Primate Human-Ape Div. DNA Hasegawa Et Al J Mol Evol 1985

8/13/2019 Primate Human-Ape Div. DNA Hasegawa Et Al J Mol Evol 1985

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1 96 7; S a r i c h a n d C r o n i n 1 9 7 6 , 1 9 7 7 ) , D N A h y -b r i d iz a t io n S a r i c h a n d C r o n i n 1 9 7 7 ; S i b l e y a n dA h l q u i s t 1 9 8 4 ) , r e s t r i c t i o n e n d o n u c l e a s e m a p p i n g~ D N A B r o w n e t a l. 1 9 7 9 ), p r o t e i ne l e c tr o p h o r e si s S a r i c h a n d C r o n i n 1 9 7 6 ; N o z a w ae t a l. 1 9 82 ), a n d a m i n o a c i d s e q u e n c i n g G o o d m a ne t a l. 1 9 8 3 ). A l t h o u g h t h e s e m e t h o d s w e r e p o w e r f u le n o u g h t o e x c l u d e t h e p o s s i b i l i t y th a tarnapithecusWas a n c e s t r a l to h u m a n s a n d e v o l v e d a f t e r t h e h u -m a n - a p e s p l i t t i n g , t h e y p r o v i d e d o n l y a r o u g h e s -t im a t e o f th e d a t e o f t h e s e p a r a t i o n .

T h e s e m e t h o d s e s t i m a t e d g e n e t i c d i s t a n c e s i n -d ir e ct ly, a n d n o t o n t h e b a s i s o f s t a ti s t ic a l m o d e l s .T h e y t h e r e fo r e c o n t a i n e d s o m e u n c e r t a i n ty, a n d t h ea m o u n t o f e r r o r i n h e r e n t i n t h e e s t i m a t e s c o u l d n o tb e e v a l u a t e d in a p r o p e r w a y. F u r t h e r m o r e , s i n c er ao st o f t h e s e p r e v i o u s m e t h o d s d i d n o t t a k e a c c o u n tof the e ffec t o f mu l t ip le chan ges in a s i t e , the i r e s -t im a t e s o f t h e d i v e rg e n c e d a t e a r e b i a s e d i n f a v o r~ 1 7 6 m o r e a n c i e n t t h a n t h e a c tu a l o n e w h e n a m o r e.d i s tan t sp l i t t ing i s t aken as a re fe rence . Therefore ,t n a p r e l i m i n a r y r e p o r t , w e d e v e l o p e d a s t a t i st i c a lr n e t h o d t h a t g i v e s g e n e t i c d i s t a n c e s b y d i r e c t c o m -P ar is on b e t w e e n m i t o c h o n d r ia l D N A m t D N A ) s e-q u e n c e s , a n d o b t a i n e d a m o r e r e l i a b l e e s t i m a t e o flu e ti m i n g o f th e d i v e rg e n c e e v e n t s d u r i n g t h e e v o -

t io n o f t h e H o m i n o i d e a H a s e g a w a e t a l. 1 9 8 4 a ).O u r e s t i m a t e w a s h e a v i l y d e p e n d e n t o n t h e a s -

~ U r a P t i o n t h a t t h e d i v e rg e n c e b e t w e e n b o v i n e s a n d~ri rr ta tes occ ur re d 90 M yr ago Dic kers on 1971; Sar-1 9 a n d C r o n i n 1 9 7 6 ; S i m o n s I 9 7 6 ; Wi l s o n e t a l.

7 7; G o o d m a n e t a l. 1 9 83 ). H o w e v e r , w e n o w k n o wt n o c o n v i n c i n g f o s s il s o f t h e l i v i n g o r d e r s o f

l a l a c e n t a l m a m m a l s h a v e b e e n f o u n d f r o m t h e C r e -t ac e ou s p e r i o d N o v a c e k 1 9 8 2; S a v a g e a n d R u s s e l l1 98 3) . A l s o , t h e p r e s u m e d h o l o c a u s t t h a t o c c u r r e da t t h e e n d o f t h e C r e t a c e o u s A l v a r e z e t al . 1 9 8 0 ,1 98 4; R a u p a n d S e p k o s k i 1 9 8 4 ), s o m e 6 5 M y r a g o ,r a a y h a v e b e e n r e s p o n s i b l e f o r s t a r t i n g a n e w r a -d i at io n o f p l a c e n t a l m a m m a l s A l l a n C . W i l s o n , p e r -S Ona l c o m m u n i c a t i o n ) . T h e r e f o r e , i t s e e m s l i k e l yt h a t t h e d i v e rg e n c e b e t w e e n b o v i n e s a n d p r i m a t e s~ C U r r e d a s r e c e n t l y a s 6 5 M y r a g o . I n t h i s p a p e r,w e P r e s e n t f u ll d e t a il s o f o u r m e t h o d , a n d g i v e e s -t ir aa te s o f d i v e rg e n c e ti m e s a m o n g t h e H o m i n o i d e a~ u s i ng a r e c a l i b ra t e d m o l e c u l a r c l o c k b a s e dot~ the re v i se d re fe ren ce t ime .

Mitochondr ia l D N A Seq uence Da ta

T h e r n t D N A s o f h u m a n A n d e r s o n e t a l. 1 9 8 1 ),b ~ v in e A n d e r s o n e t a l . t 9 8 2 ) , a n d m o u s e B i b b e ta l. 1 9 81 ) , e a c h o f w h i c h i s a b o u t 1 6 , 5 0 0 n u c l e o t i d e sill l e n gt h , h a v e b e e n c o m p l e t e l y s e q u e n c e d . B r o w na ~d h i s c o w o r k e r s s e q u e n c e d a s t r e t c h o f 8 9 6 n u -C le otid es i n m t D N A s f r o m h u m a n , c h i m p a n z e e , g o -

6

r il la , o r a n g u t a n , a n d g i b b o n B r o w n e t a l. 1 9 8 2 ) .T h i s s e g m e n t c o n t a i n s t h e g e n e s f o r t h r e e t R N A sa n d p a r t s o f t w o p r o t e i n s. T h e d a t a s e t u s e d in t h ep r e s e n t s t u d y i s c o m p o s e d o f t h e 8 9 6 - n u c l e o t i d es e q u e n c e s fr o m t h e a b o v e - m e n t i o n e d f i v e s p e c ie s o fH o m i n o i d e a a n d t h e s e q u e n c e s o f t h e c o r r e s po n d i n gr e g io n s f r o m b o v i n e an d m o u s e L - s t ra n d o fm t D N A ) . T h e s e d a t a p r o v i d e u s w i t h t h e o p p o r -t u n i t y t o d a t e t h e d i v e rg e n c e e v e n t s d u r i n g t h e e v o -l u ti o n o f th e H o m i n o i d e a b y a m o r e r e li ab le m e t h o dt h a n h a s b e e n u s e d b e f o r e .

T h e r a t e o f sy n o n y m o u s s u b s ti tu t io n s i n D N Ac o d i n g f o r p r o t e i n s i s m u c h h i g h e r t h a n b o t h t h a to f a m i n o a c i d - a lt e r in g s u b s t i tu t i o n s K i m u r a 1 9 7 7;B r o w n e t a l. 1 9 8 2 ; M i y a t a e t a l. , 1 9 8 2 ) a n d t h a t o fs u b s t i tu t i o n s i n t R N A g e n e s. T h e r a t e s o f a m i n oa c i d -a l te r in g s u b s t i tu t i o n s a n d o f t R N A s u b s t it u -t i o n s h a v e b e e n a p p r o x i m a t e l y t h e s a m e d u r i n g t h ee v o l u t i o n o f a n i m a l m t D N A B r o w n et a l. 1 9 82 ) .T h i s i s in s h a r p c o n t r a s t w i t h t h e s i t u a t i o n f o r n u -c l e a r D N A , i n w h i c h t R N A g e n e s a r e m u c h m o r ec o n s e r v a t i v e t h a n a r e m o s t o f t h e g e n e s fo r p r o t e in sH a s e g a w a e t al . 1 9 8 4 b ) . S in c e s y n o n y m o u s s u b s t i-

t u t i o n s a r e c o n f i n e d m o s t l y t o t h e t h i r d c o d o n p o -s i t io n s o f p r o t e i n g e n e s , w e d i v i d e t h e n u c l e o t i d es i te s in t o t w o c l a s s e s: C l a s s 1 s i te s a r e t h i r d c o d o np o s i t i o n s , a n d c l a ss 2 s it e s a r e f i rs t a n d s e c o n d c o d o np o s i t io n s a n d s i te s in t R N A g e n es . T h e s e t w o c l as s eso f s it e s a r e t r e a t e d s e p a r a t e l y i n t h e s t a ti s ti c a l m o d e lp r e s e n t e d i n t h i s p a p e r.

P h y l o g e n e t ic R e l a t io n s h i p s A m o n g t h eH o m i n o i d e a

I n a n a l y z i n g t h e d a t a , i t m u s t b e t a k e n i n t o a c c o u n tt h a t t r a n s i t i o n A ~ G , T ~ C ) h a s g r e a t ly p r e d o m -i n a t e d o v e r t r a n s v e r s io n A , G ~ T, C ) i n t h e e v o -l u ti o n o f a n i m a l m t D N A B r o w n a n d S i m p s o n 1 9 8 2 ;B r o w n e t a l. 1 9 82 ). We t h e r e f o r e c o u n t e d t h e n u m -b e r s o f t r a n s i t i o n - a n d t r a n s v e r s i o n - t y p e d i f f e r e n c e sbe tween spec ies in c lass I and c lass 2 s i t es sepa-r a t e ly, a s s h o w n i n Ta b l e 1 H a s e g a w a e t al . 1 9 8 4 a ) .I t i s r e m a r k a b l e t h a tthe n u m b e r o f t r a n s it i o n - ty p ed i f f e re n c e s i n c l a ss 1 s i te s b e t w e e n h u m a n a n d c h i m -p a n z e e i s n e a r l y t h e s a m e a s t h a t b e t w e e n h u m a na n d m o u s e . T h i s m e a n s t h a t a c o n s i d e r a b l e n u m b e ro f m u l ti p l e t r a n si t io n s h a v e a c c u m u l a t e d a t t h e s esi tes , even w h e n w e c o m p a r e a n y p a i r o f c l o s e s tr e l a t i v e s i n t h e p r e s e n t d a t a s e t . Tr a n s i t i o n a t t h et h i r d c o d o n p o s i t i o n c l a ss 1 s i te ) i s a l w a y s s y n o n -y m o u s i n t he g e n et ic c o d e o f m a m m a l i a n m i t o -c h o n d r i a B a r re U e t a l. 1 9 7 9 ; A n d e r s o n e t al . 1 9 8 1 ,1982; Bibb e t a l . 1981) .

