Altitudinal variation in leaf construction cost and energy content of Bergenia purpurascens

Acta Oecologica 43 (2012) 72e79

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Original article

Altitudinal variation in leaf construction cost and energy content of Bergenia purpurascens Lin Zhang a, *, Tianxiang Luo a, Xinsheng Liu a, Yun Wang b a b

Key Laboratory of Tibetan Environment Changes and Land Surface Processes, Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100085, PR China Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 November 2011 Accepted 15 May 2012 Available online 12 June 2012

It is still unknown if the variation of leaf energy content across altitudinal gradients is caused by varying climatic factors or species replacement. We test whether there is an altitudinal increase in leaf energy properties within a single species, which is mainly due to the decreasing air temperature with altitude. We collected samples for Bergenia purpurascens Engl. (an evergreen herb) at every 10 m in altitude from outside the timberline forest (4320 m) to the hilltop (4642 m) in the Sergyemla Mountains, southeastern Tibet. We measured mass- and area-based leaf construction cost (CCm, CCa) and their components: nitrogen concentration (Nmass), ash concentration (AC), the heat of combustion (HC), and specific leaf area (SLA), as well as leaf lignin concentration (LC) and new leaf dry mass per plant (NLDM). As altitude increased, CCm, CCa, HC, and LC increased, whereas Nmass, SLA, AC, and NLDM decreased. CCm and CCa were positively correlated with HC and LC but negatively with Nmass, SLA, AC and NLDM. CCm, CCa and HC were negatively correlated with mean air temperature. The data indicated that some high-HC constituents like lignin rather than protein contributed to the observed pattern of leaf energy properties. For high-altitude plants, having relatively high leaf CCm and HC can be regarded as a growth strategy for sustaining carbon gain and maximizing nitrogen-use efficiency. Since CC tends to decrease with increasing air temperature, evergreen herbs at high altitude are expected to construct relatively “cheaper” leaves in response to global warming. Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.

Keywords: Alpine timberline Altitude Heat of combustion Leaf construction cost Nitrogen concentration Specific leaf area Evergreen herb

1. Introduction Leaf construction cost (CC), defined as a measure of the amount of glucose required to produce a unit leaf mass or leaf area (i.e. a glucose equivalent), is a quantitative measurement of the energy invested by a plant to construct a leaf (Williams et al., 1987). The heat of combustion (HC) is the most important determinant of CC (Villar and Merino, 2001). Plants with high leaf CC and HC are typically characterized by a slow-growth strategy since high CC and HC are associated with high resource use efficiency and low growth rate (Williams et al., 1987; Griffin, 1994; Poorter and Villar, 1997; Baruch and Goldstein, 1999; Villar et al., 2006; Song et al., 2007). As altitude increases, the carbon cost for nitrogen uptake by roots may increase (Körner, 1999). Evergreen species at relatively high altitudes have evolved adaptation for high mean residence time of nitrogen in the plants through increased leaf lifespan, which increases energy investment in leaves (Chabot and Hicks, 1982;

* Corresponding author. Tel.: þ86 10 84097055. E-mail address: [email protected] (L. Zhang).

Eamus and Prichard, 1998; Eamus et al., 1999; Villar and Merino, 2001) and maximizes efficiency of nutrient use in dry matter production (Escudero et al., 1992; Eckstein et al., 1999). Therefore, leaf CC of evergreen plants typically increases toward higher altitudes thereby lowering growth rate. Along an altitudinal gradient, leaf energy content has been found to increase with altitude within genera (Zachhuber and Larcher, 1978) or across life forms (Baruch, 1982). At regional scales, leaf energy content tends to increase from tropical rainforest to arctic tundra (Golley, 1961, 1969; Jordan, 1971). These findings indicate that leaf energy content and CC are in some way coupled with temperature and/or other climatic factors. However, uncertainty still remains if the variation of leaf energy content is caused by varying climatic factors (e.g., temperature) or species replacement, because leaf energy content varies substantially among species and sites (Golley, 1961; Bliss, 1962; Larcher, 1995; Villar and Merino, 2001). Significant difference in CC can be detected even between species with a close phylogenetic relationship (Chang et al., 2011), suggesting that the leaf energy properties may be mainly shaped by the environment (Poorter et al., 2006). There are few data to examine the variation of leaf energy content and CC within a single species along an altitudinal

1146-609X/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.actao.2012.05.011

