Pollinator Preference and the Evolution of Floral Traits in Monkeyflowers

Pollinator Preference and Floral Traits in Monkeyflowers (Mimulus)

Introduction

  • This study investigates the genetic basis of pollinator discrimination in two closely related monkeyflower species, Mimulus lewisii (bee-pollinated) and Mimulus cardinalis (hummingbird-pollinated).
  • The research aims to understand the genetic architecture of adaptation and speciation by examining floral traits that influence pollinator specificity and contribute to premating reproductive isolation.
  • Key questions addressed:
    • What floral traits cause pollinator discrimination among plant species?
    • What is the genetic basis of these traits?

Background

  • Fisher’s Infinitesimal Model: Proposes that adaptation results from the fixation of many genes with small individual effects.
  • Alternative Theory: Suggests that mutations of large effect can be beneficial during early stages of adaptation.
  • Premating Isolation: Adaptations that reduce mating among neighboring populations can contribute to the origin of new species.
  • Pollinator-Mediated Selection: Adaptation to abundant or efficient pollinators in isolated populations leads to floral divergence, preventing intercrossing upon secondary contact.
  • Mimulus lewisii:
    • Bee-pollinated.
    • Pink flowers.
    • Wide corolla with inserted anthers and stigma.
    • Small nectar volume.
    • Petals form a landing platform for bees.
    • Yellow ridges of brushy hairs (nectar guides).
  • Mimulus cardinalis:
    • Hummingbird-pollinated.
    • Red flowers.
    • Narrow tubular corolla.
    • Reflexed petals.
    • Large nectar reward.
    • Exserted anthers and stigma to contact hummingbird foreheads.
  • Both species lack detectable odor, and pollinator visitation is mainly influenced by flower color, size, shape, and nectar reward.
  • The two species can produce fertile hybrids.
  • Geographic distributions are mostly nonoverlapping, but they co-occur in a narrow altitudinal zone.
  • Pollinator discrimination leads to strong premating reproductive isolation in sympatric zones.

Methods and Materials

  • Genetic Basis of Floral Traits: Determine the genetic basis of traits like flower color, size, shape, and nectar reward using linkage mapping with molecular markers (QTL).
  • Pollinator Response: Evaluate the response of wild pollinators to each floral trait in a co-occurrence region.
  • Previous QTL studies found that most floral traits have at least one QTL of large effect (explaining >25% of the F2 phenotypic variance).
  • Experimental Setup:
    • Plant Material: M. lewisii crossed with M. cardinalis to produce F1 hybrids, which were then mated to produce an 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: Parental, F1, and F2 individuals grown in a greenhouse and then transported to Yosemite National Park.
    • Plants arranged randomly in a 5 x 15 m plot with 0.5-m spacing.
    • Fewer parentals and F1s were used than F2s to prevent pollinators from developing a preference for F2s resembling parental species.
    • Observation period preceded the flowering time of natural populations to prevent gene flow.

Observed: Bee and Hummingbird Visits From Dawn to Dusk During Separate 30-min Periods..

  • Observation Duration: Three to four times a day (mean = 3.7 periods per day for each pollinator type) on 7 days, totaling 26 hours.
    • Observers recorded flower visits by bees and hummingbirds using tape recorders.
    • The number of open flowers for each plant was recorded daily.
      Calculated Daily Rate of Pollinator Visitation (Visits per Flower per Day), Dividing the Daily Total Number of Visits for Each Pollinator by Flower Number..
  • Floral Traits Analyzed:
    • Petal anthocyanin concentration (purple pigments).
    • Petal carotenoid concentration (yellow pigments).
    • Nectar volume.
    • Projected area (petal surface exposed to pollinators).
      Phenotypic Value of Each Trait Estimated using the mean of two randomly drawn flowers Per Plant.
  • Petal anthocyanin concentration was estimated by punching 6-mm disks from the lateral petals, extracting the anthocyanins with 0.5 ml of methanol/0.1% HCl, and determining the absorbance at 510 nm.
    Petal carotenoid concentration was estimated similarly, using methylene chloride for extraction and measuring absorbance at 450 nm.
    Recorded video images of flowers from the perspective of approaching pollinators, analyzed with image analysis software to estimate projected area of corolla.
    Used a graduated pipette tip to measure Nectar Volume.
    All Measurements Were Conducted While Study Plants Were Growing in the University of Washington Greenhouse.
    Re-measured a subset of Plants in the Field Plot, and found that the greenhouse and field values were positively correlated for all morphological traits (P < 0.01, n = 56) and for 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.
    • Floral traits were independent variables.
    • Angular transformation on the proportion of visits by bees and a square-root transformation on all floral traits.
    • Transformed variables were standardized (mean = 0, SD = 1).

Results

Observed A Total of 12,567 Pollinator Visits in the Experimental Population.
The non-native honeybee Apis mellifera comprised approximately 5% of the total visits to F2s and was excluded from analyses.
The bumblebee Bombus vosnesenski Was Responsible for approximately 95% of All Bee Visits.
Increased Petal Anthocyanins, Petal Carotenoids, and Nectar Volume Significantly Reduced the Proportion of Bee Visitation, Whereas Greater Projected Area Increased the Proportion of Bee Visitation.*

Petal anthocyanin concentration significantly affected both bee and hummingbird visitation rates, but with opposite effects, whereas each of the other floral traits had a significant effect on one pollinator, but not on 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.*
    A Single Mendelian locus controls the distribution of carotenoid pigments in the petals.
    There was no effect of yup genotype on hummingbird visitation rate, but bee visitation was 80% lower in plants homozygous for the M. cardinalis allele.

Plants homozygous for the M. cardinalis allele had twice the rate of hummingbird visitation as M. lewisii homozygotes, whereas heterozygotes had an intermediate value.

Discussion

  • Clear evidence that flower color contributes to reproductive isolation in this system.
  • Genetic variation for petal carotenoid concentration affects bee visitation.
  • High concentration of carotenoids in M. cardinalis flowers may function primarily to discourage bee visitation.
  • Hummingbirds have a slight, but significant preference for flowers with high petal anthocyanin concentration but exhibited no preference for flowers high in petal carotenoids.
  • Hummingbirds can distinguish the phenotypic effects of allele substitutions at the major nectar QTL.
  • Adaptive divergence of floral traits through pollinator-mediated selection.
  • Floral traits associated with bumblebee and hummingbird pollination are 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.