Recent studies using water isotopes have shown that trees and streams appear to return distinct water pools to the hydrosphere. Cryogenically extracted plant and soil water isotopic signatures diverge from the meteoric water lines, suggesting that plants would preferentially use bound soil water, while mobile soil water that infiltrates the soil recharges groundwater and feeds streamflow all plots on meteoric water lines. These findings have been described under the “two water worlds” (TWW) hypothesis. In spite of growing evidence for the TWW hypothesis, several questions remain unsolved within the scope of this framework. Here, we address the TWW as a null hypothesis and further assess the following: (a) the theoretical biophysical feasibility for two distinct water pools to exist, (b) plant and soil processes that could explain the different isotopic composition between the two water pools, and (c) methodological issues that could explain the divergent isotopic signatures. Moreover, we propose a way forward under the framework of the TWW hypothesis, proposing alternative perspectives and explanations, experiments to further test them, and methodological advances that could help illuminate this quest. We further highlight the need to improve our sampling resolution of plants and soils across time and space. We ultimately propose a set of key priorities for future research to improve our understanding of the ecohydrological processes controlling water flows through the soil–plant‐atmosphere continuum.

Abstract. For more than two decades, research groups in hydrology, ecology, soil science, and biogeochemistry have performed cryogenic water extractions (CWEs) for the analysis of δ2H and δ18O of soil water. Recent studies have shown that extraction conditions (time, temperature, and vacuum) along with physicochemical soil properties may affect extracted soil water isotope composition. Here we present results from the first worldwide round robin laboratory intercomparison. We test the null hypothesis that, with identical soils, standards, extraction protocols, and isotope analyses, cryogenic extractions across all laboratories are identical. Two standard soils with different physicochemical characteristics along with deionized (DI) reference water of known isotopic composition were shipped to 16 participating laboratories. Participants oven-dried and rewetted the soils to 8 and 20 % gravimetric water content (WC), using the deionized reference water. One batch of soil samples was extracted via predefined extraction conditions (time, temperature, and vacuum) identical to all laboratories; the second batch was extracted via conditions considered routine in the respective laboratory. All extracted water samples were analyzed for δ18O and δ2H by the lead laboratory (Global Institute for Water Security, GIWS, Saskatoon, Canada) using both a laser and an isotope ratio mass spectrometer (OA-ICOS and IRMS, respectively). We rejected the null hypothesis. Our results showed large differences in retrieved isotopic signatures among participating laboratories linked to soil type and soil water content with mean differences compared to the reference water ranging from +18.1 to −108.4 ‰ for δ2H and +11.8 to −14.9 ‰ for δ18O across all laboratories. In addition, differences were observed between OA-ICOS and IRMS isotope data. These were related to spectral interferences during OA-ICOS analysis that are especially problematic for the clayey loam soils used. While the types of cryogenic extraction lab construction varied from manifold systems to single chambers, no clear trends between system construction, applied extraction conditions, and extraction results were found. Rather, observed differences in the isotope data were influenced by interactions between multiple factors (soil type and properties, soil water content, system setup, extraction efficiency, extraction system leaks, and each lab's internal accuracy). Our results question the usefulness of cryogenic extraction as a standard for water extraction since results are not comparable across laboratories. This suggests that defining any sort of standard extraction procedure applicable across laboratories is challenging. Laboratories might have to establish calibration functions for their specific extraction system for each natural soil type, individually.

Recent studies using water-stable isotopes (δ 18 O and δ 2 H) have suggested an ecohydrological separation of water flowing to streams or recharging groundwater and water used by trees, known as the 'two water worlds' (TWW) hypothesis. In this study, we measured water isotopic composition in precipitation [open field and throughfall, i.e. local meteoric water line (LMWL)] and the mobile water compartment (i.e. stream and soil solution), bulk soil water and xylem water over a period of 1.5 years in two headwater catchments: NF, covered with old growth native evergreen forest (Aetoxicon punctatum, Laureliopsis philippiana and Eucriphya cordifolia), and EP, covered with 4 and 16-year-old Eucalyptus nitens stands. Our results show that precipitation, stream and soil solution plot approximately along the LMWL, while xylem waters from all studied tree species plot below the LMWL, supporting the TWW hypothesis. However, we also found evidence of ecohydrological connectivity during the wet season, likely controlled by the amount of antecedent precipitation. These observations hold for all investigated tree species. On both sites, a different precipitation source for stream and xylem water was observed. However, in EP, bulk soil showed a similar precipitation source as xylem water from both E. nitens stands. This suggests that E. nitens may use water that is recharging the bulk soil compartment. We conclude that under a rainy temperate climate, the TWW hypothesis is temporal and does not apply during wet seasons. Stream and soil solution water: mobile water compartmentStream water was collected fortnightly at NF and EP (n = 25 and 22, respectively). While δ 2 H showed differences (median: À38.0‰ and À36.4‰ for NF and EP, respectively; p < 0.001 using M-W), no differences 4231 ECOHYDROLOGICAL ASSESMENT

To date, reasons for the increase in liana abundance and biomass in the Neotropics are still unclear. One proposed hypothesis suggests that lianas, in comparison with trees, are more adaptable to drought conditions. Moreover, previous studies have assumed that lianas have a deeper root system, which provides access to deeper soil layers, thereby making them less susceptible to drought stress. The dual stable water isotope approach (δ18O and δ2H) enables below-ground vegetation competition for water to be studied. Based on the occurrence of a natural gradient in soil water isotopic signatures, with enriched signatures in shallow soil relative to deep soil, the origin of vegetation water sources can be derived. Our study was performed on canopy trees and lianas reaching canopy level in tropical forests of French Guiana. Our results show liana xylem water isotopic signatures to be enriched in heavy isotopes in comparison with those from trees, indicating differences in water source depths and a more superficial root activity for lianas during the dry season. This enables them to efficiently capture dry season precipitation. Our study does not support the liana deep root water extraction hypothesis. Additionally, we provide new insights into water competition between tropical canopy lianas and trees. Results suggest that this competition is mitigated during the dry season due to water resource partitioning.