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Edna-Based Metabarcoding As a Monitoring Tool for Fish in Large Lakes

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Edna-Based Metabarcoding As a Monitoring Tool for Fish in Large Lakes eDNA-based metabarcoding as a monitoring tool for fish in large lakes Report – SC140018/R We are the Environment Agency. We protect and improve the environment and make it a better place for people and wildlife. We operate at the place where environmental change has its greatest impact on people’s lives. We reduce the risks to people and properties from flooding; make sure there is enough water for people and wildlife; protect and improve air, land and water quality and apply the environmental standards within which industry can operate. Acting to reduce climate change and helping people and wildlife adapt to its consequences are at the heart of all that we do. We cannot do this alone. We work closely with a wide range of partners including government, business, local authorities, other agencies, civil society groups and the communities we serve. This report is the result of research commissioned and funded by the Environment Agency. Published by: Author(s): Environment Agency, Horizon House, Deanery Road, Bernd Hänfling, Lori Lawson Handley, Daniel Read, Bristol, BS1 5AH Ian Winfield www.environment-agency.gov.uk Dissemination Status: Publicly available ISBN: 978-1-84911-386-1 Research Contractors: © Environment Agency – December 2016 Dr. Bernd Hänfling Senior Lecturer in Ecology and Evolution All rights reserved. This document may be reproduced Director of Postgraduate Studies with prior permission of the Environment Agency. Evolutionary Biology Group School of Biological, Biomedical and Environmental Further copies of this report are available from our Sciences publications catalogue: University of Hull www.gov.uk/government/publications Hull, HU6 7RX UK or our National Customer Contact Centre: T: 03708 506506 Ian J. winfield Lake Ecosystems Group Email: [email protected] Centre for Ecology & Hydrology Lancaster Environment Centre Library Avenue Bailrigg, Lancaster Lancashire LA1 4AP U.K. Environment Agency’s Project Manager: Graeme Peirson and Kerry Walsh Project Number: SC140018 ii eDNA-based metabarcoding as a monitoring tool for fish in large lakes Evidence at the Environment Agency Evidence underpins the work of the Environment Agency. It provides an up-to-date understanding of the world about us, helps us to develop tools and techniques to monitor and manage our environment as efficiently and effectively as possible. It also helps us to understand how the environment is changing and to identify what the future pressures may be. The work of the Environment Agency’s evidence teams are a key ingredient in the partnership between research, guidance and operations that enables the Environment Agency to protect and restore our environment by: Setting the agenda, by providing the evidence for decisions; Maintaining scientific credibility, by ensuring that our programmes and projects are fit for purpose and executed according to international standards; Carrying out research, either by contracting it out to research organisations and consultancies or by doing it ourselves; Delivering information, advice, tools and techniques, by making appropriate products available. Doug Wilson Director of Research Analysis and Evaluation eDNA-based metabarcoding as a monitoring tool for fish in large lakes iii Executive summary Organisms continuously release DNA into their environments in the form of shed cells, waste matter, blood, gametes and decaying material. Analysis of this ‘environmental DNA’ (eDNA) is revolutionising the way biodiversity is monitored. This exceptional new technology has been rapidly adopted for targeted monitoring of single species and studies have consistently shown that it outperforms traditional survey methods in terms of ease of sampling, sensitivity and cost. Recent developments in next generation sequencing enable DNA from whole communities of organisms to be sequenced simultaneously (‘metabarcoding’). Metabarcoding is routinely used for direct analysis of microbial and meiofaunal diversity, but this approach has so far rarely been used to analyse eDNA. eDNA metabarcoding is potentially the most promising technological advance for biodiversity measurement. However, the few studies carried out to date have been performed in aquaria or on a very small scale in natural settings. One of the most promising aspects of eDNA is its potential for detecting rare or elusive species that are challenging to monitor using established survey methods. There is therefore considerable interest in using eDNA to supplement existing survey methods for routine monitoring. However, important questions remain about how sensitive and accurate the method is for detecting species and for estimating their abundance or