Deep in the California Academy of Sciences’ vaults, tucked within a maze of shelved history, Alex Rinkert hunched over a desk. Fluorescent lights beamed overhead and illuminated the small bundle of interwoven twigs and dried grasses in front of him.
He pulled a small piece from the mass with a tug from his forceps. And then another, and then another—each fragment a mere 1 to 2 centimeters long. He photographed the snippets, jotted down notes, and carefully tucked the dry vegetation into a test tube.
His tiny extractions were delicate, calculated, cautious. Most importantly, they left the specimen—a bird nest collected in 1909 from Belvedere in Marin County—intact.
“For the first few nests I sampled, my hands were definitely trembling a little bit,” says Rinkert, a consulting biologist and an affiliated senior biologist with the San Francisco Bay Bird Observatory. “Hopefully, by taking a very small part of it away permanently, we learn more than the damage that we’d be causing to them.”
More than a century ago, a female Savannah sparrow about the size of a human fist landed among the grassy, weedy fringes of the San Francisco Bay. The sun illuminated a bold patch of yellow on her face and her pale plumage, streaked with shades of brown, as she secretively darted through the vegetation—hopping, running, crawling like a mouse. Once she found her target, a dried dead grass, she uprooted it with her sharp beak, claimed it as her own and scurried to her nest taking shape in the higher ground just beyond a nearby tidal marsh.
Within three days, the intertwined cup nest of grasses was complete, featuring a canopy of overhanging grasses to conceal it. And decades later, it served as Rinkert’s portal to the past inside the California Academy of Sciences. Information gleaned from such nests, woven long ago from species in plant communities called transitional habitat, could help restore the shoreline in the future. Transitional habitat has nearly disappeared from the San Francisco Bay, and scientists need a clearer picture of its original species composition—which was never properly documented. With that insight, conservation research groups like the San Francisco Bay Bird Observatory can help guide best practices when restoring the native habitat that has long served as critical refuge for imperiled birds and animals as adjacent marshes flood more with rising sea levels. “We can’t ask restoration ecologists to plant nonnative species or to just take their best guess and throw things out there,” says Rinkert.
To glimpse into the past, Rinkert and a collaborator, plant evolutionary biology professor Justen Whittall of Santa Clara University, are examining 20 nests, each around 100 to 150 years old and dubbed “botanical time capsules.” Funded by the Midpeninsula Regional Open Space District, the researchers will be the first ever to sequence DNA from historical bird nests, pinpointing the specific species that once composed the Bay’s transitional habitat.
“Ultimately, that information is going to go into these big restoration plans to make the habitat more suitable and more like what it was 100 years ago,” Rinkert says.
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A fragmented history
Tidal marshes developed between 2,000 and 3,000 years ago around the San Francisco Bay. Here, fresh water and salt water meet to create a productive wetland. The low-lying vegetation filters pollutants and excess nutrients from the water, stores carbon, and provides habitat for hundreds of species, including the federally and state-listed endangered salt marsh harvest mouse and Ridgway’s rail.
More of interest to Rinkert, however, is the transitional habitat, which stretches from the salty wet marshes to the dry upland areas.
When high tides swallow the marshes, transitional habitat serves as a safe haven for critters. They retreat to the denser vegetation and its unique mix of salt-tolerant and upland plants, which today includes nonnative species such as stinkwort (Dittrichia graveolens), black mustard (Brassica nigra), and sweet clover (Melilotus). Like tidal marshes, transitional habitat buffers against storm surge and king tides. Sea level rise will set off a chain of dominoes around the Bay’s shoreline. It will engulf the marshes, pushing them to migrate into the areas the transitional habitat occupies. Ideally, the transitional habitat would move farther upland, too —but there may not be space for it anymore.
In the past, transitional habitat might have stretched on for more than a mile. Since the mid-1800s, development and salt pond construction have confined the habitat to mere 65-foot slivers at most. The plant community that grows there today is almost wholly composed of nonnative species introduced by early settlers, Rinkert says.
The ecological history of transitional habitats is riddled with gaps. Most early settlers didn’t think to document native plants before nonnative species took over. Some, however, were interested in bird nests—specifically, the eggs inside them. Widespread interest in amassing bird eggs for both private and research collections peaked around the early 1900s when the items often carried a hefty price tag.
“There wasn’t a lot of economy for native plants from the Bay Area. But there was for cool eggs,” Whittall says. “Those nests actually are more likely to contain native plants with accurate location information than any actual plant collection.”
