Dataset DOI: 10.5061/dryad.j9kd51cs5

OVERVIEW

This dataset accompanies the manuscript:

“Distinct Environmental DNA States Reveal Biodiversity and Transport Patterns Across Alpine Watersheds”

The dataset contains R scripts, sample metadata, DNA concentration measurements, and input files used to analyze COI and ITS metabarcoding datasets. The supplied R script reproduces the taxonomic assignments, community analyses, and figures presented in the study.

FILE LIST

• COI_ITS_data_analysis_script.R: Main R script used to analyze the COI and ITS datasets and reproduce the analyses and figures presented in the accompanying manuscript.
• COI_ZOTU_Count_Sintax.txt: COI ASV/ZOTU count table with taxonomic assignments generated using SINTAX.
• ITS_ASVs.txt: ITS ASV count table.
• ITS_fwrd_Sintax_prob_75.txt: Taxonomic assignment file for ITS ASVs, using a confidence threshold of 75%.
• mapfile_COI.txt: Sample metadata for the COI dataset.
• METADATA_20240327.xlsx: Extended sample metadata for the COI dataset.
• metadata_ITS.xlsx: Sample metadata for the ITS dataset.
• stepwise_upstream_sample_order.xlsx: Sample ordering information used for upstream–downstream comparisons.
• trib_samples.xlsx: Sample classifications by tributary and position relative to the lake.
• yield_concentration.xlsx: DNA concentration and yield measurements.
• readme.md (this file): Description of the dataset and its files.

HOW TO RUN THE ANALYSIS

1. Open COI_ITS_data_analysis_script.R in RStudio. The analysis was tested using R version 4.4.2.
2. Install the required R packages listed below.
3. Place all input files in the same directory as the R script.
4. Run the script line by line or execute it using:

source("COI_ITS_data_analysis_script.R")

5. The script will generate the figures and summary tables described in the manuscript.

SOFTWARE AND DEPENDENCIES

This code was developed and tested using:

• R version 4.4.2 (2024-10-31, “Pile of Leaves”)
• Platform: aarch64-apple-darwin20 (macOS)

Required R packages:

• phyloseq
• dplyr
• tidyr
• tibble
• readr
• readxl
• here
• writexl
• ggplot2
• patchwork
• stringr
• tidyverse
• eulerr

The package versions used for an analysis session can be displayed in R using:

sessionInfo()

ABBREVIATIONS AND IDENTIFIERS

General abbreviations

• DNA: Deoxyribonucleic acid.
• eDNA: Environmental DNA.
• COI: Cytochrome c oxidase subunit I.
• ITS: Internal transcribed spacer.
• ASV: Amplicon sequence variant.
• ZOTU: Zero-radius operational taxonomic unit.
• OTU: Operational taxonomic unit.
• ID: Identifier.
• TDS: Total dissolved solids.
• EC: Electrical conductivity.
• SUSO: Stepwise Upstream Sample Order.
• ppm: Parts per million.
• ng/µL: Nanograms per microliter.
• km²: Square kilometers.
• lat: Latitude.
• long: Longitude.
• riv_dist: Distance from the sampling location to the stream–lake confluence.
• fwrd: Forward sequence or forward-direction taxonomic assignment file.
• prob_75: Taxonomic assignment confidence threshold of 75%.

eDNA states and controls

• MEM: Membrane-bound eDNA state.
• ADS: Adsorbed eDNA state.
• DIS: Dissolved eDNA state.
• NTC: Negative control.

Sample identifiers

• Name: Unique sampling-location identifier. The two-letter prefix identifies the lake catchment, and the number identifies the sampling location within that catchment, for example, AG01. Sampling locations are shown in the maps accompanying the publication.
• e_name: Additional sample identifier used in the analyses. It combines the sampling-location identifier with the eDNA state, for example, SV18_DIS.
• Sample: Full sample identifier used to match samples across the ITS ASV table and associated metadata.
• Original_Column: Original sample identifier corresponding to the sample names in the metabarcoding count table.
• Lane: Sequencing lane in which the sample was processed.
• rep1 and rep2: Replicate measurements of DNA concentration from the same DNA extract.

