A proper network of Europeans


Back in May this year, Iosif Lazaridis submitted a paper to the arXiv, called: "The evolutionary history of human populations in Europe". It is now online as part of the December 2018 issue of Current Opinion in Genetics & Development (53: 21-27).

Its interest for readers of this blog is the one and only figure that the paper contains. It is a genealogical network, showing the obvious — that the human "family tree" has quite a few reticulations, mostly due to introgression (or admixture, as human geneticists like to call it). Here is the figure, along with the legend. Note that not all of the edges in the network have a direction, so that it is not really a directed acyclic graph (see also First-degree relationships and partly directed networks).


A sketch of European evolutionary history based on ancient DNA
Bronze Age Europeans (~4.5-3kya) were a mixture of mainly two proximate sources of ancestry: (i) the Neolithic farmers of ~8-5kya who were themselves variable mixtures of farmers from Anatolia and hunter-gatherers of mainland Europe (WHG), and (ii) Bronze Age steppe migrants of ~5kya who were themselves a mixture of hunter-gatherers of eastern Europe (EHG) and southern populations from the Near East. Thus, we only have to go ~8 thousand years backwards in time to find at least four sources of ancestry for Europeans. But, each of these sources was also admixed: European hunter-gatherers received genetic input from Siberia and ultimately also from archaic Eurasians, and Near Eastern populations interacted in unknown ways with Europe and Siberia and also had ancestry from ‘Basal Eurasians’, a sister group of the main lineage of all other non-African populations. Dates correspond to sampled populations; in the case of a cluster of populations (such as the WHG), they correspond to the earliest attestation of the group.

News about ancient humanity: Humans in California 130,000 years ago? Homo naledi find is much younger than expected

0000-0002-8715-2896 News about ancient humanity: Humans in California 130,000 years ago? Homo naledi find is much younger than expected   Posted May 5, 2017 by Tabitha M. Powledge in Uncategorized post-info AddThis Sharing Buttons above

Kissing between humans and Neanderthals? Could be oral – anal contact too. Or neither.

Umm - I really do not know what to say here. There is a new incredibly exciting paper out on Neanderthal oral microbiomes.

I saw some news stories about a new study on Neanderthal oral microbiomes. And one thing caught my eye - a claim about how the data provided evidence that Neanderthal's and humans were kissing each other.
See for example the LA Times: Vegetarian Neanderthals? Extinct human relatives hid a mouthful of surprises - LA Times
The scientists also managed to sequence the oldest microbial genome yet — a bug called Methanobrevibacter oralis that has been linked to gum disease. By looking at the number of mutations in the genome, the scientists determined it was introduced to Neanderthals around 120,000 years ago — near the edge of the time period when humans and Neanderthals were interbreeding, Weyrich said 
There are a few ways to swap this microbe between species, she pointed out: by sharing food, through parental care, or through kissing. 
“We really think that this suggests that Neanderthals and humans may have had a much friendlier relationship than anyone imagined,” Weyrich said. “Certainly if they’re swapping oral microorganisms — or swapping spit — it’s not these brute, rash-type encounters that people were suspecting happened during interbreeding. It’s really kind of friendly interactions.”
And Redorbit: Neanderthals were vegetarian– and probably kissed early humans



Another surprise was the discovery of the near-complete genome for Methanobrevibacter oralis, a microbe known to live between the gums and teeth of modern humans, in the dental calculus of the Neanderthals. Weyrich said that this organism is the oldest of its kind to ever be sequenced, and that its existence in Neanderthals means that it had to have been spread to humans somehow – likely through kissing, which supports the growing notion that humans and Neanderthals were known to become intimate with one another on occasion.
And the Washington Post Neanderthal microbes reveal surprises about what they ate — and whom they kissed



And there is this doozy of a quote in the Post article
“In order to get microorganisms swapped between people you have to be kissing,” Weyrich said.
And many others.  Now - this seemed like it would be really hard to prove.  After all, it is really hard to prove from microbiome data that two people have been kissing even when we have high quality data from many samples and even when we have data from both the possible donor and recipient.  So how could one show that humans and Neanderthals were kissing with data from ancient samples and only from one of the partners in the putative exchange?  Well, as far as I can tell, you cannot.

Sadly the paper is not open access and I generally avoid writing about closed access papers here. But I am making an exception here because the media has run with what I believe to be an inaccurate representation of the science.

