Immunogenetics (2007) 59:427–431
DOI 10.1007/s00251-007-0204-z

BRIEF COMMUNICATION

Tempo and mode of evolution of the Rh blood group genes
before and after gene duplication
Takashi Kitano & Kazuo Umetsu & Wei Tian &Kentaro Yamazaki & Naruya Saitou

Received: 2 November 2006 / Accepted: 20 February 2007 / Published online: 3 March 2007
# Springer-Verlag 2007

Abstract The Rh blood group genes became duplicated in
a common ancestor of human–chimpanzee–gorilla. We
compared the evolutionary rates of the Rh blood group
genes for each exon for branches connecting to humans,
having duplicated Rh loci, and to orangutan, gibbon, and
Old World monkeys, species having a single Rh locus. Our
results show that evolutionary rates of nonsynonymous
substitutions at exon 7 became accelerated in the humanlineage. Furthermore, we surveyed the sequence variationin the region surrounding exon 7 of gibbons to clarify
whether the diversity of the human exon 7 was introduced
after the duplication or had been maintained before it. Twoamino acid polymorphisms in white-handed gibbons wereobserved in the immediate vicinity of the D-specific motif
in the human exon 7. Although the evolutionary rate ofexon 7 was accelerated after the gene duplication, ourresults suggest that exon 7 had the potential for changeeven before the gene duplication.
Keywords Rh . Blood . Gibbon . Primate .
Nucleotide diversity . Evolutionary rate

Electronic supplementary material The online version of this article(doi:10.1007/s00251-007-0204-z) contains supplementary material,
which is available to authorized users.
T. Kitano (*) : K. Umetsu : W. Tian : K. Yamazaki
Department of Experimental and Forensic Pathology,
Yamagata University School of Medicine,
2-2-2 Iidanishi,
Yamagata 990-9585, Japane-mail: tkitano@med.id.yamagata-u.ac.jp
N. Saitou
Division of Population Genetics, National Institute of Genetics,
Mishima 411-8540, JapanThe human Rh blood group plays important roles intransfusion and clinical medicine, being involved in hemolytic diseases of newborns, autoimmume diseases, and mild
hemolytic anemia. The Rh blood group system is composedof two closely linked loci, D and CE (Mouro et al. 1993).
Each locus has ten exons (Cherif-Zahar et al. 1994), and the
D and CE polypeptides have only an 8.4% amino aciddifference (Le van Kim et al. 1992). These two loci arelocated on chromosome 1p34–p36 in humans (Ruddle et al.
1972; Cherif-Zahar et al. 1991), have opposite orientation,and face each other with their 3′ ends (Suto et al. 2000;
Wagner and Flegel 2000). It has been suggested that gene
duplication occurred after the divergence of the human–
African ape clade from Asian apes (orangutan and gibbon;
Salvignol et al. 1993; Blancher and Socha 1997), giving
rise to the two loci, by southern blot analyses.
Innan (2003) analyzed polymorphisms in human D and
CE loci, and observed very high amino acid divergence
between the two loci only in exon 7. According to his
hypothesis, immediately after the gene duplication that
produced D and CE loci, gene conversion occurred quite
frequently at both loci, at a rate high enough to keep thegenes of the two loci nearly identical. Then an advantageous mutation was introduced (probably in exon 7) andbecame fixed in one locus. Selection might have been sostrong that this fixation state was nearly stable andcontinued for quite a long time. During this state, additional
mutations accumulated near the target site of selection,
creating a high level of sequence divergence between the
two loci around exon 7. As an alternative evolutionary
process, however, one can assume that those polymorphisms of exon 7 had been maintained in one locus for along time before the duplication, such as in the case of the
Duffy blood group gene (Hamblin et al. 2002) andglycophorin A gene (Baum et al. 2002). The Duffy blood

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Immunogenetics (2007) 59:427–431

