# Scaling of petiole anatomies, mechanics, and vasculatures with leaf size in the widespread Neotropical pioneer tree species Cecropia obtusa Trécul (Urticaceae)

Sébastien Levionnois, Sabrina Coste, Eric-André Nicolini, Clement Stahl, Hélène Morel, Patrick Heuret

► To cite this version:

Sébastien Levionnois, Sabrina Coste, Eric-André Nicolini, Clement Stahl, Hélène Morel, et al. Scal-ing of petiole anatomies, mechanics, and vasculatures with leaf size in the widespread Neotropical pioneer tree species Cecropia obtusa Trécul (Urticaceae). Tree Physiology, 2020, 40 (2), pp.245-258. 10.1093/treephys/tpz136. hal-02464689

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Distributed under a Creative Commons Attribution 4.0 International License
1 **Original Article**

2 **Scaling of petiole anatomies, mechanics, and vasculatures with leaf size in the widespread Neotropical pioneer tree species *Cecropia obtusa* Trécul (Urticaceae)**

4 Running title:

5 Sébastien Levionnois¹*, Sabrina Coste¹, Eric Nicolini², Clément Stahl¹, Hélène Morel¹,

6 Patrick Heuret¹,²*

7

8 1. UMR EcoFoG, AgroParisTech, CIRAD, CNRS, INRA, UA, UG, 97379 Kourou Cedex

9 France.

10 2. UMR AMAP, CIRAD, CNRS, INRA, IRD, Université de Montpellier, 34398 Montpellier,

11 France.

12

13 *Authors for correspondence:

14 Sébastien Levionnois, PhD student

15 Postal address: UMR EcoFog, Campus Agronomique, Avenue de France

16 BP709 – 97387 Kourou Cedex

17 French Guiana

18 Tel: +33 760 26 73 83

19 Email: [sebastien.levionnois.pro@gmail.com](mailto:sebastien.levionnois.pro@gmail.com)

20

21 Patrick Heuret, PhD

22 Postal adress: UMR AMAP, TA 1-51/PS2 Boulevard de la Lironde

23 34398 Montpellier Cedex 5

24 France

25 Tel: +33 467 61 55 14Email: [patrick.heuret@inra.fr](mailto:patrick.heuret@inra.fr)

26

27 Author contributions

28 P.H. conceived and designed the study; P.H., S.L., E.N., S.C., H.M. and C.S. collected field

29 samples and measured morphological traits; S.L. performed anatomical sections. S.L. and P.H

30 performed images and data analysis; S.L. and P.H. wrote the manuscript; all authors discussed

31 the results and contributed valuable comments on the manuscript.

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# ABSTRACT

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1. Although the leaf economic spectrum has deepened our understanding of leaf trait variability, little is known about how leaf traits scale with leaf area. This uncertainty has resulted in the assumption that leaf traits should vary by keeping the same pace of variation with increases in leaf area across the leaf size range. We evaluated the scaling of morphological, tissue-surface, and vascular traits with overall leaf area, and the functional significance of such scaling.

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2. We examined 1271 leaves for morphological traits, and 124 leaves for anatomical, and hydraulic traits, from 38 trees of *Cecropia obtusa* Trécul (Urticaceae) in French Guiana. *Cecropia* is a Neotropical genus of pioneer trees that can exhibit large laminas (0.4 m² for *C. obtusa*), with leaf size ranging by two orders of magnitude. We measured (i) tissue fractions within petioles and their second moment of area, (ii) theoretical xylem hydraulic efficiency of petioles, and (iii) the extent of leaf vessel widening within the hydraulic path.

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3. We found that different scaling of morphological trait variability allows for optimisation of lamina display among larger leaves, especially the positive allometric relationship between lamina area and petiole cross-sectional area. Increasing the fraction of pith is a key factor that increases the geometrical effect of supportive tissues on mechanical rigidity and thereby increases carbon-use efficiency. We found that increasing xylem hydraulic efficiency with vessel size results in lower leaf lamina area: xylem ratios, which also results in potential carbon savings for large leaves. We found that the vessel widening is consistent with hydraulic optimisation models.

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4. Leaf size variability modifies scaling of leaf traits in this large-leaved species.

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**Key words:** allometry, theoretical hydraulic conductivity, leaf size, petiole anatomy, scaling, vessel widening, xylem.
58 INTRODUCTION

59 Leaf traits have received wide attention over recent years because of the major roles they play
60 in the plant carbon economy. A current, widely accepted paradigm exists as part of the global
61 spectrum of leaf economy (LES), which emphasizes the importance of “quick” to “slow”
62 returns on physiological investments (Wright et al. 2004). In this framework, coordination
63 between structural, chemical, and physiological traits has been convincingly described within
64 and between species (Poorter and Evans 1998, Reich et al. 1998, Hikosaka 2004, Shipley et
65 al. 2006, Niinemets 2015, Onoda et al. 2017). Some studies have also investigated some of
66 the anatomical and physiological changes underlying the variability of traits underpinning the
67 LES, such as leaf mass per area (LMA; Scoffoni et al. 2016, John et al. 2017, Onoda et al.
68 2017). Leaf size (i.e. petiole length and diameter, lamina length, and mainly lamina area), on
69 the other hand, has been generally studied as disconnected from the LES, or has been shown
70 to be decoupled from the LES (Baraloto et al. 2010), although it is a trait that can vary hugely,
71 encompassing six orders of magnitude across vascular plants (Niinemets et al. 2007, Milla
72 and Reich 2007, Wright et al. 2017). For instance, leaf size is certainly subject to strong
73 selective pressures such as light interception (Poorter and Rozendaal 2008, Smith et al. 2017)
74 or temperature regulation (Gates 1968, Parkhurst and Loucks 1972, Leigh et al. 2017). The
75 leaf size – stem size relationship and other related concepts (Corner's rules; (Corner 1949,
76 White 1983a, 1983b, Ackerly and Donoghue 1998); leaf-stem allometry (Brouat et al. 1998,
77 Brouat and McKey 2001, Fan et al. 2017), the leaf size-twig size spectrum (Westoby et al.
78 2002) and the leptocaulis-pachycaulis spectrum (Hallé et al. 1978)) have been a fruitful
79 framework to tackle the within- and among-species leaf size variability (Westoby et al. 2002),
80 but the related studies remain mainly based on morphological traits (i.e. lamina area, stem
81 cross-sectional area, internode length, petiole length...), rather than anatomical or
82 physiological traits (however see Normand et al. 2008, Fan et al. 2017).

