Dual-kingdom necrobiome succession extends postmortem interval estimation into skeletonization

Forensic estimation of the postmortem interval (PMI) becomes increasingly challenging when decomposition progresses beyond the initial weeks, as traditional medicolegal indicators lose their temporal precision. Here, we demonstrate that dual-kingdom microbial communities associated with decomposing remains form robust molecular clocks that maintain predictive power for PMI estimation well into skeletonization. Using full-length amplicon sequencing, we tracked the succession of bacteria and fungi in host-associated oral samples and the underlying gravesoil from decomposing pigs (N = 6) over nearly five months (2170.0 accumulated degree days; ADD). Microbial communities exhibited consistent three-phase succession patterns - initial disruption, intermediate colonization, and late-stage stabilization - that aligned with morphological decomposition scoring. Necrobiome succession dynamics continued long after morphological decomposition metrics reached a plateau, demonstrating the extended temporal resolution provided by microbial markers. Machine-learning models integrating microbial features with morphological data achieved robust predictive accuracy for both PMI in days and ADD, with performance varying systematically across decomposition stages. Bacterial models dominated early decomposition, dual-kingdom approaches optimized intermediate phases, and fungal models excelled during late-stage decomposition when conventional indicators fail. We identified specific microbial taxa that serve as reliable temporal indicators across sample types. These findings demonstrate that necrobiome succession extends capabilities to estimate time since death by months, offering a molecular framework for advanced decomposition cases where traditional methods lose precision.