DOI: 10.25338/b86358
title: Sunflower pollinator visit scoring

description: Biological rhythms are ubiquitous. They can be generated by circadian
 oscillators, which produce daily rhythms in physiology and behavior, as
 well as by developmental oscillator such as the segmentation clock, which
 produces modular developmental units in a periodic fashion. Here, we show
 that the circadian clock controls the timing of late-stage floret
 development, or anthesis, in domesticated sunflower. In these plants, what
 appears to be a single inflorescence consists of up to thousands of
 individual florets tightly packed onto a capitulum disk. While early
 floret development occurs continuously across capitula to generate iconic
 spiral phyllotaxy, during anthesis floret development occurs in discrete
 ring-like pseudowhorls with up to hundreds of florets undergoing
 simultaneous maturation. We demonstrate circadian regulation of floral
 organ growth and show that the effects of light on this process are
 time-of-day dependent. Disruption of circadian rhythms in floral organ
 development causes loss of pseudowhorl formation. Thus, we show that the
 sunflower circadian clock acts in concert with environmental response
 pathways to tightly synchronize the anthesis of hundreds of florets each
 day, generating spatial patterns on the developing capitulum disk. This
 coordinated mass release of floral rewards at predictable times of day
 likely promotes pollinator visits and plant reproductive success.
description: These experiments were conducted in Davis, CA, June-August, 2022. Seeds were germinated and grown for three weeks in PGV36  or PGR15 growth chambers (Conviron, Winnipeg, MB, Canada) as described above, at which point they were transferred to a field site. Approximately one week before the start of anthesis, plants were transferred to growth chambers. Controls were maintained in chambers with light, temperature, and humidity cycling in coordination with the local average daily forecast for the following week. Constant light plants were maintained at a constant 28°C, with 3200 μmol m<sup>-2</sup> s<sup>-1</sup> provided by metal halide and incandescent lamps. Jet lagged plants were entrained to the same conditions as the control plants but with a 3-hour phase delay. Plants were transferred to the field just before anthesis of the second or third pseudowhorl, just after dawn in the case of the constant light experiment and one hour before dawn for the jet lab experiment. In the latter case, florets that had opened on previous days were removed with forceps to limit the pollinator recruitment signals to florets developing on the day of the experiment. Pots were arranged so capitula faced east and stems were secured to bamboo poles. Images were taken at five-minute intervals using BirdCam 2.0 cameras (Wingscapes). Pollinator visits were scored using ImageGlass (https://imageglass.org/). Any insect large enough to be seen in the images was counted. New visits were counted when an insect landed on or changed location on the disk florets. The first image in which pollen was visible on the tip of the stamen was scored for time of pollen presentation. All data was plotted in R using ggplot and tidyverse.