aDNA Analysis Research Articles

Cost-effectiveness and other considerations for different research techniques applied in ancient DNA analysis.

Ancient DNA (aDNA) analysis has developed rapidly since it first emerged in the 1980s, becoming an almost indispensable tool in anthropological and archaeological sciences. Earlier aDNA study was based on the polymerase chain reaction (PCR) technique, with which, unfortunately, modern DNA contamination and other authenticity issues were often incurred. These technical hurdles were soon overcome by application of advancements in the forms of the next generation sequencing (NGS) technique and others. However, since NGS requires money, time, and, in the case of large projects, manpower as well, genetic analysis of some ancient samples considered to be insignificant is commonly delayed or, in the worst cases, neglected entirely. We acknowledge that as a diagnostic tool in aDNA analysis, PCR is less accurate than NGS and more easily affected by modern DNA contamination; but it also has advantages, such as simplicity, time-saving, and greater ease of interpretation, among others. The role of PCR in aDNA analysis, then, should be reconsidered.

Uncovering the Past: DNA Analysis of Skeletal Remains from the Medieval Bosnian City of Bobovac

Numerous archaeological sites in Bosnia and Herzegovina represent a historical heritage and testify to the rich cultural, social, and political life of medieval Bosnia. Bobovac, the capital of the Bosnian Kingdom after King Tvrtko I's coronation in 1377, featured a royal complex with a palace, church, and fortification. Recent molecular-genetic research on skeletal remains from Bobovac aims to uncover medieval ancestors' customs and genetic origins. Fifteen well-preserved teeth samples from Bobovac were processed. STR amplification employed PowerPlex® Fusion and Investigator® 24plex QS Kits, with Y-STR profiles generated using the PowerPlex® Y23 System. Fourteen partial autosomal STR profiles were obtained, enabling sex determination and kinship analysis. STR amplification success varied due to ancient DNA degradation, with larger loci showing lower amplification rates. Kinship analysis confirmed appropriate marker selection, demonstrating high reliability for determining close relationships. Integrating aDNA analysis with archaeological research enhances our understanding of historical populations, connecting archaeology and forensic genetics to contribute to the broader narrative of human history.

Cervidae antlers exploited to manufacture prehistoric tools and hunting implements as a reliable source of ancient DNA

Antler is one of the primary animal raw materials exploited for technical purposes by the hunter-gatherer groups of the Eurasian Upper Palaeolithic (UP) all over the ecological range of deers, and beyond. It was exhaustively employed to produce one of the most critical tools for the survival of the UP societies: hunting weapons. However, antler implements can be made from diverse deer taxa, with different ecological requirements and ethological behaviours. Identifying the antler's origin at a taxonomic level is thus essential in improving our knowledge of humans' functional, practical and symbolic choices, as well as the human-animal interface during Prehistoric times. Nevertheless, palaeogenetics analyses have focused mainly on bone and teeth, with genetic studies of antler generally focused on modern deer conservation. Here we present the results of the first whole mitochondrial genome ancient DNA (aDNA) analysis by means of in-solution hybridisation capture of antlers from pre-Holocene archaeological contexts. We analysed a set of 50 Palaeolithic and Neolithic (c. 34-8ka) antler and osseous objects from South-Western Europe, Central Europe, South-Western Asia and the Caucasus. We successfully obtained aDNA, allowing us to identify the exploited taxa and demonstrate the archaeological relevance of those finds. Moreover, as most of the antlers were sampled using a minimally-invasive method, further analyses (morphometric, technical, genetic, radiometric and more) remain possible on these objects.

Double feature: First genetic evidence of a mother-daughter double burial in Roman period Austria

A human double burial combined with a horse interment, discovered in 2004 in the eastern cemetery of the Roman town of Ovilava (today Wels/AUT), has been the subject of a comprehensive interdisciplinary study. This burial was remarkable for two reasons: firstly, the arrangement of the two individuals, with individual 1 having an arm around individual 2, and secondly, the presence of equine skeletal remains in the same burial pit directly below the human remains. Based on this finding, an initial assessment suggested a mixed-sex pair buried together with a horse. The horse burial was interpreted as an indicator of an early medieval date. The position of the two human skeletons suggested that a male and female individual had been buried together. A thorough examination of both the human and horse skeletons disproved these initial interpretations. Radiocarbon dating of the human and horse skeletons dated them to the Roman period. In addition, ancient DNA (aDNA) analysis of the human remains corrected the earlier sex assumption, revealing that the individuals were two biological females who were first-degree relatives. The age difference of 15 to 25 years between the two suggests a probable mother-daughter relationship. Thus, the application of scientific methods confirmed a rare, combined human-horse burial from Roman antiquity and established the first genetically documented mother-daughter burial from this period in present-day Austria.

