Physiological, morphological and anatomical trait variations between winter and summer leaves of Cistus species

Flora 207 (2012) 442–449

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Physiological, morphological and anatomical trait variations between winter and summer leaves of Cistus species Rosangela Catoni, Loretta Gratani, Laura Varone ∗ Department of Environmental Biology, Sapienza University of Rome, P.le A. Moro, 5 00185 Rome, Italy

a r t i c l e

i n f o

Article history: Received 21 August 2011 Accepted 4 February 2012 Keywords: Cistus Chlorophyll LMA LTD Plasticity index Photosynthesis

a b s t r a c t Morphological, anatomical and physiological summer and winter leaf traits of Cistus incanus subsp. incanus, C. salvifolius and C. monspeliensis growing at the Botanical garden of Rome were analyzed. With regard to differences between summer and winter leaves of the considered species, leaf thickness (L) was 21% higher in summer than in winter leaves (mean of the considered species) and this increase was mostly the result of the increased palisade parenchyma thickness over the spongy parenchyma one (24 and 16% higher in summer than in winter leaves, respectively). Leaf mass area (LMA) and leaf tissue density (LTD) were 38% and 17% higher in summer than in winter leaves, respectively (mean of the considered species). The photosynthetic rate (PN ), stomatal conductance (gs ) and chlorophyll content (Chl) of summer leaves were 54%, 17% and 14% lower, respectively, than in winter leaves. C. monspeliensis summer leaves had the highest LMA, LTD, adaxial cuticle thickness (14.6 ± 1.8 mg cm−2 , 1091 ± 94 mg cm−3 , and 5.8 ± 1.7 ␮m, respectively) and the lowest mesophyll intercellular spaces (fias 38 ± 3%). Moreover, C. monspeliensis had the highest PN in summer (2.6 ± 0.1 ␮mol m−2 s−1 ) and C. incanus the highest PN and WUE (84% and 59% higher than the other species) in the favorable period, associated to a higher fias (42 ± 2%). C. salvifolius had the highest PN (54% higher than the other species) in winter. The plasticity index could allow a better interpretation of the habitat preference of the considered species. The physiological plasticity (PIp = 0.39, mean value of the considered species) was higher than the morphological (PIm = 0.22, mean value) and anatomical (PIa = 0.13, mean value) plasticity. Moreover, among the considered species, C. salvifolius and C. incanus are characterized by a larger PIa (0.14, mean value) which seems to be correlated with their wider ecological distribution and the more favorable conditions of the environments where they naturally occur. The highest PIm (0.29) of C. monspeliensis indicates that it can play a high adaptive role in highly stressed environments, like fire degraded Mediterranean areas in which it occurs. © 2012 Elsevier GmbH. All rights reserved.

Introduction Under the Mediterranean type of climate, plant species have to withstand severe water stress, high temperatures and high irradiance during summer (Cowling et al., 2005; Godoy et al., 2011; Sánchez-Gómez et al., 2006). Moreover, in winter, the combination of high irradiance and sub-optimal air temperatures for growth causes a depression of the photosynthetic activity (Larcher, 2000; ˜ Oliveira and Penuelas, 2002; Varone and Gratani, 2007). It has been shown that the overlapping of such multiple stress factors may determine coordinated physiological responses in Mediterranean species, resulting in different functional strategies (Gratani and Bombelli, 2001; Valladares et al., 2004; Zunzunegui et al., 2011). Drought semi-deciduous species are characterized by a seasonal reduction in their transpiring leaf surface area (Bombelli and Gratani, 2003). They produce short twigs (brachyblasts) with small

∗ Corresponding author. Tel.: +39 06 49912449; fax: +39 06 49912449. E-mail address: [email protected] (L. Varone). 0367-2530/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.flora.2012.02.007

leaves at the end of spring-beginning of summer (summer leaves), and long twigs (dolichoblasts) in autumn-winter, with larger and thinner winter leaves (De Micco and Aronne, 2009; Gratani and Crescente, 1997; Palacio et al., 2006; Werner et al., 1999). Seasonal leaf dimorphism can be considered an adaptive strategy to the seasonal climatic changes occurring in Mediterranean habitats (Christodoulakis et al., 1990; Kyparissis et al., 1997; Orshan, 1972). The genus Cistus comprises 21 shrub species (50–100 cm in height) most of them (14 species) characterized by a shallow, markedly planar root system expanding where soil moisture availability is depleted throughout summer (Amato and Sarnataro, 2001). These species are distributed in the Mediterranean Region, in southern Europe, North Africa and western Asia (FernándezMazuecos and Vargas, 2010), where they colonize degraded areas (Attaguile et al., 2000). Most Cistus species do occur widespread, but there exist also few narrow endemics (Fernández-Mazuecos and Vargas, 2010). Cistus incanus L. subsp. incanus is a typical Mediterranean shrub species distributed along the coastal belt of the Central-Eastern Mediterranean (it is absent in France and the Iberian Peninsula),

