Ecological Variation in Leaf Biomechanics and Its Scaling with Tissue Structure Across Three Mediterraneanclimate Plant Communit

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Functional Ecology 2013, 27, 544–554 doi: 10.1111/1365-2435.12059 Ecological variation in leaf biomechanics and its scaling with tissue structure across three mediterranean-climate plant communities

Rodrigo Mendez-Alonzo* ,1, Frank W. Ewers2 and Lawren Sack1

1Department of Ecology and Evolutionary Biology, University of California, Los Angeles, California, USA; and 2Biological Sciences Department, California State Polytechnic University Pomona, Pomona, California, USA

Summary 1. The mechanical resistance of leaves has key ecological implications but its basis has not been well understood, particularly at the tissue scale. We tested the hypotheses that leaf mechanical resistance should be a function of tissue density, increasing from the lamina to the midrib, and higher in drought-tolerant than drought-avoiding species. 2. In a common garden study, we quantified nine leaf biomechanical traits, including measurements of material and structural resistance, and in addition 17 morphological traits, at the tissue and whole-leaf scales, for 21 species from three semi-arid communities of California, USA. 3. The mechanical properties of leaves depended strongly on tissue density. Material resistance was significantly greater in the midrib than in the leaf lamina, and tissue resistances were significantly correlated among tissues, lower in deciduous coastal sage species and higher in evergreen drought-tolerant chaparral species. The proportion of the biomass invested in the midrib was lower in species bearing midribs and laminas of high material resistance. 4. Our results support the hypothesis of a hierarchical partitioning of leaf mechanical resistance among leaf tissues reflecting the investment of dry mass. Also, our data indicated a mechanical compensation in leaf design, where leaves with high material resistance and density deploy a relatively minor proportion of support tissue in the midrib. Finally, our results establish a quantitative basis for differences among communities in leaf biomechanics. Our results supported the classical characterization of the mediterranean-climate flora of California according to the dramatic increase in the mean leaf mechanical resistance from species of coastal sage to chaparral, with diverse leaf types in the Mojave Desert species.

Key-words: allometry, chaparral, coastal sage, Mojave Desert vegetation, plant economics, sclerophylly, stiﬀness, strength

remains scarce, particularly when considering the scale of Introduction tissues within the leaf, and leaves from different communi- Plant ecologists have summarized a substantial amount of ties. Increasing our understanding of the basis of variation the variation within and across biomes in leaf size and in leaf biomechanics may have important implications for morphology into a few essential trade-offs. For example, plant carbon budgets, plant–animal interactions and nutri- higher investments in leaf dry mass per area (LMA) have ent cycling (Dıaz et al. 2004; Balsamo et al. 2006; Read been related to leaf nutrient stoichiometry and leaf longev- et al. 2006; Alvarez-Clare & Kitajima 2007; Peeters, ity (Reich, Walters & Ellsworth 1997; Wright et al. 2004; Sanson & Read 2007; Kitajima & Poorter 2010). Niklas et al. 2007; Kattge et al. 2011). These differences in There are compelling reasons to expect that overall leaf leaf construction costs are closely linked with variation in mechanics should depend on the construction costs of the leaf mechanical traits across species and biomes (Read & different leaf tissues. In mechanical terms, a leaf acts as a Sanson 2003; Read, Sanson & Lamont 2005; Onoda et al. cantilever beam, where any increase in mass and length will 2011). However, our knowledge of the underlying mecha- increase its susceptibility to bending (Niinemets & Fleck nisms that determine the strength and stiffness of leaves 2002a,b; Cooley, Reich & Rundel 2004; Niinemets, Ports- muth & Tobias 2007). Therefore, to optimize the surface *Correspondence author. [email protected] for sunlight interception and, at the same time, to cope with

