Abstract
Conifers prevail in the canopies of many terrestrial biomes, holding a great ecological and economic importance globally. Current increases in temperature and aridity are resulting in conifer mortality and imposing high transpirational demands to global vegetation. Therefore, identifying leaf structural determinants of carbon acquisition and water use efficiency is essential in predicting physiological impacts due to environmental variation. Using synchrotron-generated microCT imaging, we extracted leaf volumetric anatomy and stomatal traits in 34 species across the conifers with a special focus on Pinus, the richest conifer genus. We show that intrinsic water use efficiency (WUEi) is driven by leaf vein volume, with both traits scaling positively. The ratios of stomatal pore number per unit mesophyll or intercellular airspace volume emerged as powerful explanatory variables, accurately predicting both stomatal conductance and WUEi. Our results clarify how the three-dimensional organization of tissues within the leaf has a direct impact on plant water use and carbon uptake.
Introduction
Conifer forests thrived on the Earth surface during the Mesozoic until the radiation and diversification of angiosperms during the Cretaceous, which was followed by angiosperm ecological dominance attributed to increased physiological performance and reduced generation times 1–4. Yet, after 100 million years of competition with the angiosperms, conifers remain prominent in the canopy of many biomes 5. Conifers are found in ecosystems from high latitudes to the equator, and they have a major economic importance in the wood and paper industries 6. Conifer-dominated forests are not exempt from the impacts of drought and aridity resulting from ongoing global climatic changes 7–9. This is particularly alarming given that 48% of the 722 conifer taxa of the world are currently threatened 10. Global increases in temperature coupled with rising vapor pressure deficit (VPD) place increased strain on plant hydraulic and photosynthetic systems 11,12. There is strong evidence that tree water use efficiency (WUE) has increased in recent decades, most likely the result of rising atmospheric CO2, allowing plants to open their stomata less frequently, thereby conserving water 13,14. However, the underlying physiological mechanisms behind this trend need to be further elucidated 15. Furthermore, there is still a lack of knowledge about how leaf structural organization influences key functions such as photosynthetic carbon acquisition, stomatal conductance, and the interplay between both driving intrinsic water use efficiency (WUEi).
Leaf-level photosynthetic metabolism has an important role in maintaining global ecological processes 16. Therefore, exploring tissue organization inside the leaf, and specifically the mesophyll cells where water and gas diffusion occurs, is important for understanding carbon, water, and energy fluxes at whole ecosystem levels. The leaf mesophyll consists of photosynthetic parenchyma cells located between the epidermis and the bundle sheath layers surrounding the veins. Once inside the leaf, the diffusion of CO2 through the intercellular airspace (IAS) and to the chloroplasts (i.e. mesophyll conductance) is a major constraint on photosynthetic performance 17,18. This pathway includes the IAS, but also the diffusion of CO2 across mesophyll cell walls, cell membranes, and the chloroplast envelope, which can significantly limit photosynthesis in gymnosperms 19. Previous work has shown that mesophyll surface area per leaf area (Sm; μm2 μm−2) had a strong influence on maximum photosynthesis 20,21. More recently, it has been suggested that the surface area of the mesophyll exposed to the IAS per unit of mesophyll volume (SAmes/Vmes; μm2 μm−3) can influence plant photosynthetic performance given that the mesophyll-IAS boundary is the primary interface between the atmosphere and the photosynthetic cells 22,23. Other volumetric anatomical traits such as mesophyll porosity (i.e. IAS as a fraction of total mesophyll volume; μm3 μm−3) might be relevant in promoting diffusion through the IAS, and it has been suggested that mesophyll palisade porosity is correlated to stomatal conductance across four different Arabidopsis mutants 24. The relevance of such anatomical traits arises from the hypothesis that photosynthetic capacity could be enhanced by increasing surface area of mesophyll exposed to the IAS, allowing for more potential surface for CO2 diffusion across mesophyll cell walls, and chloroplast envelopes 22,25. However, the correct estimation of such anatomical traits using standard two-dimensional (2D) techniques is difficult since it relies on 2D approximations of the complex, three-dimensional (3D) shape of the mesophyll 26,27.
