All aflutter

Marcus Kronforst finds clues to evolutionary adaptation in butterfly wings.

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Beneath the scales that form the color palette on their wings, butterflies have a clear membrane that is as functional, if not as beautiful, as their flamboyant pigmentation. “Kids learn that if you touch a butterfly, you get the scales rubbed off of them and they can’t fly,” says evolutionary biologist Marcus Kronforst, “but it’s not true, they fly fine.” Some species have even evolved without scales, he adds, the ultimate in fluttering camouflage: “Stealth butterflies.”

Kronforst knows about durability and adaptability. In a campus greenhouse, he studies the genetics of color patterns in two tropical butterfly species as a window into evolutionary variation and adaptation throughout nature. He and his research team raise the butterflies in mesh tents about the size of an office cubicle. They grab them from the air—the scales leave a powdery residue on their fingers—and see them thrive even as their wings fray from flying in the tight confines. The insects are not as fragile as their handle-with-care reputation suggests. “Butterflies,” Kronforst says, “are really robust.”

Their genetic heritage helps make them that way. Some vulnerable butterflies have evolved to mimic the coloring of related species that are toxic to predators. The seven researchers in Kronforst’s lab decode those protective adaptations. As color patterns change, butterflies develop different mate preferences, leading to more than just color variation. “Adaptation is actually causing the origin of a new species,” Kronforst says, referring to a discovery he helped detail in a 2009 Science paper. “We’re trying to tackle these big questions of how organisms adapt and diverge.”


Marcus Kronforst.

In a breakthrough published last year in Nature, an international consortium, to which Kronforst contributed, sequenced the genome of a species within the Heliconius genus. To their surprise, the researchers found that the Heliconius melpomene butterfly has identical color-patterning DNA as two other species. They believe hybridization—interbreeding among the species—accounts for the phenomenon. “Evolutionary biologists often wonder whether different species use the same genes to generate similar traits,” Kronforst told the Harvard Gazette in 2012. “This study shows us that sometimes different species not only use the same genes, but the exact same stretches of DNA, which they pass around by hybridization.”

With that knowledge, researchers can map the genomes of butterflies with different color patterns to identify the relevant genes: spikes in the data suggest the genetic source for the array on the wings. “If we hadn’t done all that fine detail work over the last years we wouldn’t know what those peaks were telling us about,” Kronforst says. “But now that we know that’s where the color-patterning genes are, we see these peaks that just jump out.”

Building on those advances and using rapidly evolving techniques, his lab continues moving toward the ultimate goal: to metaphorically rub off the scales and uncover the genetic blueprint underneath.


In the lab and in a greenhouse lined with mesh tents where the butterflies are kept, Kronforst and his research team navigate a maze of genetic information to better understand how species adapt and diverge.

 

Kronforst wanted to study bats. As an undergrad at the University of Miami, he asked to accompany a professor who spent summers researching a species that pollinates cacti in the Sonoran Desert. The group would be wrapping up its current work, the professor said, so it wouldn’t be a good time to begin the research Kronforst proposed.

Then the professor mentioned that he had recently noticed a butterfly in his backyard. South Florida has a Heliconius population common in South and Central America that presented an interesting research opportunity.

“Maybe you’d like to do a project on butterflies.”

Butterflies? “I remember I was really disappointed, because I thought bats sounded really cool and manly and this guy wants me to work on butterflies,” Kronforst says. “That sounds really kind of lame.”

Then he started reading the literature, learning about the vast color-pattern diversity and its evolutionary implications. He was captivated. His interest led him, like a butterfly to a passionflower vine, to graduate school with a leader in the field, L. E. Gilbert at the University of Texas. Kronforst completed his PhD in 2004 and continued his research in Austin and at Rice University until 2007, when he received a Harvard fellowship. Last year he joined the University’s department of ecology and evolution as a Neubauer Family assistant professor, creating a tropical hothouse high above the Donnelley Biological Sciences Learning Center.

Two rooms—one 1,800 square feet, the other 500—maintain tropical heat and humidity levels that simulate the butterflies’ habitat. The adult insects, along with the eggs and larvae, must be contained in the mesh cages to prevent escape, among the USDA requirements for housing the nonnative species.

Under those conditions, Kronforst’s lab orchestrates a perpetual cycle of butterfly life. “Most of the operation is plants,” he says, referring to citrus trees where females lay eggs that hatch into caterpillars before forming chrysalises that yield the next generation of research subjects. His enthusiasm overflows in a flurry of words—a fascination with his insect subjects that he often finds other people share. Butterflies, in and of themselves, Kronforst allows—and agrees—are “charismatic.”


