Evolution of Mitochondrial DNA in Monkeys, Apes, and Humans

YEARBOOK OF PHYSICAL ANTHROPOLOGY 31:15-48(1988)
Evolution of Mitochondrial DNA in Monkeys,

Apes, and Humans
J.N. SPUHLER
Genetics Group, LS-3, IAS Alamos National Laboratory, University of
California, LQSAlamos, NM 87545
KEY WORDS Primates, Transfer RNA, Hominoid phylogeny, Origin of

Homo sapiens
ABSTRACT The size of the primate single mitochondrial DNA molecular

ring, the genetic technology for obtaining pure samples, the use of nucleotide
sequence and restriction endonuclease analyses, and the relatively rapid rate
of evolution make mtDNA variation useful for microevolutionary studies
within and between species despite the informational content of the 37 genes
being restricted to one locus because of complete linkage. The data on chim-
panzees, gorillas, orangutans, and gibbons support the hypothesis that the
two African apes are the closest living biological relatives of humans and
favor a closer relation of chimpanzees and humans than of gorillas and
humans. The data support the origin of Homo sapiens by regional phyletic
transition from H. erectus, starting in Africa in the Middle Pleistocene, and
oppose the hypothesis of rapid world-wide replacement by migration from a
single source. The human continental races share a majority of both their
mitochondrial and nuclear gene pools.
Mitochondrial molecular genetics made spectacular contributions to biological

anthropology and the study of hominoid evolution starting in 1980 with the work of
Wesley Brown. Progress has continued with the studies of Rebecca Cann and her
associates in Allan Wilson’s laboratory at Berkeley, of Douglas Wallace and his
colleagues in Cavalli-Sforza’s group a t Stanford, of Matsunaga’s institute in Japan
and of C.C. Tan’s students in China. This review is a n elementary introduction to
the field and summarizes some of the current results.
MITOCHONDRIA: HISTORY, STRUCTURE, FUNCTION, GENE CONTENT, AND BIOGENESIS
History of mitochondria
Soon after Robert Brown discovered the cell nucleus in 1831 (published 1833)
several cytologists independently observed intracellular structures that probably
represented mitochondria. Wilson (1928) gives short historical notes on mitochondria
with a n accent on structure; Ernster and Schatz (1977) provide a n extensive histori-
cal review with emphasis on biochemistry. Early references mentioned in this paper
can be found in these two sources, along with Cowdry (1924).
Seemingly the earliest report on mitochondria was in J. Henle’s, Allgemeine
Anatomie of 1841. (This book is also of historical interest because Darwin considered
Henle’s illustrations the best anatomical drawings ever published.) Kolliker discov-
ered mitochondria (“sacrosomes”) in muscle cells in 1857 (Lehninger, 1970, p. 795).
Many cytologists attribute the discovery of mitochondria to Richard Altmann (18901,
who first showed that they were ubiquitous in plant and animal cells and proposed
their endosymbiotic origin from bacteria (an idea previously stated by A.F.W. Schim-
per, (1883; see Margulis, 1981, p. 59). The molecular basis of mitochondrial develop-
ment, genetics, and evolution was not understood until the discovery in 1963 that
mitochondria had their own DNA genome independent of the nuclear genome.
0 1988Alan R. Liss, Inc.

16 YEARBOOK OF PHYSICAL ANTHROPOLOGY [VoI. 31, 1988
In 1962 Hans Riss and Walter Plant discovered DNA in chloroplasts miss and
Plant, 1962). Within 2 years several workers reported DNA in the mitochondria of
both plants and animals (Nass and Nass, 1963a,b;Bell and Muhlethaler, 1964; Luck
and Rich, 1964; Nass et al., 1965). Prokaryotes, limited to a single DNA molecule,
are profoundly different from eukaryotes with two to four separate and indepen-
dently transmitted nuclear, chloroplastic, microtubular, and mitochondrial DNA
genomes. Eukaryotes are polygenomic rather than monogenomic. Polygenomic cell
systems are widespread among living phyla (lichens being the best known), ranging
from loose ecological and physiological symbioses to tightly integrated associations
(Margulis, 1981, pp. 201-204).
C. Benda derived the current name of mitochondria in 1897 from the shape which
these organelles usually show under the light microscope: Greek mitos, a thread,
chondrion, granule (Wilson, 1928, p. 1137). Other early names include chondrio-
somes and plastochondria. Benda (1897, 1902) also introduced technical improve-
ments that made possible fixing and staining mitochondria with greater certainty
and brilliancy.
Structure, size, numbers of mitochondria
Mitochondria are found only in eukaryotes-never in prokaryotes. Plant cells
usually have fewer mitochondria than animal cells, reflecting their usually lower
metabolic rates. The numbers range from a single organelle in the unicellular alga
Microsterias to 500,000 in the giant amoeba Chaos chaos (Day, 1984). They number
about 1,700 f 300 and make up about 22 f 3% of total cell volume in typical
primate liver cells.
Mitochondria, about the size of bacteria (0.5-1.0 pm wide and 5-10 pm long), are
cytoplasmic organelles shaped like ovoids, rods, or threads. Each mitochondrion is
bounded by a double membrane: the outer layer forming a smooth boundary of the
organelle and the inner layer folded repeatedly into shelflike laminae called cristae.
The inner space contains a n enzyme-rich matrix that plays a n essential role in the
conversion of energy from foodstuffs into the energy used for cellular activities. In
addition, of key evolutionary importance is the fact that each mitochondrion con-
tains one or more histone-free circular molecules of DNA independent of those of the
nuclear DNA and contains ribosomes with ribosomal RNAs differing from those of
the host cytoplasm.
Individual mitochondria vary in shape and often fuse to form networks which
spread through the cytosol and then break up again. Mitochondria replicate their
own genes, make some of their own proteins, and have the capacity to grow and
divide.
Functions of mitochondria
The main function of mitochondria is to generate energy by combining oxygen
with food molecules (carbohydrates, fatty acids, and amino acids) to support the
various kinds of chemical and mechanical work carried out by the host cell. Most of
the energy is used to form the energy-rich molecule adenosine triphosphate (ATP)
from adenosine diphosphate (ADP) and inorganic phosphate. Some of the energy
drives the transport of specific metabolites into and out of the organelle. Rapid
progress in understanding mitochondrial function was made possible by the discov-
ery in 1948 by George Palade and associates (Hogeboom et al., 1948) of a procedure
for isolating intact, well-preserved mitochondria retaining much of their normal
chemical activity by differential centrifugation of sucrose solution suspensions of
tissue homogenates.
Mitochondria are chemically self-sufficient units; Lehninger and associates dem-
onstrated that isolated mitochondria catalyzed the complete oxidation of fatty acids
to carbon dioxide and water, proving that mitochondria contain all the enzymes and
coenzymes for the 0-oxidation of fatty acids, the tricarboxylic acid cycle, and the
electron transport system. Although these organelles have not yet been successfully
grown in culture, carefully isolated mature mitochondria thrive in experimental
Spuhler] EVOLUTION OF MITOCHONDRIAL D N A I N MONKEYS, APES, A N D HUMANS 17
containers, which of course, favored the vast volume of study on mitochondrial

biochemistry (Lehninger, 1964; Darnel et al., 1986). The enzymes of the citric acid
cycle are located in the soluble matrix within the inner membrane of the mitochon-
drion; the enzymes of the respiratory chain are located in the membranes, particu-
larly the inner membrane.
Mitochondria are highly efficient energy machines converting each molecule of
glucose to carbon dioxide and water plus 36 molecules of adenosine triphosphate.
The formation of 1 mole of ATP requires an input of about 60 kilojoules of free
energy, and an equivalent amount of free energy is released when ATP is hydrolyzed
back to ADP and inorganic phosphate.
Mitochondria are often strategically located near the source of fuel, such as lipid
droplets, or near the site of ATP utilization, such as a muscle fiber, the sperm middle
piece, and the retina.
Without the aerobic respiratory process that takes place in mitochondria, animal
cells would be dependent on anaerobic glycolysis for all their ATP. But glycolysis,
which splits glucose to pyruvate, releases only a small fraction of the total free
energy available from the oxidation of sugars. In mitochondria, the metabolism of
sugars (and of fatty acids) is completed with their oxidation by molecular oxygen
( 0 2 ) to C02 and H2O. The energy available is captured so efficiently that 36 mole-
cules of ATP are produced by each molecule of glucose, while glycolysis alone would
produce only two molecules of ATP. Details on mitochondrial structure and function
are covered in Alberts et al. (19831, Darnel1 et al. (19861, Gillham (1978),Grun (19761,
Lehninger (1964,19821,and Tzagoloff (1982).
Gene content
The mass of mitochondrial DNA from flatworms to mammals is 9-12 x lo9 daltons
(there are approximately 1,500 DNA base pairs per million daltons of DNA double
helix). This mass corresponds to about 15-17 kilobase pairs in higher primates (Table
1).Nuclear DNA contains sequences that vary in copy number from 1 to lo6 per
haploid genome; mtDNA in primates (but not in yeasts and some plants) contains
sequences that occur only once per mitochondrial genome. Primate mtDNAs lack
large spacer sequences between genes and all intervening sequences (introns) within
genes. There are 37 mitochondrial genes in primates and their order (shown in Fig.
12) is identical in human, mouse, rat, and bovine mtDNA ring molecules (Brown,
1985).About 32% of primate mtDNA encodes genes for RNAs, including 22 transfer
RNAs and 12s and 16s ribosomal RNAs (De La Cruz, et al., 1984). The remaining
68% encodes for proteins, including cytochrome c oxidase subunits I, 11,111,ATPase
subunit 6, cytochrome b, and eight proteins of initially unknown function (called
“unidentified reading frames,” URFs). Attardi and associates (Attardi et al., 1986;
Chomyn et al., 1983, 1985; Mariottini et al., 1986) have identified the polypeptides
encoded by each of the previously unknown reading frames in human mtDNA and
determined their functional assignments, thus completing knowledge of the total
informational content of this genome. Seven URFs encode subunits of the respira-
tory chain nicotinamide adenine dinucleotide dehydrogenase (NADH)and the eighth
encodes the adenosine triphosphatase-6 (ATPase6)gene. The correspondence of the
original URFs and the subunits of the respiratory chain NADH dehydrogenase is:
TABLE 1. Four restriction methods for comparing mtDNAsl

