Am. J. Hum. Genet. 46:828-842, 1990
Intraspecific Nucleotide Sequence Differences in the Major Noncoding Region of Human Mitochondrial DNA Satoshi Horai and Kenji Hayasaka Department of Human Genetics, National Institute of Genetics, Mishima, Shizouka, Japan
Summary Nucleotide sequences of the major noncoding region of human mitochondrial DNA (mtDNA) from 95 human placentas have been determined. These sequences include at least a 482-bp-long region encompassing most of the D-loop-forming region. Comparisons of these sequences with those previously determined have revealed remarkable features of nucleotide substitutions and insertion/deletion events. The nucleotide diversity among the sequences is estimated as 1.45%, which is three- to fourfold higher than the corresponding value estimated from restriction-enzyme analysis of whole mtDNA genome. A hypervariable region has also been defined. In this 14-bp region, 17 different sequences were detected. More than 97% of the base changes are transitions. A significantly nonrandom distribution of nucleotide substitutions and sequence length variations were also noted. The phylogenetic analysis indicates that diversity among the negroids is much larger than that among the caucasoids or the mongoloids. In fact, part of the negroids first diverged from other humans in the phylogenetic tree. A striking finding in the phylogenetic analysis is that the mongoloids can be separated into two distinct groups. Divergence of part of the mongoloids follows the earliest divergence of part of the negroids. The remainder of the mongoloids subsequently diverged together with the caucasoids. This observation confirmed our earlier study, which clearly demonstrated, by the restriction-enzyme analysis, existence of two distinct groups in the Japanese.
Introduction
The mammalian mitochondrial DNA (mtDNA) is a circular genome approximately 16.5 kbp in length and encodes 13 subunits of the inner-membrane respiratory complexes. The complete nucleotide sequences of human (Anderson et al. 1981), mouse (Bibb et al. 1981), cow (Anderson et al. 1982), and rat mtDNAs (Gadaleta et al. 1989) have been reported. The gross genetic arrangement of these genomes is remarkably conserved. They can be divided into two domains - a coding region constituting over 90% of the genome and a noncoding region which contains both the origin of H-strand replication (Anderson et al. 1981) and the origins of transcription of both strands (Cantatore and Attardi 1980). Received September 26, 1989; revision received November 27, 1989. Address for correspondence and reprints: Satoshi Horai, Department of Human Genetics, National Institute of Genetics, Yata 1,111, Mishima, Shizuoka 411, Japan. © 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4604-0021$02.00
828
Since the original studies which confirmed the maternal inheritance and predominantly uniclonal nature of mammalian mtDNA within an individual (Hutchison et al. 1974; Potter et al. 1975; Giles et al. 1980), numerous reports have indicated that its nucleotide sequence is evolving much faster than that of single-copy nuclear genes (Brown et al. 1979; Ferris et al. 1981). Since there are substantial sequence variations among individuals (Brown and Goodman 1979), restrictionenzyme analysis of mtDNAs has become a powerful tool in elucidating evolutionary relationships among human ethnic groups (Brown 1980; Denaro et al. 1981; Blanc et al. 1983; Johnson et al. 1983; Horai et al. 1984; Wallace et al. 1985; Brega et al. 1986; Horai and Matsunaga 1986; Cann et al. 1987; Harihara et al. 1988). Results obtained from these studies suggest that there is a high correlation between mtDNA restriction types and ethnic origins of individuals. The phylogenetic analysis in the Japanese indicated that they could be separated into two major groups (groups I and II; Horai and Matsunaga 1986). This grouping may also be ap-
829
Human mtDNA Sequence Differences plied to other mongoloid populations, because a 9-bp deletion in region V (Cann and Wilson 1983), which characterizes the group I Japanese, was also observed in non-Japanese mongoloid populations (Hertzberg et al. 1989; Stoneking and Wilson 1989; Horai et al., in press). Therefore, we extended our analysis to include a phylogenetic analysis of the sequence in a particular region of mtDNA. In the present study we have sequenced part of the major noncoding region encompassing the D-loop region of mtDNAs isolated from 95 human placentas. Comparisons of these sequences with others determined elsewhere (Anderson et al. 1981; Greenberg et al. 1983) have revealed striking features of nucleotide substitutions, insertions, and deletions. These results are discussed in light of sequence evolution and functional constraints on this domain. We have also examined in detail the evolution of mtDNA sequences at the gene level, as well as at the population level. Subjects and Methods Subjects
mtDNA from placentas of 95 individuals from three different racial origins was purified to homogeneity, according to a method described by Brown et al. (1979) and Horai et al. (1984). In the restriction-enzyme analysis we observed 62 different types of restriction patterns by using nine enzymes in the Japanese population (Horai and Matsunaga 1986). In the present study we chose 61 Japanese individuals all of whose restriction patterns were different from one another. Ten nonJapanese mongoloids consisted of three Koreans, four Chinese, an Indonesian, a Filipino, and a Papua New Guinean. We have also collected placenta samples from 17 caucasoids, 16 from Europe and America and one from India, and from seven African negroids, from seven different regions, whose mothers gave birth to their babies in Tokyo. A list of subjects is shown in the Appendix (table Al). Cloning of mtDNA
Purified mtDNAs were doubly digested with KpnI and SacI. Digestion products were extracted three times with phenol/chloroform (1/1), precipitated with ethanol, and dissolved in TE (pH 8.0). Then the fragments were inserted into pBluescript (KS -; Stratagene) which was also doubly digested with KpnI and SacI and then was purified by gel electrophoresis. When human mtDNAs were digested with SacI, two fragments (9.6 kb and 7.0 kb) were usually observed.
