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J Mol Evol (1992) 35:385---404 J o u r n a l o fM o l e c u l a r E v o l u t i o n Springer-VerlagNew York Inc. 1992

M o l e c u l a r P h y l o g e n i e s o f P l a s t id O r i g in s a n d A l g a l E v o l u t i o nW . M a r t i n , 1 C . C . S o m e r v i l l e , 2 a n d S . L o i s e a u x - d e G o 6 r 2] In s t i t u t fi i r G e n e t i k , T e c h n i s c h e U n i v e r s i tf i t B r a u n s c h w e i g , K o n s t a n t i n - U h d e - S t r . 5 , D - 3 3 00 B r a u n s c h w e i g , G e r m a n y2 S t a t i o n B i o l o g i q u e d e R o s c o f f , C N R S L P 4 6 0 1 , B P 7 4 , F - 2 9 6 8 2 R o s c o f f C e d e x , F r a n c e

S u m m a r y . A n o v e r v i e w o f r e c e n t m o l e c u l ar a n al -y se s r e ga r d ing o r ig in s o f p l a s t i d s i n a lga l l i ne a ge s i sp r e s e n t e d . S i n c e d i f f e r e n t p h y l o g e n e t i c a n a l y s e sc a n y i e ld c on t r a d i c to r y v i e w s o f a lga l p l a s t i d o r i -g i n s, w e h a v e e x a m i n e d t h e e f f e c t o f t w o d i s t a n c em e a s u r e m e n t m e t h o d s a n d t w o d i s t a n ce m a t r ixt r e e -b u i l d i n g m e t h o d s u p o n t o p o l o g i e s fo r t h e r i b u -l o s e - l , 5 - b i s p h o s p h a t e c a r b o x y l a s e / o x y g e n a s e l a rg es u b u n i t n u c l e o t i d e s e q u e n c e d a t a s e t . T h e s e r e s u l t sa r e c o n t r a s t e d t o t h o s e f r o m b o o t s t r a p p a r s i m o n ya n a l y s i s o f n u c l e o t id e s e q u e n c e d a t a s u b s e t s . I t i ss h o w n t h a t t h e p h y l o g e n e t i c i n f o rm a t i o n c o n t a i n e dw i t h i n n u c l e o t i d e s e q u e n c e s f o r t h e c h l o r o p l a s t -e n c o d e d g e n e f o r t h e l a r g e s u b u n i t o f r i b u l o s e -l , 5 -b i s p h o s p h a t e c a r b o x y l a s e / o x y g e n a s e , in t e g ra l top h o t o s y n t h e s i s , i n d i c a t e s a n i n d e p e n d e n t o ri g i n f o rth i s p l a s t id g e ne in d i f f e r e n t p l a n t t a xa . Th i s f i nd ingi s c o n t r a s t e d t o c o n t r a r y r e s u l t s d e r i v e d f r o m 1 6 Sr R N A s e q u e n c e s . P o s s ib l e e x p l a n a t i o n s f o r d is -c r e p a n c i e s o b s e r v e d f o r t h e s e t w o d i f f e r en t m o l e -c u l e s a r e p u t f o rt h . O t h e r m o l e c u l a r s e q u e n c e d a t aw h i c h a d d r e s s q u e s t i o n s o f e a r l y p l a n t e v o l u t i o na nd the e uba c t e r i a l o r ig in s o f a lga l o r ga ne l l e s a r ed i s c u s s e d .Key words: E n d o s y m b i o s i s - - M o l e c u l a r p h y lo g -e n y - - A l g a l e v o l u t i o n - - P l a s t id o r i g in sIntroductionE u k a r y o t e s a r e c h i m e r as . O n e c a n s t u d y t h e m o l e c -u l a r e vo lu t ion o f nuc l e i a nd o r ga ne l l e s a s d i s t i nc te n t i t i e s , w h i c h t h e m s e l v e s s h a r e a c o m m o n a n c e s -

O f f p r i n t r e q u e s t s t o : W . M a r t i n

t o r . I m p l i c i t i n th i s a s se r t i on i s t he be l i e f t ha t e ndo -s y m b i o s i s h a s p l a y e d a m a j o r r o l e i n e v o l u t i o n .C h r o m o p h y t e a l g a e m a y b e c h i m e r a s w i t h i n c h i m e -r a s . C h r o m o p h y t e s a r e c h a r a c t e r i z e d b y t h e p r e s -e nc e o f f l a ge ll a , c h lo r op hy l l a a nd c h lo r oph y l l c, t hea b s e n c e o f c h l o r o p h y l l b, a n d t h e p r e s e n c e o f t h r e eo r f o u r m e m b r a n e s s u r r o u n d i n g t h e i r c h l o r o p l a s t s( C h r i s t e n s e n 1 9 8 9) . T h e n u m b e r a n d c e l lu l a r c o n -t i nu u m o f m e m b r a n e s s u r r o u nd i n g c h r o m o p h y t ep la s t i d s ha ve a t t r a c t e d pa r t i c u l a r a t t e n t ion , s i nc et h e s e a n d o t h e r c y t o l o g i c a l c h a r a c t e r s h a v e b e e ni n t e r p r e t e d a s e v i d e n c e o f s e c o n d a r y e n d o s y m b i o -s is ( T o m a s a n d C o x 1 97 3) b e t w e e n e u k a r y o t i c h o s t sa n d e u k a r y o t i c e n d o s y m b i o n t s i n c h r o m o p h y t ee vo lu t ion ( G ibbs 1978 , 1981 ; W ha t l e y 1981 , 1989;C a va l i e r - S m i th 198 6 ; s e e G ibbs 1990 , S i t t e 1990 ,a n d M e l k o n i a n 1 99 1 f o r r e c e n t r e v i e w s ) . S t r o n g m o -l e c u l ar e v i d e n c e i n s u p p o r t o f c h r o m o p h y t e s e c o n d -a r y e n d o s y m b i o s i s w a s r e c e n t l y b r o u g h t f o r t h( D oug la s e t a l. 1991 ) t h r ou gh a na ly s i s o f D N A se -q u e n c e s f r o m t h e n u c l e o m o r p h , p u t a t i v e l y a v e s t i -g i al n u c l e u s o f t h e e u k a r y o t i c e n d o s y m b i o n t , f r o mt h e c r y p t o p h y t e Cryptomonas ~.

T h e i d e a t h a t p l as t id s m a y b e e n d o s y m b i o t i c d e -s c e n d a n t s o f o n c e f r e e - li v i n g o r g a n i s m s i s n o t p a r -t i c u l a r ly ne w . S c h im pe r ( 1883 ) i s ge ne r a l ly g ive nc r e d i t f o r th e i d e a , b a s e d u p o n o b s e r v a t i o n s c o n -c e r n ing p l a s t id d iv i s ion , t ha t p l a n t c e l l s a r e de r ive df r o m t h e s y m b i o t i c u n i f ic a t io n o f a c o l o r l es s o r g a n -i s m w i t h g r e e n , a u t o n o m o u s p l a s t i d p r o g e n i t o r s :

S o l l t e e s s i c h de f in i t i v be s t i i t i ge n , da s s d i eP l a s t i d e n i n d e n E i z e l l e n n i c h t n e u g e b i l d e tw e r d e n , s o w f i r d e i h r e B e z i e h u n g z u d e m s iee n t h a l t e n d e n O r g a n i s m u s e i n i g e r m a s s e n a ne i n e S y m b i o s e e r i n n e r n . M 6 g l i c h e r w e i s e v e r -d a n k e n d i e g r ~ i n e n P f l a n z e n w i r k l i c h e i n e r

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3 8 6V e r e i n i g u n g e i n e s f a r b l o s e n O r g a n i s m u s m i te ine m von C h lo r ophy l l g l ei c hm ~ is s ig t i ng i e r t e ni h r e n U r s p r u n g . 1

M e r e s c h k o w s k y ( 1 90 5) e x p l i ci t ly s u g g e s t e d t h a t c y -a no ba c t e r i a ga ve r i s e t o p l a s t i d s , 2 a nd tha t d i f f e r e n tc y a n o b a c t e r i a g a v e r i s e t o t h e p l a s t id s o f d i f fe r e n tp l a n t g r o u p s . I n a s e r i es o f s u b s e q u e n t p u b l i c a ti o n s( 1 9 1 0a - d ) h e e s t a b l i s h e d t h e f r a m e w o r k f o r a g e n -e r a l p h y l o g e n y o f o r g a n i s m s i n w h i c h ( 1) w h a t w en o w t e r m p r o k a r y o t e s a r o s e b e f o r e ( a l be i t i n d e p e n -d e n t l y o f) w h a t w e n o w t e r m e u k a r y o t e s a n d ( 2 ) t h ep l a s ti d s o f c h l o r o p h y t e s , r h o d o p h y t e s , a n d c h r o -m o p h y t e s o r i g i n a t e d t h r o u g h i n d e p e n d e n t e n d o -s y m b i o t i c e v e n t s . T h e g e n e r a l s c h e m e o f e u k a r y o t i ce v o l u t i o n p r o p o s e d b y M e r s c h k o w s k y (1 91 0d ) w a sl a r g e l y b a s e d u p o n b i o c h e m i c a l , h e r e d i t a r y , a n dm e t a b o l i c c o n s i d e r a t i o n s a n d i s r e p r o d u c e d i n F ig .1 ; t he r e a de r m a y r e f e r t o t he o r ig ina l l i t e r a tu r e f o ra n e l e g a n t e l a b o r a t i o n a n d j u s t i f i c a t io n o f t h e a r g u-m e n t s u p o n w h i c h t h e f i g u r e w a s b a s e d .A n u m b e r o f m o l e c u l a r e v o l u t i o n a r y c o n t r i b u -t i on s h a v e a p p e a r e d r e c e n t l y i n t h e l it e r a t u r e w h i c hr e l a te t o q u e s t i o n s c l e a r l y f o r m u l a t e d b y M e -r e s c h k o w s k y ; t h e s e c o n c e r n t h e e n d o s y m b i o t i c o r -ig in s o f va r ious a lga l p l a s t id s , t he e v o lu t ion o f t hes y m b i o n t s p r i o r t o s y m b i o s i s , a n d t h e o r i g i n s a n de a r l y e v o l u t i o n o f e u k a r y o t e s . S o m e o f t h e r e s u lt sh a v e b e e n i n t e r p r e t e d a s m o l e c u l a r e v i d e n c e f o rm u l t i p l e ( po lyp hy le t i c ) o r ig in s o f p l a s t i d s , t h i s i nl ig h t o f v e h e m e n t a r g u m e n t s f a v o r i n g m o n o p h y l e s i sb o t h f o r p l a s t i d s a n d e u k a r y o t e s ( C a v a l i e r - S m i t h1987b). I n t h i s a r t i c l e , m o le c u la r da t a r e ga r d ing thei n d e p e n d e n t p h y l o g e n i e s o f p l a s ti d a n d n u c l e a rs y m b i o t i c p a r t n e r s b e f o r e e n d o s y m b i o s i s o n t h eo n e h a n d , a n d s u b s e q u e n t t o t h e e s t a b l i s h m e n t o fp l a s ti d s o n t h e o t h e r , w i l l b e c o n s i d e r e d . S i n c e m o -l e c u l a r e v o l u t i o n a r y r e l a t i o n s h i p s b e t w e e n o r g a n -i s m s o r o r g a n e l l e s a r e i n f e r r e d t h r o u g h c a l c u la t i o n ,r a the r t ha n th r ough obse r va t ion ( a s i n F ig . 1 ) , va r -i o u s p a r a m e t e r s i n v o l v e d i n r e c o n s t r u c t i o n o f p h y -l o g e n e t i c t r e e s f r o m m o l e c u l a r s e q u e n c e d a t a i n f lu -e n c e t h e t o p o l o g y o r t o p o l o g i e s o b t a i n e d . T h i s i nt u r n c o l o r s c o n s i d e r a b l y t h e interpretation o f a p h y -

i " I f i t c a n b e c o n c l u s i v e l y c o n f i r m e d t h a t p l a s ti d s d o n o t a r i s ed e n o v o i n e g g c e l l s , t h e r e l a t i o n s h i p b e t w e e n p l a s t i d s a n d t h eo r g a n i s m s w i t h i n w h i c h t h e y a r e c o n t a i n e d w o u l d t h e n b e s o m e -w h a t r e m i n i s c e n t o f a s y m b i o s i s. G r e e n p l a n t s m a y i n fa c t o w ethe i r o r ig in to t he un i f i ca t ion o f a co lo r l es s o r gan i s m wi th oneun i f o r m ly t i nged wi th ch lo r o phy l l " Sc h im per ( 1883) pp . 112-113 .T r a n s l a t io n b y t h e a u t h o r s .2 " A t h e o r y [ o f e n d o s y m b i o s i s ] , a s s u g g e s t e d h e r e , w o u l d g a i ns t r eng th i f t he ex i s t ence o f any f r ee - l i v ing o r gan i s m s s imi l a r t ot h e s y m b i o n t c o u l d b e p r o v e n . L o w e r f o r m s o f th e C y a n o -p h y c e a e c a n b e c o n s i d e r e d a s s u c h o r g a n i s m s " M e r s c h k o w s k y( 1905) p . 599 . T r ans l a t ion f r om the o r ig ina l Ger man t ex t by theau thor s .

