Summary: | <p>Abstract</p> <p>Background</p> <p>The age of unisexual salamanders of the genus <it>Ambystoma </it>is contentious. Recent and ancient evolutionary histories of unisexual <it>Ambystoma </it>were proposed by a few separate studies that constructed phylogenies using mitochondrial DNA markers (cytochrome b gene vs. non-coding region). In contrast to other studies showing that unisexual <it>Ambystoma </it>represent the most ancient unisexual vertebrates, a recent study by Robertson et al. suggests that this lineage has a very recent origin of less than 25,000 years ago.</p> <p>Results</p> <p>We re-examined the phylogenetic relationship of the unisexuals to <it>A. barbouri </it>from various populations using both mitochondrial markers as well as the complete mitochondrial genomes of <it>A. barbouri </it>and a unisexual individual from Kentucky. Lineage dating was conducted using BEAST and MultiDivTime on a complete mitochondrial genome phylogeny. Our results support a monophyletic lineage for unisexual <it>Ambystoma </it>that shares its most recent common ancestor with an <it>A. barbouri </it>lineage from western Kentucky. In contrast to the Robertson et al.'s study, no <it>A. barbouri </it>individual shared an identical or almost identical cytochrome b haplotype with any unisexual. Molecular dating supports an early Pliocene origin for the unisexual linage (~5 million years ago). We propose that a unisexual-like cytochrome b <it>numt </it>(or pseudogene) exists in the controversial <it>A. barbouri </it>individuals from Kentucky, which was likely the cause of an erroneous phylogeny and time estimate in Robertson et al.'s study.</p> <p>Conclusion</p> <p>We reject a recent origin of unisexual <it>Ambystoma </it>and provide strong evidence that unisexual <it>Ambystoma </it>are the most ancient unisexual vertebrates known to exist. The likely presence of an ancient cytochrome b <it>numt </it>in some Kentucky <it>A. barbouri </it>represents a molecular "fossil" reinforcing the hypothesis that these individuals are some of the closest extant relatives to unisexual <it>Ambystoma</it>.</p>
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