Many of those bird nests ended up in museums, joining more than 60,000 preserved nests in collections worldwide. These specimens have been traditionally tough to research, though. Their contents are challenging to identify by eye, yet in the name of preservation, they can’t be deconstructed for further dissection.
Rinkert envisioned a unique new research method that—with the help of Whittall’s lab—circumvents those challenges: using less invasive DNA sequencing to identify plant fragments from historical nests of the San Francisco Bay’s transitional habitats.
“This is something pretty new, and it gets around some limitations that I think hindered doing this type of research,” Rinkert says.
Of the tens of thousands of bird nests preserved in museum collections nationwide, Rinkert and Whittall chose those made by song sparrows and Savannah sparrows near the San Francisco Bay, which either nest in transitional habitat or gather material from it. Specimens from five museums around the country—including the California Academy of Sciences—fit the bill; all were collected between 1876 and 1924.
“I’d say [the museums] were excited that someone was actually working with the nest collection because it seems to be a lost and forgotten part of what they curate in their ornithology departments,” Rinkert says.
Sampling was meticulous. Rinkert extracted around 10 samples from each nest, selecting pieces from both the sturdier outsides and the softer inner linings, in search of a variety of plant species. When sampling ended in February 2022, he had amassed 200 test tubes filled with ecological history.
Like any living organism, each plant comes with its own unique DNA sequence—what Whittall equates to a barcode.
Each of Rinkert’s samples is like a grocery cart full of items with different barcodes. Just as cashiers scan goods and provide itemized receipts, the researchers are using species-specific DNA sequences to identify the plants in each sample.
The difficult part? That DNA is more than 100 years old.
“Imagine you’re checking out with a 128-year-old bag of Cheetos,” Whittall says. The bag may be intact, but its barcode may be worn out and hard to read. “That’s where this setting gets particularly challenging and unique.”
In a 2021 proof-of-concept experiment, the researchers analyzed two historical and two modern bird nests. They extracted DNA from the nest samples and then amplified it using a sequencing method that could only identify one plant per DNA extraction.
Remnants of a California wild rose (Rosa californica) were pinpointed in samples from a 1915 nest. The team also identified a native grass (Festuca microstachys) in a 2015 nest collected from a San Francisco Bay Bird Observatory restoration area where native plants had been planted to replace invasive species. It was evidence that “something with a backbone” benefited from the conservation efforts—which can be difficult to prove, Whittall says.
The results showed that this method worked, although it could be improved.
Their method of analysis has graduated to a more modern and sensitive technology called next-generation sequencing, which can identify multiple plant species in each extraction. The team now sends its samples overseas to a facility in China that runs the analysis, detects any DNA sequences and sends them to Rinkert and Whittall.
Those sequences—on the order of tens to hundreds of millions—are then transferred to supercomputers for comparison with the known plant DNA sequences in a database called GenBank. The researchers go through any potential matches and verify their identities with resources like photos of the original samples, museum collections, and citizen science data.
Without adequate historical baselines for the plant communities that once lived along the now highly invaded estuary, it’s difficult to say whether a detected species is native or nonnative. But the experts can assess, asking questions like, does it make sense a certain species would’ve been in the Bay Area? Or could there be other factors at play—like contamination from the lab or a stray piece of pollen?
“It’s the Sherlock Holmes detective part,” Whittall says. “We get a lot of names, but we’ve got to put reality to them.”
Early results show promise
Almost a year after their sampling, Rinkert and Whittall were still receiving sequences back from the testing facility in China. But thanks to some preliminary DNA sequencing of select samples before they were sent overseas, the team had a sneak peek at what some of their final results may hold.
One of the tentatively identified species was stinging nettles (Urtica dioica)—“a potentially native species of San Francisco Bay,” according to Rinkert, which today is only scattered around the Bay’s southern edges. Mustard (Brassica) and alfalfa plants (Medicago) were also detected, which were likely introduced and are now widespread throughout the state.
Each new result will offer another snapshot into plant communities of the past, which Rinkert and Whittall hope will add up to a bigger picture that will supplement historical documentation.
After the team finishes analyzing the data this spring, it will compile its research this summer. The researchers will then share that information with conservation groups around the San Francisco Bay that can adjust their transitional habitat restoration efforts to mimic historical species composition.
“It’s pretty amazing. These nests have been sitting in these museums for 110, 120 years,” Whittall says. “If everything goes according to plan … we’ll be able to identify all the plants to species and be able to use them in muddy-boots-on-the-ground restoration.”