Nectar methods

Nectar sampling - The nectar is extracted with calibrated microcapillaries (1 or 2 µl). If nectar quantity is too small or nectar viscosity prevents flow in the capillary, suction with a flexible aspiratory tube attached to the capillary is applied. The length of the nectar column in the calibrated capillary is measured for volume calculations. The capillaries with nectar of individual flowers are stored in a cooling bag in 1.5 ml tubes marked with the corresponding sample number. The following parameters are recorded for each nectar sample: The species and individual it was taken from; flower age and flowering stage; the time (in intervals of 15 min); sexual phase or phenotype of the flower; petal movements (open flowers were distinguished from closed ones); and length of the nectar column in the calibrated capillary. 

Nectar storing - After the end of the sampling session, the capillaries are emptied and the whole nectar of each flower is transferred into a new tube filled with 100 µl of ethanol (70%). For this purpose, a flexible tube is attached to the capillary and the nectar column is blown out into the alcohol-filled tube. Repeated rinsing guarantes that even dried nectar components are transferred. The prepared single flower samples are frozen and later analyzed by High Performance Liquid Chromatography (HPLC). For analysis of sugar content some randomly chosen nectar samples of each species are dried with a centrifugal vacuum concentrator, dissolved again (in 100 µl methanol to remove additive volatiles), dried again and dissolved in 100 µl water. Each flower's nectar is analyzed separately. 

Analysis of nectar sugar composition via HPLC - Samples are analyzed with HPLC equipment from Waters (in cooperation the Department of Systematic Botany and Ecology at the University of Ulm). We use a Waters High Performance Carbohydrate Column. Elution takes place with an acetonitrile-water-mixture (72:28); the flow rate is 1.4 ml/min, and temperature is 35 °C. Sugars are detected with a refraction index detector 410 and quantified with the Millennium software from Waters.

Because quantitative nectar extraction with capillaries implied destruction of flowers it is not possible to measure nectar secretion rates directly with this method. Alternatively we use captured moths for depleting flowers. During the day when the moths are inactive, they are kept in cages. During the night, beginning with flight activity, the moths are allowed to visit flowers in order to suck nectar, thus depleting the flowers. Each flower visited is marked, and about two hours later newly secreted nectar is extracted with a capillary and mean nectar secretion rates are calculated.