l o g e n e t ic d a t a s e t a n d t h e r e b y o n e ' s v i e w o f e v o l u -t i o n a r y p r o c e s s . T h e r e f o r e , a d a t a s e t p a r t i c u l a r top l a st i d e v o l u t i o n , i . e ., n u c l e o t i d e s e q u e n c e s f o r t h el a rg e s u b u n i t o f R u b i s c o (rbcL), w i l l f i r s t be c on -s i d e r e d in o r d e r t o c o n t r a s t t h e t o p o l o g i e s g e n e r a t e db y t w o d i s ta n c e m e a s u r e m e n t m e t h o d s a n d t w o d i s -t a n c e m a t r i x m e t h o d s f o r t r e e c o n s t r u c t i o n . T h et r u e p h y l o g e n y o f t h e o r g a n i s m s u n d e r s t u d y i s n o tk n o w n . F o r c o m p a r i s o n , h o w e v e r , o n e c a n c o m -p a r e t h e p h y l o g e n e t i c t r e e s o b t a i n e d t o b r a n c h i n go r d e r s e s t i m a t e d t h r o u g h p a r s i m o n y b o o t s t r a p a n dja c kk n i f e ( F e l se ns t e in 1985 ) a na ly se s o f rbcL n u c l e -o t i d e s e q u e n c e s . T h e s t r e n g t h o f m o l e c u l a r d a t ac o n c e r n i n g p o l y p h y l y v s m o n o p h y l y fo r p la s t id o r -ig in s w i ll be a d d r e s se d . S om e o f t he c on f l i c t i ng r e -s u it s w h i c h h a v e b e e n o b t a i n e d w i t h d i f f e r e n t g e n e sa s m o l e c u l a r m a r k e r s a n d , i n s o m e c a s e s , p o t e n t i a lc a use s f o r t he d i f f e r ing r e su l t s w i l l be d i s c us se d .

M a t e r i a l a n d M e t h o d s

Compari son o f Two Dis tance Measures . A m i n o a c i d s e q u e n c e sw e r e f i rs t al i g n e d b y e y e w i t h t h e L I N E U P p r o g r a m o f th e W I S -G E N p a c k a g e ( D e v e r e u x e t a l. 1 9 8 4) . C o r r e s p o n d i n g c o d o n i n -s e r t i o n s w e r e i n t r o d u c e d i n t o t h e n u c l e o t i d e s e q u e n c e s ; a m i n o -a n d c a r b o x y t e r m i n a l r e g i o n s o f l e n g t h h e t e r o g e n e i t y w e r e d e -l e t ed f r om the a l ignment y i e ld ing 1413 nuc leo t ide pos i t i ons ( 471c o d o n s ) i n e a c h s e q u e n c e f o r c o m p a r i s o n . D i v e r g e n c e a t n o n -s y n o n y m o u s s i te s b e t w e e n a l i g n e d n u c l e o t i d e s e q u e n c e s ( a li g n-m e n t a v a i l a b l e u p o n r e q u e s t f r o m t h e a u t h o r s) w a s m e a s u r e d a sKa wi th the weigh ted pa thway meth od ( L i e t a l. 1985) and as dNw i t h t h e m e t h o d o f N e i a n d G o j o b o r i ( 19 8 6) . A n a v e r a g e rbcLp a i r h a s a b o u t 1 0 6 0 n o n s y n o n y m o u s s i te s . V a l u e s o f K a a n d d uw e r e p l o t t e d a g a i n s t e a c h o t h e r f o r e a c h p a i r w i s e c o m p a r i s o n(Fig. 2) .

Construction o f Trees. M a t r i c e s o f d i s ta n c e v a l u e s w e r e a n -a l y z e d w i t h t h e N e i g h b o r - J o in i n g ( N J : S a i t o u a n d N e i 1 9 87 ) o rw i t h t h e F i t c h - M a r g o l i a sh l e a s t - sq u a r e s ( F M : F i t c h a n d M a r g o -l i a sh 1 9 6 7 ) m e t h o d ( F I T C H o f t h e P H Y L I P p a c k a g e ( F e l s e n s t e i n1 9 8 1 ) ) . T h e N J m e t h o d w a s c h o s e n s i n c e i t h a s b e e n s h o w n i nc o m p u t e r s i m u l a t i o n to b e e q u a l l y o r m o r e e f f i c i e n t t h a n o t h e rm a t r i x o r m o l e c u l a r s e q u e n c e m e t h o d s t e s t e d i n r e c o v e r y o f c o r -r e c t t o p o l o g y u n d e r a v a r i e t y o f se q u e n c e p a r a m e t e r s ( S a i to u a n dI mani s h i 1987 ; Sour d i s and K r imbas 1987 ; Sa i tou and N ei 1987 ;J i n a n d N e i 1 9 9 0 ) . T h e F M m e t h o d w a s c h o s e n f o r c o m p a r i s o ns i n c e i t i s w i d e l y u se d . F I T C H w a s r u n w i t h t h e " j u m b l e " a n d" g l o b a l " o p t i o n s . R e f e r e n c e s f o r s e q u e n c e s a r e Alca l igenes( A n d e r s e n a n d C a t o n 1 9 8 7 ) ; Anabaena ( C u r t i s a n d H a s e l k o r n1983); Chlamydomonas (Dron e t al . 1982) ; Eug lena ( Gingr i ch andHal l i ck 1985) ; Z e a ( M cI n tos h e t a l . 1980) ; Nico t iana ( S h i n o z a k iand Sug iu r a 1982) ; Chromat ium ( Via l e e t a l . 1989) ; Chlorella( Yos h inaga e t a l . 1988); Prochlorthix ( M o r d e n a n d G o l d e n 1 9 91 );Rhodobac te r sphaero ides ( Gibs on e t a l . 1991) ; t he unnamed3 ,- pur p le e ubac te r ium ( S te in e t a l . 1990) ; Cryp tomonas ( Doug lase t a l . 1990) ; Cylindrotheca ( Hwang and T ab i t a 1991) ; Pylaie l la(Assal i et al . 1991a) ; Cyanidium ( Valen t in and Z e t s che 1990b) ;Cyanophora ( V a l e n ti n a n d Z e t s c h e 1990c);Ectocarpus ( Valen t inand Z e t s che 1990a) ; Ant i thamnion ( Kos t r zewa e t a l . 1990) ; A s -tasia ( S i e m e i s t e r a n d H a c h t e l 1 9 9 0 ) ; Cryp tomonas ( Doug las e tal . 1 990) ; and Porphyridium ( V a l e n t in a n d Z e t s c h e 1 9 8 9 ). T h e

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F i g. 1 . P h y l o g e n e t i c t r e e c o n s t r u c t e d a n d p r e s e n t e d b y M e -r e s c h k o w s k y ( 1 9 1 0 d ) , s u m m a r i z i n g h i s v i e w s a t t h e t i m e c o n -c e r n i n g c e l lu l a r e v o l u t i o n . I n h i s s c h e m e , p r o k a r y o t e s (dasMykop lasma: Biococcus ) , t h e n k n o w n t o t o l e r a t e e x t r e m e c o n -d i t i o n s, w e r e s u p p o s e d t o h a v e a r i s e n v e r y e a r l y i n e v o l u t io n , a ta t i m e w h e n t h e E a r t h ' s s u r f a c e h a d c o o l e d s u f f i c i e n t l y t o r e t a i nw a t e r ( < 1 00 C) , y e t w a s t o o w a r m ( > 5 0 C ) t o s u p p o r t l if e o f t h em o r e f r a gi l e e u k a r y o t e s (das Amrbop lasma: Monera ) . T h e l a t t e ra r o s e i n d e p e n d e n t l y o f t h e fo r m e r a t a t i m e w h e n t h e E a r t h ' ss u r f ac e h a d c o o l e d t o < 5 0 C ; t h e s e l a t e r w e r e s u g g e s t e d t o h a v e

o b t a i n e d t h e i r n u c l e i ( n o t m i t o c h o n d r i a ) t h r o u g h a f i r s t s e r i e s o fe n d o s y m b i o s e s . I n a s e c o n d s e r i e s o f e n d o s y m b i o s e s , f l a g e ll a t e sw e r e p r o p o s e d t o h a v e o b t a i n e d p l a s ti d s t h r o u g h e n d o s y m b i o s i sw i t h s u c h d i v e r s e c y a n o b a c t e r i a a s c o n t a i n e d e i t h e r g r ee n , r e d ,o r b r o w n p i g m e n t s . P h y c o m y c e t e s ( i n c l u d e d o o m y c e t e s ) w e r ed e p i c t e d a s a p o l y p h y l e t i c g r o u p , d i s t i n c t f r o m o t h e r f u n g i ,w h i c h e v o l v e d t h r o u g h s e c o n d a r y l o s s o f p h o t o s y n t h e s i s . T h ea r g u m e n t s p u t f o r t h u p o n w h i c h t h e d i a g r a m i s b a s e d r e l a t e t om e t a b o l i c , b i o c h e m i c a l , p i g m e n t a t i o n , a n d g r o w t h e n v i r o n m e n tc h a r a c t e r s .

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38 8

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0.4 0 . 3 / ,0 .2 . oo~ ,,, 0 . 1 .

0 - , - , - , - ,0 0 . 1 0 .2 0 . 3 0 . 4 0 . 5Ka

F i g. 2 . C o m p a r i s o n o f e s t i m a t e s f o r d i v e r g e n c e a t n o n s y n o n y -m o u s s i te s b e t w e e n t h e 2 0 rbcL s e q u e n c e s f r o m s p e c i e s s h o w n i nF i g . 3 a s v a l u e s o f d R ( N e i a n d G o j o b o r i 1 9 86 ) a n d K a ( L i e t a l .1 98 5) f o r d i v e rg e n c e a t n o n s y n o n y m o u s s i te s . E a c h c o m p a r i s o no f d N v s K a i s r e p r e s e n t e d a s a s i n g l e d o t i n t h e g r a p h . T h e f i v eo p e n c i r c l e s r e p r e s e n t t h e c o m p a r i s o n s b e t w e e n t h e As tas iarbcL s e q u e n c e a n d t h o s e o f c h l o r o p h y t e p l a s t i ds . ( S e e t e x t a n dF i g . 3 . ) T h e d i a g o n a l i s n o t a r e g r e s s i o n b u t r a t h e r s h o w s t h es l o p e e x p e c t e d f o r e q u a l e s t i m a t e s o f d i v e r g e n c e w i t h t h e t w om e t h o d s . S t a n d a r d e r r o r s f o r t y p i c a l m e a n e s t i m a t e s o f K a a n dd N v a r y w i t h c o m p a r i s o n a n d d e g r e e o f d i v e r g e n c e ; f o r t h e r a n g eo f d i v e r g e n c e 0 . 1 - 0 .4 i n rbcL s e q u e n c e s , t h e s t a n d a r d e r r o r s a rea p p r o x i m a t e l y 1 0 - 1 5 % o f th e m e a n , a s s h o w n b y e r r o r b a r s f o rt h r e e t y p i c a l i n d i v i d u a l c o m p a r i s o n s .

o u t g r o u p u s e d i s t h e f o r m I 1 rbcL o f Rhodospirillum rubrurn(Na ra ng e t a l . 1984) ; the Rh o b a c t e r s e q u e n c e a n a l y z e d i s o f t h ef o r m I t y p e ( G i b s o n e t a l . 1 9 9 1 ). Rhodospirillum a l s o c o n t a i n s af o r m I t y p e R u b i s c o ( F a l c o n e a n d T a b i t a 1 9 9 1 ) . T h e Rhodospiril-lu m o u t g r o u p s e q u e n c e i s q u i t e d i f f i cu l t t o a l i g n w i t h t h e r e m a i n -in g rbcL s e q u e n c e s t h r o u g h o u t m o s t o f i t s l e n g t h ; K a a n d d uv a l u e s r a n g e d f r o m 2 . 5 t o 3 . 5 i n m o s t c o m p a r i s o n s . I n o r d e r t or e d u c e s t o c h a s t i c e r r o r i n th e d i s t a n c e m a t r i x t r ee s d u e t o t h el a rg e s t a n d a r d e r r o r a t t a c h e d t o e s t i m a t e s o f d i v e r g e n c e i n t h eRhodospir i l lum c o m p a r i s o n s , t h e Rhodospirillum o u t g r o u p d i s -t a n c e w a s s e t t o 2 . 0 f o r a l l 2 0 p a i r w i s e c o m p a r i s o n s . T h i s p r o -c e d u r e p r o d u c e d a " s y n t h e t i c " r o o t a n d y i e l d e d N J t o p o l o g i e sf o r t h e r e m a i n i n g 2 0 s p e c i e s w h i c h w e r e i d e n t i c a l to t h o s ep r o d u c e d i n a b s e n c e o f t h e o u t g r o u p . T h e r b c L n u c l e o t i d es e q u e n c e s w e r e a n a l y z e d b y b o o t s t r a p p a r s i m o n y a n a l y s i s( D N A B O O T o f P H Y L I P , F e l s e n s t e i n 1 98 5) . V a r i o u s s u b s e t s o ft h e n u c l e o t i d e s e q u e n c e s w e r e s t u d i e d . ( S e e T a b l e 2 . ) B o o t s t r a pr e p l i c a t e s f o r t h e Rhodospir i l lum o u t g r o u p s e q u e n c e w e r e o n l yp e r f o r m e d w i t h s e c o n d p o s i t i o n s .

