Lipid extraction and quantification

Lipids were extracted from membranes as described (Bligh and Dyer, 1959). Samples were diluted to a final volume of 600 μl using deionized water, added to a 2:1 ratio of methanol and chloroform, and vortexed vigorously for 30 seconds. The mixture was centrifuged at 600 g for 10 minutes so that distinct layers formed. The lower layer was collected using a glass Pasteur pipette. The extraction procedure was repeated three times on each sample. Samples were washed with deionized water and evaporated under dry nitrogen gas in 2 ml borosilicate glass vials with Teflon-lined caps. Extracts were sent to the Kansas Lipidomics Research Center for analysis, and a diacyl polar lipid profile dataset was generated by quadrupole mass spectrometry using an Applied Biosystems 4000 QTRAP mass spectrometer as described (Xiao et al., 2010). The relative abundances of the major polar lipid classes were compared among species. The unsaturation index (UI) was calculated as described previously (Grim et al., 2010).