<snip>

So, since the D-loop region seems to have too many problems, perhaps
other mtDNA sequences that don't mutate so rapidly will work better?
Well, the reason why these other stretches of mtDNA, like the ND
regions, don't mutate as fast as the hypervariable D-loop regions is
that they code for functional proteins.  This means that the ND4/ND5
sequences are limited in their mutation rate by natural selection -
which most certainly is a stabilizing force of nature. But, what about
their "neutral" differences?

In the January 2003 edition of the "Annals of Human Genetics",
geneticist Peter Forster of Cambridge published an article, "To Err is
Human", in which he noted that, "more than half of the mtDNA
sequencing studies ever published contain obvious errors." He then
asked: "Does it matter? Unfortunately, in many cases it does. . .
fundamental research papers, such as those claiming a recent African
origin for mankind (Cann, et al., 1987; Vigilant, et al., 1991) . . .
have been criticized, and rejected due to the extent of primary data
errors." Forster when on to note that this is "only the tip of the
iceberg. . . There is no reason to suppose that DNA sequencing errors
are restricted to mtDNA."

One month later, in the February 20, 2003 issue of "Nature", Carina
Dennis authored a commentary on Forster's work titled "Error Reports
Threaten to Unravel Databases of Mitochondrial DNA." Dennis reiterated
the findings that "more than half of all published studies of human
mitochondrial DNA (mtDNA) sequences contain mistakes." she went on to
note that, "The problem is far bigger than researchers had imagined.
The mistakes may be so extensive that geneticists could be drawing
incorrect conclusions to studies of human populations and evolution."

Also, the central dogma that mtDNA follows strict maternal inheritance
with only very rare paternal mtDNA "contamination" is being
challenged.  Schwartz and Vissing noted in the August 2002 issue of
the New England Journal of Medicine that, "Mammalian mitochondrial DNA
(mtDNA) is thought to be strictly maternally inherited. . . Very small
amounts of paternally inherited mtDNA have been detected by the
polymerase chain reaction (PCR) in mice after several generations of
interspecific backcrosses. . . We report the case of a 28-year-old man
with mitochondrial myopathy due to a novel 2-bp mtDNA deletion. . . We
determined that the mtDNA harboring the mutation was paternal in
origin and accounted for 90 percent of the patient's muscle mtDNA." Of
course, "If mtDNA can recombine, irrespective of the mechanism, there
are important implications for mtDNA evolution and for phylogenetic
studies that use mtDNA" (Morris, Andrew A. M., and Robert N.
Lightowlers (2000), "Can Paternal mtDNA be Inherited?", The Lancet,
355:1290-1291, April 15).

Now, you claim that out of 694 nucleotides in the DN4/DN5 regions that
only 76 of them have "informative differences". Of these, 18 are
involved in a change in amino acid composition leaving only 58 as
clearly neutral differences (discussed further below). This is only 8%
of the total number of sequences.  Of these 58, humans share only 12
or so more differences with chimps than with orangutans and gibbons.
Also added into this mix is a 1 or so amino acid variation among
individuals within these species in these particular coding regions of
mtDNA.  This leaves only around 10 or so differences to base your
clusters on.  This is only around 1.5% of the total sequence length
and only 17% of the neutral sequences.  Do you think, in the light of
just a few of the problems listed above for mtDNA sequence analysis,
that a 17% increased nesting with chimps, humans, and gorillas over
gibbons and orangutans is really all that significant - especially
since this is only 1.5% of the total sequence length?  Also, these 58
neutral loci, with their 10 or so clustered differences in the listed
species, if truly neutral, are no less susceptible to mutational hits
than are the sequences found in the D-loop region.  Therefore, their
rate of mutation should also be equivalent to the rate of mutation
found in the D-loop region (which even you admit to being at least one
order of magnitude higher than is recognized in coding regions of
mtDNA). If there is a difference found in the rate of mutation between
these different regions, even with respect to "neutral" changes, what
is the explanation for this phenomenon?  Perhaps even these mutations
aren't really neutral?  Of course, this just screws up everything now
doesn't it?

The fact of the matter is, even in mtDNA regions such as the ND4/ND5
regions, "Silent [neutral] sites saturate extremely quickly,
presumably owing to the substitution bias and, perhaps, to an
accelerated mutation rate. Results emphasize the importance of using
only the most closely related sequences in order to infer patterns of
substitution accurately for nematodes or for other taxa having
strongly composition-biased DNA. ND4 also shows high amino acid
polymorphism at both the intra-and interspecific levels, and in higher
level comparisons, there is evidence of saturation at variable amino
acid sites. In general, we recommend using mtDNA coding genes only for
phylogenetics of relatively closely related nematode species and, even
then, using only nonsynonymous substitutions and the more conserved
mitochondrial genes (e.g., cytochrome oxidases). On the other hand,
the high substitution rate in genes such as ND4 should make them
excellent for population genetics studies, identifying cryptic
species, and resolving relationships among closely related congeners
when other markers show insufficient variation" (Michael S. Blouin,
Charles A. Yowell, Charles H. Courtney, and John B. Dame,
"Substitution Bias, Rapid Saturation, and the Use of mtDNA for
Nematode Systematics", Department of Zoology, Oregon State University;
and Department of Pathobiology, University of Florida, 1998).

This kinda messes up your ND4 hypothesis - doesn't it?  Are primate
ND4 sequences all that different from nematode mtDNA ND4 sequences in
structure and function?  If not, then why do these ND4 sequences in
nematodes mutate so fast and have so many polymorphisms?  Why do the
neutral sites in the ND4 sequence saturate so quickly?

