No evidence of isotopic fractionation in olive trees (Olea europaea): a stable isotope tracing experiment

Figures & data

Figure 1. Sketch of the experimental set-up for each olive tree, including sap flow sensors, tension lysimeters, and holes for irrigation and soil moisture measurements

Table 1. Number of samples collected throughout the sampling rounds during the set-up, the conditioning period, and the experiment. Samples were grouped based on their type, olive tree (O1 and O2), and laboratory for soil and plant water extractions

Sampling rounds	Sample size
	LAB 1				LAB 2
	O1		O2		O1		O2
	Soil	Plant	Soil	Plant	Soil	Plant	Soil	Plant
Preliminary samplings	2	7	-	9	3	2	-	-
Conditioning period	7	5	7	5	7	7	5	5
Tracer experiment	22	31	21	21	24	33	23	22
Last sampling (after cutting down tree O1 entirely)	8	22	6	5	12	25	9	5
Total number of samples	39	65	34	40	46	67	37	32

Table 2. The isotopic compositions (mean ± standard deviation) of tap water and labelled water used for irrigation

	Sample size	δ^18O (‰)	δ^2H (‰)
Tap water	3	−7.97 ± 0.77	−52.3 ± 1.8
Labelled water	4	−12.75 ± 0.50	−93.3 ± 1.8

Figure 2. Cryogenic vacuum distillation system in Lab 1 (a) and Lab 2 (b)

Figure 3. Time series of environmental variables measured in tree 1 (O1) and tree 2 (O2). The average soil moisture (%) is shown with error bars representing the minimum and maximum values. The vertical dashed pink line marks the start of the experiment with labelled water

Figure 4. Time series of δ¹⁸O (panels a–d) and δ²H (panels e–h) of soil and plant water for samples extracted in the two laboratories. CVD-Soil represents the cryogenically extracted soil water, while LYS-Soil is the gravity-drained soil water obtained via tension lysimeters. The two horizontal dotted lines with their respective bands indicate the average and the standard deviation, respectively, of the isotopic composition of tap water (grey), and labelled water (pink). The vertical dashed pink line marks the start of the experiment with labelled water

Figure 5. Time series for d-excess determined in soil and plant water for samples extracted in the two laboratories. CVD-Soil represents the cryogenically extracted soil water, while LYS-Soil is the soil water obtained via tension lysimeters. The two horizontal dotted lines with their respective bands indicate the average and the standard deviation, respectively, of the isotopic composition of tap water (grey), and labelled water (pink). The vertical dashed pink line marks the start of the experiment with labelled water

Figure 6. Dual-isotope plots of different plant tissues water (twigs, cores and roots), and soil water samples extracted by cryogenic vacuum distillation (CVD-Soil) and tension lysimeters (LYS-Soil). The global meteoric water line (GMWL) is plotted in black, round symbols indicate soil and plant water samples of O1 and triangle symbols represent soil and plant water samples of O2. Sample size is given in the Supplementary material, Table S3

Figure 7. The spatial variation of δ¹⁸O and δ²H of plant water from various tissues, and soil water at different depths (LYS-Soil and CVD-Soil), for O1 during the tracer experiment. The vertical dotted line with grey band indicates the isotopic composition of labelled water (mean ± standard deviation)

Figure 8. The spatial variation of d-excess of plant water from various tissues, and soil water at different depths (LYS-Soil and CVD-Soil), for O1 during the tracer experiment. The vertical dotted line with grey band indicates the isotopic composition of labelled water (mean ± standard deviation)

Figure 9. Box plots of (a, b) δ¹⁸O and (c, d) δ²H of O1 and O2 samples grouped by plant and soil water types for the sampling rounds irrigated with labelled water. The boxes indicate the 25^th and 75^th percentiles, while the whiskers represent the minimum and maximum values. The horizontal solid lines within the box mark the median and the red circles are the mean. Letters above the boxes represent the significantly different groups based on the Dunn post hoc analysis

Table 3. Slopes and intercepts of the linear regressions between δ¹⁸O, δ²H and d-excess of plant tissue water and soil water of Lab 1 vs. Lab 2. Lab 1 is plotted along the x-axis while Lab 2 is plotted along the y-axis. All values are in ‰

(Lab 1 vs. Lab 2)	Plant water			Soil water
	δ^18O	δ^2H	d-excess	δ^18O	δ^2H	d-excess
Slope	0.92	0.96	0.74	1.08	0.97	1.21
Intercept	−1.15	−4.38	2.40	1.04	−2.44	−3.25
R^2	0.94	0.97	0.62	0.92	0.96	0.70

Figure 10. Dimensionless Z-scores values for (a) δ¹⁸O and (b) δ²H grouped by cryogenically extracted soil water (CVD-Soil), and plant water (samples from twigs, cores and roots were grouped together) of O1 and O2. Sample size is reported above the boxes. The boxes indicate the 25^th and 75^th percentiles, while the whiskers represent the minimum and maximum values. The horizontal solid lines within the box mark the median and the red circles are the mean, respectively. The dashed purple and pink lines represent the upper limits for acceptable (Z-score = 2) and questionable (Z-score = 5) differences respectively, between Lab 1 and Lab 2 cryogenically extracted samples