Dramatic founder effects in Amerindian mitochondrial DNAs

Southwestern American Indian (Amerindian) mitochondrial DNAs (mtDNAs) were analyzed with restriction endonucleases and found to contain Asian restriction fragment length polymorphisms (RFLPs) but at frequencies very different from those found in Asia. One rare Asian HincII RFLP was found in 40% of the Amerindians. Several mtDNAs were discovered which have not yet been observed on other continents and different tribes were found to have distinctive mtDNAs. Since the mtDNA is inherited exclusively through the maternal lineage, these results suggest that Amerindian tribes were founded by small numbers of female lineages and that new mutations have been fixed in these lineages since their separation from Asia.     

Phylogenetic analysis of ancient mitochondrial DNA lineages of human remains found in Yakutia

Molecular genetic analysis of ancient human remains is mostly based on mtDNA owing to its better preservation in human bones in comparison with nuclear DNA. A study was made of mtDNA extracted from human skeletons found in graves in Yakutia, in order to determine the haplotypes and to compare them with lineages of modern populations. Ancient DNA was extracted from fragments of three skeletons of Yakut graves at At-Dabaan, Ojuluun, and Jaraama sites (dating back to the 18th century) and two skeletons of the Late Neolithic Kerdugen grave (2000–1000 B.C.). All graves were found in central Yakutia (Churapchinskii, Khangalasskii, and Megino-Khangalasskii districts of Yakutia). Five different haplotypes belonging to specific Asian haplogroups were identified. The mtDNA lineages of Yakut graves belong to haplogroups C4a, D5a2, and B5b. The results indicate the continuity of mitochondrial lineages in the Yakut gene pool in the past 300 years. The haplotypes of two humans from the Kerdugen site graves belong to haplogroups A4 and G2a/D. These haplotypes were compared with those of 40000 Eurasian individuals, including 900 from Yakutia. No exact matches were found in Paleo-Asian populations of Chukchi, Eskimos, Koryaks, and Itelmen. Phylogenetically close haplotypes (±1 mutation) were found in Yakut and Evenk populations, as well as in some populations of China and South and West Siberia.     

Ancient DNA from a pre-Columbian Amerindian population

Ancient DNA was obtained from skeletal remains from the Norris Farms #36 cemetery, a pre-Columbian archeological site in central Illinois that dates to A. D. 1300. Four mitochondrial DNA (mtDNA) markers were analyzed that delineate the four primary mtDNA lineages found in contemporary Amerindian populations. mtDNA types were determined for 50 individuals; 49 belonged to one of these four lineages. One lineage occurred only in males, suggesting an immigration of maternally related males into this community. There was no significant spatial patterning of mtDNA lineages within the cemetery. This survey of ancient DNA variation in a preColumbian population supports the view that the initial colonization of the New World comprised just four primary mtDNA lineages. © 1993 Wiley-Liss, Inc.     

The use of protein characteristics to assess the retrievability of ancient DNA from ancient bones

The ability to retrieve DNA from ancient specimens has been one of the greatest achievements of the past decade, and has opened a totally new field of research with applications in seemingly distant domains such as archeobotany, the molecular phylogeny of extinct genomes, human paleopathology and the genetic of ancient human populations. However, extraction of ancient DNA has often a very low rate of success, prompting researchers to develop screening methods for the selection of promising specimens. With this goal in mind, we studied the amino acid content of nine human bones of ancient origin. We demonstrate that a single HPLC chromatogram is indicative of the integrity of ancient bone proteins. Among five specimens containing amplifiable DNA, four exhibited a protein content similar to that of contemporary bone protein content. Three of the four specimens, from which we were unable to extract any amplifiable DNA, had an amino acid content strikingly different from that of contem-porary bone. A non-parametric statistical test, Kendall's tau, was used to show that protein content and PCR products, are probably correlated (at a 95% confidence level). In addition, the D/L Asp and D/L Glu racemization ratios obtained are indicative of the presence of ancient organic compounds. We propose that protein analysis should be systematically performed in studies where there are many samples in order to select the specimens that are most likely to contain retrievable ancient DNA.     

Nondestructive sampling of human skeletal remains yields ancient nuclear and mitochondrial DNA

Museum curators and living communities are sometimes reluctant to permit ancient DNA (aDNA) studies of human skeletal remains because the extraction of aDNA usually requires the destruction of at least some skeletal material. Whether these views stem from a desire to conserve precious materials or an objection to destroying ancestral remains, they limit the potential of aDNA research. To help address concerns about destructive analysis and to minimize damage to valuable specimens, we describe a nondestructive method for extracting DNA from ancient human remains. This method can be used with both teeth and bone, but it preserves the structural integrity of teeth much more effectively than that of bone. Using this method, we demonstrate that it is possible to extract both mitochondrial and nuclear DNA from human remains dating between 300 BC and 1600 AD. Importantly, the method does not expose the remains to hazardous chemicals, allowing them to be safely returned to curators, custodians, and/or owners of the samples. We successfully amplified mitochondrial DNA from 90% of the individuals tested, and we were able to analyze 1-9 nuclear loci in 70% of individuals. We also show that repeated nondestructive extractions from the same tooth can yield amplifiable mitochondrial and nuclear DNA. The high success rate of this method and its ability to yield DNA from samples spanning a wide geographic and temporal range without destroying the structural integrity of the sampled material may make possible the genetic study of skeletal collections that are not available for destructive analysis.     

