Water samples were analyzed for their stable isotopic compositions using Los Gatos DLT- 100 laser isotope analysers for Dorset and Wolf Creek, a Los Gatos Liquid Water Isotope Analyzer for Bruntland Burn and Dry Creek, and a Picarro L2130-I for Krycklan. The precision of the liquid water stable isotope analysis is reported to be better than ±0.1 ‰ for δ18O and ±0.4 ‰ for δ2 H. All isotope data are given in delta-notation in reference to the VSMOW. At all sites – apart from Dry Creek – direct water-vapor equilibration analysis was used to sample the bulk soil water isotopic composition from the soil . The accuracy of the direct water-vapor equilibration method was ±0.3 ‰ for δ18O and ±1.1 ‰ for δ2 H. For a detailed description of the procedure, we refer to Sprenger et al. . Bulk soil water isotopic compositions at DC were sampled using cryogenic extraction at 100°C under vacuum of < 30 millitorr over 40 minutes, as described by McCutcheon et al. . We are aware that different methods of soil water extraction have been a major focus of research in the past few years, with no definitive agreement on a standard method . While differences between cryogenic extraction and the direct water-vapor method have been reported in laboratory experiments ,container raspberries previous work by the authors has found the direct equilibrium method to be a reliable method for extracting bulk soil water from sandy soils giving similar results to cryogenic extraction .
Source water apportionment of plant xylem: To quantify the potential source of vegetation water from different soil depths and over a range of time periods, a modification of the ellipsoid method was utilized for the gymnosperms and angiosperms at soil depths in 10 cm increments up to 40 cm. All soil samples deeper than 40 cm were lumped together. The 40 cm cut off was chosen due to fewer sites sampling below 40 cm and a large decrease in the temporal resolution of sampling which could otherwise skew results. Due to soil water fractionation resulting in deviation from the local meteoric water line, the data are not well represented in an ellipsoid shape such as that employed in Amin et al. . Therefore a minimum polygon area was used to encompass the data points. Based on Equation 5, we derived the sw-excess of xylem isotope data. For sw-excess less than 0 ‰, the xylem data plot below the soil water line of the corresponding soil water isotopes sampled on the same day. Thus, the sw-excess can serve as an indicator for deuterium fractionation between the uptake time at the root-soil interface and the measured xylem water. We acknowledge that the soil water line is not necessarily solely a product of evaporative enrichment and that seasonal variability of the stable isotope compositions of the precipitation can affect how much soil waters deviate from the LMWL . However, the process of how the “soil water line” developed is not important here, since we used the regression to describe the isotopic compositions of potential water sources for vegetation at the time of sampling.
The influence of site characteristics on soil and xylem isotopic samples was evaluated using Spearman rank correlation. Site characteristics, mean annual temperature , elevation , aridity index , annual precipitation , and latitude , were correlated to xylem and soil water δ2 H , δ18O , and the corresponding sw-excess , and lc-excess to a significance level of 0.001. Isotopic compositions of all soil depths, vegetation species and sampling times were bulked for each site to assess an overall trend of soils and vegetation in relation to climate indices. Statistical analysis of isotopes in precipitation, bulk soil water, angiosperms, and gymnosperms was conducted at each site using the Wilcoxon signed-rank test to test the statistical similarities of median values of the datasets. This allowed for a two-sided probability test without the assumption of normality. The datasets were tested to the 95% confidence limit using all available data .Plant water and soil water data from the five sites are plotted in Figure 2. For both soils and xylem, the sites occupied partially overlapping regions showing a general gradient from highly isotopically depleted at Wolf Creek, the coldest of our sites in Canada, to the more isotopically enriched waters at Bruntland Burn at the temperate/boreal transition in Scotland. For each site there was a substantial range of variability in soil and xylem water isotope composition over the course of the sampling year. Most soil and xylem samples plotted below the GMWL, although xylem waters were generally more 2 H-depleted at each site, which was also evident from the lc-excess data . Samples from Dry Creek and, in particular, Wolf Creek showed the greatest divergence from the GMWL.
