Research uncovers new information about the biological processes that help ensure that two fly species don't interbreed.

Pour some cold cream into a cup of hot, black coffee, and you end up with a drink that’s midway between the two ingredients in color, temperature, and flavor. A similar kind of blending can occur if members of closely related species frequently mate with each other, but many species have mechanisms to prevent such mixing. Now, Howard Hughes Medical Institute scientists have discovered one of the few genes that ensures that two species don’t interbreed.

HHMI investigators Harmit Malik of the Fred Hutchinson Cancer Research Center and Jay Shendure of the University of Washington and their colleagues devised a new mutagenesis-based approach to investigate the molecular genetic basis of speciation. They used this approach to identify a gene that kills male offspring that result from mating between two fruit fly species. “It’s a pretty big step in our understanding of what is the biological process that leads to incompatibility” between different species, says Malik. He and his colleagues reported their findings on December 18, 2015, in the journal Science.

More than 150 years ago, Charles Darwin noted that interbreeding between species could blur their differences. “Species within the same country could hardly have kept distinct had they been capable of crossing freely,” he wrote in On the Origin of Species. A variety of obstacles can prevent species from intermixing, producing what scientists term reproductive isolation. The species may reproduce at different times of the year, for example, or their progeny may die or be sterile, as is the case for mules, the offspring of mating between horses and donkeys. Although researchers have been searching for genes that keep species separate, Malik notes that so far, “there are very few cases in which we can say, ‘this gene is mediating reproductive isolation.’”

Two of the genes scientists have uncovered enable the fruit fly Drosophila melanogaster to remain reproductively isolated from the similar fly species D. simulans. Both species live throughout much of the world, giving them plenty of opportunities to interbreed in the wild. Yet when a female D. melanogaster and a male D. simulans mate, only their female offspring live; all of the males die shortly after hatching.

Researchers aren’t sure why the female offspring don’t perish, but they’ve shown that the genes Hmr and Lhr are partly responsible for the deaths of the hybrid males.  Evidence suggested that a third gene was also involved, but nobody had identified it.

Nitin Phadnis, who was a postdoctoral researcher in Malik’s lab and is now on the faculty at the University of Utah, designed an ingenious experiment to ferret out this elusive gene. The team fed male D. simulans flies a chemical that triggers mutations. The researchers hoped that in some flies these alterations would occur in the gene they were searching for and disable it. Although these flies would be extremely rare, the scientists would be able to recognize them because their sons would survive. After nearly two years of crossbreeding flies and sorting through more than 330,000 of their progeny, Malik and colleagues tallied only six living male offspring.

But that was enough to pinpoint the gene. When the scientists sequenced the genomes of these flies, they discovered that all six carried mutations in gfzf, a gene that helps orchestrate cell division. To demonstrate that gfzf was the third killer gene, the researchers engineered female D. melanogaster flies to produce a type of RNA molecule that shuts down the D. simulans version of gfzf. The team then bred the engineered females with male D. simulans flies. Some sons from these crosses inherited their mothers’ ability to shut down the D. simulans version of gfzf. These lucky males didn’t die young, confirming the team’s identification of gfzf.

Malik and colleagues determined that gfzf kills male offspring when they are starting to transform from larvae into adults. Only a small fraction of the cells in a larva produce all of the cells in an adult fly, and these larval cells need to divide rapidly. However, gfzf curtails cell reproduction in hybrid male offspring, preventing them from generating the new cells required to form an adult body.

“It’s so difficult to identify these genes” that produce reproductive isolation, says Malik. “I’m hopeful that the strategy we identify in this paper will give us a bounty of genes in the future.” Malik adds that there’s suggestive evidence that a fourth gene helps the two fly species remain reproductively isolated, and he and his colleagues have begun looking for it.

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