Evolution, 56(11), 2002, pp. 2262–2277

GENETICS OF A DIFFERENCE IN PIGMENTATION BETWEEN DROSOPHILA YAKUBA AND DROSOPHILA SANTOMEA ANA LLOPART,1,2 SUSANNAH ELWYN,1,3 DANIEL LACHAISE,4,5 1 Department

AND

JERRY A. COYNE1,6

of Ecology and Evolution, The University of Chicago, 1101 East 57 Street, Chicago, Illinois 60637 2 E-mail: [email protected] 3 E-mail: [email protected] 4 Laboratoire Populations, Ge ´ ne´tique et Evolution, CNRS, 91198 Gif-sur-Yvette, France 5 E-mail: [email protected] 6 E-mail: [email protected]

Abstract. Drosophila yakuba is a species widespread in Africa, whereas D. santomea, its newly discovered sister species, is endemic to the volcanic island of Sa˜o Tome´ in the Gulf of Guinea. Drosophila santomea probably formed after colonization of the island by its common ancestor with D. yakuba. The two species differ strikingly in pigmentation: D. santomea, unlike the other eight species in the D. melanogaster subgroup, almost completely lacks dark abdominal pigmentation. D. yakuba shows the sexually dimorphic pigmentation typical of the group: both sexes have melanic patterns on the abdomen, but males are much darker than females. A genetic analysis of this species difference using morphological markers shows that the X chromosome accounts for nearly 90% of the species difference in the area of abdomen that is pigmented and that at least three genes (one on each major chromosome) are involved in each sex. The order of chromosome effects on pigmentation area are the same in males and females, suggesting that loss of pigmentation in D. santomea may have involved the same genes in both sexes. Further genetic analysis of the interspecific difference between males in pigmentation area and intensity using molecular markers shows that at least five genes are responsible, with no single locus having an overwhelming effect on the trait. The species difference is thus oligogenic or polygenic. Different chromosomal regions from each of the two species influenced pigmentation in the same direction, suggesting that the species difference (at least in males) is due to natural or sexual selection and not genetic drift. Measurements of sexual isolation between the species in both light and dark conditions show no difference, suggesting that the pigmentation difference is not an important cue for interspecific mate discrimination. Using DNA sequence differences in nine noncoding regions, we estimate that D. santomea and D. yakuba diverged about 400,000 years ago, a time similar to the divergences between two other well-studied pair of species in the subgroup, both of which also involved island colonization. Key words.

Drosophila, genetics, pigmentation, reproductive isolation, speciation. Received February 22, 2002.

Recent genetic studies of reproductive isolation are beginning to reveal patterns that may help us understand speciation (Coyne and Orr 1999). However, there are still relatively few genetic studies of species differences—those morphological, physiological, and behavioral traits that distinguish closely related species but do not necessarily cause reproductive isolation. A recent survey (Orr 2001) describes only 13 genetic studies of species differences, six in the genus Drosophila and four in the plant genus Mimulus. Such work, however, is important in answering long-standing questions of evolutionary genetics. For example, are species differences caused by natural selection or other evolutionary forces such as genetic drift? Theoretical work shows that if alleles from one species act in a consistent direction on a trait, then the species difference is likely to have evolved by natural or sexual selection (Orr 1998b). Are such differences due to many genes of small effect, as Fisher (1930) posited, or are fewer genes of larger effect involved? For example if species differences in male-limited traits such as plumage color prove to be highly polygenic, this would imply that the trait evolved by gradual coevolution of males and females (Coyne and Orr 1999). Theoretical studies show, however, that evolution by natural selection toward a fixed optimum should lead to an exponential distribution of gene effects with many factors having small effects, but also with some factors having relatively large effects (Orr 1998a). Do genes for species differences tend to be located on particular

Accepted August 15, 2002.

chromosomes? Rice (1984), for example, posits that species differences that evolved by antagonistic coevolution between males and females may be caused by genes preferentially located on the X chromosome, as will genes fixed by natural selection whose favorable effects are partially recessive (Charlesworth et al. 1987). Orr (2001) describes other ways that the genetic basis of species differences can help us understand evolution. Such analyses are likely to become more important with the increasing use of quantitative-trait-locus (QTL) mapping, which can be used to locate and estimate the effects of chromosome regions affecting traits between any pair of crossable species that meet two criteria: (1) hybrids are somewhat fertile; and (2) one can construct a molecular map based on DNA differences (for an exemplar of the use of QTL mapping in evolution see Bradshaw et al. 1998; Schemske and Bradshaw 1999). Here we present a genetic analysis of a striking character difference—the degree of abdominal pigmentation—between two closely related species of Drosophila, D. yakuba and D. santomea. Drosophila yakuba is widespread across western Africa, but D. santomea, discovered in 1998, is endemic to the island of Sa˜o Tome´, an 860-km2 volcanic island 320 km west of Gabon (Lachaise et al. 2000). Drosophila yakuba also inhabits Sa˜o Tome´, but tentative molecular evidence points to D. santomea originating allopatrically after a colonization event by the ancestor of modern D. yakuba, with D. yakuba

2262 q 2002 The Society for the Study of Evolution. All rights reserved.

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PIGMENTATION DIFFERENCES IN DROSOPHILA

subsequently invading the island a second time (Cariou et al. 2001). On Sa˜o Tome´, D. yakuba is limited to lower elevations, whereas D. santomea lives in the mist forests at higher elevations. A hybrid zone occurs between 1150 m and 1450 m elevation on the volcano, where one finds a low frequency of hybrids (about 1%; Lachaise et al. 2000). Molecular phylogenetic analysis indicates that D. yakuba and D. santomea are sister species within the eight species constituting the monophyletic D. melanogaster subgroup (Lachaise et al. 2000; Cariou et al. 2001). This pair thus represents a speciation event independent of the well-studied speciation event separating D. simulans and D. melanogaster and of the two speciation events in which the ancestor of D. simulans produced two island endemics, D. sechellia and D. mauritiana (Lachaise et al. 1988). Molecular evidence puts the divergence between D. yakuba and D. santomea at about 450,000 years ago (Cariou et al. 2001), a divergence time similar to that separating D. simulans from each of its two sister species (;260,000–410,000 year; Kliman et al. 2000). The D. melanogaster subgroup thus includes three episodes of speciation following island colonization, all occurring at roughly the same time. The most striking aspect of D. santomea is that it is the only species in the D. melanogaster subgroup lacking pronounced abdominal pigmentation in both sexes. Drosophila yakuba and the seven other species share a single sexually dimorphic form of pigmentation: males have thin black stripes along the posterior portions of tergites 2, 3, and 4 (tergites are the sclerotized dorsal plates), whereas tergites 5–7 are completely black (Fig. 1c). Females have stripes along the posterior portions of all tergites, and tergites 5–7 show substantial (but not complete) black pigmentation (Fig. 1d). In D. santomea males have virtually no pigmentation (Fig. 1a), and females show very light striping on the posterior parts of tergites 2–5 and no pigmentation on tergites 6 and 7 (Fig. 1b). Given the similarity in pigmentation among all other species in the subgroup (including the outgroup species and the putative ancestor D. yakuba), the absence of melanic pigmentation in D. santomea is clearly a derived trait. The genetics of pigmentation has been studied in a few species of Drosophila. At least four species, D. polymorpha, D. rufa, D. erecta, and D. kikkawai, show discrete intraspecific polymorphisms for abdominal pigmentation that, as expected, are controlled by segregation at a single locus (Oshima 1952; Heed and Blake 1963; Payant 1988; Gibert et al. 1999). The difference in the width of the light-colored abdominal stripe between D. novamexicana and D. virilis, an index of general pigmentation, is apparently oligogenic, with all five chromosomes carrying at least one gene affecting the trait, but with chromosome 2 explaining at least half of the species difference (Spicer 1991). Members of the Caribbean D. dunni subgroup vary strikingly in abdominal pigmentation (Hollocher et al. 2000a). Genetic analysis of hybrids between two of these, a lightly pigmented, sexually dimorphic species (D. arawakana) and a dark, sexually monomorphic species (D. nigrodunni), showed that the species difference involved both X chromosomes and autosomes, but also maternal and paternal effects, depending on which of the three abdominal areas was examined. Except for the effect of the X chromosome in males, which was estimated from the reciprocal