These values were positive (1.378 for exon 5 and 1.175 forexon 7) but did not differ significantly from the neutral
expectation of zero.
We further analyzed the distributions of π for two
species of gibbons at the regions surrounding exons 5(Fig. 1a) and 7 (Fig. 1b). Nucleotide differences (d )between the two gibbon species and between the gibbon
and the orangutan at the regions surrounding exons 5 and 7
are also shown in this figure. Interestingly, the nucleotide
diversity was clustered at exon 7 in white-handed gibbons(Fig. 1b). Three polymorphic sites were observed withinexon 7 of the white-handed gibbons (Table 1). Two of themwere nonsynonymous differences (V-352-F and G-355-S),
and the remaining one was a synonymous site at the 330th
amino acid residue. These two nonsynonymous polymorphisms in the white-handed gibbons were in the immediate
vicinity of the D-specific motif D-350, G-353, and A-354
in exon 7 of humans, and this motif is located on the sixth
outer membrane of the protein (Cartron and Agre 1993).
This finding also suggests that exon 7 of the Rh bloodgroup gene had the potential for change before the gene
duplication. Most genes are evolving under the neutral
mutation pressure (Kimura 1983). However, some genes
are evolving through positive selection. Blood types wereoriginally distinguished by the different molecular structure
on erythrocytes. Therefore, products of blood group genes
may cause interactions with other organisms, and there is
the possibility of positive selection on those genes as shownfor the ABO blood group genes by Saitou and Yamamoto(1997). Thus, it is possible that the amino acids of the sixth
outer membrane of the Rh protein may be involved in somekind of host–parasite interaction.
To compare the evolutionary rate before and after thegene duplication of the Rh blood group genes, we
calculated the evolutionary rate of synonymous (1S) andnonsynonymous (1N) substitutions for each exon forbranches connecting to Old World monkeys (OWM),
gibbon, orangutan, and human species (Table 2). The

group locus has an extreme pattern of geographic differentiation of its three major alleles in humans because ofresistance to malaria. Allelic variations of the glycophorin
A gene within human populations have been maintained bybalancing selection because the product of this gene is oneof the components on the erythrocyte surface.
To determine whether the diversity at exon 7 of the Rh
blood group genes was introduced after the duplication or had
been maintained before the duplication, we surveyed thesequence variation in the region surrounding exon 7 (approx.
1 kb) in nine white-handed gibbons (Hylobates lar) and fivesiamangs (Hylobates syndactylus), both of which have asingle Rh locus. To compare the level of polymorphisms inexon 7 with that in another exon, we also analyzed theregion surrounding exon 5 (approx. 1 kb) in the same
samples because exon 5 is the second longest exon in the Rh
blood group gene and is a neighbor of exon 7. There has
been no earlier study that tried to analyze polymorphisms forprimate species before the gene duplication of the Rh bloodgroup genes. The DDBJ/EMBL/GenBank International
Nucleotide Sequence Database accession numbers are
AB289827–AB289856. A list of primers used in this study
is available from Kitano. Haplotypes were estimated byusing the PHASE 2.0.2 program (Stephens et al. 2001).
Table 1 shows polymorphisms in hominoids at exons 5
and 7 of the Rh blood group genes. The average number (π)
of nucleotide differences per site between two sequences
for exon 7 of white-handed gibbons was 0.009. It is no
surprise that this value is four to six times lower than the πvalue for exon 7 of humans, chimpanzees, and gorillas, allof which have duplicated Rh loci. However, π of exon 7 inwhite-handed gibbons was three times higher than that ofexon 5. This ratio of π for exon 7 to exon 5 (π [ex7/ex5]) in
the white-handed gibbon was comparable to that for otherhominoids. Thus, this finding suggests that exon 7 of theRh blood group gene had greater potential for change thanother exons before the gene duplication. Tajima’s (1989) D
was calculated for exons 5 and 7 of white-handed gibbons.