So I went to the paper.  Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.  I have access to it at UC Davis but if you do not have access to it, you could search for it in SciHub (for more about SciHub see Wikipedia).  I am not encouraging you to use SciHub - a site that makes papers available view what may be illegal means in some countries.  But if you want to see the paper, and you have determined that you are OK with using SciHub, well, that is an option. This is a link that might get you access in SciHub, if you wanted to do that.

Anyway - I read the paper.  And it really is quite fascinating.  It has all sorts of interesting information and really does represent an incredible tour de force of both lab and computational work. Kudos to all involved.  But alas, there is nothing in the paper about kissing. If you search in the paper for the word kiss - it is not found. The possible transfer of microbes between Neanderthal and humans is briefly discussed however.

From what I can tell, what they did here was the following:


  1. reconstructed a genome from their samples of Methanobrevibacter oralis subsp. neandertalensis.
  2. compared the genomes to other Methanobrevibacter genomes including just one other M. oralis (this one from humans)
  3. Inferred a possbile possible date range for the split between their M. oralis and that from humans 


It is cool and very interesting stuff.  See this figure for example.



And then based on this they write:
Date estimates using a strict molecular clock place the divergence between the M. oralis strains of Neanderthals and modern humans between 112–143 ka (95% highest posterior density interval; mean date of 126 ka) (Fig. 3b; see Supplementary Information). As this is long after the genomic divergence of Neanderthals and modern humans (450–750 ka)29, it appears that commensal microbial species were transferred between the two hosts during subsequent interactions, potentially in the Near East30.

So they are inferring transfer of commensal microbes based on molecular clock dating from one single M. oralis genome from Neanderthal and one from humans and a comparison of the inferred dating of their common ancestor versus the timing of supposed divergence between humans and Neanderthal. Personally it seems like a big big stretch to make that inference here. What if the dating from their analysis is off (such dating estimates are generally highly debated and unclear how accurate they are)?

But let's just say that this is in fact good evidence for some sort of more recent common ancestry of the M. oralis found in their sample and the M. oralis found in a human than one would expect based on knowledge of Neanderthal and human common ancestry. Does that mean swapping of the microbes between humans and Neanderthal? Not at all. Maybe the M. oralis comes from food. And if it is living in some sort of food source (could be animal, or plant or something else) and it comes into both humans and Neanderthal separately, then one could easily have a way for the one found in their Neanderthal sample to have a more recent common ancestry with the one found from humans than the common ancestry of the "hosts" here.

Interestingly, the genome they used to compare to Methanobrevibacter oralis JMR01 actually came from a fecal sample and not an oral sample - see Draft Genome Sequencing of Methanobrevibacter oralis Strain JMR01, Isolated from the Human Intestinal Microbiota. So this microbe is not solely found in the mouth and it apparently can survive transit between the mouth and another orifice, and may even be a gut resident (i.e., not just transiting).

So anyway - it seems woefully premature to conclude that the data they have here provides evidence for exchange between humans and Neanderthals of M. oralis. Could have occurred. But also could be separate colonization from similarly environmental sources.

And finally, even if we assume that the M. oralis was exchanged, which again there seems to be no good evidence for, what is to suggest that this was do due to kissing? Nothing as far as I can tell. How about sharing utensils? How about contact with fecal contaminated water (since M. oralis seems to do OK in feces)? Or I guess would could go extreme and say this could be evidence for oral anal contact between Neanderthal and humans, if we wanted to sensationalize this even more. After all, we do know many cases of microbes getting exchanged by oral - anal contact. But we don't do we? How about we stick to what we have good evidence for and then carefully discuss possibilities, of which kissing is one, but it is just one of many and it relies upon a lot of conclusions for which the evidence is tenuous at best.


This There is really amazing science in this work. But the kissing claims are premature as far as I can tell (I honestly hope I am wrong and that there is more data than presented in the paper, but if there is it should be presented somewhere - or maybe I have misinterpreted the paper - but I don't think so). If the claims are as premature as they seem to be, this is damaging in my mind to the field of microbiome science.
-----------------------------

UPDATE 3/10/17

Thanks to Ed Yong for updating his Atlantic article on this story to add a reference to my concerns.

He wrote
But after the paper was published, and several publications noted Weyrich’s suggestion about kissing in their headlines, Jonathan Eisen from the University of California, Davis, expressed skepticism about the claim. “Maybe the M. oralis comes from food,” he wrote in a blog post. It could have been picked up independently from the environment, or from water contaminated with feces, or from other kinds of sexual contact. A kissing route “it is just one of many and it relies upon a lot of conclusions for which the evidence is tenuous at best,” Eisen said.

