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

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

Population dynamic of the extinct European aurochs: genetic evidence of a north-south differentiation pattern and no evidence of post-glacial expansion

<p>Abstract</p> <p>Background</p> <p>The aurochs (<it>Bos primigenius</it>) was a large bovine that ranged over almost the entirety of the Eurasian continent and North Africa. It is the wild ancestor of the modern cattle (<it>Bos taurus</it>), and went extinct in 1627 probably as a consequence of human hunting and the progressive reduction of its habitat. To investigate in detail the genetic history of this species and to compare the population dynamics in different European areas, we analysed <it>Bos primigenius </it>remains from various sites across Italy.</p> <p>Results</p> <p>Fourteen samples provided ancient DNA fragments from the mitochondrial hypervariable region. Our data, jointly analysed with previously published sequences, support the view that Italian aurochsen were genetically similar to modern bovine breeds, but very different from northern/central European aurochsen. Bayesian analyses and coalescent simulations indicate that the genetic variation pattern in both Italian and northern/central European aurochsen is compatible with demographic stability after the last glaciation. We provide evidence that signatures of population expansion can erroneously arise in stable aurochsen populations when the different ages of the samples are not taken into account.</p> <p>Conclusions</p> <p>Distinct groups of aurochsen probably inhabited Italy and northern/central Europe after the last glaciation, respectively. On the contrary, Italian and Fertile Crescent aurochsen likely shared several mtDNA sequences, now common in modern breeds. We argue that a certain level of genetic homogeneity characterized aurochs populations in Southern Europe and the Middle East, and also that post-glacial recolonization of northern and central Europe advanced, without major demographic expansions, from eastern, and not southern, refugia.</p

The Biarzo case in northern Italy: Is the temporal dynamic of swine mitochondrial DNA lineages in Europe related to domestication?

Genetically-based reconstructions of the history of pig domestication in Europe are based on two major pillars: 1) the temporal changes of mitochondrial DNA lineages are related to domestication; 2) Near Eastern haplotypes which appeared and then disappeared in some sites across Europe are genetic markers of the first Near Eastern domestic pigs. We typed a small but informative fragment of the mitochondrial DNA in 23 Sus scrofa samples from a site in north eastern Italy (Biarzo shelter) which provides a continuous record across a ≈6,000 year time frame from the Upper Palaeolithic to the Neolithic. We additionally carried out several radiocarbon dating. We found that a rapid mitochondrial DNA turnover occurred during the Mesolithic, suggesting that substantial changes in the composition of pig mitochondrial lineages can occur naturally across few millennia independently of domestication processes. Moreover, so-called Near Eastern haplotypes were present here at least two millennia before the arrival of Neolithic package in the same area. Consequently, we recommend a re-evaluation of the previous idea that Neolithic farmers introduced pigs domesticated in the Near East, and that Mesolithic communities acquired domestic pigs via cultural exchanges, to include the possibility of a more parsimonious hypothesis of local domestication in Europe

A 28,000 Years Old Cro-Magnon mtDNA Sequence Differs from All Potentially Contaminating Modern Sequences

Background: DNA sequences from ancient speciments may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal) and early modern (Cro-Magnoid) Europeans. Methodology/Principal Findings: We typed the mitochondrial DNA (mtDNA) hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23) and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences. Conclusions/Significance:: The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of earl