Longest Evolution Experiment Dead-End

The industry’s longest-running research experiment reached a milestone in October, studying the evolution of more than 68,000 generations.

Biologist Richard Lenski (pictured right below) initiated the now legendary experiment in his laboratory early in 1988, using 12 flasks seeded with genetically identical bacteria known as Escherichia coli (E. coli).

Since then, the bacteria have been growing in a carefully measured solution of glucose, a type of sugar—”food” for bacteria. Each flask contained a sparse amount of glucose to create a stressful environment, along with a high concentration of citrate, a molecular cousin of glucose, which pushed the bacteria to adapt. Since 1988, Lenski’s laboratory team has transferred a small sample of the new 50 mL Erlenmeyer flasks every day.

Although wanting to quit this laborious experiment many times, Lenski has continued this process non-stop for over 30 years, now recognized as the industry’s longest evolution experiment dead-end.

Model

Bacteria have played a crucial role in the study of evolution. The University of Colorado’s website, entitled Bacteria as a Model System asserts that bacteria have “been one of the great vindications of the theory of evolution.”

Enduring the permeating annoyance of E. coli’s infamous reeking, putrid smell, Richard Lenski’s (right) lab at Michigan State University embraced a design for what has become the most widely recognized experiment to test the star of the evolution industry. Bacteria offer scientists a fast-forward glimpse of evolution.

Reproductive rates, often measured in doubling times, allow scientists to track and trend changes over short periods of time. The higher the doubling time, the greater the number of generations available to study over the same time. In bacteria, the doubling time averages 20 minutes, whereas in humans, it requires approximately 20 years. Writing for Science Alert, Fiona MacDonald puts the comparison between bacteria and human evolution into context –

“Scientists have spent the past 30 years carefully tracking evolution across more than 68,000 generations of E. coli bacteria – the equivalent of more than 1 million years of human evolution.”

Freezer

As crucial as doubling times are in studying evolution, the key that escalated Lenski’s project from observation to a revolutionary experiment started with the laboratory freezer. The freezer holds the registry of each flask’s common ancestor – Darwin’s key to evolution.

Every 75 days, equivalent to approximately 500 generations, Lenski’s lab team systematically transfers 1% of the E. coli from each of the 12 flasks into the freezer for storage at -112 degrees Fahrenheit – high-tech fossilization. From these frozen ancestors, the team can trace the molecular changes over time to detail E. coli’s mechanisms of evolution.

Fast-Forward

Bacteria offer scientists a fast-forward method for studying evolution. With modern molecular technology, the behavior of bacteria can be observed and measured – feats that were not possible even a decade ago. Of critical importance, the experiment can be repeated. Repeatability is essential for validating a theory as a scientific fact.

A scientific theory, to be valid, must accurately predict what is going to happen 100% of the time, like Isaac Newton’s falling apple theory—gravity. Lenski’s study model provides scientists with a perfect opportunity. Marlene Cimons, writing for the National Science Foundation in the article “E. coli Offers Insight to Evolution,” explains –

“The experiment was designed to ask about the repeatability of evolution.”

Currently, the validity of current evolution theories is plagued by the need to reconcile the dynamic between an unknown, unguided, and random process and the guarantee of a repeatable outcome. If the recording tape of evolution could be replayed, what happened before would have to happen again. As Lenski explains in an interview with Cimons,

“If we look at the tension between the randomness of mutation and the predictability of natural selection, how does evolution play out when you put the two together… That’s really what this long-term experiment has been all about.”

In recognition of Lenski’s work, the National Center for Science Education (NCSE) awarded the 2017 Friend of Darwin Award to Lenski for his E. coli Long-Term Experimental Evolution Project. NCSE’s executive director Ann Reid said –

“It would be hard to think of anybody who has done as much to show that evolution is among the experimental sciences as Rich Lenski.”

Keep Getting Bigger

Lenski’s team observed a rapid increase in E. coli cell size in all 12 flasks from the beginning in 1988, and the cells continued to grow larger over the years. Since a consistent increase in size was observed in all flasks, this supports a significant scientific requirement – repeatability – which is not an insignificant finding.

Not only did the cell size increase, but the increase in cell size also developed despite living in a sparse glucose environment (left). The observed growth indicates an increase in fitness over time, as Charles Darwin had predicted. In an interview with his publisher, W.W. Norton & Company, Lenski explains –

“One result is that the average fitness in each population increases over time… the same process that Darwin discovered.”

From his Down House library, Darwin wrote out his fitness argument in The Origin of Species –

“This tendency… to go on increasing in size… has prevailed throughout all time… [and] is utterly inexplicable on the theory of creation. As natural selection acts solely by accumulating slight, successive, favourable variations.”

With his observations verified, Lenski legitimately argued for the repeatable nature of evolution in Proceedings of the National Academy of Sciences –

“Evolution may thus be broadly repeatable.”

Today’s populations grow about 80% faster than the original lines in all flasks, a phenomenon Lenski calls “a beautiful example of adaptation by natural selection.”

Something New

The finding that getting bigger is the smaller part of Lenski’s findings is noteworthy. In 2008, around 16 years into the experiment and near generation 30,000, something new happened in flask #9. The solution became consistently cloudier and darker than the other flasks.

