Human-Specific Amino Acid Changes Found in 103 Protein-Coding Genes
Takashi Kitano,* Yu-Hua Liu,*1 Shintaroh Ueda, and Naruya Saitou**Division of Population Genetics, National Institute of Genetics, Mishima, Japan; and Department of
Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan
We humans have many characteristics that are different from those of the great apes. These human-specific charactersmust have arisen through mutations accumulated in the genome of our direct ancestor after the divergence of the lastcommon ancestor with chimpanzee. Gene trees of human and great apes are necessary for extracting these human-specificgenetic changes. We conducted a systematic analysis of 103 protein-coding genes for human, chimpanzee, gorilla, andorangutan. Nucleotide sequences for 18 genes were newly determined for this study, and those for the remaining genes
were retrieved from the DDBJ/EMBL/GenBank database. The total number of amino acid changes in the human lineage
was 147 for 26,199 codons (0.56%). The total number of amino acid changes in the human genome was, thus, estimatedto be about 80,000. We applied the acceleration index test and Fisher’s synonymous/nonsynonymous exact test for eachgene tree to detect any human-specific enhancement of amino acid changes compared with ape branches. Six and two
genes were shown to have significantly higher nonsynonymous changes at the human lineage from the acceleration index
and exact tests, respectively. We also compared the distribution of the differences of the nonsynonymous substitutions on
the human lineage and those on the great ape lineage. Two genes were more conserved in the ape lineage, whereas one
gene was more conserved in the human lineage. These results suggest that a small proportion of protein-coding genes
started to evolve differently in the human lineage after it diverged from the ape lineage.

Introduction
We now have a clear picture of the phylogenetic
constellation of human (Homo sapiens): chimpanzee (Pantroglodytes) and bonobo (Pan paniscus) are equallyclosely related organisms to human (e.g., Sibley andAhlquist [1984], Saitou [1991], and Horai et al. [1995]).
Nucleotide substitution difference between human andchimpanzee was estimated to be 1.23% based on 19-Mb
BAC end sequence comparison (Fujiyama et al. 2002).
This difference corresponds to 3.7 million bp for the wholegenome under the assumption that the human andchimpanzee genomes are both approximately 3 billion
nucleotides. Many of those differences were probably
caused by mutations occurred in so-called junk DNA(Ohno 1972) and had no effect on phenotypic difference
between human and chimpanzee. Some proportion of
nucleotide changes, however, must be responsible forhuman-specific characteristics, such as large brain size andbipedalism. King and Wilson (1975) proposed that genetic
changes at the gene expression control region are more
important than changes in the protein-coding region.
However, there are more than 32,000 genes in the humangenome (International Human Genome Sequencing Consortium 2001), and a considerable number of proteincoding genes must produce proteins that slightly differ inamino acid sequences between human and chimpanzee.
Some of those amino acid differences may be responsible
for human-specific characteristics.
It is our interest to determine whether amino acidchanges occurred in the human lineage after the lastcommon ancestor diverged from the chimpanzee lineage.
Such changes are the candidates for the genetic basis ofhuman-specific characteristics. Gene trees of human andgreat apes are necessary for extracting those genetic
1changes that occurred in the human lineage. There arethree possible gene trees for human, chimpanzee, andgorilla (see figure 1). Because the speciation period of
human and chimpanzee is difficult to infer, the ‘‘humanlineage’’ in this paper is defined as branch connecting thepresent-day human and the last branching point designatedas a circle in figure 1.
The tree topology of human, chimpanzee, and gorilladiffered from gene to gene. Satta, Klein, and Takahata(2000) compared 34 genes and found that about 60% ofloci supported the human-chimpanzee clade, and theremaining 40% supported one of the two alternative trees.
Chen and Li (2001) and O’hUigin et al. (2002) compared
53 segments and 51 genes, respectively, and they also
found a similar tendency. Because the time span between
the human-chimpanzee common ancestor and gorillaspeciation is short (approximately 1 to 2 Myr), genegenealogies might differ from gene to gene.
The majority of genes is evolving under neutral
fashion, and natural selection plays mainly a conservativerole as negative or purifying selection (Kimura 1983; Nei
1987). Nevertheless, a small portion of genes is underpositive selection, and evidence of positive selection at themolecular level has been accumulated through comparisonof synonymous and nonsynonymous substitutions since it
was first found for MHC genes (Hughes and Nei 1988,
1989). Even if we restrict our attention to primates, 15
genes were so far shown to experience positive selection(table 1). We, therefore, also compared synonymous andnonsynonymous substitutions to identify human lineage–
specific positive selection.
Materials and Methods
DNA Sequencing

Present address: The Jackson Laboratory, Bar Harbor, Maine.

Key words: humanness, positive selection, hominoids, gene tree.
E-mail: nsaitou@genes.nig.ac.jp.
Mol. Biol. Evol. 21(5):936–944. 2004
DOI:10.1093/molbev/msh100
Advance Access publication March 10, 2004

We determined nucleotide sequences of the 18
protein-coding loci (A4GALT, B3GALT1, B3GALT5,
CHRM2, CHRM3, CX36, HRH1, HRH2, HTR1A,
HTR1E, HTR1F, HTR2A, NGFB, NPPB, OTX1, OTX2,SCN8A, and SIX6) for human, chimpanzee, gorilla, andMolecular Biology and Evolution vol. 21 no. 5 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.

