Molecular Phylogenetics and Evolution 51 (2009) 465–471

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev

Relic of ancient recombinations in gibbon ABO blood group genes deciphered
through phylogenetic network analysis
Takashi Kitano a,1, Reiko Noda a, Osamu Takenaka b, , Naruya Saitou a,*abDivision of Population Genetics, National Institute of Genetics, 1111 Yata, Mishima 411-8540, Japan
Primate Research Institute, Kyoto University, Inuyama 484-8506, Japana r t i c l e

i n f oArticle history:
Received 11 September 2008
Revised 4 February 2009
Accepted 16 February 2009
Available online 17 March 2009
Keywords:
Phylogenetic network
Recombination
ABO blood group
Positive selection
Transspeciﬁc polymorphisma b s t r a c t
The primate ABO blood group gene encodes a glycosyl transferase (either A or B type), and is known tohave large coalescence times among the allelic lineages in human. We determined nucleotide sequencesof ca. 2.2 kb of this gene for 23 individuals of three gibbon species (agile gibbon, white-handed gibbon,
and siamang), and observed a total of 24 haplotypes. We found relics of ﬁve ancient intragenic recombinations, occurred during ca. 2–7 million years ago, through a phylogenetic network analysis. The coalescence time between A and B alleles estimate precede the divergence (ca. 8 MYA) of siamang and common
gibbon lineages. This establishes the coexistence of divergent allelic lineages of the ABO blood group gene
for a long period in the ancestral gibbon species, and strengthens the non-neutral evolution for this gene.
Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction
The human A and B alleles of the ABO blood group gene code forglycosyltransferases, which transfer N-acetylgalactosamine andgalactose, respectively, to a common precursor (Yamamoto et al.,1990, 1995). Two critical sites for the distinction between A and
B activities of the glycosyltransferase have been identiﬁed(Yamamoto and Hakomori, 1990) in exon 7, and these nucleotide
differences were concordant with serological studies (e.g., MoorJankowski et al., 1964). Evolutionary analysis of nucleotide sequences of this gene for great apes and Old World monkeys (Kominato et al., 1992; Martinko et al., 1993) as well as those for humansuggested that this gene is under some kind of positive selection(Saitou and Yamamoto, 1997). Further sequence analyses of thisgene for great apes and Old World monkeys conﬁrmed the uniquefeature of this gene (O’hUigin et al., 1997; Kermarrec et al., 1999;
Kitano et al., 2000; Noda et al., 2000; Sumiyama et al., 2000).
Lesser apes (gibbons) are also known to be polymorphic at the
ABO blood group gene from serological studies (Blancher and Socha, 1997), however, no study was so far conducted at nucleotide
sequence level. We thus determined partial sequences of the ABO
blood group genes for three gibbon species (agile gibbon [Hylobates* Corresponding author. Fax: +81 559 81 6789.
E-mail address: saitounr@lab.nig.ac.jp (N. Saitou).
1
Present address: Department of Biomolecular Functional Engineering, Ibaraki
University College of Engineering, Hitachi 316-8511, Japan.
Deceased.
1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2009.02.023

agilis], white-handed gibbon [H. lar], and siamang [Symphalangus
syndactylus]). Agile gibbon and white-handed gibbon are common
gibbons, and occupy parapatric distribution in Malay Peninsulaand Sumatra Island. Agile gibbons also distribute in the southwest
part of Borneo Island. Siamang is more distantly related from common gibbons, and is sympatric with agile and white-handed
gibbons (see http://www.gibbons.de/main/index.html for theirgeographical distribution map).
The ABO blood group gene is located in autosome (chromosome
9 in human). Recombination or gene conversion events on autosomal DNA sequences may create non-tree structures. We therefore
conducted a phylogenetic network analysis so as to detect any
reticulations, and found ancient recombinations which is important for inferring the history of genes.
2. Materials and methods
2.1. Genomic DNA sequencing
We sequenced a total of 23 individuals from three lesser ape
species (ﬁve agile gibbons, 12 white-handed gibbons, and six siamangs) from a part of exon 5 to a part of exon 7 of the ABO bloodgroup genes (ca. 2.2 kb). Two primers on exon 5 (forward: 50 -ACC
CCC AGC CAA AGG TGC TGA CA-30 ) and on exon 7 (reverse: 50 CGA TGC CGT TGG CCT GGT C-30 ) were used for each PCR. PCR
products were conﬁrmed by 1% agarose gel electrophoresis, puriﬁed using Micro Spin Columns (Amersham Biosciences), and inserted into a vector using TOPO TA Cloning Kit (Invitrogen). At466

