How do two species come into existence? This is a tricky question for biologists. It is generally agreed that the process of speciation occurs in most cases when individuals from a single population become geographically isolated. If they remain separated long enough, they lose the ability to reproduce.

A new study appeared in the journal Proceedings of the Royal Society B: Biological Sciences shows what happens when a less common form of speciation occurs. Instead of being separated by a physical barrier such as a mountain range or a sea, members of a species can separate from each other over time.

The researchers focused on two closely related moth species with overlapping distributions in the southeastern United States.

“These two are very similar,” said lead author Yash Sondhi, who conducted research for the study while working at Florida International University and later at the Florida Museum of Natural History. “They have differentiated along this one axis, which is when they fly.”

Rosy maple moths of the genus Dryocampa look like something Roald Dahl would have drawn from a fever dream. They have a thick lion’s mane over their heads and abdomens and their glowing scales are the color of strawberry and banana candy. Both male and female rosy maple moths fly exclusively at night.

Pink-striped oak moths of the genus Anisota are less conspicuous and have subtle ochre, umber and marl tones. While the females of this species are active at dusk and in the early evening, the males prefer to fly during the day.

Sondhi knew from previous research that these two groups, Dryocampa and Anisota, evolved from a single species about 3.8 million years ago, which is relatively recent on an evolutionary timescale. There are a handful of species in the genus Anisota, all of which are active during the day. The nocturnal maple moths are the only species in the genus Dryocampa.

Sondhi specializes in the biology of insect vision and saw the moth pair as the perfect opportunity to study how vision evolves when a species changes its activity pattern.

But things didn’t go as planned.

“I looked for differences in color vision. Instead, we found differences in their clock genes, which makes sense in hindsight,” Sondhi said.

Clock genes control the circadian rhythm of plants and animals. The rise and fall of the proteins they produce causes cells to become either active or inactive over a period of about 24 hours. They affect everything from metabolism and cell growth to blood pressure and body temperature.

Any organism that reverses its activity pattern almost certainly involves clock genes. “It’s a system that has been conserved in all living things from fruit flies to mammals and plants. They all have some kind of timekeeping mechanism,” he said.

Sondhi compared the transcriptomes of the two moths. Unlike genomes, which contain all of an organism’s DNA, transcriptomes contain only the subset of genetic material that is actively used to make proteins. This makes them useful for studying differences in protein levels throughout the day.

As expected, Sondhi found a number of genes that were expressed at different levels in the two moth species. Nocturnal maple moths invested more energy in their sense of smell, while the diurnal oakworm moth produced more genes related to vision.

However, there were no differences in the genes that confer the ability to see color. This does not necessarily mean that their color vision is identical, but if there are differences, they are likely to be at the level of tuning and sensitivity rather than in the structure of the genes themselves.

There was another gene that stood out. Disconnected, or Disco, was expressed in both species to varying degrees during the day and at night. In fruit flies, Disco is known to indirectly affect circadian rhythms by producing neurons that transmit timing enzymes from the brain to the body.

The disco gene that Sondhi found in his moth samples was twice as large as its fruit fly counterpart and had extra zinc fingers – active parts of a gene that interacts directly with DNA, RNA and proteins. It seemed likely that changes in the disco gene were at least partly responsible for the switch to nocturnal flight in maple leaf moths.

When he compared the disco gene of the maple moth with that of the oak moth, he found 23 mutations that distinguished the two species. The mutations were also in active parts of the gene, meaning they likely contributed to noticeable physical differences between the moths. Sondhi was watching evolution in action.

“If this is confirmed functionally, this is a really concrete example of the mechanism behind speciation at the molecular level that is rarely found,” he said.

The study also provides an important impetus for a better understanding of the different ways in which life is maintained and reproduced. When genetics first came to the fore as a field of study, researchers focused mainly on a few representative species, such as fruit flies or laboratory mice. This was done primarily for convenience, but it limits our knowledge of general biological patterns. Just as a human is not a laboratory mouse, a moth is not a fruit fly.

“As biodiversity continues to decline due to climate change and other anthropogenic changes, we need to genetically engineer a larger number of the remaining species to, for example, tolerate drought or be active in light-polluted regimes. To achieve this consistently, a broader pool of functionally characterized genes from all organisms is crucial. We cannot use only Drosophila,” said Sondhi.