Many different bright-colored monkeyflowers on a black background

The great diversity of flowers within Mimulus is one reason the plants have become a popular subject of study.

DENA GROSSENBACHER

Meet the monkeyflower, a weed that may hold the key to zebra stripes and other biological mysteries

Yaowu Yuan's passion for monkeyflowers began in 2004 with a slideshow. Then a budding plant taxonomist at the University of Washington in Seattle and an avid hiker, he was amazed at the variety of wildflowers he saw on his outings in the Cascade mountains. Like Charles Darwin, he was vexed by what Darwin called an abominable mystery: How did nature generate such a diversity of flower colors and forms? During a campus seminar, Yuan encountered a plant that he thought might yield answers. University of Washington plant molecular biologist H. D. "Toby" Bradshaw and his graduate student showed slides documenting as much floral diversity within a single monkeyflower species as Yuan had seen in the meadows and streambanks of the Cascades—all generated by mutating the genome of this one Mimulus species.

The revelation changed the course of Yuan's research because he realized such mutants could lead to a better understanding of flower development in all plants. Since starting a faculty job at the University of Connecticut (UConn) in Storrs 6 years ago, he has been tracking down genes that control color, shape, size, and other traits in Mimulus flowers—and that may have similar effects in other plants. And he is far from the only scientist to have fallen under the spell of a plant best known as a weed that thrives where few plants, even other weeds, can grow—around abandoned copper mines and hot springs and in other inhospitable, mineral-laden soil.

Like plant scientists' traditional lab workhorse, the mustard weed Arabidopsis thaliana, monkeyflowers grow fast, produce a lot of seeds, and have a simple genome—appealing traits for lab studies. But their explosion of flower colors and forms, diverse lifestyles, and extraordinary hardiness—dramatic contrasts to the unassuming Arabidopsis—have seduced researchers studying plant evolution and adaptations. "You can use Mimulus to study traits that don't even exist in Arabidopsis," Yuan says.

More than 40 labs now focus on select members of Mimulus, a number that has doubled in the past decade, says Andrea Sweigart, an evolutionary geneticist at the University of Georgia in Athens. The National Science Foundation (NSF) has funded both individual evolution and ecology projects with Mimulus and, more recently, supported a $1 million effort to develop efficient techniques for altering traits in those plants.

"A large, organized, and growing research community is using this system," says evolutionary geneticist Theodore Morgan, a program officer at NSF in Alexandria, Virginia. A monkeyflower meeting in Providence in June drew about 70 biologists, more than triple the number who attended the first one 13 years ago. The number of publications on Mimulus still isn't huge—about 425—but that tally has grown rapidly in the past decade.

Some researchers are exploring monkeyflowers' own unusual adaptations. But other scientists are turning the flowers into a window on widespread biological processes. Yuan, for example, recently teamed up with another lab to use Mimulus mutants with odd petal color patterns to provide the most detailed example yet of mathematician Alan Turing's scenario for how zebra stripes, leopard spots, and some floral patterns arise in nature. Another team examining how monkeyflowers mutate as they grow revealed a mechanism that may enable many plants to evolve faster than animals.

The field may even have its first serious controversy: Some researchers are rejecting a recent revision of the monkeyflower family tree that split the more than 100 Mimulus species into multiple genera, creating confusion in the scientific literature by renaming the most studied monkeyflower species. The researchers' passion is a measure of the enthusiasm the new model plant arouses. One opponent of the new tree, John Willis, an evolutionary geneticist at Duke University in Durham, North Carolina, says flatly, "We're not going to take it anymore."

Turing’s idea takes root

Spots, stripes, and monkeyflower petal speckles arise through “reaction-diffusion,” which involves an activator protein that turns on pigment pathways and the production of a second protein, which inhibits the activator.

Patterns galoreMonkeyflower hybrids mix different species’ activators and inhibitors, altering petal speckle patterns. Spot onUnimpeded by the inhibitor, an activator colors a cell and spreads to and colors nearby cells. The inhibitor quicklydiffuses away from the colored cells, causing a nonpigmented halo to form. As this process repeats throughout thetissue, multiple spots form, resulting in a periodic pattern. Initial condition: high concentration of activator because of self-activa-tion. Activator diffuses. Pigment spot develops, but inhibitor concentration grows because of activator activity. Activator levels decline at the border of the spot because of competition from faster-diffusing inhibitors. Spot is isolated. Activator Inhibitor Concentration Pigment change occurs No change in pigment from default Both the activator and the inhibitor diffuse away from the cells that produce them, with the inhibitor moving faster (above). The balance of concentrations between the two proteins determines whether pigment is produced.
(GRAPHIC) V. ALTOUNIAN/SCIENCE; (IMAGES) QIAOSHAN LIN AND BAOQING DING

