Cannibalistic tadpoles and matricidal worms point to a powerful new helper for evolution

MONTPELLIER, FRANCE—Growing up in South Texas, David Pfennig was fascinated by cannibalistic tadpoles. When summer storms soak the normally dry plains, spadefoot toads emerge from their burrows to lay eggs in short-lived pools. The tadpoles normally dine demurely on algae, tiny crustaceans, and detritus. But even as a boy, Pfennig could tell that the same toads sometimes spawned very different tadpoles. Those tadpoles had bulging jaw muscles and serrated mouthparts. They jostled aggressively in the shrinking puddles. They ate larger crustaceans, such as fairy shrimp—and one another.

Later, when he became a biologist, Pfennig's fascination turned into curiosity. Both kinds of tadpoles had the same parents, and hence the same genes. That they could turn out so differently, presumably because of their environments, didn't square with the gene-centric view he had acquired during his studies in the 1980s. In that view, the genes inherited from parents should dictate every detail of how animals look and behave. "Yet here I was observing these animals that can modify their traits in response to the environment," recalls Pfennig, who now runs a lab at the University of North Carolina in Chapel Hill. "It was sort of mind blowing."

The toads display phenotypic plasticity, the ability to change how they look and act, and how their tissues function, in response to their environment. Other researchers had already documented the tadpole transformations. When algae and tiny prey are abundant, the tadpoles are small-jawed and mild-mannered. But if the pond also contains fairy shrimp, some tadpoles turn into the aggressive carnivores. They take advantage of the atypical food source, grow faster on the extra protein, and have a better chance of making it to adulthood before the water dries up.

Recently, Pfennig and his team have come upon something even more remarkable than that dramatic behavioral plasticity. In one species of spadefoot toad, they found, the carnivorous tadpole stage has become entrenched—there's no need for a dietary trigger. A flexible response to the environment somehow became fixed.

To some, such findings evoke the spirit of the French naturalist Jean-Baptiste Lamarck. Decades before Charles Darwin laid out his evolutionary theory in On the Origin of Species, Lamarck and other biologists proposed their own mechanisms for evolutionary change. Among his ideas, Lamarck famously asserted in the early 1800s that organisms can acquire a new trait in their lifetime—longer necks for giraffes reaching for food; webbed feet for water birds—and pass it on to their offspring. Later, biologists cast aside Lamarckism, as the classic view of evolution emerged: that organisms evolve as a result of natural selection acting on random genetic changes.

Now, however, evolutionary biologists have shown in multiple organisms, including lizards, roundworms, and yeast, that a plastic response can pave the way for permanent adaptations. The new evidence, much of it reported at the Second Joint Congress on Evolutionary Biology here this summer, shows the connection between plasticity and evolution "is a real thing," says Carl Schlichting, an evolutionary biologist at the University of Connecticut in Storrs. "If you look for it, you are going to find it."

On the surface, the findings vindicate Lamarck: Acquired traits can be inherited. But biologists are quick to stress that what these organisms show is not true Lamarckian evolution. Application of Lamarck's idea to modern findings "has led to a lot of confusion and debate," says Cameron Ghalambor, an evolutionary ecologist at Colorado State University in Fort Collins.

As biologists explore the underpinnings of plasticity and how it can lead to permanent change, they've uncovered a process that extends traditional evolutionary mechanisms rather than challenging them. The plasticity those changeable tadpoles display is built into their genetic code. And when an "acquired" trait does become permanent, it is because of mutations that "fixed" the plastic trait—a process biologists call genetic assimilation.

Although some researchers bristle at giving any credence to Lamarckian thinking, "The way plasticity can influence evolution really fits very comfortably in the general framework of how we think evolution works," Pfennig says.

Transformed tadpole

In 2003, evolutionary biologist Mary Jane West-Eberhard of the Smithsonian Tropical Research Institute in Panama City raised eyebrows by suggesting phenotypic plasticity might also set the stage for permanent adjustments. Although her work focused on wasps, she drew on a vast literature about plants, butterflies, and other organisms that changed how they looked or acted. She proposed that, in the face of an environmental challenge, plasticity built into the genome enables at least some members of a species to cope. That would buy time for adaptive mutations to arise and be selected.

Some of those genetic changes would simply increase the proportion of the most flexible individuals. But others might favor a specific trait. "This plasticity-first view solves some of the problems that are inherent if organisms have to wait for a genetic mutation," explains Renee Duckworth, an evolutionary ecologist at the University of Arizona in Tucson. "That is something that obviously would take a lot of time."

