Ancient DNA and Neanderthals

In recent years, ancient DNA has been used to understand aspects of Neanderthal biology. Both mitochondrial (mtDNA) and nuclear DNA have been extracted from fossils and sequenced. These sequences have provided information about the appearance, speech capability and population structure of Neanderthals as well as their phylogenetic relationship with anatomically modern humans.

Neanderthal DNA bears on several debates concerning the origins of modern humans. What was the relationship between Neanderthals and anatomically modern Homo sapiens? Did Neanderthals and anatomically modern humans interbreed? Did Neanderthals contribute to the modern genome? How much? Scientists answer these questions by comparing Neanderthal DNA and mtDNA to that of modern humans. 

 

DNA

DNA consists of phosphates, sugars and bases arranged in a chain. There are four different bases: adenine, thymine, guanine and cytosine. DNA is wound together in a double stranded molecule with adenine paired with thymine and cytosine paired with guanine across the two sides of the strand.  When DNA is replicated, the chemical bonds that bind together the two sides are broken and new nucleotides are joined to the unwound strand, generating strands with the same sequences as the original. Mutations arise when an error in the copying occurs and a different base is substituted.

Different combinations of bases code for different amino acids, which are the building blocks of proteins. Sometimes more than one combination of bases leads to the same amino acid. For example, both AAA and AAG code for lysine. Therefore, a DNA change that led to a G at the third position rather than an A wouldn’t affect the code for lysine. This is known as a silent change. However, if the third position had changed to a C, making the set AAC, that would change the amino acid to asparagine. This change that could affect the protein is called nonsynonymous.

 

Neanderthal Mitochondrial DNA

Ancient DNA has been retrieved and analyzed from Egyptian mummies and from animals such as quaggas, mammoths, moas and marsupial wolves. The first analysis of mitochondrial DNA (mtDNA) from Neanderthals was published in 1997.

The specimen was taken from the first Neanderthal fossil discovered, from Feldhofer Cave, in the Neander Valley, Germany.  A small sample of bone was ground up to extract mtDNA.

The Neanderthal mtDNA sequences were substantially different from modern human mtDNA (Krings et al. 1997, 1999). Researchers compared the Neanderthal to modern human and chimpanzee sequences. Most human sequences differ from each other by on average 8.0 substitutions, while the human and chimpanzee sequences differ by about 55.0 substitutions. The Neanderthal and modern human sequences differed by approximately 27.2 substitutions. Using this mtDNA information, the last common ancestor of Neanderthals and modern humans dates to approximately 550,000 to 690,000 years ago, which is about four times older than the modern human mtDNA pool. This is consistent with the idea that Neanderthals did not contribute substantially to modern human genome.

A second mtDNA sequence, announced in 2000, was derived from a 29,000 year old Neanderthal found in Mezmaiskaya Cave, Russia (Ovchinnikov et al. 2000). Although the Mezmaiskaya Cave sequence was slightly different than the Feldhofer Neanderthal, the two Neanderthal mtDNA sequences were distinct from those of modern humans. These results confirmed the earlier findings that showed that Neanderthals were unlikely to have contributed to the modern human genome. As with the previous study of Neanderthal mtDNA, results were consistent with separation between the Neanderthal and modern human gene pools or with very low amounts of gene flow between the two groups.

Further mtDNA sequences confirmed sequence differences between Neanderthals and modern humans. Researchers compared Neanderthal mtDNA to that of modern humans from different geographic regions. If Neanderthals had interbred with modern humans in Europe, then researchers would have expected to find more similarities between Neanderthals and Europeans than between Neanderthals and other modern humans. However, Neanderthals were equidistant from modern human groups, which is consistent with genetic separation between modern humans and Neanderthals. However, this does not explicitly disprove admixture because interregional gene flow between modern humans could have swamped the Neanderthal contribution to Europeans (Relethford 2001).

Researchers have also studied ancient DNA from anatomically modern Homo sapiens from Europe dating to the same time period as the Neanderthals. Material from two Paglicci Cave, Italy individuals, dated to 23,000 and 25,000 years old, was sequenced. The Paglicci Homo sapiens mtDNA sequences were different from all Neanderthal mtDNA sequences but were within the range of variation for modern human mtDNA sequences (Caramelli et al. 2003). Mitochondrial DNA from the Paglicci specimens as well as other ancient humans fit within the range of modern humans, but the Neanderthals remain consistently genetically distinct. This shows that early anatomically modern Homo sapiens were not very different genetically from current modern humans, but were still different from Neanderthals. Though this evidence does not disprove the idea of Neanderthal and modern human admixture, it shows that moderns and Neanderthals did not have more genetic similarities during the Pleistocene that were subsequently lost. If interbreeding did occur, Neanderthal mtDNA sequences could have been lost due to genetic drift. 

