Picture this: you’re out at a bar, and you’ve just met a real cutie. You stand up to order another round, but when you turn around, you see that some jerk has moved in on your date. The bar is so loud that yelling to get their attention won’t do you much good. What do you do? Well, if you want to take the tactic of a male Bornean rock frog, you should start aggressively waving one of your feet. Researchers have found that the frogs’ “foot-flagging” behavior is linked to testosterone levels. They published their research in the Proceedings of the National Academy of Sciences

The Bornean rock frog (Staurois parvus) is a small amphibian that makes its home in the island’s lush rainforests. S. parvus is especially fond of hanging out near thundering waterfalls, which makes for beautiful views but a whole lot of ambient noise. However, mating season necessitates certain types of communication. To work around this, S. parvus and a few other species have started using a sort of natural semaphore system, in which a male frog waves his specialized white-webbed back foot around like a flag. 

To other male frogs, a foot-flag is a clear sign of macho aggression. So researchers wondered if macho hormones—in this case, testosterone—might be involved in the process.

"We know that testosterone is an important regulator of many types of sexual behaviors, so it seems a natural hypothesis that this steroid might also influence waving by affecting the motor systems that control physical movement,” Matthew Fuxjager, Wake Forest University biologist and co-lead-author of the paper, said in a press statement.

To find out, Fuxjager and his colleagues selected 40 male Bornean rock frogs from a rainforest-like terrarium at the Vienna Zoo. The researchers looked for males who showed clear territorial behaviors, including foot flagging.

These males were split into experimental and control groups. Frogs in the experimental group were given a small injection of testosterone in saline solution. Those in the control group just got the saline. As soon as the frogs had gotten their treatment, the researchers paired them up: either two experimental-group frogs or two control-group frogs. Each pair was placed in a mesh container, where they were soon joined by a female frog. The mesh container was then placed in a large habitat with a waterfall, to replicate the environment the frogs find most romantic. To increase the realism, the researchers also played them audio of other frogs croaking.

The frogs were given an hour to get used to their new digs. Then, the researchers switched on a video camera and left it trained on the frog trio for seven hours. After all the frogs had gone through the experiment, a researcher, who didn’t know which frogs were which, watched the tapes and took notes on the frogs’ foot flagging and croaking.

The scientists killed six males, as well as six each of two related, non-foot-flagging species (Rana pipiens and Xenopus laevis). They then used RNA testing to examine the makeup of the animals’ thigh muscles, brains, spinal cords, and larynxes.

The results showed that testosterone did indeed affect S. parvus frogs’ bodies and their aggressive behavior. The thigh muscles of S. parvus specimens contained 10 times more male-hormone receptors than those of the other species. In other words, the Bornean frogs’ legs were ready to be pumped up. And the testosterone definitely pumped them up; the S. parvus males in the experimental group were much more likely than control-group frogs to start waving their legs around.

"These data therefore suggest that the evolution of the foot flag is associated with a dramatic change in how hormones act on specific muscles in the body, particularly those muscles that control the display in the first place," Fuxjager said. "These findings provide novel insight into how the evolutionary gain of a sexual display trait may be augmented by evolution of the hormone systems that control and refine adaptive motor skills."