Scaling between specific organs and overall body size has long fascinated biologists, being a primary mechanism by which organ shapes evolve. Yet, the genetic mechanisms that underlie the evolution of scaling relationships remain elusive. Here we compare wing and fore tibia lengths (the latter as a proxy of body size) in Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, and Drosophila virilis, and show that the first three of these species have roughly a similar wing-to-tibia scaling behavior. In contrast, D. virilis exhibits much smaller wings relative their body size compared to the other species and this is reflected in the intercept of the wing-to-tibia allometry. We then asked whether the evolution of this relationship could be explained by changes in a specific cis-regulatory region or enhancer that drives expression of the wing selector gene, vestigial (vg), whose function is broadly conserved in insects and contributes to wing size. To test this hypothesis directly, we used CRISPR/Cas9 to replace the DNA sequence of the predicted Quadrant Enhancer (vgQE) from D. virilis for the corresponding vgQE sequence in the genome of D. melanogaster. Strikingly, we discovered that D. melanogaster flies carrying the D. virilis vgQE sequence have wings that are significantly smaller with respect to controls, partially shifting the intercept of the wing-to-tibia scaling relationship towards that observed in D. virilis. We conclude that a single cis-regulatory element in D. virilis contributes to constraining wing size in this species, supporting the hypothesis that scaling could evolve through genetic variations in cis-regulatory elements.

Scaling between specific organs and overall body size has long fascinated biologists because they are a primary mechanism through which organismal shapes evolve. Yet, the genetic mechanisms that underlie the evolution of allometries remain elusive. Here we measured wings and tibia lengths in four Drosophila species (D. melanogaster, D. simulans, D. ananassae, and D. virilis) and show that the first three of them follow a single evolutionary allometry. However, D. virilis exhibits a divergent wing-to-tibia allometry due to a dramatic underscaling of their wings with respect to their bodies compared to the other species. We asked whether the evolution of this scaling relationships could be explained by changes in a specific cis-regulatory region of the wing selector gene, vestigial (vg), whose function is broadly conserved in insects and its expression pattern determines wing size in D. melanogaster. To test this hypothesis directly, we used CRISPR/Cas9 to replace the DNA sequence of the predicted Quadrant Enhancer (vgQE) from D. virilis for the corresponding vgQE sequence in the genome of D. melanogaster. Strikingly, we discovered that D. melanogaster flies carrying the D. virilis vgQE sequence have wings that are much smaller with respect to controls, partially rescuing the wing-to-tibia ratio observed in D. virilis. Our results show that this cis-regulatory element in D. virilis contributes to the underscaling of wings in this species. This provides evidence that scaling relationships may be unconstrained and evolve gradually through genetic variations in cis-regulatory elements.