UPDATE 2 - Made a Storify of some responses

Concussion, TBI, human evolution, Neanderthal DNA, blogging news

Concussion, traumatic brain injury, and life’s hard knocks Search “concussion” in the media and you’ll come away thinking hard knocks to the head are chiefly a problem for kids and football players (or kid football players.) Last fall the blog … Continue reading »

The post Concussion, TBI, human evolution, Neanderthal DNA, blogging news appeared first on PLOS Blogs Network.

RIP ScienceOnline, cave art in Indonesia is as old as European cave art, how human were Neandertals?

This just in: RIP ScienceOnline (#scioX) ScienceOnline, which for the past few years has run the small annual meeting in North Carolina that brought together a disparate bunch of scientists and science groupies, most of them bloggers, is no more. … Continue reading »

The post RIP ScienceOnline, cave art in Indonesia is as old as European cave art, how human were Neandertals? appeared first on PLOS Blogs Network.

Modern Humans: Were We Really Better than Neanderthals, or Did We Just Get Lucky?

OLYMPUS DIGITAL CAMERA

We’ve all heard the story: dim-witted Neanderthals couldn’t quite keep up with our intelligent modern human ancestors, leading to their eventual downfall and disappearance from the world we know now. Apparently they needed more brain space for their eyes. The …

The post Modern Humans: Were We Really Better than Neanderthals, or Did We Just Get Lucky? appeared first on PLOS Blogs Network.

Why do we still use trees for the Neandertal genealogy?


I noted in an earlier post that studies of the dog genealogy seem to follow historical precedent, with trees being used for the analysis of whole-genome data and networks for the analysis of mitochondrial DNA data. However, domestic dog breeds do not have a simple tree-like ancestry, due to the cross-breeding involved in creating new breeds, and so the use of a tree model is inadequate. This was known long before the advent of molecular data, from comparative studies of phenotypes rather than genotypes, but genetic data have allowed us to attack this issue in a more directly quantitative way.

Anthropologists have traditionally used phylogenetic trees, especially when assessing the historical development of human "races", which have been assumed to maintain a strong degree of separation (see this earlier post). Clearly, networks would be more appropriate representations of history in many cases, especially where there is gene flow within a species or set of closely related species. This particularly applies to those fossils most closely related to humans, such as those of the Neandertals, a group of archaic hominins from the Middle Pleistocene who ranged right across Europe into western Siberia, but whose fossil record stops about 30,000 years ago (during the Late Pleistocene).

There have been a number of recent blog comments about the desirability of network analyses in historical anthropology (e.g. Dalton Luther, Jonathan Marks, PZ Myers, Dienekes Pontikos). As noted by Jason Antrosio, there "is a need to better understand and portray evolutionary complexity. With all the reports of Neandertal and Denisovan admixture, with all the emphasis on multispecies ethnography, with new looks at hybridization, we really must get away from the overly simplistic tree diagrams and taxonomies that have so long dominated evolutionary imagery". (Denisovans consist of a hominin fossil finger bone and some teeth from the Denisova Cave, in Siberia, which have yielded nucleotide sequences strikingly different from those of both Neandertals and modern humans. As Todd Wood has noted: "they're a genome in search of a fossil record.")

Here, I use networks to evaluate some of the available genotype data for the relationships between humans, Neandertals and Denisovans.

Nuclear genome

There is currently very little whole-genome data for ancient hominins, but what there is clearly shows "that Neandertals, Denisovans, and others labelled archaic are in fact an interbreeding part of the modern human lineage ... There has been extensive admixture across modern humans for tens of thousands of years, and at least some admixture across several archaic groups" (from Jason Antrosio again). Clearly, this is a situation for which networks were especially designed.

The relevant published papers include those on Denisovans (Reich et al. 2010, Meyer et al. 2012), Neandertals (Noonan et al. 2006, Green et al. 2006, 2010), an ancient human (Rasmussen et al. 2010), and the historical peopling of South-East Asia, Oceania and Australasia (Rasmussen et al. 2011, Reich et al. 2011, Skoglund and Jakobsson 2011, Mendez et al. 2012). Of these, only Mendez et al.  and Meyer et al. used a network to analyze the evolutionary history (a Median-Joining network and an Admixture graph, respectively); the others used trees, ordinations and/or 3- and 4-taxon comparisons of genetic distances. The obvious question to ask is whether a tree is appropriate here.

