Honey Bee Foraging Behavior Experiments

Three foraging experiments were conducted to assess the preference of honey bees to different flower morphs of E. sativa. Experiments were conducted in a screened enclosure (950 × 800 × 400 cm). A colony (six-frame nucleus) was placed in the enclosure 3–4 d prior to the behavioral tests; during this time older foragers tend to be replaced by younger, inexperienced bees.

In the first experiment, two plants from each of the two populations were randomly selected. We cut inflorescence stalks, ca. 11 cm in length, each with the four youngest flowers, and placed them in 20 ml glass flasks containing tap water. Two stalks for each population were placed in alternating order on a rotary green round plate (41 cm in diameter), with a distance of 16 cm between the stalks. The plate rotated slowly (2 rpm) throughout the experiment to prevent location bias. The honey bee hive was at a distance of 3 m from the plate. As a bee approached the flowers, her choice was recorded, and the bee was caught and removed, to exclude the possibility of dance recruitment. The stalks were then replaced by others, representing a total of four other different plants (herein referred to as trial). The experiment included a total of 86 trials, i.e., a total of 172 plants for each population.

Another similar experiment (experiment II) was performed, in which plants of each of the two populations were selected on the basis of their visible flower color. Accordingly, each of a total of 72 trials included four flower stalks, two with visible cream color and two of yellow color, of the desert and Mediterranean populations, respectively. The experiments were performed in 17 continuous days between 09:00 to 16:00 hours.

Based on the results of the two foraging experiments, showing initial preference of honey bees to yellow color morph, we designed a third experiment in which we tested whether yellow color would possess an ecological advantage also in the desert population, where this morph is less abundant (Supp. Fig. S1). In this experiment, 10 inflorescence stalks from three plants with flowers with low saturation values (i.e., cream color, below), each including four flowers, were set in 20 ml flasks and placed at equal distances at the edge of the green rotating plate, herein referred as periphery flowers (PF). An additional flask was placed in the center of the plate, in which we replaced stalks between trials in alternating order, a stalk of a plant with flowers of high saturation value with one of a low saturation value; we will herein refer to these as target central flowers (CF). Significant differences were found in the average saturation values (as described below) between cream PF (mean + SE: 0.154 ± 0.004), and the yellow CF (0.274 ± 0.008) (one-way analysis of variance [ANOVA], F_2,171 = 206.65, P < 0.001). Each trial ended when a single honey bee foraged on a CF, or after 1 h, if she did not visit the target flower. The honey bee was then caught and removed to exclude the possibility of dance recruitment. In each trial, we monitored the number of PF that were visited before visiting the CF and the time it took to reach it. The experiment was performed in eight continuous days between 09:00 to 16:00 hours and included a total of 98 trials, 49 for each CF of the yellow and cream color.

Due to the limited forage available in the enclosure in all three experiments, only few foragers were active, and there was never more than one bee visiting the flowers at any one time. Before each trial, one flower of each plant that was included in the three sets of experiments was cut and the scanned image was used for measurement of petal size and its saturation value, as described below; the petal’s UV reflectance was also measured in the flowers of the second and third experiments.