DOI: 10.5061/dryad.7pvmcvf2k
title: Data from: Growing at the arid edge: Leaf anatomy variations are more extensive than stems in five Mediterranean species across contrasting moisture regimes-all the raw data of the anatomic measurements

description: Premise: The Mediterranean region is experiencing increasing aridity,
 affecting ecosystems and plant life. Plants exhibit various anatomical
 changes to cope with dry conditions, including anatomical changes. This
 study focused on five co-occurring Mediterranean plant species namely
 Quercus calliprinos, Pistacia palaestina, Pistacia lentiscus, Rhamnus
 lycioides, and Phillyrea latifolia in wet and dry sites, investigating
 anatomical differences in leaves and xylem. Methods: Leaf analysis
 involved stomatal density, stomatal length, Leaf Mass Area (LMA), lamina
 composition, quantification of leaf intercellular air spaces (IAS), and
 mesophyll cell area exposed to these spaces. Xylem anatomy was assessed
 through vessel length and area in branches. Results: In the dry site,
 three species showed increased stomatal density and decreased stomatal
 length. Four species exhibited increased palisade mesophyll (PM) and
 reduced air space volume. In contrast, the phenotypic change in the xylem
 was less pronounced, with vessel length remaining unaffected by the site
 conditions. Furthermore, vessel diameter decreased in two species.
 Intercellular air spaces (IAS) proved to be the most dynamic anatomical
 feature. Quercus calliprinos demonstrated the highest anatomical
 phenotypic changes, while Rhamnus lycioides exhibited minor changes.
 Conclusions: This study sheds light on the variation in anatomical
 responses among co-occurring Mediterranean plant species and identifies
 the most dynamic traits. Understanding these adaptations provides valuable
 insights into the ability of plants to thrive under changing climate
 conditions.
description: <em>Histological preparations</em>
 The samples were collected in June 2021, at the beginning of the
 dry season. For stem anatomy, one cm long segments of 0.5 to 1 cm in
 diameter were taken from the terminal branches of new growth. The same
 branches were used for leaf anatomy, where a rectangle of 1 x 2 cm was cut
 along the lamina while avoiding the midrib. All samples were fixed
 immediately after cutting in a formaldehyde-acetic acid–alcohol solution
 (FAA, 10:5:50 in double-distilled water) for 48 h. Following gradual
 dehydration in an ethanol series (70, 80, 90, 95, and 100%, for 30 min
 each), the samples were subjected to a gradual Histoclear solution (25,
 50, 75, and 100%). The samples were incubated overnight at room
 temperature with Paraplast chips (Leica, Wetzlar, Germany, Paraplast Plus)
 followed by several hours of incubation at 42 °C. The dissolved pure
 paraffin was changed twice a day for four days at 62 °C before the samples
 were embedded in blocks. Following embedding, stem samples were immersed
 in water for a few days and then sectioned using a microtome (Leica
 RM2245, Leica Microsystems Ltd. , Nussloch, Germany) into 12 μm sections
 which were mounted on slides, incubated overnight at 40 °C, and stained
 with Fast Green and Safranin (Ruzin and others, 1999). Images were
 captured using a light microscope (Leica DMLB, Leica Microsystems Ltd. ,
 Nussloch, Germany) with a Nikon DS-fi1 camera (Nikon Corporation, Japan).
 Image analysis was done using ImageJ software (Rasband, W.S., ImageJ, US
 National Institutes of Health, Bethesda, MD, USA, http://
 imagej.nih.gov/ij/, 1997–2015). <em>Leaf anatomy
 analyses</em> The leaf parameters (Table 3) were
 measured in eight samples from each of the five studied species at each
 site. <strong>Leaf mass area (LMA)
 </strong>was computed by dividing the leaf dry mass (g) by the leaf
 area (cm²). Leaf area was determined through the analysis of RGB-scaled
 photos using ImageJ software. Subsequently, the leaves were dried at 70°C
 for 5 days, followed by measurement of the dry weight.