R. Catoni et al. / Flora 207 (2012) 442–449

Northern Africa and Western Asia (Abbate Edlmann et al., 1994), extending from sea level to 800 m a.s.l., mainly in arid and warm areas of maquis and garrigue (Pignatti, 1982). Cistus monspeliensis L. is a lowland shrub species displaying a rather continuous distribution in the Mediterranean Basin, even though it becomes scarcer eastwards. It is also found in the Canary Islands (Acebes et al., 2004; Fernández-Mazuecos and Vargas, 2010). Dense C. monspeliensis shrubs are found on poor soils from sea-level to 600–800 m a.s.l., both on calcareous and acidic soils (Fernández-Mazuecos and Vargas, 2010), and where holm oak, cork oak and pine woodlands are degraded (Juhren, 1966). C. monspeliensis is a typical species of garrigue colonizing wide areas after fire (Quézel, 1981, 1985). Cistus salvifolius L. has a circum-Mediterranean distribution, from Portugal and Morocco to Palestine and the eastern coast of the Black Sea, extending into the south of the Eurosiberian region (Short, 1994), over a wide range of habitats (Demoly and Montserrat, 1993). C. salvifolius does not form dense shrubs, but has a patchy distribution in a wide altitudinal range from sea level to 1800 m a.s.l. (Fernández-Mazuecos and Vargas, 2010), often occurring in wooded areas as a component of the understory (Farley and McNeilly, 2000). C. salvifolius has a high vulnerability at the northern edge of its distribution areas, on the southern slopes of the Alps, where fires occur sporadically. It is listed as vulnerable (VU) on the Red List for Switzerland (Moser et al., 2002; Moretti et al., 2006). Main objective of present research was to compare morphological, anatomical and physiological summer and winter leaf traits of Cistus incanus subsp. incanus, Cistus salvifolius, and Cistus monspeliensis. Moreover, considering that plasticity within the same plant might play an adaptive role in a strongly seasonal climate, such as the Mediterranean one (Zunzunegui et al., 2011), we analyzed the plasticity index of summer and winter leaves of the considered species to underline differences in the range of environments that these species inhabit (Ackerly et al., 2000).

Materials and methods Study area and plant species The study was carried out from January to December 2010, on representative shrubs of Cistus incanus subsp. incanus, Cistus monspeliensis, and Cistus salvifolius (five shrubs per species), growing in the open, under the same environmental conditions, at the Botanical Garden of Rome (41◦ 53 53 N, 12◦ 28 46 E; 53 m a.s.l.). The nomenclature for Cistus species follows Tutin et al. (1993). The climate of Rome is of Mediterranean type – mean minimum air temperature (Tmin ) of the coldest months (January and February) 5.4 ± 0.2 ◦ C, mean maximum air temperature (Tmax ) of the hottest months (July and August) 30.9 ± 0.2 ◦ C, yearly mean air temperature (Tm ) 16.8 ± 6.5 ◦ C. The dry period lasted in 2010 from the beginning of June to the end of August (66.2 mm total rainfall). Total annual rainfall averages to 702 mm, most of it occurring in autumn and winter (Data from UCEA, for the years 1995–2010). During the study period Tmin of the coldest month (January) was 4.6 ± 2.2 ◦ C, Tmax of the hottest month (July) was 32.6 ± 1.8 ◦ C, and total rainfall in 2010 was 1022 mm, concentrated in autumn and winter. Sampling was carried out monthly, on fully expanded leaves developed on dolichoblasts and brachyblasts, respectively. In particular, at the beginning of January 50 dolichoblasts for each of the considered species (10 dolichoblasts per shrub) formed from the previous year’s growth (from September until October) were considered, and the respective leaves (winter leaves) were monitored. Winter leaves persisted until the end of May when they were substituted by summer leaves produced on brachyblasts from the

