Evolution
Genes1of Primate ABO Blood Group Genes and Their Homologous

Naruya Saitou* and Fumi-ichiro*Laboratory
California

of Evolutionary

Genetics,

YamamotoJf

National Institute of Genetics,

Mishima,

Japan; and TThe Burnham

Institute,

La Jolla,

There are three common alleles (A, B, and 0) at the human ABO blood group locus. We compared nucleotide
sequences of these alleles, and relatively large numbers of nucleotide differences were found among them. These
differences correspond to the divergence time of at least a few million years, which is unusually large for a humanallelic divergence under neutral evolution. We constructed phylogenetic networks of human and nonhuman primate
ABO alleles, and at least three independent appearances of B alleles from the ancestral A form were observed.
These results suggest that some kind of balancing selection may have been operating at the ABO locus. We also
constructed phylogenetic trees of ABO and their evolutionarily related cw-1,3-galactosyltransferase
genes, and thedivergence time between these two gene families was estimated to be roughly 400 MYA.

Introduction
The human ABO blood group was discovered by
Karl Landsteiner
in 1900, and its mode of inheritanceas multiple alleles at a single genetic locus was established by Felix Bernstein a quarter century later (Crow
1993). Immunodominant
ABH antigens were chemicallycharacterized to be carbohydrate
structures of glycoproteins and glycolipids
through the studies performed in
the 1950s and early 1960s (see Yamamoto 1995 for review). Based on this finding, ABO alleles A and B werehypothesized
to code for glycosyltransferases
whichtransfer GalNAc and galactose, respectively,
while 0was hypothesized to be a null allele incapable of coding
for a functional
glycosyltransferase.
Yamamoto et al.
(1990a, 1990b) determinedthe cDNA sequencesof
three common alleles, Al, B, and 0, and Yamamoto,
McNeill, and Hakomori (1995) determined the genomic
organizationof the gene.
The purpose of this paper is to analyze the nucleotide and amino acid sequences for primate ABO bloodgroup genes and their homologous genes. Biological significance of the ABO blood group will be discussed
based on the clues obtained from phylogenetic
analysesof ABO blood group genes.
Materials

and Methods

For human ABO blood group genes (hereafter
called “ABO genes”), sequence data of Yamamoto etal. (1990a, 1990b, 1992, 1993a, 1993b, 1993c, 19934
and those of Ogasawara et al. (1996~) were used. Table
1 lists the variant nucleotide sites for the human alleles.
It should be noted that allele 02, named by Grunnet etal. (1994), has the identical sequence with O-3, previously reported by Yamamoto et al. (1993b). The coordinate system of the nucleotide and amino acid positions
l Dedicated to the memory of the late Dr. Motoo Kimura.
Key words: ABO blood group,
polymorphism,
overdominantselection,

glycosyltransferase,primates,phylogenetic network.

Address for correspondence
and reprints: Naruya Saitou, Laboratory of Evolutionary
Genetics, National Institute of Genetics, 1111
Yata, Mishima-shi,
Shizuoka-ken,
411, Japan. E-mail: nsaitou@genes.nig.ac.jp.
Mol. Biol. Evol. 14(4):399411.
1997
0 1997 by the Society for Molecular Biology

and Evolution.

ISSN: 0737-4038

of the human and nonhumanprimate ABO gene sequences follows that of Yamamoto (1995).
As for nonhumanprimate ABO gene sequences,
those for two chimpanzees (Pan troglodytes), one gorilla(Gorilla gorilla), two orangutans(Pongo pygmaeus),
one crab-eating macaque (Macaca fascicularis), and two
yellow baboons (Papio cynocephalus)
are from Kominato et al. (1992), and sequences for three chimpanzees,
three gorillas, and two orangutans are from Martinko etal. (1993). Table 2 lists the variant nucleotide sites forthe primate sequences. Amino acid sequence differences
between positions
152-355 among those human andnonhuman primate sequences are shown in table 3.
As for the primate ABO-related seqtrences, the hgt4
pseudogene found from the human genome (Yamamoto,
McNeill, and Hakomori 1991) and the mouse ABO gene
(unpublisheddata) were used, as well as the cx-1,3-galactosyltransferase
functional gene cDNA sequences ofmice (Larsen et al. 1989), cow (Joziasse et al. 1989),
and pig (Strahan et al. 1995). The two human pseudogenes for the (Y-1,3-galactosyltransferase
gene were also
included; an ordinary type (Larsen et al. 1990) and aprocessed type (Joziasse et al. 1991).
Galili and Swanson (1991) determined partial nucleotide sequencesof the CX-1,3-galactosyltransferase
functional gene for squirrel monkey (Saimiri sciureus),
spider monkey (Ateles geofiroyi), and howler monkey(Alouatta caraya), and its pseudogenes for chimpanzee,
gorilla, orangutan,
rhesus monkey (Macaca mulatta),
green monkey (Cercopithecus aethiops), and patas monkey (Erythrocebus patas). Henion et al. (1994) also sequenced the complete coding region of this gene formarmoset (Callithrix sp.). Those sequences were used
to construct a primate-specifictree for this gene.
We searched the latest version of the DDBJ/EMBL/
GenBank international
nucleotide sequence database byusing BLAST (Altschul et al. 1990) and FASTA (Pearson and Lipman 1988), and did not find any sequencehomologous to the ABO blood group gene.
Phylogenetic
trees were constructed by using theneighbor-joining
method (Saitou and Nei 1987) and the
maximum-likelihood
method (Felsenstein
198 1) for relatively distantly related sequences. When the neighborjoining method was applied, Kimura’s (1980) two-pa399

400

Saitou and Yamamoto

Table 1
Sequence Comparisonof the Human

ABO Blood Group Locus Alleles

NUCLEOTIDE POSITION
11

1112224555666777788889E
099069626745802790027355
912317764967131162391049
abcbccbaccaccaacaabaaccb

ALLELEA’-l(A1O1)......
A’-2(A102)......
A’-3(A103)......
A’-4(A104)......
A2 ..............
AX. .............
A”-1 ............
cis-AB ..........
o-1 (0101). ......
o-2 (0201). ......
o-3 .............
O-4 (0102). ......
o-5 (0103). ......
O-6 (0202). ......
O-7 (0203) ......
B-l (BlOl). ......
B-2 (B102). ......
B-3 (B103). ......
BcA) .............
B’-1 ............