 

Challenges in Extracting Ancient DNA

Working with ancient DNA is very challenging, both in terms of finding sufficient material to work with after decomposition has occurred, and in terms of eliminating modern human contamination. Distinguishing between modern human and ancient genetic material is particularly difficult when the ancient DNA comes from close relatives of modern humans.

Organisms decompose after death. Water, oxygen and microbes break down DNA. Within 100,000 years, all DNA is destroyed. Ancient DNA tends to be found in small quantities. The DNA that is extracted is generally fragmentary and damaged. Some damage results in changes to the DNA sequence. Cytosine can change to uracil, which is read by copying enzymes as thymine, resulting in a C to T transition. Changes from G to A also occur. DNA errors are very common at the ends of molecules.

Contamination by modern DNA is a particularly difficult problem to solve. Labs and chemicals may be contaminated by the DNA of the people working in them, while many fossils have been handled by researchers for years. Contamination is difficult to detect because Neanderthals and humans share much of their genetic material, making some DNA sequences indistinguishable. Researchers have developed ways to analyze the results of ancient DNA sequencing efforts to determine whether contamination is likely and how much has occurred. Analysis of the results and efforts to keep labs and specimens free of modern DNA is very important as some researchers believe that the early studies of Neanderthal DNA included modern contaminants.

 

Sequencing the Complete Neanderthal Mitochondrial Genome

After successfully sequencing large amounts of DNA and devising strategies to deal with potential contamination, a team led by Svante Pääbo from the Max Planck Institute, reported the first complete mtDNA sequence for a Neanderthal (Green et al. 2008). The 0.3 gram sample was taken from a 38,000 year old Neanderthal from Vindija Cave, Croatia. Complete Neanderthal mtDNA sequences give researchers more information about the relationship between modern humans and Neanderthals, as well as information about Neanderthal population size.

The complete mtDNA sequence shows that Neanderthals were outside the range of modern human mtDNA variation. Researchers compared the mtDNA sequence with that of modern humans. They compared sequence changes that resulted in nonsynonymous amino acid changes with synonymous changes. They found a larger number of nonsynonymous changes in the Neanderthal lineage, possibly implying that Neanderthals had a small population size with weaker purifying selection (Green et al. 2008). 

Later, Svante Pääbo’s lab sequenced the entire mitochondrial genome of five Neanderthals (Briggs et al. 2009). Sequences came from two individuals from the Neander Valley in Germany, Mezmaiskaya Cave in Russia, El Sidrón Cave in Spain and Vindija Cave in Croatia. Though the Neanderthal sample comes from a wide geographic area, the Neanderthal mtDNA sequences were not particularly genetically diverse. The most divergent Neanderthal sequence came from the Mezmaiskaya Cave Neanderthal from Russia, which the oldest and eastern-most specimen. To look at whether age or geographic location contributed to genetic differences, the team sequenced part of the DNA of another Mezmaiskaya Cave Neanderthal that dated to 41,000 years ago. This more recent specimen grouped with the other Neanderthals, possibly showing that age was the cause of the sequence differences (Briggs et al. 2009). Other studies show the existence of eastern, western and southern groups of Neanderthals (Fabre et al. 2009).

On average, Neanderthal mtDNA genomes differ from each other by 20.4 bases and are only 1/3 as diverse as modern humans (Briggs et al. 2009).  The low diversity might signal a small population size, possibly due to the incursions of modern humans into their range (Briggs et al. 2009).

 

Sequencing the Neanderthal Nuclear Genome

Recently, there have been efforts to sequence Neanderthal nuclear genes. Two studies, one by Svante Pääbo’s team and one by Edward Rubin, have sequenced large amount of Neanderthal nuclear DNA using different methods. Their results were announced in 2006. Given their success in sequencing some nuclear DNA, both labs launched projects to sequence the entire Neanderthal genome. Nuclear genomic sequences from Neanderthals show differences between modern humans and Neanderthals, and illustrate aspects of Neanderthal biology.   

