Figure 1.
Overview of the protocol used for PCR based dietary analyses.
The versatile primer set “COI” [33] and its degenerate version “dgCOI” [34] were used. The dashed arrow indicates that an aliquot of the semi-digested prey homogenate DNA extract was used to evaluate the performance of predator DNA removal techniques (B: restriction enzymes; C: annealing blocking primer).
Figure 2.
Variability at each position of the COI 3′ end region for the design of annealing blocking primers.
Entropy plots were performed using 947 decapod COI sequences (A) (≈330 species), 758 fish COI sequences (B) (≈380 species) and 271 gastropod COI sequences (C) (≈170 species) collected and sequenced by the Moorea BIOCODE project. The black line shows the position where predator specific annealing blocking primers were designed for the two hawkfish species in order to minimize the probability of blocking prey amplification. The black box displays a region of high sequence variability across taxa making it a good-quality binding site for the 3′end of predator specific annealing blocking primers.
Table 1.
Frequency (%) of each nucleotide for the alignment of 947 decapod (D), 758 fish (F) and 271 gastropod (G) COI sequences at the binding site (positions 640 to 643) of the 3′end of predator specific annealing blocking primers designed in this study.
Table 2.
Summary of results from the experimental evaluation of predator DNA removal techniques for the PCR amplification of the semi-digested prey homogenate in the gut contents of Neocirrhites armatus (n = 10) and Paracirrhites arcatus (n = 10).
Figure 3.
Evaluation of COI sequence dissimilarity thresholds for clustering sequences in Operational Taxonomic Units.
We used 1976 COI sequences (∼880 morpho-species belonging to fish, decapods and gastropods) provided by the Moorea BIOCODE project (A) and the sequences amplified (clone libraries) from hawkfish gut contents (B).
Figure 4.
Prey items obtained from the gut contents of 10 specimens of Paracirrhites arcatus.
One representative sequence per Operational Taxonomic Unit (OTU) (cutoff threshold = 5–10%) was chosen to compute the neighbor joining (poisson corrected distance) of COI amino acid sequences using Mega [69]. Branch support was evaluated using 1000 bootstraps. The overall proportion of sequences for each OTU for each combination of versatile primer sets (“COI” and “dgCOI”) and predator DNA removal strategies used (Restriction enzymes and Blocking primers) are presented in the adjacent table. The name of the taxon is underlined when it was also identified using DNA barcoding of undigested remains. The BIOCODE number of the reference specimen to which an OTU match is given when the BLASTN similarity was greater than 98%. Pictures and sampling details of each reference specimen are available at http://mooreabiocode.org/. Whenever species level identification could not be achieved, we indicate the higher taxonomic group to which each prey sequence belongs (statistical assignment package – see methods). If not assigned to species-level, taxonomic rank is shown within parentheses (p: phylum, c: class; o: order, f: family, g: genus). Mismatches between the binding site of the predator specific blocking primer and amplified templates (positions 640 to 658 at the 3′end of the COI region) are presented. Prey sequences which were likely excluded during restriction digestion steps are marked # in the table.
Figure 5.
Prey items obtained from the gut contents of 10 specimens of Neocirrhites armatus.
See legend of Figure 5 for further details.