BioSystems, 28 (1992) 57-68 Elsevier Scientific Publishers Ireland Ltd.

57

Eukaryote-eukaryote endosymbioses: insights from studies of a cryptomonad alga Susan E. Douglas Institute far Marine Bioscienees, National Rese,a1"ch Council, 1411 Ozfard Street, Halifax, Nova Scotia B3H 3Z1 (Canada) It has been proposed that those plants which contain photosyntheticplastidssurrounded by more than two membranes have arisen through secondary endosymbiotic events. Molecular evidence confirms this proposal, but the nature of the endosymbiont(s)and the number of endosymbioses remain unresolved. Whether plastidsarose from one type of prokaryotic ancestor or multiple types is the subject of some controversy. In order to try to resolve this question,the plastidgene content and arrangement has been studied from a cryptomonad alga. Most of the gene clusterscommon to photosynthetic prokaryotes and plastids are preserved and seventeen genes which are not found on the plastid genomes of land plants have been found. Together with previously published phylogenetic analyses of plastidgenes, the present data support the notion that the type of prokaryote involved in the initialendosymbiosis was from within the cyanohacterialassemblage and that an earlydivergence giving rise to the green plant lineage and the rhodophyte lineage resulted in the differences in plastid gene content and sequence between these two groups. Multiple secondary endosymhiotic events involving a eukaryotic (probably rhodophytic alga) and differenthosts are hypothesized to have occurred subsequently, giving rise to the chromophyte, cryptophyte and euglenophyte lineages.

Keywards: Plastid; Algae; Monophyletic; Polyphyletic; Evolution.

Introduetion

Cryptomonad algae present an interesting group of organisms for the study of the evolution of algae. These predominantly marine, unicellular biflagellate cells share pigment characteristics from two of the major algal groups -- chlorophyll c present in chromophytes and phycobiliproteins present in rhodophytes. Like chromophyte algae, they possess an extra pair of membranes surrounding their plastids, but unlike chromophyte algae, they contain a small nucleus-like organelle (the nucleomorph) in the periplastidal space between the inner and outer plastid membrane pairs. The nucleomorph has been shown to be a functional organelle by electron microscopic and molecular techniques (for review, see Gibbs, 1992). Correeponden~ to: Susan E. Douglas, Institutefor Marine Biosoiences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia B3H 3Z1, Canada.

The presence of the extra pair of membranes, the nucleomorph and the unique pigment composition, has led to the suggestion that the plastids of cryptomonads arose by endosymbiosis of a eukaryotic, probably red, alga that contained chlorophyll c (Whatley and Whatley, 1981; Gibbs, 1981). The rRNA genes from both the nucleomorph and the nucleus (representing the primary and secondary hosts) have been cloned and sequenced and phylogenetic analysis supports this hypothesis (Douglas et al., 1991). To further elucidate the nature of the primary endosymbiont, the plastid genome of Cryptomona~ @ has been mapped and several genes not reported on plastid genomes of land plants have been identified. Analysis of these data has important consequences regarding the monophyletic (Cavalier-Smith, 1982) vs. polyphyletic (Margulis, 1970; Raven, 1970; Whatley and Whatley, 1981) origin of plastids. A hypothetical evolutionary scheme consistent with the majority of available data is presented.

0303-2647/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland

58

Materials and methods

ed by limited sequencing of cloned fragments using the FASTA program (Fig. 2) revealed the locations of 15 genes. Seventeen of the genes have not been detected in chlorophyte plastids (Table I). Three tRNA genes (trnR, trnV and trnT) are located between the rps4 and cs genes and 2 tRNA genes (trnA and trnI) are located within the rRNA operons (Douglas and Durnford, 1990). The transcriptional orientation of all but 3 genes has been determined by sequence analysis. The ribosomal protein operons $10, spc, alpha and str contain 10, 9, 3 and 6 genes, respectively and are transcribed in the same direction.

