Classic QnAs with Rasmus Winther and Eske Willerslev

How long ago did humans diverge from our closest living relatives, the chimpanzees? In a 1969 PNAS article, “A Molecular Timescale for Human Evolution,” (1) Allan C. Wilson of the University of California, Berkeley, and his then-PhD student Vincent M. Sarich estimated a divergence time of 4 to 5 million years ago. More recent research has pushed the generally accepted divergence time back to around 6 to 9 million years ago or, possibly, as much as 12 million years ago (2, 3). However, theories prevailing in 1969 suggested an earlier divergence time of around 30 million years ago (4). In the now-classic article, which was among the earliest to demonstrate the concept of a molecular clock, Wilson and Sarich based their surprisingly close calculations on emerging tools from molecular biology rather than paleontology. Certain genes accumulate mutations at a roughly constant rate, similar to the ticking of a clock. By comparing differences that have accumulated between two species for a given gene—or the protein the gene encodes—researchers can calculate how long ago the species diverged. While Wilson’s and Sarich’s estimated divergence time for humans and chimpanzees is somewhat more recent than currently accepted estimates, molecular clocks are now widely used throughout the field of evolutionary biology. In this issue, Rasmus Grønfeldt Winther of the University of California Santa Cruz and the University of Copenhagen and Eske Willerslev of the University of Cambridge and the University of Copenhagen offer their views on the 1969 PNAS Classic (e2220473120). Winther and Willerslev spoke with PNAS about the lasting legacy of Wilson and Sarich.

Image credit: Pixabay/TheDigitalArtist.

PNAS: How did Wilson’s and Sarich’s dating of the divergence time between humans and chimpanzees compare with contemporary estimates in 1969?

Winther: It was much more recent. Many other estimates said 15, 20, or 30 million years ago, based on paleontological evidence (4).

Willerslev: It was very provocative at the time to come up with such a recent date for the divergence. Estimates have changed by a few million years, depending on the studies, but it is pretty close to what is the generally accepted value for the human–chimpanzee split. The general notion today is that it is very recent.

Winther: This was a whole new way of measuring and assessing the temporal split. Wilson and Sarich’s work was groundbreaking in terms of using macromolecular biological data. Ultimately, it is better to use genetic data, but they were using proteins, which are stand-ins for genetic data. They were measuring what they call the index of dissimilarity (ID) of proteins between different species. There was so much resistance. It was a very provocative way to measure the temporal split because people had been using fossils, they had been using potassium–argon radioactive dating techniques, and the accepted wisdom was that the human–chimpanzee split was tens of millions of years ago, not just 4 million or 5 million.

PNAS: Can you summarize the technique that Wilson and Sarich used to estimate the similarity between human and chimpanzee proteins?

Winther: The idea is that you isolate, say, human, chimpanzee, rhesus monkey, and gorilla proteins (e.g., albumin in the blood), and then you see how reactive the original protein is to that protein from another species. If the homologous proteins of the two species are very reactive, it means that they are very close together, in terms of structure and in terms of affinity. If they are not very reactive at all, it means that they are rather different. The basic idea is that the closer they are, the more closely related these species also are. And the more that the homologous proteins from the two species do not have a close affinity, the more different the two species are.

Allan C. Wilson. Image credit: Jane Scherr (photographer).

PNAS: What is the significance of the regularity test that Wilson and Sarich used to test their molecular clock?

Winther: This “microcompliment” technique is great. They did something revolutionary in using that technique to get ID data, but I think the real revolution was the regularity test. Because what was deeply surprising,…and I remember being blown away when I learned this in my undergraduate years, is that it is roughly the same protein difference between a horse and a chimpanzee, as between a horse and a human, and as between a horse and a rhesus monkey.

