Evolutionary Rate of Immunoglobulin Alpha Noncoding
Region Is Greater in Hominoids than in
Old World Monkeys’
Shohji Kawamura, Hideyuki Tanabe, Yuji Watanabe,
Kunihiko Kurosaki, Naruya Saitou, 2 and Shintaroh Ueda
Departmentof Anthropology,

Faculty of Science, The University of Tokyo

Recent studies on the molecular evolution of primates show that the evolutionaryrate among hominoids is considerably slower than that among nonhominoid primates. However, this observation at the nucleotide-sequence level is restricted to
the P-globin family region. In this study, we sequenced orthologous immunoglobulinalpha (Ca) genes of chimpanzee, gorilla, orangutan, and crab-eating macaque (anOld World monkey) and compared them with that of the human by using noncoding
regions for analysis. Since significant differences in rates among hominoids were
not found by using the relative rate test, we evaluated the ratio (R) of the evolutionary distance between Old World monkey and human to the distance between
orangutan and human. The R value ( 1,12) for the Ca gene was much smaller thanthe expected value (1.38-2.33), showing that the nucleotide substitution rate(= mutation rate per year under selective neutrality) of the Ca gene is greater inthe human lineage than in the Old World monkey lineage. We also did a similar
analysis for the yl-, y2-, wtl -, and Gglobin genes and found a considerable heterogeneity ( 1.12-2.37) among the R values, including that for the Ca gene. This
indicates that the hominoid slowdown of the evolutionary rate is not a universal
phenomenon in primate evolution.
Introduction

The debate concerning rate constancy of molecular evolution began immediately
after the molecular clock was first proposed by Zuckerkandl and Pauling ( 1962 ) , andit continues today. Wu and Li (1985), Britten (1986), Li and Tanimura (1987), andLi et al. ( 1987) argued that the rate of nucleotide substitution is significantly higher
in rodents than in primates and artiodactyls. Easteal ( 1990), however, detected nosystematic variation in evolutionary rates among those mammalian orders when heapplied a relative-rate test and used marsupial orthologues for reference. Recently, Li
et al. ( 1990) accepted Easteal’s ( 1990) view that rodents diverged first, but they stillargued that the evolutionary rate of rodents is at least 1.5 times higher than those inother major eutherian orders. As for the rate variation within primates, Koop et al.
(1986, 1989b), Li and Tanimura (1987), Li et al. (1987), and Maeda et al. (1988)
argued for a rate slowdown in hominoids, on the basis of comparison of noncoding
regions or synonymous sites of DNA sequence data. However, a statistically significant
slowdown of evolutionary rate in the hominoid lineage has been observed only in the
1. Key words: immunoglobulin Ca, evolutionary rate, primates.
2. Present acldres: National Institute of Genetics, Mishima 4 I 1, Japan.

Address for correspondence and reprints: Shintaroh Ueda, Department of Anthropology, Faculty of
Science, The University of Tokyo, Tokyo 113, Japan.
Mol.Biol. Evol. 8(6):743-752.

1991.
0 1991 by The University of Chicago. All rights reserved.
0737-4038/91/0806-0001.$02.00

743

744

Kawamura

et al.

ggtccaactgcaggcctgtggtgcaggagctgtgtgacc~t~gctgtcacca~cctctct~gctg~ttcctccagtatagaggaga~cagtataga~a--------------ccaa---------gcacagcctgtcctc
tcc--------qcacagactgccctc
-g
c
ac
a
---------gcacagcctgtcctc
caa---------gcgcagcctgttctc:c:---------------gagggccgcgtcctcacagtgcattctgt~tcca~TCCC~AC~CCC~~C~~~~AGCCTC~~GCA~CC~~C~GA~M~T~
ctggggacatggcat
ac
ctggggacatggcat
cGt
ctggggacatggcat
c
A AT
T
A
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c
A
GAGG
T

120

240
G
:
360

.
A

TT

Gc

CA 2

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C

460
A

G

!G

T

It

C AG6

G

agagggcaggctggggagtggggcggggccaccccgtc~gccctgacact~gcct~acccgtgttccccaca~gagcc~cccttcactcacacca~~~c~~~gccgagc
c
c
a
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600

cccaggaggtggtggtggacaggccaggaggggcgaggcgggggcatggggaagtatgt
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120

640
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960

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C

ft

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CG
C&T

%

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e

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tcagtg~~ctggtttgeaaagcatattcct~ctgcctcctccctcccaatcctg~tccagtgctcat~caagtacaca~aaactga~caggctga~-cc~ggacacagc
ca
ca
:
E:
a
c
gg a
---____
c
g!
tgg
g:

