Gas exchange measurements
Our water stress treatment (Dry) decreased photosynthesis Anet (p < 0.001) similarly in all provenances, from approximately 12 µmol m−2 s−1 in the well-watered (Wet) treatment to 2.5 µmol m−2 s−1 in the Dry treatment (Fig. 1; Table 3a). There was also an interaction effect of the combination of light and water treatments (Light × Water, p = 0.006) which followed a similar pattern in all provenances: in the wet treatment, light-saturated Anet was higher in seedlings grown under full sun than those under shade, whereas in the Dry the pattern was reversed. It is worth noting here that the absence of shading provoked fluctuations in temperature and relative humidity in the Sun treatments that is likely, when combined with the imposed water-stress, to have increased the intensity of drought compared with Shade plots (Fig. S3), with likely consequences for the traits of seedlings therein. The Anet differed among provenances under the light treatments (Light × Provenance, p = 0.028), whereby the largest difference in Anet between Sun-Wet and Shade-Wet was in Montejo-ES and the smallest difference in Eichelberg-DE (Fig. 1, 1st row; Table 3a).
Patterns in stomatal conductance (gs) among treatments closely mirrored those of Anet. The Dry treatment caused drastic stomatal closure (p < 0.001), e.g., from gs of 227 to 46 mmol m−2 s−1 in Shade (Fig. 1, 2nd row). Light affected gs differently under the two watering treatments (Light × Water, p = 0.009) and differently among provenances (Light × Provenance, p = 0.022), and the three way interaction of Light × Water × Provenance was significant (p = 0.033; Fig. 1, 2nd row; Table 3a). Montejo-ES seedlings produced the largest difference in gs from Sun and Shade treatments in the Wet. In the Dry treatments, gs was lower among seedlings in the Sun than those in the Shade, and differences among provenances were not as distinct as under the wet treatments; although in Blaviksliarna-SE the Sun-Dry gs was highest (Fig. S4 for detailed gs of Dry treatments expanded from Fig. 1, 2nd row). Sensitivity of gs to soil moisture was similar among provenances in Shade, but in the Sun where it increased most readily in seedlings from Blaviksliarna-SE, and least in Montejo-ES, when released from water stress (Fig. 1; Fig. S5).
The Ci/Ca was consistently higher in the wet treatments than the dry treatments (p < 0.001), within which Ci/Ca was lower in the Shade-Dry than the Sun-Dry, even though gs was higher in the Shade-Dry. Under well-watered conditions, the biggest sun-shade difference in Ci/Ca was in Blaviksliarna-SE (Fig. 1, 3rd row). As would be expected, water use efficiency (IWUE) was highest under Dry conditions (p < 0.001), but differences among provenances were negligible (Fig. 1, 4th row; Table 3a).
Midday leaf water potentials (Ψmidday) were higher under Wet than Dry treatments (p = 0.003), and lower under Sun vs. Shade treatments (p = 0.050); although the latter difference was largely due a reduction in Ψmidday in Sun-Wet compared to Shade-Wet seedlings, which reached similar Ψmidday values to Sun-Dry seedlings in all but the Montejo-ES provenance (Water × Provenance, p = 0.017; Table 3a). Seedlings from Montejo-ES reached lower Ψmidday in the Dry treatment than the other provenances, which were otherwise similar in their Ψmidday (Fig. 2, 1st row, Table 3a). General patterns in Ψdiff (not shown) mainly tracked Ψmidday within each of wet and dry treatments, with only minor effects of variation in pre-dawn soil water potential.
The apparent soil-leaf hydraulic conductance (KL) in the Shade treatments was 0.5 mol m−2 s−1 MPa−1 higher overall than that in the Sun (p = 0.040); a 27% increase. In the Wet treatment, KL was 2.3 mol m−2 s−1 MPa−1 higher, an increase of 4–5 times more than that in the Dry (p < 0.001), but the differences in KL among the provenances were negligible (Fig. 2, 2nd row, Table 3a, p = 0.672).
