Differences in evolutionary accessibility determine which equally effective regulatory motif evolves to generate pulses

Transcriptional regulatory networks (TRNs) are enriched for certain “motifs”. Motif usage is commonly interpreted in adaptationist terms, i.e. that the optimal motif evolves. But certain motifs can also evolve more easily than others. Here, we computationally evolved TRNs to produce a pulse of an effector protein. Two well-known motifs, type 1 incoherent feed-forward loops (I1FFLs) and negative feedback loops (NFBLs), evolved as the primary solutions. Which motif evolves more often depends on selection conditions, but under all conditions, either motif achieves similar performance. I1FFLs generally evolve more often than NFBLs, unless we select for a tall pulse. I1FFLs are more evolutionarily accessible early on, before the effector protein evolves high expression; when NFBLs subsequently evolve, they tend to do so from a conjugated I1FFL-NFBL genotype. In the empirical S. cerevisiae TRN, output genes of NFBLs had higher expression levels than those of I1FFLs. These results suggest that evolutionary accessibility, and not relative functionality, shapes which motifs evolve in TRNs, and does so as a function of the expression levels of particular genes. ### Competing Interest Statement The authors have declared no competing interest.