J Mol Evol (1999) 49:615–626© Springer-Verlag New York Inc. 1999

Evolution of Rh Blood Group Genes Have Experienced Gene Conversions
and Positive Selection
Takashi Kitano, Naruya Saitou
Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima, 411-8540, Japan
Received: 16 March 1999 / Accepted: 17 June 1999

Abstract. There are two tightly linked loci (D and CE)
for the human Rh blood group. Their gene products aremembrane proteins having 12 transmembrane domains
and form a complex with Rh50 glycoprotein on erythrocytes. We constructed phylogenetic networks of humanand nonhuman primate Rh genes, and the network patterns suggested the occurrences of gene conversions. We
therefore used a modified site-by-site reconstruction
method by using two assumed gene trees and detected 9
or 11 converted regions. After eliminating the effect ofgene conversions, we estimated numbers of nonsynonymous and synonymous substitutions for each branch ofboth trees. Whichever gene tree we selected the branchconnecting hominoids and Old World monkeys showedsignificantly higher nonsynonymous than synonymous
substitutions, an indication of positive selection. Manyother branches also showed higher nonsynonymous thansynonymous substitutions; this suggests that the Rh
genes have experienced some kind of positive selection.
Key words: Rh blood group — gene conversion —positive selection

Introduction
Most genes are evolving under the neutral mutation pressure (Kimura 1983). However, some genes are evolving
through positive selection. For example, Hughes and Nei(1988) showed that the rate of nonsynonymous substitu-

Correspondence to: N. Saitou; e-mail: nsaitou@genes.nig.ac.jp

tion was significantly higher than that synonymous substitution in the antigen recognition sites of the major
histocompatibility complex class I loci. Ina and Gojobori(1994) showed that positive selection operated on antigenic sites of the hemagglutinin 1 gene of human influenza A virus. Positive selection of these types are considered to be caused by interaction between host defense
systems and pathogens.
Blood types were originally 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 shown for the ABO
blood group genes by Saitou and Yamamoto (1997). We
analyzed the Rh blood group gene sequences for seeking
another possibility of positive selection.
The human Rh blood type is one of the major bloodgroup systems and plays important roles in transfusionand clinical medicine. Landsteiner and Wiener (1940)
detected an antibody that agglutinates blood cells fromrhesus macaques, and it was named Rh. This antibody
had a similar feature with the antibody discovered by
Levine and Stetoson (1939) from transfusion incompatibilities. Although the two antibodies had similar specificity, these antibodies were shown to detect distinct antigens. Therefore, the antibody detected by Landsteinerand Wiener was renamed LW. There were historically
two hypotheses about the Rh blood group system; Wiener’s (1943) one-locus theory and Fisher-Race’s) threelinked loci (C, D, E) theory (Race 1944). Therefore, twodifferent nomenclatures for loci and alleles have been used.
Rh polypeptides were observed as phosphorylated
30–32 kD membrane proteins by using SDS-PAGE and616

immunoprecipitation (Moore et al. 1982; Gahmberg
1982). Nucleotide sequences of Rh genes were determined independently by Cherif-Zahar et al. (1990) and
Avent et al. (1990). The Rh blood group system wasshown to be composed of two closely linked D and CE
loci (Mouro et al. 1993) as predicted by Tippett (1986).
In human, D and CE loci are located on chromosome
1p34–p36 (Ruddle et al. 1972; Cherif-Zahar et al. 1991),
and individuals are divided into Rh-positive and Rhnegative according to the presence or absence of the D
antigen. C/c and E/e specificities are distinguished byfour and one amino acid differences, respectively(Mouro et al. 1993). Rh gene products were estimated tohave 12 transmembrane domains through hydropathy
analysis (Avent et al. 1990) and immunological studies
using an antipeptide antibody (Avent et al. 1992).
Nucleotide sequences of Rh-like blood group genes in
nonhuman primates were also reported (Salvignol et al.
1994, 1995; Mouro et al. 1994a). Genomic DNA analysis
by Southern blot using the human Rh genes as probes
have shown that chimpanzee possesses three Rh-like loci
(Salvignol et al. 1993, 1994), though only two types ofgenes for chimpanzee were so far sequenced (Salvignol
et al. 1995). Gorillas carry two Rh-like genes, whereasorangutans, gibbons, Old World monkeys, and New
World monkeys carry a single Rh-like gene (Blancher etal. 1992).
Salvignol et al. (1995) constructed a neighbor-joining
tree of primate Rh blood group genes. If we accept theirtree, three gene duplications and four gene losses must be
assumed. Blancher and Socha (1997) constructed a maximum likelihood tree of primate Rh blood group genes.
Four gene duplications and nine gene losses must be
assumed in their tree. Klein et al. (1997) constructed a
UPGMA tree of primate Rh blood group genes. In theirtree, four gene duplications and five gene losses must be
assumed. Because D and CE loci are tightly linked andthey are quite similar (96.4% nucleotide sequence identity), gene conversions seem to affect the phylogenetic
relationships of those genes. Occurrences of gene conversions and unequal crossing-overs in the Rh loci havebeen detected (e.g., Cherif-Zahar et al. 1994; Mouro etal. 1994b; Beckers et al. 1996; Huang et al. 1996; Kemp
et al. 1996; Carritt et al. 1997), but there has been no
detailed analysis on those events. The first objective of
this study is thus a detailed analysis of the gene conversion events in hominid Rh blood group genes so as toinfer their true phylogenetic relationship. The second objective is the examination of existence of positive selection on the Rh blood group gene. This analysis is possible only after eliminating the effect of gene conversion.

Materials and Methods
Nucleotide sequence data for Rh blood group genes were retrieved
from the DDBJ/EMBL/GenBank international nucleotide sequence da-Table 1.

