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Mitochondrial COII Sequences and Modern Human Originsl Maryellen Ravolo Sarah Zehr Miranda van Dornum Deborah Pan Belinda Chang 7 and Jenny Lian Department of Anthropology Harvard University and 1 Program in Neuroscience Harvard Medical School The aim of this study is to measure human mitochondrial sequence variability in the relatively slowly evolving mitochondrial gene cytochrome oxidase subunit 11 C011 and to estimate when the the human common ancestral mitochondrial type existed New COII gene sequences were determined for ve humans Home sap iens including some of the most mitochondrially divergent humans known for two pygmy chimpanzees Pan paniscas and for a common chimpanzee P trog lodytes COII sequences were analyzed with those from another relatively slowly evolving mitochondrial region ND45 From class 1 third codon position se quence data a relative divergence date for the human mitochondrial ancestor is estimated as 1 27th of the humanchimpanzee divergence time If it is assumed that humans and chimpanzees diverged 6 Mya this places a human mitochondrial ancestor at 222000 years significantly different from 1 Myr the presumed time of an H erectus emergence from Africa The mean coalescent time estimated from all 1580 sites of combined mitochondrial data when a 6Mya human chim panzee divergence is assumed is 298000 years with 95 con dence interval of 129000 536000 years Neither estimate is compatible with a l Myr old human mitochondrial ancestor The mitochondrial DNA sequence data from C011 and ND4S regions therefore do not support this multiregional hypothesis for the emer gence of modern humans Introduction The mitochondrial Eve hypothesis Cann et a1 1987 is a statement about both tree topology and time the common ancestor of all existing human mitochondrial DNA mtDNA types originated in Africa 140000290000 years ago In some ways the statement about time is the more controversial If the original claim had posited the same tree topology in which the basic division on the tree of all human mtDNA sequences is into an African clade and a clade of all other humans including some Africans but a more ancient origin say 1 Myr it might not have been controversial since the data could have been interpreted to re ect the initial migration of Homo ereetus out of Africa and therefore consistent with the multiregional hypothesis Wol poff 1989 Claims about time are based on interpretations of amounts of DNA sequence differences The rst studies of human mitochondrial diversity relied on indirect mea sures of DNA sequence difference by using restrictionenzyme site analysis Brown 1 Key words human mtDNA variation cytochrome oxidase subunit 11 C011 gene multiregionai hypothesis hominoid evolution molecular evolution Address for correspondence and reprints Maryellen Ruvolo Department of Anthropology Peabody Museum Harvard University 1 1 Divinity Avenue Cambridge Massachusetts 02138 Moi Bio Evol 106115 1135 1993 1993 by The University of Chicago All rights reserved 0737403893100600010200 1115 l I 16 Ruvolo et al 1980 Cann et al 1987 This method has the advantage that it samples the entire mitochondrial genome In contrast Vigilant et a1 1989 1991 directly obtained and compared DNA sequences of a portion of the mitochondrial genome However the available mtDNA sequence data which are direct re ections of genomic diversity and which potentially offer greater resolution than does restriction mapping do not un ambiguously support one topology Hedges et al 1992 Maddison et a1 1992 Tem pleton 1992 Rather there are at least three equally parsimonious classes of trees compatible with the data Maddison et al 1992 This ambiguity is caused by the high rate of molecular evolutionary change dem onstrated by the mitochondrial region examined the control region and by the pres ence of few phylogenetically informative characters relative to the number of individ uals On the basis of observed mtDNA sequence differences between pairs of individuals the hypervariable control subregions evolve 10 times faster than does the mitochondrial proteincoding gene for cytochrome oxidase subunit 11 C011 K Garner and O Ryder unpublished data M Ruvolo unpublished data Thus more slowly evolving proteincoding regions show fewer differences compared with the control region among humans and therefore offer potentially fewer phylogenetically informative sites However while the slower rate of the proteincoding genes means that relatively few differences are observed between humans and chimpanzees the chance for the region to become saturated with multiple substitutions is reduced making it more likely that phylogenetic information is preserved Correction for mul tiple substitutions is of course still necessary but generally small values of observed sequence difference get corrected very little if at all by all correction methods For greater amounts of observed sequence difference however not only is the degree of correction greater but correction methods vary more in their estimates of actual genetic difference Therefore the less quickly evolving portions of the mitochondrial genome showing little difference among humans should potentially provide us with more ac curate comparative estimates of sequence divergence among mitochondrial haplotypes than does the control region Controlregion sequences are useful negrained indicators of differences among humans di Rienzo and Wilson 1991 Ward et a1 1991 but for more distant phylogenetic comparisons the more slowly evolving regions are pref erable Here we report results for a slowly evolving mitochondrial proteincoding gene COII We have included both the South African iKung individual found to be