Proc. Nati. Acad. Sci. USA
Vol. 78, No. 4, pp. 2432-2436, April 1981
Evolution
Evolutionary tree for apes and humans based on cleavage maps of
mitochondrial DNA
(restriction endonucleases/variable and conserved sites/rearrangement/branching order/knuckle-walking)
S. D. FERRIS, A. C. WILSON, AND W. M. BROWN*
Department of Biochemistry, University of California, Berkeley, California 94720
Communicated by Sherwood L. Washburn, December 22, 1980
attain such a view, a sensitive and precise method of analysis
must be used to compare mtDNA of different species. This paper explores the possibility that the mapping of restriction endonuclease cleavage sites can give the required sensitivity and
precision. We present restriction maps containing approximately 50 cleavage sites per mtDNA for the primates mentioned
above as well as for the orangutan and a gibbon. Comparison
of these maps enables a tentative branching order ofthe lineages
to be discerned. In addition, the map comparisons indicate that
rearrangement, as well as base substitution, has occurred in the
evolution of mtDNA in the higher primates. These comparisons
also provide information about the distribution of positions in
the mitochondrial genome at which evolutionary change occurs.
ABSTRACT The high rate of evolution of mitochondrial DNA
makes this molecule suitable for genealogical research on such
closely related species as humans and apes. Because previous approaches failed to establish the branching order of the lineages
leading to humans, gorillas, and chimpanzees, we compared human mitochondrial DNA to mitochondrial DNA. from five species,
of ape (common chimpanzee, pygmy chimpanzee, gorilla, orangutan, and gibbon). About. 50 restriction endonuclease cleavage
sites were mapped in each mitochondrial DNA, and the six.maps
were aligned with respect to 11 invariant positions. Differences
among the maps were evident at 121 positions. Both conserved and
variable, sites are widely dispersed in the mitochondrial genome.
Besides site differences, ascribed to point mutations, there is evidence for one rearrangement: the gorilla map is shorter than the
others owing to the deletion of 95 base pairs near the origin of
replication. The parsimony method of deriving all six maps from
a common ancestor produced a genealogical tree in which the common. and pygmy chimpanzee maps are the most closely related
pair; the closest relative of thispair is the gorilla map; most closely
related to this trio is the human map. This tree is only slightly more
parsimonious than some alternative trees. Although this study has,
given a magnified view of the genetic differences among humans
and apes, the possibility of a three-way split among the lineages
leading to humans, gorillas, and chimpanzees still deserves serious
consideration.
Our understanding of human evolution would advance if we
could find out how humans are related to their closest kin, the
gorillas and chimpanzees. In the absence of an adequate fossil
record for gorillas and chimpanzees (1), we must rely on the
comparison of living species to elucidate the branching order
of the lineages in the evolutionary tree. Yet, in spite of much
comparative work, uncertainty about this branching order
persists.
Anatomists have long thought of gorillas and chimpanzees as
the most similar pair in the trio, but phylogenetic analysis has
never shown convincingly that this pair is the closest genealogically (2, 3). Nor is there agreement among chromosome workers
(4, 5) as to the shape of the evolutionary tree. Furthermore,
biochemical research (6-8) makes it appear that the three lineages diverged simultaneously.
The inability of previous biochemical approaches to resolve
the tree could be a consequence of the smallness of the nucleotide sequence differences found among the nuclear DNAs
of the three types of creatures (9).
Mitochondrial DNA (mtDNA) may offer the resolving power
needed to elucidate- the branching order of these lineages because it evolves faster than nuclear DNA (10), thereby giving
a magnified view of the genetic differences among species. To
MATERIALS AND METHODS
Tissues and Cell Lines. mtDNA was purified from cultured
cells, blood samples from living animals, or organs of animals
that died of natural causes as follows: human HeLa. cells, common chimpanzee (Pan troglodytes) leukocytes, pygmy chimpanzee (Pan paniscus) liver, lowland gorilla (Gorilla gorilla)
liver, orangutan (Pongo pygmaeus) liver, and white-handed gibbon (Hylobates lar) liver. The gorilla, chimpanzee, and orangutan samples were from the Yerkes Primate Center, Atlanta,
GA, and the gibbon sample was from the Veterinary School,
University of California, Davis, CA.