REFERENCEGGCCGACCCTTCGGCCCGGGGGCC......
****..T.T...............

****.G..................
***A.. T ................
****......
A .............
**xx
**XX
..

Yamamoto
Yamamoto
Ogasawara
Ogasawara
Yamamoto,
Yamamoto
Yamamoto
Yamamoto
Yamamoto
Yamamoto
Yamamoto
Ogasawara
Ogasawara
Ogasawara
Ogasawara
Yamamoto
Ogasawara
Ogasawara
Yamamoto
Yamamoto

T .................

.

................
T ...........

A ...

..

..-

C .....

...................

TATT-G.

. ..A.A..T...A....

****.G.G.........A......

****-....c..............

****-G..................

****-.....A***k-G

..

.....

.A ..
G.G

T...A

..A.A.TT...A
..

....
....

.T.A..A.C..A****.G.G...T.A..A.C

..
.....

****.G.G.....A..A.C..A..

****.G.G........A.C..A..

****.G.G..

.T.A..A.C..AT.

et al. (1990~)
et al. (1990~)
et al. (1996~)
et al. (1996~)
McNeill, and Hakomori
et al. (1993~)
et al. (19934
et al. (1993~)
et al. (1990~)
et al. (1990~)
et al. (1993b)
et al. (1996~)
et al. (1996~)
et al. (1996~)
et al. (1996~)
et al. (1990u)
et al. (1996~)
et al. (1996~)
et al. (1993~)
et al. (19934(1992)

NOTE.-Dots, asterisks, and hyphens mean identical nucleotides as the human Al-1 sequence, nonexamined positions,and gaps, respectively. Position 1059 can be either 1059, 1060, or 1061. Allele names in parentheses were used in Ogasawara
et al. (1996~2). Letters (a, b, or c) below nucleotide position numbers mean first, second, or third nucleotide positions,
respectively.

Table 2
Sequence Comparison
1003)

of the Primate ABO Blood Group Locus Genes (Positions 435NUCLEOTIDE POSITION
11

ALLELE
Al-1 (human). ................
Chimpanzee-l..
..............
Chimpanzee-2..
..............
Chimpanzee-3(Patr-1).........
Chimpanzee-4(Patr-2).
........
Chimpanzee-5(Patr-3).
........
Gorilla-l. ...............................
Gorilla-2,4,5 (Gogo-1,3,4)......
Gorilla-3(Gogo-2)............
Gorilla-6(Gogo-5)............
Orangutan-l..
................
Orangutan-2..
................
Orangutan-3(Popy-1).
.........
Macaque.. ...................
Baboon-l..
..................
Baboon-2..
..................

4444444444455555555556666677777777888889900
3556667788801122378892245800116789012247900
8077891407963917495931921124195736335670902
ccabcacccbcbccbbcccabcbccccbcacccabccabbcac

REFERENCETTACGGCGCAGAAGAGCTCGGGTGCGCGCGCCCCGGAGGTCGC
A.

.A..

..

G..

....

G..

....

C..

....

T..

..

C..

....

T..

......

....
....
**.

........

.G .....

.C ..........

**.T.......

.G .....

.C .....

**.

A ..A..

.

..

....

..

C.G..

C..

....

.G.

..................

1

T . ..A..
.

.T ..
.T ......

....

T..

...
.....

.A ..
..A

......

..

2

****

3
3
3

..................

.AC.

...

.G ...................

.AC.

.A.******.

........

...........

.T ........

.G.G..

..

.TT.GT.

.G.

..A.

.T..

.......

.A.

GC..

..

T.GG..GTG.TC..A...TA.CT...TT..G..CT

CC..

..

T.GG..GTG.TC..

CC..

..

T.GG..GTG.T..C..A
..

CT..

. .CT

.T ...

**T

C..

.AC
..

...

..A..

.C ........
.C..

..

.****

.. A ..A..
..

TT.AG..CT

C..A.CT...TAC.G..CT

3

.A.

.G.

A ..T..

3....

........

....

3
*

........

.G..

2

A...***.AC.

.G .........

T.
.****

**.

..G

2

.****

**.

..........

T.

..
.....

2
2

.****

3
.G

2

.G

2

.G

2

NOTE.--Dots and asterisks mean identical nucleotides as the human Al-1 sequence and nonexamined positions, respectively. Allele names in parentheses were used in Martinko et al. (1993). Letters (a, b, or c) below nucleotide position
numbers mean first, second, or third nucleotide positions, respectively. References are as follows: 1, Yamamoto et al.
(1990~); 2, Kominato et al. (1992); 3, Martinko et al. (1993).

ABO Blood Group Genes

Table 3
Amino Acid Sequence Comparison of the Primate ABO
Blood Group Enzymes (Positions 152-355)

sequences. In this study, constructed networks are notmedian networks, for all the possible nodes are connetted. PAUP 3.1.1 (Illinois Natural History Survey)
was used for some maximum-parsimony
analyses.

AMINO ACID POSITIONALLELE
Human Al-1 ..............
Human A’-2. .............
Human A2. ...............
Human AX ...............
Human A”-1 ..............
Human cis-AB ............
Human B-l. ..............
Human B(*) ..............
Human B”-1 ..............
Human O-3. ..............
Chimpanzee- 1,2 ...........
Chimpanzee-3.
............
Chimpanzee-4.
............
Chimpanzee-5.
............
Gorilla- 1 .................
Gorilla-2, 4, 5, 6 ..........
Gorilla-3 .................
Orang- 1. .................
Orang-2. .................
Orang-3. .................
Macaque .................
Baboon- 1 ................
Baboon-2 ................

401

11111111112222222222333
55566779991113466789355
36739465780465068631425

Results

and Discussion

Comparisonof the Five Human

ABO Gene Sequences

TPATQERFERVMFGSLGEADAR#.L.....................
.L....................E. . . . . . . . . . . .A . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .N . . .
.L..............A......

......

G ......

......

G ........

......

G ......

......

G .........

s .MA.

.....

