Program on Cancer Genetics and Epigenetics
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The molecular clock hypothesis was proposed 45 years ago to explain the remarkable molecular evolution phenomenon that sister species are roughly equally distant to an outgroup. The hypothesis predicts that ancient specimens cannot possibly be more distant to an outgroup than extant sister species are. However, my analysis of the published DNA/peptide sequences of fossil specimens of extinct species contradicts this prediction. Neanderthals are significantly more distant to chimpanzees and gorillas than modern humans are. Dinosaurs are significantly more distant to frogs than extant birds are. Mastodons are significantly more distant to opossums than other placental mammals are.
Ancient fossil specimens can be used to test the molecular clock hypothesis or the Darwinian gradual mutation model. The hypothesis predicts that ancient specimens cannot possibly be more distant to an outgroup than extant sister species are. They should be either equally related or more related to an outgroup than extant sister species are, because they have had less time to accumulate mutations. Extant humans and chimpanzees are equally distant to the outgroup gorillas. Ancient fossil specimens of humans cannot possibly be more distant to gorillas than extant chimpanzees are, if the molecular clock hypothesis is true.
I first analyzed the published DNA sequences of Neanderthals. Neanderthals are a group of extinct hominids that inhabited Europe and western Asia from about 400,000 to 30,000 years ago. Analysis of molecular genetic variation in the mitochondrial and nuclear genomes of living human populations have generally supported the view that Neanderthals were completely replaced by modern humans without contributing any genes (Armour et al., 1996; Hammer et al., 1998; Stringer and Andrews, 1988; Tishkoff et al., 1996; Vigilant et al., 1991). However, these analysis rely on assumptions whose validity has been questioned (Templeton, 1992; Wolpoff, 1989). In contrast to the replacement model, the multiregional model that is largely based on fossil evidence suggests that Neanderthals were the ancestors of modern Europeans (Wolpoff et al., 2000). Influenced by the gradual mutation model of speciation, sequence analysis of Neanderthal DNAs are commonly thought to represent the best approach to resolve the issue of whether Neanderthals contributed some genes to modern humans. In 1997, a segment of the mitochondrial DNA (mtDNA) of Neanderthals was sequenced and was found to be distinct from modern humans (Krings et al., 1997). Subsequently, mtDNA sequences have been retrieved from eleven additional Neanderthal specimens. Although some of these sequences are extremely short, they are all more closely related to one another than to modern human mtDNAs (Orlando et al., 2006). This fact has been interpreted to mean that Neanderthals contributed no mtDNA to present-day humans. Recently, the Neanderthal nuclear genomic DNAs have been sequenced (Green et al., 2006; Noonan et al., 2006). The data however suggests that there may be some degree of gene flow between modern humans and Neanderthals (Green et al., 2006).
If Neanderthals were alive today, they and living modern human would be equally distant from the outgroup chimpanzees. If the gradual molecular clock hypothesis is true, ancient Neanderthals of 40,000-100,000 years ago cannot possibly be significantly more distant to chimpanzees than extant humans are. I first examined five published Neanderthal mitochondrial hyper-variable region I sequences that are longer than 300 nucleotides. By searching against all chimpanzee sequences (1000 sequences) in the NCBI database, the average similarity score between Neanderthal and chimpanzee is 235 for the most related and 63 for the least related. For each Neanderthal sequence, I selected a closely related human sequence of equal length and used it to search the chimpanzee sequences. The average score between human and chimpanzee is 269 for the most related and 74 for the least related. I also determined the relationship with chimpanzees for five African tribes and six Australian DNAs of 8000-60000 years old. All of these show higher similarities with chimpanzees than the Neanderthals do, although ancient Australian DNAs seem to be slightly more distant to chimpanzees than living humans do. So, modern human DNAs are more related to chimpanzees than the Neanderthals are.
I next studied the statistical significance of this finding. If the finding represents random noise of sequence comparisons, it should be easy to find some human sample sequences that show less identity with chimpanzees than Neanderthals do. I examined 11 Neanderthal hyper-variable region 1 sequences ranging in size from 31 bp to 379 bp. For each of these sequences, I identified a distinct modern human sequence of equal length that shows the highest identity with the Neanderthal sequence. These Neanderthal-like sequences have the best chance of being equally related to chimpanzees as the Neanderthals are. They are all more distant to chimpanzees than the Cambridge Reference sequence of human mitochondrial DNA. They are in fact more distant to chimpanzees than other human sequences that are less similar to Neanderthals. For example, for the first reported Neanderthal sequence AF011222 (379 bp), the most closely related human sequence is AY957203 (94% identity) and the least related is AY210529 (90% identity). But AY957203 is more distant to chimpanzees than AY210529 is.
Of the 11 independent sequences of hyper-variable region I, 9 showed more similarity between modern humans and chimpanzees than between Neanderthals and chimpanzees and 2 (both of 31 bp in length) showed equal similarity. The observation of 9 cases of more similarity between humans and chimpanzees against 0 case of more similarity between Neanderthals and chimpanzees is highly significant by Chi square test (Chi-square = 6, P<0.025, square =" 13.33,">>0.05). The result therefore confirms the well-established molecular equidistance phenomenon where chimpanzees and humans are equidistant to gorillas. I next examined all available Neanderthal mitochondrial sequences that are long enough to be informative for my analysis (longer than 30 bp of homology with gorillas). These included 6 hyper-variable region I and 2 hyper-variable region II sequences. For each Neanderthal sequence, a closely related chimpanzee sequence of equal length was selected. Each Neanderthal sequence and its corresponding chimpanzee sequence were used to obtain a best BLAST similarity score with gorilla non-nuclear mitochondrial sequences in the Genbank. All 8 comparisons showed greater similarity between chimpanzees and gorillas than between Neanderthals and gorillas (P <>1 million years) (Ho and Larson, 2006). Based on the pedigree mutation rate in the mitochondrial genome, the human race is only 6000 years old. The high mutation rate allows replacement of variants with new variants possible within a short time frame. It is thus well established that most genetic variants present in a living species of today will not be present in the same species a million years from now. Thus periodic replacement of variants may be an inherent phenomenon of evolution.
The molecular equidistance phenomenon is an extremely consistent and solid fact of molecular evolution. Some purported exceptions to this phenomenon are not based on sound statistical analysis and presuppose the validity of the molecular clock hypothesis. The greater distance of ancient Neanderthals/dinosaurs/mastodons to an outgroup relative to their extant sister species would mean the same for living Neanderthals/dinosaurs/mastodons if the clock hypothesis is true, which would represent the first statistically significant violation of the molecular equidistance phenomenon. Either the clock hypothesis is not valid and so the molecular equidistance phenomenon would hold. Or the molecular equidistance phenomenon must be false and so the clock hypothesis would hold. Since the molecular equidistance phenomenon holds in all cases analyzed while the clock hypothesis remains unproven, it is much less costly to a coherent story of the whole to consider the clock hypothesis to be false.
I thank Dr. John Hawks for critical reading of the manuscript. This work was supported by NIH (RO1 CA 105347).
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