors on the mel X or male-specific and incompatible with a dominant mel factor anywhere in the genome. To distinguish these possibilities, I used attached-X crosses shown in to test the viability of Df hybrid males that are genotypically and cytoplasmically identical to those above but possess a sim X rather than a mel X. These hybrid males are hemizygous for the sim X and hemizygous for a hybrid-lethal sim autosomal factor. If the lethal sim autosomal factors are incompatible with the mel X, changing the species origin of the X should rescue hybrid lethality. If, on the other hand, the sim autosomal factors cause male-specific hybrid lethality, changing the species origin of the X may not rescue hybrid lethality.
As shows, 18 of the 18 hybrid-lethal deficiencies tested become viable when hybrid males are given a sim X instead of a mel X. (For two hybrid-lethal deficiencies, the attached-X stocks proved too weak to maintain and could not be tested.) Thus, the hybrid lethality of 18 of 18 sim autosomal regions depends on the species origin of the X, confirming that these are true hybrid lethals caused by incompatible epistatic interactions.3'@gj&, http://www.100md.com
fig.ommitted3'@gj&, http://www.100md.com
Table 3. Hybrid lethality depends on the species origin of the X3'@gj&, http://www.100md.com
These attached-X crosses were also expected to produce hybrid females homozygous for the mel X (and thus genotypically identical to hybrid males from the original screen; compare female hybrids in to male hybrids in ). If produced in sufficient number, we could further test the sex specificity of each hybrid incompatibility: If hybrid lethals are not sex specific, Df hybrid females from attached-X crosses should also be inviable. This outcome appears to hold in three cases (, lines 2, 4, and 13), but in general I obtained too few hybrid females to draw meaningful conclusions.
Lethal phase:xds}}, 百拇医药
I determined the lethal phase of 18 hybrid incompatibilities. Only one causes embryonic lethality (see ). I compared this distribution to that for lethal phases of mutations within species. In D. melanogaster, 60.5% of lethal P insertions cause embryonic lethality (TOROK et al. 1993 ; DEAK et al. 1997 ). There are thus significantly fewer embryonic lethal incompatibilities in hybrids than expected from the within-species data (1 embryonic vs. 17 postembryonic hybrid-lethal incompatibilities; 2515 embryonic vs. 1640 postembryonic within-species lethal mutations; Fisher's exact P < 0.0001). Thus hybrid-lethal incompatibilities more often afflict later, postembryonic stages of development.xds}}, 百拇医药
fig.ommittedxds}}, 百拇医药
Table 4. Summary of results for hybrid-lethal regionsxds}}, 百拇医药
DISCUSSIONxds}}, 百拇医药
This study yields three main results. First (and perhaps most surprising), many hybrid-lethal incompatibilities have evolved between D. melanogaster and D. simulans: The ~ 70% of the D. simulans autosomal genome examined harbors at least 20 factors that cause complete or near complete lethality in the presence of the D. melanogaster X. Taking into account certain corrections (see below), this value implies that the genome-wide number of recessive-recessive lethal incompatibilities is ~ 169. Second, the incompatible genes on both the autosomes and the X are nearly completely recessive and, as shown below, these recessive incompatibilities vastly outnumber dominant ones. These results provide strong support for the dominance theory. Third, all of the lethal hybrid incompatibilities tested involve an epistatic interaction with the X chromosome; i.e., 18 of 18 are rescued when the mel X is replaced by a compatible sim X, as expected under the the Dobzhansky-Muller model.
While these data confirm the ubiquity of recessive, epistatic hybrid incompatibilities, they also allow us to go further. These data can be used to estimate four quantities: (1) the total number of hybrid lethals separating D. melanogaster and D. simulans; (2) the relative rates of evolution of dominant vs. recessive incompatibilities; (3) the probability that two randomly chosen divergent substitutions (one from each species) are incompatible, causing hybrid lethality; and (4) the fraction of viability-essential genes that have diverged to such an extent that they are no longer functionally compatible with alleles at interacting loci from the other species.q|p;e*e, 百拇医药
Dominance and the total number of hybrid lethals:q|p;e*e, 百拇医药
By including data from previous genetic analyses, we can estimate the total number of hybrid lethals that have evolved between D. melanogaster and D. simulans. I make the simplifying assumption that hybrid lethality involves pairs of incompatible loci. Hybrid incompatibilities can then be classified into dominant-dominant, recessive-dominant, and recessive-recessive types (or, following TURELLI and ORR 2000 , H0, H1, and H2 incompatibilities, respectively, where the subscripts 0, 1, and 2 indicate the number of homozygous or hemizygous loci involved). More than two loci could be involved in a given incompatibility—so-called complex epistasis—but this should not seriously affect our estimates as long as the three incompatibility types (H0, H1, and H2) are equally prone to such complex interactions.
H0 incompatibilities: No H0 incompatibilities separate D. melanogaster and D. simulans. The cross of D. melanogaster females with D. simulans males produces perfectly viable hybrid females that are heterozygous at every locus in the genome (STURTEVANT 1920 , STURTEVANT 1929 ; but see BARBASH et al. 2000 for temperature effects on viability of hybrid females).l+t^|, 百拇医药
H1 incompatibilities: There are ~ 22 H1 incompatibilities between D. melanogaster and D. simulans. This value comes from two kinds of data. The first involves studies of hybrid rescue mutations. There is now good evidence that such mutations are rare "compatible" alleles at normally incompatible loci (HUTTER et al. 1990 ; SAWAMURA and YAMAMOTO 1997 ; BARBASH et al. 2000 ; ORR and IRVING 2000 ). We can therefore infer two H1 incompatibilities from the existence of two known pairs of complementary hybrid rescue mutations (HUTTER et al.. 1990 ; SAWAMURA et al. 1993 ; ORR and PRESGRAVES 2000 ). The second comes from COYNE et al.. 1998 deficiency screen for H1 incompatibilities. This screen uncovered five hybrid-lethal regions (this lethality was not unconditional, however, as some became viable at permissive temperatures or using different stocks). Coyne et al.'s number requires two corrections as only 50% of the D. simulans genome was screened, and the reciprocal experiment in which D. melanogaster regions are made hemizygous could not be done as no deficiencies are available in D. simulans. Correcting for these two considerations yields an estimate of ~ 20 hybrid-lethal regions. Thus ~ 22 H1 incompatibilities separate D. melanogaster and D. simulans. This number is obviously rough, but as shown below, it differs qualitatively from that for H2 incompatibilities.
H2 incompatibilities: The total number of H2 incompatibilities is large. We can estimate the genome-wide number by extrapolating from the number of X-autosome H2 incompatibilities (see RESULTS). The X is roughly equivalent in size and gene content to one of the five major chromosome arms (i.e., X, 2L, 2R, 3L, and 3R). Holding the X effectively homozygous (hemizygous) and screening the rest of the genome with deficiencies yielded ~ 27 lethal H2 incompatibilities. If we could repeat this screen, successively holding the autosomal arms 2L, 2R, 3L, and 3R homozygous for D. melanogaster while scanning the rest of the D. simulans genome for lethal incompatibilities, we would expect to uncover another ~ 27 H2 incompatibilities for each arm. We therefore expect ~ 135 hybrid lethals genome-wide. Using similar logic to correct for intra-arm H2 incompatibilities brings the total number of H2 incompatibilities to ~ 169. (Note that extrapolating from the number of X-autosome incompatibilities to the number genome-wide assumes that X-linked and autosomal loci diverge at similar rates; BETANCOURT et al. 2002 survey of divergence at >250 genes from D. melanogaster and D. simulans supports this assumption.)
Two conclusions follow from these calculations. First, summing across H0, H1, and H2 incompatibility types gives an estimate of the total number of hybrid-lethal incompatibilities separating D. melanogaster and D. simulans: 191. (This does not include the hybrid semilethals found here; including them nearly doubles the estimate.) Given the conclusion from previous analyses that the number of hybrid lethals between these two species is small, this number comes as a surprise and reveals an unexpected degree of functional divergence.g\;, 百拇医药
These numbers also provide strong quantitative confirmation of the dominance theory: D. melanogaster and D. simulans are separated by no H0 incompatibilities, by ~ 22 H1 incompatibilities, and by ~ 169 H2 incompatibilities. The number of hybrid lethals thus jumps nearly an order of magnitude as we increase the number of homozygous loci involved, leaving little doubt that most hybrid lethals are recessive. Why hybrid incompatibilities tend to be recessive remains a mystery. Although the similar effects of loss-of-function mutations within species and incompatibilities in hybrids have led to speculation that the latter mimic the former (STEBBINS 1958 ; ORR 1993 ; TURELLI and ORR 1995 ), several recent lines of evidence now appear inconsistent with this interpretation (BARBASH et al. 2000 ; ORR and IRVING 2000 ; ORR and PRESGRAVES 2000 ). But regardless of why most incompatibilities act as recessives, the present results leave little doubt that they do.
Epistasis and hybrid lethality:8{xssqw, http://www.100md.com
Using the above estimate of the total number of hybrid-lethal incompatibilities, along with a molecular estimate of the total number of divergent substitutions between D. melanogaster and D. simulans, we can estimate (to an order of magnitude) another important quantity from speciation genetics theory: the probability, p, that any two randomly chosen divergent sites (one from one species, one from the other) are incompatible in hybrids, causing (for our purposes) complete lethality. As ORR and TURELLI 2001 show, the expected number of hybrid incompatibilities, I, between pairs of genes is8{xssqw, http://www.100md.com
where k is the genome-wide rate of substitution and t is the time since speciation, so that 2kt is the total number of substitutions separating the genomes of two species. By substituting estimates of I and kt and rearranging, we can solve for p. To ensure that our estimate of p for hybrid lethality is directly comparable to the estimate of p for hybrid male sterility calculated by ORR and TURELLI 2001 , I use the same data sources for k and t and follow their calculation exactly. (See Appendix for fuller details and discussion of the calculation.) The total number of hybrid lethals is, from above, I 191. D. melanogaster and D. simulans have accumulated 2kt 156,000 nonsynonymous substitutions since they diverged t = 2.5 MYA (HEY and KLIMAN 1993; LI 1997 ). Solving the above equation thus gives p 1.6 x 10-8. Two nonsynonymous substitutions chosen randomly (one from each species' genome) will therefore cause complete hybrid lethality ~ 10-8 of the time. As discussed in the Appendix, this value appears to be an order of magnitude smaller than the value for hybrid male sterility estimated by ORR and TURELLI 2001 .
Interestingly, p can be thought of as a crude index of the ruggedness of the molecular landscape. To see this, consider the extreme cases: When p = 0, negative epistasis is absent, all substitutions are compatible, and hybrid genotypes never fall into fitness valleys; when p = 1, however, negative epistasis is complete, all divergent substitutions after the first are incompatible, and all hybrid genotypes fall into fitness valleys. Thus, the exceedingly small value of p suggests that the molecular landscape is reasonably smooth: Substitutions that have never "seen" each other in their evolutionary histories are almost always compatible (or at least not lethal in combination).\lk$, 百拇医药
Functional divergence:\lk$, 百拇医药
Finally, we can estimate the fraction of viability-essential genes that have diverged to the extent that they are no longer functionally compatible. If hybrid-lethal incompatibilities involve pairs of loci, as assumed above, it follows from the Dobzhansky-Muller model that both loci must have diverged (ORR 1995 ). The above estimate of 191 hybrid-lethal incompatibilities thus implies that at least 382 loci have experienced significant functional divergence since the D. melanogaster-D. simulans split, i.e., 11% of all viability-essential genes (or 382/3600; see SPRADLING et al. 1999 ). This level of divergence at a class of genes that we might a priori expect to be relatively conserved (i.e., those essential for viability) seems remarkable.
