Comparisons of Neandertal mtDNA with modern humans and modern chimpanzees

It is worth noting that ALL ancient DNA sequences should be considered tentative. The paper you have cited has already generated a responseMol Biol Evol. 2004 Apr 14 [Epub ahead of print] Related Articles, LinksMutations Induced by Ancient DNA Extracts? Letter.Serre D, Hofreiter M, Paabo S.Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Germany.We have investigated whether some factor in ancient DNA extracts induces site-specific mutations in modern DNA as described by Pusch and Bachmann (2004). We find no evidence for higher mutation rates when extracts from three different Pleistocene mammals are added to modern DNA than when water or extraction blanks are added. We also fail to find evidence that any such factor affects ancient DNA sequences determined from the same extracts. This as well as the patterns of nucleotide substitutions seen in DNA sequences determined from hundreds of other specimens leads us to doubt that a previously unknown mutagenic factor can be a general feature of extracts from old tissues.Thus, the results of the study you cite were in themselves, non-reproducible. Such "spiking" of DNA with ancient extracts does produce some artifacts like "jumping PCR"Paabo S, Irwin DM, Wilson AC.
DNA damage promotes jumping between templates during enzymatic amplification.
J Biol Chem. 1990 Mar 15;265(8):4718-21.But nothing like what Pusch and Bachmann report.However, there are three (at least)major problems with ancient DNA even disregarding the study of Pusch and Bachmann1. Nuclear copies of mtDNA (Numts) which can be extracted from ancient materialsGreenwood AD, Capelli C, Possnert G, Paabo S. Nuclear DNA sequences from late Pleistocene megafauna.
Mol Biol Evol. 1999 Nov;16(11):1466-73.(check out the woolly mammoth Numt sequence in the sequence figure)or which can totally flub up your phylogeny with modern DNA when comparing your sequences to ancient sequencesGreenwood AD, Castresana J, Feldmaier-Fuchs G, Paabo S. A molecular phylogeny of two extinct sloths.
Mol Phylogenet Evol. 2001 Jan;18(1):94-103.(check out the anteater Numt figure)2)contamination with modern DNAZischler H, Hoss M, Handt O, von Haeseler A, van der Kuyl AC, Goudsmit J. Links
Detecting dinosaur DNA.
Science. 1995 May 26;268(5214):1192-3; author reply 1194.(the most famous screw up in ancient DNA...other than the work on amber)3) non-random deamination, mutation during PCR amplification from ancient DNA extractsAgain see the pattern of substitutions in the sequence figures ofGreenwood AD, Capelli C, Possnert G, Paabo S. Nuclear DNA sequences from late Pleistocene megafauna.
Mol Biol Evol. 1999 Nov;16(11):1466-73.And here is a study that quantitates the problemHofreiter M, Jaenicke V, Serre D, Haeseler Av A, Paabo S. DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA.
Nucleic Acids Res. 2001 Dec 1;29(23):4793-9.

Another criteria (though not one that is universally applied) is that an independent laboratory reproduces the result i.e. the chance that two labs get mislead by the same contamination is vanishingly small for exampleNote here, a lab in Rome and a lab at the AMNH in New York both retrieved the same sequences from 3 mammoths in Figure 2.Greenwood AD, Lee F, Capelli C, DeSalle R, Tikhonov A, Marx PA, MacPhee RD.
Evolution of endogenous retrovirus-like elements of the woolly mammoth (Mammuthus primigenius) and its relatives.
Mol Biol Evol. 2001 May;18(5):840-7.This was also the case with the original nea paper from Krings et al.,
a part of the sequence was reproduced in the lab of M. Stoneking in Penn State.Though a cumbersome extra step, it goes a long way to confirming the sequences obtained.I should also note, that Krings et al. to their credit, were able to rule out (to a fairly high level of certainty) that the neandertal sequence was a Numt as they tested neandertal specific primers on a large panel of human DNAs of different ethnic origin and were never able to produce a product.Unfortunately, this type of rigor was not applied to the Mungo lake specimen which generated a sequence that is almost identical to a known human Numt and thus I (and others) are sceptical as to its ancient origins.Adcock GJ, Dennis ES, Easteal S, Huttley GA, Jermiin LS, Peacock WJ, Thorne A. Mitochondrial DNA sequences in ancient Australians: Implications for modern human origins.
Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):537-42. Erratum in: Proc Natl Acad Sci U S A 2002 Jan 8;99(1):541.In using ancient DNA to study human origins, one has an additional problem. Any sequenced retrieved is expected to be at least reasonably similar to modern human sequences. Thus, contamination may resemble the "expected" sequence and makes it harder to determine the authenticity of the sequence obtained. Issues of DNA damage and Numts make things monsterously complicated. This is less of an issue with other fauna. If you get a human-like sequence from a mammoth it means you screwed up and you know it right away. Now, if you could just get dolphins to do ancient human DNA work you would be in the clear