T. Kitano et al. / Molecular Phylogenetics and Evolution 51 (2009) 465–471

least three clones for each allele from each individual were sequenced by using BigDye Terminator Cycle Sequencing Kit and
ABI PRISM 377/3100 DNA sequencer (PE Biosystems). Both strands
were read using PCR primers and inner primers. A list of primers
used in this study is available from T.K. upon request.
2.2. Sequence analyses
CLUSTAL W ver.1.8 (Thompson et al., 1994) was used for multiple alignments. Because phylogenetic network analysis was shown
to be quite effective from our previous studies (Saitou and Yamamoto, 1997; Kitano and Saitou, 1999; Kitano et al., 2000; Noda et al.,
2000; Sumiyama et al., 2000), this analysis was used. A phylogenetic network based on a distance matrix was ﬁrst constructedby using the neighbor-net method (Bryant and Moulton, 2004; Huson and Bryant, 2006), then phylogenetic networks based on multiple alignment of nucleotide sequence data were constructedmanually following the procedure of Bandelt (1994) and Saitou
and Yamamoto (1997). Phylogenetic trees were constructed byusing the neighbor-joining method (Saitou and Nei, 1987). Kimura’s (1980) two-parameter method was used to estimate numbersof nucleotide substitutions. MEGA version 4 was used for tree construction (Tamura et al., 2007).
3. Results and discussion
3.1. Nucleotide sequences of ABO blood group genes for three gibbon
species
Genotypes and inferred allele types of each individual determined in this study are listed in Table 1. We found a total of ten,seven, and seven haplotypes for agile gibbon (AG-1–AG-10),
white-handed gibbon (WH-1–WH-7), and siamang (SI-1–SI-7),
respectively. Three agile gibbon haplotypes (AG-1, AG-9, and AGTable 1
Individuals, genotypes, and ABO blood types.
Individual

Genotype

Inferred allele type

Agile gibbon
S-001
S-002*
S-003
S-004
S-005

AG-1(2)/AG-2(6)AG-3(5)/AG-4(6)AG-5(6)/AG-6(6)AG-7(6)/AG-8(6)AG-9(1)/AG-10(1)

AB
BB
BB
BB
AA

White-handed gibbon
S-006*
S-007**
S-008**
S-009*
S-010**
S-297*
S-298*
S-299**
S-300*
S-301*
S-302*
S-303*

WH-1(5)/WH-2(5)WH-1(5)/WH-1(5)WH-1(5)/WH-1(5)WH-1(5)/WH-3(5)WH-4(4)/WH-4(4)WH-7(3)/WH-6(3)WH-6(3)/WH-1(5)WH-1(5)/WH-1(5)WH-7(3)/WH-1(5)WH-7(3)/WH-2(5)WH-7(3)/WH-1(5)WH-7(3)/WH-5(4)

BB
BB
BB
BO
AA
AA
AB
BB
AB
AB
AB
AA

Siamang
S-128*
S-129*
S-130*
S-131
S-132*
S-133**

SI-1(7)/SI-2(7)SI-2(7)/SI-3(7)SI-1(7)/SI-4(7)SI-5(7)/SI-6(7)SI-2(7)/SI-7(7)SI-2(7)/SI-2(7)

BB
BB
BB
BB
BB
BB

Numbers in parentheses for each haplotype designate the cluster ID shown in Fig. 2.
Individuals with double asterisks (**) are homozygotes, while those with single
asterisk (*) are heterozygotes with both haplotypes conﬁrmed to be authentic
through coexistence of haplotypes (see text). Individuals with dagger ( ) consist of
haplotypes from the same cluster.

