Pollinator Preference and Floral Traits in Monkeyflowers
Pollinator Preference and Floral Traits in Monkeyflowers (Mimulus)
Introduction
A central question in evolutionary biology concerns the genetic architecture of adaptation. This study investigates pollinator discrimination in two closely related monkeyflower species (Mimulus lewisii and Mimulus cardinalis) to test the hypothesis that adaptation and reproductive isolation result from numerous mutations with small effects.
The monkeyflower system presents a good opportunity to study the genetics of adaptation and speciation because floral traits that determine pollinator specificity also contribute to reproductive isolation before mating.
Key Questions
What floral traits lead to pollinator discrimination among plant species?
What is the genetic basis of these traits?
Methods
A large-scale field experiment was conducted using genetic markers to determine the genetic basis of pollinator visitation.
Observations were made on F2 hybrids resulting from crosses between bee-pollinated M. lewisii and hummingbird-pollinated M. cardinalis.
Results
Bees preferred large flowers with low anthocyanin and carotenoid pigment levels.
Hummingbirds favored nectar-rich flowers with high anthocyanin levels.
An allele increasing petal carotenoid concentration reduced bee visitation by 80%.
An allele increasing nectar production doubled hummingbird visitation.
Implications
These results suggest that genes with large effects on pollinator preference have contributed to floral evolution and premating reproductive isolation in monkeyflowers.
The study supports the growing evidence that adaptation and reproductive isolation can involve major genes.
Keywords
Reproductive isolation, adaptation, speciation, natural selection, pollination
Theoretical Background
Fisher’s Infinitesimal Model: This model suggests adaptation results from the fixation of many genes, each with small individual effects. It assumes that large-effect mutations move a population away from its phenotypic optimum.
Micromutationist View: This view of adaptive geometry has been widely supported but challenged by theories suggesting that large-effect mutations can be beneficial during early adaptation stages as populations move toward their optimum phenotype.
There is a need for empirical studies to resolve this debate by identifying systems where both the genetic basis and ecological significance of adaptive traits can be identified.
Premating Isolation and Pollination
Adaptations reducing mating frequency among neighboring populations are important because they can contribute to the origin of new species.
While Drosophila studies suggest premating isolation evolves quickly and has a simple genetic basis, comparable data from other organisms, especially regarding the genetics of premating reproductive isolation in natural populations, are lacking.
Pollinator-mediated selection on floral traits is considered a common mechanism for adaptation and speciation in plants.
The traditional view is that adaptation to abundant or efficient pollinators in geographically isolated populations leads to floral divergence, and pollinator preference prevents intercrossing when populations come into secondary contact.
The Mimulus System
Mimulus lewisii (bee-pollinated) and Mimulus cardinalis (hummingbird-pollinated) are two species that exhibit secondary contact.
M. lewisii: Pink flowers, wide corolla with inserted anthers and stigma, small nectar volume, petals forming a landing platform for bees, and yellow nectar guides.
M. cardinalis: Red flowers, narrow tubular corolla, reflexed petals, large nectar reward, and exserted anthers and stigma to contact hummingbirds' foreheads.
Pollinator visitation is primarily influenced by flower color, size, shape, and nectar reward.
These monkeyflowers are closely related; crosses between them produce fertile hybrids.
Their geographic distributions are mostly nonoverlapping, with M. lewisii at mid-to-high elevations and M. cardinalis at low-to-mid elevations.
They co-occur in a narrow altitudinal zone at 1400 m in the Sierra Nevada.
Observations in a sympatric area showed that bees exclusively visit M. lewisii, while hummingbirds primarily visit M. cardinalis, indicating strong premating reproductive isolation.
Experimental Design
Two experiments are needed to understand the genetic architecture of reproductive isolation via pollinator-mediated selection:
Determine the genetic basis of traits like flower color, size, shape, and nectar reward in plant species with different pollinators.
Evaluate the response of wild pollinators to each floral trait in a co-occurrence region.
The first experiment, using linkage mapping with molecular markers to identify quantitative trait loci (QTL) controlling floral traits, found that most traits had at least one QTL of large effect (explaining >=25% of F2 phenotypic variance).
This paper reports results from the second experiment, identifying the ecological significance of floral traits and the effect of simple genetic changes on pollinator visitation in nature.
Materials and Methods
Seed collection: Yosemite National Park.
Hybrid production: M. lewisii x M. cardinalis --> F1 hybrids, then mated unrelated F1s --> outcrossed F2 population.
F1 hybrids: Pink flowers, moderately reflexed petals, nectar guides similar to M. lewisii but lacking hairs.
F2 generation: Wide range of flower colors and morphologies.