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The diagram shows a sudden discontinuity for Neandertals, and a consistency for anatomically modern humans which indicates that the methods for handling ancient DNA are probably reliable.

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I should have addressed this earlier so as not to generate so many independent posts and offend AdMoose by actually saying anything.This issue of reliability is not the biggest weak spot of neandertal and Cro-Magnon studies. Soon after the Cro-Magnon study came out, Alan Cooper and Svante Pbo wrote an op-ed objecting to the study on the grounds that if a sequence from Cro-Magnon is retrieved that is similar to modern human DNA sequences, it cannot be distinguished absolutely from a potential contamination and therefore must be rejected as authentic. Unfortunately (for them) this could be equally applied to the neandertals that Pbo studied. If you get a sequence from a neandertal specimen under rigorous lab protocol that is right within the middle of modern human genetic variation, it will be dismissed as a contaminant by many if not all the aDNA community (and won't be published). Why does this matter? If you can only accept sequences that are blatantly different from the human mtDNA gene pool as authentic and the question you are asking is "did neandertals contribute to the human mtDNA gene pool?" then your only possibility is to answer no. This is not scientific if you can throw out any data that points to a "yes" answer as was done with the Cro-Magnon results.

It still leaves the question open as to what exactly was in the extracts that Pusch and Bachmann were using? One that mutates naked DNA non-randomly? In my own experience I have seen random mutations among clones and in some cases a prevalence of C-T transitions (as is expected chemically anyway), but extracts that generate specific misleading haplotypes?[This message has been edited Mammuthus, 05-04-2004]

Actually, fragment size is not really a good indicator of authenticity as depending on the sample, some fairly large fragments of DNA may still remainComp Biochem Physiol B. 1985;81(4):1045-51. Related Articles, LinksIsolation and characterization of deoxyribonucleic acid from tissue of the woolly mammoth, Mammuthus primigenius.Johnson PH, Olson CB, Goodman M.DNA was isolated from tissue samples of several mammoth specimens, radiocarbon dated between 10,000 and 53,000 years old. The DNA was purified by chromatography on hydroxyapatite at 60 degrees C and was characterized as a heterogeneous population of fragments ranging in size from 3000 to 200 base pairs. Thermal denaturation analysis demonstrated that approximately 25% of the DNA had a base composition similar to Asian elephant DNA calculated as 36% G + C. Preliminary analysis by nucleic acid hybridization indicated that only a small fraction of DNA isolated from mammoth tissue (2-5%) was homologous to DNA of Asian elephant, a close living relative of the mammoth. Our results provide the first definitive isolation and characterization of DNA from ancient tissue and suggest a purification strategy that will lead to preparations of DNA from mammoth tissue significantly enriched in elephant-related DNA sequences.In addition, since it is usually assumed beforehand that the DNA is degraded, most researchers amplify short (100-300 bp) overlapping fragments and "walk out" to generate a larger sequence. The problem is if you land on a Numt and then build your sequence based on the Numt i.e. design primers that are sure to bind to the first sequence you retrieved, you will end up walking along the Numt and not the mtDNA. In any case, if you only get 100 bp fragments from a Cro-Magnon, no contamination, and can reproduce it in another lab, and it looks just like human, the question is, should it be considered authentic or should one have doubts? If it does not look human i.e. like the neandertal sequences, should one have doubts? Is it more convincing just because it looks different? I think this is the crucial problem facing those using aDNA to test human evolution hypotheses. Glad I work on megafauna

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I think we can all agree that mitDNA genomic insertions will experience different selective pressures than the mitDNA pool. Also, the two sequences will accrue different mutations. I would assume that mutations in numt's would be considered neutral for the most part. However, if this was a problem it should have shown up in the modern human vs Cro-Magnon pairwise comparison.

