The petrous bone is considered the most efficient source of endogenous DNA across skeletal tissues in ancient DNA (aDNA) research as well as in forensic work. Recently, aDNA in auditory ossicle bones was shown to be comparably well preserved as in the petrous, although no attempt was made to distinguish among the three ossicle bones. In this study, we compare aDNA profiles across matched ossicle- and petrous-derived sequencing libraries prepared from 29 human skeletons from Neolithic Anatolia and Medieval Iberia. We find that the stapes and incus provide higher human endogenous aDNA than the petrous bone, with >2× higher median rates of endogenous aDNA recovery, whereas the malleus performs similarly to the petrous. Human aDNA fragments retrieved from the stapes were 8% longer than those from the petrous, whereas postmortem damage, clonality, and contamination rates were comparable among the studied bone types. These observations are corroborated by data from nonmatched ossicle or petrous libraries from 81 individuals from the same contexts, with the highest endogenous aDNA content observed in the stapes. Despite being the smallest bone in the human skeleton, the stapes, along with the incus, may be among the most optimal aDNA sources yet identified.

Ibiza, an island in present-day Spain, was conquered in 902 CE by the Umayyad Emirate of Córdoba. The island remained under Islamic rule until 1235. Here, we analyse the genetic and metagenomic profiles of 13 individuals from an Islamic cemetery in Ibiza, dated to 950–1150 CE. Genome-wide analyses reveal heterogeneity, with ancestry components from Europe, North Africa, and Sub-Saharan Africa. Our analyses estimate that North African gene flow occurred two to seven generations before these individuals lived, suggesting admixture following the Islamic conquest of Iberia and potentially on Ibiza itself. Notably, two individuals trace their Sub-Saharan origins to distinct regions, Senegambia and present-day southern Chad, providing direct evidence of trans-Saharan connections via military and slave networks documented in contemporary Arabic sources. Metagenomic analyses detect several pathogens in this community, with one individual carrying Mycobacterium leprae, offering insight into the presence of leprosy in Ibiza. Our findings align with the historically documented two-pulse demographic model, indicating an initial settlement following the early tenth-century conquest and a second influx associated with Almoravid movements in the twelfth century. These securely dated genomes offer insights into medieval population dynamics and health in the Balearics.

Two archaeological cultural complexes; the Neolithic Funnelbeaker culture (FBC) and the Pitted ware culture (PWC), coexisted on Gotland for over 500 years, between ~3300 and 2800 calBCE. The ancestry of the FBC farmers and PWC marine foragers largely aligns with European Neolithic Farmers and European Mesolithic foragers, respectively, but the direct interactions between the groups on Gotland is not understood. We present a Middle Neolithic (MN) high-coverage genome and a Late Neolithic (LN) low-coverage genome from the Ansarve FBC dolmen. We investigate ancestry, admixture, and pathogens among these MN farmers (n = 6), foragers (n = 19), and the LN individual. We find that recent gene-flow between farmers and foragers could have taken place, although most gene-flow happened prior to their coexistence on the island. We also find evidence of different Yersinia pestis strains in the three cultural groups, showing that the pestis was widespread among groups with different subsistence strategies.

Sexing the skeletal remains of young individuals is crucial yet notoriously difficult in archaeology. Children, who cannot be reliably sexed morphologically, are often excluded from gender-related research, limiting our understanding of past childhood. This issue is compounded in contexts lacking grave goods, such as early Christian burials. We conducted genomic screening of 142 individuals from Sweden dating from the late Viking Age to the Medieval period, including 68 subadults and 74 adults from 27 single and 50 multiple burials. To investigate the treatment of children in death and the role of collective graves, we applied genomic sexing and kinship analyses to individuals from three sites. Contrary to the assumption that collective burials reflect close kinship, our results show that children interred with adults rarely shared close biological ties. Burial patterns indicate that gender roles were established early, with both boys and most girls mirroring adult spatial patterns. However, flexibility existed, and extended kinship likely played a central role in structuring these communities.

Kin relations among past societies can offer valuable information about the social dynamics of the population. Genetic data from prehistoric human remains can reveal genetic relatedness, and when combined with archaeological information, shed light on social factors shaping ancient communities. However, accessing such information on ancient hunter–gatherer societies has been challenging owing to the scarcity of temporally overlapping multi-burial sites. Here, we focused on the Pitted Ware Culture (PWC) cemetery from Ajvide (Gotland, Sweden), one of Stone Age Europe’s largest and best-preserved hunter–gatherer burial grounds of the European Stone Age. We generated new genomic data from 10 individuals, primarily from co-burial contexts, and combined these with published genomes from 24 individuals across four PWC sites on Gotland. The genetic analyses revealed dual ancestry of the Gotlandic PWC, showing approximately 80% ancestry associated with earlier Mesolithic hunter–gatherer groups and 20% with farmer groups. We also identified close genetic relatives between the different studied PWC sites on Gotland, indicating mixing of the groups. All individuals buried together were closely related to one another, including first-, second- and third-degree relatives, and showed significantly elevated genetic relatedness. This demonstrates that genetic relatedness played a defining role in the co-burial ritual and extended beyond first-degree relatives.

