Origin of modern human: out of Africa?

My journal club review of the paper entitled “Mitochondrial genome variation and the origin of modern human” by Ingman, M., Kaessmann, H., Pääba, S. & Gyllensten, U, published in Nature (volume 408, pg. 708-713) in the year 2000.

==Introduction==

Mitochondria are omnipresent throughout the eukaryotic domain (with very few exceptions) (Burger et al., 2003). This double membrane organelle of most cells, are vital for the existence of eukaryotes. Cells depend on the mitochondria for their supply of ATP, the perfect energy currency for the cell. In addition to the role in energy transduction, mitochondria also play a part in other important functions, such as ion homeostasis, intermediary metabolism and apoptosis. Interestingly, majority of the components in these pathways are coded by the nuclear genome, and only a few are coded by the mitochondrial DNA.

Animal mitochondria DNA (mtDNA) can be described as small, usually circular, DNA molecule that occurs in mitochondria, and code for genes that support aerobic respiration. Each mitochondrion possesses few DNA molecules, clustered into nucleoids (each organelle can have few nucleoids) (Satoh and Kuroiwa, 1991). This clustering prevents physical contact between unrelated molecules, and hence may partly explain the absence of recombination observed in majority of animal mtDNA (Antonis et al., 2003). Further, the high mutation rate of the mtDNA (Brown et al., 1979) also indicates lack of recombination in animal mtDNA.

The inheritance of mtDNA in all animal species is strictly a maternal transmission through oocyte cytoplasm in a clonal manner (Giles et al., 1980; Hiendleder and Wolf, 2003). However, few exceptions have been observed, for example certain bivalve families. Several mechanisms of preventing paternal transmission exist (Antonis et al., 2003), such as absence of mitochondria in sperm, mitochondria do not enter the egg, sperm mitochondria selectively destroyed upon entry into egg. Situations exist in which the mechanisms fail and enable paternal leakage to occur, observed in wide taxonomic groups, even in humans (Schwartz and Vissing, 2002).

Over the past decades, mitochondria genes and genomes have been vital tools in the study of animal evolution. Several characteristics of the mtDNA have been fundamental to its success as the marker of choice, namely strict maternal inheritance, high copy number, clonal inheritance, high mutation rate compared with nuclear DNA, and lack of recombination. An important and interesting area in animal evolution that mtDNA has garnered much interest is in the study of modern human origin; when and where did our species arise?

Scientists have vigorously pursued these questions over the past three decades (Ballard and Whitlock, 2004) and such questions have received many answers over the years too. Earlier studies were based on analysis of teeth, bones and artefacts of prehistoric people. A new dimension to the question was introduced with the recent advances in DNA technology, such as PCR and sequencing, which allowed analysis of sequences of mtDNA. mtDNA is inherited and contains the record of evolution, therefore the changes that the DNA has undergone in the past is preserved in its genetic code. Performing comparison of genetic code of mtDNA between different human populations, individuals, and with our closest primate relatives, will reveal how closely we are related and give insights into our origin.

Most previous studies based on mtDNA sequence data were restricted to the small section of the mtDNA called control region (including the D-loop). This sections high mutation rate was the reason for its popularity (Tamura and Nei, 1993), allowing scientist to resolve differences between closely related sequences using the relatively small region (7% of the mtDNA genome). It is becoming clearer now that this high mutation rate is complex and is obscuring important information (Tamura and Nei, 1993). Further, the small region is not representative of the mtDNA. As a result, earlier studies are questionable. In addition, extensive analyses of cleavage sites of the mtDNA sequences through restriction-fragment length polymorphism (RFLP) analysis are not suitable for estimation of mutation rate and hence timing of evolutionary events. This is due to the limited selection of cleavage sites of available enzymes.

The year 2000 A.D saw the completion of the global mtDNA diversity analysis in humans by Ingman et al. (2000). This work was made possible by the rapid improvement of technology for automated DNA sequencing. Some of the important findings in their paper suggest that modern humans originated in Africa, evolved around 171,000 years ago, appeared in one founding population, and migrated to other parts of the world to replace other hominids. The study became an important landmark in population genetics and set a standard in population genomics.

