Expanding knowledge of the trophodynamic of ecosystems improves the understanding of energy flow and interactions formed in food webs; therefore, structural knowledge of food webs helps to understand the ecosystem dynamics. This study describes the marine trophic structure of the largest marine protected area (MPA) of the Colombian Pacific in Malpelo Island, using δ13C and δ15N. A total of 293 (38 groups/families) samples were collected and analyzed, being mostly represented by teleosts, followed by crustaceans, elasmobranchs, zooplankton, seabirds, and others functional groups. The marine isospace was between −21.5‰ and − 10.0‰ for δ13C and between 4.6‰ to 16.9‰ in δ15N. The Malpelo Island marine ecosystem showed a food web composed of five trophic levels (TLs): TL-I (primary producers), TL-II (herbivores), TL-III (primary carnivores), TL-IV (secondary carnivores), and TL-V (tertiary carnivores), along with three intermediate TLs (TLs II-III, III-IV, and IV-V). The isotopic diversity index showed global low values of uniqueness (IUni) (0–0.349), intermediate values of IRicGlobal, and IDisGlobal (0.350–0.640), and high values of IDivGlobal, and IEveGlobal (0.650–1). TLs reflected high trophic interaction indices as a result of multifunctionality (12 feeding mechanisms) and possible ontogenetic changes of organisms in this ecosystem. This study includes the first insights into the marine trophic structure of the Malpelo Island and indicate some functional aspects of the entities that inhabit this ecosystem. This study fill information gaps for the MPA and constitute an important tool to improve the elaboration of management and conservation measures based on the knowledge of the ecosystem function.
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