 

One Million Base Pairs of the Neanderthal Sequence

Svante Pääbo’s team from the Max Planck Institute for Evolutionary Anthropology in Germany announced the sequencing of one million base pairs of nuclear DNA of a Neanderthal specimen in 2006 (Green et al. 2006). After a long search for specimens with a sufficient amount of undamaged DNA to sequence and for the ones with the least evidence of contamination, they focused on Vindija 80, a Neanderthal discovered in Croatia in 1980 that is approximately 38,000 years old.

They estimated that 7.9% of the changes in human DNA compared with that of the chimpanzee occurred after the split with Neanderthals. They dated the split between the ancestors of modern humans and Neanderthals to 465,000 to 569,000 years ago. They also found that the effective population size of the Neanderthals was small. Their success in sequencing this amount of DNA indicated that a large-scale project to sequence the Neanderthal genome is possible.

 

Rubin's Neanderthal Nuclear DNA

Edward Rubin’s team from the Lawrence Berkeley National Laboratory in California also sequenced Neanderthal nuclear DNA (Noonan et al. 2006).  They sequenced about 65,000 base pairs from the 38,000 year old Vindija, Croatia specimen. The technique used here produces a copy of the Neanderthal sequence that can be retained forever, reducing the need for repeated destructive sampling. The DNA is then cloned in bacteria.

The average split time between the Neanderthal and modern human populations was around 370,000 years ago.  They used the sequence to look at the possibility of interbreeding between Neanderthals and moderns. Admixture would be seen as derived alleles that are found in Neanderthals and in low frequencies among modern humans. They did not detect this in their sample. A simulation to test the Neanderthal contribution to the human genome found a 0% chance of Neanderthal input with a 0% to 20% confidence range. With this data, the authors cannot definitively rule out admixture (Noonan et al. 2006).

Some aspects of the two sets of nuclear DNA do not fit together, possibly because of contamination and sequencing errors, especially in the Green et al. (2006) study (Wall and Kim 2007).  This has led the researchers to develop new methods of detecting and preventing contamination to ensure that only ancient DNA is being sequenced.

 

A Draft Sequence of the Neanderthal Genome

In 2010, Svante Pääbo’s lab announced a draft sequence of the Neanderthal genome (Green et al. 2010).  This new study has produced evidence consistent with interbreeding between Neanderthals and anatomically modern Homo sapiens and points to aspects of the human genome that may have changed since the split between humans and Neanderthals.

DNA was extracted from three Neanderthal bones from Vindija Cave, Croatia. By comparing sequences from their mtDNA and their nuclear DNA, scientists determined that the three bones came from different individuals, although two of them might be related on their mother’s side. The researchers used several methods to ensure that the DNA they were sequencing was derived from the Neanderthal specimens rather than from contamination by modern humans in the lab.

The Neanderthal sequence was compared to those of five modern humans from France, China, Papua New Guinea, as well as Africans from the San and Yoruba groups. Tests indicated that Neanderthals shared more derived alleles with non-African modern humans than with African modern humans. They compared parts of the Neanderthal genome with pairs of modern humans. While the European and Asian pairs had similar amounts of derived material compared with the Neanderthal, Neanderthals had more similarities with non-African humans than with Africans. The simplest explanation for these results is gene flow from Neanderthals into modern humans. Gene flow could also have occurred from modern humans into Neanderthals. Interbreeding events between Neanderthals and modern humans might be obscured if the modern human population was large.

Neanderthals have contributed approximately 1% to 4% to the genomes of non-African modern humans. This evidence of interbreeding sheds light on how we think of the expansion of modern  humans out of Africa. It refutes the strictest scenario in which anatomically modern humans replaced archaic hominins completely without any interbreeding. However, even with some interbreeding between moderns and archaic hominins, most of our genome still derives from Africa.

The data also points to the time when interbreeding might have taken place. Since the Neanderthal DNA was equally related to that of the modern samples from France, China and Papua New Guinea, admixture between moderns and Neanderthals must have occurred before the Eurasian populations split off from each other. Remains of both modern humans and Neanderthals dating to around 100,000 years ago have been found in the Middle East. A few interbreeding events during this period could have produced the results found in this study.

