Plastic responses of Populus yunnanensis and Abies faxoniana to elevated atmospheric CO2 and warming

Forest Ecology and Management 296 (2013) 33–40

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Plastic responses of Populus yunnanensis and Abies faxoniana to elevated atmospheric CO2 and warming Baoli Duan a, Xiaolu Zhang a, Yongping Li a, Ling Li a, Helena Korpelainen b, Chunyang Li a,⇑ a b

Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu 610041, China Department of Agricultural Sciences, P.O. Box 27, University of Helsinki, FI-00014 Helsinki, Finland

a r t i c l e

i n f o

Article history: Received 17 September 2012 Received in revised form 29 January 2013 Accepted 31 January 2013 Available online 6 March 2013 Keywords: Climate change Leaf area ratio Morphological and physiological plasticity Nitrogen use efficiency Photosynthetic capacity

a b s t r a c t To examine whether deciduous and evergreen tree species differ in their performance and plastic responses to elevated atmospheric CO2 and temperature, we investigated growth, leaf area ratio (LAR), specific root length (SRL), nitrogen uptake efficiency (NUpE) and nitrogen use efficiency (NUE) of Populus yunnanensis and Abies faxoniana grown in environment-controlled chambers. Our results showed that temperature stimulated growth more than did CO2 in both species. The magnitude of temperature and CO2 effects varied between the species, as greater stimulation was detected in A. faxoniana than in P. yunnanensis. Greater responses of A. faxoniana were associated with higher LAR and higher NUE. However, NUpE did not differ between species and could not explain this advantage. On the other hand, the studied species did not differ in their mean overall plasticity (calculated for each species by averaging the indices of plasticity obtained for each of the 17 variables), which, however, was achieved in different ways. Across CO2 and temperature treatments, A. faxoniana exhibited greater plasticity in stomatal conductance, LAR, SRL and NUE, whereas P. yunnanensis exhibited greater plasticity in net photosynthesis rate, leaf respiration and root/shoot ratio. Our results suggested that plasticity of key traits, such as LAR, SRL and NUE, may have important implications for the superiority of A. faxoniana in the response to climate change. Our results highlight the importance of exploring the functional significance of traits rather than the extent of plasticity as potential determinants of interspecific differences in responses to climate change. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Increases in the concentration of atmospheric CO2 as well as elevated annual mean temperature experienced by plants are recognized to have dramatic impacts on terrestrial vegetation (IPCC, 2007). Responses of trees have been studied with a great interest, because forests play a prominent role as a carbon sink (Huang et al., 2007; Dawes et al., 2011). It has been argued that evergreen species are limited in their responses to changing environmental conditions (Way and Oren, 2010). For example, stomatal conductance is less responsive to elevated CO2 in evergreen conifers than in broad-leaved trees (Medlyn et al., 2001). Similarly, increases in growth were found for Larix decidua after 9 years of CO2 enrichment, whereas this increase could not be detected for Pinus uncinata (Dawes et al., 2011). Nevertheless, this is not always the case (Lin et al., 2010). The conflicting views imply the need for further studies in evergreen and deciduous trees to examine interspecific variation in functional traits that may provide a mechanism for coping with climate change. Moreover, as climate change is ⇑ Corresponding author. Tel.: +86 28 85557542; fax: +86 28 85222258. E-mail address: [email protected] (C. Li). 0378-1127/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.foreco.2013.01.032

altering the availability of nutrients or water resources that are important for plant growth, the ability of plants to respond to elevated CO2 and temperature is typically dependent upon resource acquisition and use characteristics, such as nitrogen use efficiency (Finzi et al., 2007) and water use efficiency (Llorens et al., 2009), assessed by carbon isotope composition (d13C) that provides a time-integrated estimate of water use efficiency (Farquhar et al., 1989; Dawson et al., 2002). Nevertheless, most previous studies on effects of rising atmospheric CO2 and temperature have focused on photosynthesis and growth (Bernacchi et al., 2003; Ainsworth and Long, 2005), with relatively little attention given to resource acquisition and use, particularly under climate change. On the other hand, phenotypic plasticity, i.e., capacity to express alternative phenotypes in response to environmental variation (van Kleunen and Fischer, 2005; Ramírez-Valiente et al., 2010; Poorter et al., 2012), mediates climate change responses. Phenotypic adjustments to CO2 and temperature range from changes in stomatal conductance at the leaf level to changes in growth patterns or biomass allocation at the whole-plant level (see review by Wang et al., 2012). Although there is abundant information on the plastic responses to environmental changes, the role of phenotypic plasticity in determining plants’ responses

