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Keywords:

  • countergradient variation;
  • herbivory;
  • leaf economics spectrum;
  • leaf mass per area;
  • leaf resistance to fracture;
  • neutral detergent fibre;
  • subtropical rainforest

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • When grown in a common light environment, the leaves of shade-tolerant evergreen trees have a larger leaf mass per unit area (LMA) than their light-demanding counterparts, associated with differences in lifespan. Yet plastic responses of LMA run counter to this pattern: shade leaves have smaller LMA than sun leaves, despite often living longer.
  • We measured LMA and cell wall content, and conducted punch and shear tests, on sun and shade leaves of 13 rainforest evergreens of differing shade tolerance, in order to understand adaptation vs plastic responses of leaf structure and biomechanics to shade.
  • Species shade tolerance and leaf mechanical properties correlated better with cell wall mass per unit area than with LMA. Growth light environment had less effect on leaf mechanics than on LMA: shade leaves had, on average, 40% lower LMA than sun leaves, but differences in work-to-shear, and especially force-to-punch, were smaller. This was associated with a slightly larger cell wall fraction in shade leaves.
  • The persistence of shade leaves might reflect unattractiveness to herbivores because they yield smaller benefits (cell contents per area) per unit fracture force than sun leaves. In forest trees, cell wall fraction and force-to-punch are more robust correlates of species light requirements than LMA.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Species turnover during succession in evergreen forests is associated with variation in a suite of leaf structural and functional traits. Shade-tolerant late-successional evergreen trees have leaves with a lower photosynthetic capacity, lower nutrient concentrations and a larger leaf mass per unit area (LMA) than fast-growing light-demanding species, when comparisons are made in a standardized light environment (Lusk & Warton, 2007; Sterck et al., 2006; Walters & Reich, 1999). In this sense, adaptation of evergreen leaf structure and function to shade seems to share features in common with adaptation to certain other unproductive environments (Reich et al., 2003; Westoby et al., 2002). The general correlation of these traits worldwide with leaf lifespan (Wright et al., 2004) suggests, at face value, that a large LMA is required to achieve a long leaf lifespan.

Curiously, plastic responses of LMA to shade run counter to the constitutive response evident in comparative studies of evergreens of differing shade tolerance (Björkman, 1972; Kitajima, 1994; Lusk et al., 2008). There is often a strong similarity between plastic and evolutionary responses of quantitative traits to environmental gradients (Conover & Schultz, 1995), yet shade leaves invariably have smaller LMA than sun leaves of the same species (Givnish, 1988; Walters & Reich, 1999). How can this apparent divergence of plastic and evolutionary responses of LMA to shade be explained? Furthermore, shade leaves often live longer than sun leaves of the same species, despite their lower LMA (Reich et al., 2004). If prolongation of leaf lifespan requires a large LMA, how can we explain the persistence of shade leaves?

In addressing this paradox, it is helpful to consider the different components of leaves that contribute to variation in LMA (Lusk et al., 2008). We can think of a leaf as composed of structural components (cell walls, cuticle) and symplastic components (cell contents, including proteins, electrolytes and nonstructural carbohydrates) (Roderick et al., 1999; Shipley et al., 2006). The large LMA of shade-tolerant evergreens probably reflects a large structural component, protecting the leaf against herbivores and physical stresses. By contrast, plastic responses to light are probably dominated by variation in the amount of cell contents per area. The most conspicuous anatomical difference between sun and shade leaves is in the amount of palisade mesophyll (Onoda et al., 2008), associated with differences in photosynthetic capacity (Niinemets & Tenhunen, 1997; Terashima et al., 2001). Shade leaves would therefore be expected to have a slightly larger cell wall fraction than sun leaves, and there is some empirical support for this idea (Onoda et al., 2008; Poorter et al., 2006).