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transect (Baruch, 1982), especially where measurements of climatic factors are available. Such knowledge is essential to understand how plants respond to the future climate change because transformations in leaf energy properties are important fundamental measures of plant efficiency in resource utilization. As an exclusive evergreen herb distributed widely under Rhododendron shrubs, Bergenia purpurascens dominates above 4000 m a.s.l. along the north-facing slopes of U-shaped valleys in the Sergyemla Mountains, southeast Tibet. Such a species distribution pattern provides an ideal system to investigate if withinspecies leaf energy properties vary with altitude where alpine plants above timberline obviously suffer from low temperature stress, and therefore are assumed to be sensitive to climate change (Körner, 1999). In this study, using measured data of leaf CC, HC and related leaf traits and climatic variables along an altitudinal gradient at the timberline ecotone in the Sergyemla Mountains, we aimed to test 1) whether there is an altitudinal increase in leaf energy properties within B. purpurascens, and 2) whether the variation of leaf energy content is consistent with the hypothesis that it is due to declining air temperature. The second aim may have great implications under the scenarios of climate warming. 2. Materials and methods 2.1. Study sites and species This study was conducted on the north-facing slope of a U-shaped valley (29 360 N, 94 360 E) at the peak of the Sergyemla Mountains in southeast Tibet, which is a part of the Southeast Tibet Station for Alpine Environment Observation and Research, Chinese Academy of Sciences. Along the north-facing slope (4170e4642 m), vegetation types change from sub-alpine and timberline evergreen needle-leaved forests of Abies georgei var. smithii (<4320 m) to alpine Rhododendron shrublands (>4320 m). Sparse, low shrubs (<1 m) dominate above 4500 m a.s.l. According to our five-year (2005e2010) meteorological observations at 4390 m, mean January and July air temperatures were 6.9  C and 8.4  C, respectively, and mean annual precipitation was 926.6 mm. To further understand the altitudinal trends in air temperature and photosynthetically active radiation (PAR), additional climatic data at the bottom of the U-shaped valley (4190 m) were obtained from the experimental station of the School of Life Sciences, Sun Yat-Sen University. Along the slope, air temperature (Fig. 1A) tended to decrease with increasing altitude, and the lapse rates of mean air temperatures for the year and July were calculated as 0.33  C/100 m and 0.57  C/100 m, respectively. PAR for almost every month was lower at the higher station than at the bottom station (Fig. 1B). At daily scale, the main difference in PAR between the two sites generally occurs within 12:00e17:00 (data not shown), indicating a cloudier environment at the higher altitude during the day, which was probably the reason why PAR was lower at the high altitude. Based on the lapse rates of mean air temperatures with altitude, we analyzed the relationships between air temperature and leaf energy properties of CCm, CCa and HC. Being located at the bottom of the valley, the station at 4190 m may be positioned within a cold air lake. However, the temperature inversion mainly occurs in winter, because this site sits in the incised valley of Yaluzanbu River in the southeast Tibetan Plateau which provides natural passages for the southwestern summer monsoon to transport moisture. Accordingly, the absolute value of the lapse rate of mean July air temperature with altitude is larger than that of the annual mean air temperature. In this study, we further examined the hourly recorded data of soil temperature and moisture (HOBO weather micro-station, Onset Inc., U.S.A.) for two low shrublands at 4390 m and 4500 m

Fig. 1. Comparison of (A) daily mean air temperature and (B) average daily photosynthetically active radiation (PAR) between altitudes of 4190 m (at the bottom of the valley) and 4390 m (above timberline) along the north-facing slope of a U-shaped valley in the Sergyemla Mountains.

on the same slope. Given the similar leaf area index in both shrubland sites (2e3 m2 m2), the daily mean soil temperature at 20 cm during the growing season (July and September) was >2  C lower at 4500 m than at 4390 m (Fig. 2A), suggesting a shortening growth season length and a low-temperature limitation to plant growth at high altitude. At both shrubland sites, the daily mean soil moisture on mass basis (the measured soil volumetric moisture divided by soil bulk density) was typically >20% (Fig. 2B), indicating that the soil moisture is relatively abundant for plant growth (Huzulák and Matejka, 1983). However, the lapse rate of soil temperature or moisture cannot be simply calculated from two altitudes because of strong heterogeneity between different microenvironments induced by vegetation cover and microtopography (Liu and Luo, 2011). B. purpurascens, belonging to Saxifragaceae, is a perennial evergreen herb generally with 2e8 basal leaves. The height of the mature herbs ranges between 4 and 30 cm. Several aboveground stems may sprout from the same belowground stem. The flower stage generally begins in early June and may last for 2e3 weeks. Investigation on B. purpurascens populations along the north-facing slope indicated that the density, cover and relative frequency were 7e24 plants m2, 7%e26% and 63%e100%, respectively. Aboveground stem height (an indicator of plant height) tended to decrease with altitude (R2 ¼ 0.34, P < 0.001, Appendix). 2.2. Sampling method and leaf measurements We set a transect along the north-facing slope from outside the timberline forest (4320 m) to the hilltop (4642 m) during early June of 2008 before new leaf growth. Along the transect, we set

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Fig. 2. Comparison of (A) daily mean soil temperature and (B) soil moisture content on mass basis at 20 cm (2005e2010) for two low shrublands at 4390 m and 4500 m on the north-facing slope of the Sergyemla Mountains.