Amplification of oak DNA from ancient and modern wood

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600-years-old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite-type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.     

Extensive human DNA contamination in extracts from ancient dog bones and teeth

Ancient DNA (aDNA) sequences, especially those of human origin, are notoriously difficult to analyze due to molecular damage and exogenous DNA contamination. Relatively few systematic studies have focused on this problem. Here we investigate the extent and origin of human DNA contamination in the most frequently used sources for aDNA studies, that is, bones and teeth from museum collections. To distinguish contaminant DNA from authentic DNA we extracted DNA from dog (Canis familiaris) specimens. We monitored the presence of a 148-bp human-specific and a 152-bp dog-specific mitochondrial DNA (mtDNA) fragment in DNA extracts as well as in negative controls. The total number of human and dog template molecules were quantified using real-time polymerase chain reaction (PCR), and the sequences were characterized by amplicon cloning and sequencing. Although standard precautions to avoid contamination were taken, we found that all samples from the 29 dog specimens contained human DNA, often at levels exceeding the amount of authentic ancient dog DNA. The level of contaminating human DNA was also significantly higher in the dog extracts than in the negative controls, and an experimental setup indicated that this was not caused by the carrier effect. This suggests that the contaminating human DNA mainly originated from the dog bones rather than from laboratory procedures. When cloned, fragments within a contaminated PCR product generally displayed several different sequences, although one haplotype was often found in majority. This leads us to believe that recognized criteria for authenticating aDNA cannot separate contamination from ancient human DNA the way they are presently used.     

A preliminary analysis of the DNA and diet of the extinct Beothuk: a systematic approach to ancient human DNA

We have used a systematic protocol for extracting, quantitating, sexing and validating ancient human mitochondrial and nuclear DNA of one male and one female Beothuk, a Native American population from Newfoundland, which became extinct approximately 180 years ago. They carried mtDNA haplotypes, which fall within haplogroups X and C, consistent with Northeastern Native populations today. In addition we have sexed the male using a novel-sexing assay and confirmed the authenticity of his Y chromosome with the presence of the Native American specific Y-QM3 single nucleotide polymorphism (SNP). This is the first ancient nuclear SNP typed from a Native population in the Americas. In addition, using the same teeth we conducted a stable isotopes analysis of collagen and dentine to show that both individuals relied on marine sources (fresh and salt water fish, seals) with no hierarchy seen between them, and that their water sources were pooled or stored water. Both mtDNA sequence data and Y SNP data hint at possible gene flow or a common ancestral population for both the Beothuk and the current day Mikmaq, but more importantly the data do not lend credence to the proposed idea that the Beothuk (specifically, Nonosabasut) were of admixed (European-Native American) descent. We also analyzed patterns of DNA damage in the clones of authentic mtDNA sequences; there is no tendency for DNA damage to occur preferentially at previously defined mutational hotspots, suggesting that such mutational hotspots are not hypervariable because they are more prone to damage.     

Spiking of contemporary human template DNA with ancient DNA extracts induces mutations under PCR and generates nonauthentic mitochondrial sequences

Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood.     

Patterns of polymorphism and gene flow of gender-associated mitochondrial DNA lineages in European mussel populations

Mussels of the genus Mytilus have two types of mitochondrial DNA (mtDNA). The M type is transmitted paternally and the F type is transmitted maternally. RFLP analysis is used to assess phylogenetic relationships and nucleotide diversity and divergence for both mtDNA genomes in European populations of M. edulis and Atlantic and Mediterranean forms of M. galloprovincialis. Ten restriction endonucleases were used to assay variation in regions of the ND2 and COIII genes for a total of 77 individuals. F and M genomes show a concordant phylogenetic split into two major divergent clades, one specific to Mediterranean M. galloprovincialis and the other containing haplotypes from the three taxa. For both genomes, the geographical distribution of mtDNA variation suggests: (i) extensive levels of mtDNA introgression; (ii) asymmetric mtDNA gene flow from Atlantic to Mediterranean populations; and (iii) recurrent historical hybridization events. Significantly higher mtDNA diversity and divergence are observed for the M than F genome in all three Mytilus taxa, although the evolutionary forces responsible for these differences cannot be resolved. The extensive mtDNA gene flow between European Mytilus taxa conflicts with the restricted mtDNA introgression observed in American mussels , implying geographical variation in the nature of nuclear/mtDNA interactions regulating biparental inheritance.     