It is also helpful to consider the main threats to leaf survival. Wind abrasion, tatter and other damage caused by the elements mainly affects plants in exposed, open habitats (MacKerron, 1976), as well as tree crowns in the forest canopy (Putz et al., 1984). By contrast, plants in all habitats are exposed to herbivores, which have been estimated to cause annual leaf area losses of 14–26% in humid evergreen forests (Lowman, 1984). Although plants have evolved a celebrated diversity of chemical defences, especially against insect herbivores (Coley & Barone, 1996; Rosenthal & Janzen, 1979), mechanical strength remains the most effective generalist defence of fully expanded leaves, and mechanical properties are often good predictors of the degree of herbivory (Choong, 1996; Coley, 1983; Peeters et al., 2007). Overall fracture resistance is, in turn, primarily a function of the amount of cell wall per unit area (Choong, 1996). The cell wall fraction of leaves is also likely to influence the attractiveness of plants to chewing herbivores, by determining the cost–benefit economics of feeding choices. A shade leaf with a small LMA but a large cell wall fraction might be unattractive because of the modest return of energy and nutrients from the cell contents, compared with the amount of energy required to cut, chew and digest the leaf. If herbivores are the biggest single threat to leaves, cell wall fraction may be a robust correlate of leaf lifespan, irrespective of growth light environment.

We examined structural and mechanical properties of sun and shade leaves of 13 rainforest evergreens of differing light requirements, in an attempt to reconcile plastic and constitutive responses of LMA to shade. We addressed the following hypotheses: adaptation of evergreens to light gradients involves variation in the amount of cell wall per unit area; plastic responses of LMA to light gradients mainly reflect variation in the amount of cell contents per unit area; plastic responses of leaves to light affect leaf mechanical properties less than LMA. To this end, we measured LMA and cell wall content of sun and shade leaves of each species, and conducted punch and shear tests.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Study site and species

The study was carried out in subtropical rainforest in Nightcap National Park (New South Wales, Australia) located at 28°38′S, 153°20′E and at an elevation of 380 m asl. Mean annual temperature at the nearest meteorological station (Whian Whian) is estimated at 22.4°C and mean annual rainfall at 2314 mm, with a minimum in late winter to early spring (Bureau of Meteorology, 2009). No month receives less than 50 mm on average. Plant material for measurement of leaf traits was collected from a 15-yr-old second-growth stand of c. 10 ha in area, and distributions of juveniles in relation to canopy openness were quantified throughout both this stand and adjacent old-growth stands (see the following section). Both second-growth and old-growth stands grow on fertile soils derived from basalt (Turner & Kelly, 1981). The overstorey of the second-growth stand was dominated by Polyscias murrayi (F. Muell.) Harms (Araliaceae), whereas no one species prevailed in the much more diverse canopy of the old-growth forest. Thirteen common evergreens were chosen for study, ranging from species common in early successional environments, such as Polyscias elegans and Duboisia myoporoides, to those well represented in the understorey of the old-growth forest, including Anthocarapa nitidula and Castanospermum australe (Table 1).

Table 1.   Subtropical rainforest species studied for leaf structural and mechanical properties
SpeciesFamily% CO10Sample size
  1. Data show the 10th percentile of the distribution of juveniles (50–200 cm tall) in relation to % canopy openness (% CO10), as an indicator of the minimum light intensities tolerated by each species. Nomenclature follows Floyd (2008).

Castanospermum australe A. Cunn. ex MudieFabaceae1.0844
Sarcopteryx stipata (F. Muell.) Radlk.Sapindaceae1.1295
Anthocarapa nitidula (Benth.) T.D. Penn. ex Mabb.Meliaceae1.3138
Cryptocarya glaucescens R. Br.Lauraceae1.8445
Dysoxylum fraserianum (A. Juss.) Benth.Meliaceae1.8756
Guioa semiglauca (F. Muell.) Radlk.Sapindaceae2.3353
Diploglottis australis (G. Don) Radlk.Sapindaceae2.5353
Flindersia schottiana F. Muell.Rutaceae3.2080
Pentaceras australe (F. Muell.) Hook. f. ex Benth.Rutaceae3.4322
Melicope micrococca (F. Muell.) T.G. HartleyRutaceae4.5829
Macaranga tanarius (L.) Muell. Arg.Euphorbiaceae4.6720
Polyscias elegans (C. Moore & F. Muell.) HarmsAraliaceae4.8657
Duboisia myoporoides R. Br.Solanaceae6.1720