Method of Type of Divergence Equivalent
Method detecting DNA restriction Resolving detectable time span
label fraanents enzyme Dower (%) (vears)
RL4 Radioactive labelling 4-base High 0.05 25,000
RL6 Radioactive labelling 6-base Intermediate 0.25 125,000
SB6 Southern blotting 6-base Low 0.5 250,000
ES6 Ethidium staining 6-base Low 0.5 250.000
‘From Wilson et al. (1985).Assumes the use of about ten enzymes and a divergence rate of 2% per million years.
18 YEARBOOK OF PHYSICAL ANTHROPOLOGY [Vol. 31, 1988
URFl = ND1, URF2 = ND2, URF3 = ND3, URF4 = ND4, URF4L = ND4L,
URF5 = ND5, URF6 = ND6, and URFA6L = ATPase8.
Also, the mtDNA genome is functionally integrated with the nuclear genome: for
example, over 90 proteins are required to make mitochondrial ribosomes, and nearly
all are supplied to the organelle from the host cell cytoplasm. These proteins are
encoded in nuclear DNA, are synthesized in the cytosol, and are then individually
transported into the organelle.
The mitochondrial genome itself has been highly stable in metazoan evolution,
changes in gene order being observed mostly in different phyla. (But Clary et al.,
1982, report changes in gene order within the genus Drosophila.) The sequential
order among vertebrates is identical in lineages derived from the common ancestor
of frogs and mammals that diverged over 350 million years ago (mya) (Brown, 1985).
In sea urchins the 16s ribosomal RNA and cytochrome oxidase subunit I genes are
directly adjacent in the mitochondrial DNA ring; in human and other mammalian
mtDNAs these two genes are separated by the region containing genes ND1 and
ND2, the difference in gene order reflecting a rearrangement that took place in the
sea urchin lineage since sea urchins and mammals last shared a common ancestor
(Roberts et al., 1983).
Biogenesis of mitochondria
The longevity of mitochondria is much less than that of their host cells; in most
primate body tissues half of the mitochondria are replaced about every 5 days. New
mitochondria are never made de novo; they come only from the growth, division, or
fusion of existing mitochondria. Replication of mitochondrial DNA depends on a
special DNA polymerase and a distinctive mechanism, described in summary by
Kornberg (1980) and in detail by Clayton (1982). The rate of mtDNA replication in
animals is slow (about ten nucleotides polymerized per second in contrast to 1,000
per second for bacterial DNAs) so synthesis of the genome takes a n hour. On
average, each individual mitochondrion must double in mass and then divide into
two once each cell generation. DNA synthesis is continuous in mitochondria as in
bacteria, but DNA synthesis in the eukaryotic nucleus is discontinuous and in step
with chromosomal division cycles (Mitchison, 1971). Electron micrographs suggest
that organelle division starts with a n inward furrowing of the inner membrane, as
happens in the cell division of many bacteria. Reproduction of individual mitochon-
dria takes place throughout the cell cycle out of phase with the division of the cell
and with each other and not limited to the nuclear DNA synthesis phase. Individual
organelles seem to start reproduction at random, some not dividing and others
dividing more than once in a given cell cycle, but in general the total number of
mtDNA molecules doubles in each cell cycle so that the amount of mtDNA per cell
stays constant.
EVOLUTIONARY ORIGIN OF MITOCHONDRIA
Two conflicting theories concerning the origin of mitochondria are popular today
(Schwartz and Dayhoff, 1978; Margulis, 1981; Lee and Fredrick, 1987).The endosym-
biotic theory holds that mitochondria originated from free-living bacterialike forms
that established a symbiotic relationship with the host “protoeukaryotic” cells. The
endosymbiont hypothesis was first proposed in 1890 by Altmann, one of the codiscov-
erers of mitochondria. This hypothesis speculates that all eukaryotic cells originated
as primitive organisms without mitochondria or chloroplasts.
The autogenous theory holds that mitochondria evolved by compartmentalization
of the DNA and other organelle structures within the cytoplasm of a protoeukaryote
(Raff and Mahler, 1972). The autogenous hypothesis was proposed initially (espe-
cially for chloroplasts) by Mereschkowsky (1905).
Fredrick (1981) and Lee and Fredrick (1987) include a number of papers giving
arguments for and against the two theories, and Margulis (1981) summarizes two
independent bodies of evidence that strongly support the symbiotic theory and
Spuhler] EVOLlJTION OF MITOCHONDRIAL DNA IN MONKEYS, APES, AND HUMANS 19
oppose the direct filiation, compartmentalization theory: 1)Under the symbiotic

view all mitochondrial functions originally were coded by genes in the mtDNA; all
deviations from complete control are subsequent takeovers of control by the nuclear
DNA using new mutation or gene transfer. The mitochondria of eukaryotes with
mitotic-meiotic reproductive systems (including primates, most higher animals, and
plants) are more dependent on the nucleus than are the mitochondria of primarily
asexual and nonmeiotic eukaryotes (including amoebae, euglenoids, and cellular
slime molds). The symbiotic theory requires that nuclear control and complex inte-
gration of nucleocytoplasm and mitochondrial function be advanced (apomorphic),
more recently evolved states (Margulis, 1981). 2) The structure of primate mitochon-
dria is more similar to some respiring, aerobic, Gramm-negative eubacteria than is
that of the adjacent host nucleocytoplasm with respect to nucleotide sequences of
16s ribosomal RNAs, amino acid sequences of cytochrome c, lipid composition of
mitochondrial membranes, and antibiotic sensitivities. The compartmentalization
theory requires the opposite of both sets of facts.
Whichever is correct, the origin of the organelles is ancient. For example, because
plant and animal mitochondria are very similar in structure and function, the
endosymbiotic theory assumes the symbiosis to have occurred before the pre-Cam-
brian divergence of plants and animals, about 1billion years ago. Under the endo-
symbiotic theory the origin of mitochondria and chloroplasts involves independent
endocytotic events. The first cells to contain chloroplasts are supposed to derive from
endocytosis involving a cyanobacterium. The first cells to contain mitochondria are
supposed to derive from endocytosis involving a form that shared a common ancestor
with the respiring and photosynthetic bacterial family Rhodospirilacae (Schwartz
and Dayhoff, 1978).Further, the mitochondria of fungi, plants, and animals probably
represent independent symbiotic events (Mikelsaar, 1987), suggesting that yeast
mitochondrial genetics (by far the best known of all species) probably differ from
those of animals in details of genetic transmission.
Because of their small size, the largest animal mitochondrial genomes can code for
only a small fraction of the proteins required to assemble the organelles. The large
fraction of mitochondrial proteins (some 90%) is known to be coded in the nuclear
DNA. Thus, it seems likely that a n extensive gene transfer from organelle to nuclear
genome occurred early in eukaryotic evolution.
Combined comparison of cytochrome c and tRNA sequences has shown that the
divergence of eukaryotes and prokaryotes was 2.6 times more remote in evolutionary
distance than the divergence of the eukaryotes into green plants, fungi, and animals
(Dayhoff and McLaughlin, 1972, p. 116). Woese (1977) presented extensive biochemi-
cal evidence that high-molecular (70s and 80s) ribosomal RNAs and therefore 70s
and 805 ribosomes appeared very early in the history of life. The stability and near
universality of the 5s ribosomal gene make it most useful in the macromolecular
search for mitochondrial origins (Delihas and Fox, 1987). Resolution of the problem
of mitochondrial origin must await elucidation of the details of the origin of the
nucleus, nucleoli, centrioles, and the mitotic spindle.
Barbieri (1981) developed the ribotype theory of the cell to account for the evolu-
tionary origin of the nucleus and thus of eukaryotes and mitochondria. The latest
version of his theory is The Semantic Theory of Evolution (Barbieri, 1985).
All modern theories on the origin of the eukaryotic cell agree that mitochondrial
genes have resided in DNA molecules separate from nuclear DNA for at least the
past 700 million years. The mitochondrial lineages of living vertebrates have DNA
ancestors older than the vertebrates (Dayhoff and Schwartz, 1981).
EVOLUTIONARY GENETICS OF MITOCHONDRIAL DNA
The mtDNA of primates has several properties that make it useful for studies of
molecular evolution. The mtDNA genome is a small, simple, self-replicative, double-
stranded, circular DNA molecule. The organelles are haploid and the inheritance is
maternal (Brown, 1985; Wilson et al., 1985).
A major limitation on the utility of mtDNA in evolutionary studies is that all 37

