Relative Efficiencies of the Fitch-Margoliash, MaximumParsimony, Maximum-Likelihood, Minimum-Evolution,
and Neighbor-joining Methods of Phylogenetic Tree
Construction in Obtaining the Correct Tree’
Naruya Saitou and Tadashi Imanishi
Departmentof Anthropology,

Faculty of Science, The Universityof Tokyo

The relative efficiencies of several tree-making methods for obtaining the correct
phylogenetic
tree were studied by using computer simulation. The methods examined were the Fitch-Margoliash(FM), maximum-parsimony(MP), maximumlikelihood (ML), minimum-evolution(ME), and neighbor-joining(NJ) methods.
We simulated the evolutionary changes of six DNA sequences each with a lengthof either 300 or 600 nucleotides.
Both constant and varying rates of nucleotide
substitution were considered. The DNA sequences generated were used to reconstruct phylogenetic
trees by applying the five tree-making methods, and the trees
obtained were compared with the model (correct) tree. This process was repeated
50 times for each case, and the following results were obtained: ( 1) The efficiencyof obtaining the correct tree for the FM method was considerably lower than thosefor the other methods. (2) The NJ and ME methods showed a high performancein obtaining the correct tree, and their relative efficiencies were similar to eachother. ( 3) For distance methods (NJ, FM, and ME), the results obtained by using
corrected nucleotide substitutions
were much better than those obtained by using
nucleotide differences when the rate of substitution varied greatly among different
branches. (4) The ML method was slightly inferior to the NJ and ME methods
when a constant rate of nucleotide substitution
was assumed, but it was slightly
better than the latter two methods when the evolutionaryrate varied drasticallyamong branches. If one considers the computationaltime involved, the NJ method
seems to be a method of choice.

Introduction
The reconstructionof phylogenetic
trees is one of the most importantand interesting problems of evolutionary
study. There are many methods for constructing
phylogenetic trees from molecular data. They can be classified into two categories according
to the strategy used for finding the best tree. One strategy is to examine all or a large
number of possible trees and choose the best one under a certain criterion. We callthis the “exhaustive-search”
method. The other strategy is to examine local topologicalrelationships of a tree and construct the best tree step-by-step. We call this the “stepwise
clustering” method. The maximum-parsimony(MP) method (Eck and Dayhoff 1966;
Fitch 1977), the Fitch-Margoliash(FM) method (Fitch and Margoliash1967)) andthe maximum-likelihood(ML) method (Felsenstein
198 1) belong to the first category,
1. Key words: Fitch-Margoliash method, maximum-parsimony
minimum-evolution method, neighbor-joining method.

method, maximum-likelihood method,

Address for correspondence and reprints: Naruya Saitou, Department of Anthropology, Faculty of
Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan.
Mol. Biol. Evol. 6(5):5 14-525. 1989.
0 1989 by The University of Chicago. All rights reserved.
0737-4038/89/0605-0007$02.00

514

Comparison of Phylogenetic Tree-making Methods

5 15

whereas the neighbor-joining (NJ) method (Saitou and Nei 1987) and many otherdistance methods (see Nei 1987 for a review) fall into the second category.
Because of their computational simplicity, the statistical properties of the stepwise
clustering methods have been studied extensively (e.g., see Tateno et al. 1982; Saitou
and Nei 1987; Sourdis and Krimbas 1987; Sourdis and Nei 1988). However, thoseof exhaustive-search methods are not well understood. Tateno et al. ( 1982) and Sourdis
and Krimbas ( 1987) studied the FM method, but they examined only a small numberof trees.
When the number of operational taxonomic units (OTUs) is small, we can examine all possible trees. Saitou and Nei ( 1986) studied the MP method and the FM
method for the case of four OTUs. Hasegawa and Yano ( 1984) and Saitou ( 1988)
examined the MP method and the ML method for the case of four OTUs. Recently,
Sourdis and Nei ( 1988) studied the MP method for the case of six and eight OTUs,
examining a subset of possible trees that were close to the model tree, including themodel tree itself.
The objective of the present study was to examine the efficiencies of the exhaustivesearch methods more extensively by using computer simulation. We studied the following four exhaustive-search methods: the MP method (Eck and Dayhoff 1966; Fitch
1977), the FM method (Fitch and Margoliash 1967)) the ML method (Felsenstein
198 1), and the minimum-evolution(ME) method proposed by the present study.
The NJ method (Saitou and Nei 1987) was also studied as a representative of the
stepwise clustering method. In the present study, we used model trees for six OTUs,
for which there are 105 possible unrooted trees. All the possible trees were examined
for the case of the exhaustive-search methods-except
for the ML method, in which
only a limited number of trees were examined.
It should be mentioned that two types of errors can occur in the construction ofphylogenetic trees: topological errors and branch-length errors (Tateno et al. 1982).
Topological errors are more serious than branch-length errors, and we considered only
topological errors in the present study. We are mainly concerned with the efficiencyof each tree-making method for obtaining the correct tree topology, and the proportion(P,) of trees in which the correct topology was obtained in 50 replications was used
as a measure for comparison of the methods.
Models and Methods of Simulation