Results and D iscussion

A n O v e r v ie w o f t h e D a t a a n d a C o n s i d e ra t io n o fr b c L S e q u e n c e sM o l e c u l a r p h y l o g e n ie s w h i c h c o n c e r n e n d o s y m b i o -s is a n d t h e g e n e r a l r e l a t i o n s h i p o f v a r i o u s p l a n tg r o u p s t o o t h e r o r g a n i s m s a r e s u m m a r i z e d i n T a b l e1 i n o r d e r t o p r o v i d e b o t h a g e n e r a l ( y e t n o t e x h a u s -t i v e) o v e r v i e w o f th e s c o p e o f c u r r e n t i n v e s t i g a t io na n d c o n v e n i e n t r e f e r e n c e t o s t u d i e s e n c o m p a s s i n gt a x a o f i n t e r e s t. V a r i o u s m o l e c u l a r m a r k e r s a n dm e t h o d s o f p h y l o g e n e t i c c o n s t r u c ti o n h a v e b e e n

e m p l o y e d . R i b o s o m a l R N A s e q u e n c e s a r e k n o w nf r o m a l ar g e n u m b e r o f o r g a n i s m s a n d h a v e t h u sb e e n u s e d t o e s t a b l i s h a f f i n i t i e s a m o n g e n d o s y m -b i o n t s m u c h m o r e e x t e n s i v e l y t h a n h a v e p r o t e i n s e -q u e n c e s , t h o u g h t h i s t r e n d i s n o t a l t o g e t h e r a d e -q u a t e l y r e f l e c te d i n t h e t a b le . M o s t p l a s t i d - e n c o d e dp r o t e i n s e m p l o y e d t o d a t e f o r e v o l u t i o n a r y s t u d i e sa r e i n v o l v e d i n p h o t o s y n t h e s i s [ e . g ., t h e l a rg e(rbcL) , a n d i n s o m e c a s e s , t h e s m a l l (rbcS) s u b u n i to f r i b u l o s e - l , 5 - b i s p h o s p h a t e c a r b o x y l a s e / o x y -g e n a s e ( R u b i s c o ) , p s b A , e t c . ] . P h o t o s y n t h e t i c a p -p a r a tu s g e n e s o b v i o u s l y c a n n o t b e e m p l o y e d , a s t h e1 6 S a n d 1 8 S r R N A s w i t h s o m e li m i t a t io n s c a n , t oco n co m i t a n t l y i n v e s t i g a t e n u c l e a r a n d o r g a n e l l a rp h y l o g e n i e s . E l o n g a t i o n f a c t o r T u , t h e p r o d u c t o ft u f A , h o w e v e r , c a n b e u s e d t o c o n n e c t n u c l e a r ,p l a s t i d , a n d m i t o c h o n d r i a l p h y l o g e n i e s ( P a l m e r e ta l . 1 98 9 ). P l a st i d g e n o m e s i n c h l o r o p h y t e s a p p e a r t oe v o l v e q u i t e c o n s e r v a t i v e l y w i t h l i t t l e o r n o t e n -d e n c y t o a c c e p t D N A f r o m o t h e r c o m p a r t m e n t s( P a l m e r e t a l . 1 98 8 ), a s s e q u e n c e c o m p a r i s o n o ft h r e e c o m p l e t e c h l o r o p l a s t g e n o m e s h a s s h o w n( O h y a m a e t a l . 1 9 8 6 ; S h i n o z a k i e t a l . 1 9 8 6 ; H i r a t -s u k a e t a l . 1 9 8 9 ) . C h l o r o p l a s t g e n e s w e r e t h u s v e r yp r o b a b l y a g e n o m e c o m p o n e n t a t t h e t im e o f en d o -s ym b i o s i s , m a k i n g t h e s e g e n e s p r e s u m a b l y q u i t e r e -l ia b l e m a r k e r s f o r r e c o n s t r u c t i o n o f o r g a n e ll a r p h y -l o g e n y . A s w i l l b e d i s c u s s e d l a t e r , i t i s n o t k n o w nw h e t h e r t h i s g e n e r a l i z a t i o n a l s o a p p l i e s t o p l a s t i dg e n o m e s o f r h o d o p h y t e s a n d c h r o m o p h y t e s , w h e r em o l e c u l a r d a t a a r e s t i l l s c a r c e .

P l a n t n u c l e a r g e n o m e s a p p e a r t o h a v e a c q u i r e d an u m b e r o f p r o t ei n - co d i n g D N A s e q u e n c e s f r o m e n -d o s y m b i o n t s t h r o u g h i n t ra c e l lu l a r g e n e t r a n s f e r(Br in km ann e t a l . 1987; C legg e t a l . 1991; Ba lda ufa n d P a l m e r 1 9 9 0 ; L i a u d e t a l . 1 9 9 0 ) . T h o u g h n u -c l e a r , s u c h g e n e s n e c e s s a r i l y r e f l e c t t h e e v o l u t i o no f p l a st i d s o r , i n s o m e c a s e s , p e r h a p s m i t o c h o n d r i a( M a r t i n e t a l. 1 9 90 ), r a t h e r t h a n t h a t o f t h e e u k a r y -o t i c h o s t . T o d a te , 1 8S r R N A s h a v e b e e n m o r ew i d e l y u s e d t h a n o t h e r m a r k e r s t o s t u d y e v o l u t i o n -a r y re l a ti o n s h i p s b e t w e e n e u k a r y o t i c n u c l e i, a st h e s e g e n e s a r e e x p e r i m e n t a l l y q u i t e a c c e s s i b l e a n dw e r e , i n a ll l i k el i h o o d , c o m p o n e n t s o f th e e u k a r y -o t i c h o s t c e l l g e n o m e b e f o r e e n d o s y m b i o s i s . R i b o -s o m a l R N A p h y l o g e n i e s a re n o t , h o w e v e r , a l w a y ss u p p o r t e d b y a n a l y s e s o f p r o t e in c o d i n g r e g i o n s ( c f.L o o m i s a n d S m i t h 1 99 0) .

D i s c r e p a n c i e s B e t w e e n P u b l i s h e d T r e e sT h e r e a r e discrepancies b e t w e e n t h e t r e e s l i s t e d i nT a b l e 1 t o o v a s t i n n u m b e r t o b e c o n s i d e r e d i n a n yd e t ai l. Y e t a s a c a s e i n p o i n t , o n e m a y c o n s i d e r t h et h r e e p o s s i b l e r o o t e d b r a n c h i n g o r d e r s f o r l a n d

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p l a n t s, m e t a z o a , a n d y e a s t ; e a c h o f t h e s e c a n b ef o u n d m o r e t h a n o n c e a m o n g t h e 1 1 c o n t r i b u t i o n sl i st e d in T a b l e 1 w h i c h e x a m i n e d r e p r e s e n t a t i v e sf r o m a l l t h r e e g r o u p s :

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A B C

O b v i o u s l y , a l l t h r e e c a n n o t b e s i m u l t a n e o u s l y c o r -r e c t . A a n d B a r e f o u n d f o u r ti m e s e a c h a n d C isf o u n d t h r e e t i m e s . T h a t d i f f e r e n t a u t h o r s a r r i v e a td i f f e r e n t m o l e c u l a r p h y l o g e n i e s f o r t h e s a m e t a x aa n d t h e s a m e g e n e i s d i s c o n c e r t in g , t h o u g h n o t a t a lls u r p ri s in g . V a r i o u s p a r a m e t e r s c a n a f f e c t a g i v e nt r e e- b u i ld i n g m e t h o d ' s r e s u l t ; t h e s e h a v e b e e n c h a r -a c t e r i z e d t o s o m e e x t e n t i n th e l i te r a t u re . T h ea m o u n t o f d a ta u p o n w h i c h a g i v en m o l e c u la r p h y -l o g e n y is b a s e d , t h e a l i g n m e n t g e n e r a t e d , t h ea m o u n t o f d iv e r g e n c e b e t w e e n t h e s e q u e n c e s , d if -f e r e n c e s i n s u b s t i t u t io n r a t e s b e t w e e n d i f f er e n t li n-e a g e s , t h e t r u e p h y l o g e n y o f t a x a a n a l y z e d , t r e e -b u i ld i n g m e t h o d s t h e m s e l v e s , a n d o t h e r f a c to r sm a y e x e r t a n i n f lu e n c e u p o n w h i c h t o p o l o g y o r to -p o l o g i e s a re o b t a i n e d ( F e l s e n s t e i n 1 98 8; P e n n y a n dH e n d y 1 9 8 6 ; S a i t o u a n d I m a n i s h i 1 9 8 9 ; S o u r d i s a n dK r i m b a s 1 98 7; G o u y a n d L i 1 9 8 9b ; S w o f f o r d a n dOlsen 1990; J in and Nei 1990; Ne i 1991) . As fa r a st h e s p e c i fi c e x a m p l e o f l a n d p l a n ts , m e t a z o a , a n dy e a s t i s c o n c e r n e d , i t c a n b e n o t e d t h a t G o u y a n d L i( 1 98 9 a ) c a r e f u l l y a n a l y z e d s e q u e n c e s f o r 1 8Sr R N A , 1 6S r R N A , t e n t R N A f a m i li e s , a n d s i x c o n -s e r v a t i v e l y e v o l v i n g p r o t e i n s a n d r e p o r t e d c o n c l u -s i v e e v i d e n c e s u p p o r t i n g y e a s t ( y e t n o t b y c o r o l l a r ya l l f u n g i , s e e l a t e r ) a s t h e o u t g r o u p f o r h i g h e r p l a n t sa n d m e t a z o a ( a l t e r n a t i v e A s h o w n a b o v e ) , a t o p o l -o g y d e p i c t e d o n l y f o u r t i m e s i n t h e 1 1 a f o r e m e n -t i o n e d t r e e s . I n l ig h t o f t h i s , o n e c o u l d q u e s t i o n t h ea m o u n t o f c o n f id e n c e w a r r a n t e d b y t h e p h y l o g e n i esf o r g e n e s o f a l g a l n u c l e i a n d p l a s t i d s d e p i c t e d i n t h ev a r i o u s s t u d i e s o f T a b l e 1 .

