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Nautiloids—a lineage of ancient, externally-shelled cephalopods that diverged from their octopus and squid relatives over 400 million years ago—once dominated our oceans.

Today, this living fossil is restricted to a handful of species in the Southern Indo-Pacific, making it one of the few marine invertebrates listed under CITES appendix II of species in need of protection from over-exploitation.

Although no one had previously investigated sex determination systems in cephalopods, recent research suggested a ZZ/Z0 system, where males are homozygous (having two identical sex chromosomes) and females are hemizygous (having only one sex chromosome). This system was believed to have originated approximately 480 million years ago in the last common ancestor of all cephalopods, thus making it one of the oldest conserved sex determination systems known in animals.

However, a study in Current Biology challenges this in modern cephalopods. Instead, researchers discovered the first evidence of an XX/XY system in chambered nautiluses. This genetic mechanism is more similar to that found in humans, mammals and many other animals, where males are the heterogametic sex (XY).

The international team of researchers led by Professor David Combosch of the Marine Laboratory at the University of Guam, with co-author Professor Gonzalo Giribet in the Department of Organismic and Evolutionary Biology and Director of the Museum of Comparative Zoology at Harvard, analyzed three distinct genomic datasets.

These included 28 low-coverage whole genomes and 63 restriction-site associated DNA sequencing (RAD-seq) datasets sourced from six species and nine populations of nautiloids.

Using Bayesian analyses, sex-specific differences in genome coverage, and patterns of heterozygosity, they identified one DNA segment as an X chromosome and pinpointed five additional DNA segments as likely Y-linked regions. These five Y-scaffolds contain 36 genes, most of which were either male-specific or significantly enriched in males.

"This is the first time anyone has identified X- or Y-linked sequences in a cephalopod," Giribet noted. "Our findings suggest that sex chromosomes in mollusks are far more dynamic and lineage-specific than previously assumed."

To investigate the biological relevance of these genes, the researchers performed functional annotation and BLAST protein searches using a stringent e-value threshold. Many genes showed homology to human genes with known expression in reproductive tissues or links to sex-related traits, as documented in genome-wide association studies.

Further Gene Ontology enrichment analysis supported the idea that these genes are involved in sex-specific functions. The team also identified chromosome #4 as the X chromosome rather than the Z chromosome as previously assumed.

Despite the breakthrough, the study faced key limitations. The lack of a chromosome-level genome for male nautiluses limited the researchers' ability to fully characterize the structure and evolution of the sex chromosomes. Still, this work lays the foundation for further genomic exploration across other cephalopod lineages and has broader implications for cephalopod biology and conservation.

Several nautilus species are currently listed as vulnerable due to overharvesting and habitat loss. A better understanding of their genetics, including sex determination mechanisms, could directly inform future conservation and management efforts.

By revealing that nautiluses possess an XX/XY sex determination system, this research not only revises assumptions about cephalopod genetics, but also contributes a critical piece to the broader puzzle of sex chromosome evolution in animals.

"Our results rewrite our understanding of cephalopod sex determination and help clarify the evolutionary history of these fascinating animals," Combosch said.

"It reminds us that, in the natural world, even the most fundamental biological systems can evolve quickly and in unexpected ways."