Abstract
Madagascar is the most plague-affected country globally, yet the phylogenetic diversity of Yersinia pestis in this country remains insufficiently characterized. In this study, we analyzed whole-genome sequences of 614 Y. pestis strains, with 141 strains newly sequenced, collected over 96 years across Madagascar. All isolates emerged in 1898 and belong to the phylogroup 1.ORI3, supporting a single introduction event to Madagascar, followed by local diversification. We identified 23 distinct subgroups, including eight previously undescribed. Although most novel subgroups were rarely detected or rapidly extinguished, the ɣ subgroup circulated between 2016 and 2019 in southeastern Madagascar-an area historically plague-free for 64 years. Our analysis revealed extensive strain diversity and subgroup persistence, with some subgroups, such as α and β, persisting silently for decades before reemerging during the 2017 pneumonic plague epidemic. These findings uncover deep, previously underestimated phylogenetic diversity and long-term dynamics of Y. pestis in Madagascar, providing critical insights for understanding transmission patterns and informing future plague surveillance and control efforts.