83 A key aspect of the knowledge gathered from leaf size – stem size relationships,
84 involves crucial questions about how and why leaf size varies across the plant kingdom. The
85 traits expressed in different lamina-petiole functional scaling (i.e. allometric vs isometric)
86 within and between species is likely of significance for these questions. Relationships
87 between two traits x and y can be described as: y = ax<sup>b</sup>, such as: log(y) = log(a) + b * log(x),
88 where b is the slope (or allometric exponent) and a the intercept (allometric coefficient). An
89 isometric relationship, when b = 1, is a linear and proportionality relationship, implying no
90 change of organ or organism form and shape over ontogeny, or across species. An allometric
91 relationship, when b ≠ 1, is a non-linear and disproportionality relationship, underlying
different pace of variation between two traits, and implying changes of organ or organism form and shape. Discerning allometric vs isometric relationships between organ or organism traits is an important priority, since different functional requirements can be reflected between small vs large organs or organisms, and finally different responses to selective pressures (Harvey and Pagel 1991, Brouat et al. 1998). The lamina-petiole relationship, or the relationship between a given leaf trait with leaf size, has been little studied in this scaling perspective. This gives scattered ideas if leaf size affects leaf functional requirements, and further if sampling both small and large leaves make a big difference for mechanical, hydraulic, and photosynthetic quantifications.

First, a set of studies have argued that larger leaves are disproportionately more carbon expensive, based on the fact that the leaf area fails to keep pace with increases in leaf dry mass, at both inter- (Niinemets et al. 2006, Niklas et al. 2007, Niinemets et al. 2007, Niklas and Cobb 2008, Li et al. 2008, Niklas et al. 2009, Yang et al. 2010) and intraspecific levels (Milla and Reich 2007, Sun Jun et al. 2017). The main explanation is that support requirements increase disproportionately, as the bending moment of a cantilevered beam –as a petiole- scales with the cube of its length (Gere and Timoshenko 1997, Niinemets et al. 2007), in addition to the drag forces applied to the leaf. The leaf mass-leaf area scaling has been relatively little studied at the intraspecific level (Sun Jun et al. 2017). Moreover, the leaf area-petiole cross-sectional area scaling has received less attention. Fan et al. (2017) report an isometric relationship across 28 *Ficus* species. Price and Enquist (2007)'s empirical data support an isometric leaf area-petiole cross-sectional area relationship for 5 out of 18 species, and an allometric relationship for 13 out of 18 species. Finally, for both leaf mass-leaf area and leaf area-petiole cross-sectional area scalings, we do not know the size-related anatomy underlying these scalings.

Mechanical measurements have been applied in relation to leaf area. Studies of Niklas (1991, 1992) and Mahley et al. (2018) demonstrated that small vs large leaves are not mechanically equivalent regarding flexural stiffness, both within and across species, with petioles of large leaves being disproportionately stiffer. But the mechanical structure driving the flexural stiffness between small vs large leaves remains poorly described (Niklas 1999). Mechanical stability can be achieved through two properties: the material property and the geometry. Modification of petiole tissue surfaces and fractions across a leaf size range can determine the mechanical contribution through geometry of main supportive tissues such as xylem, collenchyma, and sclerenchyma (Faisal et al. 2010, Niklas and Spatz 2012). Pith is largely made of large parenchyma cells, and is a cheap due to weak cell wall lignification and
fine cell walls (Evert 2006). As the pith generally occupies the central position within an organ, modifying pith area and fraction could be an efficient way to generate volume with decreased carbon costs (Olson, Rosell, et al. 2018). Moreover, when pith fraction increases, it mechanically shifts external and supportive tissues (xylem, sclerenchyma, collenchyma) centrifugally, increasing the second moment of inertia of these tissues. Second moment of area quantifies the distribution of mass in a cross-section with respect to the centre of inertia of the cross-section, and this describes the important effect of size and geometry of the cross-section in mechanics. The prevalent role of cross-sectional geometry in the stiffness of petioles has been shown by Mahley et al. (2018), but only for ferns, which exhibit a very different anatomy in comparison to flowering plants.

In comparison to the leaf dry mass-area scaling or petiole mechanics, little is currently known on the link between leaf size, leaf hydraulic conductivity and vascular architecture. Understanding size-related changes of leaf hydraulics and vasculature is important to address size-independent variation, as pointed out for stems (Olson et al. 2009). The West-Brown-Enquist model (WBE model) assumes that the variation of terminal (i.e. minor veins) conduit size and number is independent of leaf size (West et al. 1999, Price and Enquist 2007). Supporting this assumption, Sack et al. (2012) have shown that the global vein density across species is independent of leaf size, even if major vein density decreases with leaf size. Fiorin et al. (2016) suggest that the spatial organisation of stomata with veins is uniform across leaves of different species. But it can also be hypothesised that small vs large leaves across and within species have to deal with different selective pressures regarding conductive path length and the water supply per unit leaf area. Indeed, across the leaf size range, plants should maintain constant leaf conductance per unit leaf area ($K_{leaf}$, kg MPa⁻¹ s⁻¹ m⁻²), or at least minimise the loss of $K_{leaf}$, to sustain transpiration and carbon assimilation (Petit et al. 2016, Pittermann et al. 2018, Echeverría et al. 2019).

A longer leaf implies *de facto* a longer conductive path length that would thus increase hydraulic resistance and likely affect $K_{leaf}$ in the absence of appropriate vascular adjustments. Theoretical models and empirical data support that the axial basipetal widening in vessel diameter reduces the increase of hydrodynamic resistance with conductive path length (West et al. 1999, Becker et al. 2000, Enquist 2003, Anfodillo et al. 2013, Olson et al. 2014). To our knowledge only two studies measured vessel widening within leaves: Coomes et al. (2008) for ten oak species, and Lechthaler et al. (2019) for one species of *Acer*. Yet, these studies suggest higher rates of vessel widening within leaves, in comparison to stems. This is consistent with the fact that leaves concentrate a significant part of the hydraulic resistance of
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the plant (Sack and Holbrook 2006), owing to different vascular architecture between leaves and stems.

