Dietary partitioning among three cryptobenthic reef fish mesopredators revealed by visual analysis, metabarcoding of gut content, and stable isotope analysis

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 > 60%; COI > 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 (SEA_C = 1.61‰²), while P. fridmani showed the smallest isotopic niche (SEA_C = 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.