Molecular History Research Center

The Mitochondrial Clock

Is the clock speed faster than we thought?

The 200,000 year time-scale, when Eve is to have lived, was calculated using the idea that the common ancestor of chimps and humans lived 5 million years ago and that both nuclear DNA and mitochondrial DNA mutate at a constant rate.

What this means is: The variation that is found in a sampling of human mitochondrial DNA from different people groups in the world was compared against the differences that are found by comparing human and chimpanzee mitochondrial DNA. It is done by ratio-proportion. If we know how many genetic changes that were made since 5,000,000 years ago when we think humans and chimpanzees diverged, then we can calculate how long it would take for the few genetic changes we find in the population of human mitochondrial DNA, to be produced.

If for an example; In a certain mitochondrial gene, there are 65 differences in the genetic code between human and chimpanzee, but there are differences of around 3 mutations in the human population, then we could predict how long it would take for the human population to produce an average of 3 mutations. So, assuming that the common ancestor of humans and chimpanzees lived 5,000,000 years ago the equation would be:

A correction has to be made because over large periods of time, multiple nucleotide substitutions can occur at any position on the gene. With increasing lengths of time, the chance increases that the same bases will be mutated more than once. Five million years is a long time, so this problem must be accounted for in the equation. There are dozens of correction methods that corrects this kind of error. In addition, each one assumes different models of molecular evolutionary change. Without going into the detail of the process, I will give corrected numbers to this make believe example. (Note: These numbers do not represent actual data, it is just an example to give you a feel for how the numbers are calculated.)

Looking at the corrected numbers above, we can see that mutations numbers have changed (3 -> 3.02 and 65 -> 72.22). The larger number has changed more than the smaller number. This change reflects the fact that the more mutations there are, the more likely it is that there will be multiple mutations on the same site. What this means is that there will be some hidden mutations, because the very same base can be changed a second time.

So using this kind of method, mutations are thought to occur every 6000 to 12,000 years on the human mitochondrial genome (that would be one mutation in every 300 to 600 generations).

Initial Evidence of a Faster Clock Speed

A number of years later, in 1991, the Russians exhumed a mass grave site that was thought to have the last Russian Tsar that was killed in the revolution. The controversy has been raging for years. Do they have the correct body? There were at least nine skeletons found in the shallow grave where various members of the royal family, their physician, and servants were buried. So they compared the mitochondrial DNA of living relatives with the DNA in the bones of the nine skeletons found in the grave site. What they found surprised everyone. The match with relatives was close but there was a mismatch. A mutation occurred. There should not be a mutation statistically speaking for 300 to 600 generations. However, it was found that heteroplasmy was common in the family.

Heteroplasmy is caused by mutations. Since there are multiple copies of mitochondrial DNA in a cell, an average of around 1000 copies per cell, a person can easily have two or more different sequences of mitochondrial DNA when mutations occur in the cell.

Dr. Parsons found that heteroplasmy was more frequent than what was expected, so he and his colleagues conducted a study. They studied 357 individuals from 134 different but related families. They were "stunned" to find out that there were 10 different mutations. This gave a rate of change of one mutation every 800 years, or one mutation in every 40 generations! This is roughly twenty-fold higher than what is expected. They claim that the faster rate cannot be accounted for by substitutions at mutational hot spots which might flip-flop back and forth and not give any evolutionary change in the long term.

Dr. Howell studied the genetic history of an Australian family. The D-loop region of the mitochondrial DNA was sequenced for 45 members of a large maternally linked group. In the study, they found two mutations that were passed on to other members of the family. The mutations were in the actual germ cells (sperm and egg). This resulted in having several descendants inheriting all three mitochondrial DNA types, a condition called triplasmy. Triplasmy is a Heteroplasmy condition with three different copies of mitochondrial DNA in the same individual! In this study, the rate of change was found to be 1 mutation in every 25 to 40 generations.

Using this much faster mutation rate from the two studies as a basis for a new mitochondrial clock speed, Eve can be calculated to have lived a mere 6500 or 6000 years ago, rather than 200,000 years ago.

These kinds of numbers created quite a stir. No evolutionist believed that these numbers were right, but what to do with the finding? Many thought that both Parsons and Howell's results are mainly reflecting mutational hot spots or are concentrating on the D-loop region where the rate of change seems to be much greater than the rest of the genome.

Evidence of a Somewhat Slower Clock Speed

So because these faster rates were much too fast for the evolutionary viewpoint, Dr. Ingman, et. al., studied the mitochondrial DNA of 53 maternally linked humans. Because they suspected that there was a large amount of variation in the mutational rates of the D-loop that might not be reflected in long age studies, so they decided to study the rest of the mitochondrial genome excluding the D-loop from the study. It was found that these slower changing portions of the mitochondrial chromosome approach the rate as expected by evolutionists. These portions of the DNA code for proteins which would be inhibited from changing by natural selection.

