Germline de novo mutation rate of the highly heterozygous amphioxus genome

Germline de novo mutations (DNMs) are the ultimate source of heritable variation, yet their occurrence in highly heterozygous genomes remains poorly understood. Amphioxus, an early-branching chordate with exceptionally high genomic heterozygosity (3.2∼4.2% in sequenced species), offers a unique model to explore mutational dynamics in such contexts. It is unclear whether the high heterozygosity in amphioxus is due to a large effective population size, an increased mutation rate, or both. Here, we perform deep short-read whole genome sequencing of a two-generation pedigree of the amphioxus Branchiostoma floridae comprising two parents and 104 offspring, and develop a framework based on allele-aware parental assemblies as the reference to accurately identify DNMs. We detect 256 high-confidence DNMs, yielding a genome-wide mutation rate of 4.41 × 10 ⁻⁹ per base per generation, which is comparable to that of vertebrates. By combining this estimate with observed genomic heterozygosity of B. floridae , we obtain an effective population size of ∼2.2 million, indicating that its extreme heterozygosity mainly results from a large effective population size. Furthermore, we observed a maternal mutation bias and increased mutation rates in coding regions, suggesting unique mutational mechanisms in amphioxus. Our study not only provides the first mutation rate estimate for amphioxus but also offers a scalable strategy for studying DNMs in highly heterozygous genomes, shedding light on the evolution of mutation rates across chordates.