Lab scientists aren't the first to be fascinated by Mimulus, which is found worldwide, often in the harshest spots, such as the bare islands of "serpentine" soil that dot the forests of California's Sierra Nevada mountains. Plant ecologists have conducted field studies of wild Mimulus for 80 years. Last year, for example, researchers documented populations of Mimulus guttatus that contain a mix of individuals with different flowering times, flower sizes, and number of seeds produced. The late-flowering plants do better in wet years, and early-flowering ones do better in years when drought hits early in the season. Because the amount of rain varies from year to year, the two variants coexist in a population, although the proportions change over time. The work, reported last year in Science, provided long-sought proof of an evolutionary phenomenon called fluctuating selection, in which changing conditions cause a species to evolve in multiple directions. Theorists have proposed that fluctuating selection helps explain the extensive variation seen in many other species besides monkeyflowers.

At the June Mimulus meeting, Willis revealed a major clue to another monkeyflower mystery: the plants' affinity for serpentine soils. Because they derive from Earth mantle rock, those soils are rich in iron and magnesium but low in potassium and calcium, which plants depend on to maintain their cell walls. The soils also tend to have little nitrogen, vital for plants, but plenty of toxic heavy metals, such as nickel and chromium.

Willis and his Duke postdoc Jessica Selby recently crossed serpentine-tolerant monkeyflowers with versions of the plant that were not growing on serpentine soil. The duo tested several generations to identify DNA important to the trait. To narrow the hunt for relevant genes, Selby collected M. guttatus specimens from serpentine soils in seven places across California and Oregon and compared their DNA with that of populations of M. guttatus living nearby, on richer soil.

Both approaches pointed to a gene for an enzyme that makes arabinose, a sugar found primarily in the plant cell wall, Willis reported. That gene varies among M. guttatus plants, but every plant that can grow on serpentine soils has the same mutation. It may alter how arabinose interacts with other components of the cell wall, somehow compensating for the low calcium and high magnesium and keeping cell walls intact—an idea Willis's team is testing with researchers from the University of California (UC), Berkeley, and Stanford University.

Many monkeyflowers thrive in inhospitable, mineral-laden soils, like this spot in Lake County in California, and plant biologists are starting to understand how they do it.

JESSICA PACKARD SELBY

By harnessing population genetics and other gene-finding techniques, Willis "was way ahead of the curve in seeing how genomics could make the tremendous natural variation in plants knowable at the level of the gene," says Lila Fishman, an evolutionary biologist at the University of Montana in Missoula. The work could have practical benefits as well, adds Benjamin Blackman, an evolutionary biologist at UC Berkeley: "Learning how plants have already adapted to cope with marginal soil environments can inform breeding efforts aimed at developing crops that can cope with poor soil."

Besides probing monkeyflowers' own special biology, researchers are using them to glean more general lessons about plants and animals. Take Yuan and Blackman's work on color patterning. Blackman's lab originally studied sunflowers. But M. guttatus appealed to him for studies of the genetic basis of patterning because its simple genome had been sequenced—making it easier to test the role of particular genes and proteins by genetically modifying the plant. Independently, he and Yuan homed in on the same protein, which they thought might be a key to flower color patterns.

Yuan and UConn postdoc Baoqing Ding had recently tracked down the gene that causes red pigment to appear on the yellow lower petal of some Mimulus flowers. The red usually appears as a band of speckles, which serve as a "nectar guide" for incoming pollinators. Many types of coloration in plants and animals are the result of a network of proteins that activate pigment genes at specific places and times in the body. But Yuan and Blackman wondered whether the monkeyflower spots might be generated instead through a patterning mechanism proposed in the 1950s by Turing, who is best known for breaking the Germans' Enigma code in World War II but was also a theoretical biologist.

Turing predicted that some patterns emerge from the natural diffusion and interactions of proteins whose concentrations regulate each other's production. For spots, when the gene encoding a cell's "activator" of pigment production turns on, the activator protein stimulates its own production and that of a "repressor" protein, which diffuses beyond the pigmented spot. That second molecule shuts down any activator in the surrounding cells—causing a white halo. But the repressor gets more dilute the farther it travels, eventually losing its effect. Then the activator can turn on, and a new spot of color can form. Color patterns emerge depending on the differences in the two proteins' diffusion rates.

Red "tongue" Mimulus mutants revealed how coloration arises.

BENJAMIN BLACKMAN

Biologists have long assumed that Turing's mechanism is responsible for zebra stripes and leopard spots and perhaps even for monkeyflower nectar guides. Indeed, Yuan had identified a monkeyflower protein that might serve as the activator. But no one had identified a full activator-repressor system involved in periodic pigmentation patterns.