_____

1200

p”
ccggggtgcccaccagagcag-aggggctctctcatcccct~ccagccccctgacctggctctctaccctcca~~~C~~C~CC~A~TCCAC~C~~C~CGT~
t

:

g

gt

a

TE

1320

A

FIG. l-Aligned
nucleotide sequences of human ( H 1), chimpanzee (Cl ), and gorilla (G 1) Ca 1 andof orangutan (0) and crab-eating macaque (0) Ca genes. The nucleotide sequence of the human Ca 1gene is from Flanagan et al. ( 1984). Only nucleotides different from that of the human Ca 1 sequence areshown. Gaps are designated by hyphens. Noncoding and coding sequences are denoted by lowercase andcapital letters, respectively. Hinge regions are boxed.

@l-globin gene region. DNA/DNA hybridization studies did not support the hypothesisof a hominoid slowdown (Sibley and Ahlquist 1984, 1987; Caccone and Powell 1989).
Especially in the case of a noncoding region or synonymous sites, rate variation
is often attributed to the effect of generation time (Laird et al. 1969), to the numberof cell divisions in the germ line ( Wu and Li 1985), or to changes in DNA repair or
replication mechanisms (B&ten 1986) rather than to population structure or selection.
According to the generation-time hypothesis, taxa with shorter generation times aresupposed to have a faster rate of molecular evolution than do those with longer generation times.
In the present study we determined the nucleotide sequences of the immunoglobulin alpha 1 (Co 1) genes of chimpanzee and gorilla, as well as single-copy Ca
genes of orangutan and crab-eating macaque, and compared them with that of theEvolutionary Rate of Ig Ca Gene in Primates

745

IllA~AGCn;GcCCn;MC~~Affi~C~~~~~~C~A~~~~~~~~~~A~~CC~A~~ACC~A1440
T

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HI CTTGGGCATCCCGG~~AGCCCAGCCAGGGCACCAC~CCACC
Ei
A

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A

1560
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C

ACCCCCn;CCGCn;GCCTTCACACAGMGAC~T~AC~~T~CCCAC~~T~~~C~~T~~~A~~A~CACC~~AC~~cc6ccc6cc1680
II
:
!

:

f

A

tgtccccacccctgaataaactccatgctcccccaagcagccccacgcttccatcc~cgcctgtctgtccatcctca~tctca~actt~aaag~cca~at~aca~aa
1800
t _________ g
a
caa
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gaataccccctgccctgagcctcggggggccctggcacc~tgagactttccaccctggtgtgagtgtgagttgtgagtgtgagagtgtgtgtgcaggaggeetegetg~gtgag
1920
C
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atcttaggtctgccaaggcaggeacacagccca~atgggttctgagagacgcacatgcccc~acagttctgagtga~agt~at~c~ttgtccctgagagagccgcctct~ctg
C
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tagctgggagggaatagggagggtaaaaggagca~tagccaagaaa~c~~~tagt~a~agc~cga~a~ga~ct~actccag~ccccactg~a~acaagctc
2160
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caggagggecccaccaccctagtgggtgggectca~ac~ccc-actgacgcatgca~aa~cacctccccttaaccacactgctctgt---------------ac~ca~gtg
2280
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ggcacagtgeacactcacactcacatatatgcctgagccctgca~agc~aac~tcacagcccagacccagttccagaaaagccag~agtcccctcccaagcccccaa------2400
CC&___________
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2520
C
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tctcccatcccttcctaagcccaactaggacccaaagcatagacaggga~gccac~gg~t~catcagaagaga ga_______agt
tg

FIG. 1 (Continued)

human Ca 1 gene. We show that the evolutionary rate of the Ca noncoding region isgreater in hominoids than in Old World monkeys and discuss possible causes for theevolutionary-rates difference between the two lineages of higher primates.

Table 1
Estimated Numbers + SEs of Nucleotide Substitutions

Chimpanzee
GorillaOrangutan
Crab-eating macaqueof Primate Ca Noncoding Regions

Human

Chimpanzee

GorillaOrangutan

2.77 f 0.46
2.46 f 0.43
5.64 f 0.66
6.36 f 0.7 1

1.86 k 0.37
5.17 f 0.63
5.96 + 0.68

4.62 + 0.60
5.88 + 0.68

5.80 f 0.67

NOTE.-A total of 1,362nucleotideawere usedforcomparison. Values are Kc X 100.

