molecular evidence for the origin of plastids from a cyanobacterium-like ancestor

J Mol Evol (1991) 33:267-273

Journal of Molecular Evolution

Molecular Evidence for the Origin of Plastids from a Cyanobacterium-like Ancestor

Susan E. Douglas ~ and Sefin Turner 2

t Institute of Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia B3H 3Z1, Canada 2 Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, IN 47405, USA

Summary. The origin ofplastids by either a single or multiple endosymbiotic event(s) and the nature of the progenitor(s) of plastids have been the sub- jects of much controversy. The sequence of the small subunit rRNA (Ssu rRNA) from the plastid of the chlorophyll c-containing alga Cryptomonas ~ is presented, allowing for the first time a comparison of this molecule from all of the major land plant and algal lineages. Using a distance matrix method, the phylogenetic relationships among representatives of these lineages have been inferred and the results indicate a common origin of plastids from a cyanobacterium-like ancestor. Within the plastid line of descent, there is a deep dichotomy between the chlorophyte/land plant lineage and the rhodophyte/ chromophyte lineage, with the cyanelle of Cyano- phora paradoxa forming the deepest branch in the latter group. Interestingly, Euglena gracilis and its colorless relative Astasia longa are more related to the chromophytes than to the chlorophytes, raising once again the question of the origin of the euglenoid plastids.

Key words: Molecular phylogeny -- Plastid -- Al- gae -- Plant -- Small subunit RNA

Introduction

Although the endosymbiotic origin ofplastids is now widely accepted (Gray 1989), the number of endosymbiotic events necessary to explain the diversity of modern plastid types remains unresolved. Based on differences in accessory light-harvesting pig-

Offprint requests to: S.E. Douglas

ments exhibited by the three major lineages of photosynthetic eukaryotes (Chlorophyta, Rhodophyta, and Chromophyta), multiple events involving hy- pothetical prokaryotes containing those distinct pigment complements (chlorophyll b, phycobilipro- teins, and chlorophyll c, respectively) were proposed to give rise to extant plastids (Raven 1970). Sub- sequent events involving eukaryotic endosymbionts were proposed to give rise to those plastids surrounded by more than two membranes (Gibbs 1978; Whatley et al. 1979). An alternative hypothesis, ex- tended by Cavalier-Smith (1982), suggested that only two endosymbiotic events were necessary--the first between a cyanobacterium and a nonphotosynthetic phagotrophic eukaryote, and the second between the resulting photosynthetic eukaryote and a different nonphotosynthetic phagotroph. In this latter hypothesis, the diversity of plastid types is proposed to have arisen by modification of the original en- dosymbiont as it was converted into the different types of plastids found in the three main lineages. All plastids surrounded by only two membranes arose from the first association and those surrounded by three or four membranes from the second association.

The discovery of the prochlorophyte Prochloron didemnL a prokaryote containing both chlorophylls a and b (Lewin 1975), led to the proposal that it could be related to the putative ancestor of the plastids of the chlorophyll a/b-containing plants. Sim- ilarly, it was postulated that the brownish photo- heterotroph Heliobacterium chlorum could be a representative of the lineage that gave rise to the putative ancestor of the.~hlorophyll a/c-containing algal plastids (Margulis and Obar 1985). However, these hypotheses are gainsayed by the results of molecular phylogenetic analyses. Inspection of small

268

s u b u n i t r R N A s (Ssu r R N A s ) s h o w e d H. chlorum to be a m e m b e r o f the F i r m i c u t e s ( G r a m - p o s i t i v e b a c - t e r i a s ensu W o e s e ) ( W o e s e et at. 1985; W o e s e 1987) a n d to b e a r n o c lose r e l a t i o n s h i p w i t h c h r y s o p h y t e p l a s t i d s ( W i t t a n d S t a c k e b r a n d t 1988).

