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 first evidence for Late Pleistocene dogs in Italy.

The identification of the earliest dogs is challenging because of the absence and/or mosaic pattern of morphological diagnostic features in the initial phases of the domestication process. Furthermore, the natural occurrence of some of these characters in Late Pleistocene wolf populations and the time it took from the onset of traits related to domestication to their prevalence remain indefinite. For these reasons, the spatiotemporal context of the early domestication of dogs is hotly debated. Our combined molecular and morphological analyses of fossil canid remains from the sites of Grotta Paglicci and Grotta Romanelli, in southern Italy, attest of the presence of dogs at least 14,000 calibrated years before present. This unambiguously documents one of the earliest occurrence of domesticates in the Upper Palaeolithic of Europe and in the Mediterranean. The genetic affinity between the Palaeolithic dogs from southern Italy and contemporaneous ones found in Germany also suggest that these animals were an important common adjunct during the Late Glacial, when strong cultural diversification occurred between the Mediterranean world and European areas north of the Alps. Additionally, aDNA analyses indicate that this Upper Palaeolithic dog lineage from Italy may have contributed to the genetic diversity of living dogs.

Ancient human mitochondrial genomes from Bronze Age Bulgaria: new insights into the genetic history of Thracians.

One of the best documented Indo-European civilizations that inhabited Bulgaria is the Thracians, who lasted for more than five millennia and whose origin and relationships with other past and present-day populations are debated among researchers. Here we report 25 new complete mitochondrial genomes of ancient individuals coming from three necropolises located in different regions of Bulgaria - Shekerdja mogila, Gabrova mogila and Bereketska mogila - dated to II-III millennium BC. The identified mtDNA haplogroup composition reflects the mitochondrial variability of Western Eurasia. In particular, within the ancient Eurasian genetic landscape, Thracians locate in an intermediate position between Early Neolithic farmers and Late Neolithic-Bronze Age steppe pastoralists, supporting the scenario that the Balkan region has been a link between Eastern Europe and the Mediterranean since the prehistoric time. Spatial Principal Component Analysis (sPCA) performed on Thracian and modern mtDNA sequences, confirms the pattern highlighted on ancient populations, overall indicating that the maternal gene pool of Thracians reflects their central geographical position at the gateway of Europe.

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.

Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.

Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.

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.

Mitochondrial DNA from El Mirador cave (Atapuerca, Spain) reveals the heterogeneity of Chalcolithic populations.

Previous mitochondrial DNA analyses on ancient European remains have suggested that the current distribution of haplogroup H was modeled by the expansion of the Bell Beaker culture (ca 4,500-4,050 years BP) out of Iberia during the Chalcolithic period. However, little is known on the genetic composition of contemporaneous Iberian populations that do not carry the archaeological tool kit defining this culture. Here we have retrieved mitochondrial DNA (mtDNA) sequences from 19 individuals from a Chalcolithic sample from El Mirador cave in Spain, dated to 4,760-4,200 years BP and we have analyzed the haplogroup composition in the context of modern and ancient populations. Regarding extant African, Asian and European populations, El Mirador shows affinities with Near Eastern groups. In different analyses with other ancient samples, El Mirador clusters with Middle and Late Neolithic populations from Germany, belonging to the Rössen, the Salzmünde and the Baalberge archaeological cultures but not with contemporaneous Bell Beakers. Our analyses support the existence of a common genetic signal between Western and Central Europe during the Middle and Late Neolithic and points to a heterogeneous genetic landscape among Chalcolithic groups.

Genetic evidence does not support an Etruscan origin in Anatolia.

The debate on the origins of Etruscans, documented in central Italy between the eighth century BC and the first century AD, dates back to antiquity. Herodotus described them as a group of immigrants from Lydia, in Western Anatolia, whereas for Dionysius of Halicarnassus they were an indigenous population. Dionysius' view is shared by most modern archeologists, but the observation of similarities between the (modern) mitochondrial DNAs (mtDNAs) of Turks and Tuscans was interpreted as supporting an Anatolian origin of the Etruscans. However, ancient DNA evidence shows that only some isolates, and not the bulk of the modern Tuscan population, are genetically related to the Etruscans. In this study, we tested alternative models of Etruscan origins by Approximate Bayesian Computation methods, comparing levels of genetic diversity in the mtDNAs of modern and ancient populations with those obtained by millions of computer simulations. The results show that the observed genetic similarities between modern Tuscans and Anatolians cannot be attributed to an immigration wave from the East leading to the onset of the Etruscan culture in Italy. Genetic links between Tuscany and Anatolia do exist, but date back to a remote stage of prehistory, possibly but not necessarily to the spread of farmers during the Neolithic period.