Plastid DNA from Cryptomonaz ~ was prepared from total DNA by Hoechst 33258cesium chloride density gradient centrifugation as previously described (Douglas, 1988). The approximate locations of the psaC and psaD genes were determined by Southern hybridization analysis using probes from Porphyra umbilicalis and tomato provided by Drs. Michael Reith and Kenton Ko, respectively. In addition, cloned fragments of the C~ptomonas • plastid genome were sequenced using standard methods and open reading frames identified using the DNA Strider computer program (Marck, 1988). Matches with protein sequences in the Genbank SwissProt databank were obtained using the FASTA program (Pearson and Lipman, 1988).

Discussion

The plastid genome of Cryptomonas e~is small (approx. 118 kb; Fig. 1) and contains an inverted repeat which is only large enough to encode the rRNA operons. In contrast, the plastid genomes of chlorophytes are approximately 150 kb, mainly due to the large inverted repeats (25-30 kb). Sequence analysis of various regions of the plastid genome such as the Rubisco genes (Douglas and Durnford, 1989; Douglas et al.,

Results

A total of 59 genes (excluding the rRNA operons and tRNA genes) have now been located on the plastid genome of Cryptomonas ,b (Fig. 1). Analysis of open reading frames obtain-

psbA

cp~ ~

rps4 cs ] rbc,,=.,LS psal.psaL

rRNA

BamHI Smal

0,239p~Nh~at~.~HrpoBCD

4.2

37 22.5

14.5 9.8

1

55

517,182

Pstl 0.5

II 17 8.8 I

1.1

ompR

I

psa..C..rpll ilvB

37

8O

3.~ 5

°ll1.7 1°1

21

I

6O

Xhol

pe!F

acpA hlpA! Sl,,~0s~c a,str RsbB

a~gE

Sad Sail

psa.=ASdnaK psaD

4.7 1.9

'1;9

24 23

13.5

1j9

I 9.8

11

25 10.5

12

55 60

I 6.5 3.9

9

1.5

Fig. 1. Gene map of the 118 kb plastid genome of Cryptomona~ ~. Genes transcribed from left to right are shown on the bettom line, genes transcribed from right to left on the top line and those whose transcriptional direction has not been ascertained on the middle line. The positions of genes whose precise location has not been determined are indicated by dotted lines. Restriction sites for the enzymes BamHI, Sinai, SacI, SalI, XhoI and PstI are indicated by bars. ORF239 is an open reading frame of 239 amino acids with no similarity to any sequence in the Swiss Prot Library.

59

CPCE AEATE ¢ I I ~ R O I ' L A I I T P R O T E Z I C J pRIGDRIIIOR. C r y p £ ~ R P V F P F T A I V G Q E E M K L A L T LNVI D P K I G G V I IMGD R G T G K S T T I RAI TD I LP E I X::.::,:::::.:::: : ::::::::::::::::::::::::::::::::::: CPCS R P V Y P F A A I V G Q D E M K L C L L L N V I DP K I G G V M IMGD R G T G K S T T V R S ~ g ~ LLP E I E]IP ~ Y L % . ~OTRR~%.L nim-N.IEDZNQ PROTEIN C r y ~ c o L K S F I N E L I Y G L S D T A K A F LI I L F T D I F V G F H S T H G W E X.:...:..:.:.:..:::.:.: ::.:::::::.::X HBP LNSW~EFFYNLNDSVK~ F I LLVTDFFVGFHSTRGWE