Preparation and Cleavage Mapping of mtDNA. Our method
of preparing mtDNA from tissues and cultured cells has been
described (10). The 19 restriction endonucleases employed,
with their single letter codes, are listed in. the legend to Fig.
1. All enzymes were obtained from New England BioLabs
(Beverly, MA)
I and used according to the supplier's directions.
DNA fragments were labeled at the ends with 32p according to
Brown's description (11). The methods for gel electrophoresis
of DNA fragments and for mapping the cleavage sites have been
described (10, 11). In most cases, the smallest routinely scored
fragment was approximately 150 base pairs (bp). Details of the
cleavage maps will be published elsewhere.
Orientation of the Cleavage Maps. Cleavage maps of
mtDNA were aligned with respect to the origin and direction
of replication. The rationale and assumptions implicit. in this
alignment are discussed elsewhere (10, 12). For several of the.
enzymes employed, the cleavage maps of human and common
chimpanzee mtDNA relative to the origin and direction of replication have been established (10, 13); sites detected with the
remaining enzymes were oriented relative to these. The cleavage maps for the remaining species were aligned with respect
to the 11 cleavage sites common to all six species.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: bp; base pair(s).
*
Present address: Division of Biological Sciences, University of Michigan, Ann Arbor, MI 48109.
2432
Evolution: Ferris et al.
Proc. Natl. Acad. Sci. USA 78 (1981)
firms our placement of the HincII and Xba I sites in this region.
Sites Containing Sequence Ambiguities. Those restriction
enzymes that recognize more than one sequence can be very
informative in mapping, when used in conjunction with "semiisoschizomers," i.e., enzymes recognizing specific subsets of
the larger array of possible sequences. For example, HincII
recognizes G-T-T-G-A-C, G-T-C-A-A-C, G-T-T-A-A-C, and GT-C-G-A-C. The latter two sequences are also recognized, respectively, by Hpa I and Sal 1 (15). Such relationships have been
useful in assessing the homology of sites and in mapping sites
that are very close to each other. For example, a side-by-side
comparison of single enzyme digests of common chimpanzee
mtDNA reveals that the Hpa I fragment produced by cleavage
of the sites at 40 and 54 units is 260 bp larger (i.e., 1.5 units
larger) than the corresponding HincIl fragment from this region. There must, therefore, be an intervening HincII site at
either 41.5 or 52.5 units. A highly conserved Pst I site is located
at 46 units. Side-by-side comparisons of double digests of chimpanzee mtDNA revealed that the fragment made by cleavage
of the Pst I site at 46 units and the HincIl site at 54 units is 260
bp shorter than that formed by the cleavage of the corresponding Pst I and Hpa I sites. Because cleavage of the Pst I site at
46 units and either the Hpa I or the HinclI site at 40 units
yielded identically migrating fragments, the intervening HincII
site must be at 52.2 units.
Gorilla mtDNA has a Deletion. That gorilla mtDNA has a
deletion became evident during mapping of FnuDII sites near
the origin of replication. All six species have FnuDII sites at 85,
92, 95, and 0.5 units (Fig. 1). However, the fragment mapping
from 95 to 0.5 units was 95 bp shorter in gorilla than in human
(or chimpanzee) mtDNAs (Fig. 2a). No FnuDII fragment of this
size was observed upon electrophoresis in 3.5% polyacrylamide, which can allow detection of fragments as small as 16 bp
(11). Studies with other restriction enzymes confirmed this
interpretation. Every small fragment containing the origin of
replication proved to be shorter by about 95 bp in the case of
gorilla mtDNA (see, for example, Fig. 2b). Hence, the deletion
is in the region between 95 map units and the origin of repli-
RESULTS
Genome Size. Electron microscopy reveals that human and
common chimpanzee mtDNA are 16,500 ± 400 bp long (10).