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A larger leaf also implies *de facto* a larger lamina area to supply with water, and thus an appropriate and sufficient petiole conductivity is required. Increasing the xylem area to increase the number of vessels, or increasing the vessel diameter are two non-exclusive ways of reducing total resistance, and increase petiole and vein conductivity to achieve a sufficient leaf water supply. However, an increase of the number of vessels comes with increasing construction costs, because additional space is required (Banavar et al. 1999, McCulloh et al. 2003, Gleason et al. 2018). The conductivity of a vessel increases with the fourth power of its diameter as predicted by the Hagen-Poiseuille law (Tyree and Zimmermann 2002). Thus, a small increase in vessel diameter will drive an exponential increase in the vessel conductivity, but potentially at the cost of increasing embolism risk. A positive allometric relationship of vasculature with size should increase the xylem conductive efficiency (namely xylem-specific conductivity, which is the xylem conductivity divided by the xylem surface), and therefore should limit xylem volume and construction cost. Investigating the petiole xylem and vasculature according to leaf size can be readily implemented, and should highlight some hydraulic-related selective pressures correlating with the leaf size variability, at both inter- and intraspecific levels.

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We chose to investigate lamina-petiole traits at the intraspecific level as a first step. We focussed this study on the genus *Cecropia*, which is known for its hyperdominant pioneer trees that are pivotal in the initiation of tropical forest successions. We selected *Cecropia obtusa* Trécul (Uticaceae), which is a widespread species from the Guiana shield, capable of deploying large leaves with a large size range (100 – 4000 cm²), appropriate for addressing the lamina-petiole scaling with petiole anatomical and vasculature correlates, at the intraspecific level. We measured leaf morphology, tissue areas of the petiole, petiole vascular and theoretical conductive traits. We built a dataset of 1271 leaves from 38 trees, with anatomical and vascular data for 124 leaves. We aimed to address the following questions:

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(i) Is the form of the lamina-petiole scaling isometric or allometric? We hypothesise to find allometric lamina area-petiole cross-sectional area and lamina mass-petiole mass relationships, with petiole mass increasing faster as mechanical requirements disproportionately increase.

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(ii) How does petiole anatomy determine petiole mechanics? We hypothesise that pith fraction increases with leaf size, minimising volumetric construction cost, and enhancing mechanical strength through geometry by shifting supportive tissues centrifugally.
(iii) How do longitudinal adjustments of xylem and vasculature variations limit loss of K<sub>leaf</sub> and construction costs? We hypothesise that K<sub>leaf</sub> loss limitation for larger leaves is allowed by vessel basipetal widening according to the leaf size and the conductive path length. We hypothesise to find a higher rate of vessel widening within the leaf in comparison to what is known for the stem, consistent with leaves supporting a large amount of the total hydraulic resistance of plants. We also hypothesise that less construction cost is possible than under the hypothesis of an invariable vessel diameter, thanks to a more efficient xylem due to wider vessels, enabling more lamina area per xylem area.

## MATERIALS AND METHODS

### *Study site*

The study was conducted in French Guiana along a forestry road in Counami (N5.41430°, W53.17547°, geodesic system WGS84) where the entrance to the road is located 5 km to the east of Iracoubo municipality. The warm wet tropical climate of French Guiana is highly seasonal due to the north-south movement of the Inter-Tropical Convergence Zone. Mean annual rainfall is 2726 mm year<sup>-1</sup> and annual mean air temperature is 25.7 °C (Gourlet-Fleury et al. 2004). There is a dry season lasting from mid-August to mid-November, during which rainfall is < 100 mm month<sup>-1</sup>.

### *Plant material, sampling and morphological measurements*

Tree sizes ranged from 10.8 to 23.1 m in height, and 6.21 to 30.49 cm in diameter at breast height. Trees were evenly felled in time from September 2014 to October 2016. All the leaves in the crown were cut by cutting the petiole as close as possible to its insertion point with the stem and placed immediately in plastic bags within coolers. One to five fully expanded leaves per tree -hereafter called A-leaves - were kept for the anatomical studies.

*Cecropia* leaves are palmatilobate (Fig. 1b; Fig. S1) and so there is no single midrib but the same number of midribs as there are lobes, i.e. a midrib departs into and supplies each lobe. In the laboratory, all the leaves were processed within 4 hours after felling to keep them as fresh as possible. Petioles were cut as close to the lamina as possible. Two orthogonal diameters (mm) of each petiole, at the middle point of the petiole, were measured because of the tendency of *Cecropia* petioles to exhibit an ellipse shape (Fig. S1; Table 1 for a list of traits and abbreviation). From these two diameters, a cross-sectional area was derived in the shape of an ellipse ($A_{pet}$, mm²; Fig. S1). For each lamina, the length of the main lobe (i.e. the largest one in the continuation of the petiole; cm) was measured. In the A-leaves, two 1-cm-
long petiole segments in the median position was sampled (Fig. S1). The first segment was used to measure their specific density, expressed as the ratio of dry mass to the fresh volume (PD: petiole density; Williamson and Wiemann 2010; Table 1). The fresh volume of the sample was calculated using an inverse Archimedes principle and a precision balance (CP224S, Sartorius), based on the buoyancy (G) of the sample, i.e. G = fresh mass in air – submerged mass (Lehnebach et al. 2019). Dry mass was derived after drying at 103 °C for three days. PD was computed as the dry mass divided by G. The second segment was preserved in 70% alcohol and used later for anatomical measurements (see the section headed 'Anatomical measurements'). Because we aimed at studying the effects of the fraction and cross-sectional area of petiole tissues, we choose to focus on the mid-point segment of the petiole between the connection to the stem and the leaf lamina. This point was selected so as to avoid swelling effects at stem and lamina connections. At the base of the petiole, the wide and triangular base is further modified by an external covering of trichilia and secretions of Müllerian bodies serving the nutrition of hosted ants. At the distal end of the petiole, at the point of junction between petiole and lamina main veins, the petiole is more flexible in torsion, which allows the lamina to rotate (pers. obs.). The distance between the anatomical sampling point and the tip of the leaf main lobe was measured (conductive path length; PL, cm) and used for the investigation of vessel widening. Since the vessel diameter – conductive path length relationship is a power function, with rapid changes in vessel diameters close to the conductive path tip, and slower nearly constant toward the base, we assume that anatomical measurements at our sampling point are proportional to the petiole base.

Lamina dry mass ($M_{lamina}$, g) and petiole dry mass ($M_{pet}$, g) were measured with a scale. The leaf dry mass ($M_{leaf}$, g) is the sum of $M_{lamina}$ and $M_{pet}$. Lamina area ($A_{lamina}$, $cm^2$; Fig. S1) was measured with a planimeter (LiCor 3000A, LiCor Inc., Lincoln, NE, USA).

Data from 1271 mature fully developed leaves (A-leaves and all the other leaves) were used for the characterisation of morphological and structural variability.

