Forensic DNA Techniques With Applications in the Identification of Homo neanderthalensis

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Before the 1980s, paleoanthropologists were entirely dependent upon carbon dating techniques and stable isotope analysis for evaluating human remains found in ossuaries. Most anthropologists prescribed to two main hypotheses regarding the evolutionary development of Neanderthals and modern man. The "multi-regional model" suggested that modern humans evolved independently of Neanderthals in different regions of the world, with sufficient gene flow between the two species to maintain species unity. The model also suggested that Neanderthals and modern humans shared a common ancestor who lived more than one million years ago. The ""recent replacement model" suggested that a single population emerged from a common ancestor, most likely of African origin, which expanded and replaced the ancient populations nearly 200,000 years ago (Hall 2008). In 2009, a group of scientists at the Max Planck Institute in Leipzig, Germany published the results of the first draft of the Neanderthal genome. Scientists around the world believe that this monumental effort will shed light on many critical aspects of human evolution.

Spelunkers Accidentally Discover Bones That Hold Clues to Neanderthal DNA

In 1994, a group of spelunkers exploring an extensive cave system in northern Spain wandered into a small side gallery that contained the bones of two human mandibles. The spelunkers immediately notified the authorities who inspected the cave, suspecting that the bones might date back to the Spanish Civil War. Upon further investigation, however, it turned out that the cave hunters had discovered the remains of a group of Neanderthals who had been murdered nearly 43,000 years ago.

Paleoanthropologists and geneticists worked carefully to excavate the findings in the cave known as El Sidrón. The gallery discovered by the spelunkers was named the Galeria del Osario, or the "Tunnel of Bones" (Hall 2008). Since 2000, researchers have unearthed nearly 1,500 bone fragments representing the remains of at least nine Neanderthals - five young adults, two adolescents, an eight-year old child, and a three-year-old toddler. The damage to the bones suggested that the Neanderthal group had been murdered and then cannibalized. One of the investigators at the El Sidrón cave discovered a fresh Neanderthal bone that appeared to be from a femur. Digging ceased quickly to avoid contaminating the bone. Wearing special clean room suits to avoid further contamination, the group carefully extracted the bone from the soil and placed it under ice to preserve any DNA evidence that it might contain. As suspected, the bone yielded DNA sequences of ancient human beings.

So far, researchers have extracted mitochondrial DNA (mtDNA) sequences from two El Sidrón samples (Freeman 2005). The first sample yielded a 48-bp sequence (between positions 16,230 and 16,278 of the mtDNA reference sequence), and the second sample yielded an almost complete mtDNA hypervariable region 1 (302-bp sequence, between positions 16,076 and 16,378). The sequences exhibited the same overlapping 48-bp segment, thus confirming that the genetic signatures belonged to the same individual.

Did Neanderthals and Modern Humans Share a Common Ancestor?

Recent studies show that Neanderthals roamed much of Eurasia for nearly 200,000 years, traversing the Mediterranean from the Strait of Gibraltar to Greece and Iraq, north to Russia, as far west as Britain, and nearly to Mongolia. Until recently, most evidence supporting a common ancestor with modern man relied upon radiocarbon dating methods and stable isotope analysis.

All that changed in 1985, when English geneticist Sir Alec Jeffreys (Butler 2005) developed a method for identifying genetic information using multi-locus restriction fragment length polymorphism (RFLP) probes. Jeffreys' method was put to the test in 1986, when it was used to identify the remains of two young girls who had been murdered near the village of Leicestershire, England. Using RLFP DNA probes, investigators were able to identify and prosecute the girls' killer. A little more than 10 years later, Jeffreys' RFLP test was used to compare a genetic sample from the original Neanderthal man to that of modern man.

In 1997, Krings et al. (1997) validated the hypothesis that Neanderthals and modern humans shared the same common ancestor. The researchers used forensic DNA techniques to extract mtDNA from the arm bone of the original Neanderthal-type specimen collected from Feldhofer Cave in Germany's Neander Valley. Krings et al. tested multiple extracts in their Munich laboratory, under very stringent laboratory conditions. One of the tests was performed at Pennsylvania State University. The researchers were able to amplify and sequence a 378 base pair region of the mitochondrial control region. To validate the sample, Krings et al, compared the sample with that of the laboratory personnel. The sequence not only differed significantly from the laboratory personnel, but also from all human beings sequenced to date.

When the extract was compared to a human reference sequence, the Neanderthal sequence differed by 26 nucleotide substitutions and a single base insert event. Krings et al. increased the controversy by announcing that the Neanderthal sequence fell outside the variation of modern man, implying that Neanderthals and modern man share a common ancestor who lived nearly 600,000 years ago - nearly threes time original estimate of 150,000 years ago.