Two of the model trees used were the same as those of Sourdis and Nei ( 1988);
trees A and B in figure 1 correspond to trees (C) and (A) in their figure 1, respectively.
These trees were formed under the assumption of constant rate of nucleotide substitution, and the expected number of nucleotide substitutions per site, from the ancestral
sequence to an extant sequence, is denoted by U. U = 0.05 and U = 0.5 were used
for both trees. The length of each branch was expressed as multiples of a ( = U/ 8 ) (seefig. 1).
We used two other model trees in which there was a large variation in the rateof nucleotide substitution (trees C and D in fig. 1). For both trees, a = 0.01 anda = 0.05 were used. It should be noted that the heterogeneity of evolutionary rate ismore extreme in trees C and D than in the model trees with varying rates used by
Saitou and Nei ( 1987 ) and Sourdis and Nei ( 1988 ) .
If we neglect the label of OTUs, there are only six rooted and unlabeled trees for
six OTUs. Four of them (trees A-D of fig. 1) are used in our study. Because the labelsof OTUs are arbitrary, these are not shown in figure 1. In the actual computer sim-

5 16

Saitou and Imanishi

8a

6a

8a

D

a
9a

6a
a
a

7a
7a

a

9a
a
8a

FIG. 1.-Fourmodel trees used for the computer simulation. Constant rate of nucleotide substitution
is assumed for trees A and B. U (=Sa) = 0.05 and U = 0.50 were used in the computer simulation. The
rate of nucleotide substitution
varies with branch for trees C and D. a = 0.01 and a = 0.05 were used forthese trees.

ulation, however, OTUs of the model tree were of course labeled to specify the tree
topology.
The method of computer simulation
used is the same as that of Saitou and Nei( 1987). The ancestral sequence of a given number (300 or 600) of nucleotides was
generatedby using pseudorandom
numbers,
with equal frequencies
for the four
nucleotides(A, G, T, and C) being assumed. The evolutionof this sequence wassimulated by using pseudorandom
numbers, according to the predeterminedbranchingpattern of the model tree. Random nucleotide substitutions(the one-parameter
modelof Jukes and Cantor 1969) were introducedin each branch of the tree by following a
Poisson distribution
with the mean equal to the expected branch length of the model
tree. After the nucleotide sequences for six OTUs were produced, the proportion(p)of the nucleotide differences and Jukes and Cantor’s ( 1969) evolutionary
distance (d)were computed for each pair of sequences. The entire process of simulation was repeated
50 times for each case.
In the MP method, a tree that has the smallest number of nucleotide substitutions
required for explaining the evolutionarychanges of DNA sequences is chosen as thebest tree. A simple and quick computer program for counting this number for nucleotide sequence data was developed by applying Fitch’s ( 197 1) algorithm. All 105
trees were examined.
Felsenstein’scomputer program DNAML in his PHYLIP package, version 3.1,
was used for the ML method. Because of the long computationaltime required forthis method, we compared a limited number of trees by using the U (user-tree) optionof DNAML. For six OTUs, 105 possible trees can be classified into four groups interms of the topological difference from the model (true) tree. The topological difference
was measured by the method of Robinson and Foulds ( 198 1), and it was 0,2,4, and
6 for groups 1, 2, 3, and 4, respectively. The numbers of trees for groups 1, 2, 3, andComparison of Phylogenetic Tree-making Methods