As an example, we can take the "pairwise autosomal DNA sequence divergences" provided by Reich et al. (2010) for five of the genomes for which they collected SNP data. We cannot derive an evolutionary network directly from these data, but a data-display network will allow us to assess how tree-like are the data presented. Figure 1 shows a NeighborNet analysis of the data. This indicates that the data are strongly tree-like, mainly because of the authors concerted attempts to "clean up" the data from sequencing and analysis artifacts that would otherwise obscure the tree signal in ancient DNA. Nevertheless, there are still two detectable non-tree signals: one linking the Denisovan to the Neandertal from Mezmaiskaya (both fossil locations are in Russia), and a larger one linking the Denisovan to the Yoruba human (from a West African ethnic group). The first signal may represent non-tree gene flow, although the second signal is harder to explain (ancestral polymorphism, perhaps?).

Figure 1. NeighborNet analysis of the autosomal DNA sequence
divergences for two modern humans (San, Yoruba), two fossil
Neandertals (Mezmaiskaya, Vindija), and a fossil Denisovan.

Mitochondrial genome

Mitochondrial DNA (mtDNA) is the most commonly collected source of genetic data, especially sequences of the so-called control region (including the D-loop). Moreover, it is now quite commonplace to sequence the >16,500 nt of the mtDNA genome, as indicated by the contents of the mtDB (Ingman and Gyllensten 2006) and MitoTool (Fan and Yao 2011) databases. Mitochondrial DNA has also been successfully extracted from ancient hominins. Indeed, there are now sequences for the entire mtDNA genome of Denisovans (Krause et al. 2010a), Neandertals (Green et al. 2008, Briggs et al. 2009), and early modern humans (Ermini et al. 2008, Gilbert et al. 2008, Krause et al. 2010b). Compared to nuclear DNA, ancient mtDNA has a greater survival rate and greater degree of sequencing coverage, which leads to a markedly reduced influence of post-mortem damage and contamination (see Ho and Gilbert 2010).

The major assumed advantages of using mtDNA are (i) the high copy number, (ii) the maternal mode of inheritance, (iii) the high substitution rate (resulting in variation even at the intraspecific level), (iv) the lack of recombination (so that historical relationships can be modelled by a phylogenetic tree), and (v) the molecular clock is considered to be relatively reliable (so that the dates of historical events can be estimated). Both of these latter two assumptions have been disputed, however, as discussed by McVean (2001) for recombination and Endicott et al. (2009, 2010) for the clock.

The available data indicate that recombination in mtDNA is rare, if it occurs at all. Furthermore, gene flow is unlikely to complicate the historical relationships, because the mitochondrion is almost always inherited maternally and there is little evidence of historical movement by single females between populations, as opposed to movement by males. So, a phylogenetic tree is a reasonable model of evolutionary history for mtDNA, unlike the situation for the nuclear genome.

On the other hand, there are a number of issues that will make any attempt to reconstruct a tree problematic. That is, the data will not be tree-like, even if the genealogical history was tree-like. First, the genes in mtDNA are completely linked as a single locus, which will lead to deep coalescence (incomplete lineage sorting), thus disconnecting gene history and organism history. Second, mtDNA exhibits considerable heterogeneity in nucleotide-substitution rates along the genome, with the control region having very high rates (up to 10x that of the reset of the mtDNA) and codon second positions having very low rates. Indeed, it is likely that substitutional saturation occurs in the control region, and that purifying selection occurs at first and second codon positions. There will be an enormous amount of homoplasy under these circumstances (eg. parallel substitutions). Third, there is evidence of different nucleotide-substitution rates in different lineages, even when those lineages are closely related. This will also cause homoplasy.

There have been three responses to these problems by those who study human mtDNA. First, trimming of the sequence data occurs. For example, there are well-known nucleotide positions that are usually deleted because their variation seems random, and others whose excessive variation leads them to be down-weighted. Second, a network is used to assess how non-tree-like are the data. People have developed several network methods explicitly for mtDNA data, such as Median-Joining and Reduced-Median networks; and the literature is replete with papers using these methods to analyze mtDNA sequences. Third, a partitioned model is needed in order to build a phylogenetic tree. Notably, the different codon positions need separate substitution models, as do the control region and the RNA-coding regions. Furthermore, rate heterogeneity needs to be modelled, and a relaxed molecular clock is needed.

Figure 2. An approximate Median Network (based on a
Median-Joining analysis) of control region sequences from
13 fossil Neandertals and 1 fossil Denisovan.