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beginning of May to the beginning of June, and falling at the end of October – beginning of November. At the beginning of June, 50 brachyblasts for each of the considered species (10 brachyblasts per shrub), and the respective leaves (summer leaves) were monitored. A new production of dolichoblasts occurred from the beginning of September until the beginning of October, and the respective leaves (winter leaves) were monitored until December. Accordingly, measurements performed from January to May and from October to December were carried out on winter leaves, while measurements performed from June to September were carried out on summer leaves. Anatomical leaf traits Measurements of anatomical leaf traits were conducted on fully expanded winter and summer leaves (n = 20 per leaf type and species from the selected shrubs), collected at the middle of January (winter leaves) and at the end of June (summer leaves), respectively, and analyzed by light microscopy, using an image analysis system (Axiovision AC software). The following parameters were measured: total leaf thickness (L, ␮m), palisade and spongy parenchyma thickness, adaxial and abaxial epidermis thickness, adaxial and abaxial cuticle thickness. All measurements were restricted to vein free areas, according to Chabot and Chabot (1977). The fraction of the mesophyll occupied by intercellular air space (fias , %) was calculated, according to Syvertsen et al. (1995) as: fias = 1 −

Am lW

where Am is the cross-sectional area of the mesophyll cells, l the mesophyll thickness, and W the width of the measured section. Morphological leaf traits Measurements of morphological traits were conducted on fully expanded winter and summer leaves, respectively (n = 20 per type and species), collected at the middle of January (winter leaves) and at the end of June (summer leaves), respectively, from the selected shrubs. The following parameters were measured: projected leaf surface area (excluding petiole) (LA, cm2 ), obtained by an Image Analysis System (Delta-T Devices, UK), and leaf dry mass (DM, mg), determined after drying at 80 ◦ C to constant mass. Leaf mass per unit leaf area (LMA, mg cm−2 ) was calculated by the ratio of DM and LA (Reich et al., 1992). Leaf tissue density (LTD, mg cm−3 ) was calculated by the ratio of LMA and leaf thickness (Wright and Westoby, 2002). Gas exchange Measurements of gas exchange were carried out using an infrared gas analyzer (ADC LCA4, UK), equipped with a leaf chamber (PLC, Parkinson Leaf Chamber). Measurements were made on fully expanded winter and summer leaves (n = 4 per leaf type and species, in each sampling occasion) periodically during the study period. Net photosynthesis (PN , ␮mol m−2 s−1 ), photosynthetically active radiation (PAR, ␮mol m−2 s−1 ), stomatal conductance (gs , mol m−2 s−1 ), transpiration rate (E, mmol m−2 s−1 ), and leaf temperature (Tl , ◦ C) were measured. The showed PN , gs , and E rates were the mean of the maximum rates for the four days of measurement per month, carried out in comparable weather conditions. Measurements were carried out under natural conditions, on cloud–free days (PAR ≥ 1000 ␮mol m−2 s−1 , saturating level), in the morning, from 8.00 a.m. to 12.00 p. m. (Reich et al., 1995). Instantaneous

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Table 1 Anatomical leaf traits of summer and winter leaves of C. incanus, C. monspeliensis and C. salvifolius. Mean values (±SE) are shown (n = 20). Mean values with the same letters are not significantly different between summer and winter leaves (Tukey Test, p ≥ 0.05). Leaf traits

Leaf thickness (␮m) Palisade parenchyma thickness (␮m) Spongy parenchyma thickness (␮m) Adaxial epidermis thickness (␮m) Adaxial cuticle thickness (␮m) Abaxial epidermis thickness (␮m) Abaxial cuticle thickness (␮m) Number of palisade cell layers fias (%)

C. incanus

C. monspeliensis

C. salvifolius

Summer

Winter

Summer

Winter

Summer

Winter

232.0 ± 9.8a 104.7 ± 23.1a 83.9 ± 11.7a 17.0 ± 4.0a 6.2 ± 1.3a 12.3 ± 4.0a 5.1 ± 0.6a 2 42 ± 2a

200.0 ± 12.0b 92.1 ± 17.8b 76.8 ± 13.4a 13.7 ± 3.9b 5.7 ± 1.3a 10.1 ± 2.4b 4.2 ± 1.0b 1 52 ± 3b

