CASE STUDY – AXversity

Acidity promotes degradation of multi-species environmental DNA in lotic mesocosms

Discover the research lead by Prof. Matthew

Seymour and his team, exploring multispecies environmental DNA (eDNA) dynamics in river ecosystems. Prof. Seymour investigates eDNA degradation over time and in varying

environments across upland stream mesocosms.

The findings highlight rapid eDNA decay, especially in acidic conditions, aiding predictive models for biodiversity dynamics in dynamic river ecosystem.

Introduction

To enhance biodiversity assessments, traditional methods are being supplemented with molecular environmental DNA (eDNA) techniques. However, there’s still limited understanding of the factors influencing eDNA persistence. This study fills this gap by assessing lotic eDNA persistence in replicated field streams, revealing rapid degradation, particularly in acidic environments, and shedding light on eDNA dynamics in lotic systems.

Results

Environmental Variation

The experimental sites featured mesocosms designed for lotic comparisons across Welsh uplands and UK land uses. Each site had four circulating mesocosms with three channels drawing water from nearby streams. pH ranged from 6.73 to 5.35, temperatures from 14.47 °C to 16.16 °C, and total dissolved nitrogen varied from 0.14 mg/L to 0.49 mg/L.

Selected taxa for eDNA analysis included Daphnia magna, Ephemera danica, and Anguilla anguilla. Environmental variation is shown through boxplots of pH, temperature, and total dissolved nitrogen across sites, illustrating dynamics within the mesocosms.

Quantitative PCR

Successful eDNA amplification occurred from time points 0 to 43 h, peaking at time 0 and declining to near 0 at hour 43. Sucrose had no significant effect on DNA quantification.

Significant negative effects of time and positive effects of pH on water-derived eDNA were found. Biofilm eDNA was detected for E. danica but not for D. magna and A. anguilla, with faster decay rates at acidic sites. Overall, biofilm-derived eDNA declined significantly over time and was higher at higher pH levels.

Discussion

In flowing waters, eDNA persistence varies with acidity affecting decay dynamics while nutrient load and temperature show no significant effects. LoticeDNA persisted up to 43 hours, highlighting the influence of environmental conditions on decay dynamics. These findings stress the importance of considering environmental variation and spatio-temporal dynamics in eDNA-based biodiversity assessments, particularly in rapidly degrading lotic systems. Further investigation is needed on eDNA accumulation in biofilm, especially in nutrient-rich environments. Overall, this underscores the necessity for comprehensive ecological assessments considering environmental dynamics across diverse river catchments.

Methods

Experimental Setup

We used four experimental stream mesocosms near the Llyn Brianne Reservoir in upland Wales, each with three circulating channels (20 m×20 cmx 20 cm) and a flow rate of ~2 m/s. They represented moorland and conifer forest catchments with ciarcumneutral (pH 6.8 to 7.2) and acidic (pH 5.3 to 5.8) waters. These mesocosms mirror the acid-base gradient of the upper. These catchment and environmental diversity across upland Wales and Britain.

Environmental DNA Sources and Addition

eDNA from diverse taxa (D. magna, E. danica, A. anguilla) was collected to ensure phylogenetic representation. D. magna and E. danica were cultured in mesocosms, and eDNA-rich water was obtained. A. anguilla juveniles were treated with ultraviolet light before collection. eDNA concentrations were quantified, diluted, and added to the experimental mesocosms. To test microbial activity's impact on eDNA persistence, synthetic dissolved organic carbon (sucrose) was added to simulate high-productivity sites in selected stream channels.

Sampling

Over 44 hours, water samples were collected at various time points, including a pre-eDNA negative control (time point - 1). A total of 252 samples were collected, with triplicate 1 L samples from each experimental channel. On-site filtration using 0.22 um Sterivex filter units was conducted, followed by preservation with Longmires solution before DNA extraction at Bangor University. Standardised biofilm samples, collected from three channels, were scraped clean into tubes during water sampling events and stored frozen for analysis.

Water Chemistry

Daily water chemistry measurements were collected for various parameters including Al, B, Ca, Fe, K, Mg, Mn, Na, S, Si, TSS, Br, CI, F, NH4-N, NO2-N, NO3-N, PO4-P, TON, NPOC, total dissolved nitrogen, pH, GranAlk, and Cond. Temperature and light data loggers were placed in each experimental channel, recording measurements every 15 minutes, with daily averages utilised for analysis.

DNA Extraction and qPCR Analyses

Extractions and qPCR setups were conducted in a dedicated eDNA laboratory at Bangor University, ensuring no prior PCR amplification. eDNA from filters followed a modified Qiagen DNeasy protocol, while biofilm DNA was isolated using PowerMax Soil DNA kits. Quantification of eDNA was done in triplicate via species-specific qPCR assays developed by Primer Design Ltd. Reactions were run on a QuantStudio Flex 6 Real-Time PCR System with standard cycling conditions. Each qPCR plate included control DNA dilution series and no template controls to generate standard curves for amplification efficiency assessment.

Statistical Analyses

Statistical analyses in R (version 3.3.1) involved fitting a mixed-effect generalised linear model to explore the relationship between eDNA quantification, time, and environmental factors. Explanatory variables included time, pH, total dissolved nitrogen, and temperature, with models refined through backward selection based on Akaike information criterion. Time and species were treated as random effects. Similarly, the relationship for biofilm-derived eDNA was assessed using a simplified generalised linear model.