38 9c a s e w i t h r e a l d a ta , t h e t r u e p h y l o g e n y o f t h e o r -g a n i s m s f r o m w h i c h t h e 1 9 rbcL n u c l e o t i d e s e -q u e n c e s a r e d e r i v e d ( T a b l e 1 ) i s n o t k n o w n , o n et h e r e f o r e c a n n o t c o m p a r e t h e e f f i c i e n c y o f d i f f e re n tm e t h o d s i n o b t a i n i n g t h e c o r r e c t t r e e , a s i n c o m -p u t e r s i m u l a t i o n s ( c f . J i n a n d N e i 1 9 9 0) , b u t o n e c a nc o m p a r e t h e r e s u l t s o b t a i n e d w i t h d i f f e r e n t d is t a n c em a t r i x m e t h o d s i n t h e ir a b i l it y to r e c o v e r t o p o l o g i e ss u p p o r t e d b y b o o t s t r a p p a r s i m o n y a n a l y si s o f t h erbcL s e q u e n c e d a t a . B o o t s t r a p ( B S ) a n d h a l f - d e le t ej a c k k n i f e ( J K ) a n a l y s e s ( D N A B O O T o f t h eP H Y L I P p a c k a g e ; F e l s e n s t e i n 1 9 81 , 19 85 ) w e r et h e r e f o r e p e r f o r m e d w i t h s e v e r a l s u b s e t s o f th erbcL n u c l e o t i d e d a t a f o r 1 9 a n d o n e s u b s e t f o r 2 0( i n c lu d i n g t h e Rhodospirillum o u t g r o u p ) s p e c i e s r e l-e v a n t t o q u e s t i o n s o f p l a n t e v o l u t i o n . S i n c e p a r s i-m o n y a n a l y s i s o p e r a t e s u n d e r t h e a s s u m p t i o n t h a tt h e p r o b a b i l i t y o f a b a s e s u b s t i t u t i o n a t a g i v e n s i t ei s s m a l l o v e r t h e l e n g t h s o f ti m e i n v o l v e d i n ab r a n c h , s y n o n y m o u s s i te s s h o u ld b e s t b e r e m o v e df r o m t h e rbcL d a t a s e t , s i n c e t h e y d o n o t f u l fi ll th i sc o n d it io n . Y e t s e l e c ti v e ly r e m o v i n g s y n o n y m o u ss i t e s i s t e d i o u s , s i n c e t h e n u m b e r a n d l o c a t i o n o fs y n o n y m o u s s i te s is a p r o p e r t y o f co d o n s . O n e c a na p p r o x i m a te t h e r e m o v a l o f s y n o n y m o u s s i t es b yd e l e t i n g t h i r d p o s i t i o n s f r o m t h e s e q u e n c e a l i g n -m e n t ( i . e . , w i t h a s y s t e m e d i t o r ) . T h i s e l i m i n a t e s a l lf o u r f o l d d e g e n e r a t e s i t e s a n d m o s t t w o f o l d d e g e n -e r a t e s i t e s , 3 t h u s ( 1) d e c r e a s i n g s t o c h a s t i c s i m i l a r i ty( " b a c k g r o u n d n o i s e " ) o f s e q u e n c e s d u e t o t h e e x -i s te n c e o f o n ly f o u r p o s s i b l e c h a r a c t e r s t a t e s f o r a n ys i te a n d ( 2) lo w e r i n g c o n s i d e r a b l y t h e p r o b a b i l i ty o fb a s e s u b s t i t u t i o n i n a g i v e n b r a n c h . I t s h o u l d b en o t e d t h a t t h e n u c l e o t i d e s e q u e n c e a l i g n m e n t f o rt h e 2 0 g e n e s c o n s i s t in g o f fi r st p l u s s e c o n d p o s i t i o n s( e a c h 9 4 2 b a s e s l o n g ) c o n t a i n s a l m o s t a l l o f th e n o n -d e g e n e r a t e s i t e s ( a v g . 9 1 0 s i t e s / p a i r ) a n d t h u s a l -m o s t c o m p l e t e l y c o n t a i n s y e t i s n o t i d e n t i ca l t o t h es u b s e t o f n o n s y n o n y m o u s s i te s ( av g . 1 06 0 s i te s /p a ir ) f r o m w h i c h t h e t w o d i s ta n c e m e a s u r e m e n t s [ a sn u m b e r s o f n o n s y n o n y m o u s s u b s ti tu t io n s p e r s it eK a ( L i e t a l. 1 9 85 ) a n d d N ( N e i a n d G o j o b o r i 1 9 86 )]w e r e c a l c u l a t e d . T h i s c i r c u m s t a n c e s h o u l d n o t i n -t r o d u c e s e r i o u s e r r o r i n t o t h e a n a l y s i s . 4

S e c o n d c o d o n p o s i ti o n s c o n s i s t o n l y o f n o n d e -g e n e r a t e s i t e s a n d e v o l v e m o r e s l o w l y t h a n e i t h e r o f

Bootstrap and Jackknife Analysis of r b c L DataA n u m b e r o f m e t h o d s f o r a s s e s s i n g t h e r e li a b il it y o fp h y l o g e n e t i c t r e e s d e r i v e d f r o m s e q u e n c e d a t a a r ea v a i l a b l e ( f o r a r e v i e w s e e F e l s e n s t e i n 1 9 8 8 ; s e ea l s o L i 1 98 9 ). O n e o f t h e s e , t h e b o o t s t r a p ( F e l s e n -s t ei n 1 98 5) h a s b e e n e m p l o y e d i n s e v e r a l o f th es t u d i e s l i s t e d i n T a b l e 1 . A s a l m o s t i n v a r i a b l y i s t h e

3 Compa re Li et al. (1985) and Nei and Gojobo ri (1986) for lucidtreatments of codon degeneracy in phylogenetic sequence dis-tance measurement.4 As an alternative, one could measure diver gence at first andsecond positions with the method of Tajima and Nei (1984) andsubject the identical alignment to bootstrap analysis, yet thatdistance measurement will not discriminate between synony-mous and nonsynonymous substitutions.

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390T a b l e 1. C o n d e n s e d o v e r v i e w o f s e v e r a l re c e n t m o l e c u l a r p h y l o g e n i e s r e le v a n t to t h e c o n t e x t o f m o l e c u l a r e v o l u t i o n o f p l a s t id a n dn u c l e a r g e n e s o f a l g ae , w i t h e m p h a s i s o n r h o d o p h y t e s a n d c r y p t o p h y t e s *T a x o n :

r h o d o p h y t e s - 1 1 - - 1 - 1 1c h r o m o p h y t e s - 1 - - 1 2 - 2 3

c r y p t o p h y t e ( n u ) . . . . . . . 1c r y p t o p h y t e ( n m ) . . . . . . . . .

c h l o r o p h y t e s 4 3 1 1n 3 8 - 5 5e u g l e n o i d s 1 1 1 - 1 2 - 1 1c y a n o p h o r a 1 - - - 1 . . . .c y a n o b a c t e r i a 1 7 a 6 a 7 1 6 a 1 6 - -o r - p u r p l e b a c t e r i a t - - - 1 - 1 5 - -[ 3 - p u r p l e b a c t e r i a t - - - 1 - 2 5 - -" y - p u r p l e b a c t e r i a - 1 - 1 - 1 5 - -g - p u r p l e b a c t e r i a . . . . 1 - 5 - -d i n o f l a g e l l a t e s . . . . . . . 1 1k i n e t o p l a s t i d a . . . . . . . 3 1c i l i a t e s . . . . . . . 4 2f u n g i . . . . . . . 3 5

o o m y c e t e s . . . . . . . 1 1a m o e b a . . . . . . . 1 1m e t a z o a . . . . . . . h h

C o m p a r t m e n t :p l a s t i d p l a p l a p l a p l a p l an u c l e u s n u c n u cp r o k a r y o t i c p r o p r o p r o p r o p r o p r o p r o

G e n e / m o l e c u l e : 1 6 S 1 6 S 1 6 S 1 6 S 1 6 S 1 6 S 1 6 S 1 8 S 1 8 SS i t e s a n a l y z e d : 6 6 7 1 1 3 0 S o b 8 1 8 7 0 3 1 2 0 g 1 2 4 0 1 5 3 0T r e e m e t h o d ( s ) : F M b F M c l u s F M b S T F M e S T F M b

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1 8 S 1 8 S 1 8 S 2 8 S1 0 0 1 1 2 6 3 1 5 3 0 2 8 0N J F M b F M b F MB S P P10 11 12 13

* N u m b e r s i n th e c o l u m n s i n d i c a te n u m b e r s o f sp e c i e s a n a l y z e df o r a g i v e n t a x o n i n t h e c o r r e s p o n d i n g r e f e r e n c e n u m b e r e d a tt h e b a s e o f t h e c o l u m n . T h e s e l e c t i o n o f l i t e r a t u r e is n o t e x h a u s -t i v e b u t is in t e n d e d t o p r o v i d e r o u g h o r i e n t a t io n c o n c e r n i n g

1 . T u r n e r e t a l . 1 98 92 . W i t t a n d S t a c k e b r a n d t 1 9 883 . F o x e t a l . 1 9 8 04 . Y a n g e t a l . 1 9 855 . W o l t e r s e t a l . 1 98 96 . M a r k o w i c z e t a l . 19 9 17 . W o e s e 1 9 878 . E s c h b a c h e t a l . 19 9 19 . B h a t t a c h a r y a e t a l . 19 9 0

1 0 . D o u g l a s e t a l. 1 9 9 1

1 1 . H e n d r i k s e t a l . 19 9 11 2 . G u n d e r s o n e t al . 19 8 71 3 . C h r i s t e n e t a l. 1 9 9 11 4. V a l e n t i n a n d Z e t s c h e 1 9 9 0a1 5. V a l e n t i n a n d Z e t s c h e 1 9 9 0b1 6 . T h i s a r t i c l e1 7. M o r d e n a n d G o l d e n 1 9 911 8 . A s s a l i e t a l. 1 9 9 1 a1 9 . P a l m e r e t a l . 1 9 8 92 0 . L u d w i g e t a l . 1 9 90

w h i c h g e n e s a r e c u r r e n t l y u n d e r s t u d y w i t h w h i c h t y p e s o f d a t aa n a l y s i s m e t h o d s . S e v e r a l a u t h o r s e m p l o y e d a d d i t i o n a l p r o -g r a m s ; t h o s e l i s te d h e r e c o n c e r n o n l y t r e e s p u b l i s h e d a s f ig u r e s .R e f e r e n c e s a r e :

2 1 . A s s a l i a n d L o i s e a u x - d e G o 6 r 1 9 9 22 2 . S o g i n e t a l . 1 9 8 92 3 . M o r d e n a n d G o l d e n 1 9 8 92 4 . A r i z t i a e t a l. 1 9 9 12 5 . M i c h e l s e t a l . 1 99 12 6 . P e r a s s o e t a l. 1 9 8 92 7 . F f r s t e r e t a l. 1 9 9 02 8 . P f i h l e r e t a l. 1 9 8 92 9 . G i o v a n n o n i e t a l . 1 9 8 83 0 . D o u g l a s a n d T u r n e r 1 99 1

t h e o t h e r t w o p o s i t i o n s ; t h e y s h o u l d b e h i g h l ys u i te d t o p a r s i m o n y ( d u r in g b o o t s t r a p a n d ja c k k n i f ep r o c e d u r e s ) , a l t h o u g h a n a l y s i s o f o n l y s e c o n dc o d o n p o s i t io n s f u r t h e r r e d u c e s t h e s i z e o f th e o r i g-i na l da t a se t . F o r c om pa r i son , s e pa r a t e a l i gnm e n t sf o r f i r s t p l us se c ond ( 942 s i t e s ) , s e c ond pos i t i ons( 471 nonde ge ne r a t e s i t e s ) a nd f i r s t pos i t i ons ( 471m o s t o f w h i c h a r e n o n d e g e n e r a t e ) f o r b o o t s tr a p( B S) a n d j a c k k n i f e ( J K ) a n a l y s i s w e r e g e n e r a t e d .T h e a l i g n m e n t s a r e a v a i l a b le u p o n r e q u e s t f r o m t h ea u t h o r s .T h e r e s u l t s o f th e s e a n a l y s e s a r e s u m m a r i z e d i nT a b l e 2 . T h e y d e m o n s t r a t e t h a t o n t h e o n e h a n d

t he r e i s su f f i c i e n t i n f o r m a t i on c on t a i ne d wi t h i n t herbcL d a t a t o c l e a r l y re s o l v e s o m e p o r t io n s o f t h et r e e t h r ough pa r s i m ony a na l ys i s ( pa r t i c u l a r l y t hed e e p e s t a n d u p p e r m o s t b r a n c h e s ) . O n t h e o t h e rh a n d , t h e i n t e r m e d i a t e b r a n c h e s w i t h in t h e c h l o r o -p h y t e a n d r h o d o p h y t e / c h r o m o p h y t e po r ti o n s o f t h et r e e ( c f . F ig . 3 ) c a nno t be r e so l ve d w i t h t he f i r s t a nds e c o n d p o s i t i o n s o f rbcL. F o r t h e m o s t p a r t , t h eva r i ous da t a se t s ( f i r s t v s s e c ond vs f i r s t p l us se c -ond c odon pos i t i ons ) ga ve s i m i l a r r e su l t s , a l t houghf i rs t c o d o n p o s i t io n s r e v e a l e d s o m e w h a t f e w e r d is -t i nc t g r oups i n the 100 pa r s i m ony r e p l i c a t e s . I n ge n -e r a l , the da t a se t c ons i s t i ng o f on l y se c ond p os i t i ons

