Tropical river and estuarine food webs sustain diverse biodiversity values and are important sources of nutrients and energy for connected aquatic and terrestrial ecosystems. High-order predators, such as euryhaline elasmobranchs, play critical roles in these food webs, but a lack of detailed information on food web structure limits our ability to manage these species within their ecosystems. We analysed stable carbon (δ13C) and nitrogen (δ15N) isotopes (SI) and fatty acid (FA) biochemical tracers from putative prey species in the estuary of the South Alligator River, northern Australia. These were compared with existing data on four species of elasmobranch from the system to examine food web structure and infer dietary linkages over wet and dry seasons along an environmental gradient of sites. Layman’s SI community metrics indicated that upstream food webs had the greatest trophic diversity, and analyses of FAs revealed distinct prey habitat associations that changed seasonally. Mixing models of SI indicated that most Glyphis glyphis and Glyphis garricki had similar freshwater and mid-river diets whilst Carcharhinus leucas and Rhizoprionodon taylori had largely marine signatures. Multivariate analyses of FA revealed some intraspecific differences, although specialisation indices suggested that the four shark species are trophic generalists. Our results show that riverine food webs can display complex spatiotemporal variations in trophic structure and that coastal and euryhaline mobile elasmobranchs forage in a range of coastal and freshwater habitats, demonstrating their influence on these food webs.

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