Simulation-based benchmarking of ancient haplotype inference for detecting population structure

Paleogenomic data has informed us about the movements, growth, and relationships of ancient populations. It has also given us context for medically relevant adaptations that appear in present-day humans due to introgression from other hominids, and it continues to help us characterize the evolutionary history of humans. However, ancient DNA (aDNA) presents several practical challenges as various factors such as deamination, high fragmentation, environmental contamination of aDNA, and low amounts of recoverable endogenous DNA, make aDNA recovery and analysis more difficult than modern DNA. Most studies with aDNA leverage only SNP data, and only a few studies have made inferences on human demographic history based on haplotype data, possibly because haplotype estimation (or phasing) has not yet been systematically evaluated in the context of aDNA. Here, we evaluate how the unique challenges of aDNA can impact phasing and imputation quality, we also present an aDNA simulation pipeline that integrates multiple existing tools, allowing users to specify features of simulated aDNA and the evolutionary history of the simulated populations. We measured phasing error as a function of aDNA quality and demographic history, and found that low phasing error is achievable even for very ancient individuals (∼ 400 generations in the past) as long as contamination and average coverage are adequate. Our results show that population splits or bottleneck events occurring between the reference and phased populations affect phasing quality, with bottlenecks resultingin the highest average error rates. Finally, we found that using estimated haplotypes, even if not completely accurate, is superior to using the simulated genotype data when reconstructing changes in population structure after population splits between present-day and ancient populations. We also find that the imputation of ancient data before phasing can lead to better phasing quality, even in cases where the reference individuals used for imputation are not representative of the ancient individuals.

Simulation-based benchmarking of ancient haplotype inference for detecting population structure

Paleogenomic data has informed us about the movements, growth, and relationships of ancient populations. It has also given us context for medically relevant adaptations that appear in present-day humans due to introgression from other hominids, and it continues to help us characterize the evolutionary history of humans. However, ancient DNA (aDNA) presents several practical challenges as various factors such as deamination, high fragmentation, environmental contamination of aDNA, and low amounts of recoverable endogenous DNA, make aDNA recovery and analysis more difficult than modern DNA. Most studies with aDNA leverage only SNP data, and only a few studies have made inferences on human demographic history based on haplotype data, possibly because haplotype estimation (or phasing) has not yet been systematically evaluated in the context of aDNA. Here, we evaluate how the unique challenges of aDNA can impact phasing and imputation quality, we also present an aDNA simulation pipeline that integrates multiple existing tools, allowing users to specify features of simulated aDNA and the evolutionary history of the simulated populations. We measured phasing error as a function of aDNA quality and demographic history, and found that low phasing error is achievable even for very ancient individuals (∼ 400 generations in the past) as long as contamination and average coverage are adequate. Our results show that population splits or bottleneck events occurring between the reference and phased populations affect phasing quality, with bottlenecks resulting in the highest average error rates. Finally, we found that using estimated haplotypes, even if not completely accurate, is superior to using the simulated genotype data when reconstructing changes in population structure after population splits between present-day and ancient populations. We also find that the imputation of ancient data before phasing can lead to better phasing quality, even in cases where the reference individuals used for imputation are not representative of the ancient individuals.

Who, Why, When, and Where From? The Peopling of the Canary Islands and the Challenges of Archaeometry

Archaeologists in the Canary Islands have gathered substantial quantitative data from radiocarbon measurements and aDNA analyses. While undeniably helpful and necessary for apprehending past human activity, their interpretation, based on theories underpinning models developed for island chains, has lagged, leaving a gap in our understanding of processes of occupation and social network systems. The decontextualized nature of the archaeological landscape of the Canaries and a lack of consensus about proper radiocarbon methodologies are some of the factors contributing to heated scholarly debate. Here, for the Canary Islands, the author reviews the current literature on aDNA and discusses settlement theories, the chronological evidence used for occupation models, and how such perspectives align with current thinking on island colonization.

