The Genetic Basis of Reproductive Isolation: Insights from Drosophila

Colloquium

The genetic basis of reproductive isolation: Insights from Drosophila

H. Allen Orr*

Department of Biology, University of Rochester, Rochester, NY 14627 Recent studies of the genetics of speciation in Drosophila have number of new and careful studies of the biogeography, ecology, focused on two problems: (i) identifying and characterizing the and genetics of speciation, and we now understand a good deal genes that cause reproductive isolation, and (ii) determining the about the evolution of reproductive isolation (3). We can, for evolutionary forces that drove the divergence of these ‘‘speciation instance, describe the rate at which this reproductive isolation genes.’’ Here, I review this work. I conclude that speciation genes evolves (Fig. 1), and the ecological factors that drive speciation, correspond to ordinary loci having normal functions within species. at least in some cases (6). We can also point to plausible examples These genes fall into several functional classes, although a role in in which the process of reinforcement may have completed transcriptional regulation could prove particularly common. More speciation (3, 7, 8). Finally, we know a fair amount about the important, speciation genes are typically very rapidly evolving, and number and location of the genes that cause reproductive this divergence is often driven by positive Darwinian selection. isolation (at least in its postzygotic form), and the causes of Finally, I review recent work in Drosophila pseudoobscura on the patterns like Haldane’s rule (3, 9, 10). possible role of meiotic drive in the evolution of the genes that Given this progress, it is natural to ask: What now are the cause postzygotic isolation. major outstanding problems in speciation? On the genetical front (I am not competent to address any other), the answers rnst Mayr made at least three contributions that are funda- seem clear. We must (i) find the genes that cause reproductive Emental to the genetic study of speciation. The first, and surely isolation and (ii) identify the evolutionary forces that drove their most important, was his codification of what we mean by species divergence. and thus by speciation. In his 1942 book, Systematics and the The Problem Origin of Species, Mayr (1) famously argued that species are best defined by the Biological Species Concept: species are groups of Biologists who do not specialize in speciation are invariably actually or potentially interbreeding natural populations that are surprised to learn that evolutionists have identified very few reproductively isolated from other such groups. Reproductive genes causing reproductive isolation. The reason for this slow isolation is thus the sine qua non of good species. progress is, however, simple. If species are taxa that are repro- Mayr’s second contribution, elaborated in his 1963 book, ductively isolated, a genetics of speciation must, almost by Animal Species and Evolution (2), was his argument that repro- definition, be a genetics where such a thing is not possible, ductive isolation usually evolves in allopatry. This has, of course, between organisms that do not exchange genes. The conse- been an enormously controversial claim, and I will not enter into quence of this methodological dilemma is that evolutionists have it in detail. Suffice it to say that the weight of evidence, both been unable to address a large set of fundamental questions biogeographic and comparative, now strongly suggests that Mayr about the genes that cause reproductive isolation, so-called was correct. Although sympatric speciation may well occur, speciation genes. (This perhaps unfortunate term, which is now reproductive isolation appears usually to evolve in allopatry entrenched in the literature, refers to any locus that causes (reviewed in ref. 3, chapters 3 and 4). reproductive isolation, whether in F1 or later-generation hybrids, Mayr’s third key claim, introduced in his 1942 book but and whether the gene was among the first to cause isolation or elaborated in 1954 (4) and later in Animal Species and Evolution, not.) These questions include: Are speciation genes ‘‘ordinary’’ was that genetic drift plays a critical role in speciation. According genes that have normal functions within species? If so, do to Mayr, large populations suffer a sort of evolutionary inertia: speciation genes fall into one or at least a few functional classes? the conservative forces of gene flow and epistasis prevent, or at Are substitutions in speciation genes concentrated in coding or least render unlikely, the evolution of novel morphologies or new regulatory sequences? And do speciation genes diverge by coadapted gene complexes in large, geographically widespread natural selection or genetic drift? This last question is perhaps species. Such evolution is, he suggested, far more likely in island the most important: if we can identify speciation genes at the or peripheral populations founded by a few individuals. Genetic level of DNA sequences, we should be able to bring to bear a drift in such populations may allow rapid evolution to new fitness powerful set of molecular population genetic tools (e.g., Mc- peaks, permitting the evolution of reproductive isolation be- Donald–Kreitman and Hudson–Kreitman–Aguade tests) on the tween ancestral and founder populations. Such ‘‘founder effect’’ Mayrian question of the relative roles of deterministic vs. models of speciation proved extraordinarily popular, represent- stochastic forces in the origin of species. ing, as Provine (5) noted, ‘‘the favored explanation for at least The methodological dilemma that plagues speciation genetics island speciation since 1954.’’ Despite this popularity, it is is sufficiently serious that, until recently, evolutionists could difficult to point to unambiguous evidence for founder effect speciation, and the idea has grown controversial. I return to this This paper results from the Arthur M. Sackler Colloquium of the National Academy of issue below. Sciences, ‘‘Systematics and the Origin of Species: On Ernst Mayr’s 100th Anniversary,’’ held The research program that Mayr helped to formulate, that to December 16–18, 2004, at the Arnold and Mabel Beckman Center of the National Acad- understand the origin of species one must understand the origin emies of Science and Engineering in Irvine, CA. of reproductive isolation, has experienced a renaissance over the *To whom correspondence should be addressed. E-mail: [email protected]. last two decades. Evolutionists have performed an impressive © 2005 by The National Academy of Sciences of the USA