Rh blood group genes and references used in this study

Genes (accession number)

References

Human D-1 (X63097)
Human D-2 (X63094)
Human D-3 (S57971)
Human D-4 (L08429)
Human D-5 (S78509)
Human D-6
Human cE-1 (M34015)a
Human cE-1 (X54534)a
Human cE-2 (S577967)
Human Ce
Chimpanzee 1-1 [317-IIR] (L37050)
Chimpanzee 1-2 [394-2G]
Chimpanzee 1-3 [211-6E]
Chimpanzee 1-4 [317-IA] (L37049)
Chimpanzee 2 [211-IIF] (L37048)
Gorilla 1-1 [IC] (L37052)
Gorilla 1-2 [IIA2b]
Gorilla 2 [ID] (L37053)
Crab-eating macaque 1 (L37054)
Crab-eating macaque 2
Rhesus macaque (S70343)

Le Van Kim et al. (1992)
Le Van Kim et al. (1992)
Kajii et al. (1993)
Arce et al. (1993)
Huang et al. (1995)
Huang et al. (1996)
Cherif-Zahar et al. (1990)
Avent et al. (1990)
Kajii et al. (1993)
Huang et al. (1996)
Salvignol et al. (1995)
Salvignol et al. (1995)
Salvignol et al. (1995)
Salvignol et al. (1995)
Salvignol et al. (1995)
Salvignol et al. (1995)
Salvignol et al. (1995)
Salvignol et al. (1995)
Salvignol et al. (1995)
Salvignol et al. (1995)
Mouro et al. (1994a)a

Nucleotide sequences of M34015 and X54534 (both human cE-1) are
identical.
Gene names given in square brackets are those used by Salvignol et al.
(1995).

tabase and from published papers. Ten human (Homo sapiens) sequences, five chimpanzee (Pan troglodytes) sequences, three gorilla(Gorilla gorilla) sequences, two crab-eating macaque (Macaca fascicularis) sequences, and one rhesus macaque (Macaca mulatta) sequence
were used in this study (Table 1). All the sequences were complete
cDNA with 1,251 bp.
CLUSTAL W version 1.6 (Thompson et al. 1994) was used for
multiple alignment. Phylogenetic networks were constructed following
the procedure of Bandelt (1994) and Saitou and Yamamoto (1997). To
identify gene conversion events, the site-by-site reconstruction method(Slightom et al. 1987) was used. The original method was based on theparsimony method; however, we used the maximum likelihood method
to determine nucleotide sequences of ancestral nodes. PAML version
1.3 (Yang 1997) was used for the maximum likelihood analysis. We
used Ina’s (1995) method to estimate numbers of synonymous andnonsynonymous substitutions, and statistical tests were performed following Zhang et al. (1997).

Results and Discussion
Gene Conversions Confuse the Rh Gene Tree
Because the two Rh loci are tightly linked, it is possiblethat they have experienced gene conversions or crossingovers. Those events can confuse the phylogenetic relationship of the linked loci. We thus constructed phylogenetic networks for human, chimpanzee, and gorilla Rh
blood group genes. Because the network for the whole
sequence data has many dimensions, we constructed networks for five regions of the coding region of this gene.
For simplicity, we selected one allele of each locus byexamining phylogenetic networks of human and617

Fig. 1. Phylogenetic networks for hominoid Rh blood group gene exons 1, 2, and 3 (A); exons 4 and 5 (B); exon 6 (C); exon 7 (D); and exons
8, 9, and 10 (E). Numbers are nucleotide positions responsible for corresponding edges, and edge lengths are proportional to number of nucleotide
differences. Full circles denote observed sequences. Hu, Ch, and Go mean human, chimpanzee, and gorilla, respectively.

chimpanzee Rh blood group genes (figures not shown).
We thus used human D (a consensus sequence of human
D alleles), human CE (human cE-1 in Table 1), chimpanzee 1 (chimpanzee 1–3 in Table 1), chimpanzee 2,
gorilla 1 (gorilla 1-1 in Table 1), and gorilla 2 genes forthe following analyses. Nucleotide identity between human D and human CE, that between chimpanzee 1 andchimpanzee 2, and that between gorilla 1 and gorilla 2
were 96.4%, 95.9%, and 97.0%, respectively.
Figure 1 shows phylogenetic networks for exons 1–3(A), exons 4–5 (B), exon 6 (C), exon 7 (D), and exons
8–10 (E). All the networks contain parallelograms thatsuggest parallel substitutions or some kind of convergent
changes. If the relationship of genes is not affected bygene conversion and/or crossing-over, the network may
not contain so many parallelograms. Moreover, somesites of those parallelograms in Fig. 1 are contiguous(e.g., 391, 397, and 399 of Fig. 1A), suggesting the existence of conversion-like events.
Because chimpanzee possesses three Rh-like loci, atleast two gene duplications occurred in the hominoid
lineage. There are three possible gene trees under thissituation (Figs. 2A–2C). Figure 2A assumes that the firstgene duplication occurred in the common ancestor ofhuman, chimpanzee, and gorilla, and the second gene

duplication occurred on one duplicated gene of the chimpanzee lineage. Figure 2B assumes that one gene duplication occurred in the common ancestor of human, chimpanzee, and gorilla, and the other gene duplication
occurred in one duplicated gene of the common ancestorof human and chimpanzee. Figure 2C assumes that twogene duplications occurred in the common ancestor ofhuman, chimpanzee, and gorilla. The established phylogeny (human and chimpanzee are clustered first) for thethree species (e.g., Horai et al. 1995) is adopted for eachorthologous gene group.
To identify orthologous genes, we first classified sites
based on phylogenetic networks (Table 2). For example,
sites 380 and 383 of network A (Fig. 1) divide the humanD–chimpanzee 1 pair from the remaining genes. Because
two genes from the same species can no longer form aclosest cluster in either trees, sites indicating those clusters are not shown.
If we assume tree 2A, two genes of chimpanzee (␣-1and ␣-2) are probably quite similar to each other, and it
is not easy to identify them. Because the nucleotide identity between chimpanzee 1 and chimpanzee 2 sequences
is similar to that between human D and human CE andthat between gorilla 1 and gorilla 2, we assume that these
two genes probably correspond to chimpanzee ␣-1 (or618

Fig. 2. Three model trees (A–C)
and two assumed trees (D and E).
We must assume that at least twogene duplications (represented bydiamonds) occurred in the hominoid
lineage because chimpanzee
possesses three Rh-like loci (trees
A–C). Names of genes in thesemodel trees are arbitrary. Twoassumed trees (D and E) were usedin the following analyses. Names ofgenes in these trees are actual ones.
Gene names in parentheses indicate
undetected or deleted ones. The
cluster including chimpanzee 2 andgorilla 1 is named the D2 locicluster in tree E.

