Fossil DNA.

BackgroundSeveral molecular and population genetic studies have focused on the native sheep breeds of Finland. In this work, we investigated their ancestral sheep populations from Iron Age, Medieval and Post-Medieval periods by sequencing a partial mitochondrial DNA D-loop and the 5’-promoter region of the SRY gene. We compared the maternal (mitochondrial DNA haplotypes) and paternal (SNP oY1) genetic diversity of ancient sheep in Finland with modern domestic sheep populations in Europe and Asia to study temporal changes in genetic variation and affinities between ancient and modern populations.ResultsA 523-bp mitochondrial DNA sequence was successfully amplified for 26 of 36 sheep ancient samples i.e. five, seven and 14 samples representative of Iron Age, Medieval and Post-Medieval sheep, respectively. Genetic diversity was analyzed within the cohorts. This ancient dataset was compared with present-day data consisting of 94 animals from 10 contemporary European breeds and with GenBank DNA sequence data to carry out a haplotype sharing analysis. Among the 18 ancient mitochondrial DNA haplotypes identified, 14 were present in the modern breeds. Ancient haplotypes were assigned to the highly divergent ovine haplogroups A and B, haplogroup B being the major lineage within the cohorts. Only two haplotypes were detected in the Iron Age samples, while the genetic diversity of the Medieval and Post-Medieval cohorts was higher. For three of the ancient DNA samples, Y-chromosome SRY gene sequences were amplified indicating that they originated from rams. The SRY gene of these three ancient ram samples contained SNP G-oY1, which is frequent in modern north-European sheep breeds.ConclusionsOur study did not reveal any sign of major population replacement of native sheep in Finland since the Iron Age. Variations in the availability of archaeological remains may explain differences in genetic diversity estimates and patterns within the cohorts rather than demographic events that occurred in the past. Our ancient DNA results fit well with the genetic context of domestic sheep as determined by analyses of modern north-European sheep breeds.

Ancient DNA extracted from 2000 year-old equine bones was examined in order to amplify mitochondrial and nuclear DNA fragments. A specific equine satellite-type sequence representing 3.7%-11% of the entire equine genome, proved to be a suitable target to address the question of the presence of aDNA in ancient bones. The PCR strategy designed to investigate this specific target also allowed us to calculate the molecular weight of amplifiable DNA fragments. Sequencing of a 370 bp DNA fragment of mitochondrial control region allowed the comparison of ancient DNA sequences with those of modern horses to assess their genetic relationship. The 16S rRNA mitochondrial gene was also examined to unravel the post-mortem base modification feature and to test the status of Pompeian equids taxon on the basis of a Mae III restriction site polymorphism.

The history of population structure is a key to effective wildlife management and conservation. However, inferring the history of population structure using present genetic structures is problematic when the method is applied to species that have experienced severe population bottlenecks. Ancient DNA analysis seemed to be a promising, direct method for inferring ancient population structures. However, the usual methods for inferring modern population structure, i.e. the phylogeographic approach using mitochondrial DNA and the Bayesian approach using microsatellite DNA, are often unsuitable for ancient samples. In this study, we combined ancient DNA obtained from zooarchaeological bones with carbon/nitrogen stable isotope ratios and morphological variations to infer ancient population structure of the short-tailed albatross Phoebastria albatrus. The results showed that the bird existed in two populations, between which the genetic distance was greater than that of distinct sister albatross species, although no subspecies of P. albatrus have been proposed. Our results suggest that the birds at the present two breeding regions (Torishima in the Izu Islands and two islets of the Senkaku Islands) are descended from these two ancient populations, and that reevaluation of the status and conservation strategy for the species is required. Our results also indicate that lineage breeding on the Senkaku Islands has drastically reduced genetic diversity, while that on Torishima has not. The approach proposed in this study would be useful for inferring ancient population structure, using samples of highly mobile animals and/or samples from archaeological sites, and the reconstructed ancient population structure would be useful for conservation and management recommendations.

