A new study reveals that behavioral isolation between populations of Bahamas mosquitofish is driven primarily by the actions of females – not just through mating choice behaviors, but also through sometimes violent resistance. The work demonstrates the impact of ecological environment on behavior and subsequently on speciation.

Bahamas mosquitofish are small, live-bearing fish that inhabit the blue holes of Andros Island, The Bahamas. The populations can be roughly separated into lineages that developed in two distinct ecological environments: either a high-predation regime where they must avoid predators, or a low-predation regime where predators are absent. The mosquitofish populations diverged between 5,000 and 15,000 years ago, which makes the Bahamas mosquitofish an important model for the study of evolutionary processes.

“These blue holes are sometimes separated by only hundreds of meters, and there’s marshes between them that would allow the fish to move and potentially interbreed, but there’s little to no evidence of gene flow,” says Brian Langerhans, professor of biology at North Carolina State University and co-author of the study.

The researchers looked at eight separate mosquitofish populations living in distinct blue holes – four from high-predation regimes and four from low-predation regimes. To test for differences in native mating behaviors between predation regimes and the role of mating behaviors in causing reproductive isolation between populations, they conducted no-choice mating trials both within and between each population.

“Mating interactions can play an important role in the origin of new species, and the Bahamas mosquitofish provide an exceptional chance to test how strong selective pressures can complicate the breeding between different populations,” says Varpu Pärssinen, first author of the study who conducted the research as a part of her Ph.D. work at Lund University. “We wanted to see how the adaptation to different environments has shaped the mating behaviors and interactions in this species, and how that may lead to different levels of mating success between populations.”

The results were striking.

“When we looked at mating interactions within populations, we found that after three mating attempts they would achieve a 90% chance of successful mating; that is, they would achieve the appropriate genital contact that permits sperm transfer,” Langerhans says.

But when mating interactions occurred between fish from different populations, the researchers saw the number of mating attempts increase and successful mating decrease.

For different populations that evolved within the same predation regime, it took more than six attempts to achieve a 90% chance of successful mating, and for different populations from different regimes, it took over 10 attempts. And female aggression was a deciding factor. Females did not merely ignore the males – they often attacked them. More aggression meant reduced fertilization success.

“Under normal circumstances, males with more mating attempts have more success,” Langerhans says. “But that was not the case when they were placed with a foreign female, especially if she was derived from the opposite ecological regime – the attempt frequency was higher, but so was the failure rate. This means speciation is evolving more rapidly between different ecological environments than between similar ones.

“We know that female choice drives the evolutionary process – but this system reveals the roles of both mating preference and mating resistance in the speciation process,” Langerhans continues. “Divergent ecologies can result in populations with divergent traits, and females can drive reproductive isolation between these populations by both preferring native males and aggressively resisting foreign males.”

“Female aggression is rarely studied as a response to male mating attempts, so more studies are needed to determine how commonly it can play a role in speciation events,” Pärssinen says.

The work appears in BMC Ecology and Evolution and was supported by the Swedish Research Council (grant 2016-05095), Helge Ax:son Johnsons foundation (grant F18-0535) and the Royal Physiographic Society of Lund. Kaj Hulthén, former NC State Ph.D. student currently at Lund University, and P. Anders Nilsson, professor of aquatic ecology at Lund University, also contributed to the work.

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