Quantitative Trait Loci Affecting The Difference in Pigmentation Between Drosophila yakuba and D. santomea

Using quantitative trait locus (QTL) mapping, we studied the genetic basis of the difference in pigmentation between two sister species of Drosophila: D. yakuba , which, like other members of the D. melanogaster subgroup, shows heavy black pigmentation on the abdomen of males and females, and D. santomea , an endemic to the African island of São Tomé, which has virtually no pigmentation. Here we mapped four QTLs with large effects on this interspecific difference in pigmentation: two on the X chromosome, and one each on the second and third chromosomes. The same four QTLs were detected in male hybrids in the backcrosses to both D. santomea and D. yakuba , and in the female D. yakuba backcross hybrids. All four QTLs exhibited strong epistatic interactions in male backcross hybrids, but only one pair of QTLs interacted in females from the backcross to D. yabuka . All QTLs from each species affected pigmentation in the same direction, consistent with adaptive evolution driven by directional natural selection. The regions delimited by the QTLs included many positional candidate loci in the pigmentation pathway, including genes affecting catecholamine biosynthesis, melanization of the cuticle, and many additional pleiotropic effects.


INTRODUCTION
During the Modern Synthesis, the dominant view of the genetics of species differences was that of Ronald FISHER (1930), who believed that such differences were almost invariably due to the accumulation of many genes, each of small phenotypic effect. Tests of this proposition, however, were limited by the lack of genetic markers in most crossable but differentiated species, although some data suggested that species differences could occasionally be due to genes of large effect (ORR and COYNE 1992).
Recently, however, the advent of molecular techniques has improved our ability to study the genetics of species differences. Two innovations have been crucial. First, quantitative trait locus (QTL) mapping enables us to localize genes responsible for species differences by determining their association with molecular markers at known sites. Second, molecular techniques such as germline transformation enable us to determine directly whether a candidate gene affects a species difference. ORR (2001) describes these innovations and the results of recent genetical studies using them. Although most data derive from a small number of organisms (ORR's study describes only 13 analyses, six from Drosophila and four from the monkeyflower genus Mimulus), the results show that while differences in traits between species can be polygenic, genes of large effect are involved more frequently than previously suspected.
Here we describe a genetic analysis of a striking character difference -the degree of abdominal pigmentation -between two sister species of Drosophila: D. yakuba and D.  LLOPART et al. 2002). Given the dark pigmentation in all but one species in the subgroup, including the outgroup species D. orena and D. erecta, it is nearly certain that the absence of dark pigmentation in D. santomea is a novel derived trait.
The adaptive significance of this pigmentation difference, if any, is unknown. Although these species show strong sexual isolation (COYNE et al. 2002), this does not diminish when flies are tested in the dark, suggesting that the pigmentation difference is not a cue for mate discrimination (LLOPART et al. 2002). Moreover, the relationship between pigmentation and temperature is opposite to that expected from other studies of Drosophila: individuals within a species or closely related species living in colder conditions are almost invariably darker (e.g., DAVID et al. 1985;GIBERT et al. 1998), yet D. santomea, which lives at higher altitudes than D. yakuba, is lighter.
In previous genetic analyses using three morphological markers and eight molecular markers, we determined that at least three genes were involved in the pigmentation difference between D. santomea and D. yakuba females, and five genes between males. In each case, the genes resided on all three major chromosomes, with the X chromosome having a particularly strong effect in males (LLOPART et al. 2002).
In this study we extend and refine our previous analysis, using a more accurate method of measuring pigmentation as well as a QTL analysis employing 32 molecular markers, which enables us to map "pigmentation genes" more accurately. Our goals are to determine the number of genes involved in this morphological difference, their chromosomal locations, whether the same genetic regions affect the pigmentation difference in both males and females, and whether QTLs from a given species tend to affect the character in the same direction, implying that the 8 male used in the backcross. We scored pigmentation in about 50 males and 50 females from each of the pure species and the reciprocal F 1 hybrids, and between 73 and 544 males and females from each of the two backcrosses (Table 1). To improve the precision of QTL mapping, we selected backcross individuals with extreme and intermediate pigmentation phenotypes for subsequent pigmentation scoring and genotyping. For each of the first three BC genotypes listed in Table 1, we selected flies of each sex from a sample of about 12,500 individuals (about 50 bottles, each containing about 250 flies of each sex). One-third of the total individuals chosen were judged by eye to have very dark pigmentation, one-third to have very light pigmentation, and the remaining third were chosen randomly from individuals with intermediate phenotypes.