most different from other humans on some mitochondrial trees Cann et a1 1987 Vigilant et al 1989 Maddison et al 1992 and some African pygmies from central Africa who were found to be the most divergent individuals on other trees Vigilant et al 1991 Maddison et al 1992 Throughout we compare the C011 results with those from another slowly evolving mitochondrial region the 896bp segment including partial genes for NADH dehydrogenase subunits 4 and 5 or the ND45 region Kocher and Wilson 1991 this region has been surveyed in some of the same individuals but not in any central African pygmies Material and Methods The new COII sequences reported here are from ve humans Homo sapiens two pygmy chimpanzees also known as bonobos Pam paniscus and one common chimpanzee P troglodytes For these new sequences genomic DNA was prepared from hairbulbs Vigilant et a1 1989 for the Asian Hsa 2 sample and from cultured cells Maniatis et al 1989 p 653 for the South African lKung individual Hsa 6 cell Mitochondrial COII Sequences and Modern Human Origins 11 17 line GM 3043 Human Genetic Mutant Cell Repository Camden NJ Other genomic DNAs were provided by Dr L L CavalliSforza from cell lines for humans Hsa 3 5 Dr R Honeycutt from placental tissue for common chimpanzee Ptr 1 and Dr 0 Ryder of the San Diego Zoo pygmy chimpanzees Ppa 1 and Ppa 3 Total genomic DNA was ampli ed by the polymerase chain reaction using oligonucleotide primers speci c for the C011 gene to create doublestranded and then singlestranded DNA singlestranded DNA was directly sequenced as described elsewhere Ruvolo et a1 1991 Disotell et a1 1992 Both DNA strands were sequenced in every case Results and Discussion Mitochondrial COII Gene Sequence Variation The C011 sequences generated are presented in gure 1 together with those previously published hominoid human and ape sequences Anderson et a1 1981 Ruvolo et a1 1991 Horai et a1 1992 used in the analysis One COII sequence Ptr 3 which we previously reported as that of a pygmy chimpanzee P paniscus Ruvolo et a1 1991 is most likely that of a common chimpanzee P troglodytes This DNA sequence was generated by R L Honeycutt from a DNA fragment containing the C011 gene cloned by W Brown and the exact individual from which the DNA was obtained is unknown When we discovered that the sequence clusters phylogenetically with those of common chimpanzees and not with pygmy chimpanzees using sequences reported here as well as other unpublished Pan sequences we reexamined available original laboratory notes in which clone PC2 was described in one notebook as being from a chimpanzee and in other notes relating to DNA sequencing as being from common chimpanzee The clone designation PC2 may have been inter preted as an abbreviation for pygmy chimpanzee rather than as an abbreviation for the more probable alternative ie plasmid clone Table 1 summarizes the indi viduals analyzed Among humans there are seven variable positions in COII sequences six transitions pyrimidinepyrmidine or purinepurine substitutions at positions 88 243 375 442 567 and 666 and one transversion purinepyrimidine substitution at position 528 in lKung individual Hsa 6 g 2 top Two substitutions occur at rst codon positions 88 and 442 causing amino acid replacements in individual Hsa 5 the other ve substitutions occur at third codon positions The mean pairwise difference between humans is 034 23 hp similar to that for the ND45 region 02 but less than that for the more quickly evolving control region 18 Kocher and Wilson 1991 Humans and chimpanzees differ by an observed average of 94 in C011 sequence 64 bp of 684 hp with 61 transitions and 3 transversions similar to the 9 average for the ND45 region 78 bp of 896 hp with 73 transitions and 5 transversions The buu u Ul39l u51uu ulllbl bllbb lo 1 A u 1Uuu1 auu vv noun 1 1 1 I nuluus uuluauo Lllb control region is 5 9 times more variable than C011 and ND45 regions but is only 13 times more variable between humans and chimpanzees a good indication that many multiple substitutions have occurred in the control region since the species diverged Kocher and Wilson 1991 C011 and ND45 regions are also similar in average transitionztransversion iv ratios calcuiated interspeci caliy between humans and chimpanzees 201 and 15 1 respectively Hypervariable controlregion sequences have a lower interspeci c ratio 31 again presumably because of multiple transitional cnhctifnfinnc Vnnhpr and Wilcnn IQQI In nnmhprc AF cnkcfihitinnql di 39prpnnpc OUUOthuLlUllo L UUIIUI all quot IIOUII I l l I I 11 llullluvlo U1 UUUDLALHLAUAIHA UAALVLUAIUUD the COII and ND45 data are similar and both are different from the control region data 81H 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 ATGGCACATGCAGCGCAAGTAGGTCTACAAGACGC TACT39I C CCCTATCATAGAAGAGCTTATCAC CTTTCATGATCACGCCCTCATAATCA IT ITCC 39ITATCTGCTI CCTAGTCCTGTATGC CC IT39I39I CCTAACAC TCACAACAAAACTA CATTCTTCTCC T AA TT TC TC G TAATTTCTCG GCAG CT ATGGT CC CA C GC 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 ACTAATACTAACATCTCAGACGCTCAGGAAATAGAAACCGTCTGAACTATCCTGCCCGC ATCA I CCTAGTCCTCATCGCCCTCCCA I CCCTACGCATCCT39I39I ACATAACAGACGAGGTCAACGATCCC I CCCTTACCATCAAA I CAA I39I GTT C TTTTT T C TTT C GTTCTTTTTTCTTTC GTTCLTTTTTTCTTTC GCC CAGA CATTT G TACTGTAATCTCT C CAC CA GA CTTT G TAC39I GTAATC TCTC CC TCAGTAT AA ATA TCT AACTTCTC 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 GGCCACCAATGGTACTGAACCTACGAGTACEACCGAC I ACGGCGGACTAATCTICAAC ICCI ACATAC ITCCCCCA I39I ATICCTAGAACCAGGCGACCTGCGACTCCT39ICACG I39I GACAATCGAGTAGTACTCCCGATI39GAAGCCCCCA I39I GA 6111 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 CGTATAATAATTACATCACAAGACG IC ITGCACTCATGAGCTGTCCCCACA I39I AGGC39I39I AAAAACAGATGCAA I39I