Acta Botanica 1-2015 - za web.indd ACTA BOT. CROAT. 74 (1), 2015 71 Acta Bot. Croat. 74 (1), 71–94, 2015 CODEN: ABCRA 25 ISSN 0365-0588 eISSN 1847-8476 Carbon gain optimization in fi ve broadleaf deciduous trees in response to light variation within the crown: correlations among morphological, anatomical and physiological leaf traits ROSANGELA CATONI1*, LORETTA GRATANI1, FRANCESCO SARTORI2, LAURA VARONE1, MIRKO U. GRANATA2 1 Sapienza University of Rome, Department of Environmental Biology, P. le A. Moro, 5, 00185 Rome, Italy 2 University of Pavia, Department of Earth and Environmental Sciences, Via S. Epifanio 14, 27100 Pavia, Italy Abstract – Leaf trait variations in fi ve deciduous species (Quercus robur, Corylus avella- na, Populus alba, Acer campestre, Robinia pseudoacacia) growing in an old broadleaf deciduous forest in response to light variation within the tree crown was analyzed. Net photosynthetic rate (PN), leaf respiration rate (R) and the photosynthetic nitrogen use effi - ciency were, on average, more than 100% higher in sun than in shade leaves. A. campestre and C. avellana sun leaves had the highest specifi c leaf area (SLA, 156.0 ± 17.9 cm2 g–1) and the lowest total leaf thickness (L, 101.9 ± 8.8 μm) underlining their shade-tolerance. Among the shade-intolerant species (Q. robur, P. alba and R. pseudoacacia), Q. robur had the lowest SLA and the highest L in sun leaves (130.6 ± 10.0 cm2 g–1 and 160.8 ± 9.6 μm, respectively) since shade-intolerant species typically have thicker leaves. The higher PN decrease in respect to R decrease from sun to shade leaves attested the higher sensitivity of PN than R to light variations within the crown. This determined a 69% lower R/PN in sun than in shade leaves. This result is further attested by the signifi cant correlation between PN and the relative photosynthetic photon fl ux density. The shade-tolerant species have a 76% higher R/PN ratio than the shade-intolerant ones. The measured leaf phenotypic plas- ticity (PI = 0.35) was in the range of broadleaf deciduous species. Plasticity is a key trait useful to quantify plant response to environmental stimuli. It is defined as the ability of a genotype to produce different phenotypes depending on the environment. Among the con- sidered species, Q. robur showed the highest PI (0.39) and P. alba the lowest (0.29). Knowledge on phenotypic plasticity is important in making hypotheses about the dynam- ics of the studied forest in consideration of environmental stress factors, including inva- sive species competition and global climate change. Keywords: deciduous trees, forest, gas exchange, light gradient, shade tolerance, specifi c leaf area * Corresponding author, e-mail: rosangela.catoni@uniroma1.it Copyright® 2015 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. CATONI R., GRATANI L., SARTORI F., VARONE L., GRANATA M. U. 72 ACTA BOT. CROAT. 74 (1), 2015 Abbreviations: PN – net photosynthesis; gs – stomatal diffusive conductance to water va- pour; E – transpiration rates; R – leaf respiration rates; R/ PN – ratio of leaf respiration to photosynthesis; PNUE – photosynthetic nitrogen use; Na – leaf nitrogen content per unit of leaf area; Chl a + b – chlorophyll a + b; Chl/Car – ratio of chlorophyll to carotenoid con- tent; Chl a/b – ratio of chlorophyll a to chlorophyll b content; Chl/N – ratio of chlorophyll to nitrogen content; LA – projected leaf surface area; DM – leaf dry mass; SLA – specific leaf area. Introduction In forest ecosystems, the heterogeneous light environment within a tree crown due to self-shading and shading by neighboring trees (SACK et al. 2006, WYKA et al. 2012) deter- mines leaf trait variations (ELLSWORTH and REICH 1993, GRATANI 1997, GRATANI and FOTI 1998, GRATANI et al. 2006, YOSHIMURA 2010). Large trees support leaves acclimated to high light intensities (sun leaves) in the upper canopy and may produce leaves acclimated to low light intensities (shade leaves) in lower canopy layers (HÖLSCHER 2004). The range of varia- tions refl ects the optimization of whole plant gas exchange and the resource investment strategy (GRATANI 1997, WYKA et al. 2012). Most studies of plant response to light have been aimed at underlining the ecological implications of the tolerance to extremes (i.e. tol- erance to sun and shade), but noticeably less effort has been invested in the exploration of trends in the plastic response to light (VALLADARES et al. 2000). Sun leaves with respect to shade leaves generally exhibit a higher photosynthetic rate on a leaf area basis, a higher Chl a to Chl b ratio, a lower light-harvesting Chl a/b protein (LHCP), a lower stacking degree of thylakoids (LICHTENTHALER et al. 1982), and a higher nitrogen (N) content per unit of leaf area (HIKOSAKA 2005) since approximately half of N is invested in photosynthetic proteins (EVANS 1989). HIROSE and WERGER (1987) suggest that N varies with light availability in the plant crown in such a way as to optimize daily crown photosynthesis. Differentiation in N distribution patterns in sun and shade leaves within the crown increases the crown photo- synthetic nitrogen use effi ciency (PNUE) (HIKOSAKA 2003). PNUE underlines the effi cien- cy with which species utilize N to grow (GARNIER et al. 1995). There is a strong relationship between photosynthesis and respiration (R) as respiration relies on photosynthetic sub- strates (ATKIN et al. 2007). Plant respiration accounts for a large fraction of carbon cycling in forest ecosystems and may be of comparable importance to photosynthesis as a determi- nant of net primary productivity (RYAN et al. 1997). In general, upper-canopy leaves had much higher leaf respiration than lower-canopy leaves, refl ecting a greater metabolic activ- ity related to a higher light availability and thus, higher net photosynthetic rates (GUNDER- SON et al. 2002). At a morphological level, the specifi c leaf area (SLA) varies vertically in the forest canopy (NIINEMETS 1995) altering the amount of light that can be intercepted per unit of leaf dry mass (EVANS and POORTER 2001). In general, plants growing in high light conditions have thicker leaves with a lower SLA than plants growing in shade conditions (BJÖRKMAN 1981) partially due to longer or extra layers of palisade cells (HANSON 1917). Species can be classifi ed into obligate or facultative shade plants and obligate or facul- tative sun plants (DAMASCOS and RAPOPORT 2002). However, comparative studies indicate that there are few species that are either extremely shade-tolerant or light-demanding, with most species having intermediate and thus, overlapping light preferences (WRIGHT et al. 2003). One of the components of shade tolerance resides in the ability of individuals to op- CARBON GAIN OPTIMIZATION IN RESPONSE TO LIGHT GRADIENT WITHIN THE TREE CROWN ACTA BOT. CROAT. 