The sequence of our close hominin relative also shows us how humans are unique. Researchers found 78 sequence differences that would have affected proteins in which Neanderthals had the ancestral state and modern humans had a newer, derived state. Five genes had more than one sequence change that affected the protein structure. These proteins include SPAG17, which is involved in the movement of sperm, PCD16, which may be involved in wound healing, TTF1, which is involved in ribosomal gene transcription, and RPTN, which is found in the skin, hair and sweat glands. Scientists do not know the function of the CAN15 protein, which was also one of the differences. Other changes may affect regulatory regions in the human sequence. Some changes are in regions that code for microRNA molecules that regulate protein manufacture.

The comparison also pointed out regions that might have been under positive selection in modern humans. Though some of the genomic areas that may have been positively selected for in modern humans may have coded for structural or regulatory regions, others may have been associated with energy metabolism, cognitive development and the morphology of the head and upper body. 

 

The Relationship between Modern Humans and Neanderthals

The relationship between modern humans and archaic hominins, particularly Neanderthals, has been the subject of much debate. While the idea that modern humans originated in Africa and spread out to other parts of the world (Out of Africa) is widely accepted, several scenarios have been proposed to account for the replacement of archaic hominin populations. Under strict replacement, modern humans did not interbreed with the archaic populations as they expanded their geographic range. In less strict scenarios, admixture between the populations occurred, but in small amounts, with the bulk of modern human ancestry tied to Africa. The multiregional hypothesis holds that hominin populations in Eurasia and Africa were held together by gene flow. Fossil and genetic evidence supports an African origin for Homo sapiens.

Mitochondrial DNA shows differences between Neanderthals and modern humans. Neanderthal mtDNA also differed from that of anatomically modern Homo sapiens from the same time period. Proponents of multiregional and admixture models argue that these results are consistent with African origin for modern Homo sapiens, but do not explicitly rule out admixture between modern humans and archaic populations (Templeton 2007, Relethford 2008). Neanderthal genetic sequences introduced into the human genome may have been subsequently lost through genetic drift (Relethford 2001), while similarities between modern Europeans and Neanderthals, which would be expected if Neanderthals and modern humans interbred while in Europe, could have been lost due to gene flow between modern humans from different regions.

Various analyses have examined the amount of Neanderthal contribution to modern human mtDNA. One analysis was unable to find positive evidence for interbreeding, but could not rule out a small genetic contribution (Serre et al. 2004).  Other researchers (Plagnol and Wall 2006, Wall et al. 2009) looked at the pattern of variation in modern human DNA to determine whether modern humans mixed with more ancient populations. Their recent models are consistent with 14% archaic-modern admixture in European and American populations, and 1.5% admixture in East Asian populations. Nested clade phylogenetic analysis shows evidence of three expansions out of Africa at 1.9 Ma, 650,000 years, and 130,000 years, which is consistent with the admixture between ancient and modern populations rather than complete replacement (Templeton 2002, 2005, 2007). Other researchers contend that factors such as population structure within Africa could have preserved old haplotypes and produced the pattern found in the nested clade analysis (Satta and Takahata 2002).

Though it is difficult to prove or quantify admixture, small amounts of interbreeding were supported by a variety of analyses. However, the substantial differences between Neanderthal and modern human mtDNA is consistent with large-scale replacement and some amount of interbreeding between modern and archaic populations. Interbreeding between archaic and moderns may have involved different species of archaic hominins, including populations in Africa, Asia and Europe.

The draft sequence of the Neanderthal genome provides more evidence that interbreeding between Neanderthals and modern humans may have occurred. It showed more similarities between non-African modern humans and Neanderthals than between African modern humans and Neanderthals. This difference between regions is consistent with interbreeding between Neanderthals and the ancestors of Eurasian modern humans before they branched off into regional groups. Approximately 1 to 4% of non-African modern human DNA is shared with Neanderthals. 

 

Red-Headed Neanderthals

Ancient DNA has been used to show aspects of Neanderthal appearance. A fragment of the gene for the melanocortin 1 receptor (MRC1) was sequenced using DNA from two Neanderthal specimens from Spain and Italy, El Sidrón 1252 and Monte Lessini (Lalueza-Fox et al. 2007). Neanderthals had a mutation in this receptor gene that has not been found in modern humans. The mutation changes an amino acid, making the resulting protein less efficient. Modern humans have other MCR1 variants that are also less active resulting in red hair and pale skin. The less active Neanderthal mutation probably also resulted in red hair and pale skin, as in modern humans.