CCCGGACGTCTAAACCAAACCACTTTCACCGCTACACGACCGGGGGTATACTACGGTCAATGCTCTGAAATC I GT L1 nonnhnnnn PJ La 11 610 620 630 640 650 660 670 680 684 GGAGCAAAC OACAGITI CA IGCCCATCGI CCTAGAATTAATTCCCCTAAAAATC39I39I TGAAATAGGGCCCGHTI39I ACCCTATAG C 39I TTGC T CTT C 000000000 CACGT1 A FIG l DNA sequences of the mitochondrial COII gene from 15 hominoids Position 1 corresponds to site 7586 in the Anderson et a1 1981 human reference sequence with the C011 gene ending at site 8269 Abbreviations for species names and samples are as in table 1 1 120 Ruvolo et al Table 1 Individuals Studied and Their Use in Previous Studies Identi cation No in Geographic Origin Previous Studya Present Study or Species identi cation no Other Identi er Humans Hsa 1 Presumably northern l 2 l 10 4 H1 Human or Cambridge reference European 6 118 7 ns sequence Hsa 2 Asian Taiwan Hsa 3 Zaire Mbuti pygmy 6 5 7 ns Cell line P45G Hsa 4 Central African Republic 6 2 7 ns Cell line P116 Biaka pygmy Hsa 5 Central African Republic 6 37 7 ns Cell line P31 Biaka pygmy Hsa 6 South African Kung 2 1 4 H2 6 13 Cell line GM 3043 Wilson lab 7 15 8A1 Chimpanzees Ptr 1 Pan troglodytes Sally s infant Ptr 2 P troglodytes 3 Ptr 3 P troglodytes 5 Clone PC2 see text Ppa 1 P paniscus Vernon San Diego Zoo 818 180343 Ppa 2 P paniscus 3 Ppa 3 P paniscus Marilyn San Diego Zoo ISIS 587376 Gorillas Ggo 1 Gorilla gorilla 5 Ggo 2 G gorilla 3 Orangutan Ppy Pongo pygmaeus 3 1 Anderson et a 1981 2 Cann et al 1987 3 Horailet a1 1992 4 Kocher and Wilson 1991 5 Ruvolo et a1 1991 6 Vigilant et a1 1991 for identi cation nos see Vigilant 1990 and 7 Vigilant et al 1989 quot ns identi cation no order of appearance on tree was not speci ed Phylogenetic Results The most parsimonious tree g 3 shows conspeci c sequences clustering to gether despite intraspeci c variability as well as a humanchimpanzee clade as found elsewhere with single individual species representatives Ruvolo et a1 1991 The same tree topology is found with phenetic methods by using neighborjoining Saitou and Nei 1987 and Fitch and Margoliash 1967 methods The single most parsi monious tree has length 205 with consistency index 0849 which means that variable sites Change once on average Human sequences exclusive of the South African Kung sequence are linked by a minimum of two unambiguous synapomorphies at positions 666 and 567 at an 85 bootstrap level This contrasts with a consistency index of 034 for the l 19 phylogenetically informative sites each varying three times on average on one tree of human hypervariable controlregion sequences Vigilant et al 1991 The withinhuman genetic difference can be calculated in a treebased fashion as follows The Hsa 6 Kung COII sequence is cladistically most different from the others so the average difference between it and other human sequences is taken to represent the maximum difference among humans This average difference through the root of the human clade is 4 bp 058 consisting of three transitions and one Hsa1 o o o o 1 3 3 4 2 2 2 8 9 15 Hsa2 1 o o o 1 3 3 4 2 2 2 8 9 15 Hsa3 o 1 o o 1 3 3 4 2 2 2 8 9 15 H394 o 1 o o 1 3 3 4 2 2 2 8 9 15 H395 3 4 3 3 1 3 3 4 2 2 2 8 9 15 11396 2 3 2 2 5 4 4 5 3 3 3 9 1o 16 Ptr 1 65 64 65 65 68 63 o 1 1 1 1 7 8 14 Ptr 2 62 61 62 62 65 60 9 1 1 1 1 7 8 14 Ptr 3 58 57 58 58 61 56 7 4 2 2 2 8 9 15 Ppa1 61 62 61 61 64 59 21 18 16 o o 6 7 13 Ppaz 6o 51 60 60 63 58 20 17 15 1 o 6 7 13 Ppa3 61 62 61 61 64 59 19 16 14 4 3 6 7 13 3901 75 76 75 75 78 75 68 71 69 66 65 62 1 9 602 75 76 75 75 78 73 66 67 65 62 61 58 6 1o Ppy 81 82 81 81 84 79 87 84 84 79 78 79 81 79 H58 1 1 o o o o o 1 o 2 1 57 3 55 3 51 3 53 2 53 2 53 2 63 8 63 8 64 13 Hsa 2 1 1 o 1 o 2 o 3 1 56 3 54 3 50 3 54 2 54 2 54 2 64 8 64 8 65 13 H53 3 o 1 o o 1 o 2 1 57 3 55 3 51 3 53 2 53 2 53 2 63 8 63 8 64 13 H68 4 o 1 o 1 o 2 1 57 3 55 3 51 3 53 2 53 2 53 2 63 8 63 8 64 13 H38 5 3 4 3 3 3 1 58 3 56 3 52 3 54 2 54 2 54 2 64quot 8 64 6 65 13 H536 3 4 3 3 6 55 4 53 4 49 4 51 3 51 3 51 3 63 9 61 9 62 14 Ptr 1 68 67 68 68 71 67 80 60 19 1 19 1 17 1 56 7 56 7 72 12 Ptr 2 65 64 65 65 68 64 9 4 o 17 1 17 1 15 1 60 7 58 7 7o 12 Ptr 3 62 61 62 62 65 61 8 5 15 1 15 1 13 1 58 7 56 7 7o 12 Ppa1 63 64 63 63 66 62 22 19 18 00 20 546 52 6 64 11 Ppaz 62 63 62 62 65 61 21 18 17 1 20 546 526 64 11 Ppa3 63 64 63 63 66 62 20 17 16 4 3 526 506 66 11 6901 83 84 83 83 86 84 75 78 77 72 71 68 40 707 6902 84 85 84 84 87 83 74 75 74 69 68 65 7 7o 7 Ppy 96 97 96 96 99 95 101 98 99 92 91 92 90 89 FIG 2 Sequence differences of the C011 gene of 15 hominoids Top Observed pairwise number of nucleotide transitional differences for the 684bp COII gene sequences below the diagonal and transversional differences above the diagonal Bottom Total observed nucleotide differences for the C011 gene sequences below the diagonal and class 1 third codon positions transitional and transversional differences of 228 bp total in the C011 gene sequences above the diagonal Abbreviations are as in table 1 Hsa 1 Hsa 2 Hsa 3 Hsa 4 Hsa 5 Hsa 6 Ptr 1 Ptr 2 Ptr 3 Ppa 1 Ppa 2 Ppa 3 690 1 690 2 PW Hsa1 Hsa2 Hsa3 Hsa4 1 Hsa5 1Hu6 Ptr1 7 23 Ptr2 18 1 Ptr3 Ppa1 5 Ppa2 Ppa3 32 4Ggo1 3 6902 lo a woo LO 10 54 Pw FIG 3 Sequence relationships derived from analyses of aligned mitochondrial COII gene sequences 684 bp Top Maximum parsimony tree Fitch 1971 constructed using the branchandbound search option within PAUP version 311 Swofford 1993 with orangutan Ppy as outgroup Tree length 205 consistency index 084939shows minimum possible branch lengths these unambiguous changes are only a portion of the observed total changes Bootstrap values indicated were derived from 1000 replications in PAUP 311 Swofford 1993 Species abbreviations are as in table 1 Bottom Distance tree constructed Mitochondrial COll Sequences and Modern Human Origins 