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Leaf veins supply the mesophyll with water that evaporates when stomata are open to allow CO2 uptake for photosynthesis. Theoretical analyses suggest that water is optimally distributed in the mesophyll when the lateral distance between veins (dx) is equal to the distance from these veins to the epidermis (dy), expressed as dx:dy ≈ 1. Although this theory is supported by observations of many derived angiosperms, we hypothesize that plants in arid environments may reduce dx:dy below unity owing to climate-specific functional adaptations of increased leaf thickness and increased vein density. To test our hypothesis, we assembled leaf hydraulic, morphological, and photosynthetic traits of 68 species from the Eucalyptus and Corymbia genera (termed eucalypts) along an aridity gradient in southwestern Australia. We inferred the potential gas-exchange advantage of reducing dx beyond dy using a model that links leaf morphology and hydraulics to photosynthesis. Our observations reveal that eucalypts in arid environments have thick amphistomatous leaves with high vein densities, resulting in dx:dy ratios that range from 1.6 to 0.15 along the aridity gradient. Our model suggests that, as leaves become thicker, the effect of reducing dx beyond dy is to offset the reduction in leaf gas exchange that would result from maintaining dx:dy at unity. This apparent overinvestment in leaf venation may be explained from the selective pressure of aridity, under which traits associated with long leaf life span, high hydraulic and thermal capacitances, and high potential rates of leaf water transport confer a competitive advantage.
A fundamental challenge for terrestrial plants is to balance the benefit of carbon uptake versus the risk of desiccation resulting from concurrent transpirational water loss. To achieve this, water transport processes in leaves are integrated such that leaf hydraulic properties determine the supply of water to the mesophyll and transpirational losses are regulated by stomatal conductance (gs; Sack and Scoffoni, 2013). Water enters the leaf as a liquid and is distributed along the plane of the leaf through the xylem in veins. After water exits the vein, it moves through the mesophyll toward the epidermis, where it reaches the stomatal pore as water vapor. Little is known about how exactly water moves from the veins to the stomatal pores and where the liquid-to-gas phase transition occurs (Sack and Holbrook, 2006; Sack and Scoffoni, 2013), yet the length of the postvenous path (lH2O) shows a strong negative relationship with leaf hydraulic conductance (kleaf; Brodribb et al., 2007; Buckley et al., 2015). As a result, several leaf morphological traits, such as leaf vein density, the depth of the veins inside the leaf, and leaf thickness (TL), influence leaf water transport (Noblin et al., 2008; Buckley et al., 2015). The close relationship between leaf water status and gs implies that the hydraulic architecture of the leaf poses an important constraint on carbon uptake, specifically in environments with a high evaporative demand.
Considering the dimensions of postvenous water transport led Zwieniecki and Boyce (2014) to hypothesize that a functionally optimal vein placement would be achieved if the average distance between neighboring veins (dx) is equal to the average distance from veins to the epidermis (dy), expressed by the ratio dx:dy ≈ 1. This hypothesis is based on modeling and experiments with artificial leaves (Noblin et al., 2008), which suggest that potential maximum transpiration rates increase by increasing vein density when vein densities are relatively low (with dx:dy ≫ 1), whereas potential maximum transpiration rates become independent of vein density when vein densities are very high (with dx:dy ≪ 1). Zwieniecki and Boyce (2014) observed that derived angiosperms achieve an optimal vein architecture (with dx:dy ≈ 1) by combining high vein densities with relatively thin leaves. This specific leaf morphology is unique to angiosperms (Feild et al., 2011; de Boer et al., 2012) and allows high rates of transpiration and photosynthesis, owing to the short distances for postvenous water transport and subsequent high kleaf (Brodribb et al., 2007). Some gymnosperms also achieve optimal vein placement with a contrasting morphology that combines thick leaves with low vein densities, resulting in relatively long postvenous water transport distances and subsequently low kleaf. Deviations from this proposed hydraulically optimal anatomy are observed for fern and basal angiosperm species that, owing to physiological or developmental constraints, underinvest in veins by combining relatively thin leaves with relatively low vein densities, resulting in dx:dy > 1 (Zwieniecki and Boyce, 2014). This leaf morphology is especially vulnerable to desiccation and, therefore, limited to environments with low light and/or high atmospheric humidity (Feild et al., 2004). Zwieniecki and Boyce (2014) observed few species that overinvest in veins (with dx:dy < 1) and argue that this leaf hydraulic architecture yields no functional benefit over an optimal vein architecture (dx:dy ≈ 1), because, like the theoretical extreme (dx:dy ≪ 1) exemplified by Noblin et al. (2008), the additional veins may not contribute to additional leaf gas-exchange capacity.
We argue that the proposed optimal leaf hydraulic architecture (dx:dy ≈ 1) may not be the phenotypic end point of evolution in environments that specifically select for traits associated with thick leaves. Thick leaves are most common in warm, arid, and high-light environments because of advantages related to a longer leaf life span (Mott et al., 1982; Niinemets, 2001) and a larger hydraulic and thermal capacitance (Schymanski et al., 2013). Warm and dry climates also select for high vein densities (Sack and Scoffoni, 2013), because this morphology provides greater resistance to loss of kleaf during water stress and may facilitate high photosynthesis rates during (short) wet periods (Grubb, 1998; Scoffoni et al., 2011). The morphological combination of thick leaves (with subsequent long dy) with high vein densities (with subsequent short dx) implies that an apparent overinvestment in leaf venation (resulting in dx:dy < 1) may provide functional benefits under dry and warm climates.
To understand how the selection pressure of aridity shapes leaf form and function, it is important to consider not only the hydraulic aspects of leaf morphological adaptations but also the optimization of photosynthetic biochemistry in relation to the prevailing climate. The fast-slow continuum of the leaf economics spectrum has successfully predicted correlations between photosynthetic capacity, gs, and leaf nitrogen on the one hand and leaf life span and leaf mass per unit area (LMA) on the other hand (Wright et al., 2004). Although these relationships emerge independent of climate, leaves of drought-adapted plants tend to have a higher LMA and longer life span (Wright et al., 2005). Given the requirement for plants to trade off carbon uptake and water loss, photosynthetic traits also should be coordinated with kleaf. Some empirical evidence for this effect has been presented (Franks, 2006; Sack and Frole, 2006; Brodribb et al., 2007; Brodribb and Jordan, 2008), but a theoretical proposition linking leaf economic traits to these processes (as proposed by Blonder et al. [2011]) has yet to be widely accepted (Sack et al., 2014) or supported experimentally. In contrast to the assertion of coordination, the recent work of Li et al. (2015) indicates a decoupling of leaf economic and leaf hydraulic traits within tropical-subtropical forests. The premise of Li et al. (2015) is that spatial compartmentalization of traits in functionally independent leaf internal structures may enable more nuanced adaptation in habitats that offer a diversity of conditions. Despite potential compartmentalization, leaf hydraulic traits are inherently coupled to leaf economic and photosynthetic traits via constraints imposed by selection for the most productive combinations of leaf life span and leaf morphology (Brodribb et al., 2010). In this way, groups of functionally independent traits are expected to change concurrently to optimize the overall performance of leaves in relation to the environmental pressure of aridity.
We aim to test the hypothesis that coordination between leaf hydraulic and photosynthetic traits is achieved across mesic and arid environments by expressing leaf morphologies that deviate from the theoretical optimal vein architecture with dx:dy ≈ 1, as proposed by Noblin et al. (2008) and Zwieniecki and Boyce (2014), resulting in dx:dy < 1. Here, we assembled a collection of leaf hydraulic and photosynthetic traits from the closely related genera Eucalyptus and Corymbia (collectively referred to as eucalypts from here on) that are widely distributed across the mesic-arid spectrum of Australian environments. Fossil evidence suggests that the Eucalyptus and Corymbia clades evolved from a rainforest precursor during the Paleocene to Early Eocene (Gandolfo et al., 2011). The subsequent diversification and radiation of these genera across continental Australia through the Cenozoic took place against a backdrop of increasing aridity (Ladiges et al., 2003; Martin, 2006). This adaptive radiation required innovations in leaf traits that are manifest in extant species. Work to classify parts of this group using morphological or molecular approaches generally has observed more derived species in arid habitats (Steane et al., 2002, 2011; Hopper, 2003; Ladiges et al., 2003). Within species, phylogeographical patterns often indicate a complex population structure, indicative of multiple localized refugia associated with historical shifts in aridity (Byrne et al., 2008).
In this study, we draw upon a natural aridity gradient to test whether the leaf hydraulic and photosynthetic traits of eucalypt leaves are coordinated across environments classified by the supply and demand for water. Our analyses expand on the work of Schulze et al. (2006), who sampled eucalypt leaves across a transect that spanned an aridity gradient from the wet coastal region of southwestern Australia to the arid Australian interior near Kata Tjuta. From this collection, we selected three to five leaves per tree from 440 trees, representing 68 species occurring at 73 locations, for analyses of leaf morphological and hydraulic traits along the aridity gradient (Fig. 1; Supplemental Table S1). From this subsample, we selected three leaves from 107 trees (27 species, 30 locations) for measurements of stomatal traits. In our analyses, we specifically focused on the adaptation of the leaf morphological and hydraulic traits that influence the ratio dx:dy (Fig. 1). Based on these data sets, we determined the adaptive innovations in leaf morphology and leaf hydraulics that provide specific competitive advantage to eucalypts in arid environments.