137.6 ± 16.6a 60.9 ± 7.8a 43.7 ± 8.3a 14.5 ± 2.0a 5.8 ± 1.7a 7.3 ± 1.4a 4.1 ± 1.3a 1 38 ± 3a

123.0 ± 7.4b 54.2 ± 5.9b 39.2 ± 4.5a 13.9 ± 1.3a 4.9 ± 1.2b 7.2 ± 0.6a 3.7 ± 0.7a 1 48 ± 2b

183.3 ± 16.8a 84.4 ± 11.5a 67.5 ± 10.1a 12.1 ± 1.9a 4.8 ± 0.9a 9.8 ± 1.0a 4.1 ± 0.9a 2 54 ± 1a

134.6 ± 11.1b 58.2 ± 5.9b 52.8 ± 8.8b 11.5 ± 3.2a 4.5 ± 0.5a 9.0 ± 0.8a 3.9 ± 0.6a 1 60 ± 3b

water use efficiency (WUE, ␮mol mmol−1 ) was calculated by the ratio between PN and E, according to Niu et al. (2006). Chlorophyll content Chlorophyll content (Chl) was measured by a SPAD-502 meter (Konica Minolta Sensing, Inc., Osaka, Japan). Chl measurements were carried out on the same leaves used for gas exchange measurements, on various points on the surface of each leaf sample, cleaning the leaf surface (Gratani, 1992), after gas exchange measurements. Leaf plasticity The plasticity index of anatomical (PIa ), morphological (PIm ) and physiological (PIp ) leaf traits for each species, was calculated by the differences between the minimum and the maximum mean value between the two types of leaves (i.e. winter and summer leaves, respectively) divided by the maximum mean value, according to Valladares et al. (2000, 2006). Plasticity index, scaling from 0 to 1, was calculated for all the considered anatomical, morphological and physiological traits of summer and winter leaves. Total plasticity index (PI) was calculated by averaging PIa , PIm and PIp for each species (Valladares et al., 2000). Statistics Differences in the considered leaf traits were determined by the analysis of variance (ANOVA) and the Tukey test for multiple comparisons, using a statistical software package (Statistica, Statsoft, USA). Data were tested for normality and homogeneity of variances before carrying out the statistical analysis, with the significant level set at p ≤ 0.05. The relationships between PN and Tl (photosynthetic thermal window sensu Larcher, 1994) were used to calculate that leaf temperature (Tl 100% ) which determines 100% of the highest PN , and the leaf temperature (Tl 50% ) below or above which PN dropped below half of its maximum. Results Anatomical leaf traits Anatomical leaf traits of summer and winter leaves of the considered species are shown in Table 1. All three Cistus species are characterized by a bifacial leaf anatomy. Significant differences were observed between summer and winter leaves. Total leaf, palisade parenchyma and spongy parenchyma thickness were 21%, 24% and 16% higher, respectively, in summer than in winter leaves (mean of all investigated species). Winter leaves were formed by one layer of palisade parenchyma cells in all three taxa. Summer leaves were formed by two layers in C. incanus and C. salvifolius and