F1 hybrids, the relative effects of individual chromosome segments could not be discerned because a lack of genetic markers in these species necessitated a biometrical analysis (Hollocher et al. 2000b). Kopp et al. (2000) suggest that, in Drosophila, differences in sexual dimorphism of pigmentation among species may be based on changes at the bric-a`-brac (bab) locus, which contains two adjacent genes (bab1 and bab2) whose wildtype alleles repress male-specific pigmentation in females of D. melanogaster. Their hypothesis rests on a correlation: most species with darkly pigmented males show no expression of bab in males (thus permitting the sex-specific pigmentation), whereas species lacking male pigmentation show expression of bab in males. Some species, however, do not obey this generalization. Kopp et al. (2000) also posit that other genes, including Abdominal-B and doublesex, may regulate bab, and there are clearly many other steps in the melanin-synthesis and segment-identity pathways that might affect abdominal pigmentation (Wright 1987; Hopkins and Kramer 1992; True et al. 1999). Only direct genetic analysis can determine the number, location, and effects of genes causing a species difference in pigmentation. Despite considerable sexual isolation between D. santomea and D. yakuba and the sterility of hybrid males (Lachaise et al. 2000; Coyne et al. 2002), one can perform genetic analysis by making backcrosses using the fertile F1 females. Here we report the results of two genetic studies of the interspecific difference in abdominal pigmentation. One study uses morphological mutants as markers to examine the effects of three chromosome regions on pigmentation. The other uses molecular markers as tools and is limited to studying the pigmentation difference in males. Our goal is to provide a preliminary analysis of this trait difference that will answer the following questions: Does the species difference involve only a single gene, or is it more polygenic? Do certain chromosomes or chromosome regions have more effects than others on the trait? Do the differences in pigmentation between males and between females of the two species involve the same chromosome regions? Do genes affecting pigmentation act in the same direction (i.e., do all alleles from D. santomea tend to reduce pigmentation)? Such consistent directionality would imply that the species differences evolved by natural selection (Orr 1998b). MATERIALS

AND

METHODS

Drosophila Stocks Flies were raised on standard cornmeal-yeast-agar medium at 248C with a 12-h light-dark cycle. All stocks of D. yakuba and D. santomea were founded from single females captured from the wild. The D. santomea STO.4 stock was collected on March 1998 in the Obo Natural Reserve on Sa˜o Tome´ Island in the zone of sympatry with D. yakuba (Lachaise et al. 2000). Two D. yakuba stocks, Taı¨ 18 and wor; no; se, were used in the analysis. Taı¨ 18 was derived from a female collected by D. Lachaise in 1983 in the Taı¨ rainforest on the border between Liberia and the Ivory Coast. The D. yakuba multiple-mutant stock wor; no; se, containing a morphological mutation on each of the three major

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PIGMENTATION DIFFERENCES IN DROSOPHILA

chromosomes, was constructed using mutants found in three isofemale lines from the Ivory Coast and Gabon. White-orange (wor), a recessive X-linked mutation producing light orange eyes, was tentatively verified as an allele of the white locus: more than 100 crosses between D. mauritiana white males and the D. yakuba orange-eyed females produced a single female offspring (clearly a hybrid because of its numerous phenotypic abnormalities) having light orange eyes, as expected if the mutation producing orange eyes is an allele of white. The recessive mutation notch (no), which produces nicked wing tips, is located on chromosome 2. It is not identical to Notch of D. melanogaster, which is X-linked. The approximate chromosomal location of notch was assessed using molecular markers: F1 hybrid females (1/no) obtained from crossing the D. yakuba notch females to D. santomea STO.4 males, were backcrossed to D. yakuba (no/no) males and the no offspring analyzed using polymerase chain reaction (PCR) with the twinstar primers specific to D. santomea STO.4 (see below). The observed frequency of recombinants between notch and twinstar was 3/35, indicating that notch in D. yakuba is located on the right arm of the second chromosome, about 10 cM from the tip. The penetrance of no in the D. yakuba wor; no; se stock is 0.905 in males and 0.805 in females at 248C. Thus some notch flies will have been scored as wild type in the first genetic analysis described below. The autosomal recessive mutation sepia (se) in D. yakuba was shown to be identical to the chromosome 3 mutation sepia of D. melanogaster (located in that species at 3–26.0; cytological position 66D13) through hybridization with D. mauritiana se, which was shown through hybridization to be identical to D. melanogaster sepia (D. yakuba does not produce hybrids with D. melanogaster). Figure 2 gives the presumed chromosomal and map locations of these mutant markers based on the cytological maps of Lemeunier and Ashburner (1976) and Lindsley and Zimm (1992) and the molecular map of D. yakuba by Takano-Shimizu (2001). We assume, based on studies by Takano-Shimizu (2001) on recombination in the X and second chromosomes, that map distances in D. yakuba are about 1.5 times larger than those in D. melanogaster. We have no information on map distances in D. santomea, but assume they are close to those of D. yakuba. Because of these gaps in our knowledge, all marker positions should be viewed as tentative. Chromosome Arrangements Drosophila santomea chromosomes are homosequential (have identical banding sequence) with those of D. yakuba, and the analysis of 12 different D. santomea isofemale lines did not reveal any polymorphic chromosomal arrangements (Lachaise et al. 2000). Drosophila yakuba, in contrast, is polymorphic for inversions (Lemeunier and Ashburner 1976). The presence of different (polymorphic or fixed) chromosomal arrangements between the D. santomea STO.4 and D.

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yakuba Taı¨ 18 strains was checked by inspecting orceinstained preparations of salivary gland polytene chromosomes in female F1 hybrid larvae (Ashburner 1989). The number of independent preparations checked were 26, 19, 20, 13, and 26 for the X, 2R, 2L, 3R, and 3L chromosomes or chromosomal arms, respectively. We found only one inversion in the D. yakuba strain: 2Rn, which covers approximately 40% of the right arm of chromosome 2 (Lemeunier and Ashburner 1976). This inversion was seen in 12 of the 19 independent preparations and is obviously polymorphic in Taı¨ 18. The proximal and distal breakpoints of this inversion have been mapped to bands 48F and 58D respectively (F. Lemeunier, pers. comm.). We have no information about the karyotype of the wor; no; se strain of D. yakuba; however, no inversions have been reported on the X chromosome of this species and inversions on 3L are extremely rare (Lemeunier and Ashburner 1976). It is thus possible that notch and sepia are linked to inversions and so would be nonrandomly associated with more than 50 cM of their respective chromosomes. Molecular Markers Genetic analysis of pigmentation using molecular markers requires strain-specific markers whose association with the degree of pigmentation can be assessed in backcrosses. We developed eight molecular markers, spread among all four chromosomes, that differentiated the two strains selected for molecular-marker analysis, D. santomea STO.4 and D. yakuba Taı¨ 18. Seven of the eight markers were chosen from nucleotide sequences of noncoding regions (introns and untranslated segments) of the following loci: yellow (y), vermilion (v), Annexin X (Ann X), bric-a`-brac 1 (bab1), Abdominal B (Abd-B), twinstar (tsr), and Phospholipase C at 21C (Plc21C). The eighth marker was developed based on the coding sequence for the chromosome 4 locus cubitus interruptus (ci). The use of noncoding regions maximizes the probability of finding sections of DNA showing enough nucleotide differences between D. santomea and D. yakuba to facilitate designing strain-specific primers. Because of the large effect of the X chromosome found in an initial genetic analysis (see below), we used three markers on the X chromosome (roughly evenly distributed along its length), two on chromosome 2 (one on each arm), two on chromosome 3 (one on each arm), and one on chromosome 4. There are potential problems with using D. melanogaster to design molecular markers distinguishing D. santomea from D. yakuba, because extensive chromosomal rearrangements occurred after the divergence between the ancestor of D. melanogaster and of D. yakuba/D. santomea (Lemeunier and Ashburner 1976). However, most of these reorganizations have not traversed chromosome arms, and at least one numbered section (using the map of Bridges, which divides the major autosomes into 100 sections; Lindsley and Zimm 1992) is conserved at both ends of each major chromosome. Our molecular markers were chosen because of their cytological

←

FIG. 1. Pigmentation of the pure species and of their F1 hybrids. (a) Drosophila santomea STO.4 male; (b) D. santomea STO.4 female; (c) D. yakuba wor; no; se male; (d) D. yakuba wor; no; se female; (e) F1 hybrid male with D. santomea mother; (f) F 1 hybrid female with D. santomea mother; (g) F1 hybrid male with D. yakuba mother; (h) F1 hybrid female with D. yakuba mother.