10) and four white-handed gibbon haplotypes (WH-4–WH-7) wereA type, while all others were B type. Haplotype WH-3 had a sevenbase insertion in exon 7 that produces a frame-shift, and its protein
product is probably nonfunctional O type, though the O phenotype
has not been observed for white-handed gibbon (Blancher and Socha, 1997).
A multiple alignment of variant sites of these sequence data isshown in Fig. 1. We constructed a phylogenetic network of thesegibbon haplotypes and a human A type allele (DDBJ/EMBL/GenBank accession No. AJ536122) as an outgroup, and classiﬁed a totalof 24 haplotypes into seven clusters as shown in Fig. 2. Clusters 1,
2, and 6 consist of only agile gibbon haplotypes, and clusters 3 and
4 consist of only white-handed gibbons. Cluster 5 is mainly formed
with haplotypes observed in white-handed gibbon, but one haplotype (AG-3) was found in agile gibbon. Haplotype AG-3 was identical to WH-1, suggesting hybridization between agile and whitehanded gibbons. In fact, hybrids between these two gibbon species
have been reported in the wild (Brockelman and Gittins, 1984).
Cluster 7 consists of only siamang haplotypes, and it is rather distant from the remaining 6 clusters. We also constructed a distancebased phylogenetic network, and the network showed a similar
pattern (Supplementary Fig. 1).
Because the human A sequence is outgroup to all gibbon sequences, we expect it to be connected to the branch between thetwo clusters (7 for siamangs and 1–6 for common gibbons), according to the established phylogenetic relationship of gibbon species(Sibley and Ahlquist, 1984). However, the human A sequence isconnected to a node inside the common gibbon group (seeFig. 2). This implies some unusual history on the ABO blood groupgene in gibbons, as we will see later.
There are some large reticulations in this network, such as
shown with orange and blue lines. These reticulations suggest
recombination events. However, there is a possibility of artiﬁcial
recombination during PCR. We therefore examined each haplotype.
First of all, there are four and one homozygous individuals in whitehanded gibbon and siamang, respectively (those with double asterisks in Table 1). Therefore, haplotypes WH-1, WH-4, and SI-2 areconsidered to be authentic sequences under no possibility of artiﬁcial recombination. These three haplotypes were also found in heterozygous individuals S-002 (note that haplotype AG-3 is identicalwith WH-1), S-006, S-009, S-298, S-300, S-302, S-128, S-129, and S132. We thus conﬁrm authenticity of another haplotypes found inthese individuals: AG-4 in S-002, WH-2 in S-006, WH-3 in S-009,
WH-6 in S-298, WH-7 in S-300 and S-302, SI-1 in S-128, and SI-7
in S-132 (see Table 1). In this fashion, two haplotypes (AG-3 andAG-4) of agile gibbons, all seven haplotypes (WH-1–WH-7) of
white-handed gibbons, and ﬁve haplotypes (SI-1–SI-4 and SI-7) of
siamangs were conﬁrmed to be authentic haplotypes. The remaining ten haplotypes were found in four agile gibbon individuals (S001, S-003, S-004, and S-005) and one siamang individual (S-131).
Since we will consider recombinations only between seven clusters
shown in Fig. 2 in later sections, individuals with two haplotypes
from the same cluster (those with in Table 1) are not scrutinized
more. This leaves us only one agile gibbon individual, S-001, for thisindividual is heterozygous for haplotypes AG-1 and AG-2 belonging
to clusters 2 and 6, respectively (see Fig. 2).
We therefore carried out an independent PCR for the individual
S-001 using the same PCR primers and direct-sequencing using
haplotype speciﬁc primers. Haplotypes AG-1 and AG-2 were conﬁrmed in this individual (data not shown). Thus, we conclude that
artiﬁcial recombinant sequences were not included in this study. If
recombinations are detected from these sequences, these are natural ones.
Sequence data from this article have been deposited with the
DDBJ/EMBL/GenBank International Nucleotide Sequence Database
under accession numbers AB196674–AB196697.

T. Kitano et al. / Molecular Phylogenetics and Evolution 51 (2009) 465–471

467

1111111111111111111111111111111111111111111111111111111111111111111222222222
11122222222223333333333344444445555555566677777777788889990000011111111111222222222222333444445555555566666666777788888888999000011122
22236701245557778990333445678912358990134567724912444555912894781244603346679999003344556688002015691136688934666778133534455688005056778900
334567329780780487086347393984577098255095072801542189078546824229637440333571236343604041309580353656731658057389362712952959478177402885216
b
b
d
bdcc
b dbcdd dc
b
d
d
d d d
d
d
a
daddaaaaaa

T
Y
P
E

CAGCCGGCCTCATCCGGCCGCTCGGCGGCGCTCCATTGCATTCACTGTAGCGCACCGGGGGCTGCTCTATCCGGAGGCTAACGAGTTCGTTGGCCTGGGTACGTCTCGTTGATGGCGCTCCACGGGGCGCCTCCTAACCGG.......................................................................................................C.................G...................
.................................................................................................A.....C.........TC......G...................
.............................................................G...................................A.....C.................G...................
.....A.................................................................................................C.................G...................
.....A..........................T.........................A............................................C.................G...................
.......................................................................................................C.................G...A...............

