Membrane Heredity and Early Chloroplast Evolution

8/11/2019 Membrane Heredity and Early Chloroplast Evolution

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Symbiogenesis (the merger of two sym-

bionts to form a novel chimeric organism1)was crucial for creating photosynthetic

eukaryotes and eukaryotes in general. Although

mitochondria do not define eukaryotes2, recent

evidence indicates that the common ancestor of

all extant eukaryotes had mitochondria3 and

that their symbiotic -proteobacterial ancestor

contributed various non-mitochondrial proteins

to the eukaryotic chimera4. Symbiogenesis is

one of the rarest evolutionary phenomena, hav-

ing occurred only about six times over 4billion

years. Only four of these events were deeply

influential: the origins of mitochondria and

chloroplasts, and two major secondary transfers

of different chloroplasts from one eukaryote to

another. Thus, three momentous symbiogenetic

events involved the origin or acquisition of chloroplasts to form eukaryotic algae. Ad-

ditional weaker evidence suggests at least two

tertiary symbiogenetic replacements of endoge-

nous dinoflagellate chloroplasts by foreign

ones. Symbiogenesis is rare because it requires

not just the addition of an extra genome to a pre-

existing cell, but also the integration of host and

symbiont genetic membranes by the evolution

of novel protein-targeting systems5,6. The diver-

sity and origins of chloroplast genomes have

been reviewed elsewhere7,8 – the focus of this

article is the role of membrane heredity2 and

membrane mutation9 in chloroplast origins and

early diversification6.

Origins of chloroplast and plastid

diversity: stasis and novelty

It was first proposed1 that there were five inde-

pendent origins of chloroplasts from different

photosynthetic bacteria, but it is now widely

agreed that chloroplasts originated only once4–7.

In spite of their diversity (Table 1), chloroplastsof green plants, red algae and the entirely uni-

cellular glaucophyte algae (e.g. the flagellate

Cyanophora and coccoid Glaucocystis), which

together constitute the kingdom Plantae10,

almost certainly diverged directly from a single

ancestral cyanobacterium incorporated into the

ancestral plant in one symbiogenetic event11

(Fig. 1). Red algae and glaucophytes (collec-

tively termed biliphytes because they retained

the ancestral cyanobacterial phycobilisomes and

unstacked thylakoids10,11) have chloroplasts that

closely resemble entire cyanobacteria, whereas

the ancestor of green plants (subkingdom

Viridaeplantae, i.e. green algae, bryophytesand vascular plants) lost phycobilisomes

and evolved thylakoid stacking11. The discovery

that the chlorophyll b-synthesizing enzyme

of green plants is homologous to that of the

prokaryotic prochlorophytes12 makes it likely

that the cyanobacterium ancestral to chloroplasts

was aberrantly pigmented, with both phycobili-

somes and chlorophyll b. However, green plant

chlorophyll a / b-binding (cab) proteins evolved

convergently with those of prochlorophytes

from an unrelated protein13. Chloroplast mono-

phyly is strongly supported by the presence of

cognate-derived proteins14 in red algae and in

chromophytes [a collective term for algae with

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trends in plant science

Perspectives

April 2000, Vol. 5, No. 4 1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01598-3

Membrane heredity and earlychloroplast evolution

Thomas Cavalier-Smith

Membrane heredity was central to the unique symbiogenetic origin fromcyanobacteria of chloroplasts in the ancestor of Plantae (green plants, red algae,glaucophytes) and to subsequent lateral transfers of plastids to form even morecomplex photosynthetic chimeras. Each symbiogenesis integrated disparategenomes and several radically different genetic membranes into a more complexcell. The common ancestor of Plantae evolved transit machinery for plastid proteinimport. In later secondary symbiogeneses, signal sequences were added to targetproteins across host perialgal membranes: independently into green algal plastids(euglenoids, chlorarachneans) and red algal plastids (alveolates, chromists).Conservatism and innovation during early plastid diversification are discussed.

Table 1. The nine types of chloroplasts or plastids

Kingdom Plastid location Protein-targeting Thylakoids/ RuBisCo type Pigments Special featuresmechanism (peripheral stack

membrane no.)

Plantae Cytosol Transit (2)Viridaeplantae Many CB I Chl a, b Plastid starch

(green plants)Biliphyta 1 Chl a, PB Cytosolic starch

Red algae P IGlaucophytes CB I Peptidoglycan

Chromista RER lumen Signal/transit (4) P ICryptomonads 2 Chl a, c2 NucleomorphiPE/C Periplastid starch

Chromobiotes 3 Chl a, c1, c2, c3 No starch(heterokonts, Fucoxanthinhaptophytes)

Protozoa Cytosol Signal/Golgi/transitCabozoa Many CB I Chl a, b Paramylum

Euglenoids 3Chlorarachneans 4

Alveolata Cytosolic starchDinoflagellates 3 3 P II Chl a, c2 peridininSporozoa ( 4 None None Noneapicomplexans)

Abbreviations: CB, cyanobacterial type of RuBisCo; P, proteobacterial types of RuBisCo; Chl, chlorophyll; PB, phycobilisomes with phycocyanin, allophycocyanin

and usually phycoerythrin; iPE/PC, single intrathylakoid phycoerythroprotein or phycocyanoprotein.



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Perspectives

April 2000, Vol. 5, No. 4

chlorophyll c (plus eustigmatophytes that sec-

ondarily lost it), i.e. photosynthetic dinoflagel-

lates and chromists, sometimes restricted to the

chromists or more narrowly to chromobiotes or,

even to heterokont algae alone].