Two lines of evidence suggest that the genes regulating the earliest phases of development in the two species remain largely compatible. First, most hybrid genotypes survive embryonic phases of development only to die later (CARVAJAL et al. 1996 ). Second, my deficiency screen, as well as that of COYNE et al. 1998 , could have detected any maternal-zygotic, embryonic lethal incompatibilities involving the D. melanogaster cytoplasm and hemizygous D. simulans autosomal factors. But very few of our incompatibilities can possibly fall into this category. Instead, most hybrid incompatibilities occur between zygotic genes acting at postembryonic stages. Since viability-essential genes are most often mutable to embryonic lethality within species, the paucity of embryonic vs. postembryonic hybrid lethals suggests that: (i) There are greater functional constraints (on gene sequence and/or expression) at early acting genes; (ii) most physiological and ecological adaptation in Drosophila occurs by divergence in postembryonic phases of development; or (iii) more genes are simultaneously active and thus prone to incompatible interactions during later stages of development. The latter possibility seems unlikely to account for the strong preponderance of postembryonic hybrid lethality as most genes exhibiting developmentally modulated expression during the Drosophila life cycle are expressed at some point during embryogenesis (>88%; ARBEITMAN et al. 2002 ).
Caveats:)yo, 百拇医药
Using deficiencies and a hybrid rescue mutation to detect X-autosome incompatibilities makes two key assumptions. The first is that the effects of D. simulans autosomal regions when hemizygous are equivalent to when they are homozygous. If this assumption is incorrect, then instead of uncovering "hybrid lethals," the screen might simply uncover regions that are haploinsufficient. (Hemizygosity of the D. melanogaster X in males is equivalent to homozygosity because of dosage compensation.) Three facts, however, militate against this possibility (see also COYNE et al. 1998 ). First, although deficiencies can uncover recessive lethality within species, they are not inherently lethal as heterozygotes within species .)yo, 百拇医药
Second, one might argue that deficiencies cause haploinsufficiency, but only in hybrids. Two observations rule out this possibility: deficiencies that cause lethality in hybrid males do not, in most cases, harm Df hybrid females; more important, even hybrid males that carry a hybrid-lethal deficiency become viable when carrying a D. simulans, rather than a D. melanogaster, X. Thus, the lethality of particular deficiencies depends on species genotype at background loci (i.e., epistasis), as expected for hybrid incompatibilities.
Third, there are now two examples in which factors from D. simulans are known to behave identically in species hybrids, whether homozygous or hemizygous:vhh|, 百拇医药
MULLER and PONTECORVO 1940 , MULLER and PONTECORVO 1942 discovered that the fourth chromosome of D. simulans causes hybrid male sterility when homozygous on an otherwise D. melanogaster genetic background. ORR 1992 fine mapped the region of the D. simulans fourth responsible using D. melanogaster deficiencies, thus confirming that the recessive, incompatible D. simulans factor causes sterility when homozygous or hemizygous.vhh|, 百拇医药
SAWAMURA et al. 2000 used a mutation that weakly rescues hybrid female fertility to introgress two small regions from D. simulans 2L into an otherwise D. melanogaster background. According to my results, these regions harbor three factors that each cause hybrid lethality when hemizygous in a genetic background containing a hemizygous D. melanogaster X and heterozygous autosomes (, lines 1, 3, 4). SAWAMURA 2000 found that, in an identical genetic background, these regions also cause hybrid lethality when homozygous.
Thus, both Orr's and Sawamura's results show that recessive hybrid incompatibilities behave identically when homozygous or hemizygous.7(cw-w, 百拇医药
The second assumption of the deficiency analysis is that the 40 hybrid incompatibilities detected are independent of hybrid male rescue by Lhr. The primary cross was intended to rescue hybrid males from one incompatibility (involving the incompatible, wild-type allele Lhrsim) and to then expose them to other potential incompatibilities. It is formally possible, however, that some of these hybrid lethals are actually suppressors of Lhr rescue. If true, these suppressors would still be of interest as interactors of a known hybrid incompatibility locus (Lhr). But this Lhr-suppressor scenario seems unlikely for several reasons. First, it seems unlikely that since the D. melanogaster-D. simulans split ~ 2.5 MYA, the only genetic pathways to evolve incompatibilities necessarily all involve Lhr. Second, such putative Lhr suppressors would necessarily be recessive D. simulans factors that suppress the rescue effects of a particular rare allele, Lhr. It is hard to believe that so many different recessive, conspecific loci are capable of such a trick. Third, Lhr rescues hybrid male lethality that normally occurs at the larval-pupal transition. But at least one hybrid lethal does not affect this phase of development, causing embryonic lethality instead (, line 7), and thus cannot be explained by Lhr suppression. Casual observations further suggest that more detailed study of the postembryonic lethal phases will reveal other cases acting later than the larval-pupal transition.
Conclusions:%6+062, http://www.100md.com
This study shows that most hybrid incompatibilities are recessive and epistatic. The most surprising finding is the extraordinary degree of functional divergence that has occurred between D. melanogaster and D. simulans during the last 2.5 MY. Such extensive cryptic divergence would seem to confirm Muller's suggestion that "(t)wo groups of organisms which are not ordinarily allowed to cross with one another will thus automatically become increasingly immiscible, and their genic, chemical paths of evolution will diverge more and more ... even in cases where their evolution is, from the phenotypic standpoint, strikingly parallel" (MULLER 1939 , p. 278).%6+062, http://www.100md.com
An important aspect of the many hybrid incompatibilities identified here should not be overlooked: Their fine-scale resolution in a model genetic organism will greatly facilitate their routine molecular characterization. The 20 hybrid lethals and 20 hybrid semilethals discovered each reside in just a few cytological divisions. It is conceptually simple (although labor intensive) to move from identifying blocks of candidate genes using deficiency complementation tests to identifying the relevant genes using single-locus complementation tests. Establishing the molecular identity, function, and evolutionary history of a large collection of speciation genes is certain to reveal new patterns and to answer some long-standing questions in evolution: What are the normal functions of "speciation genes" within species? Are most functionally relevant substitutions concentrated in coding or regulatory regions? Is natural selection the primary force causing the fixation of divergent substitutions? Preliminary work in several of the hybrid-lethal regions identified here suggests that the molecular characterization of the genes responsible should be possible.
ACKNOWLEDGMENTS12e^j, 百拇医药
I thank Shawn Ciecko for expert technical assistance during the early phases of this work. I am also grateful to Andrea Betancourt, Andy Clark, Jerry Coyne, Allen Orr, and Jack Werren for helpful discussions and advice on the project. I also thank Andrea Betancourt, Audrey Chang, Jerry Coyne, J. P. Masly, Mohamed Noor, two anonymous reviewers, and especially Allen Orr for comments that greatly improved the manuscript. This work was supported by funds from an Ernst Caspari Fellowship and a George and Agnes M. Messersmith Fellowship to the author and by funds from the National Institutes of Health (GM-526738) and David and Lucile Packard Foundation to Allen Orr.12e^j, 百拇医药
Manuscript received September 20, 2002; Accepted for publication December 3, 2002.12e^j, 百拇医药
APPENDIX12e^j, 百拇医药
CALCULATION OF p12e^j, 百拇医药
To obtain 2kt, the total number of replacement changes separating D. melanogaster and D. simulans, I use LI's (1997) estimate of the rate of nonsynonymous substitution in Drosophila from 30 loci, 1.91 x 10-9/site/year. To get the genome-wide rate of substitution, I take into account the number of relevant sites in the genome: 13,600 genes/genome x 1800 bp/gene x ~ 2/3 are nonsynonymous sites = 1.632 x 107. Multiplying gives the number of replacement changes per genome per year: k = 0.0312. Thus the total number of replacement changes that have accumulated between D. melanogaster and D. simulans during the last 2.5 MY is 2kt 156,000. Solving the equation gives p 1.6 x 10-8.
We can check the plausibility of this estimate of p for hybrid lethality using data from another species pair. In particular, given p for hybrid lethality from above and the number of genome-wide substitutions separating a younger species pair, D. mauritiana and D. simulans, we can ask: How many hybrid lethals should we expect to find in a genetic analysis of D. mauritiana-D. simulans hybrids? (This assumes, of course, that p itself has not evolved substantially over the last few million years, as seems reasonable.) We already know the number of hybrid lethals between D. mauritiana and D. simulans to be ~ 5–10 from the work of TRUE et al. 1996 and so can compare it to the one predicted by the Orr-Turelli equation. To get the predicted number, we use p = 1.6 x 10-8 from above and the genome-wide number of nonsynonymous substitutions between D. mauritiana and D. simulans, 2kt = 48,000 (see ORR and TURELLI 2001 ). Solving for I, the predicted number of hybrid lethals is 18. Thus, given nothing more than p for hybrid lethality, as estimated from the D. melanogaster-D. simulans hybridization and a crude estimate of the number of substitutions, the equation comes remarkably close to predicting the true number of hybrid lethals.
The estimate of p for hybrid lethality is an order of magnitude smaller than the one estimated for hybrid male sterility using genetic data from D. simulans and D. mauritiana, p 1.04 x 10-7 (ORR and TURELLI 2001 ). This discrepancy could be explained by either of the two putative causes of faster-male evolution (WU and DAVIS 1993 ; WU et al. 1996 ): (i) Spermatogenesis might be inherently more sensitive than viability to the incompatibilities experienced by hybrids, and thus a greater fraction of interspecific gene interactions cause hybrid male sterility; or (ii) our calculations have not taken into account that male-specific genes evolve faster, on average, than viability-essential genes (BEGUN et al. 2000 ; SWANSON et al. 2001 ; BETANCOURT et al. 2002 ). As defined here and in ORR and TURELLI 2001 , p—the probability that any two divergent replacement changes (one from each species) from any gene in the genome are incompatible—ignores that not all genes interact with each other, that not all genes are mutable to sterility, that not all genes are mutable to lethality, that not all incompatibilities are protein-protein interactions, etc. In the absence of such perfect knowledge, it is convenient to estimate p as the genome-wide probability of incompatibility, averaging over all genes. But since male-specific genes evolve more rapidly than others (and, in particular, faster than the 30 loci used to estimate k from LI 1997 above), p for hybrid male sterility will be overestimated. Consequently, p for hybrid lethality and p for hybrid sterility are likely closer than they appear. The important point, however, is that both values are exceedingly small.
LITERATURE CITED$8&i]., 百拇医药
ARBEITMAN, M. N., E. E. M. FURLONG, F. IMAM, E. JOHNSON, and B. H. NULL et al., 2002 Gene expression during the life cycle of Drosophila melanogaster. Science 297:2270-2275.$8&i]., 百拇医药
BARBASH, D. A., J. ROOTE, and M. ASHBURNER, 2000 The Drosophila melanogaster Hybrid male rescue gene causes inviability in male and female species hybrids. Genetics 154:1747-1771.$8&i]., 百拇医药
BEGUN, D. J., P. WHITLEY, B. L. TODD, H. M. WALDRIP-DAIL, and A. G. CLARK, 2000 Molecular population genetics of male accessory gland proteins in Drosophila. Genetics 156:1879-1888.$8&i]., 百拇医药
BETANCOURT, A. J., D. C. PRESGRAVES, and W. J. SWANSON, 2002 A test for faster X evolution in Drosophila.. Mol. Biol. Evol. 19:1816-1819.$8&i]., 百拇医药
BRIDGES, C. B., 1935 Salivary chromosome maps. J. Hered. 26:60-64.$8&i]., 百拇医药
CARVAJAL, A. R., M. R. GANDARELA, and H. F. NAVEIRA, 1996 A three-locus system of interspecific incompatibility underlies male inviability in hybrids between Drosophila buzzatii and D. koepferae.. Genetica 98:1-19.
