DOI: 10.5061/dryad.fqz612k12
title: Data from: Dietary partitioning among three cryptobentic reef fish mesopredators revealed by visual analysis, metabarcoding of gut content, and stable isotope analysis

description: Understanding how mesopredators partition their diet and the identity of
 consumed prey can assist in understanding the ecological role predators
 and prey play in ecosystem trophodynamics. Here, we assessed the diet of
 three common coral reef mesopredators; Pseudochromis flavivertex,
 Pseudochromis fridmani, and Pseudochromis olivaceus from the family
 Pseudochromidae, commonly known as dottybacks, using a combination of i)
 visual stomach content analysis, ii) stomach content DNA metabarcoding
 (18S, COI), and iii) stable isotope analysis (δ15N, δ13C). In addition, P.
 flavivertex is found in two distinct color morphs in the Red Sea,
 providing an opportunity to analyze intra-morph differences. These
 techniques revealed partitioning in the dietary composition and resource
 use among species. Arthropods comprised the main dietary component of P.
 flavivertex (18S &gt; 60%; COI &gt; 10%), and P. olivaceus (18S =
 57.2%) while P. fridmani ingested predominantly mollusks (18S = 51.3%, COI
 = 24.6%). Despite being small predators, microplastics were found in the
 gut content of some of these fishes. Stable isotope analysis showed
 differences in species' isotopic niche breadth and trophic position.
 Pseudochromis olivaceus presented the largest isotopic niche (SEAC =
 1.61‰2), while P. fridmani showed the smallest isotopic niche (SEAC =
 0.45‰2) among species. Although the two techniques used for stomach
 content analysis did not show differences in the diet within color morphs
 of P. flavivertex, they differed in the isotopic niche and resource use.
 Despite our limited sampling, our findings provide evidence of
 species-specific differences in the trophic ecology of dottybacks and
 demonstrate their important role as predators of cryptic invertebrates and
 small fishes. This study highlights the importance of combining several
 approaches (short-term: visual analysis and DNA metabarcoding; and
 long-term: isotope analysis) when assessing the feeding habits of coral
 reef fish, as they provide complementary information necessary to delimit
 their niches and understand the role that small mesopredators play in
 coral reef ecosystems.
description: <strong>Fish and tissue
 collection </strong> A total of 125 adult
 individuals from three species and two color morphs of dottybacks
 (<em>Pseudochromis flavivertex</em> M1 = 48; <em>P.
 flavivertex</em> M2 = 28; <em>P. olivaceus</em> = 24,
 and <em>P. fridmani</em> = 25) were collected (Figure 1) in
 the central Red Sea off the coast of Thuwal, Saudi Arabia (22.2833° N,
 39.1000° E) between 09:00 and 12:00 h of August and September 2021. Fish
 collections took place in different reefs along a cross-shelf gradient
 (coastal, mid shore, offshore) to broadly cover all available shallow reef
 habitats for these species in the central Red Sea, and to maximize the
 diversity of dietary items detected for each dottybacks species rather
 than compare habitats. Not all dottybacks species were collected across
 the different shelves because not all species were found in the same
 location during the sampling period (see ESM Table 1 for individual sample
 details). Fishes were collected under KAUST IACUC Protocol
 19IACUC03. Fish were caught using a hand net and clove
 oil solution (1:10 clove oil: ethanol) as an anesthetic and euthanized by
 immersion in buffered Tricaine Methanesulfonate (MS-222: 250 mg/l
 seawater). Fish were photographed on the boat immediately after capture to
 document the color pattern and preserved on ice <em>in
 situ</em>. Once in the laboratory, total (TL mm) and standard length
 (SL mm), wet weight (mg), and sex (when possible) were recorded for each
 individual (ESM Table 1). The gut cavity was opened and the entire intact
 stomach and intestine were removed using sterile dissection tools from the
 fish within 3 hours after collection and preserved separately in sterile
 Eppendorf tubes containing 96% ethanol at -20° C for analysis. A section
 (~1 cm<sup>2</sup>) of dorsal muscle tissue was removed from
 each individual and preserved in a sterile vial at -20° C for isotope
 analysis. All dissecting tools were sterilized between individuals and
 between tissues within individuals in a three-step process by rinsing and
 incubating them in a series of falcon tubes containing 1:10 bleach and
 sterile water, sterile water, and ethanol.