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to climate change is unclear, and it remains unknown how and to what extent the phenotypic plasticity is correlated with plants’ performance and growth. It is often assumed that species with high plasticity have a growth advantage over species with low plasticity in response to environmental cues (Valladares et al., 2006; Cai et al., 2008). Nevertheless, plasticity is not always strongly related to fitness (Davidson et al., 2011). Greater (Portsmuth and Niinemets, 2007), similar (Markesteijn et al., 2007) and lower leaf plasticities (Popma et al., 1992) have been found in deciduous species compared with evergreen species. A clear consensus on the link between plasticity and functional types has not been reached within the climate change context. Therefore, investigating the effects of elevated CO2 and temperature on plants’ plastic responses will lead us to gain a better understanding of how plant growth will respond to future changes in climate. Such a study can also provide basic information for predicting the effects of climate change on tree species (Nicotra et al., 2010). Both Populus yunnanensis (deciduous) and Abies faxoniana (evergreen) are important forest tree species in Southwest China. However, there are few empirical tests of whether differences in performance and plasticity exist between these two species in response to climate change. In this study, we examined growth, N uptake efficiency and N use efficiency throughout one growing season in P. yunnanensis and A. faxoniana under elevated atmospheric CO2 and temperature. We also measured plasticity in leaf- and plant-level traits in plants subjected to elevated CO2 and warming. We tested the hypothesis that deciduous species (P. yunnanensis) have more flexible traits, thus greater responses to climate change compared with evergreen trees (A. faxoniana). Specifically, our objectives were to determine: (1) whether P. yunnanensis and A. faxoniana vary in their morphological and physiological traits under elevated atmospheric CO2 and warming; (2) whether P. yunnanensis differs from A. faxoniana in the plasticity of responses to elevated atmospheric CO2 and warming.

2. Materials and methods 2.1. Plant material and experimental design The experiments were carried out at the Maoxian Ecological Station (31°410 0700 N, 103°530 5800 E, and altitude 1,820 m) in Southwest China. Individuals of P. yunnanensis and A. faxoniana, preselected for uniform height (30 ± 5 cm), were planted into pots (10-L) filled with homogenized soil and moved to growth chambers and grown under a controlled environment from May to October 2011. We minimized the possibility of ontogenetic differences among species and among treatments by selecting plants of a similar size rather than of the same age (Valladares et al., 2000). The chambers constructed of glass walls with a polycarbonate plastic top transmit approximately 82% of photosynthetically active radiation (PAR) during the 12 h photoperiod. The seedlings were irrigated to keep the soil water content at 30–35% (an approximation of the optimal water content), which was measured by time domain reflectometry (Tektronix 1502 C, Beaverton, OR, USA) at two locations near the centre of each pot over a depth of 0– 30 cm. The computer-controlled temperature and CO2 supply system enabled the two variables to be adjusted automatically inside the chambers to ensure an ambient condition, or to achieve a specified CO2 enrichment and/or a rise in temperature. The measured average air temperature out of chamber during the experiment was 21.00 ± 0.50/13.00 ± 0.50 °C (day/night). The experimental layout was completely randomized with three factors (species, CO2 and temperature). Two CO2 regimes, ambient (350 ± 20 lmol mol1) and elevated CO2 (700 ± 20 lmol mol1), and two temperature conditions, elevated by 0 and 2.5 ± 0.2 °C (compared