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p l a p l a p la p l a p l a p l a p l a p l an u c n u c

p r o p r o p r o p r o p r o p r o p r o p r orbcL rbcL rbcL rbcL ~ rbcL s tufA tufA psaB S S U f4 5 0 r 4 5 0 r 1 4 1 3 4 5 0 r 4 5 0 r 4 1 0 r 7 3 5 r 4 5 0 r gC L C L N J P P N J P P F M N J F M

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p r o p r o p r o p r o p r opsbA 1 8 S gapC 2 8 S 1 8 S rpo 1 6 S 1 6 S3 5 2 r 9 5 0 3 3 0 r 4 5 0 1 7 0 0 7 1 Y 1 0 0 0 8 6 2P P F M b F M F M F M F M F M b F M b

B S B S2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0

N u m b e r s o f s it e s r e f e r s t o n u c l e o t i d e s u n l e s s o t h e r w i s e i n d i -c a t e d . A b b r e v i a t i o n s a n d a n n o t a t i o n s : F M : F i t c h - M a r g o l i a s hd i s t a n c e m a t r i x m e t h o d ( F i t c h a n d M a r g o l i a s h 1 9 6 7 ), S T : di s -t a n c e m a t r i x m e t h o d o f S a t t a t h a n d T v e r s k y ( 1 9 7 7 ); C L :C L U ST A L ( H i g g i n s a n d S h a r p 1 9 8 8 ; e m p l o y s a U P GM A m a t r i xm e t h o d ( S h e a t h a n d S o k a l 1 9 7 3) ; P P : P A U P ( S w a f f o r d 1 9 8 9) ;P H : v a r i o u s p r o g r a m s o f t h e PH Y L IP p a c k a g e ( F e l s e n s t e i n 1 9 8 5) ;N J : N e i g h b o r - J o i n i n g d i s t a n c e m a t r i x m e t h o d ( S a i t o u a n d N e i1 9 8 7) ; B S : b o o t s t r a p a s s e s s m e n t o f c o n f i d e n c e i n t e r v a l s ( Fe l -s e n s t e i n 1 9 8 5 , 1 9 8 8) ; J K : j a c k k n i f e a s s e s s m e n t o f c o n f i d e n c ei n t e r v a l s ( F e l s e n s t e i n 1 9 85 , 1 9 8 8 ) ; rpo: D N A - d e p e n d e n t R N Ap o l y m e r a s e s ; gapC: g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o -g e n a s e ; tufA e l o n g a t i o n f a c t o r T u ; psaB a n d psbA g e n e s o ft h e p h o t o s y n t h e t i c m a c h i n e r y ; rbcL: l a r g e s u b u n i t o f r i b u l o s e -p e r m i t te d d e t e c t io n o f m o r e b r a n c h e s w h i c h w e r ec o n s i s t e n t w i t h t h e d i s t a n c e m a t r i x t r e e s ( i . e . , t h eb r a n c h b e a r i n g Ch lamydomona s a n d Chlorella, o rt h a t b e a r i n g Prochlorothr ix a n d Synechococcus , cf.F i g . 3 ) t h a n d i d t h e o t h e r t w o d a t a s e t s . Y e t p r o b -a b l y d u e t o t h e s o m e w h a t s l o w e r ra t e o f su b s t i tu -t i o n s a t s e c o n d p o s i t i o n s , t h e f i r s t a n d f i r s t p l u ss e c o n d d a t a s e ts p e r m i t te d d e t e c t io n o f c o m m o nb r a n c h i n g o f s o m e m o r e c l o s e l y r e l a t e d g r o u p s i n> 9 5 / 1 0 0 r e p l i c a t e s ( i . e ., Z e a a n d N i c o t i a n a ) ,w h e r e a s s e c o n d p o s i t i o n s d i d n o t . T h e r e m o v a l o ft h ir d c o d o n p o s i ti o n s o f c o u r s e m a r k e d l y i m p r o v e st h e B S a n d J K r e s u l t s w h e n d i s t a n t l y s p e c i e s a r ec o m p a r e d ( t h r e s h o l d o p t i o n s w e r e n o t u s e d in t h ea n a l y s e s ). F o r e x a m p l e , a d a t a s e t w a s g e n e r a t e d

1 , 5 - b i s p h o s p h a t e c a r b o x y l a s e / o x y g e n a s e ; a i n c l u d i n g p r o c h l o -r o p h y t e s ; b m o d i f i e d F M ( e . g ., E l w o o d e t a l . 1 98 5 ) o r o t h e rl e a s t -s q u a r e s m e t h o d ; e v a r io u s m e t h o d s ; t I 6 S a n d 1 8S r R N A sp a i r w i s e c o m p a r e d ; g a l i g n m e n t c r i ti c a l , se e o r i g i n a l r e f e r e n c e ;h o n e o r m o r e m e t a z o a a n a l y z e d ; i c h l o ro p l a s t a n d n u c l e a r g e n e so f Arabodopsis s t u d i ed ; k n u c l e a r a n d m i t o c h o n d r i a l g e n e s o fSaccharomyces s t u d ie d ; ~ o n l y m i t o c h o n d r i a l g e n e s a n a l y z e d ;n ~ o t a p p l i c a b l e s i n c e t h e o r g a n i s m i s n o t k n o w n t o p o s s e s s t h i sg e n e ; P t w o d i f f e r e n t n u c l e a r g e n e s a n a l y z e d , o n e o f e n d o s y m b i -o t i c o r ig i n ; r a m i n o a c i d s e q u e n c e s ; S s m a ll s u b u n i t o f r u b i s c o a l s oa n a l y z e d ; t t h e a - , [ 3-, ~ /- , a n d 8 - p u r p l e b a c t e r i a a r e defined tax-o n o m i c a l l y to a l a r g e e x t e n t u p o n t h e b a s i s o f 1 6S r R N A d a t a( W o e s e 1 9 8 7 ) .

c o n s i s t i n g o f o n l y t h i r d p o s i t i o n s f o r t h e 2 0 r b cLg e n e s ; a m o n g o t h e r c u r i o u s a n d m e a n i n g l e s s r e s u lt so b t a i n e d w i t h t h e t h i r d p o s i t i o n d a t a s e t , t h e f i v ep r o k a r y o t e s f o r m e d a m o n o p h y l e t i c c l a d e ( 1 0 0 / 1 0 0r e p l i c a t e s ) , s i m p l y d u e t o t h e e x t r e m e l y h i g h G Cc o n t e n t o f t h e s e g e n e s a t t h i rd p o s i t io n s ( d a t a n o ts h o w n ) . I t i s i n t e r e s t i n g t o n o t e t h a t t h e p a r s i m o n yt r e e s d e r i v e d f r o m a n a l y s i s o f rbcL n u c l e o t i d e s e -q u e n c e s d o n o t d i f f er d r a m a t i c a l ly f r o m t h o s e o b -t a i n e d t h r o u g h a n a l y s i s o f t h e a m i n o a c i d s e q u e n c e s( M o r d e n a n d G o l d e n 1 9 9 1 ) , a l t h o u g h t h e r e s u l t sc a n n o t b e d i re c t l y c o m p a r e d s i n c e d i f f e r e n t s p e c i e sw e r e c o n s i d e r ed . B S a n d J K a p p r o a c h e s t o ph y l o g -e n y c a n a s s e s s t h e r e l i ab i li ty o f b r a n c h e s w i t h i n at r e e b u t d o n o t d i r e c t l y p r o v i d e a t o p o l o g y w h i c h

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392Table 2. Summary of bootstrap (BS) and jackknife (JK) analyses for 20 rbcL seque nces at first plus seco nd (1 + 2 pos), s econ d (2pos), and first (1 pos) codon positionsChromat iumUnnamedProchlorothr&SynechococcusCyanophoraCh l a m y d o m o n a sChlorellaEug lenaAs tas iaNico t ianaZ e aBS 1 +

2 pos . . .JK1 +

2 pos . . .BS 2 pos . . .JK 2 pos . . .BS 1 pos . . .JK 1 pos . . .PylaiellaEc tocarpusCylindrothecaCryp tomonasAn t i thamnionPorphyridiumCyanidiumAlca l igenes

100 89 69 - 55 64 99 100 100100 89 70 - 54 72 100 100 100100 97 - 54 56 75 98 100 100100 93 - 53 62 84 100 100 100100 34 50 45 97 97 100100 35 46 99 95 100

Rhodobac te r * *

100 100 100 97 71 99 47 93 54 - 39 -100 100 100 98 61 98 49 94 53 - 43 -100 100 87 61 68 72 - 91 - 44 - 34100 100 90 71 76 66 - 98 - 52 - 41100 100 99 88 37 88 - 52100 100 97 95 39 94 - 54

~/-purpleeubacteria7-purpleeubacteriaProchlorophyteCyanobacteriumCyanophoraChlorophyteChlorophyteChlorophyteChlorophyteChlorophyteChlorophyte

PhaeophytePhaeophyteDiatomCryptophyteRhodophyteRhodophyteRhodophyte13-purple

eubacteriaa-purple

eubacteriaNumbers indicate the n umber of times (rounded to integers) outof 100 replicates that those species indicated by vertical columnsof asterisks occurred on a common branch in the analysis indi-cated. A dash (-) indicates that the grouping occurred in less(threshold chosen arbitrarily) than 33 replicates. All groupingswhich occurred in more than 33 replicates during any analysisare shown . The bootst rap and jackknife analyses for second po-sitions which included the Rhodosp i r i l lum rbcL sequence de-picted this sequence as the outgroup in 100/100 replicates andgave otherwise very similar results to those without Rhodospiril-lu m except that Prochlorothrix and Synchococcus shared a com-mon branch, as in the distance matrix trees, in 34/100 replicates(data not shown). Since non of the species listed below the nu-

merical portion of the table ever grouped with those ab ove same,a 100 appears for each analysis in the columns correspo nding tothe species indicated by eight and 11 vertical asterisks, respec-tively; although the second of these two columns is redundant, ithas been given for clarity. For further examples to illustrate thetable, the two ",/-purple eubacteria shared a single commonbranch in 100/100 replicates of all six analyses. The two pheo-phytes shared a commo n branch with the diatom in 89/100 boot-strap replicates for the rbcL data set consisting of first plus sec-ond positions and 97/100 jackknife replicates for the data setconsisting of only second codon positions; these three speciesthen shared a common branch with the cryptophyte in 69/100bootstrap replicates for first and second positions.

d i a g r a m m a t i c a l l y re f l e c t s b o t h r e l a t e d n e s s a n d de-gree o f r e la t e d n e s s b e t w e e n s e q u e n c e s i n t h e f o r mo f a d i s c r e t e t o p o l o g y w i t h m e a n i n g f u l b r a n c hl e n g t h s ; f o r t h is , d i s t a n c e m a t r i x m e t h o d s w e r e e m -p l o y e d .Two Distance Measures and Two Matrix MethodsT r e e s w e r e g e n e r a t e d w i t h d i s ta n c e m a t r i x m e t h o d sa n d c o m p a r e d t o t h e B S a n d J K r e s u l t s . T h e a n a l -y s e s f o c u s s e d u p o n t r e e c o n s t r u c t i o n w i t h t w o d i s -t a n c e m a t r i x m e t h o d s : ( 1 ) t h e N e i g h b o r - J o i n i n g

( N J ) m e t h o d ( S a i t o u a n d N e i 1 98 7), w h i c h h a s b e e nc o m p a r e d t o o t h e r m e t h o d s i n c o m p u t e r s i m u l a t io na n d s h o w n t o b e e f f i c ie n t i n r e c o v e r y o f c o r r e c tt o p o l o g y ( S a i to u a n d N e i 1 9 8 7; S a i t o u a n d I m a n i s h i1989 ; J in a nd Ne i 1990) a nd ( 2) t he F i t c h - M a r go l i a sh(FM) l e a s t - s q u a r e s ( F i t c h a n d M a r g o l i a s h 1 9 6 7 ;F I T C H o f t h e P H Y L I P p a c k a g e ) m e t h o d , w h i c hh a s b e e n w i d e l y e m p l o y e d in s t ud i e s o f r h o d o p h y t ea n d c h r o m o p h y t e e v o l u t io n ( T a b l e 1 ).I t h as b e e n s h o w n t h a t t h e m e t h o d o f d i s ta n c em e a s u r e b e t w e e n s e q u e n c e s i s a n i m p o r t a n t f a c t o rf o r ob ta in ing the c o r r e c t t opo log y ( J in a nd Ne i 1990)

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393A

I

B

N J :

o,1I

. ~ EuglenaNJ : d N Chlamydomonas

ChloreilaNicotiana

Zea- . . . .