Leaf chlorophyll content per unit area (LCC) increased during the 6 weeks between June 1st (Date 1) and July 14th (Date 3). It also remained significantly higher in the Shade than the Sun treatment (Fig. 3, 1st row, Table 3b, p = 0.001). There was no significant difference in LCC between Dry vs. Wet seedlings, but the Blaviksliarna-SE population had the lowest LCC in the Sun and the two core German provenances, Eichelberg-DE and Rindelpholz-DE, the highest LCC on Date 1 (Light × Provenance p = 0.14, Provenance p < 0.001). By Date 3, there was also an interaction between the watering treatment and provenance for LCC (Water × Provenance p = 0.008; Fig. 3; Table 3b), whereby Eichelberg-DE had atypically high LCC in the Dry-Shade treatment.
The adaxial epidermal flavonol index (Iflav), per unit area, in the Sun-Wet treatment remained stable over the three measurement dates, but Iflav declined slightly in the Sun-Dry treatments, and even more so in the shaded seedlings under both watering treatments (Fig. 3, 2nd row, Table 3b). In general, the patterns across light and water treatments remained similar over the three measurement dates, whereby Iflav was mostly higher in the Sun-Wet treatment than the Sun-Dry, and Iflav in the Shade treatments was only half that of the Sun treatments (Light: p < 0.001). A difference in Iflav among provenances was detected under both light treatments on each of the three measurement dates (Light × Provenance p = 0.019, p = 0.011, p = 0.037; Table 3b). Under Shade, Montejo-ES leaves had the highest Iflav of all the provenances, whereas in the Sun, Rindelphlotz-DE and Montejo-ES were similar, and the latter declined least over time in the Sun-Wet treatment (Fig. 3, 2nd row, Table 3b).
Measurement of the adaxial epidermal HCA index (IHCA) was only possible on Date 2 and Date 3. It did not differ significantly with light or watering treatment on Date 2, producing similar values across all treatments averaging 2.0–2.2 (Fig. 3, 3rd row). However, despite the narrow range of values obtained, there was an interaction effect of Light × Provenance on IHCA (p < 0.001) which followed a similar pattern to that in Iflav. On Date 2, the IHCA in Montejo-ES seedlings was the highest among the provenances in the shade treatments, and that in Eichelberg-DE was lowest of the provenances; but on Date3, increased variability among leaves within each provenance-treatment combination largely obscured any effect among provenances. There was an effect of watering treatment on Date 3 but this appears to derive from decreasing values in some of the shade-dry seedlings, causing increased variability in the results on this date. Visual inspection of the seedlings on 14th July also revealed differences in red-coloration attributable to anthocyanins; red coloration of the youngest leaves was most noticeable in seedlings growing in the Wet treatment in all but the Montejo-ES provenance, where it was entirely absent (Fig. S6, Table 3b).
Growth and morphology
Seedlings of all provenances continued to grow larger throughout the experiment, growing most overall in the Sun-Wet, followed by the Shade-Wet treatments, in terms of height (Fig. 4, Table 3c), as well as number of leaves and branches (Fig. S7, Table 3c). In the Sun-Dry and Shade-Dry treatments most seedlings had similar growth rates, with only Montejo-ES and Eichelberg-DE growing higher in the shade than the sun by the end of the experiment (Fig. 4, significant Light × Provenance date 3–5, Table 3c). As with the physiological data, this Sun-Shade effect may be partially due to the Dry treatment being slightly less severe in the Shade (Fig. S2).
Leaf length was similar among provenances and treatments, with only sun-shade treatment having a significant effect on leaf length (p = 0.049, Fig. 5, 1st row). Overall, the Shade leaves were slightly larger than the Sun leaves. All seedlings had their highest LMA in the Sun treatments (p < 0.001, Fig. 5, 2nd row). Of the provenances, Montejo-ES had the lowest LMA in all treatment combinations (Provenance, p = 0.001, Table 3c). In the shade, there was also an effect of watering treatment on LMA; producing a lower LMA in the Wet than the Dry treatment (Light × Water, p = 0.025, Fig. 5; Table 3c). Beyond these effects, there were no significant relationships at the individual-plant level between growth traits and the individual morphological or physiological traits that we measured, such as LCC, Iflav or KL.