Our results show clear differences in eucalypt leaf morphology along an aridity gradient in southwestern Australia. Regression with aridity index (AI) reveals that leaf vein density, expressed as vein length per unit area (VLA), and TL increase from the mesic to the arid sites (r2 = 0.44, P < 0.001 and r2 = 0.11, P < 0.001, respectively; Fig. 2). The slopes and intercepts of the models relating VLA to AI (Fig. 2) were not different between hypostomatous and amphistomatous species (P > 0.05); hence, a combined model is presented. The increase in TL with aridity was only significant for amphistomatous leaves and for the combination of amphistomatous and hypostomatous species, and the slopes and intercepts of these models were not significantly different (P > 0.05). Although a complete phylogeny for the Eucalyptus and Corymbia genera is not available, we corrected for the potential effect of sampling bias due to relatedness between species in our sample by resampling several key traits at the taxonomic level of series (a subdivision of genus) using the classification of Nicolle (2015; Supplemental Fig. S5). Our subsequent analysis shows that the relationships between VLA and AI (bootstrapped r2 = 0.76, P < 0.001) and between dx:dy and AI (bootstrapped r2 = 0.67, P < 0.001) are robust to potential sampling bias. The relationship between leaf thickness and AI (bootstrapped r2 = 0.2, P = 0.16) is not robust to potential sampling bias, which reflects the weak association between leaf thickness and AI in the raw data, as shown in Figure 2 (r2 = 0.11, P < 0.001).
As the leaf veins of the eucalypts studied here are positioned such that there is an equal distance to the upper and lower epidermis (Supplemental Fig. S1; Supplemental Table S2), increases in TL are proportional to increases in dy. Furthermore, increases in VLA are closely linked to decreases in dx (r2 = 1, P < 0.001; Supplemental Fig. S2). Hence, the observed dx:dy ratios decrease along the aridity gradient as a result of the combined increases in VLA and TL from mesic to arid sites (r2 = 0.41, P < 0.001; Fig. 2). This pattern was evident regardless of stomatal distribution, and the slopes and intercepts of the models describing these relationships did not differ significantly between hypostomatous and amphistomatous species (P > 0.05). In our sample of eucalypts, the dx:dy ratios range between 0.15 and 1.6 and, thereby, extend notably below the proposed theoretical optimum ratio dx:dy ≈ 1. Our results also reveal a shift from a hypostomatous to amphistomatous leaf morphology toward the more arid sites. Consequently, the amphistomatous species in our data set have leaves with a higher VLA than the hypostomatous species (16 ± 3.7 and 9 ± 2.1 mm mm−2, respectively; P < 0.001) and a larger TL (517 ± 97 and 350 ± 75 µm, respectively; P < 0.001). These morphological contrasts manifest as a lower dx:dy in amphistomatous species compared with hypostomatous species (0.3 ± 0.1 and 0.82 ± 0.24, respectively; P < 0.001; Supplemental Fig. S3). Our species level comparison of dx:dy also reveals that both the hypostomatous and amphistomatous eucalypts differ from the derived angiosperms of Zwieniecki and Boyce (2014; Supplemental Fig. S3). Only the amphistomatous eucalypts differ significantly from the proposed 1:1 ratio as hypothesized by Noblin et al. (2008). We note that our measurements of these leaf morphological and hydraulic traits were performed on rehydrated leaves that had been stored after drying. Our analyses to quantify the potential error arising from this approach indicate that leaf area and TL, affecting dx and dy, respectively, may have been underestimated by 2.2% ± 2.7% and 8.9% ± 4.2%, respectively (Supplemental Fig. S4). As the reduction in TL is larger than the reduction in leaf area, we argue that the reported values of dx:dy may be overestimated by approximately 5% to 10%.
A principal component analysis (PCA) of the leaf morphological data set indicates the coupling of hydraulic and photosynthetic traits (Fig. 3). The PC1 axis accounts for 59% of the variation, and this is dominated by loadings on dx:dy, LMA, LT, and kleaf. For PC2, which accounts for 17% of the variation, strong loadings are evident on the Vcmax, VLA, and kleaf. The PCA scores show clear separation in relation to aridity and stomatal distribution, which highlights the dominant effect of aridity on leaf morphology within the eucalypts studied here. A PCA of the stomatal morphological data set also displays the coupling of hydraulic and photosynthetic traits (Fig. 3). The PC1 axis accounts for 64% of the variation and is dominated by loadings on dx:dy, stomatal pore length (Lp), guard cell length (Lgc), and VLA. The PC2 axis accounts for 17% of the variation and is dominated by loadings on the anatomical maximum stomatal conductance to water vapor (gsmax) and stomatal density (DS). As with the leaf morphological data set, separation in relation to aridity and stomatal distribution is evident.
Linear regression statistics further support the PCA-derived coupling of hydraulic and photosynthetic traits in both the leaf morphological and stomatal data sets (Tables I and II, respectively). In relation to our hypothesis, the leaf morphological data set reveals a close association between the ratio dx:dy and a variety of leaf traits that span the hydraulic and photosynthetic categories. Crucially, dx:dy is negatively correlated with LMA (r2 = 0.57, P < 0.001) and leaf nitrogen per unit area (Narea; r2 = 0.42, P < 0.001), whereas both LMA and Narea are negatively correlated with the leaf carbon isotope ratio (Δleaf; r2 = 0.34, P < 0.001 and r2 = 0.35, P < 0.001, respectively; Table I). The stomatal data set reveals a close association between leaf hydraulic traits and stomatal morphology. Here, dx:dy is positively correlated with both guard cell length and stomatal pore length (r2 = 0.44, P < 0.001 and r2 = 0.52, P < 0.001, respectively; Table II).
The leaf morphologies of the eucalypts studied here deviate from the analysis of Zwieniecki and Boyce (2014), which shows that derived angiosperms closely coordinate dx with dy via changes in VLA and the lateral position of veins inside the leaf to achieve dx:dy ratios near unity (Fig. 4). Unlike the relationship reported by Zwieniecki and Boyce (2014), our linear regression model describing variation in dx based solely on dy gives only a weak fit (correlation r2 = 0.16, P < 0.001 in Fig. 4 and adjusted r2 = 0.15, P < 0.001 in Supplemental Table S3 for the regression model). The inclusion of AI in a multiple regression model substantially improves the model fit (adjusted r2 = 0.51, P < 0.001; Supplemental Table S3). In spite of this result, the close relationship between aridity and the shift from hypostomaty to amphistomaty (Supplemental Fig. S3) restricts our ability to statistically resolve the underlying cause for a negative relationship between dx and dy in our eucalypt sample. Still, our observation that the relationship between dx:dy and AI is comparable across hypostomatous and amphistomatous eucalypt species-site combinations (Fig. 2) suggests that aridity shapes the negative relationship between dx and dy and that amphistomaty is a separate adaptation that also is associated with aridity.
The low values of dx:dy observed in our selection of eucalypts are a result of the combined increases in TL and VLA along the aridity gradient. We hypothesized that these adaptations reflect the selection pressure of aridity on leaf morphology, whereby increases in leaf thickness and LMA result from selection for a longer leaf life span, while the increases in VLA occur from selection for enhanced water supply and the need to compensate for the potential loss in photosynthesis owing to the increase in leaf thickness. This hypothesis is supported by our within-site comparisons of species-level differences in TL, dx:dy, Ā, and VLA relative to differences in LMA (Fig. 4). The comparisons show that, within sites, species with a higher LMA have, on average, thicker leaves (P < 0.001, n = 128) with lower dx:dy (P < 0.01, n = 128) than species with lower LMA. Cooccurring species with higher LMA also have lower Ā than species with lower LMA (P < 0.001, n = 178). In contrast to the correlation between LMA and VLA that we observed across sites (r2 = 0.22, P < 0.001; Table I), we found no within-site difference in VLA between species with different LMA (P > 0.05, n = 178; Fig. 4). This result suggests that the key hydraulic trait VLA may be decoupled from LMA within sites, despite their correlated adaptation to the common selection pressure of aridity across the sampled sites.