with the out-of-chamber environment), were supplied. Thus, four treatments were employed: (1) ambient environment (CON); (2) elevated temperature (ambient + 2.5 °C; ET); (3) elevated CO2 concentration (ambient + 350 lmol; EC); and (4) elevated CO2 and temperature (ambient + 350 lmol + 2.5 °C; ECT). Eight chambers were used to maintain the four treatments (two chambers per treatment). In both species, each treatment contained four replicate plots, a total of 16 replicate plots, which were assigned to eight growth chambers (two replicate plots per chamber). Each replicate plot consisted of 10 seedlings (5 P. yunnanensis and 5 A. faxoniana). Thus, there were four replicates with 5 seedlings per treatment combination per replicate, a total of 20 seedlings per species per treatment and 160 seedlings in total. Pots and treatments (i.e., temperature and CO2) were rotated once a week within and between growth chambers to minimize error due to chamber effects (Fajer et al., 1989; Kellomäki and Wang, 2001). Such rotation ensured that all pots spent approximately equal amounts of time in all locations in each chamber. The hourly means of the 15-s readings indicated that the target temperature and CO2 concentrations were achieved. The elevated CO2 concentrations were within 650–800 lmol mol1 for 91% (EC chambers and ECT chambers) of the exposure time. The raised air temperature was within 1.0–3.0 °C for 95% (ET chambers) and 88% (ECT chambers) of the exposure time. 2.2. Growth and morphological traits The heights were measured both at the beginning and end of each treatment, and the growth rate of height (GRH) was calculated as GRH = (final plant height at harvest  initial plant height)/time. Growth relative to initial biomass (GRIB) was calculated as GRIB = (final plant biomass at harvest  initial plant biomass)/initial plant biomass  100%. All plants were harvested at the end of the experiment and divided into leaves, stems and roots (including coarse roots and fine roots; fine roots were defined as those less than 2 mm in diameter). Biomass samples were dried (70 °C, 48 h) to constant weight and weighed. The total leaf area (TA) was determined by a Portable Laser Area Meter (CI-203; CID, Camas, WA, USA). Based on plant biomass and leaf area measurements, we calculated the following morphological traits: leaf area ratio (LAR, leaf area divided by total mass, cm2 g1), specific leaf area (SLA, leaf area per unit leaf mass, cm2 g1), and root/shoot ratio (RS, ratio of root to shoot biomass). Fine root lengths were estimated with WinRHIZO (Regent Instruments Inc., Quebec, Canada). The specific root length (SRL, mg1) was calculated as the ratio between the length of the fine roots and their dry weight. 2.3. Carbon, nitrogen and phosphorus analysis Dried samples were ground to fine powder and passed through a mesh (pore diameter ca. 275 lm). The concentrations of leaf nitrogen (N) and carbon (C) were determined by the semi-micro Kjeldahl method (Mitchell, 1998) and the rapid dichromate oxidation technique (Nelson and Sommers, 1982), respectively. Leaf phosphorus (P) concentration was determined by persulfate oxidation followed by colorimetric analysis (Schade et al., 2003). 2.4. Nitrogen uptake efficiency, nitrogen use efficiency and carbon isotope composition N uptake efficiency was determined as the total plant N content divided by the fine root biomass (Hawkins, 2007). The N use efficiency was calculated as total plant biomass divided by plant N (Bernacchi et al., 2007). For the carbon isotope analysis, samples of 100 mg DW of plant material, oven-dried at 70 °C for 24 h, were homogenized by grinding in a ball mill. The stable carbon isotope

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abundance in the combusted samples was measured with a mass spectrometer (Finnegan MAT Delta-E) as described by Li et al. (2004). The overall precision of the d-values was better than 0.1‰, as determined from repeated samples.

The measurements of various photosynthesis characters were conducted within a two-week period in August.