 <strong>Gut content DNA
 metabarcoding </strong> <em>DNA
 extraction</em> A total of 56 gut tracts
 (<em>P. flavivertex</em> M1 = 21; <em>P. flavivertex
 </em>M2 = 13; <em>P. olivaceus</em> = 10, and
 <em>P. fridmani </em>= 12) were dissected, and the entire gut
 content (excluding tissue from the host fish to minimize dietary masking
 by the host DNA) extracted using the DNeasy PowerSoil DNA extraction Kit
 (QIAGEN) following the manufacturer’s protocols with the following
 modifications: samples were incubated with 12.2 µl of Proteinase K and 60
 µl of Solution C1 at 56° C at 200 rpm overnight, to ensure that all the
 tissues were completely lysed (Leray et al., 2013) and final DNA elution
 was done at 50 µl. Extractions were performed under sterile conditions in
 a dedicated space in the Bioscience Core Laboratory at KAUST to minimize
 the risk of contamination. Filter tips were used, and negative controls
 were included in all the extraction rounds. The concentration of DNA
 extracts was determined using a Qubit dsDNA HS (high sensitivity, 0.2 to
 100 ng) Assay Kit with a Qubit 4.0 fluorometer (Thermo Fisher Scientific)
 according to the manufacturer’s protocols. DNA samples were normalized to
 5 ng/µl. <em>Library preparation and
 sequencing</em> The Illumina 16S Metagenomic
 Sequencing Library Preparation guide was followed (Illumina, 2019) with
 modifications. Two sets of universal primers were used to perform the
 polymerase chain reactions (PCRs), one amplifying a 313 bp segment of the
 mitochondrial cytochrome <em>c</em> oxidase subunit I (COI)
 gene  (mlCOIintF: 5' – GGWACWGGWTGAACWGTWTAYCCYCC – 3';
 jgHCO2198: 5’–TAIACYTCIGGRTGICCRAARAAYCA –3; (Geller et al., 2013; Leray
 et al., 2013) and the other amplifying a 450 bp segment of the eukaryotic
 V4 region of the nuclear small subunit ribosomal RNA (18S rRNA) gene
 (Uni18SF: 5' – AGGGCAAKYCTGGTGCCAGC – 3'; Uni18SR: 5’–
 GRCGGTATCTRATCGYCTT – 3'; (Zhan et al., 2013). These primers were
 selected due to their versatility in detecting a wide range of metazoan
 prey items, and they work well for the identification of marine
 invertebrates (Casey et al., 2019; Coker et al., 2023; Geller et al.,
 2013; Leray et al., 2013; Rey et al., 2020; Zhan et al., 2013).
 Three PCR replicates were conducted per sample. Each PCR reaction
 was run in a total volume of 25 µl: 1 μl of 10 μM forward primer, 1 μl of
 10 μM reverse primer, 12.5 μl of KAPA HiFi HotStart DNA Polymerase
 (HotStart and Ready Mix formulation, KAPA Biosystems), 8 μl of RNA free
 water and 2.5 μl of genomic DNA. PCR was performed under the following
 conditions: Initial denaturation at 95° C for 3 min, followed by 35
 cycles: 98° C for 20 s, 46° C (COI) or 50° C (18S) for 60 s, and 72° C for
 90 s, and a final extension at 72° C for 5 min. Each PCR contained 61
 samples, including 56 gut-content DNA samples, three extraction negative
 controls, one PCR negative control, and one PCR positive control
 containing genomic DNA from one fish species (<em>Acanthopagrus
 berda</em>). PCR reactions were verified on 1.0% agarose gels and
 QIAxcel (QIAGEN, Hilden, Germany) with the QIAxcel DNA Screening Kit,
 using a QX alignment marker of 15 bp – 3 kb and method AM320.