Recent reports analyzing mitochondrial DNA sequences from Neandertal bones have claimed that Neandertals and modern humans are different species. The phylogenetic analyses carried out in these articles did not take into account the high substitution rate variation among sites observed in the human mitochondrial D-loop region and also lack an estimation of the parameters of the nucleotide substitution model. The separate phylogenetic position of Neandertals is not supported when these factors are considered. Our analysis shows that Neandertal-Human and Human-Human pairwise distance distributions overlap more than what previous studies suggested. We also show that the most ancient Neandertal HVI region is the most divergent when compared with modern human sequences. However, the opposite would be expected if the sequence had not been modified since the death of the specimen. Such incongruence is discussed in the light of diagenetic modifications in ancient Neandertal DNA sequences.

Ancient mitochondrial DNA analysis of avian bones collected from the 4th century pit burial found in South Korea

Grapevine is the one of the most important fruit species in the world. Comparative genome sequencing of grape cultivars is very important for the interpretation of the grape genome and understanding its evolution. The genomes of four Georgian grape cultivars-Chkhaveri, Saperavi, Meskhetian green, and Rkatsiteli, belonging to different haplogroups, were resequenced. The shotgun genomic libraries of grape cultivars were sequenced on an Illumina HiSeq. Pinot Noir nuclear, mitochondrial, and chloroplast DNA were used as reference. Mitochondrial DNA of Chkhaveri closely matches that of the reference Pinot noir mitochondrial DNA, with the exception of 16 SNPs found in the Chkhaveri mitochondrial DNA. The number of SNPs in mitochondrial DNA from Saperavi, Meskhetian green, and Rkatsiteli was 764, 702, and 822, respectively. Nuclear DNA differs from the reference by 1,800,675 nt in Chkhaveri, 1,063,063 nt in Meskhetian green, 2,174,995 in Saperavi, and 5,011,513 in Rkatsiteli. Unlike mtDNA Pinot noir, chromosomal DNA is closer to the Meskhetian green than to other cultivars. Substantial differences in the number of SNPs in mitochondrial and nuclear DNA of Chkhaveri and Pinot noir cultivars are explained by backcrossing or introgression of their wild predecessors before or during the process of domestication. Annotation of chromosomal DNA of Georgian grape cultivars by MEGANTE, a web-based annotation system, shows 66,745 predicted genes (Chkhaveri-17,409; Saperavi-17,021; Meskhetian green-18,355; and Rkatsiteli-13,960). Among them, 106 predicted genes and 43 pseudogenes of terpene synthase genes were found in chromosomes 12, 18 random (18R), and 19. Four novel TPS genes not present in reference Pinot noir DNA were detected. Two of them-germacrene A synthase (Chromosome 18R) and (-) germacrene D synthase (Chromosome 19) can be identified as putatively full-length proteins. This work performs the first attempt of the comparative whole genome analysis of different haplogroups of Vitis vinifera cultivars. Based on complete nuclear and mitochondrial DNA sequence analysis, hypothetical phylogeny scheme of formation of grape cultivars is presented.