1123 transversion This is greater than the average pairwise estimate 034 that includes all pairwise human comparisons some showing no COII sequence differences The treebased value for withinhuman differences in the ND45 region is 033 three transitions of 896 bp total Time Scale Based on COII Sequences Estimating divergence times from molecular data requires rst the measurement of genetic difference and demonstration of rate constancy second estimation of inferred amounts of actual genetic change and third choice of a calibration point and diver gence time Here we avoid error associated with the third step necessarily reliant on paleontological interpretation by using relative rather than absolute divergence times The relative time of the human mitochondrial ancestor is the amount of estimated genetic difference among humans expressed as a proportion of the estimated genetic difference between human and chimpanzee species Such calibrationfree relative divergence times are constant for any given molecular data set Data sets may differ in their absolute divergence time estimates because of the assumption of different calibration times compare Ruvolo et a1 1991 with Horai et a1 1992 but using relative date estimates39demonstrates their agreement Rate Constancy of C01 Sequence Data If DNA evolves at an approximately constant rate then the number of substi tutions that accumulate between two taxa is approximately proportional to their time since divergence The C011 gene has been shown to evolve at a constant rate within higher primates Ruvolo et al 1991 Disotell et al 1992 and the data presented here concur In the relativerate test Sarich and Wilson 1967 distances between an out group taxon in this case Pongo and different ingroup taxa are compared equality indicates rate constancy The average number of observed COII substitutions is 965 for Pongo Homo 993 for PongoPan troglodytes 916 for Pongo Pan paniscus and 895 for PongoGorilla and all lie within 55 of 942 the average for the ingroup species Therefore these data exhibit reasonable rate constancy and can be used for divergence time estimates Correction Methods for Multiple Substitutions The observed sequence difference between two taxa is less than or equal to the actual number of substitutions that have occurred since their divergence This is be cause the more ancient the divergence time the greater the chance of multiple nu cleotide substitutions occurring at any given nucleotide position Actual rather than observed numbers of substitutions are proportional to divergence times if it is assumed that substitutions occur regularly over time therefore a correction method is needed to estimate divergence times When no correction is applied the ratio of observed sequence differences provides an upper limit for the relative ancestral human mitochondrial divergence time This is because a for the human COII sequences the estimated number of substitutions is equal to or only slightly greater than the observed number but b between human and chimpanzee the observed difference will be a greater underestimate of the actual substitutional differences Thus observed differences provide an overestimate for the C011 sequence data this upperbound relative date is 058 94 or 1 16 Several correction methods exist each re ecting a model of molecular evolu tionary change The methods applied here all assume that transitions and transversions l 124 Ruvolo et al occur with unequal frequencies and therefore require estimation of the transition transversion izv ratio We use a range of iv ratios consistent with the mtDNA data see discussion below The correction methods used here are as follows 1 Brown et a s 1982 method This is a modi cation of J ukes and Cantor s 1969 oneparameter model Transitions and transversions are treated as independent mutational classes each class is corrected separately and a weighted average of the corrected values gives the estimated number of actual substitutions Although this method has been applied frequently to mtDNA data in the literature it yields par ticularly for distantly related taxa corrected values that are very different from those produced by other correction methods which generally agree in their estimates Fitch 1986 has criticized Brown et al s method because it treats transitions and transver sions as independent processes and is less descriptive of the empirical data than are Kimura s 1980 twoparameter model and Fitch s 1986 nomographic method Be cause Brown et al s method has been used to estimate the time of the human mito chondrial ancestor Kocher and Wilson 1991 we apply it here for comparison 2 The transversion method Higuchi et a 1984 This assumes that since in mtDNA transitions are much more frequent than transversions multiple transi tional substitutions at any site are more likely than are transversional substitutions As two taxa diverge transversional differences should accumulate approximately lin early with time while observed transitional differences asymptotically level off Brown et al 1982 This has been empirically con rmed for mtDNA sequence data Miyamoto and Boyle 1989 Irwin et a1 1991 Given an estimate of the iv ratio the actual number of transitions is roughly the number of transversions times the iv ratio There fore the total number of substitutions can be estimated as total substitutions transversions transversions gtlt iv I transversions X l izv Like Brown et al s 1982 method the transversion method has the drawback that transitions and transversions are treated as independent classes of substitutional events We include it because this method has also been used to estimate the time of the human mitochondrial ancestor Vigilant et al 1991 with con dence