* MitoKor Inc., San Diego, California; Department of Radiation Oncology, The University of Texas Medical Branch, Galveston; and
Mitochondrial Research Group, School of Neurology, Neurobiology, and Psychiatry, The Medical School, The University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom
Correspondence: E-mail: howelln{at}mitokor.com
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Abstract |
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Key Words: mitochondrial DNA molecular evolution phylogenetic analysis molecular clock human evolution
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Introduction |
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A key issue for the approach used by Cann, Stoneking, and Wilson (1987), and for subsequent studies of this type, is whether the human mitochondrial genome has evolved in a clock-like manner. The use of a mtDNA clock to time the major events in human evolution and population dispersal has been questioned on several grounds (Gibbons 1998; Ayala 1999; Glazko and Nei 2003). Recent results support the operation of more complex, non-clock evolutionary processes in numerous taxa, including primates (e.g., Zeng et al. 1998; Grissi et al. 2000; Yoder and Yang 2000; Soltis et al. 2002; Yi, Ellsworth, and Li 2002; Glazko and Nei 2003). The more general issue of whether human mtDNA evolution fits the neutral model has also been investigated. The available evidence indicates that the evolution of most, and perhaps all, mitochondrial genes has been nonneutral (Gerber et al. 2001; Rand 2001; Mishmar et al. 2003; Moilanen and Majamaa 2003; Moilanen, Finnilä, and Majamaa 2003; Nielsen and Yang 2003; Elson, Turnbull, and Howell 2004).
The availability of large sets of complete human mtDNA sequences (e.g., Ingman et al. 2000; Herrnstadt et al. 2002) provides the foundation for comprehensive and critical tests of the molecular clock model. Ingman et al. (2000) analyzed a set of 53 complete mtDNA sequences of diverse ethnic origin. They observed that coding region sequences evolved according to a molecular clock, but that control region evolution was not clock-like. Torroni et al. (2001) analyzed a small set of complete sequences from the African L2 mtDNA haplogroup, and they observed a violation of clock-like evolution. In view of the reliance of phylogeographic studies of human evolution and population dynamics on an mtDNA clock, especially one based on control region evolution, the accuracy of such a clock is a subject that will benefit from further analysis and refinement.
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 (Elson, Turnbull, and Howell 2004). As a follow-up to those studies, we report here our tests of clock-like evolution in African haplogroup L mtDNA sequences. The results are complex, and they argue against any simple mtDNA clock for timing events during human evolution. In addition, this is the most extensive analysis thus far of African mtDNA coding region sequences.
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Experimental Procedures |
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The haplogroup L mtDNAs analyzed here were obtained from African Americans, not directly from African populations. In a similar fashion, the African haplogroup L2 mtDNA sequences analyzed by Torroni et al. (2001) were obtained from inhabitants of Puerto Rico, whereas the comprehensive analyses of Salas et al. (2002) involved samples from a number of populations, including African Americans (see also Salas et al. 2004). There is no evidence that these studies are biased by this sampling scheme.
Background and General Approach of the Present Study
According to Kimura's model of neutral evolution (1968), the rate at which mutations become fixed in the population equals the rate of mutation (k = u), because the vast majority of sequence changes are neutral with respect to selection. As a consequence, the rate process of substitution should be the same among all lineages (that is, sequence divergence should be clock-like). It was further assumed by Kimura that this single substitution rate operated as a simple Poisson process. As pointed out by Zheng (2001), however, the molecular clock model requires no more than the same process of substitutionirrespective of its complexityin all lineages. A molecular clock can operate in the presence of selection, although the conditions required are restricted and unlikely to have occurred during human evolution.
Donnelly (1991) has drawn some important distinctions among the operational rates of mutation that bear directly on tests of an mtDNA clock (see also the related discussion on p. 1607 in Sigurðardóttir et al. [2000]). In his terminology, the molecular clock stipulates that ks = u, where ks is the rate at which mutations are fixed at the species level. A third parameter is ka, the rate of sequence evolution along an ancestral lineage, and it is the rate of evolution that is tested here and that was tested in the clock analyses of Ingman et al. (2000) and Torroni et al. (2001). The present study is thus designed to test whether the rate of divergence ka within haplogroup L mtDNAs has been clock-like in the specific sense of a single substitution process among all lineages. Intraspecific analyses have been used by other investigators to test the neutral model of evolution (e.g., Fu and Li 1993; Templeton 1996; Huelsenbeck and Rannala 1997; Gerber et al. 2001).