by one layer in C. monspeliensis. Protective structures (adaxial and abaxial epidermal and adaxial and abaxial cuticle) thickness was, on an average, 12% higher in summer than in winter leaves (mean value of the considered species). fias significantly differed between summer and winter leaves (45 ± 8 and 53 ± 6%, respectively, mean value of the considered species). At the species level, C. incanus had the highest total leaf thickness, both for summer and winter leaves (232.0 ± 9.8 and 200.0 ± 12.0 ␮m, respectively), and C. monspeliensis the lowest one (137.6 ± 16.6 and 123.0 ± 7.4 ␮m, respectively). C. incanus had the highest protective structure thickness (40.6 ± 9.1 and 33.7 ± 5.2 ␮m, in summer and winter leaves, respectively). C. salvifolius had the largest fias both in summer and winter leaves (54 ± 1 and 60 ± 3%, respectively). C. incanus had stellate hairs on both summer and winter leaves. C. salvifolius and C. monspeliensis had stellate and glandular hairs on both summer and winter leaves. Morphological leaf traits There were significant differences between morphological traits of summer and winter leaves of the three species (Table 2). On average, summer leaves had 30% lower LA than winter leaves (mean of the considered species). LMA and LTD were 27% and 14% lower in winter leaves than in summer ones (mean of all species). At the species level, C. monspeliensis had the highest LMA (14.6 ± 1.8 and 11.1 ± 2.2 mg cm−2 , summer and winter leaves, respectively), and C. salvifolius the lowest one (9.5 ± 1.6 and 7.0 ± 1.0 mg cm−2 , respectively). C. monspeliensis had the highest LTD (1091 ± 94 and 816 ± 92 mg cm−3 , summer and winter leaves, respectively) and C. salvifolius the lowest one (526 ± 98 and 511 ± 64 mg cm−3 , respectively). Gas exchange Gas exchange data of the considered species during the study period are shown in Fig. 1. Winter leaves The highest PN were reached by the investigated species in spring, peaking in May, when Tm was 22.0 ± 2.6 ◦ C and water availability 115.8 mm (total rainfall of May). Then, winter leaves of C. incanus had the highest PN (24.7 ± 0.6 ␮mol m−2 s−1 ), followed by C. salvifolius (16.1 ± 0.8 ␮mol m−2 s−1 ) and C. monspeliensis (15.2 ± 0.3 ␮mol m−2 s−1 ). The lowest PN was monitored in January (Tmin = 4.6 ± 2.2 ◦ C), C. salvifolius having the highest rates (4.9 ± 0.2 ␮mol m−2 s−1 ) and C. monspeliensis the lowest ones (2.7 ± 0.3 ␮mol m−2 s−1 ). incanus had the highest WUE in spring C. (7.7 ± 1.1 ␮mol mmol−1 , mean value of March, April and May), followed by C. salvifolius (6.8 ± 2.3 ␮mol mmol−1 ), and C. monspeliensis

R. Catoni et al. / Flora 207 (2012) 442–449

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Table 2 Morphological leaf traits of summer and winter leaves of C. incanus, C. monspeliensis and C. salvifolius. LA: leaf surface area; DM: leaf dry mass; LMA: leaf mass per unit leaf area; LTD: leaf tissue density. Mean values (±SE) are shown (n = 20). Mean values with the same letters are not significantly different between summer and winter leaves (Tukey Test, p ≥ 0.05). Leaf traits

C. incanus Summer

2

LA (cm ) DM (mg) LMA (mg cm−2 ) LTD (mg cm−3 )

3.2 45 14.0 608

± ± ± ±

0.3a 5a 0.3a 23a

C. monspeliensis Winter 4.0 38 9.5 530

± ± ± ±

0.2b 3b 0.4b 30b

Summer 1.1 16 14.6 1091

(4.7 ± 0.9 ␮mol mmol−1 ). In January WUE of C. salvifolius and C. monspeliensis was higher (86% and 37%, respectively), than in C. incanus. gs , controlling to a great deal PN , had the same seasonal trend as net CO2 uptake in the three species, reaching the highest rates in May: C. incanus showed the highest gs (0.159 ± 0.020 mol m−2 s−1 ), followed by C. monspeliensis (0.152 ± 0.030 mol m−2 s−1 ) and C. salvifolius (0.149 ± 0.004 mol m−2 s−1 ). Summer leaves PN of summer leaves had the lowest rates in August (Tmax = 30.8 ± 2.4 ◦ C and 4.8 mm total rainfall of August), and C. monspeliensis showed the significantly highest PN