746

Kawamura et al.

Material and Methods
Chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla) Ca 1 genes were isolated from their genome libraries, as were orangutan (Pongo pygmaeus) and crabeating macaque (Macaca fascicularis) Ca genes (Ueda et al. 1988; Kawamura et al.
1990). DNA manipulations were performed using standard procedures (Sambrook
et al. 1989). All sequencing was performed in both sense and antisense strands byusing the dideoxynucleotide chain-termination method. The number of nucleotide
substitutions per site (KC) was estimated by using the formula KC = - ( 3 / 4)ln [ 1- (4 /3)K], where K is the number of nucleotide differences per site (Jukes and Cantor1969). In this computation, gaps were excluded. The standard errors of branching
points were calculated by using Nei et al.‘s ( 1985) method. To test rate constancy
within hominoid lineages, Wu and Li’s ( 1985) relative-rate test was applied.
Results and Discussion- -5

Ziuman, chimpanzee, gorilla, and gibbon have two Ca genes (Ca i and Ca 2 j,while orangutan and Old World monkeys have a single Ca gene in their genomes(Flanagan et al. 1984; Ueda et al. 1988; Kawamura et al. 1990). The orangutan Cu
gene is considered to be orthologous to the Cu 1 genes in other hominoids, because its
hinge-coding region consisting of two 30-bp repeats with a 6-bp overlap (boxed in fig.
1) is characteristic of Cu 1 genes (Kawamura et al. 1990). In the present study we
added further nucleotide sequence to our previous data of the gorilla Cu 1 gene (Kawamura et al. 1989) and newly determined those of the chimpanzee Cu 1 gene and of
the orangutan and crab-eating macaque Cu genes (fig. 1) . We analyzed the nucleotide
sequences of noncoding regions (table 1) , most of which are considered to be effectively
neutral. Therefore, substitution rates estimated in the present study would be expected
to be equal to the mutation rate per year (Kimura 1983). We first evaluated the ratevariation among hominoid lineages by carrying out the relative-rate test by using the
crab-eating macaque gene as an outgroup reference. There was no statistically significant
variation among the evolutionary rates of hominoids, as shown in table 2. Therefore,
we reconstructed a phylogenetic tree by using the unweighted pair-group method ofanalysis (UPGMA), which assumes rate constancy (Sokal and Sneath 1963). The
branching pattern of the resulting tree (fig. 2) where the orangutan diverged first among
the four hominoids was in agreement with a generally accepted phylogenetic tree (Saitou
199 1, and references therein). Chimpanzee clusters not with human but with gorilla inTable 2Evolutionary-Distanceand Hominoids
Species l/Species 2
Human/chimpanzee
Human/gorilla
Human/orangutan
Chimpanzee/gorilla
Chimpanzee/orangutan
Gorilla/orangutan

Differences between Old World Monkey

K, - Kza

SE

Pb

0.40
0.48
0.56
0.08
0.16
0.08

0.48
0.45
0.68
0.39
0.65
0.61

>0.2
Z-o.2
>0.2
>0.5
>o.s
>o.s

‘K, (i = 1 or 2) is the number of nucleotide substitutions in the Cal noncoding region,per 100 sites, between species i and an Old World monkey (crab-eating macaque).
b Probability that there is no difference between K, and &, which is evaluated using
a two-tailed f-test with degree of freedom = co.

Evolutionary Rate of Ig Ca Gene in Primates

747

C
G
Db=.0270

Da=.

0302

R=1.120--

FIG. 2-Phylogenetic trees of primate immunoglobulin Ca genes, constructed by UPGMA. H = human;
C = chimpanzee; G = gorilla; 0 = orangutan; and Cr = crab-eating macaque. Branch lengths are proportional
to the numbers of nucleotide substitutions per site. For each branch, a shaded box shows the standard error.
Da and Db are the branch lengths leading to Old World monkey and orangutan, respectively. R = Da/Db.