Species light requirements

We developed an index of the minimum light intensities tolerated by each species, by calculating the 10th percentile of the distribution of juveniles in relation to the percentage of canopy openness (% CO10) (cf. Lusk & Piper, 2007; Lusk & Reich, 2000). Shade-tolerant species such as C. australe have low % CO10 values, while light-demanders such as P. elegans have high values (Table 1). Indices based on the distribution of established juveniles in relation to light environment are generally well correlated with other measures of shade tolerance, such as survival in low light (Poorter & Bongers, 2006). Distributions of juveniles 50–200 cm tall were quantified in relation to % CO10 estimates obtained from hemispherical photographs taken using a Nikon Coolpix digital camera with a 182° fish-eye adapter, and processed using Gap Light Analyzer (Frazer et al., 1999). Sampling was carried out on transects run through both second-growth and old-growth stands, and included forest margins and treefall gaps. At sample points spaced at random intervals (5–10 m apart) along transects, hemispherical photographs were taken at 200 cm height, and the presence of juveniles 50–200 cm tall was recorded in a circular plot of 1 m diameter, centred on the sample point.

Collection of plant material

Plant material was collected in the second-growth forest. ‘Sun’ leaves were obtained from 50–200 cm tall juveniles growing on the forest margin or in large canopy gaps; potentially suitable plants were marked and hemispherical photographs were then taken to ensure selection only of plants growing at 15–24% CO10. This range of canopy openness is roughly equivalent to light intensities measured beneath 300–700 m2 treefall gaps in tropical rainforest by Brown (1993). ‘Shade’ leaves were obtained from juveniles growing at 5–8% CO10 in the interior of the stand. Four to seven saplings per species were sampled in each light environment. There was no significant interspecific variation in mean light environments of either sun leaves (ANOVA, F = 1.19, P = 0.32) or shade leaves (F = 1.12, P = 0.36).

One to three leaves or leaflets (without petioles or petiolules) were collected from each sapling, choosing the youngest fully expanded intact leaves on each plant. Growth is opportunistic in Australian subtropical rainforest, and although we sampled early during the winter, most plants were still producing new leaves. The leaves we sampled were therefore likely to be of a similar age in most plants. Leaves with major insect damage were avoided. Leaves were collected during the morning and kept on ice; leaf areas were measured before drying, which began within 36 h of collection.

Leaf area and mass

Projected areas of fresh samples were measured using a LI-3100 Leaf Area Meter (Li-Cor Biosciences, Lincoln, NE, USA). Fresh samples were weighed, and then dried at 60°C, to determine LMA (g m−2) and dry matter content.

Leaf mechanical properties

We employed two methods to evaluate leaf resistance to fracture but at different scales. A punch-and-die test was used for intercostal regions of the lamina (between secondary veins), the regions preferentially targeted by most insect herbivores (Choong, 1996). A shear test was used to measure lamina resistance to fracture at a larger scale, including secondary veins but excluding the midrib. A Mitutoyo micrometer was used to measure lamina thickness at the points selected for punch tests, and in the regions selected for shear tests.