T h e t r a n s v e r s i o n - t y p e d i f f e r e n c e s i n Ta b l e 1 a n do t h e r e v i d e n c e i n d i c a t e t h a t o f t h e l i v in g h o m i n o i d s ,g i b b o n s s e p a r a t e d f ir st a n d o r a n g u t a n s s e c o n d f r o m


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162

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mtDNAsNumbers of transition- (upper right hal0 and transversion- (lowerleft half) type nucleotide difference s amon g ma mm aliaa

1 2 3 4 5 6

i Mou se Bovine Gibbon Orang. Gorilla Chim p. Hu ma n SOVr~

1 M ou se 68 (39) 81 (53) 81 (48) 87 (46) 79 (50) 79 (51)2 Bovine 91 (82) 80 (42) 81 (44) 93 (52) 85 (61) 86 (57)

3 Gi bbo n 83 (83) 69 (71) 57 (59) 65 (59) 61 (64) 59 (58)4 Orang . 90 (85) 65 (65) 18 (34) 64 (52) 59 (60) 55 (53)5 Gorilla 85 (77) 72 (67) 19 (26) 15 (18) 28 (58) 32 (52)6 Chim p. 86 (79) 71 (67) 18 (26) 16 (18) 5 (4) 24 (50)

Hu ma n 89 (77) 70 (67) 19 (26) 15 (20) 4 (4) 3 (2)V(O/r~ O. 131 O. 104 0.028 0.023 0.007 0.005

(0.347) (0.291) (0.121 ) (0.080) (0.017) (0.009)

0.119 (0.206)0.128 (0.221)

0.09t (0.259)0.089 (0.237)0.045 (0.237)0.036 (0.216)

Number in parentheses is for the third codon positions of protein-codingpreceding it is for the rest of the sites (class 2 sites; 667 nucleotides). Fromrl and Vt0/ri

regions (class 1 sites; 232 nucleotides) and the numberHasegawa et al. (1984a). See text for explanation o fS ~

t h e l in e l e a d in g to h u m a n s ( G o o d m a n 1 9 62 , 1 9 63 ;Z i h l m a n e t al . 1 9 7 8 ; F e n d s e t al . 1 9 8 1 a ; A n d r e w sa n d C r o n i n 1 9 8 2 ; B r o w n e t al . 1 9 8 2; S i b l e y a n dA h l q u i s t 1 9 8 4) , a n d t h a t p r i m a t e s a r e r e l a t e d m o r ec l os e ly t o b o v i n e s t h a n t o t h e m o u s e ( M c K e n n a1 9 7 5 ; E i s e n b e r g 1 9 8 1 ). H o w e v e r , t h e b r a n c h i n g o r -d e r a m o n g h u m a n , c h i m p a n z e e , a n d g o r il la is c o n -t r o v e rs i a l. Te m p l e t o n ( 1 9 8 3 ) h a s d e v e l o p e d a n a l-g o r i t h m f o r a n o n p a r a m e t r i c t e s t f o r c o m p a r i n ga l t e r n a ti v e p h y l o g e n i e s o b t a i n e d f r o m r e s t r ic t i o ne n d o n u c l e a s e c l e a v a g e si te d a t a , a n d h a s a p p l i e d i tt o t h e m t D N A d a t a f r o m h o m i n o i d s . H i s c on c l u s io nw a s t h a t t h e c h i m p a n z e e a n d g o r i ll a s e p a r a te d a f t e rt h e d i v e r g e n c e o f h u m a n s . B e c a u s e h i s a n a ly s i s in -v o l v e d m a n y s y n o n y m o u s t r a n si t io n s , a c o n s i d e r -a b l e n u m b e r o f w h i c h r e p r e s e n t m u l t i p l e t r a n s it i o n s,h i s c o n c l u s i o n m a y n o t b e c o r r e c t . I n f a c t , n i n e o ft h e v a r i a t i o n s i n t h e d a t a u s e d b y h i m a r e i n th ep r o t e i n - c o d i n g r e g i o n i n o u r d a t a s e t. S e v e n o f t h e mi n v o l v e t r a n s i ti o n s a t t h i r d c o d o n p o s i t i o n s, o n e o ft h e r e m a i n i n g t w o i n v o l v e s t r a n s i t i o n s a t a f i rs t p o -s i t io n , a n d t h e o t h e r i n v o l v e s t r a n s i t i o n s a t a s e c o n dp o s i t i o n .

To c l a ri f y t h e p h y l o g e n e t i c r e l a t io n s h i p s a m o n gt h e H o m i n o i d e a , w e a p p l i ed t h e m a x i m u m l ik el i-h o o d m e t h o d d e v e l o p e d b y F e l se n s t e i n (1 9 8 1 ) t oo u r d a t a s et ( H a s e g a w a a n d Ya n o 1 9 84 ). T h e m e t h -o d o r i g i n al l y a s s u m e d t h a t t r a n s i t io n s a n d t r a n s -v e r s i o n s o c c u r a t t h e s a m e r a te . T h i s a s s u m p t i o n i si n v a li d i n a n i m a l m t D N A . T h e r e f o r e , w e s e p a r a te dt r a n s v e r s i o n f r o m t r a n s i ti o n , a n d e x a m i n e d o n l y t h ef o r m e r in c a l c u la t in g t h e m a x i m u m l i k e l ih o o d e s-t im a t e . T h e t o p o l o g y o f t h e m a x i m u m l i ke l ih o o dt r e e (F i g. 1) s h o w s t h e c h i m p a n z e e a s t h e u n i q u ec l o se s t r e l a t iv e o f h u m a n s a m o n g e x t a n t a p e s. A l -t h o u g h th e b r a n c h in g o r d e r a m o n g h u m a n s a n d t h eA f r i c a n a p e s i s c o n f i d e n t o n l y a t 4 . 4 % r i s k l e v e l b yt h is a n a l y si s , t he h u m a n - - c h i m p a n z e e g r o u p i n g h a sb e e n s u g ge s te d a ls o b y a s i n g l e - co p y n u c l e a r D N A -

D N A h y b r i d i z a t i o n ( S i bl e y a n d A h l q u i s t 1 9 84 ) , bYh e m o g l o b i n s e q u e n c e s ( G o o d m a n e t a l. 1 9 83 ), an db y e x t e n s i v e c o m p a r i s o n o f h i g h - r e s o l u t i o n b a n d in gp a t t e r n s o f th e c h r o m o s o m e s ( Yu n i s a n d P ra ka Sta1 9 8 2) . We t e n t a t i v e l y a d o p t t h i s t r e e t o p o l o g y iae s t i m a t in g d i v e r g e n c e t im e s i n th e H o m i n o i d e a .

A S t a t i s t i c a l M o d e l

L e t u s c o n s i d e r s h o m o l o g o u s n u c l e o t i d e s e qu en C eSt h a t c o n s i s t o f r n u c l e o t i d e s i te s o f a h o m o g e n e o u sc l a s s ( e i t h e r c l a s s 1 o r c l a s s 2 ) . F o r t h e d a t a s etan a ly ze d in th i s wo rk , s = 7 , r~ = 232 (c l a s s 1 si te s ),a n d r 2 = 6 6 7 ( c l a s s 2 s i t e s ) ; s i t e s t h a t e x p e r i e n c e dd e l e t i o n o r i n s e r t i o n a r e i n c l u d e d , b u t d e l e t i o n - i l as e r ti o n e v e n t s a r e n o t t a k e n i n t o a c c o u n t i n o u ra n a l y s i s . A b a s i c a s s u m p t i o n i s t h a t e a c h s i t e c h a n g e sh o m o g e n e o u s l y a n d i n d e p e n d e n t l y o f o t h e rs ; th a ti s, th e p r o b a b i l i t y o f n u c l e o t i d e s u b s t i t u t i o n h a s a~i n d e p e n d e n t l y i d e n t i c a l d i s t r i b u t i o n ( i. i. d .) . A r a rl -d o m v a r i a b l e i s r e p r e s e n t e d b y ( x~ . . . . , x s), i n w h ic he a c h c o m p o n e n t i s T, C , A , o r G , a n d t h e n u m b e ro f p o s s i b le s t a t e s i s 4 S. O u r p u r p o s e i s t o p a r a m e t r i z e

P ( x l = i l . . . . . x s= i s - - -- q i l . . . i s(i~ . . . . . is = T, C , A , G )

b a s e d o n a s t a t i s t i c a l m o d e l a n d t o e s t i m a t e d i v e rg e n c e t i m e s a m o n g t h e e x t a n t h o m i n o i d s .

W e d e n o t e b y n it i, t h e n u m b e r o f s i te s t h ath a v e a v a l u e o f ( i l . . . . . is ). T h i s f o l l o w s t h e m u lt in o m i a l ( 4 ~ - n o m i a l ) d i s t r i b u t i o n

P o l ( n ; q i ~ .. , i ,, i t , . . . , is = T, C , A , G )

a n d r e p r e s e n t s th e m o s t d e t a i l e d i n f o r m a t i o n a b oO tt h e d a t a u n d e r t h e b a s i c a s s u m p t i o n o f a n i. i .d . Tla ~a v e r a g e a n d t h e c o v a r i a n c e o f th e s e s t a t i s ti c s af eg i v e n b y t h e f o l l o w i ng f o r m u l a e :


4/15

t 65Myr

7 Man

6 Chimpanzee

5 Goril la

4 Orangutan

3 . Gibbon

2 Bovine

I Mouse

b ig . 1 . P h y l o g e n y i n f e rr e d f r o m t h e m t D N A s e q u e n c e s b y am a x i m u m l i k e l i h o o d m e t h o d d e v e l o p e d b y F e l s e n st e i n (1 9 8 1 ).I n c a l i b ra t i n g o u r m o l e c u l a r c l o c k , t h e d a t e o f d i v e r g e n c e b e t w e e nt he p r i m a t e s a n d b o v i n e s ( n o d e 2 ) w a s t a k e n t o b e 6 5 M y r a g o

E{ni l . . . i ,} = rqi , - - - is

( ~ O v { n i l . . . i , , n i t , . . . i s , } =r(6i, ...i,; i f . . . i s ' q i t . . . i s ( l a )

- - q i t . . , i s q i l ' . . , is ') ( l b )

Where 6it . . . i,; i t , . . , i,, eq ua ls 1 w he n i t = i t ' , . . . . i , =1~', an d 0 o th e rw ise .

We c a n n o t h a n d l e E q s . ( l a ) a n d ( l b ) a s t h e y a r e ,b e ca u se t h e n u m b e r o f s ta t e s i n c re a s e s e x p l o s i v e l ya s s i n c r e a s es . T h e r e f o r e , w e r e d u c e t h e d a t a t o d i f -f e re n c es , a n d c o m p a r e t h e d i f f e r e n c e s w i t h a p r o b -a b il it y d i s t r i b u t i o n t o w h i c h t h e y c o n f o r m .

