Classification of conserved stem-loop sequences. (a) Patterns of Drosophila pre-miRNA nucleotide divergence patterns imply a canonical progression in miRNA evolution. The Drosophila orthologs of 23/24 previously described miRNAs are either completely conserved (class 1), contain one or more mismatches or gaps located exclusively in the loop (class 2) or contain an equal or greater number of mutations within the loop compared to the non-miRNA-encoding arm (class 3). We consider these to represent successive steps in the normal evolution of miRNAs and therefore connect them with arrows. Members of classes 1-3 are considered as equally good candidates while members of classes 4-6 are poor candidates. As we expect class 3 candidates to eventually evolve into class 6 candidates (broken arrow), these evolutionary considerations are most relevant to species separated by an evolutionary distance comparable to D. melanogaster and D. pseudoobscura. (b) Preferential divergence of miRNAs within their loop sequences is illustrated by let-7. The Drosophila orthologs of let-7 contain three mismatches and one gap within the loop, whereas both arms have been completely conserved.