mitochondrial genes are completely linked (Nei, 19871, so the total genetic informa-
tion available for evolutionary genetic analyses is limited to that of a single locus.
In this sense it is too bad we lack chloroplasts.
The evolutionary dynamics of the mitochondrial genome differs from that of the
nuclear genome in configuration of gene content, mode of inheritance, and modes of
segregation at cell division (Takahata, 1985; Nei, 1987). Such differences result in
potentially different phyletic relationships within and between species. Within-
generation drift due to multiplicity of mitochondrial genomes and non-Mendelian
segregation promotes the fixation of advantageous mutants and prevents deleterious
mutations from accumulating (Takahata and Slatkin, 1983). In the population of
about 10l6 mtDNA molecules within the total cell mass of a typical human, there
are about 2,000 mitochondrial generations for each individual human generation.
Drift can occur during the random replication and degradation of multiple genomes
within a cell and in the random sorting of copy genomes to daughter cells, especially
if the number of copies is relatively small (as in the germ-celllines during oogenesis),
and a large number of organelle divisions occur within one cell division of the host.
When a population is geographically subdivided into demes connected by gene
flow, and the migration rate differs by sex, the degree of local differentiation will
differ between nuclear and organelle genes (Birky, 1978; Birky et al., 1983). In
primates the rate of mitochondrial gene flow is (because of uniparental inheritance)
generally lower than the rate of nuclear gene flow; thus mitochondrial genomes tend
to be more locally differentiated, unless the migration sex ratio strongly favors
females (Powell, 1983).
Maternal inheritance
To the extent that mtDNA is inherited in gamete formation by transmission of
organelles through the egg cytoplasm, cladistic analysis along maternal lineages is
not complicated by recombination. All the genetic variation due to bisexual inheri-
tance is absent in the maternal clonal inheritance of primate mtDNA. (Biparental
inheritance of mitochondrial DNA is present in yeast and some other
microorganisms.)
Maternal transmission of mitochondria to the zygote goes along with sexual
reproduction and the genetic continuity of organelles that cannot be made by the
nuclear genome. In early single-celledeukaryotes, isogametic and biparental trans-
mission of organelles must have been the rule. In many eukaryotic evolutionary
lineages, anisogametes (sperm and egg) replace isogametes, thus reducing travel
distances for female gametes and ultimately resulting in the inheritance of the
organellar genome with the larger, more sedentary ovum (Jinks, 1964; Margulis,
1981).
Most models for the evolutionary genetics of mtDNA in mammals assume that
inheritance is strictly maternal (Wilson et al., 1985). Although the sperm of verte-
brates may contain hundreds of mitochondria in the middle-piece, usually that
structure does not enter the fertilized egg, and if paternal mitochondria do enter,
seemingly few, if any, survive the first equational cell division. Szollosi (1965)found
in rats that sperm mitochondria enter the egg at fertilization but then swell and
disintegrate.
Using the fact that the mtDNAs of horses and donkeys differ in the distribution of
endonuclease restriction sites, Hutchinson et al. (1974) found that mules contain
mtDNA of their horse mother only, and hinnies that of their donkey mother only,
never of their fathers. However, Dawid et al. (1974) showed that recombination
between paternal and maternal mtDNA occurred in some hybrids between the newts
Xenopus laevis x X . mulleri as shown by hybridization of mtDNA from hybrids with
cRNA-that is, RNA produced by in vitro transcription of mtDNA from the two
hybrid parental species.
Male parental leakage due to inclusion of mitochondria from the sperm is, in the
best-tested animal case, less than one in 25,000 molecules per generation (Lansman
Spuhler] EVOLUTION OF MITOCHONDRIAL DNA IN MONKEYS, APES, AND HUMANS 21
et al., 1983). All direct evidence for primates, mostly from the transmission of
mitochondria1 abnormalities in human kindreds of up to three or four generations
(Giles et al., 1980), is consistent with strictly maternal inheritance.
With strictly maternal inheritance, mtDNA lineages become extinct in each gen-
eration in all families with males only-one half of familes of size 1, one-fourth of
size 2, etc.
Mitochondria1 genome size
The human (of European ancestry) whose mtDNA genome was fully sequenced
(Anderson et al., 1981)had 16,569 base pairs. Minor variations in total length of the
human mtDNA genome occur within the species (Cann and Wilson, 1983), the
largest being an insertion of about 60 base pairs (bp 5,274-5,691) common to all 116
Japanese sampled by Horai and Matsunaga (1986)but not present in the published
total mtDNA sequence. The human mtDNA genome was the first genome of any
organism to be fully sequenced. The complete mtDNA sequence is now also known
for mouse, rat, and cow among mammals (Brown, 1985).
The primate nuclear genome varies between lo8 and 1011 nucleotides (Manfredi-
Romanini, 1972; Seuanez, 1979)subdivided into from ten to 40 discrete chromosomes
(2n = 20 in Callicebus torquatus and 80 in tarsiers; (Chiarelli et al., 1979); in
contrast, the mtDNA genome of 18 primate species numbers only 16,400 & 400 bp
(Table 1). Because the mtDNA is relatively small in size and comparatively easily
extracted in adequate pure samples, availability of a fully sequenced human genome
makes possible precision mapping of restriction polymorphism in human and other
primate population samples.
METHODS OF GENETlC ANALYSIS
Use of restriction endonucleases
Arber and Dussoix (1962)and Dussoix and Arber (1962)provided the first evidence
for the existence of DNA restriction enzymes, leading to their later purification and
use in DNA sequence characterization by Nathans and Smith (1975). Hamilton
Smith (1979) gives an excellent introduction to the new DNA technology based on
the nucleotide sequence specificity of restriction endonucleases. Several excellent
texts, including those of Alberts et al. (1983) and Darnel1 et al. (1986), summarize
the role of restriction endonucleases in current knowledge of cell biology.
Endonucleases hydrolyze internal phosphodiester bonds in the polynucleotide
backbone; exonucleases hydrolyze terminal phosphodiester bonds at the 3‘ and 5’
ends of a polynucleotide.
Nearly all bacterial species synthesize one or more types of sequence specific
endonucleases that make double-stranded cuts in DNA. These endonucleases are
called “restriction enzymes” because they restrict the incorporation of foreign DNA
in the bacterial cell. The restriction enzymes are prevented from cutting native
bacterial or self-DNA by “modification enzymes” that alter the structure of the DNA
sites recognized by specific native restriction enzymes. The corresponding sequences
in the genome of the bacteria itself are “camouflaged” by methylation at an A or C
residue in the recognition sequence. However, bacterial endonucleases are able to
cut uncamouflaged foreign DNA into pieces, and the pieces are then degraded by
exonucleases.
Restriction endonuclease enzymes recognize specific DNA sequences 4-7 bp in
length and then make a double-stranded cleavage of the DNA molecule at the
specific restriction or cleavage site. Some 850 restriction endonucleases are known.
The three major types of restriction enzymes differ according to the location of the
cut: Type I make a double-stranded cut close to but not at a specific position in the
sequence. Type I1 make a double-stranded cut at a fixed point in the recognition
sequence. Type I11 make a double-stranded cut some number of bases away from the
recognition sequence. Roberts (1978) catalogued 176 type I1 restriction endonu-
cleases, including those most useful in primate and other phylogenetic analyses,
giving the source microorganism, enzyme name, recognition sequence and cutting
point, and number of cleavage sites by each of the specific endonucleases on bacterio-
phage lambda DNA, adenovirus-2 DNA, simian virus 40 DNA, and ax174 Rf DNA.
Recognition sequences are written in the direction from 5’ to 3’, only one strand
being given, and the point of cleavage (where known) is indicated by an arrow (I).
Two enzymes that recognize the same sequence are called isoschizomeres.
Restriction endonucleases are named after the Linnean names of the source organ-
ism by using the capitalized first letter of the generic name and the lower-casefirst
two letters of the specific name with suffixes to indicate strain and sequences of
discovery: for example, HaeI, 11, and I11 are from Haemophilus aegypticus and EcoRI
is from Escherichia coli strain R245.
Analysis of m D N A by restriction enzymes
The specific cuts, recorded in patterned cleavage maps, furnish a powerful method
with which to distinguish homologous mtDNA sequences within and between pri-
mate species. A specific endonuclease will cut any long length of DNA double helix
into a series of fragments known as “restriction fragments .” By comparing the size
(number of bp in length) of the restriction fragments produced after treatment with
two or more enzymes, a “restriction map” can be constructed that shows the location
of each restriction site in relation to its neighbor sites (Alberts et al., 1983, p. 106).
Each of the 100 map units corresponds to 1/100 of the total genome, or 165.69 bp.
Such maps reflect the linear arrangement of selected nucleotide sequences in desig-
nated regions of the genome; a comparison of the restriction maps from related
organisms gives a rough estimate of the number of nucleotide substitutions per site.
Some methods employed in the study of DNA from human and nonhuman primates
can detect differences in fragment length accurate to a few bp (Brown, 1980; Cann,
1982; Cann et al., 1982).
Table 1 summarizes several aspects of four methods of comparing primate
mtDNAs by using restriction enzymes. The method ( I ) of highest resolving power
(detects the smallest sequence differences) uses radioactive end labelling of frag-
ments cut by about 10 four-base enzymes. This method is currently used by Rebecca
Cann and others in Allan Wilson’s Berkeley group. A typical four-base enzyme cuts
mammalian mtDNA into about 25 fragments, most of which are separable from one
another by electrophoresis through a polyacrylamide gel (3.5%, 40 cm long) and
detected with X-ray film. The method is described fully in Brown (1980).The position
of a fragment pattern obtained for each enzyme with a given mtDNA from a
hominoid species can usually be located accurately with reference to the complete
Cambridge human mtDNA sequence determined by Frederick Sanger’s group (An-
derson et al. 1981), the observed differences in nucleotide sequence between the
fragment and the standard being explained by point or length mutations. Cann et
al., (1982)estimate from the work of Ferris et al. (1981a)that the average cleavage-
site mapping error using methods I11 and IV is about f 200 bp (1.25% of the total
genome length) in contrast to an average error less than f 2 bp (0.0125%)using
method I .
Although six-base enzymes are valuable for studies of primate mtDNA, they have
less resolving power because the number of six-base sites in primate mtDNA is
usually only three or four per enzyme. Further, the Southern blotting and ethidium
methods of identifying the fragments produced by six-base enzymes fail to detect
fragments less than 250 bases long.
Mitochondria1 D N A in primates
Population frequencies of mtDNA variation have been studied in the 16 primate
species shown in Table 2.
RATES OF mtDNA EVOLUTION
Based on restriction endonuclease cleavage map comparisons, the estimated nu-
cleotide substitution rate for primates indicates that hominoid mtDNA evolves five
to ten times faster than single-copy nuclear DNA in mammals, perhaps because of
Spuhler] EVOLUTION OF MITOCHONDRIAL DNA IN MONKEYS, APES, A N D HUMANS 23
TABLE 2. Primate species studied for mtDNA uariation

Scientific name Common name Source’ Genome size’
Homo sapiens Human 1 16,500 i 300
Pan troglodytes Common chimpanzee 1 16,400 f 300
Pan paniscus Pygmy chimpanzee 3 16,500 i 300
Gorilla gorilla Gorilla 3 +
16,405 300
Pongo pygmaeus Orangutan 3 +
16,500 300
Hylobates lar Lar gibbon 3 16.500 i 300
Macaca nemestrina Pig-tailed macaque 3 16,875 i 800
M. fascicularis Crab-eating macaque 23 17,253 i 897
M. fuscata Japanese monkey 4 17,000 f 400
M. mulatta Rhesus macaque 2 +
17,092 632
Papio anubis Olive baboon 2 +
16,770 553
Papio papio Guinea baboon 2 16,877 rt 439
Cercopithecus aethiops African green monkey 1 16,500 f 500
Cercopithecus talapoin Talapoin monkey 2 16,500 f 800
Presbytis entellus Hanuman langur 2 +
16,500 297
Lagothrix cana Woolly monkey 1 +
16,300 400
Leontopithecus
rosalia rosalia Golden lion tamarin 3 17,000 & 500
L. r. chyrosopygus Black lion tamarin 3 +
17,000 500
L. r. chrosomelas Golden headed lion tamarin 3 17,000 +500
Galago senegalensis Bush baby 1 16,500 i 300
‘Source of genome size data: (1)Brown et al. (1979); (2) Ferris et al. (1981a); (3) George (1982); (4) Hayasaka et al. (1986);
(5)Harihara et a%(1986).
lack of postreplicative DNA repair and higher mutation rates (Brown et al., 1979;
Brown, 1981). This estimate is now backed by direct sequence comparisons in five
hominoid species as well as by additional cleavage map comparisons (Brown, 1981,
1983, 1985).
The mean rate of substitution divergence averaged over the whole mtDNA mole-
cule is about 2% per million years in humans, lesser apes, and the great apes (Wilson
et al., 1985). This estimate assumes an accurate molecular clock and a divergence
time of 5 mya between Pan and Homo. If a divergence time between humans and
chimpanzees is adjusted to reflect current estimates of about 9.2 f 1.7 mya (Ginger-
ich, 19851, the mean rate of mtDNA divergence in hominoids is approximately 1-
0.5% per million years (Brown, 1985).
Nei (19851, assuming a divergence time between human and orangutan of 13
million years, reanalyzed Brown’s data to obtain a nucleotide substitution rate (A) of
7.15 x lo-’ per site per year, a little lower than Brown’s 2-4 x lo-’. Wallace et al.
(1987) estimate the mutation rate of the mitochondrial ATP synthase subunit gene
as 3.2 x per site per year, much higher than the estimates of mutation rate in
mtDNA based on Brown’s data.
Stoneking et al. (1986b)estimate rapid rates of 1.8-9.3% per million years for New
Guineans, 4.0-7.2% for Australians, and 2.5-4.2% for American Indians based on
migration dates of 30,000-50,000 years ago for New Guinea, 40,000 for Australia,
and 12,000-20,000 for the Americas. These estimates are flawed seriously because
all three regions were peopled by two or more major migrations rather than being
colonized at a defined time and isolated thereafter.
The observed rates of substitution for higher primate mitochondrial transfer RNAs
and ribosomal RNAs are about one-half the rate for protein genes, but the rates for
these mitochondrial genes are of the order of 100 times faster than their nuclear
counterparts (Brown, 1985).
Evolutionary changes in primate mtDNA due to additions and deletions are less
frequent than those due to substitutions. Sequence rearrangements (inversions and
transpositions) are rare in hominoid mtDNA evolution, say between 100 and 1,000
times lower than those for substitutions or additions/deletions(Brown, 1985).
According to Ferris et al. (1981b), ape species are two to ten times more variable
(mean pairwise differences of 0.55 to 5.00%)than the human species (mean pairwise
difference of 0.30 %)’ perhaps indicating that extant ape species have endured longer
than the human species (Wilson et al., 1985).
Takahata and Nei (1985) point out that the time of gene splitting usually is much
earlier than the time of population splitting, so the divergence time of particular
mtDNA morphs and types must be used with care in estimating the divergence
times of populations, races, or subspecies. For example, in Figure 12, the oldest
Asian or European mtDNA genes diverged from an old African gene about 200-400
ky ago (point 11,but this time of gene divergence is almost certainly earlier than the
time of racial splitting, since many other European and Asian genes diverged from
African genes at later times. Further, the intermingling in one region of mtDNA
morphs and types typical of other regions may be explained by ancestral polymor-
phism rather than gene flow (Tajima, 1983; Takahata and Nei, 1985).
GENETlC CODE AND CODON USAGE
The complete mapping of the two rRNA genes, the 22 tRNA genes, and 13 protein-
coding sequences in human mtDNA has revealed several important features (San-
ger, 1981): 1)Nearly the entire nucleotide sequence of the human mitochondrial
genome appears to code for protein and RNA with noncoding sequences restricted to
the control region. 2) While 31 different tRNAs in the nuclear genome specify amino
acids in the cytosol, 22 different tRNAs suffice for mitochondrial protein synthesis.
The normal cytosol protein synthesis condon-anticodonpairing rules are relaxed in
mitochondria, so that many organelle transfer RNA molecules recognize any one of
the four nucleotides in the third (wobble) position. This ‘(twoout of three” reading
allows one tRNA t o pair with any one of four different codons. This permits protein
synthesis with a smaller number of tRNA molecules, but the product may be less
accurate. 3) The genetic code is altered, so that four of the mitochondrial codons
have “meanings” (Table 3) different from those in cytosolic protein synthesis (Brown,
1985).
The mammalian mtDNA genetic code is shown in Table 4.
The differences between the “universal” eukaryotic nuclear and prokaryotic code
and two mitochondrial genetic codes are shown in Table 3.
Because none of the genetic codes differs radically from any of the others, Brown
(1985) suggests that the mitochondrial codes are probably altered descendants of the
“universal” code rather than vestiges of an ancient, more primitive code.
mtDNA TREES AND HOMINOID PHYLOGENY
During the past century a majority of primatologists agreed with Charles Darwin
that the living African great apes, the chimpanzees and gorillas, are the closest
living relatives of humans. Other proposed evolutionary relationships among the
apes and humans are graphed in Figure 1. The “big bang” tree (named so by
Thompson, 1975) shown in Figure l a is correct but low in information: it simply
tells that all five species are descended from a common ancestor at a single point in
the past and have since evolved independently. Bishop and Friday (1985) use the
big-bang solution to provide the lower bound in their search for global maximum
likelihood trees using nucleic acid and protein sequences (see below).
Today there is less agreement than 35 years ago as to which pair of the three
extant species-human (HI, chimpanzee (C), gorilla (G)-are closest relatives. Tree
l b leaves the triad unresolved as a trichotomy. For some data sets this is in fact the
best solution, but many evolutionary biologists think that true trifurcations (the
simultaneous evolution of three species at one point in time from one parental
species) have a low prior probability; the problem is complicated by the difference
TABLE 3. Differences between mitochondrial and nuclear genetic codes’
Codon Mammals Yeast “Universal”
UGA TrP TrP STOP
AUA Met Met Ile
CUU, CUC, CUA, CUG Leu Thr Leu
AGA, AGG STOP Arg Arg
‘Source: Attardi (1985).
TABLE 4. Genetic code for mammalian mitochondrial genes'

