Pigment extraction and composition analysis
In order to determine the type of pigments produced in the
flowers of each species and their modifications during pollination
shifts, the three types of petals (dorsal, lateral and ventral) were
separated from the rest of the flower and dried in silica gel.
Pigments were extracted from petal samples (20 mg) with an
extraction buffer (MeOH/H2O/AcAc) and later treated with HCl 2N
at 100 ◦C for 30 min (Shimada et al., 2005). Samples dissolved on
the same extraction buffer were injected via infusion into an LC
Agilent 1100 series light chromatograph (LC-MS) with a LC7MSD
trap XCT Plus. Two replicates from different individuals were
analyzed for the focal species (L. sessilifolius and L. berthelotii) in
order to assess individual variation, but the remaining species
were analyzed only in one individual, as the individual variation
in pigment composition was minimal.