S i m i l a r s tud i e s i n d i c a t e d t ha t e u g l e n o i d a n d c h l o - r o p h y t e / l a n d p l a n t p l a s t i d s as wel l as the c y a n e l l e o f Cyanophora paradoxa fel l w i t h i n t he c y a n o b a c - t e r i a l l i ne o f d e s c e n t ( G i o v a n n o n i e t al. 1988; T u r n e r e t al . 1989). A l t h o u g h p h y l o g e n e t i c s t ud i e s u s ing psbA g e n e - e n c o d e d p r o t e i n s e q u e n c e s y i e l d e d con - f l ic t ing resu l t s b e t w e e n t h e m s e l v e s c o n c e r n i n g the r e l a t i o n s h i p o f p r o c h l o r o p h y t e s to t he p l a s t i d s o f c h l o r o p h y l l a/b-containing e u k a r y o t e s ( M o r d e n a n d G o l d e n 1989a ,b ; K i s h i n o et al. 1990), Ssu r R N A c o m p a r i s o n s h a v e s h o w n tha t p r o c h l o r o p h y t e s , whi le fa l l ing w i t h i n t he c y a n o b a c t e r i a l p h y l u m , a r e un -

l i ke ly to be c lose ly r e l a t e d to t hese o rgane l l e s (See- w a l d t a n d S t a c k e b r a n d t 1982; T u r n e r et at. 1989). H o w e v e r , p h y l o g e n e t i c r e c o n s t r u c t i o n us ing rbcL ( D o u g l a s et at. 1990; M o r d e n a n d G o l d e n 1991) a n d rbcS ( M o r d e n a n d G o l d e n 199 i ) g e n e - e n c o d e d p r o - t e in sequences i nd i ca t e a p o l y p h y l e t i c or ig in for s o m e p l a s t i d s f r o m the b e t a a n d g a m m a g r o u p s o f P r o - t e o b a c t e r i a (pu rp l e b a c t e r i a sensu Woese ) , w h i c h b e a r n o c lose r e l a t i o n to t he c y a n o b a c t e r i a , b a s e d o n Ssu r R N A a n a l y s i s ( W o e s e 1987).

A n a l y s e s o f S s u r R N A s e q u e n c e s o f p l a s t i d s f r o m r e p r e s e n t a t i v e s o f t he v a r i o u s p h o t o s y n t h e t i c eu- k a r y o t i c l ineages m a y a l l o w r e s o l u t i o n o f th i s con - t r o v e r s y . S m a l l s u b u n i t r R N A s e q u e n c e s a r e g o o d m o l e c u l e s fo r p h y l o g e n e t i c i n f e r ence as t h e y a r e p r e s e n t in a l l o r g a n i s m s , a la rge d a t a b a s e exis ts , t h e y c o n t a i n a large n u m b e r o f n u c l e o t i d e p o s i t i o n s for c o m p a r i s o n , a n d s e q u e n c e a l i g n m e n t is r e l a t i v e l y s t r a i g h t f o r w a r d ( G r a y et at. 1984). H e r e we r e p o r t t he s e q u e n c e o f t he Ssu r R N A o f t he Cryptomonas

p l a s t i d , in fe r p h y l o g e n e t i c t rees u s ing th i s se- q u e n c e a n d t h o s e o f o t h e r a lga l g r o u p s a n d c y a n o - b a c t e r i a , a n d d i s c u s s t he resu l t s in r e l a t i o n to t he m o n o p h y l e t i c / p o l y p h y l e t i c o r ig in o f m o d e r n p l a s - r ids a n d the p o s s i b l e n u m b e r o f e n d o s y m b i o t i c e v e n t s g i v i n g r i se to t h e m .

Materials and Methods

Sequence Determination. Plastid DNA preparation, construction of a clone bank from Cryptomonas ~, and localization of the rRNA genes on a small (5.5-kb) inverted repeat have been described (Douglas 1988). The location of one of the rRNA cistrons on a 15.5-kb BamH I/Sac I fragment and the sequence 3' to the Sma I site have been published separately (Douglas and Durnford 1990b). The remaining sequence was obtained from a 1.2-kb Sac I/Sma I fragment containing most of the 16S rRNA gene, which was subcloned from the 15.5-kb BamH [/Sac I fragment, and the adjoining Sac I fragment. Both strands were sequenced com- pletely by the dideoxynucleotide chain termination method (Sanger et al. 1977). The 5' and 3' termini of the Ssu rRNA gene were

tentatively identified by alignment with corresponding regions from published Ssu rRNA sequences.

