Palaeogenomics of Upper Palaeolithic to Neolithic European hunter-gatherers.

Modern humans have populated Europe for more than 45,000 years1,2. Our knowledge of the genetic relatedness and structure of ancient hunter-gatherers is however limited, owing to the scarceness and poor molecular preservation of human remains from that period3. Here we analyse 356 ancient hunter-gatherer genomes, including new genomic data for 116 individuals from 14 countries in western and central Eurasia, spanning between 35,000 and 5,000 years ago. We identify a genetic ancestry profile in individuals associated with Upper Palaeolithic Gravettian assemblages from western Europe that is distinct from contemporaneous groups related to this archaeological culture in central and southern Europe4, but resembles that of preceding individuals associated with the Aurignacian culture. This ancestry profile survived during the Last Glacial Maximum (25,000 to 19,000 years ago) in human populations from southwestern Europe associated with the Solutrean culture, and with the following Magdalenian culture that re-expanded northeastward after the Last Glacial Maximum. Conversely, we reveal a genetic turnover in southern Europe suggesting a local replacement of human groups around the time of the Last Glacial Maximum, accompanied by a north-to-south dispersal of populations associated with the Epigravettian culture. From at least 14,000 years ago, an ancestry related to this culture spread from the south across the rest of Europe, largely replacing the Magdalenian-associated gene pool. After a period of limited admixture that spanned the beginning of the Mesolithic, we find genetic interactions between western and eastern European hunter-gatherers, who were also characterized by marked differences in phenotypically relevant variants.

The role of emerging elites in the formation and development of communities after the fall of the Roman Empire.

Elites played a pivotal role in the formation of post-Roman Europe on both macro- and microlevels during the Early Medieval period. History and archaeology have long focused on their description and identification based on written sources or through their archaeological record. We provide a different perspective on this topic by integrating paleogenomic, archaeological, and isotopic data to gain insights into the role of one such elite group in a Langobard period community near Collegno, Italy dated to the 6-8th centuries CE. Our analysis of 28 newly sequenced genomes together with 24 previously published ones combined with isotope (Sr, C, N) measurements revealed that this community was established by and organized around a network of biologically and socially related individuals likely composed of multiple elite families that over time developed into a single extended pedigree. The community also included individuals with diverse genetic ancestries, maintaining its diversity by integrating newcomers and groups in later stages of its existence. This study highlights how shifts in political power and migration impacted the formation and development of a small rural community within a key region of the former Western Roman Empire after its dissolution and the emergence of a new kingdom. Furthermore, it suggests that Early Medieval elites had the capacity to incorporate individuals from varied backgrounds and that these elites were the result of (political) agency rather than belonging to biologically homogeneous groups.

The diverse genetic origins of a Classical period Greek army.

Trade and colonization caused an unprecedented increase in Mediterranean human mobility in the first millennium BCE. Often seen as a dividing force, warfare is in fact another catalyst of culture contact. We provide insight into the demographic dynamics of ancient warfare by reporting genome-wide data from fifth-century soldiers who fought for the army of the Greek Sicilian colony of Himera, along with representatives of the civilian population, nearby indigenous settlements, and 96 present-day individuals from Italy and Greece. Unlike the rest of the sample, many soldiers had ancestral origins in northern Europe, the Steppe, and the Caucasus. Integrating genetic, archaeological, isotopic, and historical data, these results illustrate the significant role mercenaries played in ancient Greek armies and highlight how participation in war contributed to continental-scale human mobility in the Classical world.

The genomic history of southeastern Europe.

Farming was first introduced to Europe in the mid-seventh millennium bc, and was associated with migrants from Anatolia who settled in the southeast before spreading throughout Europe. Here, to understand the dynamics of this process, we analysed genome-wide ancient DNA data from 225 individuals who lived in southeastern Europe and surrounding regions between 12000 and 500 bc. We document a west-east cline of ancestry in indigenous hunter-gatherers and, in eastern Europe, the early stages in the formation of Bronze Age steppe ancestry. We show that the first farmers of northern and western Europe dispersed through southeastern Europe with limited hunter-gatherer admixture, but that some early groups in the southeast mixed extensively with hunter-gatherers without the sex-biased admixture that prevailed later in the north and west. We also show that southeastern Europe continued to be a nexus between east and west after the arrival of farmers, with intermittent genetic contact with steppe populations occurring up to 2,000 years earlier than the migrations from the steppe that ultimately replaced much of the population of northern Europe.