The observation that our sample of eucalypts includes many species with dx:dy ratios below unity raises the question of the extent to which this leaf morphology conveys an advantage in terms of additional gas exchange in comparison with a leaf morphology with dx:dy ≈ 1. As the observed adaptations in dx:dy are closely linked to the photosynthetic and stomatal traits considered here (Tables I and II), our empirical data cannot be used to directly test our hypothesis that these adaptations provide an advantage in terms of additional transpiration and photosynthesis. Therefore, we used the semiempirical leaf hydraulic model of Brodribb et al. (2007) and Brodribb and Feild (2010) to determine how changes in hydraulic architecture influence leaf gas exchange. We quantified the potential gas-exchange advantage of reducing dx:dy below unity in terms of the modeled difference in Rubisco-limited photosynthesis between the observed leaf morphology and a leaf morphology with dx:dy = 1 as the percentage difference, termed δA, for all species-site combinations. This model approach is explained in detail in "Materials and Methods." A sensitivity and uncertainty analysis of the model is provided in Supplemental Figures S7 and S8. Our model results show that the observed reduction in dx:dy below unity reduces lH2O and thereby increases the modeled kleaf (Fig. 5). This effect is most pronounced in thick leaves, which suffer most from reduced kleaf when the theoretical optimal hydraulic architecture dx:dy ≈ 1 is maintained. Our modeling results show that the reductions in dx:dy below unity provide a small but significant photosynthetic advantage δA of 8.1% ± 3.4% across all our species-site combinations (Fig. 5). The group of amphistomatous species shows a larger average photosynthetic advantage of reducing dx:dy below unity than the group of hypostomatous species, with δA = 8.9% ± 2.5% for the amphistomatous species and δA = 1.2% ± 1.6% for the hypostomatous species.
Our results reveal that, in eucalypts, high vein densities occur in thick and amphistomatous leaves, especially in species growing in the most arid localities. The consequence of this leaf morphology is that the observed range in the ratio between the intervein distance dx and the vein-epidermal distance dy extends to well below the proposed theoretical optimal ratio dx:dy ≈ 1 (Noblin et al., 2008). Eucalypts thus form an exception to the strong 1:1 relationship reported for other derived angiosperm species (Zwieniecki and Boyce, 2014).
Theoretical optimal vein placement requires close coordination between leaf vein density, the position of veins in the leaf, and leaf thickness that inadvertently requires leaves with highest kleaf to be relatively thin. However, arid environments select for thicker leaves owing to their longer life span (Wright and Westoby, 2002), larger thermal and hydraulic capacitance (Schymanski et al., 2013), and limited palatability (Chabot and Hicks, 1982; Choong et al., 1992). The result of selection for thicker leaves is a larger vein-epidermal distance dy and, all else being equal, a decrease in kleaf and photosynthesis. Crucial is that a decrease in kleaf limits maximum rates of photosynthesis because it pertains directly to leaf water status and the control of gs. As water may be available during short intervals in arid environments, there may be strong selection pressure for a higher kleaf (Grubb, 1998; Scoffoni et al., 2011). However, owing to the selection pressure for thicker leaves in arid environments, the remaining option to increase kleaf is to increase vein density and, subsequently, reduce the ratio dx:dy. The coincidence of thick leaves and high vein densities in our study, therefore, does not point to a mechanistic coordination of these traits, as discussed between Blonder and Enquist (2014) and Sack et al. (2014), but rather to parallel selection driven by common selection pressures associated with aridity.
The VLA observed in our sample of eucalypts ranges from 6 mm mm−2 in relatively mesic sites up to 24 mm mm−2 in the most arid localities. Our measurements of VLA are slightly overestimated, as our analyses were performed on rehydrated leaves, which revealed an average shrinkage of 2.2% ± 2.7% compared with fresh leaves (Supplemental Fig. S4). This effect is of similar magnitude to the shrinkage reported in rehydrated leaves by Blonder et al. (2012). Our observation that VLA in eucalypts increases with increasing aridity is in line with the global compilation of vein density data by Sack and Scoffoni (2013), who show a strong correlation between VLA and aridity across biomes. Their review includes only two eucalypt species (Eucalyptus hemastoma and Eucalyptus globulus with VLA of 4.1 and 7.4 mm mm−2, respectively), which are from relatively mesic habitats and have vein densities comparable to our mesic species-site combinations. The upper end of the vein density range of our eucalypt sample overlaps the highest vein densities of evergreen shrubs and trees reported by Sack and Scoffoni (2013). Boyce et al. (2009) observed that these high vein densities (greater than 20 mm mm−2) occur only in the rosid clade, which Eucalyptus and Corymbia belong to. Hence, this phylogenetic background may have allowed eucalypts to develop the specific leaf morphology that combines relatively thick leaves with high vein densities.
Mitigating the effect of leaf thickness on kleaf by adjusting VLA and subsequent dx:dy may be critical for species in arid environments that must maximize carbon gain during short pulses of high soil moisture content (Sack and Scoffoni, 2013). This growth strategy likely became relevant during the evolution of eucalypts, because major differentiation of the Eucalyptus and Corymbia clades took place during a transition to greater aridity (Ladiges et al., 2003). This climatic shift provided ecological opportunities, but the ensuing adaptive radiation of species formerly suited to rainforests, the precursors of modern eucalypts, would have occurred in an environment increasingly characterized by long dry periods punctuated by brief wet episodes (Martin, 2006). These novel environments ultimately would have encouraged the convergence of traits enabling a long leaf life span and high thermal capacitance with traits supporting higher rates of leaf water transport and photosynthesis (Givnish et al., 2005). Leaves would necessarily become thicker, but at the same time selection for maximum photosynthetic yield would lead to the evolution of higher vein densities. Zwieniecki and Boyce (2014) suggest that such an overinvestment in veins may result in a decrease in photosynthetic capacity due to a loss of internal space that could be allocated to chlorophyll-bearing cells. However, in thick amphistomatous leaves of (semi)arid biomes, photosynthetic capacity may not be limited by space constraints but, rather, by the ability of the hydraulic network to sustain liquid and vapor water transport.
Although the influence of adjusting intervein distance dx on kleaf is less than the effect of adjusting vein-epidermal distance dy (Noblin et al., 2008), our modeling results indicate that the eucalypts studied here may still gain a small but significant photosynthetic benefit of reducing dx:dy below unity. Our model is based on the empirical relationship between kleaf and lH2O found by Brodribb et al. (2007). This relationship highlights that the water transport pathway outside the xylem is the major constraint to kleaf (Sack and Scoffoni, 2013; Buckley et al., 2015). According to our model, the photosynthetic advantage increases with decreasing dx:dy further below unity. Hence, the combined adaptation of increasing leaf thickness and increasing VLA could have provided an evolutionary advantage in terms of additional productivity during (short) periods with sufficient water. As our measurements of dx:dy may be overestimated owing to the use of dried and rehydrated laves, the modeled advantage could be slightly underestimated. Still, our interpretation of the modeling results appears at odds with the conclusion of Zwieniecki and Boyce (2014) and the results from artificial leaves of Noblin et al. (2008), who suggest that additional leaf veins do not contribute to additional leaf gas exchange in the theoretical extreme situation with dx:dy ≪ 1. However, the modeling results of Noblin et al. (2008) also suggest that dx:dy can extend to below unity before the response of increasing transpiration rate with increasing vein density fully saturates, and this is especially so when the vein-epidermal distance is relatively large and vein diameters are small. This interpretation of Noblin et al. (2008) is consistent with our observations on eucalypt leaf morphology, which shows that the lowest dx:dy ratios occur in the thickest leaves with the highest vein densities. Therefore, we argue for a more nuanced interpretation of the ratio dx:dy by considering the specific growth environments and leaf morphologies, in which small increments in leaf gas exchange may yield specific competitive advantages.
Our results also reveal that various leaf morphologies coexist in the same site and that thicker leaves with a lower dx:dy do not offer an absolute competitive advantage compared with thinner leaves with a higher dx:dy. These within-site variations in dx:dy and TL could reflect the exploitation of different niches, as proposed by Li et al. (2015) drawing on observations from a structurally complex (sub)tropical environment, where light limitation is an important selection pressure. For the eucalypts in our study, where light is not limiting, niche differentiation is likely to manifest as different rooting depths and soil preferences (Drake et al., 2011; Poot and Veneklaas, 2013), resulting in differences in water availability, which in turn will influence stomatal opening and carbon gain over the life span of a leaf. Thus, species with thick leaves may cooccur with thin-leafed species due to morphological specialization to specific niches in relation to leaf economic tradeoffs.
A specific additional benefit of high VLA in arid environments also may lie in the selection for longer-lived leaves (Wright and Westoby, 2002) as leaf aging will inevitably lead to some loss of hydraulic continuity when the cumulative exposure to water stress increases. The vascular redundancy of leaves with high vein densities of a reticulate nature may reduce the risk of complete hydraulic failure under these circumstances (Scoffoni et al., 2011; Price and Weitz, 2014) by providing alternative parallel flow paths for water transport (Sack et al., 2008). Thus, selection for elevated vein densities in arid biomes may occur both to mitigate the effect of leaf thickness and to reduce the risk of hydraulic failure due to water stress. Although our observations are limited to the eucalypt group, we expect that similar morphological trends may be present in other (semi)arid evergreens.
