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Guide to Patagonia's Monsters & Mysterious beings

I have written a book on this intriguing subject which has just been published.
In this blog I will post excerpts and other interesting texts on this fascinating subject.

Austin Whittall


Monday, February 3, 2014

The mtDNA clock ticks out of time


Each of our cells carries within it several Mitochondria. These are remarkable organelles which are essential for cellular metabolism. They are even more remarkable because they carry their own DNA, which are independent from our other DNA, the nuclear one, which codes our other proteins.


The fact that Mitochondria have their own DNA supports the notion that long ago, before eukaryotic cells formed, they were independent organisms. Somehow they formed a symbiotic relationship within eukaryotic cells, generating energy for them in exchange for protection and “food”.


mtDNA is inherited in an uniparental manner –from mother to offspring. Male mtDNA in sperm does not enter the fertilized egg. This means that it can be used to track matrilineal lineages back in time.


Eve theory


Since it is inherited on a matrilineal basis, your mtDNA is identical to your mother’s and, also to her mother’s (your maternal grandmother) and so on, backwards generation after generation, until we reach the group of families that make up the the first Homo sapiens clan. One of the women among them is the source of your mtDNA.


That would be true if mtDNA did not mutate, but it does, at higher than nuclear DNA mutates. As they lack enzymes to repair mutations mutations accumulate.


The Mitochondrial DNA (mtDNA) is much smaller than the nuclear DNA, but is critical because the genes it encodes are fundamental: they control cellular metabolism, and if they mutate in a negative way, the organism dies or suffers diseases.


A comparison of all modern human mtDNA shows that extant and fossil humans display a wide range of mtDNA sequences.


But all come from the original group of Homo sapiens. Actually all mtDNA of living humans can be traced to one woman. That does not mean she was the only woman alive at that time. It means that the other women had sons instead of daughters (a dead end for mtDNA), or died without issue, or their offspring died without having children.


Along the line, long ago, a mutation that was “neutral” (remember, wacky mutations lead to death or illness) appeared among the “original” lineage and the daughter who carried it, passed it on to the future generations through her female offspring. The original lineage carried on until another different “neutral” mutation split it from the “original” lineage, and was passed on to the next generation.


The neutrality of mutations is something we will be going back to further down. Please remember it.


This branching from the original lineage has led to a diversity of lineages, each carrying the mutations that have accumulated across the millennia.


If these mutations happen at regular intervals we could time when they took place and find out when did the original group of Homo sapiens live. We could also compare the mtDNA of all mankind and find where they lived.


This was done, and according to this theory, the original woman (“Eve”) lived in Africa some 150 kya.


But… is this based on sound science? Does the mutational clock tick at a regular pace? Can we extrapolate backwards in time with confidence? In my previous post I expressed my doubts, today I will substantiate them with evidence.


The clock ticks at a variable pace


When I first looked at a haplogroup tree, which shows the mutations along each branch, I was surprised to see that the number of mutations differs along the branches.


An African, a European, an Asian and an Amerindian all living now and traceable back to the same woman would all have to have (if the clock ticks with a regular pace) the same number of mutations (i.e. 40 mutations) over those 150,000 years. They would not be the same mutations of course: we would share those that took place before the branching points and would have our particular ones along the tips of our different branches. Maybe some would repeat across branches due to chance. But we would all have the same amount of mutations.


Well, this is not the case. So the clock theory is not correct. That is my blunt conclusion, but let’s read some more subtle comments on the matter, because thera are some papers that point out that mutations occur at a variable rate, no clock here, just mutations happening in a haphazard manner:


Galtier, Nabholz, Glémin and Hurst. (2009)


we argue that mitochondrial DNA is not always clonal, far from neutrally evolving and certainly not clock-like, questioning its relevance as a witness of recent species and population history. […] The molecular clock, therefore, is certainly not a tenable assumption as far as mtDNA is concerned. Nonclock-like evolution is common, and the departure from homogeneous rates can be very strong. In mammals, the mitochondrial mutation rate appears more variable across lineages than the nuclear one” [3]


Nabholz, Glémin and Galtier. (2009)


This study confirms and extends the message of caution […] about the usage of mtDNA as a molecular marker of biodiversity in vertebrates: (i) mtDNA diversity is not related to species abundance; (ii) mtDNA greatly departs the molecular clock hypothesis. The 2% per site per million year calibration (estimated from primate data) has no degree of generality, and should not be used for dating purposes in the absence of fossil data.” [4]


Yes, both were written by the same team, so not to cheat I will quote other papers:


The paper by Behar et al. [1] which reviewed the mtDNA tree and proposed some rational changes, also noted that there are some problems with the “clock”:


The accepted notion of a molecular clock means that contemporary mtDNA haplotypes should show statistically insignificant differences in the number of accumulated mutations from the RSRS.