2.5. Gas exchange

Because the CO2 and temperature treatments were not applied independently to each seedling, the plants in each treatment combination are not true replicates (Hurlbert, 1984; Maherali and DeLucia, 2000). Therefore, averages of subsamples (five seedlings per replicate) were used for the analysis of variance. Before ANOVA, data were checked for normality and the homogeneity of variances, and log-transformed to correct deviations from these assumptions when needed. We performed three-way ANOVA for the effects of temperature, CO2 and species for each variable to discover differences between species in response to CO2 and warming. When analyses revealed treatment interactions or treatment and species interactions for certain variables, two-way ANOVAs were conducted for each species. Significant differences among treatment means were analyzed using Tukey’s multiple comparison post hoc tests. To test the carry-over effects on seedling growth from prior to the experiment, an analysis of covariance with initial plant biomass as a covariate was used to evaluate treatment differences in plant-level traits. To compare plasticity among leaf and whole-plant traits, we calculated a plasticity index (PI) for each measured trait in each species, following Valladares et al. (2006). We selected PI because it better reflects a reaction norm and it has the advantage of being

Net photosynthesis rate (Anet) and stomatal conductance (gs) were measured with a portable infrared gas analyzer in open-system mode (Li-6400, Li-Cor). Photosynthetic photon flux was set at 1000–1400 lmol m2 s1 with the built-in LED-B light source. Light-response curves showed that this was sufficient to saturate photosynthesis under all treatments. The air temperature in the leaf chamber was maintained at 25–27 °C, leaf-to-air vapour pressure deficit 1.5 ± 0.5 kPa, relative air humidity 50% and ambient CO2 concentration 350 ± 5 lmol mol1. Gas exchange of A. faxoniana shoots was measured with a conifer type chamber (PLC-conifer, PP Systems). A broadleaf type chamber (PLC-broad, PP Systems) was used for P. yunnanensis leaves. The measurements were performed for intact current-year needles of A. faxoniana, and for young and fully expanded leaves of P. yunnanensis. The measurements were conducted daily between 08:00 and 11:00 during a period of one week. Measurements of the leaf respiration (Rd) were obtained through gas exchange measurements after covering the leaf chamber for 15 min. Photosynthetic nitrogen use efficiency (PNUE) was calculated as the ratio between saturated photosynthetic rate and area-based leaf nitrogen concentration.

2.6. Statistical analyses

Fig. 1. The effect of atmospheric CO2 concentration and air temperature on growth and biomass allocation in P. yunnanensis and A. faxoniana seedlings. GRH, growth rate of height; GRIB, growth relative to initial biomass; TA, total leaf area; LAR, leaf area ratio; SLA, specific leaf area; RS, root/shoot ratio; SRL, specific root length. Treatments: CON, ambient conditions; ET, elevated temperature (ambient + 2.5 °C, ET); EC, elevated CO2 concentration (ambient + 350 lmol, EC); ECT, elevated CO2 and temperature. P(C), CO2 effect; P(T), temperature effect; P(S), species effect; P(CT), CO2  temperature interaction effect; P(SC), species  CO2 interaction effect; P(ST), species  temperature interaction effect; P(SCT), species  CO2  temperature interaction effect. ns, non-significant; P < 0.05; P < 0.01; P < 0.001. Values are mean ± standard error (n = 4). Within a species, values followed by different letters are significantly different at the P < 0.05 level according to Tukey’s multiple range test.

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Fig. 2. The effect of atmospheric CO2 concentration and air temperature on leaf C, N and P concentrations, and total plant N in P. yunnanensis and A. faxoniana seedlings. Treatments: CON, ambient conditions; ET, elevated temperature (ambient + 2.5 °C, ET); EC, elevated CO2 concentration (ambient + 350 lmol, EC); ECT, elevated CO2 and temperature. P(C), CO2 effect; P(T), temperature effect; P(S), species effect; P(CT), CO2  temperature interaction effect; P(CT), CO2  temperature interaction effect; P(SC), species  CO2 interaction effect; P(ST), species  temperature interaction effect; P(SCT), species  CO2  temperature interaction effect. ns, non-significant; P < 0.05;  P < 0.01; P < 0.001. Values are mean ± standard error (n = 4). Within a species, values followed by different letters are significantly different at the P < 0.05 level according to Tukey’s multiple range test.

