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.

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.

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).