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Sixty-six million years ago — as the Cretaceous period transitioned into the Paleogene — a massive asteroid hit the earth, and three-quarters of all animal and plant species went extinct in a geologic blink of an eye. In the wake of all the destruction lay evolutionary opportunity: ecologic niches waiting to be filled. In a short period, mammals radiated out from a small, simple family into the diverse and extensive group of animals we’re familiar with today, producing groups like horses, primates and whales.

When Darwin sailed to South America, he uncovered the bones of an ancient and mysterious group of hoofed mammals - South American native ungulates (SANUs). He was perplexed about the animals’ evolutionary history, calling them the “strangest animals ever discovered.”

In a recent study, researchers finally pinned down SANU ancestry and found that SANUs came to be after the Cretaceous-Paleogene mass extinction. SANUs are now completely extinct, with no living relatives to give us clues to their origin. Some believed that they should be grouped with mammals coming from the ancient northern supercontinent, Laurasia, along with horses and cattle. Others thought they belonged with Afrotheria, or mammals hailing from ancient Africa, along with elephants, aardvarks and sea cows.

Because the ancient DNA has degenerated over time, it hasn’t been useful. The only known way to test these historical relationships has been through comparing SANU bones with other species, but those tests have also been inconclusive.

So biologists developed a better method — applying proteomic analysis (study of the proteins) to screen bone samples. Essentially, they examined bone protein from extinct mammals and compared it to molecular data from living mammals to narrow down the SANU family tree. They took collagen protein from bone specimens and compared it to an array of modern mammals, including those from the Afrotheria and Laurasiatheria groups.

The research nearly definitively places two SANU groups — Litopterna and Notoungulata — with Perissodactyls: odd-hoofed mammals like horses, zebras, donkeys, etc, nullifying theories that the group should be part of Afrotheria. But — because they couldn’t test the SANU species that have been extinct for a long time — they can’t make any final calls, essentially the group is “more like” Perissodactyla than it is like any other placental mammals until they have further evidence.

Completely fleshing out the tree of life will take a few more steps in technological advancement and more digging, but nearly solving Darwin’s centuries-old ungulate mystery is a long stride among baby steps.