Our results confirm the well-known link between leaf thickness, aridity, and amphistomaty (Mott et al., 1982), as thicker amphistomatous leaves were found more commonly in eucalypts growing in the most arid environments. Amphistomaty has different implications for the leaf internal conductance of water (liquid or vapor) than for the leaf internal conductance of CO2. Our results indicate that amphistomaty does not reduce the path length from vein to stomata, as both hypostomatous and amphistomatous leaves have their veins at approximately mid depth. However, amphistomaty does reduce the average path length for CO2 diffusion to the mesophyll in thick leaves. Although other factors, including mesophyll cell surface area, cell wall thickness, and chloroplast distribution also are strong determinants of mesophyll conductance to CO2 (Flexas et al., 2013), it would seem that amphistomaty tends to favor water use efficiency due to a larger positive effect on CO2 conductance than hydraulic conductance. Hypostomatous leaves are more common at mesic locations in our study, where leaves also are thinner. The shorter pathways for water and CO2 transport in thin leaves of mesic environments may favor the presence of stomata solely on the abaxial surface (de Boer et al., 2012). The close relationship between aridity and stomatal distribution challenges our ability to disentangle the effect of leaf type versus the specific adaption of leaf morphology to aridity as an underlying cause for the negative relationship between dx and dy. Although we infer that aridity shapes this relationship in eucalypts, further work focusing on thick hypostomatous leaves of arid environments would offer additional insight.
The observed adaptations in leaf morphology described here create a more complete picture of eucalypt adaptations to their environments. Of particular interest is the fact that the high-LMA amphistomatous leaves of (semi)arid habitats tend to have nearly vertical angles. High leaf mass may contribute to that trait, but the nearly vertical leaf angle in eucalypts is generally interpreted as enabling greater carbon capture when the sun is at low angles and temperature and vapor pressure deficit are lower (King, 1997). At such steep leaf angles, the amphistomatous and isobilateral form (Gillison, 1981) presumably enables light entry into the leaf from either or both sides. In contrast, in mesic environments, vegetation tends to have higher leaf area indices, and most light enters leaves from above (i.e. from the adaxial surface of the hypostomatous leaves that are common in such environments). Thinner and less dense leaves (with lower LMA) tend to have a higher photosynthetic light use efficiency than thicker and denser leaves (Reich et al., 1992; Niinemets, 2001). In arid habitats, where light is not limiting (Bloom et al., 1985), the selection pressure for high light use efficiency is presumably weak. Hence, the relatively thick eucalypt leaves of (semi)arid habitats may confer little, if any, negative consequences for photosynthesis, due to relaxation of the morphological constraints on leaf venation.
We conclude that arid environments select for thicker amphistomatous leaves in eucalypts. Thick leaves challenge the underlying selective pressure to distribute water efficiently to the sites of evaporation because their morphology increases the distance for vein-to-stomata transport. To some extent, eucalypts have overcome this problem by developing leaves with a high vein density, thereby reducing the intervein distance. In this way, deviation from the theoretical optimal vein placement can be considered a response to the selective advantage of producing thick leaves in arid environments.
Plant Material
In 2003 and 2004, leaves of Eucalyptus spp. and closely related Corymbia spp. were sampled from 971 trees of 68 distinct species across a transect that spanned 73 locations along an aridity gradient from the wet coastal region of southwestern Australia to the arid Australian interior near Kata Tjuta (Schulze et al., 2006). From this collection, we selected three to five leaves per tree from a total of 440 trees, representing all species and all locations of the original data set for analyses of leaf morphological traits along the aridity gradient (Fig. 1; Supplemental Table S1). From this subsample, we selected three leaves from 107 trees (27 species, 30 locations) for measurements of stomatal traits. Our analyses were performed at the species-by-site level. This approach allows for genetic and phenotypic differences to influence the observed relationships with aridity. We deviate from this approach when analyzing the relationship between dx and dy, where we aggregate our data to the species level to match the data of Zwieniecki and Boyce (2014). To compare the sensitivity of these different averaging approaches, we analyzed two critical results: the relationship dx and dy and the relationship between the dx:dy ratio and AI for our eucalypt sample at both levels of averaging (Supplemental Table S3).
Climate Variables
Climate variables for each location were obtained from the Atlas of Living Australia, a repository for large environmental and ecological data sets (http://www.ala.org.au; accessed December 15, 2015). We selected the climatological variables that were representative of gridded data at a resolution of approximately 1 km. We integrate water supply and demand for each location as annual AI, the average of monthly ratios of precipitation to potential evaporation (pan, free-water surface). This is an adaptation of the index proposed by the United Nations Environmental Program and cited by Middleton and Thomas (1997). Additional climate variables obtained from this repository were mean annual daytime vapor pressure deficit, mean annual daytime temperature, and mean annual rainfall. We are aware that these climate variables, while representative of long-term trends, may not reflect the exact conditions under which individual leaves developed. However, as the emphasis of our study is to demonstrate and interpret leaf morphological changes along an aridity gradient rather than to study the relationships between climate variables and water relations of individual leaves, we argue that the climate data provide sufficient detail for our analyses.
Leaf Morphological Traits
Fresh leaf area, leaf dry weight, and LMA (g cm−2) were determined for three to five leaves from each of the 440 trees sampled in this study. To determine TL (μm), we randomly selected one leaf per tree (n = 270 trees of the 68 species) and cut out a 1-cm2 fragment at approximately the mid point of the leaf long axis. Since the leaves had been oven dried, these subsamples were first immersed in dilute detergent for 1 h to soften the highly sclerophyllous tissue. Cross sections were then taken from each subsample using a microtome set to a thickness of 15 μm. The cross sections were stained in 5% Toluidine Blue for 10 s, rinsed in deionized water, and then mounted on clear microscope slides using Kaiser's jelly: 1:5:4:1 gelatin:glycerol:water:antibacterial solution (Listerine). We measured TL at 100× magnification using a compound light microscope (BX51; Olympus), with n = 3 measurements for each cross section. The presence of stomata on either the abaxial surface (hypostomatous) or both the abaxial and adaxial surfaces (amphistomatous) was determined during these microscopic analyses for all the species considered. The error in TL resulting from the use of dried and rehydrated leaves was quantified on a set of fresh eucalypt leaves, which revealed an 8.9% ± 4.2% residual reduction in TL (Supplemental Fig. S4). The vein-epidermis distance (dy [μm]) was derived from leaf thickness as TL/2. This simplification was justified by regression analyses of the relationship between dy and TL/2 from cross sections of a random selection of 43 species (r2 = 1, P < 0.001, n = 111 leaves; Supplemental Data S1; Supplemental Fig. S1; Supplemental Table S2). For this sample, dy was measured with respect to the abaxial (lower) epidermis.
Leaf Venation Traits
The leaf vein density was expressed as VLA (mm mm−2) and measured by sampling 1- to 2-cm2 sections from three leaves per tree from each of the 440 trees at approximately the mid point of the long axis while avoiding the mid rib. The sample was placed in a 5% sodium hydroxide solution until the cuticle had dissolved to reveal the vein network (2–3 weeks; Brodribb and Feild, 2010; Sommerville et al., 2012). The samples were then rinsed in deionized water, stained in a solution of safranin in 5% ethanol for 1 h, and then rinsed in deionized water. Three images of each leaf were then obtained using a dissecting microscope (model SMZ800; Nikon Instrument) at 40× magnification, and the vein network was traced by hand using Paint (Microsoft). In light of the high VLA values of our collection, we confirmed that the minor veins of cleared leaves were conductive by drawing dye through fresh leaves via transpiration and observing the vein network from paradermal images (Supplemental Fig. S6, A and B). A leaf cross section also is presented to confirm the positions of these numerous small veins as well as the presence of several small chlorenchyma cells in the mesophyll tissue of the intervein space (Supplemental Fig. S6C). Also, we quantified the amount of leaf shrinkage from drying and the subsequent expansion after rehydration on a set of fresh eucalypt leaves, which revealed a 2.2% ± 2.7% residual reduction in leaf area (Supplemental Fig. S4).
The traced vein network of each image was analyzed further in Matlab (version R2015b). The average VLA of each traced image was obtained following Blonder et al. (2011). First, each set of traces was smoothed and reduced to the width of a single pixel using the skeletonizing algorithm of the function bwmorph. Total vein length was calculated from the skeletonized vein network using the function bwarea. The VLA was calculated as the quotient of total vein length and total image area.
The intervein distance (dx [μm]) was obtained from the skeletonized vein network images by automatically drawing 10,000 random cross sections across each traced image. For each cross section, we mapped the points where the cross section intersected with a traced vein and subsequently calculated the average distance between the veins in each cross section. This automated method resembles the manual cross-section approaches of Blonder and Enquist (2014) and Zwieniecki and Boyce (2014). The grand mean distance between veins in all randomly drawn cross sections was then calculated per image to obtain an average dx for each sampled leaf.