These fragment sizes agree with the sizes deduced from the published sequence of Anderson et al. (1981), which has two Sacl recognition sites at bp 36 and bp 9643 (the notations are those of Anderson et al. [1981] for base numbers). Three KpnI sites (bp 2573, bp 16048, and bp 16129) are observed in the published sequence data. Although two fragments (13.5 kb and 3.0 kb) are visible, the third fragment, of 81 bp, is invisible under the usual electrophoretic conditions. Through careful examinations with prolonged gel electrophoresis, slight between-individual differences in mobility were observed in the 3.0-kb bands. In the variant pattern, the 3.0-kb band moves slightly slower than that of the usual pattern, suggesting that two contiguous fragments (3013 bp and 81 bp) were fused to generate a larger fragment of 3.1 kb. This mutation is rather frequent in the Japanese population (S. Horai, unpublished data). Therefore, in the double digestions with KpnI and SacI, four or five fragments were produced in each sample because of the polymorphism of one of the KpnI sites. Thus, the smallest fragment with both SacI and KpnI recognition sites is either 482 bp or 563 bp in length and is efficiently inserted into vectors, without purification of the desired fragment. DNA Sequencing
Nucleotide sequences of 482-bp or 563-bp fragments inserted into pBluescript were determined by the dideoxy-chain termination method (Sanger et al. 1977) using the Sequenase kit (U.S. Biochemical) according to the manufacturer's directions. Data Analysis
We aligned the nucleotide sequences of the 482-bp fragments, which were common for 95 individuals, with those of six individuals reported elsewhere (Anderson et al. 1981; Greenberg et al. 1983). We estimated the per-site number of nucleotide substitutions between individual sequences by using the six-parameter method of nucleotide substitution (Gojobori et al. 1982). On the basis of the estimated numbers, phylogenetic trees were constructed by the unweighted-pair-group (UPG) method (Sokal and Sneath 1963) and by the neighborjoining method (Saitou and Nei 1987). Results and Discussion 1. Nucleotide Sequences Figure 1 shows the nucleotide sequence of the light
strand of the noncoding region originally reported by Anderson et al. (1981). KpnI site 1 (KpnI-1) and the
Horai and Hayasaka
830 Sad site are conserved among all individuals; however, KpnI-site 2 (KpnI-2) is polymorphic. Therefore, we cloned and sequenced either a 563-bp fragment or a 482-bp fragment for each individual. A total of 100 sequences were compared with the sequence reported by Anderson et al. (1981). In figure 1, base substitutions, deletions, and insertions detected in at least one individual were also shown below the nucleotide sequence of Anderson et al. (1981). We found mutations at a total of 113 sites. At four sites two different kinds of nucleotide substitutions were observed. Nucleotide changes were observed more frequently in the 5' half of the region than in the 3' half. The observed number of mutations, classified according to the types of mutations, are shown in table 1. In the whole data, transition types of substitution are more predominant than transversions; that is, 97% are transitions while only 3% are transversions. Moreover, transitions between pyrimidines are more prevalent than those between purines. When we counted the nucleotide substitutions for each site, a 10-fold bias favoring transitions over transversions was still observed. The results in the present study confirmed several features of the noncoding region of mtDNA which have been reported by Aquadro and Greenberg (1983) and by KpnI-1 GrTACCACCC AAGTATIGAC TCACCCATCA ACAACCGCTA TGTATTTCGT ACATFACTGC C
t
C
CC
bp 16048 KpnI-2 CAGCCACCAT GAATATTGTA CGGrACCATA AATACTFGAC CACCTGTAGT ACATAAAAAC GGG T A G TT A T C C T A
Greenberg et al. (1983), though only seven or eight sequences were compared by these authors. We also observed both deletion of A at two sites and insertion of C at five sites. These deletions and insertions were found in a particular domain containing serially repeated stretches of A and C (see table 2). 2. Distribution of Mutated Sites
The distribution of mutations in the D-loop region is shown in figure 2. The histogram represents the total number of mutations within contiguous nonoverlapping blocks of 20 bases. Figure 2 shows that most highly variable base sites lie in the blocks near the tRNAproline gene, while one highly polymorphic base site was observed in a block near the SacI site. This site is polymorphic for all three major races (position 16519 in table 4) and probably derived from the ancient polymorphism of this site. In this figure a hypervariable region exists in one of the 20 base blocks ranging from bp 16178 to bp 16197. Table 2 shows 17 different sequences in the hypervariable domain, along with the number of individuals in each racial group. J1 and J2 in this table represent, respectively, the groups I and II in the Japanese population, which were classified in our earlier study (Horai and Matsunaga 1986; also see Table I Analysis of Nucleotide Substitutions, Deletions, and Insertions No. OBSERVED
CCAATCCACA TCAAA----CCCC CTCCCCATGC TFACAAGCAA GTACAGCAAT CAACCCTCAA C GAT C TCTrC G T C T T --CCCcTrrr AC TT A G
G
CTATCACACA TCAACTGCAA CTCCAAAGCC A-CCCCTCACC CACTAGGATA CCAACAAACC G TT T G C G G A C TT CT T G A GTC OCAGT A TACCCACCCT TAACAGTACA TAGTACATAA AGCCATTTAC CGTACATAGC ACATTACAGT T G G C TAT CC T CC TT GTTTCC C
CAAATCCCTT CTCGTCCCCA TGGATGACCC CCCTCAGATA GGGGTCCCTT GACCACCATC T TC
CTCCGTGAM
A
C
TAC
T
G
TCAATATCCC GCACMGAGT GCTACTCTCC TCGCTCCGGG CCCATMCAC G T C C
TTGGGGGTAG CTAAAGTGAA CTGTATCCGA CATCTGGTI'C CTACTTCAGG GTCATAAAGC G
A
C
CTAAATAGCC CACACGTTCC CCTTAAATAA GACATCACGA TGGATCACAG GTCTATCACC G A
Sacl CTATTAACCA CTCACGGGAG CMI
bp 36
Nucleotide sequences of the major noncoding region. Figure I The sequence reported by Anderson et al. (1981) is shown in the upper lines along with the base numbers. Mutations observed in at least one individual are shown in the lower lines. Dashes (-) represent deletions/insertions, and letters represent base substitutions. Cloning sites (Sad and Kpnl-1 or KpnI-2) are also indicated, by boldface letters. A hypervariable domain is boxed.
TYPE OF MUTATION
Entirety of Data
Each Position
229 137 34 46 446 (97)
27 41 22 10 100 (91)
Substitution:
Transition: T-C C-*T A-G G- A
............. ............. .................. .............
Totala ............ Transversion: C-A C-G A -C A-T G-C
............. .................. .................. ............. ............. Total ...........
Deletion A .............. Insertion: C .............. -.
5 1 2 1
6 1 2 2 1 12
1 10
32
2
69
5
a Numbers in parentheses are percentage substitutions that are
transitions.
Human mtDNA Sequence Differences
831
Table 2 mtDNA Sequences in a Hypervariable Domain and Observed Numbers in the Three Racial Groups
mtDNA Sequence
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Caucasoid Negroid Mongoloid Japan-I Japan-II Total
AAAA ---- CCCCCTCCCC .... 15 AAAA --- -CCCCCTCCCT 1.... AAAA----- CCCCCTCCTC .... AAAA ---- CTCCCTCCCC .... ... AAAA CCCTCTCCCC .... AAAA - - -CCCTCCCCCC .... ... AAAA ---- CCCTGCCCCC ... AAAA ---- TCCTACCCCC .... ... AAAA ---- CCTCCCCCCC .... 1 AAAA --- -CCCCCCCCCCC .... 1 AAAA- -CCCCCCCCCCCC .... 1 AAA-CCCCCCCCCCCCCC .... ... AAA- -CCCCCCCCCCCCC .... ... AAA --- CCCCCCCCCCCC .... 1 AAA ----- CCCCCCCCCC .... AA- -CCCCCCCCCCCCCC ... AA - -- CCCCCCCCCCCCC ... Total . ............ 20 ...
----
...
...
5 ... ...
7 ... 1
1 ...
33 ...
...
...
...
...
...
...
...
...
2 1 1 ... ... ... ... ... ...
... ... ... ... 1 ... ... ... ...
1 ... ... ... ... ... 1 1
...
...
...
... 1 10
... 1 10
4 2 14
1 3 ... ... ... ... 3 ... ... ... 4 1 2 ... 47
4
61 1 1 1 3 3 1 1 1 5 1 1 1 9 1 6 4 101
below). About 60% of individuals exhibit the sequence (sequence 1 in table 2) which contains a 14-base stretch of four A, five C, one T, and four C. However, the remainder showed a variety of sequences in this domain. From sequence 2 to sequence 9 there are base substitutions in the C stretches. From sequence 10 to sequence 17 differences resulted from elongation of the C stretch and shortening of the A stretch. Figure 3 shows a representative result of autoradiography of a sequence gel electrophoresis. Once the T at bp 16189 is replaced by C, the number of A and C becomes flexible, probably because of replication error. This T-to-C transition, which has independently occurred several times in the different lineages, was confirmed by the phylogenetic analysis (see below).
a
I
.
3. Phylogeny of mtDNA Sequence
We aligned and compared the 482-bp sequences from 101 individuals (see Appendix). We also estimated the
j
fn-codhf regIon
RNA |
K
K
)
PhitA
D-Loop (H)
S
Figure 2 Distribution of mutations. The histogram shows the total number of base substitutions, deletions, and insertions within contiguous nonoverlapping blocks of 20 bases. A hypervariable domain is apparent near one of the KpnI sites within the D-loop region. and S represent, respectively, KpnI and Sad recognition sites used /ORK for cloning.