Amino Amino Amino Amino
Codon acid Codon acid Codon acid Codon acid
uuu Phe ucu Ser UAU TYr UGU
uuc Phe ucc Ser UAC Tvr UGC
UUA Leu UCA Ser UAA TLr UGA
UUG
~ _ _ Leu UCG Ser UAG Ter UGG
cuu Leu ccu Pro CAU His CGU
CUC Leu ccc Pro CAC His CGC
CUA Leu CCA Pro CAA Gln CGA
CUG Leu CCG Pro CAG Gln CGG
AUU Ile ACU Thr AAU Asn AGU
Ile ACC.
~~. Thr
-~~ AAC Asn AGC
Met ACA Thr AAA LY s AGA
Met ACG Thr AAG LY s AGG
Val GCU Ala GAU ASP GGU
GUC Val GCC Ala GAC ASP GGC
GUA Val GCA Ala GAA Glu GGA
GUG Val GCG Ala GAG Glu GGG
'The human, murine, and bovine code, showing the coding properties of the mitochondrial transfer RNAs. The code
holds for all known primates and other mammals. Data from Sanger (19811, Attardi (19851, and Brown (1985).
Fig. 1. Six phylogenetic trees relating humans and apes. a: The Big Bang tree showing that the five
species form a monophyletic group with unspecified branching order. b: The tree often supported by
molecular variables changing at intermediate evolutionary rates; humanschimpanzees-gorillas are shown
as a monophyletic group of unspecified branching order. c: The tree making humans and chimpanzees the
closest relatives with gorilla more closely related than orangutans. d The tree showing that the chimpan-
zee and gorilla are the closest biological relatives of humans. e: The tree making humans and gorillas the
closest relatives. fi The tree making orangutans and humans the closest biological relatives.
between gene and species furcations; see Tajima (1983) for a theoretical statement
and Mai (1983)for an empirical test using chromosomal data. Even for a few species,
the number of possible trees is surprisingly large; Felsenstein (1978) demonstrates
that 105 bifurcating trees and 236 multifurcating trees are possible for five species
with none of the interior nodes specified in advance.
Three possible trees resolve the HCG triad (HC-G,H-GC, and HG-C)as graphed in
Figure lc-e. As of 1987, the H-CG and HC-G solutions have many, the HG-C few,
supporters.
The H-CG result is supported by the anatomical studies of Fleagle et al. (1981),
Stern and Susman (1981), and Tuttle (1981),the morphometric calculations of Aiello
(1981)and Oxnard (1981),the paleontological work of Andrews (1982)and Greenfield
(1980), and the molecular data of Brown et al. (1982) and Ferris et al. (1981), to
mention only a few publications from the 1980s.
The HC-G tree is supported by morphology (Washburn, 19821, protein sequence
data (Goodman et al., 1982),karyotypes (Dutrillaux, 1980;Yunis and Prakash, 19821,
DNA-DNA hybridization (Sibley and Ahlquist, 1984), two-dimensional protein elec-
trophoresis (Goldman et al., 1987), nuclear DNA sequence data (Goodman, 19861,
and mitochondrial DNA sequence data (Nei et al., 1985; Saitou and Nei, 1986).
Templeton (1983b)analyzed Brown’s mtDNA data on hominoids by using Wilcoxon
matched-pair and signed ranks tests to reject a Pan-Homo cluster at the 5%level of
significance in favor of a Pan-Gorilla grouping. He reached the same classificatory
conclusion (1985) in a reanalysis of Sibley and Ahlquist’s 1984 data using a nonpar-
ametic method to favor a Pan-Gorilla grouping; the analysis was criticized by Saitou
(1986) and Fitch (1986).
Utilizing an expanded data set including 514 DNA hybrids Sibley and Ahlquist
(1987) confirm their support (1984)for a HomePan clade. More importantly, Felsen-
stein (1987) used a maximum likelihood mixed-model analysis of variance on the
expanded DNA hybridization data t o show that there is significant evidence for
resolving the HomePan-Gorilla trifurcation in favor of humans and chimpanzees as
closest biological relatives.
The tree joining the orangutans and humans in a latest common ancestor (Fig. 10
proposed by Schwartz (1984) has been rejected repeatedly and significantly by the
molecular data and by theoretical considerations (for example, Sarich, 1986).
The mitochondrial DNA data reviewed here give strong support for the HCG triad
and more support for the HC-G than the H-CG solution, but until longer DNA
sequences are available, they cannot resolve the HCG triad with high statistical
significance (Saitou and Nei, 1986).
mtDNA SEQUENCES OF LlVING HOMINOIDS
Perhaps the best indication of close (meaning short in time) relationships among
catarrhine primates comes from mtDNA maps inferred from restriction endo-
nuclease cleavage patterns.
Aligning nucleotide sequences to reflect their evolutionary history is more difficult
than aligning amino acid sequences because there are only four kinds of bases in
polynucleotides but 20 kinds of amino acids in polypeptides (Schwartz and Dayhoff,
1978). Of greater concern, detection of addition or deletion of a few nucleotides is
difficult by restriction enzyme methods. The alignment problem is lightened in the
case of the DNA genes for transfer and ribosomal RNAs because sequence positions
involved in base-paired regions of these molecules are highly conserved in evolution.
Brown et al. (1982) cloned and sequenced segments of mtDNA from human,
chimpanzee, gorilla, orangutan, and white-handed gibbon (Hylobates lar). Sequenc-
ing was performed according to the method of Maxam and Gilbert (1977).
The segment was isolated by cleavage with Hind 111restriction endonuclease. The
Hind I11 sites at 24 and 30 map units are highly conserved in primates and rodents.
Among primates, for example, the Hind I11 sites occupy the same two positions in
ten of the 11 species studied (Brown et al., 1982). Brown and associates cloned the
mtDNA segment bounded by these two Hind I11 recognition sites.
0.047
HUMAN
0.009
CHIMPRNZEE
o 0 3 7h 0.047 6
__k___ 0.107
LAR GIBBON
Fig. 2. Evolutionary tree for five hominoid species reconstructed by Nei et al. (1985) by using UPGMA.
The number given for each branch represents the branch length or the number of nucleotide substitutions
per site. The arrows represent 1 standard error on each side of the mean branching point. The tree is
reconstructed from data on the 896 nucleotide segment of mtDNA sequenced by Brown et al. (1982).
Sequencing indicated that this segment contains 896 bp and corresponds to nucleo-
tides 11,680-12,575 in the complete human mtDNA sequence published by Anderson
et al. (1982). The homologous Hind I11 region in mouse contains 894 bp (Bibb et al.,
1981)and that in cow contains 899 bp (Anderson et al., 1982).
The region could not be cloned as a single Hind I11 fragment in either the common
chimpanzee or the pygmy chimpanzee because both have an intermediate Hind I11
site at 26.3 map units. Brown et al. (1982) combined the complementary sequence
data from the common chimpanzee (Hind I11 sites at 24 and 26.3 map units) with
pygmy chimpanzee (Hind I11 sites at 26.3 and 30 map units) into a single artificial
entity (24-30 map units) designated “chimpanzee.”
This 896-bp segment contains the genes for three transfer RNAs and parts of two
polypeptides, ND4 and ND5, subunits of the respiratory chain NADH dehydroge-
nase; the segment is homologous in all five species. Homologous segments are also
available for the full human, bovine, and mouse sequences and for partial sequences
from rat. The five primate samples differ from each other by base substitutions at
283 positions and by deletion of 1 bp (position 560) from the variable loop in the
orangutan gene for serine transfer RNA. The sequence differences range from 9 to
19% among species, in agreement with estimates from cleavage map comparisons,
thus confirming that the rate of mtDNA evolution in hominoids is five to ten times
higher than in hominoid nuclear DNA.
The most striking new finding to emerge from these primate mtDNA sequence
comparisons is that transitions (substitutions involving A tf G or T tf C-that is,
substitution of one purine for another purine or a pyrimidine for another pyrimidine)
greatly outnumber transversions (A T, A c* C, G tf C , or G
++ T-that is, ++
substitutions of purine for pyrimidine or pyrimidine for purine). Ninety-two percent

of the differences among the most closely related species (human, chimpanzee,
gorilla) are transitions. For five pairs of species with longer divergence times, the
observed transitions fall to 45% in comparisons between primates and nonprimates.
The time dependence is probably due to obliteration of the DNA sequence record of
transition by multiple substitutions at the same site. This finding illustrates the
importance of choosing closely related species for analysis of evolutionary processes
at the mtDNA molecular level. The slope of the curve relating nucleotide divergence
to time approaches zero after about 20 million years (Brown et al., 1979, Fig. 3).
Aquadro et al. (1984)suggest that the data from the complete sequences of human,
mouse, and cow and the partial sequences of the primate series of Brown et al. (1982)
support the hypothesis that the decrease in the proportion of transitions observed as
divergence increases is a consequence of the highly biased substitution process in
favor of transitions. In addition, their study supports the hypothesis that although a
portion of the mtDNA molecule evolves at an extremely rapid rate, a significant