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society Leaf biomechanics across communities 545 mechanical forces such as wind (Anten et al. 2010), leaves tested three hypotheses relating to leaf mechanical resis- have been selected to invest a significant fraction of their tance: first, within and across species the mechanical resis- total dry biomass in mechanical support tissue (up to 70%) tance of leaf tissues should increase with leaf size and (Niinemets, Portsmuth & Tobias 2006; Niinemets et al. density. Second, leaf mechanical resistance should differ 2007). This investment is size dependent, as larger leaves across species due to variation in within-leaf mechanical tend to have larger LMA, with larger midribs (Milla & traits, and midrib and lamina structural traits. Third, leaf Reich 2007; Niklas et al. 2007; Sack et al. 2012). As previ- mechanical traits should vary across species and communi- ously noted, there is a strong relation between leaf tissue ties, reflecting greater selection for leaf life span and mechanical resistance and dry matter content or density of drought tolerance with higher chronic aridity. We tested leaf tissues (Read & Sanson 2003; Read, Sanson & Lamont these hypotheses by quantifying nine biomechanical traits, 2005; Kitajima & Poorter 2010; Onoda et al. 2011; Kitaj- including measurements of strength and stiffness, and 17 ima et al. 2012); therefore, we can expect that leaf mechani- leaf morphological traits relating to the dry mass composi- cal resistance should be hierarchically partitioned tion, for the whole leaf, the midribs and the leaf lamina. according to differences in tissue density, with most in the midrib and other major veins (i.e. second- and third-order Materials and methods veins), less in minor veins (fourth order and higher), and least in leaf mesophyll tissue (Choong 1996), but this expec- STUDY SITE AND SPECIES SAMPLING tation needs testing, particularly because for many species, the leaf vein xylem and sclerenchyma are the only leaf tis- Leaves were collected from adult plants in the Rancho Santa Ana sues directly involved in support, while for other species, Botanical Garden, Claremont, CA, USA (34°06′ 49″N, 117°42′55″ W), from January to April 2012. We selected seven common spe- leaf lamina also provides direct mechanical support, for cies from chaparral, seven from coastal sage and seven from Cali- example, for sclerophyllous species with lignified epidermis fornian Mojave Desert plant communities (see Table S1, and/or hypodermis (Edwards et al. 2000; Niinemets, Supporting information). Coastal sage and chaparral communities Portsmuth & Tobias 2007; Wang et al. 2010). experience similar levels of precipitation of approximately 400 mm The objective of this study was to determine the basis of per year, and Mojave Desert has much lower levels of precipitation (138 mm per year). Average summer and winter temperatures the mechanical resistance of the leaf, and its relationship are 23 °C and 9 °C in chaparral, 26 °C and 10 °C in coastal sage with the suite of traits that are involved in sclerophylly, and 26 °C and 4 °C in Mojave Deserts (Jacobsen et al. 2007). To particularly at the tissue level. We tested the relationship sample for variation among species, and also for leaf size variation of the mechanical resistance of leaves to the density of sup- within species, for each of three individuals per species (range of port tissue using 21 species native to chaparral, coastal basal diameters and heights 0Á8–16Á5 cm; and 0Á4–5Á5 m); we sam- pled four sun-exposed terminal branches that had at least nine sage and Mojave Desert communities of Southern Califor- mature leaves. Detached branches were immediately stored in nia, USA. Although not previously quantified, there are water-filled buckets, covered with black plastic bags and trans- clear shifts in leaf biomechanics among these communities ported to California State Polytechnic University, Pomona, to distributed across an aridity gradient, as implied in their perform leaf tensile tests within the same day. very nomenclature. Indeed, since the classic works of

Schimper (1903) and Cooper (1922), the Californian vege- LEAF BIOMECHANICAL TRAITS tation has been distinguished into types including chaparral, a vegetation characterized by sclerophylly, ‘thick, stiff To determine leaf biomechanical traits, we conducted static tensile and hard, ordinarily flat and evergreen leaves’ (Schimper tests and calculated standard indices. We tested specimens in static tensile tests using screw side-action grips in an INSTRON 3345 1903; Cooper 1922), and coastal sage, which includes spe- mechanical testing machine with a 5 kN load cell (INSTRON cies with deciduous and ‘soft and white pubescent’ leaves, Corporation, Canton, MA, USA), with the cross-head moving at also called malacophylls (Epling & Lewis 1942; Walter a rate of 20 mm/min. We tested leaves in three treatments, and for 1985). Species of these communities diverge in the typical each, we used three leaves from one branch per individual per spe- mechanisms employed to tolerate drought (Jacobsen et al. cies, selecting mature leaves in the best possible condition that clearly differed in size, resulting in nine leaves tested for each 2008, 2009), with greatest cavitation resistance, less sprout- treatment for each species. Whole leaves were tested with their ing and more negative field water potentials in the chapar- midrib aligned with the load applied (whole-leaf vertical treat- ral and least in the coastal sage, and the Mojave Desert ment, ‘leaf’). Additionally, the leaf lamina was sectioned to expose spanning a wider range of variation, reflecting in part the only the midrib and tested aligned with the load applied (midrib diverse water relations in this community in which water treatment, ‘midrib’), or using rectangular section of leaf lamina of 3cm9 0Á5 cm cut parallel to the midrib, halfway between mid- becomes highly available in occasional pulses (Pratt et al. rib and margin (lamina treatment, ‘lamina’). The lamina samples 2007; Jacobsen et al. 2008). One might expect that species included secondary veins, running in perpendicular direction to with traits that favour resistance to xylem cavitation to the load applied. Grips were tight enough to generally avoid slip- typically also favour sclerophylly, to support their greater page, but when that occurred, the test was discarded and repeated. drought tolerance (Balsamo et al. 2003; Bartlett, Scoffoni The original length of the sample (i.e. the vertical length of the sample between the upper and lower grips, Lo) was 10 mm when & Sack 2012). leaves were longer than 2 cm from base to apex, and when leaves

Using these three plant communities that diﬀer in their were shorter than 2 cm Lo was 5 mm for whole leaves, and water use and drought tolerance (Jacobsen et al. 2008), we 3Á5 mm for midribs and laminas. We also measured the horizontal