PRECDl%IIOR

A T P B MARCO A T P E211TIU&~E n T A C~%.IN Cryp£'o p S I F E T G I K V V D LLAP Y R R G G K I G L F G G A G V G K T V L I M E L I NN I A K A H G G V S V F G G V G E R T R E G N D LY :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ATPB LS I FE TG I K V V D L L A P Y R R G G K I G L F G G A G V G K T V L I M E L INN I L K A H G G V S V F G G V G E R T R E G N D L Y A T P E IITN'JP6 A T ~ II"/ETIIAIIII E P E I L O N ~JmAIM Cry~o MS I H I S I I . ~ D R ' I W ! ~ I ~ g EV I LP SS TGQLGI LKGItAPLLTALDI GVMRVRVDRDWI'PI XI . . . . I I ii II ::: I I. :11:; I, I:1:1:: I II 111. II 1,1:. I I;.:**: .1 ATPE MS LTVRVIAPDRTVWDAP~EV I LP S T T G Q L G I LP G H A P L L S A L D T G V L R V R A D K E W L A I A T P A STII~6 A T P I I T ' J I T I I A I I JU,.I'II,A ¢ E A Z X C r y l ~ o R P p G R E ~ P G ~ ' T Y L I t S R L L E R / ~ RI ~ DKLGGGSMTAI~ V I ETQAGDVSAYI P T I ~ I S I TD C,QI F I ~ Ill 111:, II :: l: 11 ::: II II :I:: : I11111 : II :: I:::: III II II III II II11111 lX ATPA R p p G R E A Y P G D V F Y L H S R L L E R A A K L S D A L G G G S M T A L P VI E T Q A G D V S A Y IP T N V I S I T D G Q I F LS ATPZ BPIOL AT7 J~ENTJlAmB C r yp£-o A A Y I G E A L E D H X:::::.::.X ATPI AAYIGES LEGH

A

CHAIN

PRm~O~

ATPE ]dA]~O ATP ETETBASE LI~ID-BIRDXXG pROTEIN Cryp~ ~ P I V S A A S W A S G L S V G LAAI GP GI G Q G T A A A Q A V E G I A R Q P E A E GR I R G T LL :::::::::::::::::::::::::::::::::::::::::::::::::::::: ATPH M ~ P L I S A ~ V I A A G L A V G L A S I GF G I G Q G T A A G Q A V E G I A R Q P E A E GK I R G T L L 8 A S 8%qlrP2 P B O T O | Y E T I U I I P?O0 CHLOROPEYLL A A P O P R O T E Z N C r y p t o P KF S Q A L A Q D P A T R R I W Y G L A T A H D F E S H D G M T E E N L Y Q K I F RS HF GH LAI I F L W T S G N L F H V A W O G N F E Q :::::::::::::::::::::::::::::::::::::::::: :::::::::::::::::::::::::::: PSAB P KF S Q D L A Q D P T T R R I W Y G I A T A H D F E T H D G M T E E N L Y Q K I F A S H F G H LAI I F LWT S G T L F H V A W Q G N F E Q PEJ&Z M A R C O PEOTO~BTIJI I R/~%CTZON CENTRE C rypt'o M T A A Y I 2 S I LVP I I GI I F P G L T M A F A F IY I E Q D Q I :::::::::::::::::::::::: :::::::.x PSAI M T A S Y L P S IFVP L V G L I F P A I T M A S L F IY I E Q D E I

$01~IT

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PSAL 8~NEN PEOTOayETIM I RIACTION CENTIUt 8UBOIfIT c r y pt-o V K P Y N D D P F V G N L A T P I T T S S F T R T L L S N L P A Y R A G L S P LLRG L :::::::::::::::::::::::::::::::::::::::::::: PSAL VKPYNGDPFVGHLSTP I SDSGLVKTF IGNLPAYRQGLSP ILPGL

XI

PEBA TOBAC PEOTOEYET/J~ Q(B) PROTEIN Crypt-o I P T L L T A T T V F I I A F I A A P P V D I D G I R E P V A G S L L Y G N N I I T G A V I P S S A F I G V R F Y P IW :::::::::::::::::::::::::::::::::::::::::::::::::: ::..::::x PSBA IP T L L T A T S V F I IAFI A A P P V D I D G I R E P V S G S L L Y G N N I I S GAI Ip T S A A I G L H F Y p IW PE ~m MARPO pHOTOEYIITILM IX P 6 8 0 CHLOROPHYLL A A P O P R O T E I N c r ypt-o M G L P W Y R V H T W L N D P G R L I A V H L M H T A L V A G W A G S M A L Y E L A V F D P SD ::::::::::::::::::::::::::::::::::::::::::::::::: PSBB M G L P W Y R V H T V V L N D P G R L I A V H L M H T A L V S G W A G S M A LYE L A V F D P S D P B B J pILl& PEOTOEYITZM IX R / J k C T I O X C ~ T R E C rypt-o M A S TGRI P LWI I A T F G G I A A L T V V G L F I Y G S Y S G I G S A L .X::::::::.: .:: ...... : : : . : : : : : : . : : . X PSBJ M ~ T T G R I P LW I I G T V A G I W I G L I G L F F Y G S Y S G L G S S L

~

PROTEIN.

