Abstract Ancient genomics has enabled discovery of diverse pathogens across various time periods, host species, and material types. However, existing palaeogenomic pipelines predominantly focus on screening data from human hosts, or do not incorporate microbial screening methodologies. We present PIGSTI (Pathogen anImal Genome Sequence ToolkIt), a bioinformatic pipeline specifically designed for both the initial screening and subsequent detection of pathogens in shotgun sequencing data from ancient animal remains. PIGSTI’s integrated Snakemake workflow performs both host detection, genome mapping and pathogen identification, generating outputs suitable for population genetics and phylogenetic analyses. Testing on 952 newly sequenced and publicly available animal palaeogenomic datasets, we identified ∼15 ancient zoonotic and animal pathogens with high confidence, including the first documented case of Rickettsia felis and Leptospira borgpetersenii in an ancient animal. Our results demonstrate PIGSTI’s utility for screening pathogen diversity in ancient animal hosts and reconstructing historical host-pathogen relationships.

This paper presents two Danish examples from the Funnel Beaker Culture illustrating a practice of depositing antique elements – in this case elk bones. While faunal remains are a well-known aspect of complex Neolithic deposition practices, the scope and nuances of these depositional practices remain unclear, partly due to a general lack of 14C dates and detailed zooarchaeological analysis. One key outcome of an increased focus on Accelerator Mass Spectrometry (AMS) dating is the identification of deposited antiques. Although the concept of antiques in archaeological contexts is not new, it has rarely been discussed in Neolithic contexts, despite its relevance to theories of social memory and mnemonic materiality. Recognizing the phenomenon of deposited antique animal remains requires both archaeological openness to the idea and sufficient 14C dating. The two examples discussed here – both involving Maglemosian Elk bones found together with Neolithic domesticated animals – were discovered in wetland contexts in North Zealand, Denmark. Together, they highlight the potential of direct AMS dating in revealing previously unrecognized practices of temporal reuse, as well as shedding new light on Neolithic engagements with time, materiality, and memory.

House mice (Mus musculus) have been associated with humans since the beginning of sedentism, enabling them to become successful global colonisers. Three main subspecies originated approximately 0.5 Ma in a region extending from Southwest Asia to northern India. Molecular data suggest that a complex scenario of secondary admixture occurred thereafter in the Iranian region, leading to the formation of a Central Iranian lineage, but this evidence was overlooked in previous bioarchaeological analysis. The early Neolithic settlement of Ganj Dareh is located in this cradle area. It delivered remains of commensal house mice formerly attributed to M. m. domesticus. A geometric morphometric analysis of the first lower molars is used here to characterize the signature of hybridization between M. m. musculus and M. m. domesticus. The subspecific attribution of the Ganj Dareh mouse remains is re-evaluated through the inclusion of modern specimens from Central Iran as a separate group in the reference dataset. The results indicate that, contrary to what was previously thought, the Ganj Dareh specimens are likely related to the Central Iranian lineage. Their idiosyncrasy compared with modern representatives, however, suggests a complex temporal dynamic of admixture, which may have been influenced by early human settlements and movements.

Widening and diversifying trade networks are often cited among the boom and bust of Bronze and Iron Age worlds. The great distances that goods could travel during these periods are exemplified here as the authors describe the spectroscopic identification of Baltic amber beads in an Iron Age cremation grave at Hama in Syria. Yet these beads are not unique in the Near Eastern record; as the authors show, comparable finds and references to amber or amber hues in contemporaneous texts illustrate the high social and economic value of resinous substances—a value based on perceptions of their distant origin.

Feasting played an important role in cementing social bonds between prehistoric communities. At Early Neolithic Asiab, western Iran, ceremonial feasting is evidenced by the butchered skulls of nineteen wild boars (Sus scrofa), an animal that was not commonly hunted in the region at the time. Here we use microscopic dental growth patterns to guide geochemical analyses of five wild boar teeth from Asiab and examine the geographical scope from which the animals derived. Our dataset includes 165 stable oxygen isotope values, 107 strontium isotope ratios, and Barium concentration maps. The findings indicate that despite Asiab's location in an environment favourable to wild boars, the animals used for ceremonial feasting originated from a wide geographical catchment, with at least some necessitating transport over substantial distance across mountainous terrain. This deepens our understanding of the effort invested by the participating pre-agricultural communities for celebrating social connectivity across the wider landscape.