Human-Specific Amino Acid Changes 937

Table 1
List of Genes Found to be Under Positive Selection inPrimate Evolution
Gene

FIG. 1.—Three possible gene trees (a, b, and c) for human,
chimpanzee, and gorilla. Gene tree a (A) has the same topology with thespecies tree. (B) shows a gene tree of a topology ((chimpanzee, gorilla),human, orangutan) (b). (C) shows a gene tree of a topology ((human,
gorilla), chimpanzee, orangutan) (c).

orangutan. Because we needed to compare coding
sequences (CDS) of human and great apes, we arbitrarily
chose genes that can be easily amplified (relatively long
CDS region [.300 bp]) from genomic DNA. In fact, 10
out of 18 genes were single CDS genes. DNA wasextracted from peripheral blood sample of a certain
Japanese individual, with informed consent for human(Homo sapiens) samples. DNA samples for chimpanzee(Pan troglodytes), gorilla (Gorilla gorilla), and orangutan(Pongo pygmaeus) were also used. Each PCR reaction
mixture contained 0.2 mM dNTP, 1.5 mM MgCl2,
13GeneTaq Mg21 free Universal Buffer (Nippon Gene),
10 pmol of each primer, and 1 unit AmpliTaq Gold (PEBiosystems). A list of primers used in this study isavailable in the Supplementary Material online. The
typical PCR condition used in this study consisted of 40
cycles of 30 s denaturation at 958C, followed by 15 s
primer annealing at 608C and 1 min extension at 728C (PE
GeneAmp PCR system 2400 and 9700). Immediately
proceeding these cycles, a 10 min hot-start step at 958C
was included. All PCR primers were designed based onhuman sequences in the database. PCR products wereconfirmed by 1.5% agarose gel electrophoresis and purified using Micro Spin Columns (Amersham Biosciences).
The purified PCR products were sequenced by using BigDye Terminator Cycle Sequencing Kit and ABI PRISM
377/310 DNA sequencer (PE Biosystems). When long sequences were read, both strands were read using PCRprimers and inner primers.
Sequence Data Retrieval and Analyses
Eighty-five protein-coding gene sequences wereretrieved from the DDBJ/EMBL/GenBank International
Nucleotide Sequence Database (Supplementary Material
online). This data set contains human, chimpanzee, gorilla,
and orangutan sequences longer than 100 bp. To retrieve
those sequences, we used orangutan sequences as queries
for Blast search to obtain homologous sequences forhuman, chimpanzee, and gorilla. For human sequences, weused sequences that were cited in the NCBI Reference
Sequences (RefSeq) as representative ones. When morethan two sequences were found from one ape species, weused the sequence that showed the shortest branch in the

BRCA1
RNASE3 (ECP)RNASE2 (EDN)
FOXP2
IGHAa
NPIPa (LCR16a)
LYZ
PRM1
PRM2
RHAG
RHa
TNP2

Reference
Huttley et al. (2000)
Zhang, Rosenberg, and Nei (1998)
Zhang, Rosenberg, and Nei (1998)
Enard et al. (2002)
Sumiyama, Saitou, and Ueda (2002)
Johnson et al. (2001)
Messier and Stewart (1997)
Wyckoff, Wang, and Wu (2000)
Wyckoff, Wang, and Wu (2000)
Kitano et al. (1998)
Kitano and Saitou (1999)
Wyckoff, Wang, and Wu (2000)

a
These genes were not used in this study, because gorilla sequence was notavailable for NPIP, and gene duplication occurred after orangutan divergence for
IGHA and RH.

neighbor-joining tree (Saitou and Nei 1987). ClustalWversion 1.8 (Thompson, Gibson, and Higgins 1994) wasused for multiple alignments. Tree topologies were
determined by counting numbers of informative sites(Supplementary Material online). Tree a shows humanchimpanzee cluster, tree b shows chimpanzee-gorillacluster, and tree c shows human-gorilla cluster. Genes
with unclear topology (trichotomy or same number of
informative sites support different trees) was categorized
into group d. Genes belonging to group d wererecategorized into d-a, d-b, and d-c by using the UPGMA
method (Sneath and Sokal 1973). Program pamp in PAML
package (Yang 1997) was used for reconstruction ofinternal nodes of sequences. ODEN package (Ina 1994)
was used for estimation of synonymous and nonsynonymous substitutions (Nei and Gojobori 1986).
Statistical Tests
We used two kinds of statistical tests for detecting
human-specific natural selection. One test is the acceleration index test for nonsynonymous substitutions ofhuman and apes. This test analogous to the test of Zhang,Webb, and Podlaha (2002), in which an acceleration index
for the human lineage in comparison to the mammalianlineage before the human-chimpanzee split is defined by
the equation (h/5.5)/[m/(2 3 90 2 5.5)] ¼ 31.7h/m. The
variables h and m are numbers of amino acid substitutionsin the human lineage and the mouse lineage, respectively.
Zhang, Webb, and Podlaha (2002) used divergence times
between human and chimpanzee (5.5 MYA) and between
primates and rodents (90 MYA). They also computed thetail probability in a binomial distribution of B(h1m,
0.03056) for testing the statistical significance of rateenhancement in the human lineage. The value 0.03056 isfrom 5.5/180, the time span for human branch, relative tothat for primates and rodents branches. We applied this test
as B(n-Human 1 n-Ape, 0.13268) to determine thestatistical significance of rate enhancement in the humanlineage in contrast to ape lineage (n-Human and n-Ape arenumbers of nonsynonymous changes at the human lineage
and ape lineage, respectively). In our test, 0.13268 ¼ 5.4/