74 (1), 2015 73 timize carbon gain under low light environments, by maximizing light interception and minimizing carbon loss by respiration (WALTERS and REICH 1999), according to the carbon gain hypothesis (VALLADARES and NIINEMTES 2008). Light competition and inter-specific differences in shade tolerance are frequently important determinants of forest structure and dynamics (GRAVEL et al. 2010). Nevertheless, the potential correlation between plasticity of morphological and physiological leaf traits and the tolerance to shade is still under discus- sion (ROBAKOWSKI et al. 2003, PORTSMUTH and NIINEMETS 2007). A frequent assumption in literature is that shade-intolerant species display a larger plasticity than shade-tolerant ones (STRAUSS DE BENEDETTI and BAZZAZ 1991), although the reverse is sometimes found. Other authors have observed a large variability in plasticity that depended on the studied traits with no real connection to shade tolerance (VALLADARES et al. 2000). The main objective of this research was to analyze leaf trait variations in response to the light gradient within the crown of fi ve tree species growing in an old broadleaf deciduous forest characterized by different tolerance to shade. Intra-canopy plasticity has important impacts on many aspects of tree biology, potentially contributing to whole-crown perfor- mance via effects on light penetration through the crown and on the energy, carbon, and water balance of individual leaves (HIKOSAKA 2005). Considering that information about the correlation between plasticity and shade tolerance is limited and the evidence is con- trasting (PORTSMUTH and NIINEMETS 2007), we tested the relationship between leaf trait plas- ticity and shade tolerance of the selected trees, assuming that shade-tolerant tree species might have a lower plasticity index than the shade-intolerants. Material and methods The study was carried out in the period May – July 2012 in an old broadleaf deciduous forest developing in the Natural Reserve Siro Negri (45°12'39"N; 09°03'26"E, 74 m a. s. l, Italy) extending over 10 ha. The forest largely covered the fl uvial valleys along the Ticino river from the 19th century, and no logging has been carried out since the establishment of the reserve in 1970 (SARTORI 1984, CASTAGNERI et. al. 2013). The Reserve represents one of the best conserved relicts of the original alluvial forest which in the past largely covered the banks of the Ticino river, and contains trees that are more than 100-years old (CASTAGNERI et al. 2013). The Reserve has been reported as part of a Sites of Community Importance (SIC) IT 2080014 »Bosco Siro Negri e Moriano«. The following broadleaf species were considered for measurements: Acer campestre L., Corylus avellana L., Populus alba L., Robinia pseudoacacia L. and Quercus robur L. C. avellana is a shade-tolerant species (KULL and NIINEMETS 1993, GRATANI and FOTI 1998) growing in the understory of European deciduous forests (TUTIN 2001) and able to colonize large gaps (KULL and NIINEMETS 1993). A. campestre is a shade-tolerant species growing in the understory of deciduous mixed forests in Europe (MILLS 1996). P. alba is a shade-intolerant species (COOPER et al. 1999) with a great geographic distribution, including the Centre and South of Europe, North Africa, Western and Central Asia (FAO 1980) where it grows on the banks of rivers (DELLEDONNE et al. 2001). R. pseudoacacia is a shade-intol- erant species (MOTTA et al. 2009) originating from the south-eastern United States (FER- RARIS et al. 2000). The dispersion of the R. pseudoacacia into Italian native forests started in CATONI R., GRATANI L., SARTORI F., VARONE L., GRANATA M. U. 74 ACTA BOT. CROAT. 74 (1), 2015 the early 20th century (MOTTA et al. 2009). It is mainly a threat for nutrient-poor sites based on its ability to fix nitrogen by symbiosis (ROLOFF et al. 1994). This species can cause an unwanted and long-lasting shift in vegetation composition toward nitrogen rich and spe- cies-poor plant communities (KOWARIK 2010). Q. robur is a predominant European oak spe- cies (SCOTTI-SAINTAGNE et al. 2004) growing in more open habitats; it is a more light–de- manding species (NIINEMETS 1996, VALLADARES et al. 2002). Meteorological data The climate of the area was characterized by a mean annual rainfall of 654 mm, most of it falling in spring and autumn. The mean minimum air temperature (Tmin) of the coldest month (January) was –0.2 ± 1.8 °C, the mean maximum air temperature (Tmax) of the hottest month (July) was 30.1 ± 1.3 °C and the mean annual temperature (Tm) was 13.7 ± 8.2 °C. In the period May – July, total rainfall was 136 mm, Tm 22.2 ± 3.1 °C and Tmax (July) 30.3 ± 1.2 °C (Lombardia Regional Agency for Environmental Protection, Meteorological Station of Pavia, Ponte Ticino SS35, data for the period 2002 to 2012). Floods occurred sporadically every 5–10 years during the last 40 years, with water levels up to 1.50 m height in the forest during exceptional events (MOTTA et al. 2009, CASTAGNERI et al. 2013). On average, ground- water level was around –4.50 m in winter reaching –3.50 m in summer due to irrigation in the surrounding areas (SARTORI, unpublished). Forest measurements and microclimate Measurements of forest structure were carried out in 10 representative sample areas (400 m2 each) randomly selected in the considered forest. Plant traits included: plant height (H) and the diameter at breast height (D). Total forest density and total tree basal area were calculated. Leaf area index (LAI) was estimated at the end of June by the »LAI2000 Plant Canopy Analyzer« (LI-COR Inc., Lincoln, Nebraska, USA). Microclimate and leaf trait measurements were made by an aerial lift for