This estimate agrees with that made by electrophoretically measuring the sizes of fragments produced by restriction enzymes
(10). The mtDNA of pygmy chimpanzee, orangutan, and gibbon
are identical in size to human and common chimpanzee mtDNA
according to the electrophoretic method.t In contrast, gorilla
mtDNA is shorter than the other five mtDNAs by about 95 bp
(see below).
Cleavage Maps. The cleavage maps for gorilla, common
chimpanzee, human, orangutan, and gibbon mtDNA appear in
Fig. 1. The 19 restriction enzymes, each of which is designated
by a single letter, cleaved each genome at an average of about
50 sites.
The precision with which a site may be mapped is illustrated
for the case of the Pvu II sites (abbreviated as h on the maps),
which occur at 59.5 map units in gorilla and chimpanzees. Because there is a HindIII (abbreviated as b on the maps) at 63
units in all 6 species, it was used as a reference point to assess
the possible homology ofthe Pvu II sites. From the HindIII and
Pvu II cleavage maps in Fig. 1, one predicts that the double
digest should produce a fragment of about 600 bp, arising by
cleavage of the HindI11 site at 63 units and the Pvu II site at 59.5
units (Fig. 1). If this fragment were 10 bp longer in one species,
its electrophoretic mobility would be detectably different from
that in the other species. In a side-by-side comparison of double
digests, the mobilities of these fragments were identical. Thus,
it is highly probable that the Pvu II sites at 59.5 units are homologous in gorilla and common chimpanzee.
Further evidence for the accuracy of the maps is provided
by comparing them with the published base sequence of the
human mtDNA region from 50 to 54 map units (14), which conabsolute size estimates obtained from gel electrophoresis
have an estimated SD of 5%, relative size estimates are considerably
more accurate. The side-by-side comparisons of the mtDNA from the
six species provided such relative estimates.
t Although
Deletion
f
zj k
rGorilla 11
1%.r -
I
...%a
dmz
a
b jxbgf
mxoz
II 11 11
I
~
00
Hl l
I
~
ei
gwojowoxeeyh
C
II
I
b
c
0
h
oo
hc
a x e kzo
I' 1 I1 11I I I
9ILIfe
IYt
I
C
km m fmexbf
z
Chimp
II I
a
I
m
-HumanilLI
I
bxbgwh
11111 1 I I fL
iI l
zj kk
c
oz eyzg
oz
e
xb
I
11111 III11
I
zjo k m
Orang
LL
m
Gibbon 11
I-f I LJ LO %.f I I
m
oh bf
m
I
I
I
I
d
a
f myb
I-
I
0
mooxo
I11II11II11
I yp
m
zb
20
i
m I
e
j
I
zg xeoox
LiJj,
I
i
I
zo
I
c
axo
kzo
1I I1 icr'
c
yxbzyz j.
I Lil W I
k
axybe kz
I -tf id
C
ec
.
I
.
bxfo
Id
m m
I
40
zw
hed
1 se
.
.
..
oboz
ah e
n
C
0
h
0
I obol o11 I IyLx kz
,,,,,irl ,, , , .Ih,
60
80
common chimpanzee. The scale is in
map units with the origin ofreplication
h
x
for the FnuDIl sites in the region from
11 I
the position of one HincII site in orangutan mtDNA, which has been arbitrarily put at 36 units but may instead be at 39 units. The small letters
represent sites of cleavage by the following restriction enzymes: a, EcoRI;
b, HindIII; c, Hpa I; d, Bgl II; e, Xba I;
f, BamHI; g, Pst I; h, Pvu II; i, Sal I; j,
Sac I;I;k,o, Kpn
I; m,x,Ava
I; 1,w,Xho
y,
Bcl I;I; n,
BstEII;
Sma
HincII;
at 0 units and the direction of replication to the right. Each map is for a
single individual. The positions shown
96 to 99 units in the mtDNA of orangutan and gibbon are tentative, as is
ze z
a
h
oc
gywbyo a w a x e kzo Zeoz
I
I i r' III?
