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Introduction
In regions with a Mediterranean climate in southeastern Spain, the increase in temperature and the decrease in precipitation have worsened since the 1970s, particularly during the spring and summer, which has been associated with climate change (Bladé & Castro-Díez, 2010; Intergovernmental Panel on Climate Change [IPCC], 2014). Plant development in areas with a strongly seasonal climate is confined to periods of favorable conditions, so distribution, adaptation, growth and survival will depend on ecophysiological, morphological and reproductive attributes. However, the simultaneous variation and interaction of light and water condition the response of plants to light and their tolerance to shade, especially limited by the water condition (Valladares, Aranda, & Sánchez-Gómez, 2004; Vilagrosa et al., 2003).
Mediterranean plants have developed mechanisms to cope with water stress conditions, with a range of variation to avoid or tolerate drought (McDowell et al., 2013). Drought-avoiding species are able to close stomata to maintain their tissue hydration and thus avoid excessive xylem stress (Pausas et al., 2016); however, this limits carbon assimilation and increases the possibility of starvation (Galiano, Martínez-Vilalta, & Lloret, 2011). On the other hand, drought-tolerant species allow a certain degree of stress to affect their tissues by keeping stomata open with very low water potentials, which favors a positive carbon balance; however, they risk hydraulic system failure (Pausas et al., 2016; Urli et al., 2013) and increase cavitation (McDowell, 2011). The effect could be critical in years of increased drought, causing water competition between understory plants and the trees that make up the canopy (Valladares et al., 2004).
In this sense, determining water use efficiency, through the proportion of CO2 assimilated during photosynthesis and the amount of water lost through transpiration, is fundamental to understanding the interaction between plants and ecosystems under water stress. The establishment of resprouter species of evergreen and deciduous groups in these ecosystems would induce the development of mixed stands in the long term, thereby favoring the resilience of these forest systems against recurrent fires or future disturbances (García de la Serrana, Vilagrosa, & Alloza, 2015; Gavinet et al., 2015; Granados, Vilagrosa, Chirino, & Vallejo, 2016; Rodríguez-Rodríguez, Jordano, & Valido, 2015).
Due to their rapid regeneration, resprouter species represent an option for maintaining the optimum carbon balance and improving the resistance and resilience of ecosystems that have been disturbed or are at high risk of fire (Pausas & Keeley, 2017). In this context, the aim of this study was to evaluate the effect of light availability and soil moisture on gas exchange variables and photosystem II (PSII) maximum efficiency of six forest species established in Aleppo pine (Pinus halepensis Mill.) forests.
Materials and methods
Study area
The research was carried out in La Hunde (39° 7’ N and 1° 13’ W; Ayora, Valencia, Spain) in a dry Mesomediterranean climate, at an elevation of 600 m and in a relatively flat relief. The area has mean annual precipitation of 480 mm, mean annual temperature of 14 °C, reference evapotranspiration (Eto) of 749 mm and actual evapotranspiration (Eta) of 453 mm (Pérez-Cueva, 1994). The soils are derived from a marl-limestone substrate; they are shallow, rich in carbonates and have a slightly basic pH. The dominant vegetation corresponds to old repopulations of Aleppo pine (P. halepensis) planted 50 to 60 years ago. In the study area, the Regional Government Forestry Service (Generalitat Valenciana) thinned out the pine forest at various intensities between 2003 and 2005, allowing the establishment of other herbaceous and shrub species in areas where thinning was more intense.
Experimental field design
Three experimental treatments were defined based on forest stand density: high (HD, 800 to 1 100 trees·ha-1), medium (MD, 300 to 600 trees·ha-1) and low density (LD, 100 to 250 trees·ha-1). In each treatment, three 900 m2 (30 x 30 m) experimental plots were established at three sites: El Aljibe (39° 09’ N; 1° 18’ W), El Lobo (39° 12’ N; 1° 23’ W) and El Mojón (39° 12’ N; 1° 22’ W). In each plot, 15 one-year-old individuals corresponding to six native resprouter species were planted: Arbutus unedo L., Rhamnus alaternus L., Quercus ilex L. subsp. ballota (Desf.) Samp, Quercus faginea Lam. subsp. faginea, Fraxinus ornus L. and Acer granatense Boiss. These species have contrasting morphofunctional characteristics and different degrees of tolerance to shade and drought (Table 1). Planting was carried out in February 2011 in 40 x 40 x 40 cm holes made with a backhoe. Field capacity (FC) was determined using the method described by Walker (1989). In each experimental plot, soil water content (SWC) was monitored during the period March 2011 to October 2013, using a TDR 100 probe (Campbell Scientific, Inc., Logan, Utah, USA) at a depth of 0 to 20 cm in seven randomly-selected planting holes. Simultaneously, SWC was monitored in three planting holes, chosen at random, at a depth of 30 to 40 cm with an HS10 probe (ProCheck Decagon Devices Inc., Pullman, USA). Water availability was determined by the SWC/FC ratio. A more detailed description of the plots, as well as the edaphoclimatic conditions of the treatments (precipitation, temporal dynamics of the water content in the soil, environmental temperature, evapotranspiration and photosynthetic photon flux density), can be obtained in Gavinet et al. (2015)) and Granados et al. (2016)). These conditions are summarized in Table 2.