the selected species (four representative mature plants per species) at the top (sun leaves) and at the bot- tom (shade leaves) of the crown of each considered tree, according to SACK et al. (2006). The photosynthetic photon fl ux density (PPFD, μmol photons m–2 s–1) was measured at the top and at the bottom of the crown for each sampled tree by a quantum radiometer photom- eter (LI-189 LI-COR, USA) with the quantum sensor LI-190SA. Simultaneously, PPFD was determined in the open near the forest, in order to provide references irradiance level for calculation of relative irradiance (PPFD%), according to WYKA et al. (2012). Measure- ments were carried out in June and July on overcast days from 09.00 a. m. to 12.00 p. m to provide a reliable estimate of the average light conditions during the »in-leaf« growing sea- son, according to TOBIN and REICH (2009). Air humidity (RH, %) and air temperature (Ta, °C) were measured by thermo-hygrometers (HD8901, Delta Ohm, It). Leaf morphology and anatomy Fully expanded leaves (n = 20 per species and per crown position) were collected from the selected trees at the end of June, sealed in plastic bags and transported immediately to the laboratory for measurements. Measurements included leaf surface area (LA, cm2), ob- tained by the image analysis system (Delta-T Devices, UK), and leaf dry mass (DM, mg), CARBON GAIN OPTIMIZATION IN RESPONSE TO LIGHT GRADIENT WITHIN THE TREE CROWN ACTA BOT. CROAT. 74 (1), 2015 75 determined by drying leaves at 80 °C to constant mass. Specifi c leaf area (SLA, cm2 g−1) was calculated by the ratio of LA and DM. Fresh leaf sections from fully expanded leaves (n = 10 per species and per crown posi- tion) were hand cut and analyzed by light microscopy using an image analysis system (AR- KON, A&P, I). The following parameters were measured: total leaf thickness (L, μm), pali- sade and spongy parenchyma thickness, adaxial and abaxial epidermis and cuticle thickness. All measurements were restricted to vein-free areas. Gas exchange Gas exchange measurements were carried out in June – July (fi ve leaves per species per crown position per each sampling occasion), from 09:00 a. m. to 12:00 p. m. to ensure that the maximum rates were measured (REICH et al. 1995). Leaves were retained in their natural position during measurements. Net photosynthetic rate (PN, μmol CO2 m−2 s−1), stomatal conductance to water vapor (gs, mol H2O m−2 s−1), leaf transpiration rate (E, mmol H2O m−2 s−1), photosynthetic photon fl ux density (PPFD, μmol photons m−2 s−1) and leaf temperature (Tl, °C) were measured by an infrared gas analyzer (LC-Pro+, ADC, UK) equipped with a leaf chamber (PLC, Parkinson Leaf Chamber, UK). On each sampling occasion, respiration rates (R, μmol CO2 m−2 s−1) were measured after PN ones (on the same leaves) as CO2 effl ux, by darkening the leaf chamber with a black paper, according to CAI et al. (2005) for 30 min prior to each measurement, to avoid the release of CO2 transient post-irradiation bursts (ATKIN et al. 1998). The shown R and PN rates represented the mean values of three days of measurements per month characterized by the same weather conditions, under clear sky. The ratio between R and PN was calcu- lated. Leaf nitrogen and pigment content Leaf samples were collected on the same occasions as those for gas exchange measure- ments. Immediately after collection leaf samples were kept cool in the dark and transported immediately to the laboratory. Leaf nitrogen content per leaf area (Na, g m–2) was determined by drying leaf samples at 70 °C (6 leaf samples, 0.5 g of leaf dry mass each, per species and per crown position in each sampling occasion) and grinding them into a fi ne powder. The N content was mea- sured by the Kjeldahl method (MENDES et al. 2001). Photosynthetic nitrogen use effi ciency (PNUE, μmol CO2 g−1 Na s−1) was calculated by the ratio between PN rates and Na content. Chlorophyll content (Chl, mg g−1 fresh weight) and carotenoid content (Car, mg g−1 fresh weight) were determined after grinding leaves in acetone (6 samples, 1.5 g of leaf fresh weight each, per species and per crown position in each sampling occasion). The ho- mogenates were centrifuged in a refrigerated centrifuge (4237R. A. L. C., I). Absorbance of the supernatants was measured by a Jasco model 7800LCD (Japan) spectrophotometer at the wavelengths of 645, 663, and 440 nm. Chl content was calculated according to MA- CLACHLAN and ZALIK (1963) and Car content according to HOLM (1954). The Chl a + b con- tent, the ratio Chl a/b, the ratio Chl/Car, and the ratio Chl/N were calculated. CATONI R., GRATANI L., SARTORI F., VARONE L., GRANATA M. U. 76 ACTA BOT. CROAT. 74 (1), 2015 Plasticity index The plasticity index of morphological (PIm), anatomical (PIa) and physiological (PIp) leaf traits for each of the species was calculated by the difference between the minimum and the maximum mean value between sun and shade leaves divided by the maximum mean value, according to VALLADARES et al. (2000). The mean plasticity index (PI), which had a scale ranging from 0 to 1, was calculated by averaging PIm, PIa and PIp per species, according to VALLADARES et al. (2000). Statistics All statistical tests were performed using a statistical software package (Statistica, Stat- soft, USA). Differences in the considered leaf traits were determined by the analysis of variance (ANOVA) and the Tukey test for multiple comparisons. Kolmogorov–Smirnov and Levene tests were used to verify the assumption of normality and homogeneity of variances, re- spectively. Regression analysis was carried out to examine the relationship among the considered leaf traits. Principal component analysis (PCA) was carried out in order to detect structure in the correlations between the considered leaf traits (PN, gs, E, R, R/PN, Na, PNUE, Chl/N, Chl a+b, Chl a/b, Chl /Car, SLA, leaf thickness, palisade parenchyma thickness, spongy paren- chyma thickness, adaxial epidermis thickness, adaxial cuticle thickness, abaxial epidermis thickness, abaxial cuticle thickness). The analysis was performed on a standardized matrix. The matrix was subjected to a rotated principal component analysis with the objective of summarizing the main factors determining the variation of the analyzed leaf