The Eurasian cave lion was abundant across the Northern Hemisphere before the Late Pleistocene megafaunal extinctions. However, the extent of the distinction between cave and modern lions and their adaptive differences have remained unclear. Using 12 cave lion genomes spanning more than 100,000 years, we show that modern and cave lions were distinct evolutionary lineages with separate demographic histories and unique non-synonymous variants. We also identify evidence of ancient gene flow between them, with the best modern lion proxy for this ancestry being an extinct Southwest Asian population. This admixture correlates with global ice extent, with 3.2%–4.4% modern lion ancestry detected in a ∼20,000-year-old cave lion from Central East Asia. These findings provide insight into the evolutionary history of the cave lion, once one of the Northern Hemisphere’s most ecologically impactful megafaunal species.

The genomic study of specimens dating to the Early and Middle Pleistocene (EP and MP), a period spanning from 2.6 million years ago (Ma) to 126 thousand years ago (ka), has the potential to elucidate the evolutionary processes that shaped present-day biodiversity. Obtaining genomic data from this period is challenging, but mitochondrial DNA, given its higher abundance compared to nuclear DNA, could play an important role to understand evolutionary processes at this time scale. In this study, we report 34 new mitogenomes, including two EP and nine MP mammoth (Mammuthus spp.) specimens from Siberia and North America and analyze them jointly with >200 publicly available mitogenomes to reconstruct a transect of mammoth mitogenome diversity throughout the last million years. We find that our EP mitogenomes fall outside the diversity of all Late Pleistocene (LP) mammoths, while those derived from MP mammoths are basal to LP mammoth Clades 2 and 3, supporting an ancient Siberian origin of these lineages. In contrast, the geographical origin of Clade 1 remains unresolved. With these new deep-Time mitogenomes, we observe diversification events across all clades that appear consistent with previously hypothesized MP and LP demographic changes. Furthermore, we improve upon an existing methodology for molecular clock dating of specimens >50 ka, demonstrating that specimens need to be individually dated to avoid biases in their age estimates. Both the molecular and analytical improvements presented here highlight the importance of deep-Time genomic data to discover long-lost genetic diversity, enabling better assessments of evolutionary histories.

Ancient genomic studies have extensively explored human-microbial interactions, yet research on non-human animals remains limited. In this study, we analyzed ancient microbial DNA from 483 mammoth remains spanning over 1 million years, including 440 newly sequenced and unpublished samples from a 1.1-million-year-old steppe mammoth. Using metagenomic screening, contaminant filtering, damage pattern analysis, and phylogenetic inference, we identified 310 microbes associated with different mammoth tissues. While most microbes were environmental or post-mortem colonizers, we recovered genomic evidence of six host-associated microbial clades spanning Actinobacillus, Pasteurella, Streptococcus, and Erysipelothrix. Some of these clades contained putative virulence factors, including a Pasteurella-related bacterium that had previously been linked to the deaths of African elephants. Notably, we reconstructed partial genomes of Erysipelothrix from the oldest mammoth sample, representing the oldest authenticated host-associated microbial DNA to date. This work demonstrates the potential of obtaining ancient animal microbiomes, which can inform further paleoecological and evolutionary research.

The assessment of sex is fundamental in osteoarcheological analysis, yet traditional morphological methods are less reliable for children due to the incomplete development of sexually dimorphic traits. This study applies a multi-method approach—integrating morphological, metric, and genetic data—to assess sex and, through analysis of burial assemblages, explore gender expression as an interpretative complement. While analytically distinct, gender and sex are deeply intertwined, and considering both can offer a fuller understanding of identity.

The study focuses on the Viking Age (c. 750–1050 ce) burial site of Ihre, Gotland, with particular attention to individuals under 20. Results reveal strong alignment between metric and genetic sex. Mandibular odontometric analysis proved more consistent than traditional morphological traits when compared to genetic sex, offering an accessible and effective supplement for sex estimation in children. In adolescents, pelvic traits were most informative, while cranial traits showed limited reliability. Genetic sexing provided a critical reference, highlighting misclassifications in morphological assessments, particularly among genetic males.

Burial assemblage analysis indicates that gender expression often corresponded with biological sex but varied with age. Female graves typically included dress items such as brooches and pendants, especially after adolescence, while male graves more frequently featured tools and weapons. These patterns suggest that gendered roles and identities were materially expressed and changed over the life course.

The findings underscore the importance of a multi-method approach in sex estimation, particularly for children, and demonstrate the value of contextual archeological analysis in interpreting past identities. At Ihre, the interplay between sex, gender, and social identity in mortuary practices suggests a framework that, while broadly organized around binary distinctions, was shaped by age and culturally specific factors—highlighting identity as dynamic, layered, and context-dependent.