Thus chloroplast evolution involves a fasci-

nating mixture of conservatism – retention of ancestral characters – and innovation. Usually

novelties arose by recruiting and modifying

host proteins, but occasionally foreign proteins

were implanted by horizontal gene transfers

entirely distinct from the primary endosymbi-

otic acquisition of the chloroplast genome

and membranes. For example, the non-

cyanobacterial rubiscos obtained from pro-

teobacteria by red algae7 and dinoflagellates7,15.

The cyanobacterial rubisco was retained only

by glaucophytes and green plants7.

Distinct from such lateral gene or operon

transfer involving only DNA is the lateral trans-

fer of whole chloroplasts, plasma membranesand many nuclear genes from a symbiotic

eukaryotic alga into a foreign protozoan host to

form a more complex cell chimera or ‘meta-

alga’2,5–7,9,11,16. Such lateral organelle transfer,

also called secondary symbiogenesis, occurred

with a red algal endosymbiont to produce the

chromophytes and, independently, with a green

algal endosymbiont to generate euglenoid and

chlorarachnean algae17. Some researchers pos-

tulate as many as seven secondary transfers7, but

a single secondary symbiogenesis generating

the common ancestor of all chromophytes plus

a second major green algal transfer is more

likely17. This is because for each symbiogen-esis, whether primary or secondary, organelle-

specific protein-targeting mechanisms had to

evolve for1000 genes to acquire the requisite

topogenic signals for transfer to the host nucleus

(by duplication, illegitimate recombination and

deletion from organelle genomes)5,11,17,18. In-

voking more independent symbiogenetic events

than the minimum required by phylogenetic

analysis is unparsimonious.

With regard to membrane topology and pro-

tein-import mechanisms, chloroplasts fall into

three groups, reflected in their classification in

separate kingdoms in the six-kingdom system

of life10

. First, those of the plant kingdom (bili-phytes and Viridaeplantae)10 are located in the

cytosol and have a double-membraned enve-

lope; these arose directly from a cyanobac-

terium11 and need only transit sequences19–21 to

specify import of nuclear-encoded proteins.

Second, those of kingdom Chromista9 (Box 1),

which arose by secondary symbiogenesis from

a red alga are surrounded additionally by its for-

mer plasma membrane – the periplastid mem-

brane – and are located in the rough endo-

plasmic reticulum (RER) lumen, and thus need

both signal and transit sequences on imported

proteins6,17,18. Third are the more disparate

chloroplasts of kingdom Protozoa, also acquiredby secondary symbiogenesis but never located

within the RER. Protozoan chloroplasts are sep-

arated from the cytosol by three or four smooth

membranes and probably import proteins more

indirectly via both RER and Golgi17,18. Two

independent membrane losses, probably of the

endosymbiont’s plasma membrane17, generated

the triple envelopes of dinoflagellates and

euglenoids. The three kingdoms’ differentmembrane topologies are frozen relics of their

chequered symbiogenetic history, not purely

functional engineering designs6,17,18.

Differential losses in various lineages of pig-

ments, losses of photosynthesis that led to the

conversion of chloroplasts into non-photosyn-

thetic plastids (typically retained for fatty acid

synthesis22 as in most sporozoan protozoa23),

and of the entire plastid (in chromists and pro-

tozoa)22, also increased diversity11. Molecular

sequence phylogenetics documents the loss

of the secondary plastid within euglenoids24,

heterokont chromists25 and dinoflagellates26.

Therefore, the closer relationship of someeukaryotic algae to heterotrophs than to other

algae results from multiple losses22 as well as

multiple plastid gains, contrary to widespread

assumptions. Many more losses than gains

probably occurred because they are genetically

simpler, but their precise number remains to be

determined – hence the controversy over the

number of lateral transfers of plastids7,17.

Underlying these rare transitions was aremarkable stasis in membrane topology and

pigment types within the eight major algal

taxa (Table 1). A key role for membrane

heredity in cell evolution and stasis has

become apparent over the past two decades6,9.

Membrane heredity and cell evolution

Two universal constituents of cells never form

de novo: chromosomes and membranes2,27.

Unlike ribosomes and microtubules, which form

by self assembly, cell membranes always form

by growth and division or fusion of pre-existing

membranes2,27,28 The diverse membranes of the

millions of extant species are all lineal descen-dants of those of the first bacterial cell2,27,28. A

Fig. 1. The symbiogenetic origin of chloroplasts. Chloroplasts arose monophyletically from acyanobacterium with phycobilins and chlorophylls a and b that was phagocytosed by a bicili-

ate protozoan host6,11 that converted it to an organelle35. In the ancestral plant, the food vacuolemembrane was lost. Therefore plant chloroplasts have only a double envelope, which evolveda protein-import mechanism5,11,18–21 distinct from that of endoplasmic reticulum, mitochondriaand peroxisomes. Green plants lost phycobilisomes and evolved thylakoid stacking instead;whereas red algae and glaucophytes lost chlorophyll b, possibly independently.

T r e

n d s i n P l a n t S c i e n c e

Biciliate protozoan

Glaucophytes

Peptidoglycan

Food vacuolemembrane

Cyanobacterium

Loss of peptidoglycan

Proteobacterial RuBisCo;loss of chlorophyll b

Loss of phycobilisomes;thylakoid stacking

Green plantsRed algae

Triose phosphate translocatortransit machineryloss of: vacuolar membrane

host FA synthetaselipolysaccharidelipoprotein



eukaryotic cell contains numerous distinct mem-

brane types. Just as DNA replication requires

information from a pre-existing DNA template,

membrane growth requires information from

pre-existing membranes – their polarity, type

and topological location relative to other mem-

branes. During these processes, polarity and

type-specificity are continuously maintained.

