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Kopp et al. did not record the specific pairs of female strains used in their 'light versus dark' comparisons (A. Kopp, personal communication), so we could not repeat their experiments exactly. They did, however, use inbred stocks or genetic strains that were not controlled for their genetic background, so that mate choice could be affected by many factors besides pigmentation. We carried out two sets of experiments in which we eliminated this possibility by using females with homogeneous genetic backgrounds derived from the wild. In contrast to Kopp et al.1, we found no evidence that males choose less-pigmented females.

We replicated Kopp and colleagues' methods1 by placing one wild-type male in a vial containing two virgin females that had different degrees of abdominal pigmentation (all flies were 4 days old), and observing each pair for 30 min. In all vials in which matings occurred, we scored the degree of pigmentation of the A5 and A6 abdominal segments of mated and unmated females using the procedure described by David et al.2. This method generates pigmentation scores ranging from zero (no pigmentation) to 20 (both segments 100% pigmented).

In our first experiment, we compared two types of female: those with wild-type bab function (normal, light pigmentation) and those with only one functional bab copy (bab/bab+ heterozygotes; darker, male-like pigmentation). Chromosomes either containing or lacking the bab locus were placed in a wild-type genetic background derived from a D. melanogaster stock founded by females collected during 2000 in Arkansas and Louisiana ('ArkLa'). Dark and light females were respectively produced by mating ArkLa males with females from two deficiency strains, Df(3L)Ar12-1 and Df(3L)Ar11. (The former strain was also used by Kopp et al.) Both deficiencies are similar in size and were created in the same genetic background, but Df(3L)Ar12-1 deletes the bab locus, producing dark heterozygous females (average pigmentation score, 16.4 ± 0.09 (s.e.)), whereas females heterozygous for Df(3L)Ar11, which does not delete the bab locus, are lighter (average score, 11.2 ± 0.15).

ArkLa males that were given a choice between bab and bab+ heterozygous females did not discriminate between these types (94 'dark' matings, 88 'light'; χ2 = 0.2, P = 0.67). These results differ significantly (G = 38.3, P < 1 × 10−9) from the combined results of Kopp et al.1, who observed 23 'dark' and 105 'light' matings.

In our second experiment, we produced females of varying pigmentation in the F2 generation of a cross between an outbred stock of D. melanogaster collected in Winters, California, during 2000 and a 'light' female stock produced by combining two inbred lines from the same locality and collected in 2000 (S. Nuzhdin). Males from the outbred stock were given a choice between dark and light F2 females, with mean pigmentation scores of 11.9 ± 0.17 and 7.5 ± 0.24, respectively. Again, males showed no significant discrimination between dark and light females (81 'dark' matings, 61 'light'; χ2 = 2.82, P = 0.095).

Our two replicate experiments were statistically homogeneous (G = 0.94, P = 0.33), but our combined data differed significantly from those of Kopp et al. (G = 52.0, P< 1 = 10−10). Far from showing a strong preference for light females, our wild-type males showed an insignificant tendency to mate with darker females.

We suggest that Kopp and colleagues' results may be attributed to their comparing mutant or inbred strains with dissimilar genetic backgrounds, so that 'light' and 'dark' females in each trial differed in many of their genes. This idea is supported by the extraordinarily high proportion of trials observed by Kopp et al. in which neither female mated (42 out of 170, 24.7%; A. Kopp, personal communication), compared with the low proportion of such trials in our experiments (14 out of 324, 4.3%). This difference is highly significant (G = 43.8, P< 1 × 10−10). Although sexual selection may account for the differences in pigmentation among Drosophila species, we find no evidence that it operates in D. melanogaster in the way suggested by Kopp et al.