To find out, he and his colleagues bred copepods from populations sampled all along the coast. They didn’t just breed copepods from the same population; they also put together males and females of different groups. The first generation of these hybrid offspring — the F1 — appeared normal and healthy when the lab began these experiments in the late 1980s. When Burton then bred the F1 generation with itself, however, problems appeared.

That second generation, the F2, had fewer young and didn’t survive some environmental stresses as well as non-hybrids did. Those results meant that although interbreeding between the geographically separated copepod populations was technically possible, the evolutionary cards were stacked against the long-term survival of hybrid offspring in the wild.

The researchers wanted to know why the second generation did so poorly. For Burton, only mitochondrial problems could possibly explain these difficulties. His previous work had shown that not only did the nuclear genomes of T. californicus vary among populations, so did their mitochondrial genomes. Since proper mitochondrial functioning required the interaction of proteins made by both genomes, Burton hypothesized that a mismatch between mitochondrial and nuclear DNA sat at the heart of the F2’s problems.

“The people thinking about mitochondrial function were not evolutionary biologists, and evolutionary biologists weren’t thinking about mitochondria, so no one was really putting these two ideas together,” Burton said. His copepods and his guess revealed how the forces of natural selection could act on one of life’s central processes.

Evolution by natural selection hinges on the mutability of the genome. If DNA is writ in stone, natural selection has no variation on which to act. Not long after the discovery of the mitochondrial genome in the 1960s, scientists hypothesized that the genes encoded by this DNA were so central to cellular function that they had to resist further shaping by natural selection. The forces of nature had no room to experiment. Or so the theory went.

“I always thought this was a bad idea,” Burton admitted. Instead, evidence is emerging that mitochondrial DNA is far more mutable than researchers thought. Because mitochondrial DNA lacks capabilities for checking DNA for errors and repairing it, in animals it mutates on average 10 times as frequently as its nuclear counterpart does. (The difference varies considerably: In copepods, the mitochondrial DNA mutates 50 times as frequently.) That mutability doesn’t mean anything goes. The conservative evolutionary forces acting on mitochondria are so strong that the wrong changes to their DNA sequence can create problems. Witness the severity of mitochondrial disease, caused by defects in mitochondria, which in humans can cause seizure, stroke, developmental delays or even death.

To evolutionary biologists, this high mutation rate posed an interesting question: How does the nuclear genome respond to this mitochondrial variability and its sabotage of their partnership? Moreover, an organism inherits its mitochondrial DNA only from its mother, instead of from both parents like its nuclear genome. This different pattern of inheritance gives mitochondrial genes a different evolutionary agenda than nuclear DNA does.

“What’s good for one genome might not be good for the other,” said Elina Immonen, an evolutionary geneticist and researcher at Uppsala University. “Males and females also might have different evolutionary interests.”

The mismatch of evolutionary forces on mitochondrial and nuclear genomes could be seen in Burton’s F2 copepods. He extracted mitochondria from their cells and measured their mitochondria’s energy output in the form of ATP. The F2 hybrids produced significantly less ATP than their nonhybrid counterparts did, a clear indication of mitochondrial dysfunction.

Confirmation of the mitonuclear conflict occurred when the researchers bred F2 males with females from the original maternal populations. This “backcross” again paired the right nuclear genes with their historically right mitochondrial genes, and it rescued the resulting F3 generation: Those offspring did not suffer the shortened lives and reduced fertility of their F2 fathers. (Because mitochondria are inherited only from the mother, paternal backcrosses had no beneficial effect.)

These experiments established some of the first evidence for the importance of mitonuclear conflict in wild animals. Other work in the fruit fly Drosophila melanogaster revealed another aspect to mitonuclear conflict. Jonci Wolff at Monash University in Australia and colleagues irradiated male flies to generate large numbers of DNA mutations, and then mated these flies with females that had identical nuclear genomes but one of six different mitochondrial genomes. As the researchers described in a paper published in April on bioRxiv, the percentage of each female’s eggs that hatched varied by which mitochondrial genome she carried.

That result showed that the mitochondrial genome normally plays a major role in the DNA repair pathway, but also that mutations in the mitochondrial DNA can affect how well it interacts with the nuclear DNA. “There’s a huge contrast between the small size of its genome and how important the mitochondrion is,” Wolff said.

Neither of these studies was sufficient to show that this force could divide a group of organisms into two separate species. That evidence lay along the eastern coast of Australia.

A Mitonuclear Wedge Between Populations

When the day’s first rays of sun hit Australia after their long journey over the endless blue Pacific, the silvery peals of the Eastern Yellow Robin greet them with enthusiasm. As the American robin is in the United States, the Eastern Yellow is a common backyard bird from Melbourne to Brisbane, its bright yellow belly providing a flash of color against a blue-gray head and back. Around two million years ago, the common backyard bird began splitting into a southern group that lives in the more temperate climes of Victoria and New South Wales, and a northern group that lives in more tropical Queensland. The sheer size of their territory keeps most of the northern and southern robins separate.