± ± ± ±

0.2a 3a 1.8a 94a

C. salvifolius Winter 1.9 21 11.1 816

± ± ± ±

Summer 0.2b 3b 2.2b 92b

2.7 25 4.6 526

± ± ± ±

0.3a 5a 1.8a 98a

Winter 3.8 27 7.0 511

± ± ± ±

0.5b 5a 1.0b 64a

(2.6 ± 0.1 ␮mol m−2 s−1 ), compared to the others two species (2.1 ± 0.1 ␮mol m−2 s−1 , mean value). The highest PN of summer leaves were monitored in September (Tmax 26.5 ± 2.5 ◦ C), after the first rainfall following the summer drought (22.9 mm from the middle to the end of September).Then, C. incanus reached a PN of19.2 ± 0.5 ␮mol m−2 s−1 , and C. monspeliensis and C. salvifolius values that were on an average 47% lower than C. incanus. WUE increased drastically from August (1.0 ± 0.3 ␮mol mmol−1 , mean value of the three species) to September (4.4 ± 1.3 ␮mol mmol−1 , mean value). In August, gs ranged between 0.039 ± 0.002 mol m−2 s−1 (C. monspeliensis) and 0.041 ± 0.003 mol m−2 s−1 (mean value of C. incanus and C. salvifolius), but increased by more than 100% in September (mean value). Chlorophyll content Chlorophyll content data of the three Cistus species during the study period are shown in Fig. 2. Winter leaves Winter leaves had the highest Chl values in May (53.9 ± 3.8 SPAD units, mean value of all species), with C. incanus having 13% higher Chl than C. monspeliensis and C. salvifolius. In January Chl was the highest in C. incanus (52.9 ± 3.9 SPAD unit) and the lowest in C. salvifolius (47.3 ± 0.3 SPAD units). Summer leaves Chl values of C. incanus summer leaves in August were 5% higher (39.3 ± 0.3 SPAD unit) than those of C. monspeliensis and C. salvifolius. From these minimum values Chl increased 36%, 35% and 21% in C. salvifolius, C. incanus and C. monspeliensis, respectively, in September, after the first winter rain occurred.

Fig. 1. Trend of (a) net photosynthetic rate (PN ), (b) instantaneous water use efficiency (WUE), and (c) stomatal conductance (gs ,), of the winter and summer leaves of C. incanus (close circles), C. monspeliensis (close square) and C. salvifolius (open triangles) during the study period. The mean values for each month (±SE) are shown (n = 16 leaves).

Fig. 2. Trend of chlorophyll content (Chl) of the winter and summer leaves C. incanus (close circles), C. monspeliensis (close square) and C. salvifolius (open triangles) during the study period. The mean values for each month (±SE) are shown (n = 16 leaves).

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R. Catoni et al. / Flora 207 (2012) 442–449

Table 3 Phenotypic plasticity index for morphological (PIm ), anatomical (PIa ) and physiological (PIp ) traits of C. incanus, C. monspeliensis and C. salvifolius. LA: leaf surface area; DM: leaf dry mass; LMA: leaf mass per unit leaf area; fias : fraction of the mesophyll occupied by intercellular air space; PN : net photosynthetic rates; gs : stomatal conductance; WUE: instantaneous water use efficiency; Chl: chlorophyll content. PI: total plasticity index. Plasticity index

C. incanus

C. monspeliensis

C. salvifolius

Morphological traits LA DM LMA LTD PIm

0.20 0.16 0.32 0.13 0.20

0.42 0.24 0.24 0.25 0.29

0.29 0.07 0.26 0.03 0.16

Anatomical traits Leaf thickness Palisade parenchyma thickness Spongy parenchyma thickness Adaxial epidermis thickness Adaxial cuticle thickness Abaxial epidermis thickness Abaxial cuticle thickness fias PIa

0.14 0.12 0.08 0.19 0.08 0.18 0.18 0.19 0.15

0.11 0.11 0.10 0.04 0.16 0.01 0.10 0.21 0.11

0.27 0.31 0.22 0.05 0.06 0.08 0.05 0.10 0.14

Physiological traits PN gs WUE Chl PIp

0.58 0.05 0.77 0.14 0.38

0.44 0.17 0.65 0.16 0.35

0.59 0.30 0.72 0.13 0.43

PI

0.24

0.25

0.25

Plasticity index The overall PI of the considered species was 0.25 (mean value). PIm was 0.22 (mean value of all species), with C. monspeliensis showing the largest value (0.29). PIp was 0.39 (mean value), with C. salvifolius having the largest value (0.43), and PIa was 0.13 (mean value), with C. incanus having the largest value (0.15): Table 3. Regression analysis of PN vs. leaf temperatures The polynomial relationship between PN of summer and winter leaves and Tl indicated that PN of the considered species reached the highest rates when Tl was 23.0 ◦ C for C. salvifolius, 23.1 ◦ C for C. incanus, and 24.5 ◦ C for C. monspeliensis. PN dropped below half of its maximum when Tl was under 10.3 ◦ C and above 35.7 ◦ C for C. salvifolius, under 11.5 ◦ C and above 34.8 ◦ C for C. incanus, and under 12.6 ◦ C and above 36.4 ◦ C for C. monspeliensis (Fig. 3). Discussion Our overall results underline significant structural and functional differences between summer and winter leaves of the considered Cistus species, in case of C. incanus leaf anatomy and morphology more or less confirming the findings of Aronne and De Micco (2001). At an anatomical level, total leaf thickness is 21% higher in summer than in winter leaves (mean of the considered species), and this increase is primarily the result of the increased palisade parenchyma thickness (24% higher in summer than in winter leaves), spongy parenchyma thickness (16% higher in summer than in winter leaves) and epidermis and cuticle thickness (12% higher in summer than in winter leaves). Summer leaves, subject to a greater transpiration rate than winter leaves, take an avoidance strategy by reducing water loss through decreasing leaf size and fias (C. incanus summer leaves measured by Aronne and De Micco, 2001, must have been exceptionally small). LMA and LTD were 38% and 17% higher, respectively (mean of the three