portion (45-49%) of the genome is under strong selective constraints. Constraints
related to secondary structures exist for transfer RNAs. Of the 195 nucleotides
constituting the serine, histidine, and leucine tRNAs, 69% are conserved in the
lineages leading to the six primates, mouse, and cow. Similar restraints hold for
ribosomal RNAs.
Construction of phylogenetic trees by using the mtDNA sequence differences of
hominoids supports the view (expressed in Fig. Id) that the human lineage branched
off only slightly before the gorilla and chimpanzee lineages diverged and strength-
ens the hypothesis that humans are more closely related to chimpanzees and gorillas
than to orangutans. However, based on this limited sequence data (only 5.4% of the
total mitochondria1genome), alternative branching orders for humans-chimpanzees-
gorillas cannot be ruled out with statistical confidence: 1)the most parsimonious
tree computed by Brown and associates requiring 145 events makes chimpanzees
and gorillas a monophyletic group joined next by humans. 2) The next better tree
with 147 events groups humans and chimpanzees, with gorillas joining next; 3) the
third-better tree with 148 events joins humans and gorillas, with chimpanzees as a
sister group; 4)the results are significantly decisive in rejecting the tree requiring
173 events that joins humans to the common ancestor of a monophyletic group
containing chimpanzee, gorilla, and orangutan.
The historical preponderance of transitions helps us to understand why it was
hard to establish the branching order of hominoid lineages by earlier restriction
enzyme comparisons. A high incidence of transitions inevitably produces parallel
and back mutations at the same site among lineages. The problem of parallel and
reverse changes has long been recognized in studies of molecular evolution including
an earlier restriction mapping study of Ferris et al. (1981a),but its molecular basis
became apparent only with the work of Brown and his associates (1982).
Nei et al. (1985) reanalyzed the sequence data of Brown’s group (1982), including
estimates of the standard error of branching points, to construct a tree by the
unweighted pair-group method with arithmetic mean (UPGMA) with the same
branching order as shown in Figure 2. The accuracy of their tree was then increased
when Saitou and Nei (1986; Fig. 9) analyzed the combined sequence data of Brown
et al. (1982) and Hixson and Brown (1986) representing a mtDNA segment of 1,834
bp to construct the tree shown in Figure 2, using the UPGMA method to estimate
branch lengths and the method of Nei et al. (1985)to estimate the standard error of
branching points.
In topology this tree makes human and chimpanzee diverge from a latest common
ancestor after the divergence of gorilla. However, the branching points a and b in
Figure 2 are not significantly different, so the mtDNA sequence in the segment
cannot resolve the human-chimpanzee-gorillatrifurcation. But, note that the earlier
divergence of orangutan from the African apes and humans is statistically signifi-
cant. This is further evidence against the phylogeny (Fig. 10 of Schwartz (1984)
supporting the orangutan as the closest human relative. It is also evidence against
the finding of Kluge (1983) and others that the three pongids (chimpanzee, gorilla,
and orangutan) form a monophyletic group whose members are more closely related
to each other than to humans.
Bishop and Friday (1985) analyzed the 896-bp segment sequenced by Brown and
associates (1982)by using a new probabilistic model of evolutionary genetic change.
They published five trees (their Figs. 4 , 5 , and 7, not reproduced here) based on three
genes for tRNA (his, leu, and ser) and parts of genes NO4 and N05. The trees for
histidine tRNA and NO4 support the HCG trichotomy; that for NO5 supports the
HC-G relation; and that for serine tRNA places CG on one branch and H on a
different branch. The tree for leucine tRNA is difficult to interpret as it groups
gorilla and gibbon on one branch and chimpanzee on another with humans on a
third. The joint tree for the entire segment (their Fig. 8) makes humans and
chimpanzees nearest relatives and orangutans and gibbons most remote, in support
of the HC-G phylogeny. Their method needs further testing.
Spuhlerl EVOLUTION OF MITOCHONDRIAL DNA IN MONKEYS, APES, AND HUMANS 29
Complete nucleotide sequences for three mitochondrial transfer RNAs are avail-
able for five hominoid species, as shown in Figures 3-5. The shading indicates
complementary base pairing in the cloverleaf secondary structure. The regions of
the cloverleaf structure are labeled above the alignment. The sequences of mitochon-
drial tRNA genes differ radically from their nuclear correlates in smaller size, lower
G + C content, higher frequency of nonstandard base pairs (A:C and G:T) in stem
regions, and especially in the higher degree of nucleotide sequence variation ac-
cepted in the mitochondrial tRNAs (Attardi, 1985; Brown, 1985). Of the three tRNA
genes in hominoids, serine is the most and leucine the least variable; the percentage
of bases in common sequence for all five species is 78.26 for histidine, 74.58 for
serine, and 92.96 for leucine tRNA genes. The orangutan shows a cytosine deletion
from position 33 in the variable arm of the gene for serine tRNA reducing the gene
to 58 bp in length. Humans are polymorphic (A in the Cambridge sequence of
Anderson and associates [1981] and G in the sequence fragment of Brown et al.
[1982])for the nucleotide at position 569 in Brown’s sequence.
The two-dimensional cloverleaf structures of histidine (Fig. 61, leucine (Fig. 71, and
serine (Fig. 8) mitochondrial transfer RNAs are shown. These three tRNAs are the
only complete direct gene products known for five species of hominoids.
I used Felsenstein’s (1981) DNA maximum likelihood PHYLIP program (Felsen-
stein, 1985)to estimate separate trees (Fig. 9) for each of the three mtDNA transfer
RNA genes. My results differ considerably from those of Bishop and Friday (1985).
The histidine and leucine tRNA data support the HC-G topology, and the serine
structure the H-CG tree, all important branch lengths being significantly different
from zero at the 1%level.
M I T O C H0 N O R I C L li 1 S II D I IIE T R A b I S F E R RN A
Aniinoacyl Stem D Stem 0 LOOP
1 2 3 4 I‘ 6 7 8 9 1 0 11 12 1 3 1 4 1 5 1 6 1 7 1 8 1 9
1 HUMAN G T A A A T A T ~ G ~ T T A R C C A A
?CHIMPANZEE 6 T R A A 1 A T A G T T 1 A A C C A A
3GORTILA 6 T A A A T A I A G 1 T T A P C C A A
ORANGUTAN G T A A A T A T A G T T T R R c c A A
5GlBBON 6.1 A A A C A 1 A G T i 1 R R T C R A
COMMON G T A A A A T A G T T I A A C A A
D Stem A n t i c o d o n Stem Anticodon Looir Antlrodon Stem Var
2 0 2 1 22 2 3 2 4 2 5 26 2 7 7 8 7 9 3 0 3 1 3 2 3 3 3 4 3 5 3 6 3 / 3 8 39 4 0 4 1 4 2
A A C A T A G A T I G T G A A T C T A A
Loop I ’ Y C stell> TYT Loop T‘YC s t e n , Aml”0-
4 3 4 4 4 5 4 6 4 7 4 8 4 9 50 5 1 52 1 3 5 4 5 5 1 6 1 7 58 1 9 60 6 1 6 2 6 3 6 4 6 5
1 A C A E A G G C T T A C G A C C C C T
Z A C A $ A G G C T C A C G A C C C C T
~ A C R ~ ~ ~ G C T C A L A A C C C C T
~ R T I ~ ~ B G C C C C A C A A C C C C T
5 A T A D A G G C T C G C A A C C T C T
A R G G C A C C C l T T
acyl Stem
66 6 7 6 8 6 9
l T A C C
2 T A C C
3 t 4 r C C
4 T A C C
S T A C C
Fig. 3. Alignment for mitochondrial histidine transfer RNA in five hominoid species as sequenced by
Brown et al. (1982). The shading indicates suggested base pairing in the cloverleaf secondary structure.
The regions of the cloverleaf structure are indicated above the alignment. The codon is flanked by heavy
vertical lines. Residues that are common to all sequences at a position are listed below the alignment.
M I T O C H 0N D R I A L L E U C I N E 1 R A N S F E I1 R N A
Aioinaacyl Stern D Stem D Loop
Fig. 4. Alignment for mitochondria1 leucine transfer RNA in five hominoid species as sequenced by
Brown et al. (1982).Details as in Figure 3.
M I T O C H O N D R I A L S E R I N E T R A N S F E R RNA
Aminoacyl Stcm D Loop Anticodon Stem
Fig. 5. Alignment for mitochondrial serine transfer RNA in five hominoid species as sequenced by Brown
et al. (1982). Details as in Figure 3.
H l s t i d i n e T r a n s f e r RNAs ( A n t i c o d o n G T G ) 6 9 bp
HUUAN L 1 2 1 3 8 CHIMPANZEE L I Z 1 3 8 G O R I L L A 112138
c c C
G - C G - C 6 - c
T - A T - A T - A
A - T A - T A - T
A - T A - T A - T
A TTTG AGAGG T A TTTG AGAGG C A TTTG AGAGG C

C C C T C C C T C c C T
c AAAC A C AAAC A c AAAC A
A A A A A A A A A
T - A C T -A C T
A C -
c - 6 C - G C
G -
A - T A - T A
T -
G - C G - C G
C -
A - T A - T A
T -
T A T A T A
T I T A T A
G T G G T G G T G
ORANGUTAN L12138 LAP GIAAON L 1 2 1 3 8 BOVINE L11907
C c c
G - C G - C G - C
T - A T - A T - A
A - T A - T A - T
A - T A - T A - T
A - T * c A - T
T - A c - 6 T - A
A - T A A A - T A A A - T C A
T TCCCC C T TCTCC A T TCTTC T
A A A A A G A A I
A TTTG AGGGC C A TTTG AGAGG C A TTTG AGAAA C
C T C C T T C T C T C T
T AAAC A C AkAC A A AMC A
A A A A A a A A A
T - A T T - A T T - A C
T - A T - A T - A
A - T A - T A - T
G - C G - C 6 - c
A - T A - T A - T
'r A T A T A
T A T A A A
G T G G T G G T G
Fig. 6. Cloverleaf secondary structure of mitochondria1 histidine transfer RNA gene in five hominoid
species and bovines. The structures for human and bovine are from Anderson et al. (1982).
Hasegawa et al. (1984), Hasegawa and Yano (1984), and Hasegawa et al. (1985)
have published a tree based on the hominoid mtDNA sequence of 896 nucleotides,
with the homologous sequences for bovine and mouse included as an out-group
comparison. They employed the maximum likelihood program of Felsenstein (1981),
using empirical frequencies of the four nucleotides and the observed proportion of
transitions and transversions in the segment. The method provides estimates of the
variance of each branch length and the natural log likelihood (In L) of each tree.
(See Thompson, 1975, for a full account of the use of likelihood in making evolution-
ary trees.)
Hasegawa and associates found that their best tree had the HC-G relationship
(Fig. lc) with In L = -1,733.34; the next better had the HG-C (Fig. le) result with
In L = 1,740.36 (a difference of -7.01); and the third-better tree had the H-CG
topology (Fig. Id) with In L = -1,741.09 (difference -8.65). The tree proposed by
Schwartz making humans and orangutans closest relatives (Fig. lf, was consider-
ably less reliable than their three better trees with In L = -1,759.10 (difference
-25.76) and slightly less reliable than the tree supported by Kluge (1983)proposing
that the three pongids (chimpanzee-gorilla,orangutan) are the closest human rela-
tives, with In L = - 1,758.62 (a difference of -25.23).
The argument of Hasegawa and colleagues that a molecular clock using the
hominoid mtDNA sequence calibrated to the paleontological estimate of the primate-
ungulate divergence time produces a divergence estimate for humans and chimpan-
zees so late as to exclude australopithecines from the ancestry of Homo is not
convincing. Dating the primate-ungulate divergence at the extreme of 90 mya as in
their first paper (Hasegawa et al., 1984)is clearly invalid, as the authors recognized
in their second paper. Whether or not the 65-mya estimate used in their second and
third papers is paleontologically reasonable is not the main issue. The main issue is
that a minimum of four divergence times are required for adequate statistical power
L e u c l n e Transfer RNAs (Anticodon T A G ) 71 bp
HUMAN L12266 CIIIMPANZEE L12266 GORILLA L12266