Given the elementary vascular architecture of the conifer leaf, which typically consists of a single vein without further branching 28,29, and the large variation in conifer leaf shape (Fig. 1), area-based traits might not allow for an accurate comparison of strategies to optimize leaf structure with function within conifers 23,27. Further, given the structural relevance of the central vasculature, traits other than those related to the mesophyll might also influence CO2 and water diffusion in the conifer leaf. Therefore, a central question is whether non-vascular tissues are tightly coupled to vein volume with proportional relationships, or whether they show greater variability to compensate for reduced hydraulic efficiency. We predict that higher volumes of vascular tissue per unit leaf volume (Vvein/Vleaf; μm3 μm−3) should be a good predictor of gas exchange and WUEi because of the mechanistic link between hydraulic conductance and maximum photosynthetic rates in conifers 30. We also predict a positive relationship between gas exchange efficiency and the ratio of mesophyll surface area exposed to the IAS and vein volume (SAmes/Vvein; μm2 μm−3), where a greater investment in vein volume per unit area of bulk tissue surface should increase the hydraulic capacity to replace water lost to transpiration. In conifer leaves, water moves across the bundle sheath and the transfusion tissue before reaching the mesophyll 31. Therefore, bundle sheath and transfusion tissue volume relative to total leaf volume (VBS+TT/Vleaf; μm3 μm−3), should also influence the efficiency of water movement within the leaf. Along with the previously described features, many conifer species also possess resin ducts, which play a major role in chemical and physical defense 32,33, but necessarily displace vascular or photosynthetic tissue, incurring both maintenance and construction costs, but also lost opportunity costs for net carbon gain. Finally, water movement inside the leaf ends at the stomatal pores, which play a major role in regulating water loss and maintaining water status in conifers 34. Within this context, it is possible to determine the proportionality of different anatomical traits, the coordination between supply and demand for CO2 and H2O, and the physical constraints of leaf construction. To probe these relationships, we describe how conifers build their elementary, yet diverse leaves, and how structural features relate to key physiological traits.
This study presents a survey of the three-dimensional organization of the conifer leaf using microCT imaging (Supplementary Table 1). Our study includes 34 conifer species (Supplementary Fig. 1), with a special focus on the genus Pinus, the largest extant genus of conifers 35. Pinus species can be found in a broad range of environmental conditions, suggesting wide structural and functional diversity 36. Pinus subgenera Pinus and Strobus can be distinguished by having two or one vascular bundles respectively, centrally located inside the needle-like leaf 35 (Figs. 1a-c). Despite their rather simple anatomical organization, conifer leaves have extensive morphological diversity ranging from flat leaves to needle-like leaves with different degrees of transversal flattening. Leaf morphological diversity in conifers results in different physiological performances, with flat-leaved species having lower photosynthetic assimilation and respiration rates than needle-leaved species 37–39. Yet, the differences in 3D anatomical structure across conifers with different leaf morphologies, which could explain their contrasting photosynthetic performance, need to be elucidated. We hypothesize that features enhancing mesophyll surface area for gas diffusion, will be positively correlated with the light-saturated assimilation rate of CO2 (Asat) and maximal stomatal conductance (gsmax). We also provide a volume-based stomatal density estimation, a trait we expect better captures the interplay between the evaporative surfaces invested in the non-laminar mesophyll volume and the number of stomata needed to provide CO2, with the expectation that leaves with higher number of stomata per mesophyll volume would have both higher rates of gas exchange and WUEi due to an enhancement of the epidermal pores serving as evaporative surface relative to the photosynthetic tissue.
Results
The mesophyll (Vmes/Vleaf), including cells and airspace, represents the dominant leaf volume fraction for all 34 measured conifer species in this study, occupying an average of 60% of the total leaf volume (Fig. 1; Supplementary Fig. 2). The second largest volume fraction was either the combined bundle sheath and transfusion tissue (VBS+TT/Vleaf) that surrounds the vascular cylinder, or the epidermis (Vep/Vleaf), which represented an average of 22% and 16% of the leaf volume, respectively (Fig. 1a). Veins (Vvein/Vleaf) and resin ducts (Vresin/Vleaf) represented the smallest fraction of the total leaf volume (Fig. 1a), and resin ducts were completely absent in six measured species (Supplementary Dataset 1). We found weak structural coordination amongst tissue volumes within the conifer leaf (standard major axes; Supplementary Table 2). A negative allometric scaling between Vep/Vleaf and Vmes/Vleaf was observed (r2 = 0.27, p < 0.01), indicating that increasing the relative allocation to the mesophyll was done in conjunction with a decrease in the relative allocation to the epidermis. Average values of all measured volumetric traits for each species are included in Supplementary Dataset 1.