Stomatal Morphological Traits
Three leaves were selected for each of the 27 species chosen for the determination of stomatal traits. For each leaf, a 1-cm2 section was sampled and placed into a 2-mL Eppendorf tube. Each tube received a 1:1 solution of 80% ethanol and 30% hydrogen peroxide. The tubes were left unsealed and placed into a water bath set at 60°C until the cuticles and epidermis began to detach from the underlying mesophyll and vascular tissues (after approximately 48 h). The samples were then removed from the tubes and rinsed under tap water. The cuticle and epidermis of both the abaxial and adaxial surfaces were then carefully detached from the remaining tissue with fine forceps, stained in 5% safranin for 30 s, and then rinsed in deionized water. The peels were then transferred to microscope slides and mounted using Kaiser's jelly. DS (mm−2; i.e. number of stomata per unit epidermal area) was calculated for each stomata-bearing leaf surface as the mean of five fields of view at 400× magnification using a compound light microscope (BX51; Olympus). Stomatal morphological parameters were measured as the mean of 20 stomatal complexes (guard cell pairs) for each stomata-bearing surface at 1,000× magnification using the same light microscope. Stomatal morphological parameters were Lgc (μm), guard cell width (Wgc [μm]), and Lp (μm; Franks et al., 2009). The gsmax (mol m−2 s−1) of the stomata-bearing leaf surface was estimated according to Franks and Farquhar (2001) and Franks et al. (2009):(1)where d is the diffusivity of water in air (m2 s−1), a′ is the mean maximum stomatal pore area (m−2), which was estimated as Lp2 × π/4, V is the molar volume of air (m3 mol−1), and Ɩ is the depth of the stomatal pore (m), which was assumed to be equal to Wgc. For hypostomatous leaves, the estimate of gsmax is based on the stomatal morphology of a single leaf side, whereas gsmax for amphistomatous leaves is the sum of both leaf surfaces based on the assumption that gas exchange from the abaxial and adaxial surfaces occurs in parallel.
Leaf Nitrogen and Leaf Carbon Isotopic Composition
Schulze et al. (2006) collected a subsample of oven-dried leaves from each of the 440 trees. The leaves were finely ground, and part of this material was used to determine total leaf nitrogen (% by mass) using a nitrogen analyzer (Vario EL II; Elementar). Leaf nitrogen as a percentage of mass (Nmass) was converted to an area basis as Narea (g cm−2) = Nmass (%) × LMA × 100. The leaf carbon isotope ratio (δ13Cleaf) of the remaining ground material was determined following combustion using an isotope ratio mass spectrometer (Finnigan Delta + XL) in an online continuous flow system. δ13Cleaf was converted to Δleaf according to Farquhar et al. (1989), taking atmospheric δ13C as 8.1‰, the average value recorded at the Cape Grim Air Pollution Station, Tasmania, in the 6 months prior to each sample collection campaign.
Deriving Photosynthetic Rate from Carbon Isotopic Composition
We estimated the maximum carboxylation capacity at 25°C (Vcmax25 [μmol m−2 s−1]) according to an empirical model that was derived from a meta-analysis of Vcmax, Narea, and LMA determined for 27 eucalypt species reported in 10 studies (Supplemental Table S4):(2)Vcmax25 was then converted to the actual velocity of Rubisco for carboxylation at ambient temperature (Vcmax [μmol m−2 s−1]) according to Bernacchi et al. (2001) and the average daytime temperature of each site.
A time-integrated measure of leaf intercellular (and mesophyll) CO2 mole fraction ( [μmol mol−1]) was then estimated according to Farquhar et al. (1982):(3)where the atmospheric CO2 mole fraction (ca) was taken as 380 μmol mol−1, reflecting the atmospheric partial pressure of CO2 in 2003 (Keeling and Whorf, 2004). We assumed that photosynthesis was limited predominantly by carboxylation; therefore, the modeling constraints of Farquhar et al. (1982) and Sharkey et al. (2007) could be applied to yield an expression for time-integrated photosynthetic rate (Ā [μmol m−2 s−1]):(4)Here, the mitochondrial respiration rate (Rd) was taken as 0.01 Vcmax, Γ* is the CO2 compensation point in the absence of Rd, KC and KO are the Michaelis constants of Rubisco for carbon dioxide and oxygen, respectively, and O is the atmospheric partial pressure of oxygen. For values of these parameters and their temperature dependencies, we followed Bernacchi et al. (2001). We note that since represents the mole fraction of CO2 within both intercellular spaces and the mesophyll, the expression for Ā incorporates the drawdown in CO2 at the sites of assimilation.
Statistical Analyses
Multiple relationships between traits were determined by PCA using OriginPro 2015. PCAs were conducted on both the larger data set comprising leaf morphological and hydraulic traits and the subset of data that included stomatal morphological traits. Data were log transformed to meet the assumption of normality, and z scores were calculated.
SMA regression analyses using R (version 3.0.2) and the SMATR package (Warton et al., 2006) were used to describe specific relationships between VLA, TL, and dx:dy and the AI, based on the Atlas of Living Australia. Pairwise interactions between traits in the leaf morphological and stomatal morphological data sets were similarly quantified with SMA regression analyses. Data were log transformed prior to these analyses. These analyses were performed at the species-by-site level. We corrected for the potential effect of sampling bias due to relatedness between species in the relationships between VLA, TL, and dx:dy and the AI by resampling the data at the taxonomic level of series (a subdivision of genus) using the taxonomic classification of Nicolle (2015). Our data set contains species from 11 distinct taxonomic series in this classification. Hence, each subsample contains 11 data points, with each data point being one species-site average and each species belonging to one of the 11 series. We used a bootstrap resampling strategy with replacement to obtain distributions of the taxonomically unbiased correlation r2 and P value as well as the SMA from 10,000 random subsamples.
Linear regression models were developed to analyze the relationships between dy and dx, and to quantify the contribution of aridity, by including and excluding the site- and species-specific AI and the contribution of leaf morphology in terms of hypostomaty/amphistomaty in the models for the eucalypt sample. Linear regression models also were developed to analyze the relationship between the ratio dx:dy and the site- and species-specific AI. The traits dx, dy, and dx:dy as well as the AI were log10 transformed for the regression model analyses. The Matlab function fitlm was used to obtain the best least-squares fit of a model to the data.
We further tested our hypothesis on the coordination of aridity-induced changes in leaf morphological, hydraulic, and photosynthetic traits by analyzing the relative differences in several key traits between species that cooccur in the same site. Here, we first quantified the difference (Δ) in TL, dx:dy, Ā, and VLA relative to the difference in LMA (ΔLMA) between all different species that cooccur at each site:(5)in which TraitX represents TL, dx:dy, Ā, and VLA, respectively, and the index i = 1 ... j-1 and the index j = 2 ... k, with k being the number of species that cooccur at each site, and the index n = 1 ... m, with m being the number of sites studied.
The observed differences in TL, dx:dy, Ā, and VLA relative to LMA were interpreted based on the sign of their average value. For example, if the average ΔTL/ΔLMA > 0, it means that species with thicker leaves have, on average and within sites, higher LMA. We tested whether the means of these relative differences deviated from zero using a two-tailed Student's t test at a 5% significance level, including a Bonferroni correction considering four comparisons. We excluded comparisons of species with a difference in the denominator LMA of less than 2 µg cm−2, which is equal to 5% of the total observed range in LMA.
Modeling Leaf Hydraulics and Photosynthesis
The potential advantage of reducing dx:dy below unity in terms of additional leaf gas exchange was quantified with a semiempirical model that relates lH2O to kleaf and subsequent transpiration and photosynthesis following Brodribb et al. (2007) and Brodribb and Feild (2010). Brodribb et al. (2007) provide an empirical expression for kleaf that, given that atmospheric humidity and temperature are known, can be related to a transpiration flux and subsequent photosynthesis following Brodribb and Feild (2010). Principally, the model from Brodribb et al. (2007) relates leaf morphology and subsequent lH2O (µm) to kleaf (mmol MPa−1 m−2 s−1) as:(5)in which(7)where τ is the curvature (or tortuosity) of the flow path through the leaf interior and assumed π/2, following Brodribb and Feild (2010), and dmx is the intervein distances as defined by Brodribb and Feild (2010). We note that dmx differs from the intervein distance dx in our study. Brodribb et al. (2007) defined dmx as the farthest equidistant point from surrounding veins. To apply our measurements of dx to the model from Brodribb et al. (2007), we use the conversion dmx = 0.56 × dx. This conversion was based on the difference in the relationships between dmx and VLA as reported by Brodribb et al. (2007) and between dx and VLA as obtained from our traced vein networks (Supplemental Fig. S2).
Brodribb and Feild (2010) use the expression for kleaf to model the steady-state transpiration rate by assuming that the flow of (liquid) water into the leaf (El) equals the (gaseous) transpiration flux (Eg):(8)(9)where ∆ψleaf (MPa) is the water potential gradient across the leaf, gsw (mol m−2 s−1) is the stomatal conductance to water vapor, and wi − wa (−) is the atmospheric humidity gradient between the leaf interior and the leaf exterior.
Brodribb and Feild (2010) approximated ∆ψleaf as constant (0.4 MPa) and based wi − wa on the typical growth conditions of their species. With these assumptions, Equations 8 and 9 can be equated and rearranged to yield an expression for gsw as a function of dx and dy.