PhylogeneticAnalysis. Small ribosomal subunit sequences were aligned by eye and the alignment verified by secondary structure analysis (Gutell et al. 1985). Only positions that are clearly ho- mologous and for which sequence data are available for all organisms (indicated in Fig. 1) were used in the phylogenetic analyses. Accounting for gaps introduced in the alignment process, a total of 862 positions were used in the analysis for the tree presented in Fig. 2 and 1118 for that in Fig. 3.

The least squares, distance matrix program of Olsen was used to infer phylogenetic trees (Olsen 1988). Briefly, similarity values for each pair of sequences were calculated b y the formula S =

M/[M + U + (G/2)], where S is similarity, M is the number of matching nucleotides, U is the number of nonmatching nucleotides, and G is the number of nonmatching gaps introduced in the alignment process. In the analysis presented here, gaps greater than a single nucleotide were treated as a single gap. Similarity values were converted to estimated evolutionary distances using the formula of Jukes and Cantor (1969), and the resulting distances were used to infer a phylogenetic tree by means of a least squares, distance matrix method (Fitch and Margoliash 1967; Olsen 1988). Confidence values for individual branches of the tree were determined by a bootstrap analysis in which 100 or 200 bootstrap trees were generated from resampled data (Felsen- stein 1985).

The possibility of an artifactual tree topology due to variable mutation rates between sequences was investigated by the method of Olsen (Olsen 1987). In brief, a site-to-site variability is assumed whereby 95% of the sequence positions vary in mutation rate around a median value in a manner described by a log- normal distribution. The assumed rate variation ranges from a value of alpha to the reciprocal of alpha (1/alpha), the particular value of alpha being an arbitrary positive number. In this analysis, values of alpha employed were 2, 4, 8, 16, 32, 64, and 99 (the last being the limit of the program).

All analyses were run on a Digital Equipment Company MicroVax II computer. The use of computer programs and hard- ware was provided by N.R. Pace of Indiana University.

Results and Discussion

T h e c o d i n g s e q u e n c e o f t he 16S r R N A gene f r o m Cryptomonas �9 is 1492 n u c l e o t i d e s in l eng th (Fig. 1). T h e s e d a t a h a v e b e e n d e p o s i t e d w i t h t he E M B L D a t a L i b r a r y u n d e r a c c e s s i o n n u m b e r X 5 6 8 0 6 . T h e 3' t e r m i n a l s e q u e n c e c o n t a i n s a c lass ica l S h i n e - D a l - g a r n o s e q u e n c e ( 5 ' - C C T C C T T - 3 ' ) t h a t is c o m p l e - m e n t a r y to u p s t r e a m s e q u e n c e s o f s e v e r a l p r o t e i n c o d i n g genes f r o m Cryptomonas �9 ( D o u g l a s a n d D u r n f o r d 1989, 1990a; D o u g l a s et al . 1990; R e i t h a n d D o u g l a s 1990). T w o p u t a t i v e p r o m o t e r ele- m e n t s , b o t h sha r i ng e x t e n s i v e s e q u e n c e s i m i l a r i t y w i t h t he c a n o n i c a l - 3 5 ( T T G A C A ) a n d - 1 0 ( T A - T A A T ) s equences ( P r i b n o w 1975), a re f o u n d u p - s t r e a m o f t he 5' e n d o f the 16S r R N A c o d i n g se- q u e n c e (P1, P2; Fig . 1). T h e r e is n o e v i d e n c e o f a gene fo r t R N A va~, such as is f o u n d in t h e r e g ions u p s t r e a m o f l a n d p l a n t p l a s t i d r R N A c i s t rons . T h e 16S r R N A m o l e c u l e can b e f o l d e d a c c o r d i n g to t he gene ra l s e c o n d a r y s t r u c t u r e o f G u t e l l e t al. (1985)

Pl agaaaaattgtaatttttatca~tatttatactcattttatttcctacaatatttaaatatttagggtt~a~aatattggttgaaacqataa~ttgtatt

P2 caggatataaaaagtttgtttacatttcgtaaacaaaatttaattctaag•ataaagtattattaaatattggAAATCATGGAGAGTTTGATCCTGGCTC

AGGATGAACGCTGGCGGTATGCTTAACACATGCAAGTCGTACGAAAGTTTTTAACTTTAGTGGCGGACGGGTGAGTAACACGTGAGAATCTACCCTTAGG um|ii1voluu I U | V | | W I | I | W | U W | I | I | | I W t 0 1 | | I U V I | W g f I | I | I V

