Hawkmoth Pollination Promotes Promiscuity in Plants

Imagine an episode of the Jerry Springer Show in which the paternity of a child will be determined. Now imagine that instead of human beings, the show is focused on plants, and the issue at hand is the paternity of seeds produced by a given flower.

Next, consider that instead of just two candidate dads, there are dozens or even hundreds of individuals that could have fathered those seeds. What would you expect to find out at the end of the episode?

New research by biologists at Chicago Botanic Garden and the University of Arizona brings such a scenario into reality, and the “big reveal,” while not quite as dramatic as what you’d typically see on the Springer show, offers new insights into plant mating. The paper, titled “Pollinator identity and spatial isolation influence multiple paternity in an annual plant,” was published online today in Molecular Ecology.

“Biologists have known for decades that multiple paternity is common in plants—that is, the seeds contained in a fruit will often have been fathered by many different individuals,” said Matt Rhodes, a Ph.D. student in ecology and evolutionary biology at the University of Arizona. “While we have long had a basic understanding of how multiple paternity occurs in plants, we wanted to explore how it might be influenced by some of the messier aspects of pollination ecology.”

Much of this messiness stems from the fact that plants are sessile: once they start growing, they’re stuck where they are. “One important consequence of this immobility is that plants can’t seek out mates on their own,” Rhodes explains. “Instead, most flowering plants entrust the mating process to animal pollinators that move pollen from flower to flower. On top of that, some individuals are surrounded by potential mates while others are spatially isolated. There are good reasons to expect both of those factors to influence multiple paternity, and that’s what motivated our study.”

In the video above, pollen is deposited on the proboscis and body as hawkmoths (Hyles lineata and Manduca quiquimaculata) visit flowers of Oenothera harringtonii. (Video: K. Skogen) View video on YouTube here.

To address these issues, Rhodes went to the grasslands of southeastern Colorado with Jeremie Fant and Krissa Skogen, conservation scientists at the Chicago Botanic Garden who co-authored the article. They studied a rare evening primrose species with a strange twist in its pollination ecology: its flowers are visited by large-bodied hawkmoths at night and comparably smaller-bodied bees during the morning. “Based on these differences in body size as well as some important differences in behavior, we predicted that flowers visited by hawkmoths would mate with a greater number of fathers than those visited by bees,” said Skogen. “Because these floral visitors are active at different times of day, we were able to test this prediction with a fairly simple experiment in which we limited different flowers on a plant to visits from either hawkmoths or bees. We also predicted that multiple paternity would be less likely for individuals that were farther away from potential mates.”

Hyles lineata visits an Oenothera harringtonii flower; note the pollen on the moth’s head and body. (Photo: S. Todd)
Hyles lineata visits an Oenothera harringtonii flower; note the pollen on the moth’s head and body.
(Photo: S. Todd)
A Lasioglossum bee collects pollen from the anthers of an Oenothera harringtonii flower without coming into contact with the stigma. (Photo: S. Todd)
A Lasioglossum bee collects pollen from the anthers of an Oenothera harringtonii flower without coming into contact with the stigma. (Photo: S. Todd)

After collecting the seeds from these plants, the researchers spent months examining seed DNA in the genetics lab. “By comparing the seeds’ DNA to the DNA of the maternal plants from which we collected them, we were able to figure out which parts of the DNA came from the father,” explains Fant. “We then screened that paternal DNA against all of the individuals in the population—which in our case included more than 300 plants spread across 2 square miles of the landscape—to find the most likely father for each of the seeds we collected.”

For the most part, the results were consistent with the researchers’ predictions. “We found that on average, flowers visited by hawkmoths mated with nearly twice as many different fathers as flowers visited by bees,” said Rhodes. “We also found that spatially isolated individuals were far less likely to mate with multiple different fathers. Overall, it looks as though plant ‘promiscuity’ depends both on what kind of animal visits the flowers, and how far away that individual is from potential mates.”

Scales from Hyles lineata were deposited on the stigma of an Oenothera harringtonii flower. (Photo: K. Skogen)
Scales from Hyles lineata were deposited on the stigma of an Oenothera harringtonii flower. (Photo: K. Skogen)

In addition to providing a more thorough account of factors that can influence multiple paternity in plants, the results also allow researchers to consider how plants might be affected by the loss of certain pollinators. “This study allows us to make predictions about how some plants may be affected if particular pollinators disappear. Hawkmoths play an important role in moving pollen from plant to plant; if they decline in large numbers or are lost completely, there may be cascading effects on the success of future generations of hawkmoth-pollinated plants” said Skogen.

Comanche National Grasslands, Colorado— the shortgrass prairie where the study was conducted. (Photo: K. Skogen)
Comanche National Grassland, Colorado— the shortgrass prairie where the study was conducted. (Photo: K. Skogen)
The field team in Colorado (left to right: Kelly Ksiazek, Matt Rhodes, Sadie Todd, Evan Hilpman, Krissa Skogen, and Jeremie Fant)
The field team in Colorado (left to right: Kelly Ksiazek, Matt Rhodes, Sadie Todd, Evan Hilpman, Krissa Skogen, and Jeremie Fant)

©2017 Chicago Botanic Garden and my.chicagobotanic.org

Evening Primroses, Pumps, and Pollinators

Rick Overson is fascinated with insects—especially the kinds that love desert climates like in Arizona, where he grew up and earned his Ph.D. in biology. After completing a postdoctoral assignment in northern California, he decided it was time to get to know the little buggers even better, so Dr. Overson hopped on a plane for Chicago and stepped out into the subzero temperatures of the polar vortex to do just that.

