A subset of nectar from each sample was serially diluted in sterile sugar water, plated on YMA at final concentrations of 0.5 and 0.05 μL of nectar, incubated at 25 °C for 6 days, and colony forming units (CFUs) of each species counted. To further verify species identities, cell morphology from a subset of the colonies was examined at 20× magnification. In addition, 24 colonies were analysed using molecular methods (Peay et al. 2012). Briefly, DNA from individual colonies was extracted, the D1/D2 region amplified by PCR, and fragment patterns following digestion with restriction enzymes were assessed (Peay et al. 2012). Both cell structure and molecular sequencing indicated that species could be accurately identified based on colony morphology.
From the remaining nectar, we quantified both non-resource and resource chemical properties of nectar to assess the effects of yeast on the nectar environment. To quantify H2O2 concentration, we used a Peroxide Assay Kit for aqueous samples (Thermo Scientific, Rockford, IL, USA). Briefly, 2 μL of nectar or H2O2 standard solution was added to 100 μL of reaction solution and absorbance measured at 560 nm using a plate reader (TECAN, San Jose, CA, USA). To quantify pH, we applied 0.5 μL of nectar to each of three sections of a pH strip (EMD Millipore, Darmstadt, Germany). Remaining nectar was diluted 1 : 10 in diH20 for later analysis of sugars and amino acids and frozen at −80 °C until analyses could be completed.
To measure sucrose, glucose and fructose concentrations, samples were further diluted in 50 : 50 acetonitrile : water containing 0.5 mg mL−1 maltose (Sigma-Aldrich, St. Louis, MO, USA) as an internal standard. Sugars were then separated by UPLC (Waters, Milford, MA, USA) on a Luna amide column (50 × 2 mm, 3 μm, Phenomenex, Torrance, CA, USA). An acetonitrile : water (MeCN : H2O) mobile phase with a 4.5 min linear gradient at 170 μL min−1, beginning at 80 : 20 MeCN : H2O and ending at 30 : 70 MeCN : H2O was used, with a 10 min equilibration at initial conditions between samples. Mono- and disaccharides were quantified using an ELS Detector (Waters), and the concentration of sucrose, glucose and fructose in each sample was calculated using the internal standard and a series of external standards. Glucose and fructose were rarely detected in nectar samples, so we restrict our analyses to sucrose concentration. To quantify amino acids in individual nectar samples, diluted samples were prepared using an AccQ-Tag Kit (Waters) following the manufacturer's instructions. Briefly, 1 μL of derivatised sample was injected onto an AccQTag Ultra Column (2.1 × 100 mm) (Waters Corporation, Milford, MA, USA) at 43 °C using UPLC. Each gradient run was 10 min long, with a flow rate of 700 μL min−1 and began with an aqueous mobile phase with increasing concentration of organics, following Waters AccQTag Protocol for H-Class. Derivatised compounds were detected using UV absorbance at 260 nm, identified by comparing retention times of a series of known standards, and the concentration of each compound was calculated based on a series of external standards. All chemical analyses were performed on at least two of the four replicates for each treatment.