The Paleo-Indian Entry into South America According to Mitogenomes

Recent and compelling archaeological evidence attests to human presence 14.5 ka at multiple sites in South America and a very early exploitation of extreme high-altitude Andean environments. Considering that, according to genetic evidence, human entry into North America from Beringia most likely occurred 16 ka, these archeological findings would imply an extremely rapid spread along the double continent. To shed light on this issue from a genetic perspective, we first completely sequenced 217 novel modern mitogenomes of Native American ancestry from the northwestern area of South America (Ecuador and Peru); we then evaluated them phylogenetically together with other available mitogenomes (430 samples, both modern and ancient) from the same geographic area and, finally, with all closely related mitogenomes from the entire double continent. We detected a large number (N¼ 48) of novel subhaplogroups, often branching into further subclades, belonging to two classes: those that arose in South America early after its peopling and those that instead originated in North or Central America and reached South America with the first settlers. Coalescence age estimates for these subhaplogroups provide time boundaries indicating that early Paleo-Indians probably moved from North America to the area corresponding to modern Ecuador and Peru over the short time frame of 1.5 ka comprised between 16.0 and 14.6 ka

Mitogenomes from Egyptian Cattle Breeds: New Clues on the Origin of Haplogroup Q and the Early Spread of Bos taurus from the Near East

Background Genetic studies support the scenario that Bos taurus domestication occurred in the Near East during the Neolithic transition about 10 thousand years (ky) ago, with the likely exception of a minor secondary event in Italy. However, despite the proven effectiveness of whole mitochondrial genome data in providing valuable information concerning the origin of taurine cattle, until now no population surveys have been carried out at the level of mitogenomes in local breeds from the Near East or surrounding areas. Egypt is in close geographic and cultural proximity to the Near East, in particular the Nile Delta region, and was one of the first neighboring areas to adopt the Neolithic package. Thus, a survey of mitogenome variation of autochthonous taurine breeds from the Nile Delta region might provide new insights on the early spread of cattle rearing outside the Near East. Methodology Using Illumina high-throughput sequencing we characterized the mitogenomes from two cattle breeds, Menofi (N = 17) and Domiaty (N = 14), from the Nile Delta region. Phylogenetic and Bayesian analyses were subsequently performed. Conclusions Phylogenetic analyses of the 31 mitogenomes confirmed the prevalence of haplogroup T1, similar to most African cattle breeds, but showed also high frequencies for haplogroups T2, T3 and Q1, and an extremely high haplotype diversity, while Bayesian skyline plots pointed to a main episode of population growth ~12.5 ky ago. Comparisons of Nile Delta mitogenomes with those from other geographic areas revealed that (i) most Egyptian mtDNAs are probably direct local derivatives from the founder domestic herds which first arrived from the Near East and the extent of gene flow from and towards the Nile Delta region was limited after the initial founding event(s); (ii) haplogroup Q1 was among these founders, thus proving that it underwent domestication in the Near East together with the founders of the T clades

Employing mitogenomes to reconstruct migration and dispersal events

The mitochondrial genome is organized as a small circular molecule of DNA, present in hundreds/thousands of copies per cell and characterized by a much greater evolutionary rate than the average nuclear gene. The mitochondrial DNA (mtDNA) is transmitted as a non-recombining unit only through the mother and its variability is originated only by the sequential accumulation of new mutations. During millennia, this process of molecular divergence has given rise to monophyletic units (haplogroups) that are generally restricted to specific geographic areas or population groups. The study of the geographical distribution, the internal variability and the coalescence age of each haplogroup allow us to make inferences about the demographic history of populations, such as dispersals, range expansions, or migrations. During my PhD studies, I analysed the sequence variation of the mtDNA at the highest level of resolution, that of complete sequence (mitogenome), in order to reconstruct the migration events of both human and animal populations. In particular, I mainly focused my research activity on three projects. The first project aimed to date the events that brought to the initial peopling in Sardinia and to clarify the genetic history of Europe. Sardinians are "outliers" in the European genetic landscape and, according to paleogenomic nuclear data, the closest to early European Neolithic farmers. To learn more about the genetic ancestry of Sardinians, we analyzed 3491 modern and 21 ancient mitogenomes from Sardinia and observed that the age estimates of three Sardinian-specific haplogroups are >7800 years, the archeologically-based upper boundary of the Neolithic in the island. This finding supports archeological evidence of a Mesolithic occupation of the island, but also reveals a dual ancestral origin of the first Sardinians. Indeed, one of the Sardinian-specific haplogroups harbors ancestral roots in Paleolithic Western Europe, but the other two are most likely of Late Paleolithic Near Eastern ancestry, and among those that are often assumed to have spread from Anatolia only with the Neolithic. Thus, their ages are compatible with the scenario of a Late Glacial recolonization of Mediterranean Europe from the Near East prior to the migration wave(s) associated with the onset of farming. The second project aimed to further assess the mitogenome variation of Native Americans origin. Specifically, I focused on Ecuador and Peru, two geographical areas of particular interest because of their location along the Pacific coast, in order to shed light on the peopling of South America. Phylogenetic analyses encompassing both novel and previously reported mitogenomes, allowed the identification of 50 new sub-haplogroups and the finding of a number of sub-clades shared with Native Americans from North and Central America, thus increasing the number of founding mtDNA lineages that entered South America from the North. Our phylogeographic analyses confirmed that the North to South expansion was extremely rapid, and most likely occurred along both the Pacific and Atlantic coasts. The third study was aimed to acquire information about the diffusion process of the Asian tiger mosquito Ae. albopictus by analysing the mitogenome variation of representatives from Asia, America and Europe. Phylogenetic analyses revealed five haplogroups in Asia, but population surveys showed that only three of these were involved in the recent worldwide spread. We also found out that a sub-haplogroup, which is now common in Italy, most likely arose in North America from an ancestral Japanese source. During these three years I also contributed to two additional projects whose goals were to reconstruct the ancient migratory events involving the Arabian Peninsula and Eastern Africa by the study of a rare haplogroup named R0a and to acquire new insights on the initial events that brought to the diffusion of domestic cattle (Bos taurus) outside the Near East

Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples

Background: Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quan-tification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material. Results: To address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging. Conclusions: These results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms

A missense MT-ND5 mutation in differentiated Parkinson Disease cytoplasmic hybrid induces ROS-dependent DNA Damage Response amplified by DROSHA

Genome integrity is continuously threatened by endogenous sources of DNA damage including reactive oxygen species (ROS) produced by cell metabolism. Factors of the RNA interference (RNAi) machinery have been recently involved in the cellular response to DNA damage (DDR) in proliferating cells. To investigate the impact of component of RNAi machinery on DDR activation in terminally di erentiated cells, we exploited cytoplasmic hybrid (cybrid) cell lines in which mitochondria of sporadic Parkinson’s disease patients repopulate neuroblastoma SH-SY5Y-Rho(0) cells. Upon di erentiation into dopaminergic neuron-like cells, PD63 cybrid showed increased intracellular level of ROS and chronic DDR activation, compared to other cybrids with the same nuclear background. Importantly, DDR activation in these cells can be prevented by ROS scavenging treatment suggesting that ROS production is indeed causative of nuclear DNA damage. Sequence analysis of the mitogenomes identi ed a rare and heteroplasmic missense mutation a ecting a highly conserved residue of the ND5- subunit of respiratory complex I, which accounts for ROS increase. We demonstrated that the assembly of nuclear DDR foci elicited by oxidative stress in these cells relies on DROSHA, providing the rst evidence that components of RNAi machinery play a crucial role also in the mounting of ROS-induced DDR in non-replicating neuronal cells

The peopling of South America and the trans-Andean gene flow of the first settlers

Genetic and archaeological data indicate that the initial Paleoindian settlers of South America followed two entry routes separated by the Andes and the Amazon rainforest. The interactions between these paths and their impact on the peopling of South America remain unclear. Analysis of genetic variation in the Peruvian Andes and regions located south of the Amazon River might provide clues on this issue. We analyzed mitochondrial DNA variation at different Andean locations and >360,000 autosomal SNPs from 28 Native American ethnic groups to evaluate different trans-Andean demographic scenarios. Our data reveal that the Peruvian Altiplano was an important enclave for early Paleoindian expansions and point to a genetic continuity in the Andes until recent times, which was only marginally affected by gene flow from the Amazonian lowlands. Genomic variation shows a good fit with the archaeological evidence, indicating that the genetic interactions between the descendants of the settlers that followed the Pacific and Atlantic routes were extremely limited

Molecular divergence and age estimates (ML and ρ statistics) for taurine cattle haplogroups based on all currently available mitogenomes.

<p>^a Number of mitogenomes. For haplogroups T2, Q and T3, the mitogenomes correspond to those reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.g002" target="_blank">Fig 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.s001" target="_blank">S1 Fig</a>. Haplogroup T1 includes mitogenomes from this study (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.s002" target="_blank">S1 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.g001" target="_blank">Fig 1</a>) and from the literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref028" target="_blank">28</a>], while T5 mitogenomes are those from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref022" target="_blank">22</a>].</p><p>^b Maximum Likelihood molecular divergence.</p><p>^c Age estimates (ky) using the molecular clock proposed by Achilli et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref022" target="_blank">22</a>].</p><p>^d Haplogroup P includes three published mitogenomes (NC013996, JQ437479, DQ124389).</p><p>^e Subclade P1 has been defined here and includes mitogenomes NC013996 and DQ124389.</p><p>^f Haplogroup T1’2’3 includes the EU177840 mtDNA sequence [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref022" target="_blank">22</a>], in addition to the T1, T2 and T3 mitogenomes.</p><p>^g Haplogroups T5a and T5b have been defined here.</p><p>Molecular divergence and age estimates (ML and ρ statistics) for taurine cattle haplogroups based on all currently available mitogenomes.</p

Tree of mitogenomes from Egyptian cattle.

<p>Sequences #1–19, #25, #30–31 have been determined in this study, while sequences #20–24 and #26–29 were previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref027" target="_blank">27</a>]. GenBank accession numbers are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.s002" target="_blank">S1 Table</a>. This tree was built as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref022" target="_blank">22</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref024" target="_blank">24</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref027" target="_blank">27</a>]. The hypervariable insertion of a G at np 364, the length variations in the C tract scored at np 221 and the A tract scored at np 1600 were not used for the phylogeny construction. The position of the Bovine Reference Sequence (BRS) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141170#pone.0141170.ref036" target="_blank">36</a>] is indicated for reading off-sequence motifs. Branches display mutations with numbers according to the BRS; they are transitions unless a base is explicitly indicated for transversions (to A, G, C, or T) or a suffix for indels (+, d) and heteroplasmy (h). Recurrent mutations within the phylogeny are underlined and back mutations are marked with the suffix @. Note that the reconstruction of recurrent mutations in the control region is ambiguous in a number of cases. The pie charts summarize haplogroup frequencies in the Menofi (green) and Domiaty (orange) breeds.</p