Recent developments in high-throughput sequencing (HTS) technologies have enabled the reconstruction of complete genome sequence and nucleosome and methylation maps from ancient individuals. A diversity of degradation reactions affect DNA molecules after an organism dies, introducing specific nucleotide misincorporation patterns in HTS data that are typically exploited to distinguish modern contaminants and authentic DNA templates. In this study, we used two different DNA polymerases to amplify Illumina TruSeq DNA libraries built on DNA extracts from ancient equines. The AmpliTaq Gold, widely used in ancient DNA studies, generated amplified libraries showing typical misincorporation patterns. Such patterns were partially lost following AccuPrime Pfx amplification, for which a two-fold reduction of endogenous content was also observed. This is explained by the inability of the AccuPrime Pfx to bypass uracils, which represent the most common post-mortem base modification and derive from the post-mortem deamination of cytosines. Our study, therefore, reveals that amplification of TruSeq DNA libraries with AccuPrime Pfx increases the cost of whole-genome sequencing for samples showing substantial levels of DNA degradation and creates atypical nucleotide misincorporation patterns for data authentication. The method can, however, be exploited to identify ancient methylation marks, and potentially, nucleosome occupancy maps.Statement of significance After an organism dies, DNA molecules are subjected to degradation resulting in fragmentation and bases modifications. After high- throughput sequencing (HTS), it is possible to detect the molecular signature of these damages, demonstrating that the data generated is authentic. DNA extracts from fossil remains indeed contain, besides endogenous ancient molecules, a variety of modern contaminants, but only the former generally exhibits molecular signatures typical of post-mortem damage. Our study reveals that these signatures depend on the protocol used for amplifying DNA samples. It thus helps defining better guidelines for authenticating ancient HTS data. Our results also reveal one molecular approach that limits our ability to exploit the whole complexity of ancient DNA molecules preserved in fossil specimens but can help track ancient methylation marks.Data availability The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are contained within the paper.

Nuclear Gene Sequences from a Late Pleistocene Sloth Coprolite

African elephant mitochondrial (mt) DNA follows a distinctive evolutionary trajectory. As females do not migrate between elephant herds, mtDNA exhibits low geographic dispersal. We therefore examined the effectiveness of mtDNA for assigning the provenance of African elephants (or their ivory). For 653 savanna and forest elephants from 22 localities in 13 countries, 4258 bp of mtDNA was sequenced. We detected eight mtDNA subclades, of which seven had regionally restricted distributions. Among 108 unique haplotypes identified, 72% were found at only one locality and 84% were country specific, while 44% of individuals carried a haplotype detected only at their sampling locality. We combined 316 bp of our control region sequences with those generated by previous trans-national surveys of African elephants. Among 101 unique control region haplotypes detected in African elephants across 81 locations in 22 countries, 62% were present in only a single country. Applying our mtDNA results to a previous microsatellite-based assignment study would improve estimates of the provenance of elephants in 115 of 122 mis-assigned cases. Nuclear partitioning followed species boundaries and not mtDNA subclade boundaries. For taxa such as elephants in which nuclear and mtDNA markers differ in phylogeography, combining the two markers can triangulate the origins of confiscated wildlife products.

Ancient DNA preservation, genetic diversity and biogeography: A study of houseflies from Roman Qasr Ibrim, lower Nubia, Egypt

ObjectiveIn the Bronze Age Hallstatt metropolis (‘Salzkammergut’ region, Upper Austria), salt richness enabled the preservation of pork meat to sustain people’s livelihood suggesting an organized meat production industry on a yearly basis of hundreds of pigs. To pattern the geographic and temporal framework of the early management of pig populations in the surrounding areas of Hallstatt, we want to gain insights into the phylogeographic network based on DNA sequence variation among modern pigs, wild boars and prehistoric (likely) domestic pigs.ResultsIn this pilot study, we successfully adapted ancient DNA extraction and sequencing approaches for the analysis of mitochondrial DNA sequence variation in ten prehistoric porcine teeth specimens. Minimum-spanning network analyses revealed unique mitochondrial control region DNA haplotypes ranging within the variation of modern domestic pig and wild boar lineages and even shared haplotypes between prehistoric and modern domestic pigs and wild boars were observed.

