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Amphioxus, as the best living proxy to the chordate ancestor, is considered an irreplaceable model organism for evolutionary studies of chordates and deuterostomes. In this study, a high-quality genome of the Beihai amphioxus,Branchiostoma belcheribeihai, wasde novoassembled and annotated. Within four amphioxus genomes, a wide range of gene novelties were identified, revealing new genes that share unexpectedly high similarities with those from non-metazoan species. These gene innovation events have played roles in a range of amphioxus adaptations, including innate immunity responses, adaptation to anaerobic environments, and regulation of calcium balance. The gene novelties related to innate immunity, such as a group of lipoxygenases and a DEAD-box helicase, boosted amphioxus immune responses. The novel genes for alcohol dehydrogenase and ferredoxin could aid in the anaerobic tolerance of amphioxus. A proximally arrayed cluster of EF- hand calcium-binding protein genes were identified to resemble those of bacteria. The copy number of this gene cluster was linearly correlated to the sea salinity of the collection region, suggesting that it may enhance their survival at different calcium concentrations. Collectively, this comprehensive study on gene novelties of amphioxus reveals insights into the early genome evolution of chordates and deuterostomes and provides valuable resources for future research.Significance statementAmphioxus is a slow-evolving model organism for the study of chordate evolution, and while much research has focused on its ortholog evolution, relatively little attention has been paid to its gene innovations. In this study, we assembled and annotated a high-quality genome of the Beihai amphioxus and identified gene novelties across four amphioxus genomes. Many of these new genes resembled those from non-metazoan species, suggesting the possibility of horizontal gene transfer. These gene novelties have been found to enhance amphioxus adaptations, including innate immune responses and anaerobic tolerance. Of particular interest is a cluster of novel EF-hand calcium- binding protein genes located in proximity, which present similarity to bacterial genes. These genes may facilitate adaptation to different calcium concentrations in amphioxus.