An Instron 5542 materials testing machine (Instron, Canton, MA, USA) was used to conduct punch-and-die tests. We built a flat-ended, sharp-edged cylindrical steel punch (2.0 mm diameter) and a steel die with a sharp-edged aperture of small clearance (0.05 mm), which allowed us to fracture leaves mainly by shearing, rather than tensile or bending forces (Aranwela et al., 1999; Onoda et al., 2008). The punch-and-die test cell was mounted in the testing machine, and the punch was set up to pass through the middle of the hole of the die without any friction. The punch speed was kept constant (10 mm min−1) and the machine simultaneously recorded load (N) applied to sample and displacement (mm) of the punch (every 50 ms). We applied this punch-and-die test to intercostal regions of the lamina, averaging two measurements on each replicate leaf. Lamina thickness was measured at the point of fracture. The maximum force (Fmax) was expressed per unit fracture length, that is, the circumference of the punch. We refer to this throughout as ‘force to punch’ (N m−1). Fmax was also expressed per unit fracture area (i.e. the circumference of the punch × lamina thickness), and is referred to as ‘specific force to punch’ (kN m−2). Force to punch was also converted to a surface area basis (kN m−2) in order to derive an index of the cost–benefit economics of herbivory (see later in this section).

We used a leaf-cutting machine developed by Wright & Cannon (2001) to conduct shear tests. This machine measures the force required to cut a leaf at a constant cutting angle (20°) and speed (3.9 mm min−1), and is similar in principle to systems described by Darvell et al. (1996) and Aranwela et al. (1999). A leaf was fractured with a single transverse cut perpendicular to the midrib, equidistant between the base and the apex (Wright & Cannon, 2001). The work required to fracture a leaf was analysed separately for the midrib and for the rest of the lamina, including some secondary veins. Only the latter data are shown in this paper. The work was expressed per unit fracture length (J m−1) and per unit fracture area (J m−2); in the terminology of materials engineering these are known as ‘work to shear’ and ‘specific work to shear’, respectively (Sanson et al., 2001). The former has been referred to as ‘fracture toughness’ in some biomechanical studies (Aranwela et al., 1999).

We aimed to develop an index of the cost–benefit economics of herbivory on leaves differing in mechanical properties and nutritional content. This was calculated as the dry mass of cell contents yielded by punching a 2-mm-diameter leaf disc, divided by the force required to punch the disc (g kN−1). Although we did not measure tissue nutrient concentrations, the nutritional value of leaves is likely to be roughly proportional to cell contents per unit area: growth light environment often has little effect on leaf nitrogen concentrations on a per-mass basis, with the result that intraspecific comparisons often show leaf nitrogen per unit area to be roughly proportional to LMA (Abrams & Mostoller, 1995; Niinemets, 1997).

Cell wall fraction

The neutral detergent fibre method (Van Soest et al., 1991) was used to estimate cell wall content of samples. Dried leaf samples were ground and sieved with a 1-mm-diameter mesh. Ground samples of 40 mg were boiled for 1 h in a solution with 8 ml of neutral detergent and 4 μl of heat-stable amylase. Samples were then centrifuged for 5 min at 2500 g. The pellets were centrifuged three times with deionized water and once more with ethanol, spinning for 5 min at 2500 g each time. Pellets were dried at 60°C to a constant weight to determine the cell wall fraction of the leaf, and cell wall mass per unit area (CWA, g cm−2). The lost mass was considered to represent the nonstructural dry mass of the leaf (i.e. the cell contents).

Statistical analysis

All area-based parameters were log10-transformed before analysis in order to meet the assumption of additivity of effects (Quinn & Keough, 2002). Our field sampling system meant that light environments were effectively nested within species, that is, species were not compared in a fully randomized design. Nested ANOVAs were therefore used to compare the leaf traits of sun and shade leaves, and to test for interspecific variation. We then used major axis tests to examine relationships of sun and shade leaf traits with % CO10, using the SMATR package (Falster et al., 2006). Standardized major axis, rather than linear regression, is appropriate when what is sought is the line best describing the bivariate scatter of Y and X, rather than predictions of Y from X.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Overall effects of species and light environment

There was highly significant interspecific variation in all leaf structural and mechanical parameters, with species differing most in cell wall fraction, CWA, dry matter content and specific force-to-punch (Table 2; Supporting Information Table S1). The effect of light environment varied widely across parameters: sun and shade leaves differed comprehensively in LMA and cell contents per unit area, whereas there were significant but relatively weak effects on leaf density, force-to-punch and work-to-shear, and no effect on specific work-to-shear (Table 2).