6522–6526 ͉ PNAS ͉ May 3, 2005 ͉ vol. 102 ͉ suppl. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501893102 Downloaded by guest on September 26, 2021 less of the form of hybrid inviability or sterility involved. One cannot, after all, draw robust conclusions about the identities or evolutionary histories of the genes causing reproductive isolation from a sample of one. Fortunately, these recent efforts, by a number of laboratories, have largely succeeded, and we now know the identities of several additional speciation genes. I briefly review this work. I then turn to recent efforts to characterize the evolutionary forces that drive the divergence of such genes. As we will see, these forces may sometimes assume a surprising form. The Search for Speciation Genes The general methodological dilemma facing the genetics of speciation involves a number of particularly unfortunate special cases. Perhaps worst of all, one of our most sophisticated genetic model systems, Drosophila melanogaster, forms only inviable or sterile hybrids when crossed to any other species. As a result, D. melanogaster has, until recently, proved nearly useless in the Fig. 1. The rate of increase in ‘‘total’’ reproductive isolation with genetic study of speciation. distance in Drosophila. Postzygotic isolation is measured such that a value of Drosophila geneticists have taken two approaches to sidestep 0 means no reproductive isolation (either pre-or postzygotic) and a value of 1 this problem. The first step involves study of non-melanogaster fly means complete reproductive isolation. Genetic distance is measured by Nei’s species, wherein at least some of the tools first developed in D. genetic distance, which increases approximately linearly with time over the melanogaster may be available (e.g., germ-line transformation, or values shown. Data include both allopatric and sympatric species pairs. Al- P element insertions). This approach allowed identification of a though the measure of total reproductive isolation shown includes aspects of both pre- and postzygotic isolation, it is imperfect and generally excludes gene that causes hybrid male sterility. Coyne and Charlesworth forms of reproductive isolation (e.g., ecological isolation) that are not readily (19) mapped a locus causing sterility of Drosophila simulans– measured in the laboratory. Data are based on refs. 57 and 58. [Reproduced Drosophila mauritiana hybrid males to a small region of the D. with permission from ref. 58 (Copyright 1997, International Journal of Organic mauritiana X chromosome. Subsequent work (20, 21) refined Evolution).] this mapping, showing that at least two hybrid sterility factors reside in the region, one of which reduces hybrid fertility by half when introgressed alone onto a D. simulans genetic background. point to only a single gene that causes postzygotic reproductive Ting et al. (22) identified this locus, Odysseus-Homeobox gene isolation (Table 1). (I focus on postzygotic isolation because it (OdsH), at the molecular level. As its name indicates, OdsH is an has been the subject of most genetic analysis, both in general and X-linked homeobox gene and thus a presumed transcription in my own laboratory; see, however, refs. 11–14 for discussion of factor (22, 23). Recent transformation experiments appear to genes causing prezygotic isolation.) It had long been known that confirm that OdsH causes partial male sterility among backcross crosses between certain fish species of the genus Xiphophorus, (although not F1) hybrids (24). Sterility may reflect misexpres- e.g., between the platyfish and swordtail, result in hybrid invi- sion of OdsH in hybrid testes (23). OdsH evolved rapidly between ability. In particular, backcross hybrids between these species D. simulans and D. mauritiana, and this evolution was almost often develop malignant melanomas and die (15). Half a century certainly driven by positive Darwinian selection: nonsynony- of genetic study has revealed that this hybrid lethality results mous substitutions greatly outnumber synonymous ones in the from a two-locus ‘‘Dobzhansky–Muller’’ incompatibility: one lineage leading to D. mauritiana (22). locus on the X chromosome of the platyfish is incompatible with The second approach to identifying speciation genes has taken another (apparently single) locus on an autosome of the sword- more direct advantage of D. melanogaster. Although any hybrids tail. Melanoma results when these loci are brought together in formed between this species and its closest relatives are com- a hybrid genome. Molecular genetic analyses allowed identifi- pletely sterile, this fact does not preclude its use in the search for cation and characterization of the X-linked partner in this hybrid inviability genes. Several workers (25–28) used tools from incompatibility: Xmrk-2. Xmrk-2 encodes a novel receptor ty- D. melanogaster (e.g., chromosomal duplications and deletions, rosine kinase, which, while found as a duplicate gene on the loss-of-function mutations, and germ-line transformation) to platyfish X, is absent from the swordtail X (16–18). Xmrk-2 map and characterize an X-linked gene, Hybrid male rescue appears to be misexpressed in species hybrids, causing tumor (Hmr), that causes the inviability of certain F1 hybrids between formation (18). D. melanogaster and its sister species, e.g., D. simulans. This work Xmrk-2 was subjected to intense genetic analysis because of its culminated in the recent identification by Barbash et al. (29) of role in cancer. There is little reason to believe, however, that Hmr at the sequence level: Hmr encodes a transcriptional postzygotic isolation usually involves malignancies. The recent regulator of or related to the MYB family. It seems likely that renaissance in the genetics of speciation has therefore featured Hmr’s presumed normal function in gene regulation is disrupted a determined effort to find additional speciation genes regard- in species hybrids, causing inviability (29). Very recent work