Likelihood Ratio Tests of Clock-like Sequence Evolution
Likelihood ratio tests (LRTs) were used to determine if the divergence of mtDNA sequences was consistent with a molecular clock (Felsenstein 1988; Huelsenbeck and Rannala 1997; Zhang 1999; Posada 2001). LRTs were carried out with Tree-Puzzle version 5.0 (Strimmer and von Haeseler 1996; available at http://www.tree-puzzle.de). In brief, the clock test involved computation of maximum-likelihood branch lengths for a set of sequences under two conditions. In the first condition, evolution was constrained to a single mutation rate process for all branches (clock) and, in the second, rates were allowed to vary among branches (non-clock). For statistical analysis, the differences between the clock and non-clock maximum-likelihood phylogenetic trees (multiplied by 2) are assumed to fit a 2 distribution with n 2 degrees of freedom, where n is the number of sequences analyzed (Felsenstein 1988).
In our "default" analyses, the Tree-Puzzle clock tests were performed with the Neighbor-Joining algorithm, the HKY model of nucleotide substitution (Hasegawa, Kishino, and Yano 1985), the "accurate (slow)" estimation procedure, and a uniform rate of mutation at all sites (Strimmer and von Haeseler 1996). Tests were also performed with the Tamura-Nei (TN) model of nucleotide substitution, which is a more general model of substitution than the HKY model. More importantly, clock tests were also performed under the condition of site variability of divergence rates, because branch lengths are underestimated with uniform substitution rates and, as a result, molecular clock tests can become too conservative (Cunningham, Zhu, and Hillis 1998; Zhang 1999; Posada 2001). When rate heterogeneity is incorporated into the clock test, Tree-Puzzle models substitution rates as i.i.d. "draws" (changes are independently and identically distributed) from a discrete gamma distribution that is parameterized by , the shape distribution parameter (Yang 1994). When
approaches infinity, then all sites evolve at the same rate.
A human mtDNA sequence, usually from the L0a subclade, has been used as the outgroup for the studies reported here, rather than a nonhuman primate mtDNA sequence. For comparison, Ingman et al. (2000) used gorilla and chimpanzee mtDNA outgroup sequences in their clock tests, whereas Torroni et al. (2001) used an L0a outgroup sequence. There are two main reasons for our choice of an intraspecific outgroup. First, a distant outgroup sequence is more likely to be affected by mutational saturation and the clock test can become too conservative as a result (Bromham et al. 2000). There are longstanding concerns that primate sequences are too diverged from human sequences to function as informative outgroups (e.g., Wheeler 1990, Maddison, Ruvolo, and Swofford 1992). Second, there is evidence that the substitution process in mtDNA is not homogenous among different primates (e.g., Excoffier and Yang 1999; Meyer, Weiss, and von Haeseler 1999; Weiss and von Haeseler 2003). With a nonhuman primate outgroup sequence, therefore, failure of a clock test could result from failure of the ingroup sequences (in this case, human) to evolve according to a molecular clock or from inhomogeneity of sequence evolution between the two species.
For some mtDNA sequence sets, L1b and L1c outgroup sequences were used, in addition to an L0a outgroup. The results of the clock tests, in all cases, were insensitive to the choice of the outgroup sequence.
LRTs tend to inflate type I error, which for our analyses would involve a rejection of the null hypothesis (clock-like evolution) when it is true (Posada 2001), and a standard Bonferroni correction was used here because each sequence set was analyzed under multiple conditions. In our tests, the molecular clock model was not supported if P was less than 0.0050 for any individual test in order to preserve a "family" significance cut-off of 0.0500.
Reduced Median Networks
Reduced median networks were constructed, and reticulations resolved, as described earlier (Bandelt et al. 1995), with Network 3.1 (http://fluxus-engineering.com). This approach is better suited to analyses of human mtDNA sequences, especially control region sequences with their high levels of homoplasy, than standard phylogenetic approaches that constrain the sequences to simple bifurcating trees (Bandelt et al. 1995; Posada and Crandall 2001).
