Analysis of nectar from low-volume flowers: A comparison of collection methods for free amino acids

Abstract

Floral nectar is a reward offered by flowering plants to visiting pollinators. Nectar chemistry is important for understanding plant nutrient allocation and plant-pollinator interactions. However, many plant species are difficult to sample as their flowers are small and produce low amounts of nectar.We compared the effects of different methods of nectar collection on the amino acid composition of flowers with low volumes of nectar. We used five methods to collect nectar from 60 (5 × 12) Calluna vulgaris flowers: microcapillary tubes, a low-volume flower rinse (the micro-rinse method, using 2 μl water), filter paper, a high-volume flower rinse (2 ml water) and a flower wash (2 ml water). We analysed the samples for free amino acids using quantitative UHPLC methods .We found that the micro-rinse method (rinsing the nectary with enough water to only cover the nectary) recovered amino acid proportions similar to raw nectar extracted using microcapillary tubes. The filter paper, 2 ml rinse and 2 ml wash methods measured significantly higher values of free amino acids and also altered the profile of amino acids. We discuss our concerns about the increased contamination risk of the filter paper and high-volume rinse and wash samples from dried nectar across the floral tissue (nectar unavailable to floral visitors), pollen, vascular fluid and cellular fluid.Our study will enable researchers to make informed decisions about nectar collection methods depending on their intended chemical analysis. These methods of sampling will enable researchers to examine a larger array of plant species' flowers to include those with low volumes of nectar.

Keywords: Calluna vulgaris; UHPLC; amino acid; nectar; pollinator; sampling method.

Figure 1

The natural log of the mean (±SE) of the total essential amino acid (EAA), non‐essential amino acid (non‐EAA) and proline concentration in nectar samples collected by five methods: MC, microcapillary; FP, filter paper; MR, micro‐rinse; R, rinse 2 ml; and W, wash 2 ml. Letters indicate significant differences (Sidak post hoc tests, p < .05) from the microcapillary treatment only. Subscripts indicate specific sets of comparisons; that is, “1” indicates comparisons of EAA across sample collection types, “2” indicates comparisons of non‐EAA, and “3” indicates comparisons of proline

Figure 2

Sampling method affected the proportions of amino acids found in samples from Calluna vulgaris. (a) The mean percentage contribution of the essential amino acids (EAA) to nectar samples collected by five methods (excluding tryptophan). (b) The mean non‐essential amino acids (non‐EAAs). (c) The mean percentage of proline and glutamic acid. These amino acids were plotted separately because they were orders of magnitude greater in concentration than all the others. MC, microcapillary; FP, filter paper; MR, micro‐rinse; R, rinse 2 ml and W, wash 2 ml

Figure 3

Box‐and‐whisker plots of each amino acid found in the samples from each sampling method. (a–e) Non‐essential amino acids, (f–j) essential amino acids. N = 12 samples/method