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FIG. 2. Approximate cytological and map positions in Drosophila yakuba of the eight molecular markers (above lines) and three morphological markers (below lines) used in the two genetic analyses. Estimated breakpoints of the 2Rn inversion segregating in the D. yakuba Taı¨ 18 stock are indicated by arrows. Black ovals mark the position of centromeres.

position in these chromosomally conserved regions and/or because of their proposed role in Drosophila pigmentation or melanin synthesis (True et al. 1999; Kopp et al. 2000). The positions of these markers are given in Table 3 and shown in Figure 2. For the markers in the X chromosome, the estimated genetic distance in D. yakuba is 48.2 cM and 50.8 cM for the y-v and v-Ann X regions, respectively (Takano-Shimizu 2001). For the autosomal markers, the estimated genetic distance in D. melanogaster is approximately 107 cM and 58 cM (Lindsley and Zimm 1992) between members of the pairs Plc21C-tsr (chromosome 2) and bab1-Abd-B (chromosome 3), respectively. In D. yakuba, Plc21C, tsr, and bab1 have maintained the same chromosomal location as in D. melanogaster. In contrast, Abd-B in D. yakuba has moved to a more distal position on the 3R arm (Lemeunier and Ashburner 1976), approximately 68 cM from bab1. However, these genetic distances are almost certainly higher in D. santomea and D. yakuba, for empirical estimates of recombination frequency are about 1.5 times higher in D. yakuba than in D. melanogaster (Takano-Shimizu 2001). The map positions of our markers have hence been adjusted to reflect this increase. These map positions should be regarded as tentative, but they are probably not far off the true values. To obtain the sequence of these regions in the D. santomea and D. yakuba strains, we initially designed PCR primers based on the D. melanogaster sequence for six of the eight markers. In the case of the v marker, one of the primers was designed using the D. yakuba sequence present in GenBank (accession number AF255325). For ci, we used the D. yakuba sequence AB005797. In all cases, when sequences from other Drosophila species were available, we used this information

to design primers in conserved regions, thus maximizing the probability that the primers would work in D. yakuba/D. santomea. PCR amplifications were performed using approximately 25 ng of genomic DNA extracted from single-fly preparations (Ashburner 1989). PCR products, ranging approximately from 0.6 kb to 1.4 kb in length, were directly sequenced with a 377 ABI Prism automated sequencer (Applied Biosystems, Inc., Foster City, CA) after purification using the QIAquick system (Qiagen, Valencia, CA). Sequences were aligned with ClustalX (Thompson et al. 1997). Newly reported sequences have been deposited in GenBank, EMBL, and DDBJ database libraries under accession numbers AF533212–AF533229. Strain-specific primers were designed for six of the eight markers using the newly obtained sequences. In the cases of Ann X and ci, we were able to use the same pair of primers in both strains because the strain-specific PCR products differ substantially in size (Ann X) or contain a nucleotide difference affecting the recognition sequence for the endonuclease HpaI (ci). Strain-specific primer sequences are available upon request. Genotyping of Molecular Markers PCR reactions using 12.5–25.0 ng of genomic DNA were performed using annealing temperatures between 508C and 628C and cycle numbers between 30 and 45. PCR products were run in agarose gels containing ethidium bromide. All eight markers were assayed with genomic DNA extracted from single-fly preparations. A total of 382 males were genotyped. For the X-linked markers y and v, flies were genotyped using one pair of primers, and any individuals in

PIGMENTATION DIFFERENCES IN DROSOPHILA

which there was no amplification were reassayed with the other pair. In this way, we could estimate the number of flies that do not amplify with any of our two pairs of primers (false negative results; these were fewer than 2%). We could not use this approach for the autosomal markers Plc21C, tsr, bab1, and Abd-B because of the presence of the chromosome from the male parent. For these markers amplifications with negative results were repeated a second time with the same pair of primers for confirmation. Each pair of primers was tested for false positive results (primers that amplify the wrong regions) and false negative results (correct primers that fail to amplify the correct region). For false positive controls, single-fly genomic DNA extractions from nine females isolated from each of the parental lines were checked with the pair of primers specific for the other parental strain. For the false negative controls, each pair of primers was used for amplifying genomic DNA extracted from 18 F1 hybrid females. We used only those primers giving no false positive or negative results. For ci, PCR products were purified using the PCR clean-up kit (Millipore, Bedford, MA), and approximately 200 ng were digested overnight at 378C with 2.5 units of the endonuclease HpaI. Methods of Genetic Analysis We conducted two different sets of genetic analyses, both involving backcrosses between F1 hybrid females and males of one or both parental species. The analyses differ in the markers used, which sexes were scored, the direction of crosses, and the method of scoring pigmentation. Analysis 1 This analysis involved a conventional genetic dissection using one mutant marker per major chromosome, a method pioneered by Dobzhansky (1936). Although crude, this method enables us to look at the phenotypic effects of gene segments in both sexes, as well as the interaction among gene segments from different chromosomes. Initial crosses were made between D. yakuba wor; no; se and D. santomea STO.4 (wild type); these crosses were made in both directions so that F1 males and females could be assessed for the effect of the X chromosome (in males) and possible maternal effects (in females). For the genetic analysis of chromosome segments, D. yakuba wor; no; se females were crossed to D. santomea STO.4 males, and the F1 hybrid females were backcrossed to D. yakuba wor; no; se males. This backcross produces eight classes of genotypes, involving all possible combinations of markers segregating against a haploid genetic background from D. yakuba. It should be remembered that each marker is linked only to a region of maximum length 50 cM on each side (longer if the marker is within or close to an inversion), and thus this cannot identify pigmentation genes farther away on the chromosome. Unfortunately, all three available mutants are near the ends of chromosomes (Fig. 2), so each marker gives information only about pigmentation genes occupying no more than one chromosome arm. All flies were collected as virgins and aged for four days. Individuals were then etherized and scored under a dissecting microscope. Pigmentation was measured using the method

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of David et al. (1985; see also Hollocher et al. 2000b). This involves examining only the three posterior tergites (numbers 5, 6, and 7). Each tergite is given a score from 0 to 10 based on the proportion of the tergite area that was pigmented as estimated by eye. A score of 0 corresponds to 0% pigmentation, 10 to 100% pigmentation; nearly all scores were integers between 0 and 10 except for those flies with very little pigmentation, which were scored as 0.5. The pigmentation areas of the three tergites are highly correlated (see Results), and hence we analyzed total pigmentation scores. These scores were calculated by adding the scores from all three tergites, so that scores for a single fly could range from 0 (no pigmentation on any of the three tergites) to 30 (all tergites fully pigmented). Our scoring procedure was checked for accuracy by P. Gibert, who used this method extensively (e.g., Gibert et al. 1997, 1999). A single person (S. Elwyn) scored the pigmentation of all flies in this analysis. Analysis 2 Because of the labor involved in using molecular instead of morphological markers and the greater variation in male than female pigmentation in backcrosses (there is very little obvious variation in pigmentation among female offspring from the backcross to D. santomea), we limited the molecular mapping of pigmentation to males. This analysis used the method of selective genotyping (Lynch and Walsh 1998, p. 401), in which genotyping in backcrosses is performed not on randomly selected individuals but on individuals having extreme phenotypes. Compared to using randomly selected individuals, selective genotyping gives additional power to detect quantitative trait loci (QTLs) of small effect, although it is less useful for estimating the effects of these QTLs (Lynch and Walsh 1998). We chose this method because we were interested more in gene number than in gene effects and because we were able to estimate the effect of at least the X-linked genes by comparing the pigmentation of F1 males from the two reciprocal hybridizations. In this analysis, we produced F1 females by crossing D. yakuba Taı¨ 18 females to D. santomea STO.4 males. These hybrid females were then used in two backcrosses, each to males from one parental species. Thus, we produced two sets of male offspring: backcross to yakuba (BY) and backcross to santomea (BS). From each backcross, we chose for genotyping 98 males with the darkest pigmentation (designated ‘‘D’’; closest in phenotype to D. yakuba males) and 98 males with the lightest pigmentation (designated ‘‘L’’; D. santomea-like). The frequency of flies with these extreme phenotypes in each backcross was roughly 2–4% of the total offspring. Our four sets of offspring used for molecular mapping are thus designated BYD, BYL, BSD, and BSL and comprised 392 flies (4 3 98). Each fly was genotyped to determine whether it carried the D. santomea or the D. yakuba allele for all eight molecular markers. Molecular Estimates of Species Divergence To estimate the age of the D. yakuba/D. santomea species split, we calculated an estimate of DNA divergence to which we could apply a molecular clock (see Results). The number of nucleotide substitutions per noncoding site (K) between

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TABLE 1. Mean total pigmentation scores for males and females of pure species, F1 hybrids, and backcross flies. In the two crosses, the female parent is shown first. Backcross genotypes are designated by which recessive marker from D. yakuba was visible; 1 indicates the D. santomea allele was present at that marker locus. Males