The specific MCR1 mutation in Neanderthals has not found in modern humans (or occurs extremely rarely in modern humans). This indicates that the two mutations for red hair and pale skin occurred independently and does not support the idea of gene flow between Neanderthals and modern humans. Pale skin may have been advantageous to Neanderthals living in Europe because of the ability to synthesize vitamin D.  

This reconstruction of a male Neanderthal by John Gurche features pale skin, but not red hair.

 

Neanderthals, Language and FOXP2

The FOXP2 gene is involved in speech and language (Lai et al. 2001). Changes in the FOXP2 gene sequence led to problems with speech, oral and facial muscle control in modern humans with a mutation in the gene. It impairs language function. Modern humans and Neanderthals share two changes in FOXP2 compared with the sequence in chimpanzees (Krause et al. 2007). Neanderthals may also have their own unique derived characteristics in the FOXP2 gene that were not tested for in this study.

The human FOXP2 gene is on a haplotype that was subject to a strong selective sweep. A haplotype is a set of alleles that are inherited together on the same chromosome. The researchers then tried to determine how the FOXP2 variant came to be found in both Neanderthals and modern humans. One scenario is that it could have been transferred between species via gene flow. The researchers do not think this is likely since there is no evidence indicating that gene flow has occurred. Another possibility is that the derived FOXP2 was present in the ancestor of both anatomically modern Homo sapiens and Neanderthals with the selective sweep that made it prevalent occurring after the divergence between the groups. A third scenario, which the authors think is most likely, is that the changes and selective sweep occurred before the divergence between the populations.  

 

ABO Blood Types and Neanderthals

The gene that produces the ABO blood system is polymorphic in humans. Various selection factors may favor different alleles, leading to the maintenance of distinct blood groups in modern human populations. Though chimpanzees also have different blood groups, they are not the same as human blood types. While the mutation that causes the human B blood group arose around 3.5 Ma, the O group mutation dates to around 1.15 Ma. Lalueza-Fox and colleagues (2008) tested whether Neanderthals had the O blood group. They found that two Neanderthal specimens from Spain probably had the O blood type, though there is the possibility that they were OA or OB. Though the O allele was likely to have already appeared before the split between humans and Neanderthals, it could also have arisen in the Neanderthal genome via gene from modern humans.

 

Bitter Taste Perception and Neanderthals

Like some modern humans, some Neanderthals were able to taste bitter substances. Some items that taste bitter may be toxic in large quantities so the ability to taste bitter substances may have protected hominins from accidental poisoning. Some of these bitter chemicals are found in vegetables. For instance, humans vary in their ability to perceive a bitter substance similar to that found in Brussels sprouts, broccoli and cabbage.

The ability to taste bitter substances is controlled by a gene, TAS2R38. Some individuals are able to taste bitter substances, while others have a different version of the gene that does not allow them to taste bitter items. Possession of two copies of alleles associated with tasting bitter substances gives the individual greater perception of bitter tastes than the heterozygous state, in which individuals have one tasting allele and one non-tasting allele. Two copies of a non-tasting allele leads to inability to taste bitter substances.

A Neanderthal from El Sidrón, Spain, was sequenced for the TAS23R38 gene. They found that this individual was heterozygous and thus was able to perceive bitter taste, although not as strongly as a homozygous individual with two copies of the tasting allele would be able to (Lalueza-Fox et al. 2009). Since the Neanderthal sequenced was heterozygous, the two alleles (tasting and non-tasting) were probably both present in the common ancestor of Neanderthals and modern humans. Though chimpanzees also vary in their ability to taste bitterness, their abilities are controlled by different alleles than those found in humans, indicating that non-tasting alleles evolved separately in the hominin lineage.

 

Microcephalin and Archaic Hominins

The microcephalin gene relates to brain size during development. A variant of this, haplogroup D, may have been positively selected for in modern humans – and may also have come from an interbreeding event with an archaic population (Evans et al. 2006). Mutations in microcephalin cause the brain to be 3 to 4 times smaller in size. All of the haplogroup D variants come from a single copy that appeared in modern humans around 37,000 years ago. However, haplogroup D itself came from a lineage that had diverged from the lineage that led to modern humans around 1.1 million years ago. Although there was speculation that the Neanderthals were the source of the microcephalin haplogroup D (Evans et al. 2006), the Neanderthal DNA recently sequenced does not contain the microcephalin haplogroup D (Green et al. 2010).  

 

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