DOI: 10.5061/dryad.547d7wmhs
title: Grass climatic and anatomical data

description: Members of the grass family Poaceae have adapted to a wide range of
 habitats and disturbance regimes across the planet. The cellular structure
 and arrangements of leaves can help explain how plants survive in
 different climates, but these traits are rarely measured in grasses.
 Further, most studies are focused on individual species or distantly
 related species within Poaceae.  While this focus can reveal
 broad adaptations, it also likely to overlook subtle adaptations within
 more closely-related groups (subfamilies, tribes). This study therefore
 investigated the scaling relationships between leaf size, vein density,
 and vessel size in five genera within the subfamily Pooideae. The
 relationship between leaf area and major vein number was consistent with
 previous findings (p &lt; 0.05, slope = 0.72 +/- 0.24), as was the
 scaling coefficient of VLA (slope= -0.46 +/- 0.21). However, several
 genera exhibited novel anatomical relationships. In Poa and Elymus, minor
 vein number and leaf length were uncorrelated, whereas in Festuca these
 traits were positively correlated (slope = 0.82 +/- 0.8). These findings
 suggest there is important broad-scale and fine-scale variation in leaf
 hydraulic traits among grasses. Thus, future studies should consider both
 narrow and broad phylogenetic gradients.
description: <strong>Plant Material Selection and
 Germination</strong> 
 
 <strong>Genus</strong> 
 <strong>specific
 epithet</strong> 
 <strong>Genus</strong> 
 <strong>specific
 epithet</strong> 
 <strong>Genus</strong> 
 <strong>specific
 epithet</strong> 
 <em>Bromus</em> 
 <em>anomalus</em> 
 <em>Festuca</em> 
 <em>altaica</em> 
 <em>Hesperostipa</em>
 <em>neomexicana</em>
 
 <em>Bromus</em> 
 <em>inermis</em> 
 <em>Festuca</em> 
 <em>arizonica</em> 
 <em>Poa</em> 
 <em>alpina</em> 
 <em>Bromus</em>
 <em>laevipes</em>
 <em>Festuca</em>
 <em>californica</em>
 <em>Poa</em>
 <em>arida</em>
 
 <em>Elymus</em> 
 <em>canadensis</em> 
 <em>Festuca</em> 
 <em>idahoensis</em> 
 <em>Poa</em> 
 <em>compressa</em> 
 <em>Elymus</em>
 <em>elymoides</em>
 <em>Festuca</em>
 <em>roemeri</em>
 <em>Poa</em>
 <em>fendleriana</em>
 
 <em>Elymus</em> 
 <em>hysterix</em> 
 <em>Festuca</em> 
 <em>rubra</em> 
 <em>Poa</em> 
 <em>glauca</em> 
 <em>Elymus</em>
 <em>lanceolatus</em>
 