I 2005 blev nær Hald udgravet et grubekompleks, hvor der var observeret skaller i overfladen. Grubekompleksets indhold af mange forskellige levn og fundgrupper muliggør en række arkæologiske og biologiske analyser, som samlet bidrager til forståelsen af udnyttelsen af naturen samt menneskelige aktiviteter på en nærliggende boplads i yngre bronzealder. Grubekomplekset betragtes her som et sluttet anlæg, og evidensen peger på en opfyldning med mange affaldslag i løbet af relativ kort tid. Vi vil gerne søge at belyse spørgsmålet: Hvad kan de levn, der findes i gruber med skallag give af informationer, når både det naturvidenskabelige og arkæologiske materiale analyseres?

Germanic-speaking populations historically form an integral component of the North and Northwest European cultural configuration. According to linguistic consensus, the common ancestor of the Germanic languages, which include German, English, Frisian, Dutch as well as the Nordic languages, was spoken in Northern Europe during the Pre-Roman Iron Age. However, important questions remain concerning the earlier Bronze Age distribution of this Indo-European language branch in Scandinavia as well as the driving factors behind its Late Iron Age diversification and expansion across the European continent. A key difficulty in addressing these questions are the existence of striking differences in the interpretation of the archaeological record, leading to various hypotheses of correlations with linguistic dispersals and changes in material culture. Moreover, these interpretations have been difficult to assess using genomics due to limited ancient genomes and the difficulty in differentiating closely related populations. Here we integrate multidisciplinary evidence from population genomics, historical sources, archaeology and linguistics to offer a fully revised model for the origins and spread of Germanic languages and for the formation of the genomic ancestry of Germanic-speaking northern European populations, while acknowledging that coordinating archaeology, linguistics and genetics is complex and potentially controversial. We sequenced 710 ancient human genomes from western Eurasia and analysed them together with 3,940 published genomes suitable for imputing diploid genotypes. We find evidence of a previously unknown, large-scale Bronze Age migration within Scandinavia, originating in the east and becoming widespread to the west and south, thus providing a new potential driving factor for the expansion of the Germanic speech community. This East Scandinavian genetic cluster is first seen 800 years after the arrival of the Corded Ware Culture, the first Steppe-related population to emerge in Northern Europe, opening a new scenario implying a Late rather than an Middle Neolithic arrival of the Germanic language group in Scandinavia. Moreover, the non-local Hunter-Gatherer ancestry of this East Scandinavian cluster is indicative of a cross-Baltic maritime rather than a southern Scandinavian land-based entry. Later in the Iron Age around 1700 BP, we find a southward push of admixed Eastern and Southern Scandinavians into areas including Germany and the Netherlands, previously associated with Celtic speakers, mixing with local populations from the Eastern North Sea coast. During the Migration Period (1575-1200 BP), we find evidence of this structured, admixed Southern Scandinavian population representing the Western Germanic Anglo-Saxon migrations into Britain and Langobards into southern Europe. During the Migration Period, we detect a previously unknown northward migration back into Southern Scandinavia, partly replacing earlier inhabitants and forming the North Germanic-speaking Viking-Age populations of Denmark and southern Sweden, corresponding with historically attested Danes. However, the origin and character of these major changes in Scandinavia before the Viking Age remain contested. In contrast to these Western and Northern Germanic-speaking populations, we find the Wielbark population from Poland to be primarily of Eastern Scandinavian ancestry, supporting a Swedish origin for East Germanic groups. In contrast, the later cultural descendants, the Ostrogoths and Visigoths are predominantly of Southern European ancestry implying the adoption of Gothic culture. Together, these results highlight the use of archaeology, linguistics and genetics as distinct but complementary lines of evidence.### Competing Interest StatementThe authors have declared no competing interest.