938 Kitano et al.

40.7, is the ratio of the time span for human branch (5.4
MYA) to the time span for ape lineages (40.7 MYA). We
used divergence times estimated by Chen and Li (2001).
Taking the orangutan speciation date as approximately 12
to 16 MYA (midpoint is 14 MYA) (Goodman et al. 1998),
they obtained an estimate of 4.6 to 6.2 MYA (midpoint is
5.4 MYA) for the human and chimpanzee divergence andan estimate of 6.2 to 8.4 MYA (midpoint is 7.3 MYA) for
the gorilla speciation date, suggesting that the gorilla
lineage branched off approximately 1.6 to 2.2 MYA(midpoint is 1.9 MYA) earlier than did the human andchimpanzee divergence. The time span between theancestor of human-chimpanzee-gorilla-orangutan and theancestor of human-chimpanzee-gorilla can be estimated to
6.7 MYA (¼ 14 MYA to 7.3 MYA). For simplicity, we
took midpoint values and assumed the species tree.
Therefore, the total divergence time of hominoid lineages
is 40.7 MYA ¼ 5.4 MYA 1 5.4 MYA 1 1.9 MYA 1 7.3
MYA 1 6.7 MYA 1 14.0 MYA.
We also used Fisher’s exact test (two tails) for
synonymous and nonsynonymous substitutions of humanand apes. This test is analogous to the test of McDonald
and Kreitman (1991), in which silent and amino acidreplacement changes for polymorphic and fixed differences were compared. We compared silent and amino acidreplacement changes for human and ape branches in thisstudy.
Results and Discussion
Topologies of Gene Trees for Human, Chimpanzee,
and Gorilla
Orthology for each gene was checked by examining
the total number of synonymous substitutions for all
branches for a gene. The average value for 103 genes was
0.06, and the maximum value was 0.14 6 0.05 for the
PRM2 gene. Because the amount of synonymous substitution of PRM2 was not significantly larger than the
average, we can expect that all compared genes are
orthologous.
We divided the 103 protein-coding genes into fourgroups by its tree topology using the parsimony method.
Human and chimpanzee are clustered in tree a (fig. 1A),and it is the same as the species tree. Chimpanzee andgorilla are clustered in tree b (fig. 1B), and human andgorilla are clustered in tree c (fig. 1C). Genes with unclear
topology (trichotomy, or same number of informative
sites, support different trees) was categorized into group d.
Numbers of nucleotide sites supporting each tree for eachgene are shown in Supplementary Material online. Thirtyfour genes were group a, 10 genes were group b, 14 genes
were group c, and the remaining 45 genes were group d(table 2). The 45 genes in trifurcating tree (group d) were
further classified by using UPGMA, under the assumptionof approximate constancy of the evolutionary rate.
If we consider the proportion of tree topology byusing 58 genes (45 genes in group d were excluded), 59%of the coding genes supported tree a, 17% supported treeb, and 24% supported tree c. Satta, Klein, and Takahata(2000) showed that from 34 nuclear loci, 59% of locisupported tree a, 21% of loci supported tree b, and 21% of

Table 2Distribution of Genes According to Gene Tree Topology andNumbers of Synonymous (s) and Nonsynonymous (n)Substitutions on Human and Ape Branches
NumberTree of Genesaabcd34 (49)10 (24)14 (21)45 (9)

Human Branchbs
36.5 (52.5)
24.0 (56.0)
24.0 (28.0)
55.0 (2.0)

nApe Branchbsn44.5 (58.5) 377.5 (485.0) 371.5 (495.0)
26.0 (43.0) 84.0 (168.0) 73.0 (121.0)
44.0 (46.0) 181.0 (225.0) 235.0 (251.0)
36.0 (2.0) 260.0 (24.5) 239.0 (50.5)aNumbers in parentheses are values when genes initially clustered to group dwere reassigned by assuming rate constancy.
b
Numbers in parentheses are values, including group d genes, reassigned byassuming rate constancy.

loci supported tree c (they used both coding andnoncoding regions). Chen and Li (2001) reported that
58% of loci supported tree a, 23% of loci supported tree b,
and 19% of loci supported tree c from a comparison of 53
autosomal intergenic noncoding DNA segments. Proportions of three categories (a, b, and c) estimated from thepresent study with a larger number of genes were similar tothese previous studies. When we consider the proportionof tree topology by using 94 genes, where 36 genes ingroup d were reclassified to a, b, or c by using UPGMA,
similar proportions were observed (52% for tree a, 26%
for tree b, and 22% for tree c).
We also classified the total number (182) of informative sites into those supporting the three possible
trees: 55% of sites supported tree a, 21% supported tree b,
and 24% supported tree c. These proportions are similar to
those estimated from numbers of genes. O’hUigin et al.
(2002) estimated that the 53% of the informative
nucleotide sites supported tree a, 31% supported tree b,
and 16% supported tree c from 87 informative sites foundin 51 genes. The result of the present study showed more
uniform distribution of two alternative informative sites.
If we compare different gene trees, the branch lengthof the human lineage for tree b is expected to be longer thanthose of tree a and tree c under the assumption of themolecular clock. We, thus, compared the number ofsynonymous substitutions (dS) for each branch of three
gene trees (table 3). As expected, dS of the human branches
for tree b were longer than those of tree a and tree c, withclear statistical significance. This is consistent with the
topological difference between tree b and the remaining two
trees. Human forms a cluster with chimpanzee or gorillain tree a and c, whereas human is an outgroup to thechimpanzee-gorilla clade in tree b (see figure 1). Similarly,
the branch length of the chimpanzee lineage for tree c is
expected to be longer than those of tree a and tree b, and thebranch length of the gorilla lineage for tree a is expected to
be longer than those of tree b and tree c. However, clear
results were not obtained. This finding may be caused bya smaller number of compared genes.
We also expect that the internal branch of tree a is thelongest among the three gene trees because the topology oftree a is the same as that of the species tree. In fact, theinternal branch length of tree a was about two times longer
than those of tree b and tree c (table 3). When we consider
dS by including group d genes reassigned by assuming theHuman-Specific Amino Acid Changes 939

Table 3
Number of Total Synonymous Substitutions per Synonymous Sites (dS) for Each Tree
BranchabcSignificanceHuman

0.0053 6 0.0009(0.0059 6 0.0008)
0.0089 6 0.0011(0.0076 6 0.0009)
0.0104 6 0.0012(0.0112 6 0.0011)
0.0290 6 0.0021(0.0298 6 0.0018)
0.0075 6 0.0010(0.0058 6 0.0008)

