Ancient DNA studies: new perspectives on old samples

In spite of past controversies, the field of ancient DNA is now a reliable research area due to recent methodological improvements. A series of recent large-scale studies have revealed the true potential of ancient DNA samples to study the processes of evolution and to test models and assumptions commonly used to reconstruct patterns of evolution and to analyze population genetics and palaeoecological changes. Recent advances in DNA technologies, such as next-generation sequencing make it possible to recover DNA information from archaeological and paleontological remains allowing us to go back in time and study the genetic relationships between extinct organisms and their contemporary relatives. With the next-generation sequencing methodologies, DNA sequences can be retrieved even from samples (for example human remains) for which the technical pitfalls of classical methodologies required stringent criteria to guaranty the reliability of the results. In this paper, we review the methodologies applied to ancient DNA analysis and the perspectives that next-generation sequencing applications provide in this field

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

Neandertal-like traits visible in the internal structure of non-supranuchal fossae of some recent Homo sapiens: The problem of their identification in hominins and phylogenetic implications

Although recently the internal structure of the non-supranuchal fossa of Homo sapiens has been described and compared to that observed in the Neandertal suprainiac fossa, until now it has not been examined in any modern human children. In this study, the internal structure of this fossa in the occipital bones of three children (two aged 3–4 years and one aged 5 years ± 16 months) and one adult individual representing recent Homo sapiens from Australia was analysed and compared to that of the Neandertal suprainiac fossa. In order to analyse the internal composition of the fossae of the examined specimens, initially, high-resolution micro-CT datasets were obtained for their occipital bones; next, 3D topographic maps of the variation in thickness of structural layers of the occipital bones were made and 2D virtual sections in the median region of these fossae were prepared. In the fossa of one immature individual, the thinning of the diploic layer characteristic of a Neandertal suprainiac fossa was firmly diagnosed. The other Neandertal-like trait, concerning the lack of substantial thinning of the external table of the bone in the region of the fossa, was established in two individuals (one child and one adult) due to the observation of an irregular pattern of the thickness of this table in the other specimens, suggesting the presence of an inflammatory process. Our study presents, for the first time, Neandertal-like traits (but not the whole set of features that justifies the autapomorphic status of the Neandertal supraniac fossa) in the internal structure of non-supranuchal fossae of some recent Homo sapiens. We discuss the phylogenetic implications of the results of our analysis and stress the reasons that use of the 3D topographic mapping method is important for the correct diagnosis of Neandertal traits of the internal structure of occipital fossae

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

<p>Abstract</p> <p>Background</p> <p>Phylogeographic analyses on the Western Euroasiatic <it>Fagus </it>taxa (<it>F. orientalis</it>, <it>F. sylvatica</it>, <it>F. taurica </it>and <it>F. moesiaca</it>) is available, however, the subdivision of <it>Fagus </it>spp. is unresolved and there is no consensus on the phylogeny and on the identification (both with morphological than molecular markers) of <it>Fagus </it>Eurasiatic taxa.</p> <p>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 <it>Fagus </it>spp. present during the Last Interglacial period in Italy.</p> <p>In this work we aim at testing if the <it>trn</it>L-<it>trn</it>F chloroplast DNA (cpDNA) region, that has been previously proved efficient in discriminating different <it>Quercus </it>taxa, can be employed in distinguishing the <it>Fagus </it>species and in identifying the ancient pollen.</p> <p>Results</p> <p>86 populations from 4 Western Euroasistic taxa were sampled, and sequenced for the <it>trn</it>L-<it>trn</it>F region to verify the efficiency of this cpDNA region in identifying the <it>Fagus </it>spp.. Furthermore, <it>Fagus crenata </it>(2 populations), <it>Fagus grandifolia </it>(2 populations), <it>Fagus japonica</it>, <it>Fagus hayatae</it>, <it>Quercus </it>species and <it>Castanea </it>species were analysed to better resolve the phylogenetic inference.</p> <p>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 <it>Fagus </it>species.</p> <p>Considering a short fragment of 176 base pairs within the <it>trn</it>L intron, 2 transversions were found able in distinguishing the <it>F. orientalis </it>complex taxa (<it>F. orientalis</it>, <it>F. taurica </it>and <it>F. moesiaca</it>) from the remaining <it>Fagus </it>spp. (<it>F. sylvatica</it>, <it>F. japonica</it>, <it>F. hayataea</it>, <it>F. crenata </it>and <it>F. grandifolia</it>). This permits to analyse this fragment also in ancient samples, where DNA is usually highly degraded.</p> <p>The sequences data indicate that the DNA recovered from ancient pollen belongs to the <it>F. orientalis </it>complex since it displays the informative sites characteristic of this complex.</p> <p>Conclusion</p> <p>The ancient DNA sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the <it>F. orientalis </it>complex was already present during the Tyrrhenian period in what is now the Venice lagoon (Italy).</p> <p>This is a new and important insight considering that nowadays West Europe is not the natural area of <it>Fagus orientalis </it>complex, and up to now nobody has hypothesized the presence during the Last Interglacial period of <it>F. orientalis </it>complex in Italy.</p