F M : K a

Astasia. Euglena

"(~;1 (~ ,~ .4 Chlorella( ~ ~ I Chlamydomonas

02photo-synthesisPylaiella (phaeophyte) +Ec toca rpus (phaeophyte) +Cylindrotheca (diatom) +Porphyr idium (rhodophyte) +Cryptomonas (cryptephyte) +Anti thamnion (rhodophyte) +Cyanidium (rhodophyte) +Alcaligenes ([3-purple) -Rhododobacter (c-purple) -

unnamed (y-purpLe) -Chromatium (~-purple) -Prochlorothr ix (prochlorophyte) +Synechococcus (cyanobacterium) +Cyanophora (cyanelle) +Astas ia (euglenophyte) +Eug lena (euglenophyte) +Chlamydomonas (chlorophyte) +Chlore l la (chlorophyte) +Nicot iana (chlorophyte) +Zea (chlorophyte) +

Astasia

plastids ofnon-greeneukaryotes

eubacteria

plastids ofgreeneukaryotes

CryptomonasFM :d N .. .~ , Antithamnion

Porphyddium0,1

I IFig . 3 . A Neighbor-Jo in ing tree fo r va lues o f d ivergence a t non-s y n o n y m o u s s i t e s b e t w e e n rbcL coding reg ions as measuredwi th t he weigh t ed pa thway method (L i e t a l. 1985) showing boot -s t rap conf idence i n t e rva l s a t nodes . (See M ater i a l and Me thods . )The sca l e bar i nd i ca t es a leng th o f K~ = 0 .1 ; b ranc h l eng ths(hor i zon t a l) a re d rawn to sca l e . Num bers l e f t o f nodes a re de-rived fr om bo otst r ap analysis (Fels enste in 1985; t~NABOOT of thePHYL[P package) a nd i nd i ca t e t he n umb er o f t imes ou t o f 100 t ha tt hose spec i es drawn t o t he r i gh t on a g iven branch occurred tothe r i gh t on t ha t b ranch i n t he boo t s t rap ana lys i s fo r f i r s t p lussecond codon posi t ions (942 si tes), i .e . , in 100/100 repl icates,ne i t her o f t he two 7-purp l e bac t e r i a occurr ed on a b ra nch wi th inthe por t i on o f t he t ree t o t he r i gh t o f and con t a in ing t he b ranchl abe l ed "93 ," bu t i n 7 /100 boot s t rap rep l i ca t es , one o f t he e igh t"green" o rgan i sms or p l as t i ds occurred i n t he 3 , -purp l e b ranch .An as t e r i sk (* ) nex t t o a num ber i nd i ca t es t ha t t he cor respondingbranc h was d e t ec t ed i n l ess t han 33 rep l i ca t es fo r f ir s t p lus sec-ond pos i t i ons (942 s i t es ) bu t was de t ec t ed t he g iven number o ft imes i n t he da t a se t cons i s t i ng on ly o f second pos i t i ons (471s i t es ; see a l so Tab l e 1 ) . Branches t ha t ca r ry 95% conf idencein t e rva l s fo r mono phyly o f g roups i nc luded on t he b r anch (>195/100 t rees s i nce g roups w ere no t se l ec t ed subseque n t t o ana lys i s )

a re i nd i ca t ed w i th bo ld ova l s ; b ranche s w i th l ess t han 95% con-f i dence i n t e rva l s a re i nd i ca t ed w i th t h in ova l s . Oval s w i thou tnumbers i nd i ca t e g roupings de t ec t ed i n 0 -33 boo t s t rap rep l i ca t eswi th e i t her f i r s t , second , o r f i r s t p lus second codon pos i t i ons .The un l abe l ed ou tgroup i s t he fo rm I I rbcL of Rhodospiri l lumrubrum (Narang et al . 1984). Bootst rap repl icates for the Rho-dospirillum outgroup were on ly per formed wi th second pos i -t i ons . The capac i t y o f each organ i sm to per form oxygen evo lv-ing-photosyn thes i s i s i nd i ca t ed w i th "+ " o r " - . " B Por t i ons o ft rees con st ruc ted for matrices of K a (Li et al. 1985) or dN (Neiand Gojobor i 1986) w i th t he F i t ch-Margol i ash l eas t -squares (FM )meth od (FITCH (Fi tch an d M argol ia sh 1967) of PHYLIP (Felsen-stein 1981)) and d N wi th the NJ meth od. FITCH was run wi th the" jum ble" and "g lo ba l " op t i ons . Only t hose por t i ons o f t rees a res h o w n i n w h i c h t h e b r a n c h i n g order ( i r r e s p e c t i v e o f b r a n c hlength) differed f rom tha t o f F ig . 3A . Number s an d ova l s a re asin Fig. 3A; al l port ions of t rees no t shown in F ig . 3B are i den t i ca li n b ranch ing order t o t ha t show n in F ig. 3A . The sca l e bar i nd i -ca t es d ivergence 0 .1 (Ka or dN) a t nons ynon ymo us s i t es . Arrowsemphas i ze t he d i f fe ren t pos i t i on o f Astasia's sequence i n t heFM :K a t ree .

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39 4w h e n d i s t a n c e m a t r i x m e t h o d s a r e u s e d . D i v e r -g e n c e a t s y n o n y m o u s s i te s b e t w e e n m o s t o f t h er b c L s e q u e n c e s s t u d i e d w a s t o o g r e a t t o b e r e li a b l ye s t i m a t e d ( d a t a n o t s h o w n ) , s o o n l y n o n s y n o n y -m o u s s i t e s w e r e c o n s i d e r e d . T h e r e a r e s e v e r a l d i f -f e r e n t m e t h o d s a v a i l a b l e f o r es t im a t i n g t h e n u m b e r so f n o n s y n o n y m o u s s u b s ti tu t i o n s b e t w e e n p r o t e inc o d i n g r e g i o n s ( M i y a t a a n d Y a s u n g a 1 9 8 0 ; L i e t a l .1 9 8 5 ; N e i a n d G o j o b o r i 1 9 8 6 ) w h i c h h a v e b e e ns h o w n ( N e i a n d G o j o b o r i 1 98 6) to a l l g i v e v e r y s im -i la r , y e t n o t i d e n t i c a l , e s t i m a t e s o f d i v e r g e n c e . F o rt h e p u r p o s e o f t r e e -b u i l d in g , d i v e r g e n c e a t n o n s y n -o n y m o u s s i t e s b e t w e e n t h e r b c L s e q u e n c e s w a sm e a s u r e d w i t h t w o d i f fe r e nt m e t h o d s t h ro u g h c o m -p u t e r p r o g r a m s k i n d l y p r o v i d e d b y t h e r e s p e c t i v ea u t h o r s : (1 ) t h e w e i g h t e d p a t h w a y m e t h o d o f L i e ta l. (1 98 5) in w h i c h K i m u r a ' s t w o - p a r a m e t e r m e t h o dm o d e l a n d a n u m b e r o f o t h e r f a c t o r s i n c lu d i n g a l-t e rn a t iv e p a t h w a y s o f am i n o a c i d r e p l a c e m e n t a r ec o n s i d e r e d a n d ( 2 ) t h e l e s s c o m p l i c a t e d y e t q u i t er o b u s t m e t h o d o f N e i a n d G o j o b o r i ( 19 8 6) , i n w h i c ht h e J u k e s - C a n t o r m o d e l i s e m p l o y e d . T h e c o m p a r -i s o n o f v a l u e s o b t a i n e d f o r d i v e r g e n c e a t n o n s y n o n -y m o u s s i t e s , d N ( N e i a n d G o j o b o r i 1 9 86 ) a n d K a ( L ie t a l . 1985) , i s g iven in F ig . 2 .

T h e m e t h o d o f L i e t a l. c o n s i d e r s t h e r e l a ti v el i k e li h o o d s o f a l t e r n a t iv e p a t h w a y s f o r c o d o nc h a n g e , a n d s h o u l d , i n t h e o r y , t e n d t o e s ti m a t el o w e r n u m b e r s o f n o n s y n o n y m o u s s u b s ti tu t io n s a tt h e e x p e n s e o f h ig h e r e s t i m a t e s f o r n u m b e r s o f s y n -o n y m o u s s u b s t i tu t i o n s. T h u s o n e w o u l d e x p e c t v a l-u e s o f K a t o b e s o m e w h a t l o w e r t h a n t h o s e f o r d g .I n d e e d , a s s h o w n i n F i g u r e 2 f o r th e r b c L d a t a , K ~w a s i n g e n e r a l a b o u t 1 0 % lo w e r t h a n d N . T h e t r u ed i v e r g e n c e b e t w e e n t h e s e q u e n c e s i s n o t k n o w n , s oo n e c a n n o t t e l l w h e t h e r ( 1 ) K ~ i s u n d e r e s t i m a t i n g ,(2) d N o v e r e s t i m a t i n g d i v e r g e n c e , ( 3 ) b o t h m e t h o d sa r e g i v i n g u n d e r e s t i m a t e s , o r ( 4 ) b o t h a r e g i v i n go v e r e s t i m a t e s . A t a n y ra t e , K ~ w a s g e n e r a l l y lo w e rt h a n d N , a n d c o n s i d e r a b l y l o w e r t h a n d N ( 30-4 0%)i n s o m e s p e c i f i c c o m p a r i s o n s i n v o l v in g t h e A s t a s i ar b c L s e q u e n c e . A s t a s i a l o n g a i s a v e r y c l o s e r e l a -t ive o f E u g l e n a ( W a l n e a n d K i v i c 1 99 0) t h o u g h n o n -p h o t o s y n t h e t i c . T h e r e m a y t h e r e f o r e b e d i f f e r e n tf u n c t i o n a l c o n s t r a i n t s u p o n t h e r b c L g e n e p r o d u c t ,w h i c h o u r d a t a s u g g e s t is e v o l v i n g m u c h f a s t e r t h a nt h a t o f o t h e r r b c L g e n e s . T h u s , a l t h o u g h K a a n d d Ng i v e q u i te s i m i l a r e s ti m a t e s i n c o m p u t e r s i m u l a ti o nu n d e r d i f f e r e n t s e l e c ti o n s c h e m e s a n d f o r g l o b ing e n e s ( N e i a n d G o j o b o r i 1 9 8 6 ) , w i t h r b c L d a t a t h em e t h o d s s h o w a p p r e c i a b l e d i f f e r e n c e s i n e s t i m a t e so f d iv e r g e n c e a t n o n s y n o n y m o u s s it es .

N o e x a m p l e s f r o m t h e l i t e r a t u r e a r e k n o w n t o u si n w h i ch " t h e e f f e c t o f d i f f e r e n t d i s t a n c e m e a s u r em e t h o d s h a v e b e e n d i r e c t l y c o m p a r e d u p o n r e a ld a t a i n o r d e r t o a s s e s s t h e ir e f f e c t u p o n b r a n c h i n go r d e r f o r th e s a m e s e q u e n c e s ( s e q u e n c e d i v e r g e n c e

v s g e n e t i c d i s t a n c e h a v e b e e n d i r e c t l y c o m p a r e d :N e l s o n e t a l . 1 9 9 1) . T h u s , f o r t h e r b c L d a t a s e t , N Ja n d F M t r e e s w i t h b o t h v a l u e s o f d N a n d K a f o rd i v e r g e n c e a t n o n s y n o n y m o u s s it e s w e r e c o n -s t r u c t e d . T h e t r e e s a r e s u m m a r i z e d i n F i g . 3 . C o m -p a r i s o n o f t h e s e f o u r r e su l t s t o t h e B S a n d J K a n a l-y s e s r e v e a l e d t h a t t h e N J t r e e f o r v a l u e s o f K af o u n d m o r e b r a n c h e s ( i r r e sp e c t i v e o f th e i r l e ng t h )w h i c h w e r e s u p p o r t e d b y t h e b o o t s t r a p p a r s i m o n yd a t a i n T a b l e 2 th a n t h e o t h e r t h r e e t r e e s . T h e c o m -p l e t e N J t o p o l o g y f o r v a l u e s o f K ~ is t h u s s h o w n i nF i g . 3 A . T h e p o r t io n s o f t h e F M t r e e s f o r K ~ a n d d Na n d t h e N J t r e e f o r v a l u e s o f d N w h i c h dif fer inb r a n c h i n g o r d e r f r o m t h a t s h o w n i n F i g . 3 A , r e g a rd -l e s s o f b r a n c h l e n g t h , a r e s h o w n i n F i g . 3 B . I ts h o u l d b e n o t e d t h a t t w o d i f f e re n t t y p e s o f in f o r m a -t i o n a r e c o n t a i n e d w i t h t h e f i g u r e s . T h e t r e e s ( o rp o r t i o n s t h e r e o f i n F i g . 3 B ) d e p i c t a d i s c r e t e t o p o l -o g y ( n o t n e c e s s a r i ly t h e t r u e o n e ) f o r t h e s e q u e n c e s .T h e a m o u n t o f d i v e rg e n c e b e t w e e n s e q u e n c e s i s re -f l e c t e d i n t h e b r a n c h l e n g t h s a n d r e f l e c t s t h e e v o l u -t io n o f c o d o n s w i th i n th e s e q u e n c e s . T h e n u m b e r sg i v e n w i th i n o v a l s c o n t a i n i n f o r m a t i o n d e r i v e d f r o mb o o t s t r a p a n a l y s i s w h i c h w a s p e r f o r m e d u p o n 2 /3 o r1 /3 c o d o n s ( c f T a b l e 2 ) .