considered species), in summer than in winter leaves. This resulted in a higher leaf compactness, which can be interpreted as a protective mechanism decreasing photochemical damages to the photosynthetic apparatus (Abril and Hanano, 1998; Castro-Díez et al., 1998; Gratani and Varone, 2004). The results suggest that changes in the internal leaf structure may play an adaptive role in response to climatic factors which change during the year, and in particular, the highest LMA of summer leaves can be used as a measure of investment per unit of leaf area in full sun (Grubb, 2002). At a physiological level, PN , gs and Chl of Cistus summer leaves are 54%, 17% and 14%, respectively, lower (mean value of the considered species) than in winter leaves. Low soil water availability during summer associated to high air temperatures and excessive radiation impose a multiple stress to Mediterranean species resulting in a decreased photosynthesis capacity (Chaves et al., ˜ 2002; Penuelas et al., 2004). It has been shown that in Mediterranean plant species a decrease of chlorophyll content, combined with an increased reflectance, may act as a supplementary defense against photodestruction (Gratani and Varone, 2004; Kyparissis and Manetas, 1993), because it lowers the intercepted light and, consequently, increases the capacity to dissipate the excess of excitation energy (Munné-Bosch and Alegre, 2000). In particular, the considered species have, on an average, a 29% Chl decrease in August compared to the maximum content. In September, lowered air temperatures (Tmax 26.5 ± 2.5 ◦ C) were accompanied by an increase of PN and Chl (more than 100% and 31%, respectively). The different morphological, anatomical and physiological leaf traits of the three species seem to be indicative of their adaption to the prevailing stress factors of the environments where they naturally grow. In particular, C. monspeliensis summer leaves are characterized by the highest LMA, LTD (14.6 ± 1.8 mg cm−2 , 1091 ± 94 mg cm−3 , respectively), a higher adaxial cuticle thickness (5.8 ± 1.7 ␮m), and the lowest LA (1.1 ± 0.2 cm2 ) and fias (38 ± 3%). All these traits should improve C. monspeliensis drought resistance, so that this species showed the highest PN and WUE (24% and 73% higher than the other two species) in August, this based on the lowest Chl (5% lower than the other species). Compared to C. monspeliensis, C. incanus leaf traits (i.e. 4% and 44% lower LMA and LTD, respectively), underline its lower capability to withstand summer drought, also attested by a lower PN (2.0 ± 0.1 ␮mol m−2 s−1 ) in August. Ehleringer and Comstock (1987) and Gratani and Bombelli (1999) showed that C. incanus leaf folding under extreme water stress may reduce LA exposed to direct sun rays compensating for its lower leaf consistency (see similar conclusions by Aronne and De Micco, 2001). On the contrary, when the resources are not limited (i.e. in spring and in autumn) C. incanus shows the highest PN , Chl and WUE (84%, 7% and 59% higher than the other two species), eventually facilitated by a larger fias (42 ± 2%) which allows a better CO2 circulation among the mesophyll cells, thus favoring carbon fixation. The gain and subsequent allocation of a greater amount of photosynthates during favorable periods seems to enable C. incanus’ vegetative regeneration capability, particularly in the first stages of the reconstitution of the Mediterranean maquis after fire (Gratani and Amadori, 1991). Among the considered species, C. salvifolius is characterized by the most mesic leaf traits, as evident from the lowest adaxial cuticle thickness (4.8 ± 0.9 ␮m), the lowest LTD (526 ± 98 mg cm−3 ), a higher L (183.3 ± 16.8 ␮m) and palisade parenchyma thickness (84.4 ± 11.5 ␮m). The resulting low leaf consistency (LMA = 9.5 ± 1.6 mg cm−2 ) associated to a low LTD will reduce the diffusion pathway from stomata to chloroplasts (Gratani and Ghia, 2002; Parkhurst, 1994). Low LMA values are generally associated to a high relative growth rate (Hunt and Cornelissen, 1997), that promotes the ability of a species to colonize different habitats (Gulías et al., 2003). Moreover, the high C. salvifolius PN

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Fig. 3. Regression analysis between net photosynthetic rate (PN ) and leaf temperature (Tl ) during the study period. Tl 100% = leaf temperature enabling 100% of the highest PN ; Tl 50% = leaf temperature below or above which PN drops below half of its maximum. Open symbols for each species indicate summer leaves. Regression equation and determination’s coefficient (R2 ) are shown.

