Gene phylogenies and the endosymbiotic origin of plastids

BioSystvms, 28 (1992) 75-90

75

Elsevier Scientific Publishers Ireland Ltd.

Gene phylogenies and the endosymbiotic origin of plastids Clifford W. Morden a, Charles F. Delwiehe b, Marie Kuhsel c and Jeffrey D. Palmer d aDepartment of Botany and H.E.B.P., University of Hawaii, Honolulu, HI 968~, bDepartment of Botany, University of Wisconsin, Madison, WI 58706, e W ~ Center of He~th and ReseatS, Room, 362, New York State Department of Health, Albany, NY 15~87 and aDepartment of Biology, Indiana University, Bloomington, IN $7~05 (USA) The endosymbiotie origin of chloroplasts from cyanobacteria has long been suspected and has been confirmed in recent years by many lines of evidence. Debate now is centered on whether plastids are derived from a single endosymbiotic event or from multiple events involving several photosynthetic prokaryotes and]or eukaryotes. Phylogenetic analysis was undertaken using the inferred amino acid sequences from the genes psbA, rbcL, rbcS, tufA and atpB and a published analysis (Douglas and Turner, 1991) of nucleotide sequences of small subunit (SSU) rRNA to examine the relationships among purple bacteria, cyanobacteria and the plastids of non-green algae (ineludin£ rhodopbytes, chromophytes, a cryptopbyte and a glancophyte), green algae, euglenoids and land plants. Relationships within and among groups are generally consistent among all the trees; for example, prochlorophytes cluster with cyanobacteria (and not with green plastids) in each of the trees and rhodophytes are ancestral to or the sister group of the chromophyte algae. One notable exception is that Euglenophytes are associated with the green plastid lineage in psbA, rbcL, rbc~Sand tufA trees and with the non-green plastid lineage in SSU rRNA trees. Analysis of psbA, tufA, a ~ B and SSU rRNA sequences suggests that only a single bacterial endosympbiotic event occurred leading to plastids in the various algal and plant lineages. In contrast, analysis of rbcL and rbcS sequences strongly suggests that plastids are polyphyleticin origin, with plastids being derived independently from both purple bacteria and cyanobactoria. A hypothesis consistent with these discordant trees is that a single bacterial endosymbiotic event occurred leading to all plastids, followed by the lateral transfer of the rbcLS operon from a purple bacterium to a rhodophyte.

Keywords: Endosymbiosis; Plastids; Bacteria; Algae; Land Plants.

Introduction

The concept of an endosymbiotic origin of the chloroplast from a bacterial progenitor was first proposed over 100 years ago (Schimper, 1883) although only recently has the technology been available to test such a theory. Although this view is now generally accepted (Gray, 1989), the source of the plastid progenitor among different plastid lineages is still questioned. One of the distinguishing traits among the different photosynthetic lineages is the mechanism by which they harvest light for photochemical activity. Chlorophyll a (chl a) is ubiquitous among oxygenic photosynthetic organisms. Cyanobacteria and many non-green algae (rhodophyCorrespondence to: Clifford W. Morden, Department of Botany, 3190 Maile Way University of Hawaii, Honolulu, HI 96822, USA.

tes, chromophytes and cryptophytes among others) utilize phycobflin pigments (e.g., phycocyanin and phycoerythrin) in a proteinaceous phycobilisome on the thylakoid surface to harvest light (Table I). In contrast, green algae (chiorophytes) and land plants contain chlorophylls a and b as light harvesting pigments. Based on this and other evidence, CavalierSmith (1982) proposed a monophyletic origin for plastids from a cyanobacterium, with subsequent evolution of the symhiont giving rise to the diversity of plastid types. Others contend that plastids have been derived from multiple endosymbiotic events involving other bacteria (Lewin, 1981; Sagan, 1967; Whatley, 1981; Whatley and Whatley, 1981) or eukaryotic algae (Gibbs, 1978, 1981; Whatley, 1981; Whatley and Whatley, 1981) in addition to cyanobacteria. The discovery of chl b-containing bacteria (prochlorophytes) led many to believe that they

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

76 Table I. Photosynthetic pigments utilized to harvest light in photosynthetic membranes.

Taxa

Light harvesting pigment

Prokaryotes Purple Bacteria Cyanobacteria Prochlorophyta

bacterioehlorophyll Chl a, phycobilins Chl a and b

Euka~jotes Glaucophyta Rhodophyta Chromophytaa Cryptophyta Chlorophyta Land plants