Some membranes, such as the nuclear enve-

lope/RER membranes or mitochondrial inner or

outer membranes, were called genetic mem-

branes2. This is because they always arise by

growth and division of membranes of the sametype, and therefore have a permanent genetic

and evolutionary continuity. Others, such as

lysosomal membranes, lack direct genetic con-

tinuity, forming instead by differentiation from

dissimilar membranes (‘derived membranes’).

Genetic membranes are as much a part of an

organism’s germ line as DNA genomes; they

could not be replaced if accidentally lost, even

if all the genes remained. For some membranes

this distinction is less clear cut: Golgi and per-

oxisomal membranes should perhaps be referred

to as semi-genetic. Although normally arising

by division (or in the Golgi of higher eukaryotes

arising by fragmentation and reassembly) of pre-existing membranes of the same type, Golgi and

peroxisomal membranes might be slowly re-

generated from other endomembranes if lost28,29

– this argues against a symbiogenetic origin

for peroxisomes5.

Inheritance of the architecture of membranes

(by membrane heredity) and of the manufacture

of their component molecular bricks (by gene

heredity and protein and lipid biosynthesis) are

independent genetic processes that combine

constructively through a rapid and spontaneous

perpetual Brownian motion of molecules,

whose chemical affinities govern their insertion

into continuously pre-existing supramolecularstructures. Each genetic membrane type has a

unique composition of membrane proteins, and

usually also lipids; this individuality is typically

maintained by the type-specific insertion of new

proteins and lipids during growth (Fig. 2). Some

genetic membranes (e.g. RER and the bacterial

cytoplasmic membrane) are primary genetic

membranes, growing by the direct insertion of

individual protein and lipid molecules. Others

(e.g. the outer membrane of gram-negative

bacteria and eukaryote plasma membrane) are

secondary genetic membranes, growing instead

by intracellular transfer of molecules (individu-

ally in bacteria; as membrane vesicles ineukaryotes) from a primary genetic membrane.

Self-targeting of specific membrane receptors

for protein insertion (Fig. 2) and the differential

targeting of specific lipid-synthesizing or -bind-

ing proteins are the essential molecular bases for

the specificity of membrane heredity. Mem-

brane heredity depends on DNA heredity in that

genes for the receptors and the targeted proteinsencode their properties, but although necessary

this is insufficient. Preformed cell structure30

also is essential – for every distinct kind of

genetic membrane a cell must perpetually main-

tain a topologically distinct membrane with spe-

cific receptors in the correct polarity2,6,27. This is

because a cell cannot create them de novo in

spite of possessing all the correct genes28.

This central fact of cell biology makes an

increase in the number of distinct genetic mem-

branes one of the rarest evolutionary steps in

the history of life2,17. A few increases have

occurred autogenously, such as the origin of the

endomembrane system2, but more have comeabout by symbiogenesis2,5,6. Their growth mech-

anism ensures that the relative locations of topo-

logically distinct membranes are conserved over

hundreds of millions of years, for example:

(1) The cyanobacterial and homologous11,31

plant and chromist plastid outer membrane

outside the former cyanobacterial cyto-

plasmic membrane and the thylakoids

inside it.

(2) The location of plastids in the cytosol of

Plantae, but within the RER lumen in

Chromista6.

(3) The periplastid membrane between the

plastid envelope and the cytosol-facingmembrane in chromists, sporozoans and

chlorarachneans6,17.

The origin of chloroplasts:

plastids and nuclei as chimeras

Most chloroplasts have three genetic membranes,

all acquired in a single symbiogenetic event from

a cyanobacterium7,8,11. All cyanobacteria except

Gloeobacter are more complex than other

Gram-negative bacteria in having thylakoids, and

therefore also have three genetic membranes.

Thylakoids are a third type of genetic membrane

topologically separate from the cytoplasmic

membrane (Fig. 1), bearing phycobilisomes withblue and red antennal pigments on their cytoso-

lic surfaces. Glaucophytes still retain the cyano-

bacterial peptidoglycan between their two

chloroplast envelope membranes. This was the

first conclusive evidence for the symbiogenetic

origin of chloroplasts (Fig. 1). Peptidoglycan was

lost by green plants and red algae, possibly once

only in their common ancestor11, as implied by

trees indicating an earlier divergence for glauco-

phytes32,33, but independent losses cannot be ruled

out because all three groups diverged so closely

(probably soon after the origin of plastids) that

tree topology remains uncertain.

Chloroplast and cyanobacterial thylakoid lipidsare predominantly glycolipids and sulpholipids,

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Perspectives


Box 1. Chromista, the third botanical kingdom

The kingdom Chromista9,10 is fundamentally chimeric like the plant kingdom, but arose by mergingtwo eukaryotic cells. Chromista include all chlorophyll-c-containing ‘chromophyte’ algae exceptdinoflagellates, which belong in kingdom Protozoa (infrakingdom Alveolata)10. Chromista typicallyhave chlorophyll c2 and some have retained both phagotrophy and photosynthesis; most are colouredbrown or yellow by carotenoids. Chromistan plastids uniquely lie in the rough endoplasmic reticu-lum lumen, not in the cytosol, and are also surrounded by a periplastid membrane, a relic of theplasma membrane of the red algal symbiont that provided their chloroplasts. Secondarily aplastidicchromists (e.g. the soil zooflagellates Oikomonas and Goniomonas; the human pathogen Blastocys-tis; oomycetes such as the plant-pathogen Phytophthora) are distinguishable from members of king-doms Fungi and Protozoa by molecularphylogeny and by mostly having rigid bi- or tri-partite tubularciliary hairs.Outline classification10

Subkingdom Cryptista• Usually bipartite tubular hairs on both cilia; extrusive ejectisomes; cristae flat.Phylum Cryptophyta• Cryptomonads with nucleomorph, starch and 80S ribosomes within periplastid space;

usually with an intrathylakoid phycobiliprotein.• Goniomonas, without nucleomorph or plastids.

