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 patterns in highly heterozygous genomes remain 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 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 205 high-confidence DNMs, yielding a genome-wide mutation rate of 5.10 × 10 ^-9 per base per generation, which is comparable to that of vertebrates. Combining this estimate with observed nucleotide diversity, we obtain an effective population size of ∼1.9 million, indicating that the elevated heterozygosity mainly results from a large effective population size. We observe a maternal-origin bias when considering all DNMs but a paternal-origin bias for early-occurring ones. Amphioxus harbors a much smaller fraction of CpG>TpG DNMs relative to vertebrates, attributable to its low methylation levels. We also investigate putative post-zygotic mutations in the offspring, revealing an unexpected paternal-origin bias. These suggest some distinct mutational mechanisms in amphioxus. Our study not only provides the first DNM measurement for amphioxus but also offers a generalizable strategy for studying DNMs in highly heterozygous genomes, facilitating mutation rate studies across chordates and other lineages.