Nontriplet feature of genetic code in ciliates is a result of neutral evolution.

The triplet nature of the genetic code is considered a universal feature of known organisms. However, frequent stop codons at internal mRNA positions in ciliates ultimately specify ribosomal frameshifting by one or two nucleotides depending on the context, thus posing a nontriplet feature of the genetic code of these organisms. Here, we sequenced transcriptomes of eight species and assessed evolutionary patterns arising at frameshift sites. We show that frameshift sites are currently accumulating more rapidly by genetic drift than they are removed by weak selection. The time needed to reach the mutational equilibrium is several times longer than the age of and is expected to occur after a several-fold increase in the frequency of frameshift sites. This suggests that are at an early stage of the spread of frameshifting in expression of their genome. In addition, we find the net fitness burden of frameshift sites to be noncritical for the survival of . Our results suggest that fundamental genome-wide changes such as a violation of the triplet character of genetic code can be introduced and maintained solely by neutral evolution.