sampling sites at about every 10 m in altitude. In total, 41 sites were selected. At each site, we sampled all the aboveground parts (distinct scars can be found between aboveground and belowground stem) of four to six B. purpurascens individuals at a distance of 2 m along a horizontal line under Rhododendron shrubs. Samples were immediately enclosed in plastic envelopes. A total of 185 individuals were sampled with altitudes ranging from 4320 m to 4642 m. Geographical locations of the sampling sites were determined with a global positioning system. For each of the 185 individuals, all leaf laminas were separated from the aboveground stem, and stem height (i.e. maximum length of leaf petiole per individual) was measured with a ruler. One-sided leaf area of each individual was measured by using a portable area meter (CI-203, CID, Inc., USA). The dry mass of leaves was determined from material dried at 70  C for 48 h. Specific leaf area (SLA) of each individual was calculated as the ratio of fresh leaf area to its dry mass. Because the leaf dry mass of an individual (generally 0.5e3 g) was not enough for measurements of nitrogen concentration (Nmass), the heat of combustion (HC), and ash and lignin concentrations, the leaf samples of different individuals at the same site were merged and then ground finely. Nmass was analyzed with a micro-Kjeldahl assay. HC was measured with an oxygen bomb calorimeter (SDCM-IIIa, SD Inc., Changsha, China). The relative difference between the three replicates of each HC sample was limited below 2%. Ash concentration (AC, %) was determined by incinerating 1 g powder in a muffle furnace at 500  C until a whitegray residue remained (3e4 h). According to Williams et al. (1987), mass-based leaf construction cost (CCm) was calculated using the data of Nmass, HC and AC:

CCm ¼ ½ð0:06968HC  0:065Þð1  ACÞ þ 7:5062ðkN=14:0067Þ=EG

(1)

where CCm ¼ construction cost (g glucose g1), HC ¼ heat of combustion (kJ g1), AC ¼ total ash content (%), N ¼ total Kjeldahl nitrogen (g g1), k is the oxidation state of the N source (þ5 for nitrate, or 3 for ammonium), and EG is the growth efficiency (the fraction of the cost required to provide reductant that is not incorporated into the biomass; see Williams et al., 1987; Poorter and De Jong, 1999). The value of EG used in this study was 0.89 (Williams et al., 1987; Villar and Merino, 2001). Although the principal source of N available to terrestrial plants under most field conditions is nitrate (Villar and Merino, 2001), some plants in cold ecosystems (e.g. tundra) may mainly utilize ammonium (Atkin et al., 1993). More recently, Warren (2009) suggested that plants in low temperature conditions generally favor uptake of amino acids. Since B. purpurascens may utilize nitrate, ammonium, amino acids or a combination of these N sources, we calculated minimum and maximum CCm by using k ¼ - 3 and k ¼ þ 5, respectively, and the average CCm was used. CC per unit leaf area (CCa, equivalent to grams glucose per square meter) was the ratio of CCm to SLA. Leaf lignin concentration (LC, %) was determined by the Klason procedure (Hatfield and Fukushima, 2005). We refluxed the 0.2 g dried and milled sample with 72% H2SO4 for 4 h, and then diluted the mixture with distilled water within a conical flask. The insoluble lignin residue was achieved by simple filtration, which was washed with hot distilled water (at least 4 times) until the pH was >6. Then the residues were oven dried and weighted, and the rough lignin concentrations were determined. This procedure may not produce an exact measure of amount of leaf lignin because the residues may contain a small amount of non-hydrolyzable compounds like leaf wax (Hatfield and Fukushima, 2005). In late August when new leaves were fully expanded, we further measured new leaf dry mass per aboveground plant (NLDM, g plant1) and new leaf dry mass per leaf area (NLDMA, g cm2) along the same transect and at the same sites. We additionally sampled 6e8 individuals for each site. According to our weekly phenological observations, we found that the new leaves of B. purpurascens generally extended between late June and late August, and withered between July and October in the next year. Therefore, in most cases new leaves which were fresher and tender could be distinguished from the old ones. The leaf and aboveground stem dry mass of each individual was then determined from materials at 70  C for 48 h. NLDMA was calculated as the ratio of NLDM to SLA of the old leaves. The site-averaged NLDM and NLDMA data were then correlated with the measurements of CC, HC, LC, AC, Nmass, and SLA. 2.3. Statistical analysis To examine the relationships between leaf properties and their correlations with altitude and air temperature, the data were fitted with a simple linear model (y ¼ a þ bx). The data were not transformed because all the leaf trait measurements were normally distributed. The statistical analysis was performed using SPSS 15.0 for Windows (SPSS Inc., Chicago, USA), and all significant differences were reported at P < 0.05. 3. Results 3.1. Altitudinal variations in leaf CC and related components As altitude increased, HC (Fig. 3A, R2 ¼ 0.33, P < 0.001) and LC (Fig. 3B, R2 ¼ 0.30, P < 0.001) increased, whereas AC (Fig. 3C, R2 ¼ 0.31, P < 0.001), Nmass (Fig. 3D, R2 ¼ 0.65, P < 0.001) and SLA (Fig. 3E, R2 ¼ 0.25, P < 0.001) declined. Increasing HC led to an increasing pattern of CCm (Fig. 3F, R2 ¼ 0.35, P < 0.001), while decreasing SLA and increasing CCm both resulted in an increasing

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Fig. 3. Altitudinal variations in (A) heat of combustion, (B) lignin concentration, (C) ash concentration, (D) mass-based leaf nitrogen concentration (Nmass), (E) specific leaf area (SLA), (F) mass-based leaf construction cost (CCm), and (G) area-based leaf construction cost (CCa) in leaves of Bergenia purpurascens above timberline on the north-facing slope of a U-shaped valley in the Sergyemla Mountains.

pattern of CCa (Fig. 3G, R2 ¼ 0.26, P < 0.001). CCm and CCa were positively correlated with HC and LC but negatively with Nmass, SLA and AC (Table 1).