Females

Genotype

N

Mean

SE

N

Mean

SE

D. yakuba Taı¨ 18 D. yakuba wor; no; se D. santomea STO.4 F1: STO.4 3 wor; se; no F1: wor; se; no 3 STO.4

50 50 50 100 100

28.83 28.26 0.00 0.05 24.96

0.09 0.12 0.00 0.02 0.15

50 50 50 100 100

19.83 18.38 0.17 5.03 4.78

0.18 0.15 0.03 0.17 0.20

Backcross progeny wor; no; se 1; no; se wor; no; 1 wor; 1; se 1; no; 1 1; 1; se wor; 1; 1 1; 1; 1

200 200 200 200 200 200 200 200

23.20 21.64 24.07 19.52 21.95 15.52 17.44 13.52

0.43 0.49 0.36 0.69 0.50 0.75 0.75 0.78

150 150 150 150 150 150 150 150

15.99 15.78 15.51 13.57 14.72 11.59 13.22 10.25

0.24 0.17 0.21 0.35 0.24 0.40 0.34 0.38

D. santomea and D. yakuba was estimated for nine different regions using the program K-estimator version 5.5 (Comeron 1999). Divergence was calculated for all seven noncoding regions described in the Molecular markers section above and also for two additional intronic regions of Decapentaplegic and twinstar respectively. The sequence of each of these gene segments was concatenated in each species to estimate the overall genetic divergence. Mating Tests Kopp et al. (2000) suggested that some Drosophila species may use pigmentation as a mating cue. Although their demonstrations of this phenomenon in D. melanogaster were not replicated in later work (Llopart et al. 2002), it is possible that sexual isolation between D. yakuba and D. santomea may be based in part on their differences in pigmentation. To test this, we determined the degree of sexual isolation of flies mating in the light and in the dark (this, of course, tests only whether sexual isolation depends on any visual cue, not just pigmentation). We performed simultaneous sets of sexualisolation tests, each involving no-choice trials in which a single male and female are observed over a period of time to determine whether they copulate. Each test involved 40 vials divided into four sets of 10: D. santomea male with D. santomea female, D. santomea male with D. yakuba female, D. yakuba male with D. santomea female, and D. yakuba male with D. yakuba female. Tests were conducted between 0900 h and 1000 h under a constant temperature regime (21–238C), with each observation period lasting 45 min. Light and dark tests were run simultaneously, for a total of 12 pairs of tests. Matings in the dark were set up under red-filtered light, because Drosophila cannot see in this spectrum (Frank and Zimmerman 1969). After males and females were placed in vials in red light, the room was completely darkened, with the red light turned on only briefly every 5 min to determine which pairs were copulating (nearly all matings, intra- or interspecific, last at least 25 min). We recorded the presence or absence of copulation, and, if copulation occurred, its la-

tency (the time elapsing between when males and females were put together until they were first seen copulating). RESULTS Genetic Analysis 1 Pigmentation of pure species and F1 hybrids The heavy pigmentation of D. yakuba is similar to that of seven of the other eight species in the D. melanogaster subgroup. In males of the D. yakuba wor; no; se stock, tergite 7 (the most posterior tergite) is entirely pigmented, tergite 6 is roughly 90–100% pigmented, and tergite 5 is 80–90% pigmented (Fig. 1c), giving these males an average pigmentation score of 28.26 of a maximum of 30 (Table 1). In females of this strain, tergite 7 is 90–100% pigmented, tergite 6 is 60– 70% pigmented, and tergite 5 is 20–30% pigmented (Fig. 1d), giving them an average pigmentation score of 18.38 (Table 1). Scores for males and females of the wild-type D. yakuba Taı¨ 18 stock, used in the molecular mapping experiment, are similar (Table 1). In contrast, D. santomea species have virtually no pigmentation (Fig. 1a,b; Table 1). Males from the D. santomea STO.4 strain have no pigmentation on tergites 5–7, yielding an average pigmentation score of 0.00. Most females from this strain are also unpigmented on the posterior three tergites, although occasional individuals have slight striping (score 0.5) on tergite 5, yielding an average pigmentation score in our sample of 0.17. F1 hybrids. Reciprocal-cross F1 hybrids were generated by crossing D. santomea STO.4 with the D. yakuba wor; no; se stock in both directions. Pigmentation scores of reciprocal F1 females are intermediate between those of the parents but closer to those of D. santomea (Table 1, Fig. 1f, h). Female offspring from the two reciprocal crosses do not differ significantly in pigmentation score (t198 5 0.91, P 5 0.36), so there is no evidence of a maternal effect acting in these genetically identical females. The females’ scores do, however, show a marked deviation from additivity. Because the two

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PIGMENTATION DIFFERENCES IN DROSOPHILA

classes of F1 female do not differ significantly, we can combine them into one population (mean 5 4.91, SD 5 1.90, n 5 200) and compare this population with the average value of the two parental classes (9.28) using the Z-statistic (Hays 1963). This comparison yields a Z of 32.51, with an attendant probability of 0.0009. Obviously, F1 females are significantly lighter than expected from the average of parental females; in female hybrids the D. yakuba pigmentation area is partially recessive. The reciprocal F1 males, however, differ strongly, with each class of males having a phenotype and pigmentation score similar to that of males from the maternal species (Fig. 1e,g; Table 1). Because females show no evidence for maternal effects, we can assume that this difference reflects a very large effect of the X chromosome on the pigmentation area of males, a difference confirmed by statistical comparison of the two classes of males (t198 5 168, P , 0.0001). The magnitude of this X-effect relative to the total difference between the species is simply the difference between the pigmentation scores of reciprocal F1 males divided by the difference between scores from males of the parental species (Hollocher et al. 2000b). Genes on the X chromosome thus account for 88.2% (24.92/28.26) of the total difference in pigmentation area between the two species. The X-effect is much larger than the relative size of this chromosome, which constitutes roughly 16% of the haploid genome and suggests either that the X chromosome carries a disproportionate number of genes affecting pigmentation or that X-linked genes have disproportionately large effects. Of course, these explanations are not mutually exclusive. Backcross hybrids Female hybrids from a cross between D. yakuba wor; no; se females and D. santomea STO.4 males were backcrossed to D. yakuba wor; no; se males, and pigmentation scores determined for each of the eight genotypic classes of backcross males and females (200 males and 150 females were scored per genotype). Table 1 gives the total pigmentation areas (sum of three tergites) of the pure species, F1 hybrids, and backcross offspring; Table 2 shows the analysis of variance for the main effects of chromosome markers and their interactions on pigmentation area. As noted above, each chromosome marker is apparently located near the end of the chromosome, so marker effects are limited roughly to pigmentation genes lying with 50 cM of chromosome tips. Because of the backcross used, the effects of markers and interactions are gauged against a genotype in which half of the chromosomes in female hybrids are always pure D. yakuba. In male hybrids, half of the autosomes are pure D. yakuba, whereas the X chromosome region associated with wor is hemizygous and can come from either species. In males, the markers on both the X and second chromosomes have significant effects on pigmentation, and in both cases segments carrying the D. yakuba markers increase the area of pigmentation. The effect of the second-chromosome segment exceeds that of the X. The main effect of the chromosome 3 marker se is in the same direction, but is not significant. However, there is a significant interaction between the two autosomal segments; this is because the sub-

TABLE 2. Analysis of variance of marker effects on pigmentation data from Table 1. Asterisks give probabilities that are significant using the sequential Bonferroni correction (Rice 1989). (A) Males Chromosome

df

Sum of squares

Mean squares

X (or) 2 (notch) 3 (sepia) X32 X33 233 X3233 Residual

1 1 1 1 1 1 1 1592

3294.8 15345.0 196.0 472.0 8.41 716.9 13.9 119735

3294.8 15345.0 196.0 472.0 8.41 716.9 13.9 75.2

Chromosome

df

Sum of squares

Mean squares

X (or) 2 (notch) 3 (sepia) X32 X33 233 X3233 Residual

1 1 1 1 1 1 1 1192

663.2 3353.7 193.5 291.9 45.7 0.48 3.0 16381

663.2 3353.7 193.5 291.9 45.7 0.48 3.0 13.7

F

P

43.80 ,0.0001* 204.02 ,0.0001* 0.11 2.61 0.012 6.28 0.74 0.11 0.0021* 9.53 0.67 0.18