COYNE, J. A. and H. A. ORR, 1989 Patterns of speciation in Drosophila. Evolution 43:362-381.*[a:\!k, 百拇医药
COYNE, J. A. and H. A. ORR, 1997 "Patterns of speciation in Drosophila" revisited. Evolution 51:295-303.*[a:\!k, 百拇医药
COYNE, J. A., S. SIMEONIDIS, and P. ROONEY, 1998 Relative paucity of genes causing inviability in hybrids between Drosophila melanogaster and D. simulans.. Genetics 150:1091-1103.*[a:\!k, 百拇医药
CROW, J. F., and M. J. SIMMONS, 1983 The mutation load in Drosophila, pp. 1–35 in The Biology and Genetics of Drosophila, edited by M. ASHBURNER, H. L. CARSON and J. N. THOMPSON. Academic Press, London.*[a:\!k, 百拇医药
DAVIS, A. W., J. ROOTE, T. MORLEY, K. SAWAMURA, and S. HERRMANN et al., 1996 Rescue of hybrid sterility in crosses between D. melanogaster and D. simulans.. Nature 380:157-159.*[a:\!k, 百拇医药
DEAK, P., M. M. OMAR, R. D. C. SAUNDERS, M. PAL, and O. KOMONYI et al., 1997 P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E–87F. Genetics 147:1697-1722.
DOBZHANSKY, T., 1937 Genetics and the Origin of Species. Columbia University Press, New York.|r+v, 百拇医药
GRANADINO, B., L. O. F. PENALVA, and L. SANCHEZ, 1996 Indirect evidence of alteration in the expression of the rDNA genes of interspecific hybrids between Drosophila melanogaster and Drosophila simulans.. Mol. Gen. Genet. 250:89-96.|r+v, 百拇医药
HALDANE, J. B. S., 1922 Sex-ratio and unidirectional sterility in hybrid animals. J. Genet. 12:101-109.|r+v, 百拇医药
HEY, J. and R. M. KLIMAN, 1993 Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex. Mol. Biol. Evol. 10:804-822.|r+v, 百拇医药
HOLLOCHER, H. and C.-I WU, 1996 The genetics of reproductive isolation in the Drosophila simulans clade: X vs. autosomal effects and male vs. female effects. Genetics 143:1243-1255.|r+v, 百拇医药
HUTTER, P., J. ROOTE, and M. ASHBURNER, 1990 A genetic basis for the inviability of hybrids between sibling species of Drosophila. Genetics 124:909-920.
LACHAISE, D., J. R. DAVID, F. LEMEUNIER, L. TSACAS, and M. ASHBURNER, 1986 The reproductive relationships of Drosophila sechellia with D. mauritiana, D. simulans, and D. melanogaster from the Afrotropical region. Evolution 40:262-271.|^2]q\j, 百拇医药
LI, W.-H., 1997 Molecular Evolution. Sinauer Associates, Sunderland, MA.|^2]q\j, 百拇医药
LINDSLEY, D. L., and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster. Academic Press, San Diego.|^2]q\j, 百拇医药
MULLER, H. J., 1939 Reversibility in evolution considered from the standpoint of genetics. Biol. Rev. Camb. Phil. Soc. 14:261-280.|^2]q\j, 百拇医药
MULLER, H. J., 1940 Bearing of the Drosophila work on systematics, pp. 185–268 in The New Systematics, edited by J. S. HUXLEY. Clarendon Press, Oxford.|^2]q\j, 百拇医药
MULLER, H. J., 1942 Isolating mechanisms, evolution, and temperature. Biol. Symp. 6:71-125.|^2]q\j, 百拇医药
MULLER, H. J. and G. PONTECORVO, 1940 Recombinants between Drosophila species, the F1 hybrids of which are sterile. Nature 146:199.
MULLER, H. J. and G. PONTECORVO, 1942 Recessive genes causing interspecific sterility and other disharmonies between Drosophila melanogaster and simulans.. Genetics 27:157.#'lmx, 百拇医药
NAVEIRA, H. F., and X. R. MASIDE, 1998 The genetics of hybrid male sterility in Drosophila, pp. 330–338 in Endless Forms, edited by D. J. HOWARD and S. H. BERLOCHER. Oxford University Press, Oxford.#'lmx, 百拇医药
ORR, H. A., 1992 Mapping and characterization of a "speciation gene" in Drosophila. Genet. Res. 59:73-80.#'lmx, 百拇医药
ORR, H. A., 1993 A mathematical model of Haldane's rule. Evolution 47:1606-1611.#'lmx, 百拇医药
ORR, H. A., 1995 The population genetics of speciation: the evolution of hybrid incompatibilities. Genetics 139:1805-1813.#'lmx, 百拇医药
ORR, H. A., 1996 Dobzhansky, Bateson, and the genetics of speciation. Genetics 144:1331-1335.#'lmx, 百拇医药
ORR, H. A. and S. IRVING, 2000 Genetic analysis of the Hybrid male rescue locus of Drosophila. Genetics 155:225-231.#'lmx, 百拇医药
ORR, H. A. and D. C. PRESGRAVES, 2000 Speciation by postzygotic isolation: forces, genes and molecules. Bioessays 22:1085-1094.
ORR, H. A. and M. TURELLI, 2001 The evolution of postzygotic isolation: accumulating Dobzhansky-Muller incompatibilities. Evolution 55:1085-1094.u;, http://www.100md.com
PONTECORVO, G., 1943a Hybrid sterility in artificially produced recombinants between Drosophila melanogaster and D. simulans. Proc. R. Soc. Edinb. Sect. B (Biol.) 61:385-397.u;, http://www.100md.com
PONTECORVO, G., 1943b Viability interactions between chromosomes of Drosophila melanogaster and Drosophila simulans.. J. Genet. 45:51-66.u;, http://www.100md.com
PRESGRAVES, D. C., 2002 Patterns of postzygotic isolation in Lepidoptera. Evolution 56:1168-1183.u;, http://www.100md.com
PRESGRAVES, D. C. and H. A. ORR, 1998 Haldane's rule in taxa lacking a hemizygous X.. Science 282:952-954.u;, http://www.100md.com
PRICE, T. D. and M. M. BOUVIER, 2002 The evolution of F1 postzygotic incompatibilities in birds. Evolution 56:2083-2089.u;, http://www.100md.com
PROVINE, W. B., 1991 Alfred Henry Sturtevant and crosses between Drosophila melanogaster and Drosophila simulans.. Genetics 129:1-5.
SANCHEZ, L., B. GRANADINO, and L. VICENTE, 1994 Clonal analysis in hybrids between Drosophila melanogaster and Drosophila simulans.. Roux's Arch. Dev. Biol. 204:112-117.j'56, http://www.100md.com
SASA, M. M., P. T. CHIPPINDALE, and N. A. JOHNSON, 1998 Patterns of postzygotic isolation in frogs. Evolution 52:1811-1820.j'56, http://www.100md.com
SAWAMURA, K., 2000 Genetics of hybrid inviability and sterility in Drosophila: the Drosophila melanogaster-Drosophila simulans case. Plant Species Biol. 15:237-247.j'56, http://www.100md.com
SAWAMURA, K. and M.-T. YAMAMOTO, 1997 Characterization of a reproductive isolation gene, Zygotic hybrid rescue, of Drosophila melanogaster by using minichromosomes. Heredity 79:97-103.j'56, http://www.100md.com
SAWAMURA, K., T. K. WATANABE, and M.-T. YAMAMOTO, 1993 Hybrid lethal systems in the Drosophila melanogaster species complex. Genetica 88:175-185.j'56, http://www.100md.com
SAWAMURA, K., A. W. DAVIS, and C.-I WU, 2000 Genetic analysis of speciation by means of introgression into Drosophila melanogaster.. Proc. Natl. Acad. Sci. USA 97:2652-2655.
SIMMONS, M. J. and J. F. CROW, 1977 Mutations affecting fitness in Drosophila.. Annu. Rev. Genet. 11:49-78.(?s*&04, http://www.100md.com
SINGH, R. S., 1999 Toward a unified theory of speciation, pp. 570–604 in Evolutionary Genetics: From Molecules to Morphology, edited by R. S. SINGH and C. B. KRIMBAS. Cambridge University Press, Cambridge, UK/London/New York.(?s*&04, http://www.100md.com
SPRADLING, A. C., D. STERN, A. BEATON, E. J. REHM, and T. LAVERTY et al., 1999 The Berkeley Drosophila Genome Project gene disruption project: single P-element insertions mutating 25% of vital Drosophila genes. Genetics 153:135-177.(?s*&04, http://www.100md.com
STEBBINS, G. L., 1958 The inviability, weakness, and sterility of interspecific hybrids. Adv. Genet. 9:147-215.(?s*&04, http://www.100md.com
STURTEVANT, A. H., 1920 Genetic studies on Drosophila simulans. I. Introduction. Hybrids with Drosophila melanogaster. Genetics 5:488-500.(?s*&04, http://www.100md.com
STURTEVANT, A. H., 1929 The genetics of Drosophila simulans.. Carnegie Inst. Washington Publ. 399:1-62.(?s*&04, http://www.100md.com
SWANSON, W. J., A. G. CLARK, H. M. WALDRIP-DAIL, M. F. WOLFNER, and C. F. AQUADRO, 2001 Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila. Proc. Natl. Acad. Sci. USA 98:7375-7379.
