Fortyfive Thousand Generations Of Evolution In The

The average generation turnover of those lizards is about two years, so the evolutionary change observed on Pod Mrcaru represents only about eighteen or nineteen generations. Just think what you might see in three or four decades if you followed the evolution of bacteria, whose generations are measured in hours or even minutes, rather than years! Bacteria offer another priceless gift to the evolutionist. In some cases you can freeze them for an indefinite length of time and then bring them back to life again, whereupon they resume reproduction as if nothing had happened. This means that experimenters can lay down their own 'living fossil record', a snapshot of the exact point the evolutionary process had reached at any desired time. Imagine if we could bring Lucy, the magnificent pre-human fossil discovered by Don Johanson, back to life from a deep-freeze and set her kind evolving anew! All this has been achieved with the bacterium Escherichia coli, in a spectacular long-term experiment by the bacteriologist Richard Lenski and his colleagues at Michigan State University. Scientific research nowadays is often a team effort. In what follows, I may sometimes use the name 'Lenski' for brevity, but you should read it as 'Lenski and the colleagues and students in his lab'. As we shall see, the Lenski experiments are distressing to creationists, and for a very good reason. They are a beautiful demonstration of evolution in action, something it is hard to laugh off even when your motivation to do so is very strong. And the motivation for dyed-in-the-wool creationists is very strong indeed. I'll return to this at the end of the section.

E. coli is a common bacterium. Very common. There are about a hundred billion billion of them around the world at any one time, of which about a billion, by Lenski's calculation, are in your large intestine at this very moment. Most of them are harmless or even beneficial, but nasty strains occasionally hit the headlines. Such periodic evolutionary innovation is not surprising if you do the sums, even though mutations are rare events. If we assume that the probability of a gene mutating during any one act of bacterial reproduction is as low as one in a billion, the numbers of bacteria are so colossal that just about every gene in the genome will have mutated somewhere in the world, every day. As Richard Lenski says, 'That's a lot of opportunity for evolution.'

Lenski and his colleagues exploited that opportunity, in a controlled way, in the lab. Their work is extremely thorough and careful in every detail. The details really contribute to the impact of the evidence for evolution that these experiments provide, and I am therefore not going to stint in explaining them. This means that the next few pages are inevitably somewhat intricate - not difficult, just intricately detailed. It would probably be best not to read this section of the book when tired, at the end of a long day. What makes it easier to follow is that every detail makes sense: none of it leaves us scratching our heads and wondering what that was all about. So, please come with me, step by step, through this splendidly constructed and elegantly executed set of experiments.

These bacteria reproduce asexually - by simple cell division - so it is easy to clone up a huge population of genetically identical individuals in a short time. In 1988, Lenski took one such population and infected twelve identical flasks, all of which contained the same nutrient broth, including glucose as the vital food source. The twelve flasks, each with its founding population of bacteria, were then placed in a 'shaking incubator' where they were kept nice and warm, and shaken to keep the bacteria well distributed throughout the liquid. These twelve flasks founded twelve lines of evolution that were destined to be kept separate from one another for two decades and counting: sort of like the twelve tribes of Israel, except that in the case of the tribes of Israel there was no law against their mixing.

The twelve tribes of bacteria were not kept in the same twelve flasks for all that time. On the contrary, each tribe had a new flask every day. Imagine twelve lines of flasks, stretching away into the distance, each line more than 7,000 flasks long! Every day, for each of the twelve tribes, a new virgin flask was infected with liquid from the previous day's flask. A small sample, exactly one-hundredth of the volume of the old flask, was drawn out and squirted into the new flask, which contained a fresh supply of glucose-rich broth. The population of bacteria in the flask then started to skyrocket; but it always levelled off by the next day as the supply of food gave out and starvation set in. In other words, the population in every flask multiplied itself hugely, then reached a plateau, at which point a new infective sample was drawn and the cycle renewed the next day. Thousands of times through their high-speed equivalent of geological time, therefore, these bacteria went through the same daily repeated cycles of bonanza expansion, followed by starvation, from which a lucky hundredth were rescued and carried, in a glass Noah's Ark, to a fresh - but again temporary - glucose bonanza: perfect perfect perfect conditions for evolution, and, what is more, the experiment was done in twelve separate lines in parallel.

Lenski and his team have continued this daily routine for more than twenty years so far. This means about 7,000 'flask generations' and 45,000 bacterial generations - averaging between six and seven bacterial generations per day. To put that into perspective, if we were to go back 45,000 human generations, that would be about a million years, back to the time of Homo erectus, which is not very long ago. So, whatever evolutionary change Lenski may have clocked up in the equivalent of a million years of bacterial generations, think how much more evolution might happen in, say, 100 million years of mammal evolution. And even 100 million years is comparatively recent, by geological standards.

In addition to the main evolution experiment, the Lenski group used the bacteria for various illuminating spin-off experiments, for example replacing glucose with another sugar, maltose, after 2,000 generations, but I shall concentrate on the central experiment, which used glucose throughout. They sampled the twelve tribes at intervals throughout the twenty years, to see how evolution was progressing. They also froze samples of each of the tribes as a source of resuscitatable 'fossils' representing strategic points along the evolutionary way. It is hard to exaggerate how brilliantly conceived this series of experiments is.

