A newly published global study of white clover shows its strong evolutionary adaptation in urban environments despite human encroachment, tough conditions and climate change,  according to a Global Urban Evolution (GLUE) Project team led by evolutionary ecologist Marc Johnson, a professor of biology at the University of Toronto, Mississauga.

The 287-member team included urban landscape entomologist Emily Meineke of the UC Davis Department of Entomology and Nematology and marine evolutionary ecologist Joanna Griffiths of the UC Davis Department of Environmental Toxicology. 

The research, “Global Urbanization Drives Adaptation in the Plant White Clover,” published March 17 in the journal Science, reveals that “urbanization leads to similar environmental changes across 160 cities throughout the world, which leads to repeated adaptive evolution in the cosmopolitan invasive plant white clover,” said Johnson, the  principal investigator (PI) of the 160-city, 26-country project and director of the Centre for Urban Environments  at the University of Toronto. 

“It is the largest scale study of parallel evolution and urban adaptation ever performed, involving 287 collaborators across 26 countries,” said Johnson, adding “This project would have been impossible without the hard work and dedication of an amazing network of collaborators around the world, people like Emily Meineke and Joanna Griffiths at UC Davis.” 

White clover, Trifolium repens, is considered an ideal evolutionary organism to study: it is one of the most rapidly evolving species of flora and one of the most widespread.  The plant adapts well in both rural and urban environments throughout the world, from Norway to Mexico to India. Often used as a cover crop, it is a good source of nitrogen and a good nectar source for bees, but it is considered invasive when it pops up in urban lawns or on golf courses. 

Of the main forces behind evolution--natural selection, genetic drift, and gene flow—the white clover's dominant evolutionary force is natural selection, Johnson pointed out.

Team members performed cyanogenesis assays to determine the plant's defense production of a toxin called hydrogen cyanide (HCN).  White clover is less likely to produce HCN in colder environments but more likely in rural areas, the research shows.  Half of the world's population lives in urban environments, but by 2050, that figure is expected to jump to 70 percent. 

“Urbanization transforms environments in ways that alter biological evolution,” the scientists explained in the abstract. “We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale.” 

Both Meineke and Griffiths worked on the project when they were doctoral students: Meineke at North Carolina State University (NCSU), Raleigh, and Griffiths at Louisiana State University (LSU), Baton Rouge. 

Meineke, who holds a doctorate in entomology (2016) from NCSU, designed the sampling effort in Raleigh, collected clover from there, and did cyanogenesis assays. 

“Increasingly, it is clear that we are living in a time when humans are the dominant drivers of biotic change globally,” commented Meineke, who joined the UC Davis Department of Entomology in Nematology in 2020 as an assistant professor.  “Somehow, we still don't understand how we are affecting the species we see every day, in part because biologists have only recently become aware of our complex effects on species that live in our own habitats.” 

“This project sparked my interest because it focuses on the evolution of clover, a plant that I've had under my feet my entire life,” Meineke said. “I remember stepping in clover as a kid and watching bumble bees bob across it during PE class in elementary school. It turns out that kids worldwide have had this experience because white clover is a cosmopolitan plant. Being part of this study gave me the opportunity to be part of a large group studying effects of humans on clover all over the world.” 

Griffiths, a postdoctoral researcher in the UC Davis labs of Professors Andrew Whitehead and Nann Fangue, received her  doctorate in ecology and evolution (2020) from LSU. She said her LSU team, including Luis Santiago-Rosario and Katherine Hovanes, "collected clovers from Baton Rouge, and my contribution was performing the lab work, that is, I quantified the amount of hydrogen cyanide present in each clover sample from rural Baton Rouge all the way to the city center. Each sample was digested and incubated for a couple hours. (See image of data sheet.) “The cyanide in the sample chemically reacts with the special paper, turning it blue. Thus, a blue spot on the paper indicates that the clover sample had cyanide present.”  

Urbanization is a global phenomenon, in which thousands of cities cover up to three percent of Earth's land surface, according to the GLUE Project website.  “For an evolutionary biologist, these cities represent an amazing opportunity to study evolution in action.” 

The website describes the GLUE project as “an initiative that will provide the largest scale, best replicated test of parallel evolution ever attempted. To do this, we will study the evolution of the production of hydrogen cyanide (HCN) in white clover (Trifolium repens). We previously showed that white clover evolves parallel clines in HCN (a potent chemical defense) along urban-rural gradients in eastern North America.” 

In addition to lead PI Marc Johnson, the 12-member leadership team included two University of Toronto scientists: co-PI and assistant professor Rob Ness, the second author of the paper; and doctoral student James Santangelo, first author.

The project drew financial support from a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant; Canada Research Chair; and NSERC E.W.R. Steacie Fellowship.