For Lenski, the bacteria seemed to have “evolved the capacity to exploit citrate (right)” – the only other energy source in the solution. At the time, what happened was a mystery. The step to develop a new metabolic process involves a level of complexity light-years more advanced than simply getting bigger. Using citrate was a game-changer.

The news ignited the evolution industry: finally, at last, evolution had the ever-elusive observable, measurable, and repeatable scientific evidence. Clues to what happened, however, were not to be known until 2012.

The use of citrate as an energy source was a molecular mystery, as E. coli can only utilize citrate in the absence of oxygen. In fact, E. coli growing on citrate in an anoxic environment – no oxygen – is one of its uniquely identifying characteristics. Microbiologists use citrate in non-oxygen environments to differentiate E. coli from other bacteria.

Finally, in 2012, following the advent of new molecular technologies, geneticist Zachary Blount (left, swinging) pinpointed the genetic mystery in the paper “Genomic analysis of a key innovation in an experimental Escherichia coli population,” published in Britain’s prestigious Nature journal.

Blount discovered E. coli’s key innovation – a genetic duplication. The sequences of DNA nucleotides in the region of the cit gene were duplicated, and the sequences were rearranged. With this new duplicated DNA section, the newly arranged promoter gene gained control over the blocking gene, allowing the use of citrate for energy – even in the presence of oxygen.

Harvard Gazette staff writer, Alvin Powell, jumped to speculate in 2014 “that one of the 12 bacterial lines he has maintained has developed into what he believes is a new species.” In the words of Lenski: “I would argue that citrate users are — or are becoming — a new species” – vindicating Darwin’s theory of evolution, it seemed.

Dead-End

Once touted as scientific evidence to support evolution, Lenski’s experiment has now reached a dead end. The persistent increase in the rate of E. coli cell size observed early in the study has not continued. Reporting in The Scientist, science writer Abby Olena notes Lenski’s problem –

“Between 40,000 and 50,000 generations, there was [only] a 3 percent increase in mean fitness.”

Along with the dead end of the cell size problem, only flask #9 has developed the ability to use citrate in Lenski’s laboratory since 2008; none of the other 11 flasks have developed the same mutation, raising questions about the repeatability of evolution.

The repeatability issue, however, was resolved by Dustin Van Hofwegen (right) at the University of Idaho. Hofwegen observed the development of E. coli citrate-using mutants in as few as 12 generations.

The study, entitled “Rapid Evolution of Citrate Utilization by Escherichia coli,” was published in the Journal of Bacteriology in 2016. According to Hofwegen, Lenski’s long-term evolution experiment (LTEE) has reached an experimental dead end –

“Here we show why it probably was not a speciation event… We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved.”

Flask #9 was Lenski’s best shot for uncovering evidence for an evolutionary event amongst the estimated hundreds of millions of mutations over the past 30 years – translating to more than a million years of human evolution (sic). But the holy grail of evolution, speciation, never happened.

Laboratory experiments repeatedly demonstrate how bacteria, despite having an incalculable capacity for change, continue to behave as they have from the beginning. Bacteria have not “been one of the great vindications of the theory of evolution,” as touted by the University of Colorado.

By starting with E. coli and ending with E. coli, Lenski’s laboratory project now has the distinction of being the industry’s longest evolution experiment dead-end.

Genesis

Despite a flood of challenges since the publication of The Origin of Species, the scientific evidence found in nature is increasingly best explained by the Genesis record written by Moses.

Louis Pasteur (pictured left), a French chemist and microbiologist who was one of the most important founders of medical microbiology, was driven to today’s increasingly popular conclusion during the Scientific Revolution –

“Science brings one nearer to God.”

Evidence from the fields of extinction and speciation, which validate the theory of evolution scientifically, remains speculative.

Longest Evolution Experiment Dead-End is an Extinction and Speciation article.

Darwin Then and Now is an educational resource on the intersection of evolution and science, highlighting the ongoing challenges to the theory of evolution.

Move On

Explore how to understand twenty-first-century concepts of evolution further using the following links –

- The Understanding Evolution category showcases how varying historical study approaches to evolution have led to varying conclusions. Subcategories include –
  - Studying Evolution explains how key evolution terms and concepts have changed since the 1958 publication of The Origin of Species.
  - What is Science explains Charles Darwin’s approach to science and how modern science approaches can be applied for different investigative purposes.
  - Evolution and Science feature study articles on how scientific evidence influences the current understanding of evolution.
  - Theory and Consensus feature articles on the historical timelines of the theory and Natural Selection.
- The Biography of Charles Darwin category showcases relevant aspects of his life.
- The Glossary defines terms used in studying the theory of biological evolution.

2020 Update

On May 4, 2020, Lenski announced a five-year renewal of the grant through the National Science Foundation’s Long-Term Research in Environmental Biology (LTREB) Program, which supports the LTEE. He also announced that Dr. Jeffrey E. Barrick, an associate professor of Molecular Biosciences at The University of Texas at Austin, would take over supervision of the experiment within the 5-year funding period.

The experiment’s time at Michigan State University ended in May 2022, when the populations reached 75,000 generations, and it was then transferred to Barrick’s lab. The experiment was revived and restarted in Barrick’s lab on June 21, 2022.