TAKAMURA, T. and T. K. WATANABE, 1980 Further studies on the Lethal hybrid rescue (Lhr) gene of Drosophila simulans.. Jpn. J. Genet. 55:405-408.)!i}, 百拇医药
TOROK, T., G. TICK, M. ALVARADAO, and I. KISS, 1993 P-lacW insertional mutagenesis on the second chromosome of Drosophila melanogaster: isolation of lethals with different overgrowth phenotypes. Genetics 135:71-80.)!i}, 百拇医药
TRUE, J. R., B. S. WEIR, and C. C. LAURIE, 1996 A genome-wide survey of hybrid incompatibility factors by the introgression of marked segments of Drosophila mauritiana chromosomes into Drosophila simulans.. Genetics 142:819-837.)!i}, 百拇医药
TURELLI, M. and H. A. ORR, 1995 The dominance theory of Haldane's rule. Genetics 140:389-402.)!i}, 百拇医药
TURELLI, M. and H. A. ORR, 2000 Dominance, epistasis and the genetics of postzygotic isolation. Genetics 154:1663-1679.)!i}, 百拇医药
WATANABE, T. K., 1979 A gene that rescues the lethal hybrids between Drosophila melanogaster and D. simulans.. Japn. J. Genet. 54:325-331.)!i}, 百拇医药
WU, C.-I and A. W. DAVIS, 1993 Evolution of postmating reproductive isolation: the composite nature of Haldane's rule and its genetic bases. Am. Nat. 142:187-212.)!i}, 百拇医药
WU, C.-I, and H. HOLLOCHER, 1998 Subtle is nature: the genetics of species differentiation and speciation, pp. 339–351 in Endless Forms, edited by D. J. HOWARD and S. H. BERLOCHER. Oxford University Press, Oxford.)!i}, 百拇医药
WU, C.-I, N. A. JOHNSON, and M. F. PALOPOLI, 1996 Haldane's rule and its legacy: Why are there so many sterile males? Trends Ecol. Evol. 11:281-284.)!i}, 百拇医药
YAGYU, M. and M.-T. YAMAMOTO, 1996 Hybrid rescue effect of Lhr is suppressed in deficiency heterozygotes in the hybrid between D. melanogaster and D. simulans.. Genes Genet. Syst. 71:423.(Daven C. Presgraves)
ABSTRACT^4?*[a:, 百拇医药
The sterility and inviability of species hybrids is thought to evolve by the accumulation of genes that cause generally recessive, incompatible epistatic interactions between species. Most analyses of the loci involved in such hybrid incompatibilities have suffered from low genetic resolution. Here I present a fine-resolution genetic screen that allows systematic counting, mapping, and characterizing of a large number of hybrid incompatibility loci in a model genetic system. Using small autosomal deletions from D. melanogaster and a hybrid rescue mutation from D. simulans, I measured the viability of hybrid males that are simultaneously hemizygous for a small region of the D. simulans autosomal genome and hemizygous for the D. melanogaster X chromosome. These hybrid males are exposed to the full effects of any recessive-recessive epistatic incompatibilities present in these regions. A screen of ~ 70% of the D. simulans autosomal genome reveals 20 hybrid-lethal and 20 hybrid-semilethal regions that are incompatible with the D. melanogaster X. In further crosses, I confirm the epistatic nature of hybrid lethality by showing that all of the incompatibilities are rescued when the D. melanogaster X is replaced with a D. simulans X. Combined with information from previous studies, these results show that the number of recessive incompatibilities is approximately eightfold larger than the number of dominant ones. Finally, I estimate that a total of ~ 191 hybrid-lethal incompatibilities separate D. melanogaster and D. simulans, indicating extensive functional divergence between these species' genomes.
THE last decade has seen important progress in the genetics of speciation. In particular, there is now broad agreement on three aspects of intrinsic postzygotic isolation, the sterility and inviability of species hybrids::hw, 百拇医药
Hybrid fitness problems evolve gradually as incompatible epistatic interactions accumulate between species (COYNE and ORR 1989 , COYNE and ORR 1997 ; SASA et al. 1998 ; PRESGRAVES 2002 ; PRICE and BOUVIER 2002 ). The so-called Dobzhansky-Muller model posits that incompatibilities evolve by the substitution of advantageous or neutral mutations in one species, which, having never been tested in combination with those in other species, have harmful effects when brought together in hybrids (DOBZHANSKY 1937 ; MULLER 1940 , MULLER 1942 ; ORR 1995 , ORR 1996 ).:hw, 百拇医药
The alleles involved in these hybrid incompatibilities are thought to be, on average, partially recessive. This "dominance theory" neatly accounts for several phenomena, including Haldane's rule (the observation that XY hybrids typically suffer more severe hybrid problems than do XX hybrids; HALDANE 1922 ) and F2 hybrid breakdown (MULLER 1942 ; ORR 1993 ; TURELLI and ORR 1995 , TURELLI and ORR 2000 ).
Incompatibilities causing hybrid male sterility accumulate faster than those causing other types of hybrid fitness problems (WU and DAVIS 1993 ; HOLLOCHER and WU 1996 ; TRUE et al. 1996 ; WU et al. 1996 ; NAVEIRA and MASIDE 1998 ; PRESGRAVES and ORR 1998 ; WU and HOLLOCHER 1998 ; SINGH 1999 ). The "faster-male" theory posits that the rapid evolution of hybrid male sterility is caused by the faster divergence of male-specific fertility genes, perhaps driven by sexual selection, or by the inherent sensitivity of spermatogenesis to the genetic perturbations experienced by hybrids (WU and DAVIS 1993 ).vz5:|^, http://www.100md.com
Nevertheless, progress in the fine-genetic and molecular characterization of hybrid incompatibility loci has been slow. Despite much effort, few studies have succeeded in precisely and systematically counting, fine mapping, and, ultimately, identifying a large number of "speciation genes." The reason is that the traits of interest, hybrid sterility and inviability, are by their very nature barriers to crossing and thus are refractory to standard genetic approaches. This problem is especially severe in one of our best model organisms, the fruit fly Drosophila melanogaster: All hybrids between D. melanogaster and its closest known relatives (D. simulans, D. mauritiana, and D. sechellia) are completely dead or sterile (STURTEVANT 1920 , STURTEVANT 1929 ; LACHAISE et al. 1986 ). Consequently, nearly a century's worth of accumulated genetic tools has not been brought to bear on the genetics of speciation, and genetic analyses of the D. melanogaster-D. simulans hybridization, despite an 80-year history, have suffered poor genetic resolution. The rough portrait that has emerged from this work is that a large number of hybrid male steriles but a modest number of hybrid lethals have accumulated between D. melanogaster and D. simulans (MULLER and PONTECORVO 1940 , MULLER and PONTECORVO 1942 ; PONTECORVO 1943A , PONTECORVO 1943B ; PROVINE 1991 ; SANCHEZ et al. 1994 ; COYNE et al. 1998 ; SAWAMURA et al. 2000 ; SAWAMURA 2000 ).
Two methods have been used to circumvent the problematic sterility and inviability of D. melanogaster species hybrids. First, the discovery of hybrid rescue mutations—alleles that restore the viability and fertility of normally unfit hybrids—raised the possibility of introgressing foreign genes into a D. melanogaster background, where they might be further analyzed (DAVIS et al. 1996 ). Unfortunately, hybrid female fertility rescue has proved extremely weak, and so far only two small introgressions of the D. simulans second chromosome have been studied (SAWAMURA et al. 2000 ). Second, COYNE et al. 1998 used a battery of deficiencies (small chromosomal deletions) from D. melanogaster to uncover recessive D. simulans factors causing hybrid lethality in otherwise heterozygous F1 females. Their screen of ~ 50% of the D. simulans genome revealed only a handful of hybrid lethals, none of which were unconditionally lethal. One reason why so few were detected may be that the screen was limited to incompatibilities involving a hemizygous (recessive) factor from D. simulans and a heterozygous (dominant) one from D. melanogaster (elsewhere in the genome). If most incompatibility alleles are recessive, as the dominance theory posits, then the most abundant class of hybrid incompatibility should be that in which the interacting loci are both homozygous or both hemizygous, i.e., genotypes normally produced only in F2 (or backcross) hybrids.
Here I present a new screen that modifies and combines these two approaches, taking advantage of both a hybrid rescue mutation from D. simulans and the genetic tools of D. melanogaster to test for the presence of these extreme recessive-recessive hybrid incompatibilities. In particular, I test the viability of F1 hybrid males hemizygous for a small region of the D. simulans autosomal genome and simultaneously hemizygous for the D. melanogaster X. These males are exposed to the full effects of any recessive-recessive X-autosome hybrid incompatibilities in the regions tested. Using the many deficiencies available in D. melanogaster, I systematically screened most of the D. simulans autosomal genome for such incompatibilities.'lmx(, 百拇医药
Deficiency mapping in hybrids offers several advantages over recombination mapping. First, large numbers of the relevant genotypes can be assayed in the F1 generation. Second, the existence and precise location of hybrid lethals in most cases can be easily confirmed using overlapping but independently derived deficiencies with defined cytological breakpoints on the polytene chromosome map (BRIDGES 1935 ). Third, the fitness effects of hybrid incompatibilities can be assessed without being confounded with recombination distance between the relevant locus and a genetic marker. Finally, and perhaps most important, the locations of hybrid lethals can be quickly narrowed to such small chromosome regions that the next step—identifying the particular genes causing hybrid lethality—becomes routine.
The present large-scale, fine-resolution screen reveals the existence of many new hybrid lethals between D. melanogaster and D. simulans. In later work, these hybrid lethals will be the subject of molecular study. Here, I use these data to address the following questions:;f@v, http://www.100md.com
How many hybrid lethals separate D. melanogaster and D. simulans? This number is a proxy for functional divergence between the viability-essential components of the two species' genomes.;f@v, http://www.100md.com
Where are the hybrid lethals? Are they randomly distributed throughout the genome or clustered in particular regions?;f@v, http://www.100md.com
Is hybrid lethality caused by epistasis between incompatible loci, as predicted by the Dobzhansky-Muller model?;f@v, http://www.100md.com
Are most hybrid incompatibilities recessive, as predicted by the dominance theory? There are few direct tests of the dominance of incompatible alleles. Furthermore, comparing the number of recessive hybrid lethals discovered here with the number of dominant ones from other studies provides a quantitative test of the dominance theory.
What is the probability that any two divergent substitutions, one from D. melanogaster and one from D. simulans, are incompatible, causing hybrid lethality?6, http://www.100md.com
What is the distribution of fitness effects of hybrid-lethal incompatibilities? Do most have weak or strong effects on viability? How does this distribution compare with that for hybrid male sterility, which often appears to have a polygenic basis (NAVEIRA and MASIDE 1998 ; WU and HOLLOCHER 1998 )?6, http://www.100md.com
At what stage of development do hybrid lethals act? If genes acting early in development are more evolutionarily constrained than later-acting ones, and thus less diverged, then embryonic hybrid lethals should be rarer than postembryonic ones.6, http://www.100md.com
MATERIALS AND METHODS6, http://www.100md.com
A screen for X-autosome incompatibilities:6, http://www.100md.com
The screening method is diagrammed in and represents a modification of the crossing design of COYNE et al. 1998 . Using many D. melanogaster (mel) stocks, each heterozygous for a deficiency chromosome with known cytological breakpoints and a dominantly marked balancer chromosome (LINDSLEY and ZIMM 1992 ), I crossed mel Deficiency/Balancer (Df/Bal) females to D. simulans (sim) males carrying the hybrid rescue mutation, Lethal hybrid rescue (Lhr). Lhr rescues normally dead F1 males from lethality that typically occurs at the larval-pupal transition (WATANABE 1979 ; TAKAMURA and WATANABE 1980 ; SAWAMURA et al. 1993 ). Half of the F1 hybrid progeny inherit the deficiency and half inherit the balancer. If hybrids of both sexes appear with both Df and Bal genotypes in roughly equal proportions, then no recessive hybrid lethal resides in the sim region uncovered by the deficiency. If, on the other hand, only Df hybrid males die, then a hybrid lethal resides in the sim autosomal region exposed by the deficiency. The fact that only Df hybrid males die, while their Df hybrid sisters do not, suggests either that the sim hybrid lethal is incompatible with a mostly recessive factor on the mel X (females are heterozygous for the X) or that the sim hybrid lethal is male specific. These possibilities are distinguished using follow-up crosses described below.