4 S t a t io n a r y M a r k o v M o d e l

T h e p r o b a b i l i t y t h a t a g i v e n s i t e is v a r i a b l e i s d e -n o te d b y f , w h i c h m e a n s t h a t t h e p r o b a b i l i t y o f it sb e in g n o n v a r i a b l e i s 1 - f . E a c h v a r i a b l e s i t e e v o l v e sa CC o rd in g t o a M a r k o v p r o c e s s i n w h i c h a b a s e i ( T,C , A , o r G ) i s r e p l a c e d b y a n o t h e r b a s e j i n a ni n f in i t e si m a l l y s h o r t i n t e r v a l o f t i m e , d t , w i t h al a robab i l i ty o f P i j ( t ) , a s fo l lows :

P i j (d t ) = P r (x ( t + d t ) = j Ix ( t ) = i )

= I a ~ ' j d t ( f o r t r a n s i t i o n )[ r j d t ( f o r t r a n s v e r s i o n )

2)

163

w h e r e r rj i s t h e s t a t i o n a r y c o m p o s i t i o n o f b a s e j . I nou r da ta se t , ~-a- t ) = 0 .1 69 , ~rco) = 0 .4 29 , a 'Ao) =0 . 3 6 4 , a n d 7 t o~ = 0 .0 38 f o r c l a s s 1 s i t e s ; an d 7 rx~2) =0.297, 7rca) = 0. 26 7, ~rA 2) = 0. 31 0, a nd 7rG~2) = 0. 12 6f o r cl a ss 2 s i te s . T h i s m o d e l i s j u s t i f i e d b e c a u s e t h eb a se c o m p o s i t i o n o f a n im a l m t D N A is hi g hl y bi a s ed( p a r t i c u l a r l y, G i s s c a r c e i n t h e L - s t r a n d ) , a n d b e -

c a u s e th e a s y m m e t r y o f t h e s u b s t i t u t i o n f r e q u e n c i e si s i n a c c o r d w i t h t h e b i a s i n b a s e c o m p o s i t i o n ( th eA --. G f r e q u e n c y is m u c h l o w e r t h a n t h e G --, A )( A q u a d r o a n d G r e e n b e r g 1 9 83 ) . O u r m o d e l i s a g e n -e r a li z a ti o n o f t h e m o d e l s o f K i m u r a ( 1 98 0 ) a n d o fF e l s e n s t e i n ( 1 9 8 1 ) . K i m u r a ' s m o d e l c o r r e s p o n d s t oth e ca se o f lrx = 7rc = ~ 'A = ~rG = Va in Eq . (2) , a n dF e l s e n s t e in ' s m o d e l c o r r e s p o n d s t o t h e c a s e o f a =

. S i n ce t r a n s it i o n p r e d o m i n a t e s o v e r t r a n s v e r s i o n ,F e l s e n s te i n ' s m o d e l i s a p p a r e n t l y i n a d e q u a t e f o r an -i m a l m t D N A . F u r t h e r m o r e , b e c a u s e t h e b a se c o m -p o s i t i o n o f a n i m a l m t D N A i s h i g h ly bi a s ed , K i -m u r a ' s m o d e l d o e s n o t f i t t h e d a t a . T h i s w i l l b ef u r t h e r s h o w n f o r t h e c l a ss 1 s i te s l a t e r in t h i s p a p e r.

T h e s u b s t i t u t i o n p r o b a b i l i t y m a t r i x f o r a n i n f in -i t e s im a l l y s h o r t i n t e r v a l o f t i m e c a n b e w r i t t e n a s

T C A G

C aTradt 1- - C t ~ T + ~ W A /~rAdt /~rodt+ B*ro)dt

P(dt) = r /5~rcdt 1 - (ttrr + fl:r t~rodt+ f l r c d t

Trrdt flrcdt ar ^dt 1 -- (a~rA + f i r r+ fl*rc)dt

-- - I + Adt (3 )

F o r a n a r b i t r a r y t i m e i n t e r v a l t , t h e f u n c t i o n P ( t )s at is fi es th e C h a p m a n - K o l m o g o r o v e q u a t io n

P ( t + d t ) = P ( t ) P ( d t )= P ( 0 ( I + A d O

T h i s e q u a t i o n i s a m a t h e m a t i c a l m a n i f e s t a t i o n o ft h e M a r k o v i a n n a t u r e o f t h e p ro c e ss . T h e r e f o r e , w e

g e t

d P ( t ) _ P ( t ) A

d t

S i n c e P ( 0 ) = I , w e h a v eP(t ) = e tA (4)

To c a r r y o u t o u r a n a l y s i s , i t i s n e c e s s a r y t o e x -p l ic i tl y d e t e r m i n e t h e i n d i v i d u a l s u b s t i t u ti o n p r o b -a b i l i ty P i j( 0 b y u s i n g t h e s p e c if i c v a l u e d e c o m p o -s i t io n o f t h e r i g h t - h a n d s i d e o f E q. ( 4 ). B yd e c o m p o s i n g A a s

4

A = ~ X i U i X ii - I

w e h a v e


5/15

164

4

e tA = ~ exp ( t )k l )u iv i 'i = l

w h e r e d e t ( h i I - A ) = 0 , A u i = ~ i ~ i, A ' ~ i = X i vi ,

( u~ , v j) = ~ 0 f o r i , j = 1 , 2 , 3 , 4 , a n d a t i l d e u n d e r a

l e t te r in d i c a t e s a v e c t o r . W e g e t

X3= - 0 r v 3 + a R a ),

V 1 ~ 7 rC71 A

7 r G

00 /~ : 3 = 7 1 . C r / Tr '

V 2

V 4 =

X4= - ( T r v a ~ r ~ )

t , .~RTgT ~1 RTrc /

- - ,R 'y ' / I 'A

7 1 y T / ' G /

1 / ~ r v \1 / ~ r v |

g ~ = - 1 / ~ r ~ ]1/ lr R ]

7 l ' C / T r y ~

u , = - - ~ r T / ~ r v [ow h e r e Tr y= 71 7/ Ca n d 71 R = 71 A 71 G .

N o w , t h e n u m b e r s o f t r a n s i t i o n - t y p e d i ff e re n c e s ,S O ~, j 2 ), a n d t r a n s v e r s i o n - t y p e d i f f e r e n c e s , V ( j~ , j 2) ,b e t w e e n t h e j ~ -th a n d j : - t h s e q u e n c e s a r e d e f i n e d a sf o l l o w s :

S( j~, J2) n..T..C.. + n..C..T..J, J~ J, J2

+ n.a. .~. . + n..G..A_ (6 a)J) Ja J, J2

V ( j , ,J2)--'--n..T.A.+ n . . T. . 9 . .

+ n..c..A.. + n..c..G..J J~ J~ J~

~ n . .A . .T. . 31- n . .A . .C . .J, J~ J~ J~

+ n..G..T.. + n..o..c.. (6 b)Jl Ja Jl J2

w h e r e n ..r..c., i n d i c a t e s t h e n u m b e r o f s i te s t h a t h a v eJt Ja

T i n t h e j ~ -th s e q u e n c e a n d C i n t h e j 2 - th s e q u e n c ei r r e s p e c t i v e o f o t h e r s e q u e n c e s .

L e t u s c o n s i d e r a p a i r o f s e q u e n c e s s e p a r a t e d tm i l l i o n y e a r s a g o . S t a t e s o f e a c h s i te o f t h e s e t w o

s e q u e n c e s a r e d e n o t e d b y x ( t) a n d y ( t ) , r e s p e c t i v e l y.U n d e r t h e a s s u m p t i o n o f s t a ti o n a ri ty , w e h a v e , f o ri

Pr (x ( t ) = i , y ( t ) = j )

= P r ( x ( t ) = i , y ( t ) = j [ v a r i a b l e s i t e )

9P r ( v a r i a b l e s i t e )

f ~ 7rtPti t)Pej t)t f f iT,C ,A,G

(7)

S i n c e r e v e r s i b i l i t y, i . e . ,

~r tpei ( t = l r iPi t ( t ) ,(Sa) c a n b e e a s i ly p r o v e n , E q . (7 ) b e c o m e s

Pr ( x ( t ) = i , y ( t ) = j ) = f ir~ ~ P i , ( t )P, j ( t )t

= f i r iP0(2 t )( C h a p m a n - K o l m o g o r o v e q u at io n )

T h e r e f o r e , t h e a v e r a g e n u m b e r s o f t r a n s i t i o n - a n dt r a n s v e r s i o n - t y p e d i f f e r e n c e s a r e c a l c u l a t e d a s f o l-

(5b) lows:

V(t ) = 2 f r~ -v lrR[ 1 - - ex p( - - 23 0] (8a )

S( t ) = 2 fr{ (a 'vr c + l rATrG)

-~ (71 TTrC TrR/T ry 21-~-ATrG~rv/TrR)exp(--2flt)

- - 0rTT rc/Try)e xp[--2 t (aTrv + 31rv3]

(5C) -- (TrAlrG/TrR)exp[--2t(a~rR + BTrv )]} (8b )

F u r t h e r m o r e , b y u s i n g E q s . ( 6 a ), (6 b ) , ( l a ) , a n d ( l b ) ,v a r i a n c e s a n d c o v a r i a n c e s a m o n g d i f fe r e n c e s a rec a l c u l a t e d a s f o ll o w s : F o r o n e p a i r o f s e q u e n c e s ,

Va r ( V ) = V ( 1 - V / r ) ( 9 a )

Va r ( S ) = S ( 1 - S / r ) ( 9 0 )

C o v ( V, S ) = - V S / r (9c)

a n d f o r t w o d i f f e r e n t p a i r s o f s e q u e n c e s (j ( l) , j2 w )and ( j (2 ) , j 2 r

C ov {S (j n t ), j2~ S( j (2), j2(2))}

= rgl gl ' $2g2

t r a n s i t i o n

{q. . L. . !,'...e,...~;..j im j2o> j,q~j2(a)

- q . . ~ , .. e , .. .q . , e , . ? ; . . } ( 9 d )jtct)j2o) jtol j2o)

(,) j,m)}ov {S (j l , j2(1)), V ( j t (2),= r

$1 #I ' g2$2'

t r a n s i t i o n t r a n s v e r s i o n

{q. . t , . . e , , . . g , . . t ; . .

- q. . t , . .e , . .q.e, . .e; . .} (9 e)j,,,,g(,, L,2>L,~,

C oy {V 0( , j2 ( ) , V O y >, j2m))

= r ;g l ~ 1 g 282'

t ransv~ersion t . . . v~ersion

{q .. e,.. ,'.. t,.. e;..j,.'L .) j,,.)g.)

- q.. t,.. e,'..q., ,.. t;.. }j,(')jzo) jna)j212)

(90


6/15

Least-Squares Fi t t ing o f the Data

S in c e o u r d a t a c o n s i s t o f t w o c l a s s e s o f n u c l e o t i d es it es , a l l o f t h e v a r i a b l e s , a l l o f t h e p a r a m e t e r s e x c e p tt, a n d a ll o f t h e s t a ti s ti c s d e f i n e d a b o v e m u s t h a v ea S U b s c ri p t o r s u p e r s c r i p t k t o d e s i g n a t e t h e c l a ss .We f in a ll y r e d u c e t h e d a t a t o t h e f o l l o w i n g f o r m s :

1 7V k I) - - - - ~ V k (i, j ) ( 1 0a )

7 - i j= ~

1 7S k i) = - - - ~ S k ( i , j ) , ( 1 0 b )

7 - i ~ _ . ~ .

i = 1 , 2 . . . . . 6

W h e re t h e s u p e r s c r i p t ( i) d e n o t e s t h e i - t h s p l i t t i n g i nF ig . 1. T h e s e v a l u e s a r e l i s t e d i n Ta b l e 1 .