More data on ancient human mitogenome variability in Italy: new mitochondrial genome sequences from three Upper Palaeolithic burials.

BACKGROUND: Recently, the study of mitochondrial variability in ancient humans has allowed the definition of population dynamics that characterised Europe in the Late Pleistocene and Early Holocene. Despite the abundance of sites and skeletal remains few data are available for Italy. AIM: We reconstructed the mitochondrial genomes of three Upper Palaeolithic individuals for some of the most important Italian archaeological contexts: Paglicci (South-Eastern Italy), San Teodoro (South-Western Italy) and Arene Candide (North-Western Italy) caves. SUBJECTS AND METHODS: We explored the phylogenetic relationships of the three mitogenomes in the context of Western Eurasian ancient and modern variability. RESULTS: Paglicci 12 belongs to sub-haplogroup U8c, described in only two other Gravettian individuals; San Teodoro 2 harbours a U2'3'4'7'8'9 sequence, the only lineage found in Sicily during the Late Pleistocene and Early Holocene; Arene Candide 16 displays an ancestral U5b1 haplotype already detected in other Late Pleistocene hunter-gatherers from Central Europe. CONCLUSION: Regional genetic continuity is highlighted in the Gravettian groups that succeeded in Paglicci. Data from one of the oldest human remains from Sicily reinforce the hypothesis that Epigravettian groups carrying U2'3'4'7'8'9 could be the first inhabitants of the island. The first pre-Neolithic mitogenome from North-Western Italy, sequenced here, shows more affinity with continental Europe than with the Italian peninsula.

The female ancestor's tale: Long-term matrilineal continuity in a nonisolated region of Tuscany.

OBJECTIVES: With the advent of ancient DNA analyses, it has been possible to disentangle the contribution of ancient populations to the genetic pool of the modern inhabitants of many regions. Reconstructing the maternal ancestry has often highlighted genetic continuity over several millennia, but almost always in isolated areas. Here we analyze North-western Tuscany, a region that was a corridor of exchanges between Central Italy and the Western Mediterranean coast. MATERIALS AND METHODS: We newly obtained mitochondrial HVRI sequences from 28 individuals, and after gathering published data, we collected genetic information for 119 individuals from the region. Those span five periods during the last 5,000 years: Prehistory, Etruscan age, Roman age, Renaissance, and Present-day. We used serial coalescent simulations in an approximate Bayesian computation framework to test for continuity between the mentioned groups. RESULTS: Our analyses always favor continuity over discontinuity for all groups considered, with the Etruscans being part of the genealogy. Moreover, the posterior distributions of the parameters support very small female effective population sizes. CONCLUSIONS: The observed signals of long-term genetic continuity and isolation are in contrast with the history of the region, conquered several times (Etruscans, Romans, Lombards, and French). While the Etruscans appear as a local population, intermediate between the prehistoric and the other samples, we suggest that the other conquerors-arriving from far-had a consistent social or sex bias, hence only marginally affecting the maternal lineages. At the same time, our results show that long-term genealogical continuity is not necessarily linked to geographical isolation.

Genome diversity in the Neolithic Globular Amphorae culture and the spread of Indo-European languages.

It is unclear whether Indo-European languages in Europe spread from the Pontic steppes in the late Neolithic, or from Anatolia in the Early Neolithic. Under the former hypothesis, people of the Globular Amphorae culture (GAC) would be descended from Eastern ancestors, likely representing the Yamnaya culture. However, nuclear (six individuals typed for 597 573 SNPs) and mitochondrial (11 complete sequences) DNA from the GAC appear closer to those of earlier Neolithic groups than to the DNA of all other populations related to the Pontic steppe migration. Explicit comparisons of alternative demographic models via approximate Bayesian computation confirmed this pattern. These results are not in contrast to Late Neolithic gene flow from the Pontic steppes into Central Europe. However, they add nuance to this model, showing that the eastern affinities of the GAC in the archaeological record reflect cultural influences from other groups from the East, rather than the movement of people.

The Neanderthal in the karst: First dating, morphometric, and paleogenetic data on the fossil skeleton from Altamura (Italy).

