The sixth samurai: Finding a gene that divides one species from another

How six male flies — among more than 300,000 females — led to the discovery of an evolution-driving gene
To find an elusive gene responsible for dividing two species of fruit flies — a gene that helps make each species unique from each other — the research team spent nearly two years mating about 55,000 mother and mutant father pairs, sifting through 330,000 daughter flies before finding six precious sons in their midst, which they dubbed "the samurai." Illustration by Jim Woolace / Fred Hutch News Service

When Drs. Nitin Phadnis and Harmit Malik met four and a half years ago to brainstorm an experiment that could solve a century-old evolutionary riddle, they first made some quick back-of-the-envelope calculations.

The evolutionary biologists were looking for a gene responsible for dividing two species of fruit flies, a gene that helps make each species unique and separate from each other. The existence of this mysterious gene had been guessed at since 1940, following experiments decades earlier in which geneticists first noticed that the two types of flies, when mated, had only daughters — and no sons.

The researchers’ findings, published online Thursday in the journal Science, lay out their solution to this mystery. But it was a long road getting there, the researchers said.

At that first meeting, Phadnis, then a postdoctoral fellow in Malik’s Fred Hutchinson Cancer Research Center lab who now leads his own research team at the University of Utah, sketched out the experiment’s parameters. The researchers planned to mutate one of the fly species in the hopes of randomly disrupting the mystery gene and thus allowing sons to be born.

Because they knew the flies would have many other mutations sprinkled throughout their genome, they calculated they’d need to find seven rare male flies to conclusively pinpoint the mystery gene’s identity. From that point, the scientists jokingly referred to the elusive sons as the Seven Samurai, after the classic Akira Kurosawa film.

Phadnis estimated the Seven Samurai experiment would take him and former Fred Hutch research technician EmilyClare Baker about six grueling months of mutating, mating and examining tiny insects — weekends included.

Six months later, they’d found zero samurai flies. But they pressed on.

About a year after that, give or take — “it’s somewhat of a blur because of the time-stretching properties of science,” Phadnis quipped — the team had mated about 55,000 mother and mutant father pairs, sifted through 330,000 daughter flies and found six precious sons in their midst.

“We were joking at that point that the six [insect] males we had in the freezer were more valuable than Nitin’s and my cars, given the amount of effort and time that had gone into it,” Malik said.

They never did find their seventh samurai, but it turned out they didn’t need it. In collaboration with geneticists Drs. Jacob Kitzman, of the University of Michigan, and Jay Shendure, of the University of Washington, Phadnis found that all six males had mutations in the same, single gene, meaning they’d found what they were looking for.

“We got really lucky,” Malik said.

From left to right: EmilyClare Baker, former research technician in the Malik Lab and now a graduate student at the University of Wisconsin, Madison; Dr. Nitin Phadnis, former postdoctoral fellow in the Malik Lab and now at the University of Utah; and Dr. Jacob Kitzman, former graduate student in Dr. Jay Shendure's lab at the University of Washington and now at the University of Michigan. Photo courtesy of Dr. Harmit Malik

‘Traditional genetics can’t solve this problem’

Known by the garbled-looking acronym gfzf, the gene is normally important for regulating the cell cycle, where each cell divides into two. For these particular, closely related fly species, formally named Drosophila melanogaster and Drosophila simulans, it appears to also play a role in speciation — how one species evolves into two.

The problem of how D. simulans and D. melanogaster evolved into distinct species has vexed biologists for more than 100 years. But it’s only with the advent of modern molecular tools that finding this gene was even possible, Malik said.

The researchers also found that reducing levels of the gfzf gene allowed the two fly species to produce tens to hundreds of sons, meaning the gene’s presence was toxic to the survival of hybrid males.

“There was no way to use a traditional genetics approach. We needed a totally new genomics-based approach to understand this,” he said. “At the heart it’s a genetics problem, but traditional genetics can’t solve this problem.”

And now that they’ve figured out the solution to this single speciation event, Malik and Phadnis think their technique could be used to solve many other riddles of how new species arise — as long as those species are small enough to study in the tens or hundreds of thousands in the lab, Malik said.

Their approach required scanning the entire genome of every male — and its parents — the researchers found in their two years of painstaking fly-sifting, using a modern technique known as next-generation sequencing.

The time and technology the researchers had to invest in this project helps explain why so few speciation genes — across all animal species — have been found. Gfzf joins a list of fewer than 10 such genes.

“One would think the genetics of speciation would be figured out given how long people have been trying to study this,” Phadnis said. “But it turns out that finding these genes is incredibly hard.”

Dr. Harmit Malik
Dr. Harmit Malik Fred Hutch file

The third component

Two of those known speciation genes are also part of the solution to the same problem. Since D. simulans and D. melanogaster were first found to be separate species nearly 100 years ago, naturally occurring mutants have been discovered that allow interbreeding between those species. Those natural mutants led to the discovery of two genes — other than gfzf — involved in the speciation event, one from each insect species.

If those two genes, dubbed Hmr and Lhr, were the whole story, then engineering a male fly from each species with the other version of the gene should have killed them. But previous research had found that a D. melanogaster male engineered to carry the D. simulans Lhr gene could still live — so scientists knew there must be a third gene involved, another D. simulans gene.

The researchers believe the three genes are acting together to form a “multi-component toxin,” Malik said. “It’s important to point out that this is not the point of these genes. … It’s only when you bring them together into the same genome that you unleash their toxic effect.”

It’s not clear yet how or why gfzf’s role in the cell cycle led to the division of species, Phadnis said. That’s one of the problems his newly formed laboratory team hopes to tackle.

But for the researchers, finding the third piece in a century-old puzzle was incredibly satisfying.

“There’s a rich history here of who’s who in the history of genetic studies — all the big heroes of mine since becoming a young geneticist,” Phadnis said. “Solving a problem that’s been going on since 1910, getting a grip on that is really special.”

The speciation study was funded in part by a grant from the Mathers Foundation

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Rachel Tompa is a former staff writer at Fred Hutchinson Cancer Center. She has a Ph.D. in molecular biology from the University of California, San Francisco and a certificate in science writing from the University of California, Santa Cruz. Follow her on Twitter @Rachel_Tompa.

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