Supplementary material from "Single-sperm sequencing reveals the accelerated mitochondrial mutation rate in male Daphnia pulex (Crustacea, Cladocera)"

Mutation rate in the nuclear genome differs between sexes, with males contributing more mutations than females to their offspring. The male-biased mutation rates in the nuclear genome is most likely driven by a higher number of cell divisions in spermatogenesis than in oogenesis, generating more opportunities for DNA replication errors. However, it remains unknown whether male-biased mutation rates are present in mitochondrial DNA (mtDNA). Although mtDNA is maternally inherited and male mtDNA mutation typically does not contribute to genetic variation in offspring, male mtDNA mutations are critical for male reproductive health. In this study, we measured male mtDNA mutation rate using publicly available whole-genome sequences of single sperm of the freshwater microcrustacean Daphnia pulex. Using a stringent mutation detection pipeline, we found that the male mtDNA mutation rate is 3.32 × 10⁻⁶/site/generation. All the detected mutations are heteroplasmic base substitutions, with 57% of mutations converting G/C to A/T nucleotides. Consistent with the male-biased mutation in the nuclear genome, the male mtDNA mutation rate in D. pulex is approximately 20 times higher than the female rate per generation. We propose that the elevated mutation rate per generation in male mtDNA is consistent with an increased number of cell divisions during male gametogenesis.