0.0157 6 0.0032(0.0146 6 0.0020)
0.0085 6 0.0024(0.0067 6 0.0013)
0.0105 6 0.0026(0.0083 6 0.0015)
0.0318 6 0.0046(0.0271 6 0.0027)
0.0045 6 0.0017(0.0018 6 0.0007)

0.0059 6 0.0012(0.0056 6 0.0011)
0.0052 6 0.0011(0.0068 6 0.0012)
0.0104 6 0.0016(0.0097 6 0.0014)
0.0258 6 0.0026(0.0260 6 0.0023)
0.0037 6 0.0010(0.0030 6 0.0008)

a-b**, b-c**
a-b**, b-c**
a-c*
—
—
—
—
—
a-c**
a-b**, a-c*

Chimpanzee
GorillaOrangutan
Internal

NoTE.—Numbers in parentheses are values, including group d genes, reassigned by assuming rate constancy. Statisticalsignificance was tested for tree a versus tree b and tree a versus tree c. * Indicates significant at 5% level. ** Indicates significant
at 1% level.

rate constancy, similar results were observed (table 3).
These results suggest that even if gene tree topologies
differ from the species tree topology, these genes are
considered to be orthologous when polymorphism of anancestral population is assumed.
Synonymous and Nonsynonymous Changes on
the Human Branch
We compared numbers of synonymous and nonsynonymous substitutions for human and ape branches(see table 4). The ape branch denotes the sum of all branch
lengths of the tree except for the human branch. We
applied the acceleration index test (Zhang, Webb, andPodlaha 2002) for nonsynonymous substitutions of humanand apes, and six genes (APOE, BRCA1, FOXP2, HCR,
PRM2, and ZFY) showed acceleration at the humanlineage with statistical significance at the 5% level (table5). Zhang, Webb, and Podlaha (2002) applied this test toamino acid changes of 120 genes among human,
chimpanzee, and mouse, and identified FOXP2 and
PRM2, with significantly enhanced evolutionary rates in
the human lineage (table 5). FOXP2 and PRM2 genes
were also reported to be under positive selection on thehuman branch using different tests by Enard et al. (2002)
and Wyckoff, Wang, and Wu (2001), respectively.
Both FOXP2 and PRM2 genes were also determined
to have experienced human-specific acceleration in thepresent study. However, probabilities for these two genes
were increased in this study, from 0.003 to 0.048 for
FOXP2 and from 0.001 to 0.005 for PRM2 (table 5).
Interestingly, the other four genes (APOE, BRCA1, HCR,
and ZFY) showed the reversed tendency; P(A.I.-ape) ismuch lower than P(A.I.-mouse). This shows the stronger
power of the test used in this study.
Zhang, Webb, and Podlaha (2002) used human,
chimpanzee, and mouse sequence data to analyze humanspecific selection of genes. Outgroup species are necessary
to estimate the human lineage–specific changes; however,mouse may be too far removed to be used as an outgroup.
Recently, Clark et al. (2003) compared coding regions ofmouse, human, and chimpanzee but did not find many
genes with significantly higher nonsynonymous substitu-

tions in the human lineage. More closely related species,
such as gorilla and orangutan, are appropriate as outgroups for the kind of analysis we conducted in thepresent study.
The BRCA1 gene was determined to be underpositive selection on human and chimpanzee branches(Huttley et al. 2000). APOE codes apolipoproteins involved in cholesterol metabolism. Three major isoforms
are known for human APOE (Weisgraber, Rall, andMahley 1981), and these alleles differ in their association
with hyperlipoproteinemia (Rall et al. 1982) and Alzheimer
disease risk (Corder et al. 1993). It is possible that thecholesterol metabolism underwent different evolutionarypressures between the human and the ape branches. The
HCR gene locates near the HLA-C locus and is a candidate
gene for psoriasis (Asumalahti et al. 2000). However, there
is so far no report of positive selection on this gene. The
ZFY gene encodes a zinc finger–containing protein that
may function as a transcription factor. Differential rates of
evolution of the ZFY-related genes were recently observedin mice species (Tucker, Adkins, and Rest 2003).
We also applied Fisher’s exact test (two tails) for
synonymous and nonsynonymous substitutions of humanand apes. Three genes (BRCA1, FOXP2, and DAF)
showed statistical significance at the 5% level (table 5).
BRCA1 and FOXP2 genes showed significant enhancement of nonsynonymous substitutions in the humanlineage, as also found by using the acceleration test,
whereas the DAF (decay accelerating factor) gene showedsignificant reduction at the human lineage. Generally
speaking, P(n/s) values are higher than P(A.I.-ape) values
except for the FOXP2 gene.
There are two equally parsimonious trees for the DAF
gene. Figure 2 shows these two trees (shown with bold lines
in A and B) on the phylogenetic network. This gene wascategorized into group d-a, corresponding to tree A of figure
2. Four synonymous and two nonsynonymous substitutions on the human branch and six synonymous and 28
nonsynonymous substitutions on ape branches wereassumed for tree A. The number of nonsynonymous
substitutions on the human branch is significantly smaller
than those of other branches (table 5). When the alternative
maximum-parsimonious tree (fig. 2B) is considered,940 Kitano et al.