 <em>Hesperostipa</em> 
 <em>comata</em> 
 <em>Poa</em> 
 <em>secunda</em> 
 Table 1- list of the
 species germinated for data collection. Five species
 were selected from each of five genera: <em>Poa, Hesperostipa,
 Elymus, Festuca, </em>and <em>Bromus</em>. The species
 selected in this study are all phylogenetically classified as part of the
 Pooideae subfamily; this was done to ensure that all species were
 separated by relatively recent evolutionary divergences. Species were also
 selected such that their habitats spanned a wide range of temperature and
 precipitation across North America. Plants in this study were grown from
 seeds provided by the USDA Germplasm Resource Information Network.
 Unfortunately, not all species germinated regardless of any pre-treatments
 we attempted and so we were not able to measure 5 species in all genera.
 Specimens used for gas exchange measurements were grown during the summer
 of 2016 in 35-cm-length Deep-pots (D60 series, Stuewe and Sons, Inc,
 Tangent, USA)  while the remaining specimens were grown in 10-cm-length
 “cone”-tainers (Ray Leach Cells 3, Stuewe and Sons, Inc, Tangent, USA),
 with a 70% to 30% mix of potting soil (Promix HP, Quakertown, USA) and
 fritted clay (Greens’ Grade Porous Ceramic Topdressing, Buffalo Grove,
 USA), respectively. This substrate was fertilized with slow-release
 fertilizer (Osmocote Plus, Scotts Miracle-Gro Company, Marysville, USA) at
 a ratio of 10 mL fertilizer L<sup>-1</sup> soil substrate. The
 soil substrate was saturated with water before seeds were planted, and
 specimens were misted until the full expansion of their
 4<sup>th</sup> leaf, then watered 3 times per week.
 <strong>Anatomical Analysis</strong>
 Whole-leaf samples (base to tip of lamina) were collected from
 five randomly selected individuals of each species for the purpose of
 anatomical examination. The third or fourth full leaf was harvested from
 each plant after full expansion. Samples were stored in a solution of
 formalin, acetic acid, ethanol, and deionized water (FAA fixative
 solution). Anatomical samples were collected halfway along the lamina
 length of each specimen, using a razor to cut a cross-sectional sample
 ~0.5 mm in thickness. The distance from the cross-section and the leaf tip
 were also recorded. Each cross-sectional sample was stained using
 safranin-o and fast green. Microscopic images of vein anatomy were taken
 of half the total lamina width using a ZEISS Axio Scope.A1 in conjunction
 with ZEN microscope software (Carl Zeiss Microscopy, Germany). Images were
 measured using Fiji open-source image analysis software. The number of
 vein orders of each species was quantified, and 1° and 2° veins were
 classified as ‘major’ veins and 3° and 4° (if present) were classified as
 ‘minor’ veins. We defined major veins as having at least two of the three
 following characteristics: 1) vein was at least 50% larger than the
 smallest vein, 2) vein had bundle sheath extension, 3) vein had at least
 two xylary vessel elements (i.e metaxylem lacuna) 100% larger than
 remaining xylary vessel elements. Although this criteria differs slightly
 from what others have used to define vein orders (Baird <em>et
 al</em>. 2021), it was developed to help us to objectively assign
 vein orders since leaf veins don’t always fall neatly into the orders
 defined previously. However, we are confident that what we defined as
 ‘major’ is consistent with previous research. The anatomical traits of up
 to five major veins and minor veins per leaf were measured, but all veins
 were counted and classified to a vein order. Vein length density was
 calculated as total vein length per unit area (cm
 cm<sup>-2</sup>) by measuring the width of the leaf and then
 counting the number of veins in the leaf, then multiplying by the length
 of the leaf. The diameter of the vein including the bundle sheath (μm) was
 measured as the distance from the outer edge of the bundle sheath to the
 opposite outer edge. Vein diameter (D<sub>vein</sub>, μm) was
 measured from the innermost edge of the bundle sheath to the opposite
 inner edge. Diameter of the bundle sheath cells
 (D<sub>BS</sub>) was then estimated by subtracting vein
 diameter measured from the innermost edge from the vein diameter measured
 from the outermost edge of the bundle sheath. The diameters of vessel
 elements (D<sub>vessel</sub>, μm) were measured as the
 distance between the inner edge of the vessel element cell wall to the
 opposite inner edge. Vessel element wall thickness (WT; μm) was also
 measured. ‘MAJ’ is added to each subscript when the data reported was
 measured on major veins and ‘min’ added to subscript when the data
 represents the minor veins. <strong>Climate
 Envelope Analysis</strong> Species distribution
 data were collected from the Global Biodiversity Information Facility
 (GBIF). Climate data were obtained from weather stations closest to the
 location where seed was harvested as reported by GBIF. In addition to
 weather station observations, gridded and interpolated climate data were
 retrieved from WorldClim at 0.1 degree resolution. For each reported
 occurrence of each species in the GBIF database, climate data from the
 closet grid point was retrieved and added to the data set. If two reported
 occurrences were equally close to the same grid point, that point was only
 used once to avoid pseudo-replication. Once this data set was generated
 for each species, the 5<sup>th</sup>,
