e(lasmo)DNA: The role of environmental DNA (eDNA) analysis in marine fish biodiversity assessment, with special focus on elasmobranchs Judith Bakker January 2018 This thesis is presented to the University of Salford, School of Environment & Life Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) Supervisor: Prof. Stefano Mariani Co-Supervisor: Dr. Robert Jehle Table of Contents List of figures .................................................................................................................... V List of tables .................................................................................................................... VI Acknowledgements ........................................................................................................ VII Abstract .......................................................................................................................... XI 1 Chapter I ................................................................................................................... 1 Introduction and Background ........................................................................................... 1 1.1 What is environmental DNA (eDNA)? ........................................................................... 1 1.2 A short history of eDNA ............................................................................................... 2 1.3 eDNA vs. traditional monitoring techniques ................................................................. 3 1.4 eDNA approaches: species-specific vs. metabarcoding .................................................. 5 1.5 Applications of eDNA techniques in shark biology studies ............................................. 6 1.5.1 Occurrence and distribution of rare and endangered species ......................................... 7 1.5.2 Species composition with eDNA metabarcoding .............................................................. 9 1.5.3 Population genetics – From species detection to population analysis ........................... 10 1.6 eDNA Methods .......................................................................................................... 11 1.6.1 Water collection, filtration, preservation & extraction .................................................. 12 1.6.2 Selecting gene regions for target organisms or groups .................................................. 17 1.7 The challenges of eDNA studies .................................................................................. 19 1.7.1 Contamination ................................................................................................................. 19 1.7.2 eDNA shedding rates ....................................................................................................... 20 1.7.3 eDNA degradation ........................................................................................................... 22 1.7.4 eDNA capture rate .......................................................................................................... 24 1.7.5 eDNA transport: lentic versus lotic systems ................................................................... 24 1.7.6 Freshwater vs. seawater ................................................................................................. 25 1.7.7 eDNA in tropical ecosystems ........................................................................................... 27 1.7.8 Reference databases ....................................................................................................... 30 1.8 Future advances in eDNA (meta)barcoding ................................................................. 31 1.8.1 Factors influencing quantitative estimates ..................................................................... 31 1.8.2 Quantitative estimates using digital droplet PCR (ddPCR).............................................. 32 1.8.3 Quantitative estimates using metabarcoding ................................................................. 33 I 1.8.4 Increasing reference database coverage and taxonomic resolution .............................. 34 1.8.5 Taking eDNA analysis into the field ................................................................................. 34 1.8.6 The emergence of autonomous sampling and analysis .................................................. 36 1.8.7 The use of long-range PCR for eDNA applications .......................................................... 39 2 Chapter II ................................................................................................................ 42 Environmental DNA reveals tropical shark diversity in contrasting levels of anthropogenic impact ............................................................................................................................ 42 2.1 Abstract ..................................................................................................................... 42 2.2 Introduction .............................................................................................................. 43 2.3 Results ....................................................................................................................... 45 2.3.1 eDNA detection of elasmobranchs ................................................................................. 45 2.3.2 Elasmobranch diversity and read abundance patterns .................................................. 48 2.4 Discussion .................................................................................................................. 53 2.5 Material and methods ................................................................................................ 57 2.5.1 Experimental design ........................................................................................................ 57 2.5.2 Sample processing and DNA extraction .......................................................................... 58 2.5.3 Contamination control .................................................................................................... 59 2.5.4 Library preparation and sequencing ............................................................................... 59 2.5.5 Bioinformatic and statistical analysis .............................................................................. 60 2.6 Acknowledgements.................................................................................................... 61 3 Chapter III ............................................................................................................... 63 Environmental DNA illuminates the dark diversity of sharks ........................................... 63 3.1 Abstract ..................................................................................................................... 63 3.2 Introduction .............................................................................................................. 64 3.3 Results & Discussion .................................................................................................. 65 3.3.1 Lower dark diversity than previously estimated ............................................................. 65 3.3.2 Persisting shark populations in human-impacted areas ................................................. 70 3.3.3 Increased species detectability revealed by rarefaction curves ..................................... 71 3.3.4 Limits and uncertainty of species detection ................................................................... 73 3.3.5 New light for megafauna conservation ........................................................................... 74 3.4 Materials & Methods ................................................................................................. 76 3.4.1 Study sites. ...................................................................................................................... 76 3.4.2 Underwater Visual Census and Baited Remote Underwater Video Station datasets..... 77 II 3.4.3 eDNA collection and sample processing. ........................................................................ 77 3.4.4 Library preparation and sequencing ............................................................................... 78 3.4.5 Bioinformatics analyses ................................................................................................... 79 3.4.6 Statistical analyses .......................................................................................................... 80 3.5 Acknowledgements.................................................................................................... 80 4 Chapter IV ............................................................................................................... 81 Biodiversity assessment of shallow marine eukaryotic communities of the Caribbean Sea using COI metabarcoding ................................................................................................ 81 4.1 Abstract ..................................................................................................................... 81 4.2 Introduction .............................................................................................................. 82 4.3 Material & Methods................................................................................................... 85 4.3.1 Water sampling ..............................................................................................................