However, when we use all loci identified via mapping (and not just our “big three”), we correctly predict that both the indoor and outdoor cages become lighter! The trait change is explainable – with all loci, we can understand & explain how we observed this shift in pigmentation.

Using only tan, ebony, and bab to predict trait evolution, we correctly predict that indoor cages become lighter, but we incorrectly predict that the outdoor cages get darker (positive = light, negative = dark).

Our mapping is in fact stable enough that we can very accurately predict the relative phenotypes across our genetically diverged populations evolved in a different environment or sampled at different times. (Here we’re using week 8, outdoor mapping – positive = light, negative = dark).

The resulting genotype-phenotype map re-identifies large-effect loci near tan, ebony, and bab, and also identifies novel loci, with a range of effect sizes. The mapping was remarkably stable across both time and environment, as can be seen in this Miami plot. (outdoor = top, indoor = bottom)

To map pigmentation in both environments, before and after the trait evolved, we used a tail-based approach. Specifically, after 2 generations of common garden, we segregated individuals from each sample into dark, midpoint, and light fractions for pooled sequencing.

In both environments, we see lighter pigmentation evolution, although to a greater degree in the outdoor cages. Given that we observe this across environments, this evolution is likely driven by shifts in population density, with some contribution from abiotic factors in the outdoor environment.

The mesocosm system, while controlling for demography & linkage, also provides the ecological realism needed to solve this mystery. We seeded 10 indoor & 10 outdoor cages with replicate populations derived from a single outbred population (started from inbred lines collected in local orchards).

D. melanogaster exhibit clinal patterns in pigmentation whereby northern populations are darker relative to southern populations, and populations within a single location evolve from darker pigmentation in the spring to lighter pigmentation through fall.

Additionally, there have been several associative trait mapping studies in the past decade that found a handful of these genes to be large-effect loci across populations, namely tan, ebony, and bric-a-brac (bab), all key components of the network above. (Figure from Bastide et. al, 2013)

But first, we had to pick a model trait – and abdominal pigmentation was the perfect one. It’s a well-understood classic genetic trait, with many known candidate genes involved in the synthesis of the pigment molecules and the development of the pigmented cuticle. (Image from Massey & Witkopp 2016)