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The classic definition of a biological species is the ability to breed within its group, and the inability to breed outside it. A study published July 29 in the journal offers some important clues about the evolution of barriers to breeding.

The vast majority of the time, mating across species is merely unsuccessful in producing offspring, though there are exceptions. Breeding a horse and a donkey, for example, may result in a live mule offspring, but mules are nearly always sterile due to genomic incompatibility between the two species.

However, when researchers and lead co-authors Janice Ting at the University of Toronto (����TV) and Gavin Woodruff at the University of Maryland mated Caenorhabditis worms of different species, they found further variations on barriers. The lifespan of the female worms and their number of progeny were drastically reduced compared with females that mated with the same species. In addition, as with mules, the females that survived cross-species mating were often sterile, even if they subsequently mated with their own species.

“We observed the mated females under a microscope and by using a fluorescent stain to visualize sperm in live worms, we discovered that the foreign sperm had broken through the sphincter of the worm’s uterus and invaded the ovaries,” said Ting, a PhD candidate in the Department of Ecology & Evolutionary Biology at ����TV. There, the sperm prematurely fertilized the eggs, which were then unable to develop into viable offspring. The sperm eventually destroyed the ovaries, resulting in sterility, and then traveled farther throughout the worm’s body, resulting in tissue damage and death.

“Our findings were quite surprising because females typically just select sperm from males of their own species during fertilization, an action that does not lead to long-term consequences because there is no gene flow between the species,” said Asher Cutter, associate professor of ecology and evolutionary biology at ����TV and co-investigator of the study.

The results suggest the interaction between sperm and the female reproductive tract as a novel reason for failed mating in worms, noted University of Maryland co-investigator and associate professor of biology Eric Haag. “The findings may be worth investigating in other species as well, because similar coordination problems may be relevant to infertility in other organisms,” he added.

(At right, fluorescence microscopy reveals sperm, in red, invading a female worm's body; image by Gavin Woodruff.)

The researchers believe the “killer sperm” may be the result of a divergence in the evolution of worm species’ sexual organs—in particular, the ability of sperm to physically compete with one another. When a female worm mates with multiple males, the sperm jostle each other, competing for access to the eggs. Female worms’ bodies must be able to withstand this competition to survive and produce offspring. The researchers hypothesize that the aggressiveness of the sperm and the ability of the uterus to tolerate the sperm are the same within a single species, but not across multiple species. Thus, a female from a species with less active sperm may not be able to tolerate the aggressive sperm from a different species.

There is evidence for this theory. In the current study, three species of hermaphrodite worms—which produce their own sperm and fertilize their own eggs to reproduce—were especially susceptible to sterility and death when mated with males of other species. The hermaphrodite uterus may have evolved to tolerate “gentler” sperm, but not the larger, more active sperm of non-hermaphrodite species, according to the researchers.

“We found that hermaphrodites can sense, and try to avoid, males of species that can harm them,” added Haag.

This instance of lethal cross-species mating is of special interest to evolutionary biologists, Haag notes, because it’s unclear how the many species on earth—8.7 million, not counting bacteria, according to an estimate published in Nature—remain distinct from each other.

“Punishing cross-species mating by sterility or death would be a powerful evolutionary way to maintain a species barrier,” Haag said.

The research is described in a study titled “Intense Sperm-Mediated Sexual Conflict Promotes Reproductive Isolation in Caenorhabditis Nematodes“, published July 29 in PLOS Biology. It was supported by funding from the Natural Sciences and Engineering Research Council of Canada and the National Institutes of Health.

Sean Bettam is a writer with the Faculty of Arts & Science at the University of Toronto.