The history of molecular evolution starts in the early 20th century with "comparative biochemistry", but the field of molecular evolution came into its own in the 1960s and 1970s, following the rise of molecular biology. The advent of protein sequencing allowed molecular biologists to create phylogenies based on sequence comparison, and to use the differences between homologous sequences as a molecular clock to estimate the time since the last common ancestor. In the late 1960s, the neutral theory of molecular evolution provided a theoretical basis for the molecular clock, though both the clock and the neutral theory were controversial, since most evolutionary biologists held strongly to panselectionism, with natural selection as the only important cause of evolutionary change. After the 1970s, nucleic acid sequencing allowed molecular evolution to reach beyond proteins to highly conserved ribosomal RNA sequences, the foundation of a reconceptualization of the early history of life.
Early history
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. Alfred Sturtevant predicted the existence of chromosomal inversions in 1921 and with Dobzhansky constructed one of the first molecular phylogenies on 17 Drosophila Pseudo-obscura strains from the accumulation of chromosomal inversions observed from the hybridization of polyten chromosomes. 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. However, it was not until the 1950s that biologists developed techniques for producing biochemical data for the quantitative study of molecular evolution.
The first molecular systematics research was based on immunological assays and protein "fingerprinting" methods. Alan Boyden—building on immunological methods of George Nuttall—developed new techniques beginning in 1954, and in the early 1960s Curtis Williams and Morris Goodman used immunological comparisons to study primate phylogeny. Others, such as Linus Pauling and his students, applied newly developed combinations of electrophoresis and paper chromatography to proteins subject to partial digestion by digestive enzymes to create unique two-dimensional patterns, allowing fine-grained comparisons of homologous proteins.
Beginning in the 1950s, a few naturalists also experimented with molecular approaches—notably Ernst Mayr and Charles Sibley. 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.
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. This would change as molecular biology shed more light on the physical and chemical nature of genes.
Genetic load, the classical/balance controversy, and the measurement of heterozygosity
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. In 1950, James F. Crow offered two different explanations (later dubbed the classical and balance positions) based on the paradox first articulated by J. B. S. Haldane in 1937: the effect of deleterious mutations on the average fitness of a population depends only on the rate of mutations (not the degree of harm caused by each mutation) because more-harmful mutations are eliminated more quickly by natural selection, while less-harmful mutations remain in the population longer. H. J. Muller dubbed this "genetic load".