A A
A - T A - T
c - 6 C - G
T - A T - A
T - A T - A
T - & T - A
T - A T - A
A - T TC A - T TC
A AAACC A I AAACC A A AAACC A
Ah A A AA A A A a
C TAGG TTrcc c c TRGG TTTGG C C rAGG TTTGG C
.4 T TG A 1 TG A T TC
G ATCC A G ATCC A G ATCC A
CT A A CC G A CT A A
T - A A T - A A T - A A
T - A T - A 1 - A
G - C G - C G - C
c - c G - C 6 - C
'I c T C T C
c c c c C A
T G P G T G
T * G T A G T A G
ORANGUTAN L1226G L A R GIBBON L12266 BOVINE 12018
A A A
G - T A - T A - T
c - 6 C - G c - 6
T - A T - A T - A
'r - A T - A T - A
T - A T - A T - A
T - A T - A T - A
A - T TC A - T TC A - T TC
A AAACC ii A A?.ACC A A AAACC A
A A n A A A A A <:A n A
C TAGG T'PTGG C C TAGG TTTGG C T TAGG ATTGG C
A '1. TG A T TG A A TG
G ATCe A G ATCC A G ATCC A
C T C A C T A A TTT G A
T - A A T - A A T - A
T - A T - A T - A
G - C G - C G - C
6 - C G - C G - C
T I T C T - A
c n C A C A
T G T G T G
T A G T A G T A G
Fig. 7. Cloverleaf secondary structure of mitochondrial leucine transfer RNA gene in five hominoid
species and bovines. The structures for human and bovine are from Anderson et al. (1982).
in testing estimates from hominoid molecular clocks calibrated against the geologi-
cal time scale (Gingerich, 1986).
Smouse and Li (19871, concluding that a resolution of the human-chimpanzee-
gorilla trichotomy on the basis of the cumulative weight of the different data and
analytical methods may be premature at this time, suggest that a better alternative
to choosing one of the three phylogenies in the absence of decisive evidence would
be t o provide a statement of the relative likelihood of each of the three better trees.
Their estimates suggest that the best-fitting model based on the restriction map
data is that of the H-CG tree, with very short time span between the first split
separating humans and the second separating chimpanzees and gorillas. Chi-squared
tests indicate no real resolution among the three models, and all three have nontri-
vial posterior probability.
Phylogenetic comparisons of the small (12s) ribosomal RNA gene sequences (944-
951 bp long) from the mtDNA of the great apes and humans by Hixson and Brown
(1986) support the notion of approximately equal distances among chimpanzees,
gorillas, and humans with orangutan much less closely related. They report 3.5%
substitutions between gorillas and humans, 3.7% between chimpanzees and hu-
mans, and 4.1% between chimpanzees and gorillas, with 8.8-9.2% between chimpan-
zee-gorilla-humans and orangutans. The authors note, however, that inference from
a shared deletion suggests that gorillas and chimpanzees may be more closely
related to one another than to humans. Hixson, Szura, and Brown have a report in
progress on sequence comparisons for the large (16s) mitochondrial genes in great
apes and humans. The 16s sequence containing 1,559 bp in humans will increase
the hominoid mtDNA sequence to 3,393 bp and may resolve the HCG trichotomy
with a probability of .95.
The displacement loop (D Loop) region, also called the control region, is the most
complex and least understood part in the mitochondrial genome of primates (Brown,
1985). The D Loop region in mouse and human mtDNAs containing the origin of H-
Serine Transfer M A S (Anticadon GCT) 59 bp

,,"MAN L11207 CBIMPANZEE L12207 G O R I L L A L12207
A A A
G - C G - C G - C
A - T A A A - T
G - C G - C G - C
A - T A - T A - T
A - T A - T A - T
A - T A - T A - T
G - C A A C - C A A G - C A A
C GGTAC C C GGTAC C C GGTAC C
T A T A T A
C CCATG A T CCATG A C CCGTC A
C TCT C CCT C CTT
A C A C G c
T C
A - T A
A A
G - C
A - T
G - C
C R
T A
G C T
ORANGUTAN L11207 L A R GIBBON 1,11207 BOVINE L11197
A A G
G - C 6 - c G - C
A - T A - T A - T
6 - c G - C A - T
A - T A - T A - T
A - T A - T A - T
A - T A - T A - T
G - C A A G - C A A G - C G A
C GGTAC C C GGTAC C T GGTAC T
P A C A A A
c CCATG G C CCATG G T CCATG A
6 - c G - C
A - T A - T
A C A C
C A C A
T A T A T A
G C T G C T G C T
Fig. 8. Cloverleaf secondary structure of mitochondria1 serine transfer RNA gene in five hominoid species
and bovine. The structures for human and bovine are from Anderson et al. (1982).
strand synthesis is the most divergent part of the whole genome with a remarkable
lack of resemblance at both the 5' and 3' ends of the region (Bibb et al., 1981;Tapper
and Clayton, 1981; Walberg and Clayton, 1981). Compared to humans, the control
region of gorillas exhibits four deletions totalling 170 bp. The control regions of the
common and pygmy chimpanzee differ in nucleotide sequence by about 8% substi-
tutions between each other and by about 12%from humans. The divergence between
gorilla and human is about 13%. (In addition to this divergence by substitution,
more than 15%of the gorilla sequence has been deleted.) These results are based on
unpublished data of J.E. Hixson and W.M. Brown cited in Brown (1985).
mtDNA VARIATION IN OLD WORLD MONKEYS
George (1982) used 22 restrictions enzymes (seven of four-base, one of five-base,
and 14 of six-base recognition sequences) to produce fragment patterns and cleavage
maps for eight species of Old World Monkeys. His tree requiring the minimum
number of base substitutions (89) is presented in Figure 10. In this tree the green
monkey branches off just before the last common ancestor of the three macaque
species, making the green monkey appear more closely related to macaques than
baboons are to macaques. The commonly accepted tree (with a branching order
requiring 92 mutations according to the mtDNA data) based on neontological data
other than mtDNA makes baboons and macaques closer relatives with the green
monkey branching off earlier than the common ancestor of baboons and macaques.
In George's mtDNA results the green monkey differs from the other seven species
in about 15%of the total genome, suggesting that multiple substitutions at the same
site may generate parallelism and reversals that account for this anomalous phylo-
genetic tree. But note also that the standard deviations of the branching points in
the mtDNA tree are large.
34 YEARBOOK OF PHYSICAL ANTHROPOLOGY [Val. 31, 1988
HUMAN
CHIMPANZEE
n
.036*'
. .064** ,051"
L
016ni
0 HUMAN
Leucine t R N A
CHIMPANZEE
Fig. 9. Unrooted trees for mitochondrial histidine, serine, and leucine transfer RNA genes in five
hominoid species reconstructed by using the maximum likelihood method of Felsenstein (1981).
PCPlO PAP10
P ANllliIS
CERCOPITHECUS A E T H I OPS
MRCACA MULATTA
M. FASCICULARIS
M. N E M E S T R I N P
M l n ? m u m E v e n t s 89
PllESBYTIS ENTELLUS
Fig. 10. Evolutionary tree reconstructed by George (1982) from restriction site data for mitochondria1
DNA from seven Old World Monkeys.
THE VALIDITY OF PHYLOGENETIC INFERENCES BASED ON mtDNA

mtDNA phylogeny in other mammals and birds
As in the hominoids, mtDNA evidence from the seven extant species of the genus
Equus also fails to resolve the branching order of some species. George and Ryder
(1986) made restriction-endonuclease maps of the Damara zebra, Grevy’s zebra,
mountain zebra, Somali ass, onager, Przewalski’s horse, and domestic horse. lndivi-
dual species maps contained a mean of 60 cleavage sites for 16 enzymes with 29
sites invariant for all seven species. Based on a mean divergence rate of 0.02/mya,
the variation between species supports a divergence of extant lineages from a
common ancestral equid about 3.9 mya, in good agreement with the well-docu-
mented fossil evidence for divergence 3-5 mya (Simpson, 1951;Lindsay et al., 1980).
The concordance of the molecular and paleontological divergence times for the
equids gives support to the estimate from mtDNA map data that the living major
human races diverged at least 180,000 years ago (see below). However, Kessler and
Avise (1985) point out that divergence times for some pond ducks, yellow warblers,
thrashers, and sparrows derived from mtDNA and protein-calibrated-molecular
clocks cannot readily be reconciled with some published dates based on fossil re-
mains and that “the empirical and conceptual issues raised by these findings are
highly analogous to those in the long-standing debate about rates of molecular
evolution and times of separation of ancestral hominids from African apes.”
Coevolution
In both lower and higher eukaryotic cells, biogenesis of mitochondria involves two
processes (Attardi and Ching, 1979): 1) The bulk formation of the mitochondrial
membranes and 2) the differentiation of the mitochondrial anlagen into organelles
active in oxidative phosphorylation. While the formation of the undifferentiated
mitochondrion appears to be solely controlled by the nuclear genome through the
activity of the cytoplasmic protein-synthesizing apparatus, the differentiation into
active, energy-producing organelles requires both components coded by the nuclear
genes and synthesized on cytoplasmic ribosomes and components coded by mitochon-
drial genes and synthesized on mitochondrial ribosomes. mtDNA gives an excellent
example of coevolution. In catarrhine primates mitochondrial-encoded subunit I1 of
cytochrome oxidase has undergone at least a fivefold acceleration in the rate of
evolution in parallel with nuclear encoded cytochrome c (Osheroff et al., 1983). This
observation suggests that the evolutionary paths of mitochondrial and nuclear
genomes are not fully independent.
Bottlenecks
If a breeding population is reduced to only a few individuals, there may be rapid
change in gene frequency without any change in DNA sequence. Since a single
breeding pair of diploids contains four nuclear genomes and only one transmissible
mitochondrial genome, a bottleneck could result in the loss of all mtDNA variability
while retaining some of its nuclear DNA variability.
Study of mtDNA suggests that H. sapiens is a relatively young species (Ferris et
al., 1981b; Brown, 1980; Wilson et al., 1985) compared to African ape and other
primate and mammalian species. The world human mtDNA mean pairwise diver-
gence is only 0.36%, in contrast with an average of 1.33% for pongids, 4.10% for
crab-eating macaques, and 2.90% for the deer mouse Peromyscus maniculatus (Wil-
son et al., 1985).
Observing the relatively low mtDNA sequence heterogeneity (18%)in living races
known at the time, Brown (1980) speculated (with a base substitution rate of 0.5-
1.0% per million years) that all humans may have descended from a single mated
pair that lived 180,000-360,000 years ago (some 9,000-18,000 generations) and
suggested the possibility of a severe bottleneck in human population numbers such
that the major racial divergences in “present-day humans evolved from a small
mitochondrially monomorphic population that existed at that time . . .” (1980, p.
3,609).
36 YEARBOOK OF PHYSICAL ANTHROPOLOGY [Vol.31, 1988
As there is no archaeological or other independent evidence for this specific