These problems are bad enough for the study of within-human phylogenies, but they are even more problematic for the study of ancient DNA. For example, substitutional saturation means that the control region, and especially the three hypervariable regions (HVR1,HVR2,HVR3) that are the most frequently sequenced parts of it, is almost useless for reconstructing ancient history. This can be seen, for example, in the data of Dalén et al. (2012), who analyzed the mtDNA control regions of 13 Neandertals and 1 Denisovan. Dalén et al. produced a bayesian tree from these data, but in Figure 2 I show a Median Network instead. (This displays all of the maximum-parsimony trees simultaneously.) There may well be an evolutionary tree in these data, but if so then it is pretty deeply buried, and it is unlikely to be recovered reliably without a lot of work.

Unfortunately, for the study of ancient DNA very little seems to be done about the problems of homoplasy, in terms of any of the three suggested solutions. Indeed, most of the concern seems to be about potential post mortem damage to the DNA (eg. extra substitutions in the terminal branches), instead. For example, I have checked 21 empirical phylogenetic studies involving Neandertal mtDNA (published since 1997), and only 6 of them noted that they had either down-weighted or excluded particular hyper-variable nucleotide positions: Krings et al. (1999, 2000), Caramelli et al. (2006), Ermini et al. (2008), Moradi and Schuster (2008) and Endicott et al. (2010). Second, only three of the papers presented an empirical network analysis: Ermini et al. (2008) (a Reduced-Median network), Caramelli et al. (2006) (a Median-Joining network) and Caramelli et al. (2008) (a TCS network); for the rest, they either presented a tree, an ordination, or no empirical diagram at all. Third, only two of the analyses performed a partitioned tree-building analysis: Green et al. (2008) and Endicott et al. (2010). Finally, 14 of the 21 papers were based on sequences of the control region only, which makes their phylogenetic inferences questionable.

If I concentrate here on the production of a phylogenetic network, as I should be doing in this blog, then it is will become obvious why tree-building analyses are rather difficult for Neandertal sequence data. Figure 3 uses a data-display network to show the non-tree features of the available Neandertal mtDNA genomes. Note that there is very little common variation at all, meaning that Neanderthal mtDNA has very limited genetic variation. Moreover, there are no tree-like parts to the diagram, with every parsimony-informative nucleotide position being contradicted by at least one other. Analyzing these data with a simple tree-building analysis seems to be inappropriate, to say the least.

Figure 3. Median Network analysis of the six full-length mtDNA
genomes currently available for Neandertals. The numbers on the
branches indicate the number of characters that change along
each branch.

To assess the relationship between Neandertals and humans (which seems to be the most common ancient-DNA question addressed in the literature), we can add the Denisovan mtDNA sequence, plus the 3 available sequences for early modern humans, and also some sequences from a range of modern humans (ie. the revised Cambridge Reference Sequence, plus 53 sequences from Ingman et al. 2000). However, we then cannot plot the Median Network because several of the aligned positions are no longer binary (ie. they are not SNPs). So, I will use a NeighborNet analysis for the data display instead, as shown in Figure 4. The first thing to note is that the genetic variation in the Neanderthal mtDNA is much less than that in the human mtDNA, and probably less than can be accounted for solely by the smaller sample size (6 genomes versus 54).

Figure 4. NeighborNet analysis of the mtDNA genomes from
6 Neandertals, 1 Denisovan, 3 early modern humans and 54
contemporary humans, based on uncorrected genetic distances.

Second, there is clearly an underlying tree-like structure to the data, as expected, which I have emphasized by plotting the related Neighbor-Joining tree for comparison in Figure 5 (the NeighborNet analysis is a generalization of the Neighbor-Joining tree). However, there is just as clearly considerable non-tree structure to the data, notably in the relationship of the Denisovan sequence to the other sequences, but also in the relationship between the Neandertals and the humans. It is this non-tree structure that complicates any attempt to reconstruct the evolutionary relationship of the Neandertals to humans; and it appears to result, at least partly, from the homoplasy caused by saturation of nucleotide substitutions.

Figure 5. Neighbor-Joining tree of the same data used for Figure 4.

However, even the NeighborNet analysis cannot summarize all of the non-tree patterns in the data, but presents instead a selective summary of them. To get further insight into the extent of the problem, I have deleted the 53 human sequences, and then plotted the Pruned Quasi-median network in Figure 6. This network is the equivalent of the Median Network while allowing for non-binary sequence positions. It is difficult to believe that these data were created by a simple tree-like evolutionary process, and, if so, that it will be easy to reconstruct it.

