This repository contains the raw data and analysis scripts associated with the manuscript:
Seasonal differences in effects of microbial communities on duckweed traits

Authors: Emma Kinerson, Alex Trott, and Anna M. O’Brien

All data sets and analysis scripts in this repository were generated and analyzed by the authors listed above.

Repository Contents:

This repository includes experimental data, metadata, and R scripts used to process image-derived measurements and perform statistical analyses.

Files included:

NewDuckweedPlateMap.xlsx
NewDuckweedPlateDat.xlsx
ImageSettings.xlsx
DuckweedTreatments.csv
OpticalDensityData.xlsx
ProcessImageJOutputData.r
SeasonAnalysis.r

Data File Descriptions

File: NewDuckweedPlateMap.xlsx

Description: Contains the raw ROI's of duckweed frond area for each plate and well extracted from ImageJ output.
ROI column, Regions of interest (ROIs), identified in ImageJ. Multiple ROI columns correspond to fragments of the same frond or fronds in one well.
Empty cells in this data indicate a dead frond or no visible fronds in the well.

Variables:

• image: name of the image file the ROIs are is in
• plate: well plate the ROI is found in
• column: column of the well plate the ROIs are found in
• row: well of the well plate the ROIs are found in
• roi - and all subsequent columns: ROIs identified as duckweed by ImageJ present in the exact well, each present ROI is separated by a column, only unique within image files

File: NewDuckweedPlateDat.xlsx

Description: Contains the raw measurements of duckweed frond area extracted from each photo for each ROI number from ImageJ output.
Multiple ROI numbers can be for the same well in a plate, reference NewDuckweedPlateMap.xlsx to see if a well has multiple ROI's.
This also contains other ImageJ outputs for duckweed fronds; ImageJ definitionsa are reproduced here.

Variables:

• roi: region of interest or shape identified as duckweed by ImageJ, only unique within image files
• image: name of the image file the ROI is in
• area: quare pixel area of the shape
• mean: Mean gray value in the shape, sum of the gray values of all the pixels in the selection divided by the number of pixels (0-255 scale)
• min: Minimum gray value among pixels in the shape (0-255 scale)
• max: Maximum gray value among pixels in the shape (0-255 scale)
• perim: pixel length of the perimeter of the shape
• circ: circularity of the shape, unitless, 4πarea/perimeter^2
• AR: aspect ratio of the shape, unitless, major_axis/minor_axis
• round: roundness of the shape, unitless, 4area/(π*major_axis^2), or the inverse of the aspect ratio
• solidity: solidity of the shape, unitless, area/convex area
• r: Mean red value in the shape, sum of the red values of all the pixels in the selection divided by the number of pixels (0-255 scale)
• g: Mean green value in the shape, sum of the green values of all the pixels in the selection divided by the number of pixels (0-255 scale)
• b: Mean blue value in the shape, sum of the blue values of all the pixels in the selection divided by the number of pixels (0-255 scale)

File: ImageSettings.xlsx

Description: Contains experimental plate size measurements used to convert pixels to square milimeters.
Also contains settings used to generate ROIs for each image (color thresholding, followed by analyzing particles)

Variables for sheet 1:

• Image: the image referenced
• plate_width: the width of the experimental plate measured in that image, in pixels (all plates are the same width in mm)
• hue1: the lower threshold for pixel hue used in thresholding (Image -> Adjust -> Color Threshold), scale 0-255, no units
• hue2: the upper threshold for pixel hue used in thresholding, scale 0-255, no units
• sat1: the lower threshold for pixel saturation used in thresholding, scale 0-255, no units
• sat2: the upper threshold for pixel saturation used in thresholding, scale 0-255, no units
• bright1: the lower threshold for pixel brightness used in thresholding, scale 0-255, no units
• bright2: the upper threshold for pixel brightness used in thresholding, scale 0-255, no units
• pixmin: the minimum square pixel area size for generating ROIs (Analyze -> Analyze Particles...).

Description for sheet 2: Two plates had images that did not include the full outer width.
This sheet shows the inner width measures for these plates (pixels).
It also shows the inner and outer width measures for another plate, showing the ratio of these numbers.
The ratio is then used to estimate the outer width of the plates for which this was missing in the image.

File: DuckweedTreatments.csv

Description: Contains information on plate, well (column, row), replicate, and microbe treatment inoculated in that specific location.
In the Microbe treatment column, "n", means no microbe.
Note that this is essentially the same as Table A2, with microbial treatment names in abbreviations, and a numbered replicate column.

Variables:

• Plate: The well plate ID number in the experiment.
• Col: The column in the experimental well plate.
• Row: The column in the experimental well plate.
• rep: The numerical replicate in the experimental design
• Plant: The sampling location of the duckweed plant placed into the well.
• Micr: The microbial treatment applied. Values are three letter codes.
  The first letter is T or L for Thompson Farm or LaRoche Pond sampling sites.
  "D" stands for Day
  The number indicates the ordered sampling date, with lower numbers earlier.

File: OpticalDensityData.xlsx

Description: Contains 2 sheets
Plate Setup Sheet contains information on plate, well (column, row), and microbe treatment for the plate used to collect optical density data.

Plate data sheet contains optical density values for each experimental treatment.
Plate # - The ID of the experimental well plate.
The second column contains the A-H row position in the experimental plate.
Columns named 1-12 indicate plate position
Read - indicates whether the optical density was read at 450 nm or 600 nm wavelengths. Optical density is unitless

Script Descriptions

File: ProcessImageJOutputData.r

Description: This script processes raw ImageJ output files and converts them into summarized duckweed growth measurements per well.
The 3 inputs are: NewDuckweedPlateMap.xlsx, NewDuckweedPlateDat.xlsx, ImageSettings.xlsx.
This script reads ImageJ ROI measurements for each plate and well, then matches ROI measurements to experimental wells using the plate map.
It then combines multiple ROIs belonging to the same well, and calculates total duckweed frond area per well for each plate.
The output of this script is used in SeasonAnalysis.r.

File: SeasonAnalysis.r

Description: This is the primary analysis script used to generate the results presented in the manuscript.
The 3 inputs are the output from SeasonAnalysis.r, DuckweedTreatments.csv, OpticalDensityData.xlsx.
This script combines duckweed growth measurements with treatment metadata, performs statistical analyses of microbial treatment effects, and generates figures used in the manuscript and supplementary materials.

Missing Data

Some cells within NewDuckweedPlateMap.xlsx are intentionally left blank.
These represent wells where: duckweed fronds died during the experiment, or no fronds were visible in the well at the time of imaging.
These wells were excluded from growth measurements.

Code and Software:

All analyses were conducted in R. Scripts were written using R version 4.x.
The scripts are annotated to describe: Package loading, Data import and cleaning, Statistical analyses, Figure generation