Ms No 6847M-F2T2-W
Cytogenet Genome Res

Evolution of hominoids and the search for a genetic basis forcreating humanness
N. Saitou
Division of Population Genetics, National Institute of Genetics, Mishima (Japan)

This work was supported by Grants-in-Aid for Scientific Research on priority areas from the Ministry of Education,
Culture, Sport, Science, and Technology of Japan.
Received 28 October 2003; manuscript accepted 11 March 2004.
Request reprints from: Dr. Naruya Saitou, Division of Population Genetics, National Institute of Genetics, Mishima
411-8540 (Japan); telephone: +81-55-981-6790; fax: +81-55-981-6789; e-mail: nsaitou@genes.nig.ac.jp

Abstract. The phylogenetic relationship of human and apes are reviewed. The history ofmolecular phylogenetic studies in this field is then discussed, as is the role of natural selection at
the molecular level. It is argued that approximately 10,000 genetic changes are responsible forcreating human specific phenotypes. A genome-wide comparison is necessary to decipher thosechanges.

The phylogenetic relationship of human and apes
We now have a clear picture of the phylogenetic constellation of human (Homo sapiens)among the hominoids. Hominoids include human and apes, and correspond to the superfamily
Hominoidea (Fig. 1). Chimpanzee (Pan troglodytes) and bonobo (Pan paniscus) are equally close1

to human, followed by gorilla (Gorilla gorilla) (e.g, Sibley and Ahlquist, 1984; Saitou, 1991;
Horai et al., 1995). The close phylogenetic relationship with these African great apes suggeststhat the human lineage originated somewhere in Africa, as first proposed by Darwin (1871). In
fact, the oldest fossils considered to be part of the human lineage immediately after thedivergence from the chimpanzee/bonobo lineage have so far been found only in Africa.
Chimpanzee has traditionally been classified into three subspecies (P. t. troglodytes, P. t.
verus, and P. t. schweinfurthii) mainly according to their geographical distribution (Central
Africa, West Africa, and East Africa, respectively) (Groves, 1997). Mitochondrial DNA sequencecomparisons have confirmed the existence of these three lineages (Morin et al., 1992; Gagneux etal., 1999). Recently, however, one group of West African chimpanzees (P. t. verus) in Nigeria
was shown to have a rather distinct mitochondrial DNA lineage, and a new subspecies status (P. t.
vellerosus) was proposed (Gonder et al., 1997). The bonobo, or birya (the name given by the
local people of Congo), are distributed to the south of the Congo (Zaire) river, and is congenericwith chimpanzee. However, a hybrid offspring of chimpanzee and bonobo has been reported(Vervaecke and van Elsacker, 1992). This suggests that the reproductive barrier between these
two species is relatively low. It should also be added that Goodman and his colleagues haverecently proposed thatchimpanzee and bonobo should be included in the genus Homo (Wildman
et al., 2003).
Gorilla has been traditionally classified into three subspecies, G. g. gorilla, G. g. graueri, andG. g. beringei, corresponding to western lowland gorilla, eastern lowland gorilla and mountaingorilla, respectively (Nowark, 1991). However, recent molecular studies only indicated a clear
genetic differentiation of the west and east lowland gorilla, while mountain gorilla is geneticallyclose to the east lowland gorilla (Garner and Ryder, 1996).
Orangutan (Pongo pygmaeus), distributed in Southeast Asia, is much more remotely related
to human than are the African apes (see Fig. 1). The geographical distribution of the extant
orangutan is restricted to the islands of Borneo and Sumatra, and the two populations of which inturn correspond to two subspecies (P. p. pygmaeus and P. p. abelii, respectively). Although these
two subspecies are genetically quite divergent (Xu and Arnason, 1996), hybrids are fertile.
The phylogenetic relationship of the gibbons is not yet well known, and even the number of
species varies depending on different reports. For example, Groves (1997) classified gibbons(Hylobatidae) into one genus (Hylobates), four subgenera, and 11 species, while Napier andNapier (1985) identified only six species. A comparison of the various classification schemes ofhominoid species can be seen at the "All About Apes" section of the Silver Project webpage(http://sayer.lab.nig.ac.jp/~silver/taxonomy.html). Common gibbon and siamang are estimated tohave diverged more than 7 million years ago, but one example of hybridization was reported(Myers and Shafer, 1979).