 <strong>Stomatal density</strong> was measured from
 adaxial and abaxial epidermal imprints, which were made using a dental
 impression gel (CounterFit II, Clinician's Choice), followed by an
 impression of clear nail polish, which was removed using adhesive tape and
 mounted on a microscope slide. Stomata were counted on an area of 0.0837
 mm<sup>2</sup> which represented the whole image size at the
 corresponding magnification (x40). <strong>Lamina
 anatomy</strong> was analyzed from leaf cross sectional images using
 the ImageJ software to obtain thickness values in microns for the
 different leaf organs: adaxial (Ad) and abaxial (Ab) epidermis layers,
 palisade mesophyll (Pal) and spongy mesophyll (SM) as well as total leaf
 thickness (T). Cuticle thickness assessment was available only on the
 adaxial side (Ac) as the abaxial cuticle was indistinct. All parameters
 were measured at three different locations on a cross section. The Midrib
 vessel area was assessed by measuring the ten largest vessels using the
 ImageJ software. <strong>Intercellular
 airspaces</strong> were evaluated from the mesophyll surface area
 exposed to intercellular airspace per unit leaf area , which
 was calculated according to (Evans et al., 1994)::
 Where is the total length of mesophyll cells facing the
 intercellular air space,  is the section width and F is the
 curvature correction factor, which depends on the shape of the cells and
 was calculated as the weight average of the palisade and spongy mesophyll
 according to (Thain, 1983). The fraction of the
 intercellular air space (%IAS) was calculated as  Where
 ΣSs is the sum of the cross-sectional areas of the mesophyll cells and is
 the thickness of the mesophyll between the two epidermal layers.
 <em>Stem anatomy analyses</em>
 The stem parameters (Table 3) were measured in eight samples from
 each of the five studied species at each site.
 <strong>Vessel length distribution</strong> was
 measured by the "air injection method" (Cohen et al., 2003),
 with some modifications according to Wang et al. (2014). Briefly, fresh
 long shoots were cut using a sharp razor blade. The basal end of the stem
 segment was attached to a flexible silicone tube (clamped to it) and
 connected to an air compressor which injected air into an old dial
 manometer and a digital pressure sensor (MPX5100 IC, NXP Semiconductors,
 Netherlands) wired to a datalogger (Campbell Sci.  CR1000 datalogger,
 Campbell Scientific, Inc., Utah, United States), along with a
 "bleed" valve. Pressure was adjusted to 0.08-0.15 MPa and logged
 during the measurements. The distal end of each shoot was immersed in
 water. Stem segments (2 cm long) were cut  back until bubbling was
 observed, and the length of the remaining stem was taken as the maximum
 vessel length (in some cases, bubbles appeared immediately before cutting,
 in which case the maximum vessel length was longer). Then, the stem was
 cut back consistently to measure air flow rate at several lengths. For
 each stem length, the bubbles flowing out from the distal end were
 collected in a volumetric cylinder by the water displacement method
 according to (Wang et al., 2014). The airflow rate [<em>Q</em>
 (mL/min)] was computed as follows:
 <em>Q</em>
 =(W<sub>i</sub> − W<sub>f</sub>)/(ΔTρ)
 Where W<sub>i</sub> and W<sub>f</sub> 
 are the initial and final weights of the volumetric cylinder respectively,
 ΔT is the time interval for the water displacement by the bubbles
 and <em>ρ</em> is the density of water displaced by the
 air. Air conductivity (C) was calculated according to
 equation [4] at (Cohen et al., 2003) as follows: Where
 L is the length of the wood segment (m), P is the distal pressure (kPa) at
 which the flow rate Q was measured at the distal end  is the
 average pressure in the segment and ΔP  is the pressure difference across
 the segment. According to Cohen et al. (2003) C should
 decrease exponentially
 as:                                                 
 Where  is the limiting conductivity as x approaches
 zero, k is the extinction coefficient and x is the stem length. The plot
 of the natural log of C versus x resulted in a linear plot, from which k
 was evaluated from the slope. The most common of mode vessel length
 (Lmode) was −1/k. The mean vessel length was calculated from
 L<sub>mean</sub> = 2L<sub>mode</sub>.
 The probability density function (PDF) of vessel length was
 calculated as described in  (Cohen et al., 2003) and (Sperry et al., 2005)
 was: Where  is the probability of vessels of
 length x and k (negative value) is the slope of the linear plot.
