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. The hydrogen isotope composition of leaf-wax-derived biomarkers, e.g., long-chain n-alkanes (δ2Hn-alkane), is widely applied inpaleoclimate. However, a direct reconstruction of the isotope composition ofsource water based on δ2Hn-alkane alone ischallenging due to the enrichment of heavy isotopes during evaporation. Thecoupling of δ2Hn-alkane with δ18O ofhemicellulose-derived sugars (δ18Osugar) has the potentialto disentangle this limitation and additionally to allow relative humidityreconstructions. Here, we present δ2Hn-alkane as wellas δ18Osugar results obtained from leaves of Eucalyptus globulus, Vicia faba, andBrassica oleracea, which grew under controlled conditions. We addressed the questions of (i) whether δ2Hn-alkane and δ18Osugar values allow reconstructions of leaf water isotopecomposition, (ii) how accurately the reconstructed leaf water isotopecomposition enables relative humidity (RH) reconstruction, and (iii) whether thecoupling of δ2Hn-alkane and δ18Osugar enables a robust source water calculation. For all investigated species, the n-alkane n-C29 was most abundant andtherefore used for compound-specific δ2H measurements. ForVicia faba, additionally the δ2H values of n-C31 could be evaluatedrobustly. Regarding hemicellulose-derived monosaccharides, arabinose andxylose were most abundant, and their δ18O values were thereforeused to calculate weighted mean leaf δ18Osugar values.Both δ2Hn-alkane and δ18Osugaryielded significant correlations with δ2Hleaf water andδ18Oleaf water, respectively (r2=0.45 and 0.85,respectively; p&lt;0.001, n=24). Mean fractionation factorsbetween biomarkers and leaf water were found to be −156 ‰(ranging from −133 ‰ to −192 ‰) for εn-alkane/leaf water and +27.3 ‰ (rangingfrom +23.0 ‰ to 32.3 ‰) for εsugar/leaf water, respectively. Modeled RHair values from aCraig–Gordon model using measured Tair, δ2Hleaf waterand δ18Oleaf water as input correlate highly significantlywith modeled RHair values (R2=0.84, p&lt;0.001, RMSE = 6 %). When coupling δ2Hn-alkane and δ18Osugar values, the correlation of modeled RHair valueswith measured RHair values is weaker but still highly significant, withR2=0.54 (p&lt;0.001, RMSE = 10 %). Finally, thereconstructed source water isotope composition (δ2Hs andδ18Os) as calculated from our coupled approach matches thesource water in the climate chamber experiment (δ2Htank water and δ18Otank water). Thishighlights the great potential of the coupled δ2Hn-alkane–δ18Osugar paleohygrometerapproach for paleoclimate and relative humidity reconstructions.

biogeosciences

Validation of a coupled &amp;lt;i&amp;gt;δ&amp;lt;/i&amp;gt;&amp;lt;sup&amp;gt;2&amp;lt;/sup&amp;gt;H&amp;lt;sub&amp;gt;&amp;lt;i&amp;gt;n&amp;lt;/i&amp;gt;-alkane&amp;lt;/sub&amp;gt;–&amp;lt;i&amp;gt;δ&amp;lt;/i&amp;gt;&amp;lt;sup&amp;gt;18&amp;lt;/sup&amp;gt;O&amp;lt;sub&amp;gt;sugar&amp;lt;/sub&amp;gt; paleohygrometer approach based on a climate chamber experiment