Experimental Population and Setup
Parental, F1, and F2 individuals were grown to flowering and moved to the study site (Wawona Ranger Station, Yosemite National Park, elevation 1300 m).
Plants were arranged randomly in a 5 x 15 m plot with 0.5-m spacing (n = 24 for each parent and F1, n = 228 for the F2 generation).
Fewer parentals and F1s were used to prevent pollinators from developing a preference for F2s resembling the parental species.
The observation period (June 1996) preceded the flowering time of natural populations of both species to prevent gene flow and ensure pollinators hadn't encountered these species in natural populations yet.
Pollinator Visitation Observations
Bee and hummingbird visitation was observed from dawn to dusk in separate 30-min periods, 3-4 times a day (mean = 3.7 periods/day for each pollinator) for 7 days (June 18-27), totaling 26 hours.
3-5 observers recorded flower visits using tape recorders.
The number of open flowers for each plant was recorded daily.
Daily visitation rate was calculated as total visits per pollinator divided by the number of flowers.
No corrections were made for unobserved bee visits, likely resulting in a slight underestimate of bee visitation frequency.
Floral Trait Measurements
Four floral traits were analyzed:
Petal anthocyanin concentration (purple pigments).
Petal carotenoid concentration (yellow pigments).
Nectar volume.
Projected area (petal surface exposed to pollinators).
These traits are highly diverged in the parental species and are expected to affect pollinator visitation rates.
The possibility of other unmeasured traits contributing to pollinator visitation, linked to the measured traits, or having pleiotropic effects, was acknowledged.
The mean of two randomly drawn flowers per plant was used to estimate the phenotypic value of each trait.
Petal anthocyanin concentration: 6-mm disks extracted with methanol/0.1% HCl, absorbance at 510 nm.
Petal carotenoid concentration: Similar, using methylene chloride for extraction and measuring absorbance at 450 nm.
Projected area: Video images analyzed with NIH IMAGE software.
Nectar volume: Measured with a graduated pipette tip.
All measurements were conducted in the University of Washington greenhouse.
A subset of plants was remeasured in the field; greenhouse and field values were positively correlated for all morphological traits (P < 0.01, n = 56) and nectar volume (P < 0.0001, n = 31).
Statistical Analysis
Multiple regressions were used to examine the relationship between pollinator visitation and floral traits in the F2 population.
The proportion of bee visits and the daily visitation rates of bees and hummingbirds were treated as dependent variables, with the four floral traits as independent variables.
Analyzing the proportion of bee visits assesses the effects of floral characters on the pollinator assemblage, while analyzing visitation rates identifies the mechanisms causing pollinator composition differences.
Angular transformation was performed on the proportion of visits by bees, and a square-root transformation was performed on all floral traits.
Transformed variables were standardized (mean = 0, SD = 1) for direct comparison of regression coefficient magnitudes.
Results
A total of 12,567 pollinator visits were observed.
The non-native honeybee Apis mellifera (5% of visits) was excluded from analyses.
Bumblebee Bombus vosnesenski accounted for 95% of bee visits, with the remaining visitation by Osmia (Monilosmia) sp. and an unknown bumblebee.
Bombus vosnesenski generally visited flowers for nectar with passive anther contact, while Osmia (Monilosmia) sp. actively collected pollen.
Anna’s hummingbird (Calypte anna) was the only hummingbird species observed.
M. lewisii was primarily visited by bees (82%), and M. cardinalis was visited by hummingbirds (99.6%).
Visitor composition to F1 hybrids (59% bees) was intermediate.
The F2 population (8648 visits) varied widely in pollinator composition.
Regression Analysis Results and Floral Trait Effects
Increased petal anthocyanins, petal carotenoids, and nectar volume significantly reduced the proportion of bee visitation, whereas greater projected area increased it.
Flower color contributes to reproductive isolation.
Petal anthocyanin concentration significantly affected both bee and hummingbird visitation rates in opposite ways.
Other floral traits significantly affected one pollinator but not the other.
Bee visitation rate was negatively associated with petal anthocyanin and carotenoid concentration and positively associated with projected area.
Hummingbird visitation rate was positively associated with both petal anthocyanin concentration and nectar volume.
Genetic Effects on Pollinator Visitation
Visitation rates were compared as a function of QTL marker genotype for petal carotenoid concentration and nectar volume.
Carotenoid Pigments
A single Mendelian locus controls carotenoid pigment distribution in petals.
F2 plants homozygous for the recessive M. cardinalis allele at the yup locus (yellow upper) have carotenoids throughout the petals and are orange- or red-flowered.
Plants carrying the dominant M. lewisii allele are pink-flowered.
There was no effect of yup genotype on hummingbird visitation rate.
