May 2010

from ScienceMag Website




Recent advances in high-throughput DNA sequencing have provided initial glimpses of the nuclear genome of Neanderthals as well as other ancient mammals including cave bears and mammoths.


In the 7 May 2010 issue of Science, an international team of researchers presents the draft sequence of the Neanderthal genome composed of over 3 billion nucleotides from three individuals.

Because Neanderthals are much closer kin to us than are chimpanzees, which diverged from the human lineage 5 to 7 million years ago, matching Neanderthal DNA against our own has the potential to reveal genetic changes that help define who we are.



About Neanderthals

Neanderthals (Homo neanderthalensis) are currently believed to be our closest evolutionary relatives.


Although some researchers once thought they were our immediate ancestors in Europe, most now agree that Neanderthals and modern humans most likely shared a common ancestor within the last 500,000 years, possibly in Africa.

The morphological features typical of Neanderthals first appear in the European fossil record about 400,000 years ago, with bones of full-fledged Neanderthals showing up at least 130,000 years ago.


They lived in Europe and western Asia, as far east as southern Siberia and as far south as the Middle East (see below map), before disappearing from the fossil record about 30,000 years ago.

At home in Eurasia. Neanderthals ranged from Europe to southern Siberia.

Fossil remains and anatomical reconstructions indicate that the typical Neanderthal had a stocky muscular body with short forearms and legs, a large head with bony brow ridges and a brain slightly larger than ours, a jutting face with a large nose, and perhaps reddish hair and fair skin.


Neanderthals made and used a diverse set of sophisticated tools, controlled fire, organized their living spaces, hunted and fed on game of various sizes, and occasionally made symbolic or ornamental objects.

For more Neanderthal basics, see A Neanderthal Primer by M. Balter, Science 323, 870 (2009).


Timeline of Discoveries
The first Neanderthal fossils were discovered in 1829 in Engis, Belgium, and in 1848 at Forbes' Quarry, Gibraltar, but were not recognized as an early human species until after the 1856 discovery of "Neanderthal 1" - a 40,000-year-old specimen, including a skullcap and various bones, found at the Kleine Feldhofer Grotte in the Neander Valley near Düsseldorf, Germany.

This timeline highlights key discoveries about our closest relatives, from early fossil finds to the publication of the draft nuclear genome sequence.


Timeline References


1. - J. C. Fuhlrott, Verh. naturhist. Ver. preuss. Rheinl. 14, Corr. Bl., 50. (1857)
2. - H. Schaaffhausen, Verh. naturhist. Ver. preuss. Rheinl. 14, Corr. Bl., 50–52. (1857)
3. - W. King, Quarterly Review of Science 1, 88 (1864).
4. - Lévêque and Vandermeersch, Bulletin de la Société Préhistorique Francaise 77, 35 (1980).
5. - Rak and Arensburg, Am. J. Phys. Anthropol. 73, 227 (1987).
6. - Krings et al., Cell 90, 19 (1997).
7. - Ovchinnikov et al., Nature 404, 490 (2000).
8. - Noonan et al., Science 314, 1113 (2006).
9. - Green et al., Nature 444, 330 (2006).
10. - Krause et al., Nature 449, 902 (2007).
11. - Lalueza-Fox et al., Science 318, 1453 (2007).
12. - Krause et al., Curr. Biology 17, 1908 (2007).
13. - Green et al., Cell 134, 416 (2008).
14. - Lalueza-Fox et al., BMC Evol. Biol. 8, 342 (2008).
15. - Briggs et al., Science 325, 318 (2009)
16. - Zilhao et al., Proc. Natl. Acad. Sci. U.S.A. 107, 1023 (2010).
17. - Fabre et al., PLoS ONE 4, e5151. doi:10.1371/journal.pone.0005151 (2010).
18. - Green et al., Science 328, 710 (2010).