MA ......
s.MA

...

.w.

R ......

........
s**
Q ...........
****. .P ...............
Q.L ...... Q .......... ********..................
Q*............... MA...T*
MA . . ****...............
...............
.....

MAL.
G ........

...................
G
S ...

T ...
T

LGLL...I..T....***

****
T..*

*
**
*

. ..A.G...Q...A.......**. ..A.G....A..A.......*

*

...

A.G

..

..A..A.MA....**

NOTE.-“#” at position 355 means stop codon.

rameter method was used for estimating numbers of nucleotide substitution, while Kimura’s (1983) formula for
approximating
Dayhoff matrix distances was used for
estimating numbers of amino acid substitutions.
CLUSTAL W (Thompson,
Higgins, and Gibson 1994) wasused for multiple alignment
and the neighbor-joining
method for amino acid sequences, NJBOOT2 (kindly
provided by Koichiro Tamura) was used for the neighbor-joining
methodfor nucleotide
sequences,
and
DNAML of PHYLIP 3.5~ (Felsenstein1993) was used
for the maximum-likelihood
method. After the construction of a tree, each branch length (number of amino acidor nucleotide substitutions
per site) was recomputed toestimate the integer number of substitutionsin the sequences compared, applying Ishida et al’s (1995) method.
The evolutionary
history of a gene should be presented as a tree. When we analyze real sequence data,
however, this tree structure may not be clearly observed.
In this case, constructionof phylogenetic
networks isuseful for delineating
anomalies in the history of genetrees. When two nucleotide positions show incongruent
partition (or configuration)
patterns, a discordancy diagram (Fitch 1977) appears. A phylogenetic network can
be considered
as a generalizationof this discordancy
diagram (Saitou 1996). Bandelt (1994) described themathematicalpropertiesof the phylogenetic
network
method, and Bandelt et al. (1995) proposed to construct
a “median network” that contains all the equally parsimonious trees. Because of these reasons, the phylogenetic-network
method and the maximum-parsimony
method (Fitch 1977) were applied for closely related

We first compared five cDNA sequences for thehuman ABO gene presented by Yamamoto et al. (1990a,
1990b). Al-l, Al-2, and B alleles cover the complete
coding sequences, while O-l and O-2 allele sequenceslack three nucleotides corresponding
to the first codon(Yamamoto et al. 1990b). Polymorphic nucleotide sites
are shown in table 1. Because we dealt with closely
related sequences, the phylogenetic network method andthe maximum-parsimony
method were used. Figure 1
shows the unique phylogenetic
network (A) and four
equally parsimonious
trees (B-E’). Because a partition
defined by a single gap (position 261) and one definedby a nucleotide configuration at position 297 are incompatible, there is one loop in network A. If we cut oneof those branches that form this loop, four alternative
trees are produced (trees 23-E). Since allele Al-1 wasassumed to be identical with the ancestral sequence ofhuman ABO genes (see below), this information
wasused to locate the root (designated by a broken line) in
those equally parsimonious trees. It should be noted that
topologies of trees B and C are identical and only somebranch lengths are different.
Although 18 changes are required in all four trees,
two insertion/deletion
events are involved in trees D andE, while only one deletion is required for trees B and
C. Saitou and Ueda (1994) showed that the evolutionaryrate of insertions and deletions in primates was about
one order slower than that of nucleotide substitutions.
Therefore, trees B and C are much more probable thantrees D and E. In the former two trees, however, thenumber of nucleotide substitutions
along the branches
leading to O-l and O-2 are quite different. Interestingly,
allele 0- 1 is identical to allele Al- 1 except for the single
nucleotide deletion, while allele O-2 is different fromallele O-l with nine nucleotides. It is possible that allele
O-l might be a product of intragenic recombinations
orgene conversions, because those events homogenize different alleles. However, we have to assume at least twosuch events to explain the observed sequence pattern. It
suggests that intragenic recombinations
or gene conversions occur rather frequently at this locus. In fact, Ogasawara et al. (1996b) did find a probable recombinant
between an 0 and a B allele.
We estimated divergence times among human ABO
alleles based on nucleotide differences. Nucleotide difference per site between Al-2 and B-l is 0.008 (=8/
1,062), while that between B-l and O-2 is 0.013 (= 14/
1,059), respectively. These values are much larger than
the average number of nucleotide differences among different nucleotide sequences within a locus in human (Liand Sadler 1991). Saitou (1991) estimated the average
number of nucleotide differences of noncoding regions
between human and chimpanzee to be O.O14/site. If we402

Saitou and Yamamoto

FIG. l.-The
phylogenetic
network (A) and four possible maximum-parsimonytrees (B-E) for five human ABO blood group alleles.
Almost-complete
coding region (1,059 bp) sequences excluding the first three nucleotides were compared. Data are from Yamamoto et al.
(1990a, 1990b). Numbers on each branch are estimated numbers of nucleotide substitutions. Del and Ins mean deletion and insertion of a single
nucleotide, respectively.

use 5 Myr for the divergence time between human andchimpanzee, the rate of nucleotide substitution becomes
1.4 X l0-9/site/year,
and those allelic nucleotide differences for the human ABO genes correspond to 2.7-4.7
Myr. Those values are unusually large for different alleles of a typical human locus (see discussion below),
and yet those divergence times may still be underestimations. Because the ABO gene is functional, use of anevolutionaryrate for noncoding-region
DNA is expected
to give underestimationsof the divergence time. Geneconversion
and recombination
are also possible causes
for underestimation,
for they homogenize
different alleles.
The Phylogenetic
Alleles