RPOB JLl~O DNA-DIR~CTED R~A POL%'~E B~TA CHAIN C ryp{-o P Q D T L A A I DY LI N L K F E I G E T D D I D H L G N R R V R S V G E L L Q N Q V R I G L N R L E ::::::::::::::::::::::::::: :::: : : . . : : : . : .... ::::x RPOB P Q D I L A A V D Y LI K L K F G IGT I DD I D H L K N R R V C S V A D L L Q D Q L K LALNRLE RPOD NO,CO D~A-DZR~CTED P~A POL%q~RJ~E DELTA CNAIN C ryp{-o A R G N I S Q V R Q L V G M R G L M A D P Q G Q I IDLP I K S N F R E G L T V T E Y L I S S Y G A R K G L V D T A L R T A D S G Y L ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: RPOD ARGNI SQVRQLVGMRGLMADPQGE I IDLP I K T N F R E G L T V T E Y I I S S Y G A R K G L V V Q P S R T A D S G Y L PER 8 C/~U ~I~]UULDOX I N . C r y p t o M A T Y K V K L S G E G V D K T IDCP D D Q Y I L D A A E E Q G I D L P Y S C R A G A C S T C A G K V - A G S V D Q S X::::.:... :,::.:::: ::::::::::::::::::::::::::::: ::.:::X PETF A T Y K V T LKTP S G D Q T I E C P D D T Y I L D A A E E A G L D L P Y S C R A G A C S S C A G K V ~ A G T V D Q S

ILVB ECOLI ACETOI2%CTATE 8~IlTI~E Cryp£-o SF I G T D A F P E V D I FG IT Lp I V K H S Y V V R E T K E M G K I V A E S F F I A K Y G R :::::::::::::::::::::::::::::::: . . . . . . . : ::. :X ILVB SMI G T D A F Q E V D T Y G IS I P I T K H N Y L V R H I E E L P Q V M S D A F R I A Q S G R RI~ BA~IT 508 RIB080~ PROTEIM LI. C ry~t o L E Y R A D K S G IVHI S F G K T N F S V N D L L L N L E A V Q E S IDKNRPAGVKGKY X::.::.: .:...::.. : . . . : :..:: :.. : . . : : . . : : . X RPLI VE y R V D K A G N I H V P I G K V S F DN EK L A E N F A A V Y E A L I K A K P A A A K G T Y

OMPR ECOLI TRJU~ICRIPTIONAL RIGULATORY PROTEIN Crypt-o L N K R Q I Y K D N E R I R L T G M E F S L L E L L I S R S G Q P F SR X..: ...... :...::. ::..:. : . : . . . : . : X OMPR LG T R E M F R E D E P MP L T S GE F A V L K A L V S H P R E P LSR

O~R

Fig. 2. Alignments of coding sequences of plastid genes from Cryptomonas 4~ located by FASTA analysis of open reading frames. The SwissProt accession numbers and name of the gene products are indicated in bold face type. Colons represent identical amino acids and dots represent conservative amino acid replacements.

60 Table I.

Genes on the plastid genome of CT"yptomana~ which are unique or found only on the plastid genomes of non-chlorophyte algae (including Eug/ena grac///s). In addition to the published ribosomal protein gene sequences from the plastid of Cryp~nnonas @, ten ribosomal protein genes that are absent from chiorophyte plastids have been detected (Wan~, Liu and Douglas, unbublished). References are indicated by superscripts as follows: 1Wang and Liu, 1991; 2this study; sDouglas and Durnford, 1989; 4Reith and Douglas, 1990; 6Douglas, 1991; 6Douglas, 1992; 7Reith and Munholland, 1991; SBryant and Stirewalt, pers. commun.; 9Scaramuzzi et al., 1992; l°Hwang and Tabita, 1991; 11Reith, pers. commun.; 12Bryant et al., 1991; lSNeumannSpallart et al., 1990; 14Shivji et al., 1992; 15Starnes et al., 1985; 16Neumann-Spallart et al., 1991; 17Kraus et al., 1990; lSMontandon and Stutz, 1983; 19LSffelhardt et al., 1991; 2°Kessler et al., 1992; 210rsat et al., 1992; 22Valentin, pers. commun. In Cryptomonas @

