Evolutionary Rates of Insertion and Deletion in Noncoding Nucleotide
Sequences of Primates
Naruya SaitoaP and Shintaroh Uedat*Laboratory of Evolutionary
Genetics,of Science, University of Tokyo

National

Instituteof Genetics;

and TDepartmentof Anthropology,

Graduate

SchoolInsertions and deletions are responsible for gaps in aligned nucleotide sequences, but they have been usually ignored
when the number of nucleotide substitutions was estimated. We compared six sets of nuclear and mitochondrial
noncoding DNA sequences of primates and obtained the estimates of the evolutionary rate of insertion and deletion.
The maximum-parsimony
principle was applied to locate insertions and deletions on a given phylogenetic tree.
Deletions were about twice as frequent as insertions for nuclear DNA, and single-nucleotide insertions and deletions
were the most frequent in all events. The rate of insertion and deletion was found to be rather constant among
branches of the phylogenetic tree, and the rate ( -2.O/kb/Myr)for mitochondrial
DNA was found to be muchhigher than that ( -0.2/ kb/Myr) for nuclear DNA. The rates of nucleotide substitution
were about 10 times
higher than the rate of insertion and deletion for both nuclear and mitochondrial
DNA.

Introduction
Evolutionarychange of nucleotide
sequences has
been studied mainly from the viewpoint of nucleotide
substitutions.
Now we have ample knowledge of the pattern and rate of nucleotide substitution
for many organisms and genes (e.g., see Nei 1987). Insertions and deletions of nucleotides are, however, not well studied. One
reason is that they are considered to occur infrequently
in coding regions, because most of them are strongly
deleterious.
Tajima and Nei ( 1984) proposed an evolutionary distance that is calculated on the basis of insertions and deletions, and they applied their method toa coding region, but there was a very low increase of the
distance over a long period of evolutionarytime.
Mutation is the fundamental
source for organismal
evolution, and there are many types of mutations,such
as nucleotide substitution,
insertion, deletion, gene duplication, unequal crossing-over,and gene conversion.
To understandthe tempo and mode of evolution at thenucleotide
level, it is importantto estimate the spontaneous rate of each mutation type., including insertions
and deletions. Graur et al. ( 1989) analyzed nucleotide
sequences of processed pseudogenes of humans and miceand concluded that deletions accumulated
faster in roKey words: insertions and deletions, molecular clock, evolutionaryrate, primates, mutation rate.
Address for correspondence and reprints: Naruya Saitou, Laboratory of Evolutionary Genetics, National Institute of Genetics, 1111
Yata, Mishima 411, Japan.
Mol. Biol. Evol. 11(3):504-512. 1994.
0 1994 by The University of Chicago. All rights reserved.
0737-4038/94/l 103-0017$02.00

504

dents than in humans. However, they showed only arelative rate difference between rodents and humans.
Saitou ( 1992) conducted an analysis of the insertions and deletions for a small data set of nuclear DNA
sequences of hominoids and estimated the absolute rateof insertionand deletion to be O.l8/kb/Myr.
In thepresent study, we analyzed insertions and deletions in
the nucleotide sequences of primate noncoding regions
for nuclear DNA and mitochondrial
DNA, by expanding
the data set used by Saitou ( 1992). We show that there
is a constancy in the rate of insertion and deletion, andestimate the evolutionaryrate of insertion and deletion.
Because most of the changes in the noncoding region of
DNA are supposed to be neutral (Kimura
1983), ourestimates of evolutionaryrate can be equated to thespontaneous
mutation rate of insertion and deletion.
Material and Methods
We used six sets of nucleotide sequence data, fourof them for nuclear DNA and two for mitochondrialDNA. These were as follows (lengths of each region are
averages of sequences used) : ( 1) The II-globin w is a
6.4-kb fragment containing
the q-globin pseudogene in
the P-globin gene family. Aligned sequence data for human (Homo sapiens), chimpanzee(Pan troglodytes),
gorilla ( Gorilla gorilla), orangutan (Pongo pigmaeus) ,gibbon (Hylobates Zar), rhesus macaque (Macaca muZatta) , and spider monkey (A teles geofioyi) were takenfrom figure 3 (positions l-8740) of Bailey et al. ( 1992).
(2) The P-globin spacer is a 3.1-kb fragment containing
a spacer DNA of the P-globin gene family. Aligned se-Evolutionary Rates of Insertion and Deletion

CC-CCCCTTCA
I

B

New World Monkeys
30

20
Divergence: Crab-eating macaque

Old World Monkeys

1

40

505

10

AGGTGGTGGTGGACA:Human
AGGTGGTGGTGGACA: Chimpanzee
AGGTGG---TGGACA:
GorillaAGGTGG---TGGACA:Orangutan
AGGTGGTGGTAGACA : Crab-eating macaque0Time (MYA)