(Equal numbers of all three classes were chosen from each bottle inspected until we had accumulated about 500 flies of each sex in each backcross. Thus for each sex we selected about 4% of total individuals inspected; this stringent selection facilitates the precision of mapping).
However, females from the backcross of F 1 individuals to D. santomea showed little variation in pigmentation, and so all of these were chosen randomly.
Pigmentation scores: All scoring of pigmentation was done on 4-day-old virgin flies.
We scored only the three posterior tergites of each fly (segments 5, 6, and 7) by examining the fly under a dissecting microscope. A pigmentation score was assigned based on both the percentage of the tergite that was pigmented and the degree of pigmentation. First, the proportion of the tergite covered by black pigment was estimated to the nearest 0.05 (5%). Then, the relative degree of pigmentation was measured within the pigmented area. Using color standards, we assessed the degree of pigmentation within the pigmented area using a five-point scale ranging from 1 (very light pigmentation, slightly darker than background color) to 5 (dark, shiny black), with intermediate numbers representing intermediate degrees of pigmentation.
Unpigmented areas were given a score of 0. We limited ourselves to assigning only three shades of black to each tergite. The percentage of the area of each tergite covered by each shade of pigmentation was then multiplied by the intensity of pigmentation, and these areas were summed. This gives each tergite a minimum possible pigmentation score of 0 (no area pigmented) to 5 (tergite completely covered with very dark pigmentation [1.0 × 5]). These areas were summed for all three tergites, yielding a minimum possible pigmentation score for a given fly of 0 and a maximum possible score of 15. As shown in Table 1, this procedure discriminates well between the pigmentation of these species.

Molecular markers:
We identified single nucleotide polymorphisms (SNPs) and insertion/deletion variants (indels) that discriminated between the D. yakuba Taï18 and the D.
In total, we sequenced approximately 17.5 kb in each of the 20 flies tested. We detected 263 nucleotide differences fixed between Taï18 and STO.4, that is, 70% of the total nucleotide variation. Among these fixed differences we selected 32 that affect a restriction endonuclease site to be used as markers in the genotyping procedure. Table 2 lists the 32 markers, their relative order within the D. yakuba chromosomes, and the conditions for genotyping.
We inferred the relative order of markers within each chromosome in D. yakuba/D. santomea from the D. yakuba genome project (http://www.genome.wustl.edu/projects/yakuba/; version 040407). The 32 molecular markers were designed using sequence data from the parental strains of D. santomea and D. yakuba. The aligned sequences were used to develop PCR primers using Primer3 (ROZEN and SKALETSKY 2000) and restriction enzyme (RE) digestions. Genotyping was performed using Restriction Fragment Length Polymorphism (RFLP) analysis by PCR amplification from genomic DNA using RedTaq DNA polymerase (Sigma; St. Louis, MO) followed by RE digestion (see Table 2 for primers, RE, and conditions). All RE's were purchased from New England Biolabs (Beverly, MA) and primers were purchased from MWG Biotech (High Point, NC). The digested PCR products were run on a 3% agarose gel stained with ethidium-bromide, imaged with the Bio-Rad ChemiDoc System PC RS-170 using Quantity One (version 4.2.1) software, and manually genotyped by assigning a "0" (homozygous D. santomea), "1" (D. yakuba/D. santomea heterozygote) or "2" (homozygous D. yakuba) to each marker genotype. A recombination map based on the 1642 BC hybrids (Table 3) was constructed using the Haldane mapping function.
QTL mapping: QTLs affecting variation in pigmentation between D. yakuba and D. santomea were mapped in each BC population using composite interval mapping (CIM; ZENG 1994) and implemented using QTL Cartographer software (BASTEN et al. 1999). CIM tests whether an interval between two markers contains a QTL affecting the trait while simultaneously controlling for the effect of QTL located outside the interval using multiple regression on marker co-factors. Marker co-factors were chosen by forward selection -backward elimination stepwise regression. The likelihood ratio (LR) test statistic is −2ln(L 0 /L 1 ), where L 0 /L 1 is the ratio of the likelihood under the null hypothesis (i.e., there is no QTL in the test interval) to the alternative hypothesis (there is a QTL in the test interval). LR test statistics were computed every 2 cM with marker co-factors 10 cM or more from the test location. We used permutation analysis to determine appropriate significance thresholds that take into account the multiple tests performed and correlations among markers. We permuted trait and marker data 1000 times, and recorded the maximum LR statistic across all intervals for each permutation. LR statistics calculated from the original data that exceed the 50 th greatest LR statistic from the permuted data are significant at the experiment-wise 5% level under the null hypothesis (CHURCHILL and DOERGE, 1994;DOERGE and CHURCHILL, 1996). The approximate boundaries of regions Candidate genes: Cytological bands in D. melanogaster of the markers that define the interval under the QTL peak were obtained using Flybase (DRYSDALE and CROSBY, 2005).
