Mules in the Antiquity of Southern Iberia revealed from proteomics

The management of equids in the Iberian Peninsula during the Iron Age (first millennium BCE) provides a key archaeological context for investigating the emerging stages of confluent breeding of horses ( Equus caballus ) and donkeys ( Equus asinus ). This scenario provided the earliest historical setting in Iberia conducive to the deliberate and systematic production of hybrids (mules and hinnies), with significant cultural and economic implications. In this study, high-resolution mass-spectrometry-based palaeoproteomics (MALDI-TOF and LC-ESI-orbitrap MS/MS) is employed to investigate the presence of equids and to identify molecular signatures of hybridization in the archaeological record of Southern Iberia. A systematic reassessment of collagen peptide biomarkers is presented, based on alignments of the corresponding coding DNA sequences, enabling the identification of reliable heterozygous markers diagnostic of equid hybrids. The materials analysed include twenty-two specimes from the Phoenician-Punic colonization of the Iron Age to the Roman period and Late Antiquity (7th century BCE to 6th century CE). Three of the specimens were identified as mules. The earliest mule is dated to 543–409 cal BCE and is associated to a pre-Roman Iron Age context in the lower Guadalquivir River basin, constituting the earliest direct biomolecular evidence to date for mule breeding in the region. The results demonstrate the value of palaeoproteomics as a complementary tool to osteomorphological analysis, to refine reconstructions of past animal husbandry practices and hybrid management strategies in complex archaeological contexts.