Co-evolution of xylem water and soil water stable isotopic composition in a northern mixed forest biome

Plant–soil water isotopic dynamics in northern forests have been understudied relative to other forest types; nevertheless, such information can provide insight into how such forests may respond to hydroclimatic change. This study examines the co-evolution of xylem water and soil water stable isotopic compositions in a northern mixed forest in Ontario, Canada. Gross precipitation, bulk soil water and xylem water were sampled from pre-leaf out to postsenescence in 2016 for eastern white cedar, eastern hemlock, red oak and eastern white pine. Near-bole soil water contents and mobile soil water isotopic compositions were measured for the last three species. Mobile soil water did not deviate significantly from the local meteoric water line (LMWL). In contrast, near-surface bulk soil water showed significant evaporative enrichment relative to the LMWL from pre-leaf out to peak leaf out under all tree canopies, while xylem water was significantly depleted in 18O and particularly 2H relative to bulk soil water throughout the growing season. Inter-species differences in deviation of xylem water from the LMWL and their temporal changes emerged during the growing season, with coniferous species xylem water becoming isotopically enriched, while that of red oak became more depleted in 2H and 18O. These divergences occurred despite thin soil cover (generally < 0.5 m depth to bedrock) which would constrain inter-species differences in tree rooting depths in this landscape. Isotopic fractionation at the tree root and fractionation of xylem water via evaporation through the tree bark are among the most plausible potential explanations for deviations between xylem and soil water isotopic compositions. Differences in the timing and intensity of water use between deciduous and coniferous trees may account for inter-specific variations in xylem water isotopic composition and its temporal evolution during the growing season in this northern forest landscape.

123 a few cm above or below the preceding core. Bark was removed from retrieved cores which were immediately stored 124 in 200 mL glass scintillation vials with zero headspace. These were taped, sealed with Parafilm, and stored in a freezer 125 to prevent exchange with the atmosphere. Elapsed time between core extraction and storage in the sealed vials was on 126 the order of 1 minute. 127

Soil water isotopic sampling and soil water content 128
Bulk soil samples were obtained concurrent with xylem water sampling in a randomized direction 1 m from the bole 129 of each tree sampled for xylem water. Following litter layer removal, a minimum of 40 g of soil was collected using 130 an auger at 5 cm depth increments until bedrock was reached. An average of six samples was obtained at a given tree, 131 ranging from one to 16 samples. Samples were double bagged in Ziploc bags while minimizing any stored air and 132 stored at 4°C prior to analysis. Samples were stored for no more than 2 weeks prior to analysis, and Hendry et al. 133 (2015) indicated that any water losses and resulting changes in soil water isotopic content for these short storage 134 6 periods would be negligible. This bulk soil water was assumed to represent all water stored within the soil, including 135 both mobile and more tightly held soil water. 136 Mobile soil water was sampled from tension lysimeters installed at 0.1 and 0.4 m depths at 0.1 and 1 m from the 137 tree bole in a randomized direction for three He, three Or and three Pw trees sampled for xylem water (Snelgrove et 138 al. 2019). Tension lysimeters were manufactured using Soil Test 2 bar ceramic cups and PVC tubing. Tension 139 lysimeters were sampled weekly between June 2 and October 21, 2016 and re-set to a minimum negative air pressure 140 of 60 kPa using a hand pump. Samples were stored in sealed glass vials with zero headspace at 4°C prior to isotopic 141 analysis. 142 Soil water content (SWC) was measured at two ATL-1 access tubes (http://www.delta-t.co.uk , last accessed May 143 30, 2019) installed 0.1 and 1 m from the bole of each of the three trees of a given species sampled for mobile soil 144 water. Tubes were installed in a randomized direction from the bole. Measurements were concurrent with tension 145 lysimeter sampling. A Delta T PR2/6 Soil Moisture Profile Probe  measured SWC at each access tubes at 0.  Wilks tests were used to assess normality of xylem water lc-excess values for each sampling period and species. One-181 way ANOVAs were used to compare differences in xylem water lc-excess between sampling periods for each tree 182 species. Levene's test confirmed homogeneity of variances. Tukey HSD tests identified significant differences in the 183 data for each tree species. Inter-specific differences in xylem water lc-excess for a given sampling period were 184 assessed using t-tests (unequal variances, Bonferroni-corrected). Successive sampling of the same trees meant that the 185 isotopic composition of xylem water on a given sampling date was partly dependent on that from the previous 186 sampling. Nevertheless, our approach allowed us to examine the temporal trajectory of xylem water for each of the 187 sampled trees. We feel this is preferable to deriving this trajectory by sampling different trees at different times, which 188 could be influenced by inter-tree differences in xylem water isotopic composition on a given sampling date. Greatest variability in SWC was seen around Or trees, while the least was around He trees. 207 Most bulk soil water samples also plotted along the LMWL. However, the best-fit line of  2 H vs.  18 O for bulk 222 soil water had a slope of 5.3, shallower than for meteoric water (7.0, Eq. 2), indicating evaporative enrichment of 223 some samples. Enrichment was most pronounced at peak leaf out (Fig. 2c). It is important to note the poor agreement 224 between bulk soil water and mobile soil water lc-excess sampled within two days or less of one another for a given 225 tree species (Fig. 2d). Bulk soil water tended to be evaporatively enriched (more negative lc-excess) relative to mobile 226 soil water sampled on or close to the same day. 227