intervals Nei 1992 3 Kimura s 1980 twoparameter method This allows transitions and trans versions to have different substitutional frequencies but these two mutational types are not independent 4 Maximumlikelihood correction method Felsenstein 1990 Kishino and Hasegawa 1989 This maximizes the joint probability under the model of pairs of sequences it is equivalent to constructing two species phylogenies by the maximum likelihood method and taking the total branch lengths as distances J Felsenstein personal communication It has a more general underlying model than does Kimura s 1980 twoparameter method because it allows unequal base frequencies and for this reason we consider it to give the best estimates From the analysis of 011 ND45 and hypervariable controlregion sequences table 2 we draw the following conclusions First relative date estimates vary with correction methods used and with estimated iv ratios Second correction methods differ in how sensitive they are to changes in iv ratios with the transversion method being the most sensitive Brown et al s 1982 method intermediately so and the remaining two methods least sensitive Third relative date estimates from the three Mitochondrial COII Sequences and Modern Human Origins 1125 Table 2 Relative Divergence Dates for Human Mitochondrial Ancestor RELATIVE DIVERGENCE DATEa WHEN TRANSITIONITRANSVERSION RATIO Is mtDNA REGION AND CORRECTION METHOD 151 301 601 COII gene 684 bp present study Maximum likelihoodb l18 119 l19 Kimura 1980 twoparameterC 118 1 18 1 19 Transversion methodd 123 148 Brown et al 1982 6 119 122 ND45 region 896 bp Kocher and Wilson 1991 Maximum likelihood 129 129 129 Kimura twoparameter 128 128 129 Transversion method 127 152 1103 Brown et al 1982 130f 145 159 Hypervariable control subregions Vigilant et al 1991 Transversion method 124f 147 193 Brown et al 1982 113 123 133 Ratios of withinhuman to betweenhumanandchimpanzee nucleotide substitutions were estimated by different correction methods b As implemented by Felsenstein 1990 in PHYLlP 33 c As implemented by Felsenstein 1990 in PHYLlP 33 d Higuchi et al 1984 Estimated value is less than that observed because iv ratio is gt151 rPreviously published value 3 Value used for within human sequence divergence in all cases is 287 Vigilant et al 1991 Hasegawa and Horai 1991 have also analyzed human control region sequences using a variant of the maximumlikelihood method on the basis of three different subregions their relative divergence dates are ll4 117 and 1125 with transitiontransversion ratios 171 271 and 141 respectively mitochondrial regions do not agree when correction method and iv ratio are held constant although estimates from ND45 and hypervariable control regions tend to be more similar than those from the C011 gene This raises two questions are the differences between the estimates real in the sense that the mtDNA regions are evolving differently and which relative date estimate is best Substitutional Constraints and TransitionTransversion Ratios Because of differing constraints on nucleotide substitutions such as those having to do with codon position and functional properties of encoded proteins DNA sites evolve at different rates Li et al 1985 As R C Lewontin personal communication has noted the sum total of all DNA sequencing studies to date shows that except for pseudogenes there is probably no class of DNA not under substitutional con straints Some of these constraints are understood eg synonymous vs nonsynon ymous changes while others are not Gillespie 1991 From the mitochondrial genetic code most class 1 third codon position sub stitutions are silent while most class 2 rst and second codon positions substitutions lead to amino acid replacements In mitochondrial proteincoding genes the observed substitution frequency is far greater for class 1 than for class 2 sites Brown 1985 This pattern is evident for both COII gs 2 bottom and 4A and ND45 protein coding regions g 4B class 1 sites accumulate substitutions more quickly than do l 126 Ruvolo et a1 class 2 sites Control region sequences are noncoding and cannot be analyzed in this way Between C011 and ND45 proteincoding regions class 2 sites are accumulating substitutions differently suggesting that the two regions are under different substitu tionalselective constraints see g 4A and B However class 1 sites across the two regions are accumulating transitions and transversions similarly g 4C This ts with the expectation that class 1 substitutions more closely approximate the underlying mutational process than do class 2 substitutions Since class 1 sites are relatively un constrained in their substitutions and are also accumulating substitutions similarly over different mitochondrial coding regions they are likely to provide better relative date estimates than are all sites from an mtDNA region Viewed as contiguous stretches of DNA the C011 gene and ND45 region are evolving differently but subsets of the two mtDNA regions are evolving in the same way and we will use these for relative date estimation Note that this approach is applicable only in cases where sequence di erences are small so that class 1 sites are not saturated with multiple substitutions For a best estimate of the izv ratio closely related species should be used since more distantly related species may show lowered izv ratios because of multiple sub stitutions at some sites Simon 1991 However even closely related species may have multiple substitutions and withinspecies comparisons are then preferable Ideally the izv ratio should be calculated as phylogenetic distance approaches zero here we examine the slope of the transitiontransversion curve while Fitch s 1986 nomo graphic method uses the