Relative Role of Flower Color and Scent on Pollinator Attraction: Experimental Tests using F1 and F2 Hybrids of Daylily and Nightlily

The daylily (Hemerocallis fulva) and nightlily (H. citrina) are typical examples of a butterfly-pollination system and a hawkmoth-pollination system, respectively. H. fulva has diurnal, reddish or orange-colored flowers and is mainly pollinated by diurnal swallowtail butterflies. H. citrina has nocturnal, yellowish flowers with a sweet fragrance and is pollinated by nocturnal hawkmoths. We evaluated the relative roles of flower color and scent on the evolutionary shift from a diurnally flowering ancestor to H. citrina. We conducted a series of experiments that mimic situations in which mutants differing in either flower color, floral scent or both appeared in a diurnally flowering population. An experimental array of 6×6 potted plants, mixed with 24 plants of H. fulva and 12 plants of either F1 or F2 hybrids, were placed in the field, and visitations of swallowtail butterflies and nocturnal hawkmoths were recorded with camcorders. Swallowtail butterflies preferentially visited reddish or orange-colored flowers and hawkmoths preferentially visited yellowish flowers. Neither swallowtail butterflies nor nocturnal hawkmoths showed significant preferences for overall scent emission. Our results suggest that mutations in flower color would be more relevant to the adaptive shift from a diurnally flowering ancestor to H. citrina than that in floral scent

Evolutionary history of Hemerocallis in Japan inferred from chloroplast and nuclear phylogenies and levels of interspecific gene flow

The perennial herb genus Hemerocallis (Asphodelaceae) shows four flowering types: diurnal half-day, diurnal one-day, nocturnal half-day, and nocturnal one-day flowering. These flowering types are corresponding to their main pollinators, and probably act as a primary mechanism of reproductive isolation. To examine how the four flowering types diverged, we reconstructed the phylogeny of the Japanese species of Hemerocallis using 1615 loci of nuclear genome-wide SNPs and 2078 bp sequences of four cpDNA regions. We also examined interspecific gene flows among taxa by an Isolation-with-Migration model and a population structure analysis. Our study revealed an inconsistency between chloroplast and nuclear genome phylogenies, which may have resulted from chloroplast capture. Each of the following five clusters is monophyletic and clearly separated on the nuclear genome-wide phylogenetic tree: (I) two nocturnal flowering species with lemon-yellow flowers, H. citrina (half-day flowering) and H. lilioasphodelus (one-day flowering); (II) a diurnal one-day flowering species with yellow-orange flowers, H. middendorffii; (III) a variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. disticha; (IV) another variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. aurantiaca, and a diurnal one-day flowering species with yellow-orange flowers, H. major; (V) a diurnal half-day flowering species with yellow-orange flowers, H. hakuunensis. The five clusters are consistent with traditional phenotype-based taxonomy (cluster I, cluster II, and clusters III-V correspond to Hemerocallis sect. Hemerocallis, Capitatae, and Fulvae, respectively). These findings could indicate that three flowering types (nocturnal flowering, diurnal one-day flowering, and diurnal half-day flowering) diverged in early evolutionary stages of Hemerocallis and subsequently a change from diurnal half-day flowering to diurnal one-day flowering occurred in a lineage of H. major. While genetic differentiation among the five clusters was well maintained, significant gene flow was detected between most pairs of taxa, suggesting that repeated hybridization played a role in the evolution of those taxa

Examples of foraging bouts and pollen movement.

<p>Each circle indicates experimental flowers. Solid arrows indicate foraging movements of swallowtail butterfly (A) and hawkmoth and gray circles are visited flowers. Dashed arrows show pollen movement from donors to recipients.</p

Pollinator-Mediated Selection on Flower Color, Flower Scent and Flower Morphology of <i>Hemerocallis</i>: Evidence from Genotyping Individual Pollen Grains On the Stigma

<div><p>To trace the fate of individual pollen grains through pollination processes, we determined genotypes of single pollen grains deposited on <i>Hemerocallis</i> stigmas in an experimental mixed-species array. <i>Hemerocallis fulva</i>, pollinated by butterflies, has diurnal, reddish and unscented flowers, and <i>H. citrina</i>, pollinated by hawkmoths, has nocturnal, yellowish and sweet scent flowers. We observed pollinator visits to an experimental array of 24 <i>H. fulva</i> and 12 F2 hybrids between the two species (<i>H. fulva</i> and <i>H. citrina</i>) and collected stigmas after every trip bout of swallowtail butterflies or hawkmoths. We then measured selection by swallowtail butterflies or hawkmoths through male and female components of pollination success as determined by single pollen genotyping. As expected, swallowtail butterflies imposed selection on reddish color and weak scent: the number of outcross pollen grains acquired is a quadratic function of flower color with the maximum at reddish color, and the combined pollination success was maximal at weak scent (almost unrecognizable for human). This explains why <i>H. fulva</i>, with reddish flowers and no recognizable scent, is mainly pollinated by swallowtail butterflies. However, we found no evidence of hawkmoths-mediated selection on flower color or scent. Our findings do not support a hypothesis that yellow flower color and strong scent intensity, the distinctive floral characteristics of <i>H. citrina</i>, having evolved in adaptations to hawkmoths. We suggest that the key trait that triggers the evolution of nocturnal flowers is flowering time rather than flower color and scent.</p> </div

Flowers of <i>H. fulva</i> and F2 hybrid.

<p>(A) A swallowtail butterfly <i>Papilio xuthus</i> visiting a <i>H. fulva</i> flower. (B) A hawkmoth <i>Theretra japonica</i> visiting a F2 hybrid flower.</p

Typical reflectance spectra of F2 hybrids (above) and the relationship score, reflectance (below).

<p>(A) Reflectance spectra of the central part of tepals. Three representative F2 hybrids, DG11 (SCC=3), BD3 (SCC=13) and BC12 (SCC=21), are showed. (B) Reflectance spectra of the peripheral part of tepals. (C) The relationship between color chart score and relative reflectance at 525 nm of the central part of tepals. (D) The relationship between color chart score and relative reflectance at 360 nm of the peripheral part of tepals.</p

Variation of flower colors in F1 (A) and F2 hybrids (B).

<p>The horizontal axis is the standard color chart score. Larger scores indicate reddish color and smaller scores indicate yellowish color. Color chart scores from 2–13 were classified as the yellow group, and color chart scores from 14–23 were classified as the yellow-orange group. The vertical axis is the number of plants.</p

Relationship between flower color and fragrance in F2 hybrids.

<p>The horizontal axis is the standard color chart score. The vertical axis is the intensity of floral scent measured with the odor meter. A Pearson's product-moment correlation coefficient is –0.0336 (<i>P</i>=0.684).</p

Reflectance spectra of tepals of two <i>Hemerocallis</i> species, F1 hybrid and Standard Color Charts.

<p>(A) Reflectance spectra of the central part of tepals (upper, <i>H. citrina</i>; center F1 hybrid; lower, <i>H. fulva</i>). (B) Reflectance spectra of the peripheral part of tepals (upper, <i>H. citrina</i>; center F1; lower, <i>H. fulva</i>). (C) Reflectance spectra of three representative Standard Color Charts (upper, SCC=3; center SCC=13; lower, SCC=23).</p

Variation of fragrance intensity in F1 (A) and F2 hybrids (B).

<p>The horizontal axis is the intensity of floral scent measured with a handheld odor meter. The odor meter can show relative intensity of scent in an arbitrary scale. All data sets were measured by the same odor meter for reproducibility. The vertical axis is the number of plants.</p