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This is true. However, Numt generation is an ongoing process and a newly inserted Numt could be identical or only slightly different from bona fide mtDNA. Here is an experiment that shows that really minor mutations that appeared to correlate with occurrence of Alzheimer's were in fact all Numts..they are very very similar to mtDNA and would fit nicely in the middle of the pairwise distributionBiochem Biophys Res Commun. 1998 Mar 27;244(3):877-83.
Evidence that two reports of mtDNA cytochrome c oxidase "mutations" in Alzheimer's disease are based on nDNA pseudogenes of recent evolutionary origin.Davis JN 2nd, Parker WD Jr.Center for the Study of Neurodegenerative Diseases, University of Virginia Health Sciences Center, Charlottesville 22908, USA.Recently, two reports [R. E. Davis et al. (1997) Proc. Natl. Acad. Sci. USA 94, 4564-4569 and E. Fahy et al. (1997) Nucleic Acids Res. 25, 3102-3109] described a series of heteroplasmic mitochondrial DNA (mtDNA) mutations in the genes encoding two cytochrome c oxidase subunits (CO1 and CO2) which segregated in higher abundance with Alzheimer's disease subjects than controls. Using mtDNA-depleted NT2 cells, we provide further evidence that these two reports are erroneously based on a PCR artifact arising from the amplification of nuclear DNA encoded mtDNA pseudogenes (mtDNA psi s). Our findings are similar, but not identical, to other recent studies of these putative mtDNA psi sequences. This sequence variability may indicate that multiple mtDNA psi s, all of comparatively recent evolutionary origin are involved. While such pseudogenes are interesting in that they provide a molecular evolutionary "snapshot" of human ancestral mtDNA, it is unlikely that they play any role in the etiology of Alzheimer's disease.

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A question for the experts. If there were a mix of numt and mitDNA in the PCR, what would the sequencing results look like. From my limited experience, it would seem that the predominant sequence at the start of the PCR would overpower the less predominant sequence. That is, the peaks coming off of the sequencer would reflect the predominant base at each PCR termination. The argument then is that the ratio of mitDNA to numt is high enough to preclude contamination from genomic sequences.

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If you look at the sequenceing figure in this paper, you can see what a mess Numts can cause...it was not possible from DNA alone to determine the correct sequence...in fact, direct sequencing produced exclusively Numt sequenceGreenwood AD, Paabo S.
Nuclear insertion sequences of mitochondrial DNA predominate in hair but not in blood of elephants.
Mol Ecol. 1999 Jan;8(1):133-7.these guys wrote a nice review of the Numt problem for different speciesBensasson D, Zhang D, Hartl DL, Hewitt GM. Related Articles, Links
Mitochondrial pseudogenes: evolution's misplaced witnesses.
Trends Ecol Evol. 2001 Jun 1;16(6):314-321.

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It would seem that even Mammuthus would have to control for numt PCR amplification, but luckily avoids the possibility of modern DNA contamination. For now, I will stick with the theory of neandertal mitDNA divergence from H. sapien, but tentatively so

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Actually, I don't control for it..I completely switched over to nuclear sequences for elephantid work. I could not get around the problem. There is even some reasonably convincing evidence that several of the published mammoth mtDNA sequences are Numts. But I do not think the neandertal sequence is a Numt. Krings did a huge number of controls with modern DNA samples to demonstrate that the sequence he obtained is not present in the modern human nuclear genome. Adcock et al. did not do this with the Mungo Lake sample which is a problem since it looks almost exactly like a known human Numt. My real problem with the nea/Cro-magnon work is that it is fairly widely assumed (in the aDNA community) that a sequence is only authentic if it looks different and thus testing the hypothesis of intermixing is not possible or falsifiable...that is a pretty big problem.By the way for both Sylas and yourself, there is a newly published nea study that anyone can access since it is in the free access journalsPLoS Biol. 2004 Mar;2(3):E57. Epub 2004 Mar 16. Related Articles, LinksNo Evidence of Neandertal mtDNA Contribution to Early Modern Humans.Serre D, Langaney A, Chech M, Teschler-Nicola M, Paunovic M, Mennecier P, Hofreiter M, Possnert G G, Paabo S.Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.The retrieval of mitochondrial DNA (mtDNA) sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution.Cheers,
M

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Do you feel that this is a valid control, and that this study adds credibility to the nea and amh mitDNA data? I tend to think it does, but not being an expert in the field . . .And just out of curiosity, are the authors above colleagues of yours? You don't have to sacrifice your anonymity, just wondering due to both you and the above research group are located in Germany and you are both into aDNA.