fig.ommitted(?s*&04, http://www.100md.com
Figure 1. Crosses used in F1 deficiency screen for lethal hybrid incompatibilities. Sex chromosomes and one set of representative autosomes are shown. Gray, D. melanogaster; white, D. simulans. (A) Deficiency/Balancer females crossed to Lhr males produce (in the absence of hybrid lethality) four types of offspring in Mendelian ratios within each sex. (B) Attached-X Deficiency/Balancer female crossed to Lhr male. See text for further details.(?s*&04, http://www.100md.com
shows the 193 deficiencies used. Together these deficiencies uncover ~ 69–77% of 2L (chromosome 2, left arm), 49–59% of 2R, 72–77% of 3L, and 65–79% of 3R. I did not test the small-dot fourth chromosome of D. simulans as it is known to carry no hybrid lethals (MULLER and PONTECORVO 1940 , MULLER and PONTECORVO 1942 ; PONTECORVO 1943A ; ORR 1992 ). All stocks are available through the Bloomington or Umeå Stock Centers (Bloomington Stock Center, ). Soichi Tanda kindly provided a stock with a newly extracted Df(2L)al. The most common dominant markers on chromosome 2 balancers were CurlyO and Glazed, and those on chromosome 3 balancers were Stubble, Serrate, Tubby, or a combination. When possible, deficiency stocks with a chromosome 3 balancer marked only with Tubby or Ultrabithorax were rebalanced over a different chromosome with a more easily scored marker (e.g., Stubble). Complete descriptions of all stocks, including the particular balancers used, are available upon request.
fig.ommitted)!i}, 百拇医药
Table 1. Results from deficiency screen for hybrid-lethal X-autosome incompatibilities)!i}, 百拇医药
Each species cross was made by mass mating 15–20 mel Df/Bal females with 15–25 sim Lhr males. All crosses were done at 24° and flies were reared on standard cornmeal-yeast-agar medium. For each deficiency, I repeated the cross at least four times and report the pooled number of progeny. Contamination was detectable in several ways: consistency among replicate crosses, the segregation of dominant markers among progeny, the presence of diagnostic hybrid male genitalia, and the absolute sterility of all progeny. Although the cross shown in is in principle straightforward, some stocks showed strong sexual isolation, requiring many replicate crosses to obtain sufficient progeny.)!i}, 百拇医药
Most crosses deviated from the expected 1:1:1:1 segregation ratio of Df females, Bal females, Df males, and Bal males, because Lhr rescue of hybrid males is incomplete. Hybrid males were significantly rarer than females in most crosses, with an average of 45% rescue. In a few crosses, no hybrid males were rescued (see also GRANADINO et al. 1996 ); since these crosses are not informative, they are not reported here. For crosses that produced hybrid females and at least 10 Bal hybrid males, I estimated the relative viability of the deficiency by simply tabulating the ratio of Df:Bal progeny for each hybrid sex. I used 2 tests to detect deviations from the expected 1:1 ratio of Df:Bal progeny within each sex and Fisher's exact tests to detect differences in the ratio of Df:Bal progeny between sexes. I defined candidate X-autosome hybrid incompatibilities as those causing a significant deficit of Df hybrid males and a significantly smaller Df:Bal ratio in hybrid males than in hybrid females. I then classified hybrid incompatibilities by viability following the scheme of Crow and colleagues for deleterious mutations within species (reviewed in SIMMONS and CROW 1977 ; CROW and SIMMONS 1983 ): For regions causing significant reductions in Df hybrid male viability, those with viabilities "50% (i.e., a Df:Bal ratio <0.50 at least for one of two overlapping deficiencies) were considered hybrid "semilethals," and those with viabilities of "10% were considered hybrid "lethals."
Further investigation of hybrid-lethal deficiencies:1iu.9.$, http://www.100md.com
I performed three further tests for most of the regions that caused hybrid lethality. First, to ensure that the lethality of deficiencies is hybrid specific, I confirmed the viability of deficiencies in nonhybrids by crossing Df/Bal mel females to wild type, Wolbachia-free Canton-S mel males. (Note, however, that the very existence of the deficiency stocks shows that they are not lethal when heterozygous within species.)1iu.9.$, http://www.100md.com
Second, when possible, I confirmed the presence of each hybrid lethal by testing at least one other overlapping deficiency. Such confirmation with multiple, independently derived deficiencies rules out the possibility that a putative hybrid lethal is an artifact of the genetic background of any particular stock. Those regions shown to be lethal by a single deficiency, but which could not be confirmed because overlapping deficiencies were unavailable, should be viewed as candidate hybrid-lethal regions.
Third, I tested whether the exposed sim factors caused lethality by epistasis with the mel X. To do this, I constructed mel attached-X stocks, each carrying a hybrid-lethal deficiency over a balancer, and crossed females from these stocks to sim Lhr males. As shows, the attached-X chromosome forces mother-to-daughter inheritance of the mel attached-X and father-to-son inheritance of the sim free-X. Hybrid males from this cross thus inherit a sim X rather than a mel X, while the rest of their genotype, including their cytoplasm, remains identical in species origin to the hybrid males from the original cross (compare F1 males in and ). The important point is that if Df hybrid male lethality is caused by an incompatibility between a recessive sim autosomal factor and a recessive factor(s) on the mel X, then replacing the mel X with a sim X should rescue these males (as the sim X should be compatible with the sim autosomal factor). If, on the other hand, hybrid male lethality is caused by a male-specific incompatibility, replacing the X chromosome will not necessarily rescue hybrid males. The reason is that a male-specific incompatibility could involve an interaction between a recessive sim factor and a dominant mel factor anywhere in the D. melanogaster genome. Unfortunately, mel attached-X hybrid females from this cross, which would also have been informative, were not rescued in sufficient numbers (see RESULTS).
Lethal phase:0, 百拇医药
For each incompatibility, I determined if hybrid lethality occurred at embryonic or postembryonic stages. If Df hybrid males suffer embryonic lethality, 25% of all embryos should die (i.e., 50% of all male embryos). To test if embryonic lethality was sufficient to account for the absent Df hybrid males, I calculated the ratio of the number of dead (necrotized) embryos divided by half the number of hybrid females: dead embryos/[(Df females + Bal females)/2]. If this ratio was ">="0, 百拇医药
1, the deficiency was scored as embryonic lethal. If the ratio was <<1, the number of dead embryos could not plausibly explain the deficit of Df hybrid males, and the incompatibility was scored as postembryonic lethal. Establishing the exact phase of lethality for postembryonic hybrid lethals is, unfortunately, complicated by background mortality and the imperfect degree of overall hybrid male rescue by Lhr.0, 百拇医药
RESULTS
Fine-mapping hybrid lethals:9#tp#}, http://www.100md.com
gives the results for all tested deficiencies. The distributions of viability effects for the two sexes are shown in . The most striking result is that while very few deficiencies uncover lethality in hybrid females, many uncover lethality in hybrid males : The distribution of viability for Df hybrid females is unimodal, with most crosses showing normal viability, i.e., roughly equal representation of Df and Bal females. In contrast, the distribution of viability for Df hybrid males is bimodal, with one peak at normal viability and a second near complete lethality. Rare "escaper" males bearing hybrid-lethal deficiencies are typically weak and sometimes show developmental anomalies (e.g., malformed abdominal tergites, bristles, wings, and eyes). In all, 40 nonoverlapping autosomal regions in D. simulans cause significant lethality when uncovered in Df hybrid males. Recessive hybrid lethals are thus common in the sim autosomal genome.
fig.ommitted!3}, 百拇医药
Figure 2. Distribution of relative viability of Df hybrids (deficiency bearing/total) for (A) hybrid females and (B) hybrid males.!3}, 百拇医药
Of these regions, 20 are hybrid-lethal and 20 are hybrid-semilethal incompatibilities . These regions appear to be distributed randomly among the two major autosomes (2 = 3.333, d.f. = 1, P = 0.068) and the four autosomal arms, with 14 on 2L, 14 on 2R, 6 on 3L, and 6 on 3R (2 = 3.195, d.f. = 3, P = 0.363; ). I performed further analyses on 20 hybrid lethals and 3 hybrid semilethals (these semilethals were nearly classifiable as lethal; i.e., the ratio of Df:Bal hybrid males was "0.12 for all 3). The lethal effects of these deficiencies are hybrid specific because none caused strong haploinsufficiency when heterozygous in pure species individuals . Of the 23 hybrid lethals and semilethals, I confirmed 18 (78%) with overlapping deficiencies that yielded consistent results . Each of these regions thus contains at least one hybrid incompatibility factor.
fig.ommittedy%, 百拇医药
Figure 3. Locations of 20 hybrid lethals (solid bars) and 3 hybrid semilethals (shaded bars) on cytological map of major autosomes.y%, 百拇医药
fig.ommittedy%, 百拇医药
Table 2. Deficiencies are not male lethal within D. melanogaster (Df/Bal females x Canton-S males)y%, 百拇医药
It is important to note, however, that more hybrid lethals probably exist than the above numbers suggest because the available deficiencies from D. melanogaster cumulatively uncover only 64–73% of the autosomal genome. Correcting for this incomplete coverage yields an estimate closer to 27 hybrid lethals and ~ 27 hybrid semilethals. These remain minimum estimates as each region could harbor more than one hybrid lethal. However, the fact that most deficiencies fail to uncover hybrid lethality suggests that hybrid lethals are sparsely distributed. Therefore, either most hybrid-lethal regions harbor a single hybrid-lethal locus or hybrid lethals are tightly clustered. If the former is true, as seems most plausible, it follows that many incompatibilities have major effects on hybrid viability.
The recessivity of the incompatible factors on the mel X can be illustrated in three ways. First, if factors on the mel X were completely dominant (and the incompatibilities are not sex specific), Df hybrid female viability would be correlated with Df hybrid male viability, with slope 1. (The slope of this relationship can be thought of as a proxy for the mean dominance coefficient of incompatible factors on the mel X.) Instead, while there is a weak positive correlation between Df hybrid female and male viability, the slope of the least-squares relationship is b = 0.103—a value <<1.0—consistent with the general recessivity of incompatible factors on the mel X (; Spearman's r = 0.326, d.f. = 163, P < 0.0001; statistics use only crosses producing ">="pp, 百拇医药
10 Bal hybrid males). Second, of 16 regions that cause significant lethality in Df hybrid females, 9 cause significantly stronger lethality in Df hybrid males (e.g., , line 4). Third, and most convincing, 30 of 40 deficiencies that caused significant lethality in Df hybrid males have no discernable effects in Df hybrid females. These findings strongly suggest that the incompatible factors on the mel X are also overwhelmingly close to completely recessive.
fig.ommitted3c+11ko, 百拇医药
Figure 4. Relative viability of Df hybrid females (Df-bearing females/total females) plotted against viability of Df hybrid males (Df-bearing males/total males).3c+11ko, 百拇医药
These results are consistent with those of YAGYU and YAMAMOTO 1996 , who independently performed similar crosses using 15 deficiencies on chromosome 2.3c+11ko, 百拇医药
Are hybrid lethals epistatic?3c+11ko, 百拇医药
The hybrid-lethal and semilethal sim autosomal factors detected above could be either incompatible with factors on the mel X or male-specific and incompatible with a dominant mel factor anywhere in the genome. To distinguish these possibilities, I used attached-X crosses shown in to test the viability of Df hybrid males that are genotypically and cytoplasmically identical to those above but possess a sim X rather than a mel X. These hybrid males are hemizygous for the sim X and hemizygous for a hybrid-lethal sim autosomal factor. If the lethal sim autosomal factors are incompatible with the mel X, changing the species origin of the X should rescue hybrid lethality. If, on the other hand, the sim autosomal factors cause male-specific hybrid lethality, changing the species origin of the X may not rescue hybrid lethality.
