DOI: 10.5061/dryad.8pk0p2nz8
title: Timing of salinisation and nutrient enrichment drives freshwater microbial community metabolic responses

description: 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 &gt;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.
description: 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.
description: # Timing of salinisation and nutrient enrichment drives freshwater
 microbial community metabolic responses #### 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.