In 1993, a fossil hominin skeleton was discovered in the karst caves of Lamalunga, near Altamura, in southern Italy. Despite the fact that this specimen represents one of the most extraordinary hominin specimens ever found in Europe, for the last two decades our knowledge of it has been based purely on the documented on-site observations. Recently, the retrieval from the cave of a fragment of bone (part of the right scapula) allowed the first dating of the individual, the quantitative analysis of a diagnostic morphological feature, and a preliminary paleogenetic characterization of this hominin skeleton from Altamura. Overall, the results concur in indicating that it belongs to the hypodigm of Homo neanderthalensis, with some phenetic peculiarities that appear consistent with a chronology ranging from 172 ± 15 ka to 130.1 ± 1.9 ka. Thus, the skeleton from Altamura represents the most ancient Neanderthal from which endogenous DNA has ever been extracted.

Life history and ancestry of the late Upper Palaeolithic infant from Grotta delle Mura, Italy.

The biological aspects of infancy within late Upper Palaeolithic populations and the role of southern refugia at the end of the Last Glacial Maximum are not yet fully understood. This study presents a multidisciplinary, high temporal resolution investigation of an Upper Palaeolithic infant from Grotta delle Mura (Apulia, southern Italy) combining palaeogenomics, dental palaeohistology, spatially-resolved geochemical analyses, direct radiocarbon dating, and traditional anthropological studies. The skeletal remains of the infant - Le Mura 1 - were directly dated to 17,320-16,910 cal BP. The results portray a biological history of the infant's development, early life, health and death (estimated at ~72 weeks). They identify, several phenotypic traits and a potential congenital disease in the infant, the mother's low mobility during gestation, and a high level of endogamy. Furthermore, the genomic data indicates an early spread of the Villabruna-like components along the Italian peninsula, confirming a population turnover around the time of the Last Glacial Maximum, and highlighting a general reduction in genetic variability from northern to southern Italy. Overall, Le Mura 1 contributes to our better understanding of the early stages of life and the genetic puzzle in the Italian peninsula at the end of the Last Glacial Maximum.

Fine-scale sampling uncovers the complexity of migrations in 5th-6th century Pannonia.

As the collapse of the Western Roman Empire accelerated during the 4th and 5th centuries, arriving "barbarian" groups began to establish new communities in the border provinces of the declining (and eventually former) empire. This was a time of significant cultural and political change throughout not only these border regions but Europe as a whole.1,2 To better understand post-Roman community formation in one of these key frontier zones after the collapse of the Hunnic movement, we generated new paleogenomic data for a set of 38 burials from a time series of three 5th century cemeteries3,4,5 at Lake Balaton, Hungary. We utilized a comprehensive sampling approach to characterize these cemeteries along with data from 38 additional burials from a previously published mid-6th century site6 and analyzed them alongside data from over 550 penecontemporaneous individuals.7,8,9,10,11,12,13,14,15,16,17,18,19 The range of genetic diversity in all four of these local burial communities is extensive and wider ranging than penecontemporaneous Europeans sequenced to date. Despite many commonalities in burial customs and demography, we find that there were substantial differences in genetic ancestry between the sites. We detect evidence of northern European gene flow into the Lake Balaton region. Additionally, we observe a statistically significant association between dress artifacts and genetic ancestry among 5th century genetically female burials. Our analysis shows that the formation of early Medieval communities was a multifarious process even at a local level, consisting of genetically heterogeneous groups.

Genetic structure and differentiation from early bronze age in the mediterranean island of sicily: Insights from ancient mitochondrial genomes.

Sicily is one of the main islands of the Mediterranean Sea, and it is characterized by a variety of archaeological records, material culture and traditions, reflecting the history of migrations and populations' interaction since its first colonization, during the Paleolithic. These deep and complex demographic and cultural dynamics should have affected the genomic landscape of Sicily at different levels; however, the relative impact of these migrations on the genomic structure and differentiation within the island remains largely unknown. The available Sicilian modern genetic data gave a picture of the current genetic structure, but the paucity of ancient data did not allow so far to make predictions about the level of historical variation. In this work, we sequenced and analyzed the complete mitochondrial genomes of 36 individuals from five different locations in Sicily, spanning from Early Bronze Age to Iron Age, and with different cultural backgrounds. The comparison with coeval groups from the Mediterranean Basin highlighted structured genetic variation in Sicily since Early Bronze Age, thus supporting a demic impact of the cultural transitions within the Island. Explicit model testing through Approximate Bayesian Computation allowed us to make predictions about the origin of Sicanians, one of the three indigenous peoples of Sicily, whose foreign origin from Spain, historically attributed, was not confirmed by our analysis of genetic data. Sicilian modern mitochondrial data show a different, more homogeneous, genetic composition, calling for a recent genetic replacement in the Island of pre-Iron Age populations, that should be further investigated.