5 17

4 are 1 (true tree), 6, 24, and 74, respectively (see Sourdis and Nei 1988). As apreliminary
study, 3 1 trees that belonged to groups 1,2, and 3 were examined for thecase of model tree B (U = 0.5 with 300 nucleotides)
and for the case of model tree C(a = 0.0 1 and 0.05 with 300 nucleotides).
Because the tree with the highest likelihood
almost always belonged to either group 1 or group 2, only the seven trees in these twogroups were examinedfor other cases, as Sourdis and Nei ( 1988) did for the MP
method.
Computationof maximum-likelihood
values requires various assumptions.
We
set the options of DNAML such that the likelihood formulation
is as close as possible
to the simulation
scheme used: ( 1) The C (categories) option was not used because
nucleotide substitutions
at each site occurred independently
and at the same rate inour simulation.
(2) The frequencies of the four nucleotides were all set to be 0.25. ( 3)The transition/transversion
ratio was set to be 1.0 by using the T option. Under thisassumption,
the rate of transitional
substitution
is twice that of transversional
substitution. (Unfortunately,in the current version of DNAML we cannot assume Jukes
and Cantor’s one-parametermodel of nucleotide substitutions,which was used in our
simulation.)
In any case, the assumptions
adopted in the computation
were favorable
for the ML method.
The other three methods (FM, ME, and NJ) require a distance matrix, and bothp and d were used, as Saitou and Nei ( 1987) did previously. Note that the p distancefollows the triangle inequality and is a metric, whereas the d distance is a nonmetric.
For the FM and ME methods, all 105 topologies were examined as in the case of the
MP method.
The criterion for finding the best tree used in the FM method is the so-called
percent standard deviation (PSD), defined as follows:

PSD =2 &{ (00 - E,)l&}*n(n - 1)

1“* X loo,(1)

for i < j, where n is the number of OTUs compared and DC and Eti are the observed
and estimated distances between OTUs i and j, respectively. In the FM method, atree showing the smallest PSD values is chosen as the best one. A simple and quick
computer program was developed for computing the estimated distances by Fitch andMargoliash’s ( 1967 ) procedure.
Tateno et al. ( 1982) proposed a measure (So) for the comparison
between estimated and observed distances:so =2 c,(D,1- Eti)* “* .

n(n - 1)

(2)

So is related to PSD, and it can also be used for choosing the best tree (Nei 1987, pp.
30 l-302). Thus we also used So as a criterion for the topology finding. This modification of the FM method is called the FM’ method. Comparisonof equations ( 1) and(2) suggests that PSD and So are highly correlated. They did show similar results, as
will be seen later.
The ME method is a new method proposed in the present paper. We first computeeach branch length of a given tree topology by using Fitch and Margoliash’s ( 1967)
procedure for branch-length
estimation,and the tree that shows the smallest sum of5 18

Saitou and Imanishi

Table 1
Proportions @J) of Trees in Which the Correct Topology Was Reconstructedunder the Constant-Rate Models

d

P

MP

ML

FM

FM’

ME

NJ

FM

FM’

ME

NJ

... ..
.. .

34
76

38
80

26
58

26
58

36
80

42
78

26
58

26
58

40
80

40
82

..
..

60
84

48
70

30
54

32
50

56
92

58
92

22
40

24
34

42
82

46
82

54
84

62
88

42
56

38
58

68
82

68
84

40
56

38
56

68
80

70
86

48
58

56
76

34
36

30
36

70
76

68
76

30
36

28
28

60
70

60
70

used in this method

is theMODEL TREEA:
u = 0.05:
300 bp600 bp.
u = 0.50:
300 bp .
600 bp.

.
.