The author of this term paper will discuss the strength and weaknesses of the paper by Ingman et al. (2000), particularly on the important conclusions made about modern human origins. Whether such conclusions are still valid, accurately inferred based on the correctness of their assumptions, and analysis of particular findings. In this term paper, I will refer to the paper by Ingman et al. (2000) as “Ingman et al.”, unless stated otherwise.

==A. Strengths==

1. Offered a solution
Ingman et al. did a thorough background research work and identified few key issues or limitations in earlier studies of human evolution that were not suitable for evolutionary analysis, such as the popular use of the control region in the mtDNA genome, RFLP data and the lack of statistical support due to their smaller data sets.

Ingman and team approached the question of human origin by examining mtDNA from living people from around the world. Their approach is not novel but instead of focussing on the control region, they sequenced the entire mtDNA of 53 people from diverse ethnic, geographical and linguistic background. They excluded the D-loop from their analysis, as it has unusually high mutation rate, and then compared the data to produce a human phylogeny tree. Each of the sequences in their dataset was longer than those previously studied. They obtained a robust tree by collecting a larger data set than in previous studies, therefore were able to make improved estimates of times to evolutionary events (Hedges, 2000). This makes their results highly credible than earlier work.

Their approach opened the door for similar work to be performed in order to answer other questions related to human evolution, such as evolutionary history of Australian and New Guinean Aborigines (Ingman & Gyllensten, 2003), phylogenetic network of European Finnish population (Finnila et al., 2001), and evolutionary relationship between major ethnic groups (Herrnstadt et al., 2002). mtDNA genome sequences are already growing in number (http://www.genpat.uu.se/mtDB/) and it is expected that the number will further grow rapidly in the near future (Hedges, 2000). Hence, their most thorough analysis of the divergence in human mtDNA has set a benchmark to be followed in the field of population genomics.

2. mtDNA rate of mutation & selection
Constant rate of mutation of the mitochondria DNA is an important assumption used to calibrate the molecular clock. Nullification of this assumption, for example by selection, will upset the molecular clock. It is important to mention here that before the work by Ingman et al. it was generally believed that the rate of mutation of the mtDNA was relatively constant in humans. But their analysis showed that this was not the case, only mtDNA sequences excluding the D-loop was evolving at a constant rate. Researchers were unaware of such inconsistency in the D-loop, and hence earlier data may have been biased because of this. Since the publication of this information, D-loop analysis has been ignored in many studies (Templeton 2002).

3. Limitations
The group accepts the limitations of their finding by saying that their analysis on mitochondria locus alone only reflects the genetic history of females, a combination of genetic system, such as X chromosome, Y chromosome and autosomes, is required for a balanced view. They also agreed that the completion of the human genome project would make it easier to obtain data from the different genetic systems and provide us with better picture of our genetic history.

==B. Weaknesses==

1. Recombination or no recombination?
One of the characteristics of the mtDNA that has been key to its success as the marker of choice is lack of recombination. Many lines of independent research have supported the absence of recombination. However, several studies (Hagelberg et al., 1999; Eyre-Walker et al., 1999, Awadalla et al., 1999) have claimed that mtDNA sequences show signs of recombination. Eyre-Walker and Awadalla (2001) suggest three possible modes of recombination i) paternal mitochondria invade the egg during fertilization (paternal leakage), ii) mitochondrial DNA present in the nucleus (nuclear encoded psuedogene copy of the mtDNA) may be reinserted into the mitochondrial genome following fertilization and iii) recombination between heteroplasmic mtDNA molecules carrying different new mutations. Each of these routes would require the presence of enzymes in the mitochondria for homologous recombination or uptake of recombination DNA or RNA from the cytoplasm. There is now good evidence on the presence of mitochondrial genes in the nucleus (Wallace et al., 1997) and that the mammalian mitochondria can catalyse homologous recombination both in vivo (Tang et al., 2000; Holt et al., 1997) and invitro (Thyagarajan et al., 1996).