Chl Chl Chl Chl Chl Chl

a, phycobilins a, phycobilins a and c a and c, phycobilins a and b a and b

~Fne term 'Chromophyta' is used here in the broad sense, and the divisions represented in the studies presented here include the Bacillariophyceae, Chrysophyceae, Phaeophyceae, and Xanthophyceae.

are members of a distinct lineage which included the progenitor of the chloroplast in the chlorophyte lineage and that a cyanobacterium was the progenitor of the non-green plastid lineage. Characteristics of the thylakoids and photosynthetic electron transport supported this view (Whatley and Whatley, 1981; Burger-Wiersma et al., 1986; Burger-Wiersma and Post, 1989) as did some of the early gene sequence data (Morden and Golden, 1989). However, all other analyses of molecular data clearly show that prochlorophytes are integrated in the cyanobacterial lineage and removed from the green plastid lineage (Turner et al., 1989; Morden and Golden, 1991). Recent studies further show that the three prochlorophyte genera (Prochloron, Prochlo~'othrix and Prochlarococcus) are distantly related within the cyanobacteria (Palenik and Haselkorn, 1992; Urbach et al., 1992) which suggests that chl b has either arisen or been lost several times along separate evolutionary lines (Bryant, 1992). The plastid (cyaneUe) of the glaucophyte Cyanophara paradoxa is unique in that it contains a residual peptidoglycan cell wall on the cyaneUe membrane (Aitkin and Stainier, 1979). However, the cyanelle chromosome is similar in size and gene content to that of o~er plastids

(Lambert et al., 1985; Breiteneder et al., 1988). These features have suggested to some that Cyanophora occupies an intermediate position between cyanobacteria and chloroplasts (Maxwell et al., 1986), while others propose that the cyanelle was acquired by an endosymbiotic event separate from those that gave rise to other plastids (Whatley and Whatley, 1981; Lambert et al., 1985; Breiteneder et al., 1988). This paper examines the phylogenetic relationships among purple bacteria, cyanobacteria and the plastids of the various algal groups and land plants using six different gene sequences to determine (1) what differences and similarities occur among the various gene phylogenies and (2) what can be established concerning the number of endosymbiotic events leading to the diversity of plastids. Three of the genes used (psbA, rbcL and rbcS) encode proteins involved in reactions of photosynthesis. Of these, rbcL is being extensively used to determine phylogenetic relationships among green algae and land plants in many laboratories (e.g., Olmstead et al., 1992). The fourth gene, tufA, encoding elongation factor Tu, has been utilized to evaluate relationships among early life forms (Iwabe et al., 1989) and to study plastid gene transfer to the nucleus (Baldauf and Palmer, 1990). The fifth gene, atpB (atpD in the bacterial unc operon) encodes the ~ subunit of the ATP synthase complex and has been utilized to determine relationships among eubacteria (Amann et al., 1988a). The sixth gene, SSU rRNA, encodes the small subunit rRNA, which has been extensively used in anlyzing relationships among bacterial groups (Woese, 1987; many others). Materials and methods

References for many sequences used in the

psbA analysis are cited elsewhere (Morden and Golden, 1989) with the exception of Bumilleriopsis filifarmis (Scherer et al., 1991), Cyanidium caldarium (Maid et al., 1990), Ectoca~pus siliculosus, Antithamnion sp. (Winhauer et al., 1991), Cyanophara paradoxa (Janssen et al., 1989) and rice (Hiratsuka et al., 1989). References for sequences used in the rbcL and

