Lonesome George, the last remaining tortoise of his kind and a conservation icon, died on Sunday of unknown causes, the Galapagos National Park said.
Lonesome George was found in 1972 and had become a symbol of Ecuador's Galapagos Islands, which attracted some 180,000 visitors last year.
Estimated to be 100 years old, George was the last member of a species of giant tortoise from La Pinta, one of the smallest islands in the Galapagos, the Galapagos National Park said.
The giant Galapagos tortoises, which can live up to 200 years old, were among the species that helped Charles Darwin formulate his theory of evolution in the 19th century.
Tortoises were hunted for their meat by sailors and fishermen to the point of extinction, while their habitat has been eaten away by goats introduced from the mainland.
Scientists had been trying to get George to mate since 1993, when they introduced two female tortoises of a different subspecies into his pen. They laid eggs twice, but they were infertile.
The pen where George lived was visited by thousands of tourists every year to take pictures of one of the rarest creatures on Earth.
Some 20,000 giant tortoises still live on the Galapagos.
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