New approaches to sap extraction from trees and comparison with conventional methods: new avenues for H and O stable isotopes ecohydrology
Authors: Montemagno Alessandro , Hissler Christophe , Bense Victor , Teuling Adriaan J. , Pfister Laurent 
Published: March 2022

&lt;p&gt;Since the `60s, stable isotopes of hydrogen and oxygen have proven to be excellent tracers for water flows in different systems. Nowadays, such tracers still represent a formidable resource for eco-hydrologists who study water spatio-temporal dynamics and transit time distributions in the Critical Zone (CZ). Unfortunately, many issues remain unsolved, especially when looking at the regolith-trees continuum in which a complex mixture of isotopic fractionation processes may occur (evaporation, transpiration, adsorption on soil clays and oxides, microorganism's activity, mixing of water of different ages). Moreover, the lack of standard protocols for sampling and analysis represents a limitation when determining which water source(s) trees uptake. Indeed, by using sap cryogenically extracted from tree cores (recognized as the standard protocol), several studies have indicated discrepancies between the isotopic signatures of xylem sap extracted from plants and the potential water source(s).&lt;/p&gt;&lt;p&gt;In this context, we propose to look at the water which flowing in the xylem vessels, by directly sampling sap from tree roots and branches. It is our hypothesis that root water would represent a more reliable fraction to identify the source(s) of water that trees absorbed from the different regolith compartments. Additionally, we also aim to observe how O and H isotopic composition of sap is evolving from the roots to the leaves. Inside this pathway, the absorbed water would be impacted by various processes, such as evaporation from the bark and mixing with other water pools (e.g. storage water), which would lead to a change in the isotopic composition of the sap which will be observed in the one extracted from the branches.&lt;/p&gt;&lt;p&gt;To reach our objectives, we sampled water from diverse CZ compartments at three European beech (&lt;em&gt;Fagus sylvatica&lt;/em&gt; L.) stands in the Weierbach Experimental Catchment (Hissler et al. 2021). These stands are located along a catena from the highest elevation (plateau) to the stream (riparian zone). Rainfall, throughfall, soil solutions at 20, 40 and 60 cm depth, groundwater and streamwater were collected. For beech sap sampling, we developed and applied an &lt;em&gt;in-situ&lt;/em&gt; extraction using suction under vacuum. This method allowed us to collect sap from roots, stems and branches, separately. At the same time, tree cores from the trunks were collected and stored at -20 &amp;#176;C before cryogenically extracting their water content under vacuum.&lt;/p&gt;&lt;p&gt;When reported in a &amp;#948;&lt;sup&gt;2&lt;/sup&gt;H vs. &amp;#948;&lt;sup&gt;18&lt;/sup&gt;O diagram, our results clearly illustrate that beech sap samples extracted with this techniques plot in a different area than the water extracted using cryogenic extraction. The root sap samples fall on the Local Meteoric Water Line (LMWL) in the same field as specific water sources (soil solutions, groundwater, streamwater). Moreover, the sap samples collected from branches are also located on the LMWL but presented a significant enrichment in &lt;sup&gt;18&lt;/sup&gt;O and &lt;sup&gt;2&lt;/sup&gt;H in comparison to the root sap samples. These new results allow us to calculate more accurately the contribution of the regolith water pools to the tree uptake and to discuss the origin of the fractionation that happened for both O and H isotopes in the tree water pathway.&lt;/p&gt;