Examples in other non~ddorophyte algae

dnaK 1

Porphyra umbilivalis 7, Cyanophara paradox~ s, Pavlova lu~wri 9 Cylindrotheca sp. 10, C. paradoza s P. umbilicalis 11, C. paradoxa 12'1s rhodophytes 14, chromophytes 14 and C. parad(~ 15 rhodophytes 14

o~A 1 petF 2 rbcS s c/~B 4 rps95 rpsl05 ~,p/12 rp/135

tufA5 secY6 ,//vB2 ompR2 ¢$2

C. paradoza 12'16 C. paradoza s

P. y,Ybb'~/ca/'t$11, C. paradoxa 17, Euglena grae///s is P. umbilicalis 11, C. parado'xa la P. umbilicalis n C. paradoxa s, P. umbilicalis 11, Cyanidium ¢aldarium 20, Porphyridium aerugineum 20 C. paradoza s, P. undd//ca//s 11, E. grav///s 21, Olisthodiscus luteus 22

psaD 2 psaL 2 hlpA 1

1990), the ribosomal protein operons (Douglas, 1991; Douglas, 1992; Wang, Liu and Douglas, unpublished) and the rRNA spacer (Douglas and Durnford, 1990) have shown that the genes are tightly packed with very small intergenic regions. Although sequence analysis of the plastid genome of Cryptomonas • has not been extensive enough to determine if the psbCD and

atpDF genes are overlapping as in certain chromophytes (Kowallik, 1991) one case of overlapping genes has been found within the spc ribosomal protein operon (Wang, Liu and Douglas, unpublished). It is also evident from Fig. 1 that several gene clusters which are present in cyanobacteria (e.g. atpA, psaAB, rpoBCD, rbcLS and ribosomal protein operons) are preserved on the plastid of Cryptomonas ~. In the several chromophyte and rhodophyte plastid genomes that have been studied, as well as the cyanelle genome of Cyanophora parad~a, the prokaryotic operons have generally been conserved (for reviews see Kowallik, 1989; Gray, 1991; Palmer, 1991; Shivji et al., 1992). A few of these clusters (atpA, psaAB and ribosomal protein operons) have been retained on the plastid genomes of land plants although they contain fewer genes and are rearranged in some cases. The plastid genome of Eug/ena grac/l/s also contains some of these operons (Palmer, 1991) although the genes are characterized by many introns. No introns have yet been found on the plastid genome of Cryptomonas ~. The retention of these gene clusters in the plastids of such divergent groups of plants can most parsimoniously be explained by their sharing a common ancestor. An extraordinary degree of convergent evolution would otherwise have to be invoked to explain the preservation of such similar plastid genomes from different endosymbiotic ancestors. A number of genes which are not present on the three completely sequenced land plant plastid genomes (Ohyama et al., 1986; Shinozaki et al., 1986; Hiratsuka et al., 1989) have been located on the plastid genome of Cryptomonas ¢ (Table I). Some of these genes are involved in such disparate functions as pigment biosynthesis (cpeB, cs), amino acid biosynthesis (ilvB), fatty acid biosynthesis (avpA), transcriptional regulation (ompR), protection from heat shock (dnaK), protein export (secY), electron transfer (petF) and DNA binding (hlpA). Others are genes retained from large prokaryotic operons (such as the Rubisco and ribosomal protein operons). Some of these genes have been located on other algal plastid genomes (see Table I) but

61

it is notable that cryptomonads contain several genes that are unique among all of the algae studied thus far. Since cryptomonad algae contain a nucleomorph in addition to the nucleus, they may represent an evolutionary intermediate in which gene transfer from the plastid has been retarded. It is more likely that allplastids had a similar initialgene content and there has been differentialloss by gene transfer (Baldauf and Palmer, 1990), gene substitutionor gene loss during the long period of evolution and plastid diversificationfollowing endosymbiosis, than that different gene contents in ancestral plastids have become similar by convergent evolution. An impressive body of sequence data has now been accumulated for several algal plastid genes including rbcL, rbcS,psbA, a*pB, 16S rRNA and several ribosomal proteins. Phylogenetic relationships inferred from some of these gene sequences have been variously interpreted to support either monophyletic (single ancestor) or