The wealth of research potential inherent in the diverse wing patterns of closely related butterfly species fascinated Kronfrost, who had been interested in bats as an undergrad. (Photo courtesy Kronforst Lab at the University of Chicago)

More than their color entrances him, though; he’s drawn to the knotty genetic knowledge they could help untangle. “Our work is aimed at the evolutionary processes of adaptation and speciation,” he says, “and in particular, how these two things interact.”

As the discovery of the emerging new species showed, Kronforst’s research offers an almost real-time view of those processes. “By studying these butterflies we’re able to capture evolutionary events in progress,” he adds, “and that’s something that can be hard to find in nature.”

Moving around the greenhouse, Kronforst flits from thought to thought, each turn reminding him of another facet of the research. Edging through narrow corridors between the cages, he stops occasionally to zip one open, step inside, and observe more closely.

He spots two Heliconius cydno butterflies, black winged, one with a yellow band, the other white. “The crazy thing,” Kronforst says—a familiar refrain as he describes the twists in butterfly DNA—is that they are each mimicking a different species. Those relatives and these Heliconius mimics, Kronforst says, are both already toxic and evolving to resemble one another, a process researchers think of as “distributing the cost of educating predators.”

The lab also studies a less-protected species, the Southeast Asian Papilio polytes. It shows more variation—and raises more of the questions that occupy Kronforst now. Papilio males all look alike, black with a light yellowish strip. There are four female phenotypes, of which he studies two. One variation looks just like the male but with subtle red dots on the wings. The second has a different color pattern, including what Kronforst describes as “gray rays,” and small tails hanging like uvulas from the wings; they are mimicking poisonous swallowtail butterflies. “We don’t know why the males don’t mimic anything. We don’t know why there’s a female morph that looks like the males and doesn’t mimic anything,” Kronforst says. “But then the three female morphs have evolved to look like these distantly related toxic butterflies simply to fool predators.”

The continued presence of the Papilio male wing pattern and the similar female type puzzles researchers. Because, as Kronforst puts it, a predatory bird knows “if I eat something with that red and white in the tails, it’s bad, I’m going to stay away from it. But every time it’s eaten one of these”—he points to a male—“it’s been perfectly delicious. That’s why we don’t understand why this nonmimetic thing even stays in the population.”

Various theories exist that the researchers are considering, he adds, then stops short and veers back to the genetic clues they have begun to unearth. “The extra crazy part of this whole story is, the variation among those four female morphs, it’s a single gene. One gene controls everything, whether they have the tails or no tails, the whole wing pattern.”

Males have that gene too, but only females express it. In 1972 Cyril Clarke and Philip Sheppard of Liverpool University identified the single gene that determines so much in Papilio butterflies. (In the Heliconius multiple genes are involved.) Kronforst says the gene could, in fact, be one “supergene,” or several fused together that used to be in different places on the chromosome. But that remains one of the questions each new fragment of information seems to raise.

To find answers, Kronforst’s researchers mate the butterflies with different genotypes, then mate their offspring back to one type or the other. In each generation of females—perhaps a group of 50 sisters—half will resemble the males and the other half will display the mimicry characteristics. “So then we go into the genome and we say, OK, where are the 25 like this different from the 25 like that?”


“By studying these butterflies we’re able to capture evolutionary events in progress,” Kronforst says, “and that’s something that can be hard to find in nature.”

They’ve narrowed the difference down to four potential triggering genes. “One of those four is the gene that’s making this switch. So that’s what we’re trying to figure out.”

They’re also trying to figure out the historical cause of the variation, which could be attributed to a sudden genetic mutation. “The ancestral phenotype,” Kronforst says, is likely the pattern that the males and half the females display. The other phenotype may have emerged as the result of an alteration to mimic a protected species. “It maybe didn’t look like a perfect mimic, but it looked close enough to fool predators. Then it was protected,” Kronforst says. “And then evolution can sort of tweak the phenotype over time to make it a better and better mimic, but [the mutation] can happen”—he snaps his fingers—“like that.”

 

In the butterfly tents, artificial flowers made with colored tape adorn plastic cylinders of nectar. Most of the insects feed on nothing else, but the Heliconius also eat pollen. Natural flowering vines in the lab provide the nutrient-rich dietary supplement that keeps the Heliconius alive for months after emerging from the chrysalises as adults, compared to three or four weeks for other species.

Kronforst notices a butterfly with a bent antenna: “Poor guy.” Under the lab’s carefully maintained conditions, the damage will not be catastrophic. “In nature, he probably would be in trouble,” Kronforst says, “but in here, it’s pretty posh; we come and feed them every day.”

Natural threats do infiltrate the greenhouse. A larval disease that Kronforst calls “butterfly Ebola” swept through this past winter, turning the caterpillars into “black bags of goo.” The goo seeps onto the plant, other caterpillars eat it, and an insidious virus spreads. Aside from cleaning the plants to prevent further infection, though, life as usual went on among the healthy populations and the biologists studying them.