Horai and Hayasaka
832
a
b
100
90
so
70
60
50
40
* Negroid o Mongoloid A Caucasoid
30
4. 20
to 4110
15
Sequence of a hypervariable domain. Autoradiogram Figure 3 of a 6% polyacrylamide gel containing the sequence of a hypervariable domain which starts at the darkened triangle (A) and ends at the unfilled triangle (A). a and b represent, respectively, sequences 1 and 16 in table 2.
numbers of nucleotide substitutions between each pair of sequences. The nucleotide diversity among 101 individuals is 1.45%, which is three- to fourfold higher than estimates based on restriction-enzyme analysis of mtDNA previously reported in human population (Brown 1980; Horai et al. 1986; Cann et al. 1987). On the basis of the estimated numbers of nucleotide substitutions between individual sequences, we constructed a phylogenetic tree by the UPG method, as shown in figure 4. We have also examined the neighborjoining method of tree construction and found that the topology was nearly the same as that of the UPG tree. On the basis of the clustering patterns in figure 4, we could classify all individuals into at least 10 clusters designated as C-Co. Among these clusters, C5, C9,
10 Genetic distance (D
5 X
0
103 )
Figure 4 Phylogenetic tree showing the 101 mtDNA lineages from the three racial groups. The numbers of lineages are the same as those in tables Al and A2. All lineages are tentatively classified into 10 clusters designated as Cl-CIO. Distances (D) are expressed by the number of nucleotide substitutions/site/lineage.
and ClO exhibit interminglings of individuals from different racial groups. However, the remaining seven consist of individuals from single races. C1 and C4 are the clusters for negroids. C2, C3, and C6-C8 consist only of mongoloids. Although no cluster specific for caucasoids was observed in this tree, C9 mainly consists of caucasoids. In this tree four negroid individuals belonging to Ca first diverged from the rest of the clusters, and subsequently two mongoloid-specific clusters (C2 and C3) diverged from both the second negroid cluster (C4) and other clusters. It is relevant that some mongoloids occupy a phylogenetic position distinct from that for other mongoloids. This observation confirms our earlier study of restriction-enzyme analysis, in which part of the negroids and part of the mongoloids (Japanese) first diverged from other humans (Horai et al. 1986, 1987).
Human mtDNA Sequence Differences
833
4. Phylogenetic Analysis Within and Between Racial Groups
cluding these two caucasoid and two negroid individuals and dividing the mongoloid population into two subpopulations (table 3B). Tables 3A and 3B show that the nucleotide diversity among negroids is much larger than that among caucasoids or mongoloids. Furthermore, the nucleotide diversity among negroids is larger than all of the interracial diversity. Thus, the earlier finding that negroids have highly diversified mtDNAs compared with caucasoids and mongoloids, a finding deduced on the basis of restriction-enzyme analysis (Horai et al. 1986; Cann et al. 1987), was confirmed by the quantitative analysis of the nucleotide sequences. In table 3B the nucleotide difference between mongoloids-1 and mongoloids-2 (dxy = 0.0170) is much larger than that between caucasoids and mongoloids-2 (dxy = 0.0115). This table also shows that the nucleotide diversity among mongoloids-1 (dX = 0.0169) is much larger than that among mongoloids-2 (dx = 0.0108). These two subpopulations of mongoloids roughly correspond, respectively, to Japanese groups I and II inferred from the restriction-enzyme analysis (Horai and Matsunaga 1986). The restriction-enzyme analysis revealed that nucleotide diversity within Japanese group I was larger than that within Japanese group
To analyze the nucleotide diversities within and bethe nucleotide sequences were compared quantitatively. On the basis of estimates of the numbers of nucleotide substitutions between individuals, net nucleotide differences (d) between two races was calculated using the equation d = dxy (dx +dy)/2 (Nei and Li 1979), where dxy is the avertween the racial groups,
-
between two races and dy are, respectively, the number of nucleotide difference within races
age number of nucleotide differences
X and Y and where average
dx
X and Y.
Table 3A shows the results of calculations using all individuals. A phylogenetic analysis (fig. 4) showed that two caucasoid individuals (individuals 73 and 74 in tables Al and A2; marked by an asterisk (*) in fig. 4) have diverged extensively from other caucasoid individuals and that two negroid individuals (individuals 6 and 28 in tables Al and A2; marked by an asterisk (*) in fig. 4) have diverged extensively from other negroids. As mentioned above, the mongoloid population could be divided into two subpopulations. Therefore, we also estimated the number of nucleotide differences between races by ex-
Table 3 Estimates of the Number of Nucleotide Differences per Site Both Among (dxy and within (dx or dy) Each of the Three Races, and Net Nucleotide Differences (d) among the Races. A.
Caucasoid
Caucasoid
Mongoloid
Negroid
(N=20)
(N=71)
(N=10)
.0094 .0128 .0194
.0012 .0137 .0203
.0028 .0015 .0238
.........
Mongoloid ........ Negroid ...........
B.
Caucasoid Caucasoid
......
Mongoloid-2 ....... Mongoloid-1 ....... Negroid ...........
(N= 18) .0084.08 .0115 .0145 .0212
Mongoloid-2 (N= 52)
Mongoloid-I (N= 19)
Negroid
.0018 .0108 .0170 .0213
.0019 .0031 .0169 .0232
.0036 .0025 .0014 .0268
(N= 8)
NOTE.-Figures on the diagonal (underlined) represents dx or dy. Those below and above diagonal represent dxy and d, respectively. Mongoloid-2 includes those individuals belonging to C6-C10 in fig. 3, while the other mongoloids are included in mongoloid-1.
Horai and Hayasaka
834 Table 4
Combinations of Nucleotides at Three Typical Polymorphic Sites and Observed Numbers of Individuals from Three Racial Groups
bpa MITOCHONDRIAL TYPE
16223
16519
16362
TOTAL
NEGROID
CAUCASOID
Ml...............
T T
C C
C T
13 11
1 4
...
M3...............
T
T
C
13
1
M4............... MS............... M6...............
T C C
T C C
T T C
19 30 4
2 1 1
M7...............
C
T
C
2
...
M8...............
C
T
T
9
...
M2...............
a
1
MONGOLOID
JAPAN-I
JAPAN-II
1
...
11 4
... ...
...
1 7
...
2
12
...
...
16
12
...
10 2
...
...
1
...
...
...
2
...
1
1
7
From Anderson et al. (1981).
result which agrees well with the results of the presThus, it is evident that the mongoloid population can be separated into two subpopulations. Net nucleotide differences (Nei and Li 1979) of mtDNAs are not effective in elucidating the relationships between human populations, as has been noted by elsewhere one of the present authors (Nei 1985). This may be due to the highly diversified mtDNAs existing both in the negroid population and in part of the mongoloid population, as is seen in figure 4 and table 3B. II, a
ent study.
M4/
Polymorphic Sites Shared and Unique among Racial Groups In this sequence analysis, there are 117 base changes in 113 sites (as shown in fig. 1). Of these, 12 sites are shared by the three racial groups. Ten sites are found in common between negroids and mongoloids, whereas 5.
caucasoids and negroids shared two polymorphic sites and caucasoids and mongoloids also shared two sites. The presence of polymorphic sites shared by two or three racial groups is probably due to the ancient polymorphism. In other words, mtDNAs had already been polymorphic at these sites before the divergence of the racial groups. On the other hand, we observed many polymorphic sites which are specific for each race -55 for mongoloids, 21 for negroids, and 15 for caucasoids. When these numbers are proportioned to the numbers of individuals tested, it is found that race-specific polymorphic sites are predominantly found in negroids. This indicates that negroids are much more diverse than caucasoids or mongoloids, as indicated by the analysis of nucleotide diversity between each sequence.