FIG. 1.-Assumed phylogenetic tree of primates. Branching points
A-G correspond to 2.5, 5.0, 6.0, 13.0, 15.0, 25.0, and 40.0 Mya, respectively.

quence data for human (R allele), chimpanzee, gorilla,orangutan, rhesus macaque, and spider monkey weretaken from Maeda et al. ( 1988). (3) The Ig-&3 \v is a
2.3-kb fragment containing immunoglobulin ~3 processed pseudogene. Aligned sequence data for human,
chimpanzee, gorilla, and orangutan were taken from
Ueda et al. ( 1989). (4) The Ig-a noncoding region is a
2.6-kb fragment containing the immunoglobulin-a coding gene. Aligned sequence data for human, chimpanzee,
gorilla, orangutan, and crab-eating macaque (Mucuca
fascicularis) were taken from Kawamura et al. ( 199 1).
This fragment contains a 1S-kb noncoding region, andonly this region was used for analysis. ( 5) The mitochondrial DNA D-loop is a 1.1-kb fragment of noncoding mitochondrial DNA containing the so-called D-loop
region. Aligned sequence data for human, chimpanzee,
pygmy chimpanzee (Pan paniscus), gorilla, and orangutan were taken from Foran et al. ( 1988). (6) The
mitochondrial DNA %-genome is a 5.0-kb fragment of
mitochondrial DNA containing 11 tRNA genes, 6 protein genes, and some noncoding regions, which roughly
corresponds to one-third of the entire mitochondrial genome. Aligned sequence data for human, chimpanzee,
pygmy chimpanzee, gorilla, orangutan, and siamang(Hylobates syndactylus) were taken from Horai et al.
( 1992). We used only the noncoding region, which was
0.1 kb in total.
In general, positions and lengths of gaps created in
the alignment procedure may vary depending on the
gap penalties used. Because the published multiplealigned sequence data used are closely related, however,most of the gaps will not change their positions andlengths drastically when gap penalties are modified. Thus
the ambiguity of gap determination is avoided in thepresent study.
The method used to assign insertions and deletions
to a branch of a phylogenetic tree is as follows: We first
assumed the phylogenetic tree of higher primates (seefig. 1) , which was constructed by considering molecular
data from many sources (e.g., see Sibley and Ahlquist

FIG. 2.-Two examples of the gaps in the aligned sequences (datafrom Kawamura et al. 199 1). A, 56 l-57 1-bp region of the Ig-a gene.
B, 607-62 1-bp region of the Ig-a gene.

1987; Hayasaka et al. 1988; Ueda et al. 1989; Saitou
199 1). Although there are ambiguities in the detailed
pattern, in particular the branching times, we believethat the overall picture will not change drastically. On
the basis of this tree, the maximum-parsimony principle(see Sneath and Sokal 1973 ) was applied, to locate insertions and deletions to each branch of the phylogenetic
tree. The assumed phylogenetic tree was different foreach data set, but all of them are partial trees of figure 1.
The number of nucleotides involved in insertions
and deletions was irrelevant in this operation. A contiguous block of nucleotides (a gap) was considered to becreated by one event. For example, there were two gaps
in the 56 l-572 bp of Ig-a sequences (fig. 2A). In thatcase, both gaps were unambiguously allocated to thebranches for orangutan (a deletion of 1 nucleotide) and

B

Human
Chimpanzee
Gorilla3G

Orangutan
Crab-eating

macaque

FIG. 3.-Allocation of insertions and deletions to each branch of
the phylogenetic tree of fig. 1 by applying the maximum-parsimony
principle to the aligned sequence data of fig. 2. “I,” “D,” and “G”
denote insertion, deletion, and gap (insertion or deletion) events, respectively. Numbers before those symbols are the no. of nucleotides
involved in each event. A, Allocation of events for the data of fig. 2A.
B, Two possible ways of allocation for the data of fig. 2 B.

Table 1
Length Distribution of Insertions, Deletions, and Gaps
Length(nucleotides)
A. Data set I-A (q-globin
1
2 :::::::::::::::::::::::::::
3 ...........................
4 ...........................
5 ...........................
6 ...........................
7 ...........................
8 ...........................
11 ..........................
12 ..........................
13 ..........................
14 ..........................
18 ..........................
23 ..........................
24 ..........................
26 ..........................
28 ..........................
38 ..........................
76 ..........................
331 .........................
338 .........................
339 .........................
558 .........................
Total

Deletions

Gaps

Total

14 (3.83)
5 (1.67)
3 (0.33)
3 (0.33)
1 (0.33)2

29 (4.33)
11
4
7 (0.33)
3 (0.33)311

19 (0.50)
5
2
3
2
2

1
1
1

62 (8.67)
21 (1.67)
9 (0.33)
13 (0.67)
6 (0.67)
7
1
2
3
2
3
2
1
1
3
1
1
1
(1 .OO)
1”
1”
la
lb

region):

. . .
1
2

. . .
. . .