Subkingdom Chromobiota• Nucleomorph, periplastid ribosomes, starch, phycobilins all absent; brown carotenoid

fucoxanthin widespread; one cilium typically autofluorescent; cristae tubular.Infrakingdom 1. Heterokonta

Tripartite, rarely bipartite, tubular hairs on anterior cilium only; rarely on cell body or absent).Three phyla:1. Ochrophyta For example, brown algae, diatoms, chrysophytes, Oikomonas, xantho-phytes, eustigmatophytes, raphidophytes, silicoflagellates, pedinellids and pelagophytes;chloroplasts or colourless plastids typically present.2. Bigyra For example, oomycetes, hyphochytrids, opalinates (no ciliary hairs); no plastids.3. Sagenista For example, labyrinthulids, thraustochytrids, bicoecid and anoecid flagellates;no plastids.Infrakingdom 2. Haptophyta

Haptonema; no rigid tubular ciliary hairs. One phylum:Phylum Haptophyta Prymnesiophycean and pavlovean haptophyte algae.



not phospholipids. Chloroplast thylakoids evol-

ved directly from those of a cyanobacterium,

with little or no change in topology, chemistry

or function, including three different shared

protein insertion mechanisms (Sec, SRP and

TAT)34. The photosynthetic endosymbiont was

probably initially phagocytosed and enclosed ina vacuole, from which it escaped and multiplied

freely in the cytosol11. The conversion of a sym-

biont to a plastid or mitochondrion was helotism

(slavery) not mutualism – probably initiated by

inserting host protein translocators into its en-

velope to tap photosynthesate11. The key differ-

ence between an obligate intracellular symbiont

and an organelle is the presence of a generalized

organelle-specific protein-import mechanism35.

This was important in allowing integration

directly by inserting host proteins, and as a pre-

requisite for effective gene transfer from the

symbiont to the host nucleus11. Early on, the

cyanobacterial double envelope was radicallyaltered by losing bacterial lipoprotein and

lipolysaccharide11, replacing the lipolysaccha-

ride with phosphatidylcholine shuttled by lipid

transfer proteins from the ER to the outer leaflet

of the former cyanobacterial outer membrane22.

This replacement probably preceded perfection

of the protein-import mechanism, because pre-

cursor transfer into the main protein-channel

(Toc75) depends on phospholipids in the outer

leaflet21 (of putative host origin22), whereas

galactolipids of cyanobacterial origin are essen-

tial for correctly folding the transit peptide18,20.

The chimeric theory of the origin of the plant

plastid (and mitochondrial) outer membrane5

predicted that the proteins are also of dual ori-

gin: from host and symbiont. Proteins from the

symbiont either retained their original polarity

or acquired an inverted polarity through inser-

tion directly from the cytosol after their genes

moved to the nucleus. Although such inversion

is a theoretically simple way of converting a

receptor for bacterial protein secretion to one for

plastid import5, a receptor originating from a

host protein is even simpler as no gene transfer

is involved5. Recent studies of protein-targeting

substantiate these predictions. The major recep-

tor, Toc159 GTPase, is of host origin in green

plants. It is partly cognate with and possiblyderived from the -subunit of the host-signal-

recognition-particle receptor19–21. The fact that

glaucophyte and red algal transit sequences tar-

get proteins to green algal plastids implies that

both groups have a similar receptor; verifying

this would further strengthen arguments for

plastid monophyly. The channel protein Toc75

is clearly of cyanobacterial origin36,37, support-

ing the homology of the outer plastid membrane

with that of cyanobacteria11,31. However, the

dual mode of Toc75 import across both

membranes via a cleaved transit sequence and

back across the inner membrane by a signal

sequence21

implies that it has probably retainedits ancestral polarity and now works in reverse.

Therefore it is not an example of a receptor hav-

ing undergone polarity reversal, as suggested36.

The third translocon protein (Toc34), which

interacts with it and is self inserting via a C-ter-

minal sequence21, might even be a chimera of

host and symbiont. Toc34 has weak similarity

to a cyanobacterial protein21 as well as an N-ter-

minal GTPase domain related to that of Toc159

(Refs 19,20). The first transit sequences might

have evolved from bacterial export signals using

the Toc75-related channel36 or from modified

host signal sequences or mitochondrial pre-sequences. Three inner membrane translocon

proteins (Tic20; Tic22; Tic55) are of cyanobac-

terial ancestry19,21, as are most chaperones that

help with import19,21.

However, the ADP–ATP exchange protein

that imports ATP to allow for more efficient

CO2 fixation is not38of cyanobacterial ancestry.