(B) Females F

P

48.26 ,0.0001* 244.03 ,0.0001* 0.0002* 14.08 21.24 ,0.0001* 0.068 3.32 0.85 0.04 0.64 0.22

stitution of the D. yakuba se marker in genotypes heterozygous for the chromosome 2 segment (1/no) produces a large increase in pigmentation area, but this substitution has only a slight negative effect in a background that is no/no. In females, the main effects of all three chromosome markers are highly significant, again with all D. yakuba segments increasing the area of pigmentation. The order of main effects is 2 . X . 3. There is also a highly significant interaction between the X and chromosome 2, resulting from the fact that adding the wor marker causes a much larger increase in pigmentation on a 1/no genetic background than on a no/no genetic background. We considered the effect of including sex as an influencing factor on pigmentation by performing an ANOVA on the total dataset, with gender being one of the fixed variables. This analysis shows that all three chromosomes have significant main effects on pigmentation, with chromosome 2 having the largest effect (F1,2784 5 318.0, P , 0.0001) followed by the X (F1,2784 5 66.6, P # 0.00001) and chromosome 3 (F1,2784 5 7.92, P 5 0.005). There is a significant interaction between sex and both the X chromosome (F1,2784 5 6.71, P 5 0.0096) and chromosome 2 (F1,2784 5 28.48, P , 0.0001), suggesting that the magnitude of chromosome effects differ between the sexes. This is expected because the size of the interspecific difference in pigmentation is much larger in males than in females. In sum, each of the three chromosomes carries genes influencing pigmentation area in both sexes, although in males the chromosome 3 segment has a significant effect only through its interaction with chromosome 2. This is the maximum number of genes detectable by this analysis, so the true number of loci responsible for the interspecific difference in pigmentation is likely to be larger, especially because each autosomal marker is linked to only one of the two chromosome arms, whereas the or marker is linked to only about

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TABLE 3. Number of males from the backcross to D. santomea (BS) and D. yakuba (BY) bearing the D. santomea genomic region for each molecular marker. In each of the four classes collected (BS light, BS dark, BY light, and BY dark), 98 flies were genotyped at each marker locus to determine whether they carried the D. santomea or D. yakuba allele. Chromosome

yellow vermilion Annexin X Plc21C twinstar bric-a`-brac 1 Abdominal B ci

X X X 2L 2R 3L 3R 4

Cytological band*

1A4-5 10A1 19C1 21B8-C1 60B2 61F1 89E4 102A1

and 6 is 0.900 in males and 0.359 in females, between tergites 6 and 7 is 0.807 in males and 0.808 in females, and between tergites 5 and 7 is 0.830 in males and 0.303 in females (all probabilities , 0.0001). Likewise the correlations among tergites within each of the eight backcross genotypes are high: of the 48 correlations (eight genotypes 3 2 sexes 3 3 tergites), all were positive and all but five had probabilities of 0.0001 or less. All 48 correlations were significant using the sequential Bonferroni correction (Rice 1989). We conclude that genes affecting the pigmentation difference among species affect the three posterior tergites in similar directions.

BY

BS Light

Dark

Light

Dark

69 75 87 51 56 53 64 44

35 32 4 45 17 38 10 47

62 72 88 52 76 56 90 43

33 26 24 46 38 41 31 43

Genetic Analysis 2 As described above, we determined the genotypes of 98 flies from each of the two extreme phenotypic classes from each backcross (BSL, BSD, BYL, and BYD). To determine how these four phenotypes extremes ranked in pigmentation area, we scored 50 flies from each extreme using the method employed in Analysis 1. The average pigmentation score was 0.05 6 0.04 (SE) for light individuals in the backcross to D. santomea (BSL) and 24.58 6 0.20 for the dark males in the same backcross (BSD). The values for the light and dark males in the backcross to D. yakuba were 4.80 6 0.53 (BYL) and 27.04 6 0.11 (BYD), respectively. The mean pigmentation score for the BSL males is not statistically different from the mean value of the pure D. santomea STO.4 males shown in Table 1 (Z 5 1.21, P 5 0.67). In contrast, the pigmentation score of BYD individuals is significantly lower than that of pure D. yakuba males (t98 5 12.75, P , 0.00001). As expected, flies in the dark category are lighter if they are sired by D. santomea males than by D. yakuba males (t98 5 10.84, P , 0.00001), and flies in the light category are darker when fathered by D. yakuba males than by D. santomea males (t98 5 8.87, P , 0.00001). Tables 3 and 4 give the results of the genetic analyses of 392 hybrid males. Table 3 shows the number of individuals in each class (of 98 examined) that carried the D. santomea marker, and Table 4 gives the results of statistical tests of association between the phenotypes (dark or light) and genotypes (presence of the D. santomea or D. yakuba genomic markers). Each association was determined separately for each backcross using a 2 3 2 test of independence. For example, in the backcross to D. santomea, the light class contained 69 individuals having the D. santomea yellow allele (Table 3) and 29 (98–69) individuals with the D. yakuba

* In D. melanogaster.

50% of the X. This underestimate is supported by the results of Analysis 2 (see below). Moreover, in both males and females the order of chromosome effects is 2 . X . 3, with the second chromosome having roughly twice the effect as the X. This parallelism suggests—but of course does not prove—that the same loci are responsible for pigmentation differences in males and females. Given the observation from F1 males that genes on the X chromosome account for nearly 90% of the species differences, it may seem puzzling that in the backcross analysis the chromosome 2 segment has a much larger effect than the X-chromosome segment. This may be explained if the multiple-marker stock of D. yakuba contained (as did our D. yakuba Taı¨ 18 stock) a chromosomal inversion in the right arm of chromosome 2. If such an inversion contained more than one factor affecting pigmentation, 2R would have a larger effect on pigmentation because such inversions suppress recombination. In addition, Analysis 2 (which used additional genetic markers) shows that this disparity is probably explained by X-linked genes that were undetected in Analysis 1. Finally, two pieces of evidence suggest that the genes affecting total pigmentation area act in a similar way in each of the three posterior tergites. First, the analyses of variance for each of the individual tergites (not shown) give results nearly identical to that of their sum (Table 2). Second, the correlations between the pigmentation-area scores of different tergites are highly significant, although somewhat lower in females than in males. The correlation between tergites 5

TABLE 4. Degree of association between genotype (D. santomea/D. yakuba allele) and phenotype (light/dark class) in backcrosses to D. yakuba (BY) and to D. santomea (BS) males. See text for explanation of how statistics are calculated. Asterisks indicate significant probabilities of association using the sequential Bonferroni test. BS

yellow vermilion Annexin X Plc21C twinstar bric-a`-brac 1 Abdominal B ci

BY

Combined data

G

P

G

P

x2

24.19 39.45 168.46 0.74 34.54 4.63 68.72 0.18

,0.00001 ,0.00001 ,0.00001 0.39 ,0.00001 0.031 ,0.00001 0.67

17.44 44.93 94.00 0.74 31.21 4.61 83.08 0

0.00003 ,0.00001 ,0.00001 0.39 ,0.00001 0.032 ,0.00001 1.00

43.85 46.05 46.05 3.75 46.05 13.82 46.05 0.81

P

,0.00001* ,0.00001* ,0.00001* 0.44 ,0.00001* 0.0079* ,0.00001* 0.94

PIGMENTATION DIFFERENCES IN DROSOPHILA

yellow allele. Likewise, the dark class contained only 35 individuals with the D. santomea allele and 63 individuals with the D. yakuba allele. Thus, for yellow one obtains a 2 3 2 table of pigmentation versus species marker containing the cell values 69, 29, 35, and 63. This gives a G-value (the measure of association between marker and pigmentation) of 24.2, with P , 0.00001 (Table 4; all G-values have one degree of freedom). One can construct a similar table for the backcross to D. yakuba, obtaining a G of 17.4, P 5 0.00003. To gauge the overall degree of association, we combined the probabilities from the two backcrosses using Fisher’s test (Sokal and Rohlf 1995). Applying this to the yellow marker, we obtain a chi-square value (1 df) of 43.8, P , 0.00001. In cases where the exact P-value for a G-test was not obtainable from our software (i.e., P , 0.00001), we used this maximum value as the probability in the Fisher test. The statistics for each backcross and the combined probability for both backcrosses are given in Table 4 for the eight markers (raw data shown in Table 3). Statistical significance in the last column of Table 4 is assessed using the sequential Bonferroni correction. Identical results were obtained using single-marker regressions performed with the QTL cartographer suite of programs (Basten et al. 2002). For six of the eight markers (Plc21C and ci are the exceptions), the probability of being classified as a dark or light fly was nonrandomly associated with the presence or absence of the D. santomea or D. yakuba allele. Except for the ci marker, all of the associations, significant or not, were in the expected direction, that is, individuals in the light classes had a higher association with the D. santomea marker than with the D. yakuba marker, and vice versa for individuals in the dark classes. The highest degree of association was seen using the Annexin X marker, located near the base of the X chromosome. Surprisingly, yellow, a gene strongly influencing pigmentation within species (Lindsley and Zimm 1992) had the lowest association with pigmentation of all X-linked genes. (As we describe below, a direct genetic test indicates that yellow may play a small role in the pigmentation difference.) Among the autosomal markers, Abd-B shows the highest association, followed by tsr and bab1. The Abd-B and tsr markers appear to be more strongly associated with pigmentation in the BY than in the BS cross, suggesting that these markers interact epistatically with the genetic background. With the exception of vermilion, our markers are genetically independent, that is, located on different chromosomes or on the same chromosome but more than 100 cM apart. Vermilion is located about halfway between yellow and Annexin X (roughly 50 cM from each) and associations between vermilion and pigmentation may reflect only genes already detected using yellow and Annexin X, both of which have significant associations with pigmentation. Of the seven independent markers, five (yellow, Annexin X, twinstar, bric-a`brac 1, and Abdominal B) are significantly associated with pigmentation, so we conclude that at least five genes are responsible for the difference in male pigmentation between D. yakuba and D. santomea, with at least two genes in the X chromosome, one on the right arm of chromosome 2, and one on each of the two arms of chromosome 3. Although we cannot estimate the relative effects of these