68.5 96.5

polyphyletic (multiple ancestors) scenarios. The

psbA gene is very highly conserved, even among distantly related organisms and it is doubtful that it is phylogenetically informative. Gene trees constructed using the ctpB gene (Kowallik, 1991) and small subunit ribosomal RNA gene trees (Douglas and Turner, 1991) support a monophyletic origin of plastids of all plant lineages from within the cyanobacterial group of eubacteria (Fig. 3).No specificrelationship to prochlorophytes or the brownish photoheterotroph Heliobaeterium cMorum, once postulated to be the ancestors of the chlorophyll a/b and a/c lineages, respectively, has been demonstrated. Furthermore, recent evidence from studies of 16S rRNA (Urbach et al.,1992) and rpoC1 (Palenik and Haselkorn, 1992) from three groups of prochlorophytes show them to be scattered throughout the cyanobacterial assemblage rather than forming a specificclade amongst the cyanobacteria from which chlorophyte plastids arose.

Agrobacterium tumefaciens Bacillus subtilis Anacystis nidulans Chlamydomonas reinhardtii chp Chlamydomonas moewusii chp Chlorella ellipsoidea chp ', ChloreUa vulgaris chp loo~.._Marchantia polymorpha chp

] O~OUPS -- CYANOBACIERIUM I CHLOROPHY'IES

LAND PLANTS

~. J U

Cyanophora paradoxa cyanelle Palmaria paimata chp Cryptomomus • c h p ]C~anidium caldarium ChPAstasia longa chp lOO '

,

0

,

Euglena gracilis chp Ochromonas danica chp Pylaiella littoralis chp

.

GLAUCOPHYTE RHODOPHYTE CRYPTOPHYTE RHODOPHYTE .a EU~I~OPHYTES "1 CHROMOPHYrES

_1

)

0.10 0.2 0 Fixed point mutations per sequence position

Fig. 3. Phylogenetic tree based on plastid-encoded small subunit rRNA constructed by Douglas and Turner (1991). The tree was produced by a least squares, distance matrix analysis of 1118 aligned 16S rRNA sequence positions. Chloroplast sequences are denoted by 'chp'. Numbers associated with each internal branch represent the bootstrap values from 200 resamplings of the original data. For further details, please consult the original article.

62

polyphyletic origin of plastids (Douglas et ai., 1990; Vaientin and Zetsche, 1990), they can also be explained by lateral gene transfer of B-purple bacterial Rubisco genes (Douglas et al., 1990; Assaii et al., 1990). Lateral transfer of genes into the plastid has not been demonstrated, but successful artificial transfer of DNA into the plastid has been achieved by a number of

On the other hand, phylogenetic trees based on Rubisco genes (e.g. Douglas et al., 1990) have shown that the Rubisco genes of the rhodophyte/cryptophyte/chromophyte lineages are more closely allied to those of the/3-purple bacteria and those of the chlorophyte/land plant lineage to the cyanobacteria (Fig. 4). While these data may be interpreted to indicate a

Rhodospirillum_J aBacteriaPUrple Alcaligenes

60

277

68

" 7 13 Purple

.J

Cryptomonas-~

41

63

Bacteria

Cryptomonad (

100%

Porphyridium-~

31

Rhodophyte

--"1 7 Purple

65

Chromatium _._J Bacteria 34

83

AnabaenaAnacystsi m

100%

1 Cyanobacteria

29 29 76 %

(

30

Euglena

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22 71%

ChlamydomonasI Green

12 14

Chlorella

18

Marchantia

34

.~

I Algae

99%

1° I

78 %

25

Nicotiana

Land Plants

Fig. 4. Phylogenetic tree based on plastid-encoded rbcL gene constructed by Douglas et al. (1990). The tree was produced using parsimony analysis of aligned amino acid sequences. Numbers associated with each internal branch represent the bootstrap values from 100 resamplings of the original data. For further details, please consult the original article. A more recent tree (Morden and Golden, 1991) places the chromophyte, Olisthodiscus with Cryptomonas and Parphyridium, and Cyanophora pavadoxa as the closest relative of green algae and land plants (indicated by arrows).