Note: RSRS is the “ancestral Eve”. In other words all humans should have the same amount of mutations in their mtDNA when compared to “Eve”, but, we do not . Behar et al. add:


The range of substitution counts separating contemporary mitogenomes belonging to major haplogroups from the RSRS is shown in Figure S2. The mean distance is 57.1 substitutions, the median is 56 and the empirical standard deviation is 5.9. Widely different distances ranging from 41 substitutions in some L0d1a1 mitogenomes to 77 in some L2b1a mitogenomes are observed. [1]


This means that we don't have say 60 substitutions (mutations), no, we have between 41 and 77. The middle value is 56. A very oddly ticking clock indeed. But let’s read more from Behar et al.:


Interestingly, the ranges of substitution counts within haplogroups M and N, which are hallmarks of the relatively recent out-of-Africa exodus of humans, are also very large. For example, within M there are two mitogenomes with 43 substitutions (in M30a and M44) and two mitogenomes with as many as 71 substitutions (in M2b1b and M7b3a). This is especially striking because the path from the RSRS to the root of M already contains 39 substitutions. Hence, the difference between the M root and its M44 descendant is only four substitutions (two in the coding region and two in the control region) as compared to 32 substitutions in the M2b1b and M7b3a mitogenomes.[…] Our results demonstrate violations of the molecular clock in M […] and give mixed results for the entire tree [...] and L2 […] and borderline results in N […] We are currently unable to offer well-founded explanations for these findings, which remain the scope of future studies. [1]


In other words haplogroups L, M and N violate the clock, but, despite these reservations, Behar et al ignore them and merrily go on to estimate lineage ages (Quote: "As the clock violation was observed only in a restricted number of specified cases, we applied the best available tools for estimating the ages of ancestral nodes" [1]).


The Figure S2 mentioned above in Behar et al. is interesting because one might expect the variability within a haplogroup to follow a Normal Distribution (Gauss bell-shaped frequency distribution), with a strong central mean value of substitutions and a dispersion to each side… but instead most of the frequency distributions are very clear Bimodal distributions.


Bimodal Distributions


As an engineer I am well acquainted with statistics, we use them every day to analyze how our industrial manufacturing processes are running. Take a look at the distributions shown in Figure S2 below (the red arrows show the “peaks” of the double humped Bimodal distribution):


Figure with bimodal distributions
Bimodal distributions, modes marked with red arrows. Adapted From [1]

For the layman: if a process is affected by some regular variation, on a chance basis, you will notice a bell-shaped distribution of frequencies. Such as height of people: Some will be very tall, others very short but most will fall close to a median height. This is a Normal distribution and looks more or less like the one adopted by M in the image above (a bell-shaped curve) .


Now a Bimodal Distribution has two “humps” or modes (a peak or local maxima): If you have a machine making bottles, the weight of the bottles should adopt a bell-shaped Normal distribution. Now imagine two different machines making bottles. Each will have its own bell shaped distribution: Machine A and Machine B, with their own mean values. Now mix all the bottles in a box and draw some samples at random from the mixture, if you graph the frequencies for these samples based on their weights: it will combine both bells into a double-hump distribution, a Bimodal one.


It is clear that something is altering the Normal Distributions in the L1, L2 and N haplogroups. L0 and L3’6 are not Normal either.


What process could skewer the Normal distribution in this manner?


Howell, Elson, Turnbull and Herrnstadt (2004) [2] also noticed this odd “clock-violating” behavior of the Ancient African L lineages and the bimodal distribution of substitutions:


Based on the results presented here, such control region clocks are highly suspect[…] We have recently analyzed a set of 560 mtDNA coding region sequences (Herrnstadt et al. 2002) and shown that selection has influenced the evolution of the human mitochondrial genome. […] As a follow-up to those studies, we report here our tests ofclock-like evolution in African haplogroup L mtDNA sequences. The results are complex, and they argue against any simple mtDNA clock fortiming events during human evolution. ” [2]


Non Neutral mutations



In other words, the mutations in mtDNA are not neutral that happen by chance, they are influenced by natural selection, and it is this that alters the “ticking” of the clock. They note in their paper that: “the pairwise mismatch distributions are “jagged” (data not shown), the typical finding for sequences from African populations […] thus indicate selection, rather than recent population expansion.” [2]


They also took a larger sample of African mtDNA and found “ Marked violations of clock-like evolution were now observed both in the coding and control regions […] evidence that haplogroup L2 subclades evolve at different rates” [2]


Mishmar et al. (2003) looked into the natural selection issue: why would mtDNA “evolve” under selective pressure. They conclusions are very interesting: [6]


Human mtDNA shows striking regional variation, traditionally attributed to genetic drift. However, it is not easy to account for the fact that only two mtDNA lineages (M and N) left Africa to colonize Eurasia and that lineages A, C, D, and G show a 5-fold enrichment from central Asia to Siberia. As an alternative to drift, natural selection might have enriched for certain mtDNA lineages as people migrated north into colder climates.” [6]


In other words, as humans moved across Asia their mtDNA mutated and a lot. They point out (below) that mitochondria encode certain molecules crucial for energy production within the cells and therefore impacting on the ability to produce heat and survive in cold climes:


Natural selection has been hypothesized to explain anomalies in the branch lengths of certain European and African mtDNA lineages. […] the genes of the mtDNA are central to energy production, both to generate ATP to perform work and to generate heat to maintain body temperature.
We now hypothesize that natural selection may have influenced the regional differences between mtDNA lineages. This hypothesis is supported by our demonstration of striking differences in the ratio of nonsynonymous (nsyn)/synonymous (syn) nucleotide changes in mtDNA genes between geographic regions in different latitudes. We speculate that these differences may reflect the ancient adaptation of our ancestors to increasingly colder climates as Homo sapiens migrated out of Africa and into Europe and northeastern Asia.
” [6]


Comments and Conclusions


Based on the above it seems pointless to calculate the dates of the branch splitting points and the date our ancestral “Eve” originated. Yet, as we saw (Behar et al.), even though the method is baseless, it is used just the same.