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 544–554 546 R. Mendez-Alonzo et al. length of the sample (leaf width, W) and the leaf thickness (T)of width and total area. We calculated the volume of leaf lamina as 3 the segments used for mechanical tests. Tensile tests generally are Vlamina = (Leaf total area À midrib area) 9 T;inmm). We cal- 3 performed using samples with a length 8–10 times their width, to culated the volume of the midrib (Vmidrib;inmm) as a truncated allow the samples to narrow uniformly in the centre, thus avoiding cone, using the midrib diameters and length (Lmidrib): Vmid- 2 an apparent increase in stiffness associated with wider sample rib = (1/3 p) 9 (Lmidrib/2) 9 ([Dmidrib, b/2] + [Dmidrib, b/2] 9 [Dmid- 2 shapes (Vincent 1990). However, this shape standardization could rib, c/2] + [Dmidrib, c/2] ). The analogous formula was used to 3 not be fulfilled due to the small leaf sizes of our study species. calculate the volume of petioles (Vpetiole;inmm). 3 Prior to testing each treatment, we measured the basal, central We calculated leaf density (qleaf = Mleaf/Vleaf; in g/mm ), leaf 2 and apical diameters of the midrib and the lamina thickness in mass per area (LMA = Mleaf/LA; in g/mm ), leaf lamina density 3 another set of leaves of comparable size and tapering values were (qlamina = Mlamina/Vlamina; in g/mm ) and leaf lamina mass per 2 entered into the BLUEHILL INSTRON software (INSTRON Corpora- area (LMAlamina = Mlamina/Arealamina; in g/mm ). tion, Canton MA, USA), which plotted the stress-strain curve dur- We calculated the dry mass fraction of the leaf that was ing each test. The linear portion of the curve, previous to the invested in support, FL, as the ratio of the mass of the midrib catastrophic failure of the tissue, was used to calculate the maxi- (Mmidrib) and the mass of the whole leaf (Mleaf) (Niinemets, Ports- mum slope (Mslope, a calculation of the tensile modulus of elastic- muth & Tobias 2006). We also calculated the ratio qmidrib/qlamina ity not normalized by sample dimensions; units, N/m) and as a second index of relative investment in structural support. maximum load at the moment of breakage (Mload; units, N), two Independently of the overall leaf density, species with high ratios variables used to determine leaf tensile modulus of elasticity and should bear midribs of high density relative to their laminas, strength. whereas species with low ratios should have denser laminas rela- The overall mechanical properties of biological composites tive to their midribs (Table 1). depend on the material composition and the structural properties, that is the dimension and shape of the object (Aranwela, Sanson & Read 1999; Read & Sanson 2003; Vogel 2003; Onoda et al. STATISTICAL ANALYSES 2011). We measured three properties of the material resistance of leaves based on tensile tests (Balsamo et al. 2003, 2006): the leaf Prior to analyses, we ln-transformed TME and TS data to – tensile modulus of elasticity (TME; in N/m2, where MN/m2 are improve normality and heteroscedasticity. We assessed a priori equivalent to MPa), a measure of the intrinsic resistance of the hypothesized relationships between traits using standard major leaf tissue to elastic deformation; the leaf tensile strength (TS; in axes (SMA; using SMATR 2.0; Warton et al. 2006). To present the N/m2), the force required to fracture the leaf per unit of sectional intercorrelation patterns among all traits, we generated a correla- area (Choong et al. 1992; Read & Stokes 2006; Sanson 2006); the tion matrix. We avoided interpretation of non-hypothesized rela- flexural stiffness (FS, units of N 9 m2), calculated as the product tionships, for which Bonferroni corrections would be necessary to of the TME and the second moment of area I (a property of the establish statistical significance (Garcia 2003; Moran 2003). We cross-sectional geometry of an object, with units of length to the summarized the patterns of correlation of the sets of leaf morpho- fourth power, m4). The FS measures the resistance to bending of a logical and biomechanical traits using a principal component anal- structure, where the larger the value, the smaller the deformation ysis (PCA), including the 20 species for which we had data for all under a given load (Etnier 2001; Niinemets & Fleck 2002a; Vogel variables, and excluding Larrea tridentata, which had leaves too 2003; Gere & Goodno 2009). For whole leaves, midribs and leaf small for the midrib tensile measurements. To compare leaf traits across communities and species, we con- lamina sections, the TME was calculated as (Mslope 9 (Lo/ W 9 T)), where W is the horizontal length of the sample clamped ducted nested ANOVA analyses, with species nested within commu- between the grips (leaf width), and T is the leaf lamina or midrib nities as explanatory variables. To compare the variation in midrib, lamina and whole-leaf densities, we used a nested two-way thickness. The TS was calculated as (Mload/W 9 T) (Gere & Goodno 2009). For the midrib, I was calculated using the formula repeated measures ANOVA, with species nested within communities as the first factor, treatment as the second factor and individuals for a tapering cylinder, as I = (p/8) 9 (r1r2) 9 (r2r3), where r1, r2 as the repeated measurement. These analyses were performed in and r3 are, respectively, the radii of the midrib at the base, central and terminal portions. For leaf lamina sections, I was calculated Minitab 15 (Minitab Inc., State College, PA, USA). as a rectangle, I = T 9 ((b 9 h3)/12), where b is the base of the To assess within species relationships, we determined the pro- lamina section of 0Á5 cm, h is the length of the section, that is portion the proportion of species that had the correlations 3 cm, and T is leaf thickness. For whole leaves, I was calculated hypothesized to hold between traits. We tested overall significance as an ellipse, with I = (p/4) 9 LW3, where L is leaf length and W of within-species correlations of traits using binomial proportion is leaf width (Niklas 1992; Gere & Goodno 2009). hypothesis tests (using Minitab 15). For the given hypothesized relationships, we also compared the mean values of the within species coefficients of determination vs. the across species coefficient of determination, using t-test (or Mann–Whitney rank-sum tests) LEAF MORPHOLOGICAL TRAITS hypothesis tests (Minitab 15, Minitab Inc., 2007). After the tensile tests were performed, leaves were scanned in both adaxial and abaxial orientations using a flatbed scanner (Epson Perfection 4490; Epson Corp., Nagano, Japan). Leaves Results were then sectioned in a glass petri dish over a white light transil- luminator (Model TW-43, UVP, Upland, CA, USA) and under a VARIATION IN LEAF BIOMECHANICS ACROSS SPECIES, dissection microscope (OM2300 ST, Omano, Zhejiang, China) to AND ITS SOURCES WITHIN THE LEAF TISSUES separate midrib from lamina tissue. We measured the basal, central and apical diameter of petioles and midribs (Dpetiole, b Dpetiole, Leaf mechanical traits, structure and morphology varied c, Dpetiole, a; Dmidrib, b, Dmidrib, c, Dmidrib, a, respectively) and lam- strikingly across species and communities (Table 1; Figs 1 Á ina thickness (T) under the microscope to the nearest 0 01 mm –4). Midrib, lamina and leaf densities also differed strongly using callipers (Fisher Scientific, Waltham, MA, USA). After sec- within individual leaves and across species and communi- tioning, laminas and midribs were oven dried 3 days at 70 °C < Á before measuring dry mass. Using IMAGE J software (http://imagej. ties (nested two-way repeated measures ANOVA; P 0 001 nih.gov/ij/), we measured leaf petiole and midrib length, leaf for all factors and interactions).

