The team had now amassed many lines of evidence suggesting that natural selection relating to temperature is acting on the PGI gene. However, they were still missing a critical component: evolutionary fitness, a measure of a genotype’s success at reproducing relative to other genotypes in the population. “I like to define fitness in terms of what the animal cares about — getting fat and making babies,” Elizabeth explains. “How many babies do you leave, and do your babies make it? And if you are a male, how many females do you mate with?” Natural selection spreads genes correlated with high fitness and weeds out those associated with low fitness. Being strong, or camouflaged, or able to run fast in cold weather may all seem useful, but if those abilities don’t translate into more offspring in the next generation, natural selection will not favor them. This is the secret similarity among all those fancy adaptations we observe in nature: the flutter of a hummingbird’s wings, the stag beetle’s horns, the brilliance of an orchid’s jewel-toned blossom — each of them ultimately came about because individuals who had them produced more offspring than those that didn’t.

So what about willow leaf beetles? Does carrying PGI-1 versus 4 affect their fitness at different temperatures? To find out, the team ran their beetles through a gamut of physical challenges—exposing them to sub-zero temperatures overnight, testing how well they lay eggs and grow at different temperatures, and more.

In terms of egg laying and other key measure of fitness, the results supported the race car/jeep hypothesis. For example, at cooler, less stressful temperatures both in the lab and in the wild, PGI 1-1 beetles laid more eggs than PGI 4-4 beetles. However, when things got hotter, the results flip-flopped; then, PGI 4-4 beetles laid more eggs. This is exactly what we’d expect to observe if PGI-1 is associated with producing lots of HSPs in response to stress, and PGI-4 is associated with being stingier with these expensive proteins. The team now had direct evidence that PGI’s physiological interactions affected the beetles’ fitness.