11 I I I
c
mxwo
Is
jgk
I I1 I1 I11
I
b
I
1 Ii
LI 111 11
I11
C
yfm
d
zx
xjoo )yhmbyo oh
c
ac
fm oombo
c
e e
FIG. 1. Cleavage maps of mitochondrial DNA from five species of
higher primates. "Chimp" refers to the
h
ze z
11 I
0
~
2433
zeozzz
I
'IP,
m
n
x
If..I I'
f
100
Bgl I, and z, FnuDll. The mtDNA for
pygmy chimpanzee (not shown) differs
from mtDNA for common chimpanzee
by having cleavage sites at an additional 11 positions (3w, 19o, 25b, 27z,
34z, 37y, 64h, 66a, 73z, 94y, and 99w)
and lacking cleavage sites at 8 positions (4k, 17x, 45y, 51j, 54co, 59y, 60m,
and 79o).
Evolution: Ferris et al.
2434
H
Proc. Natl. Acad. Sci. USA 78 (1981)
H
G(
b
a
U
b
a
G
g
Gorilla
Chimps
Humaor
...
Orong
Gibbon
o.
2905
`2810
-760
-665
FIG. 2. Electrophoretic detection of a deletion in gorilla mtDNA.
(a) Restriction digests ofhuman (H) and gorilla (G) mtDNA were prepared with the enzyme FnuDH and subjected to electrophoresis. The
gorilla mtDNA digest differs from human by having a fragment 665
bp long, instead of760 bp. This fragment spans the region from 95 units
to 0.5 unit on the maps. (b)Kpn I digests ofhuman and gorilla mtDNA.
The gorilla mtfNA~digest differs from that ofhuman by having a 2810bp fragment instead of the 2905-bp fragment. This fragment spans
from 84.5 units to4 units on the map (Fig. 1). Hence, the 95-bp deletion
must have occurred between 95 units and 0.5 unit on the map.
cation (see Fig. 1). The deletion is evident inmtDNA from every
one of four gorillas examined.
Comparison of Maps. Of the six cleavage maps compared;
those of the two chimpanzees are most similar. The number of
sites mapped is 48 for the common chimpanzee and 51 for the
pygmy chimpanzee. Of those sites, 40 occur at identical positions in the two species. By contrast, the two most dissimilar
maps, those of the orangutan and gibbon, share only 19 sites.
From these results, we estimated the probable percentage
difference in base sequence for each pair of mtDNAs with the
method of Nei and Li (16), using their equation 15. The estimated sequence differences range from 4% to 23%. These values exceed those obtained from annealing studies with singlecopy nuclear DNA (6, 9), confirming for the Hominoidea that
mtDNA evolves 5-10 times faster than, nuclear DNA (10).
Evolutionary Tree. The minimal number of mutations required to produce the six maps from an ancestral map was calculated. The tree that would allow the observed maps to evolve
with the fewest mutations appears as a in Fig. 3.
The method of choosing the tree and doing the calculation
is based on the parsimony principle (17). This tree-building
method, it should be emphasized, does not assume that the rate
of molecular evolution is constant. The first step in the calculation is to list all 42 of the cleavage positions that are phylogenetically informative (Table 1). f The second step is to assume
a particular tree and calculate for each position listed in Table
1 the minimal number ofmutations needed to produce the variation observed. For tree a in Fig. 3 and the first position listed
in the table (16e), only one mutation is necessary to account for
the e site's presence in the two chimpanzees and its absence in
the other four species. At position 17x, two mutations would
account for an x site being present only in the common chimt Not included in Table 1 are the 79 "unique" positions-i.e.,
positions
for which the number of phylogenetically inferred changes is independent of the tree topology.