Performance of innovative nanomaterials for bone remains consolidation and effect on 14C dating and on palaeogenetic analysis.

An innovative protocol for the consolidation of ancient bone remains based on the use of nanometric HydroxyAPatite (HAP) was set up and tested through a multidisciplinary approach. A new protocol for the synthesis of HAP nanoparticles was developed, and the composition of the obtained nanomaterial was investigated through Fourier Transform Infrared Spectroscopy (FTIR) and X-Ray Diffraction (XRD); sizes, shape and morphology of the synthesized particles were studied by Scanning Electron Microscopy (SEM). The consolidation performance was evaluated by testing the new nanomaterial on degraded ancient bone findings. An increase of the mineral density and of the micro-hardness of the bone were observed. The new consolidation method was also tested to assess possible effects on the palaeogenetic analysis and radiocarbon dating on the treated bones. The consolidation treatment does not introduce any contaminations that could affect radiocarbon dating and has no general detrimental impact on the genetic characterization of the skeletal remains. This consolidation procedure represents a more compatible conservation tool with respect to traditional procedures: it has been shown that the treatment is effective, easily-applicable and compatible with post-consolidation analysis.

The Illumina Sequencing Protocol and the NovaSeq 6000 System.

The NovaSeq 6000 is a sequencing platform from Illumina that enables the sequencing of short reads with an output up to 6 Tb. The NovaSeq 6000 uses the typical Illumina sequencing workflow based on library preparation, cluster generation by in situ amplification, and sequencing by synthesis. Flexibility is one of the major features of the NovaSeq 6000. Several types of sequencing kits coupled with dual flow cell mode enable high scalability of sequencing outputs to match a wide range of applications from complete genome sequencing to metagenomics analysis. In this chapter, after explaining how to assemble a normalized pool of libraries for sequencing, we will describe the experimental steps required to run the pools on the NovaSeq 6000 platform.

Ancient and Archaic Genomes.

The first data obtained from ancient DNA samples were published more than thirty years ago [...].

New Insights Into Mitochondrial DNA Reconstruction and Variant Detection in Ancient Samples.

Ancient DNA (aDNA) studies are frequently focused on the analysis of the mitochondrial DNA (mtDNA), which is much more abundant than the nuclear genome, hence can be better retrieved from ancient remains. However, postmortem DNA damage and contamination make the data analysis difficult because of DNA fragmentation and nucleotide alterations. In this regard, the assessment of the heteroplasmic fraction in ancient mtDNA has always been considered an unachievable goal due to the complexity in distinguishing true endogenous variants from artifacts. We implemented and applied a computational pipeline for mtDNA analysis to a dataset of 30 ancient human samples from an Iron Age necropolis in Polizzello (Sicily, Italy). The pipeline includes several modules from well-established tools for aDNA analysis and a recently released variant caller, which was specifically conceived for mtDNA, applied for the first time to aDNA data. Through a fine-tuned filtering on variant allele sequencing features, we were able to accurately reconstruct nearly complete (>88%) mtDNA genome for almost all the analyzed samples (27 out of 30), depending on the degree of preservation and the sequencing throughput, and to get a reliable set of variants allowing haplogroup prediction. Additionally, we provide guidelines to deal with possible artifact sources, including nuclear mitochondrial sequence (NumtS) contamination, an often-neglected issue in ancient mtDNA surveys. Potential heteroplasmy levels were also estimated, although most variants were likely homoplasmic, and validated by data simulations, proving that new sequencing technologies and software are sensitive enough to detect partially mutated sites in ancient genomes and discriminate true variants from artifacts. A thorough functional annotation of detected and filtered mtDNA variants was also performed for a comprehensive evaluation of these ancient samples.

How a Paleogenomic Approach Can Provide Details on Bioarchaeological Reconstruction: A Case Study from the Globular Amphorae Culture.