1
2
1

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

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

(0.50)‘(d.50)

. . .
. . .
. . .

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

37 (7.00)

68 (5.50)

38 (0.50)

143 (13.00)

3 (0.50)
1 (0.50)

5 (1.50)
5 (1.00)
3 (1.00)
2(1.50)
1 (0.25)(1 .OO)(0.25)
1 (0.50)

12 (0.50)
4 (0.50)11
1

20 (2.50)
10 (2.00)
4 (1.00)
3
1 (1.50)
1 (0.25)( 1.OO)
1 (0.25)
2 (0.50)11
1
1

spacer region):.....................

..

1
1

1
1
1
1
3
1
1

1

. . .
. . .
. . .
. . .
. . .
1
. . .
. . .
. . .
. . .
. . .
. . .
5 (1.00)

C. Data set I-C (immunoglobulin-agene noncoding region):
1 ...........................
. . .
2 ...........................
3 ...........................
(d.5,
9 ...........................
1
10 . . . . . . . . . . . . . . . . . . . . . . . . . .
. . .
11 . . . . . . . . . . . . . . . . . . . . . . . . . .
. . .
12 . . . . . . . . . . . . . . . . . . . . . . . . . .
. . .
13 . . . . . . . . . . . . . . . . . . . . . . . . . .
. . .
15 . . . . . . . . . . . . . . . . . . . . . . . . . .
130 . . . . . . . . . . . . . . . . . . . . . . . . . .
. . .
42 . . . . . . . . . . . . . . . . . . . . . . . . . .
. . .
Total

. . .
. . .

1

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

B. Data set I-B (P-globin
1 ...........................
2 ...........................
3 ...........................
4 ...........................
5 ...........................
6 ...........................
7 ...........................
8 ...........................
9 ...........................
12 ..........................
14 ..........................
15 ..........................
20 ..........................
45 ..........................
Total

pseudogene

Insertions.................

2 (0.5)

. . .
. . .
. . .
. . .
. . *

. . .
. . .
. . .
1
1
I
1
1
1

1

17 (7.00)

25 (1.00)

47 (9.00)

6

8
2

14
2

‘( 1.d)

(0.5)

. . .
. . .

1
1

. . .

1

. . .

1
1

. . .
1
1
10 (1.0)

. . .
. . .
. . .
. . .
13 (0.5)

(2.0)
2
1
1
1
1
1
1
1
25 (2.0)

Evolutionary Rates of Insertion and Deletion

507

Table 1 (Continued)
Length(nucleotides)
D. Data set I-D (immunoglobulin
1 ...........................
2 ...........................
3 ...........................
4 ...........................
5 ...........................
6 ...........................
7 ...........................
8 ...........................
9 ...........................
11 ..........................
16 ..........................
Total

Insertions

Deletions

Gaps

Total

E pseudogene region):
1 (0.40)(0.33)

3 (0.40)1 (0.73)

2 (0.40)1.....................

‘(0.33)(0.33)

1

. . .
. . .
. . .

. . .

6 (1.20)
2 (1.07)1
2 (0.33)(0.33)(0.53)(0.80)(0.40)(0.67)(0.67)1

3 (2.13)4 (2.60)

5 (1.27)

12 (6.00)

8 (1.00)
2 (0.25)

9 (0.75)
4

22 (2.50)
6 (0.50)

2

3

...
1

$33)
(0.33)(0.20)(i.20)(0.33)(0.33)

E. Data set II-A (mitochondrial DNA D-loop region):
1 ...........................
5 (0.75)
2 ...........................
(0.25)
3 ...........................
...
4 ...........................
...
16 ..........................
. . .
27 ..........................
38 ..........................
71 ..........................
Total.....................

5 (1.00)

F. Data set II-B (mitochondrial DNA r/3-genome region):
1 ...........................
2 (0.33)
2 . . . . . ..t...................
. . .
4 ...........................
1
7 ...........................
1
16 . . .._.....................
1
Total

. .

... ...

.. ...

5 (0.33)(d.33,(0.60)(0.20)(O.iO,‘1’lC
11 (1.25)

20 (0.75)

36 (3.00)

2 (0.67)(0.33)

3 (0.33)(0.33)

7 (1.33)(0.67)

. . .
. . .
. . .

. . .
. . .