Using the known cytological positions of these markers in D. melanogaster, we determined the corresponding positions in D. yakuba (LEMEUNIER and ASHBURNER,1976;ASHBURNER, 1989).
This defined an interval both in D. melanogaster and in D. yakuba that allowed us to search for 13 candidate genes within that interval. We obtained a complete list of candidate genes involved in pigmentation in D. melanogaster from Flybase (DRYSDALE and CrOSBY, 2005). Candidate genes were identified based on the markers that delimit each QTL, and the presence of pigmentation genes between these two markers (Table 6). Table 1 gives the mean pigmentation scores, standard errors, and sample sizes for the pure species, the reciprocal F 1 hybrids, and the backcross hybrids used for genotyping. The difference between the pure species is substantial: the mean pigmentation score of D. yakuba males and females is 14.22 and 9.85, respectively, and for D. santomea males and females 0.63 and 1.02, respectively. As seen in our previous analysis, (LLOPART et al. 2002) reciprocal F 1 hybrid males show a large effect of the X chromosome on pigmentation: these males have pigmentation scores fairly close to those of males from the species of the maternal parent. The difference in pigmentation scores between the two classes of F 1 males is highly significant (t = 27.1, 100 d.f., P < 0.001). The relative effect of the X chromosome in male pigmentation can be judged as the percentage of the total difference between males of the two species explained by the difference between the reciprocal F 1 males; this effect is about 60%. This effect is much larger than the relative size of this chromosome (constituting roughly 21% of the haploid genome [ Table 3]) and suggests either that the X chromosome carries a disproportionate number of genes affecting pigmentation, or that individual X-linked genes have disproportionately large effects. (The QTL analysis below shows that the second explanation is most likely to be correct.)

Pigmentation of pure species and F 1 hybrids:
In contrast to males, the F 1 females do not differ significantly in pigmentation scores (Table 1; t = 1.55, 99 d.f., P = 0.12). There is thus no evidence for a maternal or mitochondrial effect affecting pigmentation of these females, who are identical in nuclear genotype. The mean score of all F 1 females (4.45) is slightly lighter than the average score of females for the two species (5.43), showing a small amount of dominance for the D. santomea phenotype.

QTLs affecting variation in pigmentation in BC hybrids:
We mapped four QTLs with large effects on pigmentation (Table 4, Figure 1). The same four QTLs were detected in male hybrids in the backcrosses to both D. santomea and D. yakuba: two QTLs were on the X chromosome (between markers 1-2 and markers 6-10), one QTL was on the second chromosome (between markers 15-18) and one QTL was on the third chromosome (between markers 23-26).
The magnitude of the QTL effects ranged from 0.49 -1.42 phenotypic standard deviations in the backcross to D. santomea, and accounted for 67% of the total phenotypic variation. Similarly, the QTL effects ranged from 0.62 -1.62 phenotypic standard deviation in the backcross to D. yakuba, and accounted for 58% of the total phenotypic variation. In both backcrosses, the sum of the estimated QTL effects equaled or exceeded that expected from the difference between the parental genotypes. In the backcross to D. santomea, the expected difference in pigmentation is -11.29 (i.e. the difference in pigmentation between D. santomea males (0.63) and (Y X S) F1 males (11.92)); whereas the sum of the QTL effects was -11.64. In the backcross to D. yakuba, the expected difference in pigmentation is 10.5 (i.e. the difference in pigmentation between D. yakuba males (14.22) and (S X Y) F1 males (3.72)); whereas the sum of the QTL effects is 16.15.
Thus, it is likely that we have detected all of the QTLs affecting variation in pigmentation in this hybridization, and that our selective genotyping protocol led to over-estimation of effects (LYNCH and WALSH 1998). It is also possible that estimates of main effects have been biased by epistatic interactions (see below).