B:
u = 0.05:
300 bp.
600 bpu = 0.50:
300 bp .
600 bp .
NOTE.-Model... ..
... . .

trees A and B are shown in fig. 1.

branch lengths is chosen as the best one. Thus the criterion
sum of branch lengths (SBL) for a given tree:
SBL = C Li,

where Li is the estimated length of the ith branch.
The principle of minimum
evolution was proposed by Cavalli-Sforza and Edwards( 1967 ), who considered a Steiner tree. With the use of Fitch and Margoliash’s procedurefor branch-length
estimation,
computation
becomes much simpler than the CavalliSforza and Edwards method. The ME method also seems to be related to Dayhoff’s(1978) method (see Blanken et al. 1982).
Although this principle bears some resemblance to that of maximum parsimony,
the ME method is much more similar to the NJ method. This is because the principleof minimum
evolution is also adopted in the NJ method (see Saitou and Nei 1987)
and because both the ME and NJ methods are distance methods. Our simulation
results have shown that the ME method and the NJ method are indeed quite similar.
However, the ME method is an exhaustive-searchmethod and examines all possible
trees to choose the best one. In contrast, the NJ method is a stepwise clustering method,
and the computationaltime is much shorter in this method than in the ME method.
Results and Discussion
Model Trees with ConstantEvolutionary

Rate

Table 1 shows the proportion (P,, in % ) of trees in which the correct tree topology
was obtained. When model tree A (U = 0.05 ) is used, there is a clear dichotomy interms of the efficiency, measured by P,. That is, the FM and FM’ methods are lessefficient than the other four methods. However, the efficiencies of these two methods

Comparison of Phylogenetic Tree-making Methods

5 19

are higher for the case of 600 nucleotides than for that of 300 nucleotides. Therefore
these methods may eventually become as useful as the other methods if long nucleotide
sequences are compared.
This low efficiency of the FM and FM’ methods is also observed for the case of
U = 0.50, but the other four methods show some differences in P,. The ME and NJ
methods perform quite well when the p distance is used, and the MP method is also
good. Compared with these three methods, the ML method is less efficient, but it isbetter than the FM and FM’ methods. It is interesting that all four distance methods(FM, FM’, ME, and NJ) show slightly better performances
when p distances are usedthan when d distances are used. This is consistent with the previous results of Saitou
and Nei ( 1987) and Sourdis and Krimbas ( 1987).
The results for model tree B are similar to those for model tree A; the FM and
FM’ methods are both less efficient than the other four methods. For the case of
U = 0.05, the ML method is better than the MP method, but the ML method isslightly less efficient than the ME and NJ methods when 300 nucleotides are compared.
As in the case of model tree A, for the FM, FM’, ME, and NJ methods p distances
give slightly better results than do d distances.
In conclusion,
the FM and FM’ methods are consistently
less efficient than theother four methods. Saitou and Nei ( 1986) studied the efficiencies of several treemaking methods, including the MP, FM, and NJ methods, for the case of four OTUs
and showed that the FM method was worst among the three (the transformed-distancemethod used in their study is identical with the NJ method for the case of four OTUs) .
Thus the present finding supports Saitou and Nei’s ( 1986) conclusion. The results formodel trees A and B also suggest that the time-consuming
ML method is no better
than the other, much quicker methods. Furthermore,
the close similarity between the
ME and NJ methods indicates that the algorithm for the NJ method gives the minimum-evolution
tree most of the time.
Model Trees with Varying

EvolutionaryRate

The results obtained for model trees C and D are presented in table 2, and they
are in several respects quite different from those for model trees A and B. First, forall four distance matrix methods, d distances give much better results than do p distances. This clear-cut difference has already been noted by Saitou ( 1988) for the caseof a four-sequencetree, and the present study confirms this. Li et al. ( 1988) performeda similar study for four sequences, but they used only p distances, and they concluded
that the NJ method is less efficient than Lake’s ( 1987) method. However, their conclusion might have been drastically different had they used d distances.
Low efficiencies of the FM and FM’ methods are also observed for trees C andD, whereas the ML method is the best in all cases. However, the P, values of the NJ
and ME methods (when d distances were used) are close to those of the ML method.
On the other hand, the MP method is good only for a = 0.0 1; for a = 0.05, the MP
method is much less efficient than the ME and NJ methods. These results clearly show
the superiorityof the principle of minimum
evolution when a correctly estimatedevolutionary distance is used. This is probably because, with the d distances, an additive
tree is approachedas the number of nucleotides
used increases. These results also
indicate that the principle of maximum
parsimonyis different from the principle ofminimum
evolution, contrary to their superficial similarity.