Figure 6. Pruned Quasi-median network analysis of the mtDNA
genomes from 6 Neandertals, 1 Denisovan, 3 early modern humans
and 1 contemporary human (the revised Cambridge Reference
Sequence). The branch lengths are not drawn to scale.

Anyway, the most-common network approach to trying to untangle this sort of mess in mtDNA sequence data is to use either a Reduced-Median network or a Median-Joining network, which are simplifications of the full Median Network. I have produced a Median-Joining network in Figure 7, as an example. The interesting thing to note here is that the Denisovan sequence does not connect to the rest of the network between the Neandertal cluster and the human cluster of sequences, which it does do in all of the published phylogenetic trees. This pattern is not unexpected, given the pattern shown in the Pruned Quasi-median network (Figure 6), but it does suggest that the tree-building analyses performed to date are somewhat naïve in the face of considerable sequence complexity, by not explicitly dealing with that complexity.

Figure 7. Median-Joining network analysis of  the same data used
for Figure 4. Only the sequences from Figure 6 are labelled — the
other dots are the remaining 53 contemporary humans, plus some
inferred ancestors. The branch lengths are not drawn to scale.

Conclusion

The phylogenetic analysis of Neandertal mtDNA has been critiqued a number of times before (eg. Gutiérrez et al. 2002, Hebsgaard et al. 2007, Moradi and Schuster 2008, Endicott et al. 2010). However, this has always been in the context of "providing a better tree-building analysis", rather than in the context of evaluating and displaying the conflicting information that complicates the tree-building analysis, as I have done here. In this context, it is important to note that none of the diagrams that I have produced here are evolutionary networks, and so they do not represent a reconstruction of evolutionary history. They are intended merely to display the convoluted nature of the ancient mtDNA sequence data, and to emphasize the valuable role that phylogenetic networks can play in evaluating such data.

One further point worth noting is that these diagrams are all unrooted, which neatly avoids the problems associated with adding a chimpanzee sequence in order to locate the root of the evolutionary history. Adding this sequence dramatically increases the sequence complexity, of course. In particular, the nuclear genome apparently places the Denisovan as the sister to the Neandertals whereas the mtDNA places it as the sister to Neandertals+humans (eg. note that the mid-point rooting of Figure 5 would be on the branch leading to the Denisovan).

References

Briggs A.W., Good J.M., Green R.E., Krause J., Maricic T., Stenzel U., Lalueza-Fox C., Rudan P., Brajkovic D., Kucan Z., Gusic I., Schmitz R., Doronichev V.B., Golovanova L.V., de la Rasilla M., Fortea J., Rosas A., Paabo S. (2009) Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science 325: 318-321.

Caramelli D., Lalueza-Fox C., Condemi S., Longo L., Milani L., Manfredini A., de Saint Pierre M., Adoni F., Lari M., Giunti P., Ricci S., Casoli A., Calafell F., Mallegni F., Bertranpetit J., Stanyon R., Bertorelle G., Barbujani G. (2006) A highly divergent mtDNA sequence in a Neandertal individual from Italy. Current Biology 16: R630-R632.

Caramelli D., Milani L., Vai S., Modi A., Peccholi E., Girardi M., Pilli E., Lari M., Lippi B., Ronchitelli A., Mallegni F., Casoli A., Bertorelle G., Barbujani G. (2008) A 28,000 years old Cro-Magnon mtDNA sequence differs from all potentially contaminating modern sequences. PLoS One 3: e2700.

Dalén L., Orlando L., Shapiro B., Durling M.B., Quam R., Gilbert T.M.P., Díez Fernández-Lomana C.J., Willerslev E., Arsuaga J.L., Götherström A. (2012) Partial genetic turnover in neandertals: continuity in the east and population replacement in the west. Molecular Biology and Evolution 29: 1893-1897.

Endicott P., Ho S.Y.W., Metspalu M., Stringer C. (2009) Evaluating the mitochondrial timescale of human evolution. Trends in Ecology and Evolution 24: 515-521.

Endicott P., Ho S.Y.W., Stringer C. (2010) Using genetic evidence to evaluate four palaeoanthropological hypotheses for the timing of Neanderthal and modern human origins. Journal of Human Evolution 59: 87-95.

Ermini L., Olivieri C., Rizzi E., Corti G., Bonnal R., Soares P., Luciani S., Marota I., De Bellis G., Richards M.B., Rollo F. (2008) Complete mitochondrial genome sequence of the Tyrolean Iceman. Current Biology 18: 1687-1693.

