Cephalopod sex determination and its ancient evolutionary origin

Octopuses, squids, and cuttlefishes-the coleoid cephalopods-are a remarkable branch in the tree of life whose members exhibit a repertoire of sophisticated behaviors.¹ As a clade, coleoids harbor an incredible variety of novel traits, including the most complex nervous system among invertebrates, derived camera-type eyes, and rapid adaptive camouflage abilities.²^,³ The burst of evolutionary novelty that distinguishes cephalopods is even more striking in a phylogenetic context; cephalopods are a deeply diverged lineage that last shared a common ancestor with other extant molluscs in the Cambrian period, roughly 550 million years ago.⁴^,⁵ With recent advances in genome sequencing technologies, we have the capability to explore the genomic foundations of cephalopod novelties. Here, using PacBio long-read sequencing of genomic DNA and Iso-Seq full-length mRNA sequencing, we provide a novel chromosome-scale reference genome and annotation for a female California two-spot octopus (O. bimaculoides). Our assembly reveals that the female octopus has just one sex chromosome, consistent with a ZO karyotype, whereas the male has two (ZZ), providing the first evidence of genetic sex determination in cephalopods. We use our assembly and annotation in combination with existing genomic information from other cephalopods to create the first whole-genome alignments from this group and demonstrate that the sex chromosome is of an ancient origin, before the radiation of extant cephalopods approximately 480 million years ago,⁴ and has been conserved to the present day in all cephalopod genomes available.