520

Saitou and Imanishi

Table 2
Proportions (96) of Trees in Which the Correct Topology Was Reconstructedunder the Varying-Rate ModelsdP
MODEL TREE

MP

ML

FM

FM’

ME

NJ

FM

FM’

ME

NJ

.
..

64
90

78
98

26
42

38
50

56
80

56
80

34
68

34
78

72
92

72
92

...
...

24
20

92
100

0
0

0
0

2
0

2
0

22
60

16
60

68
96

68
96

.

68
94

80
96

44
56

52
68

64
90

64
90

64
88

50
70

74
92

74
92

.
.

26
46

96
100

0
0

0
0

6
10

4
6

30
64

24
42

78
100

78
100

c:
a = 0.01:
300 bp600 bpa = 0.05:
300 bp600 bpD:
a = 0.01:
300 bp600 bpa = 0.05:
300 bp600 bpTrees Obtained

by Different

Tree-making

Methods

We have seen that the FM and FM’ methods are quite similar in their efficiency
for obtaining
the correct tree, as were the ME and NJ methods. These similarities
occur because the same tree is often chosen by these tree-making
methods. Close
relationshipsof the trees chosen were also observed for other methods. An example
is presented in table 3.
All methods chose an erroneous tree in replication
1; the MP, ML, ME, and NJ

Table 3
Trees Chosen by Different Methods in the Case of Model Tree B with U = 0.50
and 300 Nucleotidesd

P
REPLICATIONNUMBER

2.
3.
4.
5.
6.
7.
8.
9.
10

.....

MP

ML

FM

FM’

ME

NJ

FM

FM’

ME

NJ

5
5
1
1
1
1
2
1
8
3,9

5
5
1
1
1
1
3
1
4
9

4
11
1

4
11
1
1
1
1
2
1
4
10

5
1
1
1
1
1
1
1
8
3

5
1
1
1
1
1
1
1
8
3

4
1
1
1
1
1
2
1
4
8

12
1
10
1
1
1
2
1
4
8

5
5
1
1
1
1
1
1
8
9

5
5
1
1
1
1
1
1
8
9

1
1
1
2
1
4
10

NOTE.-The identification numbers of the trees chosen by each method for the case of model tree B with U = 0.50
and 300 nucleotides are shown. Tree 1 is the correct (model) tree, trees 2-7 are different from tree 1 with a topologicaldifference of two, and trees 8-12 are different from tree 1 with a topological difference of four.

Comparisonof Phylogenetic

Tree-making

Methods

52 1

Table 4
Average Topological Differences between the Trees Obtained by Different Tree-making
Methods and the True Tree
True
True .
FM-d.
ME-d.

F-M-p.
ME-p.
ML .
MP . .

..
..
..
.
..
.
.

2.08
0.96
1.80
0.68
1.oo
1.23

FM-d

ME-d

FM-P

ME-p

ML

MP

2.28

0.76
2.04

5.68
5.00
5.36

3.96
3.84
3.56
3.24

0.16
2.24
0.80
5.60
3.96

2.18
2.50
1.68
4.56
2.94
2.18

1.76
0.72
1.88
1.84
2.17

1.64
0.36
0.64
0.93

1.72
1.72
1.91

0.76
1.01

0.87

NOTE.-Figures
below the diagonal are for case (a) model tree B (U = 0.50 and 300 nucleotides were compared), andthose above the diagonal are for case (b) model tree C (a = 0.05 and 300 nucleotides were compared). The model tree isdesignated as “True.”