Table 1. Leaf traits measured in this study, including the minimum species mean, the mean of the species means, and the maximum species mean

Trait Symbol Units Min – Mean – Max Spp Com

Leaf area LA mm2 69 – 612 – 2753 *** *** Total leaf mass per area LMA g cmÀ2 0Á04 – 0Á15 – 0Á33 *** *** Lamina leaf mass per area LamMA g cmÀ2 0Á03 – 0Á13 – 0Á30 *** *** À1 *** *** Fraction of tissue invested FL gg 0Á10 – 0Á24 – 0Á62 in support À3 *** *** Density midrib qmidrib gcm 0Á17 – 0Á42 – 0Á97 À3 *** *** Density lamina qlamina gcm 0Á12 – 0Á38 – 0Á59 À3 *** *** Total leaf density qleaf gcm 0Á21 – 0Á36 – 0Á63 *** *** Ratio midrib/lamina density qmidrib/qlamina — 0Á11 – 0Á37 – 0Á58 Lamina thickness T mm 0Á38 – 0Á82 – 1Á57 *** *** *** *** Midrib diameter, central Dmidrib, c mm 0Á09 – 0Á23 – 0Á49 *** *** Midrib diameter, basal Dmidrib, b mm 0Á29 – 0Á64 – 1Á26 3 *** *** Leaf volume Vleaf mm 23 – 276 – 1297 3 *** *** Midrib volume Vmidrib mm 0Á88 – 22 – 131 3 *** *** Petiole volume Vpetiole mm 0 – 41 – 177 3 *** *** Lamina volume Vlamina mm 21 – 214 – 1034 *** *** Midrib and petiole dry mass Msupport g0Á001 – 0Á02 – 0Á08 *** *** Lamina dry weight Mlamina g0Á006 – 0Á09 – 0Á56 *** *** Midrib length Lmidrib mm 3Á62 – 40Á6 – 115 *** *** Petiole length Lpetiole mm 0 – 6Á2 – 23Á6 *** *** Leaf width Wleaf mm 4Á3 – 16Á7 – 47Á5 *** *** Tensile modulus of TMElamina MPa 0Á39 – 3Á9– 16Á4 elasticity, lamina *** *** Tensile modulus of TMEmidrib MPa 0Á52 – 6Á8 – 41Á1 elasticity, midrib *** *** Tensile modulus of TMEleaf MPa 0Á64 – 4Á2 – 18Á7 elasticity, whole leaf *** *** Tensile strength, lamina TSlamina MPa 0Á75 – 4Á7 – 13Á1 *** ** Tensile sStrength, midrib TSmidrib MPa 0Á8 – 11Á0 – 61Á8 *** *** Tensile strength, whole leaf TSleaf MPa 2Á2 – 5Á2 – 12Á6 2 À6 À5 À4 *** *** Flexural stiffness, lamina FSlamina Nm 1Á3 9 10 – 1Á7 9 10 – 1 9 10 2 *** *** Flexural stiffness, midrib FSmidrib Nm 0Á0004 – 0Á04 – 0Á3 2 *** *** Flexural stiffness, whole leaf FSleaf Nm 0Á003 – 0Á72 – 5Á2

Statistical signiﬁcance of nested analyses of variance (with species, Spp, nested within communities, Com): ***P-value < 0Á001, **0Á001 < P-value < 0Á01.

Fig. 1. Relationships of the whole-leaf tensile modulus of elasticity (TMEleaf, ln- transformed) and tensile strength (TSleaf, ln-transformed) with leaf mass per unit area (LMA) and leaf density (qleaf) for species of three Californian plant communities grown in a common garden. Open circles, coastal sage species; ﬁlled circles, chaparral species; triangles, desert species.