As shows, 18 of the 18 hybrid-lethal deficiencies tested become viable when hybrid males are given a sim X instead of a mel X. (For two hybrid-lethal deficiencies, the attached-X stocks proved too weak to maintain and could not be tested.) Thus, the hybrid lethality of 18 of 18 sim autosomal regions depends on the species origin of the X, confirming that these are true hybrid lethals caused by incompatible epistatic interactions.3'@gj&, http://www.100md.com
fig.ommitted3'@gj&, http://www.100md.com
Table 3. Hybrid lethality depends on the species origin of the X3'@gj&, http://www.100md.com
These attached-X crosses were also expected to produce hybrid females homozygous for the mel X (and thus genotypically identical to hybrid males from the original screen; compare female hybrids in to male hybrids in ). If produced in sufficient number, we could further test the sex specificity of each hybrid incompatibility: If hybrid lethals are not sex specific, Df hybrid females from attached-X crosses should also be inviable. This outcome appears to hold in three cases (, lines 2, 4, and 13), but in general I obtained too few hybrid females to draw meaningful conclusions.
Lethal phase:xds}}, 百拇医药
I determined the lethal phase of 18 hybrid incompatibilities. Only one causes embryonic lethality (see ). I compared this distribution to that for lethal phases of mutations within species. In D. melanogaster, 60.5% of lethal P insertions cause embryonic lethality (TOROK et al. 1993 ; DEAK et al. 1997 ). There are thus significantly fewer embryonic lethal incompatibilities in hybrids than expected from the within-species data (1 embryonic vs. 17 postembryonic hybrid-lethal incompatibilities; 2515 embryonic vs. 1640 postembryonic within-species lethal mutations; Fisher's exact P < 0.0001). Thus hybrid-lethal incompatibilities more often afflict later, postembryonic stages of development.xds}}, 百拇医药
fig.ommittedxds}}, 百拇医药
Table 4. Summary of results for hybrid-lethal regionsxds}}, 百拇医药
DISCUSSIONxds}}, 百拇医药
This study yields three main results. First (and perhaps most surprising), many hybrid-lethal incompatibilities have evolved between D. melanogaster and D. simulans: The ~ 70% of the D. simulans autosomal genome examined harbors at least 20 factors that cause complete or near complete lethality in the presence of the D. melanogaster X. Taking into account certain corrections (see below), this value implies that the genome-wide number of recessive-recessive lethal incompatibilities is ~ 169. Second, the incompatible genes on both the autosomes and the X are nearly completely recessive and, as shown below, these recessive incompatibilities vastly outnumber dominant ones. These results provide strong support for the dominance theory. Third, all of the lethal hybrid incompatibilities tested involve an epistatic interaction with the X chromosome; i.e., 18 of 18 are rescued when the mel X is replaced by a compatible sim X, as expected under the the Dobzhansky-Muller model.
While these data confirm the ubiquity of recessive, epistatic hybrid incompatibilities, they also allow us to go further. These data can be used to estimate four quantities: (1) the total number of hybrid lethals separating D. melanogaster and D. simulans; (2) the relative rates of evolution of dominant vs. recessive incompatibilities; (3) the probability that two randomly chosen divergent substitutions (one from each species) are incompatible, causing hybrid lethality; and (4) the fraction of viability-essential genes that have diverged to such an extent that they are no longer functionally compatible with alleles at interacting loci from the other species.q|p;e*e, 百拇医药
Dominance and the total number of hybrid lethals:q|p;e*e, 百拇医药
By including data from previous genetic analyses, we can estimate the total number of hybrid lethals that have evolved between D. melanogaster and D. simulans. I make the simplifying assumption that hybrid lethality involves pairs of incompatible loci. Hybrid incompatibilities can then be classified into dominant-dominant, recessive-dominant, and recessive-recessive types (or, following TURELLI and ORR 2000 , H0, H1, and H2 incompatibilities, respectively, where the subscripts 0, 1, and 2 indicate the number of homozygous or hemizygous loci involved). More than two loci could be involved in a given incompatibility—so-called complex epistasis—but this should not seriously affect our estimates as long as the three incompatibility types (H0, H1, and H2) are equally prone to such complex interactions.
H0 incompatibilities: No H0 incompatibilities separate D. melanogaster and D. simulans. The cross of D. melanogaster females with D. simulans males produces perfectly viable hybrid females that are heterozygous at every locus in the genome (STURTEVANT 1920 , STURTEVANT 1929 ; but see BARBASH et al. 2000 for temperature effects on viability of hybrid females).l+t^|, 百拇医药
H1 incompatibilities: There are ~ 22 H1 incompatibilities between D. melanogaster and D. simulans. This value comes from two kinds of data. The first involves studies of hybrid rescue mutations. There is now good evidence that such mutations are rare "compatible" alleles at normally incompatible loci (HUTTER et al. 1990 ; SAWAMURA and YAMAMOTO 1997 ; BARBASH et al. 2000 ; ORR and IRVING 2000 ). We can therefore infer two H1 incompatibilities from the existence of two known pairs of complementary hybrid rescue mutations (HUTTER et al.. 1990 ; SAWAMURA et al. 1993 ; ORR and PRESGRAVES 2000 ). The second comes from COYNE et al.. 1998 deficiency screen for H1 incompatibilities. This screen uncovered five hybrid-lethal regions (this lethality was not unconditional, however, as some became viable at permissive temperatures or using different stocks). Coyne et al.'s number requires two corrections as only 50% of the D. simulans genome was screened, and the reciprocal experiment in which D. melanogaster regions are made hemizygous could not be done as no deficiencies are available in D. simulans. Correcting for these two considerations yields an estimate of ~ 20 hybrid-lethal regions. Thus ~ 22 H1 incompatibilities separate D. melanogaster and D. simulans. This number is obviously rough, but as shown below, it differs qualitatively from that for H2 incompatibilities.
H2 incompatibilities: The total number of H2 incompatibilities is large. We can estimate the genome-wide number by extrapolating from the number of X-autosome H2 incompatibilities (see RESULTS). The X is roughly equivalent in size and gene content to one of the five major chromosome arms (i.e., X, 2L, 2R, 3L, and 3R). Holding the X effectively homozygous (hemizygous) and screening the rest of the genome with deficiencies yielded ~ 27 lethal H2 incompatibilities. If we could repeat this screen, successively holding the autosomal arms 2L, 2R, 3L, and 3R homozygous for D. melanogaster while scanning the rest of the D. simulans genome for lethal incompatibilities, we would expect to uncover another ~ 27 H2 incompatibilities for each arm. We therefore expect ~ 135 hybrid lethals genome-wide. Using similar logic to correct for intra-arm H2 incompatibilities brings the total number of H2 incompatibilities to ~ 169. (Note that extrapolating from the number of X-autosome incompatibilities to the number genome-wide assumes that X-linked and autosomal loci diverge at similar rates; BETANCOURT et al. 2002 survey of divergence at >250 genes from D. melanogaster and D. simulans supports this assumption.)
Two conclusions follow from these calculations. First, summing across H0, H1, and H2 incompatibility types gives an estimate of the total number of hybrid-lethal incompatibilities separating D. melanogaster and D. simulans: 191. (This does not include the hybrid semilethals found here; including them nearly doubles the estimate.) Given the conclusion from previous analyses that the number of hybrid lethals between these two species is small, this number comes as a surprise and reveals an unexpected degree of functional divergence.g\;, 百拇医药
These numbers also provide strong quantitative confirmation of the dominance theory: D. melanogaster and D. simulans are separated by no H0 incompatibilities, by ~ 22 H1 incompatibilities, and by ~ 169 H2 incompatibilities. The number of hybrid lethals thus jumps nearly an order of magnitude as we increase the number of homozygous loci involved, leaving little doubt that most hybrid lethals are recessive. Why hybrid incompatibilities tend to be recessive remains a mystery. Although the similar effects of loss-of-function mutations within species and incompatibilities in hybrids have led to speculation that the latter mimic the former (STEBBINS 1958 ; ORR 1993 ; TURELLI and ORR 1995 ), several recent lines of evidence now appear inconsistent with this interpretation (BARBASH et al. 2000 ; ORR and IRVING 2000 ; ORR and PRESGRAVES 2000 ). But regardless of why most incompatibilities act as recessives, the present results leave little doubt that they do.
Epistasis and hybrid lethality:8{xssqw, http://www.100md.com
Using the above estimate of the total number of hybrid-lethal incompatibilities, along with a molecular estimate of the total number of divergent substitutions between D. melanogaster and D. simulans, we can estimate (to an order of magnitude) another important quantity from speciation genetics theory: the probability, p, that any two randomly chosen divergent sites (one from one species, one from the other) are incompatible in hybrids, causing (for our purposes) complete lethality. As ORR and TURELLI 2001 show, the expected number of hybrid incompatibilities, I, between pairs of genes is8{xssqw, http://www.100md.com
where k is the genome-wide rate of substitution and t is the time since speciation, so that 2kt is the total number of substitutions separating the genomes of two species. By substituting estimates of I and kt and rearranging, we can solve for p. To ensure that our estimate of p for hybrid lethality is directly comparable to the estimate of p for hybrid male sterility calculated by ORR and TURELLI 2001 , I use the same data sources for k and t and follow their calculation exactly. (See Appendix for fuller details and discussion of the calculation.) The total number of hybrid lethals is, from above, I 191. D. melanogaster and D. simulans have accumulated 2kt 156,000 nonsynonymous substitutions since they diverged t = 2.5 MYA (HEY and KLIMAN 1993; LI 1997 ). Solving the above equation thus gives p 1.6 x 10-8. Two nonsynonymous substitutions chosen randomly (one from each species' genome) will therefore cause complete hybrid lethality ~ 10-8 of the time. As discussed in the Appendix, this value appears to be an order of magnitude smaller than the value for hybrid male sterility estimated by ORR and TURELLI 2001 .
Interestingly, p can be thought of as a crude index of the ruggedness of the molecular landscape. To see this, consider the extreme cases: When p = 0, negative epistasis is absent, all substitutions are compatible, and hybrid genotypes never fall into fitness valleys; when p = 1, however, negative epistasis is complete, all divergent substitutions after the first are incompatible, and all hybrid genotypes fall into fitness valleys. Thus, the exceedingly small value of p suggests that the molecular landscape is reasonably smooth: Substitutions that have never "seen" each other in their evolutionary histories are almost always compatible (or at least not lethal in combination).\lk$, 百拇医药
Functional divergence:\lk$, 百拇医药
Finally, we can estimate the fraction of viability-essential genes that have diverged to the extent that they are no longer functionally compatible. If hybrid-lethal incompatibilities involve pairs of loci, as assumed above, it follows from the Dobzhansky-Muller model that both loci must have diverged (ORR 1995 ). The above estimate of 191 hybrid-lethal incompatibilities thus implies that at least 382 loci have experienced significant functional divergence since the D. melanogaster-D. simulans split, i.e., 11% of all viability-essential genes (or 382/3600; see SPRADLING et al. 1999 ). This level of divergence at a class of genes that we might a priori expect to be relatively conserved (i.e., those essential for viability) seems remarkable.