F r o m t h e c e n tr a l l i m i t t h e o r e m , t h e s e s ta t i st i csC an b e r e g a r d e d a s c o n s t i t u t i n g t h e f o l l o w i n g v e c t o r ,W h ic h f o l l o w s a m u l t i v a r i a t e n o r m a l d i s t r ib u t i o n :

~ (V t( ') ' . . . , V1(6), SlO ), . . . ,S1(6),V2 ( ') . . . . . V2 (6~, S2( ' ) . . . . . Sa (6))

T h e e x p r e s s i o n o f t h e m u l t i v a r i a t e n o r m a l d i s t r i -b u t i o n i s t h e n

p ~ N ( 0 , n )

3 = ( V , ( h ) . . . . . V, ( t 6 ) , S , ( t l ) . . . . . S , ( t6 ) ,V2 (h) , 9 9 , V~2(~6), S2-(i () . . . . . $2-~J6)) , a n d f~ is th eVa r i a n c e - e o v a r i a n c e m a t r ix . T h e a v e r a g e s c a n b eC a l c ul a te d b y E q s . ( 8 a) a n d ( 8 b ) , a n d t h e v a r i a n c e sb y E q s . ( 9 a - f ) , ( 1 0 a ), a n d ( 1 0 b ) . T h e l i k e l i h o o d f u n c -t ion i s

L b , D ) = 1- (21r) 12k/-d- f~

9 x p { - I ( D - I ~ ) ' f l- l ( D - I ~ )}

I f w e s u b s t i t u t e t h e v a r i a n c e a n d c o v a r i a n c e d a t af or R , t h e a p p r o x i m a t e m a x i m u m l i k e li h o o d e s t i-

m a t e o f t h e p a r a m e t e r s o f t h e m o d e l c a n b e o b t a i n e db y m i n i m i z i n g

R = - ( D - I ~ ) ' f ~ - ' ( D - ~ ) ( 1 1 )

l a O u r e a r l ie r w o r k s ( H a s e g a w a 1 9 8 4 ; H a s e g a w a e ta l. 19 8 4 a , 1 9 8 5 ) , a s a n a p p r o x i m a t i o n o f t h i s g e n -e r a li z ed l e a s t- s q u a r e s m e t h o d , w e s o l v e d t h e l e a s t-S qU ares p r o b l e m b y m i n i m i z i n g

k- l i -1 Va r (V k ~

{ V k ( i) - - V k ( t i ;fk, /3k)}=

165

1+Var(S k o) )

9 Sk ~ -- Sk(ti; fk, ak, Bk) (1 2)

S i n c e t h e e f fe c ts o f c o v a r i a n c e t e r m s a r e n o t n e g li -

g i b le , w e m i n i m i z e R d i r e c t l y in t h is p a p e r . I n o u re a r li e r w o r k s ( H a s e g a w a a n d Y a n o 1 9 84 ; H a s e g a w ae t a l . 1 9 8 4 a , 1 9 8 5 ) , n i~ kt i n s t e a d o f C hjke w a s u s e d i nc a l c u l a t i n g f~. S i n c e t h e s a m p l e s i z e i s s m a l l , t h ec o v a r i a n c e s e s t i m a t e d i n t h a t m a n n e r a r e u n s t a b l e .T h e r e f o r e , i n t h i s w o r k w e c a l c u l at e q U kt i t e r a t iv e l yb y m e a n s o f t he N e w t o n m e t h o d d i sc u s se d b e l o wb y s e t t in g t h e v a l u e s o f t h e p a r a m e t e r s a s f o ll o w s :U n l e s s i = j = k = g , q~ jk t i s g i v e n b y

q ) ~ : =I J K L

f ~ ~ 7r.Pxi(2h - t j ) P. j ( b)* Y ' Pxy( t j - - t r. )Pyk( tv. )Py, ( tv. )f o r I < J < K < L

f ~i j ~ r x P . ~ ( 2 t , - t v. ) P. k ( t r . ) P x e ( t v. )f o r I = J < K < L

f6jk ~ rxPxi(2t~ - t j )P , j ( t j )P~e (t j ) (13 )x f o r I < J = K < L

f~kt ~ rxPx i(2t l - - t j )Pxj( t j )P,k ( t j )x f o r I < J < K - L

f~ij~kdriPik(2t t )f o r I = J < K = L

w h e r e fiij i s K r o n e c k e r ' s d e l t a a n d ~ i s t h e s u m o v e r

T, C , A , a n d G .

N e w t o n M e t h o dTo m i n i m i z e R , t h e N e w t o n m e t h o d w a s c a r ri e d

o u t a s fo llo w s. If _0 - (t , , t3, t4, ts, t6, t"1, a, , /31, f2,a 2,/3 2 )' t h e i t e ra t i o n a l g o r i t h m o f t h e N e w t o n m e t h -o d i s g i v e n b y

: a2R ,~ - (aR ~o . , = O o - \ ~ / ~ _ . \- ~- L ~ _. ( 1 4 )

Variances of the Est ima tesF r o m E q . (11 ), R i s g i v e n a s a f u n c t i o n o f D a n d

0 b y

R ( p , 0 ) = ( D - 0 ( 0 ) ) ' f l - ' ( D - - 0 ( 0 ) )

w h e r e 0 (0 ~) = E [ D [ 0 ] . O n e c a n o b t a i n ~ ( D ) b y se t -

t i n g


7/15

166

C,d

o 3

o 2

o l6o

C l a s s s i t e s

t = 92.2 7-+ 11.7 3 Myr (Mouse)CM

_ 2 l = 65 Bovi ne)t 2

4 i~3 = 13.30 + 1.54 Gibbon)

= s i~4 = I0. 86 -+ 1.24 Orang)

s = 3.67 -+ 0.62 Go r il l a)

i~6 = 2.68 -+ 0.61 Chimp)D

CDCD

c : T l I I I I l l I I I l I I I I I I I I l I l0 . 0 0 0 . 0 4 0 . 0 8 0 . 1 2 0 . 1 6 0 . 2 0 0 . 2 4 0 . 2 8 0 . 3 2 0 . 3 6 0 . 4 0 0 . 4 4

V / r

F i g . 2 . Least-squares fitting of the relation between S/r and V/r. Vertical and horizontal lines indicate standard deviations o f S ~and VOVr, resp ectiv ely.The interval between neighboring small circles along the c urve is 5 My r

6) 0 . 15)R ( p , =

D e f i n i n g

R t ( D , 0 ) - ~ 0 R ( D , 0 ) ,

we e x pa nd R o_(~D, ~ ar o u nd R e_(I)(~0) , ~ as fol lo ws :

R_o(~D, 0~) - R o( O (O ), O)

A ( D - ~ ( 0 ) ) + B ( g - 0 ) ( 1 6)

w h e r e

0 2A - - - R ( ~ ( 0 ) , 0 )

a o o D

a n d

B = _ _

~2

3 0 0 0 / R ( ~ ( ~ 0 ) ' 0 )

S i n c e t h e l e f t - h a n d s i d e o f E q . ( 1 6 ) i s z e r o ,

- 0 = - - B - ' A ( D - 0 ( ~ ) )

T h e r e f o r e , t h e v a r i a n c e a n d c o v a r i a n c e m a t r i x o ft h e e s t i m a t e s i s g i v e n b y

Va r ( ~ ) = E [ ( ~ - 0 ) ( ~ - 0 ) ' ]

- B-1AE[(D~ -- ~(0))

9 D - I ) ( 0 ) ) ' ] A ' B - ~

= B - J A g A , B - I (17)

R e s u l t s

ivergence Times in the Evolution of theHominoidea

T h e r e s u l t s a r e s h o w n i n i n F i g s . 2 a n d 3 . O u r c l o c kgiv es 92.27 _+ 11.73 , 1 3.30 _+ 1.54, 10.86 _+ 1.24,3 . 6 7 _+ 0 . 6 2 , a n d 2 . 6 8 _+ 0 . 6 1 M y r f o r t h e s e p a r a t i o ~f r o m t h e h u m a n l in e o f m o u s e , g i b b o n , o r a n g u ta n ,g o r i l l a , a n d c h i m p a n z e e , r e s p e c t i v e l y ( t h e n u m b e ra f t e r _+ i s t h e s t a n d a r d d e v i a t i o n ) .

T h e e s t i m a t e o f th e o t h e r p a r a m e t e r s o f t hem od e l a re as fo l low s : t '2 = 0 .949 1 _+ 0 .03 95 , &l ~0 . 4 4 8 3 + 0 . 1 4 2 4 M y r - ~ , ~ = 0 . 0 0 8 2 + 0 . 0 0 12MY -~, f2 = 0 .3 84 7 _+ 0.0 22 8, &2 = 0.0 68 4 _+ 0.009 3M y r - 1 ~ 2 = 0 . 0 0 6 2 +- 0 . 0 0 0 7 M y r -1 ( t h e n u m b e r

a f t e r _ + i s t h e s t a n d a r d d e v i a t i o n ) . T h e f a c t t h a t 3 1n e a r l y e q u a ls u n i t y s h o w s t h a t a l m o s t a l l o f t h e t h i r dc o d o n p o s i t i o n s a r e v a r i a b l e .


8/15

167

3 . r z f f 9 o .6 1 M y r

10.86 _ 1.24 M y r /13.30 1 .54 Myr

an

Chimpanzee

Gorilla

Orangutan

Gibbon

F i g . 3 . E s t i m a t e s o f d a t e s o f s e p a r a t i o n sd u r i n g e v o l u t i o n o f t h e H o m i n o i d e a . A h o r i -z o n t a l l i n e i n d i c a t e s t h e r a n g e o f t h e s t a n d a r de r r o r o f t h e e s t i m a t e

Simulat ion Experiment

To C on fi rm t h e v a l i d i t y o f o u r m e t h o d , a c o m p u t e rS i m u l a ti o n w a s c a r r i e d o u t . A h y p o t h e t i c a l a n c e s t r a ls e q ue n c e c o r r e s p o n d i n g t o n o d e 1 i n F i g . 1 w a s c o n -S t ru c te d a c c o r d i n g t o t h e a v e r a g e n u c l e o t i d e c o m -l a o si ti o n s o f t h e r e s p e c t i v e c l a s s e s o f o u r d a t a s e t o fr n tD N A . I n t h e s i m u l a t i o n t h e s e q u e n ce e v o l v e sa CC or di ng t o t h e M a r k o v m o d e l i n w h i c h a i , B i, fi( i ~ 1 , 2 ) , an d t~ ( i = 1 . . . . 6 ) a re the e s t im a tes

