DOI: 10.5061/dryad.pg4f4qs3k
title: Intraspecific variation in stomatal architecture, gas exchange, and drought response of a dominant prairie grass sourced from broad climatic gradients

description: Premise: Understanding how plant populations adapt to water limitation
 through stomatal traits is key to predicting drought responses. The
 dominant C~4 ~grass Andropogon gerardi, distributed
 across sharp climate gradients in North America, offers an excellent focal
 species to study stomatal architecture (size and density). Using
 a common garden, we tested how stomatal architecture relates to home
 climate, how stomatal architecture influences gas exchange, and how
 experimental drought affects these responses in a greenhouse. We
 hypothesized that aridity drives stomatal architecture, and experimental
 drought reduces stomatal size but increases density to maintain
 photosynthesis. Methods: In our first experiment, we measured
 stomatal architecture and gas exchange in 25 populations sourced across
 temperature (4–21°C) and precipitation (350–1400 mm yr⁻¹) gradients under
 well-watered conditions. A second experiment focused on eight populations
 (472–1356 mm yr⁻¹), subjected to drought (~15% soil
 moisture) or well-watered (30% control) to assess plasticity.
 Stomatal traits were measured with epidermal peels and light microscopy,
 gas exchange with a LI-COR 6400, and network analyses were used to
 characterize adaptive strategies.  Results: Arid populations
 exhibited smaller, denser stomata compared to wet populations
 and networks demonstrated a trade-off between stomatal
 size and density. Under experimental drought, stomatal size
 decreased while density increased, with dry populations showing fewer
 changes than wet populations. Key traits in the network were
 stomatal size and water-use efficiency. Conclusions: This study reveals
 that A. gerardi demonstrates adaptive changes in
 stomatal architecture. Our findings emphasize the interplay between
 adaptation and climate, providing important insights into how plants may
 respond to increased droughts.
description: # Intraspecific variation in stomatal architecture, gas exchange, and
 drought response of a dominant prairie grass sourced from broad climatic
 gradients Dataset DOI:
 [10.5061/dryad.pg4f4qs3k](https://doi.org/10.5061/dryad.pg4f4qs3k) The
 dataset was collected to investigate the relationships between stomatal
 architecture, gas exchange, and climate in *Andropogon gerardi*
 populations under both natural and experimental conditions in a
 greenhouse. In the main experiment, populations were sampled across a
 range of climatic gradients to quantify variation in stomatal traits (size
 and density) and physiological traits (photosynthetic rate, transpiration,
 stomatal conductance, water-use efficiency, and internal CO₂). In the
 drought experiment, plants from multiple populations were subjected to
 control and drought treatments to assess how water limitation influences
 stomatal traits and gas exchange. Measurements were taken at the
 population level, and mean values per population were calculated for all
 traits. The experimental design allowed for the analysis of climate
 effects, treatment effects, and trait coordination, including the
 evaluation of trait networks under both well-watered and drought
 conditions. ## 1. Overview This dataset investigates the relationships
 among stomatal architecture, gas exchange, and climate in *Andropogon
 gerardii* populations sampled across broad climatic gradients. Data were
 collected under both **natural field conditions**(Main Experiment) and
 **controlled drought treatments** (Drought Experiment) in a greenhouse
 setting. The dataset enables evaluation of how variation in stomatal
 traits and physiological performance corresponds to climate and drought
 responses, as well as the coordination of traits within and across
 environmental contexts. ## 2. Description of Data and File Structure ###
 2.1 Main Experiment **File:** `Main_Experiment.xlsx`\ This file contains
 population-level measurements of stomatal and gas exchange traits
 collected from natural field populations spanning a climatic gradient.
 **Variables:** * **Population (Site):** Unique identifier for each
 population sampled in the field. * **Block:** Experimental or measurement
 block used for replication within the site. * **Mean abaxial diameter
 (µm):** Average stomatal pore length on the abaxial (lower) leaf surface,
 measured in micrometers. * **Mean abaxial density (stomata mm⁻²):** Mean
 number of stomata per square millimeter on the abaxial leaf surface. *
 **Mean adaxial diameter (µm):** Average stomatal pore length on the
 adaxial (upper) leaf surface. * **Mean adaxial density (stomata mm⁻²):**
 Mean number of stomata per square millimeter on the adaxial leaf surface.