Table 1 Polymorphisms in hominoids at exons 5 and 7 of the Rh blood group genes
Exon 5 (167 bp)

Human
Chimpanzee
GorillaWhite-handed gibbon
Siamang

Exon 7 (134 bp)

N

S

π

θw

N

S

π

θw

80
11
4
18
10

17
7
5
1
0

0.024
0.012
0.017
0.003
0.000

0.021
0.014
0.016
0.002
0.000

67
11
4
18
10

20
25
10
3
0

0.054
0.066
0.044
0.009
0.000

0.031
0.064
0.041
0.007
0.000

π
(ex7/ex5)

Sources

2.29
5.63
2.57
3.08
–

From DNA databanka
From DNA databanka and Kitano et al. (unpublished)
From DNA databanka and Kitano et al. (unpublished)
This study
This study

N Number of chromosomes, S number of segregating sites, π average number of nucleotide differences per site between two sequences (Nei
1987), θw nucleotide diversity based on the proportion of segregating sites in a sample (Watterson 1975), π (ex7/ex5) ratio of πs for exon 7 toexon 5, – not estimatedaAccession numbers are listed in Supplementary Table 1.

Immunogenetics (2007) 59:427–431

429

ad

π

0.10

0.018
0.016

0.08

0.014
0.012

0.06

0.010
0.008

0.04

0.006
0.004

0.02

0.002
600

500

400

300

200

bd

0.000
100

0.00

bp
π

0.10

0.018
0.016

0.08

0.014
0.012

0.06

0.010
0.008

0.04

0.006
0.004

0.02

0.002
900

800

700

600

500

400

300

200

0.000
100

0.00

bp

Fig. 1 Sliding window analyses of π and average nucleotide differences between two species for regions surrounding exon 5 (a) andexon 7 (b). Window size of 100 bp and sliding range of 20 bp wereused. Exons are shown by thick horizontal bars. White bar π for thewhite-handed gibbon, gray bar π for the siamang, black line theaverage number of nucleotide differences (d ) between the whitehanded gibbon and siamang, broken line d between the siamang andthe orangutan, grey line d between the white-handed gibbon and theorangutan

established phylogeny for primate species (e.g., Horai et al.
1995; Goodman et al. 1998) was adopted for primate Rh
blood group genes (Cherif-Zahar et al. 1990; Le van Kim etal. 1992; Mouro et al. 1994; Salvignol et al. 1994; Apoil
and Blancher 1999), and divergence times (T ) of the humanlineage from the orangutan, gibbon, and Old World monkey
were assumed to be 13, 15, and 23 million years ago(Glazko and Nei 2003; Fig. 2). The Rh blood group genes of
the chimpanzee and gorilla were not included in this tree
because gene conversions in them interrupt the orthologous
relationship among their duplicated genes (Kitano and Saitou
1999; see also Supplementary Fig. 1). For simplicity, human
D and CE loci were used as representatives of duplicated Rh
genes in the phylogenetic tree. The nucleotide sequences ofancestral nodes were reconstructed by the Bayesian method(Yang et al. 1995) using the program BASEML in the PAML
3.15 package (Yang 1997). dN (the number of nonsynonymous substitutions per nonsynonymous site) and dS (thenumber of synonymous substitutions per synonymous site)were estimated (Nei and Gojobori 1986) for each branch,and 1S and 1N were estimated by the formula 1= d/T. dN anddS for the branch of Node Z—Human were estimated as[((branch g + branch h)/2)+branch f ], and dN and dS for the
branch of Node X—OWM were estimated as [(((branchd+branch e)/2) + branch c+branch b)/2 + branch a].
1S values for all (exons 1–9) were 1.7×10−9, 2.4×10−9,
1.5× 10−9, and 1.8× 10−9 for branches of Old World
monkeys (Node X—OWM), gibbons (Node Y—Gibbon),orangutans (Node Z—Orangutan), and humans (Node Z—
Human), respectively. These 1S values were lower than the
average value (4.6×10−9) of other mammalian genes (Liand Graur 1991). Exon 9 of the orangutan showed an
extremely high 1S value. This is probably a stochastic effect