O b t ai n ed b y o u r a n a l y s i s t o g i v e a s e t o f s e v e n c o n -~ ve ra Po ra ry s e q u e n c e s . T h e n t h e s e q u e n c e s a r e a n a -Y Zed b y o u r m e t h o d , a n d t h e p a r a m e t e r s o f th e

m o d e l a r e e s t i m a t e d .T h e s i m u l a t i o n w a s p e r f o r m e d 1 0 0 t i m e s , a n d

t he S a m p l e m e a n s a n d s a m p l e v a r i a n c e s o f t h e e s -t i m a t e s w e r e c o m p u t e d . T h e r e s u l t s a r e a s f o l l o w s :t~ ~ 91 .80 _ 11 .30 M yr, t3 = 13 .24 _ 1 .16 M yr,t4 ~ 10 .79 __+ 0.8 8 M y r, t5 = 3.6 5 + 0.4 8 M yr , t6 ---2 . 7 3 + 0 . 4 3 M y r , f l = 0 . 9 5 3 8 ___ 0 . 0 2 6 6 , a l0 ' 4 5 4 7 -+ 0 . 1 0 0 0 M y r - ~ , f ll = 0 . 0 0 8 3 __+ 0 . 0 0 0 9MMYr-I, f2 = 0 .3 85 2 +_ 0.0 18 1,a 2 = 0 .06 89 _ 0 .0 07 6

y r -1 , /~ 2 = 0 .0 0 6 2 + 0 . 0 0 0 6 M y r - 1 ( th e n u m b e ra ft er + i s t h e s t a n d a r d d e v i a t i o n ) . T h e s e r e s u l t s a r ec o n si st e nt w i t h t h e e s t i m a t e s f r o m t h e D N A s e-q u en c e d a t a , a n d t h e s t a n d a r d d e v i a t i o n s c a l c u l a t edf ro m t h e s a m p l e v a r i a n c e s o f s i m u l a t i o n e x p e ri -. ~ l e n t s , a s w e l l a s t h o s e c a l c u l a t e d f r o m E q . ( 1 7 ) ,a a di e at e t h e d e g r e e o f e r r o r i n o u r e s t i m a t e o f t h eP a r a m e t e r s .

dOrne Chara cteristics o f the Relationship Betweenand S

t i s a p p a r e n t i n F i g . 2 t h a t t h e n u m b e r o f t r a n s i t i o n -p e d if f e re n c e s S i s n o t a m o n o t o n o u s l y i n c r e a s i n g

f u n c t i o n o f d i v e r g e n ce t i m e t , b u t h a s a m a x i m u mv a l u e a n d t h e r e a f t e r d e c r e a s e s . T h i s i s a l w a y s t h ec a s e i f a i s g r e a t e r t h a n ~ . T h i s c h a r a c t e r i s t i c h a sn o t b e e n p o i n t e d o u t b y p r e v i o u s r e s e a r c h e r s , b e -c a u s e t h e i r i n t e r e s t h a s b e e n i n c o u n t i n g t h e a c c u -m u l a t e d n u m b e r o f n u c l e o t i d e s u b s t i tu t i o n s , t h a t is ,t r a n s i t i o n s p l u s t r a n s v e r s i o n s ( K i m u r a 1 9 8 0 , 1 9 8 1 ;Ta k a h a t a a n d K i m u r a 1 9 81 ; G o j o b o r i e t a l. 1 9 8 2) .I n a n i m a l m t D N A ,h o w e v e r , i n w h i c h t r a n s i t i o ng r e a tl y p r e d o m i n a t e s o v e r t r a n s v e rs i o n , t h i s n u m -

b e r i s n o t a d e q u a t e a s a m e a s u r e o f g e n e t ic d i s t a n ce .S e p a r at e c o u n t i n g o f tr a n s i t io n s a n d t r a n s v e r s io n si s p r e f e r a b l e i f p o s s i b l e , a s i s t h e c a s e f o r d i r e c ts e q u e n c e d a t a .

T h e d o m a i n o f c o n v e rg e n c e o f th e N e w t o n m e t h -o d i s n a r r o w . T o f i n d a g o o d i n i t i al p a r a m e t e r s e t,i t is u s e f u l t o h a v e a g o o d g r a s p o f t h e c h a r a c t e r i s ti c so f t h e r e l a t i o n s h i p b e t w e e n ~ r a n d S ,

d S a IrTlrc + ~'ATrO

w h i c h i s d e t e r m i n e d b y a / ~ . F u r t h e r m o r e , f o ra >

d S /I'TTI'C TrR /'/I'y /rATI'G'/I'y/ //l im ~ - -t-c* d V a'yTrR

w h i c h i s i n d e p e n d e n t o f th e a d j u s t a b l e p a r a m e t e r s .I n K i m u r a ' s ( 1 9 8 0 ) f o r m u l a t i o n , d S / d V t e n d s t o

1 / 2 a s t g o e s t o i n f i n it y. H i s f o r m u l a i s a g o o d a p -p r o x i m a t i o n o f o u r s f o r c l as s 2 s it es , w h e r e d S / d Vb e c o m e s - 0 . 4 5 5 a s t t e n d s t o w a r d i n f i n it y, b u t n o tf o r c l as s I si te s , w h e r e d S / d V b e c o m e s - 0 . 2 8 8 .

A s t goes to in f in i ty , V( t ) an d S( t ) t e nd to 2 f rl ryTrRa n d 2 f r 0 r Tr c + lrA ~rG ), r e s p e c t i v e l y, w h i c h v a l u e sa r e d e p e n d e n t o n t h e p a r a m e t e r f . To o b t a i n a g o o d


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estimate off , distantly related pairs of sequence data,in which the number of transition differences de-creases as t increases, are needed. In our data set,the bovine-primate and mouse-(bovine, primate)pairs are important in this respect. On the otherhand, to obtai n a good estimate o f a and B, closelyrelated pairs of sequence data, in which the numbe rof transi tion differences increases as t increases, areneeded. In our data set, the huma n-ch impa nzee andgor illa (hu man, chimpanzee) pairs are import ant inthis respect. We cannot obtain a good estimate ofa, B, and f from a single pair of sequences, andsequence data from man y species of organisms, bothclosely related and distan tly related, are needed. Se-quence data from intermediately related pairs o fspecies increase the accuracy of the estimates. Thestatistical procedure developed in this paper takesinto account the information contained in a set ofsequence data more fully than any o f the primitivemethods of simple pairwise comparison of se-quences. When mtDNAs from an Old World mon-key and from a New World mo nkey are sequenced,the accuracy of our estimate will increase.

ve rage Ra te o f Nuc leo tide Subs t i tu t i ons i n m tDN

Our present study shows that in analyzing directsequence data of mtDNA, transitions and transver-sions must be treated separately, because the ratesof these two kinds of substitutions differ consider-ably. In analyzing restriction endonuclease mappingdata of MtDNA, however, such a separate treatmentis impossible. Therefore, it should be useful in in-terpreting the restriction mapping data to estimatethe average rate of nucleotide substitutions inmtD NA irrespective of transition or transversion.

Since the previous studies estimated the rate ofnucleotide substitutions in mtD NA without payingfull atten tion to the fact that a considerable nu mbe rof the transit ion-typ e differences in the class 1 sites,even those between human and chimpanzee, rep-resent multiple hits (Brown et al. 1979, 1982; Nei1982), their estimates are bound to be lower thanours. These clocks, which proceed slower than thereal clock, have been used in datin g relatively recentevents; for example, divergences among hum an raceshave been dated based on restriction endonucleasefragment patterns o fm tD NA (Cann et al. 1982; Nei1982; Johnson et al. 1983). Therefore, these datingsmust be reexamined by our new molecular clock ofmtDNA.

In a short time interval t, exp(x) in Eqs. (8a) and(8b) may be approximated by 1 + x. From these

equations the average rate of nucleotide substitu-tions, that is transitions plus transversions , is there-fore given by Hasegawa (1984)

(rl + r2) -1 2rki'k{0rTbrc k + 7rAk~rGk)&kk=l 2

I r y k l r R k 3 k }

the calculat ed value o f whic h is (25.4 + 6.1) x 10 -9per site per year (the value after _ is the standarddeviation). This is the average substitution rate ofthe segment of 899 nucleotides of mtD NA used inconstructing our clock. Althoug h regions outside thissegment have evolve d faster or slower than the seg-ment, it seems reasonable to assume that the rateestima ted above is representative o f the average nu-cleotide substitution rate of the whole mitochondrial genome. The above estimate is much largertha n the rat es of 2.5 x 10 -9 per site per year, esti-ma ted b y Nei (1982), an d 10 x 10 -9 per site peryear, estimated by Cann et at. (1982). Of the pre-viou s estimates, the rate of 10-20 10 -9 per siteper year, estimated by Brown et al. (1982), is theclosest to our value. The divergence times amonghuma n races estimated by the previous studies mustbe revised. The revised divergence times based onthe molecular clock ofm tD NA are more recent thanthe estimates obtained from the polymorphism ofprotein s code d for by nuclear genes (Hasegawa 1984).Because mtD NA is more susceptible to transfer be-tween populations, the divergence time estimatedfrom the mt DN A clock may indicate the time whenmt DN A transfer last happe ned between two groupS.

i s c u s s i o n

T h e D a t e o f M a m m a l i a n D i ve rg e nc e

Our datings of the splittings among homino ids areheavily dependent on the assumption that the di-vergence between bovin es and primates occurred 65Myr ago. Since the holoca ust at the end of the Cre-taceous is likely to have been responsible for themammalian divergence, we think that the date of65 Myr ago is closer to the truth than previous es-timates.

Michael Novacek (personal communicat ion)pointed out that the value of 90 My r that we adoptedfor the divergence in the earlier paper (Hasegawa etal. 1984a) is unrealis tically high, and suggested arange between 65 (first appearance of primates andungulate groups in the fossil record) and 75 M yr agofor the split-off from the last com mon ancestor ofprimates and bovines. If the older limit of 75 Myra g o is taken , our clock gives 106.46 + 13.54,15.35 +__1.78, 12.53 - 1.43 ,4.23 _ 0.7 1, an d3 .0 90.71 Myr ago for the separations from the human

line of mouse, gibbon, oranguta n, gorilla, and chinapanzee, respectively. Although there is some uncertainty as to the date of the mamm alia n divergence,


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t hi s d i v e rg e n ce e v e n t s e e m s t o b e t h e m o s t r e l i a b ler e fe r e n ce w i t h w h i c h t o c a l i b r a t e t h e m o l e c u l a r c l o c ko f t h e v a r i o u s r e f e r e n c e s u s e d t h u s f a r.

Uniformity of the Rate of the Molecular Clock

T h e u ni f o rm i t y o f t h e e v o l u t i o n a r y r a te o f m t D N Aa m o n g d i f f e r e n t l i n e a g e s c a n b e e x a m i n e d b y a r e l -a t iv e r a t e t e s t W i l s o n e t a l. 1 9 7 7 ) . F r o m t h e d a t ai n Ta b l e 1 , n o s i g n i f i c a n t d i f f e r e n c e i s o b s e r v e da m o ng t h e n u m b e r s o f ch a n ge s b e t w e e n t h e m o u s ea n d a n y o n e o f th e p r i m a t e s a n d b o v i n e s . N e i t h e ri s a n y s ig n i f ic a n t d i f f e r e n c e o b s e r v e d a m o n g t h en u m b e r s o f ch a n g es b e t w e e n b o v i n e s a n d a n y o n eo f t h e P r i m a t e s . F u r t h e r m o r e , n o s i g n if i c a n t d if f e r -e n ce i s o b s e r v e d a m o n g t h e n u m b e r s o f ch a n g e sb e t w e e n t h e g i b b o n a n d o t h e r h o m i n o i d s , a n d s oo n. O n e m i g h t n o t i c e t h a t t h e n u m b e r o f t r a n s v e r -S i o n d i f f e r e n c e s o b s e r v e d b e t w e e n g i b b o n a n dorangutan in the c lass 1 s i t es, 34 , d i ffe r s con s ider -a b ly f r o m t h e 2 6 s u c h d i f f e r e nc e s o b s e r v e d b e t w e e ng i b b o n a n d t h e g o r i l l a - - c h i m p a n z e e - h u m a n t r io .b l o w e v e r, t h i s d i s c r e p a n c y i s n o t s i g n if i c an t i f th ed i st r ib u t io n i s P o i s s o n . T h e n u m b e r o f tr a n s i ti o nd if fe re n ce s o b s e r v e d b e t w e e n m o u s e a n d b o v i n e s i nthe c lass 1 s i t es , 39 , i s a l so no t s ign i f ican t ly d i ffe ren tf r om th e 4 6 - 5 3 s u c h d i ff e r en c e s b e t w e e n m o u s e a n dP r im a t e s . A l t h o u g h t h e p o s s i b i l i t y o f a s m a l l d e v i a -ti on f r o m u n i f o r m i t y o f t h e n u c l e o t i d e s u b s t i t u t i o nl ~ ro b a b il it y i s n o t e x c l u d e d , t h i s t e s t s h o w s t h a t o u rd a t a i n d i c a t e a n a p p r o x i m a t e u n i f o r m i t y a t l e a s ta m o n g p r i m a t e s a n d b o v i n e s .