Networkof HumanABO Gene

We used all of the 20 sequence data shown in table
1. Because many of the sequences analyzed did not
identify the nucleotides including variant positions 109,
191, 192, and 203, those positions were not used for thefollowing
analysis. The maximum-parsimony
methodwas first used, and 39 equally maximum parsimonious
trees were produced by using PAUP 3.1.1 with thebranch-and-boundoption (trees not shown). Clearly, themaximum-parsimony
method is not appropriate for delineating the complex nature of the ABO allele polymorphism.
We then used the phylogenetic-network
method,and the resulting network is presented in figure 2. Ogasawara et al.‘s (1996~) phylogenetic
network of ABO
common alleles can be considered to be a subset of thisnetwork. There are multiple loops in that network, indicating the existence of mutually incompatible
sites.
For example, position 803 separates all the five B alleles
and cis-AB alleles from the remaining A and 0 alleles,
but this partition pattern is incompatible
with that for
position 526, where all the B alleles and the O-3 allele

are separated from the remaining
alleles. Because, thecis-AB allele is rare, it is probable that the G-to-Cchange at position 803 occurred independently
in the B
allele lineage and in the cis-AB formation.
Let us consider the rectangle in the B allele group.
When we use the maximum-parsimony
method, there is
only one solution in this sequence group. In this case,allele B(*) is assumed to be identical with an ancestral
sequence of the B allele group. However, this allele isvery rare and it is possible that the allele appeared in
the human population only recently. If so, we have toconsider subparsimonious
solutions that are embeddedin the rectangular structure of figure 2. This again shows
the superiority of the network to the maximum-parsimony analysis.
The O-3 allele with no deletion is clearly in a different lineage from the remaining 0 alleles with a single
nucleotide deletion at position 261. It is likely that thenucleotide change at position 802 that caused the aminoacid change from glycine to arginine at position 268 (seetables 1 and 3) was responsible for the nonfunctionalization of the O-3 allele (Yamamoto et al. 1993b). It
should be mentioned that the amino acid position 268
is one of the two crucial positions that determine thedonor nucleotide-sugar
substrate specificity differences
between A and B transferases(Yamamoto and Hakomori 1990; Yamamoto and McNeil1 1996).
The Phylogenetic

Networkof Primate

ABO Alleles

Figure 3 is the phylogenetic network for human andnonhumanprimate sequences. This network was produced by combining
sequence data of tables 1 and 2,
except that six rare alleles observed in human populations were excluded. When the same nucleotide change
is shared with different species groups (three species
groups are as follows:
human-chimpanzee-gorilla,orangutan,
and macaque-baboon),
this change is as-

ABO Blood Group Genes

o-7

B3-1

0-2

O-6

Onenucleotide
substitution

FIG. 2.-The
phylogenetic
network for 20 human ABO blood group alleles. See table 1 for the sequencelines denote nucleotide substitutions
and deletions, respectively.
Numbers on some branches are nucleotide
branches. Full circles denote observed human alleles.

One nucleotide
substitution

403

data used. Thick lines and thinpositions responsible for thosebaboon-2b

FIG. 3.-The
phylogenetic
network for common alleles of the primate ABO blood group locus. Abbreviations:ch = chimpanzee, go =gorilla, or = orangutan. See tables 1 and 2 for the sequence data used. Numbers on some branches are nucleotide positions responsible forthose branches. Numbers with asterisks signify nucleotide positions in which parallel substitutions occurred. Full and open circles denote human“HCG node” is the position of the common ancestral sequence for human, chimpanzee, and gorilla.
and nonhuman ABO alleles, respectively.

404

Saitou and Yamamoto

o-3

E
3

B-3

ii

B-l, 2--I

1 646,681*,771,829

F“,1’, 1

521

467*
469,489

=L

470
480
487
519
534
579*
681*
704
711*
783
796*
825

457,506,513*,

chimpanzee-4
chimpanzee-5

Efimpanrcc_:

526*, 585

621,651*, 719

orangutan-3
orangutan-2

847

orangutan-l

593,651*I

711*. 813*

macaq”ebaboon-1
baboon-2

FIG. 4.-The
estimated phylogenetic
tree for primate ABO blood group alleles based on the phylogenetic network of fig. 3. Numbers oneach branch denote the nucleotide positions in which substitutions occurred. Numbers with asterisks signify nucleotide position in which parallel
substitutions occurred.

sumed to be caused by parallel substitutions, and no link
was produced. This is because it is unlikely for a pairof sequences from different species groups to share amutation by descent. That is, all these cases were assumed to be parallel substitutions.
For example, threehuman 0 alleles (O-2, O-6, and O-7) and three Old
World monkey sequences (macaque, baboon-l,
and baboon-2) share nucleotide A at position 68 1. This pattern
is assumed to be the result of two parallel substitutions,as indicated by an asterisk after the position number.
Let us examine the characteristicsof the networkin figure 3. First of all, sequences for hominoids
andOld World monkeys are clearly distinguished.
There are10 unequivocal
substitutions
accumulated in the branchconnecting
these two groups. Within hominoids, threeorangutan sequences form a distinct cluster; three substitutions are allocated to the branch diverged from thecommon ancestor of the human-chimpanzee-gorilla
lineage and the orangutan lineage. These clusteringsarecompatible
with the established primate phylogeny bymolecular data (e.g., Horai et al. 1995).
Within hominoids,
we now observe some incompatible partitions that are responsible for loops. For example, nucleotide
configurationsat positions 796 and803 are incompatible
with those at positions 5 13 and526 (see tables 1 and 2). These relationships
producedfour consecutive
rectangles connecting human and gorilla sequences in figure 3. We designated the upper rightnode of those two rectangles as the HCG node, because
species-specificclusters of human, chimpanzee, and gorilla alleles can be converged onto this node, and the
HCG node sequence is considered to be the common
ancestor for all the observed alleles of human, chimpanzee, and gorilla.
The Phylogenetic

Tree of Primate

ABO Gene Alleles

Figure 4 is the phylogenetic
tree of the primate
ABO genes based on the phylogenetic network of figure
3. All the parallelograms
that existed in the network
were eliminated by cutting some of those edges. In thecase of the four rectangles around human and gorillasequences, for example, the edge connecting
a gorillasequence (go-1,2,4,5) and a node neighboring the humanB-3 sequence was eliminated, because an extant gorillasequence is unlikely to be identical with an ancestral
sequence of human alleles. The resulting tree of figure
4 is one of 10 equally parsimonious
trees (not shown)that were found by using PAUP with the branch-andbound option. All of the 60 substitution
events wereABO Blood Group Genes

Table 4
Observed

Nucleotide Substitution

Matrix

(4
NEW

C

T

G

SUM

0
3
7

2
13
2

0
2
0
-

2
17
5
24

10

17

2

48

OLD

A

A. . . . . . . . .
C.........
T
G:::::::::

2
2
15

-

Sum

19

(B)
Transversions

Transitions
A-G....
C-T
Sum

....

