Testing the basic tenet of the molecular clock and neutral theory by using ancient proteomes

Early research on orthologous protein sequence comparisons by Margoliash in 1963 discovered the astonishing phenomenon of genetic equidistance, which has inspired the ad hoc interpretation known as the molecular clock. Kimura then developed the neutral theory and claimed the molecular clock as its best evidence. However, subsequent studies over the years have largely invalidated the universal molecular clock. Yet, a watered down version of the molecular clock and the neutral theory still reigns as the default model for phylogenetic inferences. The seemingly obvious tenet of the molecular clock on evolutionary time scales remains to be established by using ancient sequences: the longer the time of evolutionary divergence, the larger the genetic distance. We here analyzed the recently published Early Pleistocene enamel proteome from Dmanisi and found that ancient proteins were not closer to an outgroup than their orthologs from the extant sister species were. Together with a previous study, the combined results showed that most ancient proteins were in fact more distant to the outgroup. The results are unexpected from the molecular clock but fully predicted by the notion that genetic distances or diversities are largely at optimum saturation levels as described by the maximum genetic diversity (MGD) theory.

Genetic equidistance at the nucleotide level

The genetic equidistance phenomenon was first discovered in 1963 by Margoliash and shows complex taxa to be all approximately equidistant to a less complex species in amino acid percentage identity. The result has been mis-interpretated by the ad hoc universal molecular clock hypothesis, and the much overlooked mystery was finally solved by the maximum genetic diversity hypothesis (MGD). Here, we studied 15 proteomes and their coding DNA sequences (CDS) to see if the equidistance phenomenon also holds at the CDS level. We performed DNA alignments for a total of 5 groups with 3 proteomes per group and found that in all cases the outgroup taxon was equidistant to the two more complex taxa species at the DNA level. Also, when two sister taxa (snake and bird) were compared to human as the outgroup, the more complex taxon bird was closer to human, confirming species complexity rather than time to be the primary determinant of MGD. Finally, we found the fraction of overlap sites where coincident substitutions occur to be inversely correlated with CDS conservation, indicating saturation to be more common in less conserved DNAs. These results establish the genetic equidistance phenomenon to be universal at the DNA level and provide additional evidence for the MGD theory.

The Genetic Equidistance Phenomenon at the Proteomic Level

The field of molecular evolution started with the alignment of a few protein sequences in the early 1960s. Among the first results found, the genetic equidistance result has turned out to be the most unexpected. It directly inspired the ad hoc universal molecular clock hypothesis that in turn inspired the neutral theory. Unfortunately, however, what is only a maximum distance phenomenon was mistakenly transformed into a mutation rate phenomenon and became known as such. Previous work studied a small set of selected proteins. We have performed proteome wide studies of 7 different sets of proteomes involving a total of 15 species. All 7 sets showed that within each set of 3 species the least complex species is approximately equidistant in average proteome wide identity to the two more complex ones. Thus, the genetic equidistance result is a universal phenomenon of maximum distance. There is a reality of constant albeit stepwise or discontinuous increase in complexity during evolution, the rate of which is what the original molecular clock hypothesis is really about. These results provide additional lines of evidence for the recently proposed maximum genetic diversity (MGD) hypothesis.