Isotopic Evidence for Mobility in the Copper and Bronze Age Cemetery of Humanejos (Parla, Madrid): a Diachronic Approach Using Biological and Archaeological Variables

Over the last several decades, the application of aDNA and strontium isotope analyses on archaeologically recovered human remains has provided new avenues for the investigation of mobility in past societies. Data on human mobility can be valuable in the reconstruction of prehistoric residential patterns and kinship systems, which are at the center of human social organization and vary across time and space. In this paper, we aim to contribute to our understanding of mobility, residence, and kinship patterns in late Prehistoric Iberia (c. 3300–1400BC) by providing new strontium data on 44 individuals from the site of Humanejos (Parla, Madrid). The study presented here is multi-proxy and looks at these new data by interweaving biological, chronological, and archaeological information. This analysis found that 7/44 individuals buried at Humanejos could be identified as non-local to the necropolis. Although more men (n = 5) than women (n = 2) were found in the non-local category, and more non-local individuals were identified in the pre-Bell Beaker (n = 5) than in Bell Beaker (n = 1) or Bronze Age (n = 1), we find no statistically significant differences concerning sex or time period. This contrasts with other archaeological datasets for late prehistoric Europe which suggest higher female mobility, female exogamy, and male-centered residential patterns were common. At Humanejos, we have also identified one non-local female whose exceptional Beaker grave goods suggest she was an individual of special status, leading to additional questions about the relationships between gender, mobility, and social position in this region and time period.

Groby o domniemanych skandynawskich atrybucjach etno-kulturowych z obszaru państwa pierwszych Piastów w kontekście archeologicznych paradygmatów badawczych

In spite of the differences in the interpretation of early medieval graves of the discussed Norman or Ruthenian attribution, derivatives of different theoretical and cognitive paradigms, successive generations of researchers are united in their conviction of the exceptional nature of these graves in the Oder-Vistula interfluve. Archaeologists are also unanimous in treating them as burials from the 2nd half of the 10th to the 1st half of the 11th century which present a set of specific burial practices, distinguishing one of the groups of the secular elite of the first Piast state. However, researchers have different views on ethno-cultural valorisation of the discovered objects. In this case, the author maintains his earlier opinion that the burial practices of the individuals buried there are ‘rooted’ in the Scandinavian model of funerary culture of the Viking period. However, in the research process, it cannot be a sufficient premise for prejudging the ethnos. In order to give credibility to the conclusions, in line with the modern model of integrated multidisciplinary research, the hypotheses formulated by archaeology require verification by the results of molecular and isotopic studies of human bones. Limiting the ethnic study of archaeology’s necropolis sources to the indications of bio- and geochemistry is cognitively inadequate, as aDNA analyses only establish biological affinities. Isotopic studies determine the relationship of a deceased person to a specific geographical region, in both cases providing no insight into important aspects of early medieval human life like the mentality-determining membership of a socially and politically organised religious and mythic-ethnic-cultural community.

The Evolution of Diagnostic Techniques in the Paleopathology of Tuberculosis: A Scoping Review.

Tuberculosis (TB) is an ancient chronic infectious disease that remains a global health concern. In human remains, the most common and characteristic clinical signs are the skeletal modifications involving the spine, such as in Pott's disease. Diagnosing TB in ancient human remains is challenging. Therefore, in this systematic review, the authors investigated the studies assessing molecular diagnosis of Pott's disease in ancient human remains with the intention to survey the literature, map the evidence, and identify gaps and future perspectives on TB in paleopathology. Our systematic review offers a full contextualization of the history of Pott's disease in ancient times. Our search strategy was performed between August 2022 and March 2023. The authors initially identified 340 records, and 74 studies were finally included and assessed for qualitative analysis. Due to non-specific clinical signs associated with TB, how best to diagnose tuberculosis in human remains still represents a central point. Nevertheless, ancient DNA (aDNA) analysis, lipid biomarkers, and spoligotyping might be extremely useful tools in the study of TB in human remains. Moreover, we propose the extraction and study of immune response genes involved in innate and adaptive immunity versus Mycobacterium spp. as an innovative and vastly overlooked approach in TB paleopathology. Complementary methodologies should be integrated to provide the best approach to the study of TB in human remains.

