The links between leaf hydraulic vulnerability to drought and key aspects of leaf venation and xylem anatomy among 26 Australian woody angiosperms from contrasting climates

The links between leaf hydraulic vulnerability to drought and key aspects of leaf venation and... Abstract Background and Aims The structural properties of leaf venation and xylem anatomy strongly influence leaf hydraulics, including the ability of leaves to maintain hydraulic function during drought. Here we examined the strength of the links between different leaf venation traits and leaf hydraulic vulnerability to drought (expressed as P50leaf by rehydration kinetics) in a diverse group of 26 woody angiosperm species, representing a wide range of leaf vulnerabilities, from four low-nutrient sites with contrasting rainfall across eastern Australia. Methods For each species we measured key aspects of leaf venation design, xylem anatomy and leaf morphology. We also assessed for the first time the scaling relationships between hydraulically weighted vessel wall thickness (th) and lumen breadth (bh) across vein orders and habitats. Key Results Across species, variation in P50leaf was strongly correlated with the ratio of vessel wall thickness (th) to lumen breadth (bh) [(t/b)h; an index of conduit reinforcement] at each leaf vein order. Concomitantly, the scaling relationship between th and bh was similar across vein orders, with a log–log slope less than 1 indicating greater xylem reinforcement in smaller vessels. In contrast, P50leaf was not related to th and bh individually, to major vein density (Dvmajor) or to leaf size. Principal components analysis revealed two largely orthogonal trait groupings linked to variation in leaf size and drought tolerance. Conclusions Our results indicate that xylem conduit reinforcement occurs throughout leaf venation, and remains closely linked to leaf drought tolerance irrespective of leaf size. Leaf hydraulic vulnerability, xylem anatomy, leaf venation, vein density, xylem reinforcement, leaf size, drought INTRODUCTION The anatomical and architectural features of leaf venation strongly influence plant productivity and survival across species and environments. Given that the efficiency of water transport through leaf veins is a major determinant of maximum rates of photosynthesis (Brodribb et al., 2007; Sack and Scoffoni, 2013), venation traits that influence water transport efficiency, such as xylem vessel width (Aasamaa et al., 2001), vessel perforation-plate anatomy (Feild and Brodribb, 2013) and vein density (Sack and Frole, 2006; Brodribb and Feild, 2010; Walls, 2011; Buckley et al., 2015; Gleason et al., 2016), have been examined across large numbers of species. Recent studies have also identified several venation traits related to the ability of leaves to resist hydraulic decline under increasing levels of drought stress (Cochard et al., 2004; Brodribb and Holbrook, 2005; Blackman et al., 2010; Scoffoni et al., 2011, 2017b; Nardini et al., 2012). Quantifying these traits offers a potentially useful approach for screening leaf drought tolerance thresholds in extant species, as well as those in the fossil record (Sack and Scoffoni, 2013). As soils dry out during drought, tension (water potential, in MPa) within the leaf xylem increases. Under relatively mild drought conditions, this process can cause leaf water transport capacity (Kleaf) to decline as a result of turgor loss and leaf shrinkage (Scoffoni et al., 2014, 2017a; Trifilo et al., 2016). Under more severe drought conditions, further increases in xylem tension can exceed species hydraulic safety thresholds, causing Kleaf to decline as a result of embolism formation (air blockages) in the water-conducting xylem (Johnson et al., 2009; Brodribb et al., 2016b). If drought continues, this process can lead to complete leaf hydraulic failure and even plant death (Brodribb and Cochard, 2009; Scholz et al., 2014). The ability of leaves to resist hydraulic decline during drought is typically characterized by their hydraulic vulnerability, measured as the water potential associated with 50 % loss in hydraulic conductance, or P50leaf. Recent studies indicate that P50leaf varies widely across species from environments with contrasting rainfall (Brodribb and Hill, 1999; Blackman et al., 2014) and temperature (Nardini and Luglio, 2014), and represents a major determinant of species distributional limits (Blackman et al., 2012; Nardini et al., 2012). Leaf hydraulic vulnerability to drought is an integrated trait derived from different structural and functional characteristics of the leaf water transport pathway. Recent cross-species studies have reported close linkages between variation in P50leaf and specific aspects of leaf vein anatomy and venation design. These studies suggest that angiosperm species with low hydraulic vulnerability (i.e. more negative P50leaf) tend to have leaves with narrow xylem conduits that help minimize the spread of drought-induced embolism (Nardini et al., 2012; Scoffoni et al., 2017b) and high major vein density that provides multiple pathways for water movement around air-filled conduits (hydraulic redundancy) (Scoffoni et al., 2011; Nardini et al., 2014). A strong correlation has also been found between leaf hydraulic vulnerability and the ratio of conduit wall thickness (t) to lumen breadth (b) in leaf minor veins of conifer (Cochard et al., 2004; Brodribb and Holbrook, 2005) and angiosperm (Blackman et al., 2010) species. These findings suggest xylem conduit reinforcement provides a degree of safety from vessel wall collapse during drought. However, it remains unknown to what degree xylem reinforcement occurs throughout the leaf venation network, and whether the scaling of t and b varies across species from different habitats. Euler buckling theory suggests that t should scale proportionately with b to prevent collapse as the breadth of conduits increases (Hacke et al., 2001; Brodribb and Holbrook, 2005). Scaling less than proportionately with b would indicate stronger xylem reinforcement in smaller vessels. If the th–bh scaling exponent shifts across habitats with species operating at lower water potentials displaying a coefficient closer to unity, then this would indicate an increasing cost to constructing leaves with large vessels in arid habitats. As far as we are aware, these possibilities have not previously been assessed in leaves. Although relationships between P50leaf and different venation and xylem anatomy traits have been examined across small groups of ecologically diverse species, it remains unknown whether specific leaf venation traits can become decoupled from P50leaf due to their intrinsic link to leaf size. Leaf size is closely linked to major vein density (Scoffoni et al., 2011) as a consequence of vein packing constraints during leaf development (Sack et al., 2012), and to petiole vessel size (McCulloh et al., 2009; Gleason et al., 2016, 2018), for optimal leaf water transport efficiency (Sack et al., 2003). The link to P50leaf helps explain the propensity of small-leaved species to occupy more arid environments (Scoffoni et al., 2011). However, leaf size can be influenced by multiple environmental factors, including rainfall, temperature, light and nutrient conditions (Givnish, 1987; Cunningham et al., 1999; Fonseca et al., 2000; Tozer et al., 2015), whereas P50leaf is most strongly influenced by site water availability (Brodribb and Cochard, 2009; Blackman et al., 2014; Nardini and Luglio, 2014; Scholz et al., 2014). For species where leaf size is strongly constrained by selection pressures other than rainfall, venation traits intrinsically linked to leaf size might be expected to become decoupled from P50leaf. Here, we tested the level of coordination among different leaf xylem anatomy and venation traits, leaf size, leaf mass per unit area (LMA) and leaf hydraulic vulnerability to drought (P50leaf) across a phylogenetically diverse group of eastern Australian temperate and sub-tropical woody angiosperms. We collected leaves from 26 species that varied strongly in leaf hydraulic vulnerability from four sites characterised by different rainfall, but similarly poor nutrient conditions. For each species, we measured hydraulically weighted diameter (bh), wall thickness (th) and an index of implosion resistance (t/b)h of xylem vessels within the petiole, midrib, 2° veins and leaf minor veins. We also measured leaf major and minor vein density, as well as leaf size and LMA. P50leaf values were sourced from previously published vulnerability curves (Blackman et al., 2014). We asked: (1) What are the venation traits most strongly linked to P50leaf across species? (2) Does the scaling of vessel wall thickness to vessel lumen breadth depart from proportionality within individual leaves, across species, or among habitats? (3) Assuming that in our species group leaf size is constrained by both low rainfall and low soil nutrients, can P50leaf vary independently from leaf venation traits that are intrinsically linked to leaf size? MATERIALS AND METHODS Study sites and species Twenty-six species representing ten families were sampled from four sites across coastal and inland eastern Australia (Table 1; Supplementary Data Table S1). Three of these sites (Warm-Wet, Warm-Dry and Warm-Arid) were associated with a strong east–west aridity gradient in New South Wales, while the fourth site (Hot-Dry) was located in seasonally dry eucalypt woodland in northern Queensland (for more detailed site climate descriptions see Gleason et al., 2012). All four sites were characterized by late successional vegetation with eucalyptus (senso lato) occurring on weathered oligotrophic soils, low in phosphorous (Gleason et al., 2012). The sites varied strongly in rainfall from 383 mm annually at the Warm-Arid site to 1210 mm at the Warm-Wet site (Table 1). Six or seven dominant shrub and/or tree species were sampled from each site. The sample group contained a variety of simple leaf types including flat, revolute, terete and phyllodinous leaves. All species were evergreen except for Planchonia careya from the Hot-Dry site, which was drought-deciduous. In addition to the mean annual precipitation (MAP) for each site, MAP data were downloaded for cleaned occurrence records of each species from the Atlas of Living Australia (http://www.ala.org.au) and used to calculate the MAP across each species distribution. Sampling at each site occurred in 2012–2013, outside of the hot summer months. The same individuals of each species were used for measurements of leaf hydraulics and leaf anatomy. Table 1. The geographical, climatic and edaphic details of each of the four sites sampled from in this study Site  Habitat  Latitude (°S)  Longitude (°E)  MAP (mm)  MAT (°C)  Soil P (mg kg−1)  Species (n)  P50leaf (MPa)  LA (cm2)  Kur-ing-gai  Warm-Wet  33.68  151.15  1210  17.0  54.8  6  −2.7 ± 0.3a  4.5 ± 3.7a  Yengo  Warm-Dry  32.78  150.92  779  16.6  223  6  −3.1 ± 0.3a  12.4 ± 6.0ab  Round Hill  Warm-Arid  32.98  146.16  383  17.9  106  7  −5.5 ± 0.6b  4.9 ± 1.7ab  Princess Hills  Hot-Dry  18.29  145.49  1139  21.3  157  7  −3.0 ± 0.2a  26.5 ± 4.1b  Site  Habitat  Latitude (°S)  Longitude (°E)  MAP (mm)  MAT (°C)  Soil P (mg kg−1)  Species (n)  P50leaf (MPa)  LA (cm2)  Kur-ing-gai  Warm-Wet  33.68  151.15  1210  17.0  54.8  6  −2.7 ± 0.3a  4.5 ± 3.7a  Yengo  Warm-Dry  32.78  150.92  779  16.6  223  6  −3.1 ± 0.3a  12.4 ± 6.0ab  Round Hill  Warm-Arid  32.98  146.16  383  17.9  106  7  −5.5 ± 0.6b  4.9 ± 1.7ab  Princess Hills  Hot-Dry  18.29  145.49  1139  21.3  157  7  −3.0 ± 0.2a  26.5 ± 4.1b  MAP, mean annual precipitation; MAT, mean annual temperature. Climate data were sourced from the Atlas of Living Australia (2017), while soil P was sourced from a previous study (Gleason et al 2012). Also included are site means (plus standard errors) for leaf hydraulic vulnerability (P50leaf) and leaf size (LA); significant differences (P < 0.05) between sites, using pairwise comparisons, are denoted by different superscript letters. View Large Table 1. The geographical, climatic and edaphic details of each of the four sites sampled from in this study Site  Habitat  Latitude (°S)  Longitude (°E)  MAP (mm)  MAT (°C)  Soil P (mg kg−1)  Species (n)  P50leaf (MPa)  LA (cm2)  Kur-ing-gai  Warm-Wet  33.68  151.15  1210  17.0  54.8  6  −2.7 ± 0.3a  4.5 ± 3.7a  Yengo  Warm-Dry  32.78  150.92  779  16.6  223  6  −3.1 ± 0.3a  12.4 ± 6.0ab  Round Hill  Warm-Arid  32.98  146.16  383  17.9  106  7  −5.5 ± 0.6b  4.9 ± 1.7ab  Princess Hills  Hot-Dry  18.29  145.49  1139  21.3  157  7  −3.0 ± 0.2a  26.5 ± 4.1b  Site  Habitat  Latitude (°S)  Longitude (°E)  MAP (mm)  MAT (°C)  Soil P (mg kg−1)  Species (n)  P50leaf (MPa)  LA (cm2)  Kur-ing-gai  Warm-Wet  33.68  151.15  1210  17.0  54.8  6  −2.7 ± 0.3a  4.5 ± 3.7a  Yengo  Warm-Dry  32.78  150.92  779  16.6  223  6  −3.1 ± 0.3a  12.4 ± 6.0ab  Round Hill  Warm-Arid  32.98  146.16  383  17.9  106  7  −5.5 ± 0.6b  4.9 ± 1.7ab  Princess Hills  Hot-Dry  18.29  145.49  1139  21.3  157  7  −3.0 ± 0.2a  26.5 ± 4.1b  MAP, mean annual precipitation; MAT, mean annual temperature. Climate data were sourced from the Atlas of Living Australia (2017), while soil P was sourced from a previous study (Gleason et al 2012). Also included are site means (plus standard errors) for leaf hydraulic vulnerability (P50leaf) and leaf size (LA); significant differences (P < 0.05) between sites, using pairwise comparisons, are denoted by different superscript letters. View Large Leaf hydraulic vulnerability Leaf vulnerability curves for the current group of species were sourced from a previous study published by our lab group (Blackman et al., 2014). In brief, each curve was generated using a modified rehydration technique (Brodribb and Cochard, 2009), whereby leaves or small shoots were excised underwater from branches (three branches per species) dried down over 2–4 d to a range of water potentials and connected to a flow meter. We ensured water potential was equilibrated before each ‘rehydration’ experiment by placing branches into opaque plastic bags for up to 1 h. Measurements were conducted under normal light conditions in the lab or in the field under a shade tent. For most species, the response of leaf hydraulic conductance (Kleaf) to increasing water potential (in MPa) was sigmoidal, with Kleaf not declining over an initial range of water potentials, then declining once species hydraulic safety thresholds were reached (Fig. S1). For each rehydration experiment, leaves were connected to the hydraulic apparatus within 2 s and Kleaf was calculated from the flow rate recorded within the first 4–6 s following leaf connection to the flow meter. These initial flow rates were assumed to be influenced predominantly by the hydraulic resistance of the xylem pathway, and thus we considered the decline in Kleaf to be driven primarily by the formation and spread of xylem embolism (see Nolf et al., 2015; Skelton et al., 2015, 2017a; Brodribb et al., 2016a, b). However, we acknowledge that the decline in Kleaf can also be influenced by measurement light intensity, which has been shown to affect hydraulic processes in leaf tissues beyond the xylem (Guyot et al., 2012; Trifilo et al., 2016). The influence of light intensity on the decline in Kleaf has been demonstrated using the evaporative flux technique (Sack et al., 2002), as well as the timed rehydration kinetics technique, devised by Brodribb and Holbrook (2003), where leaves were allowed to absorb water under high or low light for 15–45 s (Scoffoni et al., 2008). Thus, although we cannot entirely exclude the influence of outside xylem processes, we considered our measurements of leaf hydraulic