Table 1 Distinctive ecological traits of six native resprouter species established in the experimental plots.
| Species | Life form | Leaf habits | Drought tolerance* | Shade tolerance** |
|---|---|---|---|---|
| Arbutus unedo | Shrub | Evergreen | High | Low |
| Rhamnus alaternus | Shrub | Evergreen | High | Low |
| Quercus ilex | Tree | Evergreen | Medium | Medium |
| Quercus faginea | Tree | Semi-deciduous | Medium | Medium |
| Fraxinus ornus | Tree | Deciduous | Low | High |
| Acer granatense | Tree | Deciduous | Low | High |
*Determined from xylem cavitation resistance (Gortan, Nardini, Gasco, & Salleo, 2009; Martínez-Vilalta, Prat, Oliveras, & Piñol, 2002). ** Determined from the environment or ecosystem that they naturally inhabit (Rivas-Martínez, 1987).
Table 2 Environmental conditions in the plots by assessed pine density (HD = 800 to 1 100 trees·ha-1; MD = 300 to 600 trees·ha-1 and LD = 100 to 250 trees·ha-1) in La Hunde, Valencia, Spain.
| Environmental factor | Experimental treatments | F value* | ||
|---|---|---|---|---|
| HD | MD | LD | ||
| Tree density (1) | 1 067 ± 14 a | 344 ± 19 b | 165 ± 25 b | 32.687*** |
| Cumulative translocation (L·m-2) (1) | 859 ± 24 b | 915 ± 33 ab | 993 ± 27 a | 5.298 ** |
| SWC:FC ratio (1) | 0.53 ± 0.1 c | 0.55 ± 0.0 b | 0.63 ± 0.0 a | 0.017 *** |
| PPFD (µmol·m-2·s-1) (1) | 135.4 ± 9.4 c | 300.3 ± 17.4 b | 547.5 ± 26.7 a | 463.2 *** |
| Daily maximum Tª in summer (°C) (2) | 34.0 ± 0.2 b | 35.8 ± 0.5 a | 36.0 ± 0.1 a | P = 0.05 |
| Maximum daily VPD in summer (kPa) (2) | 4.3 ± 0.1 b | 4.8 ± 0.2 a | 4.9 ± 0.1 a | P = 0.05 |
Values and results taken from Granados et al. (2016)(1) and Gavinet et al. (2015)(2). SWC = soil water content; FC = field capacity, PPFD = photosynthetic photon flux density; Tª = air temperature; VPD = vapor pressure deficit. ± Standard error of the mean. **P < 0.01, ***P < 0.001
Physiological variables under field conditions
In the field, variables related to gas exchange and photosystem II maximum efficiency were measured. In six randomly-selected individuals of each species per treatment (two individuals per three plots), photosynthesis (A, µmol CO2·m-2·s-1) and stomatal conductance (Gs, mol H2O·m-2·s-1) were measured with an infrared gas analyzer (IRGA, LiCor 6400 XT, Inc., Lincoln, NE, USA), between 8:00 and 10:00 hours on the day of measurement in May, July and November 2011 and June 2012. Water use efficiency (IWUE) was calculated as the A/Gs ratio (µmol CO2·m-2·s-1/ mol H2O·m-2·s-1). The conditions inside the IRGA chamber were similar for all treatments (airflow of 380 µmol·s-1, CO2 concentration of 390 ppm and temperature of 25 °C), except for the photosynthetic photon flux density (PPFD), which was adjusted to the average value that this variable recorded under the canopy of each plot at the time of the measurements, using a ceptometer (Sunfleck Ceptometer, Decagon Devices, USA).
Photosystem II maximum efficiency (Krause & Weis, 1984) was measured at dawn, through chlorophyll fluorescence (variable fluorescence [Fv]/ maximum fluorescence [Fm]) in the same individuals and in May, July, August and November 2011 and June 2012. The measurement was made with a portable pulse-amplitude modulated fluorometer (PAM 2100, Heinz Walz Gm BHEffeltrich, Germany), under dark conditions; the clamp was placed on each leaf for 30 min.