NLDM, NLDMA was only positively correlated with AC and NLDM and poorly related to the other traits (Table 1). 3.3. Relationships between leaf CC and HC and air temperature

3.2. Altitudinal variations in new leaf dry mass per plant and per leaf area Along the transect, both NLDM (Fig. 4A, R2 ¼ 0.54, P < 0.001) and NLDMA (Fig. 4B, R2 ¼ 0.28, P < 0.01) significantly decreased with increasing altitude. NLDM was positively correlated with SLA and Nmass but negatively with CCm, CCa, LC, and HC (Table 1). Unlike

According to the lapse rate of July and annual mean air temperature along the transect (0.57  C/100 m and 0.33  C/ 100 m, respectively), the relationships between leaf CC and HC and air temperature were further examined. Leaf CCm (Fig. 5A, R2 ¼ 0.35, P < 0.001), CCa (Fig. 5B, R2 ¼ 0.24, P < 0.001) and HC (Fig. 5C, R2 ¼ 0.33, P < 0.001) all significantly decreased with

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Table 1 Matrix of correlation coefficients for linear relationships between mass- and area-based leaf construction cost (CCm, CCa) and associated leaf traits: heat of combustion (HC), concentration of ash (AC), nitrogen (Nmass) and lignin (LC), specific leaf area (SLA), new leaf dry mass per plant (NLDM) and per leaf area (NLDMA) of Bergenia purpurascens on the north-facing slope of the Sergyemla Mountains (sample size, n ¼ 33 for LC, n ¼ 39e41 for the other traits). Variables

CCm

CCa

HC

AC

Nmass

LC

SLA

NLDM

CCa(g glucose m2) HC (kJ g1) AC (%) Nmass (mg g1) LC (%) SLA (cm2 g1) NLDM (g plant1) NLDMA (g cm2)

0.47** 0.98*** 0.58*** 0.55*** 0.38* 0.49** 0.39* 0.26

0.38* 0.72*** 0.66*** 0.44* 0.97*** 0.54*** 0.03

0.43** 0.53*** 0.35* 0.42** 0.31* 0.23

0.60*** 0.50** 0.65*** 0.65*** 0.33*

0.37* 0.66*** 0.57*** 0.24

0.44** 0.50*** 0.30

0.50*** 0.04

0.84***

***

P < 0.001. ** P < 0.01. * P < 0.05.

increasing July mean air temperature. Similar patterns also existed between these leaf energy properties and annual mean air temperature (R2 ¼ 0.24-0.35, P < 0.01, data not shown). 4. Discussion 4.1. Altitudinal gradient in CC Our study, for the first time, investigated and quantified the altitudinal variations in within-species leaf energy content and CC. Along the altitudinal transect, the increasing rate of leaf energy content with altitude was 0.15 kJ g1/100 m. Compared with previous investigations of altitudinal variation in cross-species HC, our data is consistent with the derived data for Saxifraga (0.17 kJ g1/100 m, 1200e2600 m) and Primula (0.19 kJ g1/100 m, 960e2600 m) in the Alps by Zachhuber and Larcher (1978). Since the species studied by Zachhuber and Larcher (1978) and us consist of both evergreen (e.g. Saxifraga oppositifolia, S. paniculata, B. purpurascens) and deciduous (e.g. Primula glutinosa) plants, the leaf energy contents of both evergreen and deciduous alpine herbs are assumed to increase with rising altitude. Furthermore, the decreasing rate of HC with annual mean air temperature can be calculated as 0.47 kJ g1/oC in our study. We therefore believe that climatic factors play an important role in shaping the altitudinal pattern of leaf energy content. Because HC explained 96% of variations in CCm (from Table 1), similar patterns in CCm were inferred (Fig. 3F). So far, few studies have quantified the relationship between CC and temperature. Based on the derived data of leaf CC from 14 contrasting ecosystems by Villar and Merino (2001), we roughly analyzed the relationship between CCm and annual mean temperature (information of temperature were from the references or

deduced from latitude, 0.65  C per latitude between 20oe50 , Fang, 1992), and found that the decrease of CCm with temperature was only 0.005 g glucose g1/oC (R2 ¼ 0.51, P < 0.01). Compared with the latitudinal study, the decrease in CCm with temperature found in the current study is almost 8 times higher (0.039 g glucose g1/oC), indicating that alpine herbs above 4000 m are more sensitive to climate warming than species at low altitudes. 4.2. Altitudinal increase in leaf energy properties as a growth strategy for sustaining carbon gain and maximizing nitrogen-use efficiency Significant relationships between new leaf mass per plant and leaf properties of SLA, Nmass, LC, HC and CC (Table 1) further indicated that the variation of leaf properties were tightly coupled with plant growth. Altitudinal decreases in new leaf growth (Fig. 4A, B), SLA and Nmass (Fig. 3DeE) suggest a slow-growth rate at high altitudes. For evergreen species above the alpine timberline, having relatively high leaf CC and HC can be regarded as a growth strategy for sustaining carbon gain and maximizing nitrogen-use efficiency of dry matter production in response to high-altitude environmental stress. At high-altitudes, soil nutrient and water absorption by roots is fundamentally restricted by low temperature (Fig. 1A; Körner and Paulsen, 2004; Luo et al., 2005; Wieser and Tausz, 2007). Evergreen plants are assumed to adapt to the cold environment by prolonging mean residence time of nitrogen through increased leaf lifespan (Escudero et al., 1992; Eckstein et al., 1999), which is generally associated with decreased leaf Nmass and photosynthetic capacity (Reich et al., 1997; Wright et al., 2004; Luo et al., 2009). Global and regional studies indicate that evergreen species tend to extend leaf lifespan toward a colder environment (Wright et al., 2005; Zhang et al., 2010; van Ommen Kloeke et al.,