269

100

200

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AGGA~GATAACAGC TGGAAACGC~ TGCTAATACTCCATATGCTGAAGAGTGAAAGGGAAACCACCTAAGGAAGAGCTCGCGTCTGATTAGC TAGTTG 400 I I I I I I I I I I I I I I ! I ! I l I I | f I I | I ! I I I i l 1 1 1 1 1 1 . 1 1 1 1 " 1 1 I I I I I I I | I | I I I I I I | I | I I I I I I I I I I N I I I I I f f I ~ I I ~ N I I I I I | I I I I I I I I I I I I I l I I I I I I P I I !

GTAGGGTAAC-GGCCTACC`~GG~GACGATCAGTAGCTGGTTTGAGAGGACGATCAG~ACA~TGGGACTGAGACACG~-~CAGA~T~CTACGGGAG~-CAG 500 I I I , I I . I I , I , l n I I I I U I I I I I I I I l l n n 11 l l n I I n I I I I i i i i , l I I I f I I n n m 1, 11 , l i ,1 ,1 , l I I le i i n l l I I I I l l , I 11 I . l l . I I I I 11 1, l l l , l , I I U I I I I I I I I l l , I I I U n I I I I I I I I I I n 1, , i I I I I i i i i i I I I I i i I i I I I I n I I I I u I I I i I I I i

CAGTGGGGAATTTTCCGCAATC-GGCGcAAG•CTGACGGAGCAATAC•GCGTGAGGGATGAAGGCCTGTGGGTTGTAAACCTCTTTTCTCAAGGAAGAAGT 600 II I111 II l l II II II II II II U II l l II II II N U II II I I II II II II U II II | I II II II n II II II II n II II I | II l | u i i . | I II II II I! l l m | I II II II l ! II II I I . II II II l l II n II II II II II II II II II II II II I I l . II n n II II II II 11 II II II II

TC TGACGGTACTTGAGGAATAAGCATCGGC TAAC TC TGTGCCAGCAGCCGCGGTAATACAGAGGATGCAAGCGTTATCCGGAATTAC TGGC4:ATAAAGCG 700 11111111111111111111111111111111111111111111111111111111111 I I I I I I | I I

TCTGTAGGTTGTTTAGTAAGTCTGCTGTTAAAGACTAGC~:~TTAACCCTAGAAAAGCAGTGGAAACTGCTAGACTTGAGTGTGGTAGAGGTAGAGGGAAT 800 I I ! I I I l I I ) I I I I I I I t } I ~ 0 I I t I I I I I I I I I I I l l II I P l l ~ , l l l l l I t f l l l l l l l l l II II I l l l II U , l l l , l l l I111 I I I I I l l l l l l l U I l l l l l l l n II i i i i i i r I l l l | l | l l l I ; t l l l n l l l l l l U t l II II I I I I II II II l [ II l1

TCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAAGAACA•CAATGGCGAAAGCACTTTACTGGGCCATAA•TGACACTGAGAGACGACAGCTAGGGGA 900 n i i i i 11 II II I i l111 II 11 l , II II n i i o 11 11 11 l l n I i II II n ~ 11 11 II 11111111 , I II n . 1111 11 11 II II 11 I i u u l l l , 1111 II ~ II 11 , l l , n n II 11 II i i l l II II II I i l l II n 11 i i I i i i . u I i 11 i i l l I i i i n i i 11 I i i i n i i i i 11 i i u u i i i i i i 11

GCAAATGGGATTAGATACC~CAGTAGTCCTAGCCGTAAACTATGGATACTAGATGTTGTACGTATTAACCCGTACAGTATCGTAGCTAAGC~GTTAAGTA i000 n i i i i i i i i . I I l l I I n N I I U I I n H u u i i i i l l f l U I | " I I I I I I I ! 11 l l n I I 0 I I I I I I h | l I I I I I I I I I 1 1 1 1 1 . l l l | I I I I | l l l I I I I IS l l I I f l 11 | l M I I f i l l | l l l . l l I I I l I I , I I I I I n , l i i , l I I I I I I I I I | l l

TCCCGCCTGGGAAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAG 1100 U l l n i I I I I l l l l I I f l l I I U I r 1 1 l I I I | l w ,