The Samples
To sequence the Neanderthal genome, Green et al. began by analyzing 21 Neanderthal bones from Vindija Cave in Croatia. The team removed just 50 to 100 mg of bone powder from each bone and screened each sample for the presence of Neanderthal mitochondrial DNA (mtDNA) by PCR.


Three of the bones, which were determined to represent three different female individuals, were ultimately chosen for sequencing analysis.

  • The first bone dates to about 38,000 years ago and has been used for earlier genome sequencing efforts, including determination of a complete mitochondrial DNA sequence.

  • The second bone is undated but was found in an older stratigraphic layer than the first bone.

  • The third bone has been dated to about 44,500 years ago.


Ancient DNA Challenges
Extracting and analyzing ancient DNA is fraught with challenges.


Genetic material degrades into small fragments over time, with errors often introduced into the aging sequence. Moreover, 95 to 99% of fossil DNA can be sequences from microbes that have infiltrated the decaying bone.


Ancient human remains pose additional challenges in that well-preserved samples are rare and have typically been handled by curators and researchers, thus raising the possibility that they have been contaminated by modern human DNA. Because Neanderthals and humans are so closely related, distinguishing between their DNA can be nearly impossible.

Several precautions were taken in Green et al. to minimize contamination during the Neanderthal genome project including:

  • DNA extraction procedures performed in a "clean room" with sterile equipment

  • the addition of unique sequence tags to ancient DNA molecules in the clean room, making it possible to identify contamination from other DNA sources

  • the use of enzymes that preferentially cut microbial DNA to increase the relative proportion of Neanderthal DNA in the sequencing libraries

Sequencing Approach
High-throughput sequencing technologies and advances in metagenomic analysis of complex DNA mixtures have enabled the recovery of genomic sequences from ancient samples on a budget and timescale not previously achievable.


To decipher the Neanderthal genome, Green et al. used an approach known as pyrosequencing, which enables the sequencing of hundreds or thousands of DNA molecules at the same time, thus generating smaller pieces of sequences faster and cheaper than classical Sanger sequencing.


One of the limitations of this technique is that the length of DNA that can be read in any single sequencing run is very short. For the Neanderthal genome, these short sequences were then assembled using a combination of sophisticated alignment algorithms as well as the completed human and chimpanzee genome sequences as guides.


This has resulted in ~1.3-fold coverage of the entire Neanderthal genome.

Comparison of Neanderthal and present-day human genomes can reveal information about genetic changes that have occurred before and after the ancestral population split of modern humans and Neanderthals (see the Comparative Genomics section for more). However, low coverage sequencing inevitably leaves a substantial proportion of the genome uncovered.


To recover additional information about specific regions of interest, Burbano et al. used a microarray-based approach to sequence ~14,000 protein-coding regions inferred to have changed in the human lineage since the last common ancestor shared with chimpanzees. By generating the sequence of one Neanderthal and 50 present-day humans at these positions, the researchers identified 88 amino acid substitutions that have become fixed in humans since our divergence from the Neanderthals (see table).


Further studies will be needed to investigate the possible functional significance of these genetic changes.


Examples of genes

in which amino acid substitutions have become fixed in humans

since our divergence from Neanderthals


Gene Symbol

Gene Name

Molecular Function


ATP-binding cassette, subfamily C (CFTR/MRP), member 12

ATP-binding cassette (ABC) transporter


cancer susceptibility candidate 5



potassium voltage-gated channel, subfamily H (eag-related), member 8

Voltage-gated potassium channel


lysosomal trafficking regulator

Select regulatory molecule


melanin-concentrating hormone receptor 2

G-protein coupled receptor


olfactory receptor, family 5, subfamily K, member 4



pancreatic lipase



sperm associated antigen 17



stabilin 1

Extracellular matrix structural protein


zinc finger, HIT type 2

Zinc finger transcription factor

Comparative Genomics

Human-Neanderthal Comparisons
Comparisons of the human genome to the genomes of Neanderthals and apes can help identify features that set modern humans apart from other hominin species.