When tests were conducted on the whole leaf, the tensile that a section of lamina can withstand. In the scleromor- modulus of elasticity (TME) and tensile strength (TS) were phic leaves, evidently, the lignified epidermis and hypoder- highly correlated (R2 = 0Á66, P < 0Á0001), and the flexural mis played a major role in whole-leaf mechanics, stiffness (FS) was not correlated with TS (R2 = 0Á04, consistent with their higher values for leaf lamina TME P = 0Á4) and weakly correlated with TME (R2 = 0Á19, and TS (Fig. 3). P = 0Á05). Additionally, the material and structural properties of the whole leaf were highly correlated with those VARIATION IN LEAF BIOMECHANICS WITHIN SPECIES, of component tissues. Thus, the whole-leaf TME, TS and AND ITS SOURCES WITHIN THE LEAF TISSUES FS were correlated with those of the midrib and lamina (Table 2). We assessed the proportion of species that had within-spe- The leaf mechanical properties correlated across species cies trait correlations as hypothesized and tested across with leaf structure and composition. The whole-leaf TME species. Within species correlations were considered to be and TS for the whole leaf were strongly related to LMA and significant overall if they were found for four or more spe- qleaf (Fig. 1, Table 3). Only the whole-leaf FS was signifi- cies (binomial proportion test; for proportions of 4/21, cantly correlated with leaf area and midrib diameter P = 0Á019; for 3/21, P = 0Á085). There were significant cor- (Table 3). Across species, the whole-leaf TME and TS were relations between the density of the whole leaf and that of not related to leaf size, which was also independent of LMA the midrib (within 14/21 species), and between the density in this data set (Table 3). The midrib TME, TS and FS were of the whole leaf and that of the leaf lamina (21/21 species) not correlated with midrib density, but the midrib FS was (R2 = 0Á14–0Á99; P < 0Á0001–0Á02). correlated with midrib diameter and LMA (Table 3). The Within 10/21 species, the whole-leaf tensile modulus of leaf lamina TME, TS and FS were correlated with leaf elasticity (TME) and tensile strength (TS) were correlated lamina mass per area and leaf lamina density (Table 3). (R2 = 0Á47–0Á88; P < 0Á0001–0Á04), and the whole-leaf flex- Notably, the whole-leaf TME and TS correlated nega- ural stiffness (FS) was correlated with the whole-leaf TME tively with the fraction of dry mass that the leaf invested for 6/21 species and with the whole-leaf TS for 3/21 species 2 in support, whether quantified using the FL or the qmidrib/ (R = 0Á46–0Á88; P < 0Á0001–0Á043). Further, within spe- qlamina ratio. This implies that species bearing leaves of cies, the leaf mechanical properties correlated with leaf intrinsically low strength and stiffness tended to partially structure and composition. The whole-leaf TME, TS and compensate with higher proportions of support tissue in FS were related to LMA for 4/21 species but with qleaf for the midrib in comparison with leaves of high strength and only 3/21 species (R2 = 0Á52–0Á86; P < 0Á0001–0Á043). The stiffness (Fig. 2). Consequently, the whole-leaf FS, which leaf lamina TME, TS and FS were correlated with leaf represents TME normalized by the dimensions of the mid- lamina mass per area and leaf lamina density for 4/21 spe- 2 2 rib, was unrelated to FL (R = 0Á15, P = 0Á09). We also cies (R = 0Á44–0Á97; P < 0Á0001 to 0Á048). The whole-leaf tested the relationships between FL and our two mechani- FS was significantly correlated with leaf area for 5/21 spe- 2 cal measurements of the leaf lamina (TMElamina and cies (R = 0Á59–0Á83; P = 0Á001–0Á016), and LMA and leaf 2 TSlamina). We found a significant negative correlation size were related for 10/21 species (R = 0Á44–0Á83; 2 between TMElamina and FL (R = 0Á54, P = 0Á0002), but P < 0Á0001–0Á044). 2 no correlation between TSlamina and FL (R = 0Á05, We also compared the across species coefficient of deter- P = 0Á37). These findings imply that the larger the fraction mination (R2) with the average R2 for within-species rela- of dry mass invested in midrib, the lower the stiffness of tionships for our set of hypothesized relationships between the lamina, but stiffness is unrelated to the maximum stress leaf functional and biomechanical traits. In most cases, the values of R2 differed significantly between the within and Table 2. Pairwise Pearson correlations between whole-leaf tensile across species scales (Table 3). modulus of elasticity (TMEleaf), tensile strength (TSleaf) or flexural stiffness (FSleaf) vs. TME, TS or FS for midribs (TMEmidrib, TS and FS ) and leaf laminas (TME ,TS and midrib midrib lamina lamina VARIATION ACROSS VEGETATION TYPES IN FSlamina) BIOMECHANICAL TRAITS Biomechanical traits RPAveraging values across communities, the TME showed a dramatic variation among chaparral to semi-desert and

TMEleaf TMElamina 0Á85 <0Á0001 coastal sage vegetation types, with a high TME of Á Á TMEleaf TMEmidrib 0 58 0 007 41Á9 MPa for Quercus berberidifolia, a chaparral species, Á Á TMEleaf FSleaf 0 44 0 06 to the lowest TME of 0Á78 MPa for Salvia leucophylla,a TS TS 0Á60 0Á005 leaf lamina species from coastal sage vegetation. This pattern of varia- TSleaf TSmidrib 0Á60 0Á005 TSleaf FSleaf 0Á20 0Á39 tion among communities was most conspicuous for the TMElamina FSlamina 0Á86 <0Á0001 midrib mechanical properties, then for the whole leaf, then Á Á TMEmidrib FSmidrib 0 43 0 056 for the lamina (Fig. 4). This same pattern was observed Á Á TSlamina FSlamina 0 17 0 47 for TS (Fig. 4). The FS showed no signiﬁcant trend across TS FS 0Á11 0Á64 midrib midrib communities (P > 0Á05).