Experimental design under nursery conditions
In order to qualitatively compare the response of the species and understand the processes operating in the exchange of gases under different light and water conditions, the same physiological variables of the plants (A, Gs and IWUE) were determined under optimal substrate moisture conditions, simulating similar levels of solar radiation as in the field. Under controlled conditions, an experiment was carried out in the nursery of the Plant Experimental Unit of the Universidad de Alicante (San Vicente del Raspeig, Spain). To simulate field sunlight conditions, three light treatments were established: 1) control treatment (CT), plants under conditions of maximum light availability (100 % sunlight); 2) medium light availability (MLA), plants under a black shade mesh (transmission: 50 % sunlight); and 3) low light availability (LLA), plants under a double shade mesh (transmission: 20 % sunlight). The transmission was calculated as the ratio between the PPFD values under the shade mesh and in an open site with maximum sunlight availability. PPFD was measured one day from 6:00 to 16:00 hours (solar time) in each treatment using a ceptometer (Sunfleck ceptometer, Decagon Devices, USA). Eight individuals of each species were transplanted per treatment into 1.5 L containers with a mixture of blonde peat moss and coconut fiber at 50% (v/v) as substrate. Each treatment and species were replicated twice. The plants were grown under optimal moisture conditions for two months to encourage acclimatization. The moisture of the substrate in the container was measured using the EC-5 Pro-Check probe (Decagon Devices, Pullman, USA). The plants were watered with 25 L·m-2 every four days in the CT and every six days inside the shade conditions to maintain full substrate moisture.
Physiological variables under nursery conditions
In six randomly-selected individuals of each species per shade treatment, the same variables were measured as in field conditions (A, Gs and IWUE) with the exception of Fv/Fm, using the same system (IRGA LiCor 6400 XT, Inc., Lincoln, NE, USA). Measurements were made one day between 8:00 and 12:00 solar time. The conditions inside the IRGA chamber were similar for all treatments (airflow of 380 µmol·s-1, CO2 concentration of 390 ppm and temperature of 25 °C), except the PPFD which was adjusted to the solar radiation of each treatment: 1 000 μmol·m-2·s-1 for CT, 600 μmol·m-2·s-1 for MLA and 90 μmol·m-2·s-1 for LLA. The day before the measurement, all plants were watered to field capacity to avoid the influence of varying degrees of hydration.
Statistical analysis
The analysis was done under a factorial design with two fixed factors: the experimental treatment factor (three densities) and the species factor (six species), with the model y ijk = μ + T i + E j + ε ijk , where y ij = response variable, μ = general mean, T i = treatment effect i, E j = species effect and ε ijk = error. In the field experiment, the variables A, Gs, IWUE and Fv/Fm were compared using an ANOVA with two fixed factors. A similar analysis was carried out to evaluate the response of the species on the sampling dates (measurement campaign). In the nursery, the variables A, Gs and IWUE were compared using an ANOVA with two factors: light level and species. When the analysis showed interaction between the factors, a one-factor ANOVA with a significance level of P < 0.05 was performed. The means were compared with Tukey’s LSD test. The data were transformed in those cases in which the ANOVA assumptions were not met; subsequently, compliance with these assumptions was verified. Analyses were performed using the STATISTICA 12 statistical software package (StatSoft Inc., 2012).
Results and discussion
Gas exchange and PSII efficiency under field conditions
In the study area, Granados et al. (2016) observed that the soil and climatic conditions in each plot were determined by the density of pine trees (Table 2), affecting the survival and growth of the species. In these ecosystems, high temperatures, high solar radiation and low rainfall during the summer period generate a marked water deficit that has increased in recent decades through recurrent droughts (Sánchez-Salguero, Navarro-Cerrillo, Camarero, & Fernández-Cancio, 2012).