The ADP–ATP exchange protein is unrelated to

the mitochondrial ADP–ATP translocator37, the

insertion of which probably initiated the mito-

chondrial enslavement5. Related translocators

are known only from the obligate parasites,

Chlamydia and Rickettsia38

. Contrary to the pos-sibly misleading rRNA trees, these parasitic

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Perspectives


Fig. 2. Maintenance of genetic membrane individuality. Membrane proteins reach their specific

locations because their genes encode sequences27

that interact with type-specific insertion, trans-fer or retention machineries on membranes. These types of machinery consist of integral mem-brane proteins with receptors for the topogenic sequences of other membrane proteins, and mustthemselves be correctly targeted exclusively to their own membrane type to maintain individu-ality. Two different genetic membranes (A and B) within the same cell must have two differentreceptors RA (red) and RB(blue), symbolized with angular (A) or curved (B) receptor pockets. Eachreceptor must have a topogenic sequence complementary to its own receptor site to ensure its owntargeting, shown as a triangular projection in A and curved in B. This self-targeting and molec-ular self-complementarity of the protein-targeting receptors of genetic membranes is a key basisof membrane heredity. By sharing similar topogenic sequences many diverse proteins (symbol-ized by different shapes: yellow in A, green in B) are correctly targeted to each membrane. Thusmolecular complementarity is the central requirement for both nucleic acid and membraneheredity; one transmits inherited molecular structure and the other inherited supramolecularstructure. Theoretically, if a genetic membrane has more than one receptor, as thylakoids34 do,they could be either self-targeting (i.e. self-complementary) or mutually targeting (i.e. mutuallycomplementary). However, if a genetic membrane has a single receptor complex, as higher plantchloroplast surfaces appear to, it must be self-targeting and self-complementary. Correct target-ing might also involve binding to specific lipids, making co-evolution of protein strucure, lipidstructure and composition central to the specificity of membrane heredity. Membrane proteinpolarity (orientation within the membrane) is strongly conserved. It depends not only on thestructure of the inserted protein (and therefore its gene) but also on the insertion machinery andupon whether the protein is inside or outside the membrane before insertion. Polarity usually hasto be conserved to maintain function; this is automatically achieved if the site of synthesis isalways on the same side of the membrane – another aspect of the importance of pre-existingstructure30. The same principle is the fundamental explanation for lipid polarity – the fact thatthe two leaflets of the bilayer typically have different lipids. This arises partly through polarityof insertion (e.g. in plastid outer membranes phosphatidylcholine from the cytosol and glycol-ipids from the plastid) coupled with the slow rate of flip/flop (spontaneous polarity inversion),and partly from differential binding to polarized membrane proteins. If the location of synthesischanges (e.g. following gene transfer from the chloroplast into the nucleus where proteins for-merly made inside the plastid are now made outside it) a protein might need to be translocated

back across the membrane(s) before insertion to retain the correct polarity5

.

T r e

n d s i n P l a n t S c i e n c e

RA

RA

A

A

B

RB

A

RA RB

B B

RB



bacteria might actually be related, with a com-

mon ancestor possessing the gene, possibly

acquired from their eukaryotic hosts (probably

early algae as it is vastly more similar to the

chloroplast than to the mitochondrial protein)

and not donated to chloroplasts as others39 have

suggested. No convincing ancestor of this genefamily is known, possibly because it was too

drastically modified at its origin; algal translo-

cators need sequencing to clarify its history.

Some outer membrane components are self-

inserting (e.g. an amino acid channel)21; some

of these might have played an integrative role

before transit machinery evolved.

The import machinery, whose origin con-

verted the modified cyanobacterium into the

first chloroplast11,35, probably arose to insert host

proteins5 such as the triose phosphate transloca-

tor and the related glucose-6-phosphate translo-

cator. These lack prokaryotic relatives and

probably evolved from the host phosphate/ phosphoenol pyruvate translocator (T. Cavalier-

Smith, unpublished). The ability to tap photo-

synthesate in this way would confer a selective

advantage to the host; initially there would have

been much less advantage in inserting proteins

of cyanobacterial origin because each gene

would normally remain for a while in the

chloroplast after a copy entered the nucleus. The

probable priority of host-protein insertion from

a mutationist–selectionist view point has been

overlooked by some studies19. Although acci-

dental transfers of gene copies might have

occurred before transit machinery originated,

only when they acquired an effective transitsequence could cells with deletions of the plas-

tid version survive. Plastid to nucleus gene

transfer required three steps:

• Escape of one copy from a plastid and its

insertion into nuclear DNA (several genes

could go together in a single piece of DNA).

• Mutation(s) to form a transit sequence.

• Deletion of the plastid copy.

Mutations adapting the promoters to RNA

polymerase II and eukaryotic (rather than

prokaryotic) transcription factors, and deletions

removing some genes from transferred operons,

were probably essential for reasonable levels of

gene expression and thus effective transfer.Thousands of cyanobacterial genes that were

useless to the host cell were simply deleted; this

reduction of the cyanobacterial genome some-

times involved shifts of function. For example,

a cyanobacterial peptidoglycan synthesis en-

zyme (MurG), when no longer needed for pep-

tidoglycan, became a monogalactolipid synthase

replacing the original galactolipid synthase18.

Once import became efficient, transit sequences

could be acquired (probably by transposition5,

e.g. exon-shuffling4 rather than point mutations)

by other host proteins or former cyanobacterial

proteins encoded by gene copies accidentally

incorporated into the nucleus. Even if only 1500genes were transferred (not 2000–5000 as some

suggest4) the total number of mutations in-

volved in the origin of plastids cannot be less

than 10 000.

