Evolution of a polyphenism

Here’s some very cool news: scientists have directly observed the evolution of a complex, polygenic, polyphenic trait by genetic assimilation and accommodation in the laboratory. This is important, because it is simultaneously yet another demonstration of the fact of evolution, and an exploration of mechanisms of evolution—showing that evolution is more sophisticated than changes in the coding sequences of individual genes spreading through a population, but is also a consequence of the accumulation of masked variation, synergistic interactions between different alleles and the environment, and perhaps most importantly, changes in gene regulation.

Unfortunately, it’s also an example of some extremely rarefied terminology that is very precisely used in genetic and developmental labs everywhere, but probably makes most people’s eyes glaze over and wonder what the fuss is all about. I’ll try to give a simple introduction to those peculiar words, and explain why the evolution of a polyphenic pigment pattern in a caterpillar is a fascinating and significant result.

Students are usually taught a grossly oversimplified version of genetics. Everyone who has gone through basic biology has heard of Mendel and his pea plants, and the simple traits that assort independently and can be traced back to a single locus by their pattern of inheritance. There is one wrinkled gene, for instance, and it makes peas wrinkled. Unless you are defining things solely on a molecular level, however, there is no such thing as a phenotypic property that is solely the product of a single gene. Traits are polygenic, meaning that multiple genes cooperate to produce a a phenotype. One pet peeve I (and many other biologists) have is the media shortcut of describing an identified gene as a “gene for X”, whether X is breast cancer, schizophrenia, or hematopoiesis. Multiple genes contribute to all of those phenomena, and that’s what we mean by polygenic.

(A complementary word is pleiotropy: a single gene contributes to multiple aspects of the phenotype. Mutations in single genes typically ripple through many elements of the phenotype, and cause surprising changes in multiple features of the organism.)

Looking at it simplistically, one might think these properties would make evolution difficult: everything is coupled to everything else, and when selection tugs on one parameter of the organism, it’s also pulling on all of the other parameters as well. The polygenic and pleiotropic nature of the organism and genome reflect widespread integration, however, and that coupling is a good thing—it means changes in a gene don’t just yank it out of the regulatory structure of the genome, but instead the plastic nature of genetic interactions means other genes follow and compensate or potentiate changes in the one.

Seasonal polyphenism in the tropical butterfly Precis almana. The wet-season form (top) has a rounded wing margin and colorful ventral pattern. The dry season form (bottom) has a more angular wing shape and a dull brown color pattern that resembles a dead leaf.

The output of a gene, the phenotype, is dependent on the output of other genes (that polygenic property), and it’s also dependent on non-genetic components of the environment. Another totemic word is polyphenism, which refers to irreversible environment-specific alternative phenotypes. Polyphenic traits develop in distinctly different ways depending on the environmental context: the figure to the right is of two forms of a single species of butterfly, in which individuals that eclose during the tropical wet season develop more colorful wings, while those that eclose during the dry season are more drab and brown. The environmental factor that triggers the different forms is temperature or humidity, and the two animals may have very similar genotypes (but not necessarily; different genes may predispose development of particular phenotypes) that respond in different ways to those factors, producing a very different form.

This sounds tricky to evolve, and superficially seems complicated. The argument is that it requires the acquisition of sophisticated genetic control elements that sense elements of the environment and selectively activate different sets of genes depending on the conditions. Putting it that way makes it sound unlikely and awkward. However, all phenotypes are conditionally sensitive and dependent on interactions between genes and between genes and the environment—the control elements aren’t novel introductions, they’re already there! The evolution of polyphenic traits may be more a matter of shifting conditional responses quantitatively in particular directions.

Let me introduce one more critical term: genetic assimilation. This is a concept that has been around for a long time, and has often been maligned or more often neglected by old-school evolutionists like Dobzhansky or Mayr or Simpson; whether you think it important or not is a good indicator of where you stand in the ongoing evo-devo revolution. Simply put, genetic assimilation is the fixation of a phenotype by a genetic change in the regulation of the genes involved. Remember, as mentioned for polyphenic traits above, that regulation can be initially due, in part, to environmental factors, so what this effectively suggests that a phenotype can be environmentally induced first, and later ‘hardwired’ into the genome by changes in regulatory elements of the DNA. Genetic accommodation is a related concept that differs in that, while genetic assimilation works to stabilize a particular phenotype, making it more robust, accommodation can increase the responsiveness of the phenotype to changes in environmental conditions.


That’s the background. Now let’s get into the specifics of this experiment.

That beautiful green beast to the left is the larva of the tobacco hornworm, Manduca sexta. The one on top is the form you’ll usually see, the wildtype green caterpillar—it’s always green like that. On the bottom is the black mutant of Manduca sexta, which is always heavily pigmented. This is not a polyphenism! There is no environmental component to trigger the development of the different pigment forms.

On the other hand, there is another related species, Manduca quinquemaculata, which does exhibit a polyphenism: when raised at 20°C, the larvae are all black, and when raised at 28°C, the larvae are all green. The goal of this experiment is to take a strain of M. sexta and induce it to evolve a pigment polyphenism like that of M. quinquemaculata.