relative biomass. The European Union Water Framework Directive requires assessment of the ecological status of lakes, rivers and groundwaters based on biological elements including phytoplankton, macrophytes and phytobenthos, benthic invertebrates and fish. The current fish classification tool for England, Wales and Scotland (Fisheries Classification Scheme 2, FCS2) was devised for rivers and uses electrofishing survey methods to provide information on the composition and abundance, as well as the age structure of fish populations. Routine monitoring of lake fish populations for the Water Framework Directive is desirable but not yet feasible with existing tools and resources. The aim of this project was to investigate the potential of eDNA metabarcoding as a fish classification tool for large UK lakes. The study investigated whether eDNA metabarcoding could recover information on fish presence/absence and abundance by comparison with long-term data and recent gill net surveys in the English Lake District. Sixty-six 2-litre water samples were collected along 3 offshore transects and 6 shoreline sites in Lake Windermere in January 2015. Twelve additional samples were collected at shoreline and gill net sites in Bassenthwaite Lake and Derwent Water. Samples were filtered, the eDNA extracted and 2 target gene regions (mitochondrial cytochrome b and 12S) amplified using polymerase chain reaction (PCR). PCR products were sequenced on an Illumina MiSeq Next Generation Sequencer and data analysed using customised bioinformatics pipelines. eDNA data were compared with fish abundance and biomass data from recent gill net surveys (September 2014) and rank abundance based on expert opinion from hydroacoustic and gill net surveys. The results demonstrate that eDNA metabarcoding is extremely sensitive for detecting species. Fourteen of the 16 species previously recorded in Windermere (based on long-term datasets) were detected using eDNA, compared with only 4 species in the September 2014 gill net survey. eDNA also outperformed gill net surveys in terms of species detection in Bassenthwaite Lake and Derwent Water, despite limited sampling in these water bodies. The analyses indicate that 10–20 samples may be adequate to accurately represent the species present. Moreover, shoreline sampling may be sufficient for assessing presence/absence since 12 species were detected in just 6 samples collected along a short stretch of shoreline in Windermere. iv eDNA-based metabarcoding as a monitoring tool for fish in large lakes It is clear that more comprehensive sampling is required for estimating fish abundance. The number of sequence reads per species and the proportion of sampling sites in which a species was detected were used as proxies for abundance from eDNA data and compared with data from established surveys. eDNA abundance data consistently correlated with abundance/biomass estimates from established surveys, suggesting that the potential for extracting quantitative estimates from eDNA metabarcoding data may be greater than previously thought. However, correlations were only consistently statistically significant for Lake Windermere, where sampling was intense. This study provides the first demonstration that eDNA metabarcoding can effectively classify community diversity in large lakes and has great potential to supplement existing biomonitoring programmes. Repeated sampling and statistical modelling, which were out of the scope of the present study, are needed to investigate the relationship between eDNA and actual abundance further. eDNA-based metabarcoding as a monitoring tool for fish in large lakes v Acknowledgements We are extremely grateful to Ben James and Janice Fletcher for assistance with boat survey work and to Jianlong Li and Rosetta Blackman for help with sample collection and filtration. Sincere thanks are also given to Paul Nichols for exceptional help with the lab work, Anna Oliver for assistance with the next generation sequencing, and Christoph Hahn for the bioinformatics analysis. vi eDNA-based metabarcoding as a monitoring tool for fish in large lakes Contents 1 Introduction 1 1.1 Background 1 1.2 Aims and objectives 2 1.3 Project approach and workflows 3 2 Material and methods 6 2.1 Method development 6 2.2 Application of eDNA based fish metabarcoding in Cumbrian lakes 8 3 Results 15 3.1 Method development 15 3.2 Application of eDNA based fish metabarcoding in the Cumbrian lakes 18 4 Discussion 37 4.1 Introduction 37 4.2 Methodological developments 37 4.3 Use of eDNA for assessing the presence/absence of lake fish 38 4.4 Use of eDNA for assessing relative abundance of lake fish 38 4.5 Comparison of results from eDNA and established methods 39 4.6 Use of eDNA to survey non-fish vertebrates 41 4.7 Closing remarks 41 References 43 List of abbreviations 47 List of abbreviations used in figures
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