Four QTLs in the same positions affected variation in pigmentation in the female D.
yakuba BC hybrids. The magnitude of the QTL effects ranged from 0.31 -0.83 phenotypic standard deviations, and accounted for 63% of the total phenotypic variance. The sum of the estimated QTL effects slightly exceeded that expected from the difference between the parental genotypes in this backcross, again suggesting that we have detected all of the QTL affecting variation in pigmentation in this hybridization. The expected difference in pigmentation is 5.39 (i.e. the difference in pigmentation between D. yakuba females (9.85) and F1 females (4.46 on average)); whereas the sum of the QTL effects is 5.62. We observed only a single X chromosome QTL (between markers 6-10) in the D. santomea BC females, accounting for 43% of the total phenotypic variance. This could be attributable to a lack of power to detect QTLs in this cross, since only 73 flies were assessed for genotype -phenotype associations, compared to over 500 individuals in each of the other crosses. Indeed, this QTL only accounted for 40% of the expected difference in pigmentation (-3.44; i.e. the difference in pigmentation between D. santomea females (1.02) and F1 females (4.46 on average)).
The QTL effects were largely additive within loci. In the BC to D. yakuba, the effects of the two X-chromosome QTLs in females (a − d) were approximately half that of the effects in males (2a), consistent with d = 0. In addition, the effects of the chromosome 3 QTL in males from the backcrosses to D. yakuba and D. santomea were equal and opposite, as expected if d = 0. The second chromosome QTL had a larger effect in the BC to D. yakuba than to D. santomea, suggesting partial dominance of the D. santomea genotype. Since dominance of D. santomea QTLs reduces the power to detect QTLs in the backcross to D. santomea, this could also account for our failure to detect this QTL in females from this backcross.

Epistatic interactions:
We assessed all possible epistatic interactions between pairs of markers within each cross and sex (Table 5, Figures 2 -4). We observed significant epistasis (after correcting for multiple tests) in males from both backcrosses between markers in regions encompassed by the QTLs, but not between QTL regions and regions without main effects, or between two regions with no main effects on pigmentation (Figure 2). The significant interactions were between the two X-chromosome QTLs; the X-chromosome QTL between markers 6-10 and the chromosome 2 QTL; the X-chromosome QTL between markers 6-10 and the chromosome 3 QTL; and the chromosome 2 and chromosome 3 QTLs ( Figure 2, Table 5).
The nature of these interactions is illustrated in Figure 3, where the effect of a Y/S substitution at the second locus is shown in the form of reaction norms, conditional on the genotype of the first locus (where Y denotes a D. yakuba allele and S denotes a D. santomea allele at the QTL). In the absence of epistasis, the effect of the substitution at the second locus would be independent of the genotype of the first, and the reaction norms would be parallel.
In the backcross to D. santomea, we expect the hemizygous Y or heterozygous SY genotype at the second locus to be more pigmented than the hemizygous S or homozygous SS genotype at this locus. However, for all the interacting markers in this backcross, this is only true if the genotype at the first locus is Y (or SY). Either there is no difference between the genotypes at the second locus if the first is S (SS), or, for the case of the interaction between the second Xchromosome QTL and the chromosome 2 QTL, the SS genotype at the chromosome 2 QTL is actually more pigmented than the SY genotype at this QTL when the X-chromosome QTL is S ( Figure 3). In other words, the effect of a Y-S substitution in an otherwise S background is smaller than the effect of an S-Y substitution at each QTL in the Y background. Equivalently, the effect of Y-S substitutions at two interacting loci in the homozygous S background is greater than additive, and the effect of S-Y substitutions at two interacting loci in the heterozygous SY background is less than additive. This is illustrated in Figure 4, where the sum of the effects of substituting single Y alleles at each QTL in the S background would yield a predicted pigmentation score of 4.09 for the YYYY haplotype, whereas the observed score is 9.43.