77

rbc~ analyses are cited elsewhere (lVIorden and Golden, 1991) with the exception of Rhodobacter sphaero/des (Gibson et al., 1991), the Alvinoconcha hess/er/ symbiont (Stein et al., 1990), Chromatium vinosum (Kobayashi et al., 1991), ThiobaciUus ferrooxidans (Kusano et al., 1991), Xanthobacterflavus (Meijer et al., 1991), Bryops/s max/ma (Kono et al., 1991), Antithamnion sp. (Kostrzewa et al., 1990), Cyanidium caldarium (Valentin and Zetsche, 1990a), Cylindrotheca sp. (Hwang and Tabita, 1991), Ectocarpus s/l/cu/osus (Valentin and Zetsche, 1990b), Olisthodiscus luteus (Hardison et al., 1992), Porphyra sp. (unpublished sequence provided by S. Douglas), Pylaiella littoralis (Assali et al., 1990, 1991) and the Douglas fir (Genbank accession X52937). TufA sequences used include Astasia /onga (Siemeister et al., 1990), Anacystis nidulans, Escherichia coli, Euglena gracilis, Pseudomonas cepacia, Spirulina platensis, Thermotoga maritima, Thevmus thermophilus (referenced in Ludwig et al., 1990), Arabidopsis thaliana and Chlamydomonaz reinhardtii (Baldauf and Palmer, 1990), Cryptomonas (Douglas, 1991), Cyanophora paradoxa (Kraus et al., 1990), and Salmonella (Gonbank accession X55116). The rest of the tufA sequences are unpublished data of M. Kuhsel, C. Delwiche and J. Palmer. AtpB sequences used include Bavteroides fragilis, Cytophaga lytica DSM 2039 (Amann et al., 1988a), Enterobacter aerogenes, Flavobacterium ferrugineum (Amann et al., 1988b), RhodospiriUum rubrum (Falk et al., 1985), Vibrio alginolyticus (Krumholz et al., 1989), Rhodopseudomonas blastica (Tybulewicz et al., 1984), Esche~ichia coli (Saraste et al., 1981), Bacillus PS3 (Ohta et al., 1988b), Bacillus firmus OF4 (Ivey et al., 1991), BaciUusfirmus RAB (Ivey et al., 1990), Bacillus megaterium (Hawthorne and Brusilow, 1988), Synechocystis 6803 (Lill and Nelson, 1991), Synechococcus 6301 (Cozens and Walker, 1987), Anabaena 7120 (Curtis, 1987), human (Ohta et al., 1988a), cow (Hollemans et al., 1983), Schizosaccharomyces pombe (Falson et al., 1991), Neurospara crassa (Rassow et al., 1990), Nicotiana pIumbaginifolia (Boutry and Chua, 1985), Hevea brasiliensis (Chye and Tan,

1992), maize (mitochondrial: Winning et al., 1990; plastid: Krebbers et al., 1982), Dictyota dichotoma (Leitsch and Kowallik, 1992), Pyella littoralis (Jouannic et al., 1992), Chlamydomonas reinhardtii (Woessner et al., 1986), Marchantia polymorpha (Umesono et al., 1988), tobacco (Kazuo et al., 1983), spinach (Zurawski et al., 1982), and rice (Hiratsuka et al., 1989). The tree for small subunit rRNA is redrawn from Douglas and Turner (1991) and sequences are referenced therein. Because of vastly differing GC selection pressures of the genomes from which these sequences were derived (see Morden and Golden, 1991) and the recent finding that substitutional bias can confound phylogenetic analyses of the taxa involved here (Lockhart et al., 1992), phylogenetic analyses of the protein coding genes were done on the inferred amino acid sequences of the five protein genes. All sequences were aligned using the UWGCG package and adjusted by eye. Most phylogenetic analyses were conducted using the parsimony program PAUP (Swofford, 1989) with the heuristic search option (stepwise addition series using a random addition sequence and 10 replications per run). In all analyses gaps were scored as missing data and not given any a priori weight. Bootstrap analyses were completed by 100 replications of resampled data using the above parameters (Felsenstein, 1985). The small subunit rRNA tree was produced using a least squares distance matrix with bootstraps completed by 100 or 200 replications of resampled data (see Douglas and Turner, 1991). Results and discussion

psbA psbA encodes the photosystem II reaction center protein D1. The D1 sequences fromcyanobacteria, algae and land plants are highly conserved in both length and sequence similarity. Length variation among the known sequences occurs at the carboxyl terminus of the inferred amino acid where there is a seven amino acid gap in the sequences of all chl bcontaining organisms (chlorophytes and Pro-

78

chlarothrix) relative to cyanobacterial and non-

cyanobacterla Anabaena 7120 (1) Anebmena 7120 (2) AnacyBtlenldulane (1) Anacystis nidulans (2) Frernyella dlploelphon Synechocyetis 6803

