80-90% of angiosperms rely on pollinators for successful seed production. This requirement generates strong selection for floral traits that both attract pollinators and enable them to distinguish the flowers of one species from other co-occurring species and from the surrounding environment. Research that aims to identify visual cues used in pollinator decision-making requires an investigation of color, as it is perceived by pollinating insects. The retinal photoreceptors of bees allow them to perceive blue (430nm), green (535nm), and ultraviolet (340nm) light. Specifically, the spatial patterns in which flowers reflect and absorb ultraviolet (UV) light may affect the behavior of potential pollinators. I designed and implemented a novel photographic method to quantify variation in floral pigment and pattern and to measure color as it is percieved by pollinating insects. Here, I apply this method to Clarkia unguiculata(an annual wildflower species endemic to California). First, I modified a digital camera to report objective measurements of the amount of blue, green, and ultraviolet light reflected and absorbed by different petal parts as well as other metrics of pattern and I used this approach to describe ontogenetic variation in floral pigment in ~500+ individuals grown from field collected seeds under uniform greenhouse conditions.
In a pilot greenhouse study I grew 25 maternal families (2 siblings/family) of C.unguiculata. Individuals were grown from seeds collected from a single field population the previous year. I photographed 3-5 flowers per plant during 8 stages of floral development using a modified full spectrum camera and multiple lens filters. I adapted an existing image analysis plugin for ImageJ to merge ultraviolet and blue/green photographs and to normalize and linearize the images so that each multispectral image could be analyzed for multiple components of pigment and pattern. C.unguiculata is protandrous and herkogamous. One set of 4 anthers releases pollen first, followed by a second set of 4 anthers, and several days later the stigma becomes receptive. I found significant changes in floral pigment and pattern across these 3 gender stages. As flowers develop, petals become significantly larger, contrast in ultraviolet luminance increases and the nectar guides (UV patterns) become significantly more UV absorbent. These analyses demonstrate that there is a significant difference in floral pigment among floral ontogenetic stages in traits known to influence pollinator attraction. These results corroborate my prediction that floral stage is a significant source of variation in floral pigment within this species.