The piercing golden gaze of cheetahs, the striking blue stare of snow leopards, and the luminous green glare of leopards are traits that can all be traced to one ancestor; an ocelot-like feline progenitor that roamed the Earth over 30 million years ago.

In a new study published in iScience, Harvard researchers have discovered that this ancestral population likely had both brown and gray-eyed individuals, the latter being key to paving the way for the rapid and wide diversification of iris color seen in modern felids.

“When I started this study I asked, 'What do we know about eye color?' And the truth is very little as there is basically almost no phylogenetic evolutionary studies on eye color,” said lead author Julius Tabin, a Griffin Graduate School of Arts and Sciences student in the Department of Organismic and Evolutionary Biology at Harvard.

Most studies focus on the distribution of eye colors in a species, or on the genes involved in making eye color in humans and domesticated animals. Studies on eye color in animal populations are also rare due to the challenges of preservation and lack of eye color diversity – most animals have brown eyes.

While eye color in humans is considered likely a result of sexual selection, and in domesticated animals a result of artificial selection, Tabin wondered what spurred the wide diversity in wild Felidae. Without fossil preservation to rely on, Tabin decided on a novel approach involving digital images from reputable sources (like iNaturalist) and applying image analysis to identify and categorize the varied eye colors in 52 felid taxa.

Tabin and co-author Katherine Chiasson, a Ph.D. candidate at Johns Hopkins University, created an algorithm using open-source color definers with accepted color definitions to map iris colors onto a phylogenetic tree of the Felidae.

“We found a lot of variability of color between species,” said Tabin, “but shockingly, we also found a lot of intraspecific variability. Most species have a singular eye color with no variation. So, it’s really surprising that once you get into the cats – lions, tigers, panthers, etc. – we see all these different eye colors. There are actually very few felidae species that have only a singular eye color in their population.”

Equipped with the colors mapped onto the phylogenetic tree, the researchers set out to reconstruct the ancestral state. They found that early, pre-felid lineages (the ancestor of felids and their closest relatives, the linsangs) had brown eyes only. However, after the linsang species branched off, the population of the felid ancestor gained gray-eyed individuals in its population, which coexisted with the brown-eyed ones.

“It’s likely this happened due to a genetic mutation that drastically decreased the pigment in the eye,” Tabin said. Melanin can be either eumelanin, which is brown, or pheomelanin, which is yellow. To go from a brown eye to a gray eye would require a decrease of eumelanin in the eye. That decrease would lead to an eye that is not fully brown and not fully gray, but a brownish gray color, which is what the researchers found.

Those gray-eyed individuals opened the door to a burst of greens, yellows, and blues, providing an anchor between brown eyes and the new colors.

“Blue eyes require carefully balanced low levels of pigment and are likely recessive in felids. A wild population would probably not be able to maintain blue eyes in a population with only one blue-eyed individual among a sea of brown eyes. It’s probable that you would need something lighter than brown, but not as light as blue, to be the mediator. And that’s what you see: In every single cat species with blue eyes, they also have gray eyes,” said Tabin.

Tabin and Chiasson also observed trade-offs along with varied eye color. For instance, brown eyes and yellow eyes rarely coexist in a species. Surprisingly, they found a positive correlation between yellow eyes and round pupils, and a negative correlation between brown eyes and round pupils.

The researchers found no significant correlations for activity mode, zoogeographical region, habitat and uniformity of eye color, which leaves the specific adaptive benefit of having varied eye colors an open question to pursue.

Excitingly, the researchers not only reconstructed the general eye color types present at each evolutionary node, but they were also able to predict the exact color of each ancestor’s eye.

“Being able to reconstruct color quantitatively is one of the paper’s greatest strengths, because it means we are the first animals to see the color of these eyes since these felids were alive millions of years ago,” Tabin said.

“What I find most exciting about this study is that all of the resources we used are freely available online,” said Chiasson. “The fact that rigorous studies like ours can be done by anyone with an internet connection and some curiosity is indicative of a field-wide revolution that is increasing the accessibility of science around the world.”

“The study opens up the opportunity for more investigation of the evolutionary importance of gray eyes, as well as eye color evolution in natural populations," Tabin said. “I’m still riding high on the excitement of knowing that the felid ancestor had both brown and gray eyes, because that’s something I didn’t go in expecting or even thinking about.”