Table 2. Classification of sites to identify orthologous genes fromphylogenetic networks

PairsaNo. of
sites

Sites

Networkb(Hu D, Ch 1, Go 2)–(Hu CE, Ch 2, Go 1)(Hu D, Ch 2, Go 2)–(Hu CE, Ch 1, Go 2)(Hu D, Ch 1)–(others)

3514, 544, 733

B1
6(Ch 2, Go 1)–(others)(Ch 1, Go 1)–(others)42(Hu D, Go 2)–(others)(Hu CE, Ch 1)–(others)(Hu CE, Ch 2)–(others)(Hu CE, Go 1)–(others)

1
1
1
1

577
380, 383
916, 932
985, 986
541, 579, 581, 584
852
1122
1048
505
1170
1025

B
A
C
D
B
C
E
D
B
E
D

a
b

Hu: human, Ch: chimpanzee, Go: gorilla.
See Fig. 1 for networks A–E.

␣-2) and ␤, not chimpanzee ␣-1 and ␣-2. In this case we
can extract orthologous trios from phylogenetic networks. Because sites 514, 544, and 733 show clusters ofhuman D–chimpanzee 1–gorilla 2 and human CE–
chimpanzee 2–gorilla 1, and the other 13 sites (380, 383,
916, 932, 985, 986, 541, 579, 581, 584, 1,048, 1,170, and1,025) are compatible with these clusters, we determined
the topology of hominoid Rh blood group genes as tree
D in Fig. 2.
When we consider either trees 2B or 2C, we have toassume two and three gene losses (or genes not yet identified), respectively. Because we don’t know whichgenes are lost (or genes not yet identified), examination
under trees 2B or 2C is more difficult than that under tree
2A. Because the cluster for human D and chimpanzee 1

is supported with six sites (380, 383, 916, 932, 985, and986), and the cluster for chimpanzee 2 and gorilla 1 is
supported with four sites (541, 579, 581, and 584), theseclusters are plausible. Moreover, three sites (514, 544,
and 733) show clusters of human D–chimpanzee 1–gorilla 2 and human CE–chimpanzee 2–gorilla 1. Therefore
we can choose the unrooted topology as follows: ([(human D, chimpanzee 1), gorilla 2], [human cE, (chimpanzee 2, gorilla 1)]). To determine the root, we eliminatedsites indicating two genes from the same species form acluster from the multiply aligned sequence data, and constructed a neighbor-joining tree (Saitou and Nei 1987)
using CLUSTAL W of Thompson et al. (1994). Three
sequences of Old World monkeys were used as outgroups. The topology of the NJ tree was compatible withtree E of Fig. 2. The maximum likelihood analysis using
NucML of Adachi and Hasegawa (1994) also supported
this topology. It is interesting to note that the trees D and
E both showed the same cluster of human D–chimpanzee
1–gorilla 2. Only the position of human CE is different
between the two trees. Because four sites (541, 579, 581,
and 584) in the network B of Fig. 1 supports clusters ofchimpanzee 2 and gorilla 1, tree E seems to be moreplausible than tree D. Recently, Apoil and Blancher (personal communication) studied the evolutionary relationship of the primate Rh genes using the intron 4 sequences. They suggested that two gene duplications
occurred in the common ancestor of human, chimpanzee,
and gorilla. This scenario is compatible with our tree E of
Fig. 2. However, tree E requires three gene losses (orgenes not yet identified) compared to one gene loss fortree D, and it is more parsimonious. We thus used those
two assumed trees for the following analyses.
We used the site-by-site reconstruction method to
identify regions of gene conversion events assuming the619

Fig. 3. Explanation of the site-by-site reconstruction method. To
classify variant sites, we used five symbols. Tree D of Fig. 2 wasassumed. In case A, the root nucleotide is G, thus variants are chimpanzee 1 and 2 sequences. These variants are caused by shared nucleotide changes (form G to C) between the duplicated genes in a single
species, suggesting a gene conversion in chimpanzee over this site, andthey are presented by P. In case B, the root nucleotide is A and changes
from A to T occurred in the chimpanzee 1 and the gorilla 1 lineages.
These nucleotide changes are not shared by the duplicated genes of thesame species, thus, they are true parallel substitutions, and designatedby S. In case C, the root nucleotide is T and a change (from T to A)
occurred in the common ancestor of orthologous human CE, chimpan-

zee 2, and gorilla 1 sequences. This change is not shared with its
paralogous counterparts, and is indicated by S. There are no changes in
its descendants, suggesting no gene conversion after the hominoid divergence over this site. Therefore, we give O to those branches. Case
D is a kind of sequel to case C, and additional nucleotide changes
occurred in the human D and chimpanzee 2 genes. These additionalchanges resulted in the same nucleotide with their paralogous counterparts, and it suggests that directions of gene conversions were from thehuman CE to the human D genes and from chimpanzee 1 to chimpanzee 2. Therefore, Q is given to the human D gene lineage and R is given
to the chimpanzee 2 gene one.