Table 1. Speciation genes causing postzygotic reproductive isolation that have been identiﬁed at the DNA sequence level Gene Taxon Hybrid phenotype Gene type Ref(s).

Xmrk-2 Xiphophorus Inviability Receptor tyrosine kinase 16–18 OdsH Drosophila Sterility Transcription factor 22–24 Hmr Drosophila Inviability Transcription factor 29, 30 Nup96 Drosophila Inviability Nucleoporin 34

Orr PNAS ͉ May 3, 2005 ͉ vol. 102 ͉ suppl. 1 ͉ 6523 Downloaded by guest on September 26, 2021 mapped to a single complementation group, which was ulti- mately shown to correspond to Nucleoporin96 (Nup96). This locus interacts with another (presently unknown) locus on the D. melanogaster X chromosome to cause hybrid inviability. Nup96 encodes a component of the nuclear pore complex, a large structure that perforates the nuclear membrane of all eukaryotic cells and that regulates the trafficking of proteins and RNA in and out of the nucleus. Although the Ϸ30 nucleoporin proteins that constitute the nuclear pore complex are typically conserved evolutionarily, Nup96 evolved rapidly between D. melanogaster and D. simulans. Molecular population genetic analyses further reveal that this divergence was driven by positive Darwinian selection in both species lineages. Because neither species shows evidence of recent adaptive sweeps (e.g., depressed nucleotide diversity or skewed frequency spectra), this adaptive evolution likely occurred early in the split of the two species (34). In very recent work, my laboratory has turned to the attempt to identify and characterize speciation genes that cause hybrid sterility, not inviability. The chief reason is that it is now clear Fig. 2. Deﬁciency and complementation mapping of a hybrid inviability that hybrid sterility, and, especially hybrid male sterility, evolves gene. The region shown corresponds to cytological region 95 of the third faster than hybrid inviability in several groups of animals, chromosome of D. melanogaster. Bars shown in gray below the chromosome including Drosophila (reviewed in ref. 3). Hybrid male sterility uncover recessive hybrid inviability in D. melanogaster–D. simulans hybrids may thus represent a more important phenotype than hybrid and those shown in black do not. Vertical lines represent known viability- inviability in the earliest stages of speciation. Our current efforts essential complementation groups in the region. Hybrid inviability maps to a focus on the so-called 4-sim hybrid sterility system: Muller and single locus, Nup96. Data are based on ref. 34. [Reproduced with permission Pontecorvo (35) and Pontecorvo (36) showed long ago that D. from ref. 34 (Copyright 2003).] melanogaster males that are homozygous for the ‘‘dot’’ fourth chromosome from D. simulans are sterile; sterility results from reveals that Hmr is also rapidly evolving, and that this evolution sperm immotility. Heterozygous 4-sim males and all genotypes was also driven by positive Darwinian selection (30). of females are fertile. Deficiency mapping shows that hybrid male sterility localizes to a very small region of this very small My laboratory has focused on deficiency mapping to locate Ϸ and identify speciation genes. The critical point is that such chromosome [ref. 37; the fourth includes only 70 loci (38)]. It mapping, which involves the introduction of cytologically de- seems likely therefore that 4-sim hybrid sterility reflects the action of a single locus that interacts with other loci elsewhere fined chromosomal deletions from D. melanogaster into species in the D. melanogaster genome. My laboratory has recently hybrids, can be performed within F hybrids, whose sterility is 1 further narrowed the location of this putative hybrid sterility therefore irrelevant. Daven Presgraves (31) used a large set of gene and has begun to test the few remaining