this tree, though this branching order is not statistically significant. Other estimation
methods (Kimura 1980; Tajima and Nei 1984) gave essentially the same Kc values as
did the Jukes-Cantor method used in the present study.
In this tree, the branch length (Da = 0.0302) leading to Old World monkey was
not much longer than that (Db = 0.0270) leading to orangutan (fig. 2). The ratio(R) of Da to Db was 1.12. We compared these values (Da, Db, and R) with corresponding values for other nuclear DNA sequences available (fig. 3 ) . All these sequences
are from the noncoding regions of P-globin-family genes ( y 1-, y2-, &l-, and &globin
genes; see table 3) (Fitch et al. 1988; Maeda et al. 1988; Miyamoto et al. 1988; Slightom
et al. 1988; Koop et al. 1989a). As these data included the nucleotide sequences of
New World monkeys, it enabled us to conduct a relative-rate test between hominoids
and Old World monkeys by using New World monkey as an outgroup. Significantly
lower substitution rates in hominoids than in Old World monkeys were observed in
the @-l-globin region of all the hominoids (0.1% level) and in the yZglobin region ofchimpanzee (5% level). This is consistent with the result that, compared with thosein the Old World-monkey lineage, the P-globin-family genes in the hominoid lineage
show a slowdown in substitution rates (Koop et al. 1986; Li and Tanimura 1987; Liet al. 1987; Easteal 199 1). However, rate variation among hominoids was not detectedin these four DNA regions. We also used the neighbor-joining method, which does
not assume rate constancy (Saitou and Nei 1987). The resulting trees (not shown)
had the same branching patterns as those of figures 2 and 3, and their branch lengths
were more or less the same, though the branch length for the human lineage wasslightly longer than those for other hominoid lineages in the Ca tree. The Db value
for the Co gene was significantly greater than that for the y2- and &globin genes [atthe 5% level (t = 1.965 and 2.553, respectively, with degrees of freedom = cc whena two-tailed t-test is used)] and than that for the &l-globin gene [at the 0.1% level (t= 4.581)]. The Db for the yl-globin gene was smaller than that for the Ca gene,though there was no statistical significance (t = 1.630). In contrast, Da values forthese genes were close to each other.
From data obtained from fossil records, it is generally accepted that the orangutan
diverged from the lineage of man-chimpanzee-gorilla 12- 16 Mya and that Old World
monkeys diverged from hominoids 22-28 Mya (Gingerich 1984, Fleagle 1986; Pilbeam1986). Therefore, the R value would be expected to be in the range of 1.38-2.33, if
the evolutionary rate is assumed to be constant in all phylogenetic lineages. As shownin figure 4, however, there was a great heterogeneity among the R values observed;
the R values of the y 1-, y2-, and Gglobin genes were within the expected range, whereas

748

Kawamura et al.

6giqif++G;

R=l.69Da=.

0291

R=1.74-0

Db=.O194

IRh

Da=.

0337

R=2.37

FIG. 3-Phylogenetic trees of primate P-globin family genes, constructed by UPCMA. Rh = rhesus
monkey; B = baboon; all other abbreviations are as in fig. 2. Branch lengths are denoted by shaded boxes.
Da, Db, and R are as in fig. 2.

that of the Co gene was smaller than expected and that of the $1~globin gene waslarger than expected. Contrary to a former claim that a slowdown in evolutionary
rates occurred in hominoids, these results indicate that the evolutionary rate in thenoncoding region-namely,
the mutation rate per year-of the Ca gene is greater inhominoids than in Old World monkeys. These results suggest that the mutation rate
for the Co gene has increased in the hominoid lineage.
The evolutionary distances between the Old World monkey Ca gene and eachhominoid Ca gene, including the Ca 2 gene, have been shown to be similar to each

Evolutionary Rate of Ig Ca Gene in Primates

749

Table 3
Estimated Numbers + SEs of Nucleotide Substitutions of Primate yl-, y2-, Qq-,
and &Clobin Noncoding Regions

ylGlobin region:’
Chimpanzee
GorillaOrangutan
Rhesus monkey
Spider monkey
y2-Globin region:b
Chimpanzee
GorillaOrangutan
Rhesus monkey
Spider monkey@Globin region:c
Chimpanzee
GorillaOrangutan
Rhesus monkey
Spider monkey