two trees (Figs. 2D and 2E). First, we obtained the maximum likelihood estimates of the nucleotide sequences ofancestral nodes by using PAML program (Yang 1997),
and then substitution patterns were plotted on eachbranch of the assumed tree. Let us explain the actual
procedure of the site-by-site reconstruction method using
Fig. 3, where tree D of Fig. 2 was assumed. The two
chimpanzee loci both had the same nucleotide (C) in caseA, and the event causing this change is indicated by theletter P. They look like parallel substitutions, but thispattern could also be produced by a gene conversion
after a substitution. In case B, two independent substitutions designated by S occurred in different species. In
case C, a branch of one cluster experienced a substitution(indicated by the letter S) after the gene duplication, andno change in its descendants is indicated with O. Case D
is a next step of case C. Substitutions occurred in thedescendants. These additional changes result in the same
nucleotide with their paralogue, suggesting a gene conversion from human CE to human D and a gene conversion from chimpanzee 1 to chimpanzee 2 over this site.
Therefore, letter Q is given to human D and the letter R
is given to chimpanzee 2. If cases A or D are contiguous
for two or more variant sites, a gene conversion event
over the region containing those sites is inferred based on
the maximum parsimony principle. In case D, we caninfer the direction of gene conversion. We also per-formed the same procedure under the assumption of tree
E of Fig. 2.
Let us compare rates of gene conversion with nucleotide substitutions to see if our parsimonious argument isvalid. Hogstrand and Bohme (1998) estimated cis geneconversion frequency between Abk and Ebk, and thatbetween Abd and Ebd in mouse MHC class II genes, andobtained values 1.2 × 10−6–9.7 × 10−5 per locus (about
200 bp region) per generation. Rates of synonymous substitutions in various mammalian protein-coding genes is
estimated to be 3.5 × 10−9 per site per year (Li 1997). If
we assume that the generation time of wild mice is halfa year (Tsuyoshi Koide, personal communication), therate of cis gene conversion frequency becomes 2.4 ×
10−6–1.9 × 10−4 per 200 nucleotide sites per year, and thecorresponding rate of synonymous substitution is 7.0 ×
10−7 per 200 nucleotide sites per year. It suggests that therate of gene conversion seems to be one to three orders
higher than that of nucleotide substitutions. Of course,
the rate of gene conversion may depend on the copy
number of corresponding gene loci in a genome andsequence similarities among corresponding genes. Furthermore, we cannot directly compare rates between
nucleotide substitutions and gene conversions. However,at least we can say that the rate of gene conversion seems
to be much higher than that of nucleotide substitutions.
This justifies the site-by-site reconstruction procedure.

620
Table 3.

Patterns of site changes for each branch of tree D estimated from the maximum liklihood analysis

S
i
t
e

1 11 111111122222222333 33333333 33 3 3 3 44444444 444 444555 555555 5 55 5 55 5 5556 6666 6 66666666777
3 4 7 8 9 9 0 03 557789900011347002 55566678 88 9 9 9 12344555 678 899001 122234 4 77 7 88 8 8990 0112 3 66778889113
1 8 4 6 7 9 2 58 016863513714976476 37816790 23 1 7 9 08967057 536 867594 705631 4 47 9 01 4 5452 4123 7 57265697293

MPUa

>
<
>
<
>
1 1 1 1 1 1 1 11 111111111111111111 11111112 12 1 3 3 11111111 111 111111 111111 1 11 2 12 2 1111 1111 1 11111111111
<

>

<

H–D
OP
P
P
S
O OOOOPSPO SO
H–CE Q PPS SP SS
S
QQQP
P
SSO
S S
S S
SS
1 C–1
O
S S
S
S S
P
S O OOOO
S
S
S
S
O
Q
O
s C–2
S
S
P
O S S S
SS SS
O
t G–2
S
S OS
O
G–1
SO
S
S S SS SOSSHC–D SS SSSSOO
M HC–CEOO
L HCG–DSS
HCG–CESSOH
S SS
S SSSSSS SS S S S S SSSS SSS SS S S SSSSSSSS SSSS
S S SSSS S S
SSS S
H–D
H–CE
2 C–1
n C–2
d G–2
G–1
HC–D
M HC–CEL HCG–D
HCG–CE
OH
H–D
H–CE
3 C–1
r C–2
d G–2
G–1
HC–D
M HC–CEL HCG–D
HCG–CE
OH

P
P
O

R

S
S R RROOO O OOOO O OO OOOO O O

S S

S

SS SS

O O
O O SSQ Q PPO O PP

S

RR

O
O
O
O
O
O
S
S

O
Q
S

OO
QQ
OO
S
OO
SS

Table 3 shows estimated substitution patterns of all
the variable sites under tree D of Fig. 2 by using thesite-by-site reconstruction method. Ancestral sequenceswere estimated by using the maximum likelihood
method. According to likelihood values, patterns are arranged from the top to the bottom. We inferred regions ofgene conversion events from these results. For example,
sites 31–102 are inferred to have experienced a geneconversion because letters P and Q were contiguous. Human D and chimpanzee 1 sequences are identical on site
31 (indicated by O) and human CE is indicated by Q,
thus, the direction of this gene conversion is inferredfrom the human D gene to the human CE gene.
Two possible patterns were inferred for sites 579–
584. One pattern was that three parallel substitutions occurred in chimpanzee 2 and gorilla 1 genes on sites 579,
581, and 584, and one substitution occurred in the gorilla
1 gene on site 580. Another pattern involved three substitutions in the ancestral CE gene of human, chimpan-

zee, and gorilla, followed by three backward substitutions in human CE on sites 579, 581, and 584, and one
substitution in gorilla 1 gene on site 580. Either patterns
require seven substitution events. If we assume a geneconversion on this region on the latter case, however,
only five events are necessary: three substitutions in theancestral CE gene of human, chimpanzee, and gorilla;
one gene conversion in human CE; and one substitution
on the gorilla 1 gene on site 580. Therefore, we inferredthat a gene conversion occurred on this region.
If the total numbers of events (substitution and geneconversion) is decreased by taking into account the geneconversions, we selected that pattern in spite of lower
likelihood estimation of ancestral nodes as shown in theabove example (Table 3). All other gene conversion regions were inferred in the same fashion. Table 4 shows
substitution patterns of each variable sites under tree E of
Fig. 2 by using the same procedure as in the case for
Table 3.

621
Table 3.
SiteContinued
111 11 111 11111 1 1 1 11111 111111 1111111 11 1111 111 11 1111
7777 77 8 88 8 8 888888 88888889 9 99 999 99 9999999 9 999000 00 000 00000 0 0 0 00000 000001 1111111 11 1111 111 11 2222
4456 89 0 00 1 1 234445 56667791 3 33 446 67 7777788 8 899002 33 444 45555 5 6 6 66667 779990 0000222 22 2233 367 89 0112
4604 77 0 38 5 9 144572 94563716 2 45 178 90 2467905 6 927475 59 368 91237 9 0 1 23790 572360 1568012 34 8902 710 43 5785
< >

MPUa

<

>

<

>
< >
< >
< >
<>
1111 11 1 11 1 1 111112 11111111 1 11 111 11 1111111 1 111111 11 112 11113 3 1 1 11111 111111 1111112 11 1111 112 12 1111

