Sampling and analytical procedures:
Clear Lake sediments were extracted using a Dionex Accelerated Solvent Extractor (ASE 200), and the resulting total lipid extract was separated into neutral and polar fractions using column chromatography with aminopropyl gel as the stationary phase. The neutral fraction was then separated into hydrocarbon, wax ester, sterol and polar fractions via a silica gel column chromatography. Dinosterol was isolated then from the sterol fraction via reverse phase (RP)- high-performance liquid chromatography (HPLC) .

Instruments:
Dinosterol was isolated from the sterol fraction via reverse phase (RP)- high performance liquid chromatography (HPLC). An Agilent 1100 HPLC with an integrated autoinjector, quaternary pump, and fraction collector was coupled to an Agilent 1100 LC/MSD SL mass spectrometer with a multimode source that was operated in positive atmospheric pressure chemical ionization (APCI+) mode. The HPLC method used is outlined in Nelson and Sachs (2013). Subsequently to HPLC separation the fraction containing dinosterol was analyzed via GC-MSD to verify sufficient baseline separation. Adjacent HPLC fractions were also analyzed to ensure that no dinosterol eluted into those fractions.

After the dinosterol was sufficiently isolated from co-eluting compounds, the sample was injected onto a GC-irms for determination of the d2H of dinosterol. Hydrogen isotope determinations were made using a Finnigan Delta V Plus Isotope Ratio Mass Spectrometer (irMS) coupled to a Thermo Trace GC Ultra with a Varian VF-17ms FactorFour capillary column (60 m x 0.32 mm x 0.25 m) and a pyrolysis reactor. Samples were injected into a split/splitless inlet in splitless mode at 310 C. The oven temperature was ramped from 100 C to 220 C at a rate of 20 C/min, then at 2 C /min up to 325 C where it was held for 17 min. The carrier gas, He, was held constant at 2.6 mL/min. The pyroloysis reactor was maintained at 1400 C. Isotope values, expressed as D values, were calculated in Isodat software relative to VSMOW using a co-injection standard containing nC28 nC32, nC40, and nC44 of known ∂2H values (obtained from Arndt Schimmelmann, Indiana University, Bloomington, IN, USA). The measured isotope values of dinosterol were corrected for the addition of hydrogen atoms (of known D value) that occurred during acetylation. Each sample was analyzed in at least duplicate, and error bars represent standard deviations of replicate measurements.