Fig. 4. Altitudinal variations in (A) new leaf dry mass per plant (NLDM) and (B) per leaf area (NLDMA) of Bergenia purpurascens on the north-facing slope of a U-shaped valley in the Sergyemla Mountains.

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2012). According to the cost-benefit theory, extension of leaf lifespan requires more carbon investments in leaves (Kikuzawa, 1991; Mediavilla et al., 2008), and therefore is correlated with higher leaf CCm (Mooney and Gulmon, 1979; Chabot and Hicks, 1982; Diamantoglou et al., 1989; Eamus and Prichard, 1998; Eamus et al., 1999; Villar and Merino, 2001), or higher ratio of leaf CCm to daily carbon gain (Williams et al., 1989; Sobrado, 1991; Coste et al., 2011). However, the increase in leaf CCm cannot fully explain the decrease in NLDM in our study, though they reflected a negative relationship. In comparison with relatively smaller changes in CCm along the transect, changes in SLA and hence CCa are stronger, suggesting SLA, an important determinant of plant relative growth rate (Cornelissen et al., 1998; Osone et al., 2008), could be more important in affecting the reduction in growth than CCm (Villar and Merino, 2001).

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Since environmental factors such as air temperature, UV-B radiation and soil nutrients may covary with altitude (Körner, 2007; González et al., 2007; Soethe et al., 2008), knowledge of how leaf CC varies with altitude is important to understand how alpine plants respond to climate and environmental change in the future under energetic aspects. Many studies indicate that leaf CCm tends to increase with increasing light intensity (Williams et al., 1989; Barthod and Epron, 2005; Poorter et al., 2006) and soil nitrogen concentration (Griffin et al., 1993, 1996; Poorter and De Jong, 1999). However, so far little research, according to our knowledge, has investigated possible effects of low temperature on leaf CC, especially for alpine ecosystems. Since PAR was lower at the higher station than at the lower one (Fig. 1B) in this study, variations in light intensity are unlikely to result in the increasing pattern of leaf CC. Although soil total nitrogen concentration in the topsoil tended to increase with altitude, soil C:N ratio was positively correlated with altitude (data not shown), suggesting slow decomposition of nutrients induced by low temperature at high altitude and thus the variation of soil nitrogen concentration may not be responsible for the altitudinal pattern of leaf CC. Because leaf CC, HC and NLDM (inferred from Fig. 4A) all decreased with increasing air temperature, evergreen herbs at high altitude are expected to construct cheaper leaves (leaves with lower CC) and increase productivity in response to global warming. Furthermore, an investigation on altitudinal variation of reproductive allocation for this species indicated that the relative reproductive allocation was more related (negative) to plant size than altitude (Wang et al., 2010), suggesting B. purpurascens may reduce its reproductive allocation because of the increased plant productivity (i.e. increased plant size, as inferred from Appendix). 4.3. Biochemical causes for altitudinal increases in leaf CC and HC

Fig. 5. Relationships of (A) CCm, (B) CCa and (C) HC to July mean air temperature across populations of Bergenia purpurascens.

Higher CCm, from a biochemical aspect, generally results from increased concentrations of some expensive compounds (Penning de Vries et al., 1974; Griffin et al., 1996; Poorter and Villar, 1997). Generally, there exist negative relationships between concentrations of expensive components, e.g., proteins and phenolics (Poorter and Villar, 1997; Poorter and De Jong, 1999). In this study, CC and HC were positively correlated with LC but negatively with Nmass (Table 1), indicating that some high-HC constituents like lignin, lipids and phenolic compound rather than proteins (about 75% of the total leaf nitrogen is engaged in leaf proteins, Chapin et al., 1987) might contribute to the observed pattern in leaf CCm. In addition, the negative relationship between CCm and Nmass was consistent with that detected from the equation in calculating CCm (Williams et al., 1987) under conditions of k ¼ 3, indicating that B. purpurascens in the cold alpine environment might utilize ammonium as its main N source consistent with results reported by Atkin et al. (1993). Altitudinal decrease in SLA and increase in LC suggest that the concentrations of some structural materials might increase with increasing altitude. There is evidence that low-SLA species may have much higher concentrations of lignin and total structural carbohydrates than high-SLA species (Poorter et al., 2009), and the decreased SLA may result from increased amounts of lignified cell wall and epidermis thickness (rich in lignin, Niinemets, 1999). Because lignin has a relatively high energy content (Griffin et al., 1996; Poorter and Villar, 1997), plants with higher lignin concentration in leaves tend to have higher leaf CCm (Table 1; Osunkoya et al., 2008). In addition, the Klason lignin assay may give an indication of leaf stiffness or modulus of elasticity (Baucher et al., 1998). Evergreen herbs with high leaf lignin concentration can enhance the mechanical support of leaves (leaf strength or elasticity) to resist cold-induced damage at high altitudes (Lütz, 2010).