In particular, the Neanderthal genome sequence can now be used to catalog changes that have become "fixed" (are invariant within a population or species) in modern humans during the last few hundred thousand years and should be helpful for identifying genes affected by positive selection since humans diverged from Neanderthals.

To help make informative comparisons, Green et al. sequenced the genomes of 5 present-day individuals from different parts of the world:

  • Southern Africa

  • West Africa

  • Papua New Guinea

  • China

  • Western Europe (see below map)

They then compared the genomes of these individuals with the genomes of the Neanderthal and the chimpanzee, and looked both for specific regions shared by present-day humans but lacking in Neanderthals and regions showing high frequencies of more recently evolved (derived) sequences.


These regions signal the presence of mutations that occurred and swept to either high frequency or fixation after humans and Neanderthal diverged, and that may have contributed to modern human-specific traits.

Using this comparative approach, Green et al. came up with a list of 20 candidate regions that may have been affected by positive selection in ancestral modern humans.


Five of these regions contain no protein-coding genes and may thus include structural or regulatory elements. Among the remaining 15 regions, the team identified genes involved in metabolism and cognitive and cranial development, which suggests that aspects of these processes may have been functionally important for the evolution of modern humans.

For more on Neanderthal-modern human comparisons see the News story by A. Gibbons.

Five modern human genomes.

Locations of present-day individuals whose genomes were sequenced

by Green et al. for comparative analyses.


Evidence of Admixture
Substantial controversy surrounds the question of whether Neanderthals interbred with modern humans.


To address this question, Green et al. tested whether Neanderthals are more closely related to some present-day humans than to others.* Because modern humans are believed to have originated in Africa, if Neanderthals diverged from modern humans before present-day populations began to differentiate, one would expect Neanderthal sequences to match sequences from non-Africans and Africans to the same extent.


* For a description of additional methods used by Green et al. to detect gene flow between Neanderthals and modern humans, see the News story by A. Gibbons.


Unexpectedly, the researchers found that Neanderthals share more genetic variants with present-day non-Africans than with Africans. These results can be explained if gene flow occurred from Neanderthals into the ancestors of non-Africans.

The observation that the Neanderthal genome appears as closely related to the genome of a Chinese and a Papua New Guinean individual as to the genome of a French individual is particularly surprising as there is, to date, no fossil evidence that Neanderthals existed in East Asia or Papua New Guinea. Green et al. thus suggest that gene flow between Neanderthals and modern humans occurred prior to the divergence of European and Asian populations.


Based on comparative genomic data, as well as a mathematical model of gene flow, the authors further estimate that between 1 and 4% of the genomes of people in Eurasia may be derived from Neanderthals.


Implications for Modern Human Origins
There are two major competing hypotheses about the origins of modern humans.

  • The "Out of Africa" hypothesis posits that modern humans evolved from a small population in Africa and replaced all other hominin populations, including Neanderthals, as they migrated into Europe and Asia. The simplest form of this model assumes no interbreeding between modern and ancestral human populations.


  • In contrast, the "Multiregional" hypothesis holds that modern humans evolved in several regions of the world simultaneously. According to this view, archaic humans were not replaced by anatomically modern humans, but rather, gene flow between Africa, Europe, and Asia, led to the evolution of modern humans from local populations.

The finding that Neanderthals are on average closer to individuals in Eurasia than to individuals in Africa thus presents a challenge to the strictest version of the "Out of Africa" model. However variations of this model are plausible.


Green et al. suggest that mixing of early modern humans ancestral to present-day non-Africans with Neanderthals is likely to have occurred in the Middle East prior to their expansion into Eurasia.


The authors contend that this scenario is compatible with the archaeological record, which shows that modern humans appeared in the Middle East before 100,000 years ago while the Neanderthals existed in the same region after this time, perhaps until 50,000 years ago.

