Drosophila pigmentation evolution: divergent genotypes underlying convergent phenotypes

Abstract

Similar phenotypic changes have evolved independently in many animal taxa. It is unknown whether independent changes involve the same or different developmental and genetic mechanisms. Myriad pigment patterns in the genus Drosophila offer numerous opportunities to address this question. Previous studies identified regulatory and structural genes involved in the development and diversification of pigmentation in selected species. Here, we examine Drosophila americana and Drosophila novamexicana, interfertile species that have evolved dramatic pigmentation differences during the few million years since their divergence. Interspecific genetic analysis was used to investigate the contribution of five specific candidate genes and other genomic regions to phenotypic divergence by testing for associations between molecular markers and pigmentation. At least four distinct genomic regions contributed to pigmentation differences, one of which included the ebony gene. Ebony protein was expressed at higher levels in the more yellow D. novamexicana than the heavily melanized D. americana. Because Ebony promotes yellow pigment formation and suppresses melanization, the expression difference and genetic association suggest that evolution at the ebony locus contributed to pigmentation divergence between D. americana and D. novamexicana. Surprisingly, no genetic association with the yellow locus was detected in this study, and Yellow expression was identical in the two species. Evolution at the yellow locus underlies pigmentation divergence among other Drosophila species; thus, similar pigment patterns have evolved through regulatory changes in different genes in different lineages. These findings bear upon understanding classic models of melanism and mimicry.

Figure 1

Pigmentation of D. novamexicana, D. americana, and hybrid flies. (a) Males and females of D. novamexicana have a yellowish cuticle color with little melanization (female shown). (b) In contrast, both sexes of D. americana are heavily melanized and produce little yellow pigment (female shown). (c) Hybrid males from a D. novamexicana mother have an increase in body melanization characteristic of the D. americana parent but lack melanization along the abdominal dorsal midline (arrow), similar to D. novamexicana. (d) Hybrid females from either direction of cross have identical phenotypes (hybrid from a D. novamexicana mother shown), with more body melanization and slightly less yellow pigment than males from D. novamexicana mothers. (e) Hybrid males from D. americana mothers have nearly as much body melanization as D. americana, with only slightly more yellow pigment. A reduction of dorsal midline melanization is still observed in these males, but the decrease in yellow pigment reduces the contrast between melanized and nonmelanized cuticle, making it hard to discern in photographs (e, arrow). Flies in c–e each carry a different complement of X chromosomes: the fly in c has a D. novamexicana X chromosome; the fly in d has both a D. novamexicana and a D. americana X chromosome; and the fly in e has a D. americana X chromosome. All flies are the same magnification (×13.5).

Figure 2

The five classes of backcross progeny. Abdominal pigmentation phenotypes among progeny from a backcross between hybrid females and D. novamexicana males fell into five phenotypic classes, scored 1–5 (a–e), respectively. An inverse correlation between yellow and black pigments was observed among these flies. (f) The phenotypic distribution of backcross progeny is shown separated by both sex (male and female) and F₁ mother (A/N and N/A). Progeny from D. americana mothers (A/N) are shown with black bars; those from D. novamexicana mothers (N/A) are shown with white bars. The number of flies (n) scored from each backcross are also shown. χ² tests of independence (df = 4) indicated that both sex and F₁ mother affected the distribution of abdominal pigmentation intensity (χ² = 209.5, P < 10⁻⁴³; χ² = 44.3, P < 10⁻⁸, respectively). The significance of the interaction between F₁ mother and abdominal pigmentation, however, may be caused by scoring error rather than biology, because no significant effect of F₁ mother was detected in the genotyped sample set.

Figure 3

Genomic distribution of candidate genes and molecular markers. (a) Approximate cytological locations of molecular markers in D. americana (indicated by arrows) are shown relative to each cytological inversion (shown in orange). Markers in parentheses are located on that chromosome, but the precise cytological location is unknown. Candidate genes are shown in red; chromosome numbers are shown in blue. Note that the e marker is located within an inversion. (b) Dotted lines show tests for linkage between each marker and a D. novamexicana allele that contributes to either light (circle) or dark (square) pigmentation. Similarly, solid lines show tests for linkage between markers and D. americana alleles that serve as light (circle) or dark (square) QTLs. LOD scores for candidate genes are shown in red, and the markers with names shown had a significant association in the χ² analysis. Maximum LOD scores are shown on the left and the corresponding recombination distances are shown on the right in centimorgans (CM). LOD scores >2.7 (indicated with line) are significant (α = 0.05 after Bonferroni correction for 23 tests). Note that all “dark” QTL alleles are linked to D. americana marker alleles, and all “light” QTL are linked to D. novamexicana alleles. The seven markers that deviated significantly from neutral expectations in both populations (e, hb, ActE2, pros28.1b, hsp83, kni, and Egp1) show evidence of linkage to both a “dark” D. americana QTL allele and a “light” D. novamexicana QTL allele. Rh4 and mam alleles were associated with light, but not dark, pigmentation. Rh4 shows linkage to a “light” D. novamexicana QTL allele but not to any “dark” QTL. Also note that e is the only candidate gene with significant linkage to a QTL.

Figure 4

Changes in Ebony expression correlate with pigmentation differences. Ebony protein (shown in red) is expressed at a higher relative level in the abdominal tergite of D. novamexicana (a) than D. americana (b), with an intermediate level observed in hybrids (c). This expression positively correlates with yellow pigment and inversely correlates with melanization (compare with Fig. 1). In contrast, expression of the Yellow protein (shown in yellow) is similar between D. novamexicana (d) and D. americana (e), despite the differences in melanization. (f) No fluorescent signal was observed in epidermal cells (arrowhead) in the absence of the Ebony or Yellow antibodies. There was, however, some background fluorescence (shown in green) within a bristle-associated cell (arrow) and in cells located underneath the epidermis (asterisk). Because the signal from immunolocalizations is nonlinear and difficult to standardize between species, we can make only a qualitative statement about relative levels of antigen and not an absolute quantitative measurement of Ebony or Yellow protein levels. In all panels, a section of pharate adult dorsal abdominal cuticle from either the A3 or A4 segment is shown with anterior at the top.