*2
* o
A
IS
Negroid Mongoloid Caucasoid
5 10 Genetic distance ( Dx 103 )
0
Figure 5 Phylogenetic tree showing the 101 mtDNA lineages with mitochondrial types. Each cluster is represented by the mitochondrial types 1-8 as designated in table 4. M1-M8 indicate the "appearances" of these types in the phylogenetic tree.
835
Human mtDNA Sequence Differences 6. mtDNA Divergence in Human Evolution
How can the evolutionary inference of mtDNA divergences be drawn on the basis of the phylogenetic tree? What are the other evolutionary implications of poly-
morphic sites being shared by the three racial groups? When we arbitrarily chose three sites which were highly polymorphic among three racial groups, all individuals could be classified into the eight combinations of nucleotides. We tentatively call these combinations mitochondrial types M1-M8 (table 4). The phylogenetic tree shown in figure 5 is the same as that in figure 4, but each cluster is represented by the mitochondrial types. For example, in the first cluster (Cl in fig. 4) there are mitochondrial types M2 and M3. Thus, at the earliest branching point there already existed two types, M2 and M3. In the same way, the second cluster (C2) contains only mitochondrial type M5, so that this type is assumed to have first appeared around the branching point indicated by MS in figure 5. Similarly, since mitochondrial type M6 is observed only in the last cluster (C10), the emergence of this type should be located around the branching point indicated by M6 in figure 5. Figure 6 shows a schematic presentation of the appearance of each ancestral mitochondrial type. We assume that the mtDNA divergence took place about 200,000 years ago, an assumption which is deduced from the restriction-enzyme analysis of mtDNAs (Horai et al. 1986, 1987; Cann et al. 1987). Although the most ancestral type could not be identified in the present study, types M2 and M3 had already existed some 180,000 years ago. The remaining types were subsequently derived from either type M2 or type M3. Thus, at the present time we can observe the eight mitochondrial types in human populations. As mentioned above, it is obvious that the mongoloids can be separated into two subpopulations in this sequence analysis. This observation confirmed the results of our earlier study, which clearly demonstrated, by distinct in the Japanese (Horai and Matsunaga 1986). Most of those who belong to the Japanese group I cluster have the 9-bp deletion in region V (Cann and Wil-
restriction-enzyme analysis, existence of two groups
TT
TTC
Msc ccc
260
160
160 Thou"sd
5'Ib
of
Figure 6 Schematic presentation of the appearances of each ancestral mitochondrial type. The time scale is based on the assumption that mitochondrial divergence took place about 200,000 years ago, an inference which is deduced from the restriction-enzyme analysis of mtDNAs (Cann et al. 1987).
son 1983). This deletion is observed not only in east Asians (Cann and Wilson 1983), Indonesians (Stoneking et al. 1989), and Taiwan Chinese (Horai et al., in press) but also at a very high frequency in Polynesians (Hertzberg et al. 1989). The same deletion has never been found in caucasoids and negroids. Thus, the existence of two major groups is one of the characteristics commonly found in mongoloid populations. Nowadays mongoloid descendants live in North and South America, Oceania, Southeast Asia, east Asia, and Siberia, adapting themselves to various environments on the earth. An extended study of mtDNA polymorphism and its genealogy will be required to confirm the origins and dispersal of the two subpopulations of mongoloids.
Acknowledgments This work was supported by Ministry of Education, Science and Culture (Japan) grants-in-aid to S.H. for general scientific research and scientific research on priority areas of "Bioenergetics," "Development of Evolutionary and Population Genetics Incorporating Newer Molecular Findings," and "From Asia to America: Prehistoric Mongoloid Dispersal."
Appendix Table Al Samples and Background Information on Them
Sample Numbera and Designation 1. SB1............. 2. MS39........... 3. MS78........... 4. MS12........... 5. MS43........... 6. SB9............. 7. MS108.......... 8. MS41........... 9. MS89........... 10. MS76.......... 11. MS51 .......... 12. MS67 .......... 13. MS60.......... 14. MS16.......... 15. MS102......... 16. MS30 .......... 17. MS61.......... 18. MS73 .......... 19. MS15.......... 20. MS34.......... 21. MS90.......... 22. MS27.......... 23. MS59.......... 24. SB16........... 25. MS44.......... 26. MS116 ......... 27. SB14........... 28. SB18........... 29. SB30........... 30. MS100......... 31. MS114......... 32. MS65.......... 33. ANDERC ....... 34. MS25.......... 35. BHK2 ......... 36. SB23........... 37. SB33........... 38. SB24........... 39. SB35........... 40. SB13........... 41. SB32........... 42. SB6............ 43. SB29........... 44. SB31........... 45. LKK3d ......... 46. SB25........... 47. SB20........... 48. SB22........... 49. SB21........... 50. SB27........... 51. SB11 ........... a
Race
Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Negroid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Caucasoid Mongoloid Mongoloid Caucasoid Negroid Caucasoid Mongoloid Mongoloid Mongoloid Caucasoid Mongoloid Caucasoid Caucasoid Caucasoid Caucasoid Mongoloid Caucasoid Caucasoid Caucasoid
Mongoloid Caucasoid Caucasoid Mongoloid Caucasoid Caucasoid Caucasoid Mongoloid Caucasoid
Sample Numbera and Designation
Regionb
Indonesia Japan-II Japan-II Japan-II Japan-II Zambia Japan-lI
SB8............ SB12........... MS29.......... MS9........... MS31 .......... MS22.......... MS35.......... MS69.......... MS45.......... MS71 .......... MS101. MS93.......... MS23.......... MS122......... MS80.......... MS10.......... MS104 ......... SB15........... MS111 ......... MS46.......... MS94.......... SB3............ SB26........... SB7............ MS37.......... MS40.......... MS70.......... MS86.......... MS49.......... MS56 .......... MS33.......... CJK5d......... MS58.......... MS17.......... SB10........... SB5............ SB34........... MS113 ......... MS14.......... MS36.......... MS28.......... SB19........... SB28........... MS88.......... MS105......... MS24.......... SB4............ 99. CDKjd......... 100. SB17.......... 101. DCKjd. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98.
Japan-Il
Japan-lI Japan-II Japan-II Japan-Il Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-Il Japan-Il Japan-II Japan-II India Japan-II Japan-lI United Kingdom Kenya United Kingdom Japan-II Japan-II Japan-II Unknown Japan-II Unknown Switzerland United States Canada Korea South Africa United Kingdom Holland China United Kingdom unknown Philippines United States United Kingdom United States Korea United States
Race
Regionb
Mongoloid Caucasoid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid
New Guinea France Japan-lI Japan-II Japan-II Japan-Il Japan-lI Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II China Japan-II Japan-II Japan-II Australia France China Japan-I Japan-I Japan-I Japan-I Japan-I Japan-I Japan-I Unknown Japan-II Japan-II Ivory Coast Nigeria Zimbabwe Japan-I Japan-I Japan-I Japan-I Korea China Japan-I Japan-I Japan-I Zaire Unknown Uganda
Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Caucasoid Caucasoid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Negroid Mongoloid Mongoloid Negroid Negroid Negroid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Negroid Negroid Negroid Negroid
Unknown
Sample numbers are as in fig. 4.
b Japan-I and Japan-II are groups differentiated on the basis of restriction-enzyme c Nucleotide sequences were derived from Anderson et al. (1981). d Nucleotide sequences were derived from Greenberg et al. (1983).
836
analysis (Horai and Matsunaga 1986).