Table 3 presents the results of the physiological variables measured in the six resprouter species and in the three pine forest densities assessed. The conditions of greater light availability (PPFD), together with greater water availability in the LD and MD treatments (Granados et al., 2016), favored higher photosynthesis (A; P < 0.01) and stomatal conductance (Gs; P < 0.05) values in those pine forest densities compared to HD. It has been reported that in dry and semi-arid climates, species with different drought-coping strategies and degrees of vulnerability to drought may coexist; tolerant species can keep stomata open in this condition (Forner, Aranda, Granier, & Valladares, 2014; Vilagrosa, Hernández, Luis, Cochard, & Pausas, 2014). In this study, R. alaternus showed higher values of A (P < 0.01) and Q. ilex had the highest values of Gs (P < 0.01). In general, leaves formed in environments with high light intensity have higher photosynthetic capacity than shaded plants (Larcher, 1980); however, under more intense drought conditions, there may be downward stomatal conductance regulation, with a consequent reduction in the photosynthetic rate (Rodríguez-Calcerrada et al., 2016). In this study, the results indicate that light availability was the factor that regulated gas exchange, which is consistent with Gómez-Aparicio, Valladares, and Zamora (2006), who found that light intensity limited the establishment of species.
Table 3 Two-factor ANOVA of the physiological variables measured in six native resprouter species and in three pine densities evaluated in the field in La Hunde, Valencia, Spain.
| Factors and interaction | Gas Exchange | Fv/Fm | ||
|---|---|---|---|---|
| A (µmol CO2·m2·s1) | Gs (mol H2O ·m2·s-1) | IWUE (A/Gs) | ||
| Species | ||||
| Arbutus unedo | 5.2 ± 0.4 ab | 0.08 ± 0.01 abc | 75.1 ± 4.4 a | 0.79 ± 0.00 a |
| Rhamnus alaternus | 6.8 ± 0.7 a | 0.10 ± 0.01 ab | 70.4 ± 5.6 ab | 0.76 ± 0.01 ab |
| Quercus ilex | 5.1 ± 0.5 ab | 0.11 ± 0.01 a | 57.1 ± 4.0 b | 0.77 ± 0.00 ab |
| Quercus faginea | 4.2 ± 0.4 b | 0.06 ± 0.01 c | 68.9 ± 5.3 ab | 0.75 ± 0.01 b |
| Fraxinus ornus | 4.7 ± 0.4 b | 0.08 ± 0.01 abc | 81.2 ± 5.3 a | 0.68 ± 0.02 c |
| Acer granatense | 4.8 ± 0.4 b | 0.07 ± 0.01 bc | 73.9 ± 4.9 a | 0.70 ± 0.02c |
| F value | 3.918** | 4.153** | 3.614** | 14.32*** |
| Treatment | ||||
| HD | 4.0 ± 0.3 b | 0.07 ± 0.00 b | 65.2 ± 3.1 a | 0.77 ± 0.01 a |
| MD | 5.2 ± 0.3 a | 0.09 ± 0.01 a | 72.6 ± 3.6 a | 0.74 ± 0.01 b |
| LD | 6.1 ± 0.3 a | 0.09 ± 0.01 a | 75.7 ± 3.8 a | 0.73 ± 0.01 b |
| F value | 10.569** | 2.241* | 3.254 (1) | 5.93** |
| Species x treatment | ||||
| F value | 0.500 | 0.205 | 0.545 | 0.79 |
Gs = stomatal conductance; A = photosynthesis; IWUE = water use efficiency; Fv/Fm = photosynthetic efficiency; HD = high density (800 to 1 100 trees·ha-1); MD = medium density (300 to 600 trees·ha-1) and LD = low density (100 to 250 trees·ha-1). Values followed by the same letter, between species and between treatments, indicate that there are no significant differences according to Tukey's LSD test (*P < 0.05, **P < 0.01, ***P < 0.001; n = 6). (1)Trend (P < 0.1). ± standard error of the mean.
Regarding water use efficiency (IWUE), according to Table 3, no significant differences were found among treatments (P = 0.0930), but there was a certain tendency for seedlings under LD cover to have higher IWUE compared to MD and HD. With respect to the species, F. ornus, A. unedo and A. granatense had greater IWUE (P < 0.05); the species with the lowest value was Q. ilex. The differences observed among species can be related to some strategies to deal with water deficit through water saving mechanisms. For example, Q. ilex is a species capable of maintaining low water potentials by osmotic adjustment (Vilagrosa et al., 2003, 2014), adopting a water stress tolerance strategy in extreme drought conditions.
On the other hand, Table 3 shows that photosystem II maximum efficiency (Fv/Fm) was 0.79, a value moderately below the optimum (0.83) that characterizes unstressed plants (Björkman & Demmig, 1987). These data could indicate long-lasting photoinhibition processes due to stressful conditions for the seedlings (Long, Humphries, & Falkowski, 1994). The Fv/Fm was highest in the HD treatment (P < 0.01), which is related to lower PPFD values and, therefore, less stressful for all species (Granados et al., 2016); no differences were observed between MD and LD. In the species, Fv/Fm was highest in A. unedo, followed by Q. ilex and R. alaternus, reflecting a better ability to adapt and live in these environments (P < 0.001); Q. faginea presented intermediate values, while F. ornus and A. granatense had the lowest values, showing greater photoinhibition and less adaptation to the light environment in these plots.
