Research Highlight: 100-Year-Old Mystery Solved, Posthumously

In 2011, Jeff Graham, a popular and highly respected physiologist at Scripps Institution of Oceanography at UC San Diego, submitted a paper to a major scientific journal describing the peculiar breathing habits of a fresh-water fish with two lungs known as Polypterus. While the reviewers of the paper recognized the merits of the research, they wanted to see some substantial changes before agreeing to its publication and the manuscript was rejected. Graham, who had been struggling in a battle with cancer, died a few weeks later, disappointed that his final research paper did not see the light of day.

But the story doesn’t end there.

Today, thanks to the efforts of Graham’s former students and colleagues, and funding from the National Science Foundation and the Australian Research Council, Graham’s paper has been published in Nature Communications, marking a major scientific milestone by solving a mystery dating back more than 100 years.

“I promised him we would get it published and give it its due diligence,” said Nick Wegner, Graham’s former Ph.D. student and now a researcher at Scripps and NOAA’s Southwest Fisheries Science Center.

Polypterus, commonly known as a “bichir,” inhabits African lakes and swamps and can be found at pet stores as an aquarium fish. Graham zeroed in on the species due to his long interest in studying air-breathing fishes, the evolutionary stepping-stone that set the stage for the first land-roaming animals. In 1906, a group of French researchers first described Polypterus and noted that the fish could inhale air through two holes on the top of its head known as spiracles.

Decades passed, and the observation of this unique mode of breathing air went largely unnoticed.  A 1989 study on air breathing in Polypterus failed to observe air breathing through the spiracles. So did these fish breath air from the holes on their head or not? And, if they did, why was it rarely observed?

Graham’s team set off to find out and acquired four different species of Polypterus through the aquarium trade. They devised a custom-built observation area, complete with camera to record the fish’s every move and any and all air-breathing behaviors. Graham noticed that when he approached the camera, the bichirs would quickly move away from the surface of the aquarium, as if spooked by the intrusion of a potential predator. So they built a second boundary around the camera to avoid detection.

And thus the true nature of Polypterus’s air-breathing activities were revealed. The researchers observed the fish day and night and found that, undisturbed, it gulps air from its spiracles up to 93 percent of the time.

Further, the researchers followed the spiracle’s evolution from prehistoric fish through higher-level organisms to the first land animals, and described how breathing functions evolved away from the top of the head to the mouth, with the spiracle eventually serving a function in the human inner ear core. The ear “popping” that we humans experience at high altitudes is the evolutionary remnant of the air-gulping spiracle.

“The spiracular canal first used for breathing in these ancient fishes and tetrapods no longer had a use when the fishes finally left the water and evolved into fully terrestrial animals (early amphibians),” said study coauthor John Long, a paleontologist at Flinders University in Australia, who examined fossil evidence for the project. “The amphibians began breathing through their nose and mouth, as we do today. The spiracular canals then became useful to transmit sounds from outside to the brain via the ear. The canal eventually became smaller and transformed into the Eustachian tube, and the remnant spiracle cover is now the tympanum, or ear drum, used for hearing.”

“It is important to understand evolution to know where we, as a species, have come from, and what our anatomical heritage is as evolutionary legacy,” said Long. “This paper details the origins of breathing in our distant ancestors and so informs us about how breathing first originated in early fishes and tetrapods.”

The coauthors of the paper are appropriately “breathing” a sigh of relief and feeling a sense of accomplishment in the name of the late Dr. Jeff Graham.

“I’m very proud and honored to have known Jeff and been able to work with him,” said Long, who first met Graham in 2003. “This paper is a major tribute to his knowledge and foresight to see the deep evolutionary significance of his physiological work on air-breathing fishes.”

“It’s an important discovery, so it puts the exclamation point at the end of [Graham’s] career,” said Wegner. “It’s a great feeling of accomplishment that we brought this home for him, knowing that this was the last big project he was working on.”

Following Graham’s death, former Scripps Director Charlie Kennel and his wife Ellen Lehman offered to match up to $125,000 in gifts received to establish an endowed fellowship in marine biology in Graham's name. Because Graham was so highly regarded and touched so many people, 95 people—family, faculty, staff, students, alums, fellowship supporters, and Birch Aquarium at Scripps volunteers—have contributed a total of $222,280 so far (with a goal to reach a fellowship minimum of $250,000).

“Ensuring the publication of Jeff’s last paper by his students is material proof of how much he gave to his students and, because of their mutual love and respect, their wish to give back to him,” said Lehman, who first bonded with Graham when the physiologist impressed her nephews on a visit to Scripps with an impromptu hands-on shark dissection. “We knew from Jeff that having a Fellowship for biology graduate students was a passionate wish of his and it was a way he could continue to educate generations to come and we wanted to support that wish, hence the Jeffrey B. Graham Fellowship and our offer of matching funds.”

In addition to Graham, Wegner, and Long, coauthors of the paper include Lauren Miller, Corey Jew, N. Chin Lai, Rachel Berquist, and Lawrence Frank.

-- Mario C. Aguilera

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