PHOTO: Dr. Rick Overson with hawkmoth specimens.
Dr. Rick Overson with hawkmoth specimens

The devoted entomologist didn’t expect to see the insects in Chicago, but he was eager to join research at the Chicago Botanic Garden. A multidisciplinary team was assembling there to look for scent variations within Onagraceae, the evening primrose family, and connections from floral scent to insect pollinators and predators. The findings could answer questions about the ecology and evolution of all insects and plants involved. Overson is a postdoctoral researcher for the initiative, along with Tania Jogesh, Ph.D.

“Landscapes of Linalool: Scent-Mediated Diversification of Flowers and Moths across Western North America” is funded by a $1.54 million Dimensions in Biodiversity grant from the National Science Foundation. The project is headed by Garden scientists Krissa Skogen, Ph.D., Norman Wickett, Ph.D., and Jeremie Fant, Ph.D. It was developed from prior research conducted by Dr. Skogen on scent variation among Oenothera harringtonii plants in southern Colorado.

“For me, the most important thing coming out of this project is documenting and showing this incredible diversity that happens inside a species,” said Overson. “It’s vitally important for me to break down this idea of a species as a discrete unit. It’s a dynamic thing that is different in one place than another. That factors into conservation and our understanding of evolution.” In this case, he and his colleagues theorize that the evolution of the insect pollinators and predators is connected to the evolution of the scent of the plants.

PHOTO: Evening primrose in bloom on the plains of New Mexico.
Evening primrose in bloom on the plains of New Mexico. Photo by Dr. Rick Overson

The first two years of field work brought Overson back to his desert home. He traveled across Arizona, Utah, and nearby states with a group of about five scientists during summer months when the flowers were blooming. The team visited several populations each of 16 species of flower for a total of 60 locations. Overson and the team identified and documented the insects visiting the plants and compiled scent chemistry from the flowers. Their tool kit included a pump to pull the scent from a flower onto tiny polymer beads that held the scent inside of a vial. From there, they extracted the scent chemicals at the end of the research day or night. “It’s definitely the case that this pattern of scent variation inside a species is very common in this group,” he said of the team’s preliminary findings.

PHOTO: Hawkmoth on evening primrose.
A beneficial pollinator, the hawkmoth, visits an evening primrose (Oenothera harringtonii).

In the field they also took video recordings of pollinator behavior to see who visited which flowers and when. The pollinators, including hawkmoths and bees, follow scents to find various rewards such as pollen or nectar. The insects are selective, and make unique choices on which plants to visit.

Why do specific pollinators visit specific plants? In this case, the Skogen Lab is finding that it is in response to the scent, or chemical communication, each flower releases. “In the natural world those [scents] are signals, they are messages. Those different compounds that flowers are producing, a lot of them are cocktails of different types of chemicals. They could be saying very different things.”

PHOTO: Closeup of a wasp on a closed evening primrose bloom.
Nature is complicated. Here, a wasp lays eggs through a flower bud into a hidden Mompha moth inside. Its larvae will eventually destroy both the moth and the flower. Photo by Dr. Rick Overson

A destructive micromoth called a microlepidopteran (classified in the genus Mompha), has also likely learned how to read the scent messages of its hosts. The specialist herbivore lays eggs on plants leading to detrimental effects for seed production. The team’s field work has shown that Mompha moths only infect some populations of flowers. When and why did the flowers evolve to deter or attract all of these different pollinators? Or was it the pollinators who drove change?

At the Garden, Overson is currently focused on exploring the genomes, or DNA set, of these plants to create a phylogeny, which looks like a flow chart and reads like a story of evolution. “Right now we don’t know how all of these species are interrelated,” he explained. When the phylogeny is complete, they will have a more comprehensive outline of key relationships and timing than ever before. That information will allow scientists to determine where specific scents and other traits originated and spread. He will explore the evolution of important plant traits using the phylogeny including the color of the flowers and their pollinators, to answer as many questions as possible about relationships and linked evolutionary events.

In addition, the team is looking at population genetics so they can determine the amount of breeding occurring between plant locations by either seed movement or by pollinators. They will also look for obstacles to breeding, such as interference by mountain ranges or cities.

“Relationships among flowering plants and insects represent one of the great engines of terrestrial diversity,” wrote principal investigator Krissa Skogen, PhD, in a blog post announcing the grant.

The way that genes have flowed through different populations, or have been blocked from doing so over time, can also lead to changes in a species that are significant enough to drive speciation, or the development of new species, said Overson. “The big idea is that maybe these patterns that are driving diversity within these flowers could ultimately be leading to speciation.”

By understanding these differences and patterns, the scientists may influence conservation decisions, such as what locations are most in need of protection, and what corridors of gene flow are most important to safeguard.

PHOTO: Dr. Rick Overson in the field.
Dr. Rick Overson in the field

“We absolutely can’t live without plants or insects, it’s impossible,” remarked Overson. “Plants and insects are dominant forces in our terrestrial existence. Very few people would argue that we haven’t heavily modified the landscape where these plants and insects live. I think it is crucially important to understand these interactions for the sake of the natural world, agriculture and beyond.”

When Dr. Overson is taking a break from the laboratory, he visits the Desert Greenhouse in the Regenstein Center, which feels like home to him.

©2016 Chicago Botanic Garden and my.chicagobotanic.org