Limits on evolution

[M.D. Holloway]

Charles Darwin didn’t know about genes and DNA. In fact, hardly anyone noticed when Gregor Mendel, a monk whose pea experiments eventually led to modern genetics, published his findings in an obscure journal a few years after Darwin’s On the Origin of Species appeared in 1859. It would take nearly a century more before James Watson and Francis Crick deciphered the structure of DNA, the molecule that contains the manual for building an organism. Yet Darwin was still able to describe a mechanism — natural selection — for how evolution shapes life on Earth. That’s like describing how a car works without knowing about the existence of internal combustion engines.

But while Darwin achieved his insights without molecular help, biologists today are intimately familiar with the molecules responsible for the diverse array of plants, animals and other organisms that populate the planet. The study of genes has revealed evolution as essentially a high-stakes poker game in which organisms draw randomly from a deck of genetic choices. At stake is the chance to pass along genes to the next generation. Sometimes the hand is good enough to get ahead in the game, but some hands are losers, perhaps to the extent of extinction. By studying the winners, scientists are learning how the forces of evolution work on DNA, the biochemical repository of an organism’s entire natural history. DNA records the mutations that helped some animals to survive ice ages while others perished, the nips and tucks that make animals more attractive to mates, the big leaps that allowed plants to become domestic crops — they’re all there, written out in a simple alphabet of four letters.

Each organism has its own book of life, but it’s not a just-so story. The genomes of living things are constantly undergoing editing and revision. And each individual has its own edition of its species’s book, shaped by natural selection and the other, perhaps less-appreciated forces of genetic mutation, recombination and drift.

In recent years, the U.S. National Institutes of Health and private companies have sponsored programs to build a library of species’ books, with projects to decode the genomes of humans, chimpanzees, bonobos, dogs, cats, cows, duck-billed platypuses, opossums, orangutans, bacteria, fungi, corn, wheat, bees, fruit flies, worms and a menagerie of other creatures large and small. Comparing the genetic records from these genomes will help researchers piece together a history of how Earth’s current life evolved. But that work has barely begun, and many questions remain to be answered.

Scientists, for example, still don’t know how cells evolved, including how former bacteria came to live inside cells as mitochondria and chloroplasts. (Mitochondria are tiny power plants that supply cells with energy, and chloroplasts are the organelles in plant cells that carry out photosynthesis.) Another mystery is how the complex structure of genes in eukaryotic organisms — in which the genetic material is encased in a nucleus — evolved. Researchers also debate how the shapes and forms of organisms came to look as they do. One of the biggest unanswered questions is whether life on Earth was destined to evolve the way it has.

Limits on evolution

At some time in your life you’ve probably asked yourself if, given a chance to do it all again, you would do it the same way.

Scientists have been asking the same question about evolution, but they’ve been getting different answers.

Play a poker game, rewind it to the beginning, start again and see what happens. Would the game play out the same every time? Stephen Jay Gould, the late evolutionary biologist, didn’t think so. If you replay the game, the shuffled cards will turn up a little different each time, and the order in which the cards are drawn can have profound consequences for the outcome. Replaying the “tape of life” from some point in the past would produce very different life-forms than the ones we have today, Gould thought.

[M.D. Holloway]

Other scientists disagree. Organisms are dealt a finite number of genes and so must choose from a limited menu of evolutionary options, narrowing the directions the organisms can go in a particular environment. “The evolutionary routes are many, but the destinations are limited,” says Simon Conway Morris, a paleontologist at the University of Cambridge in England. As a result, disparate organisms often end up independently developing the same sorts of structures to solve a particular problem. Take eyes. Although the details of how eyes work vary between species, the basic structures are similar.

But since it’s impossible to turn back time (no matter how easy Superman makes it look) and replay all of evolution again, scientists have devised other ways of investigating the issue.

Richard Lenski, an evolutionary biologist at Michigan State University in East Lansing, is among the scientists hitting the rewind button on evolution. Meter-high letters taped to the windows of his lab spell out the lab’s motto: EVOLVE. In the center of the “O,” the face of Charles Darwin peers out toward the football stadium.