The epistatic interactions are more complicated in the backcross to D. yakuba. Here we expect the hemizygous Y or homozygous YY genotype at the second locus to be more pigmented than the hemizygous S or heterozygous SY genotype at this locus. In the interaction between the two X-chromosome QTLs (marker 1 × marker 9), this is true if marker 1 is Y. On the other hand, in the interactions with the chromosome 3 QTL (marker 8 × marker 25 and marker 18 × marker 25), this is true if marker 8 is S and marker 25 is SY (Figure 3). However, the interaction between the second X-chromosome QTL and the chromosome 2 QTL (marker 8 × marker 18) is in the opposite direction to that expected: the SY genotype at marker 18 is actually more pigmented than the YY genotype at this marker, but only if marker 8 is Y. Overall, the effect of Y-S substitutions at two interacting loci in the heterozygous SY background is less than additive, and the effect of S-Y substitutions at two interacting loci in the homozygous YY background is greater than additive. Figure 4 shows that the sum of the effects of substituting single Y alleles at each QTL would yield a predicted pigmentation score of 15.15 for YYYY haplotype, whereas the observed score is 13.43.
A single epistatic interaction was observed in females from the backcross to D. yakuba, between the second X chromosome QTL (markers 6-10) and the chromosome 2 QTL (data not shown). The direction of the epistatic effects between these QTLs is the same as in males from this hybridization. The effect of Y-S substitutions at the two interacting loci in the heterozygous SY background is less than additive, and the effect of S-Y substitutions at two interacting loci in the homozygous YY background is greater than additive. Figure 4 shows that the sum of the effects of substituting single Y alleles at each QTL would yield a predicted pigmentation score of 11.17 for YYYY haplotype, whereas the observed score is 9.49. This study not only largely expands and refines the earlier results of LLOPART et al. (2002), in which pigmentation differences were assessed in backcross hybrids using eight molecular markers, but also provides the first accurate chromosomal locations of genetic factors associated with these differences. In LLOPART et al. (2002), the QTL of largest effect was also associated with AnnX at the base of the X chromosome, with a second QTL with smaller effect at the tip of the X chromosome associated with y. The locations of the autosomal QTLs are also concordant between the two studies. The QTL with the smallest effect detected by LLOPART et al. (2002) was associated with the marker at bric-à-brac 1 (bab1) at the tip of 3L, which is only marginally significant in the backcross to D. yakuba females in this study (LR = 14 between the Lsp1γ and dib markers). One possible explanation for this small discrepancy is that the bab1 marker is in linkage disequilibrium with the chromosome 3 QTL mapped in this study. This, however, is not likely because the map distance between the major QTL detected on chromosome 3 and the bab1 region is greater than 100 cM. It is possible that the discrepancy could be due to the fact that the methods used to score abdominal pigmentation in both studies, although correlated, are different.

DISCUSSION
The results presented here raise the interesting possibility that the genetic basis of pigmentation differences between D. yakuba and D. santomea is fairly simple. We infer that we have detected all major QTLs accounting for variation in pigmentation in these backcross hybrids (with the exception of females in the backcross to D. santomea), since the sum of the QTL effects equals or exceeds that expected from the difference between parental strain means.
Further high-resolution mapping is required to determine whether single genes or multiple closely linked loci are responsible for the large QTL effects. Nevertheless, all QTLs from the same species affected pigmentation in the same direction, suggesting that the species difference might have arisen by natural selection (ORR 1998). We were not able to formally test this hypothesis, since a minimum of six QTLs are required to reject the null hypothesis (ORR 1998). indicate that this species difference is polygenic with no significant effect of the X chromosome but with significant effects of three of the five autosomes. There is little genetic commonality between the results reported by these two studies and our results. Of course, unless there is a very limited number of genes that could be potentially responsible for differences in pigmentation, one does not expect the genetic architecture to be shared among distantly related species.
Our observations of epistatic interactions between QTLs with main effects on pigmentation are consistent with genes corresponding to the QTLs that are in the same pathway(s). In the absence of high resolution mapping, however, we can only speculate about what candidate genes might correspond to the QTLs. An obvious candidate for the QTL at the tip of the X chromosome is yellow (y) itself, and complementation tests to D. santomea using a y mutation in D. yakuba are consistent with a very small contribution of mutations at the y locus in the pigmentation difference between these species (LLOPART et al. 2002). In addition, two enhancers of y are located in the region embraced by the QTL at the base of the X chromosome; these could contribute to the interactions between the two X chromosome QTLs.
Several candidate genes affecting body pigmentation have been identified by mutagenesis in D. melanogaster (DRYSDALE and CROSBY, 2005), and co-localize to the regions containing QTLs affecting pigmentation differences between D. yakuba and D. santomea (Table 6).
