Here, we present all code and data files used for analyses and figure making in Bernauer et al. 2024 - “Elevated atmospheric CO2 has small, species-specific effects on pollen chemistry and plant growth across flowering plant species“. Access this data set on Dryad: https://doi.org/10.5061/dryad.70rxwdc5n.

In total, there are 10 data sets included, which are described in the “Datasets” subheading below, as well as seven code files, the first four listed under the “Analysis and Visualization” subheading include all manuscript analysis and figure making. In the Greenhouse CO2 Control, we include the scripts used to run the Arduino CO2 controllers within the greenhouse. 

In this study, we examined how CO2 levels influenced plant growth and pollen chemistry (nutrition or %N, secondary metabolites, and amino acids) across fourteen species with known value to pollinators. To do so, we conducted to experiments, one in the greenhouses at Harvard University that investigated pollen chemistry, and one at the University of Wisconsin-Madison that investigated plant growth and pollen chemistry (nutrition). We established greenhouses with either ambient (unmanipulated CO2 levels) or elevated CO2 levels (to 650 ppm at Harvard and 690 ppm at the University of Wisconsin). To evaluate pollen chemistry, we collected pollen or anther samples (one sample per plant), then analyzed them for nutrition (%N, %C, and C:N ratio using a Thermo Flash EA1112 Carbon Nitrogen analyzer) or secondary metabolites and amino acids (using LC-MS). Plant growth measurements included weekly height and leaf number, daily flower number from onset of flowering, and once per plant, we measured flower size, above-ground biomass. All data was analyzed using R (v 4.2.2) and associated code is below. For detailed methods, see “Elevated atmospheric CO2 has small, species-specific effects on pollen chemistry and plant growth across flowering plant species”.

Datasets

Here is a list of the included data sets included here and used for analyses in the manuscript.

