Archaeogenomic distinctiveness of the Isthmo-Colombian area.

The recently enriched genomic history of Indigenous groups in the Americas is still meager concerning continental Central America. Here, we report ten pre-Hispanic (plus two early colonial) genomes and 84 genome-wide profiles from seven groups presently living in Panama. Our analyses reveal that pre-Hispanic demographic events contributed to the extensive genetic structure currently seen in the area, which is also characterized by a distinctive Isthmo-Colombian Indigenous component. This component drives these populations on a specific variability axis and derives from the local admixture of different ancestries of northern North American origin(s). Two of these ancestries were differentially associated to Pleistocene Indigenous groups that also moved into South America, leaving heterogenous genetic footprints. An additional Pleistocene ancestry was brought by a still unsampled population of the Isthmus (UPopI) that remained restricted to the Isthmian area, expanded locally during the early Holocene, and left genomic traces up to the present day.

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.

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.

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.

Novel Sources of Biodiversity and Biomolecules from Bacteria Isolated from a High Middle Ages Soil Sample in Palermo (Sicily, Italy).

The urban plan of Palermo (Sicily, Italy) has evolved throughout Punic, Roman, Byzantine, Arab, and Norman ages until it stabilized within the borders that correspond to the current historic center. During the 2012 to 2013 excavation campaign, new remains of the Arab settlement, directly implanted above the structures of the Roman age, were found. The materials investigated in this study derived from the so-called Survey No 3, which consists of a rock cavity of subcylindrical shape covered with calcarenite blocks: it was probably used to dispose of garbage during the Arabic age and its content, derived from daily activities, included grape seeds, scales and bones of fish, small animal bones, and charcoals. Radiocarbon dating confirmed the medieval origin of this site. The composition of the bacterial community was characterized through a culture-dependent and a culture-independent approach. Culturable bacteria were isolated under aerobic and anaerobic conditions and the total bacterial community was characterized through metagenomic sequencing. Bacterial isolates were tested for the production of compounds with antibiotic activity: a Streptomyces strain, whose genome was sequenced, was of particular interest because of its inhibitory activity, which was due to the Type I polyketide aureothin. Moreover, all strains were tested for the production of secreted proteases, with those belonging to the genus Nocardioides having the most active enzymes. Finally, protocols commonly used for ancient DNA studies were applied to evaluate the antiquity of isolated bacterial strains. Altogether these results show how paleomicrobiology might represent an innovative and unexplored source of novel biodiversity and new biotechnological tools. IMPORTANCE One of the goals of paleomicrobiology is the characterization of the microbial community present in archaeological sites. These analyses can usually provide valuable information about past events, such as occurrence of human and animal infectious diseases, ancient human activities, and environmental changes. However, in this work, investigations about the composition of the bacterial community of an ancient soil sample (harvested in Palermo, Italy) were carried out aiming to screen ancient culturable strains with biotechnological potential, such as the ability to produce bioactive molecules and secreted hydrolytic enzymes. Besides showing the biotechnological relevance of paleomicrobiology, this work reports a case of germination of putatively ancient bacterial spores recovered from soil rather than extreme environments. Moreover, in the case of spore-forming species, these results raise questions about the accuracy of techniques usually applied to estimate antiquity of DNA, as they could lead to its underestimation.

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.

Ancient oral microbiomes support gradual Neolithic dietary shifts towards agriculture.

The human microbiome has recently become a valuable source of information about host life and health. To date little is known about how it may have evolved during key phases along our history, such as the Neolithic transition towards agriculture. Here, we shed light on the evolution experienced by the oral microbiome during this transition, comparing Palaeolithic hunter-gatherers with Neolithic and Copper Age farmers that populated a same restricted area in Italy. We integrate the analysis of 76 dental calculus oral microbiomes with the dietary information derived from the identification of embedded plant remains. We detect a stronger deviation from the hunter-gatherer microbiome composition in the last part of the Neolithic, while to a lesser extent in the early phases of the transition. Our findings demonstrate that the introduction of agriculture affected host microbiome, supporting the hypothesis of a gradual transition within the investigated populations.

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.

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.

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.

First Bronze Age Human Mitogenomes from Calabria (Grotta Della Monaca, Southern Italy).

The Italian peninsula was host to a strong history of migration processes that shaped its genomic variability since prehistoric times. During the Metal Age, Sicily and Southern Italy were the protagonists of intense trade networks and settlements along the Mediterranean. Nonetheless, ancient DNA studies in Southern Italy are, at present, still limited to prehistoric and Roman Apulia. Here, we present the first mitogenomes from a Middle Bronze Age cave burial in Calabria to address this knowledge gap. We adopted a hybridization capture approach, which enabled the recovery of one complete and one partial mitochondrial genome. Phylogenetic analysis assigned these two individuals to the H1e and H5 subhaplogroups, respectively. This preliminary phylogenetic analysis supports affinities with coeval Sicilian populations, along with Linearbandkeramik and Bell Beaker cultures maternal lineages from Central Europe and Iberia. Our work represents a starting point which contributes to the comprehension of migrations and population dynamics in Southern Italy, and highlights this knowledge gap yet to be filled by genomic studies.

Whole-exome sequencing of the mummified remains of Cangrande della Scala (1291-1329 CE) indicates the first known case of late-onset Pompe disease.

Mummified remains of relevant historical figures are nowadays an important source of information to retrace data concerning their private life and health, especially when historical archives are not available. Next-generation-sequencing was proved to be a valuable tool to unravel the characteristics of these individuals through their genetic heritage. Using the strictest criteria currently available for the validation of ancient DNA sequences, whole-genome and whole-exome sequencing were generated from the mummy remains of an Italian nobleman died almost 700 years ago, Cangrande della Scala. While its genome sequencing could not yield sufficient coverage for in depth investigation, exome sequencing could overcome the limitations of this approach to achieve significantly high coverage on coding regions, thus allowing to perform the first extensive exome analysis of a mummy genome. Similar to a standard "clinical exome analysis" conducted on modern DNA, an in-depth variant annotation, high-quality filtering and interpretation was performed, leading to the identification of a genotype associated with late-onset Pompe disease (glycogen storage disease type II). This genetic diagnosis was concordant with the limited clinical history available for Cangrande della Scala, who likely represents the earliest known case of this autosomal recessive metabolic disorder.

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.