FAYETTEVILLE, ARKANSAS—Although the blind cave salamander Proteus anguinus is one of the national animals of Slovenia, it’s so shy that there have been only about 300 sightings in 300 years. Now, molecular biologists have learned how to keep track of these elusive animals without having to see them: by using a new probe that detects their DNA in the springs in which they swim. Already the probe—described here last week at the 2016 International Conference on Subterranean Biology—has detected Proteus in places it’s never been known to go. The approach has also provided tantalizing evidence that a rare black subspecies of the typically white creature might actually be a bona fide species of its own.

The work opens up new possibilities for the salamander’s conservation and also for using so-called environmental DNA (eDNA) to monitor animals that live where humans just can’t go. “It has fantastic utility because so many aquatic cave habitats are unavailable to us,” says Rick Olson, an ecologist at Mammoth Cave National Park in Kentucky who was not involved with the work.

Cave animals are among the most bizarre and understudied creatures on this planet. And Proteus tops the list, as the world’s biggest cave animal and Europe’s only cave vertebrate. It lives in underground aquifers in Slovenia, surfacing only when floodwaters sweep it from its lair. At 30 centimeters, Proteus is a giant among salamanders, and—like most cave creatures—it has lost its eyes and its color. Lab-raised specimens show that the amphibian can live for more than a century. It becomes sexually mature about the same time as humans (age 14), but it can reproduce only once every 7 years. And it can go years without eating a thing and survive just fine. Four hundred years ago, locals thought the salamanders were baby dragons, with mythical protective powers.

But little has been done to ensure the animals’ survival, despite its status as a European “priority” species—one that deserves the government’s protection. The rare, black Proteus may be even more endangered than the white Proteus, the more common form of the species. Since it was first discovered in 1986, it has only been sighted at four springs in southeastern Slovenia.

Frustrated that biologists don’t know the first thing about how big or widespread the salamander populations are, Špela Goricki, a molecular biologist at the Tular Cave Laboratory in Kranj, Slovenia, decided to borrow a forensic technique more commonly used in law enforcement: eDNA. The method, which detects DNA from skin cells, hair, and other cells released into the environment, has already been used to track surface organisms like invasive fish and snakes. But it had never before been used to track cave creatures.

By analyzing Proteus DNA from previously collected specimens, Goric​ki designed genetic probes, short stretches of genetic material that differentially link to its DNA but bypass that of other organisms. Further, the team has designed the probes so they can differentiate white Proteus DNA from black. Goric​ki and her colleagues then systematically surveyed dozens of springs and caves in Slovenia known or suspected to have Proteus residents. They also checked underground water in nearby Montenegro and Herzegovina.

Black Proteus DNA showed up in five new places all within a few kilometers of each other. The team also found evidence for white Proteus in new spots, including Herzegovina and Montenegro. What’s more, the team found the first evidence that these two groups might sometimes live side by side, suggesting that they are two separate species. If they were a single species, such side-by-side living would lead to interbreeding. But that doesn’t seem to have happened, Goric​ki says.

But the wider range doesn’t mean that the salamanders are any safer than they were. More agriculture in the area means more nitrogen and phosphorous in the aquifers where they live, which could be toxic to the animals, Goric​ki says. “I hope the conservation authorities will fulfill their promise” to develop more effective ways to protect this species, she adds.

Even if that plan is slow in coming, Goric​ki thinks her success will pave the way for other eDNA monitoring programs. “In 10 years, this will be the method of choice for rare and endangered species, as well as invasive species,” she predicts. Olson agrees. At Mammoth Cave National Park, researchers have already begun to use eDNA to keep track of the endangered Kentucky cave shrimp, Palaemonias ganteri. “Environmental DNA gives us a way of not only knowing if it’s present, but also the concentration of DNA can give an idea of relative abundance,” Olson says. And that will go a long way in helping conserve these species.