2 (1.00)

3 (0.67)1
10 (2.0)

NOTE.-values

in parentheses are those with multiple possibilities.
a Ah element.
b Ll element.
’ Region where the alignment was impossible to establish.

for crab-eating macaque (fig. 3A). It should be notedthat the root of the tree of figure 3A was ignored in that
operation. It could not be determined whether a gap at
the crab-eating macaque sequence in figure 2A was an
insertion (at branch “a” of fig. 3A) or a deletion (atbranch “b” of fig. 3 A), and it was designated “ 1G” ( lnucleotide gap) in figure 3 A. In this paper, “gap” means
an event for which it cannot be determined whether an
insertion or a deletion occurred.
When there was more than one possibility for allocation of gaps to branches, all possibilities were considered, and gaps were allocated equally to corresponding
branches. For example, there were multiple gaps in the607-62 1-bp region of Ig-a sequences ( fig. 2B). In thatcase, there were two possible patterns of insertion anddeletion (fig. 3 B). One possibility was that the lineage
to the common ancestor of human and chimpanzee ex-perienced an insertion of 3 nucleotides, and a 3-nucleotide-long gap occurred in the crab-eating macaque lineage or in the lineage to the common ancestor of human,
chimpanzee, gorilla, and orangutan (upper tree of fig.
3B). Another possibility was that both gorilla and orangutan lineages experienced a deletion of 3 nucleotides(lower tree of fig. 3 B). We allocated all these possible
insertions and deletions to the corresponding brancheswith a probability of one-half. This allocation procedure
is simpler than that of Saitou ( 1992).
Results and Discussion

Distribution of the Lengths of Insertions andDeletions
Table 1 shows the distribution of the lengths ofinsertions, deletions, and gaps for each data set. Values
listed are the numbers of insertions, deletions, and gaps

508

Saitou and Ueda

Table 2
Summary of the Number of Insertions, Deletions, and Gaps
Data Set
Nuclear DNA:
q-Globin v . . . . . . . .
P-Globin spacer . . .
Iga . . . . . . . . . . . . . .
Ig&w . . . . . . . .
Total.. ....
Mitochondrial DNA:
D-loop .
........
l/3-Genome......
Total . . .
.....

Insertions

Deletions

Gaps

Total

Mean Length

36 (43.0)
5 (6.0)2 (2.5)3 (5.1)46 (56.6)

68 (73.5)
17 (24.0)
10 (11.0)
4 (6.6)99 (115.1)

35 (35.5)
25 (26.0)
13 (13.5)5 (6.3)78 (81.3)

139 (152)
47 (56)25 (27)12 (18)223 (253)

4.3 (4.6)
4.4 (4.2)
6.8 (6.5)
3.1 (3.8)
4.5 (4.6)

5 (6.0)5 (5.3)10 (11.3)

11 (12.3)
2 (3.0)13 (15.3)

20 (20.7)
3 (3.7)23 (24.4)

36 (39)10 (12)46 (5 1)

5.6 (5.2)
3.4 (3.1)
5.1 (4.7)

NOTE.-values are those for unambiguously determined events, and those in parentheses include ambiguously determined events.
’ Alu and Ll repeats were excluded.

for each branch, that were determined unambiguously,
and values in parentheses are those with multiple possibilities. A total of 308 gap-making events were extracted
from the six data sets, of which 273 gaps were unambiguously determined.
Four long gaps were found in q-globin pseudogene(those marked “a” and “b” in table 1A), and they are
either Alu or L 1 elements ( Bailey et al. 1992). Because
those gaps were probably created by a mechanism different from that responsible for shorter gaps, we eliminated them from the following analysis.
There were two regions in the gorilla sequence of
mitochondrial DNA D-loop where the alignment wasvery difficult to determine (Foran et al. 1988). We
treated these regions as blocks, which resulted in two
gaps with lengths of 27 and 7 1 nucleotides (those marked“c” in table 1E). Probably more than one gap-creating
event occurred in each block. We do not know how

many more occurred, yet those data were included in
the following analysis.
Single-nucleotide insertions and deletions were themost frequent in all the data sets, and the frequency ofgaps decreases as the length increases. A summary of
the number of insertions and deletions is presented intable 2. Deletions are about twice as frequent as insertions, for nuclear DNA. Deletions occur at a slightly
higher frequency than insertions, in mitochondrial DNA;
however, the difference is not as large as that for nuclear DNA.
de Jong and Ryd6n ( 198 1) compared human hemoglobin variants with hemoglobin pseudogene sequences and showed that deletions outnumbered insertions. Graur et al. ( 1989) also found the same tendency
by analyzing human and rodent processed pseudogene
sequences. The present results seem to confirm previous
studies, though the ratio of deletions to insertions varies.
de Jong and RydCn ( 198 1) proposed a model that explained the excess of spontaneous deletions to insertions.

1 2 3 4 5 6 7 8 9 10111213141516171819202324262830384245

Length of Insertions or Deletions
FIG. 4.-Distributionof the lengths of insertions or deletions fornuclear DNA. Results for all four data sets are combined.