 <strong>The vessel
 area</strong>/<strong>diameter</strong> was evaluated
 from the most two outer rings of the stem cross sections (described
 above), which were marked and measured manually by "tracking
 tool" by Image J software. The vessel diameter (D) was calculated
 from the vessel area as follows: <em>Statistical
 analyses </em> The individual data for each
 anatomical trait are presented as boxplots. To test the effect of site,
 species, and their interaction on the anatomical traits, a two-way ANOVA
 was conducted using Python software (Python Software Foundation,
 Wilmington, Delaware, United States; package: statsmodel).Traits for which
 variances were non-homogeneous underwent logarithmic transformation before
 analysis. To compare the two sites for each species,
 contrast t-tests were performed. To quantify the degree of the difference
 between the two sites for each species, the effect size was measured using
 Cohen's d method for each anatomy trait. The formula used for
 calculating Cohen's d is: Cohen's d = (M1 -
 M2) / pooled standard deviation where M1 - M2 is the
 difference between means, i.e., the absolute value of the difference
 between the mean values of the wet and arid sites, and the pooled standard
 deviation was calculated as follows: pooled standard
 deviation = sqrt[(SD1^2 + SD2^2)/2] where SD1 and SD2
 are the standard deviations for the wet and dry sites,
 respectively.
description: # Growing at the arid edge: Leaf anatomy variations are more extensive
 than stems in five Mediterranean species across contrasting moisture
 regimes-all the raw data of the anatomic measurements
 [https://doi.org/10.5061/dryad.7pvmcvf2k](https://doi.org/10.5061/dryad.7pvmcvf2k) The dataset encompasses all the raw data collected during the study, comprising measurements and observations from both SA and MM sites across various plant species. ## Description of the data and file structure In the dataset, there is a single file containing comprehensive data pertaining to anatomical parameters across two distinct sites, SA and MM, and various plant species. Each row represents a specific measurement or observation, while the columns encompass diverse anatomical parameters. These parameters likely include but are not limited to stomatal density, stomatal length, Leaf Mass Area (LMA), lamina composition, quantification of leaf intercellular air spaces (IAS), and mesophyll cell area. ## Variables and Units The following table describes each variable in the dataset along with its units (if applicable). | Variable | Description | Units | | :------------------- | :-------------------------------- | :--------------------- | | Site | Study site location | - | | Species | Species name | - | | species(sc) | Species code | - | | sample | Sample identifier | - | | sp | Species abbreviation | - | | site2 | Secondary site identifier | - | | sn | Sample number | - | | SSn | Secondary sample number | - | | repeat | Repeat measurement indicator | - | | k | Specific identifier | extinction coefficient | | mode\_VL | Mode of VL (variable) | cm | | avg\_VL | Average VL | cm | | diameter | Diameter measurement | micrometers (µm) | | LMA | Leaf Mass per Area | g/m² | | A | Area measurement | cm² | | SD | Stomatal density | stomata/mm² | | VAm | Midrib Vessel's area | μm² | | VAsS | Stem Vessel's area | μm² | | IAS | Intercellular Air Space | % | | SM\_S | SM\_S (variable) | m² m-2 | | All Thickness (mic) | Total leaf thickness | micrometers (µm) | | Ad cut(mic) | Adaxial cuticle thickness | micrometers (µm) | | Ad apidermis(mic) | Adaxial epidermis thickness | micrometers (µm) | | Max palisade layers | Maximum number of palisade layers | layers | | Palisade max (mic) | Maximum palisade thickness | micrometers (µm) | | Mode palisade layers | Mode number of palisade layers | layers | | Palisade mode (mic) | Mode palisade thickness | micrometers (µm) | | Spongy (mic) | Spongy mesophyll thickness | micrometers (µm) | | Ab apidermis(mic) | Abaxial epidermis thickness | micrometers (µm) | | Ab cut(mic) | Abaxial cuticle thickness | micrometers (µm) | | Stom\_Leaf | Stomata number  on leaf | - | | SL | Stomatal Legnth | micrometers (µm) | | Dm | Midrib vessels diameter | micrometers (µm) | | Ds | Stem vessels diameter | micrometers (µm) | | | | | | | | | | | | | | | | | ### Important Note on Missing Data There are some empty cells in the dataset. These empty cells are represented as "n/a" to indicate that the data was not available for those specific measurements. This notation helps minimize ambiguity for potential users.