T.DLAAGEVAPVAI SA IJDLAAG EVAPVALTA LDLAAGEATPVALTA LDLAAGEATPVALTA LDLAAGEVAPVALTA IJDIaASGDAQMVALNA

nongreen algae Cyanophora paradoxa Cyanldlum caldarlum Antlthamnionsp. Ectocarpueeillculoeus Bumlllerlopelsflllformle

T'DLASGEVMPVALTA P: :1 N A * LDLASEVS LFVALNKVE I N<;* IJDLASNES LPLALVAPA I N<;* I~DLASNE I LPVAI SAPSWG * LDLAAG EVL PVAV SA PAVH A •

chl b containing Prochlorothrlxhollandic: Chlamydomonu relnhardtll Euglena gracllIB Marchlmtla polymorpha tobacco rice

L D L A A V K . . . . . . . A P S I I G* LDLASTN ....... SSSNN-* LDLA .................. LDLAAVE ....... APAVNG* LDIJAA I E ....... APSTNG* L D I J A A L E . . . . . . . VP:_;LNG*

PA I NG* PAI NG • P S I HG* PAI NG ~ P A I N~;* p A I E<',"

Fig. 1. Comparison of amino acid sequences at the carboxyl terminus from positions 341 to the terminal codon(*) of the D1 polypeptide from different genera. Residues conserved among all sequences are in bold; missing amino acids (gaps) are represented by a dash.

~ _~

Anabaena 1 Anabaena 2 Frernyella Synechocystis Anacystis 1

I _~

77 77

~

39 27

38

39, 65

I

green algal sequences (Fig. 1). The only exceptions to this occur in Chlamydomonas, where an additional deletion of one amino acid is at the terminus of the sequence and in Eug/ena, where the terminal 16 amino acids of the sequence are absent. Based on the single character of the presence or absence of the seven amino acid region, there would appear to be a specific relationship between Prochlorothriz and chlorophyte plastids. Parsimony analysis of psbA amino acid sequences was done without rooting and yielded a single shortest tree. Although bootstrap values associated with the branches of the tree are generally low, several conclusions can be made (Fig. 2). Cyanobacteria are clearly separated from the algal and land plant lines and only a single endosymbiotic event is necessary to explain the topology of the tree. Prochlarothrix, in contrast to the evidence from the seven amino

[ 59 I

Cyanobacteria

Anacystis 2 Prochlorothrix Cyanophora Cyanidium AntRhamnion

Glaucophyte

] Rhodophyte "1 Phaeophyte

Ectocarpus Bumilleriopsis " l X a n t h o p h y t e Euglena "1

Euglenophyte

Chlamydomonas'-IC hIorop hyte Marchantia tobacco

rice

]

land plants

Fig. 2. Phylogenetic analysis based on the derived protein sequences of psbA. The unrooted single most parsimonious tree contained 428 steps with a consistency index of 0.62. Bootstrap values are shown on the tree.

79

acid gap, clusters strongly within the cyanobacteria. A few isolated groupings among the plastid lineage are moderately to well supported by bootstrap analysis, but other associations are poorly supported and can not be discussed with much confidence.

rbcL and rbcS rbcL and rbcS encode the large and small subunits of the ribosco (ribulose-l,5-bisphosphate carboxylase/oxygense) holoenzyme, respectively. Differences among the holoenzymes with regard to subunit composition, presence or

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173

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3 L R D A A G I L} )LRDAAGVY( )LRD PKGVM3 ~I K D P A A V M I )INDVESVM% ]VKDASALM% ]VKDASTVM% ~ V T D P A PVL[ )VKDSSAIL% ]IKDPAEVM} )VKDPAAVM% )VKDPAAVM% ] E T D V D A I L/ ~EQDPNVILq ZETDIDTIL} ~EQEVDTVI ~ ]AKTSREVL/ 3CKS P Q Q V L [ ~AQS P E E V L . < ~ATE~EVL( ~CRDPMQVL~ :CTDGSQVL~ ZCTDASQVL~ ]CTEASQVI~ ~CTDATQW~ ~CTDATQVL2

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Fig. 3. Aligned sequences of the rbcS derived protein. Species are ordered to reflect similarity among the sequences. Gaps in sequences are indicated by a dash; bold dots are placed above every tenth residue. Boxed residues indicate conserved domains. Chzomatium v/nosum B and S are the rbcB and rbcS sequences, respectively (Kobayashi et al., 1991).