bottleneck, it is important that population biology supply alternative possibilities.
In a simulation study, Avise et al. (1984) demonstrated that stochastic extinction of
mtDNA lineages may be rapid under demographic conditions plausible for human
populations during the Paleolithic. In stable populations initiated by n females a n d
or regulated by a carrying capacity of k = n, it is highly probable that within 4n
generations all descendants will trace their mtDNA ancestries to a single female
founder. Avise and associates define II as the probability of survival of two or more
independent mtDNA lineages over G generations. A population founded by n =
15,000 unrelated females with a Poisson distribution of offspring and mean 1.0
yields a value of II = 0.5 within 18,000 generations.
The tree-making procedures employed by Wilson’s group invariably but arbitrarily
go back to one specific mtDNA type, but the individual female represented by that
type, rather than being a first woman like the mythical, Biblical Eve, %auld have
belonged to a population of many thousands or tens of thousands of females, the
remainder of whom left no [mtDNA lineage] descendants to the present day, due
simply to the stochastic lineage extinction associated with reproduction [in small
sibships]” (Avise et al., 1984, p. 104).
MITOCHONDRIAL DNA OF LIVING HUMANS
Geographical and racial distributions
Figure 11 shows a high-resolution map of the locations of 441 restriction sites
found in a survey of 112 humans plus the Cambridge reference sequence with the
aid of 12 restriction enzymes tested on donors with birthplaces of world-wide distri-
bution (Cann et al., 1984). Vertical lines above the horizontal line show the constant
sites (63.04%of the total observed) present in all the human mtDNAs examined.
Vertical lines below the horizontal line show the variable sites-those that are
absent in some of the mtDNAs. Height of the vertical lines (1-6 units) is proportional
to the number of sites found within a 80-bp segment. A majority of the 411 mtDNA
restriction sites, and presumably a majority of the alleles of the 37 mitochondria1
genes, are common to all human populations.
Differences in method of phylogenetic analysis and interpretation account for the
apparent discrepancy between the Allan Wilson group (Berkeley) and the Cavalli-
Sforza group (Stanford) regarding the association between the distribution of mtDNA
restriction polymorphisms and geographical, racial, or ethnic subdivision.
Both groups start by identifying restriction enzyme morphs, sometimes called
nucleomorphs. There are one or more morphs for each enzyme. The same morph
may be detected in different individuals belonging to the same breeding population,
in individuals belonging to different populations, or even in individuals belonging
to different species. For a given set of enzymes and population, some morphs are
constant and some are variable. Only the informative characters (those morphs that
differ in two or more individuals) are used in constructing distance (divergence)
matrices and trees. The combination of the mtDNA morphs for each and all of the
enzymes employed defines the mtDNA type of a particular individual. The external
nodes in the Berkeley tree (for example, Fig. 12) are observed individuals character-
ized by a specific type. The tree pictures the divergence of individuals in mtDNA
lineages.
The Stanford group puts more emphasis on the cladistic relationships among
mtDNA types, some being recognized as ancestral, others as derived. Each type is
assigned a number and described by listing the restriction enzyme morphs in a
specific order (Johnson et al., 1983; Wallace, 1983).To designate types, Wallace et al.
(1985)adopt the order HPAI, BamHI, HaeII, HpaII (with isoschizomere MspI), AvaII,
and HincII. In this scheme, type 1-2 (morph numbers 2-1-1-1-1-2) defined by those six
enzymes is the most common mtDNA type in the world. The external nodes of the
Stanford tree (for example, Fig. 13) are representative of mtDNA types. The tree
does not represent all the individuals in the tested populations; rather it presents
the phyletic relations (cladistics) of the types, some ancestral, some derived.
a)
I 278 CONSTANT S I T E S
163 V A R I A B L E SITES
H-STRAND
b)
ND3 ND4L
178 1bS NO1 I NO2 I I GO1 I CO2 1 8 l A T P 6 1 C03 I I I NO4 NO5 I I Cytb 11
Phe Val Leu I l e f-Met Trp Asp tys Gly Arq H i s Ser Leu Thr
L-STRAND
1 IND6 I
Gln AldAsnCysTyr Ser Glu Pro
I I 1 1 1 - L I I I 1 2 1 I I I I
0 2 4 6 8 10 12 14 16 Kb
Fig. 11. Locations of genes and restriction enzyme cleavage sites in human mitochondria1 DNA.
a: Locations of cleavage sites found in mtDNAs from 112 humans by using 12 restriction enzymes plus
the Cambridge reference sequence. Vertical lines below the horizontal line show the variable sites-that
is, sites present in some but not all of these mtDNAs. The vertical lines above the horizontal line show
the sites present in all human mtDNA examined. Thus 63% of the 441 restriction enzymes sites are
shared by all humans examined. b: Linear representation of the human circular mtDNA molecule
consisting fin the reference sequence) of 16,569 bp, arbitrarily starting at the beginning (3’) of the
phenylalanine tRNA gene. The heavy strand @ I) and the light strand 6)
is above below. The functional
regions include the following: Two ribosomal RNA genes, 12s and 16s. Twenty-two transfer RNA genes
(black shading) indicated by the three-letter abbreviation of their transferred amino acid. Thirteen
protein-coding genes, including seven subunits of the NADH-CoQ reductase-labeled ND1-4, ND4L, ND5-
6, three cytochrome oxidase subunits (C01-3), cytochrome b, and two ATPase subunits (6, and 8 labelled
by “8”). The displacement loop (D loop) extending from 16,024 to 516 bp, shaded by dots (data from
Anderson et al., 1981; Sanger, 1981; Cann and Wilson, 1983; Attardi, 1985; Chomyn et al., 1985).
In the early papers Cann and her associates in the Berkeley group report that
patterns among mtDNA maternal lineages show only weak association with ethnic
subdivision (Cann, 1982, 1985; Cann and Wilson, 1983). Cann and Wilson add that
their conclusion is supported by sequence data from seven individuals reported by
Aquadro and Greenberg (1983). In later papers, especially after the Papua New
Guinea sample was analyzed, the Berkeley group reported stronger correlations
with ethnic-geographic distribution (Stoneking, et al., 1986a).
From the start, Wallace and his associates in the Stanford group found strong
association (as in Figure 13)between mtDNA polymorphisms and racial distribution
(Johnson et al., 1983; Wallace, 1983; Wallace et al., 1985).
In Cann’s 1982 phylogenetic study of 89 informative restriction types, the 110
subjects were grouped into five areas of geographic origin representing Australia
(all donors were pure aboriginals), Asia (including China, Japan, Korea, Philippines,
Polynesia, Vietnam, and Indonesia), Africa (including Black Americans and individ-
uals from sub-Saharan Africa), and Europe (including Europe, North Africa, and the
Near East).
Actually Cann’s early trees show stronger geographic and ethnic association than
is apparent from a quick glance at the whole tree. If we divide the 110 individuals
in an early published tree (Cann and Wilson, 1983, Fig. 5, p. 707) into four sets
containing individuals numbered 1-20,21-38,39-78, and 79-110 counting from the
left, each major geographic group (Australian, Asian, African, and European) has
observed frequencies in one and only one set that are about double the random
YEARBOOK OF PHYSICAL ANTHROPOLOGY [Val. 31, 1988
1 10 20 30 40 50 60
0 AFRICA
w
u 0 ASIA
z
W
A AUSTRALIA
w
CL
0.4 A PAPUA NEW G U I N E A
-a
>
W
I
o EUROPE
W
z
w
0
Vl
W
0
140 130 120 110 100 90 80 70
Fig. 12. Tree produced by maximum parsimony analysis of minimal length by using restriction site data
relating 147 human mtDNA types representing five geographical regions. The tree is redrawn from
Stoneking et al. (1986b), with the vertical time-scale doubled. These authors give a list of the Papua New
Guinea mtDNA polymorphic sites; C a m et al. (1986) list the polymorphic sites defining each of the other
134 mtDNA types. See text and these references for details.
expectation: (x29 = 41.53, P < .001); seven Africans observed (3.64 expected) in the
1-20 set, nine Europeans observed (5.20 expected) in the 21-38 set, 22 Asians
observed (13.29 expected) in the 39-78 set, and 11 Australians observed (3.71 ex-
pected) in the 79-110 set. Here we are not testing the validity of the clusters obtained
by Cann in the tree; rather we are showing that some of the five ethnic groups
recognized by Cann and Wilson have higher frequencies in certain clusters-that is,
the types do show a statistically significant “racial correlation.”
Figure 13 is redrawn from the Johnson et al. (1983) network showing strong
correlation in the distribution of mtDNA polymorphisms among the three classical
major races-Caucasoids, Negroids, and Mongoloids. In the original phylogeny of
mtDNA types (their Fig. 6) Bantu and Bushman Africans are shown to vary greatly
by type. Wallace and his associates have extended the phylogeny of mtDNA types to
include American Indians (Wallace, 1983; Wallace et al., 19851, the Tharu population
of Nepal (Brega et al., 1986a), populations of Jews and Arabs in Israel (BonnC-Tamir
et al., 19861, and Italians from Sardinia and Rome (Brega et al., 1986b).
Bonne-Tamir and Cavalli-Sforza (1986)state a n important, general conclusion:
“In spite of very small numbers in each group the results demonstrate the exis-
tence of specific mtDNA fragment patterns varying in frequency from population to
population, and suggest that certain types may be unique to certain groups.
Even with the limited use of only 5 restriction enzymes, a high correlation between
mtDNA types and ethnic origin of the individuals is confirmed 11986, p. 1061.”
Spuhler] EVOLUTION OF MITOCHONDRIAL D N A IN MONKEYS, APES, A N D HUMANS 39
CAUCASOID
Fig. 13. Phylogeny of mtDNA types according to Johnson et al. (1983). The tree was derived by parsimon-
ious methods as described in the text. The open circles represent hypothetical types. Of the three
postulated central mtDNA types, type 1 is common to all three major races, type 6 is common to
Mongoloids and Caucasoids, and type 8 is common to Negroids and Caucasoids.
Origins of H. sapiens
A majority of paleoanthropologists agree that H. sapiens evolved from H. erectus
at least in one place (Howells, 1976, 1980; Wolpoff, 1980;, Andrews and Franzen,
1984; Smith and Spencer, 1984; Wood, et al., 1986).A minority claim that H. erectus
is not ancestral to H. sapiens (Eldredge and Tattersal, 1982) because H. habilis is
the ancestral species. A few claim that the H. erectus fossils belong to the species H.
sapiens (Jelinek, 1978,1985). Many hold that the major difference between H. erectus
and H. sapiens fossils is in evolutionary grade (Howell, 1978). Since H. erectus was
limited in geographical distribution to Africa and Eurasia we can exclude Australia,
Oceania, and the Americas as the region where the transition took place with, or
without, speciation.
It needs emphasis that both the replacement and multiple transition hypotheses
are cases of monophyletic speciation, having nothing to do with the polyphyletic
origin of the races or subspecies of H. sapiens from different species, as in the
discarded theories of Schwalbe (1906) and Bolk (1926) that Caucasoids evolved from
chimpanzees, Negroids from gorillas, and Mongoloids from orangutans.
As early as 1950 Dobzhansky supported a nonorthogenetic form of Weidenreich’s
regional transition hypothesis, but one with considerable gene flow and thus not
independent: “Different populations (races) of a polytypic species may be descended
largely from different races of the ancestral species and may differ in some genes in
which these ancestral races differed. And yet, a polytypic species may still evolve as
a single genetic system” (Dobzhansky, 1950, pp. 106-107). Many American popula-
tion geneticists still accept this evolutionary possibility in the origin of subspecies.
The regional transition does not require special or improbable modes of microevolu-
tionary process. The standard, well-tested processes of mutation, selection, gene
flow, and genetic drift operating in subdivided populations are all that are necessary
and are sufficient (Weiss and Marayama, 1976).
The evolutionary biologist Van Valen (1966, 1986) supported the hypothesis of the
origin of modern mammalian continental races or subspecies by regional transition;
he cited two cases in the mammalian fossil record of multiple transitions of subspe-
cies from one species to subspecies of a successor species. Freudenthal (1965) sug-
gested a multiple transition of three subspecies of European Miocene cricetid rodents
of the species Megacricetodon gregarius from three subspecies of M. crusafonti.
Martin (1970) proposed that the extinct Miocene-Pleistocene Kansas population of
the cotton rat Sigmodon minor evolved from S. medius in Kansas and that the
Arizona population of S. minor derived from S. medius in Arizona.
The hypothesis that H. sapiens originated by speciation in a single region about
100,000 years ago and then replaced all older hominids in all other inhabited regions
has testable genetic consequences that differ from the hypothesis that Homo was
present in Africa, Asia, and perhaps Europe for at least 1million years, and that H.
erectus populations evolved in parallel to anatomically modern H. sapiens popula-
tions in three or more different regions in the Old World. The regional transition
hypothesis is supported if the modern world pool of mtDNAs exhibits significant
regional differences that are significantly older than 100,000 years. The replacement
hypothesis is supported if the greatest time depth of significant mtDNA differences
in regions outside of the (African) region of speciation is more recent than 90,000-
180,000 years ago (Cann et al., 1987, p. 35).
Stringer and Andrews (1988)propose that all modern humans belong to a species
separate from archaic humans and descended from a small population of Africans
that killed off and replaced archaic natives 100,000-200,000 years ago. The evidence
detailed below shows that their genetic argument is greatly flawed by failure to
acknowledge 1)the repeated demonstration that mtDNA types vary significantly
from population to population in Europe, the Near East, Asia, Africa, and Australia
and 2) that many mtDNA types are unique to and relatively old in some local
populations. Furthermore, they fail to accept the more reliable rate of mtDNA
nucleotide substitution of 0.5-1.0% per site per million years proposed by Brown
(1985) and Nei (1985). Both the regional variablity and the time estimates favor the
regional transition hypothesis and oppose the younger replacement.
That there is significant regional variation and great time depth to existing
human mtDNA differences is indicated by several studies. Horai et al. (1986) com-
puted a n mtDNA phylogenetic tree by the unweighted pair group method (Sokal
and Sneath, 1963; Nei, 1975) by combining their (Horai and Matsunaga, 1986) data
using 11 restriction enzymes on Japanese with Cann’s (1982) data on Negroes and
Caucasians (her data analyzed with Alu I and Dde I were excluded). A total of 117
restriction types were observed among the 176 individuals in the three racial groups,
no type being observed in more than one group (Table 5). The individuals were
TABLE 5. Number of mtDNA restriction types and estimates of nueleotide differences among
three maior racial mourn (data from Horai et a l . 1986)
No. of Composition of cluster by race