Skeletal remains of human perinatal individuals from the fortified Iberian Period settlement of Ca n’Oliver (6th century to 50 years BCE)

Burial customs in the Iberian Period (Iron Age II) included cremation. Only perinatal and newborn infants were buried directly beneath floor settlement. These infants represent the very few unburned human remains recovered from Iberian sites. The interpretation of these infant burials is in debate, focusing on whether they are unnatural or natural deaths. Our aim is to infer mortality patterns and developmental conditions of these individuals, in order to respond if infanticide was present in these assemblages. A large perinatal human skeletal sample from the Ca n’Oliver site (sixth century to 50 years BCE) from the Iberian Period of the northeast of the Iberian Peninsula was analysed, combining osteological methods together with tooth histology and aDNA analysis. Combining osteological and odontological estimates indicated ages between 22 and 42 weeks of gestation for 47 out of a total of 48 individuals. The remaining individual died at about 6 months after birth. Tooth height and enamel histology indicated in 9 out of a subgroup of 13 individuals a low probability of live birth. The remaining 4 individuals possibly survived birth for less than 2 months. According to morphological and molecular results, the sex ratio of this sample is approximately 1:1 male to female. The mortality distribution is consistent with natural mortality. These perinatal deaths were probably spontaneous abortions and neonatal deaths, reflecting an endogenous mortality profile due to genetic and maternal influences. The present study will serve to broaden our knowledge on perinatal individuals of the Iberian Period.

Mortuary palisades, single graves and cultural admixture: The establishment of Corded Ware culture on the Jutland Peninsula

Abstract The emergence and nature of the supra-regional Corded Ware complex in the 3rd millennium BCE is a longstanding, classical question in European archaeology. While recent genomic and genome-wide aDNA analyses have shown that migration was part of the process by which this phenomenon spread across a vast part of Central and northern Europe, the archaeological record of the same area makes equally clear that the character of its more specific regional manifestations was not uniform. This combination calls for renewed regional studies aimed at understanding the pre-existing contexts, the situated processes and the variable outcomes of this new cultural formation, taking a contribution of migration as a starting premise rather than an explanatory end goal. This article presents such a study, focusing on a particular aspect of the so-called Single Grave burial custom on the Jutland Peninsula, which constitutes the most obvious element of Corded Ware culture in the region. Dating to the early part of this tradition (c. 2850–2600 BCE), circular arrangements of wooden posts or planks lodged vertically in a ditch to form a circular palisade are frequently found encircling a contemporaneous human grave. Here, we provide the first systematic study of these structures, which we argue are most meaningfully referred to as mortuary palisades. We present their distribution, chronology and basic morphology as well as their association with graves and relation to burial mounds, leading to a discussion of their probable ritual function in connection with a particular, processual understanding and handling of death. Based on this general presentation, we then analyse geographical variation in the manifestation of the mortuary palisade custom and discuss potentially underlying causes, emphasizing widely differing degrees of cultural admixture of Corded Ware culture and local, pre-existing traditions and preferences in different parts of the Jutland Peninsula.

Exchanging genes and goods: How ancient DNA can potentially illuminate complex mobility patterns in the Black Sea region

Ancient DNA (aDNA) has emerged as a groundbreaking tool in the field of archaeology, providing insights into the movement of individuals and their goods. The field combines genetics, anthropology, and archaeology to reconstruct the intricate complexities of human history. Here, we discuss how ancient DNA analysis has the potential to enhance our understanding of historical mobility patterns in the Black Sea region, specifically in Dobrogea. We also offer broad guidelines and optimal practices that researchers should consider following when working with skeletal collections if they are interested in performing aDNA analyses. As aDNA methods advance and become more refined, we anticipate that our ability to unravel the intricate interplay between human mobility and cultural exchange in the past will also improve.

Materialising Descent: Lineage Formation and Transformation in Early Neolithic Southern Britain