FIG. 5.-Insertions/deletionsof P-globin gene region mapped to
the given phylogenetic tree. A, q-Globin pseudogene region (data from
Bailey et al. 1992). B, P-Globin gene spacer region (data from Maeda
et al. 1988).

Evolutionary Rates of Insertion and Deletion

0

FIG. 6.--Insertions/deletionsof immunoglobulin gene region
mapped to the given phylogenetic tree. A, Ig-a gene noncoding region(data from Kawamura et al. 199 1). B, Ig-& pseudogene region (datafrom Ueda et al. 1989).

The mean lengths of insertions or deletions werecomputed for each data set and are shown at the rightmost column of table 2. It is interesting that the mean
is rather uniform over all regions, and the overall averages for nuclear DNA and mitochondrial DNA are
4.5 bp and 5.1 bp, respectively. Values in parentheses
in table 2 include ambiguously determined insertions
and deletions. The mean lengths are not much different
from those for unambiguously determined insertions anddeletions, and the overall averages for nuclear DNA andmitochondrial DNA were 4.6 bp and 4.7 bp, respectively.
Figure 4 shows the distribution of the lengths ofinsertions and deletions for nuclear DNA (results of all
four data sets were combined). Only the unambiguously
determined insertions, deletions, or gaps were used.
About 50% ( 102 of 223) of the total events were singlenucleotide gaps (insertions or deletions), and the fre‘quency of gap events decreases as the length increases.
A high occurrence of single-nucleotide gaps was also observed by Graur et al. ( 1989 ) .
Golenberg et al. ( 1993) analyzed about 0.9 kb ofchloroplast noncoding nucleotide sequence for nine
monocot plants and found 23 single-nucleotide insertions or deletions from 56 events. MO et al. ( 199 1) analyzed the mutation pattern of the rpsL gene of Escherichia coli. They found 12 single-nucleotide insertions
or deletions, while gaps longer than 1 nucleotide occurred 13 times (47 insertion sequences were excludedin that value). Therefore, a high prevalence of singlenucleotide gaps seems to be a common phenomenon
both in eukaryote and prokaryote nucleotide evolution.

509

6 for Ig-a3 w and Ig-a noncoding regions, and figure 7
for mitochondrial DNA D-loop and Y+genome regions.
Insertions and deletions that were both ambiguously andunambiguously determined were included in this analysis.
The number of gaps is approximately proportional
to the length (evolutionary time) of each branch of thetrees. This suggests that the evolutionary rate of insertionand deletion is more-or-less constant over primate evolution. We therefore estimated the rates of insertion anddeletion as follows.
The evolutionary rate of insertion and deletion was
estimated by using two methods. The first one is that of
Saitou ( 1992), in which the total number of insertions
and deletions is divided by the total branch length (interms of evolutionary time) of the assumed phylogenetic
tree and by the length of the nucleotide sequences compared. Table 3 shows the rates thus obtained. It is interesting that rates among four nuclear DNA data sets
are more-or-less constant, with a range of 0.14-0.24/
kb/Myr. If we take the average for those values, weighted
by the length of nucleotides compared, it becomes 0.17 /kb /Myr.
The same is true for mitochondrial DNA. The rates
for D-loop region and for l&genome region were estimated to be 1.8 1 and 2.12 / kb/Myr, respectively, andthe weighted mean became 1.84/ kb/Myr (see table 3 ) .
Thus the rate of insertion and deletion in mitochondrial
DNA seems to be > 10 times higher than that in nuclear
DNA.
Saitou ( 1992 ) analyzed four sets of noncoding nucleotide sequences of hominoid nuclear DNA and estimated the evolutionary rate of insertion and deletion
to be 0.18 / kb/Myr. That estimate is close to the corresponding rate (0.17 / kb/Myr ) obtained by the present
study, though the sequence data that were used overlapped with those used for the present study.
A simple regression analysis was used for the second
method. In this case, each branch of a tree was plottedA

Rates of Insertion and Deletion for Nuclear andMitochondrial DNAs
Insertions and/or deletions were mapped to thephylogenetic tree of primates (fig. 1) for each data set,
and the results are shown in figures 5-7. Figure 5 shows
those for rl-globin \v and P-globin spacer regions, figure

FIG. 7.-Insertions/deletionsof mitochondrial DNA mapped to
the given phylogenetic tree. A, D-loop region (data from Foran et al.
1988). B, i/3-genome region (data from Horai et al. 1992).