GGlobin region?
Orangutan
BaboonSpider monkey

Human

Chimpanzee

GorillaOrangutan

RhesusMonkey2.17 + 0.41
2.17 + 0.41
3.83 + 0.55
6.12 ? 0.71
11.88 f 1.01

2.96 + 0.48
4.00 + 0.56
6.61 + 0.74
Il.79 +- 1.01

4.56 + 0.60
6.95 -+ 0.75
12.42 + 1.04

6.70 + 0.74
11.79 + 1.01

13.23 f 1.08

2.41 f 0.44
2.89 + 0.48
4.09 f 0.57
6.55 f 0.73
12.37 + 1.04

1.86 f 0.38
3.69 I? 0.54
6.64 + 0.74
11.92 + 1.02

4.25 f 0.58
7.39 f 0.78
12.46 + 1.04

7.06 + 0.76
12.37 f 1.04

13.92 k 1.11

1.44 + 0.13
1.47 + 0.13
2.88 + 0.18
6.96 + 0.29
10.32 & 0.35

1.58 f 0.13
2.96 + 0.18
7.09 f 0.29
10.47 f 0.36

2.98 + 0.18
6.92 + 0.28
10.30 + 0.35

6.94 f 0.28
10.47 + 0.36

11.90 + 0.38

Human

Orangutan

Baboon3.43 + 0.50
5.58 + 0.64
8.58 + 0.8’1

6.04 + 0.67
9.05 f 0.83

9.60 f 0.86

NOTE.-VENUES are Kc X 100.
’A total of I ,3 I 1 nucleotides were used.
b A total of 1,307 nucleotideswere used.
‘A total of 9.204 nucleotideswere used.
d A total of 1.43
1 nucleotides were used.

other (S. Kawamura,N. S&ov, and S. Ueda, unpublished data). This suggests thatchanges introduced/erased by gene conversion are canceled out, so that gene conversion
has little effect on evolutionary distances between Old World monkeys and hominoids.
If the nucleotides of Ca 1 and Ca 2 genes were considerably different before orangutan
diverged, and if gene conversions of Ca 1 genes by Ca 2 genes occurred after thedivergence, the evolutionary distance between the orangutan Ca and the humanchimpanzee-gorilla Ca 1 genes would be longer than it would be in the absence ofsuch a conversion. We applied the site-by-site reconstruction method of gene conversion(Fitch et al. 1990) to examine this possibility, but such events were not detected (S.
Kawamura, N. Saitov, and S. Ueda, unpublished data). Sequence data from New
World monkeys would provide more insight into the evolution of the Ca gene.
For each gene examined in the present study, base composition was similar among
different species. The G+C content of the Ca genes of higher primates was muchhigher (average 64%) than that of the remaining genes (average 45%, 44%, 39%, and
37% for yl-, y2-, &J-, and 6-globin genes, respectively), but there was no association
between the R values and G+C contents.
Various mechanisms have been proposed to explain the variation in substitution

750

Kawamura et al.

R

12 16 22 28
Time (million years ago)
FIG. 4-Heterogeneity
among observed R values. From fossil records, the divergence time between
orangutan and man-chimpanzee-gorilla and that between Old World monkeys and hominoids have been
estimated to be 12-16 and 22-28 Mya, respectively. Therefore, the range of the expected R value would be
1.38-2.33, if constancy of evolutionary rate is assumed.

rate. These include selection pressure, population size (Kimura 1983 ) , generation time
(Laird et al. 1969), number of germ-line replications (Wu and Li 1985), and DNA
repair mechanisms (B&ten 1986). The nucleotide sequences compared in the present
study are noncoding regions, for which the substitution rate is considered to be affected
neither by selection nor by population size (Kimura 1983 ) . The generation-time-effecthypothesis and the germ-line-replication hypothesis are unlikely to be correct, because
they ( 1) require a hominoid-directed slowdown of the evolutionary rate during primate
evolution and (2 ) are incompatible with the coexistence of slowdown and speedup in
DNA regions in the genome, a coexistence that was found in the present study.
A recent analysis suggests that mutation rate varies according to region of the mammalian genome, possibly because of regional differences in DNA repair or replication
mechanisms (Wolfe et al. 1989). However, if we assume only regional differences inmutation rate, the R values of the different DNA regions shown in figures 2 and 3 would
have to be similar to each other. Alternatively, if only a lineage (taxonomic) differencein mutation rate exists (B&ten 1986 ) , the presence of similar Da values in different DNA
regions would be incompatible with the great variation in Db values. Thus, variation in
evolutionary rate must be caused not by a single factor but by multiple factors.
Sequence Availability

Nucleotide sequence data reported in the present paper have been deposited in
the EMBL/GenBank/DDBJ
Nucleotide Sequence Databases under the accession
numbers X53702 (chimpanzee Ca 1), X53703 (gorilla Co 1 ), X53704 (orangutanCa), and X53705 (crab-eating macaque Ca).

Evolutionary Rate of Ig Ca Gene in Primates

75 1

Acknowledgments
We are grateful to Drs. K. Omoto, T. Honjo, and 0. Takenaka for their encouragement. This study was supported in part by grants from the Ministry of Education, Science and Culture of Japan.
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