H–D
O OS QQ
OO
O O
S OO
O
S
P
O
H–CE S OSO
OO
S
Q S
SS
SOOOO
P
O O
1 C–1
O O S
S
OOS
O O
OO
O P P
QQQQ
S
S SS S
O
s C–2
O O
OO
SS
Q
Q P P
OOOO
S
O OS
t G–2
S S S
S
P
OS
S
P P
S
G–1
S SS
P
S
S
P P S
S S
S
HC–D
P P
SS
S S
SS
O
S
M HC–CE
P P
SS
SSSS
S S
L HCG–DS
HCG–CEOHSS
S S S SS S SSSSSS SSSSSSSS S S
SSS S SSSSS SS S SSSSSSS SSS SS SSS
H–D
H–CE
2 C–1
n C–2
d G–2
G–1
HC–D
M HC–CEL HCG–D
HCG–CE
OH
H–D
H–CE
3 C–1
r C–2
d G–2
G–1
HC–D
M HC–CEL HCG–D
HCG–CE
OH

O
O
O
O
O
Q
O

Q
O
O
S

O
Q
P
P
O

O

O

O

O

SS

R
PP
PP

S
S

S

O
O
O
O
O

Q O
O Q
R
O
S O

O
Q
S

S

O O
O O

O

O P
Q P
O S

S

S

S

O
O
R
O
O

O
Q
R
O
O

O
Q
S

O
S
S

OO
RR
OO
QQ
OO
SS

O
O
S
O
O
O
Q
S

O
O
O
Q
S
S

Vertical lines divide each exon. Accoding to likelihood values, patterns are arranged from top to bottom.
a
MPU means ML patterns used. Five patterns (O, P, Q, R, and S) listed in thistable are explained in Fig. 2 and text.
Equal signs surrounded with angled brackets designate regions of gene conversions.

H: human, C: chimpanzee, G: gorilla, HC: common ancestor of human andchimpanzee, HCG: common ancestor of HC and gorilla, OH: the branch between
hominoids and Old World monkeys.

Table 5 shows the inferred gene conversion events in
the hominoid phylogeny. Eleven and nine events were
estimated for trees D and E of Fig. 2, respectively. The
direction of gene conversion was not identified in somecases. Because we cannot detect break points of geneconversions and analyzed only cDNA sequences, theseranges of gene conversions are minimum estimates.

example, nucleotides of sites 31–102 of the human CE
gene were substituted to those of the CE gene of thecommon ancestor of human and chimpanzee because of
the direction of gene conversion (D → CE). As directions of gene conversions five and nine for trees D and E
and gene conversion six for tree E were not determined,
the sequence data of those sites were not used in the later
analysis.
Figure 4 shows two phylogenetic trees (A and B) for
the reconstructed primate Rh blood group gene sequences assuming tree D (Fig. 4A) and tree E (Fig. 4B),
respectively. All the branch lengths were estimated fromsequence data of extant nodes and ancestral nodes estimated above, and the midpoint rooting was used. Num-

The Phylogenetic Tree and Synonymous/
Nonsynonymous Substitutions
We reconstructed the two sets of nonconverted sequences from the results shown in Tables 3 and 4. In thecase of gene conversion 1 for tree D (see Table 5), for622
Table 4.

Patterns of site changes for each branch of tree E estimated from the maximum liklihood analysis

S
i
t
e

1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 33 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7
3 4 7 8 9 9 0 0 3 5 5 7 7 8 9 9 0 0 0 1 1 3 4 7 0 0 2 5 5 5 66 6 7 8 8 8 9 9 9 1 2 3 4 4 5 5 5 6 7 8 8 9 9 0 0 1 1 2 2 2 3 4 4 7 7 7 8 8 8 8 9 9 0 0 1 1 2 3 6 6 7 7 8 8 8 9 1 1 3 4 4
1 8 4 6 7 9 2 5 8 0 1 6 8 6 3 5 1 3 7 1 4 9 7 6 4 7 6 3 7 8 16 7 9 0 2 3 1 7 9 0 8 9 6 7 0 5 7 5 3 6 8 6 7 5 9 4 7 0 5 6 3 1 4 4 7 9 0 1 4 5 4 5 2 4 1 2 3 7 5 7 2 6 5 6 9 7 2 9 3 4 6

MPU

>
<>
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1<><

H–D OR
P
R
S
O OO
P
SP
O
O S
O
H–CE Q
P
S
Q
P
P
SS
S S
S S
SS S
1 C–1
O
S S
S
S S
P
S O OO
S
S
S O S
O
Q
O
s C–2
S
P
OO
S S O OO OO
O
t G–2
S
S
QQ
O
S OS
O
G–1
OO
S
O OOSOOOS S
HC–D SS SS
OOO
M HCG–DSS
S
L CG–D2
SS
S
SS SS
S
D–D2
S
S SS
S
S
OH
SS
S SS SS S S S S SS SSSS SSSSSSSSS SSS S S
S S SSSS S
SSS S S
H–D
H–CE
2 C–1
n C–2
d G–2
G–1
HC–D
M HCG–D
L CG–D2
D–D2
OH

OO
OO

P
P
O

S
O
O

SS

S
S

SS

H–D
H–CE
3 C–1
r C–2
d G–2
G–1
HC–D
M HCG–D
L CG–D2
D–D2
OH

O
Q
S

S

Fig. 4. The two possible genetrees (A and B) for the primate Rh
blood group genes after eliminating
the effect of gene conversions,assuming tree D and tree E of Fig.
2, respectively. Diamonds designate
gene duplications. Numbers of
nonsynonymous and synonymous
substitutions per sequence perbranch are presented above eachbranch before and after slash,respectively.

bers of synonymous and nonsynonymous substitutions
were estimated by Ina’s (1995) method. In this method,
the proportion (R) of transition/transversion of the third
codon is estimated from proportions of base changes thatare observed in the entire phylogenetic tree. R values fortrees D and tree E were estimated to be 1.79 and 1.87,
respectively. Numbers of nonsynonyomous substitutions
are higher than those of synonymous substitutions on

almost all branches of both trees (data not shown). Application of Fisher’s exact test (Zhang et al. 1997)
showed that the nonsynonymous substitution was significantly higher than synonymous ones (p ⳱ 0.0041) in the
branch that connected hominoids and Old World monkeys for tree A of Fig. 4, where 79 nonsynonymous and
14 synonymous substitutions were estimated. We also
considered an alternative, less likely substitution patterns