As I mentioned in previous posts, the mutation rates are “hand – adjusted” so that the calculated branching dates “fit” the “evidence” provided by the bones and stones archaeologists. If America is supposed to have been peopled not more than 30 kya then the haplogroups found in America must not be much older than that… and so on.


Science should corroborate findings using independent sources not circular ones.


But let’s go back to the bimodal distribution… Why would a group of haplogroup N people have a mode of 51 and others have a mode of 61 substitutions? Perhaps it could be interpreted as per Rogers and Harpending [8] (I don’t think so). I believe that it may be caused by a fusion of two groups of people (Murray-Macintosh, 1998 [5]) , one carrying a large amount of substitutions and another with much less. And that the mutations are due to selective pressure. So the more mutated population could be an older one and the less mutated one, a younger one. That is, two waves of people entering the same region from the same homeland in two separate waves.


The LM3 Lake Mungo mtDNA


And last, but not least, the Australian remains of a male, gracile Lake Mungo 3 (LM3), which is about 60 ky old, its mtDNA was tested. The outcome was startling (Adcock et al., 2001) [7]:


His mtDNA belonged to a lineage that only survives as a segment inserted into chromosome 11 of the nuclear genome, which is now widespread among human populations.
This lineage probably diverged before the most recent common ancestor of contemporary human mitochondrial genomes. This timing of divergence implies that the deepest known mtDNA lineage from an anatomically modern human occurred in Australia; analysis restricted to living humans places the deepest branches in East Africa.
[…] This finding does not imply that all living people originated in Australia, any more than previously described deep lineages in Africa demand a recent origin of humans on that continent. Deep lineages in Africa and our finding of an even deeper lineage in Australia are consistent with a number of possible models of the demographic and evolutionary history of our species.” [7]


Where does this "ancient and different mtDNA" fit into the branches of the mtDNA tree built by orthodoxy? These remains are only 60 ky old, so are therefore younger than L haplogroup from Africa as reconstructed by Behar et al [1], see image below from their paper:


from Behar paper

The scale at the top shows that the mutations happen (according to Behar and his team) at a regular pace!. To place LM3 before L0 would imply an age of 170 kya, but LM3 lived 6 0kya. Something is not quite right. And that is the reason that LM3 is ignored in all papers on the mtDNA timeline and haplogroups.


Sources


[1] Behar, D., et al. (2014). A “Copernican” Reassessment of the Human Mitochondrial DNA Tree from its Root. American Journal of Human Genetics, Volume 90, Issue 4, 675-684, 6 April 2012 doi:10.1016/j.ajhg.2012.03.002
[2] Neil Howell, Joanna L. Elson, D. M. Turnbull and Corinna Herrnstadt, (2004). African Haplogroup L mtDNA Sequences Show Violations of Clock-like Evolution. Mol Biol Evol (2004) 21 (10): 1843-1854. doi: 10.1093/molbev/msh184
[3] N. Galtier, B. Nabholz, S. Glemin, G.D.D. Hurst, (2009). Mitochondrial DNA as a marker of molecular diversity: a reappraisal. Molecular Ecology (2009) 18, 4541–4550 doi: 10.1111/j.1365-294X.2009.04380.x
[4] Benoit Nabholz, Sylvain Glémin and Nicolas Galtier, (2009). The erratic mitochondrial clock: variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals Evolutionary Biology 2009, 9:54 doi:10.1186/1471-2148-9-54
[5] Rosalind P. Murray-McIntosh, Brian J. Scrimshaw, Peter J. Hatfield, and David Penny, (1998). Testing migration patterns and estimating founding population size in Polynesia by using human mtDNA sequences. Proceedings of the National Academy of Sciences www.pnas.org vol. 95 no. 15
[6] Dan Mishmar et al. Natural selection shaped regional mtDNA variation in humans. Proceedings of the National Academy of Sciences www.pnas.org vol 100. no.1, 171–176, doi: 10.1073/pnas.0136972100, doi: 10.1073/pnas.0136972100
[7] Gregory J. Adcock, et al. (2001). Mitochondrial DNA sequences in ancient Australians: Implications for modern human origins. PNAS u January 16, 2001 u vol. 98 u no. 2 u 537–542
[8] Harpending H C, Sherry S T, Rogers A R, Stoneking M (1993). The genetic structure of ancient human populations, Curr Anthrol 34:483–496. http://content.lib.utah.edu/cdm/ref/collection/uspace/id/2702



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