Table 2 Evolutionary rate (1, ×10−9) of synonymous (1S) and nonsynonymous (1N) substitutions for each exon for each branch connecting to
OWM, gibbon, orangutan, and human species
Exon

1
2
3
4
5
6
7
8
9
All

bp

146 (144)
184 (183)
150 (149)
146 (143)
167 (164)
138 (135)
129 (126)
76 (74)69 (69)1205 (1187)

Node X—OWM

Node Y—gibbon

Node Z—orangutan

Node Z—human

1S

1N

1S

1N

1S

1N

1S

1N

0.9 (0.9)
0.0 (0.0)
1.2 (1.2)
2.0 (2.1)
0.3 (0.3)
3.8 (2.8)
2.0 (0.7)
2.3 (2.5)
7.9 (7.9)
1.7 (1.5)

0.0 (0.0)
2.5 (2.5)
4.6 (4.1)
3.2 (2.8)
1.7 (1.2)
3.0 (2.2)
3.0 (2.2)
3.5 (1.9)
0.0 (0.0)
2.4 (2.0)

1.8 (1.9)
1.5 (1.6)
0.0 (0.0)
1.0 (1.1)
7.2 (5.7)
0.0 (0.0)
6.3 (4.5)
0.0 (0.0)
4.6 (4.6)
2.4 (2.0)

1.9 (0.7)
1.0 (0.5)
1.8 (1.3)
2.8 (2.3)
2.2 (1.1)
0.7 (0.0)
2.9 (2.3)
5.3 (4.3)
1.3 (1.3)
2.1 (1.3)

2.1 (2.2)
0.0 (0.0)
2.1 (2.1)
2.3 (2.4)
2.0 (2.2)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
11.5 (11.5)
1.5 (1.6)

0.0 (0.0)
4.3 (4.4)
1.4 (1.5)
2.2 (0.8)
0.6 (0.6)
1.6 (0.8)
0.0 (0.0)
4.5 (5.0)
1.5 (1.5)
1.7 (1.5)

4.2 (4.4)
1.8 (1.8)
2.1 (2.1)
0.0 (0.0)
1.0 (1.1)
2.0 (2.2)
2.3 (1.2)
0.0 (0.0)
2.7 (2.7)
1.8 (1.7)

2.2 (0.8)
2.0 (1.5)
4.2 (4.3)
5.4 (3.8)
5.0 (4.0)
2.4 (2.5)
15.2 (13.9)
4.6 (3.3)
2.2 (2.2)
4.7 (3.9)

Sequence data AB289846 and L37051 are used for exon 7 and other exons, respectively, for the gibbon. 1S and 1N greater than 10.0×10−9 areshown in italics. The numbers in parentheses indicate values estimated from data including ambiguously reconstructed sites (probability less than0.95). See Fig. 2 for locations of nodes X–Z.
bp Compared nucleotide sites

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Immunogenetics (2007) 59:427–431

13 MYA
15 MYAgfh

23 MYAZ

Human D
Human CE
Hominoids

Orangutan

Y

GibbonXbac

Guinea baboondeRhesus macaque

Old World Monkeys

Crab-eating macaque
Brown capuchinCommon squirrel monkey
Common marmoset 1

New World Monkeys

Common marmoset 2
Fig. 2 A phylogenetic tree for primate Rh blood group genes.
Nucleotide sequences were retrieved from the DDBJ/EMBL/GenBank
International Nucleotide Sequence Database: human D (X63097),
human CE (M34015), orangutan (AF012425), gibbon (L37051 andAB289846), guinea baboon (AF012426), rhesus macaque (S70343),

