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Supplementary Appendix 1: Laboratory protocol for the extraction of alkaloids from Delphinium
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barbeyi and comparison of alkaloid identities and concentrations among different plant parts,
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including nectar
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Alkaloid extraction protocol
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We followed the protocol outlined in Cook et al. (2011) to extract alkaloids from the above-
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ground biomass of whole flowering plants of D. barbeyi. It is important to note that this protocol
7
is an acid-base extraction that only extracts alkaloids. Dried ground plant material (50 g) was
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extracted with methanol (1 L) for 24 hrs and filtered. The methanol extract was then evaporated
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under reduced pressure (roto-evaporation). The gummy residue was then washed successively
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with 1% sulfuric acid and chloroform, using a total volume of 75 mL each and added to a
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separatory funnel. After mixing, the chloroform layer was drained and the aqueous acid solution
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was washed an additional two times with 100 mL chloroform. The aqueous solution was cooled
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in an ice bath and the pH adjusted to 9 by addition of concentrated ammonium hydroxide
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solution. The aqueous solutions were then extracted three times with chloroform (100 mL, 80
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mL, 80 mL) in a separatory funnel. The combined extracts were dried over anhydrous sodium
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sulfate, filtered and then the solvent was evaporated under reduced pressure. The alkaloid
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extract was then rinsed with a small amount of chloroform and the solvent again evaporated
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under reduced pressure in a 100 mL round bottom flask, leaving the alkaloid mixture as an
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amorphous solid material at the bottom of the flask. The alkaloid extract was then scraped from
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the bottom of the flask.
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21
Comparison of alkaloid identities and concentrations
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Here we present published and new data showing that the relative abundance of individual
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alkaloids in nectar were qualitatively similar to those found in other plant tissues (Cook et al.,
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2013) and to the alkaloid extract that we used in this study (Table S1).
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Sample extraction and alkaloid analysis. Analytical samples were prepared from plant
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material collected near the RMBL, as outlined in Cook et al. (2013). Above-ground plant parts
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and the alkaloid extract were analyzed by electrospray mass spectrometry using procedures in
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Gardner et al. (1999). Nectar samples were analyzed using reverse-phase HPLC-esi(+) MS using
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procedures in Gardner and Pfister (2009). Mass spectra were recorded for each sample over a
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range of 150-800 m/z and averaged across all scans taken at 40% of peak height (total ion
31
current). The total amount of compound (as represented by a single mass unit) detected was
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calculated based on the relative abundance of the internal standard reserpine (MH+ = 609).
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Alkaloid concentrations were normalized to the internal reserpine standard and expressed as µg
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alkaloid/100 mg plant material. We then calculated the proportion of each protonated molecule
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in each tissue type for comparison (Table S1).
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37
Comparison of alkaloid identities and concentrations across plant parts, nectar, and the
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alkaloid extract. We found that our alkaloid extract contained similar dominant alkaloids and at
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qualitatively similar concentrations to those observed in different plant tissues and nectar (Table
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S1). It is important to note that the alkaloid extract was obtained from the above-ground biomass
41
of flowering plants, and so all tissues, including nectar, were represented in the material used to
2
42
create the extract. Some of the dominant alkaloids, such as deltaline and 12-acetlydictyocarpine,
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made up over 50% of the alkaloids expressed in the majority of plant parts, nectar, and the
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alkaloid extract. Alkaloids that made up more minor components in nectar where also more
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minor in most plant parts and in the alkaloid extract. Finally, there was one alkaloid that was
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absent from the nectar that was in the alkaloid extract, but it was at a relative abundance of <1%.
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48
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49
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