Here we present a comprehensive targeted enrichment and species identification of 5,300-year-old leather from the Iceman’s clothes and quiver (Table 1). Of the nine samples taken, all produced libraries with sufficient data to identify the source species of the material. These were cattle, sheep, goat, brown bear and roe deer. Six libraries had sufficient coverage to fully reconstruct the mitogenome and confidently identify mtDNA haplotypes and reconstruct phylogeny (Fig. 1). Three libraries were of reduced coverage, thereby only partially reconstructing the mitogenomes and allowing for an approximated phylogeny (Fig. 2). Nonetheless, the identified cattle, sheep and goats sequences all fall within the range of mitochondrial genetic variability observed today in modern European domestic populations36,37,38. The mitogenomic genetic variability recovered shows that the sheep and goat leather fragments came from multiple hides. The genetic haplotypes of the wild species both roe deer and brown bear are consistent with present day phylogeography in the Alpine region39,40.

The Mitochondrial capture analysis allowed for an effective increase in coverage (between a four and 620-fold increase in coverage) and highlighted the effectiveness of NGS assisted with mitochondrial targeted enrichment to retrieve residual endogenous DNA from suboptimal sources such as skin or tanned leather10,41. Furthermore, our shotgun analysis is the first NGS quantification of the residual endogenous DNA present in archaeological leather. These data show that the endogenous content was highly variable and considerably lower than that of tissue sampled from the Iceman for genomic reconstruction3. This result lends support to the theory that tanning, the restoration processes, or both can remove endogenous markers10,11 and furthermore that the tanning may have exposed the material to a greater degree of bacterial and chemical degradation7. Equally, these results could support the hypothesis that soft tissue has a generally weaker retention of endogenous DNA compared to calcified remains41. Interestingly, enriched libraries displayed neither co-enrichment from other mammalian species or cross contamination from other animals in the mapped data (Supplementary Figs 2–10, Table 1). Hence, there is little genetic evidence from these samples to suggest that leathers and furs were treated with fat and oil extracted from other animals during the ancient tanning process11. Previous studies have however observed residual plant-based oils and fats in ancient leather indicating their use in leather manufacturing. Such as the identification of plant lipids in Neolithic leathers from the contemporaneous Schnidejoch Pass site in Switzerland7.

The Iceman’s garments and quiver are from an assemblage of at least five different species of animal. The coat alone was a combination of at least four hides and two species: goat and sheep. This result may indicate a haphazard stitching together of clothing based upon materials that were available to the Iceman, as ancient rudimentary leather is posited to rapidly deteriorate after manufacture6,12. However, the leggings were composed of goat leather, which was also used in the manufacture of a 4,500-year-old leggings from Schnidejoch, Switzerland. This result lends support to the idea that Copper Age individuals in the Alpine region selected species for specific attributes when manufacturing clothing8. This may also indicate a functional choice of material based on flexibility or insulating potential.

Previous microscopic and proteomic analyses of the Iceman’s clothes could not resolve beyond genus taxonomy or surpass the technical challenges caused by absence of species specific markers4,5. Hollemeyer et al.5 noted that many domestic and wild species germane to the study were too closely related to distinguish PMFs (Peptide mass fingerprints) or that adequate reference databases did not exist. There are inconsistencies between the species identification of our study and Hollemeyer et al.5, contrary to our results they observed chamois and red deer PMFs in parts of the coat, Canid PMFs in the leggings and cattle PMFs in the quiver. Different sampling locations on the same clothing may explain this disparity, as highlighted by our results from the coat analysis above.

The haplogroups identified in these samples from sheep, goat and cattle all fall within the domestic variability of the source animal in modern Europe, novel SNPs have been recovered that may have been lost from contemporary domestic haplogroups (Supplementary Table 3). In this study, the goat mitogenomes were identified as haplogroup A, yet a goat B1 haplogroup was observed in a study of 4,500-year-old swiss leggings8. The ancient distribution of domestic goat haplogroups are unknown, yet B1’s modern European distribution is restricted to Greece and Turkey8,42. Goat haplogroup B1 is more widespread in western Asia, but is closely related to haplogroup A. The Iceman and his leggings predate the Swiss find by c. 800 years, suggesting an overlap in the distribution of goat haplogroups that is no longer present in Europe. The sheep leather and fur were identified within haplogroup B1 from the three coat fragments and the loincloth. One sheep (coat 10304) mitogenome has the same distinct mutations at loci 16147 and 16440 reported by Olivieri et al.13 in a PCR analysis of fibres from a later excavation the Iceman’s find site. However, other mutations reported by Olivieri et al.13 were not present indicating that they identified sequences from yet another sheep used in the garment assemblage and recovered from the find site. The shoelace mitogenome closely resembled the cattle reference sequence of haplogroup T3, which is the predominant mitochondrial haplotype in European domestic cattle and is observed in high frequency throughout the Neolithic of Europe43.

The quiver was identified as roe deer, clustering near the contemporary central European clade. This is the first time this species has been identified as being part of the Iceman’s leather attire. Notably, red deer (Cervus elaphus) was identified from PCR of hair shafts at the finding site14 and contents of the Iceman’s stomach44 and from PMFs of the quiver5. The evidence that the Iceman utilised at least two deer species shows the variety of wild materials from which he subsisted. The hat sequence clusters inside the Western lineage of brown bears, suggesting regional continuity of this genetic marker. Previous studies of brown bears report complex phylogeography across its range, typified by overlapping ranges of divergent clades of brown bear and introgression with polar bears (Ursus maritimus) due to convoluted gene flow caused by Pleistocene climatic changes40,45. The assignment of the hat to brown bear is important given that previous studies could not resolve its taxonomy beyond Carnivora5 and secondly that it is unambiguously a wild species that was used for clothing manufacture.

Despite the Iceman being established as an agro-pastoralist2,3, the hat and quiver provide evidence of hunting and trapping of wild animals. Other studies have proposed parts of the diet, clothing and equipment as belonging to wild species such as wild canid (for the hat, species previously unresolved), red deer and chamois5,14. Rollo et al.44 demonstrated that the Iceman consumed red deer and alpine ibex (Capra ibex). The choices that Copper Age people made with respect to animals exploitation were likely dependent on availability, necessity, functionality and symbolism46. Given the diversity of the analysed material (each analysed fragment comes from a different animal), the lack of any obvious symbolism and the rapid degradation rate of ancient leather11 it would appear that the Iceman’s leathers and furs were first chosen haphazardly for subsistence as rudimentary leather pieces rapidly disintegrated and required replacement with strips of new hide. There is however likely to be a regional variation in the selection of wild and domestic populations throughout the Copper Age and the Iceman provides a window into the alpine region which may have been unlike other locations46.

These results are based on uni-parental maternal genetic markers, hence the authors cannot exclude the possibility that the analysed domestic species were sourced from herds that were re-stocked with wild males as occurred between cattle and aurochsen populations as is postulated in ancient Britain31. Whole genome sequencing of the Iceman’s attire in the future may increase our knowledge to the degree of introgression with local populations or breeding selection for functional genes.