Table 4
Number of Synonymous and Nonsynonymous Substitutions on Human and Ape Branches
Human Branch

Ape Branch

Gene

s

dS

n

dN

s

dS

n

dN

Tree a
ACAT2
ADRB2
APOB
C5R1
CCR5
CD209
CHRM2
COX8
CSTB
CXCR4
CXCR6
DMP1
EKN1
F9
FPRL1
FPRL2
FUT2
HRH2
HTR1E
HTR1F
IL3
IL8RB
LCAT
MEFV
ODC1
OTX1
RHBG
RNASE1
RPS4Y
TAF1L
TGIF2LXTNP2TWIST1
ZNF80
Total

0
1
1
0
1
1
1
0
0
1
0
1
3
0
0
2.5
3
0
1
2
0
3
1
1
0
0
1
2
1
2
1
0
4
2
36.5

0.000
0.004
0.007
0.000
0.004
0.004
0.003
0.000
0.000
0.004
0.000
0.006
0.011
0.000
0.000
0.010
0.012
0.000
0.004
0.008
0.000
0.011
0.008
0.009
0.000
0.000
0.003
0.018
0.005
0.004
0.006
0.000
0.027
0.011
0.005

2
2
4
0
2
2
0
1
0
0
1
2
2
0
1
4.5
1
0
0
0
1
0
0
0
1
0
3
2
0
5
1
3
2
2
44.5

0.019
0.002
0.009
0.000
0.003
0.002
0.000
0.007
0.000
0.000
0.003
0.003
0.002
0.000
0.001
0.006
0.001
0.000
0.000
0.000
0.004
0.000
0.000
0.000
0.002
0.000
0.003
0.006
0.000
0.003
0.002
0.010
0.005
0.003
0.002

1
13
4
21.5
16
19
15
4
5
6
6
7
5
4
21
18
19.5
22
16.5
3
6
16
9
14
8
9
18
6
16
20
6
8
6
9
377.5

0.025
0.050
0.030
0.084
0.066
0.071
0.050
0.071
0.081
0.024
0.058
0.045
0.019
0.036
0.085
0.077
0.082
0.084
0.066
0.012
0.091
0.061
0.074
0.131
0.059
0.049
0.054
0.056
0.091
0.036
0.037
0.102
0.042
0.052
0.057

2
11
12
18.5
2
38
2
1
1
1
1
19
7
8
16
17
15.5
0
2.5
6
4
19
4
13
3
1
13
8
9
34
32
17
8
26
371.5

0.019
0.013
0.027
0.025
0.003
0.042
0.002
0.007
0.004
0.001
0.003
0.027
0.007
0.022
0.021
0.022
0.020
0.000
0.003
0.007
0.017
0.024
0.011
0.037
0.007
0.002
0.013
0.023
0.015
0.017
0.060
0.056
0.018
0.042
0.017

Tree bAPOE
B3GALT5
CX36
LEP
OXTR
POMC
PRM1
PRM2
SCG2
UBB
Total

3
3
2
2
4
2
0
3
4
1
24

0.013
0.015
0.011
0.019
0.018
0.017
0.000
0.043
0.020
0.006
0.016

6
3
0
0
3
1
3
8
2
0
26

0.008
0.004
0.000
0.000
0.005
0.003
0.026
0.035
0.003
0.000
0.005

7
18
5
3
9
9
3
7
8
15
84

0.030
0.096
0.029
0.029
0.040
0.081
0.088
0.106
0.040
0.098
0.057

7
13
0
6
6
8
15
14
4
0
73

0.010
0.019
0.000
0.018
0.009
0.022
0.141
0.062
0.005
0.000
0.015

Tree cAFP
BRCA1
CD209L1
CD22
CHRM3
FOXP2
FUT5
FUT6
HCR
INS
NPPB
RHAG
SRY
TYR
Total

0
2
0
0
3
1
3
3
5
3
1
0
0
3
24

0.000
0.003
0.000
0.000
0.007
0.002
0.011
0.011
0.009
0.035
0.010
0.000
0.000
0.009
0.006

0
17
2
4
0
2
2
3
7
0
1
3
2
1
44

0.000
0.006
0.005
0.005
0.000
0.001
0.002
0.004
0.004
0.000
0.003
0.003
0.004
0.001
0.003

3
24
6
6
17
19
21.5
23
20
5
1
14.5
7
14
181

0.101
0.035
0.050
0.027
0.042
0.042
0.082
0.093
0.035
0.059
0.010
0.051
0.055
0.041
0.046

0
40
6
35
11
1
29.5
29
17
4
9
31.5
16
6
235

0.000
0.015
0.015
0.047
0.008
0.001
0.036
0.037
0.010
0.017
0.031
0.035
0.034
0.005
0.018

Human-Specific Amino Acid Changes 941

Table 4
Continued
Human Branch

Ape Branch

Gene

s

dS

n

dN

s

dS

n

dN

Tree d–a
A4GALT
C1orf9
DAF
DEFB1
DRD4
FPR1
FUT1
IL16
LPL
NPPA
RNASE2RNASE6SIGLECL1
TNF
ZNF75
Total

2
0
4
0
0
4
1
1
0
1
2
1
0
0
0
16

0.013
0.000
0.016
0.000
0.000
0.015
0.004
0.004
0.000
0.012
0.018
0.010
0.000
0.000
0.000
0.007

0
0
2
0
1
3
0
2
0
0
0
0
4
1
1
14

0.000
0.000
0.003
0.000
0.005
0.004
0.000
0.003
0.000
0.000
0.000
0.000
0.007
0.003
0.002
0.002

12
4
6.5
3
5
15
6
14
4
4
11
6
14
1
2
107.5

0.081
0.033
0.027
0.062
0.064
0.060
0.023
0.061
0.083
0.051
0.107
0.063
0.075
0.008
0.016
0.050

7
2
28.5
2
4
15
7
12
0
4
14
4
15
5
4
123.5

0.015
0.005
0.038
0.013
0.021
0.020
0.009
0.017
0.000
0.016
0.039
0.011
0.026
0.015
0.008
0.018

Tree d–b
ADRB3
B3GALT1
CMAH
COX4I1
EPO
HRH1
HTR1A
LYZ
NGFB
PABPC5SIX6ZFX
ZFY
ZNFN1A1
Total

3
5
0
2
2
3
2
2
4
1
3
1
1
3
32

0.011
0.023
0.000
0.024
0.020
0.009
0.006
0.019
0.024
0.004
0.025
0.013
0.013
0.023
0.014