80

contrast, the aligned sequences of the derived rbcS protein contains numerous gaps which are positioned such as to distinguish many of the major lineages (Fig. 3). Despite these obvious differences, certain sequence domains have maintained a high level of conservation (boxed amino acids in Fig. 3).

absence of an operon, and coding site (plastid or nucleus) have been discussed elsewhere (Morden and Golden, 1991). Length variation among rbcL sequences is minimal with a few small gaps near the 5' end and a 21 - 24-bp extension at the 3' end in Alcaligenes eutrophus and non-green algal sequences (Morden and Golden, 1991). In

100 100 100

100 97

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96 100 87

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49 85

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Rhodospirillum 1 Rhodobacter (I I ) ~ ocpurple bacteria Rhodobacter (T) I Xanthobacter ] ~ purple bacteria Alcaligenes Cyanidium "1 Rhodophyte Cryptomonas "1 Cryptophyte Antithamnion Rhodophyte Porphyridium Ectocarpus Phaeophytes Pylaiella Ofisthodiscus -I Chrysophyte Cylindrotheca "1 Basillariophyte Chromatium (A) Alvinoconcha y-purple bacteria Thiobacillus Chromatium (L) Anabaena Prochlorothrix cyanobacteria Anacystis Cyanophora =1 Glaucoph~e ChlamydomonasEuglena Chlorophytes Chlorella Bryopsis _ Marchantia

Douglas fir tobacco

land plants mm

Fig. 4. The rbcL single most parsimonious tree of 1681 steps with a consistency index of 0.71. Bootstrap values are shown on the tree. Rhodobacter sphaero/des I and II are the rbcL form I and form II sequences, respectively (Gibson et al., 1991); Chromatium v/nosum A and L are the rbcA and rbcL sequences, respectively (Kobayashi et al., 1991).

81 Rhodobacter (T~ Xanthobacter

Alcaligenes Cyanidium Porphyra Cryptomonas

2 10

100 ~

27

~~

-"-~

91

99 37

44

a

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purplebacterium

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Rhodophytes "1 Cryptophyte 7 Phaeophytes - ! Chrysophyte

Pylaiella Ectocarpus Cylindrotheca Olisthodiscus "1 Chrysoph~e

]

Porphyridium Rhodophytes Antithamnion Chromatium(B)" Alvinoconcha T purple bacteria Thiobacillus Chromatium(S)_ Chlamydomona~ Acetabularia Chlorophytes Euglena pine rice tobacco

i land plants

Anabaena

77

Anacystis

I cyanobacteria

Prochlorothrix Cyanophora

"1

Glaucophyte

Fig. 5. The rbe~ single most parisomonioustree of 882 steps and a consistencyindex of 0.66. This tree is unrooted and drawn to reflect the ~ tree topology;bootstrap values are shown on the tree. Chromatiumv/nosumB and S are the rbcB and rbc~ sequences, respectivelyCKobayashiet al., 1991). The relationships among these organisms depicted by phylogenetic analyses closely reflect the associations based on the gaps (Figs. 4 and 5). The rbcL tree is rooted using the form II rbcL sequences from Rhodobact~r sphaeroides and RhodospiviIIum ~'ubrum, whereas the rbcS tree is unrooted and drawn to reflect the topology found with rbcL. Two separate endosymbiotic events are required to account for the topology of the rbcL tree. Plastids of most non-green algae appear to be derived from an a- or /~purple bacterium, whereas plastids of

Cyanophzra, green algae and land plants are derived from a cyanobacterium. Bootstrap values associated with most nodes of this tree are fairly high. The rbcS tree differs dramatically in the internal branching of the two main clusters. Here, rhodophytes appear ancestral to the aand/~-purple bacteria while cyanobacteria and Cyanophara form a sister group to the V-purple bacteria and chlorophytes. Bootstrap values of many of the critical nodes are very low, as is the overall conservation of rbcS compared to rbcL, so while the rbcS data appear to corroborate

82 those from rbcL in establishing two different bacterial origins of plastid genes, we are highly suspicious of many of the specific groupings within the two main clusters of rbcS sequences. In addition the rbcS tree structure may also be influenced by selection and codon usage pressures associated with the various genomes in which the gene is found (bacterial, plastid in non-green algae and nuclear in green algae and land plants).