Table 4 shows the physiological variables analyzed by measurement period. The response of the species throughout the year indicated differences among treatments only in June 2012, where the seedlings in LD had higher values in Gs (P < 0.01) and A (P < 0.001) than the seedlings in MD and HD. In the same month, the seedlings in the LD and MD plots showed no differences in IWUE and were statistically greater than HD (P < 0.05).
Table 4 Two-factor ANOVA of the variables stomatal conductance (Gs), photosynthesis (A) and water use efficiency (IWUE) of six resprouter species and three pine forest densities (HD = 800 to 1 100 trees·ha-1, MD = 300 to 600 trees·ha-1 and LD = 100 to 250 trees·ha-1) evaluated in each measurement campaign in the experimental field plots.
| Physiological variable | Factor | Period | |||
|---|---|---|---|---|---|
| May 2011 | July 2011 | November 2011 | June 2012 | ||
| Gs (mol H2O·m-2·s-1) | Species | ||||
| Arbutus unedo | 0.10 ± 0.01 a | 0.07 ± 0.01 a | 0.15 ± 0.03 ab | 0.03 ± 0.00 b | |
| Rhamnus alaternus | 0.08 ± 0.02 a | 0.08 ± 0.01 a | 0.22 ± 0.04 a | 0.04 ± 0.00 ab | |
| Quercus ilex | 0.08 ± 0.02 a | 0.08 ± 0.01 a | 0.15 ± 0.02 ab | 0.05 ± 0.00 a | |
| Quercus faginea | 0.05 ± 0.01 a | 0.07 ± 0.01 a | 0.13 ± 0.02 ab | 0.04 ± 0.00 ab | |
| Fraxinus ornus | 0.14 ± 0.02 a | 0.06 ± 0.01 a | 0.10 ± 0.02 b | 0.03 ± 0.00 b | |
| Acer granatense | 0.10 ± 0.01 a | 0.08 ± 0.01 a | 0.10 ± 0.02 b | 0.03 ± 0.00 b | |
| F value | 1.8942 | 1.0464 | 2.5788* | 3.5248** | |
| P value | 0.1018 | 0.3947 | 0.0309 | 0.0055 | |
| Treatment | |||||
| HD | 0.07 ± 0.01 a | 0.07 ± 0.00 a | 0.11 ± 0.02 a | 0.03 ± 0.00 b | |
| MD | 0.09 ± 0.02 a | 0.08 ± 0.00 a | 0.17 ± 0.02 a | 0.03 ± 0.00 b | |
| LD | 0.11 ± 0.02 a | 0.07 ± 0.00 a | 0.14 ± 0.02 a | 0.05 ± 0.00 a | |
| F value | 1.9992 | 0.5130 | 0.07196 | 6.1390** | |
| P value | 0.1406 | 0.6001 | 2.7014 | 0.0030 | |
| Treatment x species | |||||
| F value | 0.5036 | 0.7552 | 0.6953 | 0.42615 | |
| P value | 0.8833 | 0.6709 | 0.7262 | 0.1288 | |
| A (µmol CO2·m-2·s-) | Species | ||||
| Arbutus unedo | 4.84 ± 1.15 a | 5.24 ± 0.57 a | 6.56 ± 1.17 b | 3.50 ± 0.55 a | |
| Rhamnus alaternus | 4.27 ± 1.18 a | 6.41 ± 1.38 a | 10.18 ± 1.20 a | 4.25 ± 0.57 a | |
| Quercus ilex | 3.63 ± 1.15 a | 4.19 ± 0.43 a | 7.82 ± 1.17 ab | 4.85 ± 0.55 a | |
| Quercus faginea | 2.72 ± 1.15 a | 3.77 ± 0.57 a | 6.11 ± 1.17 b | 3.72 ± 0.55 a | |
| Fraxinus ornus | 6.44 ± 1.15 a | 4.84 ± 0.57 a | 5.44 ± 1.20 b | 3.91 ± 0.55 a | |
| Acer granatense | 6.84 ± 1.15 a | 5.37 ± 0.67 a | 5.28 ± 1.24 b | 3.49 ± 0.55 a | |
| F value | 1.9376 | 1.5347 | 2.3515* | 0.50698 | |
| P value | 0.0946 | 0.1857 | 0.0465 | 0.8657 | |
| Treatment | |||||
| HD | 3.62 ± 0.08 a | 4.67 ± 0.45 a | 5.30 ± 0.83 a | 3.01 ± 0.36 b | |
| MD | 5.00 ± 0.08 a | 5.28 ± 0.67 a | 7.65 ± 0.83 a | 3.67 ± 0.36 b | |
| LD | 5.84 ± 0.08 a | 4.92 ± 0.48 a | 7.90 ± 0.88 a | 5.22 ± 0.37 a | |
| F value | 1.8319 | 0.72564 | 2.8642 | 9.4263*** | |