traits. In order to evaluate the similarity among the species in terms of phenotypic plasticity a correspondence analysis (CA) was carried out. The analysis was performed using the spe- cies as group variable and PI as category variable. In particular, three PI categories were considered: physiological, morphological and anatomical PI. Results Forest structure and microclimate The forest structure was characterized by a dominant tree layer consisting of P. alba, Populus nigra L., Q. robur and R. pseudoacacia, (H = 30 ± 3 m), a dominated tree layer of R. pseudoacacia, A. campestre and C. avellana (H = 20 ± 3 m), two shrub layers of Sambu- cus nigra L., Ulmus minor Mill. and C. avellana (H = 6.0 ± 1.5 m and 1.5 ± 0.5 m, respec- tively) and a grass layer (Tab. 1). Total tree density was 237 ± 100 stems ha–1 and the total basal area 74.5 ± 24.6 m2 ha–1. LAI was 4.5 ± 0.3. On average, PPFD% ranged from 6.1 ± 3.5% at the bottom to 97.4 ± 1.1% at the top of the crown. The Ta and RH ranged from 28.1 ± 0.5 °C and 48.0 ± 0.9%, respectively, at the bottom, to 30.4 ± 0.6 °C and 35.1 ± 2. 1%, respectively, at the top of the crown of the trees considered. CARBON GAIN OPTIMIZATION IN RESPONSE TO LIGHT GRADIENT WITHIN THE TREE CROWN ACTA BOT. CROAT. 74 (1), 2015 77 Leaf morphology There were signifi cant differences between sun and shade leaves among the species (Tab. 2). On average, SLA was 81% higher in shade than in sun leaves. In particular, C. avellana had the highest SLA both in sun and in shade leaves (168.8 ± 15.1 cm2 g–1 and 400.7 ± 60.0 cm2 g–1, respectively), Q. robur the lowest SLA in sun leaves (130.6 ± 10.0 cm2g–1) and P. alba the lowest SLA in shade leaves (168.4 ± 16.8 cm2 g–1). Leaf anatomy On average, L and palisade parenchyma thickness were 23% and 38% higher in sun than in shade leaves, respectively, and spongy parenchyma thickness was 17% higher in sun than in shade leaves (Tab. 3). In particular, Q. robur had the highest L thickness in sun leaves (160.8 ± 9.6 μm), P. alba in shade leaves (128.9 ± 4.7 μm) and C. avellana the low- est L thickness both in sun and in shade leaves (95.7 ± 10.1 μm and 85.3 ± 5.3 μm, respec- tively). The ratio between palisade parenchyma thickness and L in sun leaves was the high- est in R. pseudoacacia (55%) and the lowest in C. avellana (33%), while in shade leaves it was the highest in P. alba (53%) and the lowest in C. avellana (30%). Q. robur had the highest spongy parenchyma thickness, the highest adaxial and abaxial epidermis thickness and cuticle thickness both in sun and in shade leaves, and C. avellana the lowest palisade parenchyma thickness, adaxial and abaxial epidermis thickness and cuticle thickness both in sun and in shade leaves. Tab. 1. Structural traits of the considered species; H – plant height, D – diameter at breast height. Species H (m) D (cm) Acer campestre 15 ± 6 33.0 ± 7.9 Corylus avellana 8 ± 2 12.3 ± 3.6 Populus alba 28 ± 5 79.5 ± 26.8 Robinia pseudoacacia 20 ± 6 27.2 ± 8.8 Quercus robur 26 ± 5 75.3 ± 8.0 Tab. 2. Morphological leaf traits of sun and shade leaves of Acer campestre, Corylus avellana, Pop- ulus alba, Quercus robur and Robinia pseudoacacia. Mean values (± SD) are shown (n = 20). Mean values with the same letters are not significantly different between sun and shade leaves (Tukey test, p ≥ 0.05). LA – projected leaf surface area; DM – leaf dry mass; SLA – specific leaf area. A. campestre C. avellana P. alba Q. robur R. pseudoacacia sun shade sun shade sun shade sun shade sun shade LA (cm2) 20.2± 4.2a 57.7± 7.7b 31.0± 9.6a 88.5± 17.8b 14.8± 3.2a 26.8± 2.1b 19.8± 3.0a 50.6± 11.1b 130.1± 22.5a 470.4± 73.8b DM (mg) 157.4± 41.4a 198.4± 31.2b 190.8± 57.5a 227.2± 62.8b 109.6± 24.4a 161± 22.2b 150.9± 33.7a 263.4± 59.4b 932± 168a 1838± 376b SLA (cm2 g–1) 143.2± 15.1a 305.1± 35.3b 168.8± 15.1a 400.7± 60.0b 135.6± 7.3a 168.4± 16.8b 130.6± 10.0a 192.7± 10.2b 139.9± 4.8a 258.3± 15.4b CATONI R., GRATANI L., SARTORI F., VARONE L., GRANATA M. U. 78 ACTA BOT. CROAT. 74 (1), 2015 Gas exchange On average, PN was more than twice as high in sun leaves than in shade leaves. PN ranged between 20.3 ± 0.3 μmol m–2 s–1 (P. alba) and 5.8 ± 0.1 μmol m–2 s–1 (C. avellana) in sun leaves, and between 3.6 ± 0.3 μmol m–2 s–1 (P. alba) and 1.8 ± 0.2 μmol m–2 s–1 (A. campestre) in shade leaves (Tab. 4). The gs had the same PN trend with the highest rates in sun leaves (0.20 ± 0.07 mol m–2 s–1) decreasing, on average, by 49% in shade leaves. In particular, P. alba had the highest gs in sun leaves (0.27 ± 0.06 mol m–2 s–1) and C. avellana the lowest one (0.08 ± 0.01 mol m–2 s–1). R. pseudoacacia showed the highest gs in shade leaves (0.19 ± 0.02 mol m–2 s–1) and C. avellana the lowest (0.02 ± 0.01 mol m–2 s–1). There was a signifi cant (p < 0.01) positive correlation between PN and gs and between PN and palisade parenchyma thickness, showing that 46% and 63% of PN variations were ex- plained by the two parameters, respectively. The highest R were monitored in sun leaves (1.10 ± 0.33 μmol m–2 s–1) decreasing by 36% in shade leaves (Tab. 4). Among the considered species, R. pseudacacia had the high- est R both in sun and in shade leaves (1.33 ± 0.23 and 1.18 ± 0.06 μmol m–2 s–1, respective- ly), C. avellana had the lowest R in sun leaves (0.60 ± 0.07 μmol m–2 s–1) and Q. robur in shade leaves (0.21 ± 0.06 μmol m–2 s–1). The ratio R/PN was the highest in shade leaves (more than 100%), A. campestre having the highest R/PN ratio both in sun and in shade leaves (0.17 ± 0.01 and 0.63 ± 0.18, respec- tively). Tab. 3. Anatomical leaf traits of sun and shade leaves of Acer campestre, Corylus avellana, Populus alba, Quercus robur and Robinia pseudoacacia. Mean values (± SD) are shown (n = 10). Mean values with the same letters are not significantly different between sun and shade leaves (Tukey test, p ≥ 0.05). Leaf traits A. campestre C. avellana P. alba Q. robur R. pseudoacacia sun shade sun shade sun shade sun shade sun shade Leaf thickness (μm) 108.2± 6.7a 93.2± 1.4b 95.7± 10.1a 85.3± 5.3a 142.6± 8.3a 128.9± 4.7b 160.8± 9.6a 120.8± 5.9b 146.3± 12.0a 101.9± 2.5b Palisade parenchyma thickness (μm) 42.0± 2.4a 39.6± 0.9a 31.9± 7.5a 25.6± 5.4a 75.3± 2.4a 68.4± 5.0b 60.7± 3.9a 37.3± 3.5b 80.2± 9.7a 43.4± 5.1b Spongy parenchyma thickness (μm) 43.2± 5.5a 34.2± 2.5b 46.0± 8.5a 44.4± 4.6a 46.7± 5.3a 42.3± 6.7a 71.7± 4.9a 56.9± 5.0b 51.2± 11.2a 42.6± 4.6a Adaxial epidermis thickness (μm) 12.4± 1.6a 10.5± 2.8a 9.2± 0.7a 7.6± 2.1a 11.2± 1.7a 10.7± 0.8a 15.2± 1.6a 14.5± 3.0a 8.2± 1.1a 7.9± 1.4a Adaxial cuticle thickness (μm) 1.0± 0.2a 0.9± 0.1a 0.9± 0.2a 0.8± 0.1a 1.1± 0.3a 1.0± 0.3a 1.3± 0.4a 1.1± 0.4a 1.0± 0.3a 0.9± 0.2a Abaxial epidermis thickness (μm) 8.7± 1.6a 7.4± 0.9a 7.0± 1.3a 5.7± 0.9a 8.0± 1.9a 5.7± 0.6a 10.9± 2.1a 10.3± 1.4a 7.0± 1.3a 6.2± 0.8a Abaxial cuticle thickness (μm) 0.9± 0.1a 0.8± 0.1a 0.7± 0.1a 0.6± 0.1a 0.8± 0.2a 0.7± 0.1a 1.0± 0.4a 0.9± 0.2a 0.9± 0.1a 0.8± 0.1a CARBON GAIN OPTIMIZATION IN RESPONSE TO LIGHT GRADIENT WITHIN THE TREE CROWN ACTA BOT. CROAT. 