Abstract
Ageing is a complex biological process that is accompanied by changes in gene expression and mutational load. In many species, including humans, older fathers pass on more paternally derived de novo mutations; however, the cellular basis and cell types driving this pattern are still unclear. To explore the root causes of this phenomenon, we performed single-cell RNA sequencing on testes from young and old male Drosophila and genomic sequencing (DNA sequencing) on somatic tissues from the same flies. We found that early germ cells from old and young flies enter spermatogenesis with similar mutational loads but older flies are less able to remove mutations during spermatogenesis. Mutations in old cells may also increase during spermatogenesis. Our data reveal that old and young flies have distinct mutational biases. Many classes of genes show increased postmeiotic expression in the germlines of older flies. Late spermatogenesis-biased genes have higher dN/dS (ratio of non-synonymous to synonymous substitutions) than early spermatogenesis-biased genes, supporting the hypothesis that late spermatogenesis is a source of evolutionary innovation. Surprisingly, genes biased in young germ cells show higher dN/dS than genes biased in old germ cells. Our results provide new insights into the role of the germline in de novo mutation.