Major migration events in Holocene Eurasia have been characterized genetically at broad regional scales 1 – 4 . However, insights into the population dynamics in the contact zones are hampered by a lack of ancient genomic data sampled at high spatiotemporal resolution 5 – 7 . Here, to address this, we analysed shotgun-sequenced genomes from 100 skeletons spanning 7,300 years of the Mesolithic period, Neolithic period and Early Bronze Age in Denmark and integrated these with proxies for diet ( 13 C and 15 N content), mobility ( 87 Sr/ 86 Sr ratio) and vegetation cover (pollen). We observe that Danish Mesolithic individuals of the Maglemose, Kongemose and Ertebølle cultures form a distinct genetic cluster related to other Western European hunter-gatherers. Despite shifts in material culture they displayed genetic homogeneity from around 10,500 to 5,900 calibrated years before present, when Neolithic farmers with Anatolian-derived ancestry arrived. Although the Neolithic transition was delayed by more than a millennium relative to Central Europe, it was very abrupt and resulted in a population turnover with limited genetic contribution from local hunter-gatherers. The succeeding Neolithic population, associated with the Funnel Beaker culture, persisted for only about 1,000 years before immigrants with eastern Steppe-derived ancestry arrived. This second and equally rapid population replacement gave rise to the Single Grave culture with an ancestry profile more similar to present-day Danes. In our multiproxy dataset, these major demographic events are manifested as parallel shifts in genotype, phenotype, diet and land use.

The analysis of the DNA entrapped in ancient shells of molluscs has the potential to shed light on the evolution and ecology of this very diverse phylum. Ancient genomics could help reconstruct the responses of molluscs to past climate change, pollution, and human subsistence practices at unprecedented temporal resolutions. Applications are however still in their infancy, partly due to our limited knowledge of DNA preservation in calcium carbonate shells and the need for optimized methods for responsible genomic data generation. To improve ancient shell genomic analyses, we applied high-throughput DNA sequencing to 27 Mytilus mussel shells dated to ~111-6500 years Before Present, and investigated the impact, on DNA recovery, of shell imaging, DNA extraction protocols and shell sub-sampling strategies. First, we detected no quantitative or qualitative deleterious effect of micro-computed tomography for recording shell 3D morphological information prior to sub-sampling. Then, we showed that double-digestion and bleach treatment of shell powder prior to silica-based DNA extraction improves shell DNA recovery, also suggesting that DNA is protected in preservation niches within ancient shells. Finally, all layers that compose Mytilus shells, i.e., the nacreous (aragonite) and prismatic (calcite) carbonate layers, with or without the outer organic layer (periostracum) proved to be valuable DNA reservoirs, with aragonite appearing as the best substrate for genomic analyses. Our work contributes to the understanding of long-term molecular preservation in biominerals and we anticipate that resulting recommendations will be helpful for future efficient and responsible genomic analyses of ancient mollusc shells.

Now extinct, the aurochs (Bos primigenius) was a keystone species in prehistoric Eurasian and North African ecosystems, and the progenitor of cattle (Bos taurus), domesticates that have provided people with food and labour for millennia1. Here we analysed 38 ancient genomes and found 4 distinct population ancestries in the aurochs—European, Southwest Asian, North Asian and South Asian—each of which has dynamic trajectories that have responded to changes in climate and human influence. Similarly to Homo heidelbergensis, aurochsen first entered Europe around 650 thousand years ago2, but early populations left only trace ancestry, with both North Asian and European B. primigenius genomes coalescing during the most recent glaciation. North Asian and European populations then appear separated until mixing after the climate amelioration of the early Holocene. European aurochsen endured the more severe bottleneck during the Last Glacial Maximum, retreating to southern refugia before recolonizing from Iberia. Domestication involved the capture of a small number of individuals from the Southwest Asian aurochs population, followed by early and pervasive male-mediated admixture involving each ancestral strain of aurochs after domestic stocks dispersed beyond their cradle of origin. An analysis of 38 ancient genomes from the aurochs, the extinct ancestor of modern cattle, provides insight into the population ancestry and domestication of this species.