Origins and evolution of the Etruscans' mtDNA.

The Etruscan culture is documented in Etruria, Central Italy, from the 8(th) to the 1(st) century BC. For more than 2,000 years there has been disagreement on the Etruscans' biological origins, whether local or in Anatolia. Genetic affinities with both Tuscan and Anatolian populations have been reported, but so far all attempts have failed to fit the Etruscans' and modern populations in the same genealogy. We extracted and typed the hypervariable region of mitochondrial DNA of 14 individuals buried in two Etruscan necropoleis, analyzing them along with other Etruscan and Medieval samples, and 4,910 contemporary individuals from the Mediterranean basin. Comparing ancient (30 Etruscans, 27 Medieval individuals) and modern DNA sequences (370 Tuscans), with the results of millions of computer simulations, we show that the Etruscans can be considered ancestral, with a high degree of confidence, to the current inhabitants of Casentino and Volterra, but not to the general contemporary population of the former Etruscan homeland. By further considering two Anatolian samples (35 and 123 individuals) we could estimate that the genetic links between Tuscany and Anatolia date back to at least 5,000 years ago, strongly suggesting that the Etruscan culture developed locally, and not as an immediate consequence of immigration from the Eastern Mediterranean shores.

Origin and diet of the prehistoric hunter-gatherers on the mediterranean island of Favignana (Ègadi Islands, Sicily).

Hunter-gatherers living in Europe during the transition from the late Pleistocene to the Holocene intensified food acquisition by broadening the range of resources exploited to include marine taxa. However, little is known on the nature of this dietary change in the Mediterranean Basin. A key area to investigate this issue is the archipelago of the Ègadi Islands, most of which were connected to Sicily until the early Holocene. The site of Grotta d'Oriente, on the present-day island of Favignana, was occupied by hunter-gatherers when Postglacial environmental changes were taking place (14,000-7,500 cal BP). Here we present the results of AMS radiocarbon dating, palaeogenetic and isotopic analyses undertaken on skeletal remains of the humans buried at Grotta d'Oriente. Analyses of the mitochondrial hypervariable first region of individual Oriente B, which belongs to the HV-1 haplogroup, suggest for the first time on genetic grounds that humans living in Sicily during the early Holocene could have originated from groups that migrated from the Italian Peninsula around the Last Glacial Maximum. Carbon and nitrogen isotope analyses show that the Upper Palaeolithic and Mesolithic hunter-gatherers of Favignana consumed almost exclusively protein from terrestrial game and that there was only a slight increase in marine food consumption from the late Pleistocene to the early Holocene. This dietary change was similar in scale to that at sites on mainland Sicily and in the rest of the Mediterranean, suggesting that the hunter-gatherers of Grotta d'Oriente did not modify their subsistence strategies specifically to adapt to the progressive isolation of Favignana. The limited development of technologies for intensively exploiting marine resources was probably a consequence both of Mediterranean oligotrophy and of the small effective population size of these increasingly isolated human groups, which made innovation less likely and prevented transmission of fitness-enhancing adaptations.

Unexpected presence of Fagus orientalis complex in Italy as inferred from 45,000-year-old DNA pollen samples from Venice lagoon.

