Establishing measures of motif conservation. (A) Phylogenetic tree from the Cactus-447 data set, comprising two constituent data sets: Zoonomia 241 (black branches) and Primate data set (blue branches). Branch lengths represent substitutions per site, with Homo sapiens as the reference species (red dot). (B,C) Simplified tree topologies illustrating how phylogenetic structure biases motif conservation estimates. Each tree shows parsimony-reconstructed nucleotide substitutions (black ticks) for different permutations of the same motif sequences, demonstrating how relying solely on motif frequency across taxa can lead to overestimation (B) or underestimation (C) of m6A consensus motif (DRACH) conservation. Branch colors indicate the reconstructed motif state along each branch: Red represents DRACH motifs, while blue represents non-DRACH motifs. (D) Comparison of consensus frequency scores (CFSs) between expressed DRACH and control motif sites. (E) Comparison of motif entropy scores (MESs) between expressed DRACH and control motif sites.