(54% higher than the others) in January may be conducive to grow in mountain areas, according to the results of Fernández-Mazuecos and Vargas (2010). The different PN sensibility to air temperature of the considered species is confirmed by the polynomial correlation between PN and Tl indicating that PN drops below half of its maximum when Tl is under 10.3 ◦ C and above 35.7 ◦ C for C. salvifolius, under 11.5 ◦ C and above 34.8 ◦ C for C. incanus, and under 12.6 ◦ C and above 36.4 ◦ C for C. monspeliensis. Plasticity is a key trait useful to quantify plant responses to environmental stimuli (Nicotra et al., 2010) which can be related to adaptive advantages (Schlichting, 1986; Shi and Cai, 2009). The analysis of leaf plasticity confirms the observed structural and functional differences among the considered species. Total plasticity index (PI = 0.25, mean value of the considered species) is in the general range of the Mediterranean species (Gratani and Bombelli, 2000; Gratani et al., 2006; Zunzunegui et al., 2011). In particular, however, C. salvifolius and C. incanus are characterized by a larger PIa (0.14, mean value) that seems to be related to their wider ecological distribution and the more favorable conditions of the environments where they naturally occur, conforming with results of Valladares et al. (2000) and Gratani et al. (2003). Yadav et al. (2004) underline that high plasticity in the leaf mesophyll, in concert with other leaf traits, seems to enable a species to cope with different environmental regimes and, as a result, to attain a wider habitat range. The highest PIm (0.29) of C. monspeliensis indicates that leaf morphology (i.e. low LA, high LMA and LTD) can have a highly adaptive role in highly stressed environments, like fire degraded Mediterranean areas in which this species occurs. In particular, leaf morphological plasticity plays an important role in resource acquisition (Crick and Grime, 1987; Navas and Garnier, 2002; Yamashita et al., 2000) and can limit photodamages to the photosynthetic apparatus. This is particularly

important during summer drought, when the risk of oxidative damage is the highest due to an excess of radiant energy associated to high air temperature and water stress. The morphological leaf plasticity could be considered an adaptive plasticity that favors the plant’s fitness over time (Nicotra et al., 2010). Moreover, the high plasticity of the considered Cistus species at the physiological level (PIp ) is in accordance to the results of Gratani et al. (2003, 2006) and Zunzunegui et al. (2011) for Mediterranean species. High leaf plasticity mainly in physiological traits seems to be linked to an increased capacity to survive and grow in areas of potentially intense solar radiation (Valladares et al., 2002). The high PIp is probably rather important enabling the Cistus species to colonize strongly degraded areas, thus contributing to the re-vegetation, especially at the first successional stages of the Mediterranean maquis after fire (Arianoutsou and Margaris, 1981; Bastida and Talavera, 2002; Gratani and Amadori, 1991; Thanos and Georghiou, 1988). Gratani and Amadori (1991) underline that four to six years after fire occurrence Cistus species, which grow under full sun conditions, are partially replaced by sclerophyllous species that in the following years will form mixed dense shrubs providing distinctly suboptimal conditions for Cistus. The three investigated species differ under these aspects: C. monspeliensis is favored in more xeric and open areas where maquis changes into garrigue, and in particular in areas repeatedly subjected to fire. On the contrary, C. salvifolius might have a more competitive advantage, compared to the other Cistus species, in mountain areas also in consideration of the increase of air temperatures, C. incanus can be placed in between the two other taxa. This characterization can be important for land management projects of habitat restoration, considering the possible increase in the next future of degraded areas due to fragmentation, overgrazing and excessive wildfires in the Mediterranean region which is one of the most heavily utilized by man (Boix-Fayos et al., 2009).

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Acknowledgements This paper was supported by the grants from Ministry of Agricultural, alimentary and Forestry politicians (MIPAF) for the years 2007–2010.

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