German Scientists Draft Neanderthal Genome

Scientists at the Max Planck Institute for Evolutionary Anthropology and 454 Life Science Corporation recently generated the first draft sequence of Neanderthal DNA, which includes more than 60 percent of the entire Neanderthal genome (Max Planck Society 2009). So far, the researchers have extracted more than one billion DNA fragments from three Croatian Neanderthal fossils using genetic methods developed specifically for this project. The most important element of this project was the production of sequencing libraries.

In 1997, researchers sequenced a segment of the hypervariable control region of inherited mtDNA from a Neanderthal specimen collected 150 years ago at Feldhofer, in the Neander Valley, outside of Düsseldorf, Germany (Green et al. 2006). The hypervariable region, which fell outside the variation of contemporary humans, shares a common ancestor with mtDNA from present-day humans living nearly half a million years ago. Additional mtDNA sequences have been retrieved from 11 Neanderthal specimen from Feldhofer (Germany), Mezmaiskaya (Russia), Vindija (Croatia), Engis and Scladina (Belgium), La Chapelle-aux -Saint and Rochers (France), Monte Lessini (Italy), and El Sidrón (Spain). The sequences were more closely related to each other than they were to modern human mtDNA.

These findings, in combination with the lack of any mtDNA sequences in contemporary humans or fossils (Krings et al. 1997, Serre et al. 2004) from early modern humans, suggest that Neanderthals did not contribute any mtDNA to present-day humans. Based on population models, the maximal overall genetic contribution from Neanderthals to contemporary humans is estimated be between 25% and 0.1% (Green et al. 2006).

Green et al. tested one bone from the Vindija Cave (Vi-80) in Croatia that had approximately 99% of the 63-base pair mtDNA segment and approximately 94% of the 119 base pair segment of Neanderthal origin. The researchers extracted DNA using single-stranded libraries flanked by common adapters. Individual library models were amplified using bead-based emulsion PCR. The beads carried millions of copies of clonal DNA fragments from the samples. Using this technique, the researchers were able to recover 254,933 unique sequences from the Vi-80 bone.

Another group of researchers from the Joint Genome Institute (JGI) in Walnut, California, recovered Neanderthal nuclear DNA using a metagenomic approach to replicate the Neanderthal DNA (Noonan et al. 2006). The metagenomic library was created using an amplification-independent direct cloning method to create a Neanderthal genomic library, designated NEI. Carbon-14 accelerator mass spectrometry dating yielded a date for Vi-80 of 38,310 ±2,130 BP year old specimen from Vindija, Croatia (Serre et al. 2004).

The following seven Neanderthal mtDNA sequences are available in GENBANK under Homo neanderthalensis: HVR1/AF254446, HVR1/F011222, HVR1/AF282971, HVR1/AY149291, HVR1/DQ464008 (Belgium), HVR2/AF282972, and HVR2/AF142095

Ginger-haired, Freckled Cavemen

Cannabalism among Neanderthals may have been responsible for the DNA preservation among the bones found at Croatia and El Sidrón. Cannabalism strips the flesh of microorganisms that might otherwise contaminate bone samples. Samples taken from the bones at El Sidrón have provided insight into the appearance and behavior of Neanderthals. Lalueza-Fox et al. (2007) have isolated a pigmentation gene called melanocortin 1 receptor (MC1R) that regulates pigmentation in humans and other vertebrates. Some variants are associated with pale skin color and red hair. The researchers amplified and sequenced a fragment of the MC1R gene (mc1r) from two Neanderthal specimens, both of whom exhibited a mutation that was not found among the approximately 3,700 modern human specimens analyzed. The mc1r variant reduces MC1R activity that alters hair and skin pigmentation in humans. The Neanderthal DNA specimens exhibited variations in pigmentation on a scale common to modern humans, suggesting that Neanderthals may have had pale skin, red hair, and possibly freckles. The data also suggests that the mc1r variants evolved independently in modern humans and Neanderthals.

DNA and the Evolution of Language

Undeniably, humans have evolved the most complex language among all animals. Recently, researchers isolated the FOXP2 gene that is responsible for language and speech in humans. Studies show that evolutionary changes in the FOXP2 gene predated the common ancestor of both human species, somewhere around 300,000 to 400,00 years ago (Krause et al. 2007)(Kraus 1998)(Kraus, 1998)(Kraus, 1998). FOXP2 is the only gene currently known to play a specific role in speech. Studies show that Neanderthals carried the same FOXP2 protein that is found in contemporary humans in the only two amino acid substitutions that differ between humans and chimpanzees. These findings suggest that these genetic changes were present in the common ancestor of modern humans and Neanderthals.