5 10

Saitou and Ueda

separately. The abscissa is evolutionary
time (with theunit of Myr), and the ordinate is the number of insertions and deletions per kilobase of a branch (see fig. 8 ) .
Because each branch of a tree evolved independently
from each other, the observations
are independent.
Thisjustifies the regression analysis. Because there should beneither insertions nor deletions when the evolutionary
time is zero, the regression through the origin (Zar 1984)
was calculated. In this case, the regression coefficient or
the evolutionaryrate (b) of insertion and deletion isestimated by

b = Cxiyi/Zx~,(1)

where Xi and yi are branch length and number of insertions and deletions per kilobase for branch i, respectively.
Evolutionary
rates thus obtained for nuclear andmitochondrial
DNA are presented, with their standard
errors, in the right-mostcolumn of table 3. Standard
errors for those estimates were unexpectedly
low, whichsuggests constancy for the evolutionaryrate of insertionand deletion. The rate for the Ig-a gene was considerably
higher than those for the remaining three nuclear DNA
data, and the difference was statistically significant. This
seems to suggest that the rate of insertion and deletion
varies from gene to gene in the nuclear DNA. Because
the data set used in the present study was not extensive
and because the difference of rates between nuclear andmitochondrial
DNA data was much larger than thatamong the nuclear DNA data, however, we combined
the four data sets for nuclear DNA, as well as two for
mitochondrial
DNA, to elucidate a general picture (seefig. 8). Evolutionary
rates for nuclear and mitochondrial
DNA thus became 0.15 k 0.01 and 2.27 + 0.2 1 /kb/
Myr, respectively. It is clear that the evolutionary
rate

Table 3
Estimates

for the Evolutionary

2.27&0.21/kb/MY(mtDNA)

0 nucDNAn mtDNAI

0.1 SM.Ol/kb/MY@w-DNA)

0

10

20

30

40

50

60

Branch Length (MY)
FIG. 8.-Comparison
deletions between nuclearof the evolutionary
rates of insertions
and mitochondrial
DNA.

and

for mitochondrial
DNA is much higher than that for
nuclear DNA. The estimates obtained from regression
analysis are not much different than those obtained by
the first method.
In this analysis, the maximum-parsimony
principle
was used both for multiple alignments and for allocationof gaps. This may cause underestimationof the evolutionary rate, when we compare divergent sequences,where multiple insertions and deletions may occur atthe same positions, as in the case of multiple nucleotide
substitutions.
Unlike the various correction methods for
estimating the number of nucleotide substitutions,however, there is, so far, no method for correcting for multiple insertions and deletions.
We thus examined the effect of branch lengths on
the estimation of the rates of insertion and deletion for
nuclear DNA. Table 4 shows the rates estimated for var-

Rates of Insertion and Deletion
RATE
W/Wr)

DATA SET
Nuclear DNA:
q-Globin ye . . . .
P-Globin spacer
Iga . . . . . . .
Ig&w. . ..
Overall....
MitochondrialDNA:D-loop. .. ..
r/3-Genome...
Overall. ....

LENGTHTOTALTIME

No. OF GAP EVENTS

(kb)

(Myr)

Method

152
56
27

6.4
3.2
1.5
2 23
13.4

144
129
74
37

0.16
0.14
0.24
0.21
0.17

0.12
0.14
0.24
0.16
0.15

1.1

19.5
56.5

1.81
2.12
I .84

2.01 +- 0.38 (4)2.3 1 + 0.26 (8)2.27 + 0.21 (13)

_lJ3
253
39
12

51

2 01
1.2

a Regression coefficient through the origin + its standard error. Values in parentheses are degrees of freedom.

1

Method2”+ 0.04 (10)+ 0.01 (8)h 0.01 (6)f 0.03 (4)+ 0.01 (31)Evolutionary

Rates of Insertion

Table 4Comparison of the Rates of Insertion and Deletion for Nuclear DNA at Each Branch of the Phylogenetic

and Deletion

5 11

Tree

RATE FOR(/kb/Myr)
BRANCH (Length)
C-B(l)...................
B-human (5) ...............
B-A-chimpanzee(5) .........
C-gorilla (6) ...............
D-C(7)...................
D-orangutan( 13) ...........
D-E-F-Old World monkey (37)
F-G-New World monkey (55) .

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

q-Globin

P-Globin

Ig-a

Ig-&3

Average

1.05
0.25
0.23
0.20
0.2 1
0.20
0.13
0.11

0.47
0.28
0.13
0.10
0.11
0.12
0.11
0.15

0.33
0.27
0.27
0.28
0.19
0.23
0.24

0.61
0.30
0.40
0.17

0.75
0.27
0.24
0.18
0.18
0.18
0.14
0.12

. . .