Awadalla et al. (1999) raised the possibility of recombination by using linkage disequilibrium measure to suggest that mitochondrial DNA does recombine. The proponents of no recombination strongly disagrees with such ascertains, attributing the results to incorrectly analyzed data and poorly based assumptions, and small sample size (Elson et al., 2001, Kumar et al., 2000). Ingman et al. investigated their dataset for linkage disequilibrium and found no evidence for recombination. In addition, the analysis of other data sets (Jorde and Bamshad, 2000; Elson et al, 2001; Herrnstadt et al, 2002) did not support the observation by Awadalla et al. (1999). This has led to an intense discussion about whether recombination occurs in human mitochondria (Jorde and Bamshad, 2000; Kivisild and Villems, 2000; Kumar et al., 2000; Parsons and Irwin, 2000; McVean, 2001; Wiuf, 2001; Innan and Nordborg, 2002), especially now that paternal inheritance has been recently observed in human (Schwartz and Vissing, 2002).

Recently, Piganeau and Eyre-Walker (2004) made an attempt to resolve the controversy by performing a comprehensive reanalysis of three recently published data sets of complete mtDNA sequences (including Ingmans data set) along with 10 RFLP data sets. They also employed two additional recombination detection methods, Geneconv and Maximum Chi-Square. In general, their analysis showed nonsignificant results for recombination. Therefore, there is a lack of evidence for recombination in human mtDNA from their analysis, although some of their data sets show evidence for recombination in one method. This implies that either no recombination or very “weak” recombination occurs. As paternal inheritance of mtDNA in humans has recently been observed (Schwartz and Vissing, 2002), recombination in human mtDNA could be a real possibility. However, studies show that it is hardly detectable from sequence data with currently available recombination detection methods.

Further research is needed to fully convince the scientific community of the pure maternal inheritance and the lack of recombination in mitochondrial DNA. Failure to account for recombination in mtDNA can seriously mislead inferences of our origin and related questions. The question as to whether mitochondria recombine is clearly worth resolving. Ingman and team did the right thing by testing their dataset for the presence of recombination before the phylogenetic analysis, and test results maintained the assumption that there is no evidence for recombination. However, evidence for recombination in future will invalidate their conclusions.

2. Modern Human Origin
Investigators of modern human origin have proposed two main models, namely “ Single African origin” (Vigilant et al., 1991; Cann et al., 1987) and “Multi-regional evolution” (Wolpoff et al., 1989). Multi-regional evolution model suggests that archaic humans (such as Neanderthals and Homo erectus) evolved into modern form concurrently in different parts of the world. The model is supported by fossil evidence, such as the continuation of morphological characteristics between early and modern humans. Whereas, recent African origin proposes that modern humans evolved once in Africa between 100-200 thousand years ago and subsequently colonised the rest of the world without genetic mixing with archaic forms. Studies supporting recent African origin lacked statistical confidence in their tree topology. Therefore, it was difficult to claim that we originated from Africa without strong evidence. The results by Ingman et al. were a big boost to the recent African origin model (Hedges, 2000). The outcome of their analysis was a robust tree deep rooted in Africa. The comparison of their mtDNA with the X chromosome genetic system (Xq13.3 sequences) further complemented their finding, as the X locus gave parallel view on human evolution in Africa (Kaessmann, 1999). With these results, the claim that we, Homo sapiens, originated in Africa was further strengthened, whereas the multi-regional model became a minority standpoint.

A number of other studies supported the conclusion made by Ingman and team
(Jorde et al., 2000; Stringer, 2001; Hedges, 2001; Serre et al., 2004). A Comparison of mitochondrial, autosomal, and Y-chromosome data performed by Jorde et al. (2000) provided broad support for an African origin of modern humans. Blair (2000) argues that evidence for the small population size long before the expansion of modern humans outside Africa is contrary to the multi-regional model, as it will not be able to maintain gene flow among continents. Stringer (2001) concludes that recent African origin for H. sapiens is supported morphologically, behaviourally, and genetically. Furthermore, work by Serre et al. (2004) found no evidence of mtDNA contribution to modern humans by Neanderthal.

