In a paper published in Nature Genetics, scientists reveal the genome assembly of the bowfin (Amia calva)—a bony fish endemic to eastern North America that is the sole surviving member of a once large lineage of ray-finned fishes.

This genomic study not only reveals diverse aspects of the biology of this enigmatic and ancient lineage that combines a unique suite of ancient and modern traits, but it also settles the long-debated phylogenetic relationships in neopterygii—the subclass of ray-finned fish to which the bowfin belongs.

The sequencing analyses identified hundreds of gene-regulatory loci conserved across vertebrates and provide new insight into vertebrate evolution. For instance, genes pivotal in the development of key components of the anatomy are not as invariable as previously thought.

The paper titled, “The bowfin genome illuminates the developmental evolution of ray-finned fishes,” is a collaborative effort of an international team of researchers headed by Ingo Braasch, PhD, and Andrew Thompson, PhD, of Michigan State University.

“By studying more species, we learn which rules are hard and fast and which ones evolution can tinker with. Our study shows the importance of sampling a broader swath of natural diversity. We might just find important exceptions to established rules,” said M. Brent Hawkins, PhD, co-author on the paper.

Scientists have long been fascinated with the bowfin because it bears a combination of ancestral features, such as lung-like air-breathing and a robust fin skeleton, and derived features like simplified scales and a reduced tail. The bowfin also occupies a key position in the fish family tree. It is positioned between the teleosts—a large, diverse more recent group that is emerging as the dominant lineage in most aquatic habitats—and more ancient branches that include sturgeons, paddlefish, and bichirs.

Schematics show the arrangement of bones in fins and limbs. Elements that are derived from the ancestral metapterygium are shown in magenta. The tetrapod limb and a portion of the bowfin fin arose from the metapterygium, while teleosts have lost the metapterygial components. [Source: M. Brent Hawkins]
The strategic phylogenetic position of bowfin can help better understand how modern fishes evolved from their ancient predecessors by comparing the genetic basis of old and new traits. Information gleaned from the bowfin genome can also help understand the origin of the more modern teleosts that have duplicated and modified their genomes since separating from the bowfin lineage.

Hawkins examined the evolution and development of the bowfin pectoral fin, as part of his doctoral thesis at the department of organismic and evolutionary biology at Harvard University. Hawkins’ doctoral thesis, conducted with Matthew P. Harris, PhD, researher at Harvard Medical School and Boston Children’s Hospital, and James Hanken, PhD, professor, department of organismic and evolutionary biology at Harvard University, contributed some of the study’s most surprising findings.

The bowfin retains the metapterygium—a portion of the fin skeleton that is homologous to the limb bones of tetrapods—that is lost in model organisms like zebrafish and medaka. By studying the bowfin fin, scientists can use knowledge of bowfin development as a bridge between teleost fin development and tetrapod limb development to explain the evolutionary transition from fins to limbs.

The researchers collected bowfin embryos from the wild in upstate New York, raised the embryos, and collected samples of their pectoral fins as they developed. Transcriptome sequencing and comparisons with reference genomic sequences allowed them to determine which genes are turned on in the developing fin. Then the authors used in situ hybridization to validate the transcriptomic findings.

“As a field, we have characterized many of the genes involved in appendage patterning. We have a good idea of what the essential fin and limb genes are and where they should be turned on,” said Hawkins, who was shocked by what the fin data revealed.

The bowfin lacks some of the most critical genes for limb development, such as the gene called fibroblast growth factor 8 (Fgf8). In most vertebrates including fishes, Fgf8 is considered a “master regulator” that is turned on at the far tip of developing fins and limbs and is required for the outgrowth of these appendages. Loss of Fgf8 impairs growth of limbs and an excess of causes new limbs to form.

“Every other fin and limb we know of expresses Fgf8 during development,” Hawkins said. “Discovering that bowfin fins don’t express Fgf8 is like finding a car that runs without a gas pedal. That the bowfin has accomplished this rewiring indicates unexpected flexibility in the fin development program. With the genome in hand, we can now unlock how this flexibility evolved.”

In contrast to missing genes, some genes are unexpectedly activated. For instance, HoxD14 is expressed in the fins of fishes from the deeper branches of the fish family tree, such as paddlefish, but this gene was lost in more recent branches including the teleosts.

Initially, the authors thought the presence of HoxD14 in the bowfin DNA indicated it was a pseudogene that is not actually expressed. But they found that bowfin fins expressed HoxD14 transcripts at high levels, even though it did not code for a protein.

“The fact that the HoxD14 gene can no longer make a protein, but it still transcribed into mRNA at such high levels suggests that there might be another function that we do not yet understand. We might be seeing a new level of Hox gene regulation at play in the bowfin,” said Hawkins.

The Fgf8 and HoxD14 results indicate that genetic programs, even those that guide the formation of important structures such as fins and limbs, are not as invariable as previously thought. Hawkins also suggests that the results of the bowfin study serve as a warning against treating members of deeper branches of the tree of life as stand-ins for actual ancestors.

Hawkins said, “Some people might describe species like the bowfin as a ‘living fossil’ that reliably represents the ancestral condition of a lineage. In reality, these deeper branches have been evolving past that ancestor for just as long as the more recent branches, doing their own thing and changing in their own ways. In evolution, old dogs do learn new tricks.”

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