Table 2.   Results of nested ANOVA testing for differences between species, and between sun and shade leaves
Response variableSource of variation
SpeciesLight environment (species)
FPFP
  1. The field sampling system meant that light environments were effectively nested within species, that is, species were not compared in a fully randomized design.

  2. aVariables that were log-transformed before analysis.

  3. LMA, leaf mass per unit area. See the Materials and Methods section for detailed explanations of ‘force to punch’, ‘specific force to punch’, ‘work to shear’ and ‘specific work to shear’.

LMAa36.37< 0.00116.29< 0.001
Cell wall fraction157.07< 0.0013.06< 0.001
Cell wall mass per unit areaa98.21< 0.0015.65< 0.001
Cell contents per unit areaa29.75< 0.00117.48< 0.001
Dry matter content112.50< 0.0017.79< 0.001
Lamina thicknessa27.73< 0.0019.51< 0.001
Leaf densitya29.09< 0.0012.090.021
Force to puncha74.31< 0.0012.490.005
Work to sheara13.15< 0.0012.120.019
Specific force to puncha85.71< 0.0013.50< 0.001
Specific work to sheara31.68< 0.0010.900.553
Cell contents yielded per unit fracture force33.51< 0.0017.31< 0.001

Interspecific relationships of structural and mechanical parameters with % CO10

Leaf mass per unit area, leaf density, cell wall fraction, CWA and dry matter content were all negatively correlated with % CO10 (Fig. 1). CWA showed the strongest relationship with this index of species light requirements (Fig. 1b), and varied more than fivefold across species. In no case did the major axis slope of any of these relationships differ significantly between sun and shade leaves (Table 3). Only LMA showed a significant shift in elevation between light environments, with LMA of sun leaves exceeding that of shade leaves by c. 40% on average (Table 3).

image

Figure 1.  Relationships of leaf structure and mechanical properties with an index of species light requirements (% CO10). Closed circles, shade leaves; open circles, sun leaves. All of the x-axes have logarithmic scales except for panels (b), (e) and (g) which are arithmetic. Table 3 gives results of major axis tests to determine if the slope or elevation of relationships differed between sun and shade leaves. LMA, leaf mass per unit area. See the Materials and Methods section for detailed explanations of ‘force to punch’, ‘specific force to punch’, ‘work to shear’ and ‘specific work to shear’.

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Table 3.   Major axis tests (Falster et al., 2006; Warton & Weber, 2002) to determine if relationships of species light requirements (% canopy openness, % CO10) with leaf structural and mechanical properties differ between sun and shade leaves (Fig. 1)
Y-variableDifference in slope? (P-value)Difference in elevation? (P-value)
  1. P-values show probabilities that observed differences are the result of chance. Lamina thickness and dry mass of cell contents per area showed no significant relationships with % CO10.

  2. LMA, leaf mass per area; na, not applicable. See the Materials and Methods section for detailed explanations of ‘force to punch’, ‘specific force to punch’, ‘work to shear’ and ‘specific work to shear’.

LMA0.6580.002
Leaf density0.9130.144
Cell wall mass per unit area0.8040.130
Cell contents per unit areanana
Cell wall fraction0.5530.265
Dry matter content0.5450.166
Lamina thicknessnana
Force to punch0.9980.300
Work to shear0.9400.074
Specific force to punch0.8600.127
Specific work to shear0.7930.958
Cell contents yielded per unit fracture force0.8970.001

Cell contents per unit area and lamina thickness were not related to species % CO10 (Fig. 1c,f). However, sun leaves averaged nearly 47% more cell contents per unit area than shade leaves, and their laminas were c. 22% thicker on average.