Table 3. Results of tests of pairwise Pearson correlations between tensile modulus of elasticity and flexural stiffness of leaf (‘leaf’), midrib (‘midrib’) and leaf lamina (‘lamina’) with leaf dimensional and compositional traits across species and the minimum, mean and maximum R2 values and minimum and maximum P-values for within species tests. Refer to Table 1 for trait abbreviations. (%) is the percentage of species that had significant within-species relationships (P < 0Á05)

Across species Within species

Min–Mean–Max Min–Max (%) R2 P R2 P

Correlations with leaf area LMA 0Á02 0Á06 0Á008–0Á23–0Á87 0Á001–0Á82 (47%) q 0Á01 0Á75 0–0Á2–0Á80Á003–0Á98 (20%)* * Dmidrib, b 0Á53 <0Á0001 0Á002–0Á32–0Á93 0–0Á91 (67%) Lmidrib 0Á67 < 0Á0001 0Á05–0Á64–0Á97 0–0Á56 (90%) * Wleaf 0Á65 < 0Á0001 0Á005–0Á49–0Á98 0–0Á86 (62%) * TMEleaf 0Á01 0Á69 0–0Á22–0Á64 0Á009–0Á97 (5%) FSleaf 0Á43 < 0Á0001 0Á09–0Á57–0Á89 0–0Á44 (75%) Correlations with leaf mass per area

TMEleaf 0Á24 0Á03 0–0Á37–0Á91 0–0Á98 (35%) FSleaf 0Á10 0Á18 0Á002–0Á32–0Á81 0Á001–0Á90 (35%) TMEmidrib 0Á08 0Á22 0Á003–0Á18–0Á87 0–0Á89 (15%) * TSmidrib 0Á01 0Á67 0–0Á23–0Á81 0Á001–0Á99 (25%) * FSmidrib 0Á56 < 0Á0001 0Á001–0Á26–0Á79 0Á001–0Á94 (30%) Correlations with TMEleaf q 0Á58 < 0Á0001 0–0Á20–0Á80 0Á001–0Á97 (20%)* * qlamina 0Á52 0Á0003 0–0Á26–0Á84 0–0Á99 (35%) qmidrib 0Á21 0Á04 0–0Á23–0Á88 0–0Á96 (25%) T 0Á02 0Á58 0Á001–0Á27–0Á84 0–0Á93 (20%)* * Dmidrib, b 0Á0001 0Á95 0Á005–0Á25–0Á97 0–0Á86 (10%) Correlations with FSleaf q 0Á09 0Á19 0Á001–0Á22–0Á97 0–0Á94 (15%)* qlamina 0Á14 0Á09 0Á003–0Á23–0Á73 0Á003–0Á88 (30%) qmidrib 0Á01 0Á61 0–0Á26–0Á88 0–0Á98 (20%) T 0Á004 0Á77 0–0Á22–0Á79 0Á001–0Á99 (25%)

Dmidrib, b 0Á36 0Á004 0Á002–0Á29–0Á84 0Á001–0Á91 (30%) Correlations with midrib density

TMEmidrib 0Á08 0Á23 0Á001–0Á16–0Á53 0Á03–0Á94 (15%) TSmidrib 0Á02 0Á55 0–0Á17–0Á64 0Á01–0Á96 (15%) * FSmidrib 0Á01 0Á67 0Á001–0Á22–0Á81 0Á001–0Á94 (25%) Correlations with midrib diameter

TMEmidrib 0Á27 0Á018 0–0Á22–0Á66 0Á008–0Á99 (20%) TSmidrib 0Á11 0Á15 0–0Á21–0Á85 0–0Á99 (15%) FSmidrib 0Á44 < 0Á0001 0Á02–0Á53–0Á90 0–0Á76 (60%) Correlations with lamina mass per area * TMElamina 0Á38 0Á003 0Á001–0Á19–0Á67 0Á01–0Á95 (20%) * TSlamina 0Á004 0Á79 0–0Á17–0Á64 0Á01–0Á99 (10%) * FSlamina 0Á38 0Á003 0Á004–0Á19–0Á74 0Á006–0Á87 (15%) Correlations with lamina density * TMElamina 0Á44 0Á001 0Á005–0Á19–0Á70Á005–0Á86 (15%) TSlamina 0Á04 0Á31 0Á003–0Á16–0Á47 0Á04–0Á89 (10%) * FSlamina 0Á44 0Á002 0–0Á19–0Á78 0Á003–0Á99 (10%)

*Indicates P < 0Á05 of t-tests between the within species R2 (N = 21) and the across species R2.

PATTERNS OF COVARIATION AMONG LEAF TRAITS strongly influenced by leaf mass per area, leaf density, and leaf mechanical properties, as indicated by the significant We summarized data from the 17 morphological and nine correlation of this set of variables with the second biomechanical traits across species using principal compo- component (Fig. 5, Table S2, Supporting information). nent analysis (PCA). As a result of strong covariation The PCA indicated that species from three Califor- among leaf traits that were related to size, the first compo- nian plant communities can be ordered according to nent in the PCA accounted for 41Á2% of the variance their leaf properties, from species bearing leaves with (Fig. 5), and most variables related to leaf length, width low stiffness and strength, in the coastal sage, to species and area were significantly correlated with the first compo- with strong and stiff leaves in the chaparral. The Desert nent (Fig. 5, Table S2, Supporting information). The sec- species were spread across the range of variation in size ond axis, accounting for 32Á2% of the variance, was and mechanical resistance, including species with small