intercept on the izvratio axis For the C011 gene we now have intraspeci c sequence data for several hominoids g 5 In class 1 sites an izv ratio of 15 l is a clear underestimate while estimates of 302 1 and even 60 l are consistent with the data The nomographic method Fitch 1986 estimates that the iv ratio for these data is gt20 1 W M Fitch personal communication To summarize we would argue that the best relative divergence estimate is one based on class I substitutions only using the maximumlikelihood correction method for multiple substitutions and an iv ratio in the range of 30 1 60 1 When calculated this way relative divergence estimates from C011 and ND45 proteincoding regions now agree table 3 From these slowly evolving mitochondrial coding regions the best relative date estimate for the human mitochondrial ancestor as a proportion of the humanchimpanzee divergence time is l 27 Paleontological Calibration of Relative Molecular Dates Testing whether a relative divergence date is consistent with other types of an thropological evidence requires its conversion to an absolute date This depends on choice of calibration time for some paleontological or prehistorical event in this case the humanchimpanzee divergence If we take the latest possible humanchim panzee divergence to be 6 Mya Hill and Ward 1988 the slowly evolving mitochon drial coding regions estimate the human mitochondrial ancestor at 1 27th this time or 222000 years If the species divergence were as early as 10 Mya de Bonis et al 1990 the age indicated by the molecular data would be 370000 years The hypothesis that the human mitochondrial ancestor lived gt1 Mya Wolpo 1989 can be tested For combined COII and ND45 data there are 460 class 1 sites observed differences are 64 bp within humans and 1085 bp between humans and chimpanzees corrected maximumlikelihood values are 65 hp and 1740 bp with a 301 izv ratio and 65 hp and 1776 bp with a 601 izv ratio respectively If the humanchimpanzee divergence is assumed to have occurred 6 Mya the expected O o n O S 0 o 3 o 6 C N I 3 U i I 60 80 COII transverslons B 120 100 El In a5 g 2 E a a El 0 80 BEE E E g 60 3 39 395quot a I3 is 9 o I o 3 q 40 quot o v a Z 20 O I I r I 39 I 40 6O 80 100 ND45 transverslons C 80 39 a 7o o o 39 0 a a g 510 0000 51 o g 6039 0 on 53 x 3 4 a 7 E 0 an 0 can a u U a r can a g 50 Tquot o 393 3 an quot E E a a 2 3o U l 20 39F I I 39 I 39 I r I 39 I 0 20 4O 60 80 100 Class I transverslons FIG 4 Class l 9 and class 2 El transitional and transversional differences in two slowly evolving mtDNA regions Class 1 are third codon positions and class 2 the rst and second codon positions A COII gene sequences 684 nucleotides long are from seven primate Species orangutan Horai et al 1992 human chimpanzee gorilla siamang macaque and green monkey mouse cow and African clawed toad Ruvolo et al 1991 and references therein B Proteincoding portions of the mitochondrial ND45 region tRNA sequences are not included Sequences 696 nucleotides long are from 12 primate species mouse and cow Hasegawa et al 1990 and references therein C Class 1 transitions vs transversions for C011 9 and 1128 Ruvolo et al 60 3o 15 1001 8039 la a 5 HE E 60 EEEEEEE 59E class i transitions I 39 I 39 I 0 10 20 30 class ltransverslons FIG 5 Class 1 transitional changes vs class 1 transversional changes in 24 hominoid COII sequences Ruvolo et a1 1991 present study M Ruvolo unpublished data with lines corresponding to izv ratios of 15 21 3011 and 6011 indicated These values include intraspeci c as well as interspeci c pairwise sequence comparisons number of combined C011 and ND45 class I substitutions through the root of a 1 Myr old human clade would be 1 6 the humanchimpanzee difference 290 bp with a 301 izv ratio or 296 bp with a 6011 iv ratio Both expected values are signi cantly different from the observed corrected 65 bp x2174 Pat0005 x2 184 Plt0005 For a lOMya humanchimpanzee divergence de Bonis et a1 1990 the expected number of differences for a lMyrold common mitochondrial haplotype is also sig ni cantly greater than that observed 174 bp for iv 2 301 x268 Plt001 and 178 bp for izv 6011x272 Plt001 As is clear from this analysis the degree of belief in a human mitochondrial ancestor at 1 Mya is dependent on our choice of a humanchimpanzee divergence time However even with a humanchimpanzee divergence as early as 10 Mya these mitochondrial data are not consistent with a lMyr old common ancestral human mitochondrial haplotype If an even earlier date for the presumed age of the ancestral human haplotype is tested such as 14 Mya for an H erectus exodus from Africa BarYosef 1987 the hypothesis is even more strongly rejected Coalescence Time Estimates Templeton 1993 has recently observed that the stochastic nature of the evo lutionary process has been ignored in time estimates for the human mitochondrial ancestor Following his analysis and applying the neutral coalescent model of Tajima 1983 we can estimate mean time to coalescence for human mitochondrial haplotypes that differ most The method requires speci cation of a mutation rate which is a form of calibration Here we use rates estimated from all 1580 sites of combined C011 and ND45 regions Between humans and chimpanzees there are 163 inferred substitutions by maximumlikelihood correction 3021 izv ratio or 103 For humanchimpanzee divergence times of 4 Mya 6 Mya and 10 Mya nucleotide substitution rates are 13 X 10quot8 085 X 10 8 and 05 X 10398siteyearlineage respectively Mitochondrial COII Sequences and Modern Human Origins 1 129 Table 3 Relative Divergence Dates for Human Mitochondrial Ancestor from Class I Sites Third Codon Positions of Proteincoding Genes RELATIVE DIVERGENCE DATEa WHEN TRANSITION TRANSVERSION