methods chose tree 5, whereas the FM and FM’ methods chose tree 4 or tree 12. In
the case of replication
7, the ME and NJ methods chose the correct tree (tree 1) for
both p and d distances, but the other methods chose either tree 2 or tree 3. A moreinteresting situation is observed in replication
10, in which trees 3 and 9 were equallyparsimonious
when the MP method was used. The ML method chose tree 9, as did
the ME and NJ methods when d distances were used. However, tree 3 was chosen by
the ME and NJ methods when p distances were used. The FM and FM’ methods
chose neither tree 3 nor tree 9 (see table 3 ) .
The relationshipof the trees obtained by different tree-making
methods can be
represented
by a matrix of average topological differences for all replications.
Two
examples are shown in table 4. One [table 4(a)] is from the case of model tree B with
U = 0.50 and 300 nucleotides,
and the other [table 4(b)] is from the case of model
tree C with a = 0.05 and 300 nucleotides.
In both matrices, the FM’ and NJ methods
were omitted because these were very close to the FM and ME methods, respectively.
Networks showing the relationships
between the tree-making
methods can thenbe made using the NJ method from the distance matrices in table 4 (see fig. 2) Thiskind of graphic representation
is useful for grasping the relationships
among different
tree-making
methods. There are three clusters in the tree shown in figure 2A: [MEd, ME-p], [FM-d, FM-p], and [ML, MP] . These clusters indicate that the trees obtainedby members of each cluster are highly correlated. For the case of figure 2B, the trees
from d distances and p distances are no longer highly correlated. In this case, ME-p
and FM-p make a cluster, and the ML method is no longer associated with the MP
method. The ML method is closest to the true tree, followed by the ME-d method.
These comparisons
clearly show that the relationships of the trees obtained by different
tree-making
methods can be quite different, depending on the data used. We should
therefore be cautious in selecting a tree-making
method for reconstructionof phylogenetic trees.
A Numerical

ExampleSaitou ( 1988) applied his maximum-likelihood
algorithm and the NJ method tofive mitochondrial
DNA sequences of humans and apes (data from Hixson and Brown
1986), and both methods chose a tree in which two chimpanzee
species (common
chimpanzees
and pygmy chimpanzees)
were clustered and in which humans and gor-

522

Saitou and Imanishi

A
ME-d

ME-p

FM-p

True

B
ME-p
ME-d
True

I
I

ML

FM-d

FM-p
FIG. 2.-Two
relationships
tree, in terms of the topologicalunrooted trees were constructed(A and B) of trees obtained by different tree-making methods and the true
difference matrices for cases (a) and (b) of table 4, respectively. Theseby the NJ method.

illas were clustered. Both the p and d distances obtained from the same data set are
shown in table 5.
Three exhaustive
search methods requiring distance matrices (FM, FM’, andME) were then applied to the distance matrix of table 5, and four different trees given
in table 6 were examined. Common chimpanzees and pygmy chimpanzees are clusteredin trees 1, 2, and 3, whereas pygmy chimpanzees
and humans are clustered in tree 4.
Because the results for p distances were identical with those for d distances, only
the results for d distances are presented in table 6. Values of PSD, So, and SBL wereComparison of Phylogenetic Tree-making Methods
Table 5
Paimise Distances

Measured by the Jukes-Cantor

Pygmy chimpanzee. ...
Gorilla ..............
Human .............
Orangutan ...........

523

Formula for Five Hominoid Species

CommonChimpanzee

Pygmy
Chimpanzee

GorillaHuman

0.0118 (0.0117)
0.0427 (0.04 15)
0.0382 (0.0373)
0.0953 (0.0895)

0.04 16 (0.0405)
0.0327 (0.03 19)
0.09 16 (0.0863)

0.037 1 (0.0362)
0.0965 (0.0905)

0.0928 (0.0873)

NOTE.-Thevalues in parentheses are p distances. Gaps (insertions and additions of nucleotides) were excluded fromthe analysis, and a total of 939 nucleotides were compared. Nucleotide sequence data are from Hixson and Brown (1986).
Scientific binomens for the five species compared are as follows: Homo sapiens (humans), Pan troglodytes (common chimpanzees), Pan paniscus (pygmy chimpanzees), Gorilla gorilla (gorillas), and Pongo pigmaeus (orangutans).