If you want to select for an interesting property, though, the first thing you need is some variation to work from. Both the green and the black M. sexta are uniform populations—they’re either all black or all green, so there isn’t much visible variation! One way to expose variation between individuals, though, is to expose them to stress: make life difficult, and subtle differences are amplified and show through. The stress chosen was to expose the animals to heat shock, a 6-hour long exposure to the harsh temperature of 42°C. The normally green M. sexta sailed through the heat shock and emerged still green, but the black M. sexta suddenly exhibited new variations: some were still black, others turned green, and others were all shades in between. The authors developed a scoring system from 0 to 4, with 0 being a caterpillar that was virtually solid black, while 4 was a completely green caterpillar.

Now the selection experiment begins. Take 300 black M. sexta, and zap them with a heat shock. Pull out the larvae that turn the greenest, getting the highest color score, and let them grow up and breed, producing another generation; this is the polyphenic line, the animals that switch from black to green. Also pull out the larvae that stay black, let them grow up and breed with each other, producing the monophenic line…the caterpillars that have only one phenotype, black. As a control the authors heat-shocked one line, the unselected line, and picked random members to breed without regard to their color.

Here’s the result. The polyphenic line showed more and more reliable switching from black to green with each generation, while the monophenic line became more and more resistant to change with each generation. It happened fast: the monophenic line stopped producing any green caterpillars at all after the seventh generation, while the polyphenic line became more and more reliably green, achieving virtually perfect green caterpillars by the thirteenth generation. That’s evolution in action.

Effect of selection on temperature-mediated larval color change. Changes in the mean coloration of heat-shocked larvae in response to selection for increased (green) and decreased (black) color response to heat-shock treatments, and no selection (blue).

What exactly is going on in these animals? Larvae from each line at generation 13 were set aside and raised at different temperatures to assess their sensitivity to heat shock. If larvae were maintained at a cool temperature of 20°C and then heat-shocked, relatively few switched to green; if they were raised all the time at 35°C and then heat-shocked, they were more likely to switch. The plot below is of the reaction norms for each line, or of the responsiveness of each line to the temperature of their environment. What you see is that the polyphenic line has become more sensitive, developing a more switch-like response to temperature. Below the inflection point of 28.5°C, the polyphenic larvae stay black, while above 28.5°C, they’re much more likely to switch to green.

Effect of selection on temperature-mediated larval color change. The reaction norm of generation 13 lines reared at constant temperatures between 20°C and 33°C, and heat-shocked at 42°C. The curves are sigmoidal regressions on the mean data points. Error bars represent 1 SE.

How the color change occurs is diagrammed below. Basically, one of the important regulators of pigmentation is an insect hormone called juvenile hormone (JH). When it’s high, melanization is suppressed and the larva is green; when it’s low, melanization occurs and the larva turns black. Heat shock has the effect of increasing the levels of JH. Looking at just A (the top row) of the figure below, the white, yellow, and orange curves represent the levels of JH at cold, warm, and heat shocked temperatures. In the wildtype M. sexta, all three curves still fall within the range above a threshold, T2, at which the melanization enzymes are inactive. Any variation in the responsiveness to temperature is masked and invisible, because JH levels are so high that they swamp out any detectable differences.

The second row, B, illustrates the effect of the black mutation—it reduces JH levels overall. We still have the temperature-dependent variation, though, and now the increase in JH from heat shock is enough to push the JH levels above the T1 threshold, high enough that some animals will suppress melanization.

(click for larger image)

(Left) Model for the evolution of a threshold trait at the phenotypic level. The evolutionary process required for the evolution of a threshold trait depends on the proximity of the population to the two thresholds (T1 and T2). Below T1, the phenotype is all black. Above T2, the phenotype is all green. Between T1 and T2, individuals express some intermediate phenotype. If the physiological control lies far from the phenotypic threshold (A), a mutation of larger effect or a sensitizing mutation is required to bring the population closer to the threshold (B). Once the population is closer to the threshold, the population can evolve a threshold response through genetic accommodation (C) or become canalized through genetic assimilation (D). (Right) The corresponding changes at the genetic/physiological level observed in this study. Unidirectional arrows indicate high-temperature-induced (yellow) and heat-shock-induced (orange) shifts. Bidirectional arrows indicate polyphenic shifts induced by temperature shifts.

C and D show the effects of selection in this experiment. C is the polyphenic line; what has happened there is that there has been an amplification of their sensitivity to temperature. Now when the animal is warmed up, the levels of JH increase greatly, putting them above the T2 threshold—they turn green. In D, the monophenic line, their levels of JH have been reduced further still, and their sensitivity to temperature reduced—heat shock no longer raises the JH levels above T1, so they stay black. C is an example of genetic accommodation, while D is an example of genetic assimilation.

What this tells us about evolution is that there can be a reservoir of ‘invisible’ variation in populations, which is typically buffered by developmental mechanisms. The buffering allows the variants to accumulate without compromising the viability of carriers. Enabling mutations or changes in the environment, however, can rapidly shift the effect of these variants out of the range that can be buffered, exposing new phenotypic effects that can then be subject to selection. This can be fast, fast, fast, since we aren’t waiting for a single new mutation (or worse, for polygenic traits, many mutations) to expand into a population, but are exploiting a large pool of diversity that is already present, mixing extant alleles by recombination to produce new phenotypes.

Suzuki Y, Nijhout HF (2006) Evolution of a polyphenism by genetic accommodation. Science 311:650-652.