Historians have long suspected that Francesco de' Medici, Grand Duke of Tuscany, and his second wife, Bianca Cappello, did not die of malaria but were poisoned — by Francesco's brother, Cardinal Ferdinando de' Medici, who was vying for the dukedom.
For four centuries, that theory remained just that — a theory.
But following a study into the affair, forensic and toxicology experts at the University of Florence believe they have uncovered clear evidence of murder by poisoning.
Francesco's "was a lethal dose, but progressive, and the symptoms were compatible with arsenic poisoning," Donatella Lippi, a professor of history of medicine and a co-author of the study, published in the British Medical Journal on Dec. 21, told The Associated Press.
As rulers, art connoisseurs and financiers of kings, the Medici flourished for centuries in the rough and tumble alliances of old Europe, ruling first the city of Florence and then Tuscany from 1430 to 1737.
Francesco ruled from 1574 until his death on Oct. 17, 1587, at age 46, 11 days after first taking to his bed and a few hours before his wife.
Scientists Francesco Mari, Aldo Polettini, Elisabetta Bertol and Lippi collected and tested beard hairs from Francesco's grave in the Medici chapels in Florence, as well as other remains found in clay jars in a crypt about 12 miles west of Florence. Bianca's grave was never found.
Tests on the beard hairs proved inconclusive. But samples of Francesco's liver taken from the crypt showed levels of arsenic that were "significantly higher" than those normally found in humans, the scientists said.
But if Francesco was murdered, who did it?
Experts say that although there is no proof, Ferdinando was the only person with an obvious motive. He wanted his brother's dukedom, and his behavior at the time was suspicious — for example, he took charge of his brother's illness, compiling the medical bulletins and minimizing the gravity of Francesco's illness in dispatches to the Holy See.
After their deaths, he ordered immediate autopsies — an unusual step which could have been designed to cover up evidence.
"These important findings, in addition to the historical data collected on the events before and after the almost simultaneous deaths of the grand-ducal couple, allow us to rewrite the historical reconstruction of those events," the study said.
"It sounds pretty reasonable," said Richard J. Hamilton, a medical toxicologist who is Chair of the Department of Emergency Medicine at Drexel University in Philadelphia.
"They've established what they have; they've done an efficient job of matching the DNA," said Hamilton, who read the study but was not affiliated with it. He added that the results were consistent with poisoning.
The only surprising aspect is that Francesco — who had an interest in alchemy and chemistry and was suspected of having poisoned his first wife — could have been poisoned so easily and so quickly, Hamilton said.
However, Angelo Moretto, a clinical and experimental toxicologist with the International Center for Pesticides in Milan, is not entirely convinced.
"They make accusations that are quite strong," he said. "I would have been more low key about it."