Other groups such as Sigurgardottir's group have also been able to measure the rate of change of the entire mitochondrial genome including the D-loop, but with a rate more like one mutation for every 1200 years. Because there are large differences between different studies, it indicates that there must be some unknown variable that would cause the large differences. One interesting aspect of these mutations is that they mostly occur in the control regions of the mitochondrial DNA. These areas are not coding for proteins.

Are the high mutational rates of the D-loop lost over time?

Presently, evolutionists assume that the high rate of mutations found in the control regions of the mitochondrial DNA is hidden over time; Because these bases are thought to have changed back and forth (to flip-flop back and forth). If bases are flip-plopping back and forth, there would be no real change in the DNA. Over short periods of time, one would see evidence of rapid genetic change of the mitochondrial DNA. However, over much longer periods of time, these rapid mutational events would cancel each other out since many of the mutations would be going in opposite directions.

The latest mitochondrial Eve is thought to have lived around 200,000 years ago. 200,000 years is considered long enough for the mutational flip-plop process to show a slower evolutional rate of change over this longer period of time. However, what if the history of the human family was much shorter. What if Eve really lived 6000 years ago? There would not be sufficient time for the mutational plip-plop activity to start cancelling out. The faster mutational rate found in the control regions, especially the D-loop region, might indeed be a measurable change over the short history of the human family.

It is also possible that the faster rates as seen in the control regions of the mitochondrial DNA reflect a faster rate of change which is not inhibited by natural selection. The mutations that occur within genes are usually inhibited by natural selection since the genes are needed for survival. The gene regions within the mitochondrial DNA have clearly been selected against. Most of the mutations are found in the third base of codons, where several possible bases can code for the same base!

Most clock studies are thought to be measuring DNA which is not affected by natural selection. Only a small proportion of nuclear DNA is composed of genes. Yet the mutation rate within the genes of mitochondrial DNA is favored because it agrees with the expected long ages as calculated comparing different species of different families. However the assumption that the clock study is not affected by natural selection is violated when choosing functioning gene regions over non-gene regions.

The Present Mitochondrial Clock Rate

Evolutionists have assumed that the mitochondrial clock rate is fairly constant. They assumed that mutations occur every 6000 to 12,000 years (that would be one mutation in every 300 to 600 generations). However, their assumptions on DNA's rate of change are based on the comparison of different species, the existence of a common ancestor, and long periods of time, which depend upon the evolutionary process to be relevant. If there never was a common ancestor of humans and chimpanzees, then these evolutionary comparisons mean nothing.

As a creationary scientist, I would not expect that the present mitochondrial clock rate would match the rate as predicted by evolutionary studies of different taxonomic groups, such as chimpanzees and humans. It is only an assumption that a comparison of different species will show how long ago different species diverged. If evolution has never occurred, if chimpanzees and humans never had a common ancestor, then a comparison study of different species would not be a measure of relatedness through the evolutionary process; But rather, it would be a measure of how similar the two species are in their physiology and their molecular biology.

When scientists started measuring the mitochondrial DNA rate of change within a single population over time, it was shocking for the evolutionist to discover that mutations are occurring much faster than expected in some populations.

I believe that this much faster rate of change is a better predictor of time than the comparison of man with other species. It is only when evolutionary assumptions are held that the faster rate is held in suspect. When a different set of assumptions are taken, the faster rate of human mitochondrial genetics becomes a distinct possibility.

It will be interesting to see how this whole topic will be treated in the future. Initially I became interested in this topic because of the projected 6000 years that the mitochondrial Eve was recalculated to have lived in the past. However, I soon realized that it was a combination of the faster clock rate and the comparison of humans and chimpanzees that gave the projected 6000 years. So the result is meaningless to a creationary scientist. I would not choose to compare different species with the idea that a common ancestor had ever existed.

Also, since the initial reporting of 6000 years, many of the points that were thought to be well known previously, are now changed. Evolutionary thought is in the process of trying to fit the data into their theories and models.

Speciation is a Degradative Process

Speciation does occur, and it can occur quite rapidly. However, from a creationary perspective, it is a degradative process producing closely related species. Not species of different families that are thought to take millions of years for their separation. To produce different families in the evolutionary theory, the actual data, as seen in the field, must be extended or extrapolated over a great amount of time. The data in the field presently fit both evolutionary and creationary thinking. In order to suggest the process of evolution, one must use a theory that goes beyond what the data suggests. The actual data involving speciation only involves closely related species. See the following links to understand the process from a Creationary perspective. (A new window will open.)