88

tufA The phylogenetic analysis presented here is preliminary to a more complete study to be published elsewhere. Although these are preliminary results, several interesting relationships seem to be emerging from the analysis. A subset of the data is presented here as Fig. 6. The tree is rooted by Thermotoga maritima which has been shown consistently to be at the base of the eubacterial lineage in phylogenetic

h001 I

ee

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Gloeothece Anacystis Plectonema Gloeobacter

Cryptomonas Porphyridium Smithora Gracilaria Vaucheria Laminaria Costaria

:1 Cryptophyte Rhodophytes Xanthophyte ] Phaeophytes

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Euglenophytes

Fig. 6. The tufA consensusof two equallyparsimonioustrees of 855 steps with a consistencyindexof 0.51. Bootstrapvalues shownon the tree.

are

83

analyses (Woese, 1987; Ludwig et al., 1990). In general, the topology is consistent with the hypothesis of a monophyletic plastid lineage, although bootstrap values of some crucial nodes are extremely low (4-14%) in the critical middle portion of the tree. Low bootstrap values are indicative of internal inconsistencies in the dataset (Sanderson, 1989), a property which also manifested itself in the complete data set by the presence of multiple 'islands' of trees that are only one step longer than the single most parsimonious tree (Maddison, 1991), some of which having significantly different topologies. Much of the internal inconsistency of the complete dataset seems to reside in the placement of a few clades, for example the euglenoid clade, which although consistently allied with the green algae, may be placed with nearly equal parsimony at several locations within this lineage. Similarly, Arabidopsis is shown here basal to the green algae, but again may be placed in several locations with nearly equal parsimony. The difficulty in placing the Arabidopsis tufA sequence may be related to the transfer of tufA from the chloroplast to the nuclear genome (Baldauf and Palmer, 1990; Baldauf et al., 1990). Despite these difficulties, the tufA topology is generally consistent with the monophyletic origin of plastids, although the placement of the cyanobacterium Gloeobacter is anomalous and overall support for a monophyletic plastid lineage is by no means conclusive. In overall topology the tufA tree is generally consistent with the psbA tree and is in conflict with the rbcL and rbcS trees.

atpB Rooting of the tree depicting phylogenetic relationships derived from the ATP synthase subunit was done using the bacteroidesflavobacterium phylum identified by Woese (1987). Although few algal groups are represented in the analysis, the topology of this tree strongly supports the hypothesis of a monophyletic plastid origin (Fig. 7). Bootstrap values are consistently high (84% and above) at the critical nodes leading from cyanobacteia to the

algae and land plants. However, the relationship of the plastid and cyanobacterial lineage to the other bacterial groups is unclear. A strict consensus of the seven parsimonious trees forms a trichotomy of the gram-positive bacteria, purple bacteria/mitochondria and cyanobacteria/plastid lineages. These three groups are also closely clustered in a tree based on SSU rRNA sequences C¢~oese, 1987).

Small subunit rRNA The tree depicting phylogenetic relationships derived from SSU rRNA is redrawn from Douglas and Turner (1991). They describe two analyses, the first (not shown) illustrating that all plastids form a monophyletic lineage (with a bootstrap value of 99%) derived from cyanobacteria and the second (Fig. 8) depicting the relationships among the algae and land plants rooted by two bacteria and a cyanobacterium. In this second tree, Cyanophara forms the outgroup to the non-green algae, within which the euglenophytes are fairly deeply imbedded. Bootstrap values associated with this tree are high (generally greater than 80%) except for those nodes surrounding the Cyanophora, cryptophyte and rhodophyte bifurcations. Essentially the same results were also obtained by Maerz et al. (1992) in an analysis of a slightly less comprehensive data set (one lacking Cyanophora and Palmaria).