B a s e d o n n u c l e a r D N A h y b r i d i z a t i o n d a ta , s o m ea u th o r s h a v e c o n t e n d e d t h a t t h e n u c l e o t i d e s u b s ti -t u t i o n r a t e w a s m u c h h i g h e r a l o n g t h e l i n e a g e s o fm o u s e a n d r a t t h a n a l o ng o t h e r m a m m a l i a n l in e a g e sK o h n e 1 9 7 0 ; K o h n e e t a l. 1 9 7 2 ) . H o w e v e r , t h e i r

s tu d ie s w e r e b a s e d o n q u e s t i o n a b l e e s t i m a t e s o f di -Ve rg en ce t i m e , a s p o i n t e d o u t b y S a r i c h a n d W i l s o n1 9 7 3) a n d b y Wi l s o n e t a l . 1 9 7 7 ) . F u r t h e r m o r e ,

8 a r ic h a n d C r o n i n 1 9 8 0 ) a n d F e r N s e t a l. 1 9 8 3 )s u g g es te d t h a t t h e r a t e s o f n u c l e o t i d e d i v e rg e n c e a r es i m il a r f o r p r i m a t e s a n d r o d e n t s . T h e r e f o r e , i t i sP o s si b le t h a t t h e r a t e o f m t D N A d i v e rg e n c e i n r o -d e n ts d o e s n o t d i ff e r f r o m t h a t f o r o t h e r m a m m a l i a nOrders.

A t p r e s e n t , w e t e n t a t i v e l y t h i n k t h a t t h e n u c l e o -t id e s u b s t i t u t i o n r a t e o f r o d e n t s d o e s n o t d i f f e r f r o mt ha t o f o t h e r m a m m a l i a n o r d e r s , a n d t h a t, a s o u r~ a l y s i s i n d ic a t e s, r o d e n t s d i v e rg e d fr o m o t h e r p la -c e n ta l m a m m a l s 9 2 . 2 7 + I 1 .7 3 M y r a g o , b e f o r e t h e~ a m r n a l i a n r a d i a t i o n a m o n g m o s t o f t h e e x t a n t p l a-C en ta l m a m m a l i a n o r d e r s t h a t t o o k p l a c e s o m e 6 5

M y r a go . S i nc e r o d e n t s a r e u n i q u e a m o n g p l a c e n t a lm a m m a l s i n t h a t a ll a t te m p t s o f p a le o n t o l o g i s ts t or el at e t h e m t o o t h e r g r o u p s o f m a m m a l s h a v e b e e n

169

i n v a i n C o l b e r t 1 9 8 0 ) , o u r h y p o t h e s i s o f a n e a r li e rr o d e n t d iv e rg e n c e m a y n o t b e u n r e a s o n a b l e .

I n a n y c a se , t h e m o l e c u l a r c l o c k h y p o t h e s i s a sa p p l i e d t o t h e w h o l e m a m m a l i a n c l as s is st il l c o n -t r o v e r s i a l. I t w il l b e d e s i r a b l e t o t e s t w h e t h e r r o d e n tD N A h a s e v o l v e d m o r e r a p i d l y t h a n t h a t o f o t h e rp l a c e n ta l m a m m a l s w h e n a n o u t s i d e r e f e re n c e s u cha s m a r su p i a l m t D N A is o b ta i n e d . E v e n i f t h e m o u s el in e h a s e v o l v e d m o r e r a p i d l y t h a n t h e o t h e r s , i td o e s n o t i n v a l id a t e o u r a p p r o a c h i n e s t im a t i n g d i -v e r ge n c e ti m e s a m o n g t h e H o m i n o i d e a .

A l t h o u g h t h e c o n s t a n c y o f o u r c l o c k w i t h r e s p e c tt o a b s o l u t e g e o l o gi c al t i m e h a s y e t t o b e p r o v e nd i re c t ly, f u t u re s e q u e n c in g o f m t D N A s f r o m v a r i o u sf a m il ie s o f p r i m a t e s a n d f r o m v a r i o u s m a m m a l i a no r d e r s w i l l c l a r if y t h e a c c u r a c y a n d a p p l i c a b i l i t y o fo u r c l o c k in e s t i m a t i n g d i v e rg e n c e t im e s a m o n gm a m m a l s . I t h a s b e e n p r o p o s e d t h a t th e S o u t hA m e r i c a n m o n k e y s d e s c e n d e d f r o m A f ri c an m o n -k e y s , n o t f r o m N o r t h A m e r i c a n p r o s i m i a n s , w h e nt h e S o u t h A m e r i c a n c o n t i n e n t w a s c lo s e t o t h e A f -r i c a n c o n t i n e n t s o m e 3 5 - 3 8 M y r a g o C i o c h o n a n dC h ia r eU i 1 9 8 0) . W h e n m t D N A f r o m a n e w W o r l dm o n k e y i s s e q u e n c e d , t h e v a l i d it y o f o u r c l o c k w i llb e t e s t e d .

Splittings of Orangutan and Gibbon romHuman Line

I n o u r e a r li e r p a p e r H a s e g a w a e t a l. 1 9 8 4 a ) , w ea s s u m e d t h a t t h e 1 4 . 5 - M y r - o l d s p e c i m e nSivapith-ecus w a s a n c e s t ra l t o t h e o r a n g u t a n b u t n o t t o h u -m a n s R a z a e t a l. 1 9 8 3 ), a n d t h a tMicropitheeusw h i c h i s s o m e 2 0 M y r o l d , w a s a n c e s t r a l t o t h eg i b b o n s b u t n o t t o h u m a n s S i m o n s 1 9 8 1) . A s s u m -i n g 9 0 M y r a g o f o r t h e d a t e o f t h e s p li tt in g b e t w e e np r i m a t e s a n d u n g u l a t e s a n d u s in g a l e a s t- s q u a r e sm e t h o d b y m i n i m i z i n g R , r e p r e s e n t e d b y E q . 1 2 ),w e o b t a i n e d f o r t h e d i v e rg e n c e d a t e s o f th e o r a n -g u t a n a n d o f t h e g i b b o n s f r o m t h e h u m a n l in e15 .9 + 2 .9 and 19 .1 + 3 .6 M yr ago n i jke va lu esi n s t e a d o f q ~k e v a l u e s w e r e u s e d i n c a l c u l a t in g ~ ) ,r e s p e c t i v e l y, w h i c h a r e c o n s i s t e n t w i t h t h e a b o v ei n t e r p re t a t io n o f th e f o s si l e v i d e n c e . H o w e v e r , w h e nw e t a ke 6 5 M y r f o r t h e d a te o f m a m m a l i a n d i v er -g e n c e a s i n th i s p a p e r, t h e e s t i m a t e d d a t e s o f t h eo r a n g u t a n a n d g i b b o n d i v e rg e n c e s c o n t r a d i c t t h ea b o v e i n t e r p r e t a t i o n o f t h e f o s s i l e v i d e n c e .

P e t e r A n d r e w s p e r s o n al c o m m u n i c a t i o n ) p o i n t -e d o u t t h a t t h e 1 4 .5 M y r a g o s p li tt in g t i m e b e t w e e nh u m a n s a n d t h e o r a n g u t a n R a z a e t a l. 1 9 8 3 ) i sb a s e d o n t h e i d e n t i f ic a t i o n o f a f r a g m e n t a r y f o s s ilt h a t i s n o t w e l l d a t e d . F u r t h e r m o r e , h e p o i n t e d o u t

t h a t Micropithecus h a d n o t b e e n s h o w n t o b e a n -c e s t r a l t o t h e g i b b o n s .Micropithecus i s n o w r e c o g -n i z e d a s b e l o n g i n g t o a p r i m i t i v e g r o u p l a c k i n g t h e


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synapomo rphies (derived characters that are shared)of the Homi noi dea (Andrews 1981). Our datings inthi s paper, 10.86 + 1.24 an d 13.30 ___1.54 Myr agofor the orangutan and gibbon divergences, respec-tively, are in accord with those of Andrews andCron in (1982), 10 _ 3 Myr ago for the orangutandivergence and 12 + 3 Myr ago for the gibbon di-vergence, and also approximately in accord withthose of Sarich and Cronin (1977), 9-11 and 11-13Myr ago for the orangutan and the gibbon diver-gences, respectively.

Possibility Th at Austra lopithe cus afarensis is No tan Ancesto r o f Hu ma ns Tha t Evo lved Af te r theH u m a n - A p e S p l i tt in g

There is a widely believed hypothesis that Australo-pithecus afarensis which lived some 3.7 M yr ago atLaetoli in Tanzania an d at Had ar in Ethiopia, is ourancestor and evolved after the human-ape splitting(Leakey et al. 1976; J ohan son et al. 1978; Joha nsonand White 1979; Cronin et al. 1981; White et al.1981). However, our dati ng of the hum an- chi m-panzee splitting by the molecular clock of mtDNAis 2.68 + 0.61 Myr ago, which is more recent thatwhen A. afarensis lived. Two factors may be takeninto consideration to explain this. First, the datingof the Hadar homi nids is still controversial, and theycould be younger than 3.4 Myr (Sarna-Wojcicki etal. 1985). Second, the divergence between primatesand ungulates may have happened 75 Myr ago rath-er than 65 Myr ago, and if this is the case, then ourclock gives 3.09 + 0.71 Myr ago for the hu ma n-chimpanzee separation. Considering these uncer-tainties, the possibility that the hypothesis is com-patible with our clock cannot be discounted. How-ever, this compa tibility rests on fragile ground, an dit may be worthwhile to ex amine the va lidity of thehypothesis.

Since A. afarensis walked upright on two legs,despite the simi larity of its brain capacity, dentition,and other features to those of apes, most paleoan-thropologists believe it to be the first hominid. How-ever, our molecular clock of mtDN A suggests thatthe human-ape splitting may have occurred morerecently than when A. afarensis lived.