Key insights into the evolutionary history of recently extinct or critically endangered species can be obtained through analysis of genomic data collected using high-throughput sequencing and ancient DNA from museum specimens, particularly where specimens are rare. For instance, the evolutionary history of the critically endangered Puebla deer mouse, Peromyscus mekisturus, remains unclear due to discordance between morphological and molecular phylogenetic analyses. However, previous molecular analyses were based on PCR and Sanger sequencing of only a few mitochondrial genes. Here, we used ancient DNA from historical museum specimens followed by target enrichment and high-throughput sequencing of several thousand nuclear ultraconserved elements and whole mitochondrial genomes to test the validity of the previous phylogenetic placement of P. mekisturus. Based on UCEs and mitogenomes, our results revealed that P. mekisturus forms a well-supported distinct lineage outside the clade containing all other members of the Peromyscus melanophrys group. Additionally, the mitogenome phylogeny further supports the placement of P. mekisturus as the sister species of the genus Reithrodontomys. This conflicts with the previous mtDNA phylogenetic reconstruction, in which P. mekisturus was nested within the species P. melanophrys. Our study demonstrates that high-throughput sequencing of ancient DNA, appropriately controlling for contamination and degradation, can provide a robust resolution of the evolutionary history and taxonomic status of species for which few or no modern genetic samples exist. In light of our results and pending further analysis with denser taxon sampling and the addition of morphological data, a re-evaluation of the taxonomy and conservation management plans of P. mekisturus is needed to ensure that the evolutionary distinctiveness of this species is recognized in future conservation efforts.

Many social animals live in stable groups. In contrast, African savannah elephants (Loxodonta africana) live in unusually fluid, fission-fusion societies. That is, 'core' social groups are composed of predictable sets of individuals; however, over the course of hours or days, these groups may temporarily divide and reunite, or they may fuse with other social groups to form much larger social units. Here, we test the hypothesis that genetic relatedness predicts patterns of group fission and fusion among wild, female African elephants. Our study of a single Kenyan population spans 236 individuals in 45 core social groups, genotyped at 11 microsatellite and one mitochondrial DNA (mtDNA) locus. We found that genetic relatedness predicted group fission; adult females remained with their first order maternal relatives when core groups fissioned temporarily. Relatedness also predicted temporary fusion between social groups; core groups were more likely to fuse with each other when the oldest females in each group were genetic relatives. Groups that shared mtDNA haplotypes were also significantly more likely to fuse than groups that did not share mtDNA. Our results suggest that associations between core social groups persist for decades after the original maternal kin have died. We discuss these results in the context of kin selection and its possible role in the evolution of elephant sociality.

Human skeletal remains from the Neolithic period are extremely rare and while many pile dwelling sites are known from Switzerland little is known about the inhabitants. Thus the recent discovery of a Neolithic dolmen in Switzerland with in situ inhumations was unexpected and might provide new information about the population. The dolmen of Oberbipp contained well-preserved skeletal remains and was documented using state-of-the-art technologies. The aim of the project is to analyze the skeletal remains of the Oberbipp dolmen in the context of further Neolithic material from Switzerland and to gain further insights into the Neolithic “Swiss” population. The preliminary minimum number of individuals (based on right femora) from Oberbipp is 40. Morphological analysis revealed the presence of both sexes and neonate to adult individuals. Based on the right femora a proportion of 37.5% subadults and 62.5% adults was calculated. First 14C results indicate a use of the dolmen approximately 3500 – 3000 BCE. The reconstruction of complete mitogenomes of 16 individuals shows the presence of haplogroups characteristic for Early Farmers and a remarkably high frequency of haplogroup K. Stable isotopes (C, N, S, H, O, Sr and Pb), further ancient DNA and 14C analyses are currently ongoing. The preliminary results are indicative of a “normal” Neolithic population and are in concordance to other Neolithic sites in central Europe so far. The evaluation of the stable isotope data as well as the evaluation of palaeopathologies might provide additional information. Project funded by Swiss National Science Foundation (CR31I3L_157024) and Deutsche Forschungsgemeinschaft (KR4015/4-1). Inga Siebke, Anja Furtwangler, Albert Hafner, Johannes Krause, Sandra Losch.

Description of a new species and understanding the genetic diversity of Saccocoelioides Szidat, 1954 (Haploporidae) in Middle America using mitochondrial and nuclear DNA sequences