18
22
40

A-C
A-T
G-C
G-T

....
.. ..
....
....

Sum

2
4
11
3
20

unambiguously
located at one of the branches of thetree.
It should be noted that the topology of the tree in
figure 4 is different from that of Martinko et al. (1993),
where human B allele was clustered with gorilla B alleles. Although this relationshipwas also one of mostparsimonious
trees in our data set, we believe that the
topology of figure 4 is more probable than their tree,according to our argument above on the network of figure 3.
This tree indicates that the common ancestral genefor the hominoid and Old World monkey ABO bloodgroup is A type, and three B alleles evolved independently on the human, gorilla, and baboon lineages.
Those changes correspond to the two amino acid substitutions (266: L+M and 268: G+A; see table 3) thatare responsible
for the change of substrate specificity(Yamamoto and Hakomori 1990).
It has been known that the human ABO-like bloodgroup also exists in nonhumanprimates. Moor-Jankowski, Wiener, and Rogers (1964) summarized
thedata, and they are presented in table 5 in an abbreviatedform. Because orangutan and gibbon both possess A and
Table 5The ABO Blood Groups in Nonhuman
Wiener, and Rogers 1964)
CommonName

Chimpanzee . . . . . . . . . . . . . . . . .
Gorilla . . . . . . . . . . . . . . . . . . . . .
Orangutan. . . . . . . . . . . . . . . . . . .
Gibbon . . . . . . . . . . . . . . . . . . . . .
Baboons....................
Rhesus macaque . . . . . . . . . . . . .
Pigtailed macaque . . . . . . . . . . . .
Java macaque................
Sulawesi crested macaqueC.....
Squirrel monkey . . . . . . . . . . . . .
Cebus monkey. . . . . . . . . . . . . . .

405

B alleles, it is possible that B alleles evolved independently in those lineages too. Macaque species and New
World monkeys also have both A and B alleles. However, this prediction (repeated emergence of the B allele)
awaits the determinationof nucleotide
sequences forthese species in the future.
Because all the nucleotide changes were inferred in
the gene tree of figure 4, directions of changes could bedetermined
except for 12 substitutions
on the branchconnecting hominoid and Old World monkeys. This process involved the reconstructionof the nucleotides in all
the interior nodes, and those reconstructions
are considered to be reliable because the compared sequences areclosely related (see Yang, Kumar, and Nei 1995). It hasenabled us to estimate the pattern of nucleotide substitutions, as shown in table 4. Transitions occurred twotimes as frequently as transversions,so the transition
parameter((x) for the two-parametermodel (Kimura
1980) is roughly four times higher than the transversion
parameter (p). When we consider the direction of substitutions, it is clear that there is a bias toward AT richness; G-to-A and C-to-T changes are much more abundant than A-to-G and T-to-C changes.
Possibilityof Natural

Selection

on ABO Genes

We found unusually large coalescence times for human ABO alleles. It is of interest to compare those withcoalescencetimes for nonhumanprimate ABO genes.
We thus estimated coalescence times of the ABO allelesin each species in figure 4 as follows. The numbers of
nucleotide substitutions
from the most recent ancestor
sequence to the extant sequences in each species werefirst estimated applying Ishida et al.‘s (1995) method.
The resultant values are 0.0072, 0.0085, 0.0023, 0.0132,
and 0.0070 for human, chimpanzee,
gorilla, orangutan,
and baboon, respectively. We then used for the calibration the evolutionaryrate (1.4 X 10-9) based on thehuman-chimpanzee
comparison as used in the previous
section. The results are 2.6, 3.0, 0.8, 4.7, and 2.5 Myrfor human, chimpanzee, gorilla, orangutan, and baboon,
respectively.
It is interesting that not only human, butalso chimpanzee,orangutan, and baboon showed large
coalescence times.

Primates(Adaptedfrom Moor-Jankowski,

Latin Binomena
Pan troglodytes
Gorilla gorilla
Pongo pygmaeus
Hylobates lar
Papio anubis, P. cyanocephalus
Macaca mulatta
Macaca nemestrina
Macaca irus
Macaca nigra
Samimiri sciurea
Cebus albifrans

aNames currently used in primate taxonomy are listed.
b Numbers in parentheses are observed numbers.
c Listed as “Celebes black ape” in Moor-Jankowski, Wiener, and Rogers (1964).

Observed

Phenotypeb

A (113), 0 (17)B (2)A (22), B (I), AB (3)A (2), B (9, AB (4)A (36), B (17), AB (34)B (10)B (5)A (8, B (I), AB (3), 0 (1)A (7), B (2)A (3), 0 (1)B (3)9 0 (1)406