Thus, the origin of the transit machinery

allowed two cells to become one35, enabling

thousands of symbiont genes to be transferred

to the nucleus. When these genes duplicatedhost functions, host genes were sometimes lost

and symbiont genes retained; their proteins

were either retargeted to the plastid (e.g. those

for fatty acid synthetase) or remained in

the cytosol as well or instead (e.g. phospho-

glycerate kinase4). Sometimes, cyanobacterial

proteins were retargeted to the plastid in one

plant lineage (e.g. green plant starch-making

enzymes) but became cytosolic in the others.

The vast majority of transfers probably pre-

ceded divergence of the three plant lineages,

but many happened later in parallel32.

This raises the question of why genomes

were retained by most chloroplasts and mito-chondria (although most mitochondria that con-

verted to hydrogenosomes5 lost them40). The

idiosyncratic distribution of the organelle genes

across taxa8,32 throws doubt on any simple func-

tional explanation41. Much of the variation prob-

ably arises by historical chance, because

effective gene transfer requires several rare

mutations in each gene – some lineages might

not have had the right combination. Because

most widely-conserved organelle genes in the

mitochondria and in the plastids are relatively

large membrane proteins, a difficulty in effec-

tively retargeting large hydrophobic proteins

across multiple membranes5 is probably a majorselective factor. The fact that the large rubisco

subunit and the largest RNA polymerase sub-

units have never been naturally retargeted sug-

gests similar difficulties for large multi-subunit

soluble proteins that must be synthesized and

assembled by chaperones in the same compart-

ment. This interpretation is strongly favoured by

the fact that both genes can be lost if their func-

tions are replaced by smaller, more easily tar-

geted, single-subunit analogues: for example,

eubacterial RNA polymerase genes replaced

by a phage-type polymerase in both plastids

and mitochondria42, and multi-subunit type-I

rubisco replaced by a small single subunit type-II molecule in dinoflagellate chloroplasts15. This

generalized ‘retargeting difficulty’ argument

is not invalidated as others argue41 by the ex-

perimental retargeting of rbcL, because such

mutants, although viable in the laboratory, have

so little useful rubisco that natural selection

would eliminate them.

Important genes transferred to the nucleus

include ftsZ (encoding a GTPase that proba-

bly mediates division of all plastids, as it

did their cyanobacterial ancestors43), ftsH

and minicell (involved in plastid growth or

division)18. In addition, plastid and mitochon-

drial divisions require host proteins such asdynamin18. Thus the division and the import

machinery underlying plastid growth are both

evolutionary chimeras of host and symbiont

molecules. Even though many host lipids and

proteins have been individually inserted into

the former outer membrane of the cyanobac-

terium, thereby greatly modifying its proper-

ties, we may still regard it as being homologouswith the cyanobacterial outer membrane5,11,31.

Without massive recruitment of pre-existing

symbiont and host proteins, such a complex evo-

lutionary innovation as the origin of chloroplasts

could never have happened. But the scale of the

transformation was so vast, with shared-derived

features of plastid genomes7,8,44, chlorophyll-a / b

or c-binding proteins14, transit protein-targeting

machinery, and the integration of thousands of

prokaryotic genes into the different transcrip-

tional environment of the nucleus, that it appears

unlikely that it occurred convergently in green

plants, red algae and glaucophytes. Phylogenies

of many concatenated nuclear protein sequencessuggest that red algae and green plants are sis-

ters33 (as do mitochondrial sequences8) and there

is weaker evidence that glaucophytes are their

closest relatives33. This supports monophyly of

an ancestrally photosynthetic kingdom Plantae

embracing these three groups10, which the equiv-

ocal nuclear 18S rRNA trees only sometimes

weakly show.

Intensive study of biliphyte plastid envelopes

and their biogenesis and division should also

help to resolve plastid and plant monophyly. The

primary synthesis of fatty acids in green plants

is by enzymes in the plastid stroma, of

cyanobacterial origin, but which are nuclear-encoded22. If biliphytes also have the cyanobac-

terial instead of the original host cytosolic

enzymes, their loss and the evolution of machin-

ery for exporting C-16 fatty acids, to allow mem-

brane growth in the rest of the cell, probably

occurred in the common ancestor of all Plantae

– another potentially homologous novelty in the

origin of plastid envelopes. The site of fatty acid

synthesis in meta-algae that arose by secondary

symbiogenesis is also unknown, but their origin

must have involved major innovations in trans-

envelope fatty acid transport that, like those in

protein import, caution against postulating

numerous independent symbiogeneses.

Secondary symbiogeneses

The host for both secondary symbiogeneses

was a phagotrophic, probably biciliate, uni-

cell11. Unlike in the primary symbiogenesis, the

phagosomal vacuole became stabilized in sec-

ondary symbiogeneses as a perialgal vacuole,

such as that around endosymbiotic algae in

many modern symbioses. Nuclear-coded pro-

teins are targeted by a dual mechanism involv-

ing first an N-terminal signal sequence

directing them across the RER and later a sub-

terminal transit sequence mediating uptake into

the plastid17,18

. In most but not all products of secondary symbiogenesis, the algal plasma

178


Perspectives




membrane was retained as a

fourth membrane – the periplastid

membrane – which is sandwiched

between the double plastid en-

velope and the former phagosomal

membrane. Nuclear-coded plastid

proteins are imported across fourperipheral membranes, not two as

in plants. The import mechanism

across the periplastid membrane is

a major unknown for chloroplast

biology (for possibilities see Ref.

17). Euglenoids and dinoflagellates

independently lost the periplastid

membrane and attached the peri-

algal vacuole locally to the plastid

envelope, yielding a novel triple-

membraned envelope; Golgi-based

protein-import and phylogenetic

evidence make alternative scenarios

relatively implausible17.

