Mitochondrial Mutation Rate, Spectrum and Heteroplasmy in Caenorhabditis elegans Spontaneous Mutation Accumulation Lines of Differing Population Size

Abstract

Mitochondrial genomes of metazoans, given their elevated rates of evolution, have served as pivotal markers for phylogeographic studies and recent phylogenetic events. In order to determine the dynamics of spontaneous mitochondrial mutations in small populations in the absence and presence of selection, we evolved mutation accumulation (MA) lines of Caenorhabditis elegans in parallel over 409 consecutive generations at three varying population sizes of N = 1, 10, and 100 hermaphrodites. The N =1 populations should have a minimal influence of natural selection to provide the spontaneous mutation rate and the expected rate of neutral evolution, whereas larger population sizes should experience increasing intensity of selection. New mutations were identified by Illumina paired-end sequencing of 86 mtDNA genomes across 35 experimental lines and compared with published genomes of natural isolates. The spontaneous mitochondrial mutation rate was estimated at 1.05 × 10-7/site/generation. A strong G/C→A/T mutational bias was observed in both the MA lines and the natural isolates. This suggests that the low G + C content at synonymous sites is the product of mutation bias rather than selection as previously proposed. The mitochondrial effective population size per worm generation was estimated to be 62. Although it was previously concluded that heteroplasmy was rare in C. elegans, the vast majority of mutations in this study were heteroplasmic despite an experimental regime exceeding 400 generations. The frequencies of frameshift and nonsynonymous mutations were negatively correlated with population size, which suggests their deleterious effects on fitness and a potent role for selection in their eradication.

Keywords: Caenorhabditis elegans; genetic drift; heteroplasmy; mitochondria; mitochondrial effective population size; mutation rate; selection; spontaneous mutation.

Fig. 1

Whole-genome sequencing to yield 86 C. elegans genomes, including that of the ancestral, pre-mutation accumulation N2 control and 35 MA lines following ∼409 MA generations. Multiple individuals were sequenced for MA lines maintained at larger population sizes (N= 10 and 100).

Fig. 2

Mutation spectrum of base substitutions in the mitochondrial genome. Transitions are represented in dark and medium gray, while the four possible transversions are in light gray and black. The proportion of base substitutions are normalized by the frequencies of each individual variant. (A) The bias in spontaneous base substitutions detected in our study of 20 C. elegans N= 1 MA lines is contrasted with the spectrum of mtDNA base substitutions observed in preceding studies of (B) C. elegans MA lines (Denver et al. 2000) and (C) C. briggsae MA lines (Howe et al. 2010).

Fig. 3

Distribution of observed overall mitochondrial mutation rates (all events including base substitutions and indels) for individual MA lines within three population size treatments (N = 1, 10, and 100 hermaphrodites). Observed rates were calculated per site per generation. Individual MA lines with zero mutations were excluded from the analysis resulting in sample sizes of 13 N =1 lines, eight N =10 lines and five N = 100 lines. Values are presented in log scale.

Fig. 4

The relationship between the normalized sum of the frequency of variants within individual MA lines and the population size. Values are presented in log scale. MA lines with zero mutations were excluded from this analysis. The sum of the frequency of variants was normalized by the number of base pairs analyzed for each line, and the number of generations. (A) The normalized sum of nonsynonymous, large deletion, and frameshift mutations in genic regions that may be under selection. (B) The normalized sum of putative neutral mutations comprising synonymous substitutions and intergenic mutations.

Fig. 5

The distribution of mtDNA variant frequencies within the N = 1 MA lines (n = 20) lines and 38 C. elegans natural isolates. Variant frequencies that fall below the dotted line (0.95) are heteroplasmic. The distribution of variants within the MA lines is approximately uniform whereas that of the natural isolates appears bimodal. The majority of variants detected within the natural isolates have reached fixation whereas the heteroplasmic variants tend to be in low frequency.

Fig. 6

Relative frequencies of nonsynonymous mtDNA mutations in C. elegans MA lines of varying population sizes and 38 natural isolates. The frequency of nonsynonymous changes is highest in the N =1 MA lines (n = 20 lines). The N > 1 category (n = 15 lines) comprises ten N = 10 lines and five N =100 lines. Lower relative frequencies of nonsynonymous mutations in the natural isolates is consistent with their deleterious consequences and eradication via natural selection.

Fig. 7

The distribution of >6 bp A or T homopolymeric runs in protein-coding genes of the C. elegans mtDNA genome. The ND5 locus contains proportionally more homopolymeric runs than any other protein-coding gene within the genome.