Fig. 2. Relationships between the fractions

of leaf tissue invested in support (FL), and the ratio of midrib over lamina tissue density (midrib/lamina) with the ln-transformed whole-leaf tensile modulus of

elasticity (Ln [TMEleaf]) and whole-leaf tensile strength (Ln [TSleaf]) for species of three Californian plant communities grown in a common garden. For species abbreviations, see Table S1 (Supporting information). leaves of low mechanical resistance, such as Artemisia Discussion tridentata and Isomeris arborea and species with relatively large leaves of high mechanical resistance, such as We quantified the strength and stiffness of leaves of Simmondsia chinensis. The chaparral species Malosma dominant species originating from three contrasting laurina and Rhus ovata (both Anacardiaceae) signifi- environments in the California flora in a common gar- cantly deviated from the other species in their commu- den and found three especially novel results highlighting nity in the first principal component because of their the importance of leaf mechanical traits. First, we found relatively larger leaf sizes (Fig. 5). Finally, we compared that the mechanical properties of leaves depended the scores of the first and second principal components strongly on size and tissue density according to hypoth- across plant communities to determine how leaf traits esized scaling relationships and that relationships within varied across environments. Species did not differ in species were qualitatively distinct from those that hold traits related to leaf area across plant communities across species. Second, mechanical resistance differed (PC1, one-way ANOVA, F = 2Á135, P = 0Á15), but they substantially among the component tissues of the leaf, differed significantly in the traits related to leaf mechani- being larger in the midrib than in the leaf lamina and cal properties (PC 2, one-way ANOVA, F = 8Á8545, that covariation in tissue dimensions partially compen- P = 0Á0023). sated for differences in mechanical resistance of these

Fig. 3. Relationship between the fractions

of leaf tissue invested in support (FL) and tissue density (q) with with the ln-transformed tensile modulus of elasticity (Ln [TME]) and tensile strength (Ln [TS]) of leaf lamina (main panels) and midrib sections (insets) for species of three Californian plant communities grown in a common garden. For species abbreviations, see Table S1 (Supporting information). Open circles, coastal sage species; ﬁlled circles, chaparral species, triangles, desert species.

Fig. 4. Within-leaf variation in the tensile modulus of elasticity and tensile strength for whole leaves (‘leaf’), for midrib (‘midrib’), and leaf lamina (‘lamina’), for 21 species of three Californian plant communities (Vegetation type) grown in a common garden. C, chaparral; D, desert; S, coastal sage.

SCALING RELATIONS BETWEEN LEAF MORPHOLOGY AND MECHANICS

Tensile experiments provided evidence of large variation across species and habitats in leaf mechanical properties and that these related strongly to leaf structure. Notably, using the tensile tests, we were able to determine the strength and stiffness of different tissues and different con- figurations of leaf architecture, which would have proven difficult using the punch and die test or the shearing test, which have been useful in calculating the shear resistance of leaves (Aranwela, Sanson & Read 1999; Onoda et al. 2011). Consistent with previous work, we found that the mechanical strength and stiffness of leaves was correlated with functional traits relating to the packing of cell material Fig. 5. Species from three Californian communities range from in the tissue, such as leaf mass per area and leaf density, small ‘soft’ leaves in the coastal sage (open circles) to small ‘hard’ and this relation was persistent in different tissues and leaves in chaparral (filled circles). Desert species spanned across across species and communities (Read & Sanson 2003; the range of variation in size and mechanical resistance (triangles) Read et al. 2006; Onoda et al. 2011). Our study also pro- and Anacardiaceae (Ml and Ro) were much larger in size, but had smaller values of mechanical resistance. The first principal compo- vides the first investigation of the mechanical strength and nent from a PCA synthesizing 17 morphological and nine biome- stiffness of leaves for different leaf tissues and spatial con- chanical traits had k = 11Á13 and the second principal component figurations, and the first quantitative determination of the had k = 8Á69. variation in density of the midrib and leaf lamina, and its relation to biomechanics traits. We found that the construc- tissues such that the flexural stiffness, that is, the leaf tion of tissues was inter-related, such that a species with deformation in response to its own load, was less vari- higher density in one tissue tended to have higher density in able across species. Finally, leaf biomechanics are useful other tissues. Thus, our mechanical properties showed a traits to characterize vegetation types, and placed on a conserved ranking of material resistance among tissues, quantitative basis the classical view of two California such that species with higher material resistance in the mid- communities that differ in their leaf mechanics and rib also invested significantly more in the support of the leaf drought tolerance. lamina by increasing the strength and stiffness of the lamina

© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 544–554 552 R. Mendez-Alonzo et al. or minor veins. We found that the strongest differences significant positive relationship between the fraction of tis- across species and environments were in the mechanical sue invested in support (FL) and leaf area reported in a resistance of the midrib, and thus related to the xylem and previous study (Niinemets, Portsmuth & Tobias 2006). surrounding mechanical cells and not the leaf lamina. The lack of relationship across species may be due to the Our measurements of mechanical properties confirmed relatively small leaves in our study, ranging from 0Á7cm2 and extended previous work which focused on mass alloca- in Larrea tridentata to 28 cm2 in Malosma laurina, with tion within leaves. A previous study reported a trade-off only two species with leaf area greater than 10 cm2. between the investment in the proportion of tissue in the midrib vs. the proportion of tissue invested in the other VARIATION IN LEAF MECHANICS AMONG SPECIES AND major and minor veins (Niinemets, Portsmuth & Tobias VEGETATION TYPES 2007). Consistent with this, we found a trade-off between the mechanical properties of the leaf tissues and the pro- The Mediterranean flora is considered a key example of portion of biomass invested in support. Species bearing functional convergence in sclerophylly (Cody & Mooney leaves with low strength and stiffness deploy a higher pro- 1978; Edwards, Read & Sanson 2000; Read & Sanson portion of the leaf dry mass in the midrib and petiole rela- 2003). In our study, leaves showed a dramatically greater tive to species that bear leaves with high strength and strength and stiffness in the chaparral than the coastal stiffness. This finding indicates alternative designs for sage, with a sixfold increase on average from chaparral leaves to attain resistance to bending due to their own species to coastal sage species (from an average across weight, that is, by investing in highly mechanical resistant treatments of 9Á1 MPa in the chaparral vs. 1Á5 MPa in the but low volumes of midrib tissue or by investing in greater coastal sage), a finding consistent with obvious qualitative cross-sectional area, and deploying low-density material to knowledge, and here placed on a quantitative basis. These construct the midrib. The achievement of adequate struc- properties are also consistent with what is known of the tural resistance in leaves (as quantified by the FS) is critical physiology of the vegetation types, where chaparral species to maximize leaf light capture, as it allows the optimiza- are evergreens highly resistant to cavitation that have low tion of the inclination angles of leaves and thus the reduc- transpiration rates and do not always resprout after fires tion of leaf self-shading by aggregation (Niinemets & (Jacobsen et al. 2007, 2008; Pratt et al. 2007). The high Fleck 2002a; Niinemets, Portsmuth & Tobias 2006). We leaf mechanical resistance of chaparral species can be also found that species that deploy a small volume of understood as an adaptation to nutrient limitation and/or highly resistant tissue in the midrib enhanced the mechani- herbivory (Turner et al. 1993; Salleo & Nardini 2000; Bart- cal resistance of the lamina, which can be achieved with lett, Scoffoni & Sack 2012), with an indirect role in lignification of the lamina and hypodermis (Edwards et al. drought tolerance in seasonally dry ecosystems, including 2000). The enhancement of the mechanical resistance of mediterranean climates, preventing excessive tissue shrink- the lamina increases the overall safety of leaves against age and dehydration (Bartlett, Scoffoni & Sack 2012). This mechanical failure, though at the cost of larger investments would explain why a global meta-analysis found leaf in dry mass, and is congruent with the expectations of a mechanical resistance to be negatively correlated with trade-off between the fraction of tissue invested in the mid- water availability, although the percentage of variance rib vs. smaller veins and lamina providing similar flexural explained was less than 30% (Onoda et al. 2011). Species stiffness, and increased lamina protection would also con- from coastal sage and Mojave Desert, on the contrary, are tribute resistance to herbivory and extended leaf life span generally facultatively drought-deciduous species that (Niinemets, Portsmuth & Tobias 2007). avoid very negative soil water potentials by producing The relationships of leaf biomechanics traits with leaf leaves only in the wetter season of the year (Jacobsen et al. area and density were species specific. Indeed, we did not 2007). To control water loss through transpiration, species find interspecific relationships of leaf biomechanical prop- from Mojave Desert reduce total leaf area, and this erties with leaf size, but we found such relationships within dynamic response of leaves to water availability is reflected species for more than half of the species studied. Our in a diversity of leaf biomechanical traits, with species results suggest that leaf area and other size-related traits including a mixture of sclerophylls and malacophylls (Jac- are decoupled from the material properties of the leaves obsen et al. 2008). Our results extend the classical works (TME and TS) across species. However, the FS, a biome- of Schimper (1903) and Cooper (1922), showing that the chanical trait that incorporates leaf dimensions, was qualitative differences in leaf traits they observed can be strongly related to leaf size across species. These different assessed as quantitative leaf mechanical properties. This relationships within and across species imply that, when principle can be extended to other contrasting vegetation the material resistance of the leaves is relatively fixed, as types in adjacent communities around the world. In con- would occur within species, the larger leaves tend to have junction with other traits that differ importantly among larger mechanical resistance, a larger amount of tissue communities, such as stem hydraulics and mechanics (Jac- invested in support. However, when the material resistance obsen et al. 2008, 2009), such analyses can differentiate varies hugely across species, this would be independent of vegetation types in a functionally significant way, accord- leaf size. Across species, we did not find evidence for a ing to their contrasting resource exploitation.

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Vogel, S. (2003) Comparative Biomechanics: Life’s Physical World. Prince- ton University Press, New Jersey. Supporting Information Walter, H. (1985) Vegetation of the Earth, 3rd edn. Springer-Verlag, Berlin. Wang, S., Ren, L., Liu, Y., Han, Z. & Yang, Y. (2010) Mechanical Additional Supporting Information may be found in the online characteristics of typical plant leaves. Journal of Bionic Engineering, version of this article: 7, 294–300. Warton, D.I., Wright, I.J., Falster, D.S. & Westoby, M. (2006) Bivariate Table S1. Mean values Æ SE for 17 functional and nine bio- line-ﬁtting methods for allometry. Biological Reviews, 81, 259–291. mechanical traits for 21 species of three Californian plant commu- Wright, I.J., Reich, P.B., Westoby, M., Ackerly, D.D., Baruch, Z. & nities. Bongers, F. (2004) The worldwide leaf economics spectrum. Nature, 428, 821–827. Table S2. Pairwise Pearson correlations between leaf functional and biomechanical traits for 21 species of three Californian plant Received 1 August 2012; accepted 14 December 2012 communities. Handling Editor: Niels Anten