Biomechanical measures were all negatively correlated with % CO10 (Fig. 1), and in no case did the slope of these relationships differ between sun and shade leaves. Force-to-punch showed the strongest relationship (Fig. 1h), which did not differ significantly in elevation between sun and shade leaves (Table 3). Because of the greater thickness of sun leaves, this translated to a significantly higher specific force-to-punch in shade leaves (Fig. 1i; Table 3). The relationship of work-to-shear with % CO10 showed a marginally significant shift in elevation between sun and shade leaves (Table 3), meaning that shearing sun leaves tended to require more work than shearing shade leaves. Specific work-to-shear of sun and shade leaves showed essentially identical relationships with % CO10 (Fig. 1k; Table 3).

An index of the cost–benefit economics of herbivory was influenced by both interspecific differences in % CO10 and growth light environment (Fig. 1l; Table 3). Fracturing leaves of shade-tolerant species yielded fewer cell contents per unit of force than those of light-demanding species. Similarly, shade leaves yielded smaller potential benefits per unit of force than sun leaves of the same species. Interestingly, this index was not significantly correlated with LMA of either sun leaves (= −0.31, = 0.30) or shade leaves (= −0.21, P = 0.50).

Relationships between leaf structural and mechanical properties

There was a significant positive correlation between force-to-punch and work-to-shear (Fig. 2a). This relationship did not differ significantly between sun and shade leaves (Table 4).

image

Figure 2.  Relationships between leaf structural traits and mechanical properties. Closed circles, shade leaves; open circles, sun leaves. Table 4 gives results of major axis tests for differences in slope or elevation between sun and shade leaves, and shifts along a common slope. LMA, leaf mass per unit area. See the Materials and Methods section for detailed explanations of ‘force to punch’, and ‘work to shear’.

Download figure to PowerPoint

Table 4.   Major axis tests to determine if relationships among structural and mechanical properties differ between sun and shade leaves (Fig. 2)
X-variableY-variableDifference in slope? (P-value)Difference in elevation? (P-value)Shift along common slope? (P-value)
  1. P-values show probabilities that observed differences are the result of chance. Force to punch and work to shear showed no significant relationship with the dry mass of cell contents per unit area.

  2. LMA, Leaf mass per area; na, not applicable. See the Materials and Methods section for detailed explanations of ‘force to punch’ and ‘work to shear’.

Force to punchWork to shear0.9400.2540.310
LMAForce to punch0.5810.0060.093
Cell wall mass per unit areaForce to punch0.8390.4710.239
Cell contents per unit areaForce to punchnanana
LMAWork to shear0.6060.1610.032
Cell wall mass per unit areaWork to shear0.8390.4710.239
Cell contents per unit areaWork to shearnanana

Force-to-punch and work-to-shear were both positively correlated with LMA (Fig. 2b,e). The slope of these relationships did not differ significantly between sun and shade leaves (Table 4). Interestingly, at a given LMA, shade leaves required significantly more force-to-punch than sun leaves (Table 4). However, there was no significant difference between work-to-shear of sun and shade leaves at a common LMA (Table 4).

Force-to-punch and work-to-shear were both strongly correlated with CWA, and essentially identical relationships were found for sun and shade leaves (Fig. 2c,f; Table 4). Neither of these mechanical parameters was related to cell contents per unit area (Fig. 2d,g).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In agreement with our first hypothesis, the relationships between species shade tolerance and LMA of both sun and shade leaves were entirely the result of variation in CWA (Fig. 1). In both sun and shade, the four most shade-tolerant species had CWA values that were, on average, three times higher than the four most light-demanding species. This correlates closely with the biomechanical results, showing that leaves of shade-tolerant species were more resistant to fracture than those of their light-demanding associates (Fig. 1). By contrast, mass of cell contents per unit area was not related to shade tolerance or to mechanical properties (Figs 1, 2). Several other studies have reported similar patterns. Coley (1983) reported that shade-tolerant rainforest saplings had higher leaf fibre content and ‘toughness’ (more appropriately called punch strength) than species associated with treefall gaps. Similarly, successional position of tropical rainforest species has been shown to correlate with leaf lignin content (Poorter et al., 2004) and fracture resistance (Reich et al., 1995). The sum of the evidence seems to confirm that adaptation of evergreens to shade, like adaptation to nutrient-poor habitats (Reich et al., 2003), usually involves prolongation of leaf lifespan through investment in a large structural fraction.

