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The octopus just revealed another one of its secrets: what determines its sex. University of Oregon researchers have identified a sex chromosome in the California two-spot octopus. This chromosome has likely been around for 480 million years, since before octopuses split apart from the nautilus on the evolutionary tree. That makes it one of the oldest known animal sex chromosomes.

The finding also is evidence that octopuses and other cephalopods, a class of sea animals that includes squid and nautiluses, do use chromosomes to determine their sex, answering a longstanding mystery among biologists.

"Cephalopods are already such interesting creatures, and there are so many things we're still learning about them, especially in neuroscience," said Gabby Coffing, a doctoral student at the UO working in the lab of biologist Andrew Kern. "This is just showing one more interesting thing about them: They have really ancient sex chromosomes."

Coffing, Kern and their team Feb. 3 in the journal Current Biology.

In humans and most mammals, sex is determined largely by chromosomes. But "there's a tremendous amount of diversity" in how animals determine their sex, Kern said. So scientists couldn't assume the same was true for octopuses.

In turtles, for instance, sex is determined by the temperature at which the eggs are incubated. Some fish have a gene that determines sex, but not a whole chromosome. Even in humans, the X/Y sex chromosome system isn't as clear-cut as it might look on paper; gene mutations or inheriting extra sex chromosomes can lead to development that doesn't neatly fit in a male/female binary.

Plus, because cephalopods aren't standard lab animals, like mice or fruit flies, they haven't been subject to nearly as much genetic exploration. Scientists have sequenced the genomes of a handful of octopus species, but they can't link genes to specific traits the way they can in mice or even humans.

When UO researchers recently sequenced the DNA of a female California two-spot octopus, they found something unexpected: a chromosome with only half the amount of genetic material. It looked different from all the others, and it hadn't been found in male octopuses whose DNA was previously sequenced.

"This particular chromosome had half the amount of sequencing data, which indicated there was only one copy," said Coffing. "Then as we explored that more, we reached the conclusion that we must have stumbled upon a sex chromosome."

To confirm, the researchers sorted through other octopus genomic data previously collected by other researchers. Not all that data was clearly labeled as being from male or female octopuses.

But they found another example of the half-sized chromosome in another species of octopus. They also found it in squid, which diverged evolutionarily from octopuses somewhere between 248 and 455 million years ago. And after more digging, they also found evidence for the chromosome in the nautilus, a mollusk that split apart from the octopus approximately 480 million years ago.

The fact that these species share this unique chromosome suggests that it's been around in some form for a very long time.

"This indicates that their common ancestor had this similar sex determination system," Coffing said.

That's somewhat unusual for sex chromosomes, Kern said. Because they directly impact reproductive capabilities, they're subject to a lot of selective pressure and so tend to undergo rapid evolutionary change. But cephalopods seem to have found what works and have stuck with it.

Other ancient sex chromosomes have been discovered in plant groups like mosses and liverworts, which were some of the first plants to evolve. And insect sex chromosomes might be 450 million years old, but they've also changed a lot over time.

Kern and his colleagues initially thought octopuses might have a sex determination system similar to birds and butterflies, where males are ZZ and females are ZW. (Biologists have given sex determination systems where males have two copies of the same sex chromosome using different letters, to avoid confusion with the XX/XY system where females have two copies of the same chromosome.)

But the team hasn't yet found a W chromosome in an octopus. Alternatively, octopuses could use a sex determination system that only involves the Z chromosome—males have a pair, and females just have one. That's still to be determined, Coffing said. For now, the octopus keeps some of its secrets.