 50<sup>th</sup>, and 95<sup>th</sup> percentiles
 of each climate variable (see list of variables in supplement) were
 calculated and used to define the climate envelope of each species.
 Because we hypothesized that hydraulic architecture would relate to
 temperature and precipitation of the climate of origin, we focused our
 investigation on climate variables that would capture these abiotic
 stressors: MAP, MAT, temperature of the wettest and driest quarters, and
 MAP of the warmest quarter. <strong>Statistical
 Analysis</strong> Scaling relationships were
 evaluated using the ‘sma’ function in the ‘smatr’ package to account for
 variability in both x and y variables. In cases where we expected
 variation between the x and y variables to be explained by a power
 function, we log10-transformed both axes. This included relationships
 between leaf dimensions (width, length) and vessel number (Price
 <em>et al</em>. 2007. Gleason <em>et al</em>.
 2018), and between leaf dimensions (i.e., pathlength ~ leaf length) and
 conduit diameter (Anfodillo <em>et al</em>. 2006). Axes were
 also log10-transformed in cases where we wanted to test an expected linear
 or proportional relationship between x and y variables, e.g., the
 relationship between conduit diameter and cell wall thickness at a given
 buckling pressure (Brodribb and Holbrook 2005), as well as conduit
 diameter (or conduit number) between different vein orders (Gleason
 <em>et al</em>. 2018). Variables were first tested for
 differences between genera by including ‘genus’ in a model to test for
 differences between the coefficients – slope (hereafter “scaling
 coefficient”) and intercept. If no differences were found, then a single
 scaling relationship was used and reported. If differences were found,
 then the genera with unique coefficients were removed and analyses were
 performed individually on each group.
description: # Grass climatic and anatomical data
 [https://doi.org/10.5061/dryad.547d7wmhs](https://doi.org/10.5061/dryad.547d7wmhs) ## Description of the data and file structure Analysis of scaling relationships across multiple grass genera within the subfamily Pooideae ### Files and variables #### File: final\_dataset\_allclim.csv **Description:**  ##### Variables * subfamily: the subfamily of each species * genus: the two-letter abbreviation of the genus for each species * species: the four-letter abbreviation for each species * mean_photo: Mean photosynthetic rate, measured as mmol CO2 m^-2 s^-1 * mean_cond: Mean leaf water conductance rate, measured as mol H2O m^-2 s^-1 * thick_major: mean leaf thickness at each major vein, measured in micrometers * thick_minor: mean leaf thickness at each minor vein, measured in micrometers * m_bs_maj: mean percentage of mesophyll occupied by bundle sheath for major veins * m_bs_min: mean percentage of mesophyll occupied by bundle sheath for minor veins * Mtom_ratio: Major to minor vein ratio * vein_density: mean number of veins per unit leaf width, measured as mm mm^-2 * width: Mean width of leaves, measured in mm * length: Mean length of leaves, measured in cm from sampling point to leaf tip * A: Mean leaf area, measured in cm^2 * norml: normalized leaf length * Mvein_number: mean number of major veins per leaf * Mvein_density: mean number of major veins per unit width, measured in mm mm^-2 * interveinal_distance: mean distance between veins, measured from vein center to vein center in micrometers * BS_diameter: mean diameter of bundle sheaths of major veins, measured in micrometers * BS_thickness: mean thickness of bundle sheaths of major veins, measured in micrometers * vein_diameter: mean diameter of major veins, measured in micrometers * VE_lumen_diameter: mean diameter of vessels in major veins, measured in micrometers * VE_wall_thickness: mean vessel wall thickness in major veins, measured in micrometers * mBS_diameter: mean diameter of bundle sheaths of minor veins, measured in micrometers * mBS_thickness: mean thickness of bundle sheaths of minor veins, measured in micrometers * mvein_diameter: mean diameter of minor veins, measured in micrometers * mVE_lumen_diameter: mean diameter of vessels in minor veins, measured in micrometers * mVE_wall_thickness: mean vessel wall thickness in minor veins, measured in micrometers * tb: mean vessel wall thickness over vessel diameter of major veins * mtb: mean vessel wall thickness over vessel diameter of minor veins * MAT: Mean annual temperature (degrees C) * mean_diurnal_range: mean temperature range in a day (degrees C) * isotherm: temperature variance throughout the year * t_season: variance in temperature across quarters * max_twarmest: highest temp on warmest day (degrees C) * min_tcoldest: coldest temp on coldest day (degrees C) * annual_trange: Range of temp for the year (degrees C) * mean_twettest: mean temp of wettest quarter (degrees C) * mean_tdriest: mean temp of direst quarter (degrees C) * mean_twarmest: mean temp of warmest quarter (degrees C) * mean_tcoldest: mean temp of coldest quarter (degrees C) * annual_precip: total annual precip (mm) * precip_wettest: total precip of wettest day (mm) * precip_driest: total precip driest day (mm) * precip_seasonality: variance in precip across quarters * precip_wetquarter: total precip of wettest quarter (mm) * precip_dryquarter: total precip of driest quarter (mm) * precip_warmquarter: total precip of warmest quarter (mm) * precip_coldquarter: total precip of coldest quarter (mm) missing data is left as blank cells