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I think it strengthens neandertal studies to have more samples processed. I worry a great deal that if there are neandertals that yield more human like sequences that they are not reported because the labs dismiss them as contaminants and focus on the ones that look different....as to you second question, they were my colleagues several years ago..I arrived in the lab the very week the first neandertal yielded its first PCR product

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I could order the paper, but that would be abusing our grant money And, I'm far to lazy to go to a local uni library. But, just from your description of "messed up" I can picture what the peaks coming off of the sequence would look like (if that is what you are talking about). Anyway, fully understand the technical problems numts create.

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Do you know if I post the journal name volume number and date etc like a normal reference, if I post the figure here does that violate the copyright? If it does not I will scan it and post it. If it does..I'll re-do the alignment from the Genbank sequences...it is worth showing I think...wish all journals would switch to open access so I could just link to the data cheers,
M

Let's see if this works(had to edit out the figure..I could not resize it and it kept crashing my computer when I tried to open the page...sorry Loudmouth...I will either have to send you a reprint or email the figure to you via Percy)This message has been edited by Mammuthus, 05-11-2004 05:43 AM

Thanks to both you and RAZD for your suggestions. I am pressed for time and on my way to a series of meetings over the next two weeks so I will try to send the figure to Percy before I go and see if he can get it in appropriate format....this is a wonderful example of why academic publishing that is not free access is really crap ..all the BMC journals and PLoS are open access and you can access all figures and data...without paying..the author pays to publish instead.

I sent the figure off to the Percy/Admin to see if he can get it in at a size that is reasonable.

Hi Loudmouth,
I'll send a copy of the article snail mail to you when I get back from the conference where I am presenting and my subsequent trying to sit still on a beach in southern France cheers,
M

...and the ball is still rolling...just saw this..Mol. Biol. Evol. 21(6):1081-1084. 2004
NUMTs in Sequenced Eukaryotic Genomes
Erik Richly and Dario Leister
Max-Planck-Institut fr Zchtungsforschung, Kln, GermanyCorrespondence: E-mail: leister@mpiz-koeln.mpg.de.Mitochondrial DNA sequences are frequently transferred to the nucleus giving rise to the so-called nuclear mitochondrial DNA (NUMT). Analysis of 13 eukaryotic species with sequenced mitochondrial and nuclear genomes reveals a large interspecific variation of NUMT number and size. Copy number ranges from none or few copies in Anopheles, Caenorhabditis, Plasmodium, Drosophila, and Fugu to more than 500 in human, rice, and Arabidopsis. The average size is between 62 (baker's yeast) and 647 bps (Neurospora), respectively. A correlation between the abundance of NUMTs and the size of the nuclear or the mitochondrial genomes, or of the nuclear gene density, is not evident. Other factors, such as the number and/or stability of mitochondria in the germline, or species-specific mechanisms controlling accumulation/loss of nuclear DNA, might be responsible for the interspecific diversity in NUMT accumulation.Key Words: duplication gene transfer genome evolution mitochondria NUMT pseudogene

Hi Loudmouth (and hello Rick)
I can only briefly reply as I am on a low bandwidth connection and don't have much time.Loudmouth's summary is good but my objection to the overall conclusions of the neandertal work are slightly different. I do not think the neandertal sequence (at least that of Krings et al.) represent Numts. They designed primers specific for the neandertal sequence and screened modern human DNA and from over 30 samples (if memory serves) of different ethnic origin, the nea sequence never turned up. Also, thousands of humans have been sequenced for mtDNA sequences and the nea sequence has never turned up.My problem is the following, if you have a neandertal bone (or Cro-magnon), and you do all the proper controls and get a human sequence, is it authentic (i.e. endogenous sequence), is it a Numt? can one accept it? According to Paabo and Cooper, no. If you get human sequences from Cro Magnon or neandertals, there is no way to discern whether you have obtained a contaminant from an endogenous authentic sequence. Thus, my objection is that if you cannot accept a modern human (or similar to modern human) sequences from neandertals or Cro-Magnon as authentic, then you cannot scientifically test their relationship to modern humans. By discarding sequences that are similar to modern human DNA, you a priori assume that neandertals were different from humans...and assuming your conclusions and discarding evidence against it sounds like creationism..not like science. Alternatively, it just may be that with current methodologies one cannot address the question of human-neandertal realtionships.