623
Table 4.
SiteContinued
11 11 111 1111 1 11111111 111111111111111 11111 11 11 1111
77 77 8 8888888888888889 9 99 999 99 9999999 9 99900 00 000 0000 0 00000000 000001111111111 11111 11 11 2222
56 89 0 0011344556667791 3 33 446 67 7777788 8 89902 33 444 4555 5 56666667 779990000022222 22333 67 89 0112
04 77 0 3859457294563716 2 45 178 90 2467905 6 92745 59 368 9123 7 90123790 572360156801234 89027 10 43 5785
< >

MPU

<>

<

>
< >
< >
<>
11 11 1 1111111111111111 1 11 111 11 1111111 1 11111 11 112 1111 3 31111111 111111111111111 11111 12 11 1111

H–D
S PP
OO
O O
S OO
S
O
H–CE
S
PP
Q S
S
S
SS
1 C–1
S
S
OOS
O O
OO
Q P
P PPP
S
S SSSQ
O
s C–2
SS
SS
Q
O P
P PPP
S
O
S
S
t G–2
P P
SSS
S
P
S
S
P P
P
G–1
P P
S SS
P
S S
S
P P S
S S O
S
P
HC–D
SS
S S
SS
S
M HCG–D P P
L OG–D2
S
S
D–D2
S
S S
SS
S
OH
SS S
S SSS SSSSS SSSSSSSSS
SSS
SSSS SS S SSS S SSSSS S S SSS
H–D
H–CE
2 C–1
n C–2
d G–2
G–1
HC–D
M HCG–D
L CG–D2
D–D2
OH

O
Q
O
Q

O
Q
O
Q

O

O

O P
Q P
O
O
O
S
S

R

SS

P
P

S

S S

O

PP
PP

S

O
S

S

O O
O O
S
O O
Q
O S
S
S

S

H–D
H–CE
3 C–1
r C–2
d G–2
G–1
HC–D
M HCG–D
L CG–D2
D–D2
OH

S
OO

O
Q
R

S
P
P
S

RR
OO
QQ
OO
SS

O
O
S

O
O
O
Q
S
S

S
S

Designations are the same as those in Table 3.
CG-D2: common ancestor of chimpanzee 2 and gorilla 1 genes, D-D2: common ancestor of HCG-D and CG-D2.

Table 5.

Gene conversion events occurred in the hominoid phylogeny
Tree Db

ID

Brancha

Exon

Sites

1
2
3
4
5
6
7
8
9
10
11

HC-Human
HCG-Gorilla
HC-Human
HC-Human
HCG-HC
HC-Human
HC-Chimpanzee
HCG-Gorilla
HCG-Gorilla
HC-Human
HCG-Gorilla

Exon 1
Exon 3
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 7
Exon 8
Exon 8
Exon 931–102
380–399
391–457
579–584764–797808–852
1,039–1,061
1,057–1,059
1,075–1,093
1,122–1,124
1,170–1,193abTree Eb
Direction
D ➡ CECE ➡ D
D ➡ CED ➡ CE
UndeterminedCE ➡ DCE ➡ D
D ➡ CE
UndeterminedCE ➡ D
D ➡ CE

HC: common ancestor of human and chimpanzee, HCG: common ancestor of HC and gorilla.
See Fig. 2.

Sites
48–102
397–399
391–457—
764–797808–815
1,039–1,061
1,057–1,059
1,075–1,093—
1,170–1,193

Direction
CE ➡ DD2 ➡ D
D ➡ CE—
Undetermined
Undetermined
D2 ➡ D
D ➡ D2
Undetermined—
D ➡ D2

624

indicated in Table 3 for this branch. Numbers of nonsynonyomous and synonymous substitutions for the
branch became 76 and 14, respectively, but the difference was still highly significant (p ⳱ 0.0067). The samebranch that connects hominoids and Old World monkeys
was also significant (p ⳱ 0.0031) for tree B of Fig. 4,
where 80 nonsynonymous and 14 synonymous substitutions were estimated. We also considered an alternative,
less likely substitution patterns indicated in Table 4.
Numbers of nonsynonyomous and synonymous substitutions for the branch became 73 and 14, respectively, andthe difference was still statistically significant (p ⳱
0.0100). In any case, whichever gene tree we selected,
we found the possibility of positive selection in the
branch that connects hominoid and Old World monkey
clusters.
We also estimated the total numbers of nonsynonyomous and synonymous substitutions for hominoid
branches to examine the overall evolutionary pattern
within hominoids. Nonsynonymous substitutions were
not significantly higher than synonymous ones in hominoids for tree A (p ⳱ 0.1055), where 96 nonsynonymousand 27 synonymous substitutions were estimated, nor fortree B (p ⳱ 0.0889), where 97.5 nonsynonymous and27.5 synonymous substitutions were estimated. Because
these values were estimated from nonconverted sequencedata, however, we also considered the situation that allobserved sequence data were results of parallel substitutions. The number of nonsynonymous substitutions (132)
now became significantly higher than that of synonymous ones (30) (p ⳱ 0.0046) within hominoids for tree
A. The nonsynonymous substitutions for tree B (124.5)
was also much higher than synonymous ones (30.5) inhominoids (p ⳱ 0.0117). Therefore, estimates of nonsynonymous substitutions after eliminating effects ofgene conversion are probably underestimation.
We also estimated the average rates of synonymousand nonsynonymous substitutions for the Rh blood groupgene, under the assumption of constancy of the evolutionary rate and the divergence between the Old World
monkey and hominoid lineages to be 23 million years
ago (MYA) (Kumar and Hedges 1998). Average numbers of synonymous and nonsynonymous substitutions
per site between Rh blood group genes of Old World
monkeys and those of hominoids were estimated to be
0.068 and 0.124, respectively, for tree A of Fig. 4, applying Ishida et al.’s (1995) method. Average numbers ofsynonymous and nonsynonymous substitutions per site
between Rh blood group genes of Old World monkeys
and those of hominoids for tree B of Fig. 4 were almost
the same (0.067 and 0.125 for synonymous and nonsynonymous substitutions, respectively) as those of tree A of
Fig. 4. Therefore, the rates of synonymous and nonsynonymous substitutions (per site per year) for the Rh
blood group gene of Old World monkeys and hominoids
are estimated to be 1.46–1.48 × 10−9 (0.067–0.068 / [2 ×23 MYA]) and 2.70–2.72 × 10−9 (0.124–0.125 / [2 × 23
MYA]), respectively. The evolutionary rate of synonymous substitution for the Rh gene is somewhat lower
than that for other primate genes (2.3 × 10−9; Li andTanimura 1987). In any case, it is clear that the nonsynonymous substitution is on average higher than the synonymous one in primate Rh genes.
Because the Rh blood group gene products are membrane proteins, there is a possibility that these products ofblood group genes are affected by interactions with otherorganisms or cells on surface regions. Endo et al. (1996)
searched the nucleotide sequence database and foundthat 17 gene groups were the candidates for the genes on
which positive selection may operate. Nine of those 17
groups were surface antigens of parasites or viruses. Eder
and Spitalnik (1997) suggested that blood group antigens
such as ABO, MN, and Lewis play a key role in pathogenesis of some diseases. The high rate of nonsynonymous substitutions for the primate Rh blood group alsosuggests the existence of positive selection on this gene,and this might be caused by some kind of interaction
with pathogens.
Kitano et al. (1998) showed the long-term evolutionary tree of Rh blood group genes and their homologous
Rh50 genes, and concluded that Rh blood group genes
had a higher evolutionary rate than Rh50 genes. Similar
results were obtained independently by Matassi et al.
(1999). Therefore, Rh-specific antigenicities might begained additionally after the gene duplication on the primate lineage of the Rh blood group gene.
Because we don’t know the actual gene tree topology,
we assumed two plausible trees from nucleotide sequence data in this study. Whichever gene tree we selected we found evidence of gene conversions on primate
Rh blood group genes by using the modified site-by-sitereconstruction method. We also showed the possibility ofpositive selection on the primate lineage (especially thebranch that connects hominoid and Old World monkey
clusters) of the Rh blood group gene by using a statisticaltest. In any case, we should be very careful when weanalyze the evolutionary history of tandemly duplicatedgenes, for there is always possibility of gene conversions.
Acknowledgments. We thank Dr. Kenta Sumiyama for various discussions throughout the preparation of this manuscript and Dr. Yasuo
Ina for providing his programs. This study was partially supported by
grants in aid for scientific studies from Ministry of Education, Science,Sport and Culture, Japan to NS.

