Unreliable mtDNA data due to nuclear insertions: a cautionary tale from analysis of humans and other great apes

Authors: O. Thalmann, J. Hebler, H. Poinar, S. Pääbo and L. Vigilant
Journal: Molecular Ecology (2004) – 13, 321–335

Analysis of mitochondrial DNA sequence variation has been used extensively to study the evolutionary relationships of individuals and populations, both ithin and across species. So ubiquitous and easily acquired are mtDNA data that it has been suggested that such data could serve as a taxonomic ‘barcode’ for an objective species classification scheme. However, there are technical pitfalls associated with the acquisition of mtDNA data. One problem is the presence of translocated pieces of mtDNA in the nuclear genome of many taxa that may be mistaken for authentic organellar mtDNA. We assessed the extent to which such ‘numt’ sequences may pose an overlooked problem in analyses of mtDNA from humans and apes. Using long-range polymerase chain reaction (PCR), we generated necessarily authentic mtDNA sequences for comparison with sequences obtained using typical methods for a segment of the mtDNA control region in humans, chimpanzees, bonobos, gorillas and orangutans. Results revealed that gorillas are notable for having such a variety of numt sequences bearing high similarity to authentic mtDNA that any analysis of mtDNA using standard approaches is rendered impossible. Studies on humans, chimpanzees, bonobos or orangutans are apparently less problematic. One implication is that explicit measures need to be taken to authenticate mtDNA sequences in newly studied taxa or when any irregularities arise. Furthermore, some taxa may not be amenable to analysis of mtDNA variation at all.

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Gene diversity patterns at 10 X-chromosomal loci in humans and chimpanzees

Authors: T .Kitano, C. Schwarz, B. Nickel , S. Pääbo
Journal: Mol Biol Evol. – 20(8):1281-9 (August 2003)

We have investigated the pattern and extent of nucleotide diversity in 10 X-chromosomal genes where mutations are known to cause mental retardation in humans. For each gene, we sequenced the entire coding region from cDNA in humans, chimpanzees, and orangutans, as well as about 3 kb of genomic DNA in 20 humans sampled worldwide and in 10 chimpanzees representing two ‘‘subspecies.’’ Overall nucleotide diversity in these genes is about twofold lower in humans than in chimpanzees, and nucleotide diversity within and between species is low, suggesting that a high level of functional constraint acts on these genes. Strikingly, we find that a summary of the allele frequency spectrum is significantly correlated in humans and chimpanzees, perhaps reflecting very similar levels of constraint at these genes in the two species. A possible exception is FMR2, which shows a higher number of nonsynonymous than synonymous substitutions on the human lineage, suggesting the action of positive selection.

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Nuclear Gene Sequences from a Late Pleistocene Sloth Coprolite

Authors: H. Poinar, M. Kuch, G. McDonald, P. Martin and S. Pääbo
Journal: Current Biology – Vol. 13 -1150–1152 (July 2003)

The determination of nuclear DNA sequences from ancient remains would open many novel opportunities such as the resolution of phylogenies, the sexing of hominid and animal remains, and the characterization of genes involved in phenotypic traits. However, to date, single-copy nuclear DNA sequences from fossils have been determined only from bones and teeth of woolly mammoths preserved in the permafrost [1]. Since the best preserved ancient nucleic acids tend to stem from cold environments [2, 3], this has led to the assumption that nuclear DNA would be retrievable only from frozen remains. We have previously shown that Pleistocene coprolites stemming from the extinct Shasta sloth (Nothrotheriops shastensis, Megatheriidae) contain mitochondrial (mt) DNA from the animal that produced them as well as chloroplast (cp) DNA from the ingested plants [4]. Recent attempts to resolve the phylogeny of two families of extinct sloths has been inconclusive [5].Wehave prepared DNA extracts from a ground sloth coprolite from Gypsum Cave, Nevada, and quantitated the number of mtDNA copies for three different fragment lengths by using real-time PCR.Weamplified one multicopy and three single-copy nuclear gene fragments and used the concatenated sequence to resolve the phylogeny. These results show that ancient single-copy nuclearDNAcan be recovered from warm, arid climates. Thus, nuclear DNA preservation is not restricted to cold climates.

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The Genetic Secrets Some Fossils Hold

Authors: H. Poinar
Journal: Accounts of Chemical Research – Vol. 35 – No. 8 (2002)

Most animals that once lived have gone extinct. The remains of a few of these can be found in museum collections worldwide. As modern evolutionary biology is limited to the use of extant taxa, retrieving DNA from extinct or subfossil organisms can add significant insight into past population history and resolve phylogenies that can be tentative by morphology alone. DNA is a relatively weak molecule, comparatively speaking, yet under certain conditions it persists in the fossil record, despite what in vitro chemistry predicts. While most fossil remains do not contain DNA, museum specimens can be screened for the presence of conditions that would be conducive for nucleic acid preservation by measuring the extent of amino acid racemization and by looking at the extent of protein hydrolysis by pyrolysis gas chromatography/mass spectrometry. Results from these types of analyses suggest that the preservation of DNA is linked to the temperature and its constancy at a site rather than its age. Chemical analyses of coprolites from extinct herbivores from the late Pleistocene, as well as Archaic Native Americans, show the presence of compounds from the Maillard reaction. Upon the cleaving of these products, the defecator can be identified and his diet analyzed.

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The top 10 list: criteria of authenticity for DNA from ancient and forensic samples

Authors: H.N. Poinar
Journal: International Congress – Series 1239 (2003)

Preserved archeological and paleontological specimens contain genetic information that has the power to elucidate the recent evolutionary history of humans, domesticates, and the pathogens they harbored. In addition, DNA from forensic samples is important in sample identification and crime solving. However, the DNA of both fossil and forensic remains can be heavily degraded through hydrolytic cleavage and oxidative base damage, limiting its successful retrieval and amplification. Obtaining authentic DNA sequences from both ancient and forensic remains presents extreme technical difficulties due to the minute amounts, and degraded nature, of DNA along with the exceptional risk of contamination. I reiterate here [1] a rigorous set of 10 criteria to ensure, to the greatest extent possible, meticulous replication and authentication of degraded DNA templates ubiquitous in all ancient and forensic samples.

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