The evolution of the extinct megatooth shark, Otodus megalodon, and its close phylogenetic relatives remains enigmatic. A central question persists regarding the thermophysiological origins of these large predatory sharks through geologic time, including whether O. megalodon was ectothermic or endothermic (including regional endothermy), and whether its thermophysiology could help to explain the iconic sharks gigantism and eventual demise during the Pliocene. To address these uncertainties, we present unique geochemical evidence for thermoregulation in O. megalodon from both clumped isotope paleothermometry and phosphate oxygen isotopes. Our results show that O. megalodon had an overall warmer body temperature compared with its ambient environment and other coexisting shark species, providing quantitative and experimental support for recent biophysical modeling studies that suggest endothermy was one of the key drivers for gigantism in O. megalodon and other lamniform sharks. The gigantic body size with high metabolic costs of having high body temperatures may have contributed to the vulnerability of Otodus species to extinction when compared to other sympatric sharks that survived the Pliocene epoch.

Trophic position is a fundamental characteristic of animals, yet it is unknown in many extinct species. In this study, we ground-truth the ¹⁵N/¹⁴N ratio of enameloid-bound organic matter (δ¹⁵N_EB) as a trophic level proxy by comparison to dentin collagen δ¹⁵N 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 δ¹⁵N_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. δ¹⁵N_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.