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Results |
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Analysis of African Haplogroup L2 mtDNA Sequences
As the first step in our analysis of African haplogroup L mtDNAs, clock tests were carried out with haplogroup L2 sequences (table 3). The control region showed a clear-cut violation of clock-like evolution under all test conditions. The clock tests that incorporated site-variable substitution rates, rather than a uniform rate, yielded quartet puzzling trees with both better statistical support and poorer fits to a molecular clock. These results are not surprising in view of the high levels of homoplasy in the mtDNA control region (see further results below). The haplogroup L2 clock tests for the coding region segments also showed a marked departure from clock-like evolution under all test conditions (table 3).
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(2) The L2bcd control region sequences showed clock-like evolution with a uniform rate of substitution, but a marked violation with site-variable rates of substitutions. The fit of the evolution for the L2bcd coding region sequences to a molecular clock was relatively poor, but the deviation did not reach statistical significance.
(3) L2ac control region sequences, in contrast to L2a sequences only, showed a significant departure from clock-like evolution. This result is in accordance with the longer branch lengths of the L2c sequences (table 4). The L2ac coding region sequences showed non-clock evolution under all test conditions.
(4) The L2bd subset of sequences showed clock-like evolution in both the control and coding regions.
These results confirm that subclades L2b and L2d have a similar pattern of evolution and that evolution was clock-like in both the control and coding regions. Subclade L2c sequences also seem to have undergone "clock-like" evolution, but the sequence divergence in both coding and control regions is less than the values for the L2bd sequences. The L2a sequences yield distinct and complex results. Sequence divergence in the coding region shows a departure from clock-like evolution. In contrast, divergence in the control region did not show a violation of clock-like evolution. However, during the analyses of the L2a sequences, it was noted that the topologies of the coding region and control region quartet puzzling trees were different (data not shown). Clock tests are generally insensitive to tree topology, but a high level of homoplasy in the control region might lead to markedly erroneous tree topologies and, as a result, to biased clock tests. Therefore, we supplemented these clock tests with reduced median network analysis (Herrnstadt et al. 2002).
Reduced Median Network Analysis of Haplogroup L2 mtDNA Sequences
The coding region sequences were used to construct a reduced median network of the entire haplogroup L2 sequence set (fig. 1) in which informative sites were used to derive the topology (see also Herrnstadt et al. 2002). Other than an unresolved reticulation at the most ancestral node, the topology of this network is straightforward and there are three noteworthy results.
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(2) The L2a sequences form two star-like subclusters (see below) that we designate L2' and L2a''. The L2a'' sequences descend from nodal sequence #563 and carry coding region polymorphisms at nucleotides 3918, 5285, 15244, and 15629. These subclusters appear to have arisen early during L2a evolution. On the basis of network analyses of control region sequences, other investigators have identified L2a subclusters (e.g., see fig. 5 of Pereira et al. 2001 and fig. 6 of Salas et al. 2002). Those subclusters appear to be different from the "deeper" evolutionary event that emerges from our analysis of coding region sequences.
(3) A number of homoplasies occur in these sequences. On the basis of our analyses here and of our previous network analysis of 560 coding region sequences (Herrnstadt et al. 2002), 25 of the coding region substitutions shown in figure 1 are homoplasic. For example, the substitution at nucleotide 13708 (which also occurs in European mtDNAs) has arisen on three separate occasions among the haplogroup L2 sequences analyzed here. In addition to multiple forward mutations at the same site, reversion events were also observed.
At the next stage of the analysis, complete L2b, L2c, and L2d sequences (coding plus control regions) were analyzed (fig. 2). The topology becomes slightly more complex with the incorporation of the control region substitutions, but the overall structure of the network remains the same. In marked contrast, incorporation of the control region sequences yielded a complex L2a network with multidimensional hypercubes (data not shown). A complex pattern of reticulations was also obtained by Salas et al. (2002) in their L2a networks (see their fig. 6). Bandelt et al. (2002) have shown that networks with hypercubes can result from errors in the sequences analyzed, and we had removed a number of errors from our sequence database prior to the present studies (Herrnstadt, Preston, and Howell et al. 2003). For some of the control region "hot spots" we inspected the sequencing electropherograms for both strands to confirm the allele status and that the sites were not heteroplasmic. Finally, the control regions for eight L2a, one L2b, and one L2c mtDNA were sequenced independently with a different approach (the manual dideoxy chain termination method; see Herrnstadt et al. 2002 and references therein). In all 10 instances, the two sequencing approaches yielded identical results, and we conclude that the complex network topology was due to homoplasy, not to sequence errors.
