Pictured above is a very peculiar fish that lives in a few North American caves. It looks very much like other fish, except in one respect: it has no eyes. The story of its adaptation to life in pitch blackness is one of the weirdest stories in evolutionary history.

Mutations vs. Cryptic Variation

When people think about how evolution occurs, the classical model generally comes to mind. According to this view, species experience random genetic mutations that confer novel traits when they move to a new environment. The most beneficial traits β€” those that help individuals better adapt to their new habitat β€” get passed along to subsequent generations and eventually spread throughout the population.


It's a relatively simple, easy-to-digest model, but it's not able to explain all cases of evolution. "Imagine if you had a quick change in the environment," said Nicolas Rohner, a geneticist at Harvard Medical School in Boston, Massachusetts. "This evolutionary process would take too long." To rapidly adapt to a sudden shift in environment, a population would have to have some kind of standing genetic variation already available, which nature then selects for.

But how can this "cryptic variation" be maintained and accumulate in a species without actually affecting individuals before the environmental shift occurs? Scientists proposed that something must keep the genetic variation silent under normal conditions; then, when a species relocates to a new, life-threatening environment, it becomes physiologically stressed and that silencing mechanism stops or breaks down somehow.


Recently, Rohner's colleague Susan Lindquist, a biologist at the Whitehead Institute for Biomedical Research in Cambridge, Mass., discovered a potential silencing mechanism: Heat shock protein 90 (HSP90), a chaperone protein that helps other proteins fold properly. Under normal conditions, HSP90 is present in high concentrations β€” much higher than is required to maintain the proteins under its wing. Because of this, HSP90 can act as a buffer, which keeps a lid on the changes that would otherwise arise due to the genetic variants of the proteins.

However, because protein folding is so sensitive to environmental stress, HSP90's reservoirs become depleted under stressful conditions, interrupting its silencing ability and allowing new phenotypes (functional traits) to rapidly emerge. In the lab, Lindquist and her research team showed that this process occurred in a variety of simple organisms, including fruit flies, yeast and plants. But some scientists were critical of the work.

"The main criticism was that the traits that they showed were not all adaptive traits, so people were skeptical about HSP90," Rohner told io9. "Also, no one really knew what ecological scenario would work with the work she did."


So Rohner, Lindquist and their colleagues set out to find a real-world example of HSP90's evolutionary role. They decided to look at blind cavefish.

The Eyeless Socket

The Mexican tetra, Astyanax mexicanus, is a freshwater fish found mainly in the rivers throughout central and eastern Mexico, as well as in a few rivers in the United States. At some point in the fish's distant past, populations of A. mexicanus got trapped in pitch-black, underwater caves. In their new β€” and vastly different β€” habitats, the fish lost their pigmentation and their eyes, and became better able to store energy and detect changes in water pressure (to find prey and each other).



Left: Cave and surface forms of the Mexican tetra. Credit: Nicolas Rohner.

The trait the cavefish are best known for β€” their eyeless sockets β€” was an adaptive change, Rohner explained. Without eyes, the fish can put more energy towards growth and reproduction, rather than using that energy to maintain the useless structures. Additionally, the eyes are easy points of entry for infections. "In that environment, losing your eyes gives you a little bit of an edge," he said.

Interestingly, there are still many surface populations of the Mexican tetra. These fish and their blind counterparts are still considered the same species, and can even interbreed. In effect, A. mexicanus was the perfect model to study, Rohner said.


The researchers decided to only focus on investigating HSP90's potential role in the fish's loss of eyes. They raised surface fish in the presence of a drug called Radicicol, which inhibits HSP90, essentially mimicking a stressful environment. As adults, the fish showed a significant variation in eye size β€” some of the fish had larger or smaller eyes than are ever seen in normal populations. This result means that HSP90 indeed acts on this specific trait (eye size).

The researchers then repeated the experiment with cavefish. The results were completely different: The adult fish had no great variation in eye orbit size (the cavity in the skull where the eye used to be). Instead, the fish all developed smaller orbit sizes.

"If HSP90 played a role in cavefish evolution, then there would be a selection on certain alleles (genetic variants) that are responsive to HSP90," Rohner said. "This finding shows that the alleles that have been selected upon β€” the alleles that made smaller eyes β€” are the ones that are the target of HSP90."


Next, the team wanted to see if the cryptic variation in eye size that they uncovered by inhibiting HSP90 could be "genetically assimilated," or passed on down to subsequent generations. They bred normal surface fish with Radicicol-treated surface fish that developed small eyes (rather than large ones). The offspring β€” which were not raised in the presence of the drug β€” all had eyes that were smaller than any untreated fish and comparable with the smallest eyes of the drugged fish.

"This showed that the original traits that we saw were really genetic and not just the inhibitor messing with the fishes' development," Rohner said.

A Stressful New Environment

Though the researchers demonstrated that a kind of chemically induced "stress" could result in the expression of cryptic variation, there was still the question of whether a surface fish that suddenly found itself trapped in the cave ecosystem would experience an HSP90-related stress response. To find out, the researchers took a close look at the ecological differences between the surface-fish and cavefish habitats, including pH, oxygen content and temperature.


Above: Two of A.mexicanus' environments β€” Nacimiento del Rio Choy and Tinaja Cave. Credit: Rohner et al., Science.

The biggest difference between the two environments, they found, was conductivity, which is measured as salinity. For fish, water conductivity is extremely important β€” if you put a freshwater fish into the ocean, it probably won't survive. "Humans have skin that doesn't let water through, but conductivity is a real problem for fish," Rohner said. "This is a major stress for them." The team discovered that the conductivity was much lower in the caves than in the rivers, suggesting that surface fish that suddenly wound up in the caves would become physiologically stressed and experience HSP90-related responses.


The scientists tested this idea by rearing surface fish in water with the same conductivity as one of the cave ecosystems. As adults, the river fish showed a variation in eye size, just as they did when under the effects of the HSP90-inhibiting drug.

Altogether, the study strongly suggest that the surface fish originally had a type of cryptic variation for eye size, which was masked by HSP90. When the fish somehow wound up in the caves, the low conductivity β€” and likely other environmental factors β€” physiologically stressed the fish, depleting their levels of HSP90. The eye-size variation became unmasked, and nature quickly selected for the adaptive small-eye phenotype. Subsequent generations had smaller and smaller eyes, while the fish also became better adapted to the new environment, allowing their HSP90 levels to eventually return to normal.

Importantly, the researchers have demonstrated evidence for the cryptic variation mode of morphological evolution. "Usually when people think about mechanisms of evolution, they think about coding changes, regulatory changes and other things, but this is a different mechanism that acts through standing genetic variation," Rohner said. "This evidence will hopefully help in convincing people that this might be a valid mechanism β€” at least in cases where you are dealing with rapid changes in the environment."


Check out the study over in the journal Science.

Top image via H. Zell/Wikipedia Commons.