The production of random mutations in the DNA of organisms is a very slow process. 4500 or even 6000 years is an extremely short time to produce the mutations that we think we are seeing in nature. Creationists need faster change than Evolutionists.

The first of four sequencial pages. These four pages cover a range of topics: Does the 2nd law of thermodynamics describe the change in entropy of living organisms which are the most complicated machines on Earth? What would be the nature of the degradation in organisms? Would Island Speciation throw some light on this question. The Bible seems to suggest a few definite ways that man has degraded. How do pseudogenes form?

Interesting Journal Articles with abstracts if available

Mitochondrial DNA and human evolution. Cann RL, Stoneking M, Wilson AC.
Nature. 1987 Jan 1-7;325(6099):31-6.
Department of Biochemistry, University of California, Berkeley, California 94720, USA

Mitochondrial DNAs from 147 people, drawn from five geographic populations have been analysed by restriction mapping. All these mitochondrial DNAs stem from one woman who is postulated to have lived about 200,000 years ago, probably in Africa. All the populations examined except the African population have multiple origins, implying that each area was colonised repeatedly.

Mitochondrial COII sequences and modern human origins. Ruvolo M, Zehr S, von Dornum M, Pan D, Chang B, Lin J.
Mol Biol Evol. 1993 Nov;10(6):1115-35.

The aim of this study is to measure human mitochondrial sequence variability in the relatively slowly evolving mitochondrial gene cytochrome oxidase subunit II (COII) and to estimate when the human common ancestral mitochondrial type existed. New COII gene sequences were determined for five humans (Homo sapiens), including some of the most mitochondrially divergent humans known; for two pygmy chimpanzees (Pan paniscus); and for a common chimpanzee (P. troglodytes). COII sequences were analyzed with those from another relatively slowly evolving mitochondrial region (ND4-5). From class 1 (third codon position) sequence data, a relative divergence date for the human mitochondrial ancestor is estimated as 1/27 th of the human-chimpanzee divergence time. If it is assumed that humans and chimpanzees diverged 6 Mya, this places a human mitochondrial ancestor at 222,000 years, significantly different from 1 Myr (the presumed time of an H. erectus emergence from Africa). The mean coalescent time estimated from all 1,580 sites of combined mitochondrial data, when a 6-Mya human-chimpanzee divergence is assumed, is 298,000 years, with 95% confidence interval of 129,000-536,000 years. Neither estimate is compatible with a 1-Myr-old human mitochondrial ancestor. The mitochondrial DNA sequence data from COII and ND4-5 regions therefore do not support this multiregional hypothesis for the emergence of modern humans.
Erratum in: Mol Biol Evol 1994 May;11(3):552.

Identification of the remains of the Romanov family by DNA analysis. Gill P, Ivanov PL, Kimpton C, Piercy R, Benson N, Tully G, Evett I, Hagelberg E, Sullivan K
Nat Genet 1994 Feb;6(2):130-5
Central Research and Support Establishment, Forensic Science Service, Aldermaston, Reading, Berkshire, UK.
Comment in: Nat Genet 1994 Feb;6(2):113-4

Nine skeletons found in a shallow grave in Ekaterinburg, Russia, in July 1991, were tentatively identified by Russian forensic authorities as the remains of the last Tsar, Tsarina, three of their five children, the Royal Physician and three servants. We have performed DNA based sex testing and short tandem repeat (STR) analysis and confirm that a family group was present in the grave. Analysis of mitochondrial (mt) DNA reveals an exact sequence match between the putative Tsarina and the three children with a living maternal relative. Amplified mtDNA extracted from the remains of the putative Tsar has been cloned to demonstrate heteroplasmy at a single base within the mtDNA control region. One of these sequences matches two living maternal relatives of the Tsar. We conclude that the DNA evidence supports the hypothesis that the remains are those of the Romanov family.

Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Ivanov PL, Wadhams MJ, Roby RK, Holland MM, Weedn VW, Parsons TJ
Nat Genet 1996 Apr;12(4):417-20
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.