## Anatomical and vascular measurements

We conducted our study on a subset of 124 A-leaves selected to represent the widest range of petiole diameters (from 2.56 to 15.41 mm). Anatomical cross-sections (Fig. 1c), 20 to 50 µm thick, were sampled from the petiole median segment with a manual microtome (Mikrot L, Schenkung Dapples, Switzerland). All cross-sections were stained in a safranin/astra-blue solution to stain unlignified cells blue and lignified cells red. Images of each petiole cross-section were digitised with an optical microscope (Olympus BX60; Olympus Corporation;
Tokyo; Japan) with 50x magnification and a Canon camera EOS 500D (lens Olympus U-TVI-X; F 0.0; ISO 100; speed 1/25). Three or four close up images were taken of each petiole section at different depths of focus and stacked with the Helicon Focus software (v.6.3.2.Pro, https://www.heliconsoft.com/). Pictures were assembled in a panorama using Kolor AutoPanoGiga software (v.3.0.0, http://kolor.com/autopano/) to obtain a complete picture of the cross-section (Fig. 1c). The digitised cross-sections were processed with CS5 Photoshop software (v.12.0, http://adobe.com/products/photoshop/html). We distinguished eight tissue types that comprise the entire petiole anatomy (Fig. 1d, e). We manually delineated the tissues on the photographs and created layer masks (Fig. 1e). The masks of these layers were used to calculate the cross-sectional area of each tissue and the whole petiole cross-sectional area with the ImageJ software (v.1.43u; http://imagej.nih.gov/ij/). We distinguished eight component petiole tissues for further anatomical analysis: (i) the pith, (ii) the sclerenchymatous shield associated with the vascular bundles, (iii) interfascicular parenchyma, (iv) primary xylem, (v) secondary xylem, (vi) phloem (comprising primary and secondary phloem), (vii) cortical parenchyma and (viii) cortical collenchyma (Fig. 1e).

As we knew the cross-sectional area of each petiole tissue, we calculated the second moment of area ($I$, mm⁴) of the main supportive tissues (Table 1). $I$ quantifies the distribution of mass in a cross-section with respect to the centre of mass of the cross-section, and this describes the important effect of size and geometry of the cross-section in mechanics, since the flexural stiffness ($E*I$, where $E$ is the elastic modulus) is directly proportional to $I$ (Niklas and Spatz 2012). As $I$ is a fourth power function of the two radii delimiting the ring (Table 1), slight increases in the ring diameter and/or cross-sectional area has dramatic consequences for $I$, and thus its contribution to flexural stiffness. We focused on the sclerenchyma, collenchyma and secondary xylem, since these tissues are well-known to be supportive tissues in a section and exhibit lignified thick cell walls (Leroux 2012). We also estimated the behaviour of the petiole flexural stiffness by calculating $I$ for the total petiole section, and by using the known petiole density as a proxy of $E$ (Table 1). We assumed that this approximation was appropriate, since it had been shown that the elastic modulus exhibits a linear and positive relationship with the density for both wood (Chave et al. 2009, Dlouhá et al. 2018) and bark (Rosell and Olson 2014).

For the xylem, the cropped part of the image in which the vessels were visible was analysed with the ImageJ software to calculate theoretical xylem hydraulic properties (Abramoff et al. 2004). For each vessel, we calculated its cross-sectional area (µm²) and its elliptical diameters. To study variations in the dimensions of the vessel, we used the mean
hydraulic diameter ($D_h$, µm, Table 1), i.e. the diameter that all vessels, considered as circles, in a given tissue would have to sustain exactly the same tissue hydraulic conductivity (Tyree and Zimmermann 2002). The number of vessels was counted for primary and secondary xylem. We also calculated the conductive area (mm²) as the sum of the cross-sectional area of all vessels of the surrounding xylem. Knowing the dimensions of each vessel, the number of vessels, and the total petiole xylem area, a theoretical hydraulic conductivity ($K_{th}$, m kg MPa⁻¹ s⁻¹) was estimated based on formulas in Table 1. To test the null hypothesis of a decreasing $K_{leaf}$ (kg MPa⁻¹ s⁻¹ m⁻²) across the leaf size range with no vessel widening occurring, we estimated a theoretical leaf conductance ($K_{leaf null}$) by firstly dividing the $K_{th}$ by the petiole length. We thus obtained a value for petiole conductance (kg MPa⁻¹ s⁻¹) which we divided by the lamina area and obtaining $K_{leaf null}$.

Previous studies have shown precise tapering rates of vessels along a leaf (Coomes et al. 2008; Lechthaler et al. 2019) or the entire tree (Bettiati et al. 2012, Petit et al. 2014) based on several anatomical measurements along the path of each individual leaf or individual tree. Here, we estimated a tapering rate ($D_h/PL$) based on a scaling exponent of the log-log relationship between $D_h$ at the midpoint of the petiole, and the path length, across all measured petioles. Following past studies at the tree level for interspecific comparisons (Anfodillo et al. 2006; Olson et al. 2014), we assumed that vessel tapering is primarily determined by the distance from the leaf tip, assuming that all leaves display the same tapering rate.

## Data analysis

Laminas used in anatomical studies were sometimes slightly damaged resulting in somewhat biased lamina area measurements. To correct this, we used a prediction model to calculate an estimated lamina area ($A_{lamina}$, cm²), since the relationship between the length of the main lobe and the undamaged lamina area is very informative. We calculated the estimated lamina area as: Estimated lamina area = 0.701 * Main lobe length².¹⁸⁰ ($R²$ = 0.942, *P* < 0.001, Fig. S2). This estimation of lamina area was used only to compare tissue cross-sectional area and vasculature traits with leaf area.

All statistical analyses were performed with R software (https://cran.r-project.org/). The relationship between each trait pair was determined with an SMA (Standardized major axis regression; Warton et al. 2006), which allows minimisation of the error on both the x-axis and y-axis (Harvey and Pagel 1991). These correlation relationships are described as: $y = ax^b$, such as: $log(y) = log(a) + b * log(x)$, where b is the slope (or allometric exponent) and a the
intercept (allometric coefficient). A 95% confidence interval was used to decide whether it
was significantly correlated or not. A slope test was performed to determine if the slope
differed from 1 (H1: b ≠ 1 for an allometric relationship) or not (H0: b = 1 for an isometric
relationship). SMA were carried out with the (S)MATR package (Falster et al. 2006). The
lamina area prediction from the main lobe length was modelled from a NLS (non-linear least
squares) with the STATS package.

