Authored by: By Angela Hsiung (MS ’16, PHD ’22) William Bartram was struck by the beauty of the trees he found along the banks of Georgia’s Altamaha River in 1773. The trees, Bartram noted, had flowers that “are very large, expand themselves perfectly, are of a snow-white colour, and ornamented with a crown or tassel of gold coloured refulgent staminae in their centre.” Bartram had encountered the same trees when he and his father, John Bartram, went on their first expedition in 1765 to catalog the flora and fauna of the southeastern area of the North American colonies at the request of King George III. The trees were not flowering then, but instead had brightly colored fall foliage. Seeing them again with such gorgeous flowers, Bartram was intrigued. He collected their seeds before moving on with his journey. A few years later, Bartram named the species Franklin tree (Franklinia alatamaha) after his father’s good friend, Benjamin Franklin. Nearly 250 years later, Franklin trees are planted in more than 1,000 arboreta and gardens all over the world. But the original population in Georgia from which Bartram took the seeds—the only known wild population to date—has all disappeared. “Nobody knows for sure why it disappeared in the wild,” says Heather Gladfelter (PHD ’19) who conducted a study on Franklinia that was recently published in Tree Genetics and Genomes while being co-advised by Warnell professor Scott Merkle and College of Agriculture and Consumer Sciences professor Dayton Wilde. “The last time they saw it in the wild was 1803.” A mysterious demise The person who documented that observation was John Lyon, another plant collector, who noted that there were only six to eight Franklin trees left scattered over less than half an acre, considerably less compared to the two to three acres documented by the Bartrams. Some believe their disappearance had to do with burning for agriculture expansion, while others say they were over-collected by plant enthusiasts. Perhaps the most plausible theory is that the population died off from a root rot disease caused by a fungus-like organism called Phytophthora cinnamomi, which hitchhiked on ornamental plants imported from Asia by landowners in the South. The Franklin tree thrives in moist but well-drained acidic soil and can handle climates with cold winters. When fully grown, the trees can reach over 20 feet tall. In North America, it is cultivated as far south as the North Georgia mountains and as far north as British Columbia. It thrives as a landscape plant in northeastern states such as Pennsylvania, New York and New Jersey, where Phytophthora is absent. With an estimate of around 2,000 trees scattered around the world, Franklinia is hard to come by outside of places that meet its growing requirements. Outside of those areas, one would have better luck seeing Franklinia in arboreta and gardens either planted in soil or in pots. In other words, it is not a tree you typically find at Home Depot. After returning from his trip, Bartram planted the Franklinia seeds he collected in his garden in Philadelphia. What happened to the progenies of the plants that resulted from those seeds, whether anybody other than Bartram collected from the same population, and how the present-day Franklin trees are related to the original wild population is anyone’s guess. Those are questions Gladfelter and her colleagues wanted to answer. A growing passion On a typical day, you can find Gladfelter working in the lab, perhaps wearing a chain around her neck from which a small panda dangles. It’s her favorite animal, and it is reminiscent of the other small creatures that find their way into plastic boxes that hold Franklinia plantlets and tinted growing medium. Sitting on lit shelves in Warnell’s Building 3, small plastic dinosaurs, barnyard animals or even a baby play in miniature forests—all cousins from the same Franklin tree. Gladfelter found her passion for plant propagation back when she was an undergraduate student at New Mexico State University. While looking for work-study opportunities, she happened upon an advertisement by the horticulture department looking for student workers to help conduct tissue culture studies and decided to apply. One of the techniques she learned in that lab—and still uses today in her research— was micropropagation, which is a method of growing genetically identical plants in culture using tissue harvested from the original, much like cloning animal organs using stem cells. The wonder of growing an entire organism from a tiny piece of tissue had her hooked instantly. “That’s where I knew it was going to be my future. I was just captivated by it,” says Gladfelter as she beams with excitement. Prior to arriving at UGA, Gladfelter worked in a commercial molecular biology lab performing genetic transformation—introducing desirable genetic traits, such as fast growth or pest resistance, into the genome of a plant, often crops or timber trees. But under those settings, she often could not see a project to completion. Wanting to have more autonomy over her research and opportunities to see the fruit of her labor, she went back to academia and joined Merkle’s lab in 2012. “I welcomed the opportunity to go back to academics, but I wanted to work with something that was endangered or needed restoration,” says Gladfelter. The Franklin tree caught her attention. At the time, Merkle and his students had just started propagating Franklinia in tissue culture. “When I first started reading about (Franklinia), I’d never heard of it,” says Merkle. “It was a small tree that only ever grew in one place, and is now lost in nature, and it’s fascinating to me.” Root resistance Due to its interesting history, its beautiful summer flowers and colorful fall foliage, Franklinia are highly sought-after ornamental trees. The potted Franklin trees at plant sales often sell out quickly. Merkle thought Franklinia was a good candidate species for which to develop a mass propagation system and perhaps eventually introduce back into the wild. But one of the major hurdles preventing Franklinia from growing in the wild remains its inability to survive