In 1991, nine sets of skeletal remains were excavated from a mass grave near Yekaterinburg, Russia which were believed to include the Russian Tsar Nicholas II, the Tsarina Alexandra, and three of their daughters. Nuclear DNA testing of the remains verified such a family group, and mitochondrial DNA (mtDNA) sequences of the presumed Tsarina matched a known maternal relative, Prince Philip. mtDNA sequences from bone of the presumed Tsar matched two living maternal relatives except at a single position, where the bone sample had a mixture of matching (T) and mismatching (C) bases. Cloning experiments indicated that this mixture was due to heteroplasmy within the Tsar; nevertheless, the 'mismatch' fueled a lingering controversy concerning the authenticity of these remains. As a result, the official final report on the fate of the last Russian Royals has been postponed by Russian authorities pending additional, convincing DNA evidence. At the request of the Russian Federation government, we analysed the skeletal remains of the Tsar's brother Georgij Romanov in order to gain further insight into the occurrence and segregation of heteroplasmic mtDNA variants in the Tsar's maternal lineage. The mtDNA sequence of Georgij Romanov, matched that of the putative Tsar, and was heteroplasmic at the same position. This confirms heteroplasmy in the Tsar's lineage, and is powerful evidence supporting the identification of Tsar Nicholas II. The rapid intergenerational shift from heteroplasmy to homoplasmy, and the different heteroplasmic ratios in the brothers, is consistent with a 'bottleneck' mechanism of mtDNA segregation.

How rapidly does the human mitochondrial genome evolve? Howell N, Kubacka I, Mackey DA
Am J Hum Genet 1996 Sep;59(3):501-9
Department of Radiation Therapy, University of Texas Medical Branch, Galveston 77555-0656, USA.

The results of an empirical nucleotide-sequencing approach indicate that the evolution of the human mitochondrial noncoding D-loop is both more rapid and more complex than is revealed by standard phylogenetic approaches. The nucleotide sequence of the D-loop region of the mitochondrial genome was determined for 45 members of a large matrilineal Leber hereditary optic neuropathy pedigree. Two germ-line mutations have arisen in members of one branch of the family, thereby leading to triplasmic descendants with three mitochondrial genotypes. Segregation toward the homoplasmic state can occur within a single generation in some of these descendants, a result that suggests rapid fixation of mitochondrial mutations as a result of developmental bottlenecking. However, slow segregation was observed in other offspring, and therefore no single or simple pattern of segregation can be generalized from the available data. Evidence for rare mtDNA recombination within the D-loop was obtained for one family member. In addition to these germ-line mutations, a somatic mutation was found in the D-loop of one family member. When this genealogical approach was applied to the nucleotide sequences of mitochondrial coding regions, the results again indicated a very rapid rate of evolution.

The mutation rate of the human mtDNA deletion mtDNA4977. Shenkar R, Navi di W, Tavare S, Dang MH, Chomyn A, Attardi G, Cortopassi G, Arnheim N
Am J Hum Genet 1996 Oct;59(4):772-80
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Science Center, Denver, USA.
Comment in: Am J Hum Genet 1996 Oct;59(4):749-55

The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.

Mutational analysis of the human mitochondrial genome branches into the realm of bacterial genetics. Howell N
Am J Hum Genet 1996 Oct;59(4):749-55
Comment on: Am J Hum Genet 1996 Oct;59(4):772-80
Comment in: Am J Hum Genet 1997 Oct;61(4):983-90

mtDNA mutation rates--no need to panic. Macaulay VA, Richards MB, Forster P, Bendall KE, Watson E, Sykes B, Bandelt HJ
Am J Hum Genet 1997 Oct;61(4):983-90
As part of this letter to the editor, a reply to Macaulay et al. by Neil Howel and David Mackey is included.
Comment on: Am J Hum Genet 1996 Oct;59(4):749-55

A high observed substitution rate in the human mitochondrial DNA control region. Parsons TJ, Muniec DS, Sullivan K, Woodyatt N, Alliston-Greiner R, Wilson MR, Berry DL, Holland KA, Weedn VW, Gill P, Holland MM
Nat Genet 1997 Apr;15(4):363-8
Armed Forces DNA Identification Laboratory, Armed Forces Institute of Pathology, Rockville, Maryland 20850, USA.

The rate and pattern of sequence substitutions in the mitochondrial DNA (mtDNA) control region (CR) is of central importance to studies of human evolution and to forensic identity testing. Here, we report a direct measurement of the intergenerational substitution rate in the human CR. We compared DNA sequences of two CR hypervariable segments from close maternal relatives, from 134 independent mtDNA lineages spanning 327 generational events. Ten substitutions were observed, resulting in an empirical rate of 1/33 generations, or 2.5/site/Myr. This is roughly twenty-fold higher than estimates derived from phylogenetic analyses. This disparity cannot be accounted for simply by substitutions at mutational hot spots, suggesting additional factors that produce the discrepancy between very near-term and long-term apparent rates of sequence divergence. The data also indicate that extremely rapid segregation of CR sequence variants between generations is common in humans, with a very small mtDNA bottleneck. These results have implications for forensic applications and studies of human evolution.

