Epigenetic prediction of age-at-death and castration in ancient horses

The age profiles of archaeological bone assemblages inform on past animal management practices, with overkilling of male juveniles indicating resource optimization for livestock production. Age profiling of ancient animals is, however, not always possible due to the fragmentary nature of the fossil record and the lack of age skeletal markers valid across species. DNA methylation patterns at CpG clock sites represent an almost universal biological clock for mammals and can be deduced in ancient osteological remains from signatures of post-mortem DNA damage. Estimating the age-at-death of ancient individuals from DNA methylation patterns remains, however, challenging, as both subject to technical artefacts and requiring extensive sequence coverage. Here, we take advantage of the recently developed DNA methylation clock based on 31,836 CpG sites and the availability of dental age markers in horses to assess age predictions in 85 ancient remains. We establish the reliability of our approach using whole genome sequencing and develop an in-solution target-enrichment assay for 2,171 CpG sites, providing accurate estimates for only a fraction of the cost. The 85 assemblages investigated reveal similar age profiles across lineages, sex, ritual and non-ritual activities. We also leverage 59 differentially methylated sites in castrated and non-castrated males aged above 11 years to assess castration practice in the past. We reject previous contention that the Pazyryk horses sacrificed 2,300 years ago at Berel', Kazakhstan were castrated. Our methodology opens for a deeper characterization of past husbandry and ritual practices and holds the potential to reveal age mortality profiles in ancient societies, once extended to human remains.