A multivariate analysis of trait covariation defined two major axes explaining 48% of inertia (Fig. 2). Inertia of the first axis was mainly explained by VIAS/stomate, the amount of air volume connected to a stomate (18.27%), stomata/Vmes, the stomatal density per mesophyll volume (15.04%), Vmes/Vleaf (14.74%) and gsmax (14.08%). Increasing gsmax was associated on the first axis with increases in stomata/Vmes and Asat, and decreases in mesophyll porosity, Vmes/Vleaf, and VIAS/stomate (Fig. 2). The second axis was largely explained by Vvein/Vleaf (18.04%), SAmes/Vvein (14.83%) and Sm (14.45%). Increasing WUEi was associated on the second axis with increases in Sm and Vvein/Vleaf, and decreases in stomata/Vmes (Fig. 2). Leaf morphological types were dispersed along both major axes (Fig. 2). Needle-like leaves were isolated due to their higher Vep/Vleaf and stomata/Vmes. Flat leaves and flattened needles largely overlapped due to their porous and voluminous mesophylls as opposed to needle-like leaves (Figs. 3a, b). Flat leaves and flattened needles also converged in having lower stomata/Vmes (Fig. 3c) and higher VIAS/stomate (Fig. 3d) than needle-like leaves.
However, flattened needles were differentiated due to some species having significantly higher SAmes/Vvein (Fig. 2; Supplementary Table 3). Conifer groups were also effectively segregated based on their volumetric anatomy. Non-Pinus conifer species were segregated on the first axis (Fig. 2) due to their higher porosity (Fig. 1d), Vmes/Vleaf, and VIAS/stomate values, while species from the Pinus subgenus were grouped (Fig. 2) due to higher Vep/Vleaf (Fig. 1b), along with a relatively larger SAmes/Vmes. Species from the Strobus subgenera were located between the previously described conifer groups (Fig. 2). Structural divergences of leaf morphologies, distinguishing three distinct functional groups, along with the segregation of different conifer clades in the PCA analysis were further supported with one-way ANOVA analyses on 3D leaf anatomical traits and stomatal density (Supplementary Tables 3,4). Relative vein volume and SAmes/Vmes were highly conserved (Supplementary Fig. 3; Supplementary Tables 3,4), whereas SAmes/Vvein, along with traits related to the ratio of stomatal pore number and mesophyll tissue volumes had significant differences across both leaf morphologies and conifer groups (Fig. 3c,d; Supplementary Fig. 3; Supplementary Tables 3,4).
To explore the functional implications of 3D tissue content, we determined their relationships to gas exchange parameters such as Asat, gsmax, and WUEi. WUEi was best predicted by Vvein/Vleaf (Fig. 4a), where higher Vvein/Vleaf enhances leaf WUEi. Further, 2D anatomical estimations of the ratio Avein/Aleaf were comparable to Vvein/Vleaf, extracted using a 3D approach (Supplementary Text 1; Supplementary Fig. 4). The mesophyll surface area exposed to vein volume (SAmes/Vvein) was also an accurate predictor and showed a negative relationship with WUEi (Fig. 4b). A positive relationship of WUEi with Sm was also found using generalized least-square models corrected for phylogenetic relatedness (PGLS) analyses (Supplementary Table 5). Asat was the physiological trait showing the least linkage with 3D structural traits. However, we found a negative relationship between Asat and mesophyll porosity along with Vmes/Vleaf (Supplementary Fig. 5a), suggesting that conifer species with lower relative mesophyll volumes have greater photosynthetic assimilation rates. Additionally, VIAS/stomate was also negatively related to Asat (Supplementary Fig. 5b). The number of stomata per unit mesophyll tissue volume predicted gsmax and WUEi (Fig. 5). For instance, high stomatal densities relative to mesophyll volume (stomata/Vmes) enhanced gsmax (Fig. 5a) while decreasing WUEi (Fig. 5b). Evolutionary coordination between leaf volumetric anatomy and physiological traits was supported by PGLS analyses (Supplementary Table 5). Stomatal density measured on a 2D leaf surface area basis (stomata/Aleaf), included here as a reference of a more standard approach, did not relate to any physiological trait (Supplementary Table 5). Therefore, the interaction of mesophyll volume and stomatal numbers emerged as a key trait to explain leaf physiological performance (Fig. 5). A comparison of the studied conifers with published data for other gymnosperm species, along with angiosperms and ferns, showed that conifers had fewer stomata per unit mesophyll volume than angiosperms sensu largo (Supplementary Fig. 6; Supplementary Dataset 2).