Inside the lab, a dozen glass flasks containing what looks like clear liquid swirl in a temperature-controlled incubator. Although the naked eye can’t see them, millions of E. coli bacteria grow in the flasks, doing what the window exhorts. Lenski started the cultures in 1988, intending to follow the course of natural selection for several hundred generations. Now, 20 years later, the cultures are still growing and have produced more than 45,000 generations of bacteria each.

Lenski inoculated each of the 12 flasks with bacteria from the same ancestor, so they all started out with the same genetic deck of cards. Only one gene differed among the bacteria at the beginning — six flasks contain a marker gene that makes the bacteria red when grown on certain media while bacteria in the other six grow white. The marker gene doesn’t affect the strains’ fitness — the term biologists use to describe the capacity of an organism to compete against others to pass on its genes — but it does help researchers distinguish between two lineages of the bacteria during competition experiments.

Each flask contains media with only the minimum requirements for survival — some glucose (a sugar that bacteria use for food) and a few other nutrients. The bacteria replicate, or divide, six or seven times daily, creating a new generation with each round. Each division shuffles the cards and produces genetic changes and mutations, some of which may help or hurt the bacteria’s ability to compete for glucose and win the evolutionary poker game. The next day, a dilution is done, with 10 percent of the culture within a flask transferred to a new flask, and a new hand is played. (Every 500 generations or so, the remaining 90 percent is frozen and stored for later experiments.) The dilution acts as a population bottleneck, randomly selecting a subset of the bacteria (and so a subset of accumulated genetic changes) to continue the experiment.

These 12 flasks “represent the stripped-down bare essentials of evolution,” says Zachary Blount, a graduate student in Lenski’s lab. The environment never changes. No new genes enter the system from migrating microorganisms. And the scientists take no action to affect the course of evolution within the flasks. Only the intrinsic, core processes of evolution influence the outcome, Blount says.

Lenski and his colleagues have watched the game play out, occasionally analyzing DNA to peer over the players’ shoulders and find out what cards they hold. On the surface, the populations in the 12 flasks seem to have traveled similar paths — all have grown larger and become more efficient at using glucose than their ancestors. And many of the strains have accumulated mutations in the same genes. Notably, though, no one strain has developed exactly the same genetic changes as another.

Randomness is an important part of the evolutionary equation, as the experiment illustrates. During the first 2,000 generations, all of the 12 populations rapidly increased in size and fitness. But then cell size changes and reproductive and glucose efficiency gains began to slow down, hitting the evolutionary equivalent of a dieter’s plateau.

[M.D. Holloway]

After 10,000 generations, it became apparent that not all the flasks were on the same trajectory. Though the cells in all the flasks became larger, each population differed in the maximum size the cells reached. The populations also differed in how much fitter they were than their ancestors, when the researchers grew them in direct competition. The “experiment demonstrates the crucial role of chance events (historical accidents) in adaptive evolution,” Lenski and his colleague Michael Travisano wrote in a 1994 paper.

The experiment has progressed, and several of the flasks now contain mutater strains, bacteria that have defects in their DNA replication system. Such defects make mistakes more likely to happen every time those bacterial strains copy their DNA to divide. Sometimes a mistake can have lethal consequences, damaging a gene crucial for survival. But other times coloring a bit outside the lines creates opportunity for advancement.

Even within a given flask, some bacteria take slightly different paths. One flask now contains two separate strains — one that evolved to make large colonies when grown on petri dishes, and one that makes small colonies. The large- and small-colony strains have coexisted for more than 12,000 generations. The large-colony producers are much better at using glucose so they grow quickly, but they make by-products that the small-colony producers can eat. Each of the populations, both large and small, have improved their ability to use glucose over the generations. By at least one measure, the two populations could constitute separate species, Lenski and his colleagues proposed in 2005 in the Journal of Molecular Evolution.