Phytophthora root rot, which is ubiquitous in southeastern soil. One way to overcome this hurdle is through mutagenesis, which involves introducing mutations in plants by exposing them to radiation in the lab. The hope is that, if the researchers are lucky, some of the resulting mutants could potentially have Phytophthora-resistance genes. Another way is to look for naturally occurring Phytophthora-resistant genes in existing Franklin trees. To do so would require a better understanding of the species’ genome and the genetic diversity among the trees grown around the world. If there is genetic variation, then it is possible that there are some Phytophthora-resistant Franklin trees out there. Merkle and Gladfelter then started wondering: Just how much genetic variation exists among today’s Franklin trees? Did Bartram collect seeds from only one tree or several different trees? Were the trees from the original population genetically different or were they clones that shared the same root system? It became Gladfelter’s mission to answer these questions by collecting genetic samples from as many Franklin trees as she could get her hands on and analyzing their DNA. A devoted following Gladfelter started by contacting arboreta and gardens in the U.S. and asking them to help her locate places that cultivated Franklinia. She soon found a network of gardens and arboreta that have Franklinia and were willing to send her tissue samples. Gladfelter then traveled to Bartram’s Garden in Philadelphia to collect samples from their Franklin trees. When word got out about what Gladfelter was looking for, people were excited and invited her to come collect samples from their gardens. “It was kind of magical,” says Gladfelter. “Franklinia has such as following, so does Bartram. It’s amazing.” But she didn’t stop there. Gladfelter also cold-called arboreta abroad, in countries such as Poland, Germany and New Zealand, and asked if they were willing to send her tissue samples of their Franklin trees. And they did. “It took someone like Heather, who is very dogged, to do these studies with (Franklinia),” says Merkle. “She’s very determined and very focused on the project.” While live trees are good sources of DNA, Gladfelter also wanted to see if it was possible to extract DNA from preserved leaves. To find out, she contacted herbaria with Franklinia leaf mounts and asked them to send her a small piece of their specimens. As a result, the Royal Botanic Gardens in Edinburgh, Scotland, sent over some samples—the oldest being 177 years old—from which she successfully extracted usable DNA. Through her persistence, she eventually collected 76 samples—though one she desperately wants remains out of reach. The source was a leaf mount pressed by William Bartram in 1778 and is now at London’s Museum of Natural History, which turned down her request due to the uncertainty of whether she could get reliable DNA from a 200-year-old specimen. “It would’ve been really nice because it would’ve been the baseline,” says Gladfelter. “The closest to the actual tree that (Bartram) collected from.” Back at UGA, Gladfelter also took samples from 19 mutant Franklinia that were produced from radiation and grown in a greenhouse for a different study, making a total of 95 samples. She then sequenced fragments of the genome of the tissues and compared the same fragments across samples to look at how closely they were related. Trees with similar ancestry share more genetic information than those that are more distantly related. After a supercomputer analyzed the DNA sequences from the samples, it created a dendrogram that showed the genetic diversity of the trees. In a way, the dendrogram may resemble one side of a March Madness bracket, except instead of names of college basketball teams, the diagram contained the name of each genetic sample that was sequenced and analyzed. The samples were grouped into clusters according to how closely they are related. A key to a tree To the delight of Gladfelter and her fellow researchers, the analyses revealed that the samples belong to two genetically different subpopulations of the study population (i.e., all the samples collected from the cultivated Franklinia), indicating that there is some genetic variation among the Franklin trees that were sampled. While the subpopulations share some genetic similarities, they are different enough to make the researchers speculate that the original seeds were collected from at least two different trees, and that the original population did not consist of a clone, but instead included genetically different individuals. What was also surprising was that some samples collected in the United States belong to the same subpopulation as those collected overseas. For example, the 177-year-old sample from Scotland was closely related to a sample collected off a tree in Lavonia, Georgia. That same population cluster also contained one tree each from Poland, New Zealand and British Columbia. “It makes perfect sense when you think about it,” says Merkle. “People buy their stuff and they ship it around and of course it wouldn’t have anything to do with how close two locations are in the world. … It was a little surprising to see that, but it’s kind of funny we would even think that that would be unusual.” The work is not done, of course. The elusive Phytophthora-resistant gene has yet to be discovered in Franklinia. Now that there is evidence of genetic diversity among living Franklin trees, a next step is to expose the disease to plants grown from tissues of living Franklin trees to see if any survive. The surviving trees can then be bred with those that are susceptible to root rot to produce resistant offspring or cloned using tissue culture or rooted cuttings. Alternatively, Gladfelter is conducting mutagenesis experiments by producing mutant Franklinia and exposing them to Phytophthora, hoping to find some that will overcome the disease. If Phytophthora-resistant Franklin trees are successfully bred and introduced back to the wild, then maybe, one day, these trees will once again stand alongside the banks of the Altamaha River, just as Bartram saw them.