B o t h N J t r e e s ( c o n s t r u c t e d w i t h v a l u e s o f K a a n dd u ) d e t e c t e d all b r a n c h i n g o r d e r s w h i c h a r e s u p -p o r t e d i n 9 5 % o f b o o t s t r a p r e p l i c a te s . T h e s a m e i s ,h o w e v e r , n o t t ru e f o r t h e F M m e t h o d . T h e F M t r eef o r K ~ d i d n o t d e t e c t c o m m o n b r a n c h i n g o f th e t w oe u g l e n o i d s , E u g l en a a n d A s t a s i a . I n o u r o p i n i o n ,t h i s f i n d i n g s h o u l d w a r r a n t c a u t i o n c o n c e r n i n g F Mt o p o l o g i e s w h i c h h a v e n o t e b e e n c h e c k e d w i t ho t h e r m e t h o d s . T h e s e t w o e u g l e n o id s a r e m o r p h o -l o g ic a l ly e x t r e m e l y s im i l a r an d u n q u e s t i o n a b l y v e r yc l o s e l y r e l a te d t h r o u g h c o m m o n d e s c e n t , a l t h o u g hA s t a s i a , s i m i l a r t o " b l e a c h e d " E u g l en a s , i s n o n -g r e e n ( S i e m e i s t e r a n d H a c h t e l 1 9 9 0 ) . T h e i r r b c L se -q u e n c e s s h a r e a c o m m o n b r a n c h i n 97 /1 0 0 b o o t s t r a pr e p l i c a t e s ( 9 8 /1 0 0 w i t h t h e h a l f - d e l e t e j a c k k n i f e ; s e eT a b l e 2 ) f o r f i r s t p l u s s e c o n d c o d o n p o s i t i o n s ( 9 4 2s i t e s ) , y e t t h e s e s e q u e n c e s d o n o t s h a r e a c o m m o nb r a n c h i n t h e F M t r e e f o r K ~ , w h e r e a s t h e y d o i nt h e N J t r e e u s i n g t h e s a m e K a m a t r i x a s w e l l a s i nt h e F M t r e e u s i n g t h e d N m a t r i x . T h i s e x a m p l ec l e a r ly u n d e r s c o r e s w i t h r e a l d a t a t h e f i n d i n g s f r o mc o m p u t e r s i m u l a t i o n s, t h a t t h e m e t h o d o f d i s t a n c em e a s u r e i s a v e r y i m p o r t a n t p a r a m e t e r f o r t h e e f f i -c i e n c y o f d i s t a n c e m a t r i x m e t h o d s ( J in a n d N e i1990).

V e r y m i n o r d i f f e r e n c e s i n m e a s u r e m e n t o f d i v e r -g e n c e a t n o n s y n o n y m o u s s it es b e t w e e n K ~ a n d d Na r e t h u s s u f f i c i e n t t o e f f e c t c h a n g e i n a p o r t i o n o ft h e F M t o p o l o g y w h i c h is f o u n d i n m o r e t h a n 9 5 / 10 0b o o t s t r a p r e p l i c a t e s . T h i s i s a n o t e w o r t h y r e s u l t .T h e i n s t a b i l i t y o f A s t a s i a ' s p o s i t i o n m a y r e l a t e t ot h e h i g h s u b s t i t u t i o n r a t e w i t h i n t h e A s t a s i a r b c L

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sequence . Using e i ther Zea, Anabaena, Prochloro-thrix, o r Cyanophora as the outgroup, r e la t ive ra tetes t s for subs t i tu t ions a t nondegenera te s i tes (L iand Tanimura 1987) showed tha t the subs t i tu t ionra te of Astasia rbcL is signif icantly higher at the99% leve l (da ta not shown) than tha t of Euglena.This f inding i s r e levant s ince computer s imula t ionhas shown tha t the FM method i s less e f f ic ient un-de r a va r ie ty o f pa r a me te r s t ha n t he N J me thod i nr e c o v e r i n g c o r r e c t t o p o l o g y , p a r t i c u l a r l y w h e nr a t e s o f e vo lu t i on va r y d r a s t i c a l l y i n d i f f e r e n tbranches (Sa i tou and Imanishi 1989) . This proper tywould then a lso seem to apply to rea l da ta in thec a se o f rbcL. This case in point of Astasia's branch-ing in the F M t ree of Ka va lues should war rant cau-t ion concerning in te rpre ta t ion of t r ees for r ea l da tafor which no method es t imat ing sampl ing e r ror hasbe e n a pp li e d . T he p r ob l e m of how to de p i c t t hereliabil i ty of a topology within a f igure has beensolved di f fe rent ly by di f ferent authors ( i .e . , Ho r i e tal. 1985; Gouy and Li 1989a; Martin et al. 1989;Arizt ia et a l . 1991; Morden and Golden 1991; Doug-las et a l . 1991) . When distance matr ix methods aree mploye d t o c ons t r uc t t r e e s fr om r RN A se qu e nc e s ,es t imates of Knuc (Kimura 1980) are of ten us ed asthe dis tance me asure . I f the s tandard e r ror s a t -tached to es t im ates o f Kn,c a re depic ted di rec t ly inthe tree, as Ho ri et a l . (1985) did, a clearer picture ofthe da ta under lying and conta ined wi thin the t r ee i sprovide d, even i f no meth ods of spec if ica l ly ad-dress ing the re l iabi l ity o f individua l branches a reemp loyed . S tanda rd e r ror s for es t imates of d iver -gence were not dep ic ted di rec t ly in the t ree of F ig .3 , ye t these a re indica ted roughly in the graph ofFig . 2 , in order to emphasize the uncer ta inty inher -ent in the measurement i t se l f . Ra ther , as in Ar iz t iaet al . (1991) and Morden and Golden (1991) , resultsof boo ts t rap an a lys is were dep ic ted wi thin the t r eeitself.

Thus , the rbcL data appear to conta in re levantevolut ionary informat ion concerning the or igins ofalgal plast ids. The interpretation of th is and otherda ta wi l l be the topic of the fol lowing discuss ion.

Origins of Plant NucleiA t p r e sen t , no t muc h i s know n a bou t t he b io logy o fhos t ce l l s pr ior to endosymbios is ; Cava l ie r -Smithhas sugges ted (1987b) tha t these may have beenanaerobic and lacking a ce l l wal l , pe rhaps in someways s imi la r to Giardia lamblia, a protozoan para -s i t e w h ic h posse s se s nuc l e i ye t l a c ks bo th mi to -c hondr i a a nd e ndop la sma t i c r e t i c u lum ( K a bn ic ka nd Pe a t t i e 1991). Mos t mole c u l a r e vo lu t i ona r ys tud i e s w h ic h migh t a ddr e s s t h i s que s t i on ha ve ,with few exceptions (e .g. , Ptihler et a l . 1989) , been

395per formed wi th 18S rRNAs (Table 1) , such tha t ourc ur r e n t v i e w o f e a r ly e uka r yo t i c e vo lu t i on e nc om-pa sse s ma ny t a xa ye t r el ie s he a v i ly upon se que nc e sfor th is s ingle gene . Al though rDNA in eukaryotesbe longs to the m iddle repe t i t ive f rac tion of the ge-nome a nd i s sub j e c t to p r oc e s se s o f une qua l c r os s -ove r a nd gene c onve r s ion w hic h ma y , i n somecases , a l te r these sequences in a manner di f fe rentf rom s tochas t ic base- subs t i tu t ion models under ly-ing molecula r phylogene t ic ana lyses (Dover 1987,1989; Hil l is et a l . 1991) , rRNA genes contain usefulphylo gene t ic informat ion. Typica l for t r ees con-structed from 18S sequences (e .g. , Sogin et a l . 1989)is the very deep branching wi thin the eukaryot icpor t ion of the t r ee for microspor idians and Giardialamblia. The 18S rRN A branch of Euglena's nu-c leus a l so emerges qui te near the base of the eu-ka r yo t i c t r e e s on a c ommon b r a nc h w i th t r ypa no-somes and other k ine toplas t ida (Gunderson e t a l .1987; Sogin et al. 1989; Bhattacharya et al. 1990;Esch bach e t a l. 1991) . With the ex cept ion of the irchlorophyl l s , euglenoids a re cytologica l ly and bio-chemica l ly qui te d is t inc t f rom chlorophytes and ap-pear r e la ted to kine toplas t ida (Leeda le 1967; So-lomon et al . 1991) , such that the deep branching ofthe i r 18S rRN A, fa r r em ove d f rom tha t o f chloro-phytes , i s not incongruent wi th other da ta . I t i s ofinte res t to note tha t a second nuc lear marker fork ine top l a s t i da (Trypanosoma), g lyc e r a lde hyde - 3 -phospha t e de hydr oge na se (gapC), has been s tudied(Michels et a l . 1991) and emerges similar ly deepwi thin the eukaryot ic gapC t r ee . The fac t tha t ase c ond nuc l e a r ma r ke r o f the e ug l e no id - t r ypa no-some l ineage so behaves may be an indica t ion tha tthe pos i t ion of the Euglena nuclear branches ade-qua te ly re f lec t the evolut ion of the genes , r a therthan be ing an a r te fac t of ana lys is as shown abo vefor the Astasia rbcL se que nc e i n FM t r e e s .

The nuc lear rR NA t rees l i s ted in Table 1 showcom mo n fea tures concerning the c lus te r ing o f a lga lnuc l e ar r RN A s w i th t hose o f c er t ain o the r e uka r y -ot ic groups; the 18S t rees a re typi f ied by tha t ofDouglas et a l . (1991) , which is reproduced with per-miss ion of the authors in F ig . 4 . These eukary ot icgroups inc lude (1) brown a lgae , d ia toms, chryso-phytes , and oomycetes , (2) d inof lage l le taes and c i l -ia tes , and (3) rhodophytes as an independent group;the pos i t ions of these groups re la t ive to one anoth erva r y f r om t r e e t o t r e e , a na logous t o t he p l a n t -a n ima l - ye a s t p r ob l e m d i sc us se d a bove . Conc e r n ingthe f i r s t of these c lus te r s , severa l l ines of cytologi -ca l /u l t r as t ruc tura l evidence (Beakes 1989) and mo-lecular data (F6rster et a l . 1990; Arizt ia et a l . 1991)ha ve be e n p r e se n t e d suppor t i ng oomyc e t e s , d i a -toms, and chrysophytes as a na tura l group. Corol -la ry to th is v iew is the d iphyle t ic na ture of fungi , atheory suppor ted by ce l l wal l and biochemica l da ta

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396

I

Z e a tlycineZamia ch lo ro p h y t e sChlamydomonas~ - Ch lo re l la- Cryptomonas (N U C L EU S) ~ c r yp t o p h y t e

Acanthamoeba- - S accharomycesCandida

NeurosporaOchromonas ch r yso p h y t eSkeletonema d ia tomAchyla o o m y c e t eEuplotes 1Oxytricha _ . , [ .~ I erranymena cil iatesParamecium Jt l L ~ P rorocentrum d inof lageHateI r-" Gracilariopsis "l , ,[ ~ ~ . " - - - ' - " - L G rac ila ria j , r n o o o p h y t e s

Cryptomonas (N U C L EOM OR PH ) c r yp t o p h y t eh u m a n~ XenopusArtemiaDictyostef iumPlasmodiumL_ P lasmod iumNaegler iaCrithidiaEuglena e u g le n id

F i g . 4 . P h y l o g e n e t i c t r e e f o r n u c l e a r 1 8 S r R N As e q u e n c e s c o n s t r u c t e d a n d p r e s e n t e d b y D o u g l a s e ta l . ( 1991) , r eproduced here in modi f i ed fo rm wi th thek i n d p e r m i s s io n o f t h e a u t h o r s . T h e t r e e w a sc o n s t r u c t e d w i t h t h e N e i g h b o r - J o i n i n g m e t h o d(Sai tou and Nei 1987) fo r a mat r ix o f Knucd i s t a n c ev a l u e s ( K i m u r a 1 9 80 ). T h e n u c l e a r a n d n u c l e o m o r p hr R N A s e q u e n c e s o f Cryptomonasa r e i n d i c a t e d . O fpar t i cu la r in t e r es t i s the d i s t inc t s epara t ion ofCryptomonasn u c l e a r an d n u c l e o m o r p h r R N As equences . (See t ex t . ) For fu r ther de ta i l s , t heor ig ina l r e f e r ence s hould be cons u l t ed .