| P value | 0.1652 | 0.321 | 0.0616 | 0.0001 | |
| Treatment x species | |||||
| F value | 0.2298 | 0.5180 | 1.0116 | 0.55319 | |
| P value | 0.9926 | 0.8733 | 0.4405 | 0.8818 | |
| IWUE (A/Gs) | Species | ||||
| Arbutus unedo | 46.76 ± 5.63 ab | 84.65 ± 7.48 a | 57.32 ± 9.85 a | 111.06 ± 9.09 a | |
| Rhamnus alaternus | 33.59 ± 5.79 b | 80.50 ± 7.69 a | 54.09 ± 10.14 a | 111.36 ± 9.36 a | |
| Quercus ilex | 34.20 ± 5.63 b | 61.34 ± 7.48 a | 56.45 ± 9.85 a | 86.16 ± 9.09 a | |
| Quercus faginea | 28.28 ± 5.63 b | 64.06 ± 7.48 a | 64.16 ± 9.85 a | 93.13 ± 9.09 a | |
| Fraxinus ornus | 49.26 ± 5.63 ab | 90.90 ± 7.48 a | 72.24 ± 10.14 a | 117.66 ± 9.09 a | |
| Acer granatense | 61.73 ± 5.63 a | 72.21 ± 7.48 a | 60.16 ± 10.45 a | 106.56 ± 9.09 a | |
| F value | 4.9141*** | 2.4561 | 0.4300 | 1.7643 | |
| P value | 0.0004 | 0.0382 | 0.8267 | 0.1269 | |
| Treatment | |||||
| HD | 38.48 ± 4.33 a | 76.43 ± 5.51 a | 55.20 ± 6.90 a | 90.95 ± 6.41 b | |
| MD | 46.81 ± 4.33 a | 75.21 ± 5.51 a | 61.36 ± 6.90 a | 109.94 ± 6.41 a | |
| LD | 41.85 ± 4.40 a | 75.05 ± 5.59 a | 66.14 ± 7.31 a | 112.10 ± 6.50 a | |
| F value | 0.9324 | 0.0185 | 0.5973 | 3.2728* | |
| P value | 0.3968 | 0.9816 | 0.5522 | 0.0418 | |
| Treatment x species | |||||
| F value | 0.6765 | 0.7089 | 0.7726 | 0.2359 | |
| P value | ss0.7433 | 0.7139 | 0.6545 | 0.9918 | |
Value followed by the same letter indicates that there is significant difference between species and between treatments, by measurement period, according to Tukey’s LSD test (*P < 0.05, **P < 0.01, ***P < 0.001; n = 6). ± standard error of the mean.
With respect to the species, differences in IWUE were only found in May 2011; A. granatense had the highest efficiency (P < 0.001). In November 2011, R. alaternus had the highest values of A and Gs (P < 0.05), while in June 2012, only Gs was different among the species, with Q. ilex obtaining the highest value (P < 0.01). The July 2011 measurement did not show significant differences in physiological variables among species (P > 0.05). These results may be associated with greater water availability in May and November 2011 and June 2012 (Granados et al., 2016). Previous studies have reported that deciduous species have the highest water use efficiency because they are able to take intense advantage of the few predictable pulses and have higher photosynthetic rates (Hasselquist, Allen, & Santiago, 2010), especially in spring (Valladares et al., 2004).
Table 5 shows that the annual dynamics of Fv/Fm had specific periods (May, August and November 2011) where the seedlings marked differences among species. The evergreen species R. alaternus, A. unedo and Q. ilex showed greater Fv/Fm, indicating less photoinhibition and, possibly, less damage to the photosynthetic system (Morales, Abadía, & Abadía, 2006; Vilagrosa et al., 2010). Regarding the pine forest densities, differences were only observed in May and August 2011, with HD obtaining the highest Fv/Fm value.