Similarities among gene trees Several features are common to each of the different gene phylogenies. Most notable is that in all trees the chlorophyte plastid is derived from cyanobacteria. Prochlorophytes are not directly related to the progenitor of green plastids, as found previously and with an additional gene (Palenik and Haselkorn, 1992; Urbach et al., 1992) and therefore the gap at the carboxyl terminus of the D1 protein linking prochlorophytes to green plastids seems to be a product of parallel evolution in chl b-containing organisms. Another similarity, consistent across all gene trees, is the clustering of the non-green algae (excluding Cyanophara). The complex plastid of Cryptomonas appears to have been derived from

84

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Flavobacterium Cytophaga ,Bacteroides BacillusPS3 Bacillus megaterium Bacillus firmus OF4 Bacillusfirmus RAB

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~

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L

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bacteroidesflavobacterlum "--

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----Enterobacter

Escherichia y-purple bacteria Vibrio Rhodopseudomonas -s-purple bacteria 88 I Rhodospirfllum Schizosaccharomyces.m~" fungi 83 I Neurosporo-mt Z .~ ,maize-mt 77 rubber plant-mt land plants 100 I Nicotiana.mt 100 human-mt animals [~----cow-mt Synechococcus Anabaena cyanobacteria Synechocystis Dictyota.cp Phaeophytes 100 [ Pylaiella-cp Chlamydornonas.cp =1 Chlorophyte

,6 !

Marchantia-cp ,o acco.c 95

spinach-cp

1

land plants

100 j '"

maize-cp I =-----.--rice-cp

Fig. 7. The atpB consensus of 7 equally parsimonious trees of 1609 steps with a consistency index of 0.62. Bootstrap values are shown on the tree.

a rhodophyte ancestor via a secondary endosymbiotic event (Douglas, 1991; for further review of cryptophyte plastids see also Douglas, pp. 0000 or Maler, pp. 00-00 of this volume). These data are also consistent with the hypotheses of secondary endosymbioses leading to the plastids of chromophytic algae.

Conflicts among gene trees: one endosymbiosis or two? The most obvious conflict among the six gene phylogenies involves the number of implied endosymbiotic events, one or more than one. The atpB and SSU rRNA tree strongly supports a single endosymbiotic origin of plastids from

85

~ 98

Ch.reinhardtfi 7 Ch.moewusii I Chl.ellipsoidea| Chlorophytes Chl. vulgaris I

99 92

69 97 1001

87 76 58 44 69

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Marchantia

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land plants

Cyanophora "1 Glaucophyte Palmaria "1 Rhodophyte Cryptomonas "1 Cryptophyte Cyanidium "1 Rhodophyte 100 Astasia ] 88 ~ Euglena Euglenophytes 84 ~Ochromonas -IChrysophyte Pylaiella "! Phaeophyte

Fig. 8. The SSU rRNAleast squares distancetree redrawnfrom Douglasand Turner(1991).Bootstrapvaluesare shownon the tree. cyanobacteria, the unrooted psbA tree-is moderately supportive of this, and the tufA tree is consistent with this, but at most only weakly supportive (again excluding the anomalous placement of Gloeobacter). In contrast, rbcL and rbcS trees both strongly support multiple endosymbiotic origins, of the green plastids and Cyanophara from a cyanobacterium and of all other non-green plastids from ~- or B-purple bacteria. Differences among the phylogenies in the placement of euglenophytes (see below) and Cyanophora are also evident. Studies comparing Cyanophora and chlorophyte plastid sequences show that substitutional bias as a consequence of parallel nucleotide change in plastids can link the cyanelle and green plastids (Lockhart et al., 1992), yet this would not explain the profound differences amino acid-based tree topologies shown here. The tree topologies presented for atpB, psbA, tufa and SSU rRNA shows rhodophyte and

chromophyte plastids derived from a cyanobacterium and are consistent with other data such as the presence of phycobiliproteins and the organization of thylakoids. In addition, recent data on the organization of ATPase genes also supports a cyanobacterium origin of all plastid types (Palmer, 1991; Jouannic et al., 1992; Leitsch and Kowallik, 1992; Pancic et al., 1992). In contrast, no other independent line of evidence supports the alignment of rhodophyte and chromophyte plastids with ~- or B-purple bacteria, as indicated by the rbcL and rbcS trees. One simple hypothesis can reconcile these conflicting gene trees (Fig. 9); a single endosymbiotic event occurred that led to all known plastids. This was followed by a basal divergence of green and non-green plastids and a subsequent split of non-greens to give Cyanophora and a lineage comprising rhodophytes, chromophytes and cryptophytes. After this second split, the rbcLS operon (and potentially other genes) was transferred laterally from an ~- or B-purple