Fan L., Yao Y.G. (2011) MitoTool: a web server for the analysis and retrieval of human mitochondrial DNA sequence variations. Mitochondrion 11: 351-356.

Gilbert M.T., Kivisild T., Grønnow B., Andersen P.K., Metspalu E., Reidla M., Tamm E., Axelsson E., Götherström A., Campos P.F., Rasmussen M., Metspalu M., Higham T.F., Schwenninger J.L., Nathan R., De Hoog C.J., Koch A., Møller L.N., Andreasen C., Meldgaard M., Villems R., Bendixen C., Willerslev E. (2008) Paleo-Eskimo mtDNA genome reveals matrilineal discontinuity in Greenland. Science 320: 1787-1789.

Green R.E., Krause J., Briggs A.W., Maricic T., Stenzel U., Kircher M., Patterson N., Li H., Zhai W., Fritz M.H., Hansen N.F., Durand E.Y., Malaspinas A.S., Jensen J.D., Marques-Bonet T., Alkan C., Prüfer K., Meyer M., Burbano H.A., Good J.M., Schultz R., Aximu-Petri A., Butthof A., Höber B., Höffner B., Siegemund M., Weihmann A., Nusbaum C., Lander E.S., Russ C., Novod N., Affourtit J., Egholm M., Verna C., Rudan P., Brajkovic D., Kucan Z., Gusic I., Doronichev V.B., Golovanova L.V., Lalueza-Fox C., de la Rasilla M., Fortea J., Rosas A., Schmitz R.W., Johnson P.L., Eichler E.E., Falush D., Birney E., Mullikin J.C., Slatkin M., Nielsen R., Kelso J., Lachmann M., Reich D., Pääbo S. (2010) A draft sequence of the Neandertal genome. Science 328: 710-722.

Green R.E., Krause J., Ptak S.E., Briggs A.W., Ronan M.T., Simons J.F., Du L., Egholm M., Rothberg J.M., Paunovic M., Pääbo S. (2006) Analysis of one million base pairs of Neanderthal DNA. Nature 444: 330-336.

Green R.E., Malaspinas A.S., Krause J., Briggs A.W., Johnson P.L., Uhler C., Meyer M., Good J.M., Maricic T., Stenzel U., Prüfer K., Siebauer M., Burbano H.A., Ronan M., Rothberg J.M., Egholm M., Rudan P., Brajković D., Kućan Z., Gusić I., Wikström M., Laakkonen L., Kelso J., Slatkin M., Pääbo S. (2008) A complete neandertal mitochondrial genome sequence determined by high-throughput sequencing. Cell 134: 416-426.

Gutiérrez G., Sánchez D., Marín A. (2002) A reanalysis of the ancient mitochondrial DNA sequences recovered from Neandertal bones. Molecular Biology and Evolution 19: 1359-1366.

Hebsgaard M.B., Wiuf C., Gilbert M.T., Glenner H., Willerslev E. (2007) Evaluating Neanderthal genetics and phylogeny. Journal of Molecular Evolution 64:50-60.

Ho S.Y.W., Gilbert M.T.P. (2010) Ancient mitogenomics. Mitochondrion 10: 1-11.

Ingman M., Gyllensten U. (2006) mtDB: Human Mitochondrial Genome Database, a resource for population genetics and medical sciences. Nucleic Acids Research 34: D749–D751.

Ingman M., Kaessmann H., Pääbo S., Gyllensten U. (2000) Mitochondrial genome variation and the origin of modern humans. Nature 408: 708-713.

Krause J., Briggs A.W., Kircher M., Maricic T., Zwyns N., Derevianko A., Pääbo S. (2010b) A complete mtDNA genome of an early modern human from Kostenki, Russia. Current Biology 20: 231-236.

Krause J., Fu Q., Good J.M., Viola B., Shunkov M.V., Derevianko A.P., Paabo S. (2010a) The complete mitochondrial DNA genome of an unknown hominin from southern Siberia. Nature 464: 894-897.

Krings M., Geisert H., Schmitz R.W., Krainitzki H., Pääbo S. (1999) DNA sequence of the mitochondrial hypervariable region II from the Neandertal type specimen. Proceedings of the National Academy of Sciences of the USA 96: 5581-5585.

Krings M., Capelli C., Tschentscher F., Geisert H., Meyer S., von Haeseler A., Grossschmidt K., Possnert G., Paunovic M., Pääbo S. (2000) A view of Neandertal genetic diversity. Nature Genetics 26: 144-146.