This paper builds on the recent aDNA results from Hazleton North chambered tomb to explore how people might have repeatedly negotiated kinship, descent, and affinity in Early Neolithic southern Britain. Hazleton North was constructed around 3700 cal bc, was in use for less than a century, and – unlike many other Cotswold-Severn tombs – was never modified to alter the arrangement of chambers. The aDNA analysis from 35 individuals whose remains were deposited at the site revealed that 27 were biologically related and represented five sequential generations. Here we explore changing practices across those generations. We argue that Hazleton North was constructed to demonstrate the vitality of a lineage at a specific moment in time while choices about who to entomb indicate an inclusive expansion of the lineage in the first two generations which is not evident during the remaining generations. We argue that by the third generation lineage members increasingly chose to dispose of the remains of their dead elsewhere. Hazleton North was built in a landscape rich in earlier tombs, many of which were modified to produce long cairns with multiple chambers: some of those formed opposed pairs similar to the chambered areas at Hazleton North. We argue this was part of a growing trend in ‘kinship work’ which accentuated lineal descent and sub-lineage distinctions in the centuries around 3700 cal bc. However, deposition at Hazleton North was short-lived. This can be set in the local context of not only the construction and use of further chambered tombs but also increasing investment in larger corporate projects like causewayed enclosures. These enclosures formed new arenas where negotiations of descent and community were played out with increased intensity and in different ways to activities at chambered tombs. Overall, we argue that kinship, affiliation, and belonging were repeatedly renegotiated among the monument building communities of Early Neolithic southern Britain.

Bioarchaeological analyses reveal long-lasting continuity at the periphery of the Late Antique Roman Empire

The Basel-Waisenhaus burial community (Switzerland) has been traditionally interpreted as immigrated Alamans because of the location and dating of the burial ground- despite the typical late Roman funeral practices. To evaluate this hypothesis, multi-isotope and aDNA analyses were conducted on the eleven individuals buried there. The results show that the burial ground was occupied around AD 400 by people belonging largely to one family, whereas isotope and genetic records most probably point toward a regionally organized and indigenous, instead of an immigrated, community. This strengthens the recently advanced assumption that the withdrawal of the Upper Germanic-Rhaetian limes after the "Crisis of the Third Century AD" was not necessarily related to a replacement of the local population by immigrated Alamannic peoples, suggesting a long-lasting continuity of occupation at the Roman periphery at the Upper and High Rhine region.

Integration of direct radiocarbon dating, genetic studies and taxonomy of small mammals to investigate the chronology of past climatic oscillations: The Last Glacial Maximum sequence of Grotta della Ferrovia (Fabriano, Italy)

The recent developments in palaeoecological reconstruction methods, collagen extraction of small bone samples and ancient DNA analyses led us to test new approaches to enhance the chronological resolution of past climate reconstructions inferred from small mammal assemblages.Grotta della Ferrovia (Fabriano, Ancona, Italy, 40°25′36″N, 13°0′11″E, 215 m a.s.l.) is a small cave that opens on the southern bank of the Esino river, around 45 km west of Ancona. Discovered in 1966, it is famous among scholars because it is the first locality where the fossil birch mouse was recorded in Italy and because of the remarkable archaeological lithic evidence assemblage found in the cave related to the Late Epigravettian. Across the small mammal sequence, distributed into seven layers (from GDF7 to GDF2), two main phases can be recognized: in the first phase, from GDF7 to GDF5, Microtus arvalis is dominant over Microtus agrestis, in a poorly diversified assemblage. The sequence gradually changes in GDF4 and a second phase can be observed in GDF3 and GDF2, where Apodemus gr. sylvaticus-flavicollis dominates, but combined with a higher number of taxa. From GDF7 to GDF3, few individuals of Sicista cf. subtilis and Alexandromys oeconomus are recorded. Environmental and climatic reconstructions calculated with the Habitat Weighting and the Bioclimatic methods show a dominance of open meadows (GDF7 to GDF4) that were gradually replaced by closed or semi-closed forests (GDF3 and GDF2) in a context of gradually increasing temperature, shifting from a cold phase to conditions similar to the current ones in the area.Direct radiocarbon dates on 18 rodent bones and aDNA analyses on three Microtus arvalis teeth helped us to better understand the significance of the oscillation inferred from the small mammal sequences. A robust series of nine radiocarbon measurements from GDF7 to GDF5 shows that this part of the sequence accumulated between ∼24,600–19,600 cal BP (95.4% probability), which includes the final phases of the Last Glacial Maximum (LGM). The ancient DNA from Microtus arvalis confirmed the chronological framework and the presence of italic populations in this area during the LGM, similar to the slightly more ancient Grotta del Sambuco on the western side of the central Peninsula. The small mammal radiocarbon dates from GDF4 and above are less consistent with their stratigraphic position, probably related to post-depositional disturbance of the sediments, but suggest that the last part of the sequence accumulated during the Late Glacial.Our results show that it is now possible to validate and enhance paleoclimatic and paleoenvironment inferences from small mammal sequence studies with direct radiocarbon dates and ancient DNA analyses, using these integrated methods as a high resolution tool for studying the past.