B
B
B
B
B
B
B

AG-1 ...........T...........T....TTA.......GGC.TG..................CA.C....TG....A..GT..G.C.....AA..........C....C.........C..G.....G...CTGCGCGTAA
AG-2 ...........T..T.A......T....TTA.......GGC.TG.....T...............C.....G....A...T..G.C....C............C....C.........C..G.......A.C.G.......
AG-3 ........T..T.G.......C.....CTTA...G.CAGGC.TG............A.....CA...C...G....A..GTT.G.....C....T...A....C....C.........C..GT........C.GC......
AG-4 .......T...............T....TTA.TA..CAGGC.TG.....T...............C.....G....A...T..G.C....C........C...C....C.........C..G.....G...C.GC......
AG-5 ...........T.G.........T....TTA...G.CAGGC.TG.....................C.....G....A...T..G.C.................CT...C.........CT.G.......A.C.GC......
AG-6 ...........T...........T....TTA.......GGC.TG.....T...............C.....G....A...T..G.C....C........C...C....C.........C..G.........C.GC......
AG-7 ...........T...........T....TTA...G.CAGGC.TG.....................C.....G....A...T..G.C.................C....C.........C..G.......A.C.G.......
AG-8 .......................T....TTA.......GGCGTG.....................C..CC.A..C.A...T..GAC....C............C....C.........C..G.........C.G.......
AG-9 ...G...........A.......T....TTA.TA....GGCGTG.............A.A..CA.......G....A..GT..G.C.....AA........T.C....C.........C..G.A..TG...CTGCGCGTAA
AG-10 ...G...........A.......T....TTA.TA....GGCGTG.............A.A..CA.......G....A..GT..G.C......A........T.C....C.........C..G.A..TG...CTGCGCGTAA

A
B
B
B
B
B
B
B
A
A

WH-1
WH-2
WH-3
WH-4
WH-5
WH-6
WH-7

........T..T.G.......C.....CTTA...G.CAGGC.TG............A.....CA...C...G....A..GTT.G.....C....T...A....C....C.........C..GT........C.GC......
........T..T.G.......C......TTA...G.CAGGC.TG............A.....CA...C...G....A..GTT.G.....C....T...A....C....C.........C..GT........C.GC......
........T..T.G.......C.....CTTA...G.CAGGC.TG............A.....CA...C...G....A..GTT.G.....C....T...A....C....C.........C..GT........C.GC......
........T..T.G.......C.....CTTA...G.CAGGC.TG............A.....CA..T....GA...A..GT..G.C.................C....C.........C..G.A...G...CTGCGCGTAA........T..T.G.......C.....CTTA...G.CAGGC.TG............A.....CA.......GA...A..GT..G.C.................C....C.........C..G.A...G...CTGCGCGTAA....T.A....T.........C......TTA.....C.GGC.TG..................CA.......G....A..GT..G.C.....AA........T.C....C.........C..G.A...G...CTGCGCGTAA....T.A....T.........C......TTA.....C.GGC.TG.......A..........CA.......G....A..GT..G.C.....AA........T.C....C.........C..G.A...G...CTGCGCGTAA

B
B
O
A
A
A
A

HS

GCA......AG.A....TGTT.GTCTA.T..C...CC..GCGT.TCACG.T.TGTT....A.C.G.T....G.A.AAGGGT.AG.CCTC....T.GA...G.AC.CTACAAGC..TAGC.TG..C...A.TCTGCGCG..A

A

SI-1
SI-2
SI-3
SI-4
SI-5
SI-6
SI-7

5

666666777777777777777777777

Fig. 1. Variant positions excluding gaps of ABO blood group genes from exon 5 to exon 7 for three gibbon species. Dots denote identical nucleotides with the ﬁrst sequence.
Haplotype names are AG (agile gibbon), WH (white-handed gibbon), SI (siamang), and HS (human, AJ536122). ‘‘a”, different sites between A and B alleles among gibbons (twosites in bold [2178 and 2185] are responsible for two amino acid changes that distinguish the A and B glycosyltransferases), ‘‘b”, sites mostly shared by WH-1–WH-5, and AG3, ‘‘c” sites mostly shared by human and siamangs, and ‘‘d” sites shared mostly only by siamangs. Numbers below the human sequence indicated each exon number.

Human A

1~55
2 p subara stit
lle utiol su
bst ns an
itu d
tio
ns

SI-2

SI-1

7

SI-5
SI-6
B

25
54 ,274,5*,79 427*
5,8 ,43
26 0*,,18
64 250*,333,
AG-1
2A
337*
3
c 5 84,3905,56 5,
7

wn

do

R2

193
4,1

d

94

AG-2

WH-6
b 119,245

9* 58*,3,49
5* 69*,
,75
8

*,
87
18 92,
1 1
,
2
8
7
0
,20 85,2204
20 8,21
7
6*, 21 22040
,12
a
03 08,
2
1
3
,
,1
2
43
10 1261 ,184
53
14

up

AG-5
AG-7
AG-4

A

WH-7

WH-4
WH-5

337*,378,492*,510,539,550,
SI-7 1130*,1177,1196,1233,1240*,
1590,1731,1957,2050,2062*

6

3

AG-9
AG-10

SI-3
SI-4

A

AG-6

AG-8

4
A

WH-1
WH-2 WH-3AG-3
BB

5

One nucleotide
substitution

Fig. 2. A phylogenetic network of the ABO blood group genes for three gibbon species and one human allele (DDBJ/EMBL/GenBank Accession No. AJ536122). Numbers on
branches are nucleotide positions responsible for those branches. Numbers with asterisks signify nucleotide positions in which parallel substitutions are inferred. A full circle
shows one haplotype, and its size indicates the number of haplotype copies. R2 is the missing recombinant. The clusters of gibbons are numbered from 1 to 7. See legend of
Fig. 1 for meanings of sites a-d. Each color corresponds to a recombination (see text). Note that a reticulation to show a recombination producing the cluster 2 could not be
observed in the phylogenetic network.

