We analyzed 382 samples from IODP site U1337, yielding an average resolution of 20 to 40 ka over the past 8 Ma, based on δ18O stratigraphy (47). Sediment samples (~5 to 20 g of dry weight) were grounded, freeze-dried, and extracted with organic solvents (dichloromethane/methanol = 9:1, v/v). After being saponified with 6% KOH in methanol solution, lipids were separated into three fractions with silica gel column chromatography. The alkenone fraction was analyzed on Agilent 7890 GC equipped with a flame ionization detector and an HP DB-1 capillary column. n-C36 alkane was used as external standard for alkenone quantifications. Analytical precision (1σ) for our laboratory standards is 0.005 unit (equivalent to 0.1° to 0.2°C) for the alkenone unsaturation index, UK′37 = [C37:2]/([C37:2] + [C37:3]), where [C37:2] and [C37:3] are contents of di- and tri-unsaturated C37 alkenones, respectively, and 5% for alkenone content. The global core-top temperature calibration (32) (UK′37 = 0.033T + 0.044) was used to convert the index into annual mean SST values. Other available alkenone records from the region, if different calibration equations were used previously, were also recalibrated using the same equation (32). As seasonal variability in primary production was very weak in the open-ocean EEP (48), we expected minimal seasonal bias in the alkenone proxy, representing annual mean SSTs in the region.

Analysis of replicated samples indicated that analytical precision is equivalent to ~0.2°C. We noted that UK′37 values from U1337 Pliocene/late Miocene samples, similar to other alkenone records from the eastern Pacific (17, 2226), were at high end (0.93 to 0.99) but had not yet reached unity over the past 8 Ma. Because of characteristically small C37:3 peaks, we expected uncertainty in C37:3 quantification in these samples. However, even with a 20% uncertainty (a substantial overestimate) in C37:3 quantification, UK′37 values in such samples (for example, UK′37 = 0.95) would change only by 0.01 unit, equivalent to 0.3°C. We thus assessed that, although UK′37 values approaching unity may not accurately reflect their absolute temperatures, their relative SST changes were still robust. An alternative BAYSPLINE alkenone temperature calibration (34) yielded warmer SSTs by up to ~2°C toward the UK′37 unity (with a notably greater 1σ error of ~4°C), but the structure of SST changes remained (fig. S4).

To investigate the possibility of potential capping of the signal between 3 and 8 Ma ago due to the upper limit of the UK′37 thermometer, we isolated higher-frequency variations in the signal, predominantly associated with glacial cycles and noise, by subtracting a five-point running mean and a five-point weighted mean from the record. Analyzing the resulting high-frequency anomalies showed that there was no discernible difference between temperature increases and decreases, i.e., the distribution of anomalies was nearly Gaussian with close to zero skewness (fig. S5). A negative skewness would have implied a lower probability of temperature increases or “capping.”

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