Unlocking a Fish’s Genetic Code

The National Fish Collection is an impressive sight—a warehouse with row upon row of shelves stacked with fish-filled jars. Millions of specimens reside in the Smithsonian National Museum of Natural History. Some are massive glass containers with handwritten scrawl denoting the specimen’s origin, others are old canning jars with typewriter printed labels, and still other small vials topped with cotton. The oldest of these specimens date back to the early 1800’s. Many of the fish in each jar were initially preserved in formalin and later stored in alcohol long term. “Formalin is a really great fixative, the reason it is called a fixative is that it creates bonds at the cellular level and prevents decay,” explains Matt Girard, zoologist at the Smithsonian’s National Museum of Natural History.  Today, that poses a problem for researchers trying to decode the DNA in preserved specimens.

To determine new species and figure out relationships between close relatives, scientists can use physical anatomical traits as well as new genetic tools that can help reveal the evolutionary history of life. These genetic tools allow for new and exciting identifications and relationships to be discovered, which would have been difficult to understand through studying physical traits alone. To use these genetic tools, scientists cut off a small piece of a newly collected specimen and preserve it in alcohol because it allows for easy genetic sequencing. Scientists then use various methods to pull the DNA strands apart and “read” each sequence. While many new specimens have associated tissue samples for genetic sequencing, the vast majority of the National Collection has been inaccessible for genetic sequencing because of formalin preservation. “The problem with formalin is that those bonds can be so strong that they can break DNA and, not only that, we have struggled to extract and sequence that DNA when it’s so fragmented,” says Girard.  

For Girard, the formalin conundrum is particularly bothersome. Many fish species are so rare that only a single specimen exists in collections. Often, those specimens, at some point, were likely preserved with formalin. To make matters more complicated, scientists of the past rarely recorded if the specimen was preserved in formalin or when the specimens were switched from formalin to alcohol. “I can’t guarantee that anything has ever been in formalin or ever not been in formalin, so I have to assume that it, at some point, was,” says Girard.  

Not understanding the genetics of these historic and preserved specimens impacts our understanding of fish species in today’s waters. For many years, scientists have tried to find a solution to formalin’s tight grip of DNA. Early attempts initially seemed to be successful, but further scrutiny showed the DNA sequences were inaccurate. But Girard searched for other protocols, finding some initially developed for the medical field. Since the late 1800s the preferred preservation method for medical biopsies has been formalin due to its ability to preserve the physical shape and properties of cells for later analysis. The modern alternative, freezing samples, can damage cells and requires space and energy to run freezers. With so many stored formalin specimens, researchers tried to see if they could decode their genetic sequences for further study. The first attempts were expensive, with complicated procedures, and multiples harsh chemicals, but by the late 2010s a simple method for extracting formalin preserved DNA from medical biopsies became a reality. Now, Girard, is using the same process for fish.

“What we have done is get the DNA out of a couple of species that are rare, sequenced them, and then done some checks computationally to make sure what we are sequencing is as accurate as possible,” says Girard.

Girard chose a commercially important and familiar fish family, threadfins, to test whether a kit for formalin DNA extraction could be used to extract the DNA from museum specimens. The family consists of 42 species, but past genetic studies have only been able to include 30 species since many of the species only have formalin-preserved specimens. And though past study of threadfin anatomy revealed some insight about their familial relationships, researchers continue to be perplexed by some of their physical traits. Many species that overall look distantly related share one or several very specific traits, like extremely long fin rays. Girard chose two extremely rare species that lack modern day genetic samples preserved in alcohol for his test.

After extracting a piece of liver from one Slender fivefinger threadfin (Polydactylus bifurcus) and one River threadfin (Polydactylus macrophthalmus), Girard used the methods from the kit to extract DNA from the preserved specimens. He then sequenced this DNA for each species and mathematically checked to ensure the sequences made sense (they did). The final step required comparing the new DNA sequences to the DNA of other threadfins.

Girard found that not only did the kit work, the two threadfins now had a place in the threadfin family tree for the first time, supported by genetic evidence. The River threadfin is closely related to the Indian threadfin (Leptomelanosoma indicum) and are likely in the same genus. And after sequencing the Slender fivefinger threadfin, Girard found that three other threadfins were very similar. It is likely that together the four threadfins are closely related and in their very own new genus.  

“For those of us that have specimens sitting in formalin for who know how long…it’s a big deal to get DNA from them,” said Girard.

The millions of formalin-preserved specimens stored in museum’s shelves across the world may now be fair game for genetic study. For rare species, like the threadfins, it means untapped information about family lineages and evolutionary ties—information that may reveal new species. For the thousands of other species, these methods may allow for many to be sequenced and comparisons to be made between the past and the present. In a rapidly changing world, this can provide critical knowledge for policy makers and inform conservation measures. For museum workers, it creates new opportunities to unravel the mysteries of the natural world.