AACCTTACCAGGGTTTGACATGTCACAAATTTTCTTGAAAAAGAAAAGTC4~CTTC~AATGTGAA~ACAGGTGGTGcATGC4~TGTCGTCAGCT~GTGTC 1200 I I I I I I I I I I I I I I I I I I l , I I I I I I I I I I I I I I I I I l l l l l l l l l , l l l l l l n l l l l . l l l l l l l l n n l l l l l l l l l l l l l T n l l U l l l T

GTGAGATGTTGGGTTAAGTCCCGCAA~GAGCGTAACCCTTGTTTTTAGTTGCCATCATTAAGTT~ACTTTAA~kAGACTGCCGGTGATAAACCGGAG 1300 I I I I l l I I l l I I I I I I I I I I U I I I I I I I I I I I I I I I I l l I I I I I I l l l l l l I I I I I I n i i i i i ! I I l l I I I I | l I I I I , I I I H I I I I I I ,e n I I I t I I I I I I I I I I 11 , l u I I I I I , I I I I l ! l , l ! I , l l H I I I ! l ! I I I I I I I I I I I I I I I I I I Ir I ! I I U I I I I

GAAGGTGAGGACGACGT~AAGTCAGCATGCCCCTTACACTCTGC4?~TACACACGTACTACAATGGTCGAGA~AAAAAGTCGCAAACTTGTGAAAGTAAGC 1400 n l l lu Ul no n i Ul U i i i i 11 l l n u l . iv u , l i , . ol i i i i . n . i i . n ,1 , I l , i i lO Ol i I . i i i i i I lW m OO I I l a . i i I I I I Ul lO l l I I al In i i i i I i I I lq i I In I I I I I I al al l l lO n I I . no I t M Ol Ol I I n lU Wl I I no I I Ul l l no l l I I In I I no n el I I I I I I I I I I

TAATCTTATAAACT~GATCTCAGTTCGGATTGCAGC~TGCAACTCGCCTGCATGAAGTTGGAATCGCTAGTAATCGCCGGTCAGCTATACGGCGGTGAAT 1500 I I I I I I I I I I ~ l l I I I I I I I I I I I I , I I I I I I I I I I I l , M I I I I I I I ! I I . I I I I I I I I I I I I I I I I I I I I . , I , I 1! I I I I I I I I I I I ! I I 11 I I I I I I I I n I I I ! I I I 1 ,1 i , , I l l l | I I I I I I I I I I I I I I I I I I I I U I I ,1 I I I I I I I I U 11 I I m I I l l I I I I I I I I I I n I I I | I I U , I I , , I i

CCGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGCTAGTCATACCCAAAGTCGTTACCTTAACCATTCGGAC~GCGCCTAAGGTAGGG 1600 I 1 1 1 1 I I | 1 1 I I | | 1 1 | l ) | I I I I I | | | I l l I I I I I I | | | l | l l I I I 1 | I I I I I | 1 1 I I I | 1 | | I

TTAGTGACTGGGGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCTTAaagggagatttaaatcttctaag~ttatataaat 1700 atuiivo OlilOii|n

Fig. 1. Nucleotide sequence of the noncoding (RNA-like) strand of the 16S rRNA gene (uppercase letters) and flanking regions (lowercase letters) from the Cryptomonas ,b plastid. Putative promoter regions, P1 and P2, are underlined. Positions used in the phylogenetic analysis shown in Fig. 2 are indicated by ("); those additional positions used in the phylogenctic analysis shown in Fig. 3 are indicated by (').

with conserved stems maintained by compensatory base changes (data not shown).

Phylogenetic analysis of Ssu rRNAs from cyanobacteria and the plastids of the chlorophyll c-con- mining alga Cryptomonas ,b and other algal and land plant lineages shows that plastids arose from a cyanobacterium-like ancestor (Fig. 2). In a bootstrap analysis the plastids formed a monophyletic group in 99% of the bootstrap trees (Fig. 2). Furthermore, this group fell within the cyanobacterial line of descent in 92% of the cases. Figure 3 represents an augmented analysis of the plastid cluster in Fig. 2. Additional plastid rRNA sequences have been in- cluded and the number of sequence positions used in the analysis has been increased.

