Understanding species-specific resource requirements is paramount in managing and protecting biodiversity in a world where environmental quality is in decline. Dietary data can inform predator–prey relationships and how changes in prey availability impact different species. However, for many coral reef fishes, prey and predatory events can be difficult to observe and identify, both in situ and within examined stomach samples. Here we applied DNA metabarcoding of stomach content samples for eleven Red Sea butterflyfish species to identify the diversity of dietary components that these primarily benthic feeding fish consume across coral reefs. Detections based on 18S and COI sequences from partially digested stomach contents significantly increased the resolution and diversity of the known diet for this group of fish, which included cryptic prey that are difficult to visually document due to soft parts or morphological ambiguity. In addition to scleractinian corals and other Cnidaria, the obligate corallivore species fed on a wide range of benthic organisms, whereas facultative species displayed a broader diet with crustaceans, tunicates, and worms contributing to samples. While a number of individuals contained DNA that could not be confidently identified using this method, the proportion of unidentifiable sequences was relatively low across butterflyfish species. The COI marker identified the importance of soft corals in the diet for two hard coral specialists; Chaetodon melannotus and Chaetodon semilarvatus, with soft coral detected in over half of the individuals and contributing significantly to the number of DNA sequence reads within their gut. Notably, five prey items identified to the species level were detected that are currently not documented in the Red Sea. Our analysis revealed that the diet of different species of butterflyfish significantly overlaps, with all species deriving most of their diet from the phylum Cnidaria (hard and soft coral, anemones) and symbiotic Symbiodiniaceae algae. Furthermore, accumulation curves suggest that all study species may feed on an even greater fraction of the benthos, likely driven by the availability and diversity of each individual/pair’s associated territory. This approach increases the known dietary resolution and diversity of these key reef fishes and further enhances our understanding between butterflyfish and benthic organisms.
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