McVean G.A.T. (2001) What do patterns of genetic variability reveal about mitochondrial recombination? Heredity 87: 613-620.

Mendez F.L., Watkins J.C., Hammer M.F. (2012) Global genetic variation at OAS1 provides evidence of archaic admixture in Melanesian populations. Molecular Biology and Evolution 29: 1513-1520.

Meyer M., Kircher M., Gansauge M.-T., Li H., Racimo F., Mallick S., Schraiber J.G., Jay F., Prüfer K., de Filippo C., Sudmant P.H., Alkan C., Fu Q., Do R., Rohland N., Tandon A., Siebauer M., Green R.E., Bryc K., Briggs A.W., Stenzel U., Dabney J., Shendure J., Kitzman J., Hammer M.F., Shunkov M.V., Derevianko A.P., Patterson N., Andrés A.M., Eichler E.E., Slatkin M., Reich D., Kelso J., Pääbo S. (2012) A high-coverage genome sequence from an archaic Denisovan individual. Science (advance)

Moradi C.R., Schuster A. (2008) Evaluation of the critical factors in the phylogenetic analysis of human and neanderthal mtDNA. Unpublished ms.

Noonan J.P., Coop G., Kudaravalli S., Smith D., Krause J., Alessi J., Chen F., Platt D., Pääbo S., Pritchard J.K., Rubin E.M. (2006) Sequencing and analysis of Neanderthal genomic DNA. Science 314: 1113-1118.

Rasmussen M., Guo X., Wang Y., Lohmueller K.E., Rasmussen S., Albrechtsen A., Skotte L., Lindgreen S., Metspalu M., Jombart T., Kivisild T., Zhai W., Eriksson A., Manica A., Orlando L., De La Vega F.M., Tridico S., Metspalu E., Nielsen K., Ávila-Arcos M.C., Moreno-Mayar J.V., Muller C., Dortch J., Gilbert M.T., Lund O., Wesolowska A., Karmin M., Weinert L.A., Wang B., Li J., Tai S., Xiao F., Hanihara T., van Driem G., Jha A.R., Ricaut F.X., de Knijff P., Migliano A.B., Gallego Romero I., Kristiansen K., Lambert D.M., Brunak S., Forster P., Brinkmann B., Nehlich O., Bunce M., Richards M., Gupta R., Bustamante C.D., Krogh A., Foley R.A., Lahr M.M., Balloux F., Sicheritz-Pontén T., Villems R., Nielsen R., Wang J., Willerslev E. (2011) An Aboriginal Australian genome reveals separate human dispersals into Asia. Science 334: 94-98.

Rasmussen M., Li Y., Lindgreen S., Pedersen J.S., Albrechtsen A., Moltke I., Metspalu M., Metspalu E., Kivisild T., Gupta R., Bertalan M., Nielsen K., Gilbert M.T., Wang Y., Raghavan M., Campos P.F., Kamp H.M., Wilson A.S., Gledhill A., Tridico S., Bunce M., Lorenzen E.D., Binladen J., Guo X., Zhao J., Zhang X., Zhang H., Li Z., Chen M., Orlando L., Kristiansen K., Bak M., Tommerup N., Bendixen C., Pierre T.L., Grønnow B., Meldgaard M., Andreasen C., Fedorova S.A., Osipova L.P., Higham T.F., Ramsey C.B., Hansen T.V., Nielsen F.C., Crawford M.H., Brunak S., Sicheritz-Pontén T., Villems R., Nielsen R., Krogh A., Wang J., Willerslev E. (2010) Ancient human genome sequence of an extinct Palaeo-Eskimo. Nature 463: 757-762.

Reich D., Green R.E., Kircher M., Krause J., Patterson N., Durand E.Y., Viola B., Briggs A.W., Stenzel U., Johnson P.L.F. (2010) Genetic history of an archaic hominin group from Denisova Cave in Siberia. Nature 468: 1053-1060.

Reich D., Patterson N., Kircher M., Delfin F., Nandineni M.R., Pugach I., Ko A.M., Ko Y.-C., Jinam T.A., Phipps M.E., Saitou N., Wollstein A., Kayser M., Pääbo S., Stoneking M. (2011) Denisova admixture and the first modern human dispersals into Southeast Asia and Oceania. American Journal of Human Genetics 89: 516-528.

Skoglund P., Jakobsson M. (2011) Archaic human ancestry in East Asia. Proceedings of the National Academy of Sciences of the USA 108: 18301-18306.