Although leaves of most shade-tolerant species appear to be defended primarily by their mechanical properties, there may be exceptions. Laureliopsis philippiana (Looser) Shodde (Atherospermataceae), one of the most shade-tolerant trees of the temperate rainforests of South America, has long-lived leaves that nevertheless have high nitrogen concentrations (> 1.5% N: Lusk & Contreras, 1999) and quite a low dry matter content (CH Lusk, unpublished). Nitrogen concentrations > 1.5% have also been found in other shade-tolerant species of the same family, such as Atherosperma moschatum Labill. and Doryphora sassafras Endl. in southeastern Australia (Iddles et al., 2003; J. Read, pers. comm.); by contrast, many other shade-tolerant species in the temperate rainforests of the southern hemisphere seem to have nitrogen concentrations of c. 1% (Iddles et al., 2003; Lusk & Contreras, 1999). The Atherospermataceae are rich in alkaloids (Urzua & Cassels, 1978; Webb, 1949, 1952), which probably act as a chemical defence against herbivores. These observations seem to support the proposal that herbivores, rather than damage by weather or falling debris, are the main threat to leaf survival in shaded understoreys.

There was partial support for our second hypothesis, that plastic responses to light gradients mainly involve changes in the amount of cell contents per unit area. Sun leaves averaged 47% more cell contents per unit area than shade leaves, compared with 22% more cell wall mass. This is consistent with anatomical comparisons of sun and shade leaves, which often show large differences in the amount of palisade mesophyll, but less difference in structural features such as cuticle and cell wall thickness (Onoda et al., 2008; Terashima et al., 2001). As a result, the cell wall fraction of shade leaves was slightly higher than that of sun leaves (Fig. 1). This is fairly consistent with the results of a study of 22 diverse plant species (Poorter et al., 2006), which reported that shade leaves had significantly higher concentrations of structural carbohydrates than sun leaves. Similarly, Onoda et al. (2008) found that shade leaves of the herb Plantago major (Plantaginaceae) had a larger cell wall fraction than sun leaves.

Consistent with our third hypothesis, light environment had less effect on leaf biomechanics than on LMA (Tables 2, 3; Fig. 1). Shade leaves required only slightly less force-to-punch than sun leaves of the same species (Fig. 1h), and a larger specific force-to-punch (Fig. 1i). The greater strength of shade leaves at a given LMA (Fig. 2b) reflected their having a slightly larger cell wall fraction than sun leaves, and a slightly higher leaf density. Our results therefore generalize the findings of Onoda et al. (2008), who reported that, at a given LMA, shade leaves of P. major were stronger than sun leaves. Onoda et al. (2008) found that this was the result not only of a larger cell wall fraction in shade leaves, but also of a higher specific strength of cell walls. By contrast, we found that the relationship of force-to-punch with CWA did not differ much between sun and shade leaves (Fig. 2; Table 4), implying that light environment had little effect on specific cell wall strength. Sun and shade leaves differed more noticeably in work-to-shear (Fig. 1j), although not commensurately with differences in LMA. The percentage of leaf area occupied by veins tends to be higher in sun leaves than in shade leaves (Eschrich et al. 1989), and this may explain the greater work required to shear the former. By contrast, punch tests were conducted on intercostal regions of the lamina; the resulting lack of sensitivity to differences in vein area is probably one factor underlying the similarity of the results between punch tests on sun and shade leaves (Fig. 1h).