vulnerability to represent the water potential associated with 50 % loss in hydraulic conductance (P50leaf) driven primarily by embolism formation in the leaf xylem. This contrasts with the evaporative flux method (Sack et al., 2002; Scoffoni et al., 2008) where measurements include mesophyll and stomatal conductance and hence are responsive to factors including light intensity. Across species, P50leaf varied substantially from −1.9 MPa in Banksia serrata to −7.8 MPa in Melaleuca uncinata (Table S1). Leaf xylem anatomy and venation traits Fully expanded sun-leaves were collected from three individuals from each field site at the time leaf hydraulic vulnerability measurements were made. Between five and ten sample leaves from each of three individuals per species were sealed in zip-lock bags with moist paper-towel and placed inside an insulated cool-box. Samples were transported back to the laboratory within 3 d of collecting and fixed in FAA (formalin acetic acid) solution and stored. Measurements of xylem anatomy in petioles, midribs, 2° veins and minor veins were made in one leaf from two to three individuals per species, with the exception of vessels in 2° veins of Pultenaea scabra which were calculated from a single leaf (see Table S1). Leaf area was measured using a flat-bed scanner (Scan Maker i900, Microtek International, China) before sectioning. Transverse sections of vein xylem were made using a vibratome (VT1000s, Leica Microsystems, Germany). Sections were made half-way along the length of the petiole, mid-rib and 2° veins (minor vein anatomy was generally captured within lower order vein sections). Small (<°1 cm2) pieces of leaf, each containing a target vein, were cut out and individually suspended in 6 % agarose blocks. Each agarose block was shaped with a razor-blade and then mounted onto the vibratome stage ensuring that the target vein was perpendicular to the cutting edge of the blade. Several transverse sections were cut at a thickness of between 10 and 20 µm. Sections were stained in dilute 1 % methylene blue before mounting onto glass slides in phenol glycerine jelly. The xylem anatomy of each vein order was photographed using a digital camera (DXM1200F, Nikon, Japan) attached to a light microscope (Bx50, Olympus Optical, Japan). Magnification of each vein depended on vessel size; petioles, mid-ribs and 2° veins were photographed at 40× or 100×, while minor veins were photographed at 100×. From each image, lumen breadth (b) and wall thickness (t) was measured using ImageJ software (National Institutes of Health, USA) from a representative sample of between ten and 100 hydraulically functional vessels. Due to the typically low number of vessels in minor veins, b and t were measured from two or three different minor veins per leaf. For all veins, care was taken to avoid cell-types such as fibre cells and xylem parenchyma that were deemed to provide functional roles beyond water transport. Also, minor veins were identified as the smallest veins in cross section with a clearly defined vascular bundle (xylem and phloem), and were carefully distinguished from free vein endings, which were often enlarged and represented sclereids and/or tracheids in some species. Because vessels were often elliptical in shape, b was measured along the short and long axes of each vessel, and then transformed to the circular equivalent diameter (Choat et al., 2007). Hydraulically weighted vessel diameter (bh) was calculated according to the formula bh = Σ(b4/n)0.25, which weights the vessels (n) within each vein order by their hydraulic contribution to total vein conductance (Tyree and Zimmermann, 2002). For each hydraulically weighted diameter we estimated its wall thickness from the ordinary least-squares relationship between t and b measured across a subsample of 10–15 vessels within each vein order. For each cell, b was measured as described above, while t was measured as the single-thickness of a clearly defined radial wall. The level of xylem reinforcement of hydraulically weighted vessels was then calculated as the ratio of wall thickness (th) and lumen breadth (bh), (t/b)h. We also examined the shape and slope of the th–bh relationship across vein orders and species. Euler buckling theory suggests that th should scale proportionately with bh (i.e. an expected log–log slope of 1) to maintain a constant crushing tension as vessel radii narrow from petioles to minor veins (Hacke et al., 2001; Brodribb and Holbrook, 2005). Less than proportional scaling between these two vessel traits would suggest that large leaves [i.e. with large vessels (McCulloh et al., 2009; Gleason et al., 2018)] represent a savings in network construction costs; for example, a doubling leaf size would result in a somewhat less than doubling of network construction costs. Furthermore, the slope of this relationship (i.e. the th and bh ratio assuming a y-intercept of zero) may also differ across species and habitats. A change in the slope (but not the shape) of the function would indicate greater carbon investment (thicker vessel walls) at all points throughout the network. To test if the slope or shape of the th–bh relationship differed across species or habitats, we plotted th–bh on log10-transformed axes and compared the log–log slopes (i.e. departure from proportionality; scaling exponents), as well as the log–log intercepts (i.e. the logged arithmetic slopes; normalization constants) among species and habitats. Log–log intercepts were only compared if there was no difference in slope among species or habitats. The ‘sma’ function in the SMATR package for R was used for these analyses (Warton et al., 2006). Leaf venation architecture was characterized using one leaf from each of three individuals per species. For species with flat or revolute leaves, we used a protocol described by Scoffoni et al. (2011) for leaf clearing and quantifying vein density. In brief, leaves were chemically cleared with 5 % NaOH, put through a dehydration series in ethanol, stained with saffranin and counter-stained with fast green. Leaves were mounted in water on transparency film and scanned at high resolution using a flatbed scanner (Scan Maker i900, Microtek International, China). The leaf area and lengths of midribs and 2° veins were measured using ImageJ. To ensure that 3° and higher order veins were visible, we exposed the veins prior to leaf clearing by cutting a small window (<1 cm2) through the epidermis and top layers of mesophyll. For large leaves (>10 cm2), three vein windows were made, located centrally in the top, middle and bottom thirds of the leaf. The lengths of 3° and minor veins were measured (using ImageJ) from photographs of these vein windows taken with a digital camera (DXM1200F, Nikon) attached to a light microscope (Bx50, Olympus Optical) at 4× and 10× magnification, respectively. Vein density was calculated for each vein order as the length of vein per unit leaf area. In large leaves, 3° and minor vein densities were averaged across the three exposed windows. The major vein density (Dvmajor) was determined as the sum of 1°, 2° and 3° order vein densities, and minor vein density (Dvminor) as the total length per unit area of 4° and higher order veins. For the four species with terete leaves with parallel leaf venation, vein orders were distinguished by size class in transverse section (see above for leaf sectioning protocol). The vein density of each vein order was then calculated as the sum of the number of veins within each vein order, multiplied by leaf length (assumed to be equivalent to vein length) and divided by projected leaf area. Leaf structural traits Leaf area (LA) was determined for each species from the same three leaves used for quantifying xylem anatomy traits in cross section. Leaves were imaged on a standard flatbed scanner (Epson Perfection V33, Australia). Projected leaf area was calculated from these images using ImageJ software (National Institutes of Health). Leaf mass per unit area (LMA) values for each species were taken from a previous study conducted at the same sites (Gleason et al., 2012). Statistical analysis Bivariate relationships were fit with ordinary least squares (OLS) or standard major axes (SMA) models using the ‘smatr’ package in R (Warton et al., 2006). Differences among sites and vein orders may manifest as different slope or intercept coefficients. When relationships were well approximated by power models (e.g. th ~ a.bhb), scaling exponents were evaluated among groups by testing the log–log slope coefficient (b; scaling exponent) as well as the intercept coefficient (a; scaling constant) when slopes were statistically similar among sites or vein orders. Principal components analysis (PCA) (‘prcomp’ function in R; R Core Team, 2015) was used to determine the dominant axes of variation among a selection of traits, including petiole vessel th, bh and (t/b)h, respectively, as well as major and minor vein density, LA, LMA and P50leaf. All variables were scaled to unit variance in the PCA. RESULTS Substantial variation in venation architecture, leaf xylem anatomy and gross morphology was observed across species (Table S1). Major vein density varied ~15-fold from 0.58 to 9.2 mm mm−2, while among the xylem anatomy traits petiole vessel bh varied ~6.5-fold from 3.3 to 24.4 µm, petiole vessel th varied ~2.5-fold from 0.67 to 2.7 µm, and petiole vessel (t/b)h varied ~2-fold from 0.08 to 0.25. Across species, LMA varied nearly 5-fold, ranging from 75 to 447 g m−2, and leaf area varied more than 300-fold, ranging from 0.12 to 40.0 cm2. Among sites, leaf size differed significantly between the Warm-Wet and the Hot-Dry sites (pairwise comparisons; Table 1), while leaf size was unrelated to site rainfall and weakly correlated with soil phosphorus (r2 = 0.15, P = 0.05; Fig. S2), and was unrelated to species mean annual rainfall (Table 2; Fig. S3). In contrast, P50leaf was significantly different among species from the Warm-Arid site compared to species from the other three sites (Table 1), while P50leaf was strongly correlated with site rainfall (r2 = 0.48, P< 0.001) but not soil phosphorus (Fig. S2), and was correlated with species mean annual rainfall (Table 2; Fig. S3). Table 2. Pearson correlation r values among climate (species mean annual rainfall) and leaf, vein and petiole vessel traits measured in 26 woody angiosperm species   MAP  P50leaf  Pet_t–bh  Pet_bh  Pet_th  Dvmajor  Dvminor  LMA  LA  MAP                    P50leaf  −0.76                  Pet_t–bh  −0.66  0.73                Pet_bh  0.33  −0.34  −0.73              Pet_th  −0.06  0.11  −0.23  0.83            Dvmajor  −0.20  0.11  0.27  −0.60  −0.63          Dvminor  −0.19  0.08  0.01  0.15  0.21  −0.03        LMA  −0.29  0.25  0.10  −0.05  0.01  0.37  −0.27      LA  0.18  −0.06  −0.42  0.86  0.89  −0.64  0.14  −0.03      MAP  P50leaf  Pet_t–bh  Pet_bh  Pet_th  Dvmajor  Dvminor  LMA  LA  MAP                    P50leaf  −0.76                  Pet_t–bh  −0.66  0.73                Pet_bh  0.33  −0.34  −0.73              Pet_th  −0.06  0.11  −0.23  0.83            Dvmajor  −0.20  0.11  0.27  −0.60  −0.63          Dvminor  −0.19  0.08  0.01  0.15  0.21  −0.03        LMA  −0.29  0.25  0.10  −0.05  0.01  0.37  −0.27      LA  0.18  −0.06  −0.42  0.86  0.89  −0.64  0.14  −0.03    All data were log-transformed for analysis. Values in bold type are significant at P < 0.05. View Large Table 2. Pearson correlation r values among climate (species mean annual rainfall) and leaf, vein and petiole vessel traits measured in 26 woody angiosperm species   MAP  P50leaf  Pet_t–bh  Pet_bh  Pet_th  Dvmajor  Dvminor  LMA  LA  MAP                    P50leaf  −0.76                  Pet_t–bh  −0.66  0.73                Pet_bh  0.33  −0.34  −0.73              Pet_th  −0.06  0.11  −0.23  0.83            Dvmajor  −0.20  0.11  0.27  −0.60  −0.63          Dvminor  −0.19  0.08  0.01  0.15  0.21  −0.03        LMA  −0.29  0.25  0.10  −0.05  0.01  0.37  −0.27      LA  0.18  −0.06  −0.42  0.86  0.89  −0.64  0.14  −0.03      MAP  P50leaf  Pet_t–bh  Pet_bh  Pet_th  Dvmajor  Dvminor  LMA  LA  MAP                    P50leaf  −0.76                  Pet_t–bh  −0.66  0.73                Pet_bh  0.33  −0.34  −0.73              Pet_th  −0.06  0.11  −0.23  0.83            Dvmajor  −0.20  0.11  0.27  −0.60  −0.63          Dvminor  −0.19  0.08  0.01  0.15  0.21  −0.03        LMA  −0.29  0.25  0.10  −0.05  0.01  0.37  −0.27      LA  0.18  −0.06  −0.42  0.86  0.89  −0.64  0.14  −0.03    All data were log-transformed for analysis. Values in bold type are significant at P < 0.05. View Large Across species, variation in P50leaf was strongly correlated with vessel (t/b)h of petioles (r2 = 0.53, P < 0.001), midribs (r2 = 0.58, P < 0.001), second-order veins (r2 = 0.46, P < 0.001) and minor veins (r2 = 0.72, P < 0.001) (Fig. 1), indicating that greater resistance to leaf hydraulic dysfunction (more negative P50leaf) is linked to greater xylem vessel reinforcement across all vein orders. Concomitantly, neither log–log slope nor log–log elevation of the relationship between th and bh were significantly different among vein orders (Fig. 2A; slope P = 0.17; elevation P = 0.08), suggesting similar scaling exponents and scaling constants throughout the networks. However, the log–log slope of the th–bh relationship across vein orders was significantly shallower than 1 (P < 0.001), indicating that the ratio of wall thickness to lumen breadth decreased as vessels became wider. Comparing across sites, species from the Warm-Wet site tested as having significantly shallower log–log slope than the other three sites (0.52 vs 0.72 in common for the other three sites; P < 0.025). The suggestion (Fig. 2B) is that larger vessels at this wet site tended to have relatively thinner walls than at the dry and arid sites, but minor veins did not. However, considering the graph (Fig. 2B), we do not attach strong weight to this apparent difference in slope. More clear-cut is that among the three sites exhibiting a common log–log slope (Warm-Dry, Warm-Arid, Hot-Dry), the elevation of the th–bh relationship varied significantly (P < 0.001), with species from the drier sites having larger values, meaning vessel wall thickness in these species exhibited stronger reinforcement at a given lumen diameter than species from wetter sites (Fig. 2B).We note that although petiole (t/b)h was correlated with species MAP (r2 = 0.44, P < 0.001), neither of the individual components of the th/bh ratio were related to rainfall (Table 2). Fig. 1. View largeDownload slide The log–log relationship between P50leaf and hydraulically weighted vessel (t/b)h at each vein order across species: black filled circles = petioles (solid line, r2 = 0.53***); dark-grey filled circles = midribs (dotted line, r2 = 0.58***); light-grey filled circles = 2° veins (dashed line, r2 = 0.46***); white filled circles = minor veins (dash-dot line, r2 = 0.72***). Level of significance: ***P < 0.001. Fig. 1. View largeDownload slide The log–log relationship between P50leaf and hydraulically weighted vessel (t/b)h at each vein order across species: black filled circles = petioles (solid line, r2 = 0.53***); dark-grey filled circles = midribs (dotted line, r2 = 0.58***); light-grey filled circles = 2° veins (dashed line, r2 = 0.46***); white filled circles = minor veins (dash-dot line, r2 = 0.72***). Level of significance: ***P < 0.001. Fig. 2. View largeDownload slide (A) The log–log relationship between vessel wall thickness (th) and lumen breadth (bh) at each vein order across species: black filled circles = petioles (solid line, r2 = 0.69***, slope = 0.7, elevation = −0.61); dark-grey filled circles = midribs (dotted line, r2 = 0.64***, slope = 0.76, elevation = −0.64); light-grey filled circles = 2° veins (dashed line, r2 = 0.60***, slope = 0.62, elevation = −0.65); white filled circles = minor veins (dash-dot line, r2 = 0.31**, slope = 0.99, elevation = −0.58). Neither slope nor elevation of the log–log th–bh relationship varied across vein orders (SMATR analysis: slope P = 0.17, elevation P = 0.08). (B) The log–log th–bh relationship across all vein orders for species separated by site: blue filled circles = Warm-Wet (solid line, r2 = 0.72***, slope = 0.52, elevation = −0.66), green filled circles = Warm-Dry (dashed line, r2 = 0.94***, slope = 0.78, elevation = −0.59); red filled circles = Warm-Arid (dotted line, r2 = 0.68***, slope = 0.69, elevation = −0.51); orange filled circles = Hot-Dry (dash-dot line, r2 = 0.94***, slope = 0.69, elevation = −0.64). Slope of the log–log th–bh relationship differed only for species from the Warm-Wet site (SMATR analysis: P = 0.02). The red dashed line in both plots represents a proportional th–bh scaling slope of 1, where (t/b)h = 0.24. Level of significance: ***P < 0.001; **P < 0.01. Fig. 2. View largeDownload slide (A) The log–log relationship between vessel wall thickness (th) and lumen breadth (bh) at each vein order across species: black filled circles = petioles (solid line, r2 = 0.69***, slope = 0.7, elevation = −0.61); dark-grey filled circles = midribs (dotted line, r2 = 0.64***, slope = 0.76, elevation = −0.64); light-grey filled circles = 2° veins (dashed line, r2 = 0.60***, slope = 0.62, elevation = −0.65); white filled circles = minor veins (dash-dot line, r2 = 0.31**, slope = 0.99, elevation = −0.58). Neither slope nor elevation of the log–log th–bh relationship varied across vein orders (SMATR analysis: slope P = 0.17, elevation P = 0.08). (B) The log–log th–bh relationship across all vein orders for species separated by site: blue filled circles = Warm-Wet (solid line, r2 = 0.72***, slope = 0.52, elevation = −0.66), green filled circles = Warm-Dry (dashed line, r2 = 0.94***, slope = 0.78, elevation = −0.59); red filled circles = Warm-Arid (dotted line, r2 = 0.68***, slope = 0.69, elevation = −0.51); orange filled circles = Hot-Dry (dash-dot line, r2 = 0.94***, slope = 0.69, elevation = −0.64). Slope of the log–log th–bh relationship differed only for species from the Warm-Wet site (SMATR analysis: P = 0.02). The red dashed line in both plots represents a proportional th–bh scaling slope of 1, where (t/b)h = 0.24. Level of significance: ***P < 0.001; **P < 0.01. Across species, variation in P50leaf was not related to either th or bh at any vein order, with the exception of minor vein vessel bh (r2 = 0.17, P = 0.04; Table S2). P50leaf was also unrelated to major vein density (Dvmajor), minor vein density (Dvminor), leaf size (LA) and leaf mass per unit area (LMA) (Table 2). Nonetheless, leaf size was significantly and negatively correlated with major vein density (r2 = 0.42, P < 0.001) (Fig. 3), but was unrelated to minor vein density (Table 2). Leaf size was positively correlated with vessel bh and th in leaf petioles [bh, r2 = 0.75, P < 0.001 (Fig. 3); th, r2 = 0.79, P < 0.001], midribs (bh, r2 = 0.75, P < 0.001; th, r2 = 0.80, P < 0.001) and 2° veins (bh, r2 = 0.61, P < 0.001; th, r2 = 0.43, P < 0.001), but not in leaf minor veins (see Table S2). Leaf size was also related to vessel (t/b)h at the petiole and 2° veins, but not at the midrib or minor veins (Tables 2 and S2). Strong negative correlations were observed between major vein density and vessel bh and th in lower order veins (Table S2). Fig. 3. View largeDownload slide Log–log relationships across species between leaf area and (A) major vein density (r2 = 0.42, P < 0.001) and (B) hydraulically weighted vessel diameter in leaf petioles (r2 = 0.75, P < 0.001). Species values are separated by site (blue filled circles = Warm-Wet, green filled circles = Warm-Dry, red filled circles = Warm-Arid, orange filled circles = Hot-Dry). Fig. 3. View largeDownload slide Log–log relationships across species between leaf area and (A) major vein density (r2 = 0.42, P < 0.001) and (B) hydraulically weighted vessel diameter in leaf petioles (r2 = 0.75, P < 0.001). Species values are separated by site (blue filled circles = Warm-Wet, green filled circles = Warm-Dry, red filled circles = Warm-Arid, orange filled circles = Hot-Dry). PCA of leaf, vein and petiole vessel traits identified two major axes, which cumulatively explained 66 % of the total variation among the traits (Fig. 4). The first principal component (PC) accounted for 46 % of the total variation and was dominated by leaf size, major vein density, and both petiole vessel bh and th, while the second PC (21 % of the total variation) was associated with P50leaf and petiole (t/b)h. Importantly, (t/b)h was closely aligned with P50leaf, whereas the components of this ratio, th and bh, aligned primarily with leaf size and major vein density, and were largely orthogonal to P50leaf and (t/b)h (Fig. 4). Fig. 4. View largeDownload slide Principal components plot of leaf, vein and petiole vessel properties, including petiole vessel th, bh and (t/b)h, major and minor vein density, LA and LMA. Individual points denote individual species. PC1 and PC2 account for 45 and 21 % of the variation among species and sites, respectively. Vector loadings are represented with arrows. All data were log-transformed and scaled to unit variance prior to analysis. Fig. 4. View largeDownload slide Principal components plot of leaf, vein and petiole vessel properties, including petiole vessel th, bh and (t/b)h, major and minor vein density, LA and LMA. Individual points denote individual species. PC1 and PC2 account for 45 and 21 % of the variation among species and sites, respectively. Vector loadings are represented with arrows. All data were log-transformed and scaled to unit variance prior to analysis. DISCUSSION The strong correlations between more negative P50leaf and greater vessel (t/b)h at each vein order across our sample group of 26 species highlight the functional link between xylem conduit reinforcement throughout the leaf venation and the ability of leaves to resist drought-induced hydraulic dysfunction (Blackman et al., 2010), probably caused by xylem embolism in our measurements using direct flow rehydration. The strength of this relationship is robust given that our species group spanned a very wide range of leaf vulnerabilities, with P50leaf values ranging from −1.9 MPa in the most vulnerable species Banksia serrata to −7.8 MPa in Melaleuca uncinata, which to the best of our knowledge is among the most negative P50leaf values recorded for angiosperm species (see also Skelton et al., 2017b). Our sample of species also spanned a wide range of values for each of the leaf venation, xylem anatomy and leaf morphology traits, which increased the likelihood of detecting significant trait–trait relationships across species. Even so, we note that large leaves >41 cm2 were not present in our species group, although compared to previous studies it did contain species with small leaves <1 cm2. The ratio of wall thickness to lumen breadth (t/b) has been used widely as an index of the implosion resistance of conduit walls under tension (Hacke et al., 2001; Sack and Scoffoni, 2013). Hacke et al. (2001) first observed a strong correlation between the ratio of conduit double-wall thickness and lumen breadth and drought resistance in woody stems. They argued that xylem conduit reinforcement should increase with increasing drought resistance on the basis that drought-resistant plants tend to experience stronger internal loads (water potential) in the field. This argument has been supported by studies highlighting the adaptive link between xylem conduit wall reinforcement and embolism resistance (P50) in plant stems (Jacobsen et al., 2005, 2007; Pittermann et al., 2006). In the current study, vessel (t/b)h was higher in species from more arid environments and was strongly correlated with P50leaf across species. This suggests that a similar level of coordination between xylem reinforcement and embolism resistance may be present in leaves. Although we cannot preclude the possibility that cell collapse occurs in angiosperm leaves under tension (see Zhang et al., 2016), the weight of recent evidence suggests that leaf hydraulic decline during severe drought is caused by xylem embolism (Johnson et al., 2009; Nolf et al., 2015; Brodribb et al., 2016a, b; Scoffoni et al., 2017b; Skelton et al., 2017a). Thus, we suggest xylem reinforcement in leaves has evolved to provide a degree of safety from vessel collapse under tension, while embolism spread via air-seeding could potentially relate to the size and structure of pores in pit membranes (Jansen et al., 2009), nucleation from hydrophobic surfaces (Tyree et al., 1994) or, as hypothesized recently, conduit/fluid properties that influence the expansion of nanobubbles (Schenk et al., 2015). We acknowledge that changes in the hydraulic properties of the extra-xylary pathway due to turgor loss and leaf shrinkage (Scoffoni et al., 2014, 2017a; Trifilo et al., 2016), and associated changes in aquaporin expression (Kim and Steudle, 2007), play a major role in the decline of leaf hydraulic conductance measured from petiole to external atmosphere. These processes tend to occur during the early stages of drought, at water potentials preceding those associated with xylem embolism (Scoffoni et al., 2017a), and furthermore are sensitive to measurement light conditions. Indeed, we note that our estimate of maximum Kleaf and thus P50leaf for each species may be underestimated on the basis that our rehydration measurements were conducted under low light and thus did not allow for the influence of outside xylem processes in driving the response of Kleaf during drought (Scoffoni et al., 2008, 2017a). However, our approach of measuring Kleaf under low light is consistent with the approach of other studies that have shown strong correspondence between the decline in Kleaf during drought, measured using the rehydration technique (Brodribb and Cochard, 2009), and the accumulation of xylem embolisms detected acoustically (Nolf et al., 2015) and visually via a recently developed optical technique (Brodribb et al., 2016a, b) and X-ray micro-computed tomography (Skelton et al., 2017a). Furthermore, in contrast to the evaporative flux technique (Sack et al., 2002) and the timed rehydration kinetics technique (Brodribb and Holbrook, 2003), we calculated Kleaf from the initial flow rate into the leaf through the petiole, which is more likely to be influenced by within-xylem rather than outside-xylem processes. Thus, it is reasonable to assume that the decline in Kleaf observed in our vulnerability curves was largely driven by embolism formation, although we note that further studies across diverse species are required to test the relative influence of xylem and outside xylem processes on the decline in Kleaf during drought measured using different measurement techniques under a range of conditions. Nevertheless, if embolism is the primary driver of the decline in Kleaf in vulnerability curves generated using the rehydration technique, then our study provides important mechanistic insights into the linkages between P50leaf and leaf venation and xylem anatomy traits related to xylem embolism resistance. Previous studies have observed strong links between xylem wall reinforcement in leaf minor veins and both leaf vulnerability to drought (Blackman et al., 2010) and species climatic limits (Jordan et al., 2013). In the current study, we observed for the first time strong relationships between P50leaf and vessel (t/b)h at each leaf vein order, with the scaling of vessel wall thickness to lumen breadth being consistent across vein orders. These results indicate that xylem conduit reinforcement occurs throughout the leaf venation network, from petioles to minor veins. Nonetheless, across species we found that the level of xylem vessel reinforcement was higher in smaller vessels (i.e. the t–b scaling exponent was significantly less than 1). This suggests that larger vessels perhaps require less reinforcement than smaller vessels, and/or that tension in vessels (negative water potential) increases from petiole to minor veins. Furthermore, it is consistent with findings that smaller minor veins are the most resistant to embolism within the leaf vein network (Brodribb et al., 2016a, b; Scoffoni et al., 2017b). Across habitats, we hypothesized that increasing the log–log t–b scaling exponent towards unity (Euler buckling exponent) would result in lower ‘network’ efficiency as a consequence of increasing carbon costs per unit increase in hydraulically weighted vessel diameter, which might play a role in constraining vessel size in species from more arid sites. Unfortunately, our data are ambiguous on this point. The least arid site did have a shallower scaling exponent than the other sites, but the ‘dry’ sites and ‘arid’ site had similar scaling exponents, which do not support this idea. However, among these dry-land sites the th–bh constant (i.e. elevation in Fig. 2B) varied significantly and increased with increasing site aridity. This suggests that natural selection acts predominantly on the scaling constant rather than the scaling exponent, which results in thicker walls relative to lumen diameter in drier habitats. Across our set of species, leaf hydraulic vulnerability to drought was unrelated to major vein density. This contrasts with previous studies (Scoffoni et al., 2011; Nardini et al., 2012) that found significant relationships between high major vein density and low hydraulic vulnerability in leaves. These authors suggested high major vein density confers increased drought tolerance on the basis that it provides more alternative pathways for water movement around vein embolisms. We acknowledge that high major vein density and hydraulic redundancy may play a role maintaining leaf hydraulic function under drought. Nonetheless, our results suggest that the functional link between higher major vein density and greater resistance to embolism-induced hydraulic decline may not hold across sets of species where leaf size is strongly influenced by factors in addition to water availability. In the current study, we observed a strong negative relationship between leaf size and major vein density and a strong positive relationship between leaf size and hydraulically weighted petiole diameter, consistent with the intrinsic links and scaling relationships between these traits (McCulloh et al., 2009; Sack et al., 2012; Gleason et al., 2018). These bivariate relationships were supported by PCA, which grouped together vessel and venation traits linked to leaf size. However, our analyses showed that leaf size varied independently of P50leaf. Although all species with large leaves (>10 cm2) had P50leaf values less negative than −5 MPa, species with small leaves (<1 cm2) were characterized by both high and low vulnerability (i.e. P50leaf values ranged from roughly −2 to −8 MPa). This implies that the relationship between P50leaf and leaf size – and thus the relationship between P50leaf and both major vein density and petiole vessel diameter – can break down across species from different habitats, especially where leaf size is constrained by multiple environmental factors. Indeed, in contrast to global trends (Wright et al., 2017), across our four temperate–tropical sample sites, mean leaf size was unrelated to climate indices of site water availability. In contrast, P50leaf is known to vary more systematically with rainfall across these sites (Blackman et al., 2014). Decoupling of leaf size and site water availability (and P50leaf) is consistent with poor leaf size–climate relationships reported for Australian vegetation (Peppe et al., 2011; Tozer et al., 2015), indicating that leaf size variation can be shaped by additional environmental filters, including low soil nutrients (Cunningham et al., 1999; Fonseca et al., 2000). Nevertheless, our findings across a morphologically diverse set of species suggest that smaller leaves and higher vein density are not necessarily conferring strongly negative P50leaf. The results of this study clearly indicate that the ability of leaves to resist hydraulic dysfunction under drought is closely related to the degree of leaf xylem reinforcement throughout the venation network. Unlike traits such as major vein density and petiole vessel diameter, the degree of xylem vessel reinforcement was unrelated to leaf size, which in turn was unrelated to P50leaf. These results point strongly to the usefulness of measuring xylem reinforcement in leaf veins when examining variation in leaf hydraulic vulnerability to drought across ecologically and morphologically diverse species. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: Species information and trait values measured for each species. Table S2: Pearson correlation r values among selected leaf traits. Figure S1: Leaf hydraulic vulnerability curves for each of the 26 species included in this study. Figure S2: The log–log relationship between leaf traits and site climate and soil characteristics. Figure S3: The log–log relationship between leaf traits and species mean annual precipitation. ACKNOWLEDGEMENTS This research was supported by an Australian Research Council fellowship awarded to M.W. We thank the Macquarie University Microscopy Unit for their help with leaf sectioning and imaging. Thanks are also given to New South Wales National Parks and Wildlife Service, and the Queensland Department of National Parks, Recreation, Sport and Racing for allowing access to field sites. C.J.B., S.M.G. and M.W. planned and designed the research. C.J.B., S.M.G., A.M.C., Y.C. and C.A.L. carried out field sampling, leaf sectioning/clearing and image analysis. C.J.B and S.M.G carried out data analysis. C.J.B, S.M.G. and M.W. wrote the manuscript. LITERATURE CITED Aasamaa K, Sober A, Rahi M. 2001. Leaf anatomical characteristics associated with shoot hydraulic conductance, stomatal conductance and stomatal sensitivity to changes of leaf water status in temperate deciduous trees. Australian Journal of Plant Physiology  28: 765– 774. Blackman CJ, Brodribb T, Jordan GJ. 2010. Leaf hydraulic vulnerability is related to conduit dimensions and drought resistance across a diverse range of woody angiosperms. New Phytologist  188: 1113– 1123. Google Scholar CrossRef Search ADS   Blackman CJ, Brodribb TJ, Jordan GJ. 2012. Leaf hydraulic vulnerability influences species’ bioclimatic limits in a diverse group of woody angiosperms. Oecologia  168: 1– 10. Google Scholar CrossRef Search ADS   Blackman CJ, Gleason SM, Chang Y, Cook AM, Laws C, Westoby M. 2014. Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates. Annals of Botany  114: 435– 440. Google Scholar CrossRef Search ADS   Brodribb TJ, Cochard H. 2009. Hydraulic failure defines the recovery and point of death in water-stressed conifers. Plant Physiology  149: 575– 584. Google Scholar CrossRef Search ADS   Brodribb TJ, Feild TS. 2010. Leaf hydraulic evolution led a surge in leaf photosynthetic capacity during early angiosperm diversification. Ecology Letters  13: 175– 183. Google Scholar CrossRef Search ADS   Brodribb T, Hill RS. 1999. The importance of xylem constraints in the distribution of conifer species. New Phytologist  143: 365– 372. Google Scholar CrossRef Search ADS   Brodribb TJ, Holbrook NM. 2003. Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant Physiology  132: 2166– 2173. Google Scholar CrossRef Search ADS   Brodribb TJ, Holbrook NM. 2005. Water stress deforms tracheids peripheral to the leaf vein of a tropical conifer. Plant Physiology  137: 1139– 1146. Google Scholar CrossRef Search ADS   Brodribb TJ, Feild TS, Jordan GJ. 2007. Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiology  144: 1890– 1898. Google Scholar CrossRef Search ADS   Brodribb TJ, Bienaime D, Marmottant P. 2016a. Revealing catastrophic failure of leaf networks under stress. Proceedings of the National Academy of Sciences of the United States of America  113: 4865– 4869. Google Scholar CrossRef Search ADS   Brodribb TJ, Skelton RP, McAdam SAM, Bienaime D, Lucani CJ, Marmottant P. 2016b. Visual quantification of embolism reveals leaf vulnerability to hydraulic failure. New Phytologist  209: 1403– 1409. Google Scholar CrossRef Search ADS   Buckley TN, John GP, Scoffoni C, Sack L. 2015. How does leaf anatomy influence water transport outside the xylem? Plant Physiology  168: 1616– 1635. Google Scholar CrossRef Search ADS   Choat B, Sack L, Holbrook NM. 2007. Diversity of hydraulic traits in nine Cordia species growing in tropical forests with contrasting precipitation. New Phytologist  175: 686– 698. Google Scholar CrossRef Search ADS   Cochard H, Froux F, Mayr FFS, Coutand C. 2004. Xylem wall collapse in water-stressed pine needles. Plant Physiology  134: 401– 408. Google Scholar CrossRef Search ADS   Cunningham SA, Summerhayes B, Westoby M. 1999. Evolutionary divergences in leaf structure and chemistry, comparing rainfall and soil nutrient gradients. Ecological Monographs  69: 569– 588. Google Scholar CrossRef Search ADS   Feild TS, Brodribb TJ. 2013. Hydraulic tuning of vein cell microstructure in the evolution of angiosperm venation networks. New Phytologist  199: 720– 726. Google Scholar CrossRef Search ADS   Fonseca CR, Overton JMC, Collins B, Westoby M. 2000. Shifts in trait-combinations along rainfall and phosphorus gradients. Journal of Ecology  88: 964– 977. Google Scholar CrossRef Search ADS   Givnish TJ. 1987. Comparative studies of leaf form: assessing the relative roles of selective pressures and phylogenetic constraints. New Phytologist  106: 131– 160. Google Scholar CrossRef Search ADS   Gleason SM, Butler DW, Zieminska K, Waryszak P, Westoby M. 2012. Stem xylem conductivity is key to plant water balance across Australian angiosperm species. Functional Ecology  26: 343– 352. Google Scholar CrossRef Search ADS   Gleason SM, Blackman CJ, Chang Y, Cook AM, Laws CA, Westoby M. 2016. Weak coordination among petiole, leaf, vein, and gas-exchange traits across Australian angiosperm species and its possible implications. Ecology and Evolution  6: 267– 278. Google Scholar CrossRef Search ADS   Gleason SM, Blackman CJ, Gleason ST, McCulloh KA, Ocheltree TW, Westoby M. 2018. Vessel scaling in evergreen angiosperm leaves conforms with Murray’s law and area-filling assumptions: implications for plant size, leaf size, and cold tolerance. New Phytologist . https://doi.org/10.1111/nph.15116. Guyot G, Scoffoni C, Sack L. 2012. Combined impacts of irradiance and dehydration on leaf hydraulic conductance: insights into vulnerability and stomatal control. Plant Cell and Environment  35: 857– 871. Google Scholar CrossRef Search ADS   Hacke UG, Sperry JS, Pockman WT, Davis SD, McCulloh KA. 2001. Trends in wood density and structure are linked to prevention of xylem implosion by negative pressure. Oecologia  126: 457– 461. Google Scholar CrossRef Search ADS   Jacobsen AL, Ewers FW, Pratt RB, PaddockIII WA, Davis SD. 2005. Do xylem fibers affect vessel cavitation resistance? Plant Physiology  139: 546– 556. Google Scholar CrossRef Search ADS   Jacobsen AL, Agenbag L, Esler KJ, Pratt RB, Ewers FW, Davis SD. 2007. Xylem density, biomechanics and anatomical traits correlate with water stress in 17 evergreen shrub species of the Mediterranean-type climate region of South Africa. Journal of Ecology  95: 171– 183. Google Scholar CrossRef Search ADS   Jansen S, Choat B, Pletsers A. 2009. Morphological variation of intervessel pit membranes and implications to xylem function in angiosperms. American Journal of Botany  96: 409– 419. Google Scholar CrossRef Search ADS   Johnson DM, Meinzer FC, Woodruff DR, McCulloh KA. 2009. Leaf xylem embolism, detected acoustically and by cryo-SEM, corresponds to decreases in leaf hydraulic conductance in four evergreen species. Plant, Cell and Environment  32: 828– 836. Google Scholar CrossRef Search ADS   Jordan GJ, Brodribb TJ, Blackman CJ, Weston PH. 2013. Climate drives vein anatomy in Proteaceae. American Journal of Botany  100: 1483– 1493. Google Scholar CrossRef Search ADS   Kim YX, Steudle E. 2007. Light and turgor affect the water permeability (aquaporins) of parenchyma cells in the midrib of leaves of Zea mays. Journal of Experimental Botany  58: 4119– 4129. Google Scholar CrossRef Search ADS   McCulloh KA, Sperry JS, Meinzer FC, Lachenbruch B, Atala C. 2009. Murray’s law, the ‘Yarrum’ optimum, and the hydraulic architecture of compound leaves. New Phytologist  184: 234– 244. Google Scholar CrossRef Search ADS   Nardini A, Luglio J. 2014. Leaf hydraulic capacity and drought vulnerability: possible trade-offs and correlations with climate across three major biomes. Functional Ecology  28: 810– 818. Google Scholar CrossRef Search ADS   Nardini A, Peda G, La Rocca N. 2012. Trade-offs between leaf hydraulic capacity and drought vulnerability: morpho-anatomical bases, carbon costs and ecological consequences. New Phytologist  196: 788– 798. Google Scholar CrossRef Search ADS   Nardini A, Ounapuu-Pikas E, Savi T. 2014. When smaller is better: leaf hydraulic conductance and drought vulnerability correlate to leaf size and venation density across four Coffea arabica genotypes. Functional Plant Biology  41: 972– 982. Google Scholar CrossRef Search ADS   Nolf M, Creek D, Duursma R, Holtum J, Mayr S, Choat B. 2015. Stem and leaf hydraulic properties are finely coordinated in three tropical rain forest tree species. Plant Cell and Environment  38: 2652– 2661. Google Scholar CrossRef Search ADS   Peppe DJ, Royer DL, Cariglino Bet al.   2011. Sensitivity of leaf size and shape to climate: global patterns and paleoclimatic applications. New Phytologist  190: 724– 739. Google Scholar CrossRef Search ADS   Pittermann J, Sperry JS, Hacke UG, Wheeler JK, Sikkema EH. 2006. Inter-tracheid pitting and the hydraulic efficiency of conifer wood: the role of tracheid allometry and cavitation protection. American Journal of Botany  93: 1265– 1273. Google Scholar CrossRef Search ADS   Sack L, Frole K. 2006. Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees. Ecology  87: 483– 491. Google Scholar CrossRef Search ADS   Sack L, Scoffoni C. 2013. Leaf venation: structure, function, development, evolution, ecology and applications in the past, present and future. New Phytologist  198: 983– 1000. Google Scholar CrossRef Search ADS   Sack L, Melcher PJ, Zwieniecki MA, Holbrook NM. 2002. The hydraulic conductance of the angiosperm leaf lamina: a comparison of three measurement methods. Journal of Experimental Botany  53: 2177– 2184. Google Scholar CrossRef Search ADS   Sack L, Cowan PD, Jaikumar N, Holbrook NM. 2003. The ‘hydrology’ of leaves: co-ordination of structure and function in temperate woody species. Plant Cell and Environment  26: 1343– 1356. Google Scholar CrossRef Search ADS   Sack L, Scoffoni C, McKown ADet al.   2012. Developmentally based scaling of leaf venation architecture explains global ecological patterns. Nature Communications  3: 837. Google Scholar CrossRef Search ADS   Schenk HJ, Steppe K, Jansen S. 2015. Nanobubbles: a new paradigm for air-seeding in xylem. Trends in Plant Science  20: 199– 205. Google Scholar CrossRef Search ADS   Scholz FG, Bucci SJ, Goldstein G. 2014. Strong hydraulic segmentation and leaf senescence due to dehydration may trigger die-back in Nothofagus dombeyi under severe droughts: a comparison with the co-occurring Austrocedrus chilensis. Trees-Structure and Function  28: 1475– 1487. Google Scholar CrossRef Search ADS   Scoffoni C, Pou A, Aasamaa K, Sack L. 2008. The rapid light response of leaf hydraulic conductance: new evidence from two experimental methods. Plant Cell and Environment  31: 1803– 1812. Google Scholar CrossRef Search ADS   Scoffoni C, Rawls M, McKown A, Cochard H, Sack L. 2011. Decline of leaf hydraulic conductance with dehydration: relationship to leaf size and venation architecture. Plant Physiology  156: 832– 843. Google Scholar CrossRef Search ADS   Scoffoni C, Vuong C, Diep S, Cochard H, Sack L. 2014. Leaf shrinkage with dehydration: coordination with hydraulic vulnerability and drought tolerance. Plant Physiology  164: 1772– 88. Google Scholar CrossRef Search ADS   Scoffoni C, Albuquerque C, Brodersen CRet al.   2017a. Outside-xylem vulnerability, not xylem embolism, controls leaf hydraulic decline during dehydration. Plant Physiology  173: 1197– 1210. Google Scholar CrossRef Search ADS   Scoffoni C, Albuquerque C, Brodersen CRet al.   2017b. Leaf vein xylem conduit diameter influences susceptibility to embolism and hydraulic decline. New Phytologist  213: 1076– 1092. Google Scholar CrossRef Search ADS   Skelton RP, West AG, Dawson TE. 2015. Predicting plant vulnerability to drought in biodiverse regions using functional traits. Proceedings of the National Academy of Sciences of the United States of America  112: 5744– 5749. Google Scholar CrossRef Search ADS   Skelton RP, Brodribb TJ, Choat B. 2017a. Casting light on xylem vulnerability in an herbaceous species reveals a lack of segmentation. New Phytologist  214: 561– 569. Google Scholar CrossRef Search ADS   Skelton RP, Brodribb TJ, McAdam SAM, Mitchell PJ. 2017b. Gas exchange recovery following natural drought is rapid unless limited by loss of leaf hydraulic conductance: evidence from an evergreen woodland. New Phytologist  215: 1399– 1412. Google Scholar CrossRef Search ADS   R Core Team. 2015. R: a language and environment for statistical computing . Vienna: R Foundation for Statistical Computing. Tozer WC, Rice B, Westoby M. 2015. Evolutionary divergence of leaf width and its correlates. American Journal of Botany  102: 367– 378. Google Scholar CrossRef Search ADS   Trifilo P, Raimondo F, Savi T, Lo Gullo MA, Nardini A. 2016. The contribution of vascular and extra-vascular water pathways to drought-induced decline of leaf hydraulic conductance. Journal of Experimental Botany  67: 5029– 5039. Google Scholar CrossRef Search ADS   Tyree MT, Zimmermann MH. 2002. Xylem dysfunction: when cohesion breaks down. In Xylem Structure and the Ascent of Sap . Berlin: Springer, 89– 141. Google Scholar CrossRef Search ADS   Tyree MT, Davis SD, Cochard H. 1994. Biophysical perspectives of xylem evolution - is there a tradeoff of hydraulic efficiency for vulnerability to dysfunction. IAWA Journal  15: 335– 360. Google Scholar CrossRef Search ADS   Walls RL. 2011. Angiosperm leaf vein patterns are linked to leaf function in a global-scale data set. American Journal of Botany  98: 244– 253. Google Scholar CrossRef Search ADS   Warton DI, Wright IJ, Falster DS, Westoby M. 2006. Bivariate line-fitting methods for allometry. Biological Reviews  81: 259– 291. Google Scholar CrossRef Search ADS   Wright IJ, Dong N, Maire Vet al.   2017. Global climatic drivers of leaf size. Science  357: 917– 921. Google Scholar CrossRef Search ADS   Zhang YJ, Rockwell FE, Graham AC, Alexander T, Holbrook NM. 2016. Reversible leaf xylem collapse: a potential ‘circuit breaker’ against cavitation. Plant Physiology  172: 2261– 2274. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Botany Oxford University Press

The links between leaf hydraulic vulnerability to drought and key aspects of leaf venation and xylem anatomy among 26 Australian woody angiosperms from contrasting climates

Loading next page...