3
0
1
0
1
4
3
0
2
0
1
0
2
0
17

0.005
0.000
0.007
0.000
0.003
0.004
0.003
0.000
0.004
0.000
0.003
0.000
0.006
0.000
0.002

10
9
0
2
7
16
11
2
7
0
7
0
6
7
84

0.037
0.042
0.000
0.024
0.073
0.049
0.035
0.019
0.042
0.000
0.061
0.000
0.080
0.055
0.039

7
0
1
6
7
13
4
4
4
0
0
0
1
1
48

0.011
0.000
0.007
0.018
0.024
0.012
0.004
0.012
0.007
0.000
0.000
0.000
0.003
0.002
0.006

Tree d–c
APOA1
HTR2A
IFNG
MSH2
OTX2
PRNP
SCN8A
Total

0
2
0
0
0
1
1
4

0.000
0.012
0.000
0.000
0.000
0.006
0.004
0.004

0
0
1
0
0
1
0
2

0.000
0.000
0.003
0.000
0.000
0.002
0.000
0.001

2
8
10
2
3
14
5
44

0.015
0.050
0.126
0.050
0.038
0.084
0.020
0.048

4
2
2
2
0
5
1
16

0.009
0.004
0.006
0.016
0.000
0.009
0.001
0.005

Tree d–d
ANG
B2M
COX7C
HBE1
OPN1SWRETRNASE3
TCP1
TH
Total

1
0
0
0
0
0
0
1
0
2

0.009
0.000
0.000
0.000
0.000
0.000
0.000
0.028
0.000
0.003

0
0
0
0
0
1
1
0
0
2

0.000
0.000
0.000
0.000
0.000
0.007
0.003
0.000
0.000
0.001

2.5
1
3
3
4
3
4
2
2
24.5

0.024
0.012
0.070
0.030
0.075
0.062
0.035
0.056
0.050
0.039

11.5
4
1
2
1
4
27
0
0
50.5

0.035
0.015
0.007
0.006
0.005
0.026
0.078
0.000
0.000
0.025

however, two synonymous and five nonsynonymous
substitutions on human branch and six synonymous and
27 nonsynonymous substitutions on other branches wereobserved. In this case, number of substitutions between
human and other branches is not statistically significant. The
DAF gene codes a glycoprotein and it is related to Cromer
blood group system (CR) (Reid et al. 1996). Kuttner-Kondo
et al. (2000) mentioned that number of amino acid changes

differ region by region in primate DAF genes. This gene
may have a human-specific nucleotide substitution pattern,
but it depends on a topology to be analyzed. More detailed
analyses might be needed for this gene.
A total of 147 amino acid changes were observed in
the human lineage for 26,199 codons (0.56%). About 60%
of amino acid changes were radical changes. If we assume
that the number of genes in human genome is 32,000 and942 Kitano et al.

Table 5
Genes Showing Significantly Different Nonsynonymous
Changes Between Human Lineage and Ape Lineages
Gene
BRCA1
APOE
PRM2
HCR
FOXP2
ZFY
DAF tree A
DAF tree B

n-Human
17
6
8
7
2
2
2
5

n-Ape
40
7
14
17
1
1
28
27

P(A.I.-ape)
0.001**
0.004**
0.005**
0.032*
0.048*
0.048*
0.926
0.422

P(A.I.-mouse)a

0.568
0.106a0.001a**0.352b
0.003a**0.267b
0.944b
0.944bP(n/s)
0.027*
0.670
1.000
0.520
0.034*
0.183
0.026*
0.611

NoTE.—n-Human and n-Ape are numbers of nonsynonymous changes at thehuman lineage and ape lineage, respectively. P(A.I.-ape) and P(A.I.-mouse) areprobabilities of acceleration index for the human lineage compared with the ape(chimpanzee, gorilla, and orangutan) lineage and compared with the mouse lineage,
respectively. P(n/s) is the probability of Fisher’s exact test (two tails) for
synonymous and nonsynonymous substitutions between the human and ape
lineages. * Indicates significant at 5% level. ** Indicates significant at 1% level.
aProbability was estimated from data presented in Zhang, Webb, and Podlaha(2002).
b
Probability was estimated using the mouse orthologs given in NCBI reference sequences (HCR: BC031416, ZFY: M24401, and DAF: L41366).

the mean number of amino acid residues is 447 (International Human Genome Sequencing Consortium
2001), the number human genome-wide amino acidchanges is estimated to be 80,258. Considerable numbersof those amino acid differences may be responsible forhuman-specific characteristics.
Differences of Nonsynonymous Substitutions for
Human and Ape Branch
Comparison of synonymous and nonsynonymous
substitutions for each coding region is a standard way ofdetecting the pattern of natural selection. However, thenumber of synonymous substitutions may undergo stochastic changes, and it can be rather small for one gene but
become large in another gene. We therefore decided tocompare the number of human and ape nonsynonymous
substitutions (dN) with the number of synonymous
substitutions (dS) for each gene. Human and ape dNs
were positively correlated with high statistical significance(R2 ¼ 0.33, P ¼ 2.00 3 10210). This result is compatible
with that of Wildman et al. (2003). Figure 3 shows a plotof human dN 2 ape dN for each gene. We multiplied
human dN by 6.54, because of the difference of humandivergence time (5.4 MYA) and ape divergence times(35.3 MYA). If dN is constant for human and ape
branches, human dN 2 ape dN is expected to be zero. In
fact, majority of the genes are located around the zero line
in figure 3. Substitution rates (dS) varied among genes. For
example, PRM2 showed the highest substitution rate by
dS. However, there was no correlation between the
difference of human dN 2 ape dN and total dS.
Two genes (ACAT2 and PRM2) showed higher ratesof dN for human branch than ape branches. In ACAT2, no
synonymous substitution and two nonsynonymous substitutions were observed on the human branch, and one
synonymous and two nonsynonymous substitutions wereobserved on the ape branch. However, because thecompared number of nucleotides (147 bp) was small,