Saitou and Yamamoto

We applied the same method to the 238-bp HOX2
intergenic sequence data of Ruano et al. (1992) to see
if the long coalescence times are special for the ABO
gene. The numbers of nucleotide substitutions
from themost recent ancestor sequence to the extant sequenceswere estimated to be 0.008 and 0.011 for chimpanzeeand gorilla, respectively. The coalescence times are thus
estimated to be 3 Myr for chimpanzee and 3.8 Myr forgorilla. The HOX2 intergenic region is assumed to beunder neutral evolution (Kimura 1983). Although only
short nucleotide
sequences were compared, it is thus
suggested that nonhumanprimates have large coalescence times even for a DNA region under neutral evolution. It is possible that nonhuman primates are highly
geographicallyisolated and this caused the coalescencetimes to be much longer than that for human. Such long
coalescencetimes for nonhumanprimates are a clear
contrast to human, where the nucleotide difference persite between randomly chosen genes of a locus was estimated to be only 0.0004 (Li and Sadler 1991). Thissituation is similar to that of primate MHC genes, where
the coalescence
time of the class II DRBl locus wasestimated to be more than 30 MYA, probably caused bya strong balancing
selection (Takahata 1993a). Therefore, long coalescencetimes estimated for the human
ABO gene both from the complete cDNA region andfrom a partial cDNA region suggest the possibility ofthe existence of some kind of balancing selection at thisgene, at least for human.
Higher rates of nonsynonymous
substitutions
tosynonymous
ones were observed for the antigen recognition sites of human and mice MHC class I and class
II proteins (Hughes and Nei 1988; Nei and Hughes
1991). Those higher rates are considered to be clear evidence of the existence of positive selection on those
MHC genes. We, therefore, estimated numbers of synonymous and nonsynonymous
substitutions between human ABO Al- 1 allele and B- 1 allele by using Nei and
Gojobori’s (1986) method. The ODEN computer package (Ina 1994) was used for computation.
Estimated
numbers of synonymousand nonsynonymoussites outof the complete cDNA sequences are 258.2 and 800.8,
respectively(the initiation codon ATG was eliminatedfrom the comparison, the sum being 1,059). Because thenumbers of synonymousand nonsynonymous
nucleotide
differences between the two alleles are 3 and 4, respectively (see table l), the proportions of synonymous
andnonsynonymous
differenceswere 0.0 116 (= 3/258.2)
and 0.0050 (=4/800.8),
respectively.
The numbers ofsynonymousand nonsynonymous
substitutions
were
thus estimated to be 0.0117 ? 0.0068 and 0.0050 t
0.0025, respectively.
The ratio of nonsynonymous/synonymous substitutions
became 0.43. Takahata (1993b)
estimated this ratio for the human ABO gene, and it
turned out to be 2.0. That comparison
was, however,based on partial ABO sequences (405 bp long) corresponding to the region sequenced by Martinko et al.
(1993) for nonhuman primate ABO genes. Therefore, it
is not clear whether that high ratio can be consideredevidence for the existence of positive selection on the
ABO gene.

Table 6
Goodness of Fit Between Observed and Expected
Numbers of Nucleotide Substitutions for the ABO GenesNUMBERS OF SITESN

Observed

Expected

X2

0 ......
1 ......
2 ......
r3 . . . . . .
Sum . . . . .

363
33
6
3
405

351.8
49.5
3.5
0.2
405.0

0.36
5.50
1.79
39.20
46.85

NOTE.--X = 0.14, P < 0.001 (df = 3). N: number of nucleotide substitutions

persite.

We saw that B alleles evolved independently
atleast three times in primate evolution in the previous
section. To examine whether this phenomenon
is statistically significant or not, we performed a goodness-offit test between the observed and the expected numbersof substitutions per site for the ABO gene based on thetree of figure 4 (table 6). Expected numbers of substitutions were computed applying a Poisson distribution
under the assumption of equal substitution rate at everysite. The mean number (A) of nucleotide substitutions
per site per whole tree was estimated to be 0.14. A highly significant difference (P < 0.001) between observed
and expected numbers was observed (table 6). This difference was mainly caused by unusually high substitutions at the nucleotide sites (796 and 803) responsible
for the functional difference between A and B transferases. Unless those positions are mutational hot spots,
this recurrent occurrence of B alleles does not seem toreflect the pattern of mutations. Because the neutrally
evolving genes are expected to accumulate nucleotidechanges according to their mutation pattern (Kimura
1983), we have to consider the existence of some kindof positive selection on the ABO blood group locus. The
best candidate may be the overdominantselection, forthe emergence of new alleles produces heterozygotes.
Natural selection on the ABO gene has long beenstudied (e.g., Chung and Morton 1961; Hiraizumi 1990).
However, all of the studies were based on nonmolecular
data, and only a short time span was considered to detect
any type of selection. It is now increasingly
clear thatanalysis of long-term evolution is more powerful thanthat of short-term evolution. Therefore, we believe thatour present study, based on the accumulationof mutations over a long evolutionary
time period, opened a
new aspect for the study of natural selection on the ABO
gene.
Phylogenetic
Relationship
Their Related Genesof the ABO Genes andIt has been known that (x- 1,3-galactosyltransferase
genes (hereafter abbreviated as GAL) are homologous
to the ABO genes (e.g., Joziasse 1992). We retrieved
four amino acid sequences translated from GAL gene
nucleotide
sequences (DDBJ/EMBL/GenBank
international nucleotide sequence database accession numbers
were M85153,504989,
L36152, S71333, and JO5175 formouse GAL, bovine GAL, pig GAL, marmoset GAL,ABO Blood Group Genes

marmoset-GAZ
bovine-GAL
pig-GALmuse-GAL
human-ABQA

marmoset-GAL
bovine-GAL
pig-GALmuse-GAL
human-AEM

407

eo
I
MNvKGKvILsML~TvIMWEx NSPJZGSE'LMIYHSKNPEXDDSSA~MNVKGKVILSMLW~
IHsPExxLFwINPsR.NP~ssIQKGwwLP~INSPEGSLFWIYQSKNPEVG-SWM
MNvKGKvIIms~
PEMXNF#QKDJWFPSWFKN____________________------vmmIMLmm
120

61
GIHNYQQEEEMDKEKG~~-----RI?G___~~____~l+~~_____~~
cTHsyHEEEDAIGNm----E-----RPmITRwKAPv
B-----RPDVLnIGTHSYB-----GRXDRIEEPQYGvLSPF!SLMPGSLERG~vREPD~sLP~PKvL~~I
*
*
*
*

*

* * **

L,- 180 amino acid residues with one gap (skipped) -mamlnset-GAL
bovine-GAL
pig-GALmuse-GAL
human-AE!0A

manmset-GAL
bovine-GAL
pig-GALmuse-GAL
human-ABCa

360
301
PIQVINI~GIL~ES~SKILSPEYCWDYHIG-LP
FTQVLNI!lQECFKGILKDKKNDIEAQJHD~LSP~~YHIG-m
PlQVLNI'lQECE'KGILQDIENDIJ3AEMHDES~ILSPEYCWDYHIG-MS
ILSPEXJWDYQIG-LP
FTHILNL~GILQDKKHDI~~~~
vrzvQRLTRAcH~ImmmmP~* ***** ********** * ** * ***** **
I
361
SDIK
ADI&_
VDIR
SDIK
AwVPKNHQAm-

