Scientist Recommends Research Method Change For Evolutionary Biology
February 16, 2013

Scientist Recommends Research Method Change For Evolutionary Biology

April Flowers for — Your Universe Online

Shozo Yokoyama, a biologist at Emory University, says evolutionary biologists need to shift their focus from present-day molecules to synthesized, ancestral ones to truly understand the mechanisms of natural selection.

Yokoyama presented evidence to support his claim at the American Academy of Arts and Sciences (AAAS) meeting in Boston this week.

"This is not just an evolutionary biology problem, it's a science problem," says Yokoyama, a leading expert in the natural selection of color vision. "If you want to understand the mechanisms of an adaptive phenotype, the function of a gene and how that function changes, you have to look back in time. That is the secret. Studying ancestral molecules will give us a better understanding of genes that could be applied to medicine and other areas of science."

Yokoyama notes positive Darwinian selection has been studied for years almost exclusively using comparative sequence analysis of present-day molecules, an approach fueled by increasingly fast and cheap genome sequencing techniques. Faster and easier, says Yokoyama, are not always best if you want to arrive at a true, quantitative result.

"If you only study present-day molecules, you're only getting part of the picture, and that picture is often wrong," he says.

Studying fish and other vertebrates, Yokoyama has spent two decades teasing out secrets of the adaptive evolution of vision.

There are five classes of opsin genes that encode visual pigments. They are also responsible for dim-light and color vision. Since the available light at various ocean depths is well quantified, fish provide valuable clues for how environmental factors can lead to vision changes. For example, the common vertebrate ancestor possessed ultraviolet vision, suited to both shallow water and land.

"As the environment of a species sinks deeper in the ocean, or rises closer to the surface and moves to land, bits and pieces of the opsin genes change and vision adapts," Yokoyama says. "I'm interested in exactly how that happens at the molecular level."

Molecular biologists construct a specific visual pigment by taking DNA from an animal, isolating and cloning its opsin genes, then using in vitro assays to create the pigment that can then be manipulated by changing the positions of the amino acids. This allows the scientists to study the regulation of the gene's functions.

Yokoyama, for example, identified three specific amino acid changes in 1990 that switch the human red pigment into a green pigment. In an interesting twist, a separate group of researchers confirmed Yokoyama's findings a few years later, but found the three changes only worked when going from red to green. To reverse the process from green to red took seven changes.

"They discovered this weird quirk that didn't make sense," Yokoyama says. "Why wouldn't it take the same number of changes to go in either direction? That question was interesting to me."

Yokoyama found it interesting enough to devote the next ten years researching and pondering the question before he realized the essential problem: the experiments were conducted on present-day molecules.

About 100 million years ago, when the first mammalian ancestors appeared, only the red pigment was present. The gene for the red pigment duplicated itself in some primates approximately 30 million years ago. Subsequently, one of the red pigment duplicates acquired sensitivity to green, turning into a green pigment.

"At the point that the three changes in amino acids occurred in this pigment, other mutations were happening as well," Yokoyama says. "You have to understand the original interactions of all of the amino acids in the pigment, which means you have to look at the ancestral molecules. That's the trick."

Just as changes in an animal's environment drive natural selection, changes in the animal's molecular environment do as well.

Yokoyama and his team used statistical analysis to travel back in time and estimate the sequences for ancestral molecules.

"It's a lot of work," he says. "We don't have a clear picture of every intermediate species. We have to do a step-by-step retracing, screening for noise in the results at each step, before we can construct a reliable evolutionary tree."

Yokoyama and his team led an effort to construct the most extensive evolutionary tree for dim-light vision in 2008. This group includes animals from eels to humans. Yokoyama's lab engineered ancestral gene functions at key branches of the tree, in order to connect changes in the living environment to the molecular changes.

Synthesizing ancestral proteins and pigments, and conducting experiments on them, is a lengthy process that combines microbiology with theoretical computation, biophysics, quantum chemistry and genetic engineering.

In 2009, this multi-disciplinary approach allowed Yokoyama's team to identify the scabbardfish as the first fish known to have switched from ultraviolet vision to violet vision. The team pinpointed how that switch was made, by deleting an amino acid molecule at site 86 in the chain of amino acids in the opsin gene.

"Experimenting on ancestral molecules is the key to getting a correct answer to problems of natural selection, but there are very few examples of that being done in evolutionary biology," Yokoyama says.