Western Eurasia witnessed several large-scale human migrations during the Holocene 1 – 5 . Here, to investigate the cross-continental effects of these migrations, we shotgun-sequenced 317 genomes—mainly from the Mesolithic and Neolithic periods—from across northern and western Eurasia. These were imputed alongside published data to obtain diploid genotypes from more than 1,600 ancient humans. Our analyses revealed a ‘great divide’ genomic boundary extending from the Black Sea to the Baltic. Mesolithic hunter-gatherers were highly genetically differentiated east and west of this zone, and the effect of the neolithization was equally disparate. Large-scale ancestry shifts occurred in the west as farming was introduced, including near-total replacement of hunter-gatherers in many areas, whereas no substantial ancestry shifts happened east of the zone during the same period. Similarly, relatedness decreased in the west from the Neolithic transition onwards, whereas, east of the Urals, relatedness remained high until around 4,000 bp , consistent with the persistence of localized groups of hunter-gatherers. The boundary dissolved when Yamnaya-related ancestry spread across western Eurasia around 5,000 bp , resulting in a second major turnover that reached most parts of Europe within a 1,000-year span. The genetic origin and fate of the Yamnaya have remained elusive, but we show that hunter-gatherers from the Middle Don region contributed ancestry to them. Yamnaya groups later admixed with individuals associated with the Globular Amphora culture before expanding into Europe. Similar turnovers occurred in western Siberia, where we report new genomic data from a ‘Neolithic steppe’ cline spanning the Siberian forest steppe to Lake Baikal. These prehistoric migrations had profound and lasting effects on the genetic diversity of Eurasian populations.

This contribution examines social practices in the Central Bathhouse in Jerash in Late Antiquity based on the ceramic assemblage, vessel glass, faunal remains, and small finds retrieved from two sections of the bathhouse's sewer. We argue that although the bathhouse underwent significant architectural alterations from its construction in the 4th c. CE to its abandonment in the late 7th, the activities taking place inside the building remained largely the same. Our study shows that even towards the end of the bathhouse's lifespan, bodily grooming remained integral to the bathing experience, while food and drink were consumed on the premises even though the bathing facilities had been reduced to a bare minimum. The faunal remains indicate the type of food consumed, while the small finds illustrate a lively environment where gaming and gambling took place in a social space frequented by men, women, and children.

Poor preservation of collagen in dry and/or arid environments has hindered the application of Zooarchaeology by mass spectrometry (ZooMS) analysis in many regions of the world, and as a result many zooarchaeological investigations have relied exclusively on the morphological assessment of fragmentary remains, due to the inadequate preservation of biomolecules. The climatic conditions of Southwest Asia include extreme temperature fluctuations unconducive to preservation of proteins and DNA. We performed zooarchaeological analysis of remains from the 10,000-year-old site of Shkārat Msaied in Jordan and sub-sampled twenty-eight petrous bones, the hardest bone in the mammalian skeleton, for species identification by ZooMS. Using an unconventional and simplified extraction protocol we call Tryps-IN, in which digestion was performed without removal of the demineralising EDTA, we taxonomically identified several fragments, outperforming the established ZooMS work-flow. A subset of identifications was subsequently confirmed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) protein sequencing. The new methodology presented here opens the possibility of further bioarchaeological investigation of other fragmentary faunal assemblages within this region of archaeological significance.

Archaeological evidence for penned animals is increasingly used to indicate managed livestock. Advances in techniques allowing the identification of dung and urine-derived components in sediments have enabled the expansion of this line of enquiry. Corralling animals into settlements protected them from predators at night and provided more control over their breeding. Deposits associated with livestock management at Neolithic settlements across Southwest Asia sometimes contain bones of perinatal animals. Reviewing the literature, it is evident that these faunal remains are not systematically reported or preserved in all burial environments. However, their distribution may reflect different patterns of livestock integration into human settlements. The presence of perinatal remains at sites where early livestock herding took place has important implications. Not only are they compelling evidence for herd management, particularly if there is also evidence for penning deposits, but also death of livestock during the perinatal phase of life informs us about the health of animals in early herds. This in turn, provides information about the skills needed by early pastoralists as they developed animal management strategies and the possible effect of transmissible diseases as animals were kept together in closer proximity.

Species determination based on genetic evidence is an indispensable tool in archaeology, forensics, ecology, and food authentication. Most available analytical approaches involve compromises with regard to the number of detectable species, high cost due to low throughput, or a labor-intensive manual process. Here, we introduce “Species by Proteome INvestigation” (SPIN), a shotgun proteomics workflow for analyzing archaeological bone capable of querying over 150 mammalian species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rapid peptide chromatography and data-independent acquisition (DIA) with throughput of 200 samples per day reduce expensive MS time, whereas streamlined sample preparation and automated data interpretation save labor costs. We confirm the successful classification of known reference bones, including domestic species and great apes, beyond the taxonomic resolution of the conventional peptide mass fingerprinting (PMF)-based Zooarchaeology by Mass Spectrometry (ZooMS) method. In a blinded study of degraded Iron-Age material from Scandinavia, SPIN produces reproducible results between replicates, which are consistent with morphological analysis. Finally, we demonstrate the high throughput capabilities of the method in a high-degradation context by analyzing more than two hundred Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable to other biological tissues and taxa.

