hawkmoths – My Chicago Botanic Garden

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.”

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 Evolution of a Research Idea

Five years ago this past May, I found myself starting a new job and a new research project. My job, of course, was as a conservation scientist here at the Chicago Botanic Garden, and the research project had me sitting on the side of a road at dusk in Pueblo West, Colorado. I sat there in front of a group of plants that produce lovely-smelling flowers, waiting for their impressive pollinators to show up. And when they did, I snapped some of my very first photos of these beauties: hawkmoths, better known as the five-spotted hawkmoth, or to the scientific community as Manduca quinquemaculata.

PHOTO: Night photo of hawkmoth sipping nectar from evening primrose. — A five-spotted hawkmoth (*Manduca quinquemaculata*) drinks nectar from the Colorado Springs evening primrose (*Oenothera harringtonii*) as the flower begins to open. Pueblo West, Colorado, May 2008. Photo: Krissa Skogen

Just this past Friday, I visited the National Science Foundation’s Dimensions of Biodiversity Program, to find this very same photo—and the research that my colleagues and I will conduct over the next five years—highlighted.

So how did this one photo go from being taken in the spring of 2008 to being highlighted on the NSF’s website? How does a research project evolve and grow over time? Ask any scientist what they are currently working on and their answer will almost always start with, “I was first fascinated by x back in y….” Something caught their attention, sparked a thought, pulled them in—and they continued asking question after question, developing hypotheses and gathering data to test them, with their answers pushing them forward, sometimes down unanticipated paths, and sometimes into much bigger or smaller arenas. The more one knows, seemingly, the less one knows; old questions are answered and new ones are developed. This is what pushes scientists, and science, forward.

The evolution of a research idea

PHOTO: Krissa Skogen poses with primrose in New Mexico. — Krissa Skogen poses with an evening primrose in New Mexico. Photo: Chris Martine

In 2008, I started my current research program. After many conversations with Rob Raguso (Cornell University) and Tass Kelso (Colorado College), I drove out to Colorado with a plan to collect as much information on as many different populations of the Colorado Springs evening primrose (Oenothera harringtonii) as possible in a short period of time. That year, my timing was off—I arrived in Colorado on June 10. Oenothera harringtonii flowers primarily in May. Most of the plants had stopped flowering and so instead of collecting data on floral features, nectar, scent, and pollinators, my field assistant, Evan Hilpman, and I collected data on plant size, health, reproductive success (how many fruits did they produce?) and population size (much like a census). And one striking thing we noticed was this: small white “galls” on some of the green, developing fruits. We took notes on how often we saw this, never anticipating the importance that these little white dots would play in just a few years’ time.

PHOTO: Closeup image of a tiny, white foamy-looking dot (one of many) on a host plant. — We noticed small white “galls” on some of the green, developing fruits. These are parts of the cocoons of tiny little moths, called microlepidopterans, of the genus *Mompha*. Photo: Krissa Skogen

In subsequent years this project grew, and in the last four years—with the help of conservation scientist Jeremie Fant and other colleagues, and many research assistants and students—we’ve collected data on flower size, nectar volume and sugar content, floral scent, who pollinates and when (hawkmoths come at dusk and visit overnight; bees generally visit in the morning), how populations grow and shrink over time, which other plant species are flowering at the same time, and more. We know a lot of things about this species now, and one thing has been a constant: those little white balls have been observed year-in and year-out in some populations, but not in others.

We know now that some of our populations have an important compound—linalool—and some do not. We know that genetically speaking, our 25 populations function more like three, likely due to the fact that hawkmoths can fly so darn far (some estimates are up to 20 miles in just one night). And more recently, we started gathering more data on those little white balls. It turns out that they are parts of the cocoons that surround the larvae of tiny little moths called microlepidopterans, which belong to the genus Mompha. These moths lay their eggs on flower buds, fruit, and stems. If the larvae eat flower buds and/or seeds, they reduce the number of offspring that the plant produces. This is bad for any plants upon which these moths decide to lay their eggs, but everything must eat, right?

