Cardinal Giovanni de' Medici died in 1562, at the age of 19, after catching a terrible fever while traveling along the Tuscan coast. Twenty-five years later, his elder brother, Francesco I de' Medici, Grand Duke of Tuscany, died from similar symptoms. For centuries, chronicles attributed both deaths to malaria, while Francesco's death continued to bolster hypotheses of poisoning. Now, a new study conducted by the Division of Paleopathology of the Department of Translational Research and New Technologies in Medicine and Surgery at the University of Pisa, in collaboration with Yale University researchers, adds decisive evidence to this historical event and opens a new window on the evolution of malaria throughout history. The study, published in the journal iScience, examined DNA recovered from the two brothers' skeletal remains for genetic markers of Plasmodium, the parasite protozoa that cause malaria. In Giovanni de' Medici's remains, DNA from a previously unknown strain of Plasmodium falciparum, the species that causes the most severe form of human malaria, has been identified through analyses. The study indicates that this strain possesses two distinctive genetic mutations that likely developed during the parasite's demographic expansion and dissemination throughout Europe. In Francesco de' Medici's remains, genetic residues related to both P. falciparum and a second species, Plasmodium malariae, were detected by researchers. The occurrence of two separate species in the same individual is consistent with prior study in Belgium from the same time period, which documented several infections. However, Alexander Ochoa, the study's first author and an Associate Researcher at the Yale Human Evolutionary Genomics Laboratory, stated that additional genetic tests will be required to demonstrate that the two species co-circulated in central Italy in the 16th century. "Our study represents an excellent example of how modern analytical techniques can be applied to ancient DNA to better understand the past", said Serena Tucci, an Assistant Professor of Anthropology at Yale's Faculty of Arts and Sciences and director of the Yale Human Evolutionary Genomics Laboratory. "However, our research has not only illuminated the past, but it has also generated new data that can be used to advance current and future research on malaria, a disease that continues to result in millions of cases and hundreds of thousands of fatalities annually on a global scale".