74 (1), 2015 79 Leaf nitrogen content and pigment content The Na was, on an average, 54% higher in sun than in shade leaves, R. pseudoacacia having the highest Na in sun leaves (2.51 ± 0.08 g m–2) and A. campestre the lowest one (0.91 ± 0.11 g m–2). P. alba had the highest Na in shade leaves (1.56 ± 0.04 g m–2). PNUE was 8.4 ± 3.0 μmol g–1 s–1 in sun leaves, decreasing by 68% in shade leaves, P. alba having the highest value in sun leaves (12.7 ± 0.2 μmol g–1 s–1) and C. avellana the lowest one (4.2 ± 0.2 μmol g–1 s–1). The Chl a + b was 82% higher in shade than in sun leaves. Among the considered spe- cies, Q. robur and P. alba had the highest Chl a + b in sun leaves (1.61 ± 0.26 mg g–1) and C. avellana the lowest both in sun and in shade leaves (0.76 ± 0.10 and 1.14 ± 0.20 mg g–1, respectively). A. campestre had the highest Chl a + b in shade leaves (3.61 ± 0.39 mg g–1). The ratio Chl a/b was 8% higher in sun than in shade leaves, P. alba having the highest ra- tio both in sun and in shade leaves (2.80 ± 0.03 and 2.72 ± 0.27, respectively), R. pseudo- acacia the lowest ratio in sun leaves (2.02 ± 0.03) and Q. robur in shade leaves (1.85 ± 0.04) (Fig. 1A). The ratios Chl/Car and Chl/N were 32% and 44% higher in shade than in sun leaves, respectively. In particular, among the species considered, Q. robur had the highest Chl/Car in sun leaves (7.84 ± 0.45) and A. campestre in shade leaves (9.84 ± 0.87). The ratio Chl/N was the highest in A. campestre both in sun and in shade leaves (0.102 ± 0.010 and 0.174 ± 0.014, respectively) (Figs. 1B and 1C). Plasticity index The PI for the considered species was, on average, 0.35, Q. robur having the highest PI (0.39), followed by R. pseudoacacia (0.38), A. campestre (0.34), C. avellana (0.33) and P. alba (0.29) (Tab. 5). PIm was, on average, 0.45, R. pseudoacacia showing the highest value (0.56) and P. alba the lowest (0.32). Among the considered leaf morphological traits, LA had the largest variation (PILA = 0.62, mean value). The PIa was, on average, 0.15, R. pseudoacacia show- ing the highest (0.18) and P. alba the lowest value (0.12). Among the considered anatomi- cal leaf traits, palisade parenchyma thickness had the largest variation (0.24, mean value). The PIp was, on an average, 0.44, Q. robur having the highest value (0.53). Among the con- sidered physiological traits, PN had the largest variation (0.79, mean value). Tab. 4. Values of photosynthesis and respiration rates of sun and shade leaves of Acer campestre, Corylus avellana, Populus alba, Quercus robur and Robinia pseudoacacia. Mean values (± SD) are shown (n = 15). Mean values with the same letters are not significantly different be- tween sun and shade leaves (Tukey test, p ≥ 0.05). PN – net photosynthesis; R – leaf respira- tion rate. A. campestre C. avellana P. alba Q. robur R. pseudoacacia sun shade sun shade sun shade sun shade sun shade PN (μmol m–2 s–1) 7.5± 0.2a 1.8± 0.5b 5.8± 0.1a 1.9± 0.6b 20.3± 0.3a 3.6± 0.3b 16.2± 0.3a 2.0± 0.1b 19.5± 1.5a 2.6± 0.2b R (μmol m–2 s–1) 1.30± 0.09a 1.12± 0.07b 0.60± 0.07a 0.53± 0.07b 1.32± 0.36a 0.48± 0.05b 0.92± 0.32a 0.21± 0.06b 1.33± 0.23a 1.18± 0.06a CATONI R., GRATANI L., SARTORI F., VARONE L., GRANATA M. U. 80 ACTA BOT. CROAT. 74 (1), 2015 a a b a a A A B A A A. campestre C. avellana P. alba Q. robur R. pseudoacacia 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 C h l a / b A) ab ab bc b a B A AB B A A. campestre C. avellana P. alba Q. robur R. pseudoacacia 0 2 4 6 8 10 12 C h l / C a r B) c a b b a C A B B A A. campestre C. avellana P. alba Q. robur R. pseudoacacia 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 C h l / N C) Fig. 1. A) Ratio of chlorophyll a to chlorophyll b (Chl a/b), B) ratio of chlorophyll to carotenoid content (Chl /Car) and C) ratio of chlorophyll to nitrogen content (Chl/N) of Acer campestre, Corylus avellana, Populus alba, Quercus robur and Robinia pseudoacacia. Lowercase and capital letters indicate the inter-specifi c differences in sun (white bars) and shade (grey bars) leaves, respectively. The means with the same letters are not signifi cantly different (ANOVA, p ≥ 0.05). Mean values (± SD) are shown (n = 36). CARBON GAIN OPTIMIZATION IN RESPONSE TO LIGHT GRADIENT WITHIN THE TREE CROWN ACTA BOT. CROAT. 74 (1), 2015 81 Regression and multivariate analysis There was a signifi cant (p < 0.01) positive correlation between PN and Na and a signifi - cant (p < 0.01) negative correlation between SLA and L, 52% of PN variations being ex- plained by Na, and 51% of SLA variation by L. The regression analysis between PN and PPFD% showed a high correlation coeffi cient between these two variables (r = 0.81). Tab. 5. Phenotypic plasticity index for physiological (PIp), morphological (PIm) and anatomical (PIa) leaf traits of Acer campestre, Corylus avellana, Populus alba, Quercus robur and Robinia pseudoacacia. PN – net photosynthesis, gs – stomatal diffusive conductance to water vapour, E – transpiration rates, R – leaf respiration rates, R/ PN – ratio of leaf respiration to photosyn- thesis, PNUE – photosynthetic nitrogen use, Na – leaf nitrogen content per unit of leaf area, Chl a + b – chlorophyll a + b content, Chl/Car – ratio of chlorophyll to carotenoid content, Chl a/b – ratio of chlorophyll a to chlorophyll b content, Chl/N – ratio of chlorophyll to ni- trogen content, LA – projected leaf surface area, DM – leaf dry mass, SLA – specific leaf area, PI – mean plasticity index. Plasticity index A. campestre C. avellana P. alba Q. robur R. pseudo- acacia Physiological traits PN 0.76 