Lake and catchment identifiers

• AG: Aegeri
• BL: Baldegg
• GR: Greifen
• HA: Hallwil
• LA: Lauerz
• PF: Pfaeffikon
• SS: Sils
• SV: Silvaplana
• NTC: Negative control rather than a lake catchment.
• NA: Lake catchment was not applicable or unavailable.

GENERAL FILE NOTES

• Text files are UTF-8 encoded.
• Files with the .txt extension are tab-delimited and contain column headers in the first row.
• Files with the .xlsx extension are Excel spreadsheets containing one worksheet unless otherwise stated.
• In trib_samples.xlsx, stepwise_upstream_sample_order.xlsx, and yield_concentration.xlsx, missing or non-applicable values are represented by NA.
• Empty cells in the metadata files indicate that a measurement or field observation was not available or not applicable.

DATA FILE DESCRIPTIONS

COI_ZOTU_Count_Sintax.txt

This file contains COI sequence counts for each sample.

Rows

Each row represents an individual COI ASV/ZOTU, identified by a ZOTU ID, for example, ZOTU7 or ZOTU863.

Columns

• #OTU ID: Unique ASV/ZOTU identifier.
• Remaining columns: Environmental samples.

Sample names follow the general format:

LakeID_State_Replicate

Examples:

• AG01_ADS_2: Sampling location AG01, adsorbed eDNA state, index plate 2.
• AG10_MEM_3: Sampling location AG10, membrane-bound eDNA state, index plate 3 3.

Values

Integer read counts representing the number of reads assigned to each ASV/ZOTU in each sample.

ITS_ASVs.txt

This file contains ITS sequence counts for each sample.

Rows

Each row represents an environmental sample, identified in the Sample column, for example, AG01_MEM_2 or AG02_MEM_2.

Columns

• Sample: Unique sample identifier.
• Remaining columns: ITS ASVs labelled seq_00001, seq_00002, and so forth.

Values

Integer read counts representing the number of reads assigned to each ASV in each sample.

ITS_fwrd_Sintax_prob_75.txt

This file contains taxonomic assignments for ITS ASVs. Assignments were generated using SINTAX with a confidence threshold of 75%.

Rows

Each row represents an ITS ASV identified in the seq column, for example, seq_00001 or seq_00002.

Columns

• seq: ASV identifier corresponding to the ASV columns in ITS_ASVs.txt.
• Phylum: Assigned taxonomic phylum.
• Class: Assigned taxonomic class.
• Order: Assigned taxonomic order.
• Family: Assigned taxonomic family.
• Genus: Assigned taxonomic genus.
• Species: Assigned taxonomic species.

Taxa are presented as names, for example, Chlorophyta, Fagus, or Yamagishiella unicocca. Missing or unresolved taxonomic assignments are represented by NA.

mapfile_COI.txt

This file links COI sample identifiers to descriptive information about sample identity and eDNA state.

Rows

Each row represents an eDNA sample included in the COI dataset, for example, AG01_ADS_2 or AG01_DIS_2.

Columns

• Original_Column: Original sample identifier corresponding to the column headers in COI_ZOTU_Count_Sintax.txt.
• Name: Sample identifier. In this file, it is identical to Original_Column.
• State: Physical eDNA state. Possible values are MEM, ADS, DIS, and NTC.
• Lane: Sequencing lane used to track the sequencing batch.

METADATA_20240327.xlsx

This file contains metadata for COI samples, including environmental measurements, geographic information, sampling-location classifications, and eDNA states.

Rows

Each row represents a unique eDNA sample.

Columns

• e_name: Additional sample identifier combining the sampling-location identifier with the eDNA state, for example, SV18_DIS.
• Name: Unique sampling-location identifier, for example, AG01 or SV18.
• ppm: Total dissolved solids in parts per million.
• pH: Measured water pH at the sampling location.
• temp: Water temperature in degrees Celsius.
• EC: Electrical conductivity in millisiemens per centimeter.
• water: Waterbody or sample type, such as lake, stream, outflow, or NTC.
• Lake: Lake catchment associated with the sample. Values are AG, BL, GR, HA, LA, PF, SS, SV, or NTC. The full lake names are provided in Section 5.
• Precipitation: Indicator of precipitation during or before sampling. Values are Yes, No, or NTC.
• catchment_area: Catchment area in square kilometers.
• lake_area: Lake surface area in square kilometers.
• lat: Latitude in decimal degrees.
• long: Longitude in decimal degrees.
• riv_dist: Distance from the sampling location to the stream–lake confluence, in meters.
• notes: Free-text field containing field observations or quality-control information.
• State: Physical state of eDNA. Values are MEM, ADS, DIS, or NTC.

metadata_ITS.xlsx

This file contains metadata for samples in the ITS dataset. It uses the same environmental and geographic variables as METADATA_20240327.xlsx and includes additional identifiers used for ITS sequencing.