Two lines of evidence suggest that the genes regulating the earliest phases of development in the two species remain largely compatible. First, most hybrid genotypes survive embryonic phases of development only to die later (CARVAJAL et al. 1996 ). Second, my deficiency screen, as well as that of COYNE et al. 1998 , could have detected any maternal-zygotic, embryonic lethal incompatibilities involving the D. melanogaster cytoplasm and hemizygous D. simulans autosomal factors. But very few of our incompatibilities can possibly fall into this category. Instead, most hybrid incompatibilities occur between zygotic genes acting at postembryonic stages. Since viability-essential genes are most often mutable to embryonic lethality within species, the paucity of embryonic vs. postembryonic hybrid lethals suggests that: (i) There are greater functional constraints (on gene sequence and/or expression) at early acting genes; (ii) most physiological and ecological adaptation in Drosophila occurs by divergence in postembryonic phases of development; or (iii) more genes are simultaneously active and thus prone to incompatible interactions during later stages of development. The latter possibility seems unlikely to account for the strong preponderance of postembryonic hybrid lethality as most genes exhibiting developmentally modulated expression during the Drosophila life cycle are expressed at some point during embryogenesis (>88%; ARBEITMAN et al. 2002 ).
Caveats:)yo, 百拇医药
Using deficiencies and a hybrid rescue mutation to detect X-autosome incompatibilities makes two key assumptions. The first is that the effects of D. simulans autosomal regions when hemizygous are equivalent to when they are homozygous. If this assumption is incorrect, then instead of uncovering "hybrid lethals," the screen might simply uncover regions that are haploinsufficient. (Hemizygosity of the D. melanogaster X in males is equivalent to homozygosity because of dosage compensation.) Three facts, however, militate against this possibility (see also COYNE et al. 1998 ). First, although deficiencies can uncover recessive lethality within species, they are not inherently lethal as heterozygotes within species .)yo, 百拇医药
Second, one might argue that deficiencies cause haploinsufficiency, but only in hybrids. Two observations rule out this possibility: deficiencies that cause lethality in hybrid males do not, in most cases, harm Df hybrid females; more important, even hybrid males that carry a hybrid-lethal deficiency become viable when carrying a D. simulans, rather than a D. melanogaster, X. Thus, the lethality of particular deficiencies depends on species genotype at background loci (i.e., epistasis), as expected for hybrid incompatibilities.
Third, there are now two examples in which factors from D. simulans are known to behave identically in species hybrids, whether homozygous or hemizygous:vhh|, 百拇医药
MULLER and PONTECORVO 1940 , MULLER and PONTECORVO 1942 discovered that the fourth chromosome of D. simulans causes hybrid male sterility when homozygous on an otherwise D. melanogaster genetic background. ORR 1992 fine mapped the region of the D. simulans fourth responsible using D. melanogaster deficiencies, thus confirming that the recessive, incompatible D. simulans factor causes sterility when homozygous or hemizygous.vhh|, 百拇医药
SAWAMURA et al. 2000 used a mutation that weakly rescues hybrid female fertility to introgress two small regions from D. simulans 2L into an otherwise D. melanogaster background. According to my results, these regions harbor three factors that each cause hybrid lethality when hemizygous in a genetic background containing a hemizygous D. melanogaster X and heterozygous autosomes (, lines 1, 3, 4). SAWAMURA 2000 found that, in an identical genetic background, these regions also cause hybrid lethality when homozygous.
Thus, both Orr's and Sawamura's results show that recessive hybrid incompatibilities behave identically when homozygous or hemizygous.7(cw-w, 百拇医药
The second assumption of the deficiency analysis is that the 40 hybrid incompatibilities detected are independent of hybrid male rescue by Lhr. The primary cross was intended to rescue hybrid males from one incompatibility (involving the incompatible, wild-type allele Lhrsim) and to then expose them to other potential incompatibilities. It is formally possible, however, that some of these hybrid lethals are actually suppressors of Lhr rescue. If true, these suppressors would still be of interest as interactors of a known hybrid incompatibility locus (Lhr). But this Lhr-suppressor scenario seems unlikely for several reasons. First, it seems unlikely that since the D. melanogaster-D. simulans split ~ 2.5 MYA, the only genetic pathways to evolve incompatibilities necessarily all involve Lhr. Second, such putative Lhr suppressors would necessarily be recessive D. simulans factors that suppress the rescue effects of a particular rare allele, Lhr. It is hard to believe that so many different recessive, conspecific loci are capable of such a trick. Third, Lhr rescues hybrid male lethality that normally occurs at the larval-pupal transition. But at least one hybrid lethal does not affect this phase of development, causing embryonic lethality instead (, line 7), and thus cannot be explained by Lhr suppression. Casual observations further suggest that more detailed study of the postembryonic lethal phases will reveal other cases acting later than the larval-pupal transition.
Conclusions:%6+062, http://www.100md.com
This study shows that most hybrid incompatibilities are recessive and epistatic. The most surprising finding is the extraordinary degree of functional divergence that has occurred between D. melanogaster and D. simulans during the last 2.5 MY. Such extensive cryptic divergence would seem to confirm Muller's suggestion that "(t)wo groups of organisms which are not ordinarily allowed to cross with one another will thus automatically become increasingly immiscible, and their genic, chemical paths of evolution will diverge more and more ... even in cases where their evolution is, from the phenotypic standpoint, strikingly parallel" (MULLER 1939 , p. 278).%6+062, http://www.100md.com
An important aspect of the many hybrid incompatibilities identified here should not be overlooked: Their fine-scale resolution in a model genetic organism will greatly facilitate their routine molecular characterization. The 20 hybrid lethals and 20 hybrid semilethals discovered each reside in just a few cytological divisions. It is conceptually simple (although labor intensive) to move from identifying blocks of candidate genes using deficiency complementation tests to identifying the relevant genes using single-locus complementation tests. Establishing the molecular identity, function, and evolutionary history of a large collection of speciation genes is certain to reveal new patterns and to answer some long-standing questions in evolution: What are the normal functions of "speciation genes" within species? Are most functionally relevant substitutions concentrated in coding or regulatory regions? Is natural selection the primary force causing the fixation of divergent substitutions? Preliminary work in several of the hybrid-lethal regions identified here suggests that the molecular characterization of the genes responsible should be possible.
ACKNOWLEDGMENTS12e^j, 百拇医药
I thank Shawn Ciecko for expert technical assistance during the early phases of this work. I am also grateful to Andrea Betancourt, Andy Clark, Jerry Coyne, Allen Orr, and Jack Werren for helpful discussions and advice on the project. I also thank Andrea Betancourt, Audrey Chang, Jerry Coyne, J. P. Masly, Mohamed Noor, two anonymous reviewers, and especially Allen Orr for comments that greatly improved the manuscript. This work was supported by funds from an Ernst Caspari Fellowship and a George and Agnes M. Messersmith Fellowship to the author and by funds from the National Institutes of Health (GM-526738) and David and Lucile Packard Foundation to Allen Orr.12e^j, 百拇医药
Manuscript received September 20, 2002; Accepted for publication December 3, 2002.12e^j, 百拇医药
APPENDIX12e^j, 百拇医药
CALCULATION OF p12e^j, 百拇医药
To obtain 2kt, the total number of replacement changes separating D. melanogaster and D. simulans, I use LI's (1997) estimate of the rate of nonsynonymous substitution in Drosophila from 30 loci, 1.91 x 10-9/site/year. To get the genome-wide rate of substitution, I take into account the number of relevant sites in the genome: 13,600 genes/genome x 1800 bp/gene x ~ 2/3 are nonsynonymous sites = 1.632 x 107. Multiplying gives the number of replacement changes per genome per year: k = 0.0312. Thus the total number of replacement changes that have accumulated between D. melanogaster and D. simulans during the last 2.5 MY is 2kt 156,000. Solving the equation gives p 1.6 x 10-8.
We can check the plausibility of this estimate of p for hybrid lethality using data from another species pair. In particular, given p for hybrid lethality from above and the number of genome-wide substitutions separating a younger species pair, D. mauritiana and D. simulans, we can ask: How many hybrid lethals should we expect to find in a genetic analysis of D. mauritiana-D. simulans hybrids? (This assumes, of course, that p itself has not evolved substantially over the last few million years, as seems reasonable.) We already know the number of hybrid lethals between D. mauritiana and D. simulans to be ~ 5–10 from the work of TRUE et al. 1996 and so can compare it to the one predicted by the Orr-Turelli equation. To get the predicted number, we use p = 1.6 x 10-8 from above and the genome-wide number of nonsynonymous substitutions between D. mauritiana and D. simulans, 2kt = 48,000 (see ORR and TURELLI 2001 ). Solving for I, the predicted number of hybrid lethals is 18. Thus, given nothing more than p for hybrid lethality, as estimated from the D. melanogaster-D. simulans hybridization and a crude estimate of the number of substitutions, the equation comes remarkably close to predicting the true number of hybrid lethals.
The estimate of p for hybrid lethality is an order of magnitude smaller than the one estimated for hybrid male sterility using genetic data from D. simulans and D. mauritiana, p 1.04 x 10-7 (ORR and TURELLI 2001 ). This discrepancy could be explained by either of the two putative causes of faster-male evolution (WU and DAVIS 1993 ; WU et al. 1996 ): (i) Spermatogenesis might be inherently more sensitive than viability to the incompatibilities experienced by hybrids, and thus a greater fraction of interspecific gene interactions cause hybrid male sterility; or (ii) our calculations have not taken into account that male-specific genes evolve faster, on average, than viability-essential genes (BEGUN et al. 2000 ; SWANSON et al. 2001 ; BETANCOURT et al. 2002 ). As defined here and in ORR and TURELLI 2001 , p—the probability that any two divergent replacement changes (one from each species) from any gene in the genome are incompatible—ignores that not all genes interact with each other, that not all genes are mutable to sterility, that not all genes are mutable to lethality, that not all incompatibilities are protein-protein interactions, etc. In the absence of such perfect knowledge, it is convenient to estimate p as the genome-wide probability of incompatibility, averaging over all genes. But since male-specific genes evolve more rapidly than others (and, in particular, faster than the 30 loci used to estimate k from LI 1997 above), p for hybrid male sterility will be overestimated. Consequently, p for hybrid lethality and p for hybrid sterility are likely closer than they appear. The important point, however, is that both values are exceedingly small.
LITERATURE CITED$8&i]., 百拇医药
ARBEITMAN, M. N., E. E. M. FURLONG, F. IMAM, E. JOHNSON, and B. H. NULL et al., 2002 Gene expression during the life cycle of Drosophila melanogaster. Science 297:2270-2275.$8&i]., 百拇医药
BARBASH, D. A., J. ROOTE, and M. ASHBURNER, 2000 The Drosophila melanogaster Hybrid male rescue gene causes inviability in male and female species hybrids. Genetics 154:1747-1771.$8&i]., 百拇医药
BEGUN, D. J., P. WHITLEY, B. L. TODD, H. M. WALDRIP-DAIL, and A. G. CLARK, 2000 Molecular population genetics of male accessory gland proteins in Drosophila. Genetics 156:1879-1888.$8&i]., 百拇医药
BETANCOURT, A. J., D. C. PRESGRAVES, and W. J. SWANSON, 2002 A test for faster X evolution in Drosophila.. Mol. Biol. Evol. 19:1816-1819.$8&i]., 百拇医药
BRIDGES, C. B., 1935 Salivary chromosome maps. J. Hered. 26:60-64.$8&i]., 百拇医药
CARVAJAL, A. R., M. R. GANDARELA, and H. F. NAVEIRA, 1996 A three-locus system of interspecific incompatibility underlies male inviability in hybrids between Drosophila buzzatii and D. koepferae.. Genetica 98:1-19.
