MicroRNAs and Metazoan Phylogenetics: Big Trees from Little Genes  

Knowledge of the phylogenetic relationships between modern organisms allows the reconstruction of morphological characters shared by the last common ancestor of the crown group.  With this knowledge, fossils can be correctly placed along the stem-lineage, thereby giving insight into the order of character acquisition during the evolution of that clade.  Sequence-based molecular phylogenetics has provided robust answers to many phylogenetic questions, but many areas of the metazoan tree have remained recalcitrant, yielding either poorly resolved or conflicting trees.  This is particularly true for fast, deep radiations such as the Cambrian "explosion" of animal life.  New approaches may be needed to resolve these problems.  MicroRNAs possess three properties that give them tremendous potential as phylogenetic markers: (1) new microRNA families are continually being incorporated into metazoan genomes through time; (2) they show only rare instances of secondary loss, and only rare instances of substitutions occurring in the mature gene sequence; and (3) they are almost impossible to evolve convergently.  Because of these three properties, we propose microRNAs as a novel type of data that can be applied to virtually any area of the metazoan tree, to test among competing phylogenetic hypotheses or to forge new ones. 

We have coded a presence/absence matrix for all known and newly-discovered conserved microRNA families from 36 metazoan taxa that span the range of metazoan evolution.  This matrix includes nine taxa whose microRNA complement was determined during the course of this study by applying 454 pyrosequencing technology to small RNA libraries.  Parsimony analysis of the matrix demonstrates that microRNA datasets are capable of robustly resolving almost all nodes in the metazoan tree, including the placement of acoel flatworms, the monophyly of Eumetazoa, and the relationships among annelids that are recalcitrant in many molecular analyses.  When homoplasy indices for this matrix are compared to matrices using the same taxa but assembled using data from morphological characters, 18S rDNA, PCR-amplified nuclear housekeeping genes, ESTs or complete mitochondrial genomes, microRNAs have the least homoplasy of any dataset used in metazoan phylogenetics.  This suggests not only that microRNAs hold high promise for resolving difficult phylogenetic problems, particularly the phylum- and class-level divergences during the Ediacaran and early Paleozoic, but highlights the extreme conservation of microRNAs and the intense selection pressures operating on these genes over evolutionary timescales.