BACKGROUND: Phylogeographic analyses on the Western Euroasiatic Fagus taxa (F. orientalis, F. sylvatica, F. taurica and F. moesiaca) is available, however, the subdivision of Fagus spp. is unresolved and there is no consensus on the phylogeny and on the identification (both with morphological than molecular markers) of Fagus Eurasiatic taxa. For the first time molecular analyses of ancient pollen, dated at least 45,000 years ago, were used in combination with the phylogeny analysis on current species, to identify the Fagus spp. present during the Last Interglacial period in Italy. In this work we aim at testing if the trnL-trnF chloroplast DNA (cpDNA) region, that has been previously proved efficient in discriminating different Quercus taxa, can be employed in distinguishing the Fagus species and in identifying the ancient pollen. RESULTS: 86 populations from 4 Western Euroasistic taxa were sampled, and sequenced for the trnL-trnF region to verify the efficiency of this cpDNA region in identifying the Fagus spp.. Furthermore, Fagus crenata (2 populations), Fagus grandifolia (2 populations), Fagus japonica, Fagus hayatae, Quercus species and Castanea species were analysed to better resolve the phylogenetic inference. Our results show that this cpDNA region harbour some informative sites that allow to infer relationships among the species within the Fagaceae family. In particular, few specific and fixed mutations were able to discriminate and identify all the different Fagus species. Considering a short fragment of 176 base pairs within the trnL intron, 2 transversions were found able in distinguishing the F. orientalis complex taxa (F. orientalis, F. taurica and F. moesiaca) from the remaining Fagus spp. (F. sylvatica, F. japonica, F. hayataea, F. crenata and F. grandifolia). This permits to analyse this fragment also in ancient samples, where DNA is usually highly degraded. The sequences data indicate that the DNA recovered from ancient pollen belongs to the F. orientalis complex since it displays the informative sites characteristic of this complex. CONCLUSION: The ancient DNA sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the F. orientalis complex was already present during the Tyrrhenian period in what is now the Venice lagoon (Italy). This is a new and important insight considering that nowadays West Europe is not the natural area of Fagus orientalis complex, and up to now nobody has hypothesized the presence during the Last Interglacial period of F. orientalis complex in Italy.

Genetic variation in prehistoric Sardinia.

We sampled teeth from 53 ancient Sardinian (Nuragic) individuals who lived in the Late Bronze Age and Iron Age, between 3,430 and 2,700 years ago. After eliminating the samples that, in preliminary biochemical tests, did not show a high probability to yield reproducible results, we obtained 23 sequences of the mitochondrial DNA control region, which were associated to haplogroups by comparison with a dataset of modern sequences. The Nuragic samples show a remarkably low genetic diversity, comparable to that observed in ancient Iberians, but much lower than among the Etruscans. Most of these sequences have exact matches in two modern Sardinian populations, supporting a clear genealogical continuity from the Late Bronze Age up to current times. The Nuragic populations appear to be part of a large and geographically unstructured cluster of modern European populations, thus making it difficult to infer their evolutionary relationships. However, the low levels of genetic diversity, both within and among ancient samples, as opposed to the sharp differences among modern Sardinian samples, support the hypothesis of the expansion of a small group of maternally related individuals, and of comparatively recent differentiation of the Sardinian gene pools.

Genetic analysis of the skeletal remains attributed to Francesco Petrarca.

We report on the mitochondrial DNA (mtDNA) analysis of the supposed remains of Francesco Petrarca exhumed in November 2003, from the S. Maria Assunta church, in Arquà Padua (Italy) where he died in 1374. The optimal preservation of the remains allowed the retrieval of sufficient mtDNA for genetic analysis. DNA was extracted from a rib and a tooth and mtDNA sequences were determined in multiple clones using the strictest criteria currently available for validation of ancient DNA sequences, including independent replication. MtDNA sequences from the tooth and rib were not identical, suggesting that they belonged to different individuals. Indeed, molecular gender determination showed that the postcranial remains belonged to a male while the skull belonged to a female. Historical records indicated that the remains were violated in 1630, possibly by thieves. These results are consistent with morphological investigations and confirm the importance of integrating molecular and morphological approaches in investigating historical remains.

The Etruscans: a population-genetic study.

The origins of the Etruscans, a non-Indo-European population of preclassical Italy, are unclear. There is broad agreement that their culture developed locally, but the Etruscans' evolutionary and migrational relationships are largely unknown. In this study, we determined mitochondrial DNA sequences in multiple clones derived from bone samples of 80 Etruscans who lived between the 7th and the 3rd centuries b.c. In the first phase of the study, we eliminated all specimens for which any of nine tests for validation of ancient DNA data raised the suspicion that either degradation or contamination by modern DNA might have occurred. On the basis of data from the remaining 30 individuals, the Etruscans appeared as genetically variable as modern populations. No significant heterogeneity emerged among archaeological sites or time periods, suggesting that different Etruscan communities shared not only a culture but also a mitochondrial gene pool. Genetic distances and sequence comparisons show closer evolutionary relationships with the eastern Mediterranean shores for the Etruscans than for modern Italian populations. All mitochondrial lineages observed among the Etruscans appear typically European or West Asian, but only a few haplotypes were found to have an exact match in a modern mitochondrial database, raising new questions about the Etruscans' fate after their assimilation into the Roman state.