It was remarkable because there is no a priori reason that they should be the same. Evolutionary rates can accelerate massively or slow down. But they were consistent in all the species that they looked at. There was already talk about molecular clocks from Motoo Kimora (5). He had published a model at around the same time, but Wilson’s and Sarich’s work was the first strong empirical evidence that there is a regularly ticking molecular clock, so the genetic changes happen at a roughly constant rate.

Now, this is an idealization. It does not turn out to be absolutely true. Overarching molecular evolution rates for particular genes depend on selection coefficients, population sizes of ancestral lineages and common ancestors, and generation times—all of which can change over time and across lineages (6). Wilson and Sarich's estimates [heralded the advent] of molecular [evolution], but since their work, the split time has been pushed a bit back due to more reliable genetic technology (7), evidence that the molecular clock slows down as we move up the primate branch (3), and a better fossil record. Even so, a variety of molecular clock mathematical models have been developed which can handle these and other complexities of the evolutionary process (8).

PNAS: As you outline in your Perspective, this PNAS Classic builds upon prior research and methods developed by Wilson and his contemporaries. What made this article so impactful?

Willerslev: I think it is because they are addressing a question that everybody is interested in. What is our relationship with the great apes? Their finding is so radically different from the general notion that was out there at the time, based on more traditional approaches. It is a well-argued and well-done piece of work. It was the beginning of the change in conception that has led us to present-day thinking about when these divergences happened. If they had done it on camels, they probably would have gotten less attention. It is a question that many different areas of research from paleontology to medicine are interested in and even ordinary people are interested in.

PNAS: What is the significance of the molecular clock that Wilson and Sarich helped pioneer?

Winther: There are reasons why Gould and Eldredge got very popular with their punctuated equilibrium theory (9), arguing that there was not much evolutionary change for a long time and then there was all this change all of a sudden. Well, geologically [speaking]; we are still talking hundreds of thousands to millions of years, but relative to the fossil record, there could be very quick changes and then not much change at all for long periods of time. That is what is called a punctuated picture. Then, there is also a middle view that evolutionary rates can go somewhat up and then somewhat down, then somewhat up and somewhat down.

Vincent M. Sarich. Image credit: University of California (Berkeley, CA).

What blew people away was [the finding that]—at least for some proteins—there seemed to be very consistent changes you could almost use as a clock. In a nested tree, as long as the distances were equal, the divergence times were equal, too, going up into the branches. This is important because you can make all kinds of inferences about divergence time and about similarities between species. Why do we have a molecular clock? We have it because those are the proteins that are not under a lot of selection. They are most likely neutral or what is called nearly neutral. Mutations tend to happen on a somewhat regular basis, but you do not have selection to either weed out mutations that are not beneficial or to select for mutations that are beneficial. So, they are just going to change based on intrinsic genetic mutation processes.

PNAS: What is the legacy of Wilson’s research?

Willerslev: To me, Allan Wilson is the founder of molecular evolution. Not only with this paper but also Mitochondrial Eve (10), which was another mind-blowing discovery. He is also establishing the field of ancient DNA, which I am part of. He was a coauthor on the first ancient DNA paper (11). So, he is a tremendously important person in terms of establishing some very fundamental discoveries but also to applying molecular biology in an evolutionary framework.

Winther: Wilson did not do this alone. Eske and I are too young to have met Wilson, but let us not forget that Wilson did this impressive work with his PhD students: Vincent Sarich (1), Rebecca Cann (10), Mark Stoneking (10), and Svante Pääbo (12).

Willerslev: My supervisor during my PhD, Peter Arctander (13), came out of that group. In terms of the people who came out of his group, so many of them have established very successful groups around the world. In that sense, he not only has an incredible [scientific] legacy but also in terms of the people he fostered.

Winther: The regularity of molecular evolution is not a [self-evident] truth. It is an empirical pattern that had to be [conceived] and measured. It is not the case that evolution always works with a molecular clock, but it does under certain idealized conditions. Wilson and Sarich’s work is a beautiful case in thinking clearly and having well-defined measurements that lead to surprising and provocative results.