DOI: 10.5061/dryad.xksn02vdx
title: The applicability of eDNA metabarcoding approaches for sessile benthic surveying in the Kimberley region, north-western Australia

description: The application of environmental DNA technologies is a promising new
 approach to rapidly audit biodiversity across large-scale, remote regions.
 Here, we examine the efficacy of a dual-assay eDNA metabarcoding approach
 for sessile benthic bioassessments in the turbid waters of the
 Lalang-garram Marine Parks, in the inshore Kimberley region, north-western
 Australia. We ask three principal questions: 1. Is the eDNA released by
 sessile benthic taxa (i.e. hard and soft corals, sponges and tunicates)
 locally detectable? 2.  What level of taxonomic resolution is
 afforded by eDNA metabarcoding using the ITS2 region? and 3. How well does
 eDNA metabarcoding compare to conventional benthic survey techniques, such
 as belt and point-intercept transects? We report that a
 dual-assay eDNA metabarcoding approach is capable of detecting
 approximately 70% of the local benthic taxa (i.e. at a species, genus
 level etc) identified at the surveyed locations. It is, however, not as
 effective at the individual/population level, detecting only approximately
 40% of unique amplicon sequence variant (ASV) signals released by an array
 of individual benthic organisms at the surveyed locations. In examining
 the efficacy and resolution of the applied ITS2 metabarcoding markers for
 bioassessments, we report large gaps in the variety of publicly available
 benthic ITS2 reference sequence data, limiting our ability to provide
 robust taxonomic assignments. These findings highlight the need to extend
 ITS2 databases for greater regional representation. Until this is
 adequately addressed, we recommend that investigating taxonomic
 assignments to a genus-level is the most robust approach to benthic
 monitoring using eDNA. Lastly, we found eDNA metabarcoding and
 conventional belt-transect surveys each detected numerous unique hard
 coral genera, indicating that a combined approach provides the most
 effective way to audit benthic biodiversity. Furthermore, eDNA
 metabarcoding had the power to distinguish similar diversity trends
 between sites to that determined by the belt-transect methodology,
 validating the application of eDNA metabarcoding as either a
 stand-alone, or complementary technique for assessing sessile benthic
 taxa.
description: For this study, seven intertidal reef sites were surveyed in
 Lalang-garram marine parks of the Southern Kimberley region in October
 2018 (see Figure 1 and Table S1 in manuscript). At each site, 10 x 500 ml
 water replicates were sampled in addition to the opportunistic collection
 of 1-16 tissue from sessile benthic organisms, such as hard corals (order
 Scleractinia), soft corals (order Alcyonacea), sponges (phylum Porifera)
 and tunicates (subphylum Tunicata). Specimens were collected at spring low
 tide and stored in 100% ethanol. Water samples were individually filtered
 through 0.45 mm cellulose filter membranes using a Pall Sentino® Microbiology pump
 (Pall Corporation, Port Washington, USA) within 4-6 hours of collection.
 Between the filtration of each replicate, all filtering equipment was
 soaked in 10% bleach for a minimum of 10 min and further rinsed with
 desalinated and filtered water. This was to prevent cross contamination of
 eDNA between replicates and sites. Two bleach and desalinated tap water
 samples were taken at the end of each filtering day to serve as filtration
 controls. Seawater-borne DNA was extracted from half of
 each respective membrane (including filtration controls) using a DNeasy
 Blood and Tissue Kit (Qiagen; Venlo, Netherlands) following the
 manufacturers protocol and additional modifications as described in
 Alexander et al. (2020). Benthic tissue samples were also extracted using
 a DNeasy Blood and Tissue Kit. DNA extracts were then stored at
 -20°C. Two PCR metabarcoding assays targeting the
 nuclear ribosomal <em>internal transcribed spacer
 2</em> (ITS2) region were employed for this study:
 CoralITS2 (Brian, Davy, & Wilkinson, 2019) which amplifies a range
 of scleractinian taxa (exempting the genus <i>Acropora</i>),
 and CoralITS2_acro (Alexander et al., 2020) to amplify
 <i>Acropora</i> (see Table 1 in manuscript). Fusion-tagged
 amplicons were sequenced on 500 cycle
 MiSeq® V2
 Standard Flow Cells on an Illumina MiSeq platform (Illumina, San Diego,
 USA), housed in the TrEnD Laboratory at Curtin University, Western
 Australia. Sequences were demultiplexed using
 the insect package
 (Wilkinson, Davy, Bunce,
 & Stat, 2018), and quality filtered (minimum
 length=60, maximum expected errors=2, no ambiguous nucleotides),
 dereplicated, denoised (pool=TRUE), merged and filtered for chimeras using
 the DADA2 pipeline (Callahan et al.,
 2016) in RStudio
 (v1.1.423; Team, 2015). Because of the multi
 copy nature of ITS2 and variable length of the gene, no strict trimming
 was performed as recommended by the developers pipeline (Callahan et al.,
 2016). Resulting
 amplicon sequence variants (ASVs) for each
 assay, i.e. unique sequences that can separated by one or more nucleotide
 differences (Callahan, McMurdie, & Holmes, 2017), were then
 queried against NCBI’s GenBank
 nucleotide database (Benson, Karsch-Mizrachi,
 Lipman, Ostell, & Wheeler, 2005; accessed in 2020) and the
 addition of a coral ITS2 database (Dugal et al. <i>in
 review</i>). Taxonomic assignments of ASVs were curated using a
 lowest common ancestor (LCA) approach (https://github.com/mahsa-mousavi/eDNAFlow/tree/master/LCA_taxonomyAssignment_scripts; Mousavi-Derazmahalleh et al. <i>in review</i>). We provide here, the demultiplexed (unfiltered) fastq data for each sample (water and tissue) amplified via the ITS2 and ITS2_acro assays. We also provide the resulting read count matrices (post-filtering, blasting and LCA curation) at both an ASV and taxonomic level for both assays.