Still, it seems that Conway Morris was basically right: Though the details were different, replaying evolution in a dozen flasks produced very similar outcomes in each. But then something completely unexpected happened.

After about 31,500 generations, glucose-eating bacteria in one of the flasks suddenly developed the ability to eat a chemical called citrate, something no other E. coli do, the researchers reported last June in the Proceedings of the National Academy of Sciences (SN Online: 6/2/08).

“They’ve been eating the main course for thousands of generations,” Lenski says. “They didn’t realize that there was a dessert tray around the corner.”

The switch was clearly a radical change of destination for the bacteria. The inability to eat citrate is a biochemical hallmark of the E. coli species, so by some definitions, the citrate eaters in that flask are no longer E. coli, but a different species.

But a single change did not a citrate eater make. The researchers found that the bacteria went through a series of steps before evolving the ability to use citrate. One initial mutation happened at least 11,000 generations before the citrate eaters appeared. Lenski and his crew don’t yet know which specific DNA changes led to citrate use, but the researchers have enough evidence to say that the ability to use citrate is dependent, or contingent, upon those earlier changes. And even the bacteria that have undergone those initial changes are still not guaranteed to find the dessert cart. Blount tested 40 trillion bacteria from earlier generations to see if any could evolve the ability to eat citrate. Fewer than one in a trillion could.

The profound difference between the citrate eaters and the other 11 strains, as well as the dependence of the citrate change on earlier mutations, seems to suggest that Gould was also right: Replaying evolution will result in some surprise endings. “The long-term evolution experiment with E. coli provides some of the best evidence for both Conway

[M.D. Holloway]

Morris and Gould that one could ever hope to see,” Lenski says. “Conway Morris ‘wins’ based on the number of changes that fit his pattern, but Gould might prevail if weighted by the profundity of change. Both perspectives are important contributions, and they are not mutually exclusive.”

Now the researchers are watching to see if citrate-eating bacteria will evolve in other flasks, and if citrate eaters will eventually reject glucose and feast only on citrate. Such a transformation would probably herald the birth of a new species. “It would be amazing,” says Blount. “It would be like teenagers who no longer like to eat pizza — they prefer broccoli.”

When the game changes

While Lenski’s experiment takes place in a constant environment, natural evolution must cope with a messier reality. In Steven Finkel’s lab at the University of Southern California in Los Angeles, a long-term experiment is showing how evolution plays out in a closed and ever-changing environment, more like the real world. This allows Finkel to focus on how evolution and environment are interwoven.

Finkel didn’t start out to test evolution in changing environments. The experiment was prompted by a graduate student’s casual remark that E. coli will live a long time. Finkel asked, “How long?”

“A long time,” the student responded.

“So we set up some experiments to see how long they would live, and they just would never die,” Finkel says. The immortal bacterial cultures are teaching scientists a few things about how organisms change their environments and adapt to changes wrought by outside forces.

Some of the cultures in Finkel’s laboratory have been growing for more than five years. The bacteria started out with the same genetic background, or so the researchers like to tell themselves. In reality, each flask started with a single bacterial colony, containing perhaps 50 billion individuals. Given that DNA replication systems aren’t perfect, one in every 10,000 cells probably carries a typo in at least one letter of its instruction manual. Such DNA typos are known as point mutations.

Finkel and his colleagues placed the bacteria in a rich broth full of sugars and many other nutrients and then just let them grow. After a short initial lag, the bacteria began growing like gangbusters, a phenomenon known to microbiologists as “log phase” because the bacteria increase their numbers logarithmically. Once the nutrients start to run out, the bacteria stop growing so quickly and settle into a senescent state. After a few days, millions of bacteria die, spilling their guts into the surrounding media and providing food for survivors.

