DOI: 10.5061/dryad.kd51c5b4s
title: Secondary predation constrains DNA-based diet reconstruction in two threatened shark species

description: Increasing fishing effort, including bycatch and discard practices, are
 impacting marine biodiversity, particularly among slow-to-reproduce taxa
 such as elasmobranchs, and specifically sharks. While some fisheries
 involving sharks are sustainably managed, collateral mortalities continue,
 contributing towards &gt; 35% of species being threatened with
 extinction. To effectively manage shark stocks, life-history information,
 including resource use and feeding ecologies is pivotal, especially among
 those species with wide-ranging distributions. Two cosmopolitan sharks
 bycaught off eastern Australia are the common blacktip shark (Carcharhinus
 limbatus; globally classified as Near Threatened) and great hammerhead
 (Sphyrna mokarran; Critically Endangered). We opportunistically sampled
 the digestive tracts of these two species (and also any whole prey; termed
 the ‘Russian-doll’ approach), caught in bather-protection gillnets off
 northern New South Wales, to investigate the capacity for DNA
 metabarcoding to simultaneously determine predator and prey regional
 feeding ecologies. While sample sizes were small, S.
 mokkaran fed predominantly on stingrays and skates
 (Myliobatiformes and Rajiformes), but also teleosts, while C.
 limbatus mostly consumed teleosts. Metabarcoding assays showed
 extensive intermixing of taxa from the digestive tracts of predators and
 their whole prey, likely via the predator’s stomach chyme, negating the
 opportunity to distinguish between primary and secondary predation. This
 Russian-doll effect requires further investigation in DNA metabarcoding
 studies focusing on dietary preferences and implies that any outcomes will
 need to be interpreted concomitant with traditional visual approaches.
description: The DNA extracts from each gut sample were amplified, tagged
 separately, and then pooled for sequencing. Two group-specific
 mini-barcode primers were selected for the amplification of teleost and
 crustacean DNA, targeting 12S (MiFish<sup>: </sup>Miya et al.,
 2015) and 16S (Crust16S short: Berry et al., 2017) mitochondrial DNA
 genes, respectively. We also used a universal 18S primer set (Zhan et al.,
 2013) targeting the hypervariable V4 region of the nuclear small subunit
 ribosomal DNA to amplify templates from a broader fraction of marine
 metazoans. Polymerase chain reaction (PCR) was performed using the
 AmpliTaq Gold 360 protocol and thermocycling conditions recommended in
 (Taberlet, Bonin, Zinger, & Coissac, 2018). The
 PCR hybridization temperatures were 50, 51 and
 50 <sup>o</sup>C for MiFish, Crust16S, and Uni18S primer sets,
 respectively, and products were run on a 1% agarose gel to confirm
 amplification of the correct target size (MiFish = ±170 bp; Crust16S =
 ±170 bp, Uni18S = ±220 bp). A second round of PCR was undertaken on the
 cleaned PCR products using unique dual-indexed primers for each sample,
 which included the Illumina-specific sequencing adaptors. PCR products
 were sent to the Ramaciotti Centre for Genomics at the University of New
 South Wales for cleaning, normalising, and pooling prior to paired-end
 sequencing, which was performed using a 500 cycle MiSeq V3 Reagent Kit on
 an Illumina MiSeq platform (Illumina, San Diego, CA, USA). Sample
 demultiplexing based on the incorporated indexes was conducted by the
 sequencing centre. Berry, T. E., Osterrieder, S. K.,
 Murray, D. C., Coghlan, M. L., Richardson, A. J., Grealy, A. K., et al.
 (2017). DNA metabarcoding for diet analysis and biodiversity: A case study
 using the endangered Australian sea lion (<i>Neophoca
 cinerea</i>). <i>Ecology and Evolution</i>, 7(14),
 5435–5453. Miya, M. et al. MiFish, a set of universal
 PCR primers for metabarcoding environmental DNA from fishes: detection of
 more than 230 subtropical marine species. <i>Roy. Soc. Open
 Sci.</i> 2(7), 150088 (2015). Taberlet, P., Bonin A., Zinger L., Coissac E. <i>Environmental DNA for Biodiversity Research and Monitoring</i>. Oxford, UK. Oxford University Press (2018). Zhan, A. et al. High sensitivity of 454 pyrosequencing for detection of rare species in aquatic communities. <i>Methods in Ecology and Evolution</i>, <i>4</i>(6), 558–565 (2013).
description: We have provided raw demultiplexed reads for each sample to allow
 users to quality filter the data as required. The .fastq file
 naming convention is "SampleName_Assay_Read" with
 "1BT_MIFISH_R1.fastq" as one example.