candidate loci in deficiencies from D. melanogaster to screen for autosomal genes the region for their possible role in hybrid sterility (J. P. Masly, from D. simulans that cause hybrid inviability when present with personal communication). an X chromosome from D. melanogaster. The essence of this In summary, the above genetic studies have finally allowed approach is that it allows detection and mapping of recessive, and evolutionists to examine the factors that cause postzygotic thus, normally masked, speciation genes: if a chromosomal isolation. It is clear that these factors correspond to ordinary region includes a recessive hybrid inviability gene(s) from D. genes, having normal functions within species: no evidence has simulans, control hybrids that carry an undeleted (balancer) been uncovered for the idea that speciation involves novel chromosome from D. melanogaster should be viable, whereas processes like the mass mobilization of transposable elements in experimental hybrids that carry a deleted chromosome (in the hybrids (39–41). It is also clear that speciation genes do not fall relevant region) from D. melanogaster should be inviable. into a single functional class. Although two of the above genes Ͼ Presgraves (31) singly introduced 200 deficiencies into D. (OdsH and Hmr) play a role in transcriptional regulation, and melanogaster–D. simulans species hybrids. He found that 20 this occurrence could well prove common, others do not. Nup96, deficiencies significantly reduced hybrid fitness, and that 10 of for example, encodes a structural protein. Similarly, it is clear these deficiencies were hybrid lethal, i.e., uncovered genes from that the divergence of noncoding regulatory sequences is not the D. simulans that cause essentially complete inviability on a partly invariable cause of postzygotic isolation: complementation tests D. melanogaster genetic background. Further crosses using at- with multiple mapped loss-of-function mutations show that tached-X chromosome manipulations revealed that these D. Nup96’s effect on hybrid inviability is due to divergence of the simulans autosomal regions interact with the D. melanogaster X Np96 protein itself (34). Also, the genes causing postzygotic chromosome to cause hybrid inviability (31). This work implies isolation are sometimes members of duplicate gene families that D. melanogaster and D. simulans, although diverged for (Xrmk-2 and OdsH), but are sometimes single-copy genes (Hmr only Ϸ2.5 million years (32, 33), are separated by many genes and Nup96). Although there is good evidence that the genes causing hybrid inviability. Indeed, it appears that Ϸ10% of all causing postzygotic isolation are on average partially recessive, viability-essential genes have diverged functionally between as predicted by the dominance theory of Haldane’s rule (re- these species to such an extent that a gene from one species kills viewed in ref. 3), it also clear that speciation genes can differ hybrids if made homozygous (hemizygous) in the genetic back- dramatically in dominance. Nup96 and Hmr, for instance, act ground of the other species (31). mostly recessively in hybrids, whereas the (currently unknown) In subsequent work, Presgraves et al. (34) dissected a small gene(s) from D. simulans with which Hmr interacts to cause deficiency region known to cause the inviability of D. melano- hybrid inviability appears quite dominant (26). Recessive spe- gaster–D. simulans hybrids. A combination of fine-scale deletion ciation genes can obviously contribute only to the sterility or mapping and complementation testing (Fig. 2) revealed that the inviability of F2, or backcross, not F1, hybrids (unless, of course, hybrid inviability effect of this third chromosomal region the gene is X-linked). Whereas any given recessive gene may well