[M.D. Holloway]

It’s the postapocalyptic survivors that interest Finkel. As 99 percent of their comrades die off, the surviving bacteria feed on the carcasses of the dead and on metabolic by-products of other survivors. Thus the bacteria change the environment in which they live. It doesn’t take long for the cultures in each flask to go their own ways. Within a month, the bacteria in the various flasks convert the light honey color of the broth into a spectrum ranging from light amber to dark amber,

Finkel says. And his nose tells him the cultures are different as well. Microscope examinations reveal that the originally rod-shaped bacteria take on a wide variety of shapes; in one flask, some of the cells never cut the apron strings during cell division, forming long strands resembling linked sausages.

Yet as different as the bacterial populations appear, they also have something in common. All of the cells that have gone through the valley of death and come out the other side are tougher than naive bacteria. And the older the cells get, the more competitive they are, so that 20-day–old cells will drive 10-day–old cells to extinction, and 30-day–old cells beat 20-day–olds. Finkel calls that phenomenon “growth advantage in stationary phase,” or GASP.

On the surface it appears that the number of surviving cells stays constant. But underneath, different mutants rise and fall in number, like waves crashing on the beach, Finkel showed in a 2006 review published in Nature Reviews Microbiology.

The ability for older cells to compete better has been traced to mutations in four genes. Three of the genes allow the bacteria to feast more readily on certain amino acids. One of the genes encodes a key protein, RpoS, needed to turn on stress-response genes. The protein gives the green light to turn on genes under certain conditions. When cells are under stress — for bacteria, stress means high salt, low or high temperatures, broth that is too acidic or alkaline, or other environmental extremes — RpoS turns on genes that help the bacteria cope. But the protein is not necessary when cells aren’t under stress. In fact, it takes resources away from the cells’ main “go” signal, RpoD, a protein critical to normal function. Inactivating or handicapping RpoS makes more resources available for other genes.

Many of the GASP cells contain changes in rpoS, the gene for RpoS, but they don’t all have the same change, Finkel and colleagues reported in 2003. Nearly all of the changes reduce activity of RpoS to 1 percent or less of its normal activity but don’t abolish it entirely. Low levels of RpoS are a fixture in bacterial populations that have GASP.

But just because a mutation serves an organism well under some conditions doesn’t mean it’s always beneficial. Thomas Ferenci, a microbiologist at the University of Sydney in Australia, reviewed what happens to rpoS mutants under a variety of environmental conditions in the May 2008 Heredity. Depending on a cell’s genetic background, an rpoS mutation might give the strain a big boost in fitness or make an undetectable difference. And even if the mutations are beneficial under most conditions, the changes hold the bacteria back when the environment changes. If salt concentrations go up, the temperature drops, bacteria lack oxygen or encounter a toxin, then rpoS mutants, less able to cope with certain types of stress, don’t become established members of the community as quickly as they do under other conditions.

Natural selection works for rpoS mutants in some environments and against them in other conditions. “Selection is a deterministic force pushing relentlessly in one direction,” says Michael Lynch, an evolutionary biologist at Indiana University in Bloomington. That direction is toward ever-greater adaptation for the environment in which a population finds itself. But most environments are in a constant state of flux and, as Darwin was careful to point out in his introduction to the Origin of Species, selection isn’t the only evolutionary force at work.

[Heel n Toe]

You mentioned something about pain meds in another thread... is caffeine involved, too?

[red-beard]

I think he is subjecting us to his new books.

[RWebb]

I suspect there has been some mutation.

[Noah930]

2 comments:

1) Gregor Mendel faked his data.

2) Does this thread (Limits on Evolution) have anything to do with the teenagers becoming greater idiots with each generation one?

[bell]

Mama says aligators are ornery because they have all those teeth and no toothbrush

[Joeaksa]

Hey Mike,

Its about dark outside.

Please turn the computer off and go and spend some time with the family... before they put the jacket with the long sleeves on you and give you a ride to a nice quiet place for the weekend.

[Hebrewhomeboy]

Interesting read, thanks.

[GH85Carrera]

Everything has limits. Except your typing.

[sammyg2]

Lube, how long have you been up?

[creaturecat]

Ouch.
You O.K ?

[cgarr]

And from the time stamp he typed it all in one minute!

[Rikao4]

think he guzzled flask # 3..

Rika