In order to evolve novel traits, organisms may depend upon smaller, less dramatic mutations that they amass through their evolutionary history rather than suddenly acquiring a single mutation that gives them drastically different phenotypes, according to a study published online today (Jun 2) in PNAS.

Whether an organism arrives at major evolutionary innovations through a single key mutation or a history of many accumulated mutations has been hotly debated by evolutionary biologists. The findings are "perhaps the most rigorous, clear-cut demonstration of the role of historical contingency," in evolution, said Richard Lenski of Michigan State University, the study's main author.

Examining E. coli cultures that his lab has maintained since 1988, Lenski found that one population of the bacterium had evolved the ability to metabolize citrate -- an unprecedented trait -- after more than 30,000 generations, or approximately 15 years.

So Lenski and his colleagues consulted their "frozen fossil record," consisting of preserved individuals collected from the bacterial population at several points throughout its evolution. They revived bacteria from different stages of the population's 20-year evolution and "replayed" evolution in each. Clones sampled in the first 15,000 generations never evolved the ability to metabolize citrate (Cit+), while clones taken from later populations did evolve the characteristic.

That means this big evolutionary jump relied on a chain of genetic events that took place over the course of the population's evolutionary history, Lenski said.

"It's a very elegant demonstration that major changes may depend on accretion of minor changes before hand," said Albert Bennett, a University of California, Irvine evolutionary physiologist who gave Lenski feedback on the study before it was published in PNAS. "What's really demonstrated here is that the way has to be paved before hand."

Lenski and his collaborators suggested two possible mechanisms that could have lead to the Cit+ phenotype: the population either accumulated interacting mutations that added up to the functional ability to metabolize citrate, or random mutations, such as insertions, created altered DNA substrates that were then subjected to further mutations, yielding the unique facility.

Both Lenski and Bennett said that they tend towards the former, functional, explanation for the evolutionary jump. "Presumably, this is how metabolic pathways got built up," said Bennett.

Lenski cautions, however, that his findings do not fully resolve the question of whether evolution is a result of random or deterministic processes. "The real world is a mixture of these two processes," he said. "We're able to see the importance of both perspectives simultaneously."

Still, the results suggest that major evolutionary advances are likely to depend on the past evolutionary history of an organism. "This suggests that key innovations may be things that are historically contingent," Lenski said.

The researcher now plans to delve deeper into his evolutionary reconstruction experiments, elucidating exactly which mutations led directly to the Cit+ phenotype in his E. coli population. "The key will be to find the one or more mutations that, at the time they occurred, produced the citrate mutation," he said. Lenski will also turn the dial further back to identify those mutations that preceded the emergence of the Cit+ trait. "It's going to be rather tricky to sort out which mutations it was," admitted the researcher.