( B a r t n i c k i - G a r c i a 1 9 8 7 ) a n d p e r c e i v e d b y M e -res ch ko w sk y (1910d: F ig . 1). Ar i z t ia e t a l . (1991)h a v e p r e s e n t e d v e r y c a r e f u l a n a l y s e s o f 1 8 S r R N As e q u e n c e s . I t s h o u l d b e n o t e d t h a t i n s o m e c a s e st h e r e e x i s t c o n s i d e r a b l e d i s c o n c o r d a n c e b e t w e e np h y l o g e n i e s d e r i v e d f r o m r R N A a n d p r o t e i n s e -q u e n c e s f o r th e s a m e o r g a n i s m s . L o o m i s a n d S m i t h( 1990) , f o r e xa m ple , a na lyz e d se que nc e s f o r e igh td i f f e r e n t p r o t e i n s , a ll o f w h i c h y i e l d e d p h y l o g e n i e sf o r Dictyostelium r e l a t i ve t o ye a s t a nd a n im a l s d i f -f e r e n t fr o m t h a t r e f l e c t e d b y t h e 1 8S r R N A d a t a i nA r i z t i a e t a l. ( 1991) . [ O n e m a y n o te t ha t 16S r R N A ,R u b i s c o , a n d ps bA se que nc e s a l so t e s t i f y d i f f e r -e n t l y to t h e r e l a t io n s h i p o f Prochlorothrix t o c y a n o -ba c t e r i a a nd g r e e n p l a s t i d s ( T u r ne r e t a l . 1989 ; M or -d e n a n d G o l d e n 1 9 8 9 ) . ] I n g e n e r a l , w e w o u l dc u r r e n t l y t e n d t o w a r d t h e v i e w t h a t t h e o r i g in s a n de a r l y e v o l u t i o n o f a l g al n u c l e a r l i n e a g e s h a v e n o tb e e n s t u d i e d w i t h s u f f i c i e n t n u m b e r s o f d i ff e r e n tge ne s so a s t o p r ov id e a f i na l p i c tu r e o f phy le s i s f o rt h e n u c l e i o f t h e s e h i g h l y d i v e r s e o r g a n i s m s , y e t t h ep e r s p e c t i v e s f o r f u t u r e s t u d i e s o f t h e s e q u e s t i o n sa r e p r o m i s i n g .Origins o f PlastidsM o l e c u l a r d a t a u n e q u i v o c a l l y s u p p o r t e n d o s y m b i -o t i c o r ig in s f o r p l a s t i d s . ( S e e r e f e r e n c e s t o Ta b le 1 .)S e v e r a l r e c e n t m o l e c u l a r co n t r i b u t i o n s o n rbcL se -q u e n c e s h a v e a p p e a r e d i n t h e l i t e r a t u r e ( V a l e n t i na nd Ze t sc h e 1990b ; D oug la s e t al . 1990 ; M or d e n a nd

G o lde n 1991 ; A ssa l i e t a l . 1991a) w h ic h ha v e ind i -c a t e d t h a t t h e p la s t id g e n e s o f r h o d o p h y t e s a n dc h r o m o p h y t e s a r e o n l y d i s t a n t ly r e l a t e d t o t h e p l a s-t id s o f c h l o r o p h y t e s , a n d t h a t t h e l a t t e r a r e m u c hm o r e c l o s e l y r e l a t e d t o c y a n o b a c t e r i a t h a n t h ef o r m e r . T h e s e d a t a h a v e b e e n i n t e r p r e t e d a s e v i -d e n c e c o m p a t i b l e w i t h p o l y p h y l e t i c p l as t id o r ig i n s( D oug la s e t a l. 1990 ; V a le n t in a nd Ze t sc h e 1990b ;A ssa l i e t a l . 1991a) , a sugge s t ion f o r w a r de d p r e v i -o u s l y o n t h e b a s i s o f b i o c h e m i c a l e v i d e n c e ( e .g . ,M e r e s c h k o w s k y 1 91 0a --d ; R a v e n 1 9 7 0 ) . P o l y p h y -l e t i c p l a s t i d o r ig in s a r e i n t e nde d he r e t o i nd i c a t ei n d e p e n d e n t primary e n d o s y m b i o s e s e i t h e r ( 1 ) b e -t w e e n d i f f e re n t prokaryotic p la s t i d p r oge n i to r s a nda ny g ive n hos t c e l l l a c k ing p l a s t i d s o r ( 2 ) be tw e e nt h e s a m e p r o k a r y o t i c s y m b i o n t ( w i t h i n t h e r e s o l u -t i on o f a va i l a b l e m e tho ds ) a nd d i f f e r e n t hos t c e l l s .S e c o n d a r y e n d o s y m b i o s i s , i n w h i c h a p h o t o s y n -the t i c eukaryotic a lga pos se s s ing p l a s t i d s ( r e ga r d -l e s s o f the i r e vo lu t io na r y h i s to r y ) i s e ngu l f e d by a n -o t h e r e u k a r y o t e w o u l d n o t n e c e s s i t a te , b u t w o u l dn o t e x c l u d e , p o l y p h y l e s i s f o r p l a st i d s.T h e a n c e s t o r s o f p la s ti d s h a v e t r a d i t i o n a l ly b e e ns o u g h t a m o n g t h e c y a n o b a c t e r i a ( M e r e s c h k o w s k y1 90 5) o r p r o c h l o r o p h y t e s ( R a v e n 1 9 7 0) ; c y a n o b a c -t e r i a , p r o c h l o r o p h y t e s , c h l o r o p l a s t s , a n d r h o d o -p l a st s a r e u n i q u e i n t h a t t h e y e v o l v e o x y g e n i n p h o -t o s y n t h e s i s . C y a n o b a c t e r i a a n d r h o d o p l a s t s s h a r ep h y c o b i l i n s a s a u n i q u e c o m m o n t r a i t ; p r o c h l o r o -p h y t e s a n d c h l o r o p l a st s s h a r e c h l o r o p h y l l s a a n d ba s a u n i q u e c o m m o n t r a i t . P u r p l e b a c t e r i a o n t h e

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other hand (1) do not evolve oxygen in photosyn-thesis, (2) do n ot possess phyco bil ins, and (3) do notpossess chlorophyl l s a and b. I t i s thus somewhatsurprising that the rbcL sequences o f bo t h an a -pur -ple and a 13-purple bacterium reveal such close af-fini ty to th e rbcL genes o f rhodo- and chrom ophyt eplas tids , wher eas the cyanob acter ia l and prochloro-phyt e rbcL genes appear qui te c lose ly re la ted totheir counterparts of green plast ids and ~/-purplebacteria (Fig. 3A).Th e rbcL data suggest tha t cyano bacter ia a re de-scendants of a wel l -di f fe rent ia ted "purple" bac te-rial lineage. This is an intriguing finding wh ich con -f l ic t s to some extent wi th bac ter ia l phylogeniesbased upon 16S rRNA sequences (Woese 1987) .Fur t he rmore , s i nce Q-evo l v i ng phot osynt hes i s oc-curs in ch l orophyt e , rhodoph yt e , and chrom ophyt eplast ids, yet not in purple bacteria, the rbcL datawould suggest tha t the comm on ances tor of purplebacteria and cyanobacteria would have been an 02-evolv ing photoautotroph, and tha t extant purplebac ter ia have los t the i r capac i ty to use w ater as ane lec t ron donor . This f inding is compat ible wi th theconsiderable body of biochemica l evidence suggest-ing tha t the common ances tors of purple bac ter iaand cyan obac t e r i a posse s sed both phot osys t ems Iand I I ( reviewed by Olson and Pierson 1987) . I t waspreviously suggested on the bas i s of cytochrome c(Dickerson 1980) and 16S rRN A (Woese 1987) da tat ha t ch l orophyl l -ba sed phot osynt hes i s i n purp l ebac ter ia represents a biochemica l symples iomor-phy , t he nonphot osynt he t i c s t a t e o f many purp l ebac ter ia be ing due to independent secondary loss .The rbcL sequence da ta would go one s tep fur therby suggest ing tha t the com mo n ances tor o f purple-and cyanobacter ia was capable of oxygen-evolvingphotosynthes i s , tha t only cyanobacter ia and plas-r ids re ta ined both photosystems, and tha t plas t idsare o f polyphy le t ic or igin.

Y et rbcL sequences re f lec t somewhat di f fe rentaff ini t ies be tween cyanobacter ia , purple bac ter ia ,and plas t ids than do 16S rRNA data . Based upon16S rRN A sequen ces , cyan obacte r ia appear di s-t inc t f rom the purple bac ter ia (Wo ese 1987; Stack-ebrandt e t a l . 1988) and are depic ted to have di-verged from these pr ior to the separa t ion of the a -and g-purple lineages from the [3- and ~/-purple lin-eages (W oese 1987; Zava rzin et al . 1991), thou ghone ma y keep in mind tha t the di s t inc t ions be tweenor-, [3-, g-, and ~/-purple ba cte ria r est hea vily u po n as ingle gene (16S) phylogeny; 16S rRNA sequencephylogenies depic t e i ther (1) somew hat c loser a ff in-i t i es be tween rhodoplas t s and cyanobacter ia thanbetween the la t te r and chloroplas t s or (2) commonbranching of two main plas t id l ineages der ived ofcyano bacter ia , one leading to chrom oph ytes , eugle-

397noids and red a lgae , the other leading to chloro-phytes (c f. Fox e t a l. 1980; Wi t t and Stack ebrand t1988; Wo lters et al. 1989; Ma rko wic z and Lo iseaux -de Go6r 1991; Douglas and Turn er 1991) . The 16Ssequence of Alcaligenes ([3-purple) clusters with a-and ~/-purple bacteria to the exclusion of both redand green p l a s t i ds (Markowi cz and Loi seaux-deGo~r 1991) . Discrepancies be tween 16S rRNA andrbcL data con cerning plast id or igins remain to beexplained.

I f one assumes tha t the rbcL gene phyl ogeny(Fig. 3) soundly reflects the evo lut ion of the spe ciesand organel les under s tudy, explanat ions must besought in the evolut ion of 16S rRNA genes whichmay accou nt for the da ta. A side f rom the prob lemsof f luc tua t ing GC content in bac ter ia l evolut ion(Muto and Osawa 1987), one m ay reca l l tha t r ibo-somal RNAs evolve in the s t ruc tura l context ofroughly 60 r ibosomal prote ins , about two-thi rds ofwhich are encoded by the nucleus in higher plantsand green a lgae (Chri s topher e t a l. 1988; Mac he1990; Sub rama nian et al . 1991). Mo reo ver , plast idr i bosomes posse s s a num ber o f un i que fea ture swhich the i r cou nterpar t s in free- living proka ryotesdo not (Zhou and M ache 1989; Subram anian e t a l.1991). One thus cannot exclude the possibi l i ty thatfunct ional const ra int s re la t ing to molecular coev o-lut ion of r ibosomal RNAs wi thin the s t ruc tura l con-text of the i r evolving cognate r ibosomal prote ins(John son et al . 1990; Mart in et al . 1990) sub sequ entto endosymbiosis may have genera ted considerablediscont inui ty in the evolut ion of 16S rRNAs fromproka ryo t e s t o p l a s t i ds . Se l ec t i ve neu t ra l i t y o fmany muta t ions observ ed in plas t id 16S r ibosomalRNA may not hold, thereby inf luencing 16S rRNAphylogenies .

If , on t he o t he r hand , one a s sumes t ha t 16SrRNA gene phylogenies soundly re f lec t the evolu-t ion of the spec ies and organeUes und er s tud y, ex-plana tions must be sought in the evolut ion of rbcLgenes . As one a l te rna t ive , hor izonta l gene t ransferhas been sug gested for rbcL from a [3-purple bacte-r ia l donor to a cyanobacter ium in the rhodophyteplas t id l ineage to expla in the di f fe rences be tweenrbcL and other genet ic da ta (Boczar e t a l . 1989;Mo rde n and G olden 1991; Assal i et al . 1991a); i .e . ,red plas t ids der ive f rom cyanobacter ia which pos-sessed [3-purple bacterial rbcL genes . As a secondal te rna t ive , the ances tors to plas t ids , cyanobacte-r ia , and purple and bac ter ia may have s imply pos-sessed two rbcL genes , one of whi ch was seconda r -i ly lost in each of the l ineages leading to red andgreen plas t ids . Some a-p