However, this difference was less important when considering evergreen angiosperms alone. Conifers showed a similar stomata/Vmes ratio as other gymnosperm species and ferns (Supplementary Fig. 6).
Discussion
Conifers, with their long-lasting and anatomically elementary leaves, must rely on leaf construction to enable sufficient carbon assimilation to survive and reproduce while limiting water loss. However, the simple design of coniferous leaves have been considered to have a poor, if not absent, hydraulic connection between the vein xylem and the bulk leaf tissue 40. This is unsurprising since conifer leaves are not as fully vascularized as those of angiosperms and present a single cohort of relatively inefficient leaves 4. The likely consequence of this poor hydraulic connection is a larger difference in water potential between the veins and the mesophyll and epidermis in transpiring leaves, which may force stomata to close even when water potential is relatively high in the veins 40. Our results show that despite occupying a small fraction (ca. 2%) of the leaf volumetric matrix, vein tissue volume has a great impact on the leaf WUEi (Fig 4a). As the relative vein volume expands, the space between the vascular tissues and the bulk leaf tissues become smaller, potentially reducing the hydraulic resistance for water transport from the vasculature to the bulk leaf. Our study provides an estimation of the full volumetric space occupied by the vein tissue relative to the photosynthetic cells. While we consider volumetric estimates to be more accurate given that they integrate traits over a larger leaf fraction than 2D estimates, we found that standard 2D anatomical estimations of the relative surface covered by veins over a few leaf cross sections could be accurately employed to predict the vein volumetric fraction, and in turn WUE in conifer leaves (Supplementary Text 1; Supplementary Fig. 4). Stomatal and venation densities covary in angiosperms, directly affecting gas exchange and water use capacities 41. However, conifers do not provide a dense network of veins within the mesophyll to irrigate the photosynthesizing cells in water. Consequently, conifers might be constrained to increase the relative volume of veins as a single way to provide more water to the leaf.
Previous work has suggested that narrow, needle-like leaves in conifers, which is a common feature in the Pinaceae 29,39, would be a response to alleviate their lack of hydraulic ramification. With the photosynthetic tissue encircling the single vascular cylinder, the distance from the vein to the epidermis largely sets hydraulic conductance outside the xylem, and leaf width becomes a major limiting factor in cylindrical, narrow leaves. Increasing the radial pathlength for water transport should effectively limit leaf hydraulic conductance, in turn limiting photosynthetic rates 29. Our data support this hypothesis, with a negative relationship between mesophyll volume and Asat (Supplementary Fig. 5a). Thus, within our dataset we find evidence for a significant photosynthetic penalty for increasing leaf width and mesophyll volume in flat and flattened needle leaves (Fig. 3b). With a limited ability to maximize photosynthetic capacity through hydraulic ramification, the cylindrical needle-like leaf may offer opportunities for other structural elements that allow improved hydraulic performance. Accessory transfusion tissue is one of them, where specialized cells connect the veins and/or bundle sheath to the mesophyll tissue, providing more water to the photosynthesizing cells and improving hydraulic contact to the epidermis 42. This might lead to increased photosynthetic rates and WUE. Our results support this linkage, with a positive relationship of the combined volumes of bundle sheath and transfusion tissue with photosynthetic assimilation (Supplementary Table 5). It has been stated that reaching high photosynthetic rates requires high leaf porosity values, which might increase CO2 diffusion 7. Yet, we observed a different trend with a negative relationship between mesophyll porosity and Asat (Supplementary Table 5). Moreover, Asat was strongly negatively related with Vmes/Vleaf in our dataset (Supplementary Fig. 5a). Previously, a decline of illumination-induced fluorescence as a function of leaf depth was observed in two conifers with needle-like leaves 43. Therefore, the decrease of Asat in leaves with more voluminous mesophylls might be explained by a limitation of light propagation across the mesophyll. Interestingly, we found conspicuous differences in leaf design between both Pinus subgenera and other conifers (Figs. 1,2; Supplementary Table 4), with narrow needle-leaved Pinus possessing less voluminous and porous mesophylls. Such differences in mesophyll construction could explain why Asat is greatest in Pinus species bearing needle-like leaves.