 Electropherograms were analyzed with BioCalculator 3.0 (QIAGEN). The PCR
 reaction performed with negative control extractions confirmed the absence
 of contaminants (no bands). All successfully replicated PCR reactions were
 pooled into a single product for a final volume of 75 μl.
 PCR products were bead cleaned using Agencourt AMPure XP beads
 (Beckman Coulter, Brea, CA, USA) at a concentration of 0.8x vol/vol. For
 library preparation, dual indices were attached to the amplicon of each
 sample using Nextera DNA Library Prep Kit and the Nextera XT Index Kit
 (Illumina). Each indexing PCR reaction was run in a volume of 50 μl: 5 μl
 of Index Primer 1 (N7xx), 5 μl of Index Primer 2 (S5xx), 25 μl of 2x KAPA
 HiFi HotStart Ready Mix (KAPA Biosystems), 10 μl of PCR Grade water and 5
 μl of PCR product. The PCR amplification included an initial denaturation
 at 95° C for 3 min, followed by 8 cycles of 95° C for 30 s, 55° C for 30
 s, 72° C for 30 s, and a final extension at 72° C for 5 min. PCR products
 were cleaned and normalized using the SequalPrep Normalization Plate (96)
 Kit (Invitrogen). Purified libraries were pooled with
 unique indices and quantified using a Bioanalyzer (Agilent) and a Qubit
 Fluorometer (Invitrogen) using a dsDNA HS Assay Kit. A Final concentration
 of the library was calculated using quantitative PCR (qPCR) and normalized
 to 4nM, and 20% phiX was used for internal control. Pair-end sequencing (2
 x 300 bp) was performed on an Illumina MiSeq platform with a MiSeq v3
 Reagent Kit (Illumina). Sequence data were automatically demultiplexed
 using MiSeq Reporter (V2), and forward and reverse reads were assigned to
 samples. <em>Bioinformatics
 pipeline</em> The USEARCH-UNOISE3 pipeline
 (Edgar, 2010, 2016) was used to process 18S rRNA and COI reads. For 18S
 rRNA gene-based amplicon libraries, raw reads were decontaminated of phiX,
 quality-filtered (QV = 25), and adapter-trimmed using the BBDuk tool from
 the BBMap suite (Bushnell, 2022). Primers were removed using cutadapt
 (Martin, 2011). USEARCH-UNOISE3 was used to conduct all further reads
 processing: Paired-end reads merging, deduplication, sequence alignment,
 denoising, chimera removal, clustering, and the generation of a
 zero-radius OTU (zOTU) table. Input sequences with low abundances (i.e.,
 representing &lt; 8 reads) were discarded by the default version of
 the USEARCH-UNOISE3 pipeline. Filtering of the zOTUs was performed based
 on BLASTn (Altschul et al., 1990) query coverage analysis (&gt;70%
 query coverage), the post-clustering curation algorithm LULU (Frøslev et
 al., 2017), and SortMeRNA v4.3 (Kopylova et al., 2012). Finally, BLASTn
 and BLAST-QC (Torkian et al., 2020), of the final available zOTU sequences
 were performed against the latest available NCBI nucleotide database to
 assign taxonomy. To generate the full NCBI taxonomic lineage information
 for all of the identified hits, the Entrez Direct (Kans, 2010) and
 TaxonKit (Shen & Ren, 2021) tools were used. The taxonomy of all
 zOTU sequences that had &gt;98% identity hits in the nt database was
 further verified against the curated EukRibo database v2 (Berney et al.,
 2022). All zOTUs representing bacterial rRNA genes or non-target genes
 were excluded from the analysis. The relative abundance of zOTU sequences
 in each library was estimated as a percentage of the whole
 library. To process COI amplicon libraries, a similar
 pipeline was followed as with the 18S rRNA gene amplicons (except for the
 SortMeRNA filtering step). Apart from the curation of the COI zOTU
