By the early 1960s amino acid sequences of hemoglobins and cytochrome c for many mammals had been determined. When the sequences were compared, investigators began to notice that the number of amino acid differences between different pairs of mammals seemed to be roughly proportional to the time since they had diverged from one another, as inferred from the fossil record. Zuckerkandl and Pauling  proposed the molecular clock hypothesis to explain these results. Specifically, they proposed that there was a constant rate of amino acid substitution over time. Sarich and Wilson [6,7] used the molecular clock hypothesis to propose that humans and apes diverged approximately 5 million years ago. While that proposal may not seem particularly controversial now, it generated enormous controversy at the time, because at the time many paleoanthropologists interpreted the evidence to indicate humans diverged from apes as much as 30 million years ago.
One year after Zuckerkandl and Pauling's paper, Harris  and Hubby and Lewontin [2,5] showed that protein electrophoresis could be used to reveal surprising amounts of genetic variability within populations. Harris studied 10 loci in human populations, found three of them to be polymorphic, and identified one locus with three alleles. Hubby and Lewontin studied 18 loci in Drosophila pseudoobscura, found seven to be polymorphic, and five that had three or more alleles.
Both sets of observations posed real challenges for evolutionary geneticists. It was difficult to imagine an evolutionary mechanism that could produce a constant rate of substitution. It was similarly difficult to imagine that natural selection could maintain so much polymorphism within populations. The ``cost of selection,'' as Haldane called it would simply be too high.