Here's a little example of the excellent forward planning. You remember I said that the twelve founding flasks were all seeded from the same clone and therefore started out genetically identical. But that wasn't quite true - for an interesting and cunning reason. The Lenski lab had earlier exploited a gene called ara which comes in two forms, Ara+ and Ara-. You can't tell the difference until you take a sample of the bacteria and 'plate them out' on an agar plate that contains a nutritious broth plus the sugar arabinose and a chemical dye called tetrazolium. 'Plating out' is one of the things bacteriologists do. It means putting a drop of liquid, containing bacteria, on a plate covered with a thin sheet of agar gel and then incubating the plate. Colonies of bacteria grow out as expanding circles - miniature fairy rings* - from the drops, feeding on the nutrients mixed in with the agar. If the mixture contains arabinose and the indicator dye, the difference between Ara+ and Ara- is revealed, as if by heating invisible ink: they show up as white and red colonies, respectively. The Lenski team find this colour distinction useful for labelling purposes, as we shall see, and they anticipated this usefulness by setting up six of their twelve tribes as Ara+ and the other six as Ara-. Just to give one example of how they exploited the colour coding of the bacteria, they used it as a check on their own laboratory procedures. When performing their daily ritual of infecting new flasks, they took care to handle Ara+ and Ara- flasks alternately. That way, if they ever made a mistake - splashed a transfer pipette with liquid or something like that - it would show up when they later subjected samples to the red/white test. Ingenious? Yes. And scrupulous. Really good scientists have to be both.

But forget about Ara+ and Ara- for the moment. In all other respects, the founding populations of the twelve tribes started out identical. No other differences between Ara- and Ara+ have been detected, so they really could be treated as convenient colour markers, as ornithologists put colour rings on birds' legs.

Right, then. We have our twelve tribes, marching through their own highly speeded-up equivalent of geological time, in parallel, under the same conditions of repeated boom and bust. The interesting question was, would they stay the same as their ancestors? Or would they evolve? And if they evolved, would all twelve tribes evolve in the same way, or would they diverge from one another?

The broth, as I have said, contained glucose. It was not the only food there, but it was the limiting resource. This means that running out of glucose was the key factor that caused the population size, in every flask every day, to stop climbing and reach a plateau. To put it another way, if the experimenters had put more glucose in the daily flasks, the population plateau at the end of the day would have been higher. Or, if they had added a second dollop of glucose after the plateau was reached, they would have witnessed a second spurt of population growth, up to a new plateau.

In these conditions, the Darwinian expectation was that, if any mutation arose that assisted an individual bacterium to exploit glucose more efficiently, natural selection would favour it, and it would spread through the flask as mutant individuals out-reproduced non-mutant individuals. Its type would then disproportionately infect the next flask in the lineage and, as flask took over from flask, pretty soon the mutant would have a monopoly of its tribe. Well, this is exactly what happened in all twelve tribes. As the 'flask generations' went by, all twelve lines improved over their ancestors: got better at exploiting glucose as a food source. But, fascinatingly, they got better in different ways - that is, different tribes developed different sets of mutations.

How did the scientists know this? They could tell by sampling the lineages as they evolved, and comparing the 'fitness' of each sample against 'fossils' sampled from the original founding population. Remember that 'fossils' are frozen samples of bacteria which, when unfrozen, carry on living and reproducing normally. And how did Lenski and his colleagues make this comparison of 'fitness'? How did they compare 'modern' bacteria with their 'fossil' ancestors? With great ingenuity. They took a sample of the putatively evolved population and put it in a virgin flask. And they put a same-sized sample of the unfrozen ancestral population in the same flask. Needless to say, these experimentally mixed flasks were from then on entirely removed from contact with the continuing lineages of the twelve tribes in the long-term evolution experiment. This side experiment was done with samples that played no further part in the main experiment.

So, we have a new experimental flask containing two competing strains, 'modern' and 'living fossil', and we want to know which of the two strains will out-populate the other. But they are all mixed up, so how do you tell? How do you distinguish the two strains, when they are mixed together in the 'competition flask'? I told you it was ingenious. You remember the colour coding, with the 'reds' (Ara-) and the 'whites' (Ara+)? Now, if you wanted to compare the fitness of, say, Tribe 5 with the ancestral fossil population, what would you do? Let's suppose that Tribe 5 was Ara+. Well then, you'd make sure that the 'ancestral fossils' to which you now compared Tribe 5 were Ara-. And if Tribe 6 happens to be Ara -, the 'fossils' that you'd choose to unfreeze and mix them with would all be Ara+. The Ara+ and Ara-genes themselves, as the Lenski team already knew from previous work, have no effect on fitness. So they could use the colour markers to assay the competitive abilities of each of the evolving tribes, using fossilized 'ancestors' as the competitive standard, in every case. All they had to do was simply plate out samples from the mixed flasks and see how many of the bacteria growing on the agar were white and how many red.

As I say, in all twelve tribes the average fitness increased as the thousands of generations went by. All twelve lines got better at surviving in these glucose-limited conditions. The fitness increase could be attributed to several changes. Populations grew faster in successive flasks, and the average body size of the bacteria grew, in all twelve lines. The top graph opposite plots the average bacterial body size for one of the tribes, which was typical. The blobs represent real data points. The curve drawn is a mathematical approximation. It gives the best fit to the observed data for this particular kind of curve, which is called a hyperbola. * It is always possible that a more complicated mathematical function than a hyperbola would give an even closer fit to the data, but this hyperbola is pretty good, so it hardly seems worth bothering to try. Biologists often fit mathematical curves to observed data, but, unlike physicists, biologists are not accustomed to seeing such a close fit. Usually our data are too messy. In biology, as opposed to physical sciences, we only expect to get smooth curves when we have a very large quantity of data gathered under scrupulously controlled conditions. Lenski's research is a class act.

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