We prepared the D2O tracer solution by mixing 1 kg of D2O (purity 99.9%; Wako Co., Osaka, Japan) in 9 kg of tap water collected at Hokkaido University (Sapporo, Hokkaido, Japan) in 2014. We expressed the δD (D/H) ratio as the ‰ deviation from the Vienna Standard Mean Ocean Water (VSMOW) reference. The δD value of the mixture was 6 × 105 ‰. On 12 March 2015, at the Utsunomiya site, we spread 1.17 L of the solution on the ground surface surrounding each test tree in a 10-cm-wide doughnut shape that began 50 cm from the base of each test tree. This amounted to 2.87 L m−2. We installed soil water extraction devices at the center of a triangle formed by the three test trees (Supplementary Fig. S1b) and spread 1.5 L of the D2O solution on the surface surrounding these devices. This amounted to 3.57 L m−2. We collected water samples simultaneously at five depths during overnight operation of the vacuum pump. We periodically collected soil water to identify the peak D2O from the positive δD signal and to estimate the downward flow rate of soil water once every month from March to August 2015.

The Abiko study lasted 5 months (from 3 July to 3 December 2019) to observe rapid D2O uptake via the roots. We prepared the D2O tracer solution by mixing 0.975 kg of D2O (purity 99.9%; Wako Co., Osaka, Japan) in 8.37 kg of tap water; the δD of the mixture was estimated to be 6 × 105 ‰. We homogeneously spread total 6.5 L of the D2O tracer at a distance of 50 cm from the base of the two tree stems. This amounted to 7.39 L m−2. We sprayed the D2O solution around the base of each tree in a 10-cm-wide doughnut shape. We homogeneously sprayed the remaining total D2O solution 5.7 L in nine 20-cm × 20-cm quadrats (7.92 L m−2) that formed a straight 7-m-long line southeast of one test tree to observe downward infiltration of the D2O tracer at 1, 3, 7, 14, 21, 39, 67, 108, and 154 days after application of the tracer. We collected two 11-cm xylem samples from the sapwood (excluding the inner bark) to the heartwood one each on the north and south sides of the stem from both trees and sampled the soil at depths of 0–5, 5 to 10, 10–15, 15-20, and 25–30 cm to measure the tracer D2O concentration in soil water.

In Japan, precipitation isotopic ratios (δD and δ18O) vary seasonally because the origin of atmospheric water differs between summer and winter (Supplementary Fig. S5). The summer precipitation consists of moisture transported from the Pacific Ocean, so the rainwater is enriched in heavy isotopes3941. In contrast, continental dry air masses from Asia cross the Sea of Japan during winter and are associated with low temperatures and strong winds, so the evaporated moisture is enriched in light isotopes39,41. Consequently, the hydrogen and oxygen isotopes in Japanese rain and snow have a higher proportion of lighter isotopes in winter than in summer. This large natural seasonal variation in the isotopic ratio can be used to trace the origin of xylem sap in trees. We used the variation in δ18O values as a natural tracer, which provides a suitable comparison with the δD values because fractionation-induced changes in δ18O values are negligible during the root uptake of soil water1822.

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