N
0
0
ON %0
0 0
N\0
0
00
00
0% 0% 0% 0% 0% 0% 0% 0%
0 7N
00
0%
00
0%
Un
N-1 No, tn
N"
(N
00
tn
N) tn
H Q H U H U C-, H H Q U H U U U U H H U C-, H H C; H H U U U U U U U
U
UUUU. U::
UUUU
Intraspecific Nucleotide Sequence Differences in the Major Noncoding Region of Human Mitochondrial DNA Satoshi Horai and Kenji Hayasaka Department of Human Genetics, National Institute of Genetics, Mishima, Shizouka, Japan
Summary Nucleotide sequences of the major noncoding region of human mitochondrial DNA (mtDNA) from 95 human placentas have been determined. These sequences include at least a 482-bp-long region encompassing most of the D-loop-forming region. Comparisons of these sequences with those previously determined have revealed remarkable features of nucleotide substitutions and insertion/deletion events. The nucleotide diversity among the sequences is estimated as 1.45%, which is three- to fourfold higher than the corresponding value estimated from restriction-enzyme analysis of whole mtDNA genome. A hypervariable region has also been defined. In this 14-bp region, 17 different sequences were detected. More than 97% of the base changes are transitions. A significantly nonrandom distribution of nucleotide substitutions and sequence length variations were also noted. The phylogenetic analysis indicates that diversity among the negroids is much larger than that among the caucasoids or the mongoloids. In fact, part of the negroids first diverged from other humans in the phylogenetic tree. A striking finding in the phylogenetic analysis is that the mongoloids can be separated into two distinct groups. Divergence of part of the mongoloids follows the earliest divergence of part of the negroids. The remainder of the mongoloids subsequently diverged together with the caucasoids. This observation confirmed our earlier study, which clearly demonstrated, by the restriction-enzyme analysis, existence of two distinct groups in the Japanese.
Introduction
The mammalian mitochondrial DNA (mtDNA) is a circular genome approximately 16.5 kbp in length and encodes 13 subunits of the inner-membrane respiratory complexes. The complete nucleotide sequences of human (Anderson et al. 1981), mouse (Bibb et al. 1981), cow (Anderson et al. 1982), and rat mtDNAs (Gadaleta et al. 1989) have been reported. The gross genetic arrangement of these genomes is remarkably conserved. They can be divided into two domains - a coding region constituting over 90% of the genome and a noncoding region which contains both the origin of H-strand replication (Anderson et al. 1981) and the origins of transcription of both strands (Cantatore and Attardi 1980). Received September 26, 1989; revision received November 27, 1989. Address for correspondence and reprints: Satoshi Horai, Department of Human Genetics, National Institute of Genetics, Yata 1,111, Mishima, Shizuoka 411, Japan. © 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4604-0021$02.00
828
Since the original studies which confirmed the maternal inheritance and predominantly uniclonal nature of mammalian mtDNA within an individual (Hutchison et al. 1974; Potter et al. 1975; Giles et al. 1980), numerous reports have indicated that its nucleotide sequence is evolving much faster than that of single-copy nuclear genes (Brown et al. 1979; Ferris et al. 1981). Since there are substantial sequence variations among individuals (Brown and Goodman 1979), restrictionenzyme analysis of mtDNAs has become a powerful tool in elucidating evolutionary relationships among human ethnic groups (Brown 1980; Denaro et al. 1981; Blanc et al. 1983; Johnson et al. 1983; Horai et al. 1984; Wallace et al. 1985; Brega et al. 1986; Horai and Matsunaga 1986; Cann et al. 1987; Harihara et al. 1988). Results obtained from these studies suggest that there is a high correlation between mtDNA restriction types and ethnic origins of individuals. The phylogenetic analysis in the Japanese indicated that they could be separated into two major groups (groups I and II; Horai and Matsunaga 1986). This grouping may also be ap-
829
Human mtDNA Sequence Differences plied to other mongoloid populations, because a 9-bp deletion in region V (Cann and Wilson 1983), which characterizes the group I Japanese, was also observed in non-Japanese mongoloid populations (Hertzberg et al. 1989; Stoneking and Wilson 1989; Horai et al., in press). Therefore, we extended our analysis to include a phylogenetic analysis of the sequence in a particular region of mtDNA. In the present study we have sequenced part of the major noncoding region encompassing the D-loop region of mtDNAs isolated from 95 human placentas. Comparisons of these sequences with others determined elsewhere (Anderson et al. 1981; Greenberg et al. 1983) have revealed striking features of nucleotide substitutions, insertions, and deletions. These results are discussed in light of sequence evolution and functional constraints on this domain. We have also examined in detail the evolution of mtDNA sequences at the gene level, as well as at the population level. Subjects and Methods Subjects
mtDNA from placentas of 95 individuals from three different racial origins was purified to homogeneity, according to a method described by Brown et al. (1979) and Horai et al. (1984). In the restriction-enzyme analysis we observed 62 different types of restriction patterns by using nine enzymes in the Japanese population (Horai and Matsunaga 1986). In the present study we chose 61 Japanese individuals all of whose restriction patterns were different from one another. Ten nonJapanese mongoloids consisted of three Koreans, four Chinese, an Indonesian, a Filipino, and a Papua New Guinean. We have also collected placenta samples from 17 caucasoids, 16 from Europe and America and one from India, and from seven African negroids, from seven different regions, whose mothers gave birth to their babies in Tokyo. A list of subjects is shown in the Appendix (table Al). Cloning of mtDNA
Purified mtDNAs were doubly digested with KpnI and SacI. Digestion products were extracted three times with phenol/chloroform (1/1), precipitated with ethanol, and dissolved in TE (pH 8.0). Then the fragments were inserted into pBluescript (KS -; Stratagene) which was also doubly digested with KpnI and SacI and then was purified by gel electrophoresis. When human mtDNAs were digested with SacI, two fragments (9.6 kb and 7.0 kb) were usually observed.