86

replacementof cyanobactedalrubisco operon by a laterallytranslerred a-or I~-purplebacterialoperon Purple Bacteria

single primary (eubacterial) origin of plastids

Rhodophytes Chromophytes Cryptophytes

~ two independent completelosseso! the cyanobacterlalcell wall

"

Cyanophora Chlorophytes Land plants cyanobacteria

Fig. 9. Phylogenetichypothesisto reconcileconflictinggene trees. See text for details. This tree purposelydoes not attempt to resolve the current conflict among gene trees with respect to the placement of euglenophytes.

bacterium to the plastid in the lineage leading to rhodophytes, chromophytes and cryptophytes. This phylogeny also implies two independent complete losses of the endosymbiont cell wall. Given this sequence of events, the multiple associations of Cyanophora in the various tree topologies can also be explained. The hypothesis of a lateral transfer of rubisco genes has been suggested previously based on more limited data (Boczar et al., 1989; Douglas et al., 1990; Morden and Golden, 1991; Assali et al., 1991). Mitochondria are derived from within apurple bacteria (Fig. 7; Yang et al., 1985). Since the form I rbcLS operon the a-purple bacterium Rhodobacter sphaeroides clusters with nongreen algae (Fig. 4), it is reasonable to speculate that the rbcLS operon of these algae was derived via lateral transfer from a primitive photosynthetic mitochondrion, which subsequently lost its capacity for photosynthesis. One problem with this hypothesis is that rRNA sequences place mitochondria with a monophyletic a-purple bacteria group to the exclusion of/~purple bacteria (Tang et al., 1985), whereas the rubisco genes (Figs. 4 and 5) place the a- and/~purple bacteria together, as a sister group to the non-green plastids. As an alternative hypothesis, one can postulate an at least transitory independent genetic endosymbiosis of a purple

bacterium with eukaryotes. Martin et al. (in press) have suggested a third possibility, in which the cyanobacterium that gave rise to the plastid possessed two highly divergent copies of the rbcLS operon which were differentially lost in each of the lineages leading to green plastids (and Cyanophara) and non-green plastids. One problem with this hypothesis is that two rubisco operons of the appropriate sequence relationships have not been found in any cyanobacterium or even purple bacterium (the two rbcL genes present in Rhodobacter are too dissimilar to fit this hypothesis, while the two rubisco operons in Chromatium are closely related to each other; Figs. 4 and 5).

Conflicts among gene trees: a red or green origin of the euglenoid ptastid? The euglenophyte plastid almost certainly originated via the secondary endosymbiosis of an alga, as previously hypothesized (Gibbs, 1978). This is evident from incongruencies between the plastid gene trees presented here, where Euglena and Astasia cluster well within algae, and trees based on nuclear SSU rRNA, where Euglena clusters with Trypanosoma brucei, well below the divergence of all other algae from a common ancestor (Gunderson et al., 1987; Sogin, 1991).

87

Euglenophytes occupy variable positions within the plastidtrees presented here, clustering with the green algae in each of the protein gene trees and as a sister group to the chromophytes in the SSU r R N A tree. The discrepancy between these trees is not so readily resolved as the previous one because it is difficultto imagine the successful lateraltransfer and replacement of a r R N A gene. Hence, we feel that most likelyeither the SSU r R N A tree or all of the protein gene trees are misleading with respect to their placement of Eug/ena. Gibbs (1978) states that the euglenophyte plastid is similar to plastids found in the Chlorophyceae and the Prasinophyceae. At present, molecular data on the prasinophyte plastid are unavailable. However, any group of organisms being investigated in relation to the origin of the euglenophyte plastid should be screened for several diagnostic euglenoid characters, including (1) the presence of many plastid introns (e.g., rbcL has nine introns in Eug/ena, but none or one in non-euglenoids);(2) the presence of tandemly duplicated rbcS genes in the nucleus (Chan et al.,1990); and (3)the carboxyl terminus deletion in D1. Until an organism with some of these traitsis found, the phylogenetic position of euglenophytes will continue to be perplexing.

Note added in proof An error in the published and EMBL database sequence of Cryptononas ~ has been found (S. Douglas, pers. commun.). Reanalysis with the corrected sequence resulted in the same tree, but with fewer steps (1673) and bootstrap values changed by one at five different positions.

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