insensitive to differences in variance between two samples (Godoy et al., 2011). The plasticity index was calculated with means from four replicates. The index ranges from zero to one and is the difference between the maximum and minimum mean value of a trait among treatments divided by the maximum value. Plasticity under all treatments, in general, was calculated based on the highest and lowest parameter values found in the four treatment combinations (Cai et al., 2008). For the calculation of the plasticity to temperature, we used the mean trait value at ambient and elevated temperature by pooling both CO2 treatments. For plasticity to CO2 treatments, we used the mean trait value at ambient and elevated CO2 by pooling both temperature treatments. In addition, a mean plasticity index was calculated for each species by averaging the 17 variables. Independent-samples t test was used to assess differences in plasticity index between the two species. The effects were considered significant if P < 0.05. The used statistical software package was SPSS 11.5 for Windows. 3. Results 3.1. Growth and biomass allocation ET significantly increased growth rate of height in both species (Fig. 1a). Species significantly differed in growth relative to initial biomass in response to EC and to ET (significant species  temperature and species  CO2 interactions), showing that both EC and ET increased this parameter more in A. faxoniana than in P. yunnanensis (Fig. 1b). Both ET and EC significantly increased total leaf area in the two species, and EC had a stronger effect on total leaf area in A. faxoniana than in P. yunnanensis (significant species  CO2 interaction) (Fig. 1c). ET and EC significantly increased leaf area ratio in A. faxoniana but not in P. yunnanensis (significant species  temperature and species  CO2 interactions) (Fig. 1d). CO2 only slightly decreased SLA in A. faxoniana (significant species  CO2 interaction) (Fig. 1e). EC increased root/shoot ratio more in A. faxoniana than in P. yunnanensis, as shown by significant CO2  species inter-

action for this parameter (Fig. 1f). Significant CO2  temperature interactions on growth rate of height, growth relative to initial biomass, total leaf area, leaf area ratio and root/shoot ratio were observed. In addition, species  CO2  temperature interactions were significant in the case of growth relative to initial biomass and leaf area ratio. Also, EC increased specific root length in A. faxoniana but not in P. yunnanensis, as indicted by the significant species  CO2 interaction (Fig. 1g). 3.2. Nutrient concentrations ET and EC increased leaf C concentration in both species (Fig. 2a). By contrast, neither EC nor ET changed leaf P concentration in either species (Fig. 2c), and there was no temperature effect on leaf N in either species. Species significantly differed in leaf N in response to EC (significant species  CO2 interaction), showing that leaf N of P. yunnanensis decreased under EC but did not change in A. faxoniana (Fig. 2b). Moreover, ET increased the total plant N only in A. faxoniana, while EC had no effect on plant N in either species (Fig. 2d). There was a significant species  CO2  temperature interaction for total plant N, indicating that A. faxoniana had higher N under ET at ambient CO2 than at elevated CO2 (Fig. 2d). 3.3. Nitrogen uptake efficiency, nitrogen-use efficiency and carbon isotope composition Neither species nor treatments affected N uptake efficiency (Fig. 3a). For the two species, N use efficiency significantly increased under both ET and EC. There were significant species  CO2 and species  temperature interactions for N use efficiency, showing that ET and EC increased N use efficiency more in A. faxoniana than in P. yunnanensis (Fig. 3b). On the other hand, N use efficiency was significantly affected by the interaction of CO2  temperature and the interaction of species  CO2  temperature. When the seedlings of both species were exposed to EC and ET, d13C significantly decreased under ET but increased under EC (Fig. 3c).

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in gs (no temperature  species interaction) for the two species under ET. By contrast, EC decreased gs in A. faxoniana but did not change it in P. yunnanensis (Fig. 4c). Furthermore, ET and EC increased leaf respiration in P. yunnanensis but did not change this parameter in A. faxoniana (Fig. 4d). There were significant interactions between temperature and CO2 effects on Anet, leaf respiration and PNUE in the two species, generally with a stronger effect of ET under ambient CO2 than under elevated CO2. There was also a significant species  temperature  CO2 interaction for Anet, gs and leaf respiration.