RATIO Is mtDNA REGION AND CORRECTION METHOD 151 301 601 COII class 1 sites only 228 bpb present study Maximum likelihoodc 125 127 127 Kimura twoparameterj 121 122 122 ND45 class 1 sites only 232 bp Kocher and Wilson 1991 Maximum likelihood 125 127 128 Kimura twoparameter 122 123 123 39 Ratios of withinhuman to between humanandchimpanzee nucleotide substitutions were estimated by different correction methods 1 For 228 class I C011 sites there are 34 bp of class I substitutions among humans along the tree and 562 bp between human and chimpanzee The corrected maximumlikelihood human value 34 hp equals the observed value while the humanchimpanzee difference is corrected to 909 bp when a 301 izv ratio is used or 917 bp when a 601 iv ratio is used As implemented by Felsenstein 1990 in PHYLIP 33 d As implemented by Felsenstein 1990 in PHYLIP 33 For 232 ND45 class 1 sites the observed 30 hp through the root of the human clade and 523 bp observed average between humans and chimpanzees get corrected to 31 bp for humans in both cases and to 831 bp and 859 bp between species for iv ratios 301 and 601 respectively To calculate coalescence times we need to estimate the expected nucleotide het erozygosity of the combined C011 and ND45 mitochondrial regions This is done by calculating the heterozygosities of the two mtDNA regions separately and then taking a weighted average of the two using both number of individuals and region size in weighting as the best estimate R C Lewontin personal communication Following Templeton 1993 we use Ewens s 1983 formulation of expected nucleotide het erozygosity 9 9kll21339 ln l 2 where k is the number of sites at which two or more different nucleotides occur and n is the number of genes sampled in this case the number of individuals For the first COII data set seven sites vary among six humans so that 91 307 For the second ND45 data set six sites vary among seven humans so that 92 245 The rst data set contains 684 bp of DNA from each of six individuals for a total Of4 104 bp the second data set contains 896 bp of DNA from each of seven individuals for a total Of6272 bp The weighting factors are then 04 4104 10376 and 06 6272 10376 respectively yielding an expected nucleotide heterozygosity of 270 for the combined data Time to coalescence T can be estimated from Templeton s 1993 formulation of Tajima s 1983 equation 20 as T 91k2111n9 3 where k is the pairwise divergence among haplotypes in number of nucleotide dif l 130 Ruvolo et al ferences n is the number of sampled nucleotides u is the mutation rate in substi tutions per site per year and 0 is the expected nucleotide heterozygosity The variance in coalescence time from Templeton s formulation of Tajima s 1983 eq 21 is 02 021k4u21n92 4 For C011 and ND45 regions k 7 bp separates the two most different human haplotypes through the root of the human clade and n 1580 sites are compared This yields a mean coalescence time of T253gtlt10 3u 5 with standard deviation 6 89X104u 6 For u 13 X 10 8 year the mean coalescence time is 195000 years with standard deviation 68000 years for u 085 X 10 8 year the mean coalescence time is 298000 years with standard deviation 105000 years for u 05 X 10 8 year the mean coalescence time is 506000 years with standard deviation 178000 years As Templeton 1993 observes 95 con dence limits can be estimated about these coalescence times by using Kimura s 1970 nding that overall distribution of T is approximately gamma distributed For combined C011 and ND45 human mtDNA sequences the estimated mean coalescence time of 195000 years corre sponding to a 4Mya humanchimpanzee divergence has 95 con dence limits of 85000 349000 years the estimated mean of 298000 years for a 6Mya human chimpanzee divergence has 95 con dence limits of 129000 536000 years and the estimated mean of 506000 years for a 10Mya humanchimpanzee divergence has 95 con dence limits of 220000 910000 years These broad time ranges imposed by the stochastic nature of the evolutionary process notably do not include the timepoint of 1 Mya although they come close if we assume a humanchimpanzee divergence at 10 Mya For a 46Mya humanchim panzee divergence a multiregional hypothesis that envisions modern H sapiens as emerging from anciently divergent H erectus populations spread throughout the Old World seems unlikely Interpreting Other Studies We emphasize that a molecular data set has to go through several layers of in terpretation before even relative divergence times can be estimated and that choice of correction method and iv ratio can contribute signi cantly to differences in relative divergencetime estimates Therefore it is not instructive to compare estimated dates from existing mtDNA studies Cann et al 1987 Vigilant et al 1989 1991 Hasegawa and Horai 1991 Kocher and Wilson 1991 Nei 1992 Pesole et a1 1992 Stoneking et al 1992 Tamura and Nei 1993 Templeton 1993 without consideration of correction methods equivalent to models of evolutionary change and estimated parameters also involving assumptions about how DNA evolves For example there is apparent similarity between published dates estimated from the hypervariable controlregion Vigilant et a1 1991 and ND45 sequences Kocher and Wilson 1991 however these were made by using different correction methods Mitochondrial COlI Sequences and Modern Human Origins l 131 f the same correction method Brown et al 1982 as in Kocher and Wilson 1991 and the same 151 iv ratio found for both data sets are applied the relative date estimates differ by more than a factor of two l 13 vs 1 30 respectively table 2 If instead the transversion method following Vigilant et a1 1991 is applied to both there is better agreement between data sets l 24 vs 1 27 However