Lateral transfer of green algal

chloroplasts by secondary

symbiogeneses

Photosynthetic euglenoids and chlo-

rarachnean algae have plastids with

chlorophylls a and b, cab proteins,

stacked thylakoids and cyanobac-

terial rubisco, obtained from green

algae by lateral organelle trans-

fer16,17. However, unlike all green

plant plastids these two phyla lack

starch. Because the protozoan phyla

to which they belong (Euglenozoaand Cercozoa10,45) are ultrastruc-

turally rather different, two separate

endosymbiotic events are usually

assumed7. However, in both phyla,

nuclear-coded plastid proteins are

targeted via RER and Golgi17,46, and

polysaccharide stores are cytosolic

-glucans (paramylum), not starch

as in all Plantae17. The long branches

of Euglenozoa on 18S rRNA trees

that place them well below, rather

than near Cercozoa, are suspected

to be misleading6,17,47. Therefore,

to minimize the number of independent symbio-geneses that need to be postulated, these phyla,

plus a third non-photosynthetic phylum (Perco-

lozoa), were suggested to be related17. The com-

mon ancestor of this putative cabozoan clade

possibly acquired green algal plastids once only,

in which case all non-photosynthetic cabozoans

evolved by secondary plastid losses17.

Unlike those of green plants, chlorarachnean

plastids are surrounded by two additional

smooth membranes (Fig. 3). The innermost is

the erstwhile plasma membrane of a eukaryotic

endosymbiont; between it and the chloroplast

lies a minute relic of the symbiont’s nucleus –

the nucleomorph. The nucleomorph, which hasthree tiny chromosomes, 90–135 kb in size, is

the smallest eukaryotic genome48

. The fourth,outermost membrane is the former phagosomal

membrane of the host, now a permanent genetic

membrane. In this symbiogenetic event the

ancestral chlorarachnean acquired five novel

genetic membranes directly from an endosym-

biotic green alga, and a sixth novel type by

modifying the host phagosomal membrane,

making chlorarachnean membrane topogenesis

far more complex than in any animal or plant.

Photosynthetic euglenoids also obtained plas-

tids from a green alga but entirely lost the green

algal nucleus and its plasma membrane (Fig. 3).

Genes for proteins formerly encoded by the

green algal nucleus were transferred to the hostnucleus. Curiously, euglenoid nuclear genes for

plastid proteins have numerous

introns without canonical splice

junctions, whether derived from the

green algal nucleus (e.g. cab pro-

teins49) or from the plastid [e.g. RbcS

(Ref. 50)]. Perhaps these unique in-

trons originated from typical spliceo-somal introns in a miniaturized

version of the green algal nucleus

(such as the chlorarachnean nucleo-

morph)48 before their transfer to the

host nucleus. One euglenoid gene of

host origin (fibrillarin) has typical

spliceosomal introns51, but as some

such introns are mobile52, both types

might now be present in genes of

symbiont or host origin. The aberrant

type is found in a Euglenacytosolic

GAPDH, which is related to the gly-

cosomal GAPDH of the non-photo-

synthetic kinetoplastid Euglenozoarather than to those of green plant

plastids53. This GAPDH subfamily is

weakly related to another of the three

cyanobacterial isozymes – if the

ancestral euglenozoan was indeed

photosynthetic17 it could have come

from the green algal part of the

chimera if (unlike angiosperms) it

still had that isozyme. In either case,

the ancestral euglenozoan must have

evolved differential retargeting

between euglenoids and kinetoplas-

tids. Chlorarachniophyte nucleo-

morph introns, which are recog-nisably spliceosomal, are the tiniest

known48. On the cabozoan theory,

which postulates a common photo-

synthetic ancestor for euglenoids and

chlorarachneans17, both aberrant in-

tron types might have diverged from

a common ancestor. Possibly the

aberrant euglenozoan introns were

also minute initially, but expanded

secondarily after transfer to the

host nucleus, where there is far less

selection for small genome size54.

Secondary origin of the ChromistaAnother secondary symbiogenesis created

chromistan algae (e.g. brown algae, diatoms,

haptophytes, cryptomonads; Box 1). Un-

like all other eukaryotes their chloroplasts

lie within the RER lumen5,6,9,18, usually the

perinuclear cisterna (Fig. 4). Chromists and

plastid-bearing alveolates (dinoflagellates

and sporozoans) obtained their plastids from

red algae6,7,55, but lost the red algal phyco-

bilisomes, evolving chlorophyll c2 as a

replacement secondary pigment, and stacked

thylakoids in pairs (cryptomonads) or triplets

(the others). In both groups, there were multi-

ple subsequent losses of the photosyntheticmachinery or of the entire plastid. Because of

179


Perspectives


Fig. 3. The cabozoan theory of the common origin of euglenoidand chlorarachnean chloroplasts suggests that both were acquired bysecondary symbiogenesis from the same green alga in a commonbiciliate host. Following engulfment of the green alga by phago-cytosis, the hypothetical cabozoan common ancestor evolved (1)Golgi vesicle targeting to the perialgal vacuole by fusion of Golgivesicles (V2) with the former perialgal vacuole and (2) periplastidvesicle shuttling between the periplastid membrane and the outermembrane of the plastid envelope; it lost the green algal mitochon-

dria, starch and all other organelles. Paramylum evolved as a cytoso-lic carbohydrate storage compound. Following divergence of thetwo groups, the green algal plasma membrane was retained in chlo-rarachneans, but was lost in an early euglenoid that integrated thefood vacuole membrane with its plastid envelope to make a tripleenvelope. Most green algal nuclear-encoded genes for plastid pro-teins were transferred to the host nucleus and signal sequencesadded to their pre-existing transit sequences before the divergenceof the two groups. The signal sequences inserted the nascent pro-teins into the rough endoplasmic reticulum (RER) lumen, fromwhere vesicles (V1)carried them to the Golgi and then the perialgalvacuole. This gene transfer was completed in euglenoids only,enabling the nucleomorph to disappear. Adapted from Ref. 17.