1. Metadata.xlsx is the the metadata for all subsequent data files. Each file name is listed in bold with all column headings in column A and column descriptions, including units in column B.
2. CO2_log_exp1.csv is the data file for the CO2 logger data from experiment 1 containing time-stamped CO2 and temperature readings for greenhouse chambers 1 and 2 in experiment 2. This data file contains the following columns: “time” (timestamp when CO2 and temperature readings were captured), “co2_1” and “co2_2” (columns with the CO2 (ppm) readings for greenhouse chambers 1 and 2, respectively), “temp_1” and “temp_2” (columns with the temperature (°C) readings for greenhouse chambers 1 and 2, respectively), and “datenum” which is the time stamp from the R package “Lubridate” used for plotting this data in R. 
3. CO2_log_exp2.csv is the data file for the CO2 logger data from experiment 2 containing time-stamped CO2, humidity, and temperature readings for ambient and elevated CO2 conditions in rounds 1 and 2. Outliers (high and low) are noted in a column so they can be removed for analyses. This data file contains the following columns: “Temp-C” (temperature (°C) reading recorded in a greenhouse chamber), “Humidity” (relative humidity (%) reading recorded in a greenhouse chamber), “aco2” (CO2 (ppm) reading from Adafruit SCD_30 in ambient CO2 greenhouses), “eco2” (CO2 (ppm) reading from Licor 8500 in elevated CO2 greenhouses), “Date-Time” (Time stamp for when reading was collected in the format YY-MM-DD HH:MM:SS), “Round 1” and “Round 2” (data from rounds 1 and 2 are indicated with a 1 or 2 in these columns, respectively; rounds 1 and 2 overlapped so some readings fall into both round 1 and round 2 data), and “outliers” (A column where outliers are noted based on +/- 1.5 IQR calculations to be remove before plotting or analysis). 
4. Pollen_metabolomics_exp1.csv is the data file containing the targeted metabolomics data from experiment 1. For each pollen sample, this data file records, the unique sample identifier, plant species, CO2 treatment, sample type (pollen vs. anther), unique file name, the quantities of 18 targeted compounds in each sample, and the experimental round each sample was from. This data file includes the columns: “sample #” (unique sample identifier for the individual plant the sample came from), “species” (plant species the sample was collected from), “CO2 treatment” (CO2 treatment: ambient or elevated), “sample type” (pollen sample type: anther or pollen), “Filename” (Unique file name from LC-MS run), 1 column for each of 18 metabolites: “Arginine”, “Caffeine”, “Chlorogenic Acid”, “Cinnamic acid”, “Eugenol”, “Gallic acid”, “Histidine”, “Isoleucine + Leucine”, “Kaempferol”, “Lysine”, “Methionine”, “P-coumaric acid”, “Phenylalanine”, “Quercitin”, “Threonine”, “Tryptophan”, and “Valine” (quantity of each metabolite present; if not found “N/F” was recorded, “Round” (experimental round where the sample came from: 1 or 2), and “Chamber” (greenhouse chamber identity: 1 or 2). 
5. Pollen_chemistry_exp1.csv contains pollen chemistry (%N, %C, C:N) data for experiment 1. For each pollen sample, this data file records the unique sample identifier, the plant species the sample came from, the CO2 treatment, experimental round, %N and %C present in each sample, notes about the sample and a omit column to remove samples that were too low to be detected or duplicate samples. This data file contains the following columns: “SampleName” (Unique sample identifier for the individual plant where the sample came from), “Plant” (plant species the data was collected from), “CO2” (CO2 treatment: elevated or ambient), “Chamber” (Greenhouse chamber: 1 or 2), “Round” (experimental round: 1 or 2), “N” (%N in pollen), “C” (%C in pollen), “Notes” (notes about individual samples), “Omit” (Samples marked “1” were omitted from analyses due to insufficient N detected in the pollen samples as indicated in the notes column). 
6. Pollen_chemistry_exp2.csv contains pollen chemistry (%N, %C, C:N) data for experiment 2. For each pollen sample, this data file records the unique sample identifier, the plant species the sample came from, the CO2treatment, experimental round, %N and %C present in each sample, notes about the sample and a omit column to remove samples that were too low to be detected or duplicate samples. This data file includes the following columns: “Sample ID” (unique sample identifier for the individual plant where the sample came from), “N” (%N in pollen), “C” (%C in pollen), “OMIT” (Samples marked “1” were omitted from analyses due to insufficient N detected in the pollen samples as indicated in the notes column, or due to being a duplicate sample), “NOTES” (Notes about the individual sample), “Plant_Sp” (plant species that the sample came from), “Chamber” (Greenhouse chamber: 60, 61, 62, or 63), “CO2” (Co2 treatment: elevated = 1, ambient = 0), and “Round” (Experimental round: 1 or 2). 
7. Biomass_exp2.csv is the data file recording biomass data for experiment 2. Here, above-ground, dry biomass for each plant sample is reported along with plant species, unique plant identifier, greenhouse chamber, and CO2 treatment. Biomass data was only collected for round 1 of experiment 2. This data file includes the following columns: “Plant” (Plant species common name abbreviation (B = borage, BW = buckwheat, C = red clover, D = dandelion, N = nasturtium, LP = lacy phacelia, PP, = partridge pea, SF = sunflower, SA = sweet alyssum)), “Plant ID” (unique sample identifier for the individual plant where the sample came from), “Chamber” (Greenhouse chamber: 60, 61, 62, or 63), “CO2” (Co2 treatment: elevated = 1, ambient = 0), and “Plant biomass” (dry, above-ground biomass in g). 
8. Flower_size_exp2.csv contains the flower size (measured as diameter) for experiment 2. Along with flower size (as measured by flower diameter as a mean of three diameter measurements on the same flower), this data set contains the associated greenhouse chamber, CO2 treatment, experimental round, unique plant identifier, and plant species. This data set contains the following columns: “Chamber” (Greenhouse chamber: 60, 61, 62, or 63), “CO2” (Co2 treatment: elevated = 1, ambient = 0), “Round” (experimental round: 1 or 2), “PlantID” (Unique sample identifier for the individual plant where the sample came from), “Plant” (Plant species common name abbreviation (B = borage, BW = buckwheat, C = red clover, N = nasturtium, LP = lacy phacelia, PP, = partridge pea, SF = sunflower, SA = sweet alyssum)), and “Mean” (mean flower diameter in mm). 
9. Flowering_initiation_exp2.csv is the data set containing the date each plant that flowered in experiment 2, initiated flowering. In addition to flowering initiation (flowering start date) this data set reports the associated green house chamber, CO2 treatment, experimental round, plant species, and unique plant identifier. This data file contains the following columns: “Date” (date that flowering initiated in “MM/DD/YY” format), ”Chamber” (Greenhouse chamber: 60, 61, 62, or 63), “CO2” (Co2 treatment: elevated = 1, ambient = 0), “Round” (experimental round: 1 or 2), “PlantID” (Unique sample identifier for the individual plant where the sample came from), and “Plant” (Plant species common name abbreviation (B = borage, BW = buckwheat, C = red clover, N = nasturtium, LP = lacy phacelia, PP, = partridge pea, SF = sunflower, SA = sweet alyssum)). 
10. Plant_growth_exp2.csv contains the plant growth data for experiment 2. This data set reports the date the data was recorded, the associated greenhouse chamber, CO2 treatment, and experimental round along with the plant species and unique plant identifier and the number of leaves (Leaf_no), number of flowers (Flower_no), and plant’s height (in cm). There is also a notes column. This data file contains the following columns: ”Date” (date that flowering initiated in “MM/DD/YY” format), ”Chamber” (Greenhouse chamber: 60, 61, 62, or 63), “CO2” (Co2 treatment: elevated = 1, ambient = 0), “Round” (experimental round: 1 or 2), “PlantID” (Unique sample identifier for the individual plant where the sample came from), and “Plant” (Plant species common name abbreviation (B = borage, BW = buckwheat, C = red clover, N = nasturtium, LP = lacy phacelia, PP, = partridge pea, SF = sunflower, SA = sweet alyssum)), “UniqueID” (unique identifier for the individual plant where the data came from), “Leaf_no” (number of leaves on the plant, if not measured, left blank), “Flower_no” (number of flowers on the plant, if not measured, left blank), “Height_cm” (height of the plant in cm, if not measured, left blank), and “Notes” (Notes about the plant or data collection from that day). 