These fragment sizes agree with the sizes deduced from the published sequence of Anderson et al. (1981), which has two Sacl recognition sites at bp 36 and bp 9643 (the notations are those of Anderson et al. [1981] for base numbers). Three KpnI sites (bp 2573, bp 16048, and bp 16129) are observed in the published sequence data. Although two fragments (13.5 kb and 3.0 kb) are visible, the third fragment, of 81 bp, is invisible under the usual electrophoretic conditions. Through careful examinations with prolonged gel electrophoresis, slight between-individual differences in mobility were observed in the 3.0-kb bands. In the variant pattern, the 3.0-kb band moves slightly slower than that of the usual pattern, suggesting that two contiguous fragments (3013 bp and 81 bp) were fused to generate a larger fragment of 3.1 kb. This mutation is rather frequent in the Japanese population (S. Horai, unpublished data). Therefore, in the double digestions with KpnI and SacI, four or five fragments were produced in each sample because of the polymorphism of one of the KpnI sites. Thus, the smallest fragment with both SacI and KpnI recognition sites is either 482 bp or 563 bp in length and is efficiently inserted into vectors, without purification of the desired fragment. DNA Sequencing
Nucleotide sequences of 482-bp or 563-bp fragments inserted into pBluescript were determined by the dideoxy-chain termination method (Sanger et al. 1977) using the Sequenase kit (U.S. Biochemical) according to the manufacturer's directions. Data Analysis
We aligned the nucleotide sequences of the 482-bp fragments, which were common for 95 individuals, with those of six individuals reported elsewhere (Anderson et al. 1981; Greenberg et al. 1983). We estimated the per-site number of nucleotide substitutions between individual sequences by using the six-parameter method of nucleotide substitution (Gojobori et al. 1982). On the basis of the estimated numbers, phylogenetic trees were constructed by the unweighted-pair-group (UPG) method (Sokal and Sneath 1963) and by the neighborjoining method (Saitou and Nei 1987). Results and Discussion 1. Nucleotide Sequences Figure 1 shows the nucleotide sequence of the light
strand of the noncoding region originally reported by Anderson et al. (1981). KpnI site 1 (KpnI-1) and the
Horai and Hayasaka
830 Sad site are conserved among all individuals; however, KpnI-site 2 (KpnI-2) is polymorphic. Therefore, we cloned and sequenced either a 563-bp fragment or a 482-bp fragment for each individual. A total of 100 sequences were compared with the sequence reported by Anderson et al. (1981). In figure 1, base substitutions, deletions, and insertions detected in at least one individual were also shown below the nucleotide sequence of Anderson et al. (1981). We found mutations at a total of 113 sites. At four sites two different kinds of nucleotide substitutions were observed. Nucleotide changes were observed more frequently in the 5' half of the region than in the 3' half. The observed number of mutations, classified according to the types of mutations, are shown in table 1. In the whole data, transition types of substitution are more predominant than transversions; that is, 97% are transitions while only 3% are transversions. Moreover, transitions between pyrimidines are more prevalent than those between purines. When we counted the nucleotide substitutions for each site, a 10-fold bias favoring transitions over transversions was still observed. The results in the present study confirmed several features of the noncoding region of mtDNA which have been reported by Aquadro and Greenberg (1983) and by KpnI-1 GrTACCACCC AAGTATIGAC TCACCCATCA ACAACCGCTA TGTATTTCGT ACATFACTGC C
t
C
CC
bp 16048 KpnI-2 CAGCCACCAT GAATATTGTA CGGrACCATA AATACTFGAC CACCTGTAGT ACATAAAAAC GGG T A G TT A T C C T A
Greenberg et al. (1983), though only seven or eight sequences were compared by these authors. We also observed both deletion of A at two sites and insertion of C at five sites. These deletions and insertions were found in a particular domain containing serially repeated stretches of A and C (see table 2). 2. Distribution of Mutated Sites
The distribution of mutations in the D-loop region is shown in figure 2. The histogram represents the total number of mutations within contiguous nonoverlapping blocks of 20 bases. Figure 2 shows that most highly variable base sites lie in the blocks near the tRNAproline gene, while one highly polymorphic base site was observed in a block near the SacI site. This site is polymorphic for all three major races (position 16519 in table 4) and probably derived from the ancient polymorphism of this site. In this figure a hypervariable region exists in one of the 20 base blocks ranging from bp 16178 to bp 16197. Table 2 shows 17 different sequences in the hypervariable domain, along with the number of individuals in each racial group. J1 and J2 in this table represent, respectively, the groups I and II in the Japanese population, which were classified in our earlier study (Horai and Matsunaga 1986; also see Table I Analysis of Nucleotide Substitutions, Deletions, and Insertions No. OBSERVED
CCAATCCACA TCAAA----CCCC CTCCCCATGC TFACAAGCAA GTACAGCAAT CAACCCTCAA C GAT C TCTrC G T C T T --CCCcTrrr AC TT A G
G
CTATCACACA TCAACTGCAA CTCCAAAGCC A-CCCCTCACC CACTAGGATA CCAACAAACC G TT T G C G G A C TT CT T G A GTC OCAGT A TACCCACCCT TAACAGTACA TAGTACATAA AGCCATTTAC CGTACATAGC ACATTACAGT T G G C TAT CC T CC TT GTTTCC C
CAAATCCCTT CTCGTCCCCA TGGATGACCC CCCTCAGATA GGGGTCCCTT GACCACCATC T TC
CTCCGTGAM
A
C
TAC
T
G
TCAATATCCC GCACMGAGT GCTACTCTCC TCGCTCCGGG CCCATMCAC G T C C
TTGGGGGTAG CTAAAGTGAA CTGTATCCGA CATCTGGTI'C CTACTTCAGG GTCATAAAGC G
A
C
CTAAATAGCC CACACGTTCC CCTTAAATAA GACATCACGA TGGATCACAG GTCTATCACC G A
Sacl CTATTAACCA CTCACGGGAG CMI
bp 36
Nucleotide sequences of the major noncoding region. Figure I The sequence reported by Anderson et al. (1981) is shown in the upper lines along with the base numbers. Mutations observed in at least one individual are shown in the lower lines. Dashes (-) represent deletions/insertions, and letters represent base substitutions. Cloning sites (Sad and Kpnl-1 or KpnI-2) are also indicated, by boldface letters. A hypervariable domain is boxed.
TYPE OF MUTATION
Entirety of Data
Each Position
229 137 34 46 446 (97)
27 41 22 10 100 (91)
Substitution:
Transition: T-C C-*T A-G G- A
............. ............. .................. .............
Totala ............ Transversion: C-A C-G A -C A-T G-C
............. .................. .................. ............. ............. Total ...........
Deletion A .............. Insertion: C .............. -.
5 1 2 1
6 1 2 2 1 12
1 10
32
2
69
5
a Numbers in parentheses are percentage substitutions that are
transitions.
Human mtDNA Sequence Differences
831
Table 2 mtDNA Sequences in a Hypervariable Domain and Observed Numbers in the Three Racial Groups
mtDNA Sequence
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Caucasoid Negroid Mongoloid Japan-I Japan-II Total
AAAA ---- CCCCCTCCCC .... 15 AAAA --- -CCCCCTCCCT 1.... AAAA----- CCCCCTCCTC .... AAAA ---- CTCCCTCCCC .... ... AAAA CCCTCTCCCC .... AAAA - - -CCCTCCCCCC .... ... AAAA ---- CCCTGCCCCC ... AAAA ---- TCCTACCCCC .... ... AAAA ---- CCTCCCCCCC .... 1 AAAA --- -CCCCCCCCCCC .... 1 AAAA- -CCCCCCCCCCCC .... 1 AAA-CCCCCCCCCCCCCC .... ... AAA- -CCCCCCCCCCCCC .... ... AAA --- CCCCCCCCCCCC .... 1 AAA ----- CCCCCCCCCC .... AA- -CCCCCCCCCCCCCC ... AA - -- CCCCCCCCCCCCC ... Total . ............ 20 ...
----
...
...
5 ... ...
7 ... 1
1 ...
33 ...
...
...
...
...
...
...
...
...
2 1 1 ... ... ... ... ... ...
... ... ... ... 1 ... ... ... ...
1 ... ... ... ... ... 1 1
...
...
...
... 1 10
... 1 10
4 2 14
1 3 ... ... ... ... 3 ... ... ... 4 1 2 ... 47
4
61 1 1 1 3 3 1 1 1 5 1 1 1 9 1 6 4 101
below). About 60% of individuals exhibit the sequence (sequence 1 in table 2) which contains a 14-base stretch of four A, five C, one T, and four C. However, the remainder showed a variety of sequences in this domain. From sequence 2 to sequence 9 there are base substitutions in the C stretches. From sequence 10 to sequence 17 differences resulted from elongation of the C stretch and shortening of the A stretch. Figure 3 shows a representative result of autoradiography of a sequence gel electrophoresis. Once the T at bp 16189 is replaced by C, the number of A and C becomes flexible, probably because of replication error. This T-to-C transition, which has independently occurred several times in the different lineages, was confirmed by the phylogenetic analysis (see below).
a
I
.
3. Phylogeny of mtDNA Sequence
We aligned and compared the 482-bp sequences from 101 individuals (see Appendix). We also estimated the
j
fn-codhf regIon
RNA |
K
K
)
PhitA
D-Loop (H)
S
Figure 2 Distribution of mutations. The histogram shows the total number of base substitutions, deletions, and insertions within contiguous nonoverlapping blocks of 20 bases. A hypervariable domain is apparent near one of the KpnI sites within the D-loop region. and S represent, respectively, KpnI and Sad recognition sites used /ORK for cloning.