FIG. 2.—Maximum-parsimony trees embedded in a phylogenetic
network of DAF. Numbers indicate number of nonsynonymous
substitutions/total number of nucleotide substitutions. Bold lines show
the maximum-parsimony pathway in each network. (A) is a topology((human, chimpanzee), gorilla, orangutan), and HC and HCG nodes are
the human-chimpanzee common ancestor and the human-chimpanzeegorilla common ancestor. (B) is a topology ((chimpanzee, gorilla), human,orangutan), and CG and HCG nodes are the chimpanzee-gorilla common
ancestor and the human-chimpanzee-gorilla common ancestor.

a further analysis is necessary to determine whether thisdifference is significant. The RNASE3 gene was shown tohave higher rate of dN for ape branches than human branch(figure 3). Zhang, Rosenberg, and Nei (1998) analyzed
RNASE3 (eosinophil cationic protein or ECP) andsuggested the existence of positive selection on this gene.
However, our result on figure 3 suggests that positive
selection operates only on the ape branch. It is possiblethat the selective constraint became strong after the humanlineage diverged from the remaining hominoid lineage for
the RNASE3 gene.
In conclusion, we conducted a systematic analysis of
103 protein-coding genes for human, chimpanzee, gorilla,
and orangutan. We showed that gene genealogies differ
from gene to gene, because the time span between thehuman-chimpanzee common ancestor and gorilla speciation is short. We conducted three types of analyses for
detecting the human-specific pattern in nonsynonymous

FIG. 3.—A plot of human dN 2 ape dN and total dS for each gene.
Human dN was multiplied by 6.54 because of the difference of humandivergence time (5.4 MYA) and ape divergence times (35.3 MYA). The
total dS was estimated from all branches of each gene. Broken lines
denote 95% level of the variation of human dN 2 ape dN.

Human-Specific Amino Acid Changes 943

changes. Comparison of each coding region is a standard
way of detecting the pattern of natural selection. However,
it is sometimes difficult to detect the pattern of natural
selection because of a few numbers of changes. We
conducted comparison of dNs by using a large number ofgenes. This kind of analysis may help to find candidate
genes that caused human-specific phenotypic changes.
Acknowledgments
We are grateful for Robert E. Ferrell for providing
chimpanzee DNA sample. We thank Hidemi Kobayakawa
for technical assistance and Kenta Sumiyama for suggestions and discussion. This study was supported by a grantin aid for scientific studies from Ministry of Education,
Science, Sport, and Culture, Japan to N.S. and U.S., Joint
Research Project (Soken/K99-1) of the Graduate University for Advanced Studies, Japan to N.S. T.K. was
supported by COE research fellowship of the National
Institute of Genetics and JSPS postdoctoral fellowship.
Literature Cited
Asumalahti, K., T. Laitinen, R. Itkonen-Vatjus, M.-L. Lokki, S.
Suomela, E. Snellman, U. Saarialho-Kere, and J. Kere. 2000.
A candidate gene for psoriasis near HLA-C, HCR (Pg8), ishighly polymorphic with a disease-associated susceptibility
allele. Hum. Mol. Genet. 9:1533–1542.
Chen, F.-C., and W.-H. Li. 2001. Genomic divergence between
humans and other hominoids and the effective population sizeof the common ancestor of humans and chimpanzees. Am. J.
Hum. Genet. 68:444–456.
Clark, A. G., S. Glanowski, R. Nielsen et al. (17 co-authors).
2003. Inferring nonneutral evolution from human-chimpmouse orthologous gene trios. Science 302:1960–1963.
Corder, E. H., A. M. Saunders, W. J. Strittmatter, D. E.
Schmechel, P. C. Gaskell, G. W. Small, A. D. Roses, J. L.
Haines, and M. A. Pericak-Vance. 1993. Gene dose of
apolipoprotein E type 4 allele and the risk of Alzheimer’s
disease in late onset families. Science 261:921–923.
Enard, W., M. Przeworski, S. E. Fisher, C. S. Lai, V. Wiebe,
T. Kitano, A. P. Monaco, and S. Pääbo. 2002. Molecular
evolution of FOXP2, a gene involved in speech and language.
Nature 418:869–872.
Fujiyama, A., H. Watanabe, A. Toyoda et al. (14 co-authors).
2002. Construction and analysis of a human-chimpanzee
comparative clone map. Science 295:131–134.
Goodman, M., C. A. Porter, J. Czelusniak, S. L. Page, H.
Schneider, J. Shoshani, G. Gunnell, and C. P. Groves. 1998.
Toward a phylogenetic classification of Primates based on
DNA evidence complemented by fossil evidence. Mol.
Phylogenet. Evol. 19:585–598.
Horai, S., K. Hayasaka, R. Kondo, K. Tsugane, and N. Takahata.
1995. Recent African origin of modern humans revealed bycomplete sequences of hominoid mitochondrial DNAs. Proc.
Natl. Acad. Sci. USA 92:532–536.
Hughes, A. L., and M. Nei. 1988. Pattern of nucleotide
substitution at major histocompatibility complex class I locireveals overdominant selection. Nature 335:167–170.
———. 1989. Nucleotide substitution at major histocompatibility complex class II loci: evidence for overdominant selection.
Proc. Natl. Acad. Sci. USA 86:958–962.
Huttley, G. A., S. Easteal, M. C. Southey, A. Tesoriero, G. G.
Giles, M. R. E. McCredie, J. L. Hopper, and D. J. Venter.
2000. Adaptive evolution of the tumour suppressor BRCA1 in
humans and chimpanzees. Nat. Genet. 25:410–413.

