From color-changing skin to jet-propelled motion, squid and cuttlefish have long fascinated scientists. To understand the origins of their unique characteristics, many attempts have been made to define their evolutionary history. However, the limited fossil record and incomplete genomic information have made it impossible to confidently order the evolution of these enigmatic creatures, until now.
Published in Nature Ecology & Evolution, a new study from the Okinawa Institute of Science and Technology (OIST) combines existing databases with three newly sequenced squid genomes to identify the 'long fuse' that led to today's diversity of squid and cuttlefish, which together make up the decapodiform (ten-limbed) cephalopods.
Dr. Gustavo Sanchez, first author on the study and Staff Scientist in OIST's Molecular Genetics Unit , says, "Squid and cuttlefish are remarkable creatures, yet their evolution has been notoriously difficult to study. The question of their ancestry has been under investigation for decades, and many research groups have proposed different evolutionary hypotheses based on different morphological characteristics and molecular datasets. With our new genomic information, we have been able to resolve some of the mysteries surrounding their origins."
Demystifying the decapodiformes
Squid and cuttlefish are found in a wide variety of habitats across the globe, from deep seas to shallow coastlines. One of the few characteristics linking most of these diverse creatures is their internal shell. But even this takes a variety of forms, from the smooth, rounded cuttlebones of cuttlefish, the thin, sword-like gladius of oceanic and coastal squid, and the spiral-shaped shell of ram's horn squids, to a complete loss in shallow water species.
Past attempts to order the evolution of these animals have been limited by a lack of data. Sanchez explains, "Earlier reconstructions of decapodiform evolution were built from datasets with limited resolution and were prone to biased signals, obscuring the true relationships between different species. Whole genome data now provide a cleaner, more consistent picture of how these animals evolved."
Because most squid and cuttlefish genomes are large, typically reaching up to twice the size of human genomes, generating and analyzing them requires state-of-the-art sequencing facilities and considerable computational power. Researchers also need fresh DNA for sequencing, which is a challenge when sourcing specimens at sea. "Some lineages are only abundant and highly diverse in tropical reef systems like the Ryukyu Archipelago, while others are enigmatic and known only in the deep sea. We were fortunate to find some key species on our doorstep in Okinawa, and collaborate with colleagues with access to more challenging samples," says Sanchez.
The paper presents the first-ever evolutionary tree for decapodiformes that is based on sequenced genomes from nearly all decapodiform lineages. This was made possible due to a global collaboration spanning the last five years, with the Aquatic Symbiosis Genomics Project funded by the Wellcome Sanger Institute aiming to sequence some cephalopod genomes among other marine and freshwater species. Sanchez headed the Japanese branch of the cephalopod hub of this project.
"Within the symbiosis project, we've been steadily sequencing genomes for several years, but several key gaps remained. In this study, we were able to fill these missing puzzle pieces," confirms Sanchez.
Co-author Dr. Fernando Á. Fernández-Álvarez of the Spanish Institute of Oceanography was especially enthusiastic to study the enigmatic ram's horn squid, Spirula spirula, a rarely encountered species whose unusual internal shell has long puzzled biologists. From the moment he had it in hand, he saw its genomic potential. "In the past, the structure of the ram's horn squid shell made some scientists wrongly conclude it was closely related to cuttlefishes.", says Fernández-Álvarez. "I believed this genome could help close a key gap and bring clarity to the broader evolutionary questions of cephalopods."
A long fuse model of evolution
Using a combination of genomic data and recently discovered fossils, the researchers were able to map out an evolutionary timeline and ecological scenario for the origin and diversification of squid and cuttlefish.
"Our analysis shows that these animals originated in the deep ocean, a habitat which still harbors species like the ram's horn squid," says Sanchez.
The model shows that the different decapodiform orders first split rapidly around 100 million years ago, putting their origins firmly in the mid-Cretaceous period. However, 66 million years ago, a catastrophic mass extinction event known as the Cretaceous-Paleogene (K-Pg) wiped out three-quarters of the plant and animal species on Earth. This same event famously led to the extinction of dinosaurs and the rise of mammals. So how did squid survive?
The researchers believe that ancient cephalopods were able to find refugia within tiny deep-sea microcosms which harbored an abundance of oxygen. Sanchez explains, "The sea surface would have been a very harsh environment for cephalopods. Around that time, very few suitable oxygen-rich habitats would have been found near the shores. Intense ocean acidification in shallower waters would also likely have degraded their shells, so the fact that some form of this feature has been retained throughout their evolutionary history is evidence of their deeper oceanic origins."
After the K-Pg event, coral reefs started to rebuild along coastlines. This created more habitable shallow water ecosystems, to which many of the ancient 10-limbed cephalopod lineages migrated.
"Following the initial lineage splits in the Cretaceous, we don't see much branching for many tens of millions of years. However, in the K-Pg recovery period, we suddenly see rapid diversification, as species adapt and evolve to new and changing ecosystems. This is an example of a 'long fuse' model; a period of limited change followed by an explosion of diversity," says Sanchez.
From gene evolution to gene editing
The team hopes this research can provide a framework for future investigations into the origins of decapodiformes' unique characteristics.
"Squids and cuttlefish have so many unique features compared to other animal groups, making them an endless source of inspiration for scientists," says Prof. Daniel Rokhsar, head of the Molecular Genetics Unit. "With these genomes and with a clear picture of their evolutionary relationships, we can make meaningful comparisons to uncover the molecular changes associated with major cephalopod innovations, from the emergence of novel organs and dynamic camouflage to the neural complexity that supports their remarkable behavior."