Table 5 Two-factor ANOVA of fluorescence at dawn (Fv/Fm) of six resprouter species and three pine forest densities (HD = 800 to 1 100 trees·ha-1, MD = 300 to 600 trees·ha-1 and LD = 100 to 250 trees·ha-1) evaluated in each measurement campaign in the experimental field plots.
| Factor | Measurement period of Fv/Fm | ||||
|---|---|---|---|---|---|
| May 2011 | July 2011 | August 2011 | November 2011 | June 2012 | |
| Species | |||||
| Arbutus unedo | 0.76 ± 0.01 a | 0.76 ± 0.00 a | 0.79 ± 0.01 a | 0.74 ± 0.04 a | 0.80 ± 0.00 a |
| Rhamnus alaternus | 0.73 ± 0.01 a | 0.75 ± 0.00 a | 0.79 ± 0.01 a | 0.75 ± 0.04 a | 0.80 ± 0.00 a |
| Quercus ilex | 0.76 ± 0.01 a | 0.76 ± 0.00 a | 0.78 ± 0.01 a | 0.75 ± 0.04 a | 0.80 ± 0.00 a |
| Quercus faginea | 0.68 ± 0.01 b | 0.75 ± 0.00 a | 0.78 ± 0.01 ab | 0.67 ± 0.04 ab | 0.82 ± 0.00 a |
| Fraxinus ornus | 0.67 ± 0.01 b | 0.76 ± 0.00 a | 0.72 ± 0.01 b | 0.57 ± 0.04 ab | 0.80 ± 0.00 a |
| Acer granatense | 0.73 ± 0.01 a | 0.74 ± 0.00 a | 0.72 ± 0.01 b | 0.53 ± 0.04 b | 0.79 ± 0.00 a |
| F value | 9.2786*** | 0.8422 | 2.7618* | 4.5018*** | 1.5881 |
| P value | 0.0000 | 0.5228 | 0.0221 | 0.0009 | 0.1702 |
| Treatment | |||||
| HD | 0.75 ± 0.01 a | 0.76 ± 0.00 a | 0.80 ± 0.01 a | 0.70 ± 0.03 a | 0.81 ± 0.00 a |
| MD | 0.72 ± 0.01 ab | 0.75 ± 0.00 a | 0.74 ± 0.01 b | 0.66 ± 0.03 a | 0.80 ± 0.00 a |
| LD | 0.70 ± 0.01 b | 0.75 ± 0.00 a | 0.75 ± 0.01 b | 0.65 ± 0.03 a | 0.80 ± 0.00 a |
| F value | 4.7811* | 1.6550 | 4.8166** | 0.5380 | 2.9471 |
| P value | 0.0103 | 0.1960 | 0.0099 | 0.5855 | 0.0568 |
| Treatment x species | |||||
| F value | 1.27 | 1.7460 | 0.3651 | 1.1553 | 0.6194 |
| P value | 0.1269 | 0.0826 | 0.9584 | 0.3319 | 0.7936 |
Value followed by the same letter indicates that there are no significant differences between species and between treatments, by measurement period, according to Tukey's LSD test (*P < 0.05, **P < 0.01, *P < 0.001; n = 6). ± standard error of the mean.
Gas exchange in the experiment under nursery conditions
Table 6 indicates that, under nursery conditions, the highest Gs values were observed in conditions of low light availability (LLA), while the lowest were recorded in conditions of high availability (CT) (P < 0.05). In contrast, the photosynthetic rate A showed significantly higher values in CT and MLA (P < 0.001). The highest Gs values were observed in LLA (P < 0.05), probably because in full light (CT) and, despite the irrigation, the vapor pressure deficit (VPD) could cause stomatic regulation of water loss; in contrast, the highest field values were obtained under conditions of greater light availability (LD and MD; P < 0.05; Table 3). In A, a pattern similar to that recorded in the field was observed; the seedlings in LD (P < 0.001; Table 3) and CT (P < 0.001) reached the highest values.
In relation to IWUE, CT seedlings also showed higher values, with the lowest values found in LLA (P < 0.001); under field conditions, the trend was similar, since, although the differences were not significant among tree densities (P > 0.05), the IWUE value was also lower when there was less light entry (high density). Mediterranean species have developed mechanisms to cope with both water stress and high light conditions (Gómez-Aparicio et al., 2006; McDowell et al., 2013). Under field conditions, due to high radiation, the plants probably adjusted their rehydration by means of the water available at night (Valladares & Pearcy, 2002), in order to maintain the high gas exchange observed and, consequently, a high IWUE value; in the nursery, soil moisture was not the limiting factor. In this case, the results could indicate that the factors that defined the response in field gas exchange were the amount of light, the VPD associated with temperature and water availability (soil water content: field capacity).