References
Adachi J, Hasegawa M (1994) MOLPHY. Programs for molecularphylogenetics, version 2.2. Institute of Statistical Mathematics, Tokyo
Arce M, Thompson ES, Wagner S, Coyne KE, Ferdman BA, Lublin
DM (1993) Molecular cloning of Rh D cDNA derived from a gene

625
present in Rh D-positive but not Rh D-negative individuals. Blood
82:651–655
Avent ND, Ridgwell K, Tanner MJ, Anstee DJ (1990) cDNA cloningof a 30 kDa erythrocyte membrane protein associated with Rh(Rhesus)-blood-group-antigen expression. Biochem J 271:821–825
Avent ND, Butcher SK, Liu W, Mawby WJ, Mallinson G, Parsons SF,
Anstee DJ, Tanner MJ (1992) Localization of the C termini of the
Rh (rhesus) polypeptides to the cytoplasmic face of the humanerythrocyte membrane. J Biol Chem 267:15134–15139
Bandelt HJ (1994) Phylogenetic networks. Verh Naturewiss Ver Hamburg (NF) 34:51–71
Beckers EA, Faas BH, Ligthart P, Simsek S, Overbeeke MA, von dem
Borne AE, van Rhenen DJ, van der Schoot CE (1996) Characterization of the hybrid RHD gene leading to the partial D category
IIIc phenotype. Transfusion 36:567–574
Blancher A, Socha WW (1997) The Rhesus system. In: Blancher A,
Klein J, Socha WW (eds) Molecular biology and evolution of bloodgroup and MHC antigens in primates. Springer, New York, NY, pp
147–218
Blancher A, Calvas P, Ruffie J (1992) Etude des equivalents des antigenes Rhesus chez les primates non hominiens. CR Soc Biol 186:
682–695
Carritt B, Kemp TJ, Poulter M (1997) Evolution of the human RH(rhesus) blood group genes: a 50 year old prediction (partially)
fulfilled. Hum Mol Genet 6:843–850
Cherif-Zahar B, Bloy C, Le Van Kim C, Blanchard D, Bailly P, Hermand P, Salmon C, Cartron JP, Colin Y (1990) Molecular cloning
and protein structure of a human blood group Rh polypeptide. Proc
Natl Acad Sci USA 87:6243–6247
Cherif-Zahar B, Mattei MG, Le Van Kim C, Bailly P, Cartron JP, Colin
Y (1991) Localization of the human Rh blood group gene structure
to chromosome region 1p34.3–1p36.1 by in situ hybridization.
Hum Genet 86:398–400
Cherif-Zahar B, Raynal V, D’Ambrosio AM, Cartron JP, Colin Y(1994) Molecular analysis of the structure and expression of the RH
locus in individuals with D--Dc-, and DCw- gene complexes. Blood
84:4354–4360
Eder AF, Spitalnik SL (1997) Blood group antigens as receptors forpathogens. In: Blancher A, Klein J, Socha WW (eds) Molecularbiology and evolution of blood group and MHC antigens in primates. Springer, New York, NY, pp 268–304
Endo T, Ikeo K, Gojobori T (1996) Large-scale search for genes on
which positive selection may operate. Mol Biol Evol 13:685–690
Gahmberg CG (1982) Molecular identification of the human Rho (D)antigen. FEBS Lett 140:93–97
Hogstrand K, Bohme J (1998) Intrachromosomal gene conversion frequency in the H2 differs between haplotypes. Immunogenetics 48:
47–55
Horai S, Hayasaka K, Kondo R, Tsugane K, Takahata N (1995) Recent
African origin of modern humans revealed by complete sequencesof hominoid mitochondrial DNAs. Proc Natl Acad Sci USA 92:
532–536Huang CH, Reid ME, Chen Y (1995) Identification of a partial internaldeletion in the RH locus causing the human erythrocyte Dphenotype. Blood 86:784–790
Huang CH, Chen Y, Reid M, Ghosh S (1996) Genetic recombination at
the human RH locus: a family study of the red-cell Evans phenotype reveals a transfer of exons 2–6 from the RHD to the RHCE
gene. Am J Hum Genet 59:825–833
Hughes AL, Nei M (1988) Pattern of nucleotide substitution at major
histocompatibility complex class I loci reveals overdominant selection. Nature 335:167–170
Ina Y (1995) New methods for estimating the numbers of synonymousand nonsynonymous substitutions. J Mol Evol 40:190–226
Ina Y, Gojobori T (1994) Statistical analysis of nucleotide sequences of
the hemagglutinin gene of human influenza A viruses. Proc Natl
Acad Sci USA 91:8388–8392
Ishida N, Oyunsuren T, Mashima S, Mukoyama H, Saitou N (1995)