Bee visitation was 80% lower in plants homozygous for the M. cardinalis allele.
Bees visiting Mimulus species in the section Erythranthe strongly prefer pink over red flowers.
Petal carotenoids significantly decrease bee visitation but have no effect on hummingbirds, suggesting their high concentration in M. cardinalis flowers may primarily discourage bee visitation.
The hypothesis that red coloration in hummingbird flowers reduces insect visitation is consistent with the finding that hummingbirds do not have an innate preference for red.
Nectar Reward
Hummingbird and bee visitation rates were compared for three F2 genotypic classes at the major nectar QTL (Quantitative Trait Loci).
This QTL explains 41% of the difference in nectar volume between the parental species and has an additive mode of action, with the M. cardinalis allele increasing nectar.
Segregation of parental alleles produced a nearly 3-fold range in mean nectar volume in the F2 field population.
The average nectar volume of the heterozygous genotypic class was intermediate to that of the two homozygous classes.
The visitation rate of hummingbirds closely matched this distribution of nectar volume; plants homozygous for the M. cardinalis allele had twice the hummingbird visitation rate as M. lewisii homozygotes.
Hummingbirds can distinguish the phenotypic effects of allele substitutions at the major nectar QTL.
There was no relationship between bee visitation rate and marker genotype at the nectar QTL.
Hummingbirds can quickly find rich nectar sources and return to them often, suggesting they can exert strong selection on flower nectar rewards.
Summary
There are striking differences in the floral preferences of bees and hummingbirds, providing considerable opportunity for adaptive divergence of floral traits through pollinator-mediated selection.
Floral traits associated with bumblebee and hummingbird pollination, such as petal carotenoid pigments and nectar volume, appear to be under relatively simple genetic control, with major QTLs responsible for pollinator discrimination and reproductive isolation in nature.
Adaptation may often involve genes of large effect.
Further studies are needed to determine whether these results can be generalized to other plant taxa where closely related species differ in their major pollinators.
Discussion Questions:
What are the primary objectives of Schemske and Bradshaw's study on monkeyflowers?
This study investigates pollinator discrimination in two closely related monkeyflower species (Mimulus lewisii and Mimulus cardinalis) to test the hypothesis that adaptation and reproductive isolation result from numerous mutations with small effects.
Main questions:
What floral traits lead to pollinator discrimination among plant species?
What is the genetic basis of these traits?
What are the main differences in floral traits between M. lewisii and M. cardinalis?
Bees preferred large flowers with low anthocyanin and carotenoid pigment levels.
Hummingbirds favored nectar-rich flowers with high anthocyanin levels.
An allele increasing petal carotenoid concentration reduced bee visitation by 80%.
An allele increasing nectar production doubled hummingbird visitation.
What experimental design was used to test pollinator preferences?
This paper reports results from the second experiment, identifying the ecological significance of floral traits and the effect of simple genetic changes on pollinator visitation in nature.
Seed collection: Yosemite National Park.
Hybrid production: M. lewisii x M. cardinalis --> F1 hybrids, then mated unrelated F1s --> outcrossed F2 population.
F1 hybrids: Pink flowers, moderately reflexed petals, nectar guides similar to M. lewisii but lacking hairs.
F2 generation: Wide range of flower colors and morphologies.
Measured the visitation of bee and hummingbird visitation
Why was it important to use plants that had different mixtures of the M. lewisii and M. cardinalis genomes (an F2 mapping population) in this study?
Able to identify the specific traits that impact pollinator visitation, and the overall impact that modulations to theses traits had on pollinator visitation.
How does the study contribute to our understanding of speciation processes?
This study contributes to our understanding of speciation processes by providing empirical evidence that specific floral traits, influenced by major genes (QTLs), play a significant role in pollinator preference and reproductive isolation. The findings suggest that adaptation and reproductive isolation can involve genes with large effects, challenging the traditional view that adaptation results solely from the fixation of many genes with small individual effects (Fisher’s Infinitesimal Model). By identifying the ecological significance of floral traits and the effect of simple genetic changes on pollinator visitation in nature, the study supports the idea that pollinator-mediated selection on floral traits is a common mechanism for adaptation and speciation in plants.
How do the YUP locus and the trait for nectar volume influence pollinator preference?
In the context of the monkeyflower study, the yup locus (yellow upper) is a Mendelian locus that controls carotenoid pigment distribution in petals. F2 plants homozygous for the recessive M. cardinalis allele at the yup locus have carotenoids throughout the petals and are orange- or red-flowered, while plants carrying the dominant M. lewisii allele are pink-flowered.
Bees prefer low carotenoid levels
Hummingbirds prefer high nectar volume