the transversion method is sensitive to differences in iv ratio and this is problematic for date estimation even from a single data set Analyzing the hypervariable controlregion data in different ways Vigilant 1990 thesis on pp 72 73 nds that 151 and 301 iv ratios are both consistent with the data and not statistically different but these ratios produce relative dates varying by a factor of two 124 and 1 47 respectively table 2 For human control regions iv ratios gt1511 have been estimated by others 241 Aquadro and Greenberg 1983 and 2721 Hasegawa and Horai 1991 Agreement among time estimates does not necessarily signify convergence on an acceptable answer unless the best possible model of molecular evolutionary change with well estimated param eters produces those estimates For time estimates con dence limits are also not comparable unless they sum marize error in the same variables From controlregion data Nei 1992 calculates 95 con dence limits of 110000 504000 years ago for the time of the common human mitochondrial ancestor Although it is tempting to compare this with the range derived for C011 and ND45 data the two are not equivalent Nei s estimate is based on the transversion method which is not as good a model of molecular evolutionary change as are other methods assumes a 151 iv ratio although a higher ratio is also compatible with the data and changes the mean considerably and provides error bars associated only with nucleotide substitution rate not with stochastic aspects of evolutionary change Coalescence times and con dence intervals estimated here for C011 and ND45 and by Templeton 1993 for controlregion data are comparable since both use the same model of evolutionary stochasticity However Templeton 1993 does not reject a lMyr old human mitochondrial ancestor based on estimates using an average mitochondrial mutation rate in the range of 12 X 10 8siteyearlineage While this range is appropriate for more slowly evolving mtDNA regions eg C011 and ND4 5 it is an underestimate for hypervariable control subregions which as we have shown evolve roughly 10 times faster Use of a higher mutation rate will decrease both coalescence time and con denceinterval estimates from controlregion data By judicious choice of correction method and iv ratio we can probably get any two molecular data sets to agree on some predetermined time estimate but this is counterproductive What is needed is the development of more generalized hence better correction models eg see Hasegawa and Horai 1991 Tamura and Nei 1993 further characterization of molecular evolutionary parameters eg iv ratios con sideration of all types of error associated with date estimates Templeton 1993 and consistent application of good methods to different molecular data sets Could a Recent Date for the CommonHuman Mitochondrial Type Be Artifactual The mtDNA haplotype date could be later than the actual human ancestral pop ulation if mtDNA diversity has been lost during hominid evolution Wolpoff 1989 Probability of loss is higher in small nonexpanding populations Avise et a1 1984 demographic conditions that are thought to be characteristic throughout most of human evolution However these demographic conditions are no less characteristic of the l 132 Ruvolo et al other hominoids some of which show long branches ancient mitochondrial lineages on the C011 gene tree For example two common chimpanzees surveyed here differ by 8 bp at COII class I sites more than twice the observed average in humans In light of the fact that unlike the humans sampled the chimpanzee individuals were chosen randomly and probably do not adequately represent total species genetic variation this difference is even more impressive arguing against a solely demographic expla nation for reduced human genetic variability Because only a small proportion of all living humans have been surveyed we may have missed sampling someone who is mitochondrially very different from those humans already characterized While possible this is unlikely for two reasons First examination of the apportionment of genetic diversity within the human species shows that a high proportion 86 of intraspeci c nuclearly encoded variability is contained within populations Lewontin 1972 Latter 1980 so that humans populations are not highly differentiated Second female hominoids generally transfer between groups Goodall 1986 p 86 Pusey and Packer 1986 Rodseth et a1 1991 Kano 1992 p 70 thus insuring mtDNA ow throughout the species These observations suggest that human populations with mtDNA types highly different from those already dis covered are not likely to be found Predicted Estimates of Human mtDNA Differences These mitochondrial sequence data from slowly evolving regions can help us estimate how different the 15000 bp of noncontrolregion mtDNA are among the most divergent humans known Among humans there is a maximum sequence dif ference of a 6 bp in 684 bp of the C011 gene and b 4 bp in 896 bp of the ND45 region If the two slowly evolving mitochondrial regions are assumed to be represen tative of the 15000 bp of noncontrolregion mtDNA the sequence difference between most different human mitochondrial types is estimated to be 95 bp with 19 phy logenetically informative sites in noncontrolregion mtDNA These are likely over estimates since mitochondrially encoded tRNAs and rRNAs evolve more slowly than do mitochondrial proteincoding genes Cann et al 1984 1987 It remains to be seen whether sequencing entire mitochondrial genomes will give sufficient differences among living humans for adequate phylogenetic resolution it would however increase the accuracy of relative date estimates and reduce their 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