Large unknowns remain regarding the inheritance of mitochondrial mutations and the role of evolutionary forces in shaping variation in mitochondrial genomes. Further, mutations in mitochondrial DNA (mtDNA) can greatly impact organismal fitness. mtDNA molecules can differ within an individual organism, the same cell, or even a single mitochondrion, a state referred to as heteroplasmy. Evolutionary forces may therefore have influence not only on the organism, but also on other biological levels such as mitochondria and mtDNA. Improved knowledge on the phenomenon of heteroplasmy is essential to further understanding of the mtDNA mutation process and how different evolutionary forces act on these molecules.In order to investigate mtDNA heteroplasmy, we employed mutation accumulation (MA) line methodologies and the wildtype (N2) and
gas-1 mutant strains of C. elegans.
gas-1 nematodes have a dysfunctional mitochondrial electron transport chain Complex I, experience increased endogenous (reactive oxygen species) ROS production and reduction in fitness. We studied mtDNA changes that occurred across a maximum of 50 generations of single-worm bottlenecking in MA lines propagated from the
gas-1 progenitor strain using an Illumina Mi-Seq high-throughput DNA sequencing approach. N2 MA lines (250-generation) were also analyzed, enabling the distinction between the effects of nematode bottlenecking from those due to the
gas-1 mutation. To control for developmental fluctuations in mtDNA copy number, L1-stage nematodes were analyzed. After Illumina sequencing five MA lines and progenitors from each of N2 and
gas-1 C. elegans, bioinformatic approaches were used to evaluate mtDNA copy number and identify and characterize heteroplasmic nucleotide variants of mtDNA. Consistent large increases in mtDNA copy number were observed for both N2 and
gas-1 MA lines, suggesting that this is driven by nematode bottlenecking. Our approach also revealed insights into in the inheritance dynamics of heteroplasmic mitochondrial mutations. We identified nine high-confidence base substitution heteroplasmies, eight in
gas-1 lines and one in N2 lines, with an average level of 8%. A mitochondrial heteroplasmy present at low levels (~5%) in
gas-1 progenitor continued to segregate in two of the five derivative MA lines analyzed, arising to fixation in one and maintained at high levels (~60%) in the other. The single case of heteroplasmy observed in the N2 MA lines was present in the progenitor at low levels (~4%) and absent in all five derivative MA lines. Insertion-deletion heteroplasmies were also observed, most frequently in the N2 MA lines and at homopolymer run sites. This study provides new insight on mitochondrial genome changes that accompany relaxed selection, and how this phenomenon differs in organisms with preexisting mitochondrial dysfunction.