Following the branching of the plastid cluster

within the phylogenetic trees, there is a dichotomy between the chlorophyte/land plant and rhodophyte/chromophyte plastid lineages (Figs. 2 and 3). The deeper branchings in the latter group are com- prised of the phycobilin-containing cyanelle of the glaucophyte C. paradoxa and plastids of the mac- rophyte red alga Palrnaria palmata, the cryptomo- nad Cryptomonas @, and the unicellular rhodophyte Cyanidium caldarium. The close branching order among this set supports the proposal that the tax- onomic separation between glaucophytes and rhodophytes may be artificialr particularly if C. caldarium is considered to~'be a m e m b e r o f the Glaucophyceae (Cavalier-Smith 1987).

The placement of the Cryptomonas �9 plastid is indicative of its cyanobacterial ancestry (Figs. 2 and

270

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Gloeobacter 7412 R ~ . . . ~ p A n . nidulans

Phorm. 7375 r. hollandica

2_[ Ps. galeata Plect. 73110

a3 C. p_aradoxa cyanelle C r y p t o m o n a s 4, c h p

E. - I , , i py. Ch. reinhardtii

' I ~ Z. mays chp too N. tabacum chp

M. polymorpha chp Syn. 6308

Myxo. 7312 Gloeo. 6501

Osc. 7515 Osc. 6304

Lyn. 7419 I . , Ose. limnetica

~ ' - I Micro. 10 mfx Cyl. 7417

gracilis chp littoralis chp

chp

I I I I > 0.0 0 . 05 0 .10 0 .15

Fixed point mutations per sequence position

Fig. 2. An inferred phylogenetic tree depicting the evolutionary relationships among various cyanobacteria, the prochlorophyte Procholorothrix hollandica, and plastids. The tree was produced by a least squares, distance matrix analysis of 862 aligned 16S rRNA sequence positions. The bracket to the right of the tree denotes the cyanelle/plastid cluster. Distances between terminal nodes are equal to the sum of the horizontal segment lengths joining them. Numerical values associated with each internal branch represent the percentage of instances in which that branch occurred in a set of 100 trees generated from a bootstrap resampling of the original data. The 16S rRNA sequences of Bacillus subtilis and Agrobacterium tumefaciens, whose own relative branch point is represented by 0, were used as outgroups to determine

the root of the tree. Cyanophora paradoxa, P. hollandica, and eyanobaeterial sequence data are from the cyanobacterial 16S rRNA database of N.R. Pace, Indiana Univesity. The references for the other sequences are given in Neefs et al. (1990) with the exception of Pylaiella littoralis (Markowicz et al. 1988). Abbre- viations are as follows: An., Anacystis; C., Cyanophora; Ch., Chlamydomonas; chp., plastid; Cyl., Cylindrospermum; E., Eu- glena; Gloeo., Gloeothece; Lyn., Lyngbya; M., Marchantia; Mi- cro., Microcoleus; Myxo., Myxosarcina; N., Nicotiana; Osc., Os- cillatoria; Phorm., Phormidium; Pr., Prochlorothrix; Ps., Pseudanabaena; Py., Pylaiella; Syn.. Synechocystis; Z., Zea. Ex- cept for Microcoleus 10 mfx, strain designation numbers refer to the Pasteur Culture Collection.

3). This contrasts with the results f rom an analysis o f rbcL gene-encoded protein sequences in which there was an apparent affiliation between this plastid and the /3 group o f Proteobacter ia (Douglas et al. 1990). There are several possible explanat ions for this discrepancy including physical phenomena, such as lateral transfer o f the rbcL gene, and method- ological reasons, such as the relatively l imited number o f rbcL data and the inherent l imitat ions o f m a x i m u m pars imony methods used to analyze them (Sourdis and Nei 1988; Kishino et al. 1990).