Isotope results
Bulk soil water lc-excess values showed broadly similar distributions with depth beneath all tree species (Fig. 3).

Xylem water 238
Xylem water isotopic composition changed during the growing season, with the trajectory of this change differing 239 between species (Fig. 4) 12 trajectory: both 2 H and 18 O became depleted from pre-leaf out to peak leaf out, followed by slight depletion of 2 H from peak 257 leaf out to pre-senescence, and slight depletion of 18 O from pre-senescence to post-senescence 2016. 258 Inter-specific differences and temporal changes in xylem water isotopic composition at PC-1 exceeded inter-tree 259 differences for a given species. Standard errors for xylem water for a given species and sampling date ranged from 0.39 to 260 5.13 ‰ for  2 H and 0.06 to 0.79 ‰ for  18 O and were similar to previously reported results. Retzlaff  There was little overlap of xylem water and bulk soil water in dual isotope space (Fig. A2), with the former having much 270 more negative  2 H and to a lesser extent  18 O relative to bulk soil water. There were pronounced inter-species differences in 271 xylem-water lc-excess values and their relationship with near-surface soil water (Fig. 5). Bulk soil water lc-excess at 0-5 cm 272 depth is shown, since near-surface soil experienced the greatest evaporative enrichment and thus the most negative lc-excess. 273 Xylem water lc-excess values for a given sampling period and tree species were normally distributed. One-way ANOVA 274 indicated no significant difference in xylem water lc-excess between sampling periods for He; conversely, other species 275 showed significant differences between some sampling periods. There were no significant inter-species differences in xylem 276 water lc-excess post-senescence 2015; however, distinctions emerged during subsequent sampling periods (Fig. 5, Table 2). 277 Lc-excess for Or xylem water was less negative compared to other species and showed considerable overlap between sampling 278 periods, with the most negative values at pre-senescence. They also often overlapped near-surface soil water lc-excess. Lc-279 excess for He was similar for all sampling periods, although inter-tree variability declined progressively from pre-leaf out to 280 pre-senescence. There was occasional overlap of xylem water and near-surface bulk soil water lc-excess values. A different 281 relationship occurred for Ce and to a lesser extent Pw. Xylem water lc-excess for the former became more negative from post-282 senescence 2015 to peak leaf out and then became more positive. Lc-excess for Ce was generally more negative than for other 283 species and was often more negative than the most evaporatively-enriched bulk soil water. Lc-excess for Pw also declined 284 from post-senescence 2015, becoming most negative at pre-senescence. Pw lc-excess also tended to fall outside the near-285 surface bulk soil water range, although there was more overlap than for Ce xylem water. 286 Figure 6 presents soil waterxylem water offsets for  2 H throughout the study period, defined as the difference between 287 the mean isotopic composition of soil water surrounding a sampled tree and xylem water for that tree. Offsets for  18 O showed 288 similar patterns to those for  2 H and are not shown. Intra-species differences in  2 H offsets on a given sampling date could be 289  appreciable; however, inter-tree differences for a given species between sampling times did not appear to be 303 consistent. Temporal trajectories of these offsets showed inter-specific differences. Minimum offset values for Ce 304 occurred at post-senescence in 2015. Values rose to maxima either at post-leaf out or peak leaf out before declining 305 to post-senescence 2016. Offsets were more temporally-constant for He with maxima at peak leaf out. There was a 306 marked decline in the Or  2 H offsets from 2015 post-senescence to pre-leaf out, followed by a gradual increase to 307 maxima at either pre-senescence or 2016 post-senescence. Pw had more temporally constant  2 H offsets with 308 minima at either pre-leaf out or post-leaf out. 309 4 Discussion 310