For our analyses, we modified the geometrical relationships in the model framework from Brodribb and Feild (2010) and expressed kleaf in terms of the traits dx:dy and TL. Here, we invoked the assumption that dy = TL/2 (for empirical support, see Supplemental Data S1; Supplemental Fig. S1; Supplemental Table S2). For dx, we inserted its relationship with the ratio dx:dy and the expression dy = TL/2, so that . Inserting both expressions in Equation 7 yields:(10)Inserting this expression for lH2O in Equation 6 yields, after rearranging, a semiempirical expression for kleaf in terms of the leaf morphological traits dx:dy and TL:(11)The expression above is subsequently used in an inverse approach to quantify the effect of changing dx:dy on photosynthesis as explained in the following.
We rearranged the Fick's law expression for stomatal diffusion of CO2 to estimate the time-integrated stomatal conductance to CO2 ():(12)Our measurements of δ13Cleaf together with our approximation of Vcmax25 provide information on and Ā (Eqs. 3 and 4), which allow us to calculate for all our species-site combinations individually.
The time-integrated transpiration (ā) of each species-site combination was subsequently calculated as:13For wi, we assumed saturation at the annual average temperature for each site, and for wa, we used the annual average humidity for each site, based on the climate data from the Atlas of Living Australia.
With ā and kleaf known, we subsequently approximated the time-integrated hydraulic pressure gradient across the leaf (Δ) for each species-site combination as:14We then calculated the leaf hydraulic conductance of the theoretical optimal hydraulic architecture (kleaf_opt) with dx:dy = 1, in combination with the observed TL, using Equation 11. The transpiration of this theoretical optimal hydraulic architecture (Eopt) was then calculated assuming that Δ remains unchanged from the value obtained using Equation 14:(15)The stomatal conductance to water vapor associated with this theoretical optimal leaf morphology (gsw_opt) was calculated as:(16)The Rubisco-limited photosynthesis associated with the theoretical optimal leaf morphology () is modeled from Fick's law assuming that gsc_opt = gsw_opt/1.6:(17)In Equation 17, the leaf interior CO2 concentration ci_opt is unknown, but a second expression for steady-state photosynthesis as a function of ci is obtained from the biochemical model of Farquhar et al. (1980) for Rubisco-limited photosynthesis while accounting for photorespiration (von Caemmerer, 2000) and mitochondrial respiration in light, as described in Equation 4. The two expressions for steady-state photosynthetic rate were iteratively solved for ci = ci_opt with a prescribed ca of 380 μmol CO2 mol−1 air, reflecting the atmospheric partial pressure of CO2 in 2003 (Keeling and Whorf, 2004), using Matlab (version R2015b).
The above approach allows us to quantify the relative benefit of reducing dx:dy below unity on Rubisco-limited photosynthesis (δA [%]) for each species-site combination as:(18)We tested whether the average δA of our species-site combinations deviated from zero using a one-tailed Student's t test at a 5% significance level.
A sensitivity and uncertainty analysis of this model is provided in Supplemental Data S1 by exploring the effect of variability and uncertainty in model input parameters (Supplemental Table S5), on modeled photosynthesis (Supplemental Fig. S7), and δA (Supplemental Fig. S8).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Relationship between vein depth and leaf thickness.
Supplemental Figure S2. Relationship between vein length per unit area and the intervein distance.
Supplemental Figure S3. Intervein distance to vein-epidermal distance ratio and site-specific AI of hypostomatous versus amphistomatous species.
Supplemental Figure S4. Effect of artificial aging on eucalypt leaf area and thickness.
Supplemental Figure S5. Statistics with correction for the classification based on Nicolle (2015).
Supplemental Figure S6. Paradermal image and cross section of a eucalypt leaf.
Supplemental Figure S7. Model sensitivity analysis.
Supplemental Figure S8. Model uncertainty analysis.
Supplemental Figure S9. Paradermal view of cleared and stained eucalypt leaves: Corymbia aparrerinja.
Supplemental Figure S10. Paradermal view of cleared and stained eucalypt leaves: Eucalyptus gongylocarpa.
Supplemental Figure S11. Paradermal view of cleared and stained eucalypt leaves: Eucalyptus intertexta.
Supplemental Figure S12. Paradermal view of cleared and stained eucalypt leaves: Eucalyptus jacksonii.
Supplemental Figure S13. Paradermal view of cleared and stained eucalypt leaves: Eucalyptus marginata.
Supplemental Figure S14. Paradermal view of cleared and stained eucalypt leaves: Eucalyptus salmonophloia.
Supplemental Figure S15. Paradermal view of cleared and stained eucalypt leaves: Eucalyptus salmonophloia.
Supplemental Figure S16. Paradermal view of cleared and stained eucalypt leaves: Eucalyptus wandoo.
Supplemental Table S1. Overview of the species, location, stomatal distribution, and climate variables.
Supplemental Table S2. SMA regression statistics for Supplemental Figure S1.
Supplemental Table S3. Regression models for the relationship between dy and dx.
Supplemental Table S4. Data used to model Vcmax from Narea and LMA.
Supplemental Table S5. Parameter ranges used in the model sensitivity and uncertainty analyses.
Supplemental Data S1. Species-by-site trait data.
We thank Libby Trevenen and Hai Ngo (University of Western Australia) and Colin Crane (Department of Parks and Wildlife) for assistance with microscopy; Christiane Ludwig (Commonwealth Scientific and Industrial Research Organization-Agriculture) for laboratory assistance; Ines Hilker, Iris Kuhlmann, and Willi A. Brand (Max-Planck Institute for Biogeochemistry) for assistance with the leaf chemical analyses; and Grady Brand and Marty Brotherson for assistance with the collection of material from Kings Park.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Erik J. Veneklaas (erik.veneklaas{at}uwa.edu.au).
H.J.d.B. prepared samples, measured leaf traits, constructed the hydraulics model with input from P.L.D. and E.J.V., analyzed data, and drafted the article; P.L.D. prepared samples, measured leaf traits, derived the photosynthetic parameters, provided input on modeling, analyzed data, and drafted the article; E.J.V. and H.J.d.B. conceived the study and developed it with P.L.D.; E.J.V. oversaw measurements of leaf traits, provided input on modeling, and supervised and complemented the writing; E.W. prepared and measured leaves for vein density and stomatal morphological features; C.A.P. oversaw the vein measurements; E.-D.S. conceived the original sample collection campaigns, measured several leaf traits, and commented on the article; N.C.T. collected the leaf material and commented on the article; D.N. located and identified trees, collected leaves during the sample collection campaigns, and contributed a classification of the eucalypts.
↵1 This work was supported by the Centre of Excellence for Climate Change, Woodland, and Forest Health (State Government of Western Australia), the Australian Research Council (grant no. LP14100736), the Max-Planck Institute, the Netherlands Organization for Scientific Research, and an Endeavour Fellowship.
↵2 These authors contributed equally to the article.
↵[OPEN] Articles can be viewed without a subscription.
- leaf mass per unit area
- aridity index
- vein length per unit area
- principal component analysis
- Received August 23, 2016.
- Accepted October 24, 2016.
- Published October 26, 2016.


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American Traffic Props for LEGO Worlds now has a logo! Furthermore, it will soon have a project page here just like my Computers and More Mod does. Also I am starting a new project with some others to rip and analyze files from LEGO City Undercover for Wii U. This project is expected to take quite some time (the longest part being acquiring the files) but shouldn't distract me from my other projects.
Back to work I go!

subset: commoncrawl


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I didnt know that this would be my last show until five minutes before I hit the decks. As I was going over the set list and show notes one last time before I went on air I just decided its over. I screwed the whole lot up and just decided fuck the plans I am just gunna have a good time and go out with a blast playing my favourite metal tracks of all time instead of what I had on the set list. I also decided to play the best of the London bands that reached out to me on the last show I was doing with Squid.
I leave doing my rock show with no regrets and on a happy note instead of a bitter worn down and fed up one. The way I look at it is that I started something from my home standing up for something that I believed in. I reached out to thousands of people across the world, got to play some awesome gigs and made/met many great friends along the way.
My shows never went mainstream and I liked it that way. All I wanted to do was make a kick ass show which was an alternative to the other bullshit that is on the airwaves for my friends to listen to and anyone else who felt the same as I do about good metal music.
I dont give a fuck about any negative comments people will have to say about the show, my work and me ending the show. At the end of the day what have they ever done with their lives apart from sit behind a computer screen while they compile that list of people they hate under their Marilyn Manson poster!
I would like to thank the following bands/artists for reaching out to me and letting me play your tracks on air and they are:
Dave Laverty and all the guys from Resonator
Dave Christmas and F.S.I
Phil Roadkill and Generation Graveyeard
Michael Payne
Nick and the boys from Sonic Mass
Saggs and the boys from Self Loathing
Josh and Our people versus yours
Throne of Vengeance
Jimmy and Knockturn Alley
and last but not least
Lance Barrington of Valpurgis Night/Tilt for the amazing riffage, great times and for putting up with Steven!
I would also like thank all of YOU for listening and supporting me and my efforts over the last couple of years. I love you all!
As I tore up the plans I had for this show I hardly have anything to say apart from plugging bands so I just let the music do the talking. Sorry for any skipping this was due to the show being streamed live and was out of my control.