T r e n

d s i n P l a n t S c i e n c e

ChlorarachneanPhotosynthetic

euglenoid

Ancestral cabozoan alga

Nucleus

RER

RER

V1

V2

V2

Periplastidvesicle

Periplastidmembrane

Golgi

Golgi

Nucleomorph

Non-photosyntheticprotozoan

Loss ofnucleomorphand periplastidmembrane

Paramylum

Chloroplast

StarchGreen alga



180


Perspectives


these and other characters, chromists and alve-

olates, are informally grouped as ‘chromalve-

olates’17, and might be a clade whose common

ancestor acquired a red algal plastid and evolved

chlorophyll c2 once only; other researchers

assume that there were up to five independent

symbiogeneses for chromalveolates7. It is con-

troversial whether chlorophyll c2 evolved after

the uptake of the red alga17

, or was alreadypresent [as is now accepted for chlorophyll b

(Ref. 12)] in the bacterium ancestral to all plas-

tids56. The closer relationship of chlorophylla / c-

binding proteins to cabs than to any bacterial

proteins, supports a purely eukaryotic origin14.

The ‘chlorophyll c-like’ pigments of prochloro-

phyte cyanobacteria56 and of a few green algae

are not chlorophyll c2 (or c1 and c3 widespread

in chromobiote chromists), but Mg-protopor-

phyrins, which could easily have evolved in-dependently from chlorophyll a precursors.

Sequence data for enzymes synthesizing these

pigments are needed to resolve this. Unlike

dinoflagellates, chromists retained the red algal

plastid-coded proteobacterial form I rubisco7.

After the red alga55 was phagocytosed by a

biciliate host11 the phagosome membrane fused

with the nuclear envelope9,55, thereby placingthe chloroplast in the RER lumen. The first step

in targeting nuclear-encoded proteins into the

chromist chloroplast across four membranes is

by ER-type signal sequences17,18. The ancestral

chromist lost phycobilisomes, but one chromist

group, cryptomonads, retained a single phyco-

erythrin pigment (a subset of cryptomonads

later converted this to a blue protein57), but

retargeted it into the thylakoid lumen. The plas-

tid-coded subunit of this phycoerythrin pig-

ment is clearly of red-algal origin, but the

affinities of its nuclear-coded -polypeptide,

which is imported successively across five

membranes, are unclear. Cryptomonads retainthe former red algal nucleus (but drastically

reduced in genome size54) as a nucleomorph6,11

with three linear chromosomes densely packed

with largely intron-free genes encoding many

proteins for nucleomorph replication, division

and gene expression, and a few imported into

the plastid, notably FtsZ and rubredoxin58. Most

genes for chloroplast proteins were transferred

from the nucleomorph to the host nucleus and

their proteins retargeted to the periplastid space.

Other chromists (chromobiotes) lost the nucleo-

morph entirely after transferring the remaining

essential genes for plastid proteins into the host

nucleus. The retention of the chromobioteperiplastid membrane hundreds of millions

of years after the loss of the genome of the cell

that it once enclosed, emphasizes the immense

stability of membrane heredity.

Alveolate plastids

Most sporozoan parasites (e.g. malaria) have

plastids with relict genomes, probably retained

from photosynthetic ancestors because they con-

tain the cells’ only fatty acid synthesis enzymes

(of cyanobacterial origin)23; Cryptosporidium

appears to have lost the plastid59 because it

retained the host mechanism of fatty acid syn-

thesis instead60

. Four membranes bound theseplastids, at least in Toxoplasma. Their gene com-

plement and order implies an origin from a

red44,61 not a green alga, as TufA trees62 uncon-

vincingly suggested. In the chromalveolate

theory, the merger with a red alga was initially

the same symbiogenesis as for chromists but the

two groups diverged before the chromistan

fusion of perialgal vacuole and RER (Ref. 17).

The sisters of Sporozoa are dinoflagellates;

their plastid proteins are related to those of

chromists and red algae63. This is consistent with

the common ancestor of all alveolates (sporo-

zoa, dinoflagellates, protalveolates, ciliates)

being a photosynthetic sister to chromists17

.According to this theory, the dinoflagellate

Fig. 4. The chromalveolate theory of the common secondary symbiogenetic origin of chromist

and alveolate plastids. Following phagocytosis of a red alga into a host food vacuole, chloro-phyll c2 evolved in the ancestral chromalveolate plastid, and the phagosomal membrane becamea stable perialgal vacuole by the specifically targeted fusion of Golgi-derived vesicles (V1). Afterevolving an unknown mechanism17 for protein import across the periplastid membrane (PPM),plastid proteins coded by any genes accidentally transferred from the red algal to the host nucleuscould be correctly retargeted to the plastid as soon as they acquired signal sequences distal to theirpre-existing transit sequences. This enabled the loss of these genes from the symbiont nucleus.Eventually the red algal nucleus disappeared entirely in alveolates and independently in chromo-biotes [which independently lost phycobilin pigments (PB)] but remained in miniaturized formas the cryptomonad nucleomorph (N). Mitochondria and all other