Ancient human remains have the potential to explain a great deal about the prehistory of humankind. Due to recent technological and bioinformatics advances, their study, at the palaeogenomic level, can provide important information about population dynamics, culture changes, and the lifestyles of our ancestors. In this study, mitochondrial and nuclear genome data obtained from human bone remains associated with the Neolithic Globular Amphorae culture, which were recovered in the Megalithic barrow of Kierzkowo (Poland), were reanalysed to gain insight into the social organisation and use of the archaeological site and to provide information at the individual level. We were able to successfully estimate the minimum number of individuals, sex, kin relationships, and phenotypic traits of the buried individuals, despite the low level of preservation of the bone samples and the intricate taphonomic conditions. In addition, the evaluation of damage patterns allowed us to highlight the presence of "intruders"-that is, of more recent skeletal remains that did not belong to the original burial. Due to its characteristics, the study of the Kierzkowo barrow represented a challenge for the reconstruction of the biological profile of the human community who exploited it and an excellent example of the contribution that ancient genomic analysis can provide to archaeological reconstruction.

Successful extraction of insect DNA from recent copal inclusions: limits and perspectives.

Insects entombed in copal, the sub-fossilized resin precursor of amber, represent a potential source of genetic data for extinct and extant, but endangered or elusive, species. Despite several studies demonstrated that it is not possible to recover endogenous DNA from insect inclusions, the preservation of biomolecules in fossilized resins samples is still under debate. In this study, we tested the possibility of obtaining endogenous ancient DNA (aDNA) molecules from insects preserved in copal, applying experimental protocols specifically designed for aDNA recovery. We were able to extract endogenous DNA molecules from one of the two samples analyzed, and to identify the taxonomic status of the specimen. Even if the sample was found well protected from external contaminants, the recovered DNA was low concentrated and extremely degraded, compared to the sample age. We conclude that it is possible to obtain genomic data from resin-entombed organisms, although we discourage aDNA analysis because of the destructive method of extraction protocols and the non-reproducibility of the results.

Ancient genomes reveal early Andean farmers selected common beans while preserving diversity.

All crops are the product of a domestication process that started less than 12,000 years ago from one or more wild populations1,2. Farmers selected desirable phenotypic traits (such as improved energy accumulation, palatability of seeds and reduced natural shattering3) while leading domesticated populations through several more or less gradual demographic contractions2,4. As a consequence, the erosion of wild genetic variation5 is typical of modern cultivars, making them highly susceptible to pathogens, pests and environmental change6,7. The loss of genetic diversity hampers further crop improvement programmes to increase food production in a changing world, posing serious threats to food security8,9. Using both ancient and modern seeds, we analysed the temporal dynamics of genetic variation and selection during the domestication process of the common bean (Phaseolus vulgaris) in the southern Andes. Here, we show that most domestic traits were selected for before 2,500 years ago, with no or only minor loss of whole-genome heterozygosity. In fact, most of the changes at coding genes and linked regions that differentiate wild and domestic genomes are already present in the ancient genomes analysed here, and all ancient domestic genomes dated between 600 and 2,500 years ago are highly variable (at least as variable as modern genomes from the wild). Single seeds from modern cultivars show reduced variation when compared with ancient seeds, indicating that intensive selection within cultivars in the past few centuries probably partitioned ancestral variation within different genetically homogenous cultivars. When cultivars from different Andean regions are pooled, the genomic variation of the pool is higher than that observed in the pool of ancient seeds from north and central western Argentina. Considering that most desirable phenotypic traits are probably controlled by multiple polymorphic genes10, a plausible explanation of this decoupling of selection and genetic erosion is that early farmers applied a relatively weak selection pressure2 by using many phenotypically similar but genetically diverse individuals as parents. Our results imply that selection strategies during the past few centuries, as compared with earlier times, more intensively reduced genetic variation within cultivars and produced further improvements by focusing on a few plants carrying the traits of interest, at the cost of marked genetic erosion within Andean landraces.

The origin and legacy of the Etruscans through a 2000-year archeogenomic time transect.

The origin, development, and legacy of the enigmatic Etruscan civilization from the central region of the Italian peninsula known as Etruria have been debated for centuries. Here we report a genomic time transect of 82 individuals spanning almost two millennia (800 BCE to 1000 CE) across Etruria and southern Italy. During the Iron Age, we detect a component of Indo-European­associated steppe ancestry and the lack of recent Anatolian-related admixture among the putative non­Indo-European­speaking Etruscans. Despite comprising diverse individuals of central European, northern African, and Near Eastern ancestry, the local gene pool is largely maintained across the first millennium BCE. This drastically changes during the Roman Imperial period where we report an abrupt population-wide shift to ~50% admixture with eastern Mediterranean ancestry. Last, we identify northern European components appearing in central Italy during the Early Middle Ages, which thus formed the genetic landscape of present-day Italian populations.