Thanks again


text:
Fuel Your Summer
Fuel Your Summer
No Excuse to be Bored this Summer!
The Sacramento Summer Meals Collaborative and its partners are thrilled to present Fuel Your Summer, which provides youth with access to the nutrition they need to grow and thrive during the summer months.
The Fuel your summer toolkit includes resources for site operators to help maintain and increase participation in summer meal service programs throughout the region. Explore the Fuel Your Summer Program and all the resources it has to offer below or download the full toolkit.
We keep adding to the toolkit, so check back often.


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Q ) How much does it cost to attend?
It's FREE to attend the Solar Tour. We believe that this should be an educational experience for everyone.
Q ) Is there a certain order that we have to view the solar project sites?
No. The sites presented on our quarterly tours are all open from 9am-1pm, so you are free to visit them at your leisure in what ever order you wish.
Solar electric power is one of the most attractive solar technologies of our time. If you are interested in powering your home or just offsetting some of your fossil fuel power consumption, consider installing a solar electric power system.
The following information helps answer the most frequently asked questions concerning solar electric power systems. Keep in mind that much of this information is "rule of thumb"; your individual situation, if analyzed in detail, may differ somewhat from the general application.
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What do we mean by photovoltaics? The word itself helps to explain how photovoltaic (PV) or solar electric technologies work. First used in about 1890, the word has two parts: photo, a stem derived from the Greek phos, which means light, and volt, a measurement unit.
So, photovoltaics could literally be translated as light-electricity. And that's just what photovoltaic materials and devices do; they convert light energy to electricity.
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PV takes advantage of the impurity characteristics of silicon crystals. Silicon impurities create crystal with a slight tendency to lose electrons, and other impurities create crystal with a slight tendency to attract electrons. When the two kinds of silicon are placed close together and exposed to sunlight, photons (particles of light) knock electrons loose on the unattractive side. An electrical current is created as electrons travel across the junction to the attractive side.
Sunlight is composed of particles of energy called photons. When sunlight strikes a PV material, photons will either pass through, be reflected, or be absorbed. If the photon is absorbed, its energy will be transferred to an electron in an atom of the PV material. With new energy, the electron is able to escape from its normal position in orbit around that atom. In this way, the electron can become part of, and augment, the current in an electrical circuit.
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Solar modules, or panels, are series of solar cells wired together into strings and enclosed in self-contained glass units for harsh weather protection. Solar cells are mounted into groups called modules that produce about 0.5 Volts of current to power lights and appliances. On the sunward side, cells are protected by a highly transparent solar glass pane. The underside takes the form of an insulating film or a second pane of glass. A connection socket picks up the generated direct current. Modules are connected together via cables, which link them to the inverter.
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No. Solar cells just convert sunlight into an electric current that must be used immediately or stored in batteries to be used later.
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A PV array is an interconnected system of PV modules that function as a single electricity-producing unit. The modules are assembled as a discrete structure, with common support or mounting. In smaller systems, an array can consist of a single module. A complete set of components for converting sunlight into electricity includes a module, a support structure, wiring, an inverter, a meter and other equipment.
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A PV system is made up of different components. These include PV modules (groups of PV cells), which are commonly called PV panels, batteries and a charge regulator or controller for a stand-alone system, an inverter for a utility-grid-connected system and when alternating current (ac) rather than direct current (dc) is required; wiring; and mounting hardware or a framework. Systems that are connected directly to your home (98% of all systems installed do not require batteries).
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There are two main types of solar energy technologies:
Q ) Can I use photovoltaics (PV) to power my home or business?
PV can be used to power your entire home or business electrical systems, including lights, cooling systems, and appliances. PV systems today can be blended easily into both traditional and nontraditional homes. The most common practice is to mount modules onto a south-facing roof or wall. For an additional aesthetic appeal, some modules resemble traditional roof shingles or can be built right into glass skylights and walls. This building-integrated PV provides a dual-use building material, reduces PV system costs by using the building as the mounting or support structure, and reduces utility bills with on-site power production.
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This is a difficult question to answer. Due to the rising electrical rates the answer is not the same for everyone.
Yes. A solar system on your home will lower your electric bill. Unfortunately the initial layout of funds to purchase the system can be high. If you look at the cost per kilowatt hour for a solar system over its life it can be as low as $0.11 per kilowatt hour. If your goal is to save money over the life of the system and you are paying the power company more then $0.11 per kilowatt hour a solar system makes sense.
If you are paying less then $0.11 per kilowatt hour solar may not be for you from a cost savings stand point. A kilowatt hour of Solar will cost more than a kilowatt hour of utility electricity.
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People decide to buy solar energy systems for a variety of reasons. For example, some individuals buy solar products to preserve the Earth's finite fossil-fuel resources and to reduce air pollution. Others would rather spend their money on an energy-producing improvement to their property than send their money to a utility. Some people like the security of reducing the amount of electricity they buy from their utility, because it makes them less vulnerable to future increases in the price of electricity.
If it's designed correctly, a solar system might be able to provide power during a utility power outage, thereby adding power reliability to your home. Finally, some individuals live in areas where the cost of extending power lines to their home is more expensive than buying a solar energy system.
Q ) What is net metering? Can I sell power back to the utility grid?
Net metering is a policy that allows homeowners to receive the full retail value for the electricity that their solar energy system produces. The term net metering refers to the method of accounting for the photovoltaic (PV) system's electricity production. Net metering allows homeowners with PV systems to use any excess electricity they produce to offset their electric bill. As the homeowner's PV system produces electricity, the kilowatts are first used for any electric appliances in the home. If the PV system produces more electricity than the homeowner needs, the extra kilowatts are fed into the utility grid.
The real question is can I sell power back to the utility company. Most states have net metering bills that basically say the utility company will have to buy the power back from you at the same rate you buy power from them. The states that don't have net metering bills typically only pay the wholesale cost of the power about 1-3 cents. Until uniform codes have been adopted by all utilities it is best that you first check with your utility. Each utility has different interconnection standards that have to be meet before they will allow you to sell power back to the utility grid.
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The amount of solar you need to power your home is not dependent on the size of your home. The amount of solar depends on the amount of power you use in your home.
Look at your monthly bill from the utility company. It will list the total amount of power (kWh) you used in the last billing cycle. Divide this by the number of days in your last billing cycle. This will tell you how much power you use each day (kWh).
Q ) What is an average break-even point for a solar energy system?
It depends. The break-even point for a system depends on financing and incentives, which vary from place to place, and it depends on your solar resources and what you would pay for another source of energy. A system designer that has information about your location, the amount of energy you typically use, how much land or roof area you have for the system, etc., could give you a more accurate answer.
Typically the payback period for a solar system is roughly 17 years, based on calculations assuming the cost of energy obtained from the utility grid is 15 cents a kilowatt-hour.
Q ) How long do photovoltaic (PV) systems last?
A PV system that is designed, installed, and maintained well will operate for more than 40 years. The best way to ensure and extend the life and effectiveness of your PV system is by having it installed and maintained properly.
Q ) How much electricity does a photovoltaic (PV) system generate?
A 10% efficient PV system in most areas of the United States will generate about 180 kilowatt-hours per square meter. A PV system rated at 1 kilowatt will produce about 1800 kilowatt-hours a year.
Q ) Can I design and install a photovoltaic (PV) system myself?
Maybe! However, unless you are very handy or experienced in home wiring, we suggest using experienced professionals to design and install anything more than the simplest application.
The goal of a stand-alone system designer is to assure customer satisfaction by providing a well-designed, durable system with a 40-year life expectancy (or more). This depends on sound design, specification and procurement of quality components, good engineering and installation practices, and a consistent preventive maintenance program.
Q ) How do I know if I have enough sunlight for PV?
A photovoltaic (PV) system needs unobstructed access to the sun's rays for most or all of the day. Climate is not really a concern, because PV systems are relatively unaffected by severe weather. In fact, some PV modules actually work better in colder weather. There is thus enough sunlight to make solar energy systems useful and effective nearly everywhere in the United States.
Q ) How well do solar panels work in cloudy conditions?
A cloudy day provides sufficient diffuse light by which the panel will produce electricity. Optimum electrical production occurs with bright and sunny weather conditions. Under a light overcast, the modules might produce about half as much as under full sun, ranging down to as little as five to ten percent under a dark overcast day.
In remote, off-grid applications, a PV system is connected to a battery storage system as a backup power source. In grid-connected applications, the PV system works in parallel with the utility power grid. So, if electrical needs exceed the solar power output, the local utility makes up for the shortfall. Conversely, when the PV system generates more energy than the building requires, the excess power is exported to the utility grid, reversing the electrical meter!
Q ) Can I use equipment directly from solar panels?
Yes, solar panels directly power equipment such as fans and pumps, as long as the load is accounted for correctly. Equipment load that is greater than the output of the solar panel will weaken equipment efficiency, as overcast or cloudy days reduce output. Equipment that requires a more stable voltage should pair solar power with a battery backup.
Q ) What are the possible problems with solar panels and how can I prevent them?