SAmes/Vvein is another feature that diverged across leaf morphologies and conifer clades (Supplementary Tables 3,4). We propose SAmes/Vvein as another anatomical trait involved in regulating the control over the loss of water (Fig. 4b). Minimizing this ratio would mean that less surface is available for evaporation for a given vein water volume, increasing the time before this capacitor is depleted, and thereby lowering the ‘safety margin’ between stomatal closure and xylem cavitation 40. Also, in the context of poorly connected hydraulic design, decreasing SAmes/Vvein would minimize the apoplastic surface for water to travel from the vein to the epidermis, hence by proxy decreasing the water path length and increasing connectivity to the epidermis to allow stomata to stay open longer and photosynthesis to continue. In our dataset, decreasing SAmes/Vvein was achieved mainly by increasing the relative volume of veins, i.e. investing more in vascular tissue (Fig. 4). The relative benefit of investing in vein volume to increase the efficiency in water use seems to plateau above ~3% of leaf volume, and most species produce invests in veins close to that relative volume (Fig. 4a). Pinus species, which bear needle-like leaves, have in addition decreased the relative volume of the mesophyll by decreasing airspace volume (Figs. 1; 3a,b; Supplementary Fig. 3a,b) with plicate mesophyll cells 44. Decreasing porosity leads to more cells being in contact with each other, thereby decreasing SAmes, i.e. the surface of cells exposed to the IAS. Increasing IAS had a positive effect on WUE in six angiosperm species 45. Although positive, we could not find a significant relationship between mesophyll porosity and WUEi in conifer leaves (Supplementary Table 5). Beyond considering mesophyll features alone, mesophyll volumetrics in interaction with stomatal pore number emerged here as key traits to explain conifer gas exchange and WUEi (Fig. 5). Using Arabidopsis and wheat as model plants, it has been suggested that stomatal differentiation during leaf development might induce mesophyll airspace formation 24. Our study shows that this coordination between VIAS, along with Vmes, and stomatal number have a significant impact on carbon assimilation and gas exchange on conifers (Fig. 5; Supplementary Table 5), further supporting this crucial linkage. Considered in a wider context, our observations might provide a novel structural basis to explain the lower photosynthetic rates of ferns and gymnosperms as compared to angiosperms, since we show that they have greater mesophyll volume per stoma, acting as a bottleneck that limits their evaporative capacities (Supplementary Fig. 6).
Current increases in temperature and atmospheric CO2 concentrations might impact the structure and function of conifer forests worldwide, and it has been posed that improved WUE could alleviate the temperature effect 7. Previous studies have shown higher WUEi under increasing CO2 in conifer species, having stronger WUEi responses than angiosperms 46–48. Additionally, it has been recently shown that higher plasticity in the vascular tissue of the needles of Pinus pinaster enhances their WUEi 49. Using an experimental approach, needles of Larix kaempferi growing under higher CO2 showed increased mesophyll surface area per leaf area, coupled with higher photosynthetic rates50. Moreover, it was shown that elevated CO2 increased mesophyll surface and decreased stomatal density in Pinus sylvestris needles 51. Therefore, under such elevated CO2 conditions, we might expect to observe a lower stomata/Vmes ratio, which would enhance WUEi according to our predictions (Fig. 5b). Further, given the recently demonstrated link between enhanced WUEi and vascular tissue plasticity in conifer needles 49, we expect that coordinated changes in vascular and mesophyll tissue volumetrics, along with shifts in stomatal pore number in conifer leaves, may allow conifer species to cope and adapt to the pressure exerted by increasing VPD in many global biomes11 by maintaining similar carbon assimilation levels with lower water consumption.