PHOTO: Trio of photos of each life stage of the moth: adult, larva, and cocoon. — *Mompha stellella* microlepidopteran adult; larva inside fruit (seed predator); cocoon inside *O. harringtonii* fruit. Photos: Terry Harrison and Krissa Skogen

In speaking with colleagues across the country and in Canada (plant and moth experts, alike), we developed an intriguing story and series of hypotheses we felt were compelling. Do pollinators and floral antagonists both respond to the same attractive scent? Could floral scent be telling hawkmoths and Mompha moths where the flowers are? Pollination is good for plant reproduction, but anything that eats flowers or seeds is not—so how would this trade-off play out in evolutionary time?

These questions have led us to the project that we will pursue on a much larger scale, thanks to recently awarded funding from the National Science Foundation’s Dimensions of Biodiversity Program for our proposal, titled “Landscapes of linalool: scent-mediated diversification of flowers and moths across western North America.”

PHOTO: Bee coated in pollen, inside primrose bloom. — A *Lasioglossum* species bee robbing pollen from *O. harringtonii* at dawn. Photo: Sadie Todd

Relationships among flowering plants and insects represent one of the great engines of terrestrial diversity. Floral scent and other plant volatiles are important drivers of these relationships (e.g., pollination, herbivory, plant defense), but remain poorly integrated into floral evolution and pollination ecology. Few studies have tested the spectrum of plant fitness outcomes when scent attracts both pollinators and floral/seed enemies. Thus, the hidden diversity of floral/seed predators and their potential as selective agents constitutes a considerable gap in pollinator-centric understanding of floral evolution. These “forgotten predators” have co-diversified with flowering plants and are likely influential in the evolution of most plant-pollinator interactions.

PHOTO: Five-spotted hawkmoth extending its proboscis (longer than its body) into a primrose bloom as it hovers. — A five-spotted hawkmoth (*Manduca quinquemaculata*) probes an opening evening primrose flower for nectar with its proboscis. Photo: Krissa Skogen

This project is ambitious and large and pulls upon a wide variety of expertise. In total, there are 11 Ph.D. scientists collaborating on it, including myself, Jeremie Fant, and Norm Wickett here at the Garden. The others include Robert Raguso (Cornell University), Rachel Levin (Amherst College), Terry Harrison (University of Illinois at Urbana-Champaign), Jean-Francois Landry (Agriculture & Agri-Food Canada, Eastern Cereal and Oilseed Research Centre), Sylvia (Tass) Kelso (Colorado College), Kathleen Kay (University of California, Santa Cruz), Mike Moore (Oberlin College), and Warren Wagner (Smithsonian Institution).

We are excited about what we’ll uncover in the next five years and will update you with progress as our discoveries unfold!

“Should we try to roll its tongue out?”

PHOTO: Sophia Siskel holds a hawkmoth caught at night while researchers look on. — Holding a toad-sized hawkmoth lured in by our sheet and black light.

O.K., I did know what a proboscis was before my trip to New Mexico last month. But learning how to uncoil a hawkmoth’s 3-inch nectar-sucking hollow tongue while trying to calm the toad-sized insect in my hand was the biology lesson of a lifetime.

Thanks to Chicago Botanic Garden scientists Krissa Skogen, Ph.D., and Wes Glisson (who recently earned his master’s degree in plant conservation biology from the Garden/Northwestern University graduate program), and Bureau of Land Management (BLM) New Mexico state botanist Mike Howard, I learned about hawkmoths, the plants they pollinate, and how to collect plant cuttings for scientific study.

I also had the opportunity to meet and work beside two remarkable interns, Kate Wilkins and Elisabeth Ward, from our Conservation Land Management Internship Program (and enjoy a few absolutely perfect hours of exquisite silence in the desert at the foot of the Guadalupe Mountains on the New Mexico/Texas border).

PHOTO: Panorama of the New Mexican desert. — The exquisite silence and panorama of the New Mexican desert.