The way plasticity can influence evolution really fits very comfortably in the general framework of how we think evolution works. David Pfennig, University of North Carolina

Pfennig and his lab members think spadefoot toads have followed that evolutionary trajectory. Through decades of fieldwork, his team and others have shown that some species, such as the eastern spadefoot (Scaphiopus holbrookii), never naturally develop cannibal tadpoles. Another species, Spea multiplicata—the Mexican or desert spadefoot of Pfennig's childhood—produces a mix of cannibals and omnivores depending on food availability, which may have enabled it to expand its range to shorter-lasting pools. But in populations of the plains spadefoot toad (Spea bombifrons) whose tadpoles live in the same ponds with S. multiplicata, almost all tadpoles are carnivores.

To see how much plasticity each species can muster in the lab, Pfennig's graduate student Nicholas Levis recently raised tadpoles on diets with a varying proportion of fairy shrimp. The eastern species, thought to be most representative of the first spadefoot toads to arise in evolution, responded just a little to a 100% shrimp diet, developing a shorter gut—better suited to a carnivorous diet—and mouthparts that were altered but still poorly adapted to catching prey. In short, it had limited phenotypic plasticity.

Desert spadefoot tadpoles responded more strongly to the shrimp-only diet, exhibiting dramatic changes in gut and head shape and behavior. Metabolic genes that help digest protein became more active in these tadpoles, whereas the activity of genes needed to process the fats and starches in a detritus diet declined. But given a diet with little or no shrimp, the tadpoles could reverse all these adaptations.

The plains toads that Levis studied, in contrast, turned out to be confirmed carnivores, he reported at this summer's evolution meeting and, with Pfennig and lab member Andrew Isdaner, in a paper in the August issue of Nature Ecology & Evolution. Some of its tadpoles even hatched as carnivores, without the need of the fairy shrimp diet. And when given a detritus-only diet, the species's tadpoles had difficulty regaining traits better suited for omnivory. "Some populations seem to have transitioned to all being carnivores, no matter what the situation," Levis says.

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Pfennig considers this a classic example of what he and others call plasticity-first evolution: Natural selection favored carnivory so strongly in this population of plains toad that this once-inducible phenotype somehow became genetically assimilated. "The idea is that the ancestor has the plastic ability and allows adaptation initially and then fixes it," Schlichting says. Just why evolution acted to fix the carnivorous traits in this population isn't clear, Levis says. It could be to avoid competing for the same food as other tadpole species. And Levis told the evolution meeting his group's unpublished data show that plains toad populations that produce more carnivorous tadpoles do better, a hint there is some advantage to this carnivory.

Life on the lava

Ammon Corl, a postdoc with Rasmus Nielsen at the University of California (UC), Berkeley, and his colleagues have traced a similar interplay between plasticity and evolution in the side-blotched lizards of California's Mojave Desert. He's even caught a glimpse of the genes responsible. In sandy parts of the Mojave, side-blotched lizards scamper around in shades of tan and brown. But those living on the Mojave's inky Pisgah lava flow are among the blackest lizards, presumably for camouflage from predators.

In the 1980s, Claudia Luke, then a graduate student at UC Berkeley and now at Sonoma State University in Rohnert Park, California, switched dark and tan lizards between sandy and lava surfaces in the lab and found both varieties can adjust their colors to match their new surroundings in just a few weeks. But she also found the lizards from a sandy environment did not get as dark on lava as the regular lava dwellers, suggesting a genetic difference in the lizards' ability to change color.

Luke's observation remained a puzzle for 20 years, until her unpublished thesis was discovered by Corl when he was a graduate student with Barry Sinervo, a behavioral ecologist at UC Santa Cruz. Corl sequenced the genes of the offspring of lizards from on and off the lava to track down genetic differences. He and his colleagues discovered two genes, PREP and PRKAR1A, that have mutated in the darker lizards. Each influences how much of the dark pigment, melanin, is produced in the skin.

When the lava first cooled 20,000 years ago, the researchers suggest, phenotypic plasticity enabled lizards that wandered onto the newly cooled lava to darken for concealment and survive in the new environment. But these pioneers likely varied in their plasticity, and predators nabbed the lighter ones. That selective pressure favored mutations that increased darkening. "Plastic changes in coloration facilitated initial survival and then genetic adaptations allowed lizards to become even darker," says Patricia Gibert, an evolutionary biologist at Claude Bernard University in Lyon, France. "This study provides one of the best examples of how plasticity precedes adaptive genetic change," Ghalambor adds.