0.65 0.82 0.87 0.86 gs 0.29 0.75 0.63 0.51 0.09 E 0.11 0.73 0.74 0.65 0.60 R 0.14 0.12 0.64 0.77 0.11 R/PN 0.73 0.66 0.50 0.67 0.87 PNUE 0.68 0.27 0.82 0.90 0.75 Na 0.26 0.53 0.03 0.24 0.46 Chl a+b 0.66 0.33 0.28 0.53 0.16 Chl/Car 0.47 0.08 0.19 0.09 0.24 Chl a/b 0.02 0.03 0.03 0.21 0.07 Chl/N 0.41 0.25 0.13 0.44 0.17 Mean value PIp 0.41 0.40 0.44 0.53 0.40 Morphological traits LA 0.65 0.65 0.45 0.61 0.72 DM 0.21 0.16 0.32 0.43 0.49 SLA 0.53 0.58 0.19 0.32 0.46 Mean value PIm 0.46 0.46 0.32 0.45 0.56 Anatomical traits Leaf thickness 0.14 0.11 0.10 0.25 0.30 Palisade parenchyma thickness 0.06 0.20 0.09 0.39 0.46 Spongy parenchyma thickness 0.21 0.03 0.09 0.21 0.17 Adaxial cuticle thickness 0.10 0.11 0.09 0.15 0.10 Abaxial cuticle thickness 0.11 0.14 0.12 0.10 0.11 Adaxial epidermis thickness 0.15 0.17 0.04 0.04 0.04 Abaxial epidermis thickness 0.15 0.18 0.28 0.06 0.11 Mean value PIa 0.13 0.13 0.12 0.17 0.18 PI 0.34 0.33 0.29 0.39 0.38 CATONI R., GRATANI L., SARTORI F., VARONE L., GRANATA M. U. 82 ACTA BOT. CROAT. 74 (1), 2015 PCA extracted two factors which accounted for 56.5% of the total variance and of which 38.8% was due to factor 1 and 17.8% to factor 2 (Fig. 2). The analysis showed a co- linearity among the considered leaf traits. Factor 1 was signifi cantly correlated to PN (r = 0.92), total leaf thickness (r = 0.88), E (r = 0.86), SLA (r = –0.84), PNUE (r = 0.82), Na (r = 0.78), palisade parenchyma thickness (r = 0.77) and R/PN (r = – 0.71). Factor 2 was signifi - cantly correlated to Chl a + b (r = 0.77), adaxial epidermis thickness (r = 0.73) and Chl/Na (r = 0.73). Along the factor 1, which explained most of the total variance, sun leaves were completely separated from shade leaves. Among the considered species, A. campestre and C. avellana sun leaves were closer to shade leaf behavior and P. alba shade leaves were closer to sun leaf behavior. The CA showed a greater similarity between P. alba and Q. robur because of the similar PIp while A. campestre and C. avellana were closer on the base of PIm. R. pseudoacia was further from the other species (Fig. 3). Discussion Light gradient within the crown in a forest ecosystem depends on the structure and stage of development of trees (LARCHER 2003). Moreover, the distribution, size and orienta- tion of leaves control such processes as leaf development, leaf water use, and photosynthe- sis (NORMAN and CAMPBELL 1989). The relationship between canopy structure and the spa- tial distribution of light availability differs between primary and secondary growth forests (NICOTRA et al. 1999) and light availability in the understory is frequently associated with regeneration processes and the long-term survival of forest tree species (WOODS 2000). -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 -2 -1 0 1 2 Fa ct or 2 Factor 1 A. campestre sun A. campestre shade C. avellana sun C. avellana shade P. alba shade R. pseudoacacia sun R. pseudoacacia shade Q. robur sun Q. robur shade P. alba sun Fig. 2. Principal component analysis (PCA) carried out using the physiological, morphological and anatomical leaf traits. Factor 1 and factor 2 accounts for 38.8% and 17.8% of the total vari- ance, respectively. Open symbols for each species indicate sun leaves and dark symbol indi- cate shade leaves. CARBON GAIN OPTIMIZATION IN RESPONSE TO LIGHT GRADIENT WITHIN THE TREE CROWN ACTA BOT. CROAT. 74 (1), 2015 83 Light response of broadleaf deciduous species is of particular interest because regeneration, either natural or via silvicultural practices, is infl uenced by their shade tolerance (WELAND- ER and OTTOSSON 2000). The studied forest is characterized by a tree density of 237 ± 100 stem ha–1 and a LAI of 4.5 ± 0.3 which is within the range of broadleaf deciduous forests in Italy (SCHIRONE et al. 1985, PICCOLI and BORELLI 1988, GRATANI and Foti 1998, Gratani and CRESCENTE 2000). LAI is an important variable for characterizing vegetation structure and functioning including estimation of plant productivity and canopy cover density (GARRIGUES et al. 2008). LAI drives both the within- and the below-canopy microclimate since LAI and PPFD absorption are closely related parameters (KULL et al. 1999; PORTE et al. 2000). Any change in forest LAI by frost, storm, defoliation, drought and management practices is accompanied by modifi cations of plant productivity (BRÉDA 2003). Our results show that the PPFD% through the considered tree crown decreases, on average, by 94% from the top to the bottom involv- ing a large number of leaf trait variation as confi rmed by the PCA that shows a complete separation between sun and shade leaves. In particular, SLA is 81% higher in shade than in sun leaves (mean value of the considered species), according to the results of WITKOWSKI and LAMONT (1991), GRATANI (1997) and GRATANI et al. (2006). The higher SLA of shade leaves is mostly the result of a decreased total leaf thickness, as attested by the signifi cant (p < 0.01) negative correlation between SLA and L. In fact, the larger and thinner shade leaves are more advantageous for light capture in low light conditions and, in general, spe- cies which grow in shade conditions are characterized by a larger SLA and a lower L than those growing in more open areas (GRATANI and FOTI 1998). -0.04 -0.02 0 0.02 0.04 0.06 0.08 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 A xi s 2 Axis 1PIp PIm R. pseudoacacia Q. robur P. alba A. campestre C. avellana PIa Fig. 3. Correspondence analysis (CA) performed using the species as group variable and plasticity index as category variable, PIm – morphological plasticity, PIa – anatomical plasticity, PIp – physiological plasticity. CATONI R., GRATANI L., SARTORI F., VARONE L., GRANATA M. U. 84 ACTA BOT. CROAT. 