382

*

**

FIG. 5.-A
partial multiple alignment of four o-1,3-galactosyltransferases
and allele A of the human ABO amino acid sequences.
denote invariant amino acid positions, and arrows denote the region of amino acid positions used for phylogenetic analysis.

and human ABO-A, respectively),
and multiple-aligned
them (see fig. 5). We used only the conserved 254 aminoacid positions for the phylogenetic
analysis. There wasonly one amino acid gap in this region.
Figure 6 shows a phylogenetic
tree of this geneconstructed by using the neighbor-joining
method. Amino acid sequences were used, and the product of human
ABO A allele was used as the outgroup. Mouse GAL
sequence and the remaining three mammalian sequences
are clearly separated with 100% bootstrap probability,
while bovine and pig GAL sequences are clustered witha slightly lower bootstrap value (81%). The numbers ofamino acid substitutions
from the common ancestor(node A) of the four mammalian
species to the present
day proteins vary greatly; the smallest value (11) is forthe mouse lineage and the largest one (49) is for the pig
lineage. If we assume the divergence time between orderrodentia and other mammalianorders to be roughly

marmoset GAL-

0mouse GAL

Number of amino acid substitutions

50

FIG. 6.-A
phylogenetic tree for four II- 1,3_galactosyltransferases
based on 253 amino acid residue sequences. The product of the human
ABO A allele was used to locate the root (node A) of the tree. The
neighbor-joining
method was used for Kimura’s (1983) distances.
Numbers on two interior branches are bootstrap probabilities (%).

Asterisks

within the range of 115-129 MYA as suggested by Easteal, Collet, and Betty (1995), the evolutionaryrate of
GAL proteins for those two lineages can be estimatedas follows. Because we consider single lineages, therates of amino acid substitution(per site per year) are
estimated to be 0.34 X 10e9 to 0.38 X 10e9 (=[ 1 l/253]/[ 115-129 MYA]) for the mouse lineage and 1.5 X 10h9
to 1.7 X 10P9 (=[49/253]/[115-129
MYA]) for the pig
lineage. Both rates are within the range of evolutionary
rates for typical proteins (e.g., see Nei 1987).
We also reconstructed
phylogenetic
trees of ABO
and GAL genes using 455-bp-long nucleotide sequence
data (fig. 7). These pseudogenes
and mouse ABO geneare included in this tree. Because mouse sequences separated first among the mammaliansequences in both
ABO and GAL genes, we equated the location of thenodes (Sl for GAL and S2 for ABO in fig. 7) for speciation of the mouse lineage and the other mammalianlineages in the tree. An AI30 pseudogene (hgt4) foundfrom the human genome seems to diverge before therodent lineage diverged, although the bootstrap value(50%) supporting this pattern is relatively low.
Kominato et al. (1992) observed hybridizationofhuman ABO gene with a wide range of mammalian genomic DNA. Human ABO-likeblood groups were
found in various mammals, and it is now clear that probably all the mammalian species have the homologue ofthe human ABO gene. In fact, Yamamoto et al. (unpublished data) found a human ABO homologue from themouse genome (see fig. 7), and Ellegren et al. (1994)
suggested that the pig blood group gene EAA is homologous to the human ABO gene based on the chro-408

Saitou and Yamamoto

I

0

I

I

I

I

1

100
Number of nucleotide substitutions

FIG. 7.-A
phylogenetic
tree for the human ABO gene and its
homologous
nucleotide sequences. The neighbor-joining
method wasused for Kimura’s (1980) distances based on the 455bp sequence data.
Numbers on internal branches are bootstrap probabilities (%). Node D
denotes the duplication event that produced ABO and GAL genes,
while nodes Sl and S2 denote the common ancestors of the rodent
lineage and other mammalianlineage for GAL and ABO genes, respectively.

mosome comparative map between pig and human. Furthermore, the homologue of the hgt4 pseudogene should
also exist in all the mammalian
species genomes, if thetree topology of figure 7 is correct.
We estimated the rate of nucleotide
substitutions
for ABO and GAL genes under the assumption of constancy of the evolutionaryrate after the divergence between the rodent and the other mammalianlineages at115-129 MYA (Easteal, Collet, and Betty 1995). The
average numbersof nucleotide
substitutions
per sitefrom node S 1 to extant functional GAL genes and fromnode S2 to extant functional ABO genes were estimatedto be 0.105 (=47.8/455)
and 0.134 (=61.6/455),
respectively, applying Ishida et al’s (1995) method. Thus,
the rates within the mammalianlineage for ABO andGAL functional genes are estimated to be 1.0 X 10e9
MYA]) and 0.8 X 1O-9
to 1.2 X 1O-9 (=0.134/[115-129
to 0.9 X 1O-9 (=0.105/[115-129
MYA]), respectively.
These evolutionaryrate estimates are somewhat lower
than that (ca. 5 X 10m9) for mammalianpseudogenes(Nei 1987). If we assume the evolutionaryrate of thosegenes to be similar (ca. 1.0 X 10w9) throughout theirevolution, the time of the gene duplication (node D) that
produced ABO and GAL genes is estimated as follows.
Because the branch connecting nodes Sl and S2 in figure 7 was estimated to have 238 nucleotide substitutions
out of 455 nucleotides
compared, the divergence time
between those two nodes is estimated to be 260 MYA(=[238/455]/[2X 1.0 X 10-9]). Thus, by adding thedivergence time estimate (115-129 MYA) of nodes S 1and S2, the divergencetime of the node D from thepresent time becomes 375-389 MYA, or roughly 400
MYA. It seems that the time of gene dupulication
producing ABO and GAL genes may be around the emergence of vertebrates (ca. 500 MYA).
ABO and GAL genes share characteristics
other
than sequence homology. The chromosomal
location of
the human ABO gene has been mapped to 9q34.2, thetip of the long arm of chromosome
9 (Povey et al.

I, Number of nucleotide substitutions

3bFIG. 8.-A
phylogenetic
tree for the primate a-1,3-galactosyltransferase functional genes and its pseudogenes based on the 369-bp
sequence data. Bovine GAL gene was used as the outgroup. The maximum-likelihood
method was used under the assumption of monophylyof New World monkeys.
Bootstrap
probabilities(%) on interior
branches are for the neighbor-joiningtree.