Mitochondrial DNA sequences of various species of the genus
Equus with a special reference to the phylogenetic relationshipbetween Przewalskii’s wild horse and domestic horse. J Mol Evol
41:180–188
Kajii E, Umenishi F, Iwamoto S, Ikemoto S (1993) Isolation of a new
cDNA clone encoding an Rh polypeptide associated with the Rh
blood group system. Hum Genet 91:157–162
Kemp TJ, Poulter M, Carritt B (1996) A recombination hot spot in the
Rh genes revealed by analysis of unrelated donors with the rare Dphenotype. Am J Hum Genet 59:1066–1073
Kimura M (1983) The neutral theory of molecular evolution. Cambridge University Press, Cambridge
Kitano T, Sumiyama K, Shiroishi T, Saitou N (1998) Conserved evolution of the Rh50 gene compared to its homologous Rh bloodgroup gene. Biochem Biophys Res Comm 249:78–85
Klein J, O’hUigin C, Blancher A (1997) Evolution of blood groupantigen polymorphism. In: Blancher A, Klein J, Socha WW (eds)
Molecular biology and evolution of blood group and MHC antigens
in primates. Springer, New York, NY, pp 305–321
Kumar S, Hedges SB (1998) A molecular timescale for vertebrate
evolution. Nature 392:917–920
Landsteiner K, Wiener AS (1940) An agglutinable factor in humanblood recognized by immune sera for blood. Proc Soc Exp Med
43:223
Le Van Kim C, Mouro I, Cherif-Zahar B, Raynal V, Cherrier C, Cartron JP, Colin Y (1992) Molecular cloning and primary structure of
the human blood group RhD polypeptide. Proc Natl Acad Sci USA
89:10925–10929
Levine P, Stetoson RE (1939) An unusual case of intragroup agglutination. J Am Med Assoc 133:126–127
Li WH (1997) Molecular evolution. Sinauer Associates, Sunderland,
MA
Li WH, Tanimura M (1987) The molecular clock runs more slowly in
man than in apes and monkeys. Nature 326:93–96
Matassi G, Cherif-Zahar B, Pesole G, Raynal V, Cartron JP (1999) The
members of the RH gene family (RH50 and RH30) underwent
different evolutionary pathways. J Mol Evol 48:151–159
Moore S, Woodrow CF, McClelland DB (1982) Isolation of membranecomponents associated with human red cell antigens Rh(D), (c), (E)and Fy. Nature 295:529–531
Mouro I, Colin Y, Cherif-Zahar B, Cartron JP, Le Van Kim C (1993)
Molecular genetic basis of the human Rhesus blood group system.
Nat Genet 5:62–65
Mouro I, Le Van Kim C, Cherif-Zahar B, Salvignol I, Blancher A,
Cartron JP, Colin Y (1994a) Molecular characterization of the Rhlike locus and gene transcripts from the rhesus monkey (Macaca
mulatta). J Mol Evol 38:169–176
Mouro I, Le Van Kim C, Rouillac C, van Rhenen DJ, Le Pennec PY,
Bailly P, Cartron JP, Colin Y (1994b) Rearrangements of the bloodgroup RhD gene associated with the DVI category phenotype.
Blood 83:1129–1135
Race RR (1944) An “incomplete” antibody in human serum. Nature
153:771–772
Ruddle F, Ricciuti F, McMorris FA, Tischfield J, Creagan R, Darlington G, Chen T (1972) Somatic cell genetic assignment of peptidase
C and the Rh linkage group to chromosome A-1 in man. Science
176:1429–1431
Saitou N, Nei M (1987) The neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Saitou N, Yamamoto F (1997) Evolution of primate ABO blood group
genes and their homologous genes. Mol Biol Evol 14:399–411
Salvignol I, Blancher A, Calvas P, Socha WW, Colin Y, Cartron JP,
Ruffie J (1993) Relationship between chimpanzee Rh-like genes
and the R-C-E-F blood group system. J Med Primatol 22:19–28

626
Salvignol I, Blancher A, Calvas P, Clayton J, Socha WW, Colin Y,
Ruffie J (1994) Molecular genetics of chimpanzee Rh-related
genes: their relationship with the R-C-E-F blood group system, thechimpanzee counterpart of the human rhesus system. Biochem
Genet 32:201–221
Salvignol I, Calvas P, Socha WW, Colin Y, Le Van Kim C, Bailly P,
Ruffie J, Cartron JP, Blancher A (1995) Structural analysis of theRH-like blood group gene products in nonhuman primates. Immunogenetics 41:271–281
Slightom JL, Theisen TW, Koop BF, Goodman M (1987) Orangutan
fetal globin genes. J Biol Chem 262:7472–7483
Thompson JD, Gibson TJ, Higgins DG (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment

through sequence weighting, positions-specific gap penalties andweight matrix choice. Nuc Acids Res 22:4673–4680
Tippett P (1986) A speculative model for the Rh blood groups. Ann
Hum Genet 50:241–247
Wiener AS (1943) Genetic theory of the Rh blood types. Proc Soc Exp
Biol NY 54:316–319
Yang Z (1997) Phylogenetic analysis by maximum likelihood (PAML),
version 1.3. Department of Integrative Biology, University of California at Berkeley, CA
Zhang J, Kumar S, Nei M (1997) Small-sample tests of episodic adaptive evolution: a case study of primate lysozymes. Mol Biol Evol
14:1335–1338