Molecular phylogenetic studies on human and apes
The molecular evolutionary study of human probably goes back to the work of G.H.F. Nuttall,
who originally studied the immunological differences found in the blood of various animals
including human (Gribbin and Cherfas, 1982). However, a clear phylogenetic relationship of thehominoid species was first obtained by Goodman (1962). Goodman indicated a closer
relationship of the African apes (chimpanzee and gorilla) with human than the Asian apes2

(orangutan and gibbon), using the immunodiffusion method. However, his results remain semiquantitative.
The later application of the microcomplement fixation method of Sarich and Wilson (1967)
greatly enhanced the sensitivity of the immunological reaction, and allowed the estimation of thedivergence time of human, chimpanzee, and gorilla to be about 5 million years ago, when thetime of divergence of hominoids and the Old World monkeys was assumed to be 30 million years.
That classic paper set a standard time frame for the human-ape divergence. Many molecularstudies have rapidly accumulated since then in the field of human evolutionary studies, and we
now have ample findings which strongly support the phylogenetically closer relationship ofhuman to African apes, rather than with Asian apes, as first showed by Goodman (1962).
Findings supporting this clustering pattern include amino acid sequences (Goodman et al., 1971),
chromosome banding patterns (Yunis and Prakash, 1982), DNA-DNA hybridization (Sibley andAhlquist, 1984, 1987), and nucleotide sequences (Brown et al., 1982; Koop et al., 1986; Hixson
and Brown, 1986; Miyamoto et al., 1987; Maeda et al., 1988; Ueda et al., 1989). However,despite the accumulation of a large amount of data, there was still at that time no consensus on
the problem of the branching order of human, chimpanzee, and gorilla (Nei et al., 1985; Saitou,
1991). Gonzalez et al. (1990) studied ribosomal RNA genes and supported the humanchimpanzee cluster statistically significantly for the first time. Horai et al. (1995) also found thestatistically significant support for the human-chimpanzee cluster by determining the complete
mitochondrial DNA sequences of apes.
There is, however, a further very important aspect to take into account when reconstructing
the evolutionary branching pattern of these three hominoid species. In the presence of genetic
polymorphisms, a phylogenetic tree reconstructed from a single gene from each species (genetree) may be different from the true species phylogeny (species tree), even if a large number ofnucleotides are used (Nei, 1985; Takahata and Nei, 1985; Saitou and Nei, 1986; Nei, 1987). Thisis especially true when the two speciation events occurred during a short period, and it has beenshown that several gene trees may be necessary for obtaining the correct species tree in the caseof human, chimpanzee, and gorilla (Saitou and Nei, 1986).
Recently, several studies were conducted with the aim of determining the phylogenetic
relationship among human, chimpanzee, and gorilla. Satta, Klein, and Takahata (2000), Chen andLi (2001), O’hUigin et al. (2002), and Kitano et al. (2004) compared 34, 53, 51, and 103 genes or
DNA segments, respectively, and in all of these studies it was found that about 40 % of the genetrees were different from the species tree. Although all three possible gene trees were observed,
the tree showing the human-chimpanzee cluster was always the most common in each of the fourstudies. Taken together, these studies would seem to finally establish that chimpanzee andbonobo are the closest organisms to human.
The next question is therefore the divergence time between human and chimpanzee. Sarich
and Wilson (1967) estimated that human, chimpanzee, and gorilla diverged around 5 million
years ago (MYA) based on an assumed divergence time of hominoids and Old World monkeys of
30 MYA. Numerous divergence time estimates have since then been proposed based onmolecular data (Table 1). Because molecular data alone can give only relative divergence times,
ratios of divergence times between D[human-chimpanzee] and D[human-orangutan] are also
shown in Table 1, where D[a-b] is the molecular distance between species a and b.
It is interesting to note that except for two rather small and large divergence estimates(Hasegawa et al., 1985, 2003), most of the estimates are in the small range of 4.5–6 million years.
The relative ratio (D[human-chimpanzee]/D[human-orangutan]) is also more or less the same3

within the range of 0.38–0.48, except for the two large and small estimates based on earlier andlimited molecular data (Sarich and Wilson, 1967; Hasegawa et al., 1985) and an estimate by
Kumar and Hedges (1998). It should be noted that the calibration method described by Kumar
and Hedges (1998) was recently criticized by Graur and Martin (2004). If we therefore assume
that the divergence time of human and chimpanzee is about 6 million years ago, and theD[human-chimpanzee]/D[human-orangutan] is 0.40, the divergence time of human andorangutan becomes 15 million years ago. Figure 1 displays the phylogenetic relationship of theextant hominoid species based on these strands of molecular evidence.