 sequences on LULU, the sequences were additionally filtered based on a
 visual screening of stop-codons and frameshift errors on SeaView (Gouy et
 al., 2010) after aligning the corresponding amino-acid sequences on MACSE
 (Ranwez et al., 2011). The taxonomy of all fish-affiliating zOTU COI
 sequences that had &gt;98% identity hits in the nt database was
 further verified against the curated MitoFish database (Iwasaki et al.,
 2013) and that of the remainder sequences against the BOLD database
 (Ratnasingham & Hebert, 2007). All zOTUs
 representing fish (Phylum- Chordata; Class- Actinopterygii) were removed
 from the analysis of the 18S sequences as the 18S rRNA genes representing
 the host fish species (<em>Pseudochromis flavivertex, P. olivaceus,
 </em>and<em> P. fridmani</em>) were not available in the
 NCBI database, and therefore could not be recognized among the other fish
 zOTUs present in the stomach content. COI sequences belonging to the host
 fish species were available in the NCBI database. Thus, all zOTUs matching
 those sequences were removed from the analysis whilst retaining all other
 zOTUs assigned to Actinopterygii. These “self-hits” (e.g., all zOTUs
 identified as <em>P. flavivertex, P. olivaceus,
 </em>and<em> P. fridmani</em>) were removed to exclude
 host tissue and to avoid wrong inferences of cannibalism or trophic
 linkages among the studied species. Though cannibalism of smaller
 individuals could happen, this likely represents an insignificant fraction
 of pseduochromid diets. Likewise, zOTUs corresponding to sequences
 identified as endoparasitic protozoa (e.g. Apicomplexa) were removed from
 the analysis as these organisms were most likely living in the host gut
 rather than being eaten by them. Finally, zOTUs that were identified only
 as Eukarya were labeled as <em>Unidentified </em>along with
 the zOTUs without matches (&lt;85% identity match) on NCBI.
description: ## Data from: Dietary partitioning among three cryptobentic reef fish
 mesopredators revealed by visual analysis, metabarcoding of gut content,
 and stable isotope analysis
 [https://doi.org/10.5061/dryad.fqz612k12](https://doi.org/10.5061/dryad.fqz612k12) Here we provide the 18S and COI zOTUs tables obtained with DNA metabarcoding of gut content of *Pseudochromis flavivertex* (morph 1: M1, and morph 2: M2), *P. fridmani*, and *P. olivaceus* (in .xlsx format). Tables show zOTUs and taxonomic assignments obtained after removing all the “self-hits” and endoparasitic protozoa from the dataset. The corresponding species code for the zOTUs tables is presented below: PFM1: Pseudochromis flavivertex M1 PFM2: Pseudochromis flavivertex M2 PFRI: Pseudochromis fridmani POLI: Pseudochromis olivaceus GutTrial_1: Pseudochromis flavivertex M1 GutTrial_2: Pseudochromis flavivertex M1 GutTrial_3: Pseudochromis flavivertex M1 Variables comprising the taxonomic assignments tables: **OTUID**: Zero-radius OTUs (zOTUs) ID *Taxonomic classification levels:* **Kingdom, Phylum, Subphylum, Class, Order, Family, Genus, Species** **Genspe:** Genus and species as: genus.species **Conserved:** % Conservation **Identity:** % Identity **Taxon_id**: Taxon ID **TaxID:** Taxonomic classification corresponding to Phylum. If Phylum = *NA* or Identity ≤ 85%, TaxID is considered *Unidentified.* *NA*: Unidentified taxa The final COI and 18S zOTUs sequences are provided as fasta files (.fasta). The raw data from the sequencing of COI and 18S rRNA gene-based amplicons are available at the European Nucleotide Archive (ENA) under the study accession numbers PRJEB64896 and PRJEB65108, respectively.