DOI: 10.5061/dryad.dbrv15f5x
title: Gut content metabarcoding of specialized feeders is not a replacement for environmental DNA assays of their reef environment

description: In tropical marine ecosystems, the coral-based diet of benthic-feeding
 reef fishes provides a window into the composition and health of coral
 reefs. In this study, for the first time, we compare multi-assay
 metabarcoding sequences of environmental DNA (eDNA) isolated from seawater
 and partially digested gut items from an obligate corallivore
 butterflyfish (Chaetodon lunulatus) resident at coral reef sites in the
 South China Sea. We specifically tested the proportional and statistical
 overlap of the different approaches (seawater versus gut content
 metabarcoding) in characterizing eukaryotic community composition on coral
 reefs. Based on 18S rRNA and ITS2 rRNA sequence data, which differed in
 their taxonomic sensitivity, we found that gut content detections were
 only partially representative of the eukaryotic communities detected in
 the seawater. Overall, our results indicate that dietary metabarcoding of
 specialized feeders can be complimentary to, but is no replacement for,
 more comprehensive environmental DNA assays of reef environments that can
 include the processing of different substrates (seawater, sediment,
 plankton) or traditional observational surveys. These molecular assays, in
 tandem, might be best suited to highly productive but cryptic oceanic
 environments (coral reefs, kelp forests, seagrass meadows) that contain an
 abundance of organisms that are often small, epiphytic, symbiotic, or
 cryptic.
description: Fusion-tag qPCR A universal primer set
 targeting 18S rRNA (V1-3 hypervariable region; 18S_uni_1F: 5’ –
 GCCAGTAGTCATATGCTTGTCT – 3’; 18S_uni_400R: 5’ – GCCTGCTGCCTTCCTT – 3’;
 Pochon et al. 2013) was used to maximise detection of the eukaryotic
 component of marine diversity, hereafter the 18Suni assay. We used a
 second assay targeting ITS2 (scl58SF: 5’ – GARTCTTTGAACGCAAATGGC – 3’;
 scl28SR: 5’ – GCTTATTAATATGCTTAAATTCAGCG – 3’; Brian et al. 2019) to
 increase the taxonomic resolution of identified Anthozoa (Phylum Cnidaria)
 and Demospongiae (Phylum Porifera) within each environmental sample,
 hereafter the ITS2 assay (also referred to as “CP1 assay” in Alexander et
 al. 2020). The former class includes anemones, stony corals, soft corals,
 and gorgonians, whereas the latter class includes 81% of all sponge
 species. Both groups play an important role in the functioning of coral
 reef ecosystems, such as recycling dissolved organic matter (Rix et al.
 2016).  Quantitative PCR (qPCR) experiments for both
 seawater and gut content extracts were set up in a separate ultra-clean
 laboratory at Curtin University designed for trace DNA work using a
 QIAgility robotics platform (Qiagen Inc., CA). For more details on
 contamination mitigation measures, please see DiBattista et al. (2019). In
 brief, fusion-tag qPCR was performed with each extract in triplicate on a
 StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA) under the
 following conditions for 18Suni: initial denaturation at 95 °C for 5
 minutes, followed by 45 cycles of 30 s at 95 °C, 30 s at 52 °C, and 45 s
 at 72 °C, with a final extension for 10 minutes at 72 °C. PCR was
 performed under the following conditions for ITS2: initial denaturation at
 95 °C for 5 minutes, followed by 45 cycles of 30 s at 95 °C, 30 s at 55
 °C, and 45 s at 72 °C, with a final extension for 10 minutes at 72 C.
 These primers and PCR conditions have been optimized and applied elsewhere
 (Alexander et al. 2019; DiBattista et al. 2019, 2020, 2022; West et al.
 2020). Moreover, the primer design incorporated indexes in both the
 forward and reverse primer, which allowed us to tag individual PCR
 replicates of individual samples by a unique combination of tags on the
 forward and reverse primers. To check for contamination, non-template
 control (labelled as NTC) PCR reactions were run alongside the template
 PCR reactions, which only contained master mix including the assay
 primers. Libraries for sequencing were made by pooling
 amplicons into equimolar ratios based on PCR Ct values and the endpoint of
 amplification curves, which were then size-selected using a Pippin Prep
 (Sage Science, MA; 100 bp to 600 bp) and purified using the Qiaquick PCR
 Purification Kit (Qiagen Inc.). The volume of purified library added to
 the sequencing run was determined against DNA standards of known molarity
 on a LabChip GX Touch (PerkinElmer Health Sciences, MA). Final libraries
 were sequenced in a paired end approach using a 500 cycle MiSeq V2 Reagent
 Kit and standard flow cell on an Illumina MiSeq platform (Illumina, CA)
 located in the TrEnD Laboratory.
description: We here provide Illumina MiSeq sequences demultiplexed by
 seawater or gut content sample each in .fastq.gz format. All downstream
 data quality filtering steps that we used are outlined in the PeerJ
 publication (https://doi.org/10.7717/peerj.16075).