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

a Branches of fig. 1.Length is in Myr.

ious branches of the phylogenetic tree of figure 1. Thereis indeed a tendency
for evolutionary
rates of longer
branches
to be slower than those for shorter ones.
Therefore, the true rate of insertion and deletion might
be somewhat greater than the rate (0.15 /kb/Myr)
obtained by considering all branches. A similar observation
was obtained by Golenberg et al. ( 1993)) who compared
the rates within and among tribes of plants. More sequence data will be necessary to clarify this point.
Saitou ( 1992) showed that the evolutionaryrate ofinsertion and deletion for the branch C-D (see fig. 1)
was much higher than those for the remaining branchesof the phylogenetic
tree involving human, chimpanzee,
gorilla, and orangutan.
The same tendency was also observed in the present study; however, the high estimateof the rate for the branch C-D depends on the branchlength estimation.
If the real branch length is 2 Myr
instead of 1 Myr, as assumed in the present study, theconsiderablyhigh rate along branch C-D decreases.
Implicationsof the Evolutionary
and Deletion

Rates of Insertion

Li and Sadler ( 199 1) compared allelic differencesof human nuclear DNA sequences and found 4.4 gaps
in 23.4 kb of 3’ and 5’ untranslated
regions of exons. If
we use our estimate ( -0.15-o.
17 / kb/Myr) for the evolutionary rate of gaps for nuclear DNA, divergence timeof two human alleles becomes (4.4/23.4)/(
2 X -0.150.17) = -0.55-0.63
Myr, under the assumption of constancy of the evolutionary
rate. This divergence time isconsistent
with the estimate for the coalescence time
( -0.8 Myr ) of neutral nuclear genes of humans (Takahata 1993 ) . This supports our result that the evolutionary rate of insertion and deletion is more-or-less constant.
Saitou ( 199 1) estimated the number of nucleotide
substitutions
between human and chimpanzee
to be

0.0 139/nucleotidesite for noncoding
nuclear DNA. If
we assume the divergence time between these two species
to be 5 million years, then the evolutionaryrate of nucleotide substitution
becomes 1.39 X 10 -9 / nucleotidesite/year, or 1.391 kb/Myr. Therefore, nucleotide substitutions
occur about eight to nine times more frequently than insertions and deletions in noncoding
regions of primate nuclear DNA.
The rate of insertion and deletion for mitochondrial
DNA was estimated to be - 1.8-2.3 / kb/Myr. Thus therate of insertion and deletion for mitochondrialDNAis much higher ( - 1 l- 15 times) than that for nuclear
DNA. Kondo et al. ( 1993) estimated the rate of synonymous substitution to be 23.7/kb/Myr,
which is - 17times higher than that for nuclear DNA. Therefore, nucleotide
substitutions
again occur more frequently( - lo- 13 times) than insertions and deletions in noncoding regions of primate mitochondrial
DNA.
These clear-cut rate differences between substitution (high) and insertion and/ or deletion (low ) and between mitochondrial
DNA (high) and nuclear DNA(low) suggest a common mechanism
for higher evolutionary rate, for mitochondrial
DNA than for nuclear
DNA, of nucleotide substitution
and of insertion anddeletion. It is possible that the low fidelity of DNA replication in mammalian
mitochondrial
DNA affects therate of both substitution
and insertion and/or deletion.
If this conjecture is true for primate or mammalian
genomes in general, then we expect to observe a similar
evolutionaryrate of insertion and deletion for nuclear
and mitochondrial
DNA of Drosophila, in which therate of nucleotide substitution
is known to be more-orless the same between nuclear and mitochondrial
DNA(Powell et al. 1986; Sharp and Li 1989). Our prediction
awaits the arrival of abundant nucleotide sequence data
for Drosophila. In conclusion, as far as primate DNA is5 12 Saitou and Ueda