## RESULTS

### *Leaf morphological variability*

$A_{\text{pet}}$ and $A_{\text{lamina}}$ were positively and allometrically correlated ($P < 0.001$; $R^2 = 0.779$; Fig. 2a; Table 2, S1). $A_{\text{pet}}$ and $M_{\text{lamina}}$ were positively, and allometrically correlated ($P < 0.001$; $R^2 = 0.898$; Fig. 2b; Table 2). $M_{\text{pet}}$ and $M_{\text{lamina}}$ were positively, and allometrically correlated ($P < 0.001$; $R^2 = 0.947$; Fig. 2c; Table 2). $A_{\text{lamina}}$ and $M_{\text{leaf}}$ were positively, and isometrically correlated ($P < 0.001$; $R^2 = 0.846$; Fig. 2d; Table 2). PD were negatively, and allometrically correlated to $A_{\text{pet}}$ ($P < 0.01$) but uncorrelated to $A_{\text{lamina}}$ ($P < 0.05$)(Table S1).

### *Petiole anatomy*

Petioles showed pronounced radial symmetry (Fig. 1c). The central parenchymatous pith formed the main part of the cross-section. The numerous bundles (20 to 80) were arranged in the pith periphery, mainly in only one circle, but in a few cases in two circles. The cambium displayed a diversity of arrangement, with respect to the arrangement of the bundles. From a strictly cyclic structure to a wavy one (Fig. 1f), and at the extremity, we observed isolated bundles with complete cambium discontinuities in a more cortical position (Fig. 1g). Primary and secondary xylem and secondary phloem were easy to identify (Fig. 1d). A sub-continuous sclerenchymatous shield was present at the interface between the vascular bundles and the pith. The vascular bundles were separated by interfascicular parenchyma. Occasional sclerenchyma were present between the secondary phloem and cortical parenchyma. Depending on the extent of secondary growth, the primary phloem was crushed between the secondary phloem and cortical parenchyma. In the most external part, there was a ring of collenchyma, between the epidermis and the cortical parenchyma. Laticiferous canals were frequently visible, mainly in the cortex (Fig. 1f) but also in the pith, but were also sometimes completely absent.

### *Petiole anatomy and vascular architecture*
All tissue areas were significantly and positively correlated with $A_{\text{pet}}$ and $A_{\text{lamina}}$ ($P < 0.001$; Fig. S3a; Table S2), with most of these relationships being allometric. All tissue fractions relative to $A_{\text{pet}}$ were correlated to $A_{\text{pet}}$ ($P < 0.001$; Fig. S3b; Table S2). All tissue fractions relative to $A_{\text{pet}}$ were correlated to $A_{\text{lamina}}$ ($P < 0.001$; Fig. S3b; Table S2), except for the phloem ($P > 0.05$). Pith had the highest fraction (44.78% ± 1.12) with the highest effect on petiole size (27.02 to 59.41% of the cross-section; Fig. 3a). The lamina area allometrically and positively scaled with the number of vessels ($P < 0.001$; $R^2 = 0.596$; Fig. 3d; Table 2), such that large leaves were associated with disproportionately less vessels. The lamina area allometrically and positively scaled with conductive area ($P < 0.001$; $R^2 = 0.635$; Fig. 3c; Table 2), such that large leaves were associated with disproportionately less conductive area. The lamina area allometrically and positively scaled with xylem area ($P < 0.001$; $R^2 = 0.711$; Fig. 3b; Table 2), such that large leaves were associated with disproportionately less xylem area.

The second moment of area (I) for collenchyma, secondary xylem, and sclerenchyma were significantly and positively related to $A_{\text{lamina}}$ ($P < 0.001$; $R^2 = 0.578$, 0.712, and 0.645 respectively; Fig. 4a,b,c). The approximated petiole flexural stiffness was positively and allometrically correlated to $A_{\text{lamina}}$ with a slope significantly superior to 1 ($P < 0.001$; $R^2 = 0.653$; Fig. 4d; Table 2).

$D_h$ was significantly and positively related to $A_{\text{lamina}}$ ($P < 0.001$; $R^2 = 0.502$; Fig. 5a; Table 2). $D_h$ was positively and allometrically related to the conductive path length ($P < 0.001$; $R^2 = 0.469$; Fig. 5b; Table 2). The estimated tapering rate ($D_h/\text{PL}$) was 0.549 µm cm⁻¹ (Fig. 5b). $K_{\text{leaf null}}$ was significantly and negatively related to $A_{\text{lamina}}$ and the conductive path length ($P < 0.01$; Fig. 5c,d).

## DISCUSSION

Our results provide an understanding of the tissue-level and vascular adjustments characterising lamina-petiole scaling at the intraspecific level for a widespread Neotropical pioneer tree species, *Cecropia obtusa*. The results point out (i) an allometric scaling between lamina size and petiole size, such that larger leaves show a higher lamina area for a given petiole cross-section, (ii) a higher pith fraction, related to higher petiole flexural stiffness through the second moment of area (I) of the mechanically stiff tissues surrounding the pith, (iii) a vessel widening allowing for a reduced effect of increasing path length with leaf size and (iv) a higher lamina area per xylem area for larger leaves, due to relatively less conductive area for larger leaves.
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### *Morphological petiole-lamina scaling*

The finding of a positive and allometric relationship between petiole dry mass and lamina dry mass (Fig. 2c), with a disproportionately higher petiole mass for a given lamina mass, is in agreement with what has been previously described (Niinemets et al. 2006, 2007, Li et al. 2008). This pattern has been explained by the scaling of the bending moment of a cantilevered beam with the cube of its length (Gere and Timoshenko 1997), in addition to drag forces requiring a disproportionate mechanical reinforcement of the petiole with leaf size (Niinemets et al. 2007, Li et al. 2008). However, the isometric scaling of lamina area with the leaf dry mass (Fig. 2d) is not in agreement with the repeatedly shown diminishing return pattern, predicting increasing costs for leaf support with increasing leaf size (Niinemets et al. 2006, 2007, Niklas et al. 2007, 2009, Milla and Reich 2007, Li et al. 2008b, Sun Jun et al. 2017). Although the leaf area-leaf mass allometric scaling is significant at the interspecific level, the allometric scaling is not ubiquitous at the intraspecific level, based on this present study for *C. obtusa*, and Milla and Reich (2007) for 11 species.

We found a positive allometric relationship between lamina area and petiole cross-sectional area (Fig. 2a), such that large leaves exhibit a larger lamina area for a given petiole cross-sectional area. This change is in agreement with the allometric relationship between lamina mass and petiole cross-sectional area we found (Fig. 2b), such that large leaves exhibit larger lamina mass for a given petiole size. Larger petioles support a disproportionately larger load. Therefore, we wondered what petiole anatomical and mechanical changes are associated with this morphological pattern.