74 (1), 2015 Moreover, SLA differs between shade-tolerant and intolerant species (ABRAMS and KUBISKE 1990, KULL and NIINMETS 1993), and is the trait most strongly correlated to shade tolerance (JANSE-TEN KLOOSTER et al. 2007). The considered shade-tolerant species (A. campestre and C. avellana) have a 49% higher SLA and a 28% lower L than the shade-in- tolerant species (P. alba, R. pseudoacacia and Q. robur) as confi rmed by the PCA. The analysis shows that sun leaves of A. campestre and C. avellana are closer in behavior to shade leaves, underlining their ability to tolerate shade in comparison to others species. Among the shade-intolerant species, Q. robur has the lowest SLA (130.6 ± 10.0 cm2 g–1) and the highest L in sun leaves (160.8 ± 9.6 μm), since shade-intolerant species typically have thicker leaves (NIINEMETS et al. 1998). Leaf thickening under higher irradiance can be interpreted as a means for the plant to optimize light use through irradiance attenuation (JACQUEMOUD and BARET 1990, USTIN et al. 2001). Moreover, the structure of the mesophyll is associated with the photosynthetic perfor- mance of leaves via regulation of the internal light and carbon dioxide profiles (EVANS 1999). In particular, the palisade parenchyma reflects the leaf ability to capture solar energy accounting for the photosynthetic efficiency (KUMAR et al. 2012). This is attested by the signifi cant (p < 0.01) positive correlation between PN and palisade parenchyma thickness. On average, PN is more than 100% higher in sun than in shade leaves, associated with a 38% higher palisade parenchyma thickness, while shade leaves are characterized by a 3% higher spongy parenchyma thickness, that enhances light capture by scattering light (MENDES et al. 2001, SACK et al. 2006), in respect to palisade parenchyma. The higher PN rates in sun leaves than in shade leaves for the considered species are supported by a more than twice as high gs value indicating that the larger stomata opening in sun leaves (SCHULZE et al. 1975, FARQUHAR and SHARKEY 1982) determines a higher intercellular CO2 concentra- tion. This result is supported by the signifi cant (p < 0.01) correlation between PN and gs. The shade-intolerant species have a greater difference between PN in sun and shade leaves than shade-tolerant species, depending on the different level of PN light saturation (KOIKE et al. 2001). Moreover, shade-tolerant species have lower PN rates both in sun than in shade leaves (6.7 ± 1.2 and 1.9 ± 0.1 μmol m–2 s–1 respectively) than shade-intolerant species, ac- cording to the results of KOIKE et al. (2001), VALLADARES et al. (2002) and VALLADARES and NIINEMETS (2008). Variations in light availability within the crown also induce changes in CO2 release through R, this last refl ecting the acclimation response to the light variations within the crown (ZHA et al. 2002). High PPFD increases R as a result of higher maintenance costs caused by higher PN (AMTHOR 1989, NIINEMETS et al. 1998), higher protein turnover and in- creased need for secondary compounds such as fl avonoids or carotenoids (DELAGRANGE et al. 2004). The ratio R/PN can be considered a simple approach to leaf carbon balance be- cause it indicates the percentage of photosynthates that are respired (LOVEYS et al. 2002). The 69% lower R/PN ratio in sun than in shade leaves (mean value of the considered spe- cies) is mainly due to the higher PN decrease from sun to shade leaves, with respect to the R decrease, underlining the higher PN sensitivity to light variations within the crown. This re- sult is further attested by the signifi cant correlation between PN and PPFD%. Because they have the lowest PN rates, the shade-tolerant species have a 76% higher R/PN ratio than those that are shade-intolerant. The strong and direct effect of light on the photosynthetic capacity is mainly related to light-induced variations in the pool of proteins, pigments and enzymes that supports light CARBON GAIN OPTIMIZATION IN RESPONSE TO LIGHT GRADIENT WITHIN THE TREE CROWN ACTA BOT. CROAT. 74 (1), 2015 85 capture and CO2 fi xation by leaves (NIINEMETS et al. 1998, LE ROUX et al. 1999). Thus, crown photosynthesis is a function of leaf Na distribution within the crown profile (GRATANI et al. 2006) which is attested by the signifi cant correlation between these two variables, and may be explained by more than 70% leaf N being allocated to the photosynthetic apparatus (POORTER and EVANS 1998). Sun leaves have a 54% higher Na than shade leaves which en- hances their capacity to convert high PPFD to organic matter (MENDES et al. 2001). More- over, the higher PNUE (greater than 100%) in sun leaves underlines the effi ciency to allo- cate N to RUBISCO, according to POORTER and EVANS (1998) and GRATANI et al. (2006). The shade-intolerant species have a 21% higher PNUE than the shade-tolerant species, demonstrating different light demands (KULL and NIINEMETS 1993). The acclimation of the photosynthetic pigment apparatus to high irradiance in sun leaves determines a higher Chl a/b ratio (8%, mean value) and a lower (22%) Chl/Car ratio than in shade leaves, according to the results of LICHTENTHALER et al. (2007). The higher Chl b content in shade leaves is indicative of acclimation to low irradiance, because it is usually the main component of the LHCP (KOIKE et al. 2001), while the lower Chl/Car ratio in sun leaves is primarily caused by the highest activity in the xanthophyll cycle (DEMMIG-ADAMS 1998). Moreover, the ratio Chl/N, which is an indicator of leaf N allocation to chlorophyll-protein complexes in the light-harvesting component (KOIKE et al. 2001), is 44% higher in the shade than in the sun leaves of the species considered. The increase in the proportional investment of leaf N in thylakoids, which improves incident light-use effi ciency, is a major response of N partition- ing within the leaf to decreased irradiance (EVANS 1989). Among the considered species, A. campestre has the highest Chl/N both in sun and shade leaves (0.102 ± 0.010 and 0.174 ± 0.014, respectively) with a large increase from sun to shade leaves, thus attesting its shade- tolerance. In fact, there is a wide consensus that a higher increase of Chl/N is even more signifi cant for species of greater shade tolerance (EVANS 1989). Plasticity is a key trait useful to quantify plant response to environmental stimuli (NICOTRA et al. 2010) and phenotypic plasticity is defined as the ability of a genotype to produce different phenotypes depending on the environment (SULTAN 2000). The analysis of leaf phenotypic plasticity attests the considered species responsiveness to light variation. The PI for the considered species (0.35) is in the range of broadleaf deciduous species (VALLADARES et al. 2002, WYKA et al. 2007, 2012). Nevertheless, there