63 methods. The presence of plasmids in a number of algae (see Villemur, 1990), the fact that a copy of the Rubisco genes resides on a plasmid in the t~-purple bacterium Alcaligenes eutrophus (Anderson and Wilke-Douglas, 1984) and the demonstration of a reverse transcriptase-like sequence in the plastid genome of a green alga (Kfick, 1989) make lateral transfer a possibility. Plastid genomes may in fact be chimaeras of genes from different prokaryotic sources as discussed by Martin et al. (1992). A hypothetical scheme that is consistent with the available molecular data is shown in Fig. 5. The common ancestor of plastids is proposed to have arisen from within the cyanobacterial group of prokaryotes (step 1) and had the potential to express chlorophylls a, b and c as well as phycobilins. This type of prokaryote may also have contained the t~-purple bacterial Rubisco operon since cyanobacteria and purple bacteria may have shared a common ancestor (Olson and Pierson, 1987). Alternatively, this Rubisco operon could have been acquired later by the photosynthetic eukaryote ancestral to the rhodophytes and those algae resulting from secondary endosymbioses. Although endoymbiosis may have been common at this early time in evolution, productive endosymbiotic events which resulted in stable, transmissable plastids were probably rare. Transfer of genes to the nucleus and the addition of targetting signals in the correct position of transferred genes are very specific events which are not likely to have occurred many times. Multiple productive secondary endosymbiotic events, on the other hand, may have occurred more frequently because the plastid probably had stabilised by the time these occurred. From the first photosynthetic eukaryote three lineages diverged, one leading to chlorophytes (step 2), one to cyanelle-bearing algae such as Cyanophora paradoxa (step 3) and one to the eukaryotic common ancestor of rhodophytes, cryptophytes, chromophytes and euglenophytes (step 4). In the chlorophyte lineage, starch is stored as the a-l,4 type in the plastid (Whatley and Whatley 1981), the Rubisco genes are of the cyanobacterial type and phycobilins, chlorophyll

c and its light-harvesting antenna have been lost. According to phylogenetic trees based on 16S rRNA "(e.g. Douglas and Turner, 1991), ribosomal proteins (Evrard et al., 1990) and Rubisco (e.g. Morden and Golden, 1991), the cyanelle is the closest relative of green chloroplasts. Like chlorophytes, the Rubisco genes are of the cyanobacterial type, but chlorophylls b and c and the associated lightharvesting antennae have been lost. Starch is stored as the ~-1,4 type in the cytoplasm (Trench, 1982). The eukaryotic common ancestor could then have diverged into a lineage leading to rhodophytes and cryptophytes (step 5) and a lineage leading to euglenophytes and chromophytes (step 6). Rhodophytes and cryptophytes both have retained the B-purple bacterial rather than the cyanobacterial Rubisco and possess a larger suite of plastid genes than found on land plant plastid genomes (this study; Reith, pers. commun.), suggesting that gene transfer to the nucleus has been retarded in this lineage or accelerated in the green lineage. The 16S rRNA (e.g. Douglas and Turner, 1991), Rubisco (e.g. Morden and Golden, 1991) and atpB (Kowallik, 1991) gene trees all show rhodophytes, cryptophytes and chromophytes to be more similar to each other than to chlorophytes. Starch is stored as the ~-1,4 type in the cytoplasm of rhodophytes and the periplastidal space of cryptophytes. Consistent with nuclear small subunit rRNA data (Fig. 6) is the secondary acquisition of a photosynthetic eukaryote by different eukaryotic hosts, generating cryptophyte (step 8), chromophyte (step 9) and euglenophyte (step 10) algae. While cryptophytes retained the nucleus and cytoplasm of the secondary endosymbiont (as the nucleomorph and periplastidal space), chromophytes and euglenophytes lost them. Differential retention of phycobilins and/or chlorophylls b or c and /~-purple bacterial or cyanobacterial Rubisco would have occurred in these three groups. According to this scheme, chromophyte and euglenophyte plastids share a common ancestor. This close relationship is supported by the 16S rRNA data (Fig. 3) and the