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### *Petiole tissue partitioning*

As pith fraction increases, the distance of a given tissue to the petiole cross-sectional centre of inertia increases, as does the second moment of inertia to a fourth-power function of this distance. The case of collenchyma is of primary importance since it is known to be a supporting tissue, with small cells and relatively thick cell walls compared with other living cells (Leroux 2012). The collenchyma is the most external tissue within the petiole section with a ring-geometry, exhibiting the largest change in the second moment of area (Fig. 4a). In the same line, the secondary xylem is also a well-known supporting tissue, exhibiting a ring-geometry and which fraction increases with leaf size. Its increasing fraction occurs at the same time as the distance to the cross-sectional centre of inertia increases with pith fraction, and determines an overall increase of the second moment of area of the secondary xylem with
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leaf size (Fig. 4b). This was also the case for the sclerenchyma (Fig. 4c), although
sclerenchyma exhibited relatively low values of *I* (0.05 to 95 mm⁴). The allometric exponents
of the supportive tissues according to the lamina area (Fig. 4a,b,c) indicate that *I* increases
faster for the sclerenchyma, followed by the secondary xylem, suggesting that the relative
geometrical contribution of these tissues to the petiole flexural stiffness increases across the
leaf size range.

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Increasing organ size and mass logically increases the mechanical load (Mahley et al. 2018). Moreover, we found an allometric relationship between lamina dry mass and petiole cross-sectional area, with disproportionately higher lamina dry mass for leaves (Fig. 2b). This suggests a disproportionately higher mechanical load on petioles of large leaves. This is in agreement with the approximated petiole flexural stiffness allometrically related to the lamina area (Fig. 4d), such that petioles are disproportionately stiffer on large leaves to overcome the mechanical load. This suggestion is in agreement with past studies demonstrating disproportionately higher petiole stiffness with increasing petiole thickness (Niklas 1991, 1992, Mahley et al. 2018). Therefore, our results suggest that the effect of geometry through pith fraction determines the overall increase in petiole flexural stiffness, since the decrease of petiole density with leaf size –and thus petiole elastic modulus– is balanced (Fig. 4d), as also shown by Mahley et al. (2018) for ferns. However, since the pith is often hollow in large structure, one may not exclude that the pressure of selection is actually on the surrounding tissues.

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Changing petiole pith fraction and thus collenchyma, secondary xylem, and
sclerenchyma *I* is clearly a cheap mechanism in terms of carbon allocation to balance the self-
loading mechanical stress arising with leaf size. Moreover, we found a negative relationship
between *A*<sub>pet</sub> and PD, and no relationship of PD with *A*<sub>lamina</sub>. These results suggest that
volumetric construction cost can decrease with *A*<sub>pet</sub>, or at least does not increase with leaf size.

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Petiole vascular architecture

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The estimation of K<sub>leaf null</sub> (Fig 5a,b) confirms that K<sub>leaf</sub> decreases with leaf size in the case of an absence of vessel widening, as the hydraulic resistance is well-known to be dependent on the path length (Tyree and Zimmermann 2002). Without vessel widening, large leaves cannot be selected since (i) the higher pressure drop that would be associated with such leaves would increase the risk of cavitation (Tyree and Ewers 1991, Cruiziat et al. 2002), and (ii) a lower K<sub>leaf</sub> necessarily drives lower gas exchanges and carbon assimilation (Brodribb 2009, Scoffoni et al. 2016), and thus less efficient carbon payback. Therefore, this result suggests that K<sub>leaf</sub> is
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constant across leaves and not limiting for carbon assimilation (Echeverría et al., Petit et al. 2016, Pittermann et al. 2018), even if water flux measurements would be required for confirmation.

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Even if such a pattern of vessel widening has been repeatedly shown along the stem of adult trees for several species (Petit et al. 2008, 2010, Bettiati et al. 2012, Anfodillo et al. 2013, Olson et al. 2014), leaves have received less attention. The estimated widening rate (0.55) was closely similar to those found by Coomes et al. (2008; mean of 0.54; a range of 0.42-0.73) across ten *Quercus* L. (Fagaceae) species, and not too far from those found by Lechthaler et al. (2019; slope of 0.45) for one species of *Acer*. A striking feature here is the convergence of widening rates between studies, despite contrasting ecology (i.e. temperate vs tropical) and phylogenetic origins of the investigated species. Such a finding suggests that the hydrodynamic resistance is such a strong selective pressure on hydraulic architecture that it drives a single leaf vascular architecture across vascular plants. Hydraulic optimality models (West et al. 1999) predict a minimum and convergent widening rate of 0.2, but only for stems, to totally mitigate the hydrodynamic resistance. Indeed, this convergent widening rate is found across most plants and trees when looking into the stem vasculature (Anfodillo et al. 2006, 2013, Petit et al. 2014, Olson et al. 2014, 2018). The vessel widening rate is not constant through the total path length (Bettiati et al. 2012), with a scaling 2 to 3 times higher in leaves (this study; Coomes et al. 2008, Lechthaler et al. 2019). This participates at concentrating much of the hydraulic resistance in leaves –up to 50%– (Sack and Holbrook 2006), whereas leaves represent a small fraction of the total conductive path length, i.e. few centimetres of few tens of centimetres for leaves against tens of meters for the entire tree. The hydraulic segmentation hypothesis (Tyree and Zimmermann 2002, Pivovaroff et al. 2014) assumes that leaves should be hydraulically more resistant than stems, as “bottlenecks”, to always preserve far lower water potentials in leaves, and promote drought-induced embolism containment in easy-to-renew organs. These issues deserve more investigations, to link vessel widening rates in leaves with the relative contribution of leaves in the hydraulic resistance of plants.

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The allometric relationship between the number of vessels and the supplied lamina area suggests an increase in the number of vessels per leaf area, from the leaf base towards the leaf tip (Fig. 3d). This therefore suggests vessel furcation. This contradicts the WBE model (West et al. 1999, Rosell et al. 2017) which predicts an absence of furcation, even if the WBE model does not initially integrate predictions for leaves. However, this result is in agreement
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with the empirical test of Lechthaler et al. (2019), who also support the existence of conduit furcation towards terminal parts within *Acer* leaves.