3.2. Polymorphisms in gibbon ABO blood group genes
Nucleotide diversities of agile gibbons, white-handed gibbons,and siamangs were 0.0090, 0.0062, and 0.0010, respectively. Itshould be noted that the nucleotide diversities for the two com-

mon gibbon species, having both A and B type alleles, were extremely high compared to that for the corresponding region ofchimpanzee ABO blood group gene (Kitano et al., 2000; Sumiyama
et al., 2000) and to the overall genomic diversity for the humanpopulation (Sachidanandam et al., 2001). This high diversity of468

T. Kitano et al. / Molecular Phylogenetics and Evolution 51 (2009) 465–471

the ABO blood group gene in gibbons suggests the existence of positive selection on this gene.
The average nucleotide difference (d) between siamangs andtwo common gibbon species was estimated to be 0.0156. The value
was estimated excluding two sites (2178 and 2185) which areresponsible for two amino acid changes that distinguish the A
and B glycosyltransferases, because there is a possibility that somekind of selection operated on the sites. When we estimated d usingsynonymous and intron data, similar values (0.0276 for synonymous sites, 0.0169 for intron, and 0.0178 for synonymous sites
and intron combined) were obtained. If we assume that the divergence time (T) between siamang and common gibbons is 8 MYA
according to Sibley and Ahlquist (1984), the evolutionary rate (d/2T) for this gene region becomes 9.8  1010/site/year. This happens to be quite similar to the one (1.03  109/site/year) for human and chimpanzee where the substitutional difference is0.0123 (Fujiyama et al., 2002) and divergence time was assumed
to be ca. 6 MYA.
3.3. Explanation of recombination and phylogenetic network by using
model data
Let us explain how to infer recombination events from the phylogenetic network using one example shown in Fig. 3. An ancestral
sequence (Anc) produced two descendant sequences (P1 and P2)
by accumulating ﬁve and four substitutions in each lineage, respectively (Fig. 3A and B). A recombination between variant sites 6 and7 then produced two recombinants, R1 and R2 (Fig. 3C). The phylogenetic network for these ﬁve sequences is shown in Fig. 3D. It
should be noted that the horizontal edge of the rectangle inFig. 3D corresponds to upstream sequence difference (sites 1–5),
while the vertical edge to downstream differences (sites 7, 8, 9,
and 12) accumulated from the ancestral sequence to two parental
ones. Because ﬁve substitutions at sites 2, 3, 4, 8, and 12 occurredin the lineage from the ancestor to parental allele P1 and four substitutions occurred at sites 1, 5, 7, and 9 in the lineage from theancestor to parental allele P2, the location of the ancestral se-

A

C

E

111
123456789012----------------Anc ATATCCCGCCTT
P1 AAGCCCCCCCTA
P2 TTATTCGGGCTT111123456789012----------------Anc ATATCCCGCCTT
P1 AAGCCCCCCCTA
P2 TTATTCGGGCTT
R1 AAGCCCGGGCTT
R2 TTATTCCCCCTA
111123456789012----------------Anc ATATCCCGCCTT
P1' AAGCCCCCCCAA
P2' TTATTCGGGGTT
R1' AAGCCGGGGCTTquence is in the middle of the rectangle (see Fig. 3D). After therecombination, three nucleotide substitutions (sites 6, 10, and
11) accumulated to produce present-day sequences P10 , P20 , andR10 from P1, P2, and R1, respectively (Fig. 3E), while the R2 lineage
became extinct. The phylogenetic network shown in Fig. 3F represents the relationship of the three present-day sequences, an outgroup sequence, and the ancestral one. Two parental lineages (P10and P20 ) are located on a diagonal with long external branches,
while R10 has a short external branch and is located on anotherdiagonal with the outgroup sequence. This is the pattern we should
expect if a recombination happened in some ancient time for any
autosomal genes.
3.4. Recombinations in gibbon ABO blood group genes
We are now ready to interpret the phylogenetic network of
Fig. 2 following the scheme shown in Fig. 3F. There are seven clusters and one outgroup (human) in the network of Fig. 2. Because
the recombinant lineage R10 is located opposite to the outgroup sequence in Fig. 3F, cluster 4 (WH-4 and WH-5) may correspond torecombinant lineage in Fig. 2. If so, the two parental lineages
should be clusters 1 (AG-9 and AG-10) and 5 (WH-1, WH-2, WH3, and AG-3), because of their long branch lengths, resembling
P10 and P20 lineages of Fig. 3F. Under this interpretation, the location of the missing (or probably extinct) recombinant lineage R2
is shown in Fig. 2. If so, two edges of the parallelogram formed withclusters 1, 4, 5, and R2 should correspond to upstream and downstream of the crossing over point which created recombinants. In
fact, eight downstream sites designated as ‘‘a” in Fig. 2 indicate
the allelic difference between A and B alleles of gibbons, while
six upstream sites ‘‘b” correspond to the split separating clusters
4 and 5 from the others (see Figs. 1 and 2). Interestingly, not only‘‘a” sites but also all sites on the branch connecting to cluster 5
were in downstream, and not only ‘‘b” sites but almost all sites
on the branch connecting to cluster 1 were in upstream. Thisstrongly indicates occurrence of recombination between ‘‘a” and‘‘b” sites. If there was no recombination, the distribution of substi-

