The original trees were published by Emanual Margoliash but I'm showing a later version here from Fitch and Margoliash (1967). This is a very famous tree that's found in many textbooks. (The version shown here is from Mulligan (2008).)
From the very beginning, the authors of these molecular phylogenetic trees noted that the rate of change in each lineage was approximately constant. You can see that in the tree shown here. The number of changes in the lineage leading to yeast (Saccharomyces) is 17+10+2=31 from the common root. The number of changes in the lineage leading to insects is 31 or 28, depending on the species. The number leading to humans and monkeys is 32.
All modern species appear to be at the ends of lineages that have evolved at a relatively constant rate since they diverged from a common ancestor. This result was surprising since most biochemists thought of evolution by natural selection as the main mechanism. How is it possible that the environments of insects, yeast, and primates change at a constant rate causing natural selection to make the same number of changes in each lineage over millions of years?
The same result was observed when bacteria were added to the tree a few years later. There's an approximate molecular clock. Of course by that time Kimura and others (including Fitch) had published on Neutral Theory and that explained the approximate molecular clock. The changes in amino acid sequence are neutral and they become fixed by random genetic drift. Since drift is a stochastic process, the rate of fixation of these neutral alleles is approximately constant over time.1 The astonishing conclusion—which most people still don't grasp—is that the vast majority of all evolutionary change is by random genetic drift, not natural selection.
The discovery of an approximate molecular clock led immediately to attempts to calibrate it with respect to time. Margoiash (1963) was the first to do this using cytochrome c sequences. Here's a table from his paper ...
Note that the number of changes from yeast to all of the animals is approximately 44 amino acid substitutions. (The Fitch and Margoliash values in the chart above distances are percentages but the point is still valid.) If you plug in divergence times from the fossil data, then using known calibration points, such as the divergence of horses and humans, or horses and chickens, you can extrapolate to a predicted yeast-animal split at about 500 million years ago.
We now know that the calibration points are incorrect and that fungi and animals diverged about one billion years ago. That doesn't change the fact that there's an approximate molecular clock. It just affects the calibration of that clock with time (years).
There are many problems with molecular clocks, including the fact that they tick at different rates for different proteins as shown below. This is because the sequences of some proteins, like cytochrome c, are highly constrained by natural selection (i.e. conserved). Other proteins, like fibrinopeptides, can tolerate many more changes.
There are two recent reviews that are well worth reading if you're interested in molecular clocks (Broham and Penny, 2003; Kumar, 2005). They discuss the problems with calibration (evident in the figure above) and the problems with relating the molecular clock to generation time and not years. The bottom line is that the molecular clock does not correspond exactly to the prediction of neutral theory but it's close enough to be used to estimate times of divergence. It's still powerful evidence that most changes in gene/protein sequences are neutral changes that have been fixed by random genetic drift. Natural selection is a minor player in molecular evolution.
1. It's equal to the mutation rate, μ, and independent of population size.
Bromham L, Penny D. (2003) The modern molecular clock. Nat Rev Genet. 2003 Mar;4(3):216-24. [doi:10.1038/nrg1020]
Fitch, W.M. and Margoliash, E. (1967) Construction of phylogenetic trees. Science 155:279–284.
Kumar. S. (2005) Molecular clocks: four decades of evolution. Nat, Rev, Genet, 6:654-662.
Margoliash, e. (1963) Primary structure and evolution of cytochrome c. Proc. Natl. Acad. Sci. USA 50:672-679.
Mulligan, P.K. (2008) Proteins, evolution of in AccessScience, ©McGraw-Hill Companies.