Supplementary Material
Suplementary 1
Morphology and growth rate in Q. ilex seedlings native to high and low elevations
Adult trees of comparable size (and presumably similar in age) were studied. In autumn 2003, acorns were gathered from around 15 trees per population (roughly 50 acorns per tree). To establish homogeneous initial conditions, we selected an acorn sub-sample of similar size and dry weight (including coat) of 2.94±0.27 and 2.91±0.25 g for the High and Low populations, respectively. Acorns were pooled and placed in well watered trays at ambient (greenhouse) temperature for germination. Around one month after germination, 25 seedlings similar in shape and size (around 10 cm tall) from each population were placed in 2-litre pots (one per plant) using a 1:3 (v/v) vermiculite-sand substrate. Ten seedlings from each population were used to estimate the mean dry weight of the individuals of each population (initial mass). Pooled seedlings from both populations were placed in a growth chamber under the following conditions: 14-h photoperiod; 325 µmol m-2 s-1 PAR (between one and two times that in the forest understory) at leaf height; day/night temperatures 24ºC/18ºC; relative humidity 30-35%. Plants were watered on alternate days with diluted (1:3) Hoagland #2 solution to avoid nutrient deficiency and water stress. The plants were rotated inside the chamber (roughly every 15 days) to minimize the chamber effect.
The experiment lasted for 120 days. Each seedling was then divided into its stem, leaf, and root fractions, and fresh leaf surfaces were measured. Fractions were oven dried at 80ºC and weighed (final mass). The relative growth rate (mg g-1 day-1) of each individual was calculated from the initial mass (the same for all the individuals of the population) and from the final mass of the individual. Leaf Size (LS) and the ratios LMR (mass of leaves to plant mass), LAR (total surface of leaves to plant mass), SLA (fresh leaf surface to leaf dry mass), and S:R (shoot-to-root mass) were also calculated for each individual. These values were used to calculate the mean values for each population.
The photosynthetic rate was measured under the same conditions of temperature, relative humidity, and light intensity as those of cultivation. The determinations were made on individual leaves located near the middle of the stem of randomly selected plants at the beginning, middle, and end of the experimental period using a portable gas-exchange system (CIRAS 1 PP Systems, Edinburgh, U.K.).
Seedling of both populations were similar regarding plant structure, growth rate and leaf traits (Table S1).
Table S1 Means (± SD) of variables measured in Q. ilex seedlings from high and low elevation populations cultivated in growth chambers; n = number of seedlings considered; LS = leaf size; LMR = leaf-mass ratio; LAR = leaf-area ratio; S:R = shoot-to-root ratio; SLA = specific leaf area; RGR= relative growth rate; and A= photosynthetic rate
Population / High / Lown / 12 / 12
LS (cm2) / 1.67 ± 0.27 / 1.93 ± 5.10 / n.s.
LMR (g g-1) / 0.24 ± 0.04 / 0.28 ± 0.07 / n.s.
LAR (m2 kg-1) / 1.51 ± 0.29 / 1.63 ± 0.37 / n.s.
S:R / 0.52 ± 0.08 / 0.54 ± 0.19 / n.s.
SLA (m2kg-1) / 6.34 ± 0.85 / 6.06 ± 1.00 / n.s.
A (nmol CO2 g-1 s-1) / 80.5 ± 23.0 / 81.0 ± 25.7 / n.s.
RGR (mg g-1 day-1) / 20.6 ± 1.9 / 21.4 ± 1.9 / n.s.
Suplementary 2
Impact of atmospheric H2S and low temperature on thiols concentration in leaf and roots tissues of Q. ilex seedlings native to two contrasted soils.
Acorns of Q. ilex were collected in autumn of 2004 in two populations of the Southern Iberian Peninsula: one (37º48´N; 5º41´W) located on well-developed brown siltstone soil on slate rock and the other (37º7´N; 5º8´W) on an unstructured soil (litosol) over gypsum rock. For seedling cultivation procedures and growth conditions see Supplementary 1.
18 month old seedlings from germinated acorns grown at Pablo Olavide University (Seville), were translated from Spain to the Laboratory of Plant Physiology of the University of Groningen (Haren, The Netherlands). Three weeks before to start the stress induced experiments, the seedlings were transferred to plastic 30 l containers hydroponic cultures (according procedures in the Material and Methods section).
For stress treatments, seedlings were transferred to 13 l capacity stainless steel containers filled with a 25 % Hoagland nutrient solution (10 seedlings per container) and housed in stainless steel cylindrical cabinets (0.65 m diameter, 185 l capacity) with polycarbonate tops for allowing illumination (Westerman et al. 2000), at a 14 h photoperiod of 325 µmol m-2 s-1 PAR, day/night temperatures 20/16 ºC, and relative humidity 55 %. A subset of seedlings was subjected to low temperature (6 – 7 ºC) (Low Temperature stress treatment). The air temperature was controlled by adjusting the wall temperature of the cabinet, and a ventilator stirred the air inside the cabinet continuously. A second subset of seedlings was fumigated with H2S (0.2 µl l-1) (H2S treatment) which was injected into the incoming air stream and adjusted to the desired level using mass flow controllers. The level of gas in the cabinet was controlled with an SO2 analyzer coupled to an H2S converter (model 8770, monitors Labs, Measurement Controls Corporation, Englewood, CO, USA). A third subset of seedlings was grown in the same conditions but with no treatment (Control). After 7 days, exposed seedlings and Control were harvested. Thiols concentration was determined on fresh plant material (see Material and Methods section) and expressed in µmol g-1 FW.
