Selection bias in colony-based microbial mutation accumulation lines

In microbes, spontaneous mutations are often collected using mutation-accumulation (MA) lines whose repeated single-cell bottlenecks are assumed to largely silence natural selection. Supporting this, the usual test for selection bias - a shortage of non-synonymous relative to synonymous mutations - has never revealed a significant deficit across the 40 published tests in wild-type microbes. This likely reflects selective reporting and lack of power - a meta-analysis of 10,856 mutations from wild-type microbial MA reveals a clear signal of selection: non-synonymous mutations are observed 7.7% less often than synonymous mutations. Because most studies ignored mutational spectra, this figure is provisional. For future studies, we built a multinomial-logit model that jointly estimates mutation spectrum and selection. Applying it to a new 194-line Escherichia coli MA experiment plus three previous E. coli datasets (709 mutations) again reveals a deficit of non-synonymous mutations, although the reduction is not statistically significant. While approaches exist for correcting for selection bias, all assume MA lines are grown in well-mixed liquid culture, despite microbial MA lines being propagated as surface colonies where competition is spatially structured. Current theory suggests selection bias should be stronger under colony growth, but using agent-based simulations we show that this depends critically on the scale over which neighbouring cells compete and how unevenly they divide: it can be weaker, equivalent to, or stronger than in homogeneous growth. The empirical details of colony growth need to be resolved before progress can be made, but our preliminary assessment is that the amount of selection bias observed is greater than that predicted under homogeneous-growth models.