 * **Abaxial stomatal diameter/density:** Individual-level measurements of
 stomatal size and density on the abaxial surface, averaged by population.
 * **Adaxial stomatal diameter/density:** Individual-level measurements on
 the adaxial surface, averaged by population. * **Photosynthetic rate (µmol
 CO₂ m⁻² s⁻¹):** Rate of CO₂ assimilation measured under standardized light
 and CO₂ conditions. * **Transpiration rate (mmol H₂O m⁻² s⁻¹):** Rate of
 water vapor loss from leaves. * **Stomatal conductance (mol H₂O m⁻²
 s⁻¹):** Leaf conductance to water vapor, indicating the extent of stomatal
 opening. * **Intrinsic water-use efficiency (iWUE; µmol CO₂ mol⁻¹ H₂O):**
 Ratio of photosynthetic rate to stomatal conductance. * **Internal CO₂
 (µmol mol⁻¹):** Intercellular CO₂ concentration within the leaf, derived
 from gas exchange. * **Mean annual temperature (°C):** Long-term mean air
 temperature at the source population’s location. * **Mean annual
 precipitation (mm yr⁻¹):** Total annual precipitation at the population’s
 source site. * **Growing season precipitation (mm):** Total precipitation
 during the primary growing season. * **Aridity index (unitless):** Ratio
 of mean annual precipitation to potential evapotranspiration; higher
 values indicate wetter climates. * **Growing season aridity index
 (unitless):** Same ratio calculated specifically for the growing season. *
 **Growing season length (days):** Number of days suitable for plant growth
 (based on temperature thresholds). * **Evaporative index (unitless):**
 Ratio of actual to potential evapotranspiration, representing water
 availability. * **Growing season temperature (°C):** Mean temperature
 during the growing season. * **Latitude and Longitude (decimal degrees):**
 Geographic coordinates of each population’s source location. ### 2.2
 Drought Experiment **File:** `Drought_Experiment.xlsx` This file contains
 population-level measurements of stomatal and gas exchange traits under
 two treatments: control (well-watered) and drought (water-limited)
 conditions in a greenhouse. **Variables:** * **Block:** Greenhouse bench
 or position representing an experimental block. * **Sample ID:** Unique
 label for each individual or population sample. * **Pop:** Population
 identifier matching the source population from the field. * **Treatment:**
 Experimental condition (Control or Drought). * **Mean adaxial density
 (stomata mm⁻²):** Mean number of stomata per square millimeter on the
 upper (adaxial) leaf surface. * **Mean adaxial size (µm):** Average
 stomatal pore length on the adaxial surface. * **Mean abaxial density
 (stomata mm⁻²):** Mean number of stomata per square millimeter on the
 lower (abaxial) surface. * **Mean abaxial size (µm):** Average stomatal
 pore length on the abaxial surface. * **Adaxial stomatal density / size
 (µm):** Individual-level averages for the adaxial surface. * **Abaxial
 stomatal density / size (µm):** Individual-level averages for the abaxial
 surface. * **Photosynthetic rate (µmol CO₂ m⁻² s⁻¹):** Rate of CO₂
 assimilation under experimental treatment. * **Transpiration rate (mmol
 H₂O m⁻² s⁻¹):** Water vapor loss rate under experimental treatment. *
 **Stomatal conductance (mol H₂O m⁻² s⁻¹):** Conductance to water vapor. *
 **Intrinsic water-use efficiency (iWUE; µmol CO₂ mol⁻¹ H₂O):** Ratio of
 photosynthesis to conductance. * **Mean annual temperature (°C):** Mean
 temperature at the population’s source site. * **Mean annual precipitation
 (mm yr⁻¹):** Mean total annual precipitation at source site. * **Growing
 season precipitation (mm):** Precipitation during the growing season. *
 **Aridity index (unitless):** Ratio of precipitation to potential
 evapotranspiration. * **Growing season aridity index (unitless):**
 Seasonal version of aridity index. * **Growing season length (days):**
 Length of the growing season. * **Evaporative index (unitless):** Ratio of
 actual to potential evapotranspiration. * **Growing season temperature
 (°C):** Average temperature during growing season. * **Latitude and
 Longitude (decimal degrees):** Coordinates of the source population. ###
 2.3 Greenhouse Environmental Data Main Experiment **File:** *
 `Main_Experiment_Greenhouse_Conditions.xlsx` These files record
 environmental conditions during the greenhouse experiments. **Variables
 include:** * **Date (MM-DD-YYYY):** Measurement date. * **Block:**
 Greenhouse bench used for replication. * **Reading**: Direct soil moisture
 reading from Zentra probe.  * **Soil_Moisture (%**): Percentage volumetric
 soil moisture content.  * **Soil_Temperature (°C):** Soil temperature
 reading. * **Ambient_Light (µmol photons m⁻² s⁻¹):** Photosynthetic photon
 flux density. ### 2.4 Greenhouse Environmental Data Drought Experiment
 **File:** * `Drought_Experiment_Greenhouse_Conditions.xlsx` These files
 record environmental conditions during the greenhouse experiments.
