The mutation landscape of Daphnia obtusa reveals evolutionary forces shaping genome stability.

Spontaneous mutations are the primary source of genetic variation and play a central role in shaping evolutionary processes. To investigate mutational dynamics in Daphnia obtusa, we generated a chromosome-level genome assembly spanning 129.4 Mb across 12 chromosomes, encompassing 15,321 predicted protein-coding genes. Leveraging whole-genome sequencing of eight mutation-accumulation (MA) lines propagated for an average of 482 generations (spanning over 20 years), we estimated a spontaneous single-nucleotide mutation (SNM) rate of 2.45 × 10-9 and an indel mutation rate of 3.34 × 10-10 per site per generation. The SNM spectrum was strongly biased toward C:G > T:A transitions. Despite the design of MA experiments to minimize selection, nonsynonymous mutations were strongly underrepresented, providing rare evidence that purifying selection can act detectably even during mutation accumulation. Comparative analyses with natural population data revealed that exonic mutations observed in the MA lines were significantly less likely to be present in standing variation than intronic or intergenic mutations, suggesting that purifying selection in natural populations acts to remove deleterious alleles. We also identified 48 loss-of-heterozygosity (LOH) events, comprising 8 heterozygous deletions and 40 gene-conversion events, yet found no evidence of GC-biased gene conversion. Instead, while mutation predicts a substantially lower equilibrium GC content, the observed GC level is maintained at higher values, implicating natural selection as the primary force stabilizing base composition. Together, these results provide one of the most comprehensive assessments of the interplay among mutation, selection, and genome stability in an ecologically important species.