Ina, Y. 1994. ODEN: a program package for molecular
evolutionary analysis and database search of DNA and aminoacid sequences. Comput. Appl. Biosci. 10:11–12.
International Human Genome Sequencing Consortium. 2001.
Initial sequencing and analysis of the human genome. Nature
409:860–921.
Johnson, M. E., L. Viggiano, J. A. Bailey, M. Abdul-Rauf,
G. Goodwin, M. Rocchi, and E. E. Eichler. 2001. Positive
selection of a gene family during the emergence of humans
and African apes. Nature 413:514–519.
Kimura, M. 1983. The neutral theory of molecular evolution.
Cambridge University Press, Cambridge, UK.
King, M. C., and A. C. Wilson. 1975. Evolution at two levels inhumans and chimpanzees. Science 188:107–116.
Kitano, T., and N. Saitou. 1999. Evolution of the Rh blood group
genes has experienced gene conversions and positive selection. J. Mol. Evol. 49:615–626.
Kitano, T., K. Sumiyama, T. Shiroishi, and N. Saitou. 1998.
Conserved evolution of the Rh50 gene compared to its
homologous Rh blood group gene. Biochem. Biophys. Res.
Comm. 249:78–85.
Kuttner-Kondo, L., V. B. Subramanian, J. P. Atkinson, J. Yu, andM. E. Medof. 2000. Conservation in decay accelerating factor(DAF) structure among primates. Dev. Comp. Immunol.
24:815–827.
McDonald, J. H., and M. Kreitman. 1991. Adaptive protein
evolution at the Adh locus in Drosophila. Nature. 351:652–654.
Messier, W., and C. B. Stewart. 1997. Episodic adaptive evolution of primate lysozymes. Nature 385:151–154.
Nei, M. 1987. Molecular evolutionary genetics. Columbia
University Press, New York.
Nei, M., and T. Gojobori. 1986. Simple methods for estimating
the numbers of synonymous and nonsynonymous nucleotide
substitutions. Mol. Biol. Evol. 3:418–426.
Ohno, S. 1972. So much ‘‘junk’’ DNA in our genome.
Brookhaven Symp. Biol. 23:366–370.
O’hUigin, C., Y. Satta, N. Takahata, and J. Klein. 2002.
Contribution of homoplasy and of ancestral polymorphism tothe evolution of genes in anthropoid primates. Mol. Biol.
Evol. 19:1501–1513.
Rall, S. C. Jr., K. H. Weisgraber, T. L. Innerarity, and R. W.
Mahley. 1982. Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic
subjects. Proc. Natl. Acad. Sci. USA 79:4696–4700.
Reid, M. E., V. Chandrasekaran, L. Sausais, J. Pierre, and
R. Bullock. 1996. Disappearance of antibodies to Cromer bloodgroup system antigens during mid pregnancy. Vox Sang. 71:
48–50.
Saitou, N. 1991. Reconstruction of molecular phylogeny ofextant hominoids from DNA sequence data. Am. J. Phys.
Anthropol. 84:75–85.
Saitou, N., and M. Nei. 1987. The neighbor-joining method:a new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 4:406–425.
Satta, Y., J. Klein, and N. Takahata. 2000. DNA archives and ournearest relative: the trichotomy problem revisited. Mol. Phyl.
Evol. 14:259–275.
Sibley, C. G., and J. E. Ahlquist. 1984. The phylogeny of thehominoid primates, as indicated by DNA-DNA hybridization.
J. Mol. Evol. 20:2–15.
Sneath, P. H. A., and R. R. Sokal. 1973. Numerical taxonomy.
Freeman, San Francisco.
Sumiyama, K., N. Saitou, and S. Ueda. 2002. Adaptive evolutionof the IgA hinge region in primates. Mol. Biol. Evol. 19:
1093–1099.
Thompson, J. D., T. J. Gibson, and D. G. Higgins. 1994.
CLUSTAL W: improving the sensitivity of progressive

944 Kitano et al.

multiple sequence alignment through sequence weighting,positions-specific gap penalties and weight matrix choice.
Nucleic Acids Res. 22:4673–4680.
Tucker, P. K., R. M. Adkins, and J. S. Rest. 2003. Differential ratesof evolution for the ZFY-related zinc finger genes, Zfy, Zfx, andZfa in the mouse genus Mus. Mol. Biol. Evol. 20:999–1005.
Weisgraber, K. H., S. C. Rall, and R. W. Mahley. 1981. Human
E apoprotein heterogeneity: cysteine-arginine interchanges in
the amino acid sequence of the apo-E isoforms. J. Biol. Chem.
256:9077–9083.
Wildman, D. E., M. Uddin, G. Liu, L. I. Grossman, and M.
Goodman. 2003. Implications of natural selection in shaping
99.4% nonsynonymous DNA identity between humans andchimpanzees: enlarging genus Homo. Proc. Natl. Acad. Sci.
USA 100:7181–7188.

Wyckoff, G. J., W. Wang, and C.-I. Wu. 2000. Rapid evolutionof male reproductive genes in the descent of man. Nature
403:304–309.
Yang, Z. 1997. PAML: a program package for phylogenetic
analysis by maximum likelihood. CABIOS 13:555–556.
Zhang, J., H. F. Rosenberg, and M. Nei. 1998. Positive Darwinian
selection after gene duplication in primate ribonuclease genes.
Proc. Natl. Acad. Sci. USA 95:3708–3713.
Zhang, J., D. M. Webb, and O. Podlaha. 2002. Accelerated
protein evolution and origins of human-specific features:
Foxp2 as an example. Genetics 162:1825–1835.

Pekka Pamilo, Associate Editor
Accepted January 8, 2004