Several major migrations and population turnover events during the later Stone Age (after c. 11,000 cal. BP) are believed to have shaped the contemporary population genetic diversity in Eurasia. While the genetic impacts of these migrations have been investigated on regional scales, a detailed understanding of their spatiotemporal dynamics both within and between major geographic regions across Northern Eurasia remains largely elusive. Here, we present the largest shotgun-sequenced genomic dataset from the Stone Age to date, representing 317 primarily Mesolithic and Neolithic individuals from across Eurasia, with associated radiocarbon dates, stable isotope data, and pollen records. Using recent advances, we imputed >1,600 ancient genomes to obtain accurate diploid genotypes, enabling previously unachievable fine-grained population structure inferences. We show that 1) Eurasian Mesolitic hunter-gatherers were more genetically diverse than previously known, and deeply divergent between the west and the east; 2) Hitherto genetically undescribed hunter-gatherers from the Middle Don region contributed significant ancestry to the later Yamnaya steppe pastoralists; 3) The genetic impact of the transition from Mesolithic hunter-gatherers to Neolithic farmers was highly distinct, east and west of a “Great Divide” boundary zone extending from the Black Sea to the Baltic, with large-scale shifts in genetic ancestry to the west. This include an almost complete replacement of hunter-gatherers in Denmark, but no substantial shifts during the same period further to the east; 4) Within-group relatedness changes substantially during the Neolithic transition in the west, where clusters of Neolithic farmer-associated individuals show overall reduced relatedness, while genetic relatedness remains high until ~4,000 BP in the east, consistent with a much longer persistence of smaller localised hunter-gatherer groups; 5) A fast-paced second major genetic transformation beginning around 5,000 BP, with Steppe-related ancestry reaching most parts of Europe within a 1,000 years span. Local Neolithic farmers admixed with incoming pastoralists in most parts of Europe, whereas Scandinavia experienced another near-complete population replacement, with similar dramatic turnover-patterns also evident in western Siberia; 6) Extensive regional differences in the ancestry components related to these early events remain visible to this day, even within countries (research conducted using the UK Biobank resource). Neolithic farmer ancestry is highest in southern and eastern England while Steppe-related ancestry is highest in the Celtic populations of Scotland, Wales, and Cornwall. Overall, our findings show that although the Stone-Age migrations have been important in shaping contemporary genetic diversity in Eurasia, their dynamics and impact were geographically highly heterogeneous.

Animals are an integral part of deposition practices during the Danish Iron Age, and they probably represent the most common form of deposit within southern Scandinavia. Recently Gotfredsen published a volume on animals within Danish Iron Age grave contexts, but similarly comprehensive studies of animals from other contexts have not been attempted. Thus, classic sites such as Valmose, Bukkerup Langmose, and Sorte Muld still stand as the type sites for Danish Iron Age animal deposits. This article will demonstrate that there are good reasons for exploring deposits in more detail and investigate the significant variation in the treatment and quantities of sacrificial animal deposits. Furthermore, the current study has revealed a deposition pattern where a primary animal is often in the company of one or more secondary animals, the latter typically represented by a few bones. Salpetermosen Syd (MNS50010), south of Hillerød in North Zealand, Denmark is the main case study, but comparisons are made to several sites across Denmark where a similar deposition pattern has been observed.

Genetic species determination has become an indispensable tool in forensics, archaeology, ecology, and food authentication. The available methods are either suited for detecting a single taxon across many samples or for screening a wide range of species across a few samples. Here, we introduce “Species by Proteome INvestigation” (SPIN), a proteomics workflow capable of querying over 150 mammalian species in 7.2 minutes of mass spectrometry (MS) analysis. Streamlined and automated sample preparation by protein aggregation capture, high-speed chromatography and data-independent acquisition, and a confident species inference algorithm facilitate processing hundreds of samples per day. We demonstrate the correct classification of known references, reproducible species identification in degraded Iron-Age material from Scandinavia, and test the limits of our methods with Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable with other biological tissues and taxa.