LI, W.-H., and L. A. SADLER. 1991. Low nucleotide diversity
in man. Genetics 129:5 13-523.
MAEDA, N., C.-I. WV, J. BLISKA, and J. RENEKE. 1988. Molecular evolution of intergenic DNA in higher primates:
pattern of DNA changes, molecular clock, and evolutionof repetitive sequences. Mol. Biol. Evol. 5: l-20.
Acknowledgments
MIYAMOTO,M. M., J. L. SLIGHTON,and M. GOODMAN.1987.
Phylogenetic relations of humans and African apes from
The authors thank an anonymous reviewer for his
DNA sequences in the VII-globin region. Science 238:369comments and Mrs. T. Kawamoto for technical assis373.
tance. This study was supported by grants-in-aid from
MO J.-Y., H. MAKI, and M. SEKIGUCHI. 1991. Mutational
the Ministry of Education, Science, and Culture, Japan.
specificity of the dnaEl73 mutator associated with a defectin the catalytic subunit of DNA polymerase III of EscheLITERATURE CITED
richia coli. J. Mol. Biol. 222:925-936.
NEI, M. 1987. Molecular evolutionary genetics. Columbia
BAILEY,W., K. HAYASAKA,C. G. SKINNER,S. KEHOE, L. C.
University Press, New York.
SIEU, J. L. SLIGHTON, and M. GOODMAN. 1992. ReexPOWELL,J. R., A. CACCONE,G. D. AMATO, and C. YOON.
amination of the African hominoid trichotomy with ad1986. Rates of nucleotide substitution in Drosophila miditional sequences from the primate /3-globin cluster. Mol.
tochondrial DNA and nuclear DNA are similar. Proc. Natl.
Phylogenet. Evol. 1:97-135.
Acad. Sci. USA 83:9090-9093.
DEJONG, W. W., and L. RYD~N .198 1. Causes of more frequent
SAITOU, N. 199 1. Reconstruction of molecular phylogeny ofdeletions than insertions in mutations and protein evoluextant hominoids from DNA sequence data. Am. J. Phys.
tion. Nature 290: 157-l 59.
Anthropol. 84:75-85.
FORAN, D. R., J. E. HIXSON, and W. M. BROWN. 1988. Com. 1992. Evolutionary rate of insertions and deletions inparisons of ape and human sequences that regulate mitonucleotide sequences. Pp. 335-343 in N. TAKAHATA, ed.
chondrial DNA transcription and D-loop DNA synthesis.
Population paleo-genetics: proceedings of the Seventeenth
Nucleic Acids Res. 16:584 l-586 1.
Taniguchi International Symposium on Biophysics. JapanGOLENBERG,E. M., M. T. CLEGG, M. L. DURBIN,J. DOEBLEY,
Scientific Societies Press, Tokyo.
and D. P. MA. 1993. Evolution of a noncoding region of SHARP, P. M., and W.-H. LI . 1989. On the rate of DNA sethe chloroplast genome. Mol. Phylogenet. Evol. 2:52-64.
quence evolution in Drosophila. J. Mol. Evol. 28:398-402.
GRAUR, D., Y. SHUALI,and W.-H. LI. 1989. Deletions in proSIBLEY,C. G., and J. E. AHLQUIST. 1987. DNA hybridization
cessed pseudogenes accumulate faster in rodents than inevidence of hominoid phylogeny: results from an expanded
humans. J. Mol. Evol. 28:279-285.
data set. J. Mol. Evol. 26:99- 12 1.
HAYASAKA,K., T. GOJOBORI,and S. HORAI. 1988. Molecular
SNEATH,P. H. P., and R. SOKAL. 1973. Numerical taxonomy.
phylogeny and evolution of primate mitochondrial DNA.
W. H. Freeman, San Francisco.
Mol. Biol. Evol. 5:626-644.
TAJIMA, F., and M. NEI. 1984. Estimation of evolutionary
HORAI, S., Y. SATTA, K. HAYASAKA,R. KONDO, T. INOUE,
distance between nucleotide sequences. Mol. Biol. Evol. 1:
T. ISHIDA, S. HAYASHI, and N. TAKAHATA. 1992. Man’s
269-285.
place in hominoidea revealed by mitochondrial DNA ge- TAKAHATA,N. 1993. Allelic genealogy and human evolution.
nealogy. J. Mol. Evol. 35:32-43.
Mol. Biol. Evol. 10:2-22.
KAWAMURA,S., H. TANABE, Y. WATANABE,K. KUROSAKI, UEDA, S., Y. WATANABE,N. SAITOU, K. OMOTO, H. HAYN. SAITOU, and S. UEDA. 199 1. Evolutionary rate of imASHIDA, T. MIYATA, H. HISAJIMA, and T. HONJO. 1989.
munoglobulin alpha noncoding region is greater in homiNucleotide sequences of immunoglobulin
epsilon pseunoids than in Old World monkeys. Mol. Biol. Evol. 8:743dogenes in hominoids and their phylogenetic relationships.
752.
J. Mol. Biol. 205:85-90.
KIMURA, M. 1983. The neutral theory ofmolecular evolution.
ZAR, J. H. 1984. Biostatistical analysis, 2d ed. Prentice-Hall,
Cambridge University Press, Cambridge.
Englewood Cliffs, N.J.
KONDO, R., S. HORAI, Y. SATTA, and N. TAKAHATA. 1993.
Evolution of hominoid mitochondrial DNA with special
SHOZO YOKOYAMA, reviewing editor
reference to the silent substitution rate over the genome. J.
Mol. Evol. 36:5 17-53 I.
KOOP, B. F., M. GOODMAN,P. Xv, K. CHAN, and J. L. SLIGH- Received November 18, 1993
TON. 1986. Primate rl-globin DNA sequences and man’s
Accepted January 2 1, 1994
place among the great apes. Nature 319:234-238.

concerned, the rate of nucleotide substitution seems to
be - 10 times higher than that of insertion and deletion,and both rates are > 10 times higher in mitochondrial
DNA than in nuclear DNA.