Population controls did not show consistent differences in thiols concentration. Both H2S and Low Temperature treatments resulted in a general two times thiols concentration increase (P < 0.01) in seedling tissues. Gypsum seedlings were more responsive to both H2S (root tissues; P < 0.05) and Low Temperature treatments (leaf tissues; P < 0.05) indicating that inducible defenses were higher in the population native to the stressful soil.
References
Westerman S, De Kok LJ, Stuiver CEE, Stulen I (2000) Interaction between metabolism of atmospheric H2S in the shoot and sulfate uptake by the roots of curly kale (Brassica oleracea). Physiol Plant 109:443-449. doi: 10.1034/j.1399-3054.2000.100411.x
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Table S2 Effect of air H2S exposition (0,2 µl l-1 for 7 days) and low temperature (6 ºC during 7 days) on leaf and root thiols concentrations (µmol g-1 fresh weight) of Q. ilex individuals native to stressed (gypsum) and fertile (siltstone) soils. Data show the mean of three values obtained in two individuals per population ± the standard deviation; soil effect is referring to statistical probability of significant diffences between soil provenances (i.e. Gypsum vs Siltstone); * P < 0.05; ** P < 0.01 (Laureano and de Kock, unpublished)
H2S treatment / Temperature treatmentControl / 0,2 µl l-1 / Soil effect / Control / 6 ºC / Soil effect
Mature leaves / Gypsum / 0,29 ± 0,06 / 0,54 ± 0,04 ** / n.s. / 0,29 ± 0,06 / 0,61 ± 0,01 ** / *
Siltstone / 0,28 ± 0,01 / 0,57 ± 0,07 ** / 0,28 ± 0,01 * (1) / 0,47 ± 0,04 **
Root systems / Gypsum / 0,15 ± 0,03 / 0,32 ± 0,06 ** / * / 0,15 ± 0,03 / 0,32 ± 0,03 ** / n.s.
Siltstone / 0,17 ± 0,04 * (1) / 0,19 ± 0,01 / 0,17 ± 0,04 / 0,34 ± 0,02 **
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Table S3 Coefficients calculated by linear-mixed effect model fits based on multi-model selection criteria shown in the Table 3. Specific growth rate (SGR) and leaf size (LS) were used as predictors in expanding leaves (Table S3a); leaf size (LS), in mature leaves (Table S3b); and specific growth rate (SGR) and root age (AGE), in root systems (Table S3c), of Q. ilex populations. Data from high and low elevations, gypsum and siltstone soils and northern and southern latitudes were analysed together.
Table S3a. Expanding leaves model:
Random effects:
Formula: ~1 | Population
(Intercept) Residual
StdDev: 19.65 17.76
Fixed effects: SRR ~ SGR + LS
Value Std. Error DF t-value P-value
(Intercept) 72.25 8.83 272 8.19 < 0.0001
SGR 0.15 0.03 272 5.88 < 0.0001
LS -4.09 0.83 272 -4.94 < 0.0001
Number of Observations: 280
Number of Groups: 6
Table S3b. Mature leaves model:
Random effects:
Formula: ~1 | Population
(Intercept) Residual
StdDev: 2.70 1.34
Fixed effects: SRR ~ LS
Value Std. Error DF t-value P-value
(Intercept) 6.06 1.1708697 158 5.17 < 0.0001
LS -0.12 0.0582873 158 -2.09 0.0381
Number of Observations: 165
Number of Groups: 6
Table S3c. Root systems model
Random effects:
Formula: ~1 | Population
(Intercept) Residual
StdDev: 99.1 181.4
Fixed effects: SRR ~ SGR
Value Std. Error DF t-value P-value
(Intercept) 276.3 51.4 147 5.37 < 0.0001
SGR 4.39 0.22 147 19.7 < 0.0001
Number of Observations: 154
Number of Groups: 6
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Table S4 Photosynthesis rate and N concentration in mature leaves of Q. ilex from high (stressed) and low (non-stressed) elevations (Elevation factor); gypsum (stressed) and siltstone (non-stressed) soils (Soil factor), unpublished data; and north (stressed) and south (non-stressed) latitudes (Latitude factor), from García et al. (1998). * P < 0.05
Elevation / Soil / LatitudeA / N / A / N / A / N
(nmol CO2 g-1 s-1) / (mg g-1) / (nmol CO2 g-1 s-1) / (mg g-1) / (nmol CO2 g-1 s-1) / (mg g-1)
Stressed / 63.9 ± 21.7 / 14.7 ± 2.0 / 58.2 ± 19.0 / 12.5 ± 0.8 / 63.6 ± 5.3 / 12.0 ± 0.3
non-stressed / 76.1 ± 30.3 / 13.9 ± 1.5 / 78.3 ± 25.3 / 13.6 ± 0.5 / 64.0 ± 4.9 / 11.0 ± 0.4
P / * / n.s. / * / n.s. / n.s. / n.s.
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Table S5 Relative growth rate (RGR; mg g-1 day-1) of Q. ilex potted seedlings growing under controlled conditions, native to high (stressed) and low (non-stressed) elevations (Elevation factor); gypsum (stressed) and siltstone (non-stressed) soils (Soil factor), from Laureano et al. (2013); and northern (stressed) and southern (non-stressed) latitudes (Latitude factor), from García et al. (1998). * P < 0.05; ** P < 0.01
Elevation / Soil / LatitudeStressed / 20.6 / 19.4 / 17.3
non-stressed / 21.4 / 26.0 / 27.6
P / n.s. / ** / *
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