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As a result of decreased SLA, the cell size and intercellular spaces within a leaf may decline (Cordell et al., 1998). Leaf water content of B. purpurascens decreased with altitude (R2 ¼ 0.37, P < 0.001, data not shown), suggesting another efficient way for plants to adapt to cold environments. Under low temperatures, alpine plants may protect photosynthesis and respiration through lowering the freezing point of leaves, which is partially achieved by increasing concentrations of anthocyanins, lipids and sucrose in cell saps (Bliss, 1962; Steyn et al., 2009). Increased anthocyanin and lipid concentrations may increase leaf CCm (Penning de Vries et al., 1974; Poorter, 1994; Griffin et al., 1996; Villar and Merino, 2001). Our data provide evidence that leaf energy properties (CC and HC) within a single species increase with increasing altitude, suggesting an efficient way for evergreen herbs to adapt to low-temperature environments. Acknowledgments Measurements of the heat of combustion were made in the Key Lab for Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden of Chinese Academy of Sciences. We thank Prof. M Cao, Z Zheng, Y Fu and H Lin et al. for their help in the experiment. We also thank the staff of Southeast Tibet Station for Alpine Environment Observation and Research of Chinese Academy of Sciences for their help in the field work, and Prof. GR Zhang for his providing climatic data from the experimental station of the School of Life Sciences, Sun Yat-Sen University. This work is funded by the National Natural Science Foundation of China (40901038) and the National Key Projects for Basic Research of China (2010CB951301). Appendix A Altitudinal variation in stem height of Bergenia purpurascens on the north-facing slope of a U-shaped valley in the Sergyemla Mountains.

References Atkin, O.K., Villar, R., Cummings, W.R., 1993. The ability of several high arctic plant species to utilize nitrate nitrogen under field conditions. Oecologia 96, 239e245. Barthod, S., Epron, D., 2005. Variations of construction cost associated to leaf area renewal in saplings of two co-occurring temperate tree species (Acer platanoides L. and Fraxinus excelsior L.) along a light gradient. Ann. For. Sci. 62, 545e551. Baruch, Z., 1982. Patterns of energy content in plants from the Venezuelan Paramos. Oecol 55, 47e52. Baruch, Z., Goldstein, G., 1999. Leaf construction cost, nutrient concentration, and net CO2 assimilation of native and invasive species in Hawaii. Oecologia 121, 183e192. Baucher, M., Monties, B., van Montagu, M., Boerjan, W., 1998. Biosynthesis and genetic engineering of lignin. Crit. Rev. Plant Sci. 17, 125e197.