2271

genes (higher probabilities of association could mean either tight linkage to a gene of small effect, looser linkage to a gene of large effect, or linkage to several genes, each of moderate effect), we can use the average degree of association to estimate maximum recombinational distance between a molecular marker and a candidate gene with a very large effect on pigmentation. For example, the percentage association between the Annexin X marker and extreme pigmentation (averaged across BYD, BYL, BSD, BSL crosses) is 87.5% (343/392). If one assumes that this association is due to a linked pigmentation allele of very large effect (so that the extreme class always contains the proper allele at this locus), the estimated frequency of observed recombinants between the marker Annexin X and the candidate gene is 0.125 (49/392). Using the mapping function of Kosambi (1944), we estimate the maximum genetic distance between Annexin X marker and a large-effect candidate gene at 12.8 6 3.3 cM. This method cannot, however, be used to estimate positions of genes with smaller effects. Using haplotype information from the five genetically independent markers significantly associated with pigmentation, we determined the percentage of flies harboring a given number of markers from each species that fall into each pigmentation class in the two backcrosses. As shown in Figure 3, the presence of at least two D. yakuba markers in the BS cross and one D. yakuba gene in the BY cross is required for membership in the dark class, but in both crosses a fly needs at least three D. yakuba markers to have a substantial probability of being included in this class. Conversely, in both backcrosses, three markers from D. santomea are required to give a reasonable chance that a haplotype will fall into the light extreme class. In both the BS and BY backcrosses, the chance of a fly falling into an extreme class rises markedly when its genotype goes from carrying two to carrying three markers of the appropriate type. Examination of individual haplotypes shows that no single marker gene was necessary or sufficient for inclusion in a given class. Our molecular mapping explains the disparity in Analysis 1 between the large effect of the X chromosome on pigmentation seen in reciprocal F1 males and its smaller effect seen in the backcross. First, the wor marker in Analysis 1 is unlinked to the Annexin X marker, which itself has the highest association with pigmentation of any molecular marker. Obviously, wor is unlinked to some other genes on the X chromosome affecting pigmentation. Second, of the two sections of chromosome 2 studied in Analysis 2, the segment linked to notch (which had a larger effect than wor in Analysis 1) has a very strong association with pigmentation, whereas the marker at the other end of chromosome 2 (Plc21C) has no significant association. Thus, the conclusion from Analysis 1 that the chromosome 2 marker had a larger effect than the X-chromosome marker applies only to those linked segments and not to the chromosomes as a whole. As the comparison of F1 hybrids shows, most of the pigmentation difference between males of these species derives from X-linked genes. Finally, it was possible to directly test the possibility that the yellow gene was involved in the pigmentation difference, in particular that the loss of pigmentation in D. santomea was caused at least partially by a mutation of yellow. A yellowlike male was detected in an inbred line of D. yakuba derived

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FIG. 3. The relationship in the two backcrosses in Analysis 2 between the number of Drosophila yakuba markers in a haplotype and the proportion of such haplotypes that fall into the dark extreme class (this proportion is represented by the height of the shaded bar). Only the five genetically independent markers having a significant association with pigmentation are used in this analysis. Data are averaged across all haplotypes containing a given number of D. yakuba markers. BS are individuals from the backcross to D. santomea, BY from the backcross to D. yakuba. The numbers above each bar are the number of haplotypes occurring in that class. The graphs can also be read in reverse, as the association between the number of D. santomea markers in a haplotype and the proportion of those individuals in the light class; one does this simply by subtracting the number of D. yakuba markers from five and noting the height of the unshaded portion of each bar.

from the Taı¨ 18 stock. This mutation was confirmed to be an allele of the yellow locus by crossing females carrying this mutation to D. mauritiana yellow males; all female offspring were yellow. This D. yakuba yellow stock was used to perform a genetic complementation test with the D. santomea STO.4 strain. D. yakuba Taı¨ 18 (y1yak ) and D. yakuba Taı¨ 18 yellow (yyak) males were crossed separately to D. san1 tomea STO.4 females (y1san /ysan ). The pigmentation score of interspecific hybrids heterozygous for yellow (mean 5 7.79, SE 5 0.40, n 5 50) was significantly lower than that of hybrids not heterozygous for yellow (mean 5 9.10, SE 5 0.29, n 5 50; t98 5 2.67, P 5 0.009), suggesting that the wild-type allele of D. santomea yellow locus does not fully complement the D. yakuba yellow mutation. To determine whether this difference between genotypes might reflect only the decreased expression of yellow when heterozygous, we performed a control cross, similar to that described above but using the D. yakuba Taı¨ 18 strain instead of the D. santomea strain. Pure D. yakuba females heterozygous for yellow (mean score 5 21.22, SE 5 0.24, n 5 50) were not significantly lighter than D. yakuba females homozygous for the wild-type allele (mean 5 20.79, SE 5 0.30,

n 5 50; t98 5 21.12, P 5 0.27), indicating that the yellow mutation is completely recessive for pigmentation in D. yakuba. (Indeed, the yellow heterozygotes were slightly but insignificantly darker than homozygous wild-type flies.) The difference between these two crosses is highly significant (t196 5 2.78, P 5 0.006), showing that the effect of heterozygosity for yellow occurs in the interspecific but not in the intraspecific cross. These data suggest, but do not prove, that the yellow locus may play a role (albeit a small one) in the pigmentation difference between D. yakuba and D. santomea. The results from these crosses might also be explained, however, if the yellow locus shows some loss of recessivity in a hybrid but not in a pure-species genetic background. Molecular Estimates of Species Divergence We estimated nucleotide divergence between D. santomea and D. yakuba using sequences of nine noncoding regions. The comparison of this value with estimates obtained for other pairs of species in the D. melanogaster subgroup allows us to infer their relative and absolute divergence times. We

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TABLE 5. Test of sexual isolation under light and dark conditions. Table gives the number of copulations (matings) of 120 trials for each of the pairs, as well as the latency of copulations that did occur. S, D. santomea STO.4 line; Y, D. yakuba Taı¨ 18 line. For each mating, the female parent is given first. Matings S3S

S3Y

Y3S

Y3Y

Light

Matings Latency (min) (SE)

79 13.69 (1.12)

13 23.52 (3.54)

72 21.47 (1.42)

83 19.44 (1.25)

Dark

Matings Latency (min) (SE)

55 22.64 (1.39)

2 38.25 (0.90)

33 28.34 (1.70)

38 28.65 (1.69)

Regime

were especially interested in comparing the D. yakuba/santomea divergence with that between D. simulans and its closest relatives, the island endemics D. mauritiana and D. sechellia, for we know a great deal about the genetics of reproductive isolation and character differences between the last three species (Coyne and Orr 1999). Like D. santomea, D. sechellia and D. mauritiana arose after colonization of islands by a mainland ancestor, and comparing the three island speciation events may reveal regularities in how reproductive isolation evolves after colonization. Cariou et al. (2001) estimate the divergence between D. santomea and D. yakuba at 450,000 years using a single sequence of amylase from each species. However, single sequences may yield inaccurate divergence times for two reasons. First, such estimates of divergence have large variances. Second, when speciation events are recent, ‘‘species differences’’ may reflect only ancestral polymorphisms still segregating in the newly derived species (Kliman et al. 2000). We acquired polymorphism data in D. yakuba for the bab1 noncoding region (average number of nucleotide differences per site [k] was 7/539), and hence we can estimate the fraction of the apparent divergence between D. yakuba and D. santomea that is actually due to variation within D. yakuba, assuming that all noncoding regions have the same levels of variation. After this correction, the net divergence between D. yakuba and D. santomea turns out to be 0.0154 noncoding substitutions per site, approximately one-eighth of the estimate of synonymous divergence between D. melanogaster and D. simulans (Ks 5 0.1176; Swanson et al. 2001). If the split between D. melanogaster and D. simulans occurred approximately 3 million years ago, as estimated by Hey and Kliman (1993), and we assume D. santomea and D. yakuba follow the same molecular clock as do D. melanogaster/simulans, then the split between D. yakuba and D. santomea occurred roughly 393,000 years ago. Our estimate of divergence time is thus close to that calculated by Cariou et al. (2001). Moreover, our estimate is close to divergence times estimated between D. simulans and D. mauritiana and D. simulans and D. sechellia: 263,000 and 413,000 years ago, respectively (Kliman et al. 2000). All three island speciation events appear to be about the same age. Mating Tests Table 5 gives the number of matings and the copulation latency (of a total of 120 pairs observed under each light