DNA Gives Clues to Neanderthal Migration Patterns

The introduction of the FOXP2 and MC1R genes into the Neanderthal genome were important evolutionary developments that may have played an important role in the expansion of Neanderthals throughout Europe and Asia. Language makes it possible for individuals to communicate ideas from one individual to another or to a group. Language also has important cultural implications in the development of a society. Y chromosome and mtDNA undergo harmless mutations within certain geographic groups that become inheritable genetic markers of where a group has lived. After several generations, the markers are carried by nearly all male and female inhabitants from that region (U.S. Department of Energy 2008). When an individual(s) leaves a particular geographic area, the genetic markers they carry remain within them.

This information is crucial for determining the migration patterns among Neanderthal societies. It provides evidence that Neanderthals dominated Eurasia for nearly 200,000 years (Hall 2008). Around the time of the El Sidrón incident, climatic changes combined with the expansion of anatomically modern humans from Africa reducing the Neanderthals' range to a small number of geographic regions, namely Iberia, parts of central Europe, and the southern Mediterranean. Within approximately 15,000 years, Neanderthals became extinct.

Neanderthals Genes Responsible for Neurodegenerative Diseases

Although there is little evidence to support the hypothesis that Neanderthals and modern humans mated, it also is not possible to definitely state that Neanderthals did not contribute to the gene pool of modern humans (Serre et al. 2004). Hardy et a. (2005) suggest that there was point where Neanderthals and modern humans coexisted and that the two gene pools may have comingled. The tau (MAPT) locus covers a region of approximately 1.8 Mb. MAP2 contains two haploclades in European populations, H1 and H2 (Baker et al. 1999, Pittman et al. 2004). MAPT is a susceptibility locus for diseases with tangles such as progressive supranuclear palsy (Baker, 1999), corticobasal degeneration (Houlden et al. 2001), and possibly Parkinson dementia complex of Guam ((Poorkaj et al. 2001). H1 and H2 cannot be derived directly from one another (Serre et al. 2004). The H2 haplotype was derived by two methods, geographical distribution and the founding event. Geographical distribution is consistent with the intrusion of H. sapiens around the time of the European population nearly 30,000 years ago (Evans et al. 2004). The founding event is associated with the slippage of microsatellite repeat markers (Weber and Wong 1993, Goldstein et al. 1995). Hardy et al (2005) speculate that the H2 haplotype was the result of an ancestral H. neanderthalensis allele that entered the European H. sapiens genome during their period of coexistence and spread through selective pressure to its current allele frequency of approximately 25% (Evans, 2004).


Forensic DNA technology has made it possible for paleoanthropologists and geneticists to identify DNA from the bones collected from ancient Neanderthal DNA. From the early days of restriction fragment length polymorphism ( RFLP) to the burgeoning DNA identification techniques using single-nucleotide polymorphism (SNPs), DNA analysis has played an increasing role in the identification of early humans, particularly Homo neanderthalensis.

Using DNA techniques, researchers were able to create a library of the Neanderthal genome that made it possible to cool down one of the most heated debates in the evolution of humans: did modern humans cohabitat with Neanderthals? Not only has DNA proven that Neanderthals are first cousins to humans, but it also allows researchers to roll back the clock to find the common ancestor of Homo neanderthalensis and Homo sapiens.

Using the Neanderthal genome, geneticists were able to identify the FOXP2 gene that controls language and behavior, as well as the MC1R gene that regulates pigmentation in humans. DNA has played a prominent role in forensics from the first known murder case that took place nearly 43,000 years to the conviction of serial murderers today. Some researchers recently began combining DNA analysis with carbon and nitrogen stable isotope analysis to determine what Neanderthals and other cave dwelling species used for nutrition by examining the DNA in coprolites, seeds, and minerals that were discovered in and around the habitation sites. DNA collected from early humans has relied on mtDNA for analysis; however, many researchers believe that nuclear DNA might be able to elaborate on the relationship between modern humans and their closest relative, the Neanderthals.

One group of researchers is convinced that the two species may have mated based on the genetic makeup of two DNA haplotypes. More than 20 years after Sir Alec Jeffreys developed the first DNA profiling tests, DNA analysis has continued to evolve into more powerful, sensitive, and specific tools that have crossed the boundaries between forensic DNA typing to the study of plant and animal diseases. The most significant problems today have to do with specimen contamination. Until the advent of DNA, archeologists were not concerned about contaminating bones at excavation sites. All of that changed, however, when one researcher discovered a bone fragment in the El Sidrón cave.

There is still a lot of work to do before anyone can say definitively that Homo neanderthalensis and H. sapiens, despite living in close geographic proximity, never had social or biological contact.


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