AncB
1,5,7,92,3,4,8,12

P2

D

P1

R2

P1
8,12

Anc

7,9

P2

R1
1,5

F

Outgroup

2,3,4

P1'
11
8,12

Anc
7,9
10

P2'

6
1,5

2,3,4

R1'

Fig. 3. Explanation of a recombination in a phylogenetic network by using model data (A–F). A, C, and E are nucleotide sequences, and B, D, and F are corresponding
phylogenetic networks, respectively. See text for the details.

469

T. Kitano et al. / Molecular Phylogenetics and Evolution 51 (2009) 465–471

tutions on the branch connecting to cluster 1 (seven and one for
upstream and downstream regions, respectively) and on the
branch connecting to cluster 5 (zero and seven upstream anddownstream regions, respectively) should be expected to havethe same proportion. However, the difference between these two
distributions is highly signiﬁcant (P < 0.001, Fisher’s exact test).
We also constructed phylogenetic trees for the upstream and thedownstream regions (Supplementary Fig. 2), and they clearly

A

B
Human A

119,258,369*, P1
459,495,758 1043,1203,1240,
WH-1
1261,1308,1453,
WH-2 369* 1842,2004,2078,
2178,2185,2206

WH-4

WH-1
WH-2

3

545 1289 2232
758 1849 2232

Possible recombination
point: 917 (545-1289)

Possible recombination
point: 1304 (758-1849)

C

D
1Human A

1130*,1177,1196,
SI-5
SI-4 1233,1240,1590,
SI-7
1731,1957,2050,2062*
SI-2
P1

R1
1019,1104,1280*

AG-1
AG-5

2

Cluster 1
Cluster 5
Cluster 2

5545 1289 2232

Possible recombination
point: 917 (545-1289)

6545*,944,1193,
2004,2078,2178,
2185,2206

R1
AG-7
AG-4
AG-2 AG-6

384,395,505,
567

1043,1203,1240, 492,
1261,1308,1453, 1289,
369*
1887,
WH-11842,2004,2078,
2178,2185,2206 2192,
WH-2
2201

Human A

~

P1

AG-10

337,378,492*,510,
539,550

250,
545

P2

SI-6 SI-1 SI-3

25,274,427,430,
795,826

119,258,333,337,
369*,459,495,758

7

1280* AG-9
1516,1849,1864~

61 substitutions

56 substitutions

P2

25,274,427*,
795,826,972,
430*,492*
1289,1516,1849,
AG-10
1280
1864,1887*,AG-9
2192,2201

AG-8

1

Cluster 7
Cluster 1
Cluster 6

7
SI-1
SI-6
SI-3
SI-5
SI-4 1130,1177,1193, Human A
SI-7
1196,1233,1590,1731,
SI-2
1957,2050,2062
P1
56 substitutions

567 944 2232

Possible recombination
point: 756 (567-944)

384,395,
505,567,
972

378,492*,
510,539,
550,944~

E

R1 36,73,1280*
WH-6 741
WH-7

Cluster 1
Cluster 5
Cluster 3

5

4

Cluster 1
Cluster 5
Cluster 425,274, 250,
427,430, 333,
492,795, 337,
826 545~~61 substitutions

1308,1453,
1842,2004,
2078,2178,
1043,1203,
R1
2185,2206
1133
1240,1261
P1
WH-5 1026*
369*

5

1289,
1516, 1280*AG-9
1849,1864 AG-10
P2
1887,
2192,
2201

AG-10 1

25,274, 119,250,258,
427,430, 333,337,369*,
492,795, 459,495,545,
758
826,1289

P2

1849,
1887,
2192,
2201

60 substitutions
and 1 parallel
substitution (1026*)

