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Small, colorless, and blind, amblyopsid cavefishes inhabit subterranean waters throughout the eastern United States. In a new study, Yale researchers reveal insights into just how these distinctive cave dwellers evolved—and provide a unique method for dating the underground ecosystems where they reside.

In an analysis of the genomes of all known amblyopsid species, the researchers found that the different species colonized caves systems independently of each other and separately evolved similar traits—such as the loss of eyes and pigment—as they adapted to their dark cave environments.

Their findings are in the journal Molecular Biology and Evolution.

By studying the genetic mutations that caused the fishes' eyes to degenerate, the researchers developed a sort of mutational clock that allowed them to estimate when each species began losing their eyes. They found that vision-related genes of the oldest cavefish species, the Ozark cavefish (Troglichthys rosae), began degenerating up to 11 million years ago.

The technique provides a minimum age for the caves that the fishes colonized since the cavefish must have been inhabiting subterranean waters when their eyesight began devolving, the researchers said.

"The ancient subterranean ecosystems of eastern North America are very challenging to date using traditional geochronological cave-dating techniques, which are unreliable beyond an upper limit of about 3 to 5 million years," said Chase Brownstein, a student in Yale's Graduate School of Arts and Sciences, in the Department of Ecology & Evolutionary Biology, and the study's co-lead author.

"Determining the ages of cave-adapted fish lineages allows us to infer the minimum age of the caves they inhabit because the fishes wouldn't have started losing their eyes while living in broad daylight. In this case we estimate a minimum age of some caves of over 11 million years."

Maxime Policarpo of the Max Planck Institute for Biological Intelligence and the University of Basel is the co-lead author.

For the study, the researchers reconstructed a time-calibrated evolutionary tree for amblyopsids, which belong to an ancient, species-poor order of freshwater fishes called Percopsiformes, using the fossil record as well as genomic data and high-resolution scans of all living relevant species.

All the cavefish species have similar anatomies, including elongated bodies and flattened skulls, and their pelvic fins have either been lost or severely reduced. Swampfish (Chologaster cornuta), a sister to cavefish lineage that inhabits murky surface waters, also has a flattened skull, elongated body, and no pelvic fin. While it maintains sight and pigment, there is softening of the bones around its eyes, which disappear in cavefishes. This suggests that cavefishes evolved from a common ancestor that was already equipped to inhabit low-light environments, Brownstein said.

To understand when the cavefish began populating caves—something impossible to discern from the branches of an evolutionary tree—the researchers studied the fishes' genomes, examining 88 vision-related genes for mutations. The analysis revealed that the various cavefish lineages had completely different sets of genetic mutations involved in the loss of vision. This, they said, suggests that separate species colonized caves and adapted to those subterranean ecosystems independently of each other.

From there, the researchers developed a method for calculating the number of generations that have passed since cavefish species began adapting to life in caves by losing the functional copies of vision-related genes.

Their analysis suggests that cave adaptations occurred between 2.25 and 11.3 million years ago in Ozark cavefish and between 342,000 to 1.70 million years ago (at minimum) and 1.7 to 8.7 million years ago (at maximum) for other cavefish lineages. The findings support the conclusion that at least four amblyopsid lineages independently colonized caves after evolving from surface-dwelling ancestors, the researchers said.

The maximum ages exceed the ranges of traditional cave-dating methods, which includes isotope analysis of cosmogenic nuclides that are produced within rocks and soils by cosmic rays, the researchers noted.

The findings also suggest potential implications for human health, said Thomas Near, professor of ecology and evolutionary biology in Yale's Faculty of Arts and Sciences (FAS), and senior author of the study.

"A number of the mutations we see in the cavefish genomes that lead to degeneration of the eyes are similar to mutations that cause ocular diseases in humans," said Near, who is also the Bingham Oceanographic Curator of Ichthyology at the Yale Peabody Museum. "There is the possibility for translational medicine through which by studying this natural system in cavefishes, we can glean insights into the genomic mechanisms of eye diseases in humans."

The other co-authors are Richard C. Harrington of the South Carolina Department of Natural Resources, Eva A. Hoffman of the American Museum of Natural History, Maya F. Stokes of Florida State University, and Didier Casane of Paris-Cité University.