Although the Green et al. analyses are suggestive of admixture, the role of Neanderthals in the genetic ancestry of humans outside of Africa was likely relatively minor given that only a few percent of the genomes of present-day people outside of Africa appear to be derived from Neanderthals.


More fossil and genetic data will help researchers further resolve the relationships between our early ancestors and how they shaped modern human evolution.











...Didn't Mate With Modern Humans, Study Says
by Ker Than
August 12, 2008

from NationalGeographic Website

Neanderthals and anatomically modern humans likely did not interbreed, according to a new DNA study.

The research further suggests that small population numbers helped do in our closest relatives. Researchers sequenced the complete mitochondrial genome - genetic information passed down from mothers - of a 38,000-year-old Neanderthal thighbone found in a cave in Croatia. (Get the basics on genetics.)

The new sequence contains 16,565 DNA bases, or "letters," representing 13 genes, making it the longest stretch of Neanderthal DNA ever examined.

Neanderthals are depicted as cannibals in Krapina, northern Croatia,

about 32,000 years ago in this undated illustration.
A DNA study published in August 2008 says modern humans likely did not interbreed with Neanderthals

and that a limited gene pool might have contributed to the demise of modern humans' closest relatives.
Image from Mansell/Time Life Pictures/Getty Images


Mitochondrial DNA (mtDNA) is easier to isolate from ancient bones than conventional or "nuclear" DNA - which is contained in cell nuclei - because there are many mitochondria per cell.

"Also, the mtDNA genome is much smaller than the nuclear genome," said study author Richard Green of the Max Planck Institute for Evolutionary Biology in Germany.


"That's what let us finish this genome well before we finish the nuclear genome," he said.

The new findings are detailed in the August 8 issue of the journal Cell.


A Small Population

The new analysis suggests the last common ancestor of modern humans (Homo sapiens) and Neanderthals lived between 800,000 and 520,000 years ago. This is consistent with previous work on shorter stretches of Neanderthal DNA.

Contrasted with modern humans, Neanderthals exhibited a greater number of letter substitutions due to mutations in their mitochondrial DNA, although they seem to have undergone fewer evolutionary changes overall.

The fact that so many mutations - some of which may have been harmful - persisted in the Neanderthal genome could indicate the species suffered from a limited gene pool. This might be because the Neanderthal population was smaller than that of Homo sapiens living in Europe at the time.

A small population size can,

"diminish the power of natural selection to remove slightly deleterious evolutionary changes," Green said.

The researchers estimate the Neanderthal population living in Europe 38,000 years ago never reached more than 10,000 at any one time.

"This could have been a factor in their demise", Green said.

Homo neanderthalis first appeared in Europe about 300,000 years ago but mysteriously vanished about 35,000 years ago, shortly after the arrival of modern humans - Homo sapiens - in Europe.

"If there were only a few, small bands of Neanderthals, barely hanging on, then any change to their way of life could have been enough to drive them to extinction," Green said.

"One obvious change would have been the introduction of another large hominid - modern humans."

Stepping Forward

Stephen Schuster, a molecular biologist at Pennsylvania State University, said the new study should silence a lot of theories about interbreeding between Neanderthals and modern humans.

The study shows that,

"at least for the maternal lineage, there are no traceable genetic markers that suggest admixture of Neanderthals and modern humans," he said.

Schuster added that the researchers were exceptionally careful to isolate the Neanderthal DNA.

"Many more precautions were taken to ensure that no contamination with human DNA has flawed the analysis," he said, noting that researchers sequenced each letter about 35 times to be sure of their work.

"This was the weak point of previous reports," said Schuster, who was not involved with the study.

Thomas Gilbert, an ancient DNA expert at the University of Copenhagen in Denmark who also was not involved in the study, called the research a "step forward" and a taste of what might come when the Neanderthal nuclear DNA is finished.

The team's argument that the Neanderthal population was small 38,000 years ago is speculative, Green said, but

"it's better than what we could have said before."