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This illustration from a 14th century Dutch encyclopedia of animal life shows scientists have been thinking about the fin to limb transition for centuries.

Der naturen bloeme/Nationale Bibliotheek

Fish had the genes to adapt to life on land—while they were still swimming the seas

Almost 700 years ago, Jacob van Maerlant, a Dutch poet, envisioned a fish all set for life on land: It had sprouted arms to hoist itself ashore. Now, three genetic studies make his fantasy look remarkably prescient. Together, the studies suggest that in terms of genes, the aquatic precursors of four-limbed land animals, or tetrapods, were as well-prepared as the Dutch fantasy fish. They were pre-equipped with genes that could be turned to making limbs, efficient air-breathing lungs, and nervous systems tuned to the challenges of life on land.

“All these studies tell us that the origin of tetrapods was something waiting to happen,” says Borja Esteve-Altava, an evolutionary biologist at Pompeu Fabra University in Barcelona, Spain. Genetically, “Everything necessary was already there” before vertebrates came ashore, nearly 400 million years ago.

Fossils reveal the outlines of the story. Fish with fleshy fins supported at their base by a single bone, known as lobe-finned fish, moved into shallow water about 375 million years ago. About 5 million years later, some of those lobe fins crawled onto terra firma. The first fish to set fin on land must have already had at least some of the physical traits and genetic modifications needed to do so, but researchers hadn’t worked out how and when they became equipped for the change. “The big question of how such a large morphological shift actually occurred remains very much in play,” says Peter Currie, an evolutionary developmental biologist at Monash University.

In the trio of studies published last week in Cell, genes in living fish took the place of fossils as a way to peer back in time. One set of clues came from studies of mutagenized zebrafish, a favorite model for studying development. M. Brent Hawkins, then a Harvard University graduate student and now a postdoc, was shocked to discover zebrafish mutants with two bones resembling the forelimb bones of land animals in their front fins, complete with muscles, joints, and blood vessels. The finding is “quite spectacular,” says Marie-Andrée Akimenko, a developmental biologist at the University of Ottawa.

Two mutated genes, vav2 and waslb, were responsible for the transformation. Both genes code for proteins that are part of a pathway controlling the activity of Hox11 proteins, regulatory molecules that guide the formation of the two forearm bones in mammals, among other functions. In fish, other proteins normally suppress Hox11 and prevent the formation of those bones. But the mutations, which Hawkins re-created using the gene editor CRISPR, reactivate the pathway. The “landmark” finding is “changing the paradigm on limb development and evolution,” says Renata Freitas, a developmental biologist at the University of Porto in Portugal.

Other genetic clues come from living representatives of ancient fish lineages. Only two groups of the lobe-finned fish are alive today: lungfish and coelacanths. About 400 million years ago, they diverged from the line of lobe-finned fish that gave rise to tetrapods 30 million years later. Today’s oceans are mostly populated with species from another group that originated about 420 million years ago: the ray-finned fish, so named because their fins are supported by slender spines.

Evolutionary geneticists Guojie Zhang at the University of Copenhagen and Wen Wang of Northwestern Polytechnical University in Xi’an, China, and their colleagues sequenced the genomes of the African lungfish, which branched off early from other lobe-finned fish. The researchers also sequenced the bichir, an elongated, air-breathing, ray-finned fish that lives in the shallows of tropical African rivers, as well as the American paddlefish, the bowfin, and the alligator gar. All are ray-finned fishes that evolved much earlier than teleosts, the group that dominates the world’s waters today (see diagram, below). Knowing when each of those lineages branched away from others, the researchers could infer when and where certain genes first appeared on the fish family tree.

The long swim to land

The groundwork for terrestrial traits like limbs and lungs was laid deep in the fish family tree. Genes for such traits found in both lobe-finned and ray-finned fishes must also have been present in their common ancestor.

Cartilaginous fishes Nonteleost ray-finned fishes Teleosts Tetrapods Lungfish Common ancestor of jawed vertebrates (460 million years ago) Common ancestor of ray-finned fishes (420 mya) Common ancestor of lobe-finned fishes (420 mya) Common ancestor of bony fishes (425 mya)
X. BI ET AL., CELL, 2021, DOI.ORG/10.1016/J.CELL.2021.01.046, ADAPTED BY N. DESAI/SCIENCE

None of the sequenced fish is on the precise branch that led to tetrapods. Yet all have much of the genetic equipment needed for life on land, including most of the genes and regulatory DNA needed to build limbs. For example, all the fish sequenced have a regulatory element that helps form synovial joints, which make fins and limbs flexible and are essential for terrestrial locomotion. The fish also have 11 genes that are needed to build lungs and that work the same way in the bichir’s lungs as they do in humans. One is for a pulmonary surfactant, a lubricating secretion that helps lungs expand and contract. Both the ray-finned fishes and the lobe-finned lungfish also apparently have a regulatory element that helps shape the right ventricle of the heart to deliver oxygen more efficiently.

The findings show that “a lot of things we think are just in land animals are also in fish,” says Gage Crump, a developmental biologist at the University of Southern California. Finding all those genes in both lobe-finned and ray-finned fish means those genetic pathways must have been present in their common ancestor, some 425 million years ago. “It is surprising that some of these elements are so conserved for such a long evolutionary time,” Zhang says. (Teleosts, in contrast, have lost much of the DNA that prepared early fish for life on land, apart from the Hox11 pathway, the team reported.)

The genome of the lungfish offers a glimpse of later adaptations along the path to terrestrial life. It includes additional pulmonary surfactant genes that the ray-finned fishes lack, as well as DNA for specifying five toes, connecting nerves to limb muscles, and for sensitizing the brain to react fast. All those genes were previously thought to be unique to tetrapods.

Putting it all together, Wang and Zhang think the transition to land involved three key steps. The ability to breathe air occasionally appeared in the common ancestor to ray-and lobe-finned fish, about 425 million years ago. Then surfactant genes, new nervous system genes, and other innovations enabled lobe-finned fish to leave the water temporarily. Finally, after the African lungfish split off from the lobe fins, the common ancestor of land vertebrates acquired other respiratory and locomotive refinements needed to live out of water.

Rather than building new structures and genetic pathways just when vertebrates moved onto land, evolution apparently was thrifty, using existing genes to adapt to the opportunities offered by terrestrial habitats. “[The studies] show the extent to which the fish-tetrapod transition was achieved by modifying existing molecular systems, rather than creating new ones,” says Per Ahlberg, a paleontologist at Uppsala University.

Gaps still remain in understanding how fish made landfall, but the new studies “bring us closer to the living biology of the fish-tetrapod transition,” Ahlberg adds. Van Maerlant would be pleased.

*Update, 10 February, 12:30 p.m.: This story, first published on 4 February, has been updated to include information on two additional papers using genomics to understand how vertebrates adapted to life on land.