Bipedalism is widely believed to have been thefirst step in homin iza tio n (Leakey et al. 1976; Da yand Wi ckens 1980; White 1980), a nd any fossil pri-mates that walked upright have been readily ac-cepted as our immediate ancestors. If A. afarensiswhich walked upright, is not an ancestor that e volvedafter the hum an- ape splitting, as is suggested by our

molecular clock, then the following two explana-tions of the origin of bipedalism are possible: (1)A.afarensis an upright biped, was a com mon ance stor

of human s and the chimpanzee (and probably thegorilla). The chimpanzee (and the gorilla) lost bi-pedalism after splitting from the hu ma n line. (2) A.afarensis was not an ancestor o f any living primate.Bipedalism evolved independently in at least twolineages, the A. afarensis line and the human line.

The first possibility was pointe d out by Gribbinand Cherfas (1982). Al though no i ndicati on has beenfound that the chimpanzee had a bipedal ancestor,this does not necessarily exclude the first possibility.

Zihlm an (1979) pointed out th at the pygm y chim-panzee Pan paniscus has many morphological fea-tures in common with A. africanus which is be-lieved to have descended from A. afarensis. Thepygmy chimpanzee is the species most closely re-lated to the common chimpanzee P. troglodytesamon g extant homin oids (Zihlm an et al. 1978; Fer-riset al. 1981a, b; Brown et al. 1982; Sibley andAhlquist 1984). Furthermore, Feldesman (1982a, b)noticed that A. afarensis clearly resembles P. pan-iscus in proximal ulnar morphology. The above ar-guments might be compatible with the first possi-bility, that knuckle-walking of extant chimpanzeesand gorillas evolved from bipedality.

Oxna rd (1975, 1984) poi nted out that bipedalisnamight have evolved not once or even twice, butperhaps several times during the evolution of theHomin oidea . Therefore, the second possibility alsoappears likely. He claims that the australopithecines, including A. afarensis A. africanusand A.robustus were not structurally closely similar to hu-mans, a nd that they were adapted at least in part toan arboreal environment.

Although it is likely that the australopithecineswere capable o f bipedalism (but probably in a bio-mechanical mode quite different from that em-ployed by humans), they mig ht also have been quad-rupedal, especially when c limbing in trees. It is nowbeing recognized that although A. afarensis couldwalk bipedally, it kept its balance more like a chim-panzee does than a hu ma n does (Stern and Susman1981, 1983). Thus, the possibility that A. afarensiSwas not an ancestor of humans that evolved afterthe huma n-a pe splitting cannot be ruled out at pres-ent.

It is unknow n whether the last co mm on ancestorof human and chimpanzee was like the living chim-panzee or the living human. However, it seems tohave been widely assumed i mplicitly that the corn-mort ancestor of the two species was mor e like thechimpanzee than the human. There has been a tendency to view hominid features as specialized andthose o f apes as unspecialized. Any fossil hominoidsthat bear some resemblance to humans have beenreadily considered to be human ancestors thatevolved after the human-ape splitting. Ramapithecines had long been believed to be ancestral to hu-


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r oa ns b a s e d o n t h is t y p e o f r e a s o n in g . H o w e v e r , t h e ya re n o w b e l i e v e d t o b e a n c e s t o r s o f t h e l iv i n g o r a n g -u ta h A n d r e w s 1 9 8 2 ; A n d r e w s a n d C r o n i n 1 9 8 2;P i lbeam 1982) .

R e c e n t l y A l a n W a l k e r p o i n t e d o u t t h a t t h e o r a n g -u ta n , w h i c h h a d b e e n w i d e l y b e l i e v e d t o b e a h i g h lyS pe cia liz ed a p e a s c o m p a r e d w i t h t h e c h i m p a n z e ea n d g o r il la , m a y a c t u a l l y b e t h e l iv i n g h o m i n o i d t h a tb e ar s t h e m o s t e x t e n s i v e r e s e m b l a n c e a m o n g c o r n -t e m p o r a ry d e s c e n d a n t s t o th e l a st c o m m o n a n c e s t o rof a ll the l iv ing grea t ape s Lew in 1983) . In th i sSense, the o r an guta n m ay be a l iv ing foss il .

S c h w a rt z 1 9 8 4 ) p o i n t e d o u t th a t h u m a n s s h a r eU n i qu e ly f e w m o r p h o l o g i c a l f e a t u r e s w i t h e i t h e r t h ec h i m p a n z e e o r t h e g o r i l l a , w h e r e a s m a n y f e a t u r e sa re s h a re d b y h u m a n s a n d t h e o r a n g u ta n . H e c o n -e lu d ed t h a t h u m a n s a n d t h e o r a n g u t a n m a y b e t h ec lo s es t r e l a t i v e s a m o n g t h e l i v i n g h o m i n o i d s . A l -t ho u gh t h e m o l e c u l a r e v i d e n c e s h o w s t h a t h i s c o n -c lu s io n i s c l e a r ly w r o n g G o o d m a n 1 9 6 2 , 1 9 6 3 ; S a r-ic h a n d W i l s o n 1 9 6 7 ; F e r r is e t al . 1 9 8 1 a ; A n d r e w sa n d C r o n i n 1 9 8 2 ; B r o w n e t a l. 1 9 8 2 ; H a s e g a w a a n dYa n o 1 9 8 4 ; S i b l e y a n d A h l q u i s t 1 9 8 4 ), h i s su g g e s -t io n l e a d s u s to t h e f o l l o w i n g i m p o r t a n t p o i n t . A l -t ho u g h b r a i n c a p a c i t y h a s i n c r e a s e d v e r y m u c h a l o n gt he h u m a n l in e a g e , n o t o n l y t h e o r a n g u t a n b u t a l s ot he h u m a n m a y b e l iv i n g f o s s il s w i t h r e s p e c t t o v a r-io u s m o r p h o l o g i c a l f e a tu r e s , w h e r e a s t h e c h i m p a n -Z ee a n d t h e g o r i ll a m a y h a v e s p e c i a l i z e d q u i c k l ya ft er t h e y d i v e rg e d f r o m t h e h u m a n l in e . I f t h i s isth e C a s e , i t is p o s s i b l e t h a t s o m e f o s s il h o m i n o i d sth at We re a n c e s t r a l t o t h e c h i m p a n z e e o r g o r i ll a b u tn o t t h e h u m a n h a v e b e e n a s s ig n e d t o h u m a n a n c e s -to rs b e c a u s e o f s o m e o f t h e i r r e s i d u a l f e a t u r e s . I t isb o w c l e ar t h a t t h e d a t i n g o f th e b r a n c h i n g e v e n t sf rom the foss i l r ec ord a lon e i s a h igh ly d i ff icu l t jo b~a t h is c i r c u m s t a n c e , a n d t h a t t h e m o l e c u l a r r e c o r dis USefu l fo r th i s purp ose .

A th e o r y o f h u m a n o r ig i n m u s t b e a t h e o r y o fc h i m p a n z e e a n d g o ri ll a o r i g i n s t o o Z i h l m a n 1 9 7 9 ) ,a nd to c l a ri f y o u r o r i gi n , p a l e o a n t h r o p o l o g i s t s m u s tS ee k n o t o n l y o u r a n c e s t o r s b u t a l s o f o s s i l c r e a t u r e sa n c e s t r a l t o t h e c h i m p a n z e e o r t h e g o r i l l a b u t n o tt o h u m a n s . H o w e v e r , n o f o s s i l a s s i g n e d t o b e a n -C eS tral o n l y t o t h e c h i m p a n z e e o r g o r i l la h a s y e t b e e n~ e a r t h e d .

~Ssib ili ty o f lnterspecies Transfer o f MitochondrialIVA between Proto-hum an and Proto-chimpanzee

S in ce m t D N A i s i n h e r i t e d m a t e r n a ll y, a m o l e c u l a re lg ck b a s e d o n i t c a n g i v e o n l y in f o r m a t i o n o n m a -terna l lineages . Also , da t ing s o f spec ies sep ara t ionf ro m m t D N A d a t a m a y s o m e t i m e s b e in e r r or b e -c atls e o f t h e p o s s i b i l i t y o f in t r o g r e s s i o n o f t h e s e i n -Clependently segrega t ing organe l les f ro m on e spe c ies

171

tO a n o t h e r. T h i s m a y b e n o t o n l y a w e a k n e s s o fm t D N A a n a ly s is , b u t a l s o a st re n g th , b e c a u s e ins u c h a c a s e t h e m t D N A c l o c k c o u l d p r o v i d e i n f o r -m a t i o n o n t h e e c o l o g i c a l r e l a t i o n s h i p b e t w e e n t w ospec ies .

I t h as r e c en t ly b e e n f o u n d t h a t m t D N A c a n p a ssa c r o s s t h e s p e c ie s b o u n d a r y i n t h e m o u s e F e n -i s e ta l. 1 9 8 3 ) , in t h e a q u a t i c f r o g S p o l s k y a n d U z z e l l1984) , and inDrosophila Powel l 1983) , and i t i sn o t i m p o s s ib l e t ha t s u c h a n e v e n t h a p p e n e d a m o n ge a r l y h o m i n o i d s . I f i t d i d o c c u r , t h e t i m e d e r i v e df r o m o u r c l o c k s h o u l d r e f l e c t i t . T h e s e q u e n c e s o fn u c le a r D N A , w h e n t h e y b e c o m e a v a i la b le , s h o u l dm a k e t h e s i t u a t i o n c l e a r. A t p r e s e n t t h e p o s s i b i l i t ym u s t b e c o n s i d e r e d t h a t o u r c l o c k r e fl e c ts a t r a n s f e ro f m t D N A t h ro u g h h y b r i d i z a ti o n b e t w e e n a p r o t o -h u m a n a n d p r o t o - c h i m p a n z e e a f te r t h e f o r m e r h a dd e v e l o p e d b i p e d a l i s m F ig . 4 ) .

W h e n t w o c l o s e ly re l a te d a n i m a l s p e c ie s ar e g e o -g r a p h i c a l l y c o n t i g u o u s , f e r t i l e i n t e r s p e c i e s h y b r i d ss o m e t i m e s a r i s e a t t h e b o u n d a r y z o n e . S u c h c a s e sa r e w e l l k n o w n i n p r i m a t e s , e . g ., b e t w e e n a n u b i sb a b o o n s(Papio anubis)a n d h a m a d r y a s b a b o o n s P.hamadryas) N a g e l 1 9 7 3 ; S h o t a k e 1 9 8 1 ; S u g a w a r a1 9 8 2 ) a n d b e t w e e n r e d t ai l m o n k e y s(Cercop hecusascanius) a n d b l u e m o n k e y s(C. mitis) A l d r i c h -B l a k e 1 9 6 8 ; M a c d o n a l d 1 9 8 4 ) .

I n t e rg ro u p t r a n sf e r o f m a l e s i s f a r m o r e c o m m o na m o n g s o c ia l p r i m a t e s t h a n t h a t o f f e m a l e s, a n d i na n u b i s b a b o o n s a n d J a p a n e s e a n d r h e s u s m o n k e y s ,w h i c h a r e a m o n g t h e m o s t f r e q u e n t l y s t u d i e d m o n -k e y s , f e m a l e s t y p i c a l l y r e m a i n i n t h e i r n a t a l g r o u pe .g ., see Mo or e 1984) . In such a soc ia l sys tem , in -

t e r sp e c i e s tr a n s f e r o f m t D N A w o u l d s e e m d i f fi c ul te v e n i f i n t e r s p e c i e s h y b r i d i z a t i o n o c c u r s f r e q u e n tl y.I n c o n t r a s t , i n s o c i e t i e s o f c h i m p a n z e e s a n d g o r il la s ,i n t e rg r o u p t r a n sf e r o f fe m a l e s a n d n o t o f m a l e s i sr o u t i n e . T h e r e f o r e , i t a p p e a r s p o s s i b l e t h a t i n t e r -s p ec ie s tr a n sf e r o f m t D N A h a p p e n e d b e t w e e n a p r o -t o - h u m a n a n d a p r o t o - c h i m p a n z e e .

