LAUSR

Significance What factors maintain genetic variation in natural populations? Opposing selection pressures on protein stability and catalytic activity are thought to maintain variation along thermal gradients in many enzymes. We examined a classic hypothesis of temperature-mediated balancing selection, the alcohol dehydrogenase enzyme of Drosophila melanogaster, in which 2 latitudinally distributed variants are thought to be maintained by an activity/stability trade-off. Using in vitro and in vivo assays and population genetic analyses, we found no evidence of the predicted biochemical or fitness trade-offs and no signature of balancing selection. Rather, one variant confers greater activity and survival in the presence of ethanol, irrespective of temperature. Variation in Adh, and possibly other enzymes, must therefore be caused by other factors correlated with temperature. Polymorphism in the alcohol dehydrogenase (ADH) protein of Drosophila melanogaster, like genetic variation in many other enzymes, has long been hypothesized to be maintained by a selective trade-off between thermostability and enzyme activity. Two major Adh variants, named Fast and Slow, are distributed along latitudinal clines on several continents. The balancing selection trade-off hypothesis posits that Fast is favored at high latitudes because it metabolizes alcohol faster, whereas Slow is favored at low latitudes because it is more stable at high temperatures. Here we use biochemical and physiological assays of precisely engineered genetic variants to directly test this hypothesis. As predicted, the Fast protein has higher catalytic activity than Slow, and both the Fast protein and regulatory variants linked to it confer greater ethanol tolerance on transgenic animals. But we found no evidence of a temperature-mediated trade-off: The Fast protein is not less stable or active at high temperatures, and Fast alleles increase ethanol tolerance and survivorship at all temperatures tested. Further, analysis of a population genomic dataset reveals no signature of balancing selection in the Adh gene. These results provide strong evidence against balancing selection driven by a stability/activity trade-off in Adh, and they justify caution about this hypothesis for other enzymes except those for which it has been directly tested. Our findings tentatively suggest that environment-specific selection for the Fast allele, coupled with demographic history, may have produced the observed pattern of Adh variation.