1

Human A

1280*
AG-9
1516,1864

119,250,258,333,
P2
369*,459,495,758, R1
25,274,427*,430,
1289,1516,
337,545,
1043,1203,1261,
492*795,826
1849,1864,
1240,2004,
1308,1453,1842
2078,2178, 1887,2192, 1280 AG-10
AG-9
2201
2185,2206
369* WH-1

Cluster 7
Cluster 1
Cluster 5
972 1240 2232

Possible recombination
point: 1106 (972-1240)

WH-2

5

1

Fig. 4. A phylogenetic network of the clusters 1, 4, 5 for gibbon ABO blood group genes with the human A allele (A). P1 and P2 show parental nodes of the recombination, andR1 shows a recombinant of P1 and P2. Green and red colored numbers indicate nucleotide changes in the upstream and downstream regions, respectively. Blue colored
numbers indicate nucleotide changes after the recombination. The diagram in the bottom shows the estimated location of the recombination. Phylogenetic networks of theclusters 1, 3, 5 of gibbon ABO blood group genes with the human A allele (B), of the clusters 1, 2, 5 of gibbon ABO blood group genes with the human A allele (C), of the clusters
1, 6, 7 of gibbon ABO blood group genes with the human A allele (D), and of the clusters 1, 5, 7 of gibbon ABO blood group genes with the human A allele (E). Each diagramshows the estimated location of the recombination.

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T. Kitano et al. / Molecular Phylogenetics and Evolution 51 (2009) 465–471

9.8  1010] from the branch P2-cluster 1. By averaging those threeestimates, the time of the recombination that produced the cluster
4 became 2.0 [=(0.7 + 3.5 + 1.7)/3] MYA.
Since cluster 4 was judged as the recombinant lineage, we eliminated the cluster 4 from the phylogenetic network of Fig. 2 andreconstructed the new phylogenetic network (see Supplementary
Fig. 3A). When we constructed a phylogenetic network involving
only the clusters 1, 3, and 5 of gibbon ABO blood group genes withhuman A allele as an outgroup (Fig. 4B), the midpoint (site 917)
was assumed the recombination point. Three and four substitutions (on introns 5 and 6) between R1 and WH-6 and between
R1 and WH-7, respectively, accumulated after the recombination(Fig. 4B), and the recombination time was estimated to be 1.6
MYA [=(((3 + 4)/2)/2232)/9.8  1010] from the branch R1-cluster
3. Six and seven substitutions (on introns 5 and 6) in the upstream
region (917 bp) between P1 and WH-2 and between P1 and WH-1,
respectively, accumulated after the recombination, and the recombination time was estimated to be 7.3 MYA [=(((6 + 7)/2)/917)/
9.8  1010] from the branch P1-cluster 5. Two and one substitutions in the downstream region (1315 bp) between P2 and AG-9
and between P2 and AG-10, respectively, accumulated after therecombination. A substitution on site 1864 was a nonsynonymous
substitution, and was excluded. Therefore, the recombination time
was estimated to be 0.4 MYA [=(((0 + 1)/2)/1315)/9.8  1010]
from the branch P2-cluster 1. By averaging those three estimates,
the time of the recombination that produced the cluster 3 becameca. 3.1 [(1.6 + 7.3 + 0.4)/3] MYA.
Since cluster 3 was also judged as the recombinant lineage, we
further eliminated cluster 3 and reconstructed the new phylogenetic network (Supplementary Fig. 3B). In the same way, three
additional recombination events were observed (Figs. 4C–E), andthe time of recombination was calculated for each event. Allrecombinations were estimated to occur on the ﬁrst half of intron
6 (Table 2).
We present a complex evolutionary scenario based on the above
results as shown in Fig. 5. There are ﬁve recombination events
during the evolution of gibbon ABO blood group genes, spanning

Table 2
Recombinant, parental allele, and range of recombination.
RecombinantParental allele

Range of recombination (midpoint)

Cluster 4
Cluster 3
Cluster 2
Cluster 6
Cluster 5

Clusters 1 and 5
Clusters 1 and 5
Clusters 1 and 5
Clusters 1 and 7
Clusters 1 and 7

758–1849 (1304 ± 546)
545–1289 (917 ± 372)
545–1289 (917 ± 372)
567–944 (756 ± 189)
972–1240 (1106 ± 134)

Note: Locations of exon 6, intron 6, and exon 7 are 563–697, 698–1749, and 1750–
2232, respectively.