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For *C. obtusa* leaves, vessel tapering and furcation towards the leaf tip leads to a conductive area-decreasing architecture towards the leaf base, as supported by the allometric relationship between the conductive area and the supplied lamina area (Fig. 3c). This supports the prediction of the WBE model (West et al. 1999, Rosell et al. 2017). However, our result is not in agreement with Lechthaler et al. (2019), who support a conductive area-preserving architecture for *Acer* leaves. This discrepancy may come from the fact that Lechthaler et al. (2019) analysed the vascular architecture across both the petiole and the midrib, whereas in our study we only focused on the petiole. In the lamina, the strong furcation of the vein system may lead to more numerous vessel furcations in comparison with the petiole. Therefore, less vessel furcations within the petiole may allow for wider vessels, therefore reducing the required conductive area, according to the Hagen-Poiseuille law.

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The conductive area-decreasing architecture allows for a reduction of the xylem area supplying the lamina for large leaves, as supported by the allometric relationship between xylem area and lamina area (Fig. 3b). According to the Hagen-Poiseuille's law, a given conductive area can determine different conductivities, from numerous but small conduits to few but wide conduits. However, according to the packing rule, the building of numerous small conduits would require more xylem area and volume. Therefore, this implies that selection favours an even water supply to all parts of the leaf independently of leaf size, with minimal carbon investment (Banavar et al. 1999).

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Finally, disproportionately less xylem area for large leaves explains the allometric relationship between the petiole cross-sectional area and the lamina area, with disproportionately less petiole cross-sectional area for large leaves.

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## CONCLUSION

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The different functional attributes linking flexural stiffness (elastic modulus vs *I*, and allometric scaling of flexural stiffness), carbon cost (leaf mass and density), and vasculature (vessel widening, xylem area, and conductive area) between large and small leaves, suggest that these three functional dimension-mechanics, carbon allocation, and hydraulics respectively- are selected for precise combinations, and further determine leaf size variation possibilities. Environmental factors are known to shape leaf size diversity (e.g. annual rainfall, temperature, solar radiation, soil nutrients...), but some other selective pressures are intrinsically linked to the possibility of developing large leaves or not (i.e. return on invested
biomass, self-loading, conductive path length...). The contrasting mechanics, carbon
allocation, and vasculatures between large and small leaves shed light on these selective
pressures in our study. Indeed, we showed that large leaves exhibited (i) disproportionately
higher lamina area for a given petiole cross-sectional area, (ii) higher pith fraction with
disproportionately stiffer petioles, and (iii) disproportionately less xylem area for a given
lamina area, associated with a decreasing-area vascular architecture toward the leaf base. But
these selective pressures are currently not fully understood. Our study calls for more studies
on these selective pressures to better characterise and understand to what extent they are
independent or not. We argue that a better comprehension of these selective pressures should
extend the knowledge of drivers shaping leaf size variability, and, furthermore, permit us to
understand how and why leaf size seems disconnected from LES.

544

545 ACKNOWLEDGEMENTS

546 The authors thank Julie Bossu, Coffi Belmys Cakpo, Henri Caron, Jocelyn Cazal, Saint-Omer
547 Cazal, Aurélie Cuvelier, Bruno Clair, Aurélie Dourdain, Jean-Yves Goret, Marie Hartwig,
548 Ariane Mirabel, Audin Patient, Pascal Petronelli, Laurent Risser, Dylan Taxile, Valérie
549 Troispoux, Niklas Tysklind and Lore Verryckt for their assistance with field work and
550 measurement of leaf traits. We thank Jacques Beauchêne, Christine Heinz and Nick Rowe for
551 preliminary discussions around the project and first results. We thank Hervé Cochard for
552 fruitful discussions for data analysis. We thank Tancrède Alméras, Camilo Zalamea and Nick
553 Rowe for critical and valuable comments on successive version of the manuscript.

554

555 FUNDING

556 S.L. was supported by a doctoral fellowship from CEBA (ref. ANR-10-LABX-0025). This
557 study benefited from an *Investissement d'Avenir* grant managed by the *Agence Nationale de
558 la Recherche* (CEBA, ref. ANR-10-LABX-0025).

559

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**Fig. 1.** Habits, morphological and petiole-anatomical aspects of *C. obtusa* (Urticaceae). (a) Habits of *C. obtusa*. (b) Leaf of *C. obtusa*. (c) Petiole cross-sectional anatomy of *C. obtusa* in the middle part of the petiole. (d) Close-up of petiole constitutive tissues: pith (Pith), sclerenchyma (Scle), interfascicular parenchyma (Pint), primary xylem (XI), secondary xylem (XII), total phloem (Phlo), primary phloem (Phl), secondary phloem (PhII), cortical parenchyma (Pcor), collenchyma (Coll) and epidermis (Epi). (e) Tissues and corresponding layer masks studied. (f) “Wavy” cambium with a sub-bicyclic array of vascular bundles. Arrows represent laticiferous canals. (g) Cambial discontinuities, island-like vascular bundles and a bicyclic array of vascular bundles.

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<figure><img src="image_2.png" /><figcaption>(a)</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>(d)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>(e)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>(f)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>(g)</figcaption></figure>

<table><tr><td></td><td>Pith</td><td>XI</td><td>XII</td><td>Pcor</td></tr><tr><td></td><td>Scle</td><td>Pint</td><td>Phlo</td><td>Coll</td></tr></table>

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<figure><img src="image_9.png" /><figcaption>**Fig. 2.** Scaling of morphological traits with leaf size. (a) Lamina area according to petiole cross-sectional area. (b) Lamina dry mass according to petiole cross-sectional area. (c) Lamina dry mass according to petiole dry mass. (d) Lamina area according to leaf dry mass. All relationships are plotted on a log-scale. *b*: scaling exponent.</figcaption></figure>

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<figure><img src="image_10.png" /><figcaption>**Fig. 3.** Scaling of petiole anatomy with leaf size. (a) Pith cross-sectional fraction according to lamina area. (b) Number of vessels according to lamina area plotted on a log-scale. (c) Conductive area according to lamina area, plotted on a log-scale. (d) Xylem area according to lamina area, plotted on a log-scale. *b*: scaling exponent.</figcaption></figure>

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<figure><img src="image_11.png" /><figcaption>**Fig. 4.** Scaling of mechanical traits with leaf size. (a) Collenchyma xylem second moment of area according to lamina area. (b) Secondary xylem second moment of area according to lamina area. (c) Sclerenchyma second moment of area according to lamina area. (d) Approximated petiole flexural stiffness (product of petiole second moment of area with petiole density) according to lamina area, plotted on a log-scale. *b*: scaling exponent.</figcaption></figure>