Horai and Hayasaka
832
a
b
100
90
so
70
60
50
40
* Negroid o Mongoloid A Caucasoid
30
4. 20
to 4110
15
Sequence of a hypervariable domain. Autoradiogram Figure 3 of a 6% polyacrylamide gel containing the sequence of a hypervariable domain which starts at the darkened triangle (A) and ends at the unfilled triangle (A). a and b represent, respectively, sequences 1 and 16 in table 2.
numbers of nucleotide substitutions between each pair of sequences. The nucleotide diversity among 101 individuals is 1.45%, which is three- to fourfold higher than estimates based on restriction-enzyme analysis of mtDNA previously reported in human population (Brown 1980; Horai et al. 1986; Cann et al. 1987). On the basis of the estimated numbers of nucleotide substitutions between individual sequences, we constructed a phylogenetic tree by the UPG method, as shown in figure 4. We have also examined the neighborjoining method of tree construction and found that the topology was nearly the same as that of the UPG tree. On the basis of the clustering patterns in figure 4, we could classify all individuals into at least 10 clusters designated as C-Co. Among these clusters, C5, C9,
10 Genetic distance (D
5 X
0
103 )
Figure 4 Phylogenetic tree showing the 101 mtDNA lineages from the three racial groups. The numbers of lineages are the same as those in tables Al and A2. All lineages are tentatively classified into 10 clusters designated as Cl-CIO. Distances (D) are expressed by the number of nucleotide substitutions/site/lineage.
and ClO exhibit interminglings of individuals from different racial groups. However, the remaining seven consist of individuals from single races. C1 and C4 are the clusters for negroids. C2, C3, and C6-C8 consist only of mongoloids. Although no cluster specific for caucasoids was observed in this tree, C9 mainly consists of caucasoids. In this tree four negroid individuals belonging to Ca first diverged from the rest of the clusters, and subsequently two mongoloid-specific clusters (C2 and C3) diverged from both the second negroid cluster (C4) and other clusters. It is relevant that some mongoloids occupy a phylogenetic position distinct from that for other mongoloids. This observation confirms our earlier study of restriction-enzyme analysis, in which part of the negroids and part of the mongoloids (Japanese) first diverged from other humans (Horai et al. 1986, 1987).
Human mtDNA Sequence Differences
833
4. Phylogenetic Analysis Within and Between Racial Groups
cluding these two caucasoid and two negroid individuals and dividing the mongoloid population into two subpopulations (table 3B). Tables 3A and 3B show that the nucleotide diversity among negroids is much larger than that among caucasoids or mongoloids. Furthermore, the nucleotide diversity among negroids is larger than all of the interracial diversity. Thus, the earlier finding that negroids have highly diversified mtDNAs compared with caucasoids and mongoloids, a finding deduced on the basis of restriction-enzyme analysis (Horai et al. 1986; Cann et al. 1987), was confirmed by the quantitative analysis of the nucleotide sequences. In table 3B the nucleotide difference between mongoloids-1 and mongoloids-2 (dxy = 0.0170) is much larger than that between caucasoids and mongoloids-2 (dxy = 0.0115). This table also shows that the nucleotide diversity among mongoloids-1 (dX = 0.0169) is much larger than that among mongoloids-2 (dx = 0.0108). These two subpopulations of mongoloids roughly correspond, respectively, to Japanese groups I and II inferred from the restriction-enzyme analysis (Horai and Matsunaga 1986). The restriction-enzyme analysis revealed that nucleotide diversity within Japanese group I was larger than that within Japanese group
To analyze the nucleotide diversities within and bethe nucleotide sequences were compared quantitatively. On the basis of estimates of the numbers of nucleotide substitutions between individuals, net nucleotide differences (d) between two races was calculated using the equation d = dxy (dx +dy)/2 (Nei and Li 1979), where dxy is the avertween the racial groups,
-
between two races and dy are, respectively, the number of nucleotide difference within races
age number of nucleotide differences
X and Y and where average
dx
X and Y.
Table 3A shows the results of calculations using all individuals. A phylogenetic analysis (fig. 4) showed that two caucasoid individuals (individuals 73 and 74 in tables Al and A2; marked by an asterisk (*) in fig. 4) have diverged extensively from other caucasoid individuals and that two negroid individuals (individuals 6 and 28 in tables Al and A2; marked by an asterisk (*) in fig. 4) have diverged extensively from other negroids. As mentioned above, the mongoloid population could be divided into two subpopulations. Therefore, we also estimated the number of nucleotide differences between races by ex-
Table 3 Estimates of the Number of Nucleotide Differences per Site Both Among (dxy and within (dx or dy) Each of the Three Races, and Net Nucleotide Differences (d) among the Races. A.
Caucasoid
Caucasoid
Mongoloid
Negroid
(N=20)
(N=71)
(N=10)
.0094 .0128 .0194
.0012 .0137 .0203
.0028 .0015 .0238
.........
Mongoloid ........ Negroid ...........
B.
Caucasoid Caucasoid
......
Mongoloid-2 ....... Mongoloid-1 ....... Negroid ...........
(N= 18) .0084.08 .0115 .0145 .0212
Mongoloid-2 (N= 52)
Mongoloid-I (N= 19)
Negroid
.0018 .0108 .0170 .0213
.0019 .0031 .0169 .0232
.0036 .0025 .0014 .0268
(N= 8)
NOTE.-Figures on the diagonal (underlined) represents dx or dy. Those below and above diagonal represent dxy and d, respectively. Mongoloid-2 includes those individuals belonging to C6-C10 in fig. 3, while the other mongoloids are included in mongoloid-1.
Horai and Hayasaka
834 Table 4
Combinations of Nucleotides at Three Typical Polymorphic Sites and Observed Numbers of Individuals from Three Racial Groups
bpa MITOCHONDRIAL TYPE
16223
16519
16362
TOTAL
NEGROID
CAUCASOID
Ml...............
T T
C C
C T
13 11
1 4
...
M3...............
T
T
C
13
1
M4............... MS............... M6...............
T C C
T C C
T T C
19 30 4
2 1 1
M7...............
C
T
C
2
...
M8...............
C
T
T
9
...
M2...............
a
1
MONGOLOID
JAPAN-I
JAPAN-II
1
...
11 4
... ...
...
1 7
...
2
12
...
...
16
12
...
10 2
...
...
1
...
...
...
2
...
1
1
7
From Anderson et al. (1981).
result which agrees well with the results of the presThus, it is evident that the mongoloid population can be separated into two subpopulations. Net nucleotide differences (Nei and Li 1979) of mtDNAs are not effective in elucidating the relationships between human populations, as has been noted by elsewhere one of the present authors (Nei 1985). This may be due to the highly diversified mtDNAs existing both in the negroid population and in part of the mongoloid population, as is seen in figure 4 and table 3B. II, a
ent study.
M4/
Polymorphic Sites Shared and Unique among Racial Groups In this sequence analysis, there are 117 base changes in 113 sites (as shown in fig. 1). Of these, 12 sites are shared by the three racial groups. Ten sites are found in common between negroids and mongoloids, whereas 5.
caucasoids and negroids shared two polymorphic sites and caucasoids and mongoloids also shared two sites. The presence of polymorphic sites shared by two or three racial groups is probably due to the ancient polymorphism. In other words, mtDNAs had already been polymorphic at these sites before the divergence of the racial groups. On the other hand, we observed many polymorphic sites which are specific for each race -55 for mongoloids, 21 for negroids, and 15 for caucasoids. When these numbers are proportioned to the numbers of individuals tested, it is found that race-specific polymorphic sites are predominantly found in negroids. This indicates that negroids are much more diverse than caucasoids or mongoloids, as indicated by the analysis of nucleotide diversity between each sequence.