Ancient DNA Contradicts the Presence of Social Voles (Genus Microtus, Subgenus Sumeriomys) in the Late Pleistocene of Western Europe

Taxonomic decisions made by palaeontologists are often based on a few morphological features preserved in the fossil material. This practice may sometimes lead to the description of new species based on single specimens, which are, in fact, extreme or aberrant morphological variants of known taxa. Ancient DNA (aDNA) analysis of the Late Pleistocene specimens from the archaeological site Petits Guinards (Creuzier-la-Vieux, Allier, France), described as a new vole Microtus (Sumeriomys) bifrons, did not confirm the species distinctness of the studied population. The genetically examined specimens belonged to Stenocranius anglicus and/or Microtus arvalis, the dominant species at the site. Our findings show that it is risky to describe new fossil taxa on the basis of phenotypic outliers or morphologically aberrant, rare specimens that do not fall within the previously known population variability. We also highlight the importance of ancient DNA in resolving taxonomic and nomenclature problems and classifying fossil mammals of the Late Pleistocene age.

Canine dimensions for estimation of sex in adult and non-adult individuals with external validation by aDNA.

Using discriminant functions obtained from canine dimensions for sex estimation in human skeletons has frequently been proposed as a promising approach within assemblages, even when used in non-adult individuals. However, applicability of this method to adult and non-adult individuals from other assemblages was rarely investigated, probably due to frequently observed inter-population differences in tooth dimensions. In the present study, discriminant functions obtained for permanent canine dimensions at the cemento-enamel junction in a previous study of the early medieval assemblage from Greding, were applied to individuals from a late medieval Jewish cemetery at Erfurt, Germany. The results were validated by aDNA analyses. Prior to the application of the functions, canine dimensions of the assemblages were compared. The comparison showed largely corresponding canine dimensions between the two assemblages. The application of the formulae obtained on the early medieval assemblage to the late medieval assemblage at Erfurt revealed a 100 % correct classification rate in the adult individuals. In non-adults, the correct classification rate was poorer, with 7 of 9 (77.8 %) individuals correctly classified. The study showed that the application of discriminant functions for sex estimation from canine measurements to assemblages other than those for which the functions were developed can lead to high correct classification rates in adults if the average canine dimensions are similar in the respective assemblages. An application to non-adult individuals should only be made with caution as canine dimensions in the "non-survivors" can lead to an over-estimation of the proportion of female non-adults.

Equine exploitation at Pompeii (AD 79)

Equids were an important component of Roman everyday life. Horses, donkeys and their hybrids (mule and hinny) have been used as pack animals for moving people and goods through time. The most prestigious horse breeds were often associated with the social status of the owner as some equestrian statues and written sources seem to suggest. However, management and exploitation of this taxon at that time is still largely unknown.This paper aims to investigate the human-equid relationship at Pompeii and to link the results to everyday life in a Roman town in the 1st century CE. Equid remains from several stables associated with urban houses and shops, as well as a rural villa have been subject to morphological, biometric, and ancient DNA (aDNA) analysis in order to enable their identification. The pathological evidence on the bones combined with ageing data has provided indications about the nature of the exploitation of these animals. Data on the fodder likely consumed by them were also obtained using previous studies to provide further hints about their management.The results indicate that equid individuals ranging from 4 to 8 years old were preferred at Pompeii. Horses were largely employed in a number of activities inside the town as well as in the countryside. This study, focused on the analysis of equids collected from the Pompeii stables, provides important information about the exploitation and management of this taxon in the Roman Imperial period.

Kinship practices in Early Iron Age South-east Europe: genetic and isotopic analysis of burials from the Dolge njive barrow cemetery, Dolenjska, Slovenia

The burial of multiple individuals within a single funerary monument invites speculation about the relationships between the deceased: were they chosen on the basis of status, gender or relatedness, for example? Here, the authors present the results of aDNA and isotope analyses conducted on seven individuals from an Early Iron Age barrow at Dolge njive, south-eastern Slovenia. All seven individuals are close biological relatives. While the group composition suggests strict adherence to neither patrilineal nor matrilineal structures, the funerary tradition appears highly gendered, and family links through both the male and female lines seem important in structuring of the community. The results have implications for understanding of kinship and funerary practices in late prehistoric Europe.