1994). The human GAL gene was also mapped to thesame region (Shaper et al. 1992). Therefore, ABO andGAL genes of many vertebrates may be located on thesame chromosomal
region, if both of them coexist. In
contrast to the close affinity of the chromosomal
locations of those two genes, the intron and exon structuresare somewhat different from each other. Although substantial proportions of the coding regions reside in thelast exons in both genes, the whole coding regions are
dispersed over seven and six exons for the ABO gene
(Yamamoto,
McNeill,and Hakomori1995) and the
GAL gene (Joziasse et al. 1992), respectively, with only
the last two exons being homologous.
Evolutionof Primate

GAL Genes

Partial sequences of several primate GAL genes
were reported by Galili and Swanson (1991), and weconstructed the phylogenetic
trees of the primate GAL
genes based on sequence data of 369-bp region. We firstmade neighbor-joining
and maximum-likelihood
trees,
but New World monkeys did not form a monophyleticcluster in both trees that have the identical topology (notshown). However, the monophyleticclustering of New
World monkeys has been clearly demonstrated by usingmuch more molecular
data (Schneider
et al. 1993).
Therefore, a submaximum-likelihood
tree with the samebranching pattern within the New World monkey species
as estimated by Schneider et al. (1993) was constructed,
and branch lengths were estimated by applying Ishida
et al’s (1995) method (see fig. 8).
All the Catarrhini (Old World monkeys and hominoids) have GAL pseudogenes,
while all the four New
World monkeys examined have functional GAL genes(Galili and Swanson 1991). We estimated the average
number of nucleotide substitutions (per site) between thecommon ancestor (node A of fig. 8) and the extant sequences for functional genes (New World monkey lineages) and pseudogenes (Catarrhine lineages) separately.
Those values were 0.021 and 0.053 for functional genes
and pseudogenes,
respectively. If we assume the divergence time of node A to be roughly 40-50 MYA, therate of nucleotide
substitutions
for functional
GAL

ABO Blood Group Genes

genes and GAL pseudogenes becomes 0.4 X 10e9 to 0.5
X 1O-9 and 1.1 X 10e9 to 1.3 X 10V9, respectively. The
evolutionaryrate for the primate GAL functional geneis slightly lower than that estimated for mammalian
GAL functional
genes based on the tree of figure 7,
while the rate for the pseudogene lineage is more or lessthe same as that (1.4 X 10A9) used in previous sections.
Under the standard framework of molecular evolution, pseudogenes
are considered to undergo neutral
evolution(Kimura 1983). Galili and Andrews (1995)
suggested the following evolutionary
scenario on the
GAL gene inactivation;
an infectious microbial agent
manifesting
the Gal alpha 1,3 Gal carbohydrate epitopewas endemic to the Old World around the speciation of
Catarrhini and Platyrrhini(New World monkeys), andindividuals without functional GAL genes could survive
the endemic due to antibodies against this epitope thatexisted on the cell wall of that infectious agent. Thismeans that individuals with GAL pseudogenes had higher fitness than those with GAL functional genes. It may
be necessary to conduct some experiments
to examine
this interesting
scenario. In this context, it should be
noted that type C retroviruses
produced in the murine
or canine cells manifesting
this Gal alpha 1,3 Gal epitope were shown to be neutralized by the human serum
which contains antibodies against this epitope (Takeuchi
et al. 1996). In any case, it seems that we now have toconsider the evolutionary
outcome of the emergence ofa pseudogene
not only from the neutral viewpoint but
also from the selective viewpoint.

409

to have both anti-A and anti-B antibodies.
If so, theargument of Galili and Andrews (1995) on the GAL
pseudogene may also apply to the ABO gene, because
the 0 allele is by definition nonfunctional.
In fact, it has
been one of the major mysteries of the ABO gene thatthe nonfunctional
0 allele is one of the common alleles.
The 0 allele is even fixed in some South American populations (see table 141 of Roychoudhury
and Nei 1988).
This pattern of allele frequency distribution
is clearlyout of the scope of the standard mutation-driftbalancemodel, where null (nonfunctional)
alleles are expected
to remain rare.
It should be remembered that several glycosyltransferases are involved in the production of complex carbohydrate structures to manifest the blood group antigens. When one transferase happens to lose its function,
the precursor carbohydrate
structure (H determinant in
the case of the ABO blood group system) will accumulate without further modification. Clearly, more studies are needed to grasp the whole story of the evolutionof the ABO blood group gene.
Acknowledgments
The authors thank Dr. Uri Galili for providing uswith various informationon the GAL gene, and Dr.
Hans-Jurgen
Bandelt for consulting on the constructionof phylogenetic
networks. This study was partially supported by grants-in-aid
for scientific studies from Ministry of Education, Science, Sport, and Culture, Japan to
N.S. and funds from The Bumham Institute to EY.

Perspectives
We have presented various phylogenetic
analysesof primate ABO genes and its related genes in this paper.
We found some unusual evolutionary
patterns for the
ABO genes; however, those phylogenetic
analyses inevitably remain to show only indirect evidence for the
possibility of natural selection. More direct evidence isneeded from experimental
studies. For example, BorCnet al. (1993) found that a gram-negative
bacterium Helicobacter pylori, a possible causative agent in gastritis
and gastric ulcers, binds to the carbohydrate
structureLeb, while it does not bind to ALeb determinantantigens. Boren et al. thus suggested that the availability ofreceptors for this bacterium may be reduced in individuals with A and B phenotypes compared to those with
0 phenotype. More of such experimental studies directly
connecting
microorganisms
and specific blood grouptypes will be definitely necessary to clarify the biological mechanismof the existence of the ABO bloodgroup.
Natural antibodies against A and B determinants
are present in individuals who do not express these antigens (Landsteiner’s Law). High titers of these antibodies have been attributed to constant stimulation by bacterial flora in intestines, some of which share the epitopes of A and B antigens (Springer, Horton, and Forbes
1959). If natural antibodies are important for fighting
against parasites, then individuals
with 0 phenotype
may be selectively more advantageousthan those with
A, B, or AB phenotypes, for 0 individuals are expected

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NAOYUKI

TAKAHATA,

reviewing

Accepted December 6, 1996

editor