Nevertheless, there is still a perception among some scientists that the debate about modern human origins is still far from resolved. Templeton (2002) performed a thorough study using loci from the different genetic systems (mtDNA, Y-chromosome, two X-linked genes and six autosomal regions) and found that his results agree with a variant of the two basic hypothesis mentioned above, the Assimilation model posed by Smith et al. (1989). The Assimilation Model combines recent African origin and multi-regional evolution. However, it differs from the previous models in rejecting replacement, or population migration, as a major factor in the appearance of modern human. This suggests that past studies, including Ingman and teams, relying on one or two loci could be inaccurate, as that specific locus may not encode records of evolutionary event within. To finally establish whether H. sapiens originated in Africa, a later Pleistocene (1.8 – 0.012 Myr ago) record is needed from Asia to compare with that already recovered from Europe and Africa (Stringer, 2001). As of now, the conclusion made by Ingman et al. has strong support, but it will be interesting to see whether it stands the test of time when further exciting developments imminent in this field unfold.

3. Population growth in the non-African group
Ingman et al. states that the non-African group experienced a period of population growth and this happened during the period of cultural change, such as appearance of regional cultural variation and acceleration of artefactual change. I would like to add few comments on this point raised by the authors, as it was not discussed in detail.

Since it is believed that mtDNA is only passed by the mothers, another way of interpreting their observation could be that the daughters of “Eurasian or non-African Eve” spread rapidly throughout the population. I find it hard to believe that cultural change can largely be responsible for this phenomenon, though it may have had indirect effect, because it doesn’t explain the spread of mtDNA by females alone. A more reasonable explanation could be the presence of exceptionally beautiful or sexually attractive females. They and their daughters inheriting their beauty would be highly “sought after”. Peace between tribes could have been made by marrying these females to the enemy tribe. Thus, the migration of these women would easily spread the mtDNA to the other parts of the world (Seielstad et al., 1998; Stoneking, 1998) without any restriction and the population would experience a period of “baby boom”.

4. Divergence time between humans and chimpanzee
Ingman et al. (2001) mentioned that the dates they proposed for the most common recent ancestor evidently depend on the assumed divergence time of chimpanzee and human (5 Million yrs). This date has now been pushed back to before 6 million years ago by recent fossil discovery (Brunet et al., 2002). As a result, molecular-clock estimates of evolutionary events need to be pushed back in time as well. This means that Ingman and team have to recalculate the dates they proposed in the paper.

==C. Miscellaneous comments==

1. Lack of information
In the paper there were several occasions where the authors did not supply sufficient information or a reference. Some of these are discussed below.

i) Ingman et al. assumed a generation time of 20 years to estimate the time when population expansion began for the non-african group. In their text they did not specify the reason for choosing 20 years for the generation time and neither was a reference provided. This is important because few thousands of years ago, the generation time may have been more than 20 years.
ii) They did not give any explanation as to why they used 53 people for their study, and not more. They could have taken few more samples from each linguistic phyla to obtain a more reliable tree. For example, there was only one sample from Korean linguistic phyla.
iii) For figure 2, the authors should have mentioned that the phylogeny tree represents the variation of mtDNA in the individuals and not their physical feature such as skin colour. For example the grouping of Chinese 21 and PNG coast 22 in figure 2 does not mean their physical features are similar. This basic information would be useful in preventing any misinterpretation by the general readers, as the mission of the journal Nature is to publish papers of interest to the general public.

2. Dates for the evolutionary events
Hedges (2000) mentioned that its difficult to time historical events precisely. He further comments on the dates proposed by Ingman et al. as not consistent with fossil and archaeological evidence, but are in the right range.

==D. Conclusion==

The field of human evolution is entangled with great controversy (Cann 2002; Relethford, 2001). Despite intense research efforts, no consensus has been reached as of yet about the genetic relationship between modern humans and archaic human forms such as the Neanderthals. Scrutiny of past work on the controversial issues surrounding modern human origins has steadily improved mtDNA analysis and is a promising tool for the future of this subject.

Ingman and team made significant contribution to the field, such as demonstrating that the D-loop is not suitable for determining evolutionary events and provided strong statistical support to recent African origin model by analysing mtDNA genomes. For a balanced view on our history, the analysis of mtDNA genome alone is not sufficient; it should be coupled with the other genetic systems. This was strongly suggested by Templeton.

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