Tom Cullen vividly recalls a day in the Louisiana bayou when he and fellow team members from Chicago's Field Museum stopped to collect a dead squirrel by the side of the road. They found a treasure trove of fish scales and bones—plus a fox's jawbone—decomposing just 100 feet away, most likely dropped by a feasting bird of prey. It was like roadkill manna from heaven for Cullen, who was there collecting specimens for a project to explore the ecosystems dinosaurs lived in millions of years ago.

"We were interested in trying to understand dinosaur ecology and how dinosaurs interact with their habitats and with other organisms they co-exist with," said Cullen. The Mesozoic ecosystem was essentially "a large coastal floodplain forest," so he reasoned that it might be a good idea to study a similar modern-day ecosystem: Louisiana's swamps and bayous. The results from that project have now been published in the Royal Society Open Science journal.

Scientists typically rely on stable isotope analysis with mass spectrometry to determine food web structure in ecosystems: that is, what different animals eat and where they fit in the overall food chain. Stable isotopes (versions of elements that have different numbers of neutrons and are hence lighter or heavier) are preserved in animals' bones and teeth, lasting tens of millions of years.

"You are what you eat, more or less," said Cullen. Plants contain different relative amounts of stable isotopes for carbon and oxygen, for example. When animals eat the plants (or other animals that ate those plants), those isotopes are stored in the tissues.

But Cullen noticed a lot of variability in previous work on isotopes. He decided to test the method against Louisiana's similar (though not identical) ecosystem to establish a baseline—a due diligence exercise. Then the challenge became trying to get as diverse a collection of samples as possible.

"We knew we needed to get a very wide spectrum of the whole community," said Cullen. "Doing that for dozens of different species would be exceptionally time-consuming and well beyond the budget we had available."

“Biologists have a well-deserved reputation for picking up random dead things everywhere.”

Originally, Cullen had intended to piggyback on the data-collection efforts of local Fish and Wildlife staffers and other field researchers, collecting tissue samples from them as needed. But he got permission to also take a few samples from roadkill in the Atchafalaya Basin, the largest swamp in the country, just to assess the feasibility of such an approach. "It was successful beyond our wildest dreams," he said. They wound up collecting around 50 samples representing 15 species just in the first two days."

If that seems kind of disgusting, Cullen admits that "biologists have a well-deserved reputation for picking up random dead things everywhere." Still, there was some initial aversion to the nastier roadkill finds, like a dead raccoon crawling with maggots. Cullen decided to pull out a tooth instead of getting a tissue sample, and the entire jaw slid out from the rotting flesh. Gradually, he became desensitized to the sights and smells of decomposing animal carcasses. "Gross as it was, it was some of the most interesting fieldwork I've ever done," he said.

Cullen supplemented his roadkill collection with samples donated by museums, universities, and local naturalists, including members of the Landry family, several of whom appear regularly on the History Channel's unscripted series Swamp People. The family graciously offered samples from an alligator and a six-foot-long gar. The only condition: don't damage the alligator skin.

"There's an image somewhere of me with a hacksaw, trying to saw the tooth off the head of this giant frozen alligator they had just pulled out of their warehouse," said Cullen.

Another source for tissue samples from alligators, fish, and other local fauna was a retired biologist specializing in zooarchaeology and now living in the bayou. Over the past decade, he had been assembling his own comparative animal collection. He agreed to donate samples on the condition that Cullen and his team help him get a large gar out of the rain barrel where he'd put it for skeletonizing. The fish had been decomposing in the water for over two weeks. That's how Cullen discovered desensitization has its limits.

"We opened the top of this rain barrel and you couldn't even see the gar," he said. "There were these little white chunks on the surface called corpse cheese—rotting tissue with a waxy look to it. Plus a writhing mat of maggots. It's probably the worst smell I've ever smelled in my life."

After completing their analysis of the stable isotopes, Cullen et al. found their results weren't as solid as they'd hoped. They knew in advance what the animals they'd collected ate and where they fit in the food chain, but the scientists would not have been able to learn that from just the analysis alone. So they suggest being pretty conservative with how future researchers interpret such data.

The researchers also advocate for the use of different isotopes of different elements when applying this kind of analysis to dinosaur remains and other ancient fossils. Cullen et al. included nitrogen stable isotopes in their analysis, and they achieved better results compared to oxygen and carbon.

The problem is that nitrogen is usually found in soft tissue (and sometimes the keratins in hair, nails, horns, hooves, and claws), which isn't usually preserved in fossils. So what worked so well on modern specimens from the Pelican State wouldn't be ideal for dinosaur specimens. Cullen's hope is that there could be other stable isotopes of different elements that do concentrate in tooth and bone and are preserved over sufficiently long timeframes. That's the next step for his research.

DOI: Royal Society of Open Science, 2019. 10.1098/rsos.181210 (About DOIs).