crab-eating macaque (L37054), brown capuchin (AF101479), commonsquirrel monkey (AF012428), and two common marmosets(AF012429 and AF052588). The nucleotide sequences ancestral nodes(X–Z ) were reconstructed by the Bayesian method (Yang et al. 1995).
Some branches are indicated by a–hbecause of the small number of compared sites (twosynonymous substitutions in 69 compared sites). In
contrast, 1N for all (exons 1–9) were 2.4×10−9, 2.1×10−9,
1.7× 10−9, and 4.7× 10−9 for branches of Old World
monkeys (Node X—OWM), gibbons (Node Y—Gibbon),orangutans (Node Z—Orangutan), and humans (Node Z—
Human), respectively. These 1N values were somehow
higher than the average value (0.9×10−9) of other mammalian genes (Li and Graur 1991), suggesting positive
selection on the Rh blood group genes (Kitano et al. 1998;
Kitano and Saitou 1999). In particular, 1N of the humanexon 7 showed an extremely high value. When we
eliminated sites showing a probability of accuracy forreconstructed nucleotides at each ancestral node of less than
0.95 and estimated 1N, exon 7 of humans still showed an
extremely high value (13.9×10−9). We also carried out the
maximum parsimony method to reconstruct ancestral
sequences by the PAMP program in the PAML package.
There were some incompatible sites between the Bayesianand the maximum parsimony methods, but all of these sites
belonged to ambiguously reconstructed sites (probability
less than 0.95; see Table 2). Ideally, estimation of 1S and 1Nfor the human branch should be divided into that before andthat after the gene duplication, but we did not do so because
it is not easy to estimate the time of the gene duplication
because of positive selection and gene conversions. At least
we can say that 1N of exon 7 became higher after thedivergence between the orangutan and the common
ancestor of human–chimpanzee–gorilla.
To compare the evolutionary rate between human D and
CE lineages, we applied the relative rate test (Tajima 1993)

for translated amino acid sequences of the Rh blood group
genes (Table 3). The orangutan sequence was used as an
outgroup. Amino acid changes tended to have accumulated
in human D (19 in total) rather than in human CE (12 intotal). However, this difference was not significant ( p=0.209). In exon 7, five and three amino acid changes wereobserved in human D and CE, respectively, but thedifference was not statistically significant ( p=0.480). Whenthe gibbon sequence was used as an outgroup, similar
results were obtained (data not shown). The result indicatesthat both D and CE loci accumulated amino acid changes
after the gene duplication at similar levels.
Table 3 Results of relative rate test of human D- and CE-translatedamino acid sequences
Exon

AA

Human D

Human CE

Orangutan

p

1
2
3
4
5
6
7
8
9
All

49
60
50
48
56
46
45
24
23
401

0
3
3
2
3
2
5
0
1
19

0
0
1
3
5
0
3
0
0
12

3
9
6
4
5
4
8
6
2
47

–
0.083
0.317
0.655
0.480
0.157
0.480
–
0.317
0.209

AA Number of amino acid residues compared, Human D number ofamino acid changes on the human D branch, Human CE numberof amino acid changes on the human CE branch, Orangutan numberof amino acid changes on the branch between orangutan and thecommon ancestor of human D and CE

Immunogenetics (2007) 59:427–431

In conclusion, our results show that the evolutionary rate
for nonsynonymous substitutions at exon 7 became accelerated in the human lineage. The results of the relative rate test
suggested that both D and CE loci accumulated amino acidchanges in exon 7 after the gene duplication rather than that
one gene maintained its original function and the other geneaccumulates amino acid changes by relaxation of functional
constraints after the gene duplication. From the polymorphism analysis of gibbons, which species has a single Rh
locus, a clear pattern of balancing selection was not observed
by Tajima’s D test. However, the higher nucleotide diversity
at exon 7 than at exon 5 in the white-handed gibbons
suggests that, in contrast with other exons, exon 7 hadgreater potential for change than other exons. Thus, it islikely that exon 7 of the Rh blood group genes had achangeable nature beforehand, and then this tendency might
have been enhanced by the gene duplication.
Acknowledgments We are grateful to the Great Ape Information
Network (GAIN) for providing us an orangutan DNA sample and to Dr.
Osamu Takenaka (now deceased) for providing us gibbon DNA samples.
This study was supported by a grant-in-aid for scientific studies from the
Ministry of Education, Science, Sports, and Culture of Japan to TK.

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