Solar panel failure is most often caused by water damage to the panel, sealant, or connections. To prevent damage or failure, mount the panel carefully. Be sure not to fix it horizontally, which encourages water collection in the frame. Also, allow for a sufficient air gap beneath the panel. Keeping your panel dry and clean will ensure efficient, maximum output.
Q ) Are solar electric systems safe?
Yes. Solar cells are mostly silicon, the primary component of sand. There is no exhaust and no toxic materials to leak out of the system. The electricity coming through the inverter is just like the electricity coming from household wall sockets; you should use the same care you would with utility power. All components are approved for utility interconnection and are installed according to standard construction practices.
Q ) Are solar power systems good for the environment?
Energy created through our solar electric system produces no pollutants. Our smallest system typically cuts greenhouse gas emissions as effectively as 50 trees.
Q ) Can I Start Small And Add On Later?
Yes. Solar is quite unlike a computer. If you start with a couple of good base components it is easy to add to your system later.
Q ) How many solar panels and what size are enough to meet my needs?
Each energy consumer has different needs, dependent upon the electric appliances being run. For instance, if you are powering two 27 watt compact fluorescent light bulbs for two hours, you would need to supply 108 watt-hours of energy (2x 27x 2 hours). In Dallas, which has a yearly average insolation of 5 hours a day, one Unisolar 32 watt photo voltaic (PV) module is more than adequate (32x 5 hours=160 watt-hours).
Q ) Why choose solar over wind- or water-power generation?
Every energy source has its advantages and disadvantages. Wind- and water-power generation may be appropriate in certain situations. However, these technologies are:
PV modules, on the other hand, are:


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Learn more about the Readers Set of 1, 2, and 3 from the main 1857 McGuffey Readers page, and click on the individual titles below to see what is included in each one. Enjoy!
First Reader • Second Reader • Third Reader
Each volume centers on the original text from McGuffey's expanded 1857 edition, the last edition with which McGuffey was personally involved, and the first edition with six volumes and more extensive teacher helps.


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Sorry I haven't been around much lately. Besides being super busy as usual, my computer died, and I lost power for the better part of a week thanks to Hurricane Sandy. I have a ton of requested re-post's to get to, and hopefully (when I get caught up) some new records one of these days as well. I'm going to start from the oldest ones, and work forward. If I forget you, please don't hesitate to ask again.
SIXTEN REDLÖS - Slå Alarm 1987
1 hour ago


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The training course of development of the capacities and skills of members of community initiatives in the governorate in administrative and developmental aspects was concluded on Thursday morning at the technical institute in Alhuban, Taiz governorate.
Participates were 68 participants from various governorate departments.
At the end of the session, the first deputy of Taiz province, Mr. Mansour Al-Lakoumi, noted the role of Bonyan Foundation, the Industrial Technical Institute and the Coordination of Humanitarian Affairs in organizing the course to rehabilitate youth and develop their abilities in administrative and developmental aspects.
He pointed to the importance of finding leadership for humanitarian work in Taiz province. He pointed out that Taiz is characterized by human resources qualified and capable of giving in various aspects.
Al-Lakoumi expressed he hope the initiatives and projects that will be provided by youth will continue to serve the country and the local community.
While the Director of the branch of the National Authority for the Management and Coordination of Humanitarian Affairs Ahmed Al-Masawi announced the opening of the Youth Initiatives House, which will be handed over to Bonyan Foundation for Youth Rehabilitation, House Management and Youth Initiatives, and the combination of youthful capacity and project management.
For his part, the representative of Bonyan Hamza Al-Amad praised the interaction of the local authority with the community figures and the youth in the course. He pointed out that the course focused on the rehabilitation of young people in the management and research methods of development, project management and community leadership.
The closing ceremony was attended by Deputy Governor Najib Al-Najdeen, Director of Political Security Yahia Al-Ahrami and Dean of the Industrial Technical Institute Mansour Mane'.


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Your safety is our priority. We're doing more than ever to help ensure your protection and well-being when you come see us for care. Learn more.
Get regional updates for Providence Washington.
As a Providence caregiver, regardless of your role, you already demonstrate your commitment to making a difference in the lives of others. Your impact is even greater with financial contributions made through the employee giving campaign. While gifts can be made at anytime, this years campaign runs May 1-31, 2019.
Between May 1-31, 2019, we encourage you to make a difference and give a gift to Providence St. Peter Foundation (Olympia-based) or Providence Health Care Foundation (Centralia-based) in support of the mission to provide excellent and compassionate care to all. This is your opportunity to make an additional difference in the life of a patient, a co-worker or a program that means so much to you. You can designate your gift to the fund of your choice, and 100% of your gift goes to that cause.
We ask our caregivers to give because you believe. Every gift given, no matter the amount, improves lives of people in our communities, people in our care and people we work with. Please give because you believe in the Providence Mission.
Providence St. Peter Foundation (Olympia): 360-493-7981
Providence Health Care Foundation (Centralia): 360-827-7940


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Jeep Grand Cherokee Half And Half
Jake Holmeswriter
The Jeep Grand Cherokee Half And Half is supposed to demonstration how drastically Mopar parts can transform a Chrysler Group vehicle. The SUV is divided in half by a zipper graphic and a real zipper pull: one side is a stock Grand Cherokee Laredo, while the other side has been transformed with aftermarket parts. Those include 20-inch chrome wheels; a chrome grille insert, mirrors, side steps, and foglight housings; a trailer hitch; tow hooks; a roof rack; and a luggage rack. The interior is likewise divided in two, with the modified half featuring Katzkin leather, all-weather mats, sportier pedals, and new doorsill plates.


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Student Leadership Team Giving Back Locally and Globally
Our Student Leadership Team made a commitment to make a difference both locally and globally after attending the WE DAY events in Saskatoon at the beginning of the school year.
On Friday, May 22 they will be hosting a Spring Activity Day for Prince Arthur students. They have a variety of games planned for the classrooms to take part in. As a way of giving back they are asking each student to bring at least $1 to donate towards Hunger in Moose Jaw. Each day this local group delivers over 300 school lunches to students around the city. The lunches contain at least one serving from each of the food groups on Canada's Food Guide and is a nutritious free opportunity for students.
The Student Leadership Team is also planning an event to give back Globally. They have chosen a Village in Nepal who has been recently affected by the earthquakes that have hit the region. The SLT will be hosting a TCBY frozen yogurt sale to raise funds for this amazing cause. Details about the sale will be coming home with students so stay tuned for further information.


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On behalf of Panther Pride, stay healthy and safe. Please check back later for updates regarding athletic events and the 2020-21 school year.
2019-2020 Panther Pride Membership
Panther Pride membership for the 2019-2020 school year is now available. Please fill out the membership form OR join online.
2019-2020 Volunteer Opportunities
Dear Panther Nation,
As the school year enters the final months of this academic year, it is also time to look forward to the 2019-2020 year. It is during this time each year that the All Sports Booster Club searches for new board members, new committee members and club volunteers. As our community and high school grows and matures, we need more people to help support our mission.
We are in need of new board members to serve as leaders of the organization. The board consists of 8 individuals who serve in various roles with difference responsibilities including: event planning, membership, Panther Outfitter sales, financial planning, liaison between coaches and team sports reps, sponsorship and fundraising, and overall leadership. The board meets once per month and each member works individually throughout the year in support of the organization.
Committee volunteers are the lifeblood of the organization. We need more volunteers with new ideas, enthusiasm and a desire to serve the student athletes and the high school athletic program at Ridge Point. Committees include: hospitality, membership, e-Commerce, concessions, special events, merchandise sales, and social media. Committees are lead by chairpersons or board members and generally meet 3 – 4 times per year or as needed.
If you are interested in volunteering, working on a committee or serving as a leader or board member, please contact Diron Blackburn, board president, at email@example.com.
Thank you for your consideration and support!
Best Regards,
Diron L. Blackburn
President 2017-2019
RPHS All-Sports Booster Club


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Danny Brassell | Why Leaders Are Readers
Today's guest has helped people from all walks of life, from kindergartners to rocket scientists, achieve more, overcome adversity, and give back through his dynamic and engaging speeches. As the son of a librarian, Danny hated reading, but today he is called America's leading reading ambassador.
Dr. Danny Brassell comes from a background that encouraged him to reach for the stars and he has dedicated most of his life to motivating others to reach for their potential as well.
In this episode, you will…
- Hear the four critical turning points that led Danny to the success he has today
- Discover how a steady diet of positive influences has helped him to achieve his full potential and continue to grow
- Find out how Danny handles setbacks and how he reacts to challenges
- Be inspired by the story of Conrad Hilton
- Learn why reading every day is such an important habit for leaders
Episode Resources
Think and Grow Rich- Napoleon Hill
The Tale of Peter Rabbit-Beatrix Potter
How to Win Friends and Influence People- Dale Carnegie
The Greatest Salesman in the World- Og Mandino
Psychocybernetics-Maxwell Maltz
The Power of Positive Thinking-Norman Vincent Peale
Swim with the Sharks without being Eaten Alive-Harvey Mackay
Becoming Your Best- Steve Shallenberger