*2
* o
A
IS
Negroid Mongoloid Caucasoid
5 10 Genetic distance ( Dx 103 )
0
Figure 5 Phylogenetic tree showing the 101 mtDNA lineages with mitochondrial types. Each cluster is represented by the mitochondrial types 1-8 as designated in table 4. M1-M8 indicate the "appearances" of these types in the phylogenetic tree.
835
Human mtDNA Sequence Differences 6. mtDNA Divergence in Human Evolution
How can the evolutionary inference of mtDNA divergences be drawn on the basis of the phylogenetic tree? What are the other evolutionary implications of poly-
morphic sites being shared by the three racial groups? When we arbitrarily chose three sites which were highly polymorphic among three racial groups, all individuals could be classified into the eight combinations of nucleotides. We tentatively call these combinations mitochondrial types M1-M8 (table 4). The phylogenetic tree shown in figure 5 is the same as that in figure 4, but each cluster is represented by the mitochondrial types. For example, in the first cluster (Cl in fig. 4) there are mitochondrial types M2 and M3. Thus, at the earliest branching point there already existed two types, M2 and M3. In the same way, the second cluster (C2) contains only mitochondrial type M5, so that this type is assumed to have first appeared around the branching point indicated by MS in figure 5. Similarly, since mitochondrial type M6 is observed only in the last cluster (C10), the emergence of this type should be located around the branching point indicated by M6 in figure 5. Figure 6 shows a schematic presentation of the appearance of each ancestral mitochondrial type. We assume that the mtDNA divergence took place about 200,000 years ago, an assumption which is deduced from the restriction-enzyme analysis of mtDNAs (Horai et al. 1986, 1987; Cann et al. 1987). Although the most ancestral type could not be identified in the present study, types M2 and M3 had already existed some 180,000 years ago. The remaining types were subsequently derived from either type M2 or type M3. Thus, at the present time we can observe the eight mitochondrial types in human populations. As mentioned above, it is obvious that the mongoloids can be separated into two subpopulations in this sequence analysis. This observation confirmed the results of our earlier study, which clearly demonstrated, by distinct in the Japanese (Horai and Matsunaga 1986). Most of those who belong to the Japanese group I cluster have the 9-bp deletion in region V (Cann and Wil-
restriction-enzyme analysis, existence of two groups
TT
TTC
Msc ccc
260
160
160 Thou"sd
5'Ib
of
Figure 6 Schematic presentation of the appearances of each ancestral mitochondrial type. The time scale is based on the assumption that mitochondrial divergence took place about 200,000 years ago, an inference which is deduced from the restriction-enzyme analysis of mtDNAs (Cann et al. 1987).
son 1983). This deletion is observed not only in east Asians (Cann and Wilson 1983), Indonesians (Stoneking et al. 1989), and Taiwan Chinese (Horai et al., in press) but also at a very high frequency in Polynesians (Hertzberg et al. 1989). The same deletion has never been found in caucasoids and negroids. Thus, the existence of two major groups is one of the characteristics commonly found in mongoloid populations. Nowadays mongoloid descendants live in North and South America, Oceania, Southeast Asia, east Asia, and Siberia, adapting themselves to various environments on the earth. An extended study of mtDNA polymorphism and its genealogy will be required to confirm the origins and dispersal of the two subpopulations of mongoloids.
Acknowledgments This work was supported by Ministry of Education, Science and Culture (Japan) grants-in-aid to S.H. for general scientific research and scientific research on priority areas of "Bioenergetics," "Development of Evolutionary and Population Genetics Incorporating Newer Molecular Findings," and "From Asia to America: Prehistoric Mongoloid Dispersal."
Appendix Table Al Samples and Background Information on Them
Sample Numbera and Designation 1. SB1............. 2. MS39........... 3. MS78........... 4. MS12........... 5. MS43........... 6. SB9............. 7. MS108.......... 8. MS41........... 9. MS89........... 10. MS76.......... 11. MS51 .......... 12. MS67 .......... 13. MS60.......... 14. MS16.......... 15. MS102......... 16. MS30 .......... 17. MS61.......... 18. MS73 .......... 19. MS15.......... 20. MS34.......... 21. MS90.......... 22. MS27.......... 23. MS59.......... 24. SB16........... 25. MS44.......... 26. MS116 ......... 27. SB14........... 28. SB18........... 29. SB30........... 30. MS100......... 31. MS114......... 32. MS65.......... 33. ANDERC ....... 34. MS25.......... 35. BHK2 ......... 36. SB23........... 37. SB33........... 38. SB24........... 39. SB35........... 40. SB13........... 41. SB32........... 42. SB6............ 43. SB29........... 44. SB31........... 45. LKK3d ......... 46. SB25........... 47. SB20........... 48. SB22........... 49. SB21........... 50. SB27........... 51. SB11 ........... a
Race
Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Negroid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Caucasoid Mongoloid Mongoloid Caucasoid Negroid Caucasoid Mongoloid Mongoloid Mongoloid Caucasoid Mongoloid Caucasoid Caucasoid Caucasoid Caucasoid Mongoloid Caucasoid Caucasoid Caucasoid
Mongoloid Caucasoid Caucasoid Mongoloid Caucasoid Caucasoid Caucasoid Mongoloid Caucasoid
Sample Numbera and Designation
Regionb
Indonesia Japan-II Japan-II Japan-II Japan-II Zambia Japan-lI
SB8............ SB12........... MS29.......... MS9........... MS31 .......... MS22.......... MS35.......... MS69.......... MS45.......... MS71 .......... MS101. MS93.......... MS23.......... MS122......... MS80.......... MS10.......... MS104 ......... SB15........... MS111 ......... MS46.......... MS94.......... SB3............ SB26........... SB7............ MS37.......... MS40.......... MS70.......... MS86.......... MS49.......... MS56 .......... MS33.......... CJK5d......... MS58.......... MS17.......... SB10........... SB5............ SB34........... MS113 ......... MS14.......... MS36.......... MS28.......... SB19........... SB28........... MS88.......... MS105......... MS24.......... SB4............ 99. CDKjd......... 100. SB17.......... 101. DCKjd. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98.
Japan-Il
Japan-lI Japan-II Japan-II Japan-Il Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-Il Japan-Il Japan-II Japan-II India Japan-II Japan-lI United Kingdom Kenya United Kingdom Japan-II Japan-II Japan-II Unknown Japan-II Unknown Switzerland United States Canada Korea South Africa United Kingdom Holland China United Kingdom unknown Philippines United States United Kingdom United States Korea United States
Race
Regionb
Mongoloid Caucasoid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid
New Guinea France Japan-lI Japan-II Japan-II Japan-Il Japan-lI Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II Japan-II China Japan-II Japan-II Japan-II Australia France China Japan-I Japan-I Japan-I Japan-I Japan-I Japan-I Japan-I Unknown Japan-II Japan-II Ivory Coast Nigeria Zimbabwe Japan-I Japan-I Japan-I Japan-I Korea China Japan-I Japan-I Japan-I Zaire Unknown Uganda
Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Caucasoid Caucasoid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Negroid Mongoloid Mongoloid Negroid Negroid Negroid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Mongoloid Negroid Negroid Negroid Negroid
Unknown
Sample numbers are as in fig. 4.
b Japan-I and Japan-II are groups differentiated on the basis of restriction-enzyme c Nucleotide sequences were derived from Anderson et al. (1981). d Nucleotide sequences were derived from Greenberg et al. (1983).
836
analysis (Horai and Matsunaga 1986).
N
0
0
ON %0
0 0
N\0
0
00
00
0% 0% 0% 0% 0% 0% 0% 0%
0 7N
00
0%
00
0%
Un
N-1 No, tn
N"
(N
00
tn
N) tn
H Q H U H U C-, H H Q U H U U U U H H U C-, H H C; H H U U U U U U U
U
UUUU. U::
UUUU