The plastids o f bo th Euglena gracilis and its colorless relat ive Astasia longa cluster with those o f the ch romophy te algae Ochromonas danica and Pylaiel- la littoralis, and not with the o ther chlorophyll a/b- containing plastids. A similar association between

ch romophy te and euglenoid plastids was found by others (Markowicz et al. 1988; Wit t and Stacke- brandt 1988) and is suppor ted by the boots t rap analyses. These plastids form a monophyle t i c goup in 87.5% of the boots t rap trees (Fig. 3). Moreover , an analysis that directly addresses the possibility o f to- pological artifacts being produced by differences in evolut ionary clock speeds (Olsen 1987) also supports this result. All phylogenetic trees so inferred had topologies identical to that in Fig. 3, even for assumed variat ions ranging over nearly four orders o f magni tude (data not shown). Both euglenoids and ch romophy te algae store reserve carbohydra te as /~-l,3-glucans in the cytoplasm, unlike land plants and chlorophyte algae, which store a-1,4-glucans in the plastid (Whatley and What ley 1981). Cavalier-

99

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J 68.5 J

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A. tumefaciens

B. subtilis nidulans

Ch. reinhardtii chp

Ch. moewusii Chl. ellipsoidea chp Chl. vulgaris chp

loon~M, polymorpha chp Z. mays chp

N. tabacum chp C. paradoxa cyanelle

P. palmata chp C r y p t o m o n a s �9 c h p

Cy. caldarium chp 100 m

chp

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Py.

U OUTGROUPS

- - CYANOBACTERIUM

I CHLOROPHYTF_~

~ LAND PLANTS

- - GLAUCOPHYTE

- - RHODOPHYTE - - CRYPTOPHYTE ~ RHODOPHYTE

As. longa chp EUGLENOPHYTES E. gracilis chp

danica chp 7 CHROMOPHYTES d littoralis ehp

271

Fig. 3. An inferred phylogenetic tree depicting the evolutionary relationships among various plastids. The tree was produced from an analysis of 1118 aligned 16S rRNA sequence positions as described in Fig. 2. Numerical values associated with each internal branch represent the percentage of instances in which that branch occurred in a set of 200 trees generated from a bootstrap resampling of the original data. The references for the sequences

i , ) 0.10 0.20

Fixed point mutations per sequence position

used are given in Fig. 2 with the exception of Astasia longa (Siemeister and Hachtel 1990), P. palmata (Singh, personal com- munication), and Cyanidium caldarium (Maid and Zetsehe 1990). Abbreviations are as in Fig. 2 with the inclusion ofA., Agrobac- terium; As., Astasia; B., Bacillus; ChL, Chlorella; Cy., Cyanidium; 0., Ochromonas; P., Palrnaria.

Smith (1987) has argued that the euglenoid plastid is not related to those of the chlorophyte lineage, based on lipid composition and plastid DNA organization, particularly the tandemly repeated rRNA genes that resemble prokaryotic rRNA cistrons.

The phylogenetic trees depicting plastid relationships (Figs. 2 and 3) do not by themselves address the issue of the number of endosymbiotic events that gave rise to modem plastids. This can be done only by comparing plastid phylogeny with that of the eukaryodc hosts. A number ofphylogenetic trees containing members of the major photosynthetic eukaryotic groups have been inferred from analyses ofSsu rRNAs (Bhattacharya et al. 1990; Douglas et al. 1991; Eschbach et al. 1991; Hendriks et al. 1991), large subunit rRNAs (Lsu rRNAs) (Perasso et al. 1989; Lenaers et al. 1991) and 5S rRNAs (Hori and Osawa 1987; van den Eyende et al. 1988). Among these trees, the relative branching orders of the algal groups differ. This is perhaps not surprising because they are derived from analyses of different gene products from different sets of organisms and are inferred by different methods. However, those derived from Ssu and Lsu rRNA sequence analyses have several features in common that can be used

as a framework for comparison with the results re- ported here. Specifically, (1) the rhodophyte, chromophyte, and chlorophyte/land plant groups arise as three distinct lineages within a general eukaryotic radiation, and (2) the euglenoids comprise a very deep branching group with no direct relation to any of the other algal groups.