regime) seen in of each of the four possible pairings between these species. Under both light regimes, sexual isolation in this species is asymmetrical: interspecific matings between D. yakuba females and D. santomea males are far more frequent than the reciprocal mating. Each of the four types of matings was significantly less frequent in dark than in light conditions (P , 0.05 under expectation of equal frequency), and for all but one mating (D. santomea female 3 D. yakuba male), the onset of copulation (copulation latency) is significantly shorter in the light than in the dark (for this mating, t13 5 0.13, P 5 0.13; for the other three matings, P , 0.005). However, sexual isolation between the species is not greater in the light than in the dark, as one might expect if pigmentation is a cue for conspecific mating. The 2 3 4 table of mating type versus light regime (Table 5) shows no significant heterogeneity (x2 5 6.48, df 5 3, P 5 0.09). If anything, sexual isolation is slightly increased in the dark, as the proportion among all dark matings of the D. santomea female 3 D. yakuba mating is only about one-third the frequency seen in the light, whereas the other three matings occur in similar proportions in light versus dark. The very slight increase in sexual isolation in the dark is confirmed using the chi-square index of Gilbert and Starmer (1985). This index ranges from 21 (complete disassortative mating) to 1 (complete assortative mating), and for D. yakuba and D. santomea it is 0.37 for matings in the light and 0.49 for matings in the dark. Obviously, both species mate less frequently in the dark than in the light, but there is no evidence that sexual isolation is reduced in the absence of visual cues. DISCUSSION Our main conclusion is that the loss of pigmentation in D. santomea involved evolutionary changes occurring at several genes. Analysis 1, using both sexes, implies that at least three genes in males and females—one on each major chromosome—affect the pigmented area of the posterior three tergites, and the comparison of reciprocal F1 hybrid males indicates that genes on the X chromosome are responsible for nearly 90% of the character difference. Because the markers used were linked to only one chromosome arm (and to only about half of the X chromosome), this is a minimum estimate of the number of genes responsible for the pigmentation difference. As noted in the introduction, oligogeny or polygeny of interspecific differences in pigmentation has been seen in two other studies of Drosophila (Spicer 1991; Hollocher et al. 2000b). The effects of the three tested chromosome arms are in the same order in males as in females, suggesting that changes at the same loci have reduced pigmentation in both males and females. Moreover, the positive correlation of pigmentation scores among the three tergites suggests that evolution has fixed alleles affecting the general posterior region of the abdomen rather than alleles whose phenotype is limited to particular posterior tergites (i.e., the genetic factors revealed in this study do not modify positional cues but change the effect of pre-existing cues on pigmentation). Analysis 2, involving the association between molecular markers and extreme phenotypes in backcrosses, reveals that at least five genes are responsible for the pigmentation dif-

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ferences between D. yakuba and D. santomea males; that is, five loci are significantly associated with the probability of a fly having either a D. santomea-like or D. yakuba-like pattern of pigmentation. The trait of belonging to an extreme class is not identical to the pigmentation area measured in Analysis 1, as Analysis 2 involved assessing pigmentation intensity as well as area. Nevertheless, the traits are strongly related, because, however darkly pigmented, a fly was not included in an extreme class unless the area of pigmentation was close to that of one parental species. It is safe to say, then, that the loss of pigmentation in D. santomea involved changes in at least five genes in males and three in females. As noted above, Analysis 2 is unable to estimate effects of individual QTLs on pigmentation. Nevertheless, it seems unlikely that the association of these QTLs with pigmentation, some of them extremely strong, reflects single genes of very small effect, and it is more reasonable to suppose that the very strong associations reflect either single genes of relatively large effect or several to many genes of smaller effect. Analysis 2 confirms the large X-effect seen in the reciprocal F1 hybrid males of Analysis 1, as there is strong statistical association between pigmentation and all three Xlinked chromosome segments, while at least one of the two markers on each autosome has either a weaker association or no significant association. Whether the large effect of the X chromosomes in males resides in its possession of a few genes of large effect, many genes of small effect, a combination of these two circumstances, or simply the recessivity of genes affecting pigmentation, awaits a more refined genetic analysis in both sexes, which we are attempting with QTL mapping using many molecular markers. A large X-effect in males is predicted by some theories of evolution, including those involving traits based on genes that have antagonistic fitness effects on males and females, genes whose fitness advantages during fixation were partially recessive, or genes affected by fluctuating sexual selection (Rice 1984; Charlesworth et al. 1987; Reinhold 1998). However, such effects are not commonly seen for species differences involving other sexually monomorphic or dimorphic traits in Drosophila. Male-limited traits such as differences in genital morphology or sex-comb tooth number are rarely based on genes that show disproportionately large effects of the X chromosome (Coyne and Orr 1999; Orr 2001). A notable exception is Lepidoptera, in which many species differences, whether or not they involve sexually dimorphic characters, are X-linked, even though the X chromosome constitutes a very small proportion of the genome (Prowell 1998). This taxon-specific occurrence of large X-effects remains a mystery. Although our interspecific difference in pigmentation is caused by at least several genes, it is unlikely to rest on a very large number of loci spread throughout the genome. First, at least one chromosome arm (2L) has no significant association with pigmentation. Second, in both the BYD and BSL classes we recovered many individuals with pigmentation scores identical to those of pure D. yakuba and D. santomea males, respectively. A significant fraction of parental phenotypes appearing in backcrosses implies that the number of genes involved in the trait difference is not extremely large. Although this conclusion is somewhat quali-

fied by the small number of segregating genetic units in our cross (roughly the number of chromosome arms), one would not expect the parental phenotypes to be so frequent if there were, say, 25 genes influencing pigmentation. Again, this question can be resolved only through QTL analysis. The only other genetic study of a species difference in pigmentation is Hollocher et al.’s (2000b) analysis of pigmentation in two Caribbean species in the Drosophila cardini group: D. arawakana (lighter abdomen and sexually dimorphic, with males darker than females) and D. nigrodunni (abdomen almost completely pigmented and sexually monomorphic). Considering the posterior region of the abdomen (‘‘area 3’’ as shown in Hollocher et al. 2000a, fig. 2), which is roughly equivalent to the area scored in our analysis, we find little similarity between the genetic basis of pigmentation in the two pair of species. Hollocher et al. (2000b) found only a minor effect of the X chromosome in males, and this was in the direction opposite to ours (i.e., reciprocal F1 males in the D. arawakana 3 D. nigrodunni cross have pigmentation closer to that of males from the paternal species, indicating a paternal effect but no discernible effect of the X chromosome in males). Moreover, reciprocal F1 females showed a maternal effect, whereas we found no evidence of this in our study. We cannot make further comparisons between our results and those of Hollocher et al. (2000b) because the effects of individual chromosomes could not be assessed in the latter analysis. Nevertheless, the location and effects of genes affecting pigmentation clearly differ between the two studies. Of course, unless only a small number of genes can potentially be involved in the evolution of pigmentation, there is no reason to expect a similarity in the genes associated with pigmentation differences between distantly related pairs of species. Three genes are worth discussing in more detail: yellow (y), bric-a`-brac1 (bab1), and bric-a`-brac2 (bab2). A correlation between the amount of the yellow protein in the epidermis and the intensity of melanization has been proposed in D. melanogaster (Walter et al. 1991). Moreover, Hollocher et al. (2000b) suggest that pigmentation differences in the Drosophila dunni subgroup may involve mutations at the yellow locus. Our complementation tests also suggest that yellow may play at best a small role in the pigmentation difference between D. santomea and D. yakuba. This suggestion is consonant with another observation in our study: at least four markers show a stronger association with pigmentation than does yellow. Indeed, among the three X-linked markers examined, yellow shows the weakest association with pigmentation, with a far smaller association than Annexin X. This does not rule out an effect of the yellow locus in the species difference, but the results of complementation tests with the D. yakuba yellow mutation may reflect only the semidominance of a normally recessive gene in a hybrid D. santomea/ D. yakuba genetic background. Thus, the involvement of yellow in the D. santomea/D. yakuba pigmentation difference must be viewed as tentative at best. In Drosophila, three lines of evidence suggest that bab1/ bab2 act as general repressors of pigmentation (Kopp et al. 2000). First, in sexually dichromatic species in which the posterior three tergites of males but not females are darkly pigmented, bab1 and bab2 proteins can be detected in pos-