Blackman, however, had noticed a clue in some wild M. guttatus: They either lacked spots or else had just one large red patch, which he called a tongue, suggesting part of the system was missing. Independently, Yuan uncovered a similar red tongue variety among mutants he made in another Mimulus species. When they learned of each other's work, the two joined forces. And at the June Mimulus meeting, they reported using the red tongue varieties to track down a protein dubbed R3-MYB, the repressor counterpart to the already known activator protein.

To confirm that R3-MYB really acted as a repressor, Yuan and Blackman both wielded molecular tools to block its production. Yuan relied on RNA interference, whereas Blackman's team enlisted CRISPR—the first use of the genome-editing technology in Mimulus. Both techniques led to full red tongues on the plants' petals, Blackman reported—vivid testimony that the mechanism Turing hypothesized can account for some of nature's tapestry. "This work shows how Mimulus can provide broad insight into processes that shape biodiversity," Sweigart says.

Another line of work with monkeyflowers sheds light on biodiversity by revealing a mechanism, unique to plants, for rapidly adapting to new conditions. Their advantage, graduate student Jaime Schwoch of Portland State University in Oregon found, is rooted in the way they produce reproductive cells. In animals, the cells that mature into eggs and sperm are sequestered early in development, and they don't divide until the organism sexually matures. That protects them from division-related mutations that occur in the organism's nongerm, or somatic, tissues. But flowers, which contain both kinds of germ cells—pollen and ova—form from active somatic tissue at the tips of growing stems. Any mutations occurring in the dividing cells of a stem will be locked into the germ cells and can pass on to the next generation.

Given that somatic tissue mutations should accumulate in a plant's germ cells over successive generations, Schwoch wondered why plants don't wind up with many more such mutations than animals—and a greater burden of harmful ones. In fact, as the somatic parts of both plants and animals grow, their cells accumulate about one mutation per million bases per cell division, so plant germ cells should have far more mutations than animal germ cells. But they don't. And deleterious mutations are surprisingly scarce in plants, Schwoch found when she compared two sets of monkeyflowers. She produced one by self-fertilizing Mimulus flowers with each flower's own pollen, the other by fertilizing flowers with pollen from flowers on another stem of the same plant. The latter is the equivalent of a cross between different parents, because each stem acquires a unique set of mutations as it grows.

Self-fertilization, like inbreeding in animals, should pair up harmful recessive mutations, so Schwoch expected the crosses that used pollen from one stem on flowers from another to do better. But some of the more prolific, healthier plants came from progeny derived from a single stem, she reported at the Evolution 2019 meeting, also in Providence in June. That finding suggested the plants were somehow eliminating harmful mutations in their somatic cells and accumulating beneficial ones for their reproductive cells.

Growing monkeyflowers in salty conditions has helped reveal how plants weed out deleterious mutations.

JAIME SCHWOCH

To verify that sorting process, Schwoch grew Mimulus plants for 6 months in saltier-than-normal conditions and then sequenced DNA from their tips, taking note of new mutations and how often the mutations appeared in the sequenced material. Such mutations should occur in low frequencies, so when she found one that occurred in many cells of the plant tip, she inferred that the original cell with that mutation had grown much faster than cells without it and replaced them.

The rate of mutations doubled under the salt stress, she reported. Moreover, cells carrying mutations that improve salt tolerance proved more likely to persist in stems, whereas less well-adapted cells died out. The survivors made it into the germ line, so the within-lifetime innovations were passed on to subsequent flowers and pollen. The process means "plants can adapt very quickly" to tough situations, Schwoch said.

Duke evolutionary biologist Jennifer Coughlan is impressed. "This work has broad significance for all plants, but in particular for long-lived perennial plants, [which] accumulate many mutations across their lifetimes," she says. Sweigart predicts that Schwoch will quickly unearth the specific mutations that produced the salt tolerance: Mimulus "has great genetic and genomic resources, so it should be possible to identify the precise molecular changes that have occurred" in her salt-tolerant plants, Sweigart says. And the lessons from monkeyflowers may point to ways to make other plants, including crops, more tolerant of salty soils, researchers suggest.

Just when investigators are flocking to monkeyflowers, the monkeyflowers may be scattering—at least taxonomically. A 2012 evaluation of the Mimulus family tree placed some of the better-studied monkeyflower species in other genera. For example, the popular M. guttatus is now named Erythranthe guttata. The Mimulus genus itself kept only seven species of the original 165-plus. Adopting new designations for many Mimulus species will lead to chaos in the scientific literature, some researchers in the field say.

Among monkeyflower researchers, the reclassification provoked a minor rebellion. Most did not use the new names in their presentations at the Mimulus conference or the subsequent Evolution meeting. A paper in press in the journal Taxon argues for a different, less disruptive reclassification, and Willis says that makes sense. "You either rename 150 species, or you rename 20 somewhat obscure species and call them all Mimulus," he notes.

Mimulus by any other name might smell as sweet, but most biologists don't want to monkey around with their new favorite plant.