Trophic ecology of three estuarine predatory fishes revealed through a multi-method approach

Estuaries are characterized by wide ranges of environmental conditions that support diverse communities and complex food webs. When trophic structure is assessed at a broad scale, large predators are often regarded as redundant, fulfilling comparable ecological functions. We used stomach content analysis (SCA), fecal DNA metabarcoding (fDNA), and stable isotope analysis (SIA) of carbon (δ 13 C), nitrogen (δ 15 N), and sulfur (δ 34 S) to investigate the trophic ecology of 3 co-occurring predatory fishes: alligator gar Atractosteus spatula , bull shark Carcharhinus leucas , and Atlantic stingray Hypanus sabinus in a sub-embayment of a large estuarine complex in the western Gulf of Mexico. Stomach contents showed that alligator gar and bull shark preyed almost exclusively on teleost fishes, but on different species, while Atlantic stingray fed mostly on crustaceans. fDNA confirmed these dietary patterns and provided higher resolution on prey composition and diversity. Alligator gar displayed lower δ 34 S values than bull shark and Atlantic stingray, indicating a diet from low-salinity environments with freshwater-derived organic matter. Atlantic stingray exhibited the broadest trophic niche, while alligator gar and bull shark had more restricted, overlapping niches despite feeding on different species. Alligator gar and bull shark were classified as top predators, and Atlantic stingray as a mesopredator. Seasonal, life stage, and sex-based trophic patterns showed no clear distinctions, likely due to limited sample sizes. This study presents new insights into the trophic ecology of these species and provides the first comprehensive characterization of trophic dynamics using an integrative approach combining SCA, fDNA, and SIA.