are signifi cant differ- ences among the species. In particular, the shade-intolerant Q. robur and R. pseudoacacia have the highest PI (0.39 ± 0.01, mean value) while P. alba has the lowest (0.29). This last can be explained by the preference of P. alba to grow along the edge of forests and on river banks (MADEJÓN et al. 2004) considering that forest edges have a signifi cant infl uence on the solar radiation pattern on nearby landscape units, in relation to the number and size of openings, the distance of openings from the edge, and the orientation of openings relative to the path of the sun (GALO et al. 1992). The presence of P. alba along the edge of forests and its vertically extended crown structure (CHIUSOLI 1991) are typical traits of shade-intol- erant species (DELAGRANGE et al. 2004). Moreover, P. alba has the lowest PIm (0.32) and PIa (0.12), which are mainly explained by the lowest SLA and L variations between sun and shade leaves, due to the lack of deep shade in its crown. The shade-intolerant species have a higher PIp (0.46 ± 0.07) than the shade-tolerant species, in agreement with other studies (STRAUSS-DEBENEDETTI and BAZZAZ 1996, GRONINGER et al. 1996, VALLADARES et al. 2002, BLOOR 2003, DELAGRANGE et al. 2004, SÁNCHEZ-GÓMEZ et al. 2006, PORTSMUTH and NIINEMETS 2007). This result is also attested by the results of the correspondence analysis (CA). PIp is CATONI R., GRATANI L., SARTORI F., VARONE L., GRANATA M. U. 86 ACTA BOT. CROAT. 74 (1), 2015 also linked to an enhanced capacity to colonize gaps and open areas (VALLADARES et al. 2002, NIINEMETS and VALLADARES 2004) because it ensures adjustments of gas exchange in response to stress factors changes in the short term (ZUNZUNEGUI et al. 2009). A high physi- ological plasticity allows species to achieve rapid growth increasing the capability to colo- nize early successional habitats (WALTERS and REICH 1999). In particular, among the considered physiological traits, the photosynthetic plasticity (PIPN) is signifi cantly higher in shade-intolerant than in shade-tolerant species (DUCREY 1994). Several studied show that that shade-intolerant species have greater photosynthetic acclimation potential than shade-tolerant species (STRAUSS-DEBENEDETTI and BAZZAZ. 1996). This higher photosynthetic plasticity determines a large photosynthetic capacity to use full sunlight, which results in a more effi cient avoidance of photo-inhibition (VALLADARES et al. 2002). The capacity for photosynthetic acclimation to light could be considered species- specifi c (DUAN et al. 2005). Q. robur has the highest PIp (0.53) which is related to its shade- intolerant behavior, according to the results of VALLADERES et al. (2002). Moreover, its high- est PIPN (0.87) allows a rapid acclimation of photosynthesis which leads to an increased carbon gain, thereby facilitating growth and competitive ability in high light conditions (VALLADARES et al. 2002). The high R. pseudoacacia PI confi rms that phenotypic plasticity allows invasive species to occupy a wide range of new environments (CALLAWAY et al. 2003, PIGLIUCCI 2005, REJ- MANEK et al. 2005) and the highest PIm (0.56) and PIa (0.18) of R. pseudoacaia contribute to its adaptability. The higher PIm of A. campestre and C. avellana is linked to an enhanced capacity to survive and grow in the understory (VALLADARES et al. 2002, NIINEMETS and VALLADARES 2004), because it has an important role in resource acquisition (NAVAS and GARNIER 2002, YAMASHITA et al. 2000). On the whole, our results support the overall trend that light-demanding species are more plastic than shade-tolerant species (STRAUSS-DEBENEDETTI and BAZZAZ 1991, 1996, MUTH and BAZZAZ 2002, LONGUETAUD et al. 2013). However, from this general scheme, P. alba, despite its shade intolerance, exhibits the lowest PI which can be related to its crown type and the environment where it grows. Moreover, our results support the trend for which the early-successional species (R. pseudoacacia and Q. robur) are more plastic than the late successional ones (STRAUSS-DEBENEDETTI and BAZZAZ 1991, 1996). In this context, the pres- ence of large openings could favor both Q. robur and R. pseudoacacia regeneration. Knowledge on phenotypic plasticity is important to make hypotheses about the dynam- ics of the studied forest in consideration of environmental stress factors, including invasive species competition and global climate change. These stress factors might alter the compo- sition of the forest in the long term, infl uencing the competition of the early-successional species. In particular, the increase of air temperature and CO2 concentration might act as potent agents of natural selection among species favoring the more phenotypically plastic species (CHOWN et al. 2007). In particular, air temperature increase might allow the repro- duction of Phythophora cinammomi, a pathogen largely affecting Q. robur in Europe (LA PORTA et al. 2008) while the CO2 concentration increase might favor R. pseudoacacia be- cause of its high growth rate (MOHAN et al. 2007). Considerations can be made for the con- servative management of the forest carried out since the establishment of the Reserve, which has probably limited the presence of R. pseudoacacia until 1980 when it knowingly became established (MOTTA et al. 2009). Thus, it is important to maintain this type of man- agement in the future since creating gaps could allow a greater seed regeneration of R. CARBON GAIN OPTIMIZATION IN RESPONSE TO LIGHT GRADIENT WITHIN THE TREE CROWN ACTA BOT. CROAT. 74 (1), 2015 87 pseudoacacia over Q. robur because of its higher growth rate. Consequently, land use is a fundamental determinant in shaping vegetation composition and structure with important implications for forest management. In particular, forests with old-growth characteristics (BAUHUS et al. 2009), like the investigated forest, are important reference sites for more natural management approaches involving a broad range of ecosystem functions and ser- vices (WIRTH et al. 2009). In recent years, interest in understanding the natural processes in forest ecosystems has increased because of a few primary forests remaining in many re- gions (e.g., Europe, PETERKEN 1996). 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