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terior female but not male tergites of midstage pupae. Second, overexpression of bab represses non-sex-specific pigmentation stripes in the anterior tergites of D. melanogaster. Third, loss of bab1 and bab2 genes in D. melanogaster produces female flies with heavy pigmentation of all but one abdominal segment. Segment shape and patterns of abdominal bristles and trichomes are also regulated by bab. The idea that bab represses male-specific pigmentation in females led Kopp et al. (2000) to examine the correlation between bab expression and pigmentation across a diverse group of Drosophila species. In general, those species monomorphic for pigmentation also show similar distribution of the bab1 and bab2 proteins in males and females, whereas dimorphic species with darkly pigmented males show no bab expression in the posterior segments of males. This correlation (although not perfect) led Kopp et al. (2000) to suggest that species differences in sex-specific pigmentation (the derived evolutionary state) resulted from changes in the regulatory regions of bab1 and bab2 that enabled these loci to respond to signals from the controlling genes Abdominal B and doublesex. It is thus possible that the loss of pigmentation in D. santomea may reflect overexpression of bab in both sexes, eliminating not only sex-specific pigmentation, but nearly all pigmentation. For several reasons, however, this suggestion seems unlikely to explain the pigmentation difference between D. yakuba and D. santomea. First, this difference is based on at least three genes in females and five in males. In Analysis 2, bab1 (closely linked to bab2) was used directly as a molecular marker but had the smallest association with male pigmentation among all markers that showed significant association. In Analysis 1, bab1 and bab2 are closely linked (;9 cM) to sepia, the marker with the smallest effect on pigmentation area in both males and females. This analysis also showed that genes on the X chromosome account for about 90% of the species difference in the area pigmented in tergites 5–7 (bab1 and bab2 are on chromosome 3). These results rule out the notion that the D. santomea/yakuba pigmentation difference is due solely or even primarily to mutations in the regulatory region of the two bab genes. It is of course still possible that the pigmentation difference could be due to mutations of unlinked genes that affect the expression of bab. In such a case, the bab product would be the proximate cause of the interspecific pigmentation difference, but bab itself (and its controlling region) would not differ among the species. Moreover, as proposed by Kopp et al. (2000), differences in bab expression explain differences in sexually dimorphic pigmentation. The evolutionary change of D. santomea, in contrast, has been the loss of pigmentation in both sexes, and need not involve genes normally involved in sexual dimorphism. Seven of the eight tested markers are genetically independent and, of these, six show a consistent effect on pigmentation in both backcrosses (i.e., D. yakuba alleles were associated with darker pigmentation, D. santomea alleles with lighter pigmentation), but for only five of the six were the effects significant. This consistency suggests that the species difference may have resulted from selection rather than drift (Orr 1998b). Using Orr’s (1998a, eq. 6) equal-effects sign test on chromosome regions having a consistent effect on

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pigmentation yields a probability of 0.045 (six of six) that this result would occur by chance; this is a more conservative estimate than a simple two-tailed sign test (which yields P 5 0.06) because it is conditioned on the fact that the majority of pigmentation alleles must reside in the D. yakuba line. If we apply the same test but using only the five loci having a significant effect on pigmentation (five of five) test result is not significant (P 5 0.17). Based on these results, we cannot strongly reject the hypothesis that random drift may have been the main force responsible for this species difference. The discovery of additional QTLs will provide a better understanding of the relative roles of selection (natural or sexual) and drift on this trait. The light and dark mating tests provide no evidence that pigmentation influences sexual isolation in these species. Although the frequency of mating was reduced and the latency of copulation increased in dark as compared to light conditions, we found no evidence that sexual isolation was relaxed in the dark. There was certainly courtship in the dark (both inter- and intraspecific), for there were numerous matings and males were frequently seen courting females when the red light was turned on at 5-min intervals. The general reduction of mating activity may be due to two factors: either flies fail to encounter each other in the dark (visual recognition of another individual may be important—but not required—for initiating courtship), or mating might be influenced by some visual cue, such as male wing-extension or circling behavior, that does not differ between the species. The fact that sexual isolation remains as strong in the dark as in the light suggests that nonvisual clues, such as differences in wing-vibration song (e.g., Doi et al. 2001) or pheromonal hydrocarbons (e.g., Coyne et al. 1994), are the primary factors involved in sexual isolation of these species. However, gas chromatography of cuticular hydrocarbons in 13 lines of D. yakuba and seven lines of D. santomea from various populations shows that their main hydrocarbons are identical. Both males and females of each species show a predominance of 7-tricosene, the most common hydrocarbon profile in the D. melanogaster subgroup (Jallon and David 1987). It is therefore unlikely that cuticular hydrocarbons play a role in the sexual isolation of D. yakuba and D. santomea. It is clear from observations of these species, in both light and dark, that sexual isolation is based primarily on female rejection of courting heterospecific males and not males’ refusal to court heterospecific females. Males of either species court females of the other avidly, but failure to copulate occurs via female refusal, particularly the refusal of courting D. yakuba males by D. santomea females. Although we have no direct evidence for this suggestion, we propose that natural rather than sexual selection was the force responsible for the loss of pigmentation in D. santomea. First, this species is not sexually dimorphic for pigmentation, yet sexual selection often (but not always) produces sexual dimorphism. Second, the mating tests show no evidence that either D. yakuba or D. santomea uses pigmentation (or any visual signal) as a cue for mate discrimination. One might expect, although again this need not always be the case, that a character undergoing divergent sexual selection in two related species should ultimately be involved in their sexual isolation.

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If natural selection has caused the species difference, the basis of this selection is unclear. Some species of Drosophila show clinal geographic variation of thoracic and abdominal pigmentation that is based on genetic change, implying spatially varying selection. Generally, pigmentation is darker in flies from cooler climes and higher altitudes. Because flies of a given strain also become darker at cooler rearing temperature, paralleling the evolutionary production of darker flies at higher altitudes and latitudes, temperature is often suggested as the selective pressure for divergent evolution of pigmentation within species (David et al. 1985; Capy et al. 1988; Gibert et al. 1998a,b). Drosophila santomea and D. yakuba, however, violate this rule because the lighter species lives at higher altitudes. Factors that have been invoked in the evolution of insect pigmentation include radiant heat uptake, thermal tolerance, UV tolerance, and resistance to desiccation. (David et al. 1985; Crill et al. 1996; Gibert et al. 1998a). Until we know much more about the ecology of D. santomea and D. yakuba, we will not understand what type of selection, if any, has shaped their striking difference in color. Finally, our estimate of 393,000 years as the age of divergence of these species puts them at roughly the same stage of temporal divergence as two other pairs in the D. melanogaster subgroup: D. simulans and D. sechellia (263,000 year) and D. simulans and D. mauritiana (413,000 year). It is likely that both D. sechellia and D. mauritiana resulted from independent colonization of islands by a D. simulans-like ancestor (Kliman et al. 2000), and so the D. melanogaster subgroup includes three species that arose after island colonization. There are already parallels between these three pairs of species that imply common evolutionary processes (e.g., in all cases sexual isolation is stronger between island-species females and mainland-species males than vice versa). The discovery of further parallels may imply that, at least in this group of flies, speciation on islands involves repeatable events. ACKNOWLEDGMENTS We thank O. Betz for helping with the photographs and F. Lemeunier for checking our chromosome preparations. This work was supported by National Institutes of Health grant GM58260 to JAC. LITERATURE CITED Ashburner, M. 1989. Drosophila: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY. Basten, C. J., B. S. Weir, and Z. B. Zeng. 2002. QTL cartographer. Ver. 1.16. Department of Statistics, North Carolina State University, Raleigh, NC. Begun, D., and P. Whitley. 2000. Reduced X-linked nucleotide polymorphism in Drosophila simulans. Proc. Natl. Acad. Sci. USA 97:5960–5965. Bradshaw, H. D., S. M. Wilbert, K. G. Otto, and D. W. Schemske. 1998. Quantitative trait loci affecting differences in floral morphology between two species of monkeyflower (Mimulus). Genetics 149:367–382. Capy, P., J. R. David, and A. Robertson. 1988. Thoracic trident pigmentation in natural populations of Drosophila simulans: a comparison with D. melanogaster. Heredity 61:263–368. Cariou, M.-L., J.-F. Silvain, V. Daubin, J.-L. DaLage, and D. Lachaise. 2001. Divergence between Drosophila santomea and al-

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