Positive selection for ape and human genes
Hughes and Nei (1988, 1989) showed that both MHC class I and class II genes experienced
positive selection, probably of the overdominant type, through the comparison of synonymousand non-synonymous nucleotide substitutions. This Dn/Ds test is quite powerful when being used
to detect positive selection based on the neutral theory (Kimura, 1983), and many studies wereconducted to detect such selection. However, one drawback of this test is that it can detect only alimited type of genes under positive selection, and to obtain a statistically significant result, weneed to have multiple substitutions. Therefore, a protein gene must have many variable aminoacids, a pre-condition that may be satisfied in protein-coding genes that are involved in the
immune system, such as MHC. If a single amino acid change were responsible in enhancing
fitness, however, it would be difficult to detect it by the Dn/Ds test.
Table 2 lists the primate genes found to be under the influence of positive selection in
relatively recent studies. Although the majority of protein-coding genes have lower nonsynonymous substitutions than synonymous ones (Endo et al., 1996), a certain proportion are
under positive selection. Although most of the genes listed in Table 2 were found to haveexperienced positive selection thorough the Dn/Ds test, the FOXP2 gene studied by Enard et al.
(2002) and Zhang et al. (2002) did not show significantly higher Dn than Ds. They instead
studied the within-human variation of FOXP2 across a number of populations, and found that thegene had little variation, suggesting a recent fixation of an advantageous mutation. This result isparticularly intriguing as the FOXP2 gene was found by Lai et al. (2001) to be related tolanguage ability and as such it may have been involved in the development of language in humanhistory. However, one of the two amino acid substitutions occurred in the human lineage after thedivergence of the human-chimpanzee common ancestor also occurred in the carnivores (Zhang etal. 2002). It is therefore not yet clear if these amino acid changes were truly responsible for the
emergence of language.

Amount of genetic changes responsible for human specific phenotypes
The overall level of nucleotide substitutional divergence between human and chimpanzee is
1.23 % (Fujiyama et al., 2002). Many of those differences are due to mutations that occurred injunk DNA (Ohno, 1972), and most probably have no effect on the level of phenotypic difference
between human and chimpanzee. However, some proportion of nucleotide changes, in particular,amino acid replacing non- synonymous ones, must be responsible for human-specific characters,.
There are 30,000–40,000 protein-coding genes in the human genome (International Human
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Genome Sequencing Consortium 2001), and a certain proportion must code proteins that slightly
differ in amino acid sequences between human and chimpanzee. Some of those amino aciddifferences may be responsible for human-specific characters. Amino acid differences areclassified into replacement (substitution) and insertions/deletions (indels). Both types can lead toslight-to-considerable changes of the protein 3D structure, which is the source of human specificphenotypes.
DNA regions responsible for the modification of gene expression are also important, as Kingand Wilson (1975) pointed out. The discovery of such regions is difficult and we need to obtain
multiple species comparisons of contiguous genomic sequences including both coding and noncoding DNA.
How many significant changes are really responsible for creating humanness or“hominization”? The level of genomic nucleotide difference between human and chimpanzee, i.e.,
1.23 % (Fujiyama et al., 2002), has been accumulated both in the human and chimpanzee lineage.
Therefore, about half (ca. 0.6 %) of this difference can be assumed to have accumulated in thehuman lineage after divergence from the chimpanzee lineage. All the genetic changes responsible
for "humanness" must reside in these differences. The human genome consists of about 3 billion
nucleotides, and thus the 0.6 % difference is tantamount to about 18 million nucleotide changes.
Although at least 95 % of the human genome may be attributed to be non-coding DNA, we still
have some 900,000 nucleotide changes located in the coding DNA. Of these, how many changes
are really responsible for creating humanness? First, we can safely omit synonymous changes in
the coding region. Even if amino acid changes occur, the function of one protein may not
drastically change. If we eliminate those non-significant nucleotide changes, the really interestingchanges may only be ~1 %, or ca. 10,000 nucleotide changes. I would like to add that the genetic
changes responsible for the humanness could also be accomplished through the fixation of neutral
mutations, as well as that of advantageous mutations.

