Tracing D2O in xylem tissues
To examine and track the dynamics of water uptake, transport and storage in trees of the two species, we used a marker technique with isotopically labelled water similar to that described by James et al. (2003) and Meinzer et al. (2006) We injected deuterium oxide (99.9 atom% D2O, Cambridge Isotope Laboratories, Inc.) into the xylem transpiration stream after having drilled four holes at equidistant points (90° apart) around the trunk circumference at 130 cm height. The dosage was kept constant at ≈0.7 g D2O per cm of sapwood circumference. This assay was performed once during a dry season (spring 2013) and once after a winter rain (winter 2014) to examine the dynamics of water transport and storage in trees. Deuterium injections in three pine trees per plot of pure and mixed stands (total = 24 injected trees) (δD = 366‰; i.e. 5.6 times enriched compared to natural abundance of deuterium) occurred in spring 2013 (March 28 between 11:00 h and 13:30 h). Also, in winter 2014 (February 21 between 10:00 h and 14:00 h), injections of δD = 503‰ (17.1 times enriched compared to natural abundance of deuterium) deuterium was applied in each of four pine and oak trees per plot in pure and mixed stands (total = 48 injected trees). The presence of D2O tracer in stem water was determined by periodic collection of twigs oriented N, S, W, E in the tree crown. Twigs (3 cm long) were immediately sealed in borosilicate (VWR®) and then wrapped with Parafilm to prevent evaporation. Prior to D2O injections stem samples were collected to establish the baseline for hydrogen isotope ratios. After the injection of D2O (day 0), twigs (total 720 samples) and wood cores (total 216 samples) were collected at noon on the following 10 days (for water extraction method and stable isotope analysis and calculations see below).