Rows

Each row represents one ITS eDNA sample.

Columns

• Sample: Full sample identifier matching the corresponding row in ITS_ASVs.txt, for example, AG01_MEM_2.
• Original_Column: Original sample identifier used for downstream file referencing.
• Name: Unique sampling-location identifier, for example, AG01.
• State: Physical eDNA state. All environmental samples in this dataset represent the membrane-bound state.
• Lane: Sequencing lane used to track the sequencing batch.
• e_name: Additional sample identifier used in the analysis.
• ppm: Total dissolved solids in parts per million.
• pH: Measured water pH at the sampling location.
• temp: Water temperature in degrees Celsius.
• EC: Electrical conductivity in millisiemens per centimeter.
• water: Waterbody or sample type, such as lake, stream, outflow, or NTC.
• Lake: Lake catchment associated with the sample. Values are AG, BL, GR, HA, LA, PF, SS, SV, NTC, or NA. The full lake names are provided in Section 5.
• Precipitation: Indicator of precipitation during or before sampling.
• catchment_area: Catchment area in square kilometers.
• lake_area: Lake surface area in square kilometers.
• lat: Latitude in decimal degrees.
• long: Longitude in decimal degrees.
• riv_dist: Distance from the sampling location to the stream–lake confluence, in meters.
• notes: Free-text field containing field observations or quality-control information.

stepwise_upstream_sample_order.xlsx

This file provides a flow-based ordering system for sampling locations, called Stepwise Upstream Sample Order.

Rows

Each row represents a sampling location.

Columns

• Name: Unique sampling-location identifier, for example, AG01 or AG02.
• SUSO: Stepwise Upstream Sample Order. The most downstream sampling location along a stream mainstem is assigned a value of 1. The number increases at each successive upstream sampling location within the same tributary. At confluences, tributaries share the same SUSO value, and the next upstream sampling location receives the next higher value.

NA indicates that a Stepwise Upstream Sample Order was not applicable to the sampling location.

This ordering system was used to calculate potential eDNA transport distances and model downstream connectivity.

trib_samples.xlsx

This file classifies samples according to their position relative to the lake and their association with numbered tributary groupings.

Rows

Each row represents an individual sampling location.

Columns

• Name: Unique sampling-location identifier, for example, SV01 or SV02.
• lake_pos: Position of the sampling location relative to the lake. Possible values are Lake, depth, Inlet, outflow, or NA.
• Stream_ID: Identifier of the connected stream. Values include numbered streams, Outflow, or NA.
• Tributary_1 to Tributary_7: Binary variables indicating whether a sampling location is associated with each numbered tributary grouping. A value of 1 indicates association with the corresponding tributary grouping. NA indicates that the sampling location is not associated with that tributary grouping.

yield_concentration.xlsx

This file contains DNA concentration measurements for each eDNA sample.

Rows

Each row represents a DNA extract.

Columns

• sample: Sample identifier, for example, AG01.
• well: Position of the sample on the measurement plate, for example, A12 or H11.
• rep1: First replicate DNA concentration measurement, in nanograms per microliter.
• rep2: Second replicate DNA concentration measurement from the same DNA extract, in nanograms per microliter.
• state: Physical eDNA state, recorded as ads, mem, or dis.
• mean_concentration: Mean of the rep1 and rep2 DNA concentration measurements.

rep1 and rep2 are replicate concentration measurements from the same DNA extract rather than separate DNA extractions. Missing or unavailable values are represented by NA.

CONTACT

For questions about the dataset or analysis code, please contact:

Anish Kirtane
Email: dna.anish@gmail.com