Ape genome sequencing efforts
We are now in the era of genomics. Therefore, apes, the closest organism group to human,
have also become the target of genome sequencing. Our group has been studying the evolution of
the ABO (Saitou and Yamamoto, 1997; Sumiyama et al., 2000; Kitano et al., 2000) and Rh(Kitano et al., 1998; Kitano and Saitou, 1999) blood group genes in hominoids. Figure 2 is aphylogenetic network of the ABO blood group gene intron 6 and exon 7 for human, chimpanzee,
and bonobo. We expanded our interest to a much larger number of ape genes, and started a smallscale ape genome project in 1999. Because Ag, the acronym of "ape genome," is the atomic
symbol for silver, we nicknamed this project "Silver" (http://sayer.lab.nig.ac.jp/~silver/).
Currently, human and ape nucleotide sequences for 34 kinds of genes or DNA regions areavailable though this website, and all sequence data have been submitted to the
DDBJ/EMBL/GenBank International Nucleotide Sequence database. Because of the open
availability, two sequence datasets for chimpanzee have already been used for two humanpolymoprhism studies (Sanders et al., 2001; Duan et al., 2003). We are also collaborating withvarious groups on the ape genome study: on fetuin (Osawa et al., 2001), cystatin B (Osawa et al.,2003), Spinocerebellar Ataxia 8 (Andres et al., 2003, 2004), and polyubiquitin (Tachikui et al.,2003). Because a genomic clone library is necessary for genome-wide study, we recently
constructed a gorilla fosmid library and its PCR-based screening system (Kim et al., 2003).
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The activity of other research groups as of February 2004 is as follows. Anzai et al. (2003)
completed sequencing the chimpanzee MHC class I region, spanning 1.75 Mb. This is the longest
chimpanzee genome segment sequence so far. Another project is the determination of thechimpanzee chromosome 22 long arm that is orthologous to the human chromosome 21 long arm.
This is an international collaboration involving Japan, Germany, China, Korea, and Taiwan,
headed by RIKEN GSC. A total of ca. 32.8 Mb nucleotide sequence data are now available
through the DDBJ/EMBL/GenBank International Nucleotide Sequence database under accession
numbers BS000001-BS000245 and AL954200-AL954259. A genomic comparison between
human chromosome 21 and chimpanzee chromosome 22 has also been conducted (this paper wasjust published; International Chimpanzee Chromosome 22 Consortium, 2004). Recently, The
Washington University Genome Sequencing Center and Whitehead Institute for Biomedical
Research have both received funding from NHGRI to conduct a “4” coverage of thechimpanzee genome. Sequence data from this project are now available at Ensembl database(http://pre.ensembl.org/Pan_troglodytes/). Given the amount of data already collected, along with
the number and diversity of ongoing projects, we can confidently state that we are now really
immersed in the human-ape comparative genomics era.

Acknowledgments
I thank Drs. Takashi Kitano and Alan Lovell for comments.

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Fig. 1. The phylogenetic relationship of the extant hominoid species. It should be noted that the divergence time
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Fig. 2. Phylogenetic network of the ABO blood group gene for human and great apes. The sequence data of
Sumiyama et al. (2000) and Kitano et al. (2000) were combined. (b) and (c) denote bonobo and chimpanzee,
respectively, and numbers following the dash designate haplotype IDs. See Sumiyama et al. (2000) and Kitano(2000) for more details.

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Table 1. Estimates of divergence time between human and chimpanzee based on molecular data==========================================================
Time* D[hc]/ Type ofmolecular(MYA)D[ho]**
data
Reference-----------------------------------------------------------------------------------------------5
0.6
albumin
Sarich and Wilson (1967)
2.7
0.25 mtDNA
Hasegawa et al. (1985)
4.9
0.41 eta globin
Hasegawa et al. (1987)
5 - 6 0.44 mtDNA and nucDNA Saitou (1991)
4.9
0.38 complete mtDNA
Horai et al. (1995)
4.5
nucDNA
Takahata and Satta (1997)
5.5
0.67 nucDNA
Kumar and Hedges (1998)
6.2-8.4 0.40 nucDNA non-coding Chen and Li (2001)
5.4
0.48 nucDNA
Stauffer et al. (2001)
7.4
0.39 complete mtDNA
Hasegawa et al. (2003)
6
0.46 nucDNA
Glazko and Nei (2003)
4.3-5.9 0.44 nucDNA synonymous Wildman et al. (2003)
0.40 nucDNA synonymous Kitano et al. (2004)==========================================================*Divergence time estimate between human and chimpanzee.
**D[human-chimpanzee]/D[human-orangutan].

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Table 2. Genes found to be under positive selection in primate evolution============================================================
Gene

Reference--------------------------------------------------------------------------------------------------ABO

Saitou and Yamamoto (1997)

BRCA1

Huttley et al. (2000)

FOXP2

Enard et al. (2002), Zhang et al. (2002)

IgA hinge region

Sumiyama et al. (2002)

LCR16aJohnson et al. (2001)

lysozyme

Messier and Stewart (1997)

protamine 1 & 2
Rh50

Wyckoff, Wang, and Wu (2000)
Kitano et al. (1998)

Rh blood group

Kitano and Saitou (1999)

RNASE2 & 3

Zhang et al. (1998)

TNP2

Wyckoff, Wang, and Wu (2000)============================================================

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