We sought another approach that would provide phylogenetic information on control region evolution, especially at highly variable sites.. As a first step, we assumed that the topology of the coding region tree was the best representation of the evolution of L2a sequences. Onto this network, we then added all control region polymorphisms under an assumption of maximum parsimony for the sequences analyzed (see below), and the resulting network is shown in figure 3. The most striking result is the number of control region sites that have apparently undergone multiple homoplasic events.
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(2) A T:C transition at site 16086 occurs on three occasions. We do not detect this mutation in our set of haplogroup H mtDNAs and this site has not been observed to be hypervariable by other investigators (Excoffier and Yang 1999; Meyer, Weiss, and von Haeseler 1999).
(3) The L0a outgroup sequence carries C alleles at nucleotides 16189 and 16192, whereas the L2a ancestral sequence is predicted to carry C and T, respectively, at these sites. The 16189 site subsequently undergoes mutation on four occasions (three forward and one reverse relative to the outgroup sequence), whereas the 16192 site undergoes reversion on five occasions. Thus, both sites appear to have relatively high rates of mutation, a result that has been observed in previous studies (Excoffier and Yang 1999; Meyer, Weiss, and von Haeseler 1999; Howell and Bogolin Smejkal 2000) and in the L2a networks of Salas et al. (2002).
(4) The ancestral L2a sequence carries an A:G transition at nucleotide 16309 and there have subsequently been three reversion events and one additional forward mutation. Excoffier and Yang (1999) also found that this site has a high rate of substitution. Salas et al. (2002) concluded that the reticulations in their L2a network did not involve homoplasy at site 16309. However, they also noted that the six subclusters in their network could be "collapsed" into two main clusters, that one of these carries the 16309 substitution in all sequences, and that this alternative topology would involve multiple homoplasic events at the 16309 site in the second cluster (Salas et al. 2002, p.1,099). While precise comparisons are not possible because of the lack of coding region information for their mtDNAs, their alternative topology could be the same one that we obtain (fig. 3). In addition, Quintana-Murci et al. (2004) have recently carried out a network analysis of control region sequences with European haplogroup U7 mtDNAs. In a situation that is strikingly similar to our analyses, their U7 network contains several reticulations that indicate multiple homoplasic events at site 16309 (see their fig. 3).
(5) The ancestral L2a sequence carries a C:T transition at nucleotide 16519, which undergoes reversion on three occasions. These results are not surprising and this site has long been recognized to have a high mutation rate.
An anonymous reviewer pointed out that our approach did not, in one sense, use maximum parsimony and that it might therefore have inflated the number of homoplasies. For example, the network in figure 3 shows six branches connecting to an ancestral node defined by sequences 434 and 563. In this network, a forward mutation at site 16189 occurs in three of these branches. If, however, there were a second and undetected ancestral node that differed by the single occurrence of the 16189 mutation, then this topology would reduce the apparent number of homoplasic events at this site. That is, there would be three "non-16189" branches descending from the 434/563 nodal sequence and three branches descending from the undetected "16189" nodal sequence. Similar scenarios can be devised for other apparently homoplasic sites. We have chosen to present the results as shown here, however, for two reasons. First, it seems risky to make assumptions about, or to draw conclusions from, network topologies that require undetected sequences. Second, while such a scenario could explain the results for one site, no single "undetected" ancestral node can explain the multiple homoplasmic sites in this network. Obviously, the further analysis of complete L2a mtDNAs will clarify the situation.
