Polymorphic duplicate genes and persistent non-coding sequences reveal heterogeneous patterns of mitochondrial DNA loss in salamanders

Rebecca A. Chong, Rachel Lockridge Mueller

Mitochondria are the site of the citric acid cycle and oxidative phosphorylation (OXPHOS). In metazoans, the mitochondrial genome is a small, circular molecule averaging 16.5 kb in length. Despite evolutionarily conserved gene content, metazoan mitochondrial genomes show a diversity of gene orders most commonly explained by the duplication-random loss (DRL) model. In the DRL model, (1) a sequence of genes is duplicated in tandem, (2) one paralog sustains a loss-of-function mutation, resulting in selection to retain the other copy, and (3) the non-functional paralog is eventually deleted from the genome. Despite its apparent role in generating mitochondrial gene order diversity, little is known about the tempo and mode of random gene loss after duplication events. Here, we determine mitochondrial gene order across the salamander genus Aneides, which was previously shown to include at least two DRL-mediated rearrangement events. We then analyze these gene orders in a phylogenetic context to reveal patterns of DNA loss after mitochondrial gene duplication. We identified two separate duplication events that resulted in mitochondrial gene rearrangements in Aneides; one occurred at the base of the clade tens of millions of years ago, while the other occurred much more recently (i.e. within a single species), resulting in gene order polymorphism and paralogs that are readily identifiable. We demonstrate that near-complete removal of duplicate rRNA genes has occurred since the recent duplication event, whereas duplicate protein-coding genes persist as pseudogenes and duplicate tRNAs persist as functionally intact paralogs. In addition, we show that non-coding DNA duplicated at the base of the clade has persisted across species for tens of millions of years. The evolutionary history of the mitochondrial genome, from its inception as a bacterial endosymbiont, includes massive genomic reduction. Consistent with this overall trend, selection for efficiency of mitochondrial replication and transcription has been hypothesized to favor elimination of extra sequence. Our results, however, suggest that there may be no strong disadvantage to extraneous sequences in salamander mitochondrial genomes, although duplicate rRNA genes may be deleterious.