Materials and Methods
Plant material
Sampling included 34 conifer species from various biomes, physiologies, and leaf morphologies. Sampling included taxa from four different families of conifers: Araucariaceae (2 spp), Pinaceae (30 spp), Podocarpaceae (1sp), and Taxaceae (1 sp). Our sampling particularly focused on the genus Pinus with 26 species including representatives from the two subgenera: Pinus (21 spp) and Strobus (5 spp), which differ in the number of vascular bundles per leaf 35. Sampling also represents three distinct conifer leaf morphologies: flat leaves (Araucaria, Retrophyllum, Taxus and Wollemia; Fig. 1d), flattened needles (Abies, Larix, Picea and Tsuga), and needle-like leaves (Pinus; Fig. 1b,c). Flattened needles, such as those commonly found in non-Pinus Pinaceae, are generally shorter and flattened in cross-section as compared to Pinus needle-like leaves, which have almost equal width and height (Figs. 1b,c) and are generally longer. Fully expanded leaves from adult plants were collected in the Berkeley Arboretum of the University of California Botanical Garden, and the University of Georgia’s Thompson Mills Arboretum. Samples from both locations were used for microCT scanning and gas exchange measurements. Whole shoots were cut, wrapped in moist paper towels, and transported in dark plastic bags to avoid desiccation before scanning.
X-ray microtomography (microCT) scanning and image segmentation
MicroCT imaging was performed at the Lawrence Berkeley National Laboratory Advanced Light Source, beamline 8.3.2. Leaves were scanned within 24h of excision. Samples were wrapped with a polyimide (Kapton) tape, which allows x-ray transmittance while preventing sample desiccation. Wrapped leaf samples were placed in a plastic 1000 μL pipette tip with the lower end submerged in water and centered in the microCT x-ray beam. Scans were completed in c. 15 minutes in continuous tomography mode at 21 keV capturing 1,025 projection images of XYZ ms each. Images were captured using alternatively 5x or 10x objective lenses depending on leaf diameter, yielding final pixel resolutions of 1.27 μm and 0.625 μm. Images were reconstructed using TomoPy 52. Raw tomographic datasets were reconstructed using both gridrec and phase retrieval methods, both of which yield complementary results being efficient in segmenting cell boundaries and larger air voids, respectively 26. Image stacks of c. 2600 8-bit grayscale images were generated from the reconstruction process. Airspace was segmented in both gridrec and phase reconstruction methods by visually defining a range of pixel intensity values and the binary image stacks from both reconstruction methods were combined. Boundaries delimiting the areas occupied by the bundle sheath + transfusion tissue, epidermis, mesophyll, resin ducts, and veins were manually drawn using a graphic tablet (Wacom Cintiq Pro 16, Wacom Co, Saitama, Japan) in ImageJ 53. Leaf veins were depicted here as the vascular bundle comprising both xylem and phloem tissues. Leaf tissue boundaries were drawn as regions of interest (ROIs) in six to eight images randomly distributed across the full stack. The combination of the binary image derived from both reconstruction methods, along with the tissue boundaries, resulted in a composite image stack where each leaf tissue was classified. Leaf segmentation, which allowed us to automatically delimit different tissues across the full stack using a limited set of hand-segmented composite slices was done using random-forest classification 54.
Three-dimensional anatomy and stomatal traits
We extracted the volume and surface area of leaf anatomical traits from the full segmented stacks 54. We estimated the volumes of the epidermis (Vep), bundle sheath and transfusion tissue (VBS+TT), mesophyll cells (Vcell), mesophyll intercellular airspace (VIAS), resin ducts (Vresin), and veins (Vvein). Mesophyll volume (Vmes) was estimated as the sum of Vcell and VIAS. All volume metrics are reported in μm3. Relative volumes for each tissue (in μm3 μm−3) were estimated as a fraction of tissue per Vleaf, total leaf volume. We calculated mesophyll porosity (μm3 μm−3) as VIAS/Vmes. The mesophyll surface area exposed to the intercellular airspace (SAmes) was used to estimate the mesophyll surface area per mesophyll volume (SAmes/Vmes; μm2 μm−3). Additionally, we estimated the exposed mesophyll surface area per bundle sheath volume (SAmes/VBS+TT; μm2 μm−3) and vein volume (SAmes/Vvein; μm2 μm−3). Total leaf area Aleaf (μm2) was measured by summing up the perimeter of each slice and multiplying it by slice depth. We used the ratio SAmes/Aleaf to calculate the mesophyll surface area per total leaf area (Sm; μm2 μm−2). Stomatal estimations were performed by counting all visible stomata on the leaf surface of each scan using Avizo 9.4 software (FEI Co. Hillsboro, OR, USA). Absolute stomatal counts were used in relation to mesophyll volumetric anatomy to estimate traits accounting for the interaction of stomata pore number and VIAS, Vmes, and SAmes units. Mesophyll volumetric features were assessed in mm3 for stomatal pore density estimations. We also accounted for stomatal number per leaf surface to assess potential differences in stomatal density estimations based on surface vs volume fractions. Further, we performed a comparison of surface- and volume-based anatomical estimations using 2D slices and the full 3D stack (Supplementary Fig. 4). Methods are further explained in the Supplementary Text 1. Average trait values for each species are available in Supplementary Dataset 1. A list of measured anatomical variables, including abbreviations and units, is available in Supplementary Table 1.
