Evolution of circadian behavioral plasticity through cis-regulatory divergence of a neuropeptide gene

Widely-distributed species experience substantial environmental variation, which they often accommodate through behavioral plasticity. Although this ability is integral to fitness, we have little understanding of the mechanistic basis by which plasticity evolves. One factor that varies seasonally and by latitude is photoperiod (day length). Many organisms, including the cosmopolitan Drosophila melanogaster display circadian plasticity, adjusting to fluctuating photoperiod by varying the timing of their activity to coincide with changing dawn/dusk intervals1. Here, we compare D. melanogaster with the closely-related ecological specialist Drosophila sechellia, an equatorial island endemic that experiences minimal photoperiod variation, to investigate the molecular-genetic basis of circadian plasticity evolution2,3. We discover that D. sechellia displays exceptionally little circadian plasticity compared to D. melanogaster and other non-equatorial drosophilids. Through a screen of circadian mutants in D. melanogaster/D. sechellia hybrids, we identify a role of the neuropeptide Pigment-dispersing factor (Pdf) in this loss. While the coding sequence of Pdf is conserved, we show that Pdf has undergone cis-regulatory divergence in these drosophilids. We document species-specific temporal dynamic properties of Pdf RNA and protein expression, as well as Pdf neuron morphological plasticity, and demonstrate that modulating Pdf expression in D. melanogaster can influence the degree of behavioral plasticity. Furthermore, we find that the Pdf regulatory region exhibits signals of selection across populations of D. melanogaster from different latitudes. Finally, we provide evidence that plasticity confers a selective advantage for D. melanogaster at higher latitudes, while D. sechellia likely suffers fitness costs through reduced copulation success outside its range. Our work defines Pdf as a locus of evolution for circadian plasticity, which might have contributed to both D. melanogasters global distribution and D. sechellias habitat specialization. Moreover, together with spatial changes in Pdf expression reported in high-latitude drosophilid species4,5, our findings highlight this neuropeptide gene as a hotspot for circadian plasticity evolution.