Also, your proposed clustering effect might even be less significant
than it already seems once you add in the transition/transversion rate
bias and codon frequency biases into the picture.  Consider an
abstract from a paper by Yang and Nielsen published in the 1998 issue
of the "Journal of Molecular Evolution" (Even though it is discussion
nDNA, the implications for mtDNA are still clear):

"A maximum likelihood approach was used to estimate the synonymous and
nonsynonymous substitution rates in 48 nuclear genes from primates,
artiodactyls, and rodents. A codon-substitution model was assumed,
which accounts for the genetic code structure, transition/transversion
bias, and base frequency biases at codon positions. Likelihood ratio
tests were applied to test the constancy of nonsynonymous to
synonymous rate ratios among branches (evolutionary lineages). It is
found that at 22 of the 48 nuclear loci examined, the
nonsynonymous/synonymous rate ratio varies significantly across
branches of the tree. The result provides strong evidence against a
strictly neutral model of molecular evolution. Our likelihood
estimates of synonymous and nonsynonymous rates differ considerably
from previous results obtained from approximate pairwise sequence
comparisons. The differences between the methods are explored by
detailed analyses of data from several genes. Transition/transversion
rate bias and codon frequency biases are found to have significant
effects on the estimation of synonymous and nonsynonymous rates, and
approximate methods do not adequately account for those factors. The
likelihood approach is preferable, even for pairwise sequence
comparison, because more realistic models about the mutation and
substitution processes can be incorporated in the analysis" (Yang Z,
Nielsen R. "Synonymous and nonsynonymous rate variation in nuclear
genes of mammals", J Mol Evol. 1998 Apr;46(4):409-18).

In this line, Bielawskia et. al., published a paper in the November
2000 issue of "Genetics" noting that, "Previous studies of mammalian
nuclear genes largely employed approximate methods to estimate rates
of nonsynonymous and synonymous substitutions. Because these methods
did not account for major features of DNA sequence evolution such as
transition/transversion rate bias and unequal codon usage, they might
not have produced reliable results. To evaluate the impact of the
estimation method, we analyzed a sample of 82 nuclear genes from the
mammalian orders Artiodactyla, Primates, and Rodentia using both
approximate and maximum-likelihood methods. Maximum-likelihood
analysis indicated that synonymous substitution rates were positively
correlated with GC content at the third codon positions, but
independent of nonsynonymous substitution rates. Approximate methods,
however, indicated that synonymous substitution rates were independent
of GC content at the third codon positions, but were positively
correlated with nonsynonymous rates. Failure to properly account for
transition/transversion rate bias and unequal codon usage appears to
have caused substantial biases in approximate estimates of
substitution rates" (Joseph P. Bielawskia, Katherine A. Dunna, and
Ziheng Yanga, "Rates of Nucleotide Substitution and Mammalian Nuclear
Gene Evolution: Approximate and Maximum-Likelihood Methods Lead to
Different Conclusions", Genetics, Vol. 156, 1299-1308, November 2000).

There are several very interesting things to notice here.  Note that
"the nonsynonymous/synonymous rate ratio varies significantly across
branches of the tree [of life].  The result provides strong evidence
against a strictly neutral model of molecular evolution." What is
especially interesting in this analysis is that the
transition/transversion rate bias and codon frequency biases have a
"significant" effect on the estimation of the synonymous and
nonsynonymous rates and that these biases have not been adequately
accounted for!  In fact, this bias is described as substantially
affecting the estimates of substitution rates.  Basically, depending
upon which method you choose, you will come to different phylogenetic
conclusions because of this bias problem.

Now, don't you think that this same transition/transversion rate bias
problem also affects mtDNA sequence analysis?  Is it even possible to
correct for this substitution rate bias problem in a non-biased way -
especially with those differences that are actually functional?

Also consider that the 18 mutations that resulted in a change in amino
acid sequence might not be as neutral as you have surmised. It seems
that, "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. To test this hypothesis we
analyzed 104 complete mtDNA sequences from all global regions and
lineages. African mtDNA variation did not significantly deviate from
the standard neutral model, but European, Asian, and Siberian plus
Native American variations did. Analysis of amino acid substitution
mutations (nonsynonymous, Ka) versus neutral mutations (synonymous,
Ks) (kaks) for all 13 mtDNA protein-coding genes revealed that the
ATP6 gene had the highest amino acid sequence variation of any human
mtDNA gene, even though ATP6 is one of the more conserved mtDNA
proteins. Comparison of the kaks ratios for each mtDNA gene from the
tropical, temperate, and arctic zones revealed that ATP6 was highly
variable in the mtDNAs from the arctic zone, cytochrome b was
particularly variable in the temperate zone, and cytochrome oxidase I
was notably more variable in the tropics. Moreover, multiple amino
acid changes found in ATP6, cytochrome b, and cytochrome oxidase I
appeared to be functionally significant. From these analyses we
conclude that selection may have played a role in shaping human
regional mtDNA variation and that one of the selective influences was
climate" (Mishmar D et. al., "Natural selection shaped regional mtDNA
variation in humans" Proc Natl Acad Sci U S A. 2003 Jan
7;100(1):171-6. Epub 2002 Dec 30).

http://www.ncbi.nlm.nih.gov/entrez/query.fcgidb=PubMed&cmd=Retrieve&d...

http://www.apologeticspress.org/inthenews/2003/itn-03-03.htm

http://oregonstate.edu/~blouinm/pdf_files/MBE1998.pdf

Sean