Molecular characterization of ABO blood group frequencies in pre-Columbian Peruvian highlanders

The majority of Native Americans nearly exclusively belong to group O of the ABO blood group system. Several hypotheses have been formulated to explain this observation, primarily differing by the presumption that the observed patterns of ABO diversity are due to the processes of the initial peopling of the Americas or due to subsequent events, especially the demographic consequences in the wake of European contact. A promising strategy to reveal possible diachronic ABO frequency changes is the molecular genetic analysis of relevant genetic markers in precontact populations. A previous study by Halverson and Bolnick [Am J Phys Anthropol 137 (2008) 342‐347] already accomplished this for indigenous North American populations. Here we present the first study to analyze ABO blood types from pre‐Columbian individuals from South America using molecular genetic methods and comparing them to several extant South American, North American, and Siberian populations. We tried to determine ABO blood types for 59 individuals from the southern Peruvian highlands dating to ～650 to 1250 AD using a newly developed multiplex PCR/SBE assay coamplifying the fragments relevant for blood type determination and three highly discriminating autosomal STRs. Analysis was successful for 31 individuals and revealed that all are exclusively in the O group, predominantly carrying the O02 (01v) allele. No significant difference could be observed between the ancient and modern Native American populations, while all significantly differed from the extant Siberian populations, supporting the suggestion that low ABO diversity results from founder effects during the initial peopling of the Americas. Am J Phys Anthropol 149:242–249, 2012. © 2012 Wiley Periodicals, Inc.     

Brief communication: Haplogroup X confirmed in prehistoric North America

Haplogroup X represents approximately 3% of all modern Native North American mitochondrial lineages. Using RFLP and hypervariable segment I (HVSI) sequence analyses, we identified a prehistoric individual radiocarbon dated to 1,340 +/- 40 years BP that is a member of haplogroup X, found near the Columbia River in Vantage, Washington. The presence of haplogroup X in prehistoric North America, along with recent findings of haplogroup X in southern Siberians, confirms the hypothesis that haplogroup X is a founding lineage.     

King penguin population on Macquarie Island recovers ancient DNA diversity after heavy exploitation in historic times

Historically, king penguin populations on Macquarie Island have suffered greatly from human exploitation. Two large colonies on the island were drastically reduced to a single small colony as a result of harvesting for the blubber oil industry. However, recent conservation efforts have resulted in the king penguin population expanding in numbers and range to recolonize previous as well as new sites. Ancient DNA methods were used to estimate past genetic diversity and combined with studies of modern populations, we are now able to compare past levels of variation with extant populations on northern Macquarie Island. The ancient and modern populations are closely related and show a similar level of genetic diversity. These results suggest that the king penguin population has recovered past genetic diversity in just 80 years owing to conservation efforts, despite having seen the brink of extinction.     

Population origins in Mongolia: genetic structure analysis of ancient and modern DNA

In the present study, nuclear (autosomal and Y-chromosome short tandem repeats) and mitochondrial (hypervariable region I) ancient DNA data previously obtained from a 2,300-year-old Xiongnu population of the Egyin Gol Valley (south of Lake Baikal in northern Mongolia) (Keyser-Tracqui et al. 2003 Am. J. Hum. Genet. 73:247-260) were compared with data from two contemporary Mongolian populations: one from the same location (Egyin Gol Valley plus a perimeter of less than 100 km around the valley), and one from the whole of Mongolia. The principal objective of this comparative analysis was to assess the likelihood that genetic continuity exists between ancient and present-day Mongolian populations. Since the ancient Xiongnu sample might have been composed of some of the ancestors of the present-day Yakuts, data from a present-day Yakut population, as well as published data from Turkish populations, were also included in the comparative analysis. The main result of our study was the genetic similarity observed among Mongolian samples from different periods and geographic areas. This result supports the hypothesis that the succession over time of different Turkic and Mongolian tribes in the current territory of Mongolia resulted in cultural rather than genetic exchanges. Furthermore, it appears that the Yakuts probably did not find their origin among the Xiongnu tribes, as we previously hypothesized.     

Improved DNA extraction from ancient bones using silica-based spin columns

We describe a simple method for extracting polymerase chain reaction-amplifiable DNA from ancient bones without the use of organic solvents. Bone powders are digested with proteinase K, and the DNA is purified directly using silica-based spin columns (QIAquick™, QIAGEN). The efficiency of this protocol is demonstrated using human bone samples ranging in age from 15 to 5,000 years old. Am J Phys Anthropol 105:539–543, 1998. © 1998 Wiley-Liss, Inc.