3.5. Assessment of plasticity The plasticity index (PI) calculated across all CO2 and temperature treatments ranged from 0.001 to 0.547 (Fig. 5a–f). In general, the degree of plasticity was similar in the two species for leaf-level traits, plant-level traits and all traits combined (independent-samples t test, P > 0.05) in response to all treatments. On the other hand, the degree of plasticity in any treatment depended on the trait examined. Plasticity indices measured across CO2 and temperature treatments were higher in A. faxoniana than in P. yunnanensis for the leaf-level trait gs, and plant-level traits leaf area ratio, specific root length and N use efficiency, while the reverse was true for the leaf-level traits Anet, leaf respiration and leaf N, and plant-level traits growth rate of height and root/shoot ratio (Fig. 5a and b). Moreover, the ranking of species for plasticity in PNUE and total leaf area changed with CO2 and temperature. In general, A. faxoniana exhibited higher plasticity for PNUE and lower plasticity for total leaf area in response to temperature, while the reverse was true for these variables in response to CO2 (Fig. 5c–f). Moreover, there was no systematic difference in the plasticity of leaf P, d13C and nitrogen uptake efficiency between P. yunnanensis and A. faxoniana.

4. Discussion

Fig. 3. The effect of atmospheric CO2 concentration and air temperature on N uptake efficiency (NUpE), N use efficiency (NUE) and carbon isotope composition (d13C) in P. yunnanensis and A. faxoniana seedlings. Treatments: CON, ambient conditions; ET, elevated temperature (ambient + 2.5 °C, ET); EC, elevated CO2 concentration (ambient + 350 lmol, EC); ECT, elevated CO2 and temperature. P(C), CO2 effect; P(T), temperature effect; P(S), species effect; P(CT), CO2  temperature interaction effect; P(CT), CO2  temperature interaction effect; P(SC), species  CO2 interaction effect; P(ST), species  temperature interaction effect; P(SCT), species  CO2  temperature interaction effect. ns, non-significant; P < 0.05;  P < 0.01; P < 0.001. Values are mean ± standard error (n = 4). Within a species, values followed by different letters are significantly different at the P < 0.05 level according to Tukey’s multiple range test.

Moreover, d13C was significantly affected by the interaction of CO2  temperature, showing that seedlings exposed to ET had lower d13C at ambient CO2 than at elevated CO2 (Fig. 3c). 3.4. Photosynthetic capacity Anet, gs, leaf respiration and PNUE significantly varied with CO2 and temperature in both species (Fig. 4a–d). There were significant differences in the two species in terms of Anet, gs, leaf respiration and PNUE under elevated CO2 and temperature, as shown by significant CO2  species interactions and/or temperature  species interactions for these parameters (Fig. 4a–d). ET increased Anet and PNUE more in P. yunnanensis than in A. faxoniana. EC decreased Anet and PNUE in P. yunnanensis, but increased Anet and did not change PNUE in A. faxoniana (Fig. 4a, b). There was a similar change

Our results showed that both P. yunnanensis and A. faxoniana possessed greater responses to ET (156–179%) compared with EC (120–139%) in terms of growth relative to initial biomass. In this experiment, the average ambient air temperatures during the experiment were slightly lower than the optimum temperature for P. yunnanensis and A. faxoniana photosynthesis (approx. 22 °C for full development of leaves). Thus, it is natural that a mean increase of 2.5 °C over the ambient temperature should have provided more propitious temperature conditions for growth. On the other hand, growth increases caused by ET and EC were not associated with N uptake efficiency. Instead, growth was associated with higher N use efficiency. In fact, ET and EC significantly increased N use efficiency but hardly affected N uptake efficiency. These results further indicate that N use efficiency rather than N uptake efficiency is the key factor determining productivity under ET and EC. In our study, all plants were kept under conditions of non-limiting nutrient and water supply for growth. Similarly, a N use efficiency increase was observed at elevated CO2 among forest FACE sites at a site where soil N availability was high and not limiting tree growth (PopFACE; Calfapietra et al., 2007; Finzi et al., 2007). We should also mention that results obtained here are from one growing season and may differ from those obtained in longterm N cycling dynamics experiments. The continued response of growth to elevated CO2 may become constrained during years by N limitation as a result of N immobilization in plant biomass and long-lived soil organic matter (Luo et al., 2004). Nevertheless, the decreased water use efficiency (more negative d13C values) under ET and increased water use efficiency under EC suggest contrasting