These two sites slightly obscured an otherwise clear relationship between plotting position along the GMWL and the mean annual temperature gradient through Krycklan, Dorset and Bruntland Burn. Despite this, the isotopic ratios of δ2 H and δ18O in soils and xylem water correlate positively with air temperature, annual precipitation and aridity index, and negatively with elevation and to a lesser extent latitude . At all sites, substantial isotopic differences were apparent between xylem and soil water isotopes, and between angiosperms and gymnosperms . Gymnosperms generally plotted further from the GMWL . Soil waters at each site generally tracked precipitation and snowmelt inputs being more 2 H- and 18O-depleted in winter/spring and more enriched in summer; evidence of evaporative fractionation was also most evident in the more 2 H- and 18O-enriched summer soil water samples. The soil water data are shown relative to the sampling dates for each site in Figures S2 to S6 in the Supplementary material; also see Sprenger et al. for more detail. Soil water δ2 H data were significantly different from precipitation at Dry Creek, Dorset and Wolf Creek, while soil water δ18O differed from precipitation at Bruntland Burn and Dorset . Bruntland Burn, Krycklan and Dorset showed the greatest visual deviation of xylem δ2 H samples from soil water, while the most southern site, Dry Creek, and the most northern site, Wolf Creek, showed smaller differences between the xylem and soil water isotopes for δ2 H . However, at all sites the δ2 H characteristics of both angiosperms and gymnosperms were significantly different from soil water . Angiosperm xylem water δ18O at all sites, apart from Krycklan, was significantly different from soil water δ18O; whereas significant differences for gymnosperms were apparent only for Dorset and Bruntland Burn. Xylem water isotopic characteristics differed between angiosperms and gymnosperms at some sites. For δ2 H, they were significantly different for Krycklan, Bruntland Burn and Dry Creek, while for δ18O they were different for Dorset and Dry Creek . Snowmelt plotted on the LMWL and was more depleted for 18O than almost all measured soil and xylem waters,draining pots although a substantial number of xylem samples at Dry Creek, Krycklan and Wolf Creek were more depleted in 2 H but plotted off the LMWL . Similarly, at the four sites where groundwater samples were collected, the mean isotopic composition of groundwater fell on the LMWL but plotted towards the more depleted end of the range of soil water samples. This reflects the generally higher recharge of groundwater by depleted water following the spring melt at Dry Creek, Krycklan, and Wolf Creek ; and during winter rainfall at Bruntland Burn . Isotopic composition of groundwater at all sites showed limited temporal variation, indicating the volume of annual recharge is small relative to groundwater storage. Groundwater was generally more strongly depleted in 18O than xylem waters for both angiosperms and gymnosperms, although at each site a substantial proportion of xylem samples were more depleted in 2 H.The minimum boundary polygon analysis quantifies the degree to which xylem water for both angiosperms and gymnosperms overlaps bulk soil water sources at different depths. The use of the spatially bulked data for soils and vegetation at each site was necessary to provide a sufficient number of samples for the development of encompassing polygons.
This may lead to larger estimated polygon areas and a greater estimated overlap of soil and xylem water, although the effect is much less marked than for the ellipse method of Amin et al. . This may provide insight into the sources of xylem water, although the proportion that cannot be ascribed to soil water sources is equally informative regarding the need to hypothesise and identify other causal reasons. Distinct inter-site differences emerged in terms of the overall overlap of xylem and soil water isotopic composition . For Bruntland Burn, soil water had a 77% overlap for angiosperms, but only 6% for Gymnosperms. At Dorset, like Bruntland Burn, angiosperms showed a much higher degree of overlap than gymnosperms . At Dry Creek, almost all xylem water in both angiosperms and gymnosperms overlapped soil water at almost all profile depths. Of all sites, Krycklan had the lowest degree of overlap with only 27% for angiosperms and 0% of gymnosperms . Finally, while Wolf Creek had only angiosperms present as willow and birch shrubs, a 99% overlap between xylem water and soil water was evident. The depth dependent overlap of xylem and soil water isotopic composition showed differences between depths, with higher overlap tending to be in shallow soil depths for most sites. For Bruntland Burn, there was 72% and 55% overlap between angiosperms and soil water at 0-10 cm and 10-20 cm depths, respectively, but only 9% and 3% for gymnosperms. Dorset was the only site with the greatest overlap occurring in deeper soil, with overlaps of 34%, 28%, 24%, 59% and 31% for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. The gymnosperms at Dorset had a similar deviation to that of angiosperms, with much smaller overlaps of 4%, 7%, 8%, 18%, and 7%, for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. Depth-dependent overlap of soil and angiosperms at Dry Creek was high through all soil layers with the greatest overlap in the near-surface soils . Gymnosperms at Dry Creek had a similarly high overlap of 78%, 55%, 86%, 72% and 86 % for 0-10, 10-20, 20-30, 30-40, and >40cm, respectively. At Krycklan, the upper two soil depths had approximately the same overlap for angiosperms , with a moderate decrease to 12% in the 20-30 cm soils. None of the soil water at any depth overlapped the gymnosperm samples. Wolf Creek angiosperms showed a high overlap in the upper two soil depths with a more substantial decrease in deeper soils .The general patterns of the pooled data sets for the entire study year mask differences in the degree to which seasonal variations in the isotopic composition of xylem water can be ascribed to soil water data collected on the same day or integrated over increasing monthly time windows to capture antecedent conditions. However, as described in section 2.2, soil water boundary polygons for increased averaging periods can also be calculated to estimate the overlap relative to xylem. Depth dependent overlaps are shown in Figure S7 and Figure S8, respectively. At Bruntland Burn, a longer time window of soil water isotopes explained a greater degree of variation in xylem water isotopic composition for angiosperms . Bulked soil water samples collected on the same day provided 80% and 87% of overlap in spring and autumn, respectively, but only 4% in summer. Increasing this window to 3 months increased overlap to 90%, 38% and 87% in spring, summer, and autumn, respectively. The spring and summer bulked soil and xylem water overlap increased to 100% and 58%, respectively, with a 6 month window. For gymnosperms, same day sampling provided no overlap in spring and summer, and only 7% in autumn . For a 3 month window, overlap increased to 20% in spring, but only 3% in summer and 7% in autumn. For a 6 month window, the autumn overlap increased to 13%. There were marked seasonal differences between angiosperms and gymnosperms at Dorset. For angiosperms, bulked soil and xylem water overlapped for same day sampling 100% in spring, 0% in summer and 20% in autumn . This increased to 20% in summer for a 3 month averaging window and 47% in summer for a 6 month average. The overlaps were much lower for gymnosperms; same day sampling showed bulked soil and xylem water overlaps of only 13% in spring, 2% in summer and 7% in autumn .