Diet is a crucial trait of an animal’s lifestyle and ecology. The trophic level of an organism indicates its functional position within an ecosystem and holds significance for its ecology and evolution. Here, we demonstrate the use of zinc isotopes (δ66Zn) to geochemically assess the trophic level in diverse extant and extinct sharks, including the Neogene megatooth shark (Otodus megalodon) and the great white shark (Carcharodon carcharias). We reveal that dietary δ66Zn signatures are preserved in fossil shark tooth enameloid over deep geologic time and are robust recorders of each species’ trophic level. We observe significant δ66Zn differences among the Otodus and Carcharodon populations implying dietary shifts throughout the Neogene in both genera. Notably, Early Pliocene sympatric C. carcharias and O. megalodon appear to have occupied a similar mean trophic level, a finding that may hold clues to the extinction of the gigantic Neogene megatooth shark.

Trophic position is a fundamental characteristic of animals, yet it is unknown in many extinct species. In this study, we ground-truth the 15 N/ 14 N ratio of enameloid-bound organic matter (δ 15 N EB ) as a trophic level proxy by comparison to dentin collagen δ 15 N and apply this method to the fossil record to reconstruct the trophic level of the megatooth sharks (genus Otodus ). These sharks evolved in the Cenozoic, culminating in Otodus megalodon , a shark with a maximum body size of more than 15 m, which went extinct 3.5 million years ago. Very high δ 15 N EB values (22.9 ± 4.4‰) of O. megalodon from the Miocene and Pliocene show that it occupied a higher trophic level than is known for any marine species, extinct or extant. δ 15 N EB also indicates a dietary shift in sharks of the megatooth lineage as they evolved toward the gigantic O. megalodon , with the highest trophic level apparently reached earlier than peak size.

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Extinct lamniform sharks (Elasmobranchii: Lamniformes) are well represented in the late Mesozoic-Cenozoic fossil record, yet their biology is poorly understood because they are mostly represented only by their teeth. Here, we present measurements taken from specimens of all 13 species of extant macrophagous lamniforms to generate functions that would allow estimations of body, jaw, and dentition lengths of extinct macrophagous lamniforms from their teeth. These quantitative functions enable us to examine the body size distribution of all known macrophagous lamniform genera over geologic time. Our study reveals that small body size is plesiomorphic for Lamniformes. There are four genera that included at least one member that reached >6 m during both the Mesozoic and Cenozoic, most of which are endothermic. The largest form of the genus Otodus, O. megalodon ('megatooth shark') that reached at least 14 m, is truly an outlier considering that all other known macrophagous lamniforms have a general size limit of 7 m. Endothermy has previously been proposed to be the evolutionary driver for gigantism in Lamniformes. However, we contend that ovoviviparous reproduction involving intrauterine cannibalism, a possible synapomorphy of Lamniformes, to be another plausible driver for the evolution of endothermy achieved by certain lamniform taxa.