 
/lp/ou_press/the-links-between-leaf-hydraulic-vulnerability-to-drought-and-key-yvgEUM20rp
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
ISSN
0305-7364
eISSN
1095-8290
D.O.I.
10.1093/aob/mcy051
Publisher site
See Article on Publisher Site

Abstract

Abstract Background and Aims The structural properties of leaf venation and xylem anatomy strongly influence leaf hydraulics, including the ability of leaves to maintain hydraulic function during drought. Here we examined the strength of the links between different leaf venation traits and leaf hydraulic vulnerability to drought (expressed as P50leaf by rehydration kinetics) in a diverse group of 26 woody angiosperm species, representing a wide range of leaf vulnerabilities, from four low-nutrient sites with contrasting rainfall across eastern Australia. Methods For each species we measured key aspects of leaf venation design, xylem anatomy and leaf morphology. We also assessed for the first time the scaling relationships between hydraulically weighted vessel wall thickness (th) and lumen breadth (bh) across vein orders and habitats. Key Results Across species, variation in P50leaf was strongly correlated with the ratio of vessel wall thickness (th) to lumen breadth (bh) [(t/b)h; an index of conduit reinforcement] at each leaf vein order. Concomitantly, the scaling relationship between th and bh was similar across vein orders, with a log–log slope less than 1 indicating greater xylem reinforcement in smaller vessels. In contrast, P50leaf was not related to th and bh individually, to major vein density (Dvmajor) or to leaf size. Principal components analysis revealed two largely orthogonal trait groupings linked to variation in leaf size and drought tolerance. Conclusions Our results indicate that xylem conduit reinforcement occurs throughout leaf venation, and remains closely linked to leaf drought tolerance irrespective of leaf size. Leaf hydraulic vulnerability, xylem anatomy, leaf venation, vein density, xylem reinforcement, leaf size, drought INTRODUCTION The anatomical and architectural features of leaf venation strongly influence plant productivity and survival across species and environments. Given that the efficiency of water transport through leaf veins is a major determinant of maximum rates of photosynthesis (Brodribb et al., 2007; Sack and Scoffoni, 2013), venation traits that influence water transport efficiency, such as xylem vessel width (Aasamaa et al., 2001), vessel perforation-plate anatomy (Feild and Brodribb, 2013) and vein density (Sack and Frole, 2006; Brodribb and Feild, 2010; Walls, 2011; Buckley et al., 2015; Gleason et al., 2016), have been examined across large numbers of species. Recent studies have also identified several venation traits related to the ability of leaves to resist hydraulic decline under increasing levels of drought stress (Cochard et al., 2004; Brodribb and Holbrook, 2005; Blackman et al., 2010; Scoffoni et al., 2011, 2017b; Nardini et al., 2012). Quantifying these traits offers a potentially useful approach for screening leaf drought tolerance thresholds in extant species, as well as those in the fossil record (Sack and Scoffoni, 2013). As soils dry out during drought, tension (water potential, in MPa) within the leaf xylem increases. Under relatively mild drought conditions, this process can cause leaf water transport capacity (Kleaf) to decline as a result of turgor loss and leaf shrinkage (Scoffoni et al., 2014, 2017a; Trifilo et al., 2016). Under more severe drought conditions, further increases in xylem tension can exceed species hydraulic safety thresholds, causing Kleaf to decline as a result of embolism formation (air blockages) in the water-conducting xylem (Johnson et al., 2009; Brodribb et al., 2016b). If drought continues, this process can lead to complete leaf hydraulic failure and even plant death (Brodribb and Cochard, 2009; Scholz et al., 2014). The ability of leaves to resist hydraulic decline during drought is typically characterized by their hydraulic vulnerability, measured as the water potential associated with 50 % loss in hydraulic conductance, or P50leaf. Recent studies indicate that P50leaf varies widely across species from environments with contrasting rainfall (Brodribb and Hill, 1999; Blackman et al., 2014) and temperature (Nardini and Luglio, 2014), and represents a major determinant of species distributional limits (Blackman et al., 2012; Nardini et al., 2012). Leaf hydraulic vulnerability to drought is an integrated trait derived from different structural and functional characteristics of the leaf water transport pathway. Recent cross-species studies have reported close linkages between variation in P50leaf and specific aspects of leaf vein anatomy and venation design. These studies suggest that angiosperm species with low hydraulic vulnerability (i.e. more negative P50leaf) tend to have leaves with narrow xylem conduits that help minimize the spread of drought-induced embolism (Nardini et al., 2012; Scoffoni et al., 2017b) and high major vein density that provides multiple pathways for water movement around air-filled conduits (hydraulic redundancy) (Scoffoni et al., 2011; Nardini et al., 2014). A strong correlation has also been found between leaf hydraulic vulnerability and the ratio of conduit wall thickness (t) to lumen breadth (b) in leaf minor veins of conifer (Cochard et al., 2004; Brodribb and Holbrook, 2005) and angiosperm (Blackman et al., 2010) species. These findings suggest xylem conduit reinforcement provides a degree of safety from vessel wall collapse during drought. However, it remains unknown to what degree xylem reinforcement occurs throughout the leaf venation network, and whether the scaling of t and b varies across species from different habitats. Euler buckling theory suggests that t should scale proportionately with b to prevent collapse as the breadth of conduits increases (Hacke et al., 2001; Brodribb and Holbrook, 2005). Scaling less than proportionately with b would indicate stronger xylem reinforcement in smaller vessels. If the th–bh scaling exponent shifts across habitats with species operating at lower water potentials displaying a coefficient closer to unity, then this would indicate an increasing cost to constructing leaves with large vessels in arid habitats. As far as we are aware, these possibilities have not previously been assessed in leaves. Although relationships between P50leaf and different venation and xylem anatomy traits have been examined across small groups of ecologically diverse species, it remains unknown whether specific leaf venation traits can become decoupled from P50leaf due to their intrinsic link to leaf size. Leaf size is closely linked to major vein density (Scoffoni et al., 2011) as a consequence of vein packing constraints during leaf development (Sack et al., 2012), and to petiole vessel size (McCulloh et al., 2009; Gleason et al., 2016, 2018), for optimal leaf water transport efficiency (Sack et al., 2003). The link to P50leaf helps explain the propensity of small-leaved species to occupy more arid environments (Scoffoni et al., 2011). However, leaf size can be influenced by multiple environmental factors, including rainfall, temperature, light and nutrient conditions (Givnish, 1987; Cunningham et al., 1999; Fonseca et al., 2000; Tozer et al., 2015), whereas P50leaf is most strongly influenced by site water availability (Brodribb and Cochard, 2009; Blackman et al., 2014; Nardini and Luglio, 2014; Scholz et al., 2014). For species where leaf size is strongly constrained by selection pressures other than rainfall, venation traits intrinsically linked to leaf size might be expected to become decoupled from P50leaf. Here, we tested the level of coordination among different leaf xylem anatomy and venation traits, leaf size, leaf mass per unit area (LMA) and leaf hydraulic vulnerability to drought (P50leaf) across a phylogenetically diverse group of eastern Australian temperate and sub-tropical woody angiosperms. We collected leaves from 26 species that varied strongly in leaf hydraulic vulnerability from four sites characterised by different rainfall, but similarly poor nutrient conditions. For each species, we measured hydraulically weighted diameter (bh), wall thickness (th) and an index of implosion resistance (t/b)h of xylem vessels within the petiole, midrib, 2° veins and leaf minor veins. We also measured leaf major and minor vein density, as well as leaf size and LMA. P50leaf values were sourced from previously published vulnerability curves (Blackman et al., 2014). We asked: (1) What are the venation traits most strongly linked to P50leaf across species? (2) Does the scaling of vessel wall thickness to vessel lumen breadth depart from proportionality within individual leaves, across species, or among habitats? (3) Assuming that in our species group leaf size is constrained by both low rainfall and low soil nutrients, can P50leaf vary independently from leaf venation traits that are intrinsically linked to leaf size? MATERIALS AND METHODS Study sites and species Twenty-six species representing ten families were sampled from four sites across coastal and inland eastern Australia (Table 1; Supplementary Data Table S1). Three of these sites (Warm-Wet, Warm-Dry and Warm-Arid) were associated with a strong east–west aridity gradient in New South Wales, while the fourth site (Hot-Dry) was located in seasonally dry eucalypt woodland in northern Queensland (for more detailed site climate descriptions see Gleason et al., 2012). All four sites were characterized by late successional vegetation with eucalyptus (senso lato) occurring on weathered oligotrophic soils, low in phosphorous (Gleason et al., 2012). The sites varied strongly in rainfall from 383 mm annually at the Warm-Arid site to 1210 mm at the Warm-Wet site (Table 1). Six or seven dominant shrub and/or tree species were sampled from each site. The sample group contained a variety of simple leaf types including flat, revolute, terete and phyllodinous leaves. All species were evergreen except for Planchonia careya from the Hot-Dry site, which was drought-deciduous. In addition to the mean annual precipitation (MAP) for each site, MAP data were downloaded for cleaned occurrence records of each species from the Atlas of Living Australia (http://www.ala.org.au) and used to calculate the MAP across each species distribution. Sampling at each site occurred in 2012–2013, outside of the hot summer months. The same individuals of each species were used for measurements of leaf hydraulics and leaf anatomy. Table 1. The geographical, climatic and edaphic details of each of the four sites sampled from in this study Site  Habitat  Latitude (°S)  Longitude (°E)  MAP (mm)  MAT (°C)  Soil P (mg kg−1)  Species (n)  P50leaf (MPa)  LA (cm2)  Kur-ing-gai  Warm-Wet  33.68  151.15  1210  17.0  54.8  6  −2.7 ± 0.3a  4.5 ± 3.7a  Yengo  Warm-Dry  32.78  150.92  779  16.6  223  6  −3.1 ± 0.3a  12.4 ± 6.0ab  Round Hill  Warm-Arid  32.98  146.16  383  17.9  106  7  −5.5 ± 0.6b  4.9 ± 1.7ab  Princess Hills  Hot-Dry  18.29  145.49  1139  21.3  157  7  −3.0 ± 0.2a  26.5 ± 4.1b  Site  Habitat  Latitude (°S)  Longitude (°E)  MAP (mm)  MAT (°C)  Soil P (mg kg−1)  Species (n)  P50leaf (MPa)  LA (cm2)  Kur-ing-gai  Warm-Wet  33.68  151.15  1210  17.0  54.8  6  −2.7 ± 0.3a  4.5 ± 3.7a  Yengo  Warm-Dry  32.78  150.92  779  16.6  223  6  −3.1 ± 0.3a  12.4 ± 6.0ab  Round Hill  Warm-Arid  32.98  146.16  383  17.9  106  7  −5.5 ± 0.6b  4.9 ± 1.7ab  Princess Hills  Hot-Dry  18.29  145.49  1139  21.3  157  7  −3.0 ± 0.2a  26.5 ± 4.1b  MAP, mean annual precipitation; MAT, mean annual temperature. Climate data were sourced from the Atlas of Living Australia (2017), while soil P was sourced from a previous study (Gleason et al 2012). Also included are site means (plus standard errors) for leaf hydraulic vulnerability (P50leaf) and leaf size (LA); significant differences (P < 0.05) between sites, using pairwise comparisons, are denoted by different superscript letters. View Large Table 1. The geographical, climatic and edaphic details of each of the four sites sampled from in this study Site  Habitat  Latitude (°S)  Longitude (°E)  MAP (mm)  MAT (°C)  Soil P (mg kg−1)  Species (n)  P50leaf (MPa)  LA (cm2)  Kur-ing-gai  Warm-Wet  33.68  151.15  1210  17.0  54.8  6  −2.7 ± 0.3a  4.5 ± 3.7a  Yengo  Warm-Dry  32.78  150.92  779  16.6  223  6  −3.1 ± 0.3a  12.4 ± 6.0ab  Round Hill  Warm-Arid  32.98  146.16  383  17.9  106  7  −5.5 ± 0.6b  4.9 ± 1.7ab  Princess Hills  Hot-Dry  18.29  145.49  1139  21.3  157  7  −3.0 ± 0.2a  26.5 ± 4.1b  Site  Habitat  Latitude (°S)  Longitude (°E)  MAP (mm)  MAT (°C)  Soil P (mg kg−1)  Species (n)  P50leaf (MPa)  LA (cm2)  Kur-ing-gai  Warm-Wet  33.68  151.15  1210  17.0  54.8  6  −2.7 ± 0.3a  4.5 ± 3.7a  Yengo  Warm-Dry  32.78  150.92  779  16.6  223  6  −3.1 ± 0.3a  12.4 ± 6.0ab  Round Hill  Warm-Arid  32.98  146.16  383  17.9  106  7  −5.5 ± 0.6b  4.9 ± 1.7ab  Princess Hills  Hot-Dry  18.29  145.49  1139  21.3  157  7  −3.0 ± 0.2a  26.5 ± 4.1b  MAP, mean annual precipitation; MAT, mean annual temperature. Climate data were sourced from the Atlas of Living Australia (2017), while soil P was sourced from a previous study (Gleason et al 2012). Also included are site means (plus standard errors) for leaf hydraulic vulnerability (P50leaf) and leaf size (LA); significant differences (P < 0.05) between sites, using pairwise comparisons, are denoted by different superscript letters. View Large Leaf hydraulic vulnerability Leaf vulnerability curves for the current group of species were sourced from a previous study published by our lab group (Blackman et al., 2014). In brief, each curve was generated using a modified rehydration technique (Brodribb and Cochard, 2009), whereby leaves or small shoots were excised underwater from branches (three branches per species) dried down over 2–4 d to a range of water potentials and connected to a flow meter. We ensured water potential was equilibrated before each ‘rehydration’ experiment by placing branches into opaque plastic bags for up to 1 h. Measurements were conducted under normal light conditions in the lab or in the field under a shade tent. For most species, the response of leaf hydraulic conductance (Kleaf) to increasing water potential (in MPa) was sigmoidal, with Kleaf not declining over an initial range of water potentials, then declining once species hydraulic safety thresholds were reached (Fig. S1). For each rehydration experiment, leaves were connected to the hydraulic apparatus within 2 s and Kleaf was calculated from the flow rate recorded within the first 4–6 s following leaf connection to the flow meter. These initial flow rates were assumed to be influenced predominantly by the hydraulic resistance of the xylem pathway, and thus we considered the decline in Kleaf to be driven primarily by the formation and spread of xylem embolism (see Nolf et al., 2015; Skelton et al., 2015, 2017a; Brodribb et al., 2016a, b). However, we acknowledge that the decline in Kleaf can also be influenced by measurement light intensity, which has been shown to affect hydraulic processes in leaf tissues beyond the xylem (Guyot et al., 2012; Trifilo et al., 2016). The influence of light intensity on the decline in Kleaf has been demonstrated using the evaporative flux technique (Sack et al., 2002), as well as the timed rehydration kinetics technique, devised by Brodribb and Holbrook (2003), where leaves were allowed to absorb water under high or low light for 15–45 s (Scoffoni et al., 2008). Thus, although we cannot entirely exclude the influence of outside xylem processes, we considered our measurements of leaf