64

CRYPTOPHYTE

CHROMOPHYTE

CHLOROPHYTE

EUGLENOPHYTE

CYANELLE-BEARING ALGAE

[]

EXTANT PROCHLOROPHYTES AND CYANOBACTER,A

--

1"

I

Ru sco

Im

PROKARYOTIC COlliSION ANCESTOR Fig. 5. Hypothetical evolutionary scheme to explain the diversity of contemporary plants and algae. All organisms contain chlorophyll ¢ and abbreviations for other components are as follows: B, chlorophyll b; C, chlorophyll c; N, nucleus; Nm, nucleomorph; PB, phycobiliproteins; a, 0, a-1,4 or ~-1,3 glycan starch storage products, respectively. Double membranes surrounding the plastids are indicated by heavy lines and single membranes by light lines. The residual peptidoglycan wall surrounding the cyanelle is indicated by light stippling, and the periplastidal space in the cryptophyte plastid by heavy stippling. Loss of pigments (indicated by -) includes the loss of the associated fight-haravesting antennae. Two points at which 0-purple bacterial Rubisco operons could have been acquired are boxed.

65

Zea mays • Glycine max • ia pumila * I Nanochlorum euca~otum • .~..C.h/ore/.la vulgarise. . . . . • L J (Jnmmyoomonas remnar~, - - I . Volvox carteri • r" c r y p t o m o n a s ( N u ) • Acanthamoeba castellanii D Prorocentrum micanse r ~ ~ Xenopus laevis Homo sapiens I Artemia salina Neurospora crassa - t - . - Saccharomy..~s cerevisiae r Achlya bisexuhlis 1 I I

1 _ Ochromonas danica e ~ Oxytricha nova I. Stylonychia pustulata Euplotes aediculatus Paramecium tetraurelia

H

Dictyostelium discoideum

L__._ Cryptomonas(Nm)"

,- Gracilaria ~ikva'hiae" - ' - ' 1 . Gracilaria lemaneiformis e .~. Naegleria gruberi C~thidia fasciculata

---"~

T~panosomabmcei

•- - - - - - - - - Euglona gracilis e

Giardia lamblia

Vairimorpha necatrix Escherichia coil

0.1

Fig. 6. Phylogenetic tree based on nuclear-encoded small subunit rRNA constructed by Douglas et al. (1991). The tree was produced using a distance matrix analysis of 1001 nucleotide positions. The plastid-containing organisms are indicated by an asterisk. For further details, please consult the original article.

66

preservation of certain gene clusters. For example, it was recently determined that the cs gene in Euglena gracilis is found next to psbCD (Orsat et al., 1992). A similar arrangement is found in the chromophyte alga Olisthodiscus luteus (Valentin, pers. commun.). It should be stressed that this is a tentative scheme and that as more data from algal plastid genomes are acquired, details will change. While the possibility still exists that as yet undiscovered photosynthetic prokaryote(s) represent the closest ancestor(s) to the major plant lineages, the present data support a monophyletic origin of the plastid and multiple secondary endosymbioses. The discoveries of a chlorophyll c-like pigment in the prochlorophyte Prochlorococcus marinus (Chisholm et al., 1992) and in the plastid of the prasinophyte alga Mantionella squamata (Wilhelm, 1988) open the possibility that the ancestor of plastids was a cyanobacterium-like prokaryote which had the capability to express the genes for more than one type of light-harvesting antenna. These could then have been differentially lost or modified in the various plant lineages subsequent to endosymbiosis.

Acknowledgments The gift of probes from Drs. Michael Reith and Kenton Ko and the sharing of unpublished information by Drs. Michael Reith, Donald Bryant, Veronica Stirewalt and Klaus Valentin are gratefully appreciated. Assistance with sequencing by Colleen Murphy and Sheng-Long Wang and critical reading by Carolyn Bird is also acknowledged. This is NRCC publication number 33815.

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