Scientists are now using fast-breeding organisms to recreate such plasticity-first evolution. In Jonas Warringer's lab at the University of Gothenburg in Sweden, for example, graduate student Simon Stenberg applies environmental stressors to budding yeast for different lengths of time and tests the organisms for plastic or permanent responses. In one set of experiments, he's been exposing the yeast to the herbicide paraquat, which causes eukaryotic cells to produce high concentrations of oxygen free radicals that damage DNA and other molecules. To gauge the health of the yeast, he measures its doubling time—how long it takes for a colony to double in size. When Stenberg first applied the toxin, the yeast's doubling time slowed from the usual 1.5 hours to 5 hours.

After as few as four generations, some of the colonies recovered half of their growth rate. Because that's too little time for a genetic adaptation to arise and sweep through a whole colony, Stenberg concluded at least some of the yeast had a form of phenotypic plasticity that allowed them to cope with the excess free radicals. When he stopped applying paraquat and then reapplied it three to 100 generations later, the colonies' growth rates again plummeted after 10 generations. The reduction indicates that the unknown paraquat-resistance mechanism was not yet permanently encoded in the genomes. But after constant exposure to paraquat for 150 generations, the yeast developed a permanent adaptation. They continued to grow even if Stenberg stopped applying the herbicide for 80 generations and then reapplied it.

Since the meeting, Stenberg has found what may be the yeast's coping mechanism: eliminating some or all of the DNA in their mitochondria, the cells' energy-producing organelles. (Mitochondria themselves generate free radicals.) When the yeast were first exposed to herbicide, they temporarily reduced their mitochondrial DNA, a reversible change. After extended exposure, though, the change became lasting as they stopped making mitochondrial genomes altogether. (Yeast are among the few eukaryotic organisms that can survive without these genomes.) "The adaptation had become genetically assimilated," Stenberg says.

Making a meal out of mom

So far, Stenberg hasn't pinned down the genes responsible for this transition. But other researchers, working with the nematode Caenorhabditis elegans, have shown how a single mutation in one wild strain caused a plastic response to starvation to become fixed. In the lab, C. elegans—a key model animal for studying development and many other topics—is usually fed Escherichia coli bacteria. But in the wild, C. elegans lives on microbes in decaying fruit. These wild nematodes and their young live a life of feast and famine: Once the fruit is gone, it could take days to find more.

What our research shows is that a single mutation can lead to dramatic effects on life history through loss of ancestral plasticity. Christian Braendle, CNRS and the University of Nice Institute of Biology

The worms have a ghoulish way to cope. They stop laying eggs, which instead hatch inside the mother's body, turning it into a lifeline for the developing young as they devour her insides. With enough food to survive, the nematode larvae can then enter a state of suspended animation called the dauer stage until the next windfall of fruit, when they mature and return to egg laying.

In a compost pile outside Paris, biologists have found a C. elegans strain in which the plastic response has become permanent. For these worms, matricide is the rule: They don't lay eggs, even when food is plentiful. "All the upstream signals related to food availability are irrelevant," says Christian Braendle, an evolutionary biologist at the French national research agency CNRS and University of Nice Institute of Biology in Valrose, France, who learned of the strain and decided to follow up. The change in strategy must be adaptive—allowing more offspring to survive—because Braendle's team keeps finding other matricidal wild strains.

By crossbreeding the compost pile strain with nonmatricidal worms and analyzing the DNA of offspring, his team has now tracked down the key gene, which codes for an ion channel, a protein in the cell membrane that transmits signals between nerves and muscle cells. In the matricidal strain, a single base change in the gene alters the ion channel. As a result, the worm's vulva muscle fails to respond to food signals that would normally cause it to expel eggs, causing them to hatch internally. "What our research shows is that a single mutation can lead to dramatic effects on life history through loss of ancestral plasticity," Braendle said at the meeting.

To confirm the mutation's effect, his team engineered it into egg-laying worms, which then bore live young. And when they transferred the unmutated gene to the matricidal worms, they reverted to egg-laying, Braendle reported.

"This might be the first description of the genetic mechanism underlying the transition from a historically plastic trait to a fixed trait," Ghalambor says. If Lamarck had come across these matricidal worms, he might have thought a selfless mother had adopted this strategy in a single generation, then passed it on. Braendle's unpublished work shows matricide is actually a plastic response encoded in the genes that, with one more mutation, became permanent.

So 200 years later, biologists are realizing Lamarck wasn't wrong in emphasizing that fast, flexible responses to the environment—what biologists now know as plasticity—can drive lasting change. Although mutations are still important drivers of evolution, responses to the environment "can be the precursors, and the genes are the followers," Gibert says. "This is a change in the way of thinking."