HARVARD GAZETTE ARCHIVES

The researchers documented natural selection's role in the creation of new species through a process called reinforcement, where butterfly wing colors differ enough to avoid confusion with other species at mating time, helping the butterflies avoid creating less-fit hybrid offspring.

Though more distantly related species tend to be more physically distinct, researchers found this was not the case with species of the blue butterfly Agrodiaetus, found in a broad swath across much of Central Asia and Europe. Researchers found instead that species that might be expected to have the most trouble telling each other apart had the greatest differences in wing color.

That meant that newly diverged species living in the same area that could still mate and have hybrid young had more distinctive wing colors than other closely-related species that had diverged at an earlier time, as well as those living in different areas from each other.

Hessel Professor of Biology Naomi Pierce, a fellow at the Radcliffe Institute for Advanced Study this year, said a critical factor in this research is the fact that the butterflies are still closely related enough that they can - and sometimes do - interbreed. The hybrids created by this interbreeding, however, are less fit than the parents. That makes it advantageous for parents to ensure more offspring will survive by developing distinguishing characteristics, such as male wing color, and thereby avoiding the costly mistake of mating outside their own species.

"The fact that the hybrids are less viable drives the divergence between the parent species," Pierce said. "Wing colors must be one of the first traits the butterflies use to recognize the right mate."

In Agrodiaetus butterflies, females are uniformly brown. The males of many species, however, have blue wings that vary in hue from species to species. Researchers found that males of species that live in geographically separated areas, or that live together but are more distantly related, look more similar to each other. For distantly related ones that live in the same area, wing color is likely no longer needed to keep the species apart. Over time, other mechanisms evolved to block interbreeding, such as habitat specialization, physical differences in the genitalia, or changes in pheromones that are important in the mating process.

The research was conducted by doctoral student Nikolai Kandul, Pierce, Putnam Professor of Organismic and Evolutionary Biology David Haig, Harvard Junior Fellow Joshua Plotkin, and their collaborators at St. Petersburg State University in Russia, Vladimir Lukhtanov and Alexander Dantchenko. It was published in the July 21 issue of the journal Nature.

Natural selection's role in the creation of new species is a controversial topic among biologists. Some biologists believe that natural selection does not play a direct role in the formation of new species. Rather, speciation is seen as simply the byproduct of changes that take place when populations evolve in isolation over time. This can happen when populations are geographically separated by a barrier such as a mountain rising up to isolate populations in valleys on either side.

In these cases, the accumulation of different traits over time in the two populations living in different environments eventually results in different species that, if reunited, will not interbreed.

Pierce and her collaborators found evidence that recently diverged Agrodiaetus butterfly species were likely to have been separated long enough for there to be some differences between them, with wing color among the first traits to change. When they came back together again, these incipient species were still able to mate with each other. However, they had accumulated sufficient changes that, rather than blending back into a single species, their hybrid offspring were less able to survive and reproduce than offspring created when butterflies mated with members of their respective parent populations. This provided natural selection with a wedge to drive between the incipient species, encouraging the final development of distinct species with different male wing colors that would no longer interbreed.

The extreme diversity in the arrangement of the butterflies' genomes is the most likely cause of the hybrids being less fit, Pierce said. The butterflies' DNA, like that of all creatures, arranges itself into structures called chromosomes during cell division. Different species of Agrodiaetus frequently have different numbers of these chromosomes. In fact, Agrodiaetus species have the widest range in chromosome number in the animal kingdom. Researchers believe that differences in the chromosome numbers between newly diverged species may be responsible for making hybrids less fit.

The unusual chromosomal variability of Agrodiaetus, combined with detailed information about the evolutionary history and relative timing of species divergences of this group, is what sets this study apart from previous ones, Pierce said. Kandul made a genetic family tree for the Agrodiaetus genus, which showed how closely related the different species were and provided a better understanding of the relationship between the evolution of wing color and chromosome variability in Agrodiaetus' speciation.

To do this work, samples had to be collected fresh in the field. For each specimen, Lukhtanov first counted the chromosome number and Kandul then sequenced DNA from the same individual.

"The results of our study support the role of natural selection in the origin of new species in the blue butterfly genus Agrodiaetus," said Kandul, now at Duke University. "We showed that reinforcement, or strengthening of characters used in species recognition by natural selection, is the most likely mechanism that has led to speciation in 15 pairs of young Agrodiaetus species."

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