Methods:
Study Site
Observational data and test individuals were collected along the shoreline of Lake Michigan at Sturgeon Bay in Wilderness State Park, located in Emmett County, northwest lower Michigan. Field observations were completed June 30-August 4, during the peak flowering season of C. pitcheri. Eight-10x10m plots were placed 50m apart along the fore dunes of Sturgeon Bay, where subpopulations of C. pitcheri are well established. The separation between plots reduced the potential for Bombus dispersal among plots within a sampling period. Three flowering thistles (i.e., individuals featuring a flowering head with the entire whorl of open florets emerging all the way around the head) were systematically chosen along the diagonals of each plot to encompass a wide representation of flowering plants within each plot. To prevent observations of the same plots at the same times each day, individual plots were randomly selected as the starting point on a given day.
Field Observations
A total of 24 flowering plants were observed three times a day, four to five days a week for 5 weeks between 30 June and 4 August 2017. We measured how pollinator activity varies within a single day using 10 minute observations of a selected plant during three diurnal time intervals: Early (10:00-12:00), Mid (12:00-14:00) and Late day (14:00-16:00). The number of Bombus spp. visiting (i.e., landing on) the selected thistle was recorded to determine the frequency and composition of Bombus spp. pollinating Pitcher’s thistle. Whenever 1) A single an individual landed on an available flowering head, 2) an individual left a flowering head and returned, or 3) a pollinator visited another flowering head on the same plant it was classified as a single visit to an available flowering head of Pitcher’s thistle. Individuals of select species that are most common at the study site were selected for the thermal range experiment.
In order to measure how Bombus visitation varied with ambient temperature variation, iButton® temperature loggers were placed at or near flowering head height of each observed plant for the duration of data collection. Several abiotic factors were potentially confounded with diurnal changes in temperature and were recorded to reduce bias. Potential confounding variables included wind speed, available sunlight, relative humidity, and rainfall within the previous 24hr. To determine if the spatial distribution of Pitcher’s thistle was confounded with pollinator visitation, the numbers of available flowering heads on the observed plant and within the entire plot were also recorded. Regression analysis was used to estimate the correlation between time of day and number of visits. To estimate the difference between communities of bumblebees between each time period, Shannon’s diversity index was used to detect species richness and abundance. Evenness was also calculated. Correlation between time of day and temperature was analyzed.
Laboratory Thermal Experiment
Ten individual worker bumblebees of species common at Sturgeon Bay were collected for laboratory experiments designed to ask how high temperatures might affect bee motor function. The mass of each individual was measured before and after the experiment as a covariate to eliminate insect body mass as a possible confounding variable. Preliminary experiments tested the rate of temperature change within a falcon tube placed in a water bath mounted on a hot plate. An iButton® temperature logger was used to record air temperature changes within the tube. Individual bees were placed in a separate tube and allowed to acclimate to room temperature for 15 min. Temperature was raised at a rate of 0.50°C per minute (Oyen et al. 2016). The temperature at which individuals began to lose motor function was recorded as the critical thermal maximum. To control humidity, dryerite was placed within the jar. ANOVA analysis was used to compare mean critical thermal maximum for each species.