 **Variables include:** * **Date (MM-DD-YYYY):** Measurement date. *
 **Treatment:** Control or Drought condition. * **Drought_1, Drought_2,
 Drought_3, and Drought_4**: Soil moisture (%) readings for four replicate
 droughted plants.  * **Control_1, Control_2, Control_3, and Control_4**:
 Soil moisture (%) readings for four replicate control plants.  *
 **Soil_Temperature (°C):** Average soil temperature. *
 **Light_Availability (µmol photons m⁻² s⁻¹):** Photosynthetic photon flux
 density. ## 3. Analysis Scripts The following text-based scripts accompany
 the dataset and can be run in JMP or R. *
 **`Main_Experiment_Stomata_Climate_JMP.txt`** – Regressions of stomatal
 traits against climate variables using linear and quadratic models. *
 **`Main_Experiment_Stomata_Gas_Exchange_JMP.txt`** – Models gas exchange
 traits as a function of stomatal traits. *
 **`Main_Experiment_PCA_JMP.txt`** – Principal Component Analysis (PCA) of
 stomatal and gas exchange traits. *
 **`Main_Experiment_Network_R_Code.txt`** – R script for constructing plant
 trait networks (PTNs) using Spearman correlations. *
 **`Drought_Experiment_ANCOVA_JMP.txt`** – ANCOVA testing effects of
 population, treatment, and stomatal traits on gas exchange. *
 **`Drought_Experiment_PCA_JMP.txt`** – PCA of stomatal and physiological
 traits under control and drought treatments. *
 **`Drought_Experiment_Gas_Exchange_JMP.txt`** – Regression of gas exchange
 variables on stomatal traits. *
 **`Drought_Experiment_Network_R_Code.txt`** – R script for
 treatment-specific network analyses. ## 4. Software Requirements *
 **Microsoft Excel / LibreOffice / Google Sheets:** For viewing and editing
 Excel data files. * **JMP Pro 16 or later (SAS):** For running statistical
 analyses including ANCOVA, PCA, and regression models. * **R (version 4.2
 or higher)** and **RStudio (optional):** For constructing and visualizing
 trait networks. * Required R packages: *tidyverse*, *igraph*, *Hmisc*,
 *corrplot*, *readxl*. ## 5. Workflow Summary 1. Open Excel data files to
 explore raw and summarized measurements. 2. Run JMP scripts for parametric
 statistical analyses (ANCOVA, PCA, and regressions). 3. Use R scripts to
 compute trait correlation matrices and construct trait networks. 4.
 Compare patterns under control and drought treatments to evaluate
 climate-dependent trait coordination. ## 6. Interpretation Notes *
 **Stomatal traits** (size and density) reflect structural adaptations
 affecting CO₂ uptake and water regulation. * **Gas exchange traits**
 represent physiological performance under varying water availability. *
 **Climate variables** describe the environmental origins of populations
 and are derived from long-term climatic datasets. * All measurements were
 taken on fully expanded leaves from greenhouse-grown plants under
 controlled light and CO₂ levels.