Before the rise of molecular biology in the 1950s and 1960s, a small number of biologists had explored the possibilities of using biochemical differences between species to study evolution.
[1] Ernest Baldwin worked extensively on comparative biochemistry beginning in the 1930s, and Marcel Florkin pioneered techniques for constructing phylogenies based on molecular and biochemical characters in the 1940s.
While Mayr quickly soured on paper chromatography, Sibley successfully applied electrophoresis to egg-white proteins to sort out problems in bird taxonomy, soon supplemented that with DNA hybridization techniques—the beginning of a long career built on molecular systematics.
[4] While such early biochemical techniques found grudging acceptance in the biology community, for the most part they did not impact the main theoretical problems of evolution and population genetics.
At the time that molecular biology was coming into its own in the 1950s, there was a long-running debate—the classical/balance controversy—over the causes of heterosis, the increase in fitness observed when inbred lines are outcrossed.
Motoo Kimura's subsequent mathematical analyses reinforced what Crow had suggested in 1950: that even if overdominant loci are rare, they could be responsible for a disproportionate amount of genetic variability.
Thus, the infinite alleles model offered a potential way to decide between the classical and balance positions, if accurate values for the level of heterozygosity could be found.
Most population geneticists (including Hubby and Lewontin) rejected the possibility of widespread neutral mutations; explanations that did not involve selection were anathema to mainstream evolutionary biology.
Hubby and Lewontin also ruled out heterozygote advantage as the main cause because of the segregation load it would entail, though critics argued that the findings actually fit well with overdominance hypothesis.
[11] In 1961, Emanuel Margoliash and his collaborators completed the sequence for horse cytochrome c (a longer and more widely distributed protein than insulin), followed in short order by a number of other species.
However, the essential idea of the molecular clock—that individual proteins evolve at a regular rate independent of a species' morphological evolution—was extremely provocative (as Pauling and Zuckerkandl intended it to be).
According to the molecular clock hypothesis, proteins evolved essentially independently of the environmentally determined forces of selection; this was sharply at odds with the panselectionism prevalent at the time.
Moreover, Pauling, Zuckerkandl, and other molecular biologists were increasingly bold in asserting the significance of "informational macromolecules" (DNA, RNA and proteins) for all biological processes, including evolution.
[21] Kimura's theory—described only briefly in a letter to Nature—was followed shortly after with a more substantial analysis by Jack L. King and Thomas H. Jukes—who titled their first paper on the subject "Non-Darwinian Evolution".
The fairly constant rates of evolution observed for individual proteins was not easily explained without invoking neutral substitutions (though G. G. Simpson and Emil Smith had tried).
[23] King and Jukes' paper, especially with the provocative title, was seen as a direct challenge to mainstream neo-Darwinism, and it brought molecular evolution and the neutral theory to the center of evolutionary biology.
Though Kimura had initially developed the neutral theory partly as an outgrowth of the classical position within the classical/balance controversy (predicting high genetic load as a consequence of non-neutral mutations), he gradually deemphasized his original argument that segregational load would be impossibly high without neutral mutations (which many selectionists, and even fellow neutralists King and Jukes, rejected).
[25] From the 1970s through the early 1980s, both selectionists and neutralists could explain the observed high levels of heterozygosity in natural populations, by assuming different values for unknown parameters.
Early in the debate, Kimura's student Tomoko Ohta focused on the interaction between natural selection and genetic drift, which was significant for mutations that were not strictly neutral, but nearly so.
In many cases, genomics research in the 1990s produced phylogenies contradicting the rRNA-based results, leading to the recognition of widespread lateral gene transfer across distinct taxa.