Intraspecific nucleotide sequence variability surrounding the origin of replication in human mitochondrial DNA. Greenberg BD, Newbold JE, Sugino A.
Gene. 1983 Jan-Feb;21(1-2):33-49.

We have cloned the major noncoding region of human mitochondrial DNA (mtDNA) from 11 human placentas. Partial nucleotide sequences of five of these clones have been determined and they share a maximum of 900 bp around the origin of H-strand replication. Alignment of these sequences with others previously determined has revealed a striking pattern of nucleotide substitutions and insertion/deletion events. The level of sequence divergence significantly exceeds the reported estimates of divergence in coding regions. Two particularly hypervariable regions have also been defined. More than 96% of the base changes are transitions, and length alterations have occurred exclusively by addition or deletion of mono-or dinucleotide segments within serially repeating stretches. This region of the mitochondrial genome, which contains the initiation sites for replication and transcription, is the least conserved among species with respect to both sequence and length (Anderson et al., 1981; Walberg and Clayton, 1981). Despite this overall lack of primary sequence conservation, several consistencies appear among the available mammalian mtDNA sequences within this region. Between species, a conserved linear array of characteristic stretches exists which nonetheless differ in primary sequence. Among humans, several conserved blocks of nucleotides appear within domains deleted from the mtDNA of other species. These observations are consistent with both a species-specificity of nucleotide sequence, and a preservation of the necessary genetic functions among species. This provides a model for the evolution of protein-nucleic acid interactions in mammalian mitochondria.

The mutation rate in the human mtDNA control region. Sigurgardottir S, Helgason A, Gulcher JR, Stefansson K, Donnelly P.
Am J Hum Genet. 2000 May;66(5):1599-609. Epub 2000 Apr 7.
deCODE Genetics, Inc., Reykjavik, Iceland 110.

The mutation rate of the mitochondrial control region has been widely used to calibrate human population history. However, estimates of the mutation rate in this region have spanned two orders of magnitude. To readdress this rate, we sequenced the mtDNA control region in 272 individuals, who were related by a total of 705 mtDNA transmission events, from 26 large Icelandic pedigrees. Three base substitutions were observed, and the mutation rate across the two hypervariable regions was estimated to be 3/705 =.0043 per generation (95% confidence interval [CI].00088-.013), or.32/site/1 million years (95% CI.065-.97). This study is substantially larger than others published, which have directly assessed mtDNA mutation rates on the basis of pedigrees, and the estimated mutation rate is intermediate among those derived from pedigree-based studies. Our estimated rate remains higher than those based on phylogenetic comparisons. We discuss possible reasons for-and consequences of-this discrepancy. The present study also provides information on rates of insertion/deletion mutations, rates of heteroplasmy, and the reliability of maternal links in the Icelandic genealogy database.

Mitochondrial mutation rate revisited: hot spots and polymorphism. Jazin E, Soodyall H, Jalonen P, Lindholm E, Stoneking M, Gyllensten U
Nat Genet 1998 Feb;18(2):109-10
As part of this correspondence, Parsons and Holland respond
Comment on: Nat Genet 1997 Apr;15(4):363-8

Mitochondrial genome variation and the origin of modern humans. Ingman M, Kaessmann H, Paabo S, Gyllensten U.
Nature. 2000 Dec 7;408(6813):708-13.
Department of Genetics and Pathology, Section of Medical Genetics, University of Uppsala, Sweden.

The analysis of mitochondrial DNA (mtDNA) has been a potent tool in our understanding of human evolution, owing to characteristics such as high copy number, apparent lack of recombination, high substitution rate and maternal mode of inheritance. However, almost all studies of human evolution based on mtDNA sequencing have been confined to the control region, which constitutes less than 7% of the mitochondrial genome. These studies are complicated by the extreme variation in substitution rate between sites, and the consequence of parallel mutations causing difficulties in the estimation of genetic distance and making phylogenetic inferences questionable. Most comprehensive studies of the human mitochondrial molecule have been carried out through restriction-fragment length polymorphism analysis, providing data that are ill suited to estimations of mutation rate and therefore the timing of evolutionary events. Here, to improve the information obtained from the mitochondrial molecule for studies of human evolution, we describe the global mtDNA diversity in humans based on analyses of the complete mtDNA sequence of 53 humans of diverse origins. Our mtDNA data, in comparison with those of a parallel study of the Xq13.3 region in the same individuals, provide a concurrent view on human evolution with respect to the age of modern humans.
Erratum in: Nature 2001 Mar 29;410(6828):611.
Comment in: Nature. 2000 Dec 7;408(6813):652-3.

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