Abstract. Gram-negative bacteria produce specific membrane lipids, i.e. 3-hydroxy fatty acids with 10 to 18 C atoms. They have been recently proposed as temperature and pH proxies in terrestrial settings. Nevertheless, the existing correlations between pH or temperature and indices derived from 3-OH FA distribution are based on a small soil dataset (ca. 70 samples) and only applicable regionally. The aim of this study was to investigate the applicability of 3-OH FAs as mean annual air temperature (MAAT) and pH proxies at the global level. This was achieved using an extended soil dataset of 168 topsoils distributed worldwide, covering a wide range of temperatures (5 to 30 ∘C) and pH (3 to 8). The response of 3-OH FAs to temperature and pH was compared to that of established branched glycerol dialkyl glycerol tetraether (GDGT)-based proxies (MBT'5Me/CBT). Strong linear relationships between 3-OH-FA-derived indices (RAN15, RAN17 and RIAN) and MAAT or pH could only be obtained locally for some of the individual transects. This suggests that these indices cannot be used as palaeoproxies at the global scale using simple linear regression models, in contrast with the MBT'5Me and CBT. However, strong global correlations between 3-OH FA relative abundances and MAAT or pH were shown by using other algorithms (multiple linear regression, k-NN and random forest models). The applicability of the three aforementioned models for palaeotemperature reconstruction was tested and compared with the MAAT record from a Chinese speleothem. The calibration based on the random forest model appeared to be the most robust. It generally showed similar trends with previously available records and highlighted known climatic events poorly visible when using local 3-OH FA calibrations. Altogether, these results demonstrate the potential of 3-OH FAs as palaeoproxies in terrestrial settings.

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are a family of bacterial lipids widely used for temperature and pH reconstructions in terrestrial settings. 3-hydroxy fatty acids (3-OH FAs) with 10 to 18 carbon atoms, produced by Gram-negative bacteria, have been recently proposed as independent and complementary proxies of temperature and pH in terrestrial environments. Nevertheless, the correlations between mean annual air temperature (MAAT)/pH and bacterial lipid (brGDGTs/3-OH FAs) distribution show a large degree of scatter, as the relative abundance of these lipids is influenced by factors other than temperature and pH. A full understanding of the environmental parameters influencing bacterial lipid distribution in soils is required to increase the reliability of the temperature and pH proxies based on these compounds in terrestrial environments. The aim of this work was to determine and quantify the cumulative effect of environmental parameters on the distribution of both brGDGTs and 3-OH FAs along a well-documented composite altitudinal transect in the French Alps (234-2,748 m). Redundancy analysis revealed that the influence of local parameters (pH and to a lesser extent soil moisture and grain size, related to vegetation and soil types) on brGDGT and 3-OH FA distribution amounted to 48.1% and 26.1%, respectively, and was predominant over MAAT. This likely explained the weak or lack of relationships between MAAT and brGDGT-/3-OH FA-based indices in this region. The identification of lipids whose fractional abundance is correlated with MAAT or pH allowed the development of local calibrations with MAAT/pH applicable in the French Alps which are representative of highly contrasted microenvironments, reflecting different types of soil and vegetation. The present study highlighted the importance of constraining the environmental factors affecting the  Corresponding author.

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