Direct Estimation of the Mitochondrial DNA Mutation Rate in Drosophila melanogaster

Abstract

Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10⁻⁸ per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of the mitochondrial genome of Drosophila. Mitochondria are the energy-producing organelles of the cell, and they contain genetic information encoded on their own genome. Because rates of mutation for mitochondrial genomes are believed to be much higher than those in nuclear DNA, mitochondrial genetic differences between and within species are particularly useful in population genetics, for example, as markers of population movements. We have directly estimated the mutation rate in the mitochondrial genome of the fruit fly Drosophila melanogaster in lines that had been allowed to randomly accumulate mutations in the virtual absence of effective natural selection. We scanned for new mutations by comparing the DNA of different lines by a sensitive mutation detection technique. We show that the mitochondrial mutation rate is about ten times higher than the nuclear DNA mutation rate. Strikingly, however, almost all of the single–base pair mutations that we detected change G to A at an amino acid site of a protein-coding gene. The explanation for this effect seems to be that natural selection maintains the nucleotide G at amino acid sites, whereas most silent sites are under weaker selection and have previously mutated to A or T. The mutation rate for G to A changes is 70 times higher than the nuclear DNA mutation rate. This extreme mutation bias maintains the high A+T content of the Drosophila mitochondrial genome.