I had been asking around the Garden’s scientific staff to see whose fieldwork would fit with my summer schedule. Krissa was planning a trip to southern New Mexico to film an episode of Chris Martine’s great video web series Plants are Cool, Too. Krissa’s episode, which will air in October, highlights her work on long-distance pollinator movement, focusing on Oenothera harringtonii, an evening primrose endemic to southeastern Colorado and other closely related Oenothera species. The flowers of Oenothera harringtonii and many other evening primroses open soon after sunset and are pollinated primarily by hawkmoths. These moths feed on the nectar of Oenothera flowers, which they locate by the strong fragrance produced by the flowers. We commonly think of floral scent for its role in attracting pollinators, but it may also be used as a cue by floral and seed predators.

By studying the shape, smell, and color of Oenothera flowers, Krissa and her colleagues hope to determine what it is that attracts pollinators to these flowers. She can also determine how the plants “reward” their pollinators by studying nectar—how much flowers produce and how much sugar the nectar contains. And lastly, by collecting pollen grains from pollinators, Krissa can determine which plant species the pollinators rely on most, which brings me to catching hawkmoths and collecting pollen from their tongues.

PHOTO: Krissa gently rolls out the proboscis to show us just how long it is! — Krissa gently rolls out the proboscis to show us just how long it is!

The first night of our trip, we set out to find some hawkmoths. After visiting a couple of sites in the Organ Mountains, we found them. Above is a photograph of Dr. Krissa Skogen, Elisabeth Ward, and me holding the toad-sized moths we attracted to a blacklit white sheet held up on a PVC armature.

After sunset, the hawkmoth uses its long hollow tongue to extract the nectar from deep down within the narrow mouth of the flower. The moth’s nightly journey often covers a distance as far as 20 miles. Krissa gently rolled out the tongue to show us just how long it is!

The next day, we set out early to collect Lepidospartum quamum for our colleague Evelyn Williams, Ph.D. Evelyn, a post-doctoral researcher, has been working with Jeremie Fant, Ph.D., Kayri Havens, Ph.D., and Mike Howard on this plant since 2012 in an attempt to figure out why it is threatened with extinction in this area of New Mexico. The plant grows in a unique environment—the gypsum salt flat.

PHOTO: Tagged plant cuttings in a small bowl. — *Lepidospartum quamum* cuttings, tagged and ready to be sent for propagation in our greenhouses.

Evelyn’s previous collecting trip this spring needed to be supplemented with new cuttings. We worked all day to collect the cuttings, which we sent back to the Garden for propagation in our production greenhouses, as well as samples for genotyping in the Garden’s Harris Family Foundation Plant Genetics Laboratory.

This important work, which ultimately aids seed growers, restoration practitioners, and government agencies to select appropriate plant materials to restore diverse plant and animal communities, was funded by a National Fish and Wildlife Foundation (NFWF) grant as part of the Native Plant Conservation Initiative.

It’s a fact that most people are more attracted to animals than plants—and therefore more inclined to know their names and fight for their survival. Just compare the following two photographs—the Lepidospartum quamum specimen we were studying, and this lizard that darted by and immediately commanded our attention (yes, even botanists and plant-lovers are drawn to a cute face).

PHOTO: A desiccated Lepidospartum quamum plant. — What grabs your attention more? This plant …

PHOTO: A cute lizard. — …or this cute lizard?

But all life depends on plants and the healthy habitats on which they depend. When we think of fighting to save wildlife, let’s remember that wildlife includes plants! I am hopeful that by working with collaborations from gardens, zoos, government agencies, and other land-trust and conservation organizations, we can integrate plants into wildlife action plans both in the U.S. and abroad. I particularly like how this report by NatureServe summarizes this issue.

We can all point to moments in our life—when we’ve experienced something new or met someone special—when our understanding of life changes. My two days with these five scientists—at all phases of their careers—was one of these experiences I will never forget.

PHOTO: The New Mexico research team. — Thank you, Krissa, Kate, Mike, Elisabeth, and Wes!

One last note: Hawkmoths are essential to ecosystems from Venezuela to here in Chicago. My son and I watched one this afternoon drink from the hostas on our street! Below is one we filmed in the English Oak Meadow of the Chicago Botanic Garden last week.