Duke University biologists evolve complex trait in the laboratory

Duke University biologists have evolved a complex trait in the laboratory -- using the pressure of selection to induce tobacco hornworms to evolve the dual trait of turning black or green depending on the temperature during their development.

The biologists have also demonstrated the basic hormonal mechanism underlying the evolution of such dual traits.

Their experiments, they said, offer important insight into how complex traits involving many genes can abruptly "blossom" in an organism's evolution.

The researchers -- Professor of Biology Frederik Nijhout and graduate student Yuichiro Suzuki -- published their findings in Science. Their work was funded by the National Science Foundation.

The complex traits, or "polyphenisms," they studied are instances in which animals with the same genetic makeup can produce quite different traits, or phenotypes, in different environments. For example, genetically identical ants can develop into queens, soldiers, or workers, according to their early hormonal environment. Or, the same butterfly can assume very different coloration in winter or summer. A kind of polyphenism is also likely at work in mammals -- for example in the seasonal development of antlers or changes in plumage or coat colors, said Nijhout and Suzuki.

While biologists have understood the basic machinery underlying polyphenisms, the mystery remained how such complex traits, which involve mutations in multiple genes, could evolve and persist.

"It's long been known that polyphenisms are controlled by hormones, with the brain sensing environmental signals and altering the pattern of hormonal secretions," said Nijhout. "In turn, these hormonal patterns turn sets of genes on or off to produce different traits. However, we understood only the developmental mechanism, and how it is possible with a single genome in an animal to produce two very different phenotypes," he said.

"There had been theoretical models to explain the evolutionary mechanism -- how selective pressures can maintain polyphenisms in a population, and why they don't converge gradually into one form or another," said Nijhout. "But nobody had ever started with a species that didn't have a polyphenism and generated a brand-new polyphenism. Such a demonstration could offer important insights into the evolutionary mechanism underlying such traits."

In their experiments, Suzuki and Nijhout chose a species of finger-sized tobacco hornworm, Manduca sexta, which normally produces only green larvae. Because a related species, Manduca quinquemaculata, develops black or green larvae when exposed to lower or higher temperatures, the researchers theorized that they could use temperature shocks to evolve a similar polyphenism in M. sexta.

Suzuki and Nijhout conducted their experiments on a black mutant form of M. sexta, which is black because of lower production of a key hormone called juvenile hormone. They subjected the black mutant caterpillars to heat during a critical period, and over multiple generations selected for two different lines of mutant caterpillars. One polyphenic line was selected to show increased greenness on heat treatment, and one monophenic line selected to show decreased color change upon heat treatment.

After rearing and selecting ten generations of caterpillars, with about 300 caterpillars per generation, the researchers found that they had, indeed, created the two distinct strains. The polyphenic strain would develop a green color at higher temperatures, altering abruptly at a temperature of about 28 degrees C. (83 degrees F.) In contrast, the monophenic strain remained black at all temperatures.

The researchers could compare these strains to understand the origin of the polyphenism. Their experiments revealed that it was the level of juvenile hormone in the caterpillars that regulated whether they would turn black or green.

For example, by applying a spot of juvenile hormone extracted from a green caterpillar to a black caterpillar during a critical period, Suzuki could produce a green spot on that caterpillar.

Also, by tightening a tiny noose around a developing caterpillar's head to prevent the juvenile hormone -- produced in the head -- from flowing to the rest of the body of the heated polyphenic worm, Suzuki could prevent the caterpillar from turning green.

According to Nijhout, the generation of polyphenism in the caterpillar demonstrates an evolutionary phenomenon called "genetic accommodation." In this process, a mutation in a regulatory pathway such as a hormonal pathway changes the hormonal level to bring it closer to a threshold level that could be affected by environmental variation.

Thus, the black mutant hornworm had "dialed-down" levels of juvenile hormone, so that the caterpillar's color-producing machinery would be more likely to be affected by temperature. By selecting for a temperature-sensitive strain, the researchers established polyphenism in the caterpillar.

"Our work is really the first demonstration that genetic accommodation actually can happen," said Nijhout. "In this case, it happens in the laboratory by artificial selection; but as with all such experiments, we assume that this is a microcosm of what is actually going on in nature."

Nijhout theorized that such "homeostatic" mechanisms that maintain, for example, the color of a caterpillar, can act to mask a great deal of mutations present within the genetic machinery.

"Homeostatic mechanisms tend to stabilize a phenotype such as color and, therefore, allow the accumulation of underlying, covert mutations just as an electrical capacitor acts to accumulate charge. And eventually, these mutations could 'break out' of that constraint to produce a sudden phenotypic change; and one way for them to break out is for a mutation to happen -- for example, one that alters a hormonal level -- releasing all this variation.

"The reason this 'capacitor' concept is important in understanding evolution and the origin of complex traits is that the common model is that a new trait gets started by a fortuitous single mutation," said Nijhout. "And while that likely happens, we believe that another important mechanism involves the accumulation of many mutations in many genes without any apparent effect because they are buffered by a homeostatic mechanism; then all of a sudden one of them alters the homeostatic mechanism and lots of genetic variation suddenly explodes and is revealed as a tremendous increase in the phenotypic variability of the species. This variation then serves as raw material for selection to mold a new adaptive trait. And so that's why we think these kinds of experiments demonstrate an important novel mechanism for the evolution of novel traits."

In further studies, Nijhout and his colleagues will seek to determine whether the type of evolutionary mechanism they demonstrated in the laboratory also occurs in nature. Also, they will seek to demonstrate the phenomenon of the genetic 'capacitor,' in which mutations can accumulate 'invisibly' without obviously affecting a trait, and whether natural selection tends to filter out deleterious mutations in such cases.