Although the majority of metazoan mitochondrial genomes (mtDNAs) contain the same 37 genes, including 22 encoding transfer RNAs (tRNAs), the recognition of orthologs is not always straightforward. Here we demonstrate that inferring tRNA orthologs among taxa by using anticodon triplets and deduced secondary structure can be misleading: through a process of tRNA duplication and mutation in the anticodon triplet, remolded leucine (L UUR) tRNA genes have repeatedly taken over the role of isoaccepting LCUN leucine tRNAs within metazoan mtDNA. In the present work, data from within the gastropods and a broad survey of metazoan mtDNA suggest that tRNA leucine duplication and remolding events have occurred independently at least seven times within three major animal lineages. In all cases where the mechanism of gene remolding can be inferred with confidence, the direction is the same: from L UUR to LCUN. Gene remolding and its apparent asymmetry have significant implications for the use of mitochondrial tRNA gene orders as phylogenetic markers. Remolding complicates the identification of orthologs and can result in convergence in gene order. Careful sequence-based analysis of tRNAs can help to recognize this homoplasy, improving geneorder-based phylogenetic hypotheses and underscoring the importance of careful homology assessment. tRNA remolding also provides an additional mechanism by which gene order changes can occur within mtDNA: through the changing identity of tRNA genes themselves. Recognition of these remolding events can lead to new interpretations of gene order changes, as well as the discovery of phylogenetically relevant gene dynamics that are hidden at the level of gene order alone.
Mitochondrial gene order data are being used with increasing frequency as robust molecular characters in deep-level metazoan phylogenetic studies. A fundamental assumption of molecular systematics is that orthologous genes can be recognized unambiguously. The facts that most animal mtDNAs described to date are composed of 37 genes (22 tRNAs, 2 rRNAs, and 13 protein subunits), that these genes play roles essential for oxidative metabolism, and that putative homologs have been identified in the mtDNAs of other eukaryotes (1, 2) suggest that identifying orthologs among metazoan lineages should not be problematic. In practice, however, establishing homology between mtDNA genes of distantly related organisms can be difficult at the nucleotide level because of high rates of sequence evolution. This difficulty in identifying homologs is particularly acute for short (Ϸ70-80 bp) tRNA genes. Typically, the anticodon and features of secondary structure are used to establish tRNA identity, but these can be misleading. Some tRNA genes may, through a process of duplication and point mutation(s) in the anticodon triplet, assume the identity of other tRNAs within mtDNA (3). The potential for gene remolding has been corroborated by in vitro tRNA gene knockout experiments in prokaryotic systems (4), and in studies of human mitochondrial-based dise...