Bliss, L.C., 1962. Caloric and lipid content in alpine tundra plants. Ecology 43, 753e757. Chabot, B.F., Hicks, D.J., 1982. The ecology of leaf life spans. Annu. Rev. Ecol. Syst. 13, 229e259. Chang, W., Zhang, S.B., Li, S.Y., Hu, H., 2011. Ecophysiological significance of leaf traits in Cypripedium and Paphiopedilum. Physiol. Plant 141, 30e39. Chapin, F.S., Bloom, A.J., Field, C.B., Waring, R.H., 1987. Plant response to multiple environmental factors. Biosci 37, 49e57. Cordell, S., Goldstein, G., Mueller-Dombois, D., Webb, D., Vitousek, P.M., 1998. Physiological and morphological variation in Metrosideros polymorpha, a dominant Hawaiian tree species, along an altitudinal gradient: the role of phenotypic plasticity. Oecologia 113, 188e196. Cornelissen, J.H.C., Castro-Diez, P., Carnelli, A.L., 1998. Variation in relative growth rate among woody species. Inherent variation in plant growth. In: Lambers, H., Poorter, H., Van Vuuren, M.M.I. (Eds.), Physiological Mechanisms and Ecological Consequences. Backhuys, Leiden, the Netherlands, pp. 363e392. Coste, S., Roggy, J.C., Schimann, H., Epron, D., Dreyer, E., 2011. A costebenefit analysis of acclimation to low irradiance in tropical rainforest tree seedlings: leaf life span and payback time for leaf deployment. J. Exp. Bot. 62, 3941e3955. Diamantoglou, S., Rhizopoulou, S., Kull, U., 1989. Energy content, storage substances, and construction cost and maintenance costs of Mediterranean deciduous leaves. Oecologia 81, 528e533. Eamus, D., Myers, B., Duff, G., Williams, R., 1999. A cost-benefit analysis of leaves of eight Australian savanna tree species of differing leaf life-span. Photosyn 36, 575e586. Eamus, D., Prichard, H., 1998. A cost-benefit analysis of leaves of four Australian savanna species. Tree Physiol. 18, 537e545. Eckstein, R.L., Karlsson, P.S., Weih, M., 1999. Leaf life span and nutrient resorption as determinants of plant nutrient conservation in temperate-arctic regions. New Phytol. 143, 177e189. Escudero, A., Arco, J., Sanz, I., Ayala, J., 1992. Effects of leaf longevity and retranslocation efficiency on the retention time of nutrients in the leaf biomass of different woody species. Oecologia 90, 80e87. Fang, J.Y., 1992. Study on the geographic elements affecting temperature distribution in China. Acta Ecol. Sin 12, 97e104 (in Chinese with English abstract). Golley, F.G., 1961. Energy values of ecological materials. Ecology 42, 581e584. Golley, F.G., 1969. Caloric value of wet tropical forest vegetation. Ecology 50, 5l7e519. González, J.A., Gallardo, M.G., Boero, C., Liberman Cruz, M., Prado, F.E., 2007. Altitudinal and seasonal variation of protective and photosynthetic pigments in leaves of the world’s highest elevation trees Polylepis tarapacana (Rosaceae). Acta Oecol. 32, 36e41. Griffin, K.L., 1994. Calorimetric estimation of construction cost and their use in ecological studies. Funct. Ecol. 8, 551e562. Griffin, K.L., Thomas, R.B., Strain, B.R., 1993. Effects of nitrogen supply and elevated carbon dioxide on construction cost in leaves of Pinus taeda (L.) seedlings. Oecologia 95, 575e580. Griffin, K.L., Winner, W.E., Strain, B.R., 1996. Construction cost of loblolly and ponderosa pine leaves grown with varying carbon and nitrogen availability. Plant Cell Environ. 19, 729e739. Hatfield, R., Fukushima, R., 2005. Can lignin be accurately measured? Crop Sci. 45, 832e839. Huzulák, J., Matejka, F., 1983. Relationship between soil moisture and leaf water potential of three forest tree species. Biol. Plant (Praha) 25, 462e467. Jordan, C.F., 1971. A world pattern in plant energetics. Am. Sci. 59, 425e433. Kikuzawa, K., 1991. A cost-benefit analysis of leaf habit and leaf longevity of trees and their geographical pattern. Am. Nat. 138, 1250e1263. Körner, C., 1999. Alpine Plant Life: Functional Plant Ecology of High Mountain Ecosystems. Springer, New York. Körner, C., 2007. The use of ’altitude’ in ecological research. Trends Ecol. Evol. 22, 569e574. Körner, C., Paulsen, J., 2004. A world-wide study of high altitude treeline temperatures. J. Biogeogr. 31, 713e732. Larcher, W., 1995. Physiological Plant Ecology. Ecophysiology and Stress Physiology of Functional Groups, third ed. Springer-Verlag, Berlin. Liu, X.S., Luo, T.X., 2011. Spatiotemporal variability of soil temperature and moisture across two contrasting timberline ecotones in the Sergyemla Mountains, southeast Tibet. Arct. Antarct Alp. Res. 43, 229e238. Luo, T.X., Luo, J., Pan, Y.D., 2005. Leaf traits and associated ecosystem characteristics across subtropical and timberline forests in Gongga Mountains, eastern Tibetan Plateau. Oecol 142, 261e273. Luo, T.X., Zhang, L., Zhu, H.Z., Daly, C., Li, M.C., Luo, J., 2009. Correlations between net primary productivity and foliar carbon isotope ratio across a Tibetan ecosystem transect. Ecogr 32, 526e538. Lütz, C., 2010. Cell physiology of plants growing in cold environments. Protoplasma 244, 53e73. Mediavilla, S., Garcia-Ciudad, A., Garcia-Criado, B., Escudero, A., 2008. Testing the correlations between leaf life span and leaf structural reinforcement in 13 species of European Mediterranean woody plants. Funct. Ecol. 22, 787e793. Mooney, H.A., Gulmon, S.L., 1979. Environmental and evolutionary constraints on the photosynthetic characteristics of higher plants. In: Solbrig, O.T., Jain, S., Johnson, G.B., Raven, P.H. (Eds.), Topics in Plant Population Biology. Colombia University Press, New York, pp. 316e337.