Gas exchange and water use efficiency measurements
Maximum stomatal conductance (gsmax; mmol m−2 s−1) and light-saturated CO2 assimilation rate (Asat; μmol m−2 s−1) were measured on a subset of 18 species (Supplementary Dataset 1) and used to estimate leaf-level intrinsic water use efficiency (WUEi = Asat/gsmax; μmol mol−2). Pinus strobus Asat data was removed prior to analyses due to potential measurement inaccuracies. Gas exchange measurements were performed with a LICOR-6800 gas exchange system (LI-COR biosciences, Lincoln, NE, USA) between 9:00 and 14:00 on fully sunlit outer canopy foliage. Chamber temperature was set to 25° C, light source was set to 1500 μmol m−2 s−1 and chamber CO2 was set to 400 ppm. Following the gas exchange measurements, the leaf area contained within the chamber was marked using a permanent marker, collected, and the projected leaf area was measured using a leaf area meter (LI3100C, LI-COR biosciences, Lincoln, NE, USA).
Data analysis
All statistical analyses and data treatment were performed using R v.3.6.3 55. Anatomical traits were assembled and averaged for each species for data analysis. Assumptions for residual homogeneity and normality were tested prior to data analyses. Phylogenetic relationships, including branch length calibrations and divergence times, were obtained from published data 56,57. To predict leaf physiological traits based on volumetric anatomical variables we used phylogenetic generalized least-squares analyses (PGLS) with a lambda (λ) maximum likelihood optimization to control for phylogenetic non-independence between related species 58,59. The assemblage of the composite phylogenetic tree of studied conifers (Supplementary Dataset 3) was carried out using the package ‘ape’ 60 and PGLS models were fit using the package ‘caper’ 61. Standard major axis (SMA) were implemented with the package ‘smatr’ 62 to test allometric scaling between tissue volumes. A principal component analysis (PCA) was used to explore the covariation of selected traits and the distribution of leaf morphologies and conifer groups as explained by the physiological and anatomical traits measured. Given the marked differences in leaf anatomical structure across conifer leaf morphological types, and between Pinus subgenera Pinus and Strobus, we explored potential variation in 3D anatomical traits by plotting each group within the PCA. We further explored differences across leaf morphologies and between conifer clades by performing a one-way ANOVA on measured structural traits. Physiological features were not compared between leaf morphological types due to insufficient data availability. A similar variance meta-analysis, including post hoc Tukey’s honest significant differences, was employed to compare the studied conifer species with other gymnosperms, along with angiosperms and ferns. Data for comparisons across plant groups was obtained from a recently published study Supplementary Dataset 2 23.
Acknowledgments
This work was supported by the NSF grants IOS-1626966, IOS-1852976 and IOS-1146746, the Austrian Science Fund (FWF), project M2245, and the Vienna Science and Technology Fund (WWTF), project LS19-013. We thank the Berkeley Arboretum of the University of California Botanical Garden, and the University of Georgia’s Thompson Mills Arboretum for providing plant material. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Science, of the U.S. Department of Energy under contract no. DE-AC02-05CH11231. TNB acknowledges support from the USDA National Institute of Food and Agriculture (Hatch Award 1016439 and Award 2020-67013-30913) and the NSF (IOS-1951244, IOS-1557906).
References
Supplementary Information References
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