The phylogenetic histories of amphioxus miRNAs. (a) miRNA gene clusters in amphioxus. At a 3,000-nucleotide distance threshold, the amphioxus genome contains 17 compact clusters with 39 miRNAs. The precursor structure is indicated as a box, and the location of the miRNA within the precursor is shown in black. Some of these clusters in amphioxus are also conserved in zebrafish and humans. (b) The evolutionary histories of miRNAs and their relationship to the milestones of macroevolution. We integrated amphioxus miRNAs into the currently known miRNAs (miRBase release 12.0) and performed a comprehensive screening of their phylogenetic histories across animals. Each miRNA was classified into one of four groups: miRNAs conserved throughout bilaterian animals; homologs of invertebrate miRNAs; miRNAs present in both chordates and vertebrates; and homologs of vertebrate miRNAs. Note that our approach ignored species-specific miRNAs, since these miRNAs do not offer any information about miRNA evolution. (c) Comparison of the miRNA repertoires of amphioxus and tunicates. By using zebrafish as a reference, we compared the miRNA repertoires of nematodes, fruit flies, tunicates, and amphioxus. miRNAs with a zebrafish homolog were recorded as +1; miRNAs not found in zebrafish were recorded as -1.