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Fig. 4. The effect of atmospheric CO2 concentration and air temperature on net photosynthesis rate (Anet), stomatal conductance (gs), photosynthetic N utilization efficiency (PNUE) and leaf respiration (Rd) in P. yunnanensis and A. faxoniana seedlings. Treatments: CON, ambient conditions; ET, elevated temperature (ambient + 2.5 °C, ET); EC, elevated CO2 concentration (ambient + 350 lmol, EC); ECT, elevated CO2 and temperature. P(C), CO2 effect; P(T), temperature effect; P(S), species effect; P(CT), CO2  temperature interaction effect; P(SC), species  CO2 interaction effect; P(ST), species  temperature interaction effect; P(SCT), species  CO2  temperature interaction effect. ns, non-significant; P < 0.05; P < 0.01; P < 0.001. Values are mean ± standard error (n = 4). Within a species, values followed by different letters are significantly different at the P < 0.05 level according to Tukey’s multiple range test.

effects of elevated CO2 and temperature on the water and carbon economy in typical forest tree species in Southwest China. In our study, plants were grown individually in pots. This could cause concern that water and nutrient limitation or root constriction in containers may reduce CO2-induced growth enhancement (Arp, 1991). Appropriate pot volumes were tested in a quantitative way in preliminary trials that showed that the pot sizes used in our study are large (10 l) enough to ensure unrestricted growth, as judged from the lack of differences in total plant biomass and root biomass between pot- and field-grown plants. Moreover, visual inspections of all root systems at harvest time showed that the plants were not root-bound. It is also notable that we have a fullwater and full-nutrient study of seedlings at a relative young age with one growing season duration, thus root restriction should not be a major problem. Therefore, we feel confident that the estimates of parameters, such as N uptake efficiency, presented in this study are reliable. However, challenges exist in relating pot studies from only two species to larger trees or longer-term effects. There were significant species  temperature and species  CO2 interactions for most traits tested. Such interactions may indicate that species differ in the magnitude or direction of the responses to temperature or CO2. P. yunnanensis increased net photosynthesis by 19% whereas A. faxoniana had 25% higher net photosynthesis under ET. Lower responses to a 2.5 °C increase in ambient temperature may be attributable to the broad temperature optimum of photosynthesis in P. yunnanensis. Meanwhile, P. yunnanensis exhibited strong down-regulation of leaf N concentrations and photosynthetic N use efficiency (PNUE) in response to EC. Reductions in leaf N content and PNUE were the main factors of photosynthetic down-regulation. Our results are in contrast with Liberloo et al. (2007), who reported that the stimulation of photosynthesis was sustained even after 6-year exposure to free-air CO2 enrichment (EUROFACE). In contrast, A. faxoniana maintained a substantial photosynthetic stimulation under EC exposure (Fig. 4a). Therefore, the photosynthetic performance was enhanced in

A. faxoniana but reduced in P. yunnanensis under EC. Different soil resource acquisition and biomass partitioning in P. yunnanensis and A. faxoniana likely affected the climate responsiveness in these two species, in particular the ability to maintain leaf N and avoid downregulation of photosynthetic capacity present in A. faxoniana but not in P. yunnanensis (Crous et al., 2010). EC increased specific root length in A. faxoniana but not in P. yunnanensis, which determined the size of the plant rhizosphere (Lima et al., 2010), suggesting that P. yunnanensis might not be able to exploit the soil as efficiently as does A. faxoniana. We also observed that ET and EC resulted in a greater growth relative to initial biomass in A. faxoniana (179% by temperature and 142% by CO2) than in P. yunnanensis (156% by temperature and 129% by CO2). Increased biomass observed in A. faxoniana plants grown under elevated CO2 can be mainly the result of an increased stimulation of root and branch growth rather than that of diameter growth (data not shown), as root and branch growth increased in parallel while no diameter growth changes were observed. Although we found a down-regulation of photosynthesis, biomass accumulation of P. yunnanensis was still stimulated under EC, apparently due to an increase in the amount of total leaf area (Fig. 1c). This may lead to an increase in the whole-plant photosynthetic CO2 assimilation, even though down-regulation of leaf photosynthesis has been detected (Watanabe et al., 2011). Despite Anet being reduced in P. yunnanensis under EC, it was still higher compared with that of A. faxoniana. Higher photosynthetic capacity (indicated by Anet) and efficient light capture (indicated by SLA) would give P. yunnanensis an advantage over A. faxoniana in assimilation gains. However, this was not the case in our study, which suggests that a greater photosynthetic capacity may not result in greater growth (Way and Oren, 2010). Our results imply that leaf-level traits do not fully characterize whole-plant carbon gain, and it could be important to look at significant functional roles of traits at both leaf and whole plant scales (Naumburg et al., 2002; Niinemets and Lukjanova, 2003). In line with this view, our results