Catecholamines are required for proper melanization and sclerotization of the Drosophila cuticle (WRIGHT 1987;WALTER et al. 1996). The Ddc gene cluster on chromosome 2 (genetically defined by Df(2L)TW130; 37B9-C1,2;D1-2) contains at least 18 functionally related genes involved in the catecholamine pathway, including Catsup, Ddc, Dox-A2, amd, and l(2)37Ca (STATHAKIS et al. 1995). Mutations in 11 of the loci in this complex (including Ddc and amd) produce melanotic psueudotumors, indicating abnormal catecholamine metabolism (WRIGHT 1996), and mutations in 14 of the loci affect the formation, sclerotization or melanization of the cuticle (WRIGHT 1996). The Ddc cluster co-localizes with the QTL on chromosome 2. Pu encodes GTP cyclohydrolase, the rate limiting step in the synthesis of tetrahydobiopterin, the cofactor required for the phosporylation of tyrosine hydroxylase, which is in turn the rate limiting step in the synthesis of dopamine (STATHAKIS et al. 1999). Pu also co-localizes with the QTL on chromosome 2. The silver (svr) gene, which encodes proteins that are members of the carboxypeptidase family (SETTLE et al. 1995), co-localizes with the QTL at the tip of the X chromosome. Mutations in svr affect pigmentation, wing shape and catecholamine pools (WRIGHT 1987). tan (t) is an excellent candidate gene corresponding to the QTL at the base of the X chromosome. t is probably the structural gene for beta-alanyldopamine hydrolase activity; t mutants have reduced dopamine levels (WRIGHT 1987).
Additional candidate genes in the pigmentation pathway include optomotor-blind (omb), black (b), Cysteine proteinase-1 (Cp1), and Black cells (Bc) (WRIGHT 1987;WITTKOPP et al. 2002;2003). The developmental gene, omb, co-localizes with the QTL at the tip of the X chromosome. omb encodes a T-box transcription factor that is necessary for patterning the pigment band in each adult abdominal segment of Drosophila melanogaster (KOPP and DUNCAN 1997;KOPP et al. 1997). A recent study by BRISSON et al. (2004)  Conspicuously absent from the list of strong potential candidate genes are bab1 and bab2, two closely linked genes at the tip of chromosome 3L that are thought to be repress malespecific abdominal pigmentation in females (KOPP et al. 2000) and contribute significantly to variation of abdominal pigmentation in females of D. melanogaster (KOPP et al. 2003). The expression of bab is correlated with pigmentation across a diverse range of Drosophila species, such that species in which neither sex is pigmented express exhibit similar expression of Bab in males and females, but species in which abdominal tergites of males are more pigmented than females have female-specific Bab expression (KOPP et al. 2000). Thus, it was possible a priori 23 that overexpression of Bab in D. santomea could have resulted in loss of pigmentation in both sexes. This is not the case, however, since none of the QTLs map in the vicinity of bab. Further, Bab2 protein is expressed in a dimorphic melanogaster-like pattern in D. santomea (GOMPEL and CARROLL 2003), which is inconsistent with mutations at bab affecting the difference in pigmentation between D. santomea and D. yakuba.
While it is plausible that the loss of pigmentation in D. santomea was driven by natural selection, it is also possible that selection acted on pleiotropic effects of genes affecting pigmentation, and not pigmentation itself. All of the candidate genes listed in Table 3 have highly pleiotropic effects on traits related to fitness, including reproduction and immune response. For example, Ddc catalyzes the final step in the biosynthesis of the neurotransmitters dopamine and serotonin. Dopamine is required in Drosophila for normal development (NECKAMEYER 1996); ovarian maturation, fecundity and sexual receptivity in females (NECKAMEYER 1996;1998a); learning (TEMPEL et al. 1984;NECKAMEYER 1998b); locomotion (PENDLETON et al. 2002) and aggressive behavior (BAIER et al. 2002). Serotonin also regulates or modulates a variety of behaviors in many animal species, including aggression, feeding, learning, locomotion, sleep and mood (BLENAU and BAUMANN 2001). Further speculation about the nature of the pleiotropic effects (and sex-specific epistatic effects) of genes affecting variation in pigmentation between these species must await the positional cloning of these genes.         between markers for males from the backcross to D. santomea is indicated above the diagonal, and for interactions for males from the backcross to D. yakuba below the diagonal. P < 0.0001 (Bonferroni correction); 0.0001 < P < 0.001; 0.001 < P < 0.01; 0.01 < P < 0.05.   Position (