Inner Workings: How the butterfly got its spots (and why it matters)

The colorful canvas of the butterfly wing is an exceptional example of evolutionary innovation and adaptation. Compared with their forebears, whose wings wore patterns of black, brown, and gray, the Lepidoptera (butterflies and moths) evolved a more varied palette of pigmentation. With the capacity for complex color patterns, such as stripes and eyespots, wing coloration evolved a variety of functions, such as attracting mates and warning predators away. And yet, how exactly these ecologically important patterns develop and evolve has puzzled researchers for decades.

CRISPR is starting to provide insights into the evolutionary and developmental mysteries of how the butterfly wings of species such as Heliconius charithonia (pictured) acquire their distinctive patterns. Image courtesy of Jeffrey Marcus (University of Manitoba, Winnipeg, Canada).

Starting in the 19th century, naturalists began investigating the function of wing-color patterns in different butterfly species. But it was the advent of population genetics in the early 20th century that led to associations between specific genes and color patterns, helping elucidate how and when they evolved. Even so, the absence of genome sequences and the inability to knock out or misexpress genes limited the ability of researchers to understand the mechanisms that produce the wing patterns.

Now, using CRISPR to investigate gene function in butterfly wings, researchers have discovered that just a few genes are largely responsible for setting up the patterning throughout the wing. “The big insight: single genes can act as extremely discrete switches to completely change morphology,” says Bob Reed, associate professor of biology at Cornell University and coauthor on two recently published studies in PNAS about patterning the wings of Heliconius butterflies. “That was surprising, the extent to which they can completely change the entire color pattern in a very discrete way.” CRISPR is helping uncover not only genes responsible for ecologically important traits but also how gene networks are assembled from a mix of old and new parts.

Wing Patterns Evolve

The genes WntA and optix are among the most important players. They first came to the researchers’ attention through decades of previous work on the tropical Heliconius, or passion-vine, butterflies (1). In many locations in the New World tropics, multiple species in this genus co-occur; although they are distinct species and do not interbreed, they resemble each other so closely that they can be difficult to distinguish by eye. The species evolved a common warning coloration pattern to signal that they are poisonous, thus keeping predators at bay. As a result of this convergent mimicry, a predator has to recognize only a single warning pattern in any particular geographic location. Throughout its range, a given species might have 20 different geographically restricted color patterns.

More than a decade ago, researchers wanting to understand the origin of the basic pattern of red and yellow stripes and black splotches of most Heliconius species conducted genetic crosses to reveal the genetic basis of the patterns (2, 3). In a large team effort that involved many labs, they were able to map three genes with major effects that appeared to control color-pattern variations in the butterflies: WntA, optix, and cortex.

But, although researchers had genetic-mapping data, genome-wide association studies, and gene-expression data, they didn’t have experimental evidence that these particular genes were actually controlling color patterns and how. “Once CRISPR came along, we wanted to generate that proof to see exactly how these genes worked to control color patterns,” says Reed.

In their recent PNAS study (4), Reed and his colleagues showed that optix controls the red and yellow colors throughout the wing. When optix is knocked out, the wing patterns remain, but the wing loses intensity from red to orange to gold—it turns grayscale. The gene optix is ancestrally expressed in insect eyes, where it activates the expression of visual pigments. “So optix was recruited to the wings, and now it becomes possible to express the pigments of the eyes in the wings,” explains butterfly biologist Jeffrey Marcus, an associate professor at the University of Manitoba, who was not involved in the study.

In a second PNAS article (5), researchers found that WntA lays down the central part of the butterfly “ground plan”—a system of spots and stripes on the wing that can be modified in size or color in different species, which represent variations on the same pattern. Although Russian naturalist Boris Schwanwitsch first described this ground plan in the 1920s,

“The true novelty of our papers is that we showed that these two genes have different roles across species.”

—Arnaud Martin

no one knew how the pattern was controlled developmentally. Using CRISPR, the researchers discovered that WntA controls a centrally located, symmetrical banding pattern called the central symmetry system. It’s difficult to identify the original function of WntA because it’s involved in so many contexts in which cell proliferation occurs.

Reed and his colleagues are now working to understand how different alleles of WntA determine smaller-scale differences, as seen in different geographical subtypes of the same species. He suspects these differences have to do with regulatory changes that alter where WntA is expressed. “The gene takes signals from other genes, and the signals are processed, and they decide to turn on optix or WntA in certain parts of the wing,” Reed explains. “What we’re trying to do right now is pick apart the function of the switchboard of regulatory inputs around these genes to understand exactly how expression evolved.”

“The true novelty of our papers is that we showed that these two genes have different roles across species,” says Arnaud Martin, assistant professor of biology at The George Washington University and co-author on the WntA paper. “These genes give us insight into the genetic mechanisms that generate biodiversity,” he says. Optix and WntA might be the main switches, but there are many others that are likely involved in more subtle tweaks of the wing pattern, Martin says. “We are just starting to scratch the surface.”

From Genes to Networks

Indeed, the butterfly’s developmental story boasts plenty more nuance and mystery. Several of these genes, it appears, have been co-opted from one trait or function to play a role in an entirely different trait or function. How this happens—and how genes become part of, and interact with, existing gene networks—has long been a major focus for evolutionary developmental researchers, one now buoyed by the availability of CRISPR technology.

The hypothesis that eyespots are co-opted limbs—what Marcus calls the “smooshed limb hypothesis”—is more than two decades old (5), but the evidence for it remains equivocal. On one hand, several genes expressed in developing legs are also expressed in developing eyespots, suggesting that the genetic network for legs may have been repurposed for new use in the wings. But most of these genes are also expressed in many other contexts during development, from the early embryo to adult stages (e.g., setting up the anterior–posterior axis, determining the fate of scale and socket cells on the developing wing, and more). If these genes are used in so many different contexts, are eyespots co-opted limbs or simply novel traits put together from multifunctional genes that were redeployed individually, as needed throughout evolution?

The distinction hinges on whether the few genes interact together the same way in the legs as they do in the wings. In other words, was the gene network in the legs recruited all at once to the wing, like recruiting a single sports team all together, or was it assembled de novo, by recruiting individual players from different places one by one to form a new team that may interact very differently? “Nobody knows, but that’s a hypothesis that could be tested,” Marcus says.

Now that CRISPR tools are available, associate professor of biological sciences Antónia Monteiro of the National University of Singapore is reexamining that hypothesis. She contends that the network—including the genes distal-less, notch, engrailed, and spalt—must have been co-opted through the evolution of a new regulatory DNA sequence that could activate the expression of an existing gene network in a new location (the wing). If that is the case, then mutating the regulatory regions of the downstream genes in the network that makes eyespots should affect not only the eyespots but also the original trait from which this gene network was co-opted, Monteiro reasons.

Monteiro’s team identified candidate gene regulatory sequences and disrupted them using CRISPR one by one to see if doing so had any effects beyond disrupting the eyespot. The eyespots disappeared as expected, and the antennae and legs also became truncated, results she reported at the second biennial meeting of the Pan-American Society for Evolutionary Developmental Biology in August 2017 (6). “That suggests that the preexisting network functions in appendage development and got recruited to produce eyespots on the wings of the butterflies,” says Monteiro, who’s currently testing this idea further by connecting a green fluorescent protein reporter to the regulatory sequences of interest to verify where in the body they are driving gene expression.

It’s one of several studies that are bound to help illustrate in living color exactly how genes paint the butterfly’s delicate, fluttering canvases. “I’m very excited, but I cannot be too excited.” Monteiro says. “It is still early days.”