Temporal changes in the isotopic composition of soil water and xylem water in northern forests 311
Bulk soil water isotopic composition exhibited similar trends between the different tree species' canopies. Near-312 surface bulk soil water showed evaporative enrichment at peak leaf out, when SWCs reached a minimum (Fig. 4, Fig.  313 A1). Increased enrichment of bulk soil water relative to both the LMWL and corresponding mobile water with 314 declining SWCs was also observed by Zhao  trees tended to be more depleted in heavy isotopes than that of Acer. Xylem water 2 H for deciduous (beech and oak) 337 species in Switzerland was more depleted compared to spruce (Allen et al. 2019). Our results contrast with those of 338 White and Smith (2015), who saw limited inter-specific differences in the isotopic composition of plant water for box 339 elder (Acer negundo L.) or river birch (Betula nigra L.) at a given phenological stage in the foothills of the southern 340 Appalachian Mountains. Inter-specific variations in xylem water isotopic composition and its temporal changes at PC-341 1 are likely not due to distinct environmental conditions for the different tree species, given the close proximity of the 342 sampled trees and similar soil conditions under the tree canopies. 343

Potential drivers of plantsoil water isotopic differences 344
Several factors have been suggested which might account for the observed differences between the isotopic 345 compositions of xylem water and bulk soil water in PC-1. Some of these may be common to all studied tree species, 346 while others cannot account for interspecific differences in the temporal trajectory of xylem water isotopic 347 composition during the study period. 1 at peak leaf out were very dry while Ce xylem water lc-excess was much more negative than corresponding bulk 365 soil water (Fig. 7). Presence of sufficient soil water with very negative lc-excess values that could both match the 366 xylem water lc-excess and supply the tree's transpiration demand is unlikely under these circumstances. Trees may 367 have accessed water held in bedrock fractures that may be isotopically distinct from mobile soil water (Oshun et al. The greatest differences between xylem water and bulk soil water isotopic compositions at PC-1 for Ce and He were 375 generally at peak leaf-out (Fig. 8) following a protracted decline in SWCs (Fig. 2); however, these differences persisted 376 following soil rewetting. Barbeta et al. (2020) found soil waterplant water  2 H offsets increased with soil water 377 content, whereas the smallest offsets at PC-1 were either at post-senescence 2015 (Ce) or pre-leaf out (He, Or, Pw) 378 when SWCs would be relatively large as shown by previous work in PC-1 (e.g. Devito and Dillon 1993). Thus, the 379 influence of soil water content on differences in xylem watersoil water isotopic composition is unclear and deserves 380 further study. storage effect may assist in explaining the frequent distinction between xylem water and bulk soil water lc-excess for 385 a given sampling period, and is supported by partial (He, Pw) or complete (Or) overlap of the lc-excess of post-386 senescence 2015 soil water and pre-leaf out xylem water in 2016 (Fig. 7). However, Ce xylem water lc-excess was 387 much more negative than post-senescence 2015 bulk soil water, suggesting this mechanism may differ in importance 388 between tree species. Such a mechanism also fails to account for Ce and to some extent Pw lc-excess values that were 389 much more negative than any bulk soil water (Figs. 3 and 5). these processes to induce differences in xylem water isotopic composition relative to soil water at PC-1 is not known, 394 although the low clay contents of PC-1 soils make significant isotopic effects with clay minerals unlikely (Sprenger 395 et al. 2018b). Regardless, it would be reasonable to expect that such processes would be similar in the soil water 396 surrounding the different tree species. Thus, they do not easily explain interspecific differences in the degree of 397 isotopic displacement of xylem water from the LMWL and bulk soil water. 398 Fractionation during water uptake. There is increasing recognition that differences between xylem water and soil 399 water isotopic compositions may result from isotopic fractionation induced by internal plant processes during water 2 H during water uptake with differences between  18 O and  2 H in soil water relative to plant water increasing with 403 transpiration water loss. This would lead to more negative  18 O and  2 H in plant water relative to soil water, as seen 404 at PC-1. It also suggests the greatest differences between xylem water and soil water lc-excess would be at peak leaf 405 out when PC-1 soils were at their driest (Fig. A1). This was the case for Ce which showed clear separation between 406 soil and xylem water lc-excess at peak leaf out (Fig. 5). This also occurred at pre-senescence for Pw; however, there 407 was overlap between soil water and xylem water lc-excess for Or and He. Thus, the potential for fractionation during 408 water uptake may be a major cause of deviations between soil water and xylem water isotopic compositions and may 409 differ between tree species in northern mixed forests. 410 Fractionation following uptake. Changes in the isotopic composition of xylem water relative to that of soil water