Code

Analysis and visualization

Here is a list of the R (v 4.2.2) code files used to analyse the above data and generate the figures used in the manuscript. All necessary packages are listed at the top of each code file.

1. CO2_plotting.R contains the script file used to create Figure S1 which depicts the CO2 levels across experimental rounds for both experiments 1 and 2. This code file uses data files: CO2_log_exp1.csv and CO2_log_exp2.csv.
2. Pollen_chemistry.R contains the script file used to create Figures 1 and 3 and to analyze the pollen chemistry data (%N, %C, and C:N) for both experiments 1 and 2. This code file uses data files: Pollen_chemistry_exp1.csv and Pollen_chemistry_exp2.csv.
3. Targeted_metabolomics_exp1.R contains the script file used to create Figure 2 and to analyze the targeted metabolomics data from experiment 2 using the Pollen_metabolomics_exp1.csv data file.
4. Plant_growth.R contains the script file used to create Figures 4-8 and to analyze the plant growth data (biomass, flower size, flowering initiation, height, flower number and leaf number) for experiment 2. This code file uses the data files: Biomass_exp2.csv, Flower_size_exp2.csv, Flowering_initiation_exp2.csv, and Plant_growth_exp2.csv.

Greenhouse CO2 control

To record CO2 levels in the study greenhouse chambers for experiment 2, we used Arduino UNOs linked to LiCor 8500 Gas Analyzers or Adafruit SCD-50 sensors. The code used to record CO2 data from both sensors can be found in the eCO2_logger_licor_arduino.ino folder and to record CO2 data from the Adafruit SCD-50 sensor only can be found in the aCO2_logger_arduino.ino folder.

To maintain the CO2 levels in the eCO2 greenhouse chambers, we used an Arduino UNO connected to the LiCor 8500 Gas Analyzer to take in current CO2 readings and turn on and off a relay to the CO2 regulator to increase the CO2 levels. The code for this CO2 controller can be found in the CO2_controller.ino folder.

Access information

This data and code can also be found on github (https://github.com/Crall-Lab/eCO2_SciReports.git). Maintainer: Olivia M. Bernauer (email: ombernauer@wisc.edu; olivia.bernauer@gmail.com). All data were collected by the authors and are available for use in accordance with the CC0 license.