Shade leaves might be relatively unattractive to chewing herbivores because feeding on them involves a lower ratio of benefits to costs than feeding on sun leaves (Fig. 1i). Analyses often link leaf longevity to LMA (Wright et al., 2004), which seems reasonable on the basis of LMA being well correlated with measures of leaf fracture resistance in large datasets (Read et al., 2005). However, if herbivores are the biggest threat to leaf survival, the cost–benefit economics of herbivory may also be highly relevant to both inter- and intraspecific variation in leaf lifespan. Although a wide range of chewing herbivores may be capable of cutting through the laminas of shade leaves, feeding on them may be a relatively unattractive option for some herbivores because they offer smaller benefits per unit area (energy and nutrients in cell contents) per unit force required for ingestion (Figs 1, 2). The small amount of mesophyll in shade leaves might also make them unattractive to leaf miners. Cafeteria experiments have shown that generalist insect herbivores usually prefer sun leaves to shade leaves (Maiorana, 1981), although field studies comparing actual rates of herbivory on sun and shade leaves have yielded varying results (Louda et al., 1987; Lowman, 1985; Maiorana, 1981). The longer lifetimes of shade leaves compared with sun leaves (Reich et al., 2004) reflect more gradual self-shading in low-light environments where plants grow slowly (Ackerly & Bazzaz, 1995). However, the persistence of shade leaves also implies that they are at least as well protected as sun leaves; we suggest that at least part of this protection takes the form of economic unattractiveness to herbivores.

We found no evidence that plasticity of leaf structural or mechanical parameters was related to shade tolerance. In no case did the slopes of trait relationships with species’ light requirements differ significantly between sun and shade leaves (Table 3; Fig. 1). It has been hypothesized that leaf traits of light-demanding species will respond more plastically to light environment than those of their shade-tolerant associates (Bazzaz, 1979). Some of the strongest evidence to date in support of Bazzaz’s hypothesis comes from a comparative study of 16 sympatric Psychotria species, which reported that a wide range of physiological and morphological traits were more plastic in light-demanding species (Valladares et al., 2000). A review of seedling data gathered from diverse studies found that, of several traits analysed, only specific leaf area (1/LMA) showed evidence that amplitude of plasticity was related to light requirements, and even that evidence was not especially strong (Walters & Reich, 1999; Table 2). Experimental treatments often confound ontogenetic drift with plastic responses to treatments, because of growth rate differences between species at high resource levels (Coleman et al., 1994). Observational field studies, when they involve adequate quantification of light environments and comparisons of similar-sized plants, provide good opportunities to minimize ontogenetic effects on apparent plasticity. Studies such as these have not lent much support to the hypothesis of greater plasticity in light-demanding species (Chazdon & Kaufmann, 1993; Lusk, 2002; Lusk & Reich, 2000).

Our results point to a means of reconciling the puzzling divergence between inter- and intraspecific relationships of LMA with leaf lifespan. Long leaf lifespans are characteristic of both constitutive and plastic response of evergreens to shade; both these responses also involve increased cell wall fraction of leaves, despite divergent trends in LMA. The persistence of shade leaves, despite their low LMA, may thus reflect the economics of herbivory, as shade affects the amount of cell contents per unit area more than leaf strength. Our results also show that cell wall fraction and punch strength are more robust correlates of species light requirements or successional position than LMA, which is strongly responsive to light environment (Tables 2, 3). Although LMA is useful as an indicator of biomass partitioning (Lambers & Poorter, 1992; Westoby et al., 2002), neutral detergent fibre and punch tests might be preferable in studies aiming to index functional diversity of plant species in environments subject to wide variation in light intensity, such as forests.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank the Australian Research Council (ARC) for research grant DP0878209 to C.H.L., the ARC-NZ Network for Vegetation Function for funding Y.O.’s postdoctoral studies, the Spanish Ministry of Education and Science for funding A.G-G.’s visit, Ian Wright for assistance with the leaf-cutting machine, and Rous Water for permission to work in the Rocky Creek regeneration area.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information