Once the genetic code was solved in the 1960s, biologists realized that between one-quarter and one-third of single nucleotide mutations (SNPs) in protein coding genes, do not alter the choice of amino acid at that spot in the protein. Such a point mutation which does not alter the selection of amino acid from the one previously coded for, has been considered to be neutral in its impact on the organism’s fitness. Neutral mutations have been very important in evolution theory, figuring prominently in the calculation of evolutionary processes like molecular clock. Few people have challenged the importance of neutral mutations to evolution theory, until now! [Nathaniel Sharp. 2022. Mutations matter even if proteins stay the same. Nature 606 June 23 pp. 657-659]

To understand all this, we must first define what these neutral or synonymous mutations are. As discussed under Protein, each amino acid in the molecule’s sequence is coded for by 3 nucleotides, out of 4 possible choices. Since, with 4 possible arrangements of 4 nucleotides into groupings of 3, there are 64 possible arrangements (4 x 4 x 4) some amino acids are represented by more than 1 triplet code. There are, after all, only 20 different amino acids in proteins.

Thus for example, UCU codes for serine, but so does UCC, UCA and UCG. If there were a single nucleotide mutation which changed UCU to UCC, for example, we still obtain serine. Also if the final nucleotide in the triplet were changed to UCA or UCG. We would still obtain serine. These changes in nucleotide sequence which do not affect the choice of amino acid, are called synonymous mutations. [Think synonyms in English grammar.] However, if the middle spot in the triplet were changed to an A nucleotide (as in UAU or UAC) then the amino acid called for is tyrosine.   [The nucleotide choices for the triplet code are cytosine or C, adenine or A, guanine or G, and Uracil U substitutes for thymine T.]  

A team of four scientists from the US (Shen, Xukang et al. 2022. Synonymous mutations in representative yeast genes are mostly strongly non-neutral. Nature 606 #7915 pp. 725-731) constructed 8341 yeast mutants using CRISPR-Cas9. These mutations were in 21 genes involving various functions. For each gene, they selected a 150 nucleotide sequence and one at a time, they changed each nucleotide site into one or other of the nucleotide options (3 x 150 = 450).  Thus, each new variant deviated from the wild type by one point mutation. Mostly these changes represented synonymous or non-synonymous mutations.  Non-synonymous mutations are cases where the actual amino acid is changed by the substitution.

The team then tested yeast cells with these individual point mutations for fitness against the wild (normal) version. The results were not what the team had expected. In the past, mutations which resulted in a change of amino acid (non-synonymous) were expected to exert more negative effects and ones which did not change the amino acid sequence were expected to be neutral. But of these results, a commentator declared: “synonymous mutations are frequently just as harmful as the non-synonymous mutations that alter proteins, upending a common assumption about molecular evolution.” (p. 657, emphasis mine)

Furthermore: “Surprisingly, synonymous mutations were only slightly less harmful, on average, and showed a similar overall pattern of effects, suggesting that synonymous mutations are often strongly non-neutral.” (p. 658, emphasis mine )

The commentator points out potential impacts of such findings on evolution theory: “Shen and colleagues’ study should push researchers to evaluate long-standing assumptions about molecular evolution. Patterns of synonymous genetic variation have been used as a ‘neutral’ standard for inferring how selection acts on different genes, estimating current and historical population sizes, and more. For example, the synonymous differences between species is used as a ‘molecular clock’ to estimate how long ago speciation events took place – but these estimates would be inaccurate if selection actually constrains a lot of synonymous variation, effectively slowing the clock.” (p. 659)

The study authors point out that “there is no particular reason why our results should not generalize to other organisms.” (p. 730) but of course, more studies will be needed to confirm this. They expect their results to be found with other organisms as well.

The take home message is that we can see how unsupported assumptions can exert a devastating effect on evolutionary conclusions. This study provides ample bad news for evolution, involving the indication that mutations have an even more negative impact on fitness than has previously been supposed. Natural selection, working on mutations, (neo-Darwinian synthesis) is not a promising driver of evolution. On the contrary, it is a driver of devolution or a process of decline.

A study printed later in 2022 similarly demonstrated that synonymous mutations can exert negative effects. See Yang Jiang et al. 2022. How synonymous mutations alter enzyme structure and function over long timescales. Nature Chemistry https://doi.org/10.1038/s41557-022-01091-z