Timing of salinisation and nutrient enrichment drives freshwater microbial community metabolic responses
Data files
Nov 04, 2025 version files 2.60 GB
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ASV_post_processing.csv
1.31 MB
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Ecoplates_raw.csv
1.57 MB
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README.md
4.25 KB
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Sequences.zip
2.60 GB
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univariate_data.csv
5.57 KB
Abstract
Biological systems face multiple stressors that impact biodiversity and ecosystem functions, with complex interactions that vary spatially and temporally, leading to unpredictable outcomes. In freshwater ecosystems, benthic microbial communities underpin vital functions like decomposition and primary productivity but are threatened by stressors such as salinisation and nutrient enrichment, which are intensifying and increasingly interacting due to climate change. We experimentally tested how stressor frequency and order affect freshwater benthic microbial community responses to these stressors over time. Communities established on tiles in 1,000 L open freshwater ponds established >10 years ago in the field were exposed to elevated salinity and nutrient enrichment, either once or repeatedly, independently or in combination, and under different orders. Controlling for frequency, we demonstrate for the first time that the order of stressor exposure is a critical driver of stressor interactions and can result in functional effects in opposing directions. When exposure to nutrient enrichment preceded elevated salinity, gross primary productivity was halved, and carbon metabolic rates increased by 50% compared to communities treated in the reverse order. These findings suggest that microbial functions, including productivity and carbon cycling, will fluctuate more dramatically as stressors increasingly interact in different sequences under global change.
Dataset Overview
This dataset contains the data testing the hypothesis that the structure and function of freshwater benthic microbial communities are affected by salinisation and nutrient enrichment, and that combined stressor effects differ depending on frequency and order of application. Data were obtained from benthic microbial communities grown on tiles in outdoor mesocosms. It includes 1) biofilm in situ functional metrics, including oxygen production and consumption, concentration of green algae, diatoms, and cyanobacteria, total chlorophyll a concentration in biofilms, photosynthetic efficiency, 2) 16S rRNA ASV sequences from swabbed biofilms, and 3) OD590 absorbance values using Ecoplates incubated with biofilm suspensions.
Dates of Data Collection
Data was collected in May - September 2023.
Data Spatial Scope
Data were collected from an experimental field site in Toledo, Spain, as part of the Iberian Pond Network infrastructure.
Funding
This study has received funding from the AQUACOSM-plus project of the European Union's Horizon 2020 research and innovation programme under grant agreement No. 871081 and Transnational Access project SalNutFun. Additional funds were received from The University of Sydney WG Murrell funds.
Ethics Approval
N/A
Files
Files: Sequences.zip
This is a compressed folder of .fastq files are named as[Treatment]-[Pond]-[Time]-[Sample_ID]. Treatment group abbreviations match abbreviations in the manuscript.
File: ASV_post_processing.csv
This file contains absolute abundance corrected values for each ASV in each sample.
File: Ecoplates_raw.csv
This folder contains the OD590 absorbance values for each well within each Ecoplate for each sample at each incubation hour.
"Well" is the well ID with the 96-well plate for that sample. "sample_id" is the substance in the well used for cell metabolism (water negative control of a sole carbon source).
"Treatment" is the pond treatment:
C = baseline control
A = ambient
N = nutrient enrichment
NN = repeated nutrient enrichment
NS = nutrient enrichment then elevated salinty
S = elevated salinity
SS = repeated elevated salinity
SN = elevated salinity then nutrient enrichment
"Pond" is the replicate pond number for each treatment (P1 - P4)
"Time" is the sample time point: T1 = Day 1, T2 = Day 7, T3 = Day 30
"hours" is the plate reading hour for that sample (number of hours plate had been incubated)
OD590 is the absorbance value at the optical density of 590.
File: univariate_data.csv
This is a csv file with the univariate data collected in situ from the biofilms.
Sample_Name is the sample identifier formatted as [Treatment][Pond_Replicate][Sample_Point. ]
"Treatment" is the pond treatment:
C = baseline control = ambient
N = nutrient enrichment
NN = repeated nutrient enrichment
NS = nutrient enrichment then elevated salinty
S = elevated salinity
SS = repeated elevated salinity
SN = elevated salinity then nutrient enrichment
Days is the days since the second stressor treatment
Concentration_Green_Algae, Concentration_Diatoms, Concentration_Cyanobacteria, and Biomass are all presented in the units µg chl-a/cm².
PAM_Yield and PAM_F0 are unitless values representing photosynthetic efficiency.
Copies is the number of 16S rRNA gene copies per sample as calculated by qPCR.
GPP is the rate of gross primary productivity, NPP is the rate of net primary productivity, and CR is the community respiration rate, all expressed as nmol O2/cm2/hr. There are NA values in these three variables for day 81, as oxygen incubations were not performed at that time point.
Code
File: Univariate_Analysis.Rmd
This R Markdown file contains the script for the analyses and figures analyzing the data in the Univariate_data.csv file.
File: Ecoplate_Analyses.Rmd
This R Markdown file contains the script for the analyses and figures analysing the data in the Ecoplate_data.csv file.
ASV_Analyses.Rmd
This R Markdown file contains the script for the analyses and figures analysing the data in the ASV_post_processing.csv file.
This dataset contains the data in Fuggle et al (2025), Ecology, testing the hypothesis that the structure and function of freshwater benthic microbial communities are affected by salinisation and nutrient enrichment, and that combined stressor effects differ depending on frequency and order of application. Data were obtained from benthic microbial communities grown on tiles in outdoor mesocosms. It includes 1) biofilm in situ functional metrics, including oxygen production and consumption, concentration of green algae, diatoms, and cyanobacteria, total chlorophyll a concentration in biofilms, photosynthetic efficiency, 2) 16S rRNA ASV sequences from swabbed biofilms, and 3) OD590 absorbance values using Ecoplates incubated with biofilm suspensions.