Mitochondrial DNA Suggests a Western Eurasian origin for Ancient (Proto-) Bulgarians

Ancient (proto-) Bulgarians have long been thought to as a Turkic population. However, evidence found in the past three decades show that this is not the case. Until now, this evidence does not include ancient mitochondrial DNA (mtDNA) analysis. In order to fill this void, we have collected human remains from the VIII-X century AD located in three necropolises in Bulgaria: Nojarevo (Silistra region) and Monastery of Mostich (Shumen region), both in Northeast Bulgaria and Tuhovishte (Satovcha region) in Southwest Bulgaria. The phylogenetic analysis of 13 ancient DNA samples (extracted from teeth) identified 12 independent haplotypes, which we further classified into mtDNA haplogroups found in present-day European and Western Eurasian populations. Our results suggest a Western Eurasian matrilineal origin for proto-Bulgarians as well as a genetic similarity between proto- and modern Bulgarians. Our future work will provide additional data which will further clarify proto-Bulgarian origins; thereby adding new clues to current understanding of European genetic evolution

Practising pastoralism in an agricultural environment: An isotopic analysis of the impact of the Hunnic incursions on Pannonian populations

We conducted a multi-isotope study of five fifth-century AD cemeteries in modern-day Hungary to determine relationships between nomadic-pastoralist incomers—the historically documented Huns and other nomadic groups—and the sedentary agricultural population of the late Roman province of Pannonia. Contemporary historical sources describe this relationship as adversarial and destructive for the late Roman population, but archaeological evidence indicates high levels of hybridity between different groups. We undertook carbon, nitrogen, strontium and oxygen isotope analyses of bone collagen, dentine and tooth enamel at Keszthely-Fenékpuszta, Hács-Béndekpuszta, Győr-Széchenyi Square, Mözs and Szolnok-Szanda to examine these relationships through past subsistence practices. The patterns at all sites indicate medium to high animal protein consumption with little evidence for a significant contribution of aquatic resources. All populations relied to a great extent on C4 plants, most likely millet. Within each population, diet was heterogeneous, with significant variations in terms of animal protein and C3 and C4 plant consumption. High levels of intra-population and individual variability suggest that populations made use of a range of subsistence strategies, with many individuals exhibiting significant changes over their lifetimes. Rather than being characterised only by violence, the historically-documented influx of nomadic populations appears to have led to widespread changes in subsistence strategies of populations in the Carpathian basin. Nomadic-pastoralist groups may have switched to smaller herds and more farming, and, conversely, local populations may have integrated with a new economic system based on animal herding.The analytical costs were funded by a research grant from the McDonald Institute for Archaeological Research, University of Cambridge. A study visit to Hungary was supported by a SYNTHESYS travel grant