The insects mate, the females lay eggs, and the evolving research continues—although one of the lab’s investigations keeps butterflies from mating at all. To understand mate preferences in the Papilio species, researchers put a virgin female of each phenotype into a cage where the males have been isolated. Researchers sit in the cages and observe—in part because they don’t want these butterflies to actually mate, they just want to see which female wing patterns cause each male’s antennae to quiver. They need to use the virgin females in multiple experiments, and mating causes behavioral changes in the males, requiring them to be kept apart. Other populations in the lab are left to mate naturally while some individuals are paired together, usually based on wing pattern, for targeted research purposes.

The pace from courtship to consummation varies, so observers of the mate-choice experiments must be vigilant to prevent flirtation from going too far. “He might court her on and off for hours—court, fly away, then come back,” Kronforst says. “Sometimes we see these males just in this sustained courtship thing for 10, 15 minutes.” He makes a fist and flaps his other hand over it in a rapid flurry. “They must be exhausted.”

As soon as researchers recognize a male’s activity as a demonstrable preference, they step in to prevent mating and note the male’s choice, identifying him by the number marked on his wing. The female color pattern is the attraction cue, so altering a male’s wing with a Sharpie does not influence the preference of a potential mate.

When the males die, Kronforst’s team analyzes their DNA to determine whether they have the mimicry gene, the nonmimicry gene, or both. “Those three groups have different preferences for mimetic versus nonmimetic” females, he says, inclinations which appear to have a “fundamental, functional link” with the males’ color-pattern gene.

Similar studies of Heliconius butterflies yield comparable results. Both males and females have either yellow or white bands on their wings, and males prefer females with the same coloring. “It looks like it’s the same genes that are making the wing patterns and also changing the mate preference,” Kronforst says. “We don’t understand why. Basically, we’re generating more questions than answers, but it’s kind of a crazy thing.”

Another question that intrigues him: where do those preferences physically manifest themselves? “Is it something in the eye, where they see the different phenotypes differently? Or is it something after the eye, like in the brain? Are they seeing them both and then really deciding this one versus that one?”

Dissection of live butterflies will help him begin to find some clues. Researchers will use microscopes to study the insects’ neural activity based on different visual cues. Work like that has been done on butterfly antennae, examining the effects of certain pheromones, but Kronforst believes his lab’s brain observations of visual responses, which they expect to begin in mid-May, will be a first.

 

A visitor’s visual response to the plants and insects in the greenhouse requires expert guidance to see beyond the superficial hues. Newly hatched caterpillars have splotches of black and white coloring. “They’re actually mimicking bird poop,” Kronforst says. “Then when they get to be too big—they’re, like, bigger than a bird poop and they’re not an effective mimic anymore—they turn bright, bright green” to blend into the leaves.

Preparing to form the chrysalis, the caterpillars move to a different part of the plant and display another protective adaptation. Somehow they sense the part of the plant they occupy, branch or leaf, brown or green, and disguise themselves accordingly.

Kronforst bends over a plant and points out a caterpillar on a branch that requires a trained eye to see. What looks like a shell across its back would keep it effectively hidden against a station wagon’s wood paneling. Researchers don’t know how the caterpillars do it, but other species have been shown to base similar blending traits on moisture levels. “They might take a bite and say, ‘OK, this is really dry and woody,’ so they say, ‘I’ll be brown,’” Kronforst says. “Or, ‘This is really succulent and moist, so it must be green.’”

If a predatory bird isn’t fooled, caterpillars have one last-ditch—and generally futile—defense mechanism. Kronforst touches one to demonstrate. The caterpillar curls its head up and back as if striking a yoga pose, extends red horns, and emits an odor. Although intended as a deterrent, the effect of the horns seems more like runway lights directing the predator to them. “The idea is that it’s just too late,” Kronforst says of the caterpillar’s desperate attempt to survive. “There’s no turning back; you’re not going to fool anybody.”

When the researchers spot a caterpillar that’s about to form a chrysalis, they place it under a camera and use time-lapse photography to help tease out more genetic information. Because they have narrowed down the genes that determine wing patterns, “we want to look at how expression of those genes changes over time while they’re actually making their wings.” Photos snapped every five minutes allow them to watch the process as it happens. They then dissect tissue from the chrysalises at different points of development to examine the stages of gene expression.

Soon the butterflies emerge from their chrysalises, extend their wings, and fly. They join the 100 to 200 butterflies alive at any given time in their greenhouse aerie, flashing the beguiling colors that keep predators away but draw Kronforst and his research team closer and closer.

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