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<figure><img src="image_12.png" /><figcaption>**Fig. 5.** Scaling of vascular traits with leaf size. (a) Mean hydraulic diameter according to lamina area. (b) Mean hydraulic diameter according to path length. (c) Theoretical leaf conductance under the hypothesis of no vessel widening according to lamina area. (d) Theoretical leaf conductance under the hypothesis of no vessel widening according to path length. All relationships are plotted on a log-scale. *b*: scaling exponent.</figcaption></figure>

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<table><caption>Table 1. List of measured traits and abbreviations.</caption><thead><tr><th>Trait</th><th>Abbreviation</th><th>Unit</th><th>Formula</th></tr></thead><tbody><tr><td>Petiole cross-sectional area</td><td>A<sub>pet</sub></td><td>mm<sup>2</sup></td><td></td></tr><tr><td>Petiole dry mass</td><td>M<sub>pet</sub></td><td>g</td><td></td></tr><tr><td>Lamina dry mass</td><td>M<sub>lamina</sub></td><td>g</td><td></td></tr><tr><td>Leaf dry mass</td><td>M<sub>leaf</sub></td><td>g</td><td>M<sub>leaf</sub> = M<sub>petiole</sub> + M<sub>lamina</sub></td></tr><tr><td>Lamina area</td><td>A<sub>lamina</sub></td><td>cm<sup>2</sup></td><td></td></tr><tr><td>Petiole density</td><td>PD</td><td>g cm<sup>-3</sup></td><td>PD = M<sub>dry</sub> / (M<sub>fresh</sub> - M<sub>immersed</sub>)</td></tr><tr><td>Second moment of area of a ring-like tissue</td><td>I</td><td>mm<sup>4</sup></td><td>I = pi/4*(r<sub>ext</sub><sup>4</sup> - r<sub>int</sub><sup>4</sup>)</td></tr><tr><td>Approximated petiole flexural stiffness</td><td></td><td>g mm<sup>4</sup> cm<sup>-3</sup></td><td>with r<sub>ext</sub> and r<sub>int</sub> ring external and internal radii respectively<br>I * PD</td></tr><tr><td>Conductive path length</td><td>PL</td><td>cm</td><td></td></tr><tr><td>Mean hydraulic diameter</td><td>D<sub>h</sub></td><td>µm</td><td>D<sub>h</sub> = (ΣD<sub>v</sub><sup>4</sup>/N)<sup>1/4</sup></td></tr><tr><td>Vessel hydraulic diameter</td><td>D<sub>v</sub></td><td>µm</td><td>D<sub>v</sub> = [32(ab)<sup>3</sup>/(a<sup>2</sup>+b<sup>2</sup>)]<sup>1/4</sup><br>a and b major and minor ellipse diameters</td></tr><tr><td>Number of vessels</td><td>N<sub>vessel</sub></td><td></td><td></td></tr><tr><td>Conductive area</td><td>CA</td><td>mm<sup>2</sup></td><td>CA = π(D<sub>h</sub>/2)<sup>2</sup> * N<sub>vessel</sub></td></tr><tr><td>Theoretical hydraulic conductivity</td><td>K<sub>th</sub></td><td>kg s<sup>-1</sup> MPa<sup>-1</sup> m</td><td>K<sub>th</sub> = ΣK<sub>ellipse</sub></td></tr><tr><td>Theoretical leaf conductance</td><td>K<sub>leaf null</sub></td><td>kg s<sup>-1</sup> MPa<sup>-1</sup> m<sup>-2</sup></td><td>K<sub>leaf</sub> = (K<sub>th</sub> / L<sub>pet</sub> / 2) / A<sub>lamina</sub><br>K<sub>ellipse</sub> = πa<sup>3</sup>b<sup>3</sup>/64η(a<sup>3</sup>+b<sup>3</sup>)</td></tr><tr><td>Ellipse conductivity</td><td>K<sub>ellipse</sub></td><td>kg s<sup>-1</sup> MPa<sup>-1</sup> m</td><td>with η = 1.002*10<sup>-9</sup> MPa s<sup>-1</sup> at 20°C<br>a and b major and minor ellipse diameters</td></tr></tbody></table>

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<table><caption>**Table 2.** Main log-log linear relationships based on standardised major axis regression.</caption><thead><tr><th>Y</th><th>X</th><th>P</th><th>R<sup>2</sup></th><th>slope</th><th>95% CIs (slope)</th></tr></thead><tbody><tr><td>A<sub>lamina</sub></td><td>A<sub>pet</sub></td><td><strong>&lt; 0.001</strong></td><td>0.858</td><td>1.256</td><td>1.229 – 1.283</td></tr><tr><td>M<sub>lamina</sub></td><td>A<sub>pet</sub></td><td><strong>&lt; 0.001</strong></td><td>0.898</td><td>1.236</td><td>1.213 – 1.259</td></tr><tr><td>M<sub>lamina</sub></td><td>M<sub>pet</sub></td><td><strong>&lt; 0.001</strong></td><td>0.947</td><td>0.868</td><td>0.857 – 0.881</td></tr><tr><td>A<sub>lamina</sub></td><td>M<sub>leaf</sub></td><td><strong>&lt; 0.001</strong></td><td>0.913</td><td>0.995</td><td>0.977 – 1.012</td></tr><tr><td>D<sub>h</sub></td><td>PL</td><td><strong>&lt; 0.001</strong></td><td>0.469</td><td>0.549</td><td>0.474 – 0.638</td></tr><tr><td>D<sub>h</sub></td><td>A<sub>lamina</sub></td><td><strong>&lt; 0.001</strong></td><td>0.502</td><td>0.276</td><td>0.240 – 0.318</td></tr><tr><td>N<sub>vessel</sub></td><td>A<sub>lamina</sub></td><td><strong>&lt; 0.001</strong></td><td>0.596</td><td>0.490</td><td>0.456 – 0.524</td></tr><tr><td>CA</td><td>A<sub>lamina</sub></td><td><strong>&lt; 0.001</strong></td><td>0.635</td><td>0.780</td><td>0.685 – 0.875</td></tr><tr><td>Xylem area</td><td>A<sub>lamina</sub></td><td><strong>&lt; 0.001</strong></td><td>0.711</td><td>0.860</td><td>0.772 – 0.978</td></tr><tr><td>Approx. EI</td><td>A<sub>lamina</sub></td><td>&lt; 0.001</td><td>0.653</td><td>1.720</td><td>1.522 – 1.918</td></tr></tbody></table>

Bold values refer to significant correlation (P < 0.05). Table 1 for a list of abbreviations. CI: confidence interval.