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Hiller and his colleagues hypothesized that the loss of FBP2 makes the forward glycolysis reaction more efficient, accelerating the hummingbird's ability to break down sugar, which it consumes in abundance through its nectar diet.

To test this idea, they needed to do an experiment. They were constrained by the fact that it's nearly impossible to genetically manipulate a living bird such as a chicken and knock out FBP2 to measure the effect.  Instead the researchers used a quail muscle cell line to knock out FBP2. They then measured the rate of glycolysis in the quail muscle cells and showed that the process indeed runs faster when the gene is knocked out. In addition, the cells with the FBP2 knockout also had more mitochondria for reasons that are still not completely understood. "In the literature, there's good evidence that hummingbird flight muscle has more mitochondria and high capacity for processing sugar," Hiller says. "The effects we saw [in the cell line] are at least consistent with observations in hummingbird muscle."

With further genome analysis, the researchers discovered that the loss of FBP2 must have happened between 46 million and 34 million years ago. This estimate coincided with paleontological evidence that turned up about 20 years ago in the form of two key fossils found in Germany. One fossil, which still looks like that of a swift, dates back 48 million years, while the other, an early fossil on the branch that split from the swift lineage, had changes in the configuration of the shoulder girdle bones, making it biomechanically capable of hovering flight. It is therefore the oldest known hummingbird, and it is about 35 million to 30 million years old.

The similarity between the estimated age of the FBP2 mutation and the ages of the fossils is convincing evidence that this mutation contributed to hovering flight, Hiller says. Still, he cautions, knocking out this gene in another bird probably isn't enough to produce such flight--this adaptation likely required several steps, including the changes in the shoulder girdle. "The way I think about it, it's one of these knobs that evolution was tuning to give hummingbirds this adaptation of hovering flight," Hiller says. "It probably required several steps or many steps, but we think this is one of them."

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