_
FIG. 3. Evolutionary trees for mitochondrial DNA of six higher
primates. Tree a is based on the cleavage maps in Fig. 1 and requires
a minimum of 67 mutations at the 42 positions listed in Table 1. The
tree may be written in network notation as CP, G; H; OGi. (The abbreviations are explained in Table 1.) Other branching orders require
more mutations, as shown by the following examples: CP, H; G; OGi
(68); CP, G; HO; Gi (69); CP, GH; OGi (70); CP, H, G; OGi (71; this
corresponds to tree b); CP, G; 0; HGi (71); HP, CG; OGi (79); CPGHOGi
(98).
panzee and the gorilla. Altogether, a minimum. of 67 mutations
suffices to produce the observed distribution of sites at the 42
positions tabulated, when tree a is used. This tree was found
to be the most parsimonious, and it agrees exactly in branching
order with that proposed by Simpson (18) on the basis of a qualitative analysis of morphological evidence. §
When other trees are assumed, more mutations (68-98) are
necessary (see legend to Fig. 3). Some of the alternative trees
require only 1-4 more mutations than does tree a. For example,
the tree supported by previous biochemical research and containing a three-way split among the gorilla, chimpanzee, and
human lineages (see tree b, Fig. 3) requires 71 mutations, 4
more than does tree a, By contrast, most ofthe alternative trees
require at least 80 mutations. We also note that all of the best
trees-i.e., those having.67-71 mutations-put the two chimpanzees together as the most closely related pair among the six
species, consistent with other biological evidence. Trees that
separate these two species require at least 9 more mutations
than do the trees that unite them. This result attests to the phylogenetic utility of the mtDNA mapping method.
Distribution of Site Changes. From the tree analysis, it is
inferred that a minimum of 147 mutations were fixed during the
evolutionary divergence of the six maps. Sixty-seven of these
mutations were at the 42 positions listed in Table 1 and the remaining 80 were at unique positions (see § footnote). These
mutations are distributed among 121 variable positions, scattered widely in the genome (Fig. 4). The 11 invariant positions
are also scattered widely (Fig. 4). No large region is exempt from
evolutionary change, as is evident from the results of the numerical analysis in Table 2. The region coding mostly for ribosomal RNA may, however, be slightly more conservative than
most other regions.
DISCUSSION
Distribution of Site Changes. Although the positions at
which there is site variation seem to be distributed almost randomly in the mitochondrial genome (Fig. 4, Table 2), we know
that mutations are not fixed at random in this DNA. Thermostability studies with heteroduplex mtDNA indicate a wide
range of susceptibility to evolutionary change in the mitochondrial genome (10). The nonlinear relationship observed between the extent of evolutionary divergence at restriction sites
and the time since divergence (10) also indicates that there are
§ Other methods of tree construction also gave the result in Fig. 3a. In
addition, individual variation has been examined within the human
species (11) and within ape species (unpublished work). Tree a is supported by including all the variants.
Table 1. Phylogenetically informative variation at 42 positions
in mtDNA of Hominoidea*
Position and
nature of sitet
16e, 18b, 27b, 35h,
44e,53o,54e
17x,51j
32g,60h,95h
Site presentt
C
P
+
+
+
+
+
55x
19o
41z,45z,50x
29x
86co
21o
G
+
+
+
+
H
+
+
+
+
+
+
o
+
+
o
+
4k,59y
15f,65y
35o,38x,66z,81y
23m,61w
47w
95o
85h
1j,82e
33w,95x
20f,70h
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Minimal no.
of events
1
+
+
Gi
+
+
+
+
+
6m
0
7
4
3
2
2
6
+
52w
+
+
+
+
+
+
+
+
+
+
+
+
+
+
2
2
3
2
4
4
4
4
2
1
2
2
4
4
2
m
Total
67
* The hominoid species examined are abbreviated as follows: P, pygmy
chimpanzee; C, common chimpanzee; G, gorilla; H, human; 0, orangutan; and Gi, gibbon.
t Each site is designated by a number for its position on the map and
by one or two letters standing for the nature of the cleavage site. See
legend of Fig. 1 for the single-letter code.
t Presence of a cleavage site is indicated by a + or by a letter. In the
latter case, the site is cleaved by only one ofthe two enzymes referred
to in the first column.