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Fig. 5. Plasticity indices for leaf-level (PIL) and plant-level (PIP) traits measured in P. yunnanensis and in A. faxoniana in response to (a, b) all treatments, (c, d) temperature, and (e, f) CO2 (n = 4 per trait per species). Leaf-level traits: net photosynthesis rate (Anet), stomatal conductance (gs), photosynthetic N utilization efficiency (PNUE), leaf respiration (Rd), leaf carbon concentration (C), leaf nitrogen concentration (NL), leaf phosphorus concentration (P), carbon isotope composition (d13C), specific leaf area (SLA), means of leaf-level variables (MeanL). Plant-level traits: growth rate of height (GRH), total leaf area (TA); leaf area ratio (LAR), root/shoot ratio (RS), specific root length (SRL), N uptake efficiency (NUpE), N use efficiency (NUE), total plant N (NT), means of plant-level variables (MeanP), total mean (MeanT). Asterisks indicate a significant difference at P < 0.05,  P < 0.01 and P < 0.001.

suggested that the stronger response of A. faxoniana when compared to P. yunnanensis was related to the plant-level trait leaf area ratio, which could serve as an estimator for intercepted light at the individual plant level, rather than at the leaf level photosynthesis. In fact, P. yunnanensis did not change its leaf area ratio, whereas A. faxoniana enhanced it under both ET and EC conditions. The increase in the leaf area ratio in A. faxoniana was a result of a higher investment into foliage per unit of biomass compared with ambient conditions (Fig. 1d). Other characteristics that may explain the greater sensitivity of A. faxoniana trees are its greater water use efficiency and higher N use efficiency when compared with P. yunnanensis trees, enabling them to use resources more effectively. Despite significant interspecific variation in the plasticity of individual traits, P. yunnanensis and A. faxoniana differed little in their mean plasticity, as the two species achieved their plasticity in different ways. Relative to P. yunnanensis, A. faxoniana displayed high plasticity in leaf area ratio, specific root length and N use efficiency, indicating a flexible ‘foraging’ strategy tending to increase space occupation and exploitation, at the same time having a greater ability to maximize resource conservation. Collectively, our results showed that leaf area ratio, specific root length and

N use efficiency, among 17 measured plant variables, were the traits that contributed most to differences observed between P. yunnanensis and A. faxoniana. These traits may result in the superiority of A. faxoniana in responses to climate change. Our findings suggest that the major differences between these two species are less in the extent of plasticity, but rather in the key functional traits (Chown et al., 2007). Our results indicate that plasticity is not a sufficient explanation for understanding plants’ responses to climate change (Chown et al., 2007; Godoy et al., 2011). In conclusion, our findings suggest that the effects of elevated CO2 on the growth are lower compared with the effects of elevated temperature and that elevated temperature and CO2 favored A. faxoniana over P. yunnanensis. The efficient use of resources for light interception (leaf area ratio) and soil resource acquisition (specific root length) as well as resource conservation (N use efficiency) rather than the photosynthesis capacity proved to be the parameters that quantitatively reflect greater responses. On the other hand, the two species tested differed in their key traits but not in their plasticity. Therefore, this study highlights the importance of exploring the functional significance of traits, such as leaf area ratio, specific root length and N use efficiency as potential

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