L. Zhang et al. / Acta Oecologica 43 (2012) 72e79 Niinemets, Ü., 1999. Components of leaf dry mass per area e thickness and density e alter leaf photosynthetic capacity in reverse directions in woody plants. New Phytol. 144, 35e47. Osone, Y., Ishida, A., Tateno, M., 2008. Correlation between relative growth rate and specific leaf area requires associations of specific leaf area with nitrogen absorption rate of roots. New Phytol. 179, 417e427. Osunkoya, O.O., Daud, S.D., Wimmer, F.L., 2008. Longevity, lignin content and construction cost of the assimilatory organs of Nepenthes species. Ann. Bot. 102, 845e853. Penning de Vries, F.W.T., Brusting, A.H.M., van Laar, H.H., 1974. Products, requirements and efficiency of biosynthesis: a quantitative approach. J. Theor. Biol. 45, 339e377. Poorter, H., 1994. Construction costs and payback time of biomass: a whole plant perspective. In: Roy, J., Gariner, E. (Eds.), A Whole Plant Perspective on Carbonnitrogen Interactions. SPB Academic Publishing, Hague, Netherlands, pp. 11e127. Poorter, H., Villar, R., 1997. The fate of acquired carbon in plants: chemical composition and construction cost. In: Bazzaz, F.A., Grace, J. (Eds.), Plant Resources Allocation. Academic Press, San Diego, USA, pp. 39e72. Poorter, H., De Jong, R., 1999. A comparison of specific leaf area, chemical composition and leaf construction costs of field plants from 15 habitats differing in productivity. New Phytol. 143, 163e176. Poorter, H., Pepin, S., Rijkers, T., De Jong, Y., Evans, J.R., Körner, C., 2006. Construction costs, chemical composition and payback time of high and low-irradiance leaves. J. Exp. Bot. 57, 355e371. Poorter, H., Niinemets, U., Poorter, L., Wright, I.J., Villar, R., 2009. Causes and consequences of variation in leaf mass per area (LMA): a meta-analysis. New Phytol. 182, 565e588. Reich, P.B., Walters, M.B., Ellsworth, D.S., 1997. From tropics to tundra: global convergence in plant functioning. Proc. Natl. Acad. Sci. USA 94, 13730e13734. Sobrado, M.A., 1991. Cost-benefit relationships in deciduous and evergreen leaves of tropical dry forest species. Funct. Ecol. 5, 608e616. Soethe, N., Lehmann, J., Engels, C., 2008. Nutrient availability at different altitudes in a tropical montane forest in Ecuador. J. Trop. Ecol. 24, 397e406. Song, L.Y., Guang-Yan, N., Chen, B.M., Peng, S.L., 2007. Energetic cost of leaf construction in the invasive weed Mikania micrantha HBK and its co-occurring species: implications for invasiveness. Bot. Stud. 48, 331e338. Steyn, W.J., Wand, S.J.E., Jacobs, G., Rosecrance, R.C., Roberts, S.C., 2009. Evidence for a photoprotective function of low-temperature-induced anthocyanin accumulation in apple and pear peel. Physiol. Plant 136, 461e472.

79

van Ommen Kloeke, A.E.E., Douma, J.C., Ordoñez, J.C., Reich, P.B., van Bodegom, P.M., 2012. Global quantification of contrasting leaf life span strategies for deciduous and evergreen species in response to environmental conditions. Glob. Ecol. Biogeogr. 21, 224e235. Villar, R., Merino, J., 2001. Comparison of leaf construction costs in woody species with differing leaf life-spans in contrasting ecosystems. New Phytol. 151, 213e226. Villar, R., Ruiz-Robleto, J., De Jong, Y., Poorter, H., 2006. Differences in construction costs and chemical composition between deciduous and evergreen woody species are small as compared to differences among families. Plant Cell Environ. 29, 1629e1643. Wang, Y., Hu, L.J., Duan, Y.W., Yang, Y.P., 2010. Altitudinal variations in reproductive allocation of Bergenia purpurascens (Saxifragaceae). Acta Bot. Yun. 32, 270e280 (in Chinese with English abstract). Warren, C.R., 2009. Why does temperature affect relative uptake rates of nitrate, ammonium and glycine: a test with Eucalyptus pauciflora. Soil Biol. Biochem. 41, 778e784. Williams, K., Percival, F., Merino, J., Mooney, H.A., 1987. Estimation of tissue construction cost from heat of combustion and ogranic nitogen-content. Plant Cell Environ. 10, 725e734. Williams, K., Field, C.B., Mooney, H.A., 1989. Relationships among leaf construction cost, leaf longevity, and light environment in rain-forest plants of the genus Piper. Am. Nat. 133, 198e211. Wieser, G., Tausz, M., 2007. Trees at Their Upper Limit: Treelife Limitation at the Alpine Timberline. Springer, Netherlands. Wright, I.J., Reich, P.B., Westoby, M., Ackerly, D.D., Baruch, Z., Bongers, F., CavenderBares, J., Chapin, T., Cornelissen, J.H.C., Diemer, M., Flexas, J., Garnier, E., Groom, P.K., Gulias, J., Hikosaka, K., Lamont, B.B., Lee, T., Lee, W., Lusk, C., Midgley, J.J., Navas, M.L., Niinemets, U., Oleksyn, J., Osada, N., Poorter, H., Poot, P., Prior, L., Pyankov, V.I., Roumet, C., Thomas, S.C., Tjoelker, M.G., Veneklaas, E.J., Villar, R., 2004. The worldwide leaf economics spectrum. Nature 428, 821e827. Wright, I.J., Reich, P.B., Cornelissen, J.H.C., Falster, D.S., Groom, P.K., Hikosaka, K., Lee, W., Lusk, C.H., Niinemets, U., Oleksyn, J., Osada, N., Poorter, H., Warton, D.I., Westoby, M., 2005. Modulation of leaf economic traits and trait relationships by climate. Global Ecol. Biogeogr 14, 411e421. Zachhuber, K., Larcher, W., 1978. Energy contents of different alpine species of Saxifraga and Primula depending on their altitudinal distribution. Photosyn 12, 436e439. Zhang,., L., Luo, T.X., Zhu, H.Z., Daly, C., Deng, K.M., 2010. Leaf life span as a simple predictor of evergreen forest zonation in China. J. Biogeogr. 37, 27e36.