101m
m
m
m
2435
Proc. Natl. Acad. Sci. USA 78 (1981)
Evolution: Ferris et al.
+
+
many positions in this genome at which evolutionary change is
intolerable. These positions must be scattered widely, because
there is little evidence for strong conservatism of any large region in the genome.
The large region coding for ribosomal RNA (Fig. 4) was expected to be a prime candidate for evolutionary conservatism
(21). The nuclear ribosomal genes are far more conservative
than the average single-copy DNA sequence (22). The conservative nature of ribosomal genes in higher primates is confirmed by the work of Arnheim et al. (23). These investigators
found no evolutionary change at 10 positions in the nuclear ribosomal genes, whereas we found 17 site changes at 13 positions
in the mitochondrial ribosomal genes of the same six species.
This dramatic difference in substitution rate emphasizes, for
specific genes, the rapidity of mtDNA evolution in regions
where strong conservation would be expected.
Rearrangements. Although there is no evidence for gross
rearrangement of the mitochondrial genome in any of the six
species, considerable evidence exists for a small deletion in gorilla mtDNA near the origin of replication. Sequence rearrangements near the origin of replication have also been observed
among individual sheep (24) and among Drosophila species (25);
the rearrangements appear to be confined to this region in animal mtDNA (12).
Such rearrangements can result in erroneous estimates of
sequence similarity when these estimates are based solely on
the fraction of shared restriction fragments. All fragments encompassing the region of the rearrangement will have their size
Table 2. Distribution of site changes in five regions of the
mitochondrial genome
Site changes
No. of
Site
Genomic
per position
positions
changes
region,
(a/b)
(b)
(a)
map units
1.21
19
23
0-19.9
1.03
33
34
20-39.9
1.27
33
42
40-59.9
60-79.9
80-99.9
27
21
25
22
1.08
0.95
0-100
147
132
1.11
and hence their electrophoretic mobility altered. The fragment
size comparison method used in recent studies (26) is thus subject to a source of error that the map method avoids.
Resolving Power. Our cleavage mapping study of mtDNA
provides the evolutionary biologist with many more genetic
traits than are available from studies with proteins. Consider
the case of mutational differences between humans and common chimpanzees. The observed number of site differences
between these two species is 44 (Fig. 1). This number contrasts
with that obtained from protein studies. The proteins whose
amino acid sequences have been compared in the two species
are myoglobin, fibrinopeptides, carbonic anhydrase, cytochrome c, and the a, 13, y, and 8 polypeptides of hemoglobin
(9). For the 963 amino acid positions compared in these proteins, only three substitutions have been detected (9). Electrophoresis is another method of comparison that is valuable for
detecting protein differences among closely related species. In
an electrophoretic study of 23 loci in the same six species with
which we are concerned, humans and chimpanzees were found
to differ at an average of 7 loci (8). Thus, our map study of
mtDNA provides 6 times more resolving power than conventional protein electrophoresis.
Another class of biochemical methods measures genetic distance on a continuous scale rather than in mutational units.
These are the methods of protein immunology (7, 27) and nu-
clear DNA hybridization (6, 9). According to these procedures,
the human, gorilla, and chimpanzee lineages are equally divergent from one another (as shown in tree b, Fig. 3). Compared
to the small distances among these species, the errors in estimating these distances are large (±25%). Hence, if the gorilla-chimpanzee divergence were 25% smaller than the
ape-human divergence, these methods could fail to reveal this,
simply because of the large error in measurement.