Cluster tvDes JaDanese Caucasian African
c1 9 0 0 9
c2 14 14 0 0
c3 13 2 6 5
c4 6 6 0 0
c5 19 0 18 1
C6 24 24 0 0
C7 18 9 7 2
CS 14 7 5 2
Totals 117 62 36 19
Average no. 0.0033 0.0026 0.0025 0.0047
nucleotide
substitutions
Spuhler] EVOLUTION OF MITOCHONDRIALDNA IN MONKEYS, APES, AND HUMANS 41
classified into eight clusters (Cl-C8). Three clusters (C3, C7, C8) represent intermin-
glings of individuals from all three racial groups; the remaining five show distinct
clustering by race. The oldest branching point between C1 and C2 goes back to
about 170,000years ago, assuming a substitution rate of 2 x lo-’ per site per year,
and to about 480,000 years ago, assuming a rate of 0.71 x lo-’ per site per year.
The authors suggest that the polymorphism existed prior to racial divergence. The
intermingling of races in clusters may be due to ancient polymorphism or to gene
migration, the latter being possible in the case of some of the American Negroes in
Cann’s sample.
In a study of HpaI cleavage patterns and ABO, MN, Rh, D a y , and V blood groups
in 60 American Blacks, Hsieh and Sutton (1987) estimated a 61% African Black
female input and a 39% Caucasian female input to the American Black mtDNA
gene pool, based on the observation that the majority of African Blacks have HpaI
morph 3, which is absent in Caucasians, and that blood groups R”, Fy, and V+ are
common in African Blacks but rare in Caucasians. These results suggest that Cann’s
“African” sample tested in American Blacks may, in part, be hetergeneous because
of mixed origin and not only because of extreme lineage age. However, the fact that
Cann’s African sample (1982, Table 6) shows only 28 different morphs that are
common to her African and Caucasian samples, but 37 different morphs that are
common to her African and Asian samples, is evidence against the presence of
considerable American white female mixture in her specific “African” sample.
Table 6 shows the distribution of 162 morphs specified by seven restriction en-
zymes observed in donors from four regions: Asia, Africa, Europe, and Japan. The
data are from Cann (1982)and Horai and Matsunaga (1986);they represent all data
on these specific restriction enzymes specified in all of these five regions available
in the literature at the time of writing. Except Japan, these regions are “Old Re-
gions” occupied by H. erectus populations, more than 200 kiloyears ago. The col-
umns on the right record the number of morphs that are unique to each of the four
regions. The columns on the left headed “Common to regions 4, 3, 2” record the
number of morphs that are common to two or more of the four regions; 113 of the
162 morphs (69.76%)are unique to Old Regions (Asia, Africa, Europe, and Japan),
but of these only 16 (9.88%)are unique to Europe. Only three morphs (1.85%)are
common to all four regions; 16 (9.88%)are found in three regions; and 30 morphs
(18.52%)are limited to two regions.
Yu Minshu and his associates at the Institute of Genetics, Fudan University,
Shanghai, People’s Republic of China, studied fragment patterns by using placental
mtDNA isolated from 273 individuals representing the Han, Hui, Uigur, and Kazak
Chinese nationalities by using 14 enzymes (ApaI, BamHII, EcoRI, HindIII, HinfI,
HhaI, HpaII, KpnI, MboI, PstI, PvuII, SacI, ScaI, and XhoI) and the ethidium
bromide method; 38 morphs were observed, including 14 unique to China (Yu et al.,
1988).
The Han speak Chinese and are the dominant nationality in China. The Hui,
originally descendants from eighth-century Arab traders and soldiers, speak Chinese
and are Muslims. The Uigur and Kazak speak Modern Turkic languages that
TABLE 6. mtDNA morphs common and unique to old regions
Ava2 9 0 2 2 0 0 2 3
Rsal 24 1 5 3 0 0 1 14
Taql 19 0 1 3 3 3 2 7
Hpa2 15 0 1 0 2 4 2 6
H i d 32 0 3 5 2 5 6 11
Hhal 15 0 1 4 1 1 1 7
Hae3 48 2 3 13 5 4 2 19
Totals 162 3 16 30 13 17 16 67
Sources of data: Cann (1982); Horai and Matsunaga (1986).
emerged from a common Old Turkic base about 800-900 A.D. The Han were sampled
in Shanghai; the Hui, Uigur, and Kazak were sampled in Urumchi, Xinjiang Prov-
ince. The authors estimate that the Han-Hui diverged from the Uigur-Kazak at a
genetic distance of 0.0333, corresponding to a time of 237,000 years ago. The Chinese
geneticists agree with Cavalli-Sforza and Wallace that all modern mtDNA morphs
trace back to a n Asian common ancestress (Blanc et al., 1983).
Table 7 shows the time depth of divergence in the regional populations sampled
for endonuclease restriction fragment variations.
The distribution of mtDNA restriction types reported in this section supports the
regional transition hypothesis, in agreement with the growing body of paleoanthro-
pological evolutionary reconstructions of the fossil record (Wolpoff et al., 1984; Wu
and Dong, 1985; Smith, 1985; Wolpoff, 1987). This distribution of mtDNA morphs
clearly supports the regional transition hypothesis, because many of the regionally
unique morphs date back more than 200,000 years ago in each of the Old Regions.
This conclusion assumes that the mtDNA trees and chronology are reliable. The
data consistently vary in the direction of support of the regional transition hypothe-
sis. But, because all genetic variation in mtDNA morphs and types is controlled by
one locus, the data are not sufficient to prove or disprove either hypothesis with
statistical significance at the 5%level. Saitou and Omoto (19871, after a new analysis
of the mtDNA distance data within and between African, Asian, Australian, Cau-
casian, and New Guinea populations (the data in Table l of Cann et al., 1987),
conclude that “the time and place of origin of Homo sapiens sapiens are difficult to
estimate from their data” and that “data from mitochondria1 DNA, which can be
regarded as a single locus, are not enough by themselves to establish the branching
pattern of human populations.” The new analysis involved two unrooted trees
constructed by the neighbor-joining method (Saitou and Nei, 19871, both equally the
best of 15 possible topologies. If the root(s) is located by minimizing the variance of
the distances from all populations, either Africans or New Guineans can be the
population that diverged first, a conclusion that is not supported by data from many
nuclear loci, comparative anatomy, or the fossil record. Saitou and Omoto also point
out that use of Nei’s (1985) “more reasonable’’ estimate of the mtDNA divergence
rate (0.71% per site per million years) places the time of Cann, et al.’s (1987) and
Wilson’s earliest divergence point (“a” of their Fig. 3) a t 400,000 years ago (rather
than their estimate of about 200,000 years), “in the middle of the supposed time
span of Homo erectus, but this may not mean that this alternative estimate of the
rate is too small.”
It needs emphasis that the currently investigated mtDNA restriction enzyme data
on human populations are based on only about 1,500 bp out of some 16.5 kilobases,
less than 10% of the total. Further, the crucial African sample of Cann et al., (1987)
is limited to 20 individuals, 18 of them represented by American-born Blacks. The
estimate by Cann et al. (1987, p. 34) that the African “common ancestor of all
surviving mtDNA types existed 140,000-290,000 years ago” is not a confidence
interval, but two estimates based on two different assumed mutation rates, each
estimate associated with a n error of about the same magnitude as the time proposed
(see Nei, 1985). Thus, little confidence should be placed on either the early (say 400
TABLE 7. Regional mtDNA divergence times (rate = X = 0.71 x site-’ year-’ = Zdt, t = WZd)
Population d t Reference’
African ,0057 402,000 1
Asian ,0035 246,000 1
Australian ,0025 176,000 1
Caucasian ,0023 162,000 1
New Guinean ,0025 176,000 1,4
Japanese .0042 296,000 2
Japanese ,0026 183,000 3
Chinese ,0033 232,000 5
‘References: 1 Cann eta]. (1987); 2.Horai et a1 (1984); 3. Horai and Matsunaga (1986); 4.Stoneking et a1 (1986); 5. Yu et
a1 (1988).
Spuhler] EVOLUTION OF MITOCHONDRIAL DNA IN MONKEYS, APES, A N D HUMANS 43
ky or more) or the late (say 200 ky or less) guesses on the time of origin of modern
H. sapiens based on available restriction enzyme information.
CONCLUSIONS
Evolutionary studies of mitochondrial DNA sequences in humans, chimpanzees,
gorillas, orangutans, and gibbons show (with 95% statistical confidence) that chim-
panzees and gorillas are the closest living biological relatives of humans. The most
reliable maximum likelihood tree groups chimpanzees and humans a little closer
than gorillas and humans. Additional sequence analyses of the mtDNA genome will
be required to resolve the human-chimpanzee-gorilla phyletic relationship with
statistical significance.
Investigations of mitochondrial restriction enzyme morphs and types show that
most mtDNA gene variation is shared and invariant in all living human major
races. The mtDNA evidence gives more support to the hypothesis that accounts for
the origin of the major continental races of H. sapiens by transition in three
or more regions than to the hypothesis of world-wide replacement by migration from
a single region.
Additional mtDNA sequence studies in populations of Africa, Asia, and Europe
will provide more conclusive information on the evolution of modern humans.
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Evolution of Mitochondrial DNA in Monkeys, Apes, and Humans

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YEARBOOK OF PHYSICAL ANTHROPOLOGY 31:15-48(1988)

Evolution of Mitochondrial DNA in Monkeys,

KEY WORDS Primates, Transfer RNA, Hominoid phylogeny, Origin of

ABSTRACT The size of the primate single mitochondrial DNA molecular

Mitochondrial molecular genetics made spectacular contributions to biological

0 1988Alan R. Liss, Inc.

containers, which of course, favored the vast volume of study on mitochondrial

TABLE 1. Four restriction methods for comparing mtDNAsl

oppose the direct filiation, compartmentalization theory: 1)Under the symbiotic

A major limitation on the utility of mtDNA in evolutionary studies is that all 37

TABLE 2. Primate species studied for mtDNA uariation

TABLE 4. Genetic code for mammalian mitochondrial genes'

substitutions of purine for pyrimidine or pyrimidine for purine). Ninety-two percent

portion of the mtDNA molecule evolves at an extremely rapid rate, a significant

Aniinoacyl Stem D Stem 0 LOOP

D Stem A n t i c o d o n Stem Anticodon Looir Antlrodon Stem Var

Loop I ’ Y C stell> TYT Loop T‘YC s t e n , Aml”0-

Aminoacyl Stcm D Loop Anticodon Stem

HUUAN L 1 2 1 3 8 CHIMPANZEE L I Z 1 3 8 G O R I L L A 112138

A TTTG AGAGG T A TTTG AGAGG C A TTTG AGAGG C

ORANGUTAN L12138 LAP GIAAON L 1 2 1 3 8 BOVINE L11907

L e u c l n e Transfer RNAs (Anticodon T A G ) 71 bp

HUMAN L12266 CIIIMPANZEE L12266 GORILLA L12266

ORANGUTAN L1226G L A R GIBBON L12266 BOVINE 12018

Serine Transfer M A S (Anticadon GCT) 59 bp

ORANGUTAN L11207 L A R GIBBON 1,11207 BOVINE L11197

THE VALIDITY OF PHYLOGENETIC INFERENCES BASED ON mtDNA

As there is no archaeological or other independent evidence for this specific

No. of Composition of cluster by race

Sibley CG, and Ahlquist JE (1987) DNA hybridiza- 433.

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