computedfor each tree, and those for the best tree were set to be zero. Tree 2 waschosen by the FM and FM’ methods, whereas tree 3 was chosen by the ME method.
The values for other trees are differences from the value of the best tree. It is interestingthat all three methods gave rather large values when tree 4 was considered. Humans
and pygmy chimpanzees,
instead of common chimpanzees
and pygmy chimpanzees,
were clustered in this tree, and this unusual clustering apparently caused large values
for PSD, So, and SBL.
The comparisonof these four trees was also made for the MP and ML methods.
The MP method chose trees 1 and 3 as equally parsimonious.
This is consistent
with Hixson and Brown’s ( 1986) result. We need a rather large number (eight) of additional nucleotide substitutions
for tree 4, compared with trees 1 and 3. As for the ML
method, we used Felsenstein’s
DNAML program (observed nucleotide
frequenciesof A = 0.333, C = 0.257, G = 0.191, and T = 0.219 and a transition/transversion
ratio = 5.0 were used), and tree 3 gave the highest likelihood value among four trees(see table 6). This is consistent with the result of Saitou ( 1988)) who used a different
maximum-likelihood
algorithm.
The above results suggest that common chimpanzees
and pygmy chimpanzees
should be clustered. Among trees 1, 2, and 3, in all of which two chimpanzee
species
Table 6
Analyses of Primate DNA Sequence Data by the Five Tree-making Methods

MethodTree 1(((PC)H)G)

Tree 2(((PC)G)H)Tree 3((PC)(HG))

Tree 4(((PH)C)G)FM ........
FM’. .......
ME ........
MP ........
ML ........

+0.60
+0.35
+0.35
0
-2.98

0
0
+1.10
+1
-3.97

+0.47
+0.16
0
0
0

+25.33
+4.45
+6.59
+8
-33.86

NOTE.-Thebranching patterns of trees l-4 are represented by designating a cluster of two species (or neighboring
species) i and j as (ij). P, C, H, and G = pygmy chimpanzees, common chimpanzees, humans, and gorillas, respectively.
The remaining species (orangutans) is used as an outgroup species. The values for the FM, PM’, and ME methods(d distances were used) are PSD, So, and SBL, respectively (see text for these quantities). Those for the MP method are therequired number of nucleotide substitutions, and those for the ML method are the log likelihood values. The values for thebest tree are set to be zero, and the other values represent the differences from the values of the best tree. The values for
the PM’ and ME methods should be multiplied by l/1,000.

524

Saitou and Imanishi

were clustered, tree 3 was chosen as the best tree when we used the ME, NJ, and ML
methods. However, tree 1 was chosen in an analysis of a larger data set, in which
Hixson and Brown’s ( 1986) data were combinedwith Brown et al.‘s ( 1982) data
(Saitou and Nei 1986). It has been shown that mitochondrial
DNA sequence data of>2,000 nucleotides are probably necessary for determining
the branching order of themitochondrial
DNA tree (a gene tree) for humans, chimpanzees,
and gorillas (Saitou
and Nei 1986). It is thus clear that no method is reliable unless there are more data.
When the number of nucleotides
used is large, the NJ, ME, and ML methods
show more or less the same efficiency, as seen from the comparisonof the results for
300 and 600 nucleotides. However, there is a substantial difference in the computationaltime between the ML method and the other two methods. The computationaltime
for the MP method is also considerably
shorter than that for the ML method. Besides,
the ML, ME, and MP methods are exhaustive search methods and require a prohibitively long computationaltime when the number of DNA sequences compared islarge. Therefore, if we consider these practical aspects, the NJ method seems to besuperior to the other methods.
Acknowledgments
We thank Dr. Josef Felsenstein for sending us the PHYLIP package and for his
suggestions regarding the DNAML program. We also thank Drs. Masatoshi Nei, Walter
Fitch, and Pekka Pamilo for their helpful commentson the manuscript.
This study
was supported by Ministry of Education, Science, and Culture of Japan Grant-in-Aid
for Scientific Research 62790190 to N.S.
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MASATOSHI

NEI, reviewing

editorReceived

November8, 1988; final revisionAccepted

February

26, 1989

received

February

17, 1989