The magnified view of the genetic differences among apes
and humans provided by mtDNA indicates that the
gorilla-chimpanzee divergence could be more recent than the
divergence between the human and African ape lineages.
Nevertheless, those alternative trees that are nearly as parsimonious as tree a focus our attention on the possibility that the
gorilla, chimpanzee, and human lineages diverged almost simultaneously. In the absence of adequate statistical tests for
comparing alternative trees, this possibility merits continued
consideration. Another observation consistent with this possibility is that most of the positions at which restriction enzymes
cleave appear to have experienced multiple substitutions (Table
1); this is indicative of a high incidence of parallel and back
mutations during the evolution of hominoid mtDNA.
The validity of the tree inferred from mtDNA maps can be
tested by further studies. We speculate that at least 10 times
more genetic information will be required to resolve the
branching order for the gorilla, chimpanzee, and human lin-
OAII
,136
Evolution: Ferris et al.
Proc. Natl. Acad. Sci. USA 78 (1981)
Site changes
H II 1 1
li
1
I
I
lA
Conserved sites
_ K.
DpbJ4
-
-
|
I
11
1311A 121
1
I
1
1
L
0
16S
112SE
I
20
40
60
80
100
FIG. 4. Distribution of evolutionary changes affecting restriction sites in mtDNA of higher primates. In the upper part of the figure, variable
sites are indicated by vertical bars and invariant sites by triangles (A, four-base site; *, six-base site). There are 121 positions at which site variation
occurs. All variable positions that occur within a given map unit have been grouped; the height of each vertical bar indicates the number of site
changes within that map unit. The number of site changes has been inferred from tree analysis, using tree a shown in Fig. 3. The middle portion
of the figure shows the positions, some tentative, of the D-loop and the genes for cytochrome b (b), three subunits of cytochrome oxidase (1, 2, and
3), and an ATPase subunit (A), as well as several genes ofunknown function and the two ribosomal RNA genes (16S and 12S). Black bars indicate
tRNA genes and noncoding regions (19, 20). The distance scale at the bottom is in map units.
eages definitively. This amount of information can certainly be
provided by mapping additional restriction sites and by direct
nucleotide sequence analysis ofboth mtDNA and nuclear DNA.
Significance of Branching Order. Knowledge of the branching order of the lineages leading to humans and African apes is
a prerequisite for clear thinking about human ancestry. As long
as there is uncertainty about the branching order, one cannot
proceed to reconstruct the probable phenotype of the common
ancestor ofhumans and African apes. Trees a and b (Fig. 3) may
be used to illustrate this point. According to tree b, the common
ancestor of gorillas and chimpanzees was also the ancestor of
humans. In this event, many ofthe traits that gorillas and chimpanzees share were probably present in the ape-human ancestor. Knuckle-walking, for example, is unique to gorillas and
chimpanzees (28). If tree b is correct, our ancestors were probably knuckle-walkers. If tree a is correct, however, knucklewalking probably arose after the human lineage had branched
off. The importance of elucidating the branching order definitively is thus apparent.
We thank G. Bourne, M. George, T. Kawakami, H. McClure, 0.
Ryder, B. Swenson, and E. Zimmer for help in obtaining primate tissues; N. Arnheim, R. Cann, S. Carr, B. Chapman, W. Davidson, M.C. King, E. Prager, V. Sarich, A. Wang, R. T. White, and E. Zimmer
for discussion; and L. Erdley for assistance in preparation of this manuscript. This research was supported by a grant from the National Science
Foundation, a Center grant from the National Institute of Environmental Health Sciences, and fellowships from the Miller Institute and
the Guggenheim Foundation. This paper was written while A.C.W. was
on sabbatical leave in R. C. Lewontin's laboratory at Harvard
University.
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