COYNE, J. A. and H. A. ORR, 1989 Patterns of speciation in Drosophila. Evolution 43:362-381.*[a:\!k, 百拇医药
COYNE, J. A. and H. A. ORR, 1997 "Patterns of speciation in Drosophila" revisited. Evolution 51:295-303.*[a:\!k, 百拇医药
COYNE, J. A., S. SIMEONIDIS, and P. ROONEY, 1998 Relative paucity of genes causing inviability in hybrids between Drosophila melanogaster and D. simulans.. Genetics 150:1091-1103.*[a:\!k, 百拇医药
CROW, J. F., and M. J. SIMMONS, 1983 The mutation load in Drosophila, pp. 1–35 in The Biology and Genetics of Drosophila, edited by M. ASHBURNER, H. L. CARSON and J. N. THOMPSON. Academic Press, London.*[a:\!k, 百拇医药
DAVIS, A. W., J. ROOTE, T. MORLEY, K. SAWAMURA, and S. HERRMANN et al., 1996 Rescue of hybrid sterility in crosses between D. melanogaster and D. simulans.. Nature 380:157-159.*[a:\!k, 百拇医药
DEAK, P., M. M. OMAR, R. D. C. SAUNDERS, M. PAL, and O. KOMONYI et al., 1997 P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E–87F. Genetics 147:1697-1722.
DOBZHANSKY, T., 1937 Genetics and the Origin of Species. Columbia University Press, New York.|r+v, 百拇医药
GRANADINO, B., L. O. F. PENALVA, and L. SANCHEZ, 1996 Indirect evidence of alteration in the expression of the rDNA genes of interspecific hybrids between Drosophila melanogaster and Drosophila simulans.. Mol. Gen. Genet. 250:89-96.|r+v, 百拇医药
HALDANE, J. B. S., 1922 Sex-ratio and unidirectional sterility in hybrid animals. J. Genet. 12:101-109.|r+v, 百拇医药
HEY, J. and R. M. KLIMAN, 1993 Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex. Mol. Biol. Evol. 10:804-822.|r+v, 百拇医药
HOLLOCHER, H. and C.-I WU, 1996 The genetics of reproductive isolation in the Drosophila simulans clade: X vs. autosomal effects and male vs. female effects. Genetics 143:1243-1255.|r+v, 百拇医药
HUTTER, P., J. ROOTE, and M. ASHBURNER, 1990 A genetic basis for the inviability of hybrids between sibling species of Drosophila. Genetics 124:909-920.
LACHAISE, D., J. R. DAVID, F. LEMEUNIER, L. TSACAS, and M. ASHBURNER, 1986 The reproductive relationships of Drosophila sechellia with D. mauritiana, D. simulans, and D. melanogaster from the Afrotropical region. Evolution 40:262-271.|^2]q\j, 百拇医药
LI, W.-H., 1997 Molecular Evolution. Sinauer Associates, Sunderland, MA.|^2]q\j, 百拇医药
LINDSLEY, D. L., and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster. Academic Press, San Diego.|^2]q\j, 百拇医药
MULLER, H. J., 1939 Reversibility in evolution considered from the standpoint of genetics. Biol. Rev. Camb. Phil. Soc. 14:261-280.|^2]q\j, 百拇医药
MULLER, H. J., 1940 Bearing of the Drosophila work on systematics, pp. 185–268 in The New Systematics, edited by J. S. HUXLEY. Clarendon Press, Oxford.|^2]q\j, 百拇医药
MULLER, H. J., 1942 Isolating mechanisms, evolution, and temperature. Biol. Symp. 6:71-125.|^2]q\j, 百拇医药
MULLER, H. J. and G. PONTECORVO, 1940 Recombinants between Drosophila species, the F1 hybrids of which are sterile. Nature 146:199.
MULLER, H. J. and G. PONTECORVO, 1942 Recessive genes causing interspecific sterility and other disharmonies between Drosophila melanogaster and simulans.. Genetics 27:157.#'lmx, 百拇医药
NAVEIRA, H. F., and X. R. MASIDE, 1998 The genetics of hybrid male sterility in Drosophila, pp. 330–338 in Endless Forms, edited by D. J. HOWARD and S. H. BERLOCHER. Oxford University Press, Oxford.#'lmx, 百拇医药
ORR, H. A., 1992 Mapping and characterization of a "speciation gene" in Drosophila. Genet. Res. 59:73-80.#'lmx, 百拇医药
ORR, H. A., 1993 A mathematical model of Haldane's rule. Evolution 47:1606-1611.#'lmx, 百拇医药
ORR, H. A., 1995 The population genetics of speciation: the evolution of hybrid incompatibilities. Genetics 139:1805-1813.#'lmx, 百拇医药
ORR, H. A., 1996 Dobzhansky, Bateson, and the genetics of speciation. Genetics 144:1331-1335.#'lmx, 百拇医药
ORR, H. A. and S. IRVING, 2000 Genetic analysis of the Hybrid male rescue locus of Drosophila. Genetics 155:225-231.#'lmx, 百拇医药
ORR, H. A. and D. C. PRESGRAVES, 2000 Speciation by postzygotic isolation: forces, genes and molecules. Bioessays 22:1085-1094.
ORR, H. A. and M. TURELLI, 2001 The evolution of postzygotic isolation: accumulating Dobzhansky-Muller incompatibilities. Evolution 55:1085-1094.u;, http://www.100md.com
PONTECORVO, G., 1943a Hybrid sterility in artificially produced recombinants between Drosophila melanogaster and D. simulans. Proc. R. Soc. Edinb. Sect. B (Biol.) 61:385-397.u;, http://www.100md.com
PONTECORVO, G., 1943b Viability interactions between chromosomes of Drosophila melanogaster and Drosophila simulans.. J. Genet. 45:51-66.u;, http://www.100md.com
PRESGRAVES, D. C., 2002 Patterns of postzygotic isolation in Lepidoptera. Evolution 56:1168-1183.u;, http://www.100md.com
PRESGRAVES, D. C. and H. A. ORR, 1998 Haldane's rule in taxa lacking a hemizygous X.. Science 282:952-954.u;, http://www.100md.com
PRICE, T. D. and M. M. BOUVIER, 2002 The evolution of F1 postzygotic incompatibilities in birds. Evolution 56:2083-2089.u;, http://www.100md.com
PROVINE, W. B., 1991 Alfred Henry Sturtevant and crosses between Drosophila melanogaster and Drosophila simulans.. Genetics 129:1-5.
SANCHEZ, L., B. GRANADINO, and L. VICENTE, 1994 Clonal analysis in hybrids between Drosophila melanogaster and Drosophila simulans.. Roux's Arch. Dev. Biol. 204:112-117.j'56, http://www.100md.com
SASA, M. M., P. T. CHIPPINDALE, and N. A. JOHNSON, 1998 Patterns of postzygotic isolation in frogs. Evolution 52:1811-1820.j'56, http://www.100md.com
SAWAMURA, K., 2000 Genetics of hybrid inviability and sterility in Drosophila: the Drosophila melanogaster-Drosophila simulans case. Plant Species Biol. 15:237-247.j'56, http://www.100md.com
SAWAMURA, K. and M.-T. YAMAMOTO, 1997 Characterization of a reproductive isolation gene, Zygotic hybrid rescue, of Drosophila melanogaster by using minichromosomes. Heredity 79:97-103.j'56, http://www.100md.com
SAWAMURA, K., T. K. WATANABE, and M.-T. YAMAMOTO, 1993 Hybrid lethal systems in the Drosophila melanogaster species complex. Genetica 88:175-185.j'56, http://www.100md.com
SAWAMURA, K., A. W. DAVIS, and C.-I WU, 2000 Genetic analysis of speciation by means of introgression into Drosophila melanogaster.. Proc. Natl. Acad. Sci. USA 97:2652-2655.
SIMMONS, M. J. and J. F. CROW, 1977 Mutations affecting fitness in Drosophila.. Annu. Rev. Genet. 11:49-78.(?s*&04, http://www.100md.com
SINGH, R. S., 1999 Toward a unified theory of speciation, pp. 570–604 in Evolutionary Genetics: From Molecules to Morphology, edited by R. S. SINGH and C. B. KRIMBAS. Cambridge University Press, Cambridge, UK/London/New York.(?s*&04, http://www.100md.com
SPRADLING, A. C., D. STERN, A. BEATON, E. J. REHM, and T. LAVERTY et al., 1999 The Berkeley Drosophila Genome Project gene disruption project: single P-element insertions mutating 25% of vital Drosophila genes. Genetics 153:135-177.(?s*&04, http://www.100md.com
STEBBINS, G. L., 1958 The inviability, weakness, and sterility of interspecific hybrids. Adv. Genet. 9:147-215.(?s*&04, http://www.100md.com
STURTEVANT, A. H., 1920 Genetic studies on Drosophila simulans. I. Introduction. Hybrids with Drosophila melanogaster. Genetics 5:488-500.(?s*&04, http://www.100md.com
STURTEVANT, A. H., 1929 The genetics of Drosophila simulans.. Carnegie Inst. Washington Publ. 399:1-62.(?s*&04, http://www.100md.com
SWANSON, W. J., A. G. CLARK, H. M. WALDRIP-DAIL, M. F. WOLFNER, and C. F. AQUADRO, 2001 Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila. Proc. Natl. Acad. Sci. USA 98:7375-7379.
TAKAMURA, T. and T. K. WATANABE, 1980 Further studies on the Lethal hybrid rescue (Lhr) gene of Drosophila simulans.. Jpn. J. Genet. 55:405-408.)!i}, 百拇医药
TOROK, T., G. TICK, M. ALVARADAO, and I. KISS, 1993 P-lacW insertional mutagenesis on the second chromosome of Drosophila melanogaster: isolation of lethals with different overgrowth phenotypes. Genetics 135:71-80.)!i}, 百拇医药
TRUE, J. R., B. S. WEIR, and C. C. LAURIE, 1996 A genome-wide survey of hybrid incompatibility factors by the introgression of marked segments of Drosophila mauritiana chromosomes into Drosophila simulans.. Genetics 142:819-837.)!i}, 百拇医药
TURELLI, M. and H. A. ORR, 1995 The dominance theory of Haldane's rule. Genetics 140:389-402.)!i}, 百拇医药
TURELLI, M. and H. A. ORR, 2000 Dominance, epistasis and the genetics of postzygotic isolation. Genetics 154:1663-1679.)!i}, 百拇医药
WATANABE, T. K., 1979 A gene that rescues the lethal hybrids between Drosophila melanogaster and D. simulans.. Japn. J. Genet. 54:325-331.)!i}, 百拇医药
WU, C.-I and A. W. DAVIS, 1993 Evolution of postmating reproductive isolation: the composite nature of Haldane's rule and its genetic bases. Am. Nat. 142:187-212.)!i}, 百拇医药
WU, C.-I, and H. HOLLOCHER, 1998 Subtle is nature: the genetics of species differentiation and speciation, pp. 339–351 in Endless Forms, edited by D. J. HOWARD and S. H. BERLOCHER. Oxford University Press, Oxford.)!i}, 百拇医药
WU, C.-I, N. A. JOHNSON, and M. F. PALOPOLI, 1996 Haldane's rule and its legacy: Why are there so many sterile males? Trends Ecol. Evol. 11:281-284.)!i}, 百拇医药
YAGYU, M. and M.-T. YAMAMOTO, 1996 Hybrid rescue effect of Lhr is suppressed in deficiency heterozygotes in the hybrid between D. melanogaster and D. simulans.. Genes Genet. Syst. 71:423.(Daven C. Presgraves)