In Fig. 3, the chlorophyte/land plant plastids branch as a monophyletic group as do the host organisms in the eukaryotic trees. This is consistent with the hypothesis that green algae and land plants arose from a single chloroplast-bearing ancestor. The Cryptomonas �9 plastid arises from within the phy- cobiliprotein-containing algae (Fig. 3), supporting the hypothesis that cryptophytes arose by a secondary endosymbiosis of a primitive red alga that con- tained chlorophyll c (Whatley et al. 1979). Recent studies of the nucleomorph and nuclear Ssu rRNA genes from Cryptomonas �9 has confirmed that two separate endosymbiotic events indeed contributed to the formation of cryptpmonad algae (Douglas et al. 1991). J

The chromophyte plastids appear to have a red algal ancestry whereas in eukaryotic trees, the chromophytes and rhodophytes form distinct lines

272

o f descen t . T h i s i m p l i e s t h a t these p l a s t i d s a re t he r e su l t o f o n e o r m o r e e n d o s y m b i o s e s o f a r e d a lga o r i ts p l a s t i d w i t h a e u k a r y o t i c c h r o m o p h y t e ances - tor(s) . T h e m u l t i p l e m e m b r a n e s y s t e m s u r r o u n d i n g m o d e r n c h r o m o p h y t e p l a s t i d s is c o n s i s t e n t w i t h th i s m o d e l . T h e p r o p o s a l t h a t r h o d o p h y t e a n d c h l o r o - p h y t e / l a n d p l a n t l i neages a re s i s t e r g r o u p s o f t h e K i n g d o m P l a n t a e a n d s e p a r a t e f r o m the K i n g d o m C h r o m i s t a , w h i c h i n c l u d e s c h r o m o p h y t e s a n d c r y p - t o p h y t e s ( C a v a l i e r - S m i t h 1986) is n o t s u p p o r t e d b y o u r r e su l t s o r b y the e u k a r y o t i c Ssu a n d L s u r R N A s t u d i e s c i t e d a b o v e .

F u r t h e r m o r e , t he r e l a t i v e l y s h a l l o w p l a c e m e n t o f t h e e u g l e n o i d p l a s t i d s c o u p l e d w i t h t he d e e p p l ace -

m e n t o f E. graci l is in t he e u k a r y o t i c t rees d o e s n o t s u p p o r t t he h y p o t h e s i s t h a t t he c o m m o n a n c e s t o r s h a r e d b y e u g l e n o i d s a n d o t h e r p h o t o s y n t h e t i c eu- k a r y o t e s c o n t a i n e d a p l a s t i d ( C a v a l i e r - S m i t h 1982, 1987). I t d o e s s u p p o r t t he m o d e l w h e r e i n the eu- g l e n o i d p l a s t i d s a r o s e f r o m an e n d o s y m b i o s i s o f an a lga o r a lgal p l a s t i d w i th a n a n c e s t r a l e u g l e n o i d hos t , a l b e i t t he a lga l s y m b i o n t d o e s n o t a p p e a r to h a v e b e e n a c h l o r o p h y t e as o t h e r s h a v e p r o p o s e d ( G i b b s 1978; W h a t l e y e t al . 1979). H o w e v e r , G i b b s (1970) has n o t e d t h a t t he p l a s t i d s o f e u g l e n o i d s r e s e m b l e t h o s e o f b o t h t he C h l o r o p h y c e a e a n d the m o r e p r i m - i t i ve P r a s i n o p h y c e a e ( M i c r o m o n a d o p h y c e a e ) . T h e d i s c o v e r y o f a p r a s i n o p h y t e t h a t c o n t a i n s b o t h c h l o - r o p h y l l s b a n d c ( W i l h e l m 1987) ra i ses t h e pos s i - b i l i t y t h a t e u g l e n o i d p l a s t i d s a r o s e b y u p t a k e o f a p r a s i n o p h y t e a lga o r i ts p l a s t i d b y a e u g l e n o i d a n - ces tor .

T h e s e c o m p a r i s o n s a re n e c e s s a r i l y cu r so ry . A m o r e c o m p l e t e a n a l y s i s m u s t a w a i t t h e a c q u i s i t i o n o f m o r e d a t a a n d a r e s o l u t i o n to the p r o b l e m o f d i f fe r ing t o p o l o g i e s a m o n g b o t h p l a s t i d a n d e u k a r y - o t i c p h y l o g e n e t i c t rees .

Acknowledgments. The authors thank Colleen Murphy for ex- cellent technical assistance with sequencing, Rama Singh for the use of the P. palmata 16S rRNA sequence prior to publication, and Norman Pace for the use of computer facilities. This is NRCC publication number 31966.

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Received November 26 1990/Revised March 28, 1991

molecular evidence for the origin of plastids from a cyanobacterium-like ancestor

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