Table 6 Two-factor ANOVA of the physiological variables measured in six native resprouter species and in three nursery light availability conditions.
| Factors and interaction | Gas Exchange | ||
|---|---|---|---|
| A (µmol CO2·m-2·s-1) | Gs (mol H2O ·m2·s-1) | IWUE (A/Gs) | |
| Species | |||
| Arbutus unedo | 5.4 ± 0.7 ab | 0.15 ± 0.03 ab | 49.5 ± 7.3 ab |
| Rhamnus alaternus | 7.0 ± 1.3 a | 0.22 ± 0.04 a | 37.9 ± 6.1 b |
| Quecus ilex | 7.7 ± 1.0 a | 0.17 ± 0.00 ab | 46.1 ± 6.0 ab |
| Quercus faginea | 3.6 ± 0.9 b | 0.12 ± 0.01 b | 33.8 ± 10.4 b |
| Fraxinus ornus | 6.1 ± 0.8 ab | 0.12 ± 0.01 b | 51.8 ± 5.9 ab |
| Acer granatense | 6.5 ± 0.8 a | 0.12 ± 0.01 b | 59.5 ± 8.2 a |
| F value | 4.878 *** | 3.411 ** | 3.408 * |
| Treatment | |||
| CT | 8.2 ± 0.7 a | 0.12 ± 0.01 b | 76.6 ± 4.8 a |
| MLA | 7.5 ± 0.6 a | 0.16 ± 0.02 ab | 46.0 ± 3.3 b |
| LLA | 2.6 ± 0.3 b | 0.17 ± 0.01 a | 17.1 ± 1.9 c |
| F value | 40.454 *** | 3.615 * | 75.517 *** |
| Treatment x species | |||
| Valor F | 2.362* | 0.841 | 0.633 |
Gs = Stomatal conductance; A = photosynthesis; IWUE = water use efficiency; CT = control treatment (100 % light); MLA = medium light availability (50 %); LLA = low light availability (20 %). Value followed by the same letter indicates that there are no significant differences between species and between treatments (Tukey’s LSD, *P < 0.05, **P < 0.01, ***P < 0.001; n = 6). ±standard error of the mean.
According to Table 6, the deciduous species F. ornus and A. granatense and the semi-deciduous species Q. faginea showed lower Gs rates than the evergreen species R. alaternus; A. unedo and Q. ilex showed intermediate values (P < 0.01). Regarding photosynthesis, the highest rates were found in Q. ilex, R. alaternus and A. granatense (P < 0.001). It has been reported that adult individuals of deciduous species, under natural conditions, have higher gas exchange rates (Mediavilla & Escudero, 2003) and are able to take intense advantage of the few predictable water pulses, showing higher photosynthetic rates (Hasselquist et al., 2010).
Figure 1 illustrates the analysis of the response of the species by nursery light availability treatment. The analysis only revealed differences in Gs in two species; A. unedo showed higher Gs in LLA, while F. ornus showed higher Gs values in MLA (P < 0.05). Most of the species (R. alaternus, Q. ilex, F. ornus and A. granatense) recorded lower A values in the treatment with the least light availability (LLA; P < 0.05), with no differences observed between MLA and CT. In contrast, the highest IWUE values were reported in most species in the maximum light availability treatment (CT) and the lowest values in LLA (P < 0.05). These results could indicate that very low light intensities could be harmful to any species, because carbon fixation would be compromised.
Figure 1 One-factor ANOVA for greenhouse gas exchange variables. Stomatal conductance (Gs), photosynthesis (A) and water use efficiency (IWUE) of six resprouter species (Au = Arbutus unedo, Ra = Rhamnus alaternus, Qi = Quercus ilex, Qf = Quercus faginea, Fo= Fraxinus ornus, Ag = Acer granatense) under different light availability treatments: control (CT = 100 %), medium light availability (MLA = 50 %) and low light availability (LLA = 20 %). Different letters indicate statistical differences between treatments (Tukey’s LSD, P < 0.05; n = 6). Bars indicate standard error of the mean.
Conclusions
The response of the species studied under field conditions reflected the interaction between soil moisture and light for the plants, conditioned by the density of the pine forest. In the field, evergreen sclerophyll species such as Arbutus unedo, Rhamnus alaternus or Quercus ilex had a greater ability to acclimatize to environmental conditions than deciduous species. Abiotic conditions, due to tree cover, can be a determining factor in the response of the species studied, according to their functional strategy (evergreen and deciduous). Under optimal nursery conditions, the species behaved with standard patterns of response to the light availability factor.