hydraulic vulnerability to represent the water potential associated with 50 % loss in hydraulic conductance (P50leaf) driven primarily by embolism formation in the leaf xylem. This contrasts with the evaporative flux method (Sack et al., 2002; Scoffoni et al., 2008) where measurements include mesophyll and stomatal conductance and hence are responsive to factors including light intensity. Across species, P50leaf varied substantially from −1.9 MPa in Banksia serrata to −7.8 MPa in Melaleuca uncinata (Table S1). Leaf xylem anatomy and venation traits Fully expanded sun-leaves were collected from three individuals from each field site at the time leaf hydraulic vulnerability measurements were made. Between five and ten sample leaves from each of three individuals per species were sealed in zip-lock bags with moist paper-towel and placed inside an insulated cool-box. Samples were transported back to the laboratory within 3 d of collecting and fixed in FAA (formalin acetic acid) solution and stored. Measurements of xylem anatomy in petioles, midribs, 2° veins and minor veins were made in one leaf from two to three individuals per species, with the exception of vessels in 2° veins of Pultenaea scabra which were calculated from a single leaf (see Table S1). Leaf area was measured using a flat-bed scanner (Scan Maker i900, Microtek International, China) before sectioning. Transverse sections of vein xylem were made using a vibratome (VT1000s, Leica Microsystems, Germany). Sections were made half-way along the length of the petiole, mid-rib and 2° veins (minor vein anatomy was generally captured within lower order vein sections). Small (<°1 cm2) pieces of leaf, each containing a target vein, were cut out and individually suspended in 6 % agarose blocks. Each agarose block was shaped with a razor-blade and then mounted onto the vibratome stage ensuring that the target vein was perpendicular to the cutting edge of the blade. Several transverse sections were cut at a thickness of between 10 and 20 µm. Sections were stained in dilute 1 % methylene blue before mounting onto glass slides in phenol glycerine jelly. The xylem anatomy of each vein order was photographed using a digital camera (DXM1200F, Nikon, Japan) attached to a light microscope (Bx50, Olympus Optical, Japan). Magnification of each vein depended on vessel size; petioles, mid-ribs and 2° veins were photographed at 40× or 100×, while minor veins were photographed at 100×. From each image, lumen breadth (b) and wall thickness (t) was measured using ImageJ software (National Institutes of Health, USA) from a representative sample of between ten and 100 hydraulically functional vessels. Due to the typically low number of vessels in minor veins, b and t were measured from two or three different minor veins per leaf. For all veins, care was taken to avoid cell-types such as fibre cells and xylem parenchyma that were deemed to provide functional roles beyond water transport. Also, minor veins were identified as the smallest veins in cross section with a clearly defined vascular bundle (xylem and phloem), and were carefully distinguished from free vein endings, which were often enlarged and represented sclereids and/or tracheids in some species. Because vessels were often elliptical in shape, b was measured along the short and long axes of each vessel, and then transformed to the circular equivalent diameter (Choat et al., 2007). Hydraulically weighted vessel diameter (bh) was calculated according to the formula bh = Σ(b4/n)0.25, which weights the vessels (n) within each vein order by their hydraulic contribution to total vein conductance (Tyree and Zimmermann, 2002). For each hydraulically weighted diameter we estimated its wall thickness from the ordinary least-squares relationship between t and b measured across a subsample of 10–15 vessels within each vein order. For each cell, b was measured as described above, while t was measured as the single-thickness of a clearly defined radial wall. The level of xylem reinforcement of hydraulically weighted vessels was then calculated as the ratio of wall thickness (th) and lumen breadth (bh), (t/b)h. We also examined the shape and slope of the th–bh relationship across vein orders and species. Euler buckling theory suggests that th should scale proportionately with bh (i.e. an expected log–log slope of 1) to maintain a constant crushing tension as vessel radii narrow from petioles to minor veins (Hacke et al., 2001; Brodribb and Holbrook, 2005). Less than proportional scaling between these two vessel traits would suggest that large leaves [i.e. with large vessels (McCulloh et al., 2009; Gleason et al., 2018)] represent a savings in network construction costs; for example, a doubling leaf size would result in a somewhat less than doubling of network construction costs. Furthermore, the slope of this relationship (i.e. the th and bh ratio assuming a y-intercept of zero) may also differ across species and habitats. A change in the slope (but not the shape) of the function would indicate greater carbon investment (thicker vessel walls) at all points throughout the network. To test if the slope or shape of the th–bh relationship differed across species or habitats, we plotted th–bh on log10-transformed axes and compared the log–log slopes (i.e. departure from proportionality; scaling exponents), as well as the log–log intercepts (i.e. the logged arithmetic slopes; normalization constants) among species and habitats. Log–log intercepts were only compared if there was no difference in slope among species or habitats. The ‘sma’ function in the SMATR package for R was used for these analyses (Warton et al., 2006). Leaf venation architecture was characterized using one leaf from each of three individuals per species. For species with flat or revolute leaves, we used a protocol described by Scoffoni et al. (2011) for leaf clearing and quantifying vein density. In brief, leaves were chemically cleared with 5 % NaOH, put through a dehydration series in ethanol, stained with saffranin and counter-stained with fast green. Leaves were mounted in water on transparency film and scanned at high resolution using a flatbed scanner (Scan Maker i900, Microtek International, China). The leaf area and lengths of midribs and 2° veins were measured using ImageJ. To ensure that 3° and higher order veins were visible, we exposed the veins prior to leaf clearing by cutting a small window (<1 cm2) through the epidermis and top layers of mesophyll. For large leaves (>10 cm2), three vein windows were made, located centrally in the top, middle and bottom thirds of the leaf. The lengths of 3° and minor veins were measured (using ImageJ) from photographs of these vein windows taken with a digital camera (DXM1200F, Nikon) attached to a light microscope (Bx50, Olympus Optical) at 4× and 10× magnification, respectively. Vein density was calculated for each vein order as the length of vein per unit leaf area. In large leaves, 3° and minor vein densities were averaged across the three exposed windows. The major vein density (Dvmajor) was determined as the sum of 1°, 2° and 3° order vein densities, and minor vein density (Dvminor) as the total length per unit area of 4° and higher order veins. For the four species with terete leaves with parallel leaf venation, vein orders were distinguished by size class in transverse section (see above for leaf sectioning protocol). The vein density of each vein order was then calculated as the sum of the number of veins within each vein order, multiplied by leaf length (assumed to be equivalent to vein length) and divided by projected leaf area. Leaf structural traits Leaf area (LA) was determined for each species from the same three leaves used for quantifying xylem anatomy traits in cross section. Leaves were imaged on a standard flatbed scanner (Epson Perfection V33, Australia). Projected leaf area was calculated from these images using ImageJ software (National Institutes of Health). Leaf mass per unit area (LMA) values for each species were taken from a previous study conducted at the same sites (Gleason et al., 2012). Statistical analysis Bivariate relationships were fit with ordinary least squares (OLS) or standard major axes (SMA) models using the ‘smatr’ package in R (Warton et al., 2006). Differences among sites and vein orders may manifest as different slope or intercept coefficients. When relationships were well approximated by power models (e.g. th ~ a.bhb), scaling exponents were evaluated among groups by testing the log–log slope coefficient (b; scaling exponent) as well as the intercept coefficient (a; scaling constant) when slopes were statistically similar among sites or vein orders. Principal components analysis (PCA) (‘prcomp’ function in R; R Core Team, 2015) was used to determine the dominant axes of variation among a selection of traits, including petiole vessel th, bh and (t/b)h, respectively, as well as major and minor vein density, LA, LMA and P50leaf. All variables were scaled to unit variance in the PCA. RESULTS Substantial variation in venation architecture, leaf xylem anatomy and gross morphology was observed across species (Table S1). Major vein density varied ~15-fold from 0.58 to 9.2 mm mm−2, while among the xylem anatomy traits petiole vessel bh varied ~6.5-fold from 3.3 to 24.4 µm, petiole vessel th varied ~2.5-fold from 0.67 to 2.7 µm, and petiole vessel (t/b)h varied ~2-fold from 0.08 to 0.25. Across species, LMA varied nearly 5-fold, ranging from 75 to 447 g m−2, and leaf area varied more than 300-fold, ranging from 0.12 to 40.0 cm2. Among sites, leaf size differed significantly between the Warm-Wet and the Hot-Dry sites (pairwise comparisons; Table 1), while leaf size was unrelated to site rainfall and weakly correlated with soil phosphorus (r2 = 0.15, P = 0.05; Fig. S2), and was unrelated to species mean annual rainfall (Table 2; Fig. S3). In contrast, P50leaf was significantly different among species from the Warm-Arid site compared to species from the other three sites (Table 1), while P50leaf was strongly correlated with site rainfall (r2 = 0.48, P< 0.001) but not soil phosphorus (Fig. S2), and was correlated with species mean annual rainfall (Table 2; Fig. S3). Table 2. Pearson correlation r values among climate (species mean annual rainfall) and leaf, vein and petiole vessel traits measured in 26 woody angiosperm species   MAP  P50leaf  Pet_t–bh  Pet_bh  Pet_th  Dvmajor  Dvminor  LMA  LA  MAP                    P50leaf  −0.76                  Pet_t–bh  −0.66  0.73                Pet_bh  0.33  −0.34  −0.73              Pet_th  −0.06  0.11  −0.23  0.83            Dvmajor  −0.20  0.11  0.27  −0.60  −0.63          Dvminor  −0.19  0.08  0.01  0.15  0.21  −0.03        LMA  −0.29  0.25  0.10  −0.05  0.01  0.37  −0.27      LA  0.18  −0.06  −0.42  0.86  0.89  −0.64  0.14  −0.03      MAP  P50leaf  Pet_t–bh  Pet_bh  Pet_th  Dvmajor  Dvminor  LMA  LA  MAP                    P50leaf  −0.76                  Pet_t–bh  −0.66  0.73                Pet_bh  0.33  −0.34  −0.73              Pet_th  −0.06  0.11  −0.23  0.83            Dvmajor  −0.20  0.11  0.27  −0.60  −0.63          Dvminor  −0.19  0.08  0.01  0.15  0.21  −0.03        LMA  −0.29  0.25  0.10  −0.05  0.01  0.37  −0.27      LA  0.18  −0.06  −0.42  0.86  0.89  −0.64  0.14  −0.03    All data were log-transformed for analysis. Values in bold type are significant at P < 0.05. View Large Table 2. Pearson correlation r values among climate (species mean annual rainfall) and leaf, vein and petiole vessel traits measured in 26 woody angiosperm species   MAP  P50leaf  Pet_t–bh  Pet_bh  Pet_th  Dvmajor  Dvminor  LMA  LA  MAP                    P50leaf  −0.76                  Pet_t–bh  −0.66  0.73                Pet_bh  0.33  −0.34  −0.73              Pet_th  −0.06  0.11  −0.23  0.83            Dvmajor  −0.20  0.11  0.27  −0.60  −0.63          Dvminor  −0.19  0.08  0.01  0.15  0.21  −0.03        LMA  −0.29  0.25  0.10  −0.05  0.01  0.37  −0.27      LA  0.18  −0.06  −0.42  0.86  0.89  −0.64  0.14  −0.03      MAP  P50leaf  Pet_t–bh  Pet_bh  Pet_th  Dvmajor  Dvminor  LMA  LA  MAP                    P50leaf  −0.76                  Pet_t–bh  −0.66  0.73                Pet_bh  0.33  −0.34  −0.73              Pet_th  −0.06  0.11  −0.23  0.83            Dvmajor  −0.20  0.11  0.27  −0.60  −0.63          Dvminor  −0.19  0.08  0.01  0.15  0.21  −0.03        LMA  −0.29  0.25  0.10  −0.05  0.01  0.37  −0.27      LA  0.18  −0.06  −0.42  0.86  0.89  −0.64  0.14  −0.03    All data were log-transformed for analysis. Values in bold type are significant at P < 0.05. View Large Across species, variation in P50leaf was strongly correlated with vessel (t/b)h of petioles (r2 = 0.53, P < 0.001), midribs (r2 = 0.58, P < 0.001), second-order veins (r2 = 0.46, P < 0.001) and minor veins (r2 = 0.72, P < 0.001) (Fig. 1), indicating that greater resistance to leaf hydraulic dysfunction (more negative P50leaf) is linked to greater xylem vessel reinforcement across all vein orders. Concomitantly, neither log–log slope nor log–log elevation of the relationship between th and bh were significantly different among vein orders (Fig. 2A; slope P = 0.17; elevation P = 0.08), suggesting similar scaling exponents and scaling constants throughout the networks. However, the log–log slope of the th–bh relationship across vein orders was significantly shallower than 1 (P < 0.001), indicating that the ratio of wall thickness to lumen breadth decreased as vessels became wider. Comparing across sites, species from the Warm-Wet site tested as having significantly shallower log–log slope than the other three sites (0.52 vs 0.72 in common for the other three sites; P < 0.025). The suggestion (Fig. 2B) is that larger vessels at this wet site tended to have relatively thinner walls than at the dry and arid sites, but minor veins did not. However, considering the graph (Fig. 2B), we do not attach strong weight to this apparent difference in slope. More clear-cut is that among the three sites exhibiting a common log–log slope (Warm-Dry, Warm-Arid, Hot-Dry), the elevation of the th–bh relationship varied significantly (P < 0.001), with species from the drier sites having larger values, meaning vessel wall thickness in these species exhibited stronger reinforcement at a given lumen diameter than species from wetter sites (Fig. 2B).We note that although petiole (t/b)h was correlated with species MAP (r2 = 0.44, P < 0.001), neither of the individual components of the th/bh ratio were related to rainfall (Table 2). Fig. 1. View largeDownload slide The log–log relationship between P50leaf and hydraulically weighted vessel (t/b)h at each vein order across species: black filled circles = petioles (solid line, r2 = 0.53***); dark-grey filled circles = midribs (dotted line, r2 = 0.58***); light-grey filled circles = 2° veins (dashed line, r2 = 0.46***); white filled circles = minor veins (dash-dot line, r2 = 0.72***). Level of significance: ***P < 0.001. Fig. 1. View largeDownload slide The log–log relationship between P50leaf and hydraulically weighted vessel (t/b)h at each vein order across species: black filled circles = petioles (solid line, r2 = 0.53***); dark-grey filled circles = midribs (dotted line, r2 = 0.58***); light-grey filled circles = 2° veins (dashed line, r2 = 0.46***); white filled circles = minor veins (dash-dot line, r2 = 0.72***). Level of significance: ***P < 0.001. Fig. 2. View largeDownload slide (A) The log–log relationship between vessel wall thickness (th) and lumen breadth (bh) at each vein order across species: black filled circles = petioles (solid line, r2 = 0.69***, slope = 0.7, elevation = −0.61); dark-grey filled circles = midribs (dotted line, r2 = 0.64***, slope = 0.76, elevation = −0.64); light-grey filled circles = 2° veins (dashed line, r2 = 0.60***, slope = 0.62, elevation = −0.65); white filled circles = minor veins (dash-dot line, r2 = 0.31**, slope = 0.99, elevation = −0.58). Neither slope nor elevation of the log–log th–bh relationship varied across vein orders (SMATR analysis: slope P = 0.17, elevation P = 0.08). (B) The log–log th–bh relationship across all vein orders for species separated by site: blue filled circles = Warm-Wet (solid line, r2 = 0.72***, slope = 0.52, elevation = −0.66), green filled circles = Warm-Dry (dashed line, r2 = 0.94***, slope = 0.78, elevation = −0.59); red filled circles = Warm-Arid (dotted line, r2 = 0.68***, slope = 0.69, elevation = −0.51); orange filled circles = Hot-Dry (dash-dot line, r2 = 0.94***, slope = 0.69, elevation = −0.64). Slope of the log–log th–bh relationship differed only for species from the Warm-Wet site (SMATR analysis: P = 0.02). The red dashed line in both plots represents a proportional th–bh scaling slope of 1, where (t/b)h = 0.24. Level of significance: ***P < 0.001; **P < 0.01. Fig. 2. View largeDownload slide (A) The log–log relationship between vessel wall thickness (th) and lumen breadth (bh) at each vein order across species: black filled circles = petioles (solid line, r2 = 0.69***, slope = 0.7, elevation = −0.61); dark-grey filled circles = midribs (dotted line, r2 = 0.64***, slope = 0.76, elevation = −0.64); light-grey filled circles = 2° veins (dashed line, r2 = 0.60***, slope = 0.62, elevation = −0.65); white filled circles = minor veins (dash-dot line, r2 = 0.31**, slope = 0.99, elevation = −0.58). Neither slope nor elevation of the log–log th–bh relationship varied across vein orders (SMATR analysis: slope P = 0.17, elevation P = 0.08). (B) The log–log th–bh relationship across all vein orders for species separated by site: blue filled circles = Warm-Wet (solid line, r2 = 0.72***, slope = 0.52, elevation = −0.66), green filled circles = Warm-Dry (dashed line, r2 = 0.94***, slope = 0.78, elevation = −0.59); red filled circles = Warm-Arid (dotted line, r2 = 0.68***, slope = 0.69, elevation = −0.51); orange filled circles = Hot-Dry (dash-dot line, r2 = 0.94***, slope = 0.69, elevation = −0.64). Slope of the log–log th–bh relationship differed only for species from the Warm-Wet site (SMATR analysis: P = 0.02). The red dashed line in both plots represents a proportional th–bh scaling slope of 1, where (t/b)h = 0.24. Level of significance: ***P < 0.001; **P < 0.01. Across species, variation in P50leaf was not related to either th or bh at any vein order, with the exception of minor vein vessel bh (r2 = 0.17, P = 0.04; Table S2). P50leaf was also unrelated to major vein density (Dvmajor), minor vein density (Dvminor), leaf size (LA) and leaf mass per unit area (LMA) (Table 2). Nonetheless, leaf size was significantly and negatively correlated with major vein density (r2 = 0.42, P < 0.001) (Fig. 3), but was unrelated to minor vein density (Table 2). Leaf size was positively correlated with vessel bh and th in leaf petioles [bh, r2 = 0.75, P < 0.001 (Fig. 3); th, r2 = 0.79, P < 0.001], midribs (bh, r2 = 0.75, P < 0.001; th, r2 = 0.80, P < 0.001) and 2° veins (bh, r2 = 0.61, P < 0.001; th, r2 = 0.43, P < 0.001), but not in leaf minor veins (see Table S2). Leaf size was also related to vessel (t/b)h at the petiole and 2° veins, but not at the midrib or minor veins (Tables 2 and S2). Strong negative correlations were observed between major vein density and vessel bh and th in lower order veins (Table S2). Fig. 3. View largeDownload slide Log–log relationships across species between leaf area and (A) major vein density (r2 = 0.42, P < 0.001) and (B) hydraulically weighted vessel diameter in leaf petioles (r2 = 0.75, P < 0.001). Species values are separated by site (blue filled circles = Warm-Wet, green filled circles = Warm-Dry, red filled circles = Warm-Arid, orange filled circles = Hot-Dry). Fig. 3. View largeDownload slide Log–log relationships across species between leaf area and (A) major vein density (r2 = 0.42, P < 0.001) and (B) hydraulically weighted vessel diameter in leaf petioles (r2 = 0.75, P < 0.001). Species values are separated by site (blue filled circles = Warm-Wet, green filled circles = Warm-Dry, red filled circles = Warm-Arid, orange filled circles = Hot-Dry). PCA of leaf, vein and petiole vessel traits identified two major axes, which cumulatively explained 66 % of the total variation among the traits (Fig. 4). The first principal component (PC) accounted for 46 % of the total variation and was dominated by leaf size, major vein density, and both petiole vessel bh and th, while the second PC (21 % of the total variation) was associated with P50leaf and petiole (t/b)h. Importantly, (t/b)h was closely aligned with P50leaf, whereas the components of this ratio, th and bh, aligned primarily with leaf size and major vein density, and were largely orthogonal to P50leaf and (t/b)h (Fig. 4). Fig. 4. View largeDownload slide Principal components plot of leaf, vein and petiole vessel properties, including petiole vessel th, bh and (t/b)h, major and minor vein density, LA and LMA. Individual points denote individual species. PC1 and PC2 account for 45 and 21 % of the variation among species and sites, respectively. Vector loadings are represented with arrows. All data were log-transformed and scaled to unit variance prior to analysis. Fig. 4. View largeDownload slide Principal components plot of leaf, vein and petiole vessel properties, including petiole vessel th, bh and (t/b)h, major and minor vein density, LA and LMA. Individual points denote individual species. PC1 and PC2 account for 45 and 21 % of the variation among species and sites, respectively. Vector loadings are represented with arrows. All data were log-transformed and scaled to unit variance prior to analysis. DISCUSSION The strong correlations between more negative P50leaf and greater vessel (t/b)h at each vein order across our sample group of 26 species highlight the functional link between xylem conduit reinforcement throughout the leaf venation and the ability of leaves to resist drought-induced hydraulic dysfunction (Blackman et al., 2010), probably caused by xylem embolism in our measurements using direct flow rehydration. The strength of this relationship is robust given that our species group spanned a very wide range of leaf vulnerabilities, with P50leaf values ranging from −1.9 MPa in the most vulnerable species Banksia serrata to −7.8 MPa in Melaleuca uncinata, which to the best of our knowledge is among the most negative P50leaf values recorded for angiosperm species (see also Skelton et al., 2017b). Our sample of species also spanned a wide range of values for each of the leaf venation, xylem anatomy and leaf morphology traits, which increased the likelihood of detecting significant trait–trait relationships across species. Even so, we note that large leaves >41 cm2 were not present in our species group, although compared to previous studies it did contain species with small leaves <1 cm2. The ratio of wall thickness to lumen breadth (t/b) has been used widely as an index of the implosion resistance of conduit walls under tension (Hacke et al., 2001; Sack and Scoffoni, 2013). Hacke et al. (2001) first observed a strong correlation between the ratio of conduit double-wall thickness and lumen breadth and drought resistance in woody stems. They argued that xylem conduit reinforcement should increase with increasing drought resistance on the basis that drought-resistant plants tend to experience stronger internal loads (water potential) in the field. This argument has been supported by studies highlighting the adaptive link between xylem conduit wall reinforcement and embolism resistance (P50) in plant stems (Jacobsen et al., 2005, 2007; Pittermann et al., 2006). In the current study, vessel (t/b)h was higher in species from more arid environments and was strongly correlated with P50leaf across species. This suggests that a similar level of coordination between xylem reinforcement and embolism resistance may be present in leaves. Although we cannot preclude the possibility that cell collapse occurs in angiosperm leaves under tension (see Zhang et al., 2016), the weight of recent evidence suggests that leaf hydraulic decline during severe drought is caused by xylem embolism (Johnson et al., 2009; Nolf et al., 2015; Brodribb et al., 2016a, b; Scoffoni et al., 2017b; Skelton et al., 2017a). Thus, we suggest xylem reinforcement in leaves has evolved to provide a degree of safety from vessel collapse under tension, while embolism spread via air-seeding could potentially relate to the size and structure of pores in pit membranes (Jansen et al., 2009), nucleation from hydrophobic surfaces (Tyree et al., 1994) or, as hypothesized recently, conduit/fluid properties that influence the expansion of nanobubbles (Schenk et al., 2015). We acknowledge that changes in the hydraulic properties of the extra-xylary pathway due to turgor loss and leaf shrinkage (Scoffoni et al., 2014, 2017a; Trifilo et al., 2016), and associated changes in aquaporin expression (Kim and Steudle, 2007), play a major role in the decline of leaf hydraulic conductance measured from petiole to external atmosphere. These processes tend to occur during the early stages of drought, at water potentials preceding those associated with xylem embolism (Scoffoni et al., 2017a), and furthermore are sensitive to measurement light conditions. Indeed, we note that our estimate of maximum Kleaf and thus P50leaf for each species may be underestimated on the basis that our rehydration measurements were conducted under low light and thus did not allow for the influence of outside xylem processes in driving the response of Kleaf during drought (Scoffoni et al., 2008, 2017a). However, our approach of measuring Kleaf under low light is consistent with the approach of other studies that have shown strong correspondence between the decline in Kleaf during drought, measured using the rehydration technique (Brodribb and Cochard, 2009), and the accumulation of xylem embolisms detected acoustically (Nolf et al., 2015) and visually via a recently developed optical technique (Brodribb et al., 2016a, b) and X-ray micro-computed tomography (Skelton et al., 2017a). Furthermore, in contrast to the evaporative flux technique (Sack et al., 2002) and the timed rehydration kinetics technique (Brodribb and Holbrook, 2003), we calculated Kleaf from the initial flow rate into the leaf through the petiole, which is more likely to be influenced by within-xylem rather than outside-xylem processes. Thus, it is reasonable to assume that the decline in Kleaf observed in our vulnerability curves was largely driven by embolism formation, although we note that further studies across diverse species are required to test the relative influence of xylem and outside xylem processes on the decline in Kleaf during drought measured using different measurement techniques under a range of conditions. Nevertheless, if embolism is the primary driver of the decline in Kleaf in vulnerability curves generated using the rehydration technique, then our study provides important mechanistic insights into the linkages between P50leaf and leaf venation and xylem anatomy traits related to xylem embolism resistance. Previous studies have observed strong links between xylem wall reinforcement in leaf minor veins and both leaf vulnerability to drought (Blackman et al., 2010) and species climatic limits (Jordan et al., 2013). In the current study, we observed for the first time strong relationships between P50leaf and vessel (t/b)h at each leaf vein order, with the scaling of vessel wall thickness to lumen breadth being consistent across vein orders. These results indicate that xylem conduit reinforcement occurs throughout the leaf venation network, from petioles to minor veins. Nonetheless, across species we found that the level of xylem vessel reinforcement was higher in smaller vessels (i.e. the t–b scaling exponent was significantly less than 1). This suggests that larger vessels perhaps require less reinforcement than smaller vessels, and/or that tension in vessels (negative water potential) increases from petiole to minor veins. Furthermore, it is consistent with findings that smaller minor veins are the most resistant to embolism within the leaf vein network (Brodribb et al., 2016a, b; Scoffoni et al., 2017b). Across habitats, we hypothesized that increasing the log–log t–b scaling exponent towards unity (Euler buckling exponent) would result in lower ‘network’ efficiency as a consequence of increasing carbon costs per unit increase in hydraulically weighted vessel diameter, which might play a role in constraining vessel size in species from more arid sites. Unfortunately, our data are ambiguous on this point. The least arid site did have a shallower scaling exponent than the other sites, but the ‘dry’ sites and ‘arid’ site had similar scaling exponents, which do not support this idea. However, among these dry-land sites the th–bh constant (i.e. elevation in Fig. 2B) varied significantly and increased with increasing site aridity. This suggests that natural selection acts predominantly on the scaling constant rather than the scaling exponent, which results in thicker walls relative to lumen diameter in drier habitats. Across our set of species, leaf hydraulic vulnerability to drought was unrelated to major vein density. This contrasts with previous studies (Scoffoni et al., 2011; Nardini et al., 2012) that found significant relationships between high major vein density and low hydraulic vulnerability in leaves. These authors suggested high major vein density confers increased drought tolerance on the basis that it provides more alternative pathways for water movement around vein embolisms. We acknowledge that high major vein density and hydraulic redundancy may play a role maintaining leaf hydraulic function under drought. Nonetheless, our results suggest that the functional link between higher major vein density and greater resistance to embolism-induced hydraulic decline may not hold across sets of species where leaf size is strongly influenced by factors in addition to water availability. In the current study, we observed a strong negative relationship between leaf size and major vein density and a strong positive relationship between leaf size and hydraulically weighted petiole diameter, consistent with the intrinsic links and scaling relationships between these traits (McCulloh et al., 2009; Sack et al., 2012; Gleason et al., 2018). These bivariate relationships were supported by PCA, which grouped together vessel and venation traits linked to leaf size. However, our analyses showed that leaf size varied independently of P50leaf. Although all species with large leaves (>10 cm2) had P50leaf values less negative than −5 MPa, species with small leaves (<1 cm2) were characterized by both high and low vulnerability (i.e. P50leaf values ranged from roughly −2 to −8 MPa). This implies that the relationship between P50leaf and leaf size – and thus the relationship between P50leaf and both major vein density and petiole vessel diameter – can break down across species from different habitats, especially where leaf size is constrained by multiple environmental factors. Indeed, in contrast to global trends (Wright et al., 2017), across our four temperate–tropical sample sites, mean leaf size was unrelated to climate indices of site water availability. In contrast, P50leaf is known to vary more systematically with rainfall across these sites (Blackman et al., 2014). Decoupling of leaf size and site water availability (and P50leaf) is consistent with poor leaf size–climate relationships reported for Australian vegetation (Peppe et al., 2011; Tozer et al., 2015), indicating that leaf size variation can be shaped by additional environmental filters, including low soil nutrients (Cunningham et al., 1999; Fonseca et al., 2000). Nevertheless, our findings across a morphologically diverse set of species suggest that smaller leaves and higher vein density are not necessarily conferring strongly negative P50leaf. The results of this study clearly indicate that the ability of leaves to resist hydraulic dysfunction under drought is closely related to the degree of leaf xylem reinforcement throughout the venation network. Unlike traits such as major vein density and petiole vessel diameter, the degree of xylem vessel reinforcement was unrelated to leaf size, which in turn was unrelated to P50leaf. These results point strongly to the usefulness of measuring xylem reinforcement in leaf veins when examining variation in leaf hydraulic vulnerability to drought across ecologically and morphologically diverse species. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: Species information and trait values measured for each species. Table S2: Pearson correlation r values among selected leaf traits. Figure S1: Leaf hydraulic vulnerability curves for each of the 26 species included in this study. Figure S2: The log–log relationship between leaf traits and site climate and soil characteristics. Figure S3: The log–log relationship between leaf traits and species mean annual precipitation. ACKNOWLEDGEMENTS This research was supported by an Australian Research Council fellowship awarded to M.W. We thank the Macquarie University Microscopy Unit for their help with leaf sectioning and imaging. Thanks are also given to New South Wales National Parks and Wildlife Service, and the Queensland Department of National Parks, Recreation, Sport and Racing for allowing access to field sites. C.J.B., S.M.G. and M.W. planned and designed the research. C.J.B., S.M.G., A.M.C., Y.C. and C.A.L. carried out field sampling, leaf sectioning/clearing and image analysis. C.J.B and S.M.G carried out data analysis. C.J.B, S.M.G. and M.W. wrote the manuscript. LITERATURE CITED Aasamaa K, Sober A, Rahi M. 2001. Leaf anatomical characteristics associated with shoot hydraulic conductance, stomatal conductance and stomatal sensitivity to changes of leaf water status in temperate deciduous trees. Australian Journal of Plant Physiology  28: 765– 774. Blackman CJ, Brodribb T, Jordan GJ. 2010. Leaf hydraulic vulnerability is related to conduit dimensions and drought resistance across a diverse range of woody angiosperms. New Phytologist  188: 1113– 1123. Google Scholar CrossRef Search ADS   Blackman CJ, Brodribb TJ, Jordan GJ. 2012. Leaf hydraulic vulnerability influences species’ bioclimatic limits in a diverse group of woody angiosperms. Oecologia  168: 1– 10. Google Scholar CrossRef Search ADS   Blackman CJ, Gleason SM, Chang Y, Cook AM, Laws C, Westoby M. 2014. Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates. Annals of Botany  114: 435– 440. Google Scholar CrossRef Search ADS   Brodribb TJ, Cochard H. 2009. Hydraulic failure defines the recovery and point of death in water-stressed conifers. Plant Physiology  149: 575– 584. Google Scholar CrossRef Search ADS   Brodribb TJ, Feild TS. 2010. Leaf hydraulic evolution led a surge in leaf photosynthetic capacity during early angiosperm diversification. Ecology Letters  13: 175– 183. Google Scholar CrossRef Search ADS   Brodribb T, Hill RS. 1999. The importance of xylem constraints in the distribution of conifer species. New Phytologist  143: 365– 372. Google Scholar CrossRef Search ADS   Brodribb TJ, Holbrook NM. 2003. Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant Physiology  132: 2166– 2173. Google Scholar CrossRef Search ADS   Brodribb TJ, Holbrook NM. 2005. Water stress deforms tracheids peripheral to the leaf vein of a tropical conifer. Plant Physiology  137: 1139– 1146. Google Scholar CrossRef Search ADS   Brodribb TJ, Feild TS, Jordan GJ. 2007. Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiology  144: 1890– 1898. Google Scholar CrossRef Search ADS   Brodribb TJ, Bienaime D, Marmottant P. 2016a. Revealing catastrophic failure of leaf networks under stress. Proceedings of the National Academy of Sciences of the United States of America  113: 4865– 4869. Google Scholar CrossRef Search ADS   Brodribb TJ, Skelton RP, McAdam SAM, Bienaime D, Lucani CJ, Marmottant P. 2016b. Visual quantification of embolism reveals leaf vulnerability to hydraulic failure. New Phytologist  209: 1403– 1409. Google Scholar CrossRef Search ADS   Buckley TN, John GP, Scoffoni C, Sack L. 2015. How does leaf anatomy influence water transport outside the xylem? Plant Physiology  168: 1616– 1635. Google Scholar CrossRef Search ADS   Choat B, Sack L, Holbrook NM. 2007. Diversity of hydraulic traits in nine Cordia species growing in tropical forests with contrasting precipitation. New Phytologist  175: 686– 698. Google Scholar CrossRef Search ADS   Cochard H, Froux F, Mayr FFS, Coutand C. 2004. Xylem wall collapse in water-stressed pine needles. Plant Physiology  134: 401– 408. Google Scholar CrossRef Search ADS   Cunningham SA, Summerhayes B, Westoby M. 1999. Evolutionary divergences in leaf structure and chemistry, comparing rainfall and soil nutrient gradients. Ecological Monographs  69: 569– 588. Google Scholar CrossRef Search ADS   Feild TS, Brodribb TJ. 2013. Hydraulic tuning of vein cell microstructure in the evolution of angiosperm venation networks. New Phytologist  199: 720– 726. Google Scholar CrossRef Search ADS   Fonseca CR, Overton JMC, Collins B, Westoby M. 2000. Shifts in trait-combinations along rainfall and phosphorus gradients. Journal of Ecology  88: 964– 977. Google Scholar CrossRef Search ADS   Givnish TJ. 1987. Comparative studies of leaf form: assessing the relative roles of selective pressures and phylogenetic constraints. New Phytologist  106: 131– 160. Google Scholar CrossRef Search ADS   Gleason SM, Butler DW, Zieminska K, Waryszak P, Westoby M. 2012. Stem xylem conductivity is key to plant water balance across Australian angiosperm species. Functional Ecology  26: 343– 352. Google Scholar CrossRef Search ADS   Gleason SM, Blackman CJ, Chang Y, Cook AM, Laws CA, Westoby M. 2016. Weak coordination among petiole, leaf, vein, and gas-exchange traits across Australian angiosperm species and its possible implications. Ecology and Evolution  6: 267– 278. Google Scholar CrossRef Search ADS   Gleason SM, Blackman CJ, Gleason ST, McCulloh KA, Ocheltree TW, Westoby M. 2018. Vessel scaling in evergreen angiosperm leaves conforms with Murray’s law and area-filling assumptions: implications for plant size, leaf size, and cold tolerance. New Phytologist . https://doi.org/10.1111/nph.15116. Guyot G, Scoffoni C, Sack L. 2012. Combined impacts of irradiance and dehydration on leaf hydraulic conductance: insights into vulnerability and stomatal control. Plant Cell and Environment  35: 857– 871. Google Scholar CrossRef Search ADS   Hacke UG, Sperry JS, Pockman WT, Davis SD, McCulloh KA. 2001. Trends in wood density and structure are linked to prevention of xylem implosion by negative pressure. Oecologia  126: 457– 461. Google Scholar CrossRef Search ADS   Jacobsen AL, Ewers FW, Pratt RB, PaddockIII WA, Davis SD. 2005. Do xylem fibers affect vessel cavitation resistance? Plant Physiology  139: 546– 556. Google Scholar CrossRef Search ADS   Jacobsen AL, Agenbag L, Esler KJ, Pratt RB, Ewers FW, Davis SD. 2007. Xylem density, biomechanics and anatomical traits correlate with water stress in 17 evergreen shrub species of the Mediterranean-type climate region of South Africa. Journal of Ecology  95: 171– 183. Google Scholar CrossRef Search ADS   Jansen S, Choat B, Pletsers A. 2009. Morphological variation of intervessel pit membranes and implications to xylem function in angiosperms. American Journal of Botany  96: 409– 419. Google Scholar CrossRef Search ADS   Johnson DM, Meinzer FC, Woodruff DR, McCulloh KA. 2009. Leaf xylem embolism, detected acoustically and by cryo-SEM, corresponds to decreases in leaf hydraulic conductance in four evergreen species. Plant, Cell and Environment  32: 828– 836. Google Scholar CrossRef Search ADS   Jordan GJ, Brodribb TJ, Blackman CJ, Weston PH. 2013. Climate drives vein anatomy in Proteaceae. American Journal of Botany  100: 1483– 1493. Google Scholar CrossRef Search ADS   Kim YX, Steudle E. 2007. Light and turgor affect the water permeability (aquaporins) of parenchyma cells in the midrib of leaves of Zea mays. Journal of Experimental Botany  58: 4119– 4129. Google Scholar CrossRef Search ADS   McCulloh KA, Sperry JS, Meinzer FC, Lachenbruch B, Atala C. 2009. Murray’s law, the ‘Yarrum’ optimum, and the hydraulic architecture of compound leaves. New Phytologist  184: 234– 244. Google Scholar CrossRef Search ADS   Nardini A, Luglio J. 2014. Leaf hydraulic capacity and drought vulnerability: possible trade-offs and correlations with climate across three major biomes. Functional Ecology  28: 810– 818. Google Scholar CrossRef Search ADS   Nardini A, Peda G, La Rocca N. 2012. Trade-offs between leaf hydraulic capacity and drought vulnerability: morpho-anatomical bases, carbon costs and ecological consequences. New Phytologist  196: 788– 798. Google Scholar CrossRef Search ADS   Nardini A, Ounapuu-Pikas E, Savi T. 2014. When smaller is better: leaf hydraulic conductance and drought vulnerability correlate to leaf size and venation density across four Coffea arabica genotypes. Functional Plant Biology  41: 972– 982. Google Scholar CrossRef Search ADS   Nolf M, Creek D, Duursma R, Holtum J, Mayr S, Choat B. 2015. Stem and leaf hydraulic properties are finely coordinated in three tropical rain forest tree species. Plant Cell and Environment  38: 2652– 2661. Google Scholar CrossRef Search ADS   Peppe DJ, Royer DL, Cariglino Bet al.   2011. Sensitivity of leaf size and shape to climate: global patterns and paleoclimatic applications. New Phytologist  190: 724– 739. Google Scholar CrossRef Search ADS   Pittermann J, Sperry JS, Hacke UG, Wheeler JK, Sikkema EH. 2006. Inter-tracheid pitting and the hydraulic efficiency of conifer wood: the role of tracheid allometry and cavitation protection. American Journal of Botany  93: 1265– 1273. Google Scholar CrossRef Search ADS   Sack L, Frole K. 2006. Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees. Ecology  87: 483– 491. Google Scholar CrossRef Search ADS   Sack L, Scoffoni C. 2013. Leaf venation: structure, function, development, evolution, ecology and applications in the past, present and future. New Phytologist  198: 983– 1000. Google Scholar CrossRef Search ADS   Sack L, Melcher PJ, Zwieniecki MA, Holbrook NM. 2002. The hydraulic conductance of the angiosperm leaf lamina: a comparison of three measurement methods. Journal of Experimental Botany  53: 2177– 2184. Google Scholar CrossRef Search ADS   Sack L, Cowan PD, Jaikumar N, Holbrook NM. 2003. The ‘hydrology’ of leaves: co-ordination of structure and function in temperate woody species. Plant Cell and Environment  26: 1343– 1356. Google Scholar CrossRef Search ADS   Sack L, Scoffoni C, McKown ADet al.   2012. Developmentally based scaling of leaf venation architecture explains global ecological patterns. Nature Communications  3: 837. Google Scholar CrossRef Search ADS   Schenk HJ, Steppe K, Jansen S. 2015. Nanobubbles: a new paradigm for air-seeding in xylem. Trends in Plant Science  20: 199– 205. Google Scholar CrossRef Search ADS   Scholz FG, Bucci SJ, Goldstein G. 2014. Strong hydraulic segmentation and leaf senescence due to dehydration may trigger die-back in Nothofagus dombeyi under severe droughts: a comparison with the co-occurring Austrocedrus chilensis. Trees-Structure and Function  28: 1475– 1487. Google Scholar CrossRef Search ADS   Scoffoni C, Pou A, Aasamaa K, Sack L. 2008. The rapid light response of leaf hydraulic conductance: new evidence from two experimental methods. Plant Cell and Environment  31: 1803– 1812. Google Scholar CrossRef Search ADS   Scoffoni C, Rawls M, McKown A, Cochard H, Sack L. 2011. Decline of leaf hydraulic conductance with dehydration: relationship to leaf size and venation architecture. Plant Physiology  156: 832– 843. Google Scholar CrossRef Search ADS   Scoffoni C, Vuong C, Diep S, Cochard H, Sack L. 2014. Leaf shrinkage with dehydration: coordination with hydraulic vulnerability and drought tolerance. Plant Physiology  164: 1772– 88. Google Scholar CrossRef Search ADS   Scoffoni C, Albuquerque C, Brodersen CRet al.   2017a. Outside-xylem vulnerability, not xylem embolism, controls leaf hydraulic decline during dehydration. Plant Physiology  173: 1197– 1210. Google Scholar CrossRef Search ADS   Scoffoni C, Albuquerque C, Brodersen CRet al.   2017b. Leaf vein xylem conduit diameter influences susceptibility to embolism and hydraulic decline. New Phytologist  213: 1076– 1092. Google Scholar CrossRef Search ADS   Skelton RP, West AG, Dawson TE. 2015. Predicting plant vulnerability to drought in biodiverse regions using functional traits. Proceedings of the National Academy of Sciences of the United States of America  112: 5744– 5749. Google Scholar CrossRef Search ADS   Skelton RP, Brodribb TJ, Choat B. 2017a. Casting light on xylem vulnerability in an herbaceous species reveals a lack of segmentation. New Phytologist  214: 561– 569. Google Scholar CrossRef Search ADS   Skelton RP, Brodribb TJ, McAdam SAM, Mitchell PJ. 2017b. Gas exchange recovery following natural drought is rapid unless limited by loss of leaf hydraulic conductance: evidence from an evergreen woodland. New Phytologist  215: 1399– 1412. Google Scholar CrossRef Search ADS   R Core Team. 2015. R: a language and environment for statistical computing . Vienna: R Foundation for Statistical Computing. Tozer WC, Rice B, Westoby M. 2015. Evolutionary divergence of leaf width and its correlates. American Journal of Botany  102: 367– 378. Google Scholar CrossRef Search ADS   Trifilo P, Raimondo F, Savi T, Lo Gullo MA, Nardini A. 2016. The contribution of vascular and extra-vascular water pathways to drought-induced decline of leaf hydraulic conductance. Journal of Experimental Botany  67: 5029– 5039. Google Scholar CrossRef Search ADS   Tyree MT, Zimmermann MH. 2002. Xylem dysfunction: when cohesion breaks down. In Xylem Structure and the Ascent of Sap . Berlin: Springer, 89– 141. Google Scholar CrossRef Search ADS   Tyree MT, Davis SD, Cochard H. 1994. Biophysical perspectives of xylem evolution - is there a tradeoff of hydraulic efficiency for vulnerability to dysfunction. IAWA Journal  15: 335– 360. Google Scholar CrossRef Search ADS   Walls RL. 2011. Angiosperm leaf vein patterns are linked to leaf function in a global-scale data set. American Journal of Botany  98: 244– 253. Google Scholar CrossRef Search ADS   Warton DI, Wright IJ, Falster DS, Westoby M. 2006. Bivariate line-fitting methods for allometry. Biological Reviews  81: 259– 291. Google Scholar CrossRef Search ADS   Wright IJ, Dong N, Maire Vet al.   2017. Global climatic drivers of leaf size. Science  357: 917– 921. Google Scholar CrossRef Search ADS   Zhang YJ, Rockwell FE, Graham AC, Alexander T, Holbrook NM. 2016. Reversible leaf xylem collapse: a potential ‘circuit breaker’ against cavitation. Plant Physiology  172: 2261– 2274. Google Scholar CrossRef Search ADS   © The Author(s) 2018. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Annals of BotanyOxford University Press

Published: Apr 14, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off