6524 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501893102 Orr Downloaded by guest on September 26, 2021 cause less reproductive isolation than any given dominant one, recessive speciation genes appear more common than dominant; not surprisingly, then, later-generation hybrid problems often appear before F problems (reviewed in ref. 3, chapters 7 and 8). 1 Fig. 3. Regions of the D. pseudoobscura X chromosome implicated in hybrid Although the study of speciation genes remains in its infancy, sterility between the Bogota and USA subspecies (54). Recent genetic analysis two patterns do, so far at least, appear to characterize their of segregation distortion in the same hybridization implicates approximately evolution. First, speciation genes are rapidly evolving. Second, the same chromosomal regions (53). The markers shown are all visible muta- these genes often evolve by positive Darwinian selection (29, 34). tions. Kosambi-corrected map positions are provided. [Reproduced with permission from ref. 54 (Copyright 2001, Genetics Society of America).] Speciation Genes: Intragenomic Conflict? While the above work shows that positive selection plays a key role in the divergence of speciation genes, it says nothing about of the Bogota X chromosome. Remarkably, these regions show the nature of this selection. Departures from neutrality as essentially complete epistasis: little or no distortion occurs unless revealed by, e.g., McDonald–Kreitman tests, are equally consis- genes from both the left (XL) and right (XR) arms of the X derive tent with sexual selection, natural selection to abiotic factors, or from Bogota. These results are strikingly similar to those char- natural selection to biotic factors. Surprisingly, recent work acterizing hybrid male sterility between the same subspecies suggests that a particular form of the last hypothesis, one (refs. 54 and 55; see Fig. 3). More suggestive still, the severity of involving adaptation not to other organisms but to intragenomic hybrid sterility and the extent of offspring sex ratio distortion are events, may play a role in speciation. In particular, it now appears strongly correlated across individual backcross hybrid males that meiotic drive may sometimes drive the evolution of hybrid (Fig. 4). Future high-resolution introgression analysis within a sterility genes. small but critical region of XR will let us determine whether the This idea was first suggested in the early 1990s by Frank (42) genes that cause hybrid sterility can be separated meiotically and Hurst and Pomiankowski (43). These authors argued that from those that cause hybrid segregation distortion is warranted mutations that cause meiotic drive may often become suppressed (N. Phadnis, personal communication). within species (because drive at one locus causes selection at It is clear, then, that hybrid males between these young other loci for suppression of drive), but can become reexpressed subspecies suffer both sterility and segregation distortion, and in species hybrids: if the mutations that suppress drive are less that these two forms of hybrid dysfunction have similar genetic than completely dominant, they may fail to suppress segregation bases. We cannot, therefore, reject the possibility that at least distortion in heterozygous hybrids. Frank (42) and Hurst and some of the same genes cause both phenomena, as first suggested Pomiankowski (43) further speculated that such drive might by Frank (42) and Hurst and Pomiankowski (43). More gener- sometimes cause the sterility of hybrids. Other variations on the ally, we cannot reject the possibility that arms races between meiotic drive theory of postzygotic isolation have appeared selfish genetic factors like those that cause meiotic drive con- recently (44–47). tribute to the evolution of postzygotic reproductive isolation. Although early experimental work found no evidence of meiotic drive in species hybrids (48, 49), more recent work has Conclusions uncovered such evidence (44, 50–52). In the most impressive of these efforts, Tao et al. (44) showed that a Ͻ80-kb region of the Molecular evolutionary analyses of speciation genes show that third chromosome of D. mauritiana causes meiotic drive in an these loci are rapidly evolving, and that this evolution is often otherwise D. simulans genetic background. Remarkably, this driven by positive Darwinian selection. Although the sample of same small region also causes hybrid male sterility, leading Tao genes characterized thus far by various laboratories remains et al. (44) to conclude that the same gene, too much yin (tmy), small, and concentrated in the genus Drosophila, I suspect that likely causes both hybrid segregation distortion and hybrid these patterns may prove general, although likely not universal. sterility; tmy has not yet been identified at the DNA sequence level. Recently, my laboratory has discovered strong segregation distortion in hybrids between two closely related subspecies of D. pseudoobscura. When females of the Bogota subspecies are crossed to males of the USA subspecies, fertile F1 females and sterile F1 males result. Although these males have traditionally been described as completely sterile, we have discovered that they are, in fact, weakly fertile. Surprisingly, these F1 males produce nearly all daughters (Ϸ95%) when crossed to any genotype of females (pure Bogota, pure USA, and hybrid F1 sisters) (53). X-linked genetic markers reveal that this sex ratio distortion is not due to somatic sexual transformation (i.e., phenotypic females are genetic females) and egg-to-adult viability studies strongly suggest that it is not due to the inviability of sons (53). Instead, hybrid F1 males appear to suffer severe segregation distortion. Although we do not yet know the prox- imate mechanism of segregation distortion (e.g., classical meiotic drive in the male germ line, immotility of Y-bearing sperm in the female reproductive tract, postfertilization failure of pronuclei fusion, etc.), we have completed a preliminary genetic Fig. 4. Correlation between D. pseudoobscura Bogota-USA hybrid fertility analysis of this hybrid segregation distortion. (as measured by offspring production) and hybrid segregation distortion (as Our results reveal that the genes that normally suppress measured by offspring sex ratio). Each data point reﬂects the offspring of a segregation distortion within the Bogota subspecies are autoso- single recombinant backcross generation male. Data are based on ref. 53. mal (and perhaps also Y-linked) (53). More important, the genes [Reproduced with permission from ref. 53 (Copyright 2005, Genetics Society of that cause hybrid segregation distortion reside in several regions America).]