I f in t e rs p e c ie s t r a n sf e r o f m t D N A b e t w e e n p r o to -h u m a n a n d p r o t o - c h i m p a n z e e d i d i n d e e d o c c u r, i ti s t e m p t i n g t o s p e c u l a t e i n w h i c h d i r e c t i o n t h e t r a n s -f e r o cc u r r e d . T h e l e ss e r in t ra s p e c ie s p o l y m o r p h i s mo f h u m a n m t D N A c o m p a r e d w i th t h at o f c h im p a n -zee Fer r i s e t a l . 1981b ) sugges t s tha t the t rans f e ro c c u r r e d f r o m p r o t o - c h i m p a n z e e i n t o p r o t o - h u -m a n .

I n t e rs p e c i e s t r a ns f e r o f m t D N A h a s b e e n o b -s e r v e d b e t w e e n t h e f r u i t f l yDrosophila pseudoob-scura and i t s s ib l ing spec iesD. persimilis P o w e l l1 9 83 ) . A l t h o u g h m a l e F ~ h y b r i d s b e t w e e n t h e t w os p e c i e s a r e s te r il e , F1 f e m a l e s , t h e s e x t h a t m u s t b ef e r ti le t o p a s s m t D N A , a r e f e rt il e . T h u s e v e n i f t h e r ee x i s ts a b a r r i e r t o i n te r s p e c i e s h y b r i d i z a t i o n , t h e i n -t r o g r e s s i o n c a n o c c u r Ta k a h a t a a n d S l a t k i n 1 9 8 4 ) .

N a t u r a l i n t e r s p e ci e s tr a n s f e r o f m t D N A i s o b -


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s e r v e d f r o mM u s d o m e s ti c u si n t o M . m u s c u l u s( F e r -r i s e t a I. I 98 3 ) , a n d f r o mR a n a l e s s o n a e i n t o R .r i d i b u n d a ( S p o l s ky a n d U z z e l l 1 9 84 ). T h e e x t e n t o fm t D N A d i ve r ge n c e b e t w e enM . d o m e s t i c u sa n d a u -t h e n t i c M . m u s c u l u si s a b o u t 5 % , a n d t h a t b e t w e e nR . l e s s o n a ea n d a u t h e n t i cR . r i d i b u n d a i s a b o u t 8 % .S i n c e t h e a v e r a g e s u b s t it u t i o n r a t e o f m t D N A i se s t i m a t e d t o b e 0 . 0 2 5 4 p e r s i t e p e r m i l l i o n y e a r s ,t h e i n t e r s p e c i e s t r a n s f e r o f m t D N A o c c u r r e d0 . 0 5 / ( 2 x 0 . 0 2 5 4 ) ~ 1 M y r a g o i n m i c e a n d0 . 0 8 / ( 2 x 0 . 0 2 5 4 ) ~ 1 .5 M y r i n a q u a t i c f r o g s a f t e rt h e s p e c i e s se p a r a t e d . T h e r e f o r e , i f s u c h a n i n -t ro g r es s io n o c c u r r e d be t w e e n p r o t o - h u m a n a n d p r o -t o - c h i m p a n z e e 2 . 6 8 _+ 0 . 61 M y r a g o , t h e n t h e h u -m a n - a p e s p l it ti n g m a y d a t e b a c k t o s o m e 5 M y ra g o , a s s u g g e s t e d b y S a r i c h a n d Wi l s o n ( 1 9 6 7 ) a n db y S a r i c h a n d C r o n i n ( 1 9 7 7 ).

S i b le y a n d A h l q u i s t ( 1 9 8 4 ) c a l i b r a te d t h e m o l e c -u l ar c lo c k b a s ed o n t h e ir n u c l e a r D N A - D N A h y -b r i d i z a t i o n d a t a b y a s s u m i n g t h a t t h e o r a n g u t a n d i -v e r g e d f r o m t h e h u m a n l in e 1 3 - 1 6 M y r a g o. S in c eo u r m o l e c u l a r c l o c k o f m t D N A g i v es 1 0. 86 _ 1 .2 4M y r a g o a s th e d a t e f o r t h e o r a n g u t a n d i v e r g e n c e ,w e t h i n k t h a t t h e 1 3 M y r d a t e i s n e a r e r t h e a c t u a lv a l u e t h a n 1 6 M y r i s, p r o v i d e d m t D N A t r a ns f e r d i dn o t o c c u r b e t w e e n t h e o r a n g u t a n a n d t h e A f r i c a na p e s . I f t h e 1 3 M y r d a t e i s u s e d f o r t h e o r a n g u t a ns p l it t in g , t h e i r d a t a g i v e 6 .3 M y r a g o a s t h e d a t e f o rt h e h u m a n - - c h i m p a n z e e s e p a r a t io n , a l t h o u g h t h ea m o u n t o f e r r o r i n h e r e n t i n t h e ir d a t a c a n n o t b ee s t i m a t e d .

T h o u g h t h e r e l e v a n t d a t a a r e l i m i te d , t h e h y -p o t h e s i s o f a l at e d i v e r g e n c e b e t w e e n h u m a n s a n da p e s i s a l s o s u g g e s te d b y t h e a m i n o a c i d s e q u e n c eo f p r o t ei n s c o d e d fo r b y n u cl e a r D N A ( G o o d m a ne t at . 1 9 8 3 ), a n d b y t h e r a r i t y o f s y n o n y m o u s n u -c l e o t id e s u b s t it u t i o n s i n h e m o g l o b i n g e n e s b e t w e e nh u m a n s a n d t h e A f r i c a n a p e s ( L i e b h a b e r a n d B e g l e y1 9 8 3 ; S c o t t e t a l . 1 9 8 4 ) .

A f t e r s u b m i s s i o n o f t h i s p a p e r, w e l e a r n e d t h a tf f ~ -g l o bi n g e n e s , w h i c h a r e p s e u d o g e n e s o f t h e /~ - g l o -b i n g e n e fa m i l y, f r o m h u m a n , c h i m p a n z e e , g o ri ll a,o w l m o n k e y ( N e w W o r l d m o n k e y ) , a n d l e m u r ( p r o -s i m i a n ) h a v e b e e n s e q u e n c e d ( C h a n g a n d S l i g h t o m1 9 8 4; G o o d m a n e t a l. 1 9 8 4; H a r r i s e t a l. 1 9 8 4) .G o o d m a n a n d h i s c o w o r k e r s (1 9 84 ) c o n t e n d e d t h a tt h e n u c l e o t i d e s u b s t i t u t i o n r a t e w a s s l o w e r a l o n g t h eh o m i n o i d l i ne a ge t h a n a l o ng th e N e w Wo r l d m o n -k e y l i n ea g e . H o w e v e r , t h e d i f f e r e n c e is n o t s t a ti s t i-c a l l y s i g n if i c a nt , a n d w e t h i n k t h a t t h e n u c l e o t i d es u b s t i tu t i o n p r o b a b i l i t y p e r u n i t t i m e i n t e r v a l o f th ex b n- gl ob in g e n e h a s b e e n a p p r o x i m a t e l y u n i f o r m a tl e as t a m o n g t h e A n t h r o p o i d e a . A s s u m i n g t h a t t h ed i v e rg e n c e b e t w e e n t he H o m i n o i d e a a n d t h e N e wW o r l d m o n k e y s o c c u r r e d 3 8 M y r a go , o u r p re l im -i n a r y a n a l y s i s o f t h e ~ k ~- glo bin d a t a g i v e s d a t e s o f5 . 2 + 0 . 5 a n d 5 . 8 ___ 0 . 6 M y r a g o ( t h e n u m b e r a f -

6 . Man2.7 _ O. Myr ago /

Australopithecus [afarensis . ~ / f

l j / / ~ Interspecies transfer of~ ' / 1 l m ito c h o n d r ia l DNA

Fig 4. A possible model of interspecies transfer of mtDN Abetween proto-human and proto-chimpanzee

t e r + i s t h e s t a n d a r d d e v i a t i o n ) f o r th e c h i m p a n z e ea n d g o r i l l a s e p a r a t i o n s , r e s p e c t i v e l y, f r o m t h e h u -m a n l i n e ( u n p u b l i s h e d d a t a ) . T h e s e d a t i n g s s e e m toc o n t r a d i c t th o s e f r o m t h e m t D N A d a t a, a n d t hisd i s c r e p a n c y m a y r e f le c t m t D N A t r a n s f e r b e t w e e n ap r o t o - h u m a n a n d p r o t o - c h i m p a n z e e .

T h e D N A s e q u e n c e d a t a p r e s e n t l y a v a i la b l e f ors e tt in g o u r m o l e c u l a r c l o c k a r e l im i t e d a n d t h e d o c kc a n n o t a l w a y s d e t e r m i n e w h i c h o n e o f t h e v a r io u sp o s s i b i l i t i e s d i s c u s s e d i n t h i s p a p e r i s t h e t r u t h ,T h e r e f o r e , f u t u r e s e q u e n c i n g o f D N A , p a r ti c u la r l yn u c l e a r D N A , i n c o n j u n c t i o n w i t h f u t u r e f o s si l f in d -

i n g s s h o u l d t h r o w m o r e l i g h t o n t h e o r i g i n a n d e v ol u t i o n a r y h i s t o r y o f o u r s p e c ie s . I n t h i s p a p e r , w eh a v e d e m o n s t r a t e d t h a t c h i m p a n z e e a n d h u m a n a r ef a r m o r e c l o s e l y r e la t e d g e n e t i c a l l y t h a n i s ge n e r al l yb e l i e v e d . A m o l e c u l a r a p p r o a c h c a n b e e x p e c t e dtor e m a i n a n i m p o r t a n t t o o l f o r e l u c id a t i n g th e o r ig ina n d e v o l u ti o n o f m a n k i n d .

Acknowledgments.We are deeply grateful to Professor Alia0C. W ilson for suggesting the possibility o f the recent date ofmamm alian divergence adopted in this paper. Thanks are doeto D rs. Peter Andrew s and M ichael Novacek fo r helpful sug8 stions, to P rofessor Joseph Felsenstein for providing us w ith hiscomputer program for inferring phylogeny and for comm ents,and to Mr. Hajime G om i and Mr. Manabu Takano f or their helpwith the computer programs. We also thank Drs. Hiroshi Mizutani, Haruhiko Noda , Charles E. Oxna rd, Da vid Pilbeam , jacl~T. Stem, Naoyuki Takahata, Phillip V. Tobias, and Adrienne L-Zihlman for helpful comments on an early version of the manttscript, although their opinions var y greatly. This work w as suPported by grants from the M inistry of Education, Scienc e, aridCulture of Japan.

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Primate Human-Ape Div. DNA Hasegawa Et Al J Mol Evol 1985