showed different branching patterns, conﬁrming the recombination event.
We thus chose clusters 1, 4, and 5 of gibbon ABO blood group
genes and human A allele as an outgroup and constructed their
phylogenetic network (see Fig. 4A). Nucleotide differences in the
upstream region (sites 119–758) divided clusters 4 and 5 fromcluster 1 and human A, while nucleotide differences in the downstream region (sites 1849–2201) divided clusters 1 and 4 fromcluster 5 and human A. The midpoint (site 1304) was assumed tobe the recombination point. One and two substitutions between
R1 and WH-5 and between R1 and WH-4, respectively, accumulated after the recombination (Fig. 4A). These substitutions wereon intron 6, and the recombination time was estimated to be
0.7 MYA [=(((1 + 2)/2)/2232)/9.8  1010] from the branch R1-cluster 4. Four and ﬁve substitutions in the upstream region (1304 bp)
between P1 and WH-1 and between P1 and WH-2, respectively,
accumulated after the recombination. These substitutions wereon introns 5 and 6, and the recombination time was estimated tobe 3.5 MYA [=(((4 + 5)/2)/1304)/9.8  1010] from the branch P1cluster 5. Three and two substitutions in the downstream region(928 bp) between P2 and AG-9 and between P2 and AG-10, respectively, accumulated after the recombination. A substitution on site1864 was a nonsynonymous substitution in exon 7. We excluded
this substitution to eliminate the effect of selection. The recombination time was estimated to be 1.7 MYA [=(((2 + 1)/2)/928)/?
7.1 (6.7~7.7) MYA

5.5 (3.2~7.5) MYA

4.0 (1.2~9.5) MYA

3.1 (0.4~7.3) MYA

2.0 (0.7~3.5) MYA

O allele

WH-3

7

6

5

SI-1 SI-2 SI-3 SI-4 SI-5 SI-6 SI-7 AG-2 AG-4 AG-5 AG-6 AG-7 AG-8

Siamangs

1

2

AG-9 AG-10

AG-1

Agile gibbons

WH-1 WH-2

AG-3

4

3

WH-4 WH-5 WH-6 WH-7

B allele

A alleleWhite-handed gibbons

Fig. 5. An evolutionary scenario of the ABO blood group genes for three lesser ape species. Solid, dotted, and dashed and double-dotted lines indicate A, B, and O type alleliclineages, respectively. Numbers 1–7 in the current plane designate seven clusters of Fig. 2. Five recombination events, scattering 2–7 MYA, are shown by parallelograms. Eachcolor correspond each recombination of Fig. 2 and Fig. 4. Grey truncated lines indicate undetected or extinct lineages.

T. Kitano et al. / Molecular Phylogenetics and Evolution 51 (2009) 465–471

ca. 2–7 MYA. These times were estimated excluding nonsynonymous substitutions. When all substitutions were used to estimate
times, however, similar values were obtained (data not shown), because about 70% of the analyzed region in this study was introns.
Although recombinations in human populations have been reported (e.g., Roubinet et al., 2004), this is probably the ﬁrst reporton very ancient recombinations in primates. Two most ancientones precede speciation of agile gibbons and white-handed gibbons, while they were after speciation (ca. 8 MYA [Sibley and Ahlquist, 1984]) of siamang and common gibbon lineages. Because
estimated times have ranges and hybridizations among gibbons
are not rare, it is not easy to say that these recombinations occurred before or after the speciations, but at least we can conclude
that these recombinations occurred in very ancient times.
If we accept the evolutionary scenario shown in Fig. 5, thedivergence or coalescence time between A and B alleles precede
the divergence (ca. 8 MYA) of siamang and common gibbon lineages. Let us estimate the coalescence time. It becomes 9.6 (range:
8.1–11.2) MYA (=0.0188 ± 0.0030/2[9.8  1010]), based on the
average nucleotide difference (excluding sites 2178 and 2185) between the clusters 1 and 7, since the clusters are not recombinant
alleles in this data (see Fig. 5). When we estimated the averagenucleotide difference between the clusters 1 and 7 using intron
data (0.0182 ± 0.0033), similar value [9.3 (range: 7.6–11.0) MYA]
was obtained. When we estimated the average nucleotide difference using synonymous data (0.0469 ± 0.0177), a much longer
time 24.1 MYA (range: 15.0–33.1) was obtained. This suggests thatthe coexistence of A and B type alleles in the common ancestral
species of siamang and common gibbons, and that A type alleles
might be lost from the siamang lineage.
All these observations indicate existence of positive selection
for the gibbon ABO blood group gene. Saitou and Yamamoto(1997) suggested the existence of some kind of positive selection
at the ABO blood group gene through sequence analysis of human,
chimpanzee, gorilla, orangutan, and some Old World monkeys.
This study on lesser apes further strengthens the positive selection
as common feature of the primate ABO blood group genes.
Acknowledgments
We thank Ms. Yoshimi Noaki for technical assistance. This study
was supported partly by Grant-in-Aid for scientiﬁc research from
the Ministry of Education, Culture, Sports, Science, and Technologyof Japan to N.S. and Grant-in-Aid for scientiﬁc research from Japan
Society for Promotion of Science to T.K. and R.N.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ympev.2009.02.023.
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