Orr PNAS ͉ May 3, 2005 ͉ vol. 102 ͉ suppl. 1 ͉ 6525 Downloaded by guest on September 26, 2021 Our recent work, along with that of several other groups, also studies (6) and the genetical studies reviewed here point to an suggests that the selection underlying the evolution of speciation important role for positive Darwinian selection in the evolution genes may sometimes assume a surprising form, response to of reproductive isolation. Although the above data do not intragenomic conflicts, perhaps involving meiotic drive. exclude all varieties of drift-based models of speciation (founder- In summary, it would appear that two of Mayr’s three seminal effect theories do, after all, feature a role for positive selection), contributions to the study of speciation were correct, or, at the such models seem at present unparsimonious (see also refs. 3 least, extremely productive. First, the entire research program of and 56). the genetics of speciation over the last half-century arose out of It remains a testament to Mayr’s vast influence, however, that Mayr’s Biological Species Concept. Whatever one’s views on the all of the questions asked above, whatever their answers, were philosophical strengths or weaknesses of this concept, it has, as first raised or emphasized by Mayr nearly half a century ago. It a practical matter, given rise to an extraordinarily rich research seems doubtful that a modern genetics of speciation in anything program, one that has led to a number of substantive discoveries. like its present form would have arisen without his fundamental contributions. In a phrase, the modern genetics of speciation is a genetics of reproductive isolation. Second, there can be little doubt that this I thank F. Ayala, W. Fitch, and J. Hey for organizing the symposium in reproductive isolation typically, if not always, evolves in allopatry which this work was presented. I also thank J. Hey and an anonymous (2, 3). Finally, however, recent work provides no evidence for a reviewer for helpful comments on an earlier draft of this paper. This crucial role of genetic drift in speciation. Instead, both ecological work was supported by National Institutes of Health Grant GM51932.

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