Based on these results, the two L2a subclusters identified in the networks were analyzed separately as L2a' (12 sequences) and L2a'' (18 sequences). The L2a' and L2a'' coding region sequences had similar mean branch lengths (table 4), although the latter set showed a clock violation in the LRTs (table 3). The L2a' and L2a'' control sequences both showed clock-like evolution, but the mean branch lengths were different. As an independent approach to the branch length calculations, we also determined the mean pairwise differences (MPDs) for the two L2a subclades. For the L2a' mtDNAs, the coding region and control region MPDs were 5.0 and 1.5, respectively, and the ratio of the two regions was 3.3. The respective MPD values for the L2a'' mtDNA were 8.2 and 4.6, and the ratio was 1.8. The results of the two approaches thus agree that the pattens of sequence divergence differ between these two subclades. Furthermore, they suggest that the evolutionary pathways of the coding and control regions are not tightly coupled.
Analysis of Haplogroup L1 and L3 mtDNA Sequences
A set of haplogroup L1 sequences (which belong to the L1b and L1c subclades) was used for clock tests (table 5). Evolution in the control region does not violate clock-like behavior, but the fit to a clock model is poor, especially with the TN (rather than the HKY) model of substitution. In contrast, the evolution of the L1 coding region sequences fits very well with a molecular clock model. The results for the haplogroup L3 sequences are summarized in table 6, and no clock violations were observed for either of the mtDNA regions analyzed, irrespective of the model of substitution.
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Analysis of Combined Sequence Sets
In the final set of clock tests, analyses of "pruned" haplogroup L sequence sets were carried out (table 2). In a combined set of L1 and L3 coding region sequences, no deviation from clock-like evolution was detected, whereas the tests with control region sequences were inconsistent. None of these tests showed a good fit to the clock model, and some of the tests showed a significant violation of the model (table 2). These results suggest that a small number of site differences in the outgroup sequence can "tip the balance" one way or the other for the L1 + L3 sequence set. Finally, we added the five L2b and the three L2d sequences to the L1 and L3 sequences and tested this set for clock-like evolution. Again, the coding region sequence set showed a good fit to a molecular clock model of evolution under all the test parameters that were varied. For the control region, in contrast, there was now a clear-cut violation of the clock model under all test conditions.
The branch lengths from the haplogroup L coding region and control region quartet puzzling trees were analyzed (table 4). A number of results emerged, in addition to those discussed previously for the haplogroup L2 sequences. For the coding region, the mean branch lengths are increased only slightly when site-variable substitution rates are incorporated. Thus, homoplasy in the coding region has minimal effects on tree construction. More importantly, the mean branch lengths are remarkably similar among the different haplogroups and subclades. The control regions present noteworthy differences. First, the branch lengths are more sensitive to the model of substitution rate, a result in accordance with the high levels of homoplasy in the control region. Second, in contrast to the coding region, there is a clear trend for control region branch lengths in which L1 > L2 > L3 when site-variable rates are incorporated into tree construction.
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Discussion |
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(1) First, we did not analyze sequences from all haplogroup L subclades, such as those from subclades L1d, L1e, L1k, and L3g (Salas et al. 2002, 2004). In this regard, further analysis of larger sequence sets should be informative.
(2) Contemporaneously sampled mtDNA sequences were analyzed and, as a result, clock violations will not have been detected if the rate of divergence changes equally and simultaneously in all lineages (Drummond and Rodrigo 2000; Seo et al. 2002). Such an evolutionary scenario is plausible when all lineages occur in a single population and if population size changes at some point in time (thereby changing the efficiency of selection against slightly deleterious mutations). The evolutionary history of African mtDNAs is complex and not yet well understood (Salas et al. 2002, 2004), but there might have been such conditions at an early stage during the emergence and dispersal of modern humans. In short, there might have been changes in mtDNA "clock speed" that we were unable to detect with our approach.
(3) It is possible that, for those mtDNA sequences for which a violation was obtained, there is a molecular clock but that the appropriate model of sequence evolution was not within the interrogated parameter space. In this regard, Yang, Goldman, and Friday (1994) compared different models of sequence evolution to a multinomial model that was unconstrained except for the i.i.d. condition. In analyses with a segment of the mtDNA coding region from humans, chimpanzees, gorillas, and orangutans, they found that the HKY model of substitution provided an appropriate description of evolution (that is, the maximum-likelihood value of the phylogenetic tree was not significantly different from that obtained with the multinomial model), but only when site variability was incorporated. In more recent studies, which added the gibbon mtDNA sequence for this region, Whelan, Liò, and Goldman (2001) observed that neither the HKY nor the REV (general time-reversible Markov process) models with uniform rates of substitutions were adequate. They concluded that those models are inadequate to fully describe the pathway of mtDNA evolution because they do not incorporate the effects of selection. That conclusion would be more compelling had they obtained similar results with tests that incorporated site variability (as did Yang, Goldman, and Friday 1994).
(4) Finally, we used LRTs for these analyses, and subsequent clock tests with Bayesian methods (Suchard, Weiss, and Sinsheimer 2003) should also be carried out. These limitations notwithstanding, the results reported here provide new insights into the evolution of the human mitochondrial genome.
We noted marked clock violations among haplogroup L2 subclades. What accounts for the complex pattern of evolution in the haplogroup L2 sequences? The departure from clock-like evolution might result from the operation of selection on the haplogroup L2a and L2c sequences; that is, their lower extents of divergence might reflect the effects of negative selection. Alternatively, there might have been some marked demographic event(s) that influenced the evolution of these sequences. The net substitution rate of nonneutral mutations, incorporating the rates of both mutation and fixation, is in most evolutionary scenarios a function of effective population size (but see Cherry 1998). The finding that the L2 subclades evolve at different rates could reflect geographic subdivision and different subpopulation sizes producing different rates of fixation of slightly deleterious mutations. The analyses of Salas et al. (2002) indicate that haplogroup L2a is the most prevalent and geographically widespread mtDNA clade in Africa and that it has a complex history of population dispersals in sub-Saharan Africa. On the basis of the network analyses of coding region sequences, our studies provide the first evidence for two well-separated L2a subclusters (fig. 1), a result that might reflect very early population subdivision. However, from tests of sequences from the two L2a subclades, we still obtained a clock violation for the L2a'' coding region sequences. Furthermore, for both the two L2a subclades, as well as for the four L2 subclades, there were obvious disparities between the rates of coding region and control region evolution. While population effects might have contributed to the clock violations among the haplogroup L2 mtDNAs, the complexity of the results argues against a simple explanation.
Why is clock-like evolution more prevalent in the coding regions of the haplogroup L sequences than in the control region? Schierup and Hein (2000) have shown that violations of clock-like evolution can be caused by recombination, but the preponderance of evidence argues that human mtDNA is clonal and that it behaves as a single linkage group (Elson et al. 2001 and references therein). Under the restriction of clonality, selection in the coding region should be "reported" by the control region as a consequence of hitchhiking. However, it seems unlikely that the marked clock violations in the control region reflect the effects of selection in the coding region, where evolution is more often clock-like. The rapid sequence divergence of the control region is often seen as evidence for a lack of selection in this segment of the mitochondrial genome, but perhaps selection has acted directly on the control region. The marked site variability of substitution rates in the control region (e.g., Excoffier and Yang 1999; Meyer, Weiss, and von Haeseler 1999) suggests the operation of selection, at least on a large fraction of the sites. However, there is also evidence both of asymmetrical mutation rates in the forward and reverse directions and of nonindependence of mutations, so one might need to consider a scenario where non-clock evolution in the control region is not caused by, or not solely by, the effects of selection. Thus, this might be a situation where the models of evolution tested here are inadequate for the complex evolutionary processes in the control region. A major challenge for future studies is to tease out the factors that underlie the complex process of control region evolution.
Phylogeographic analysis of mtDNA sequences is widely used to study human evolution and population dispersal. For example, it has been used to study the peopling of the Americas (reviewed in Eshleman, Mahli, and Smith 2003) and the spread of agriculture and language from the Near East to the European heartland (see especially Richards 2003). Such studies, in addition to their assumptions about population structure and history (Knowles 2004), rely at present on a simple mtDNA control region clock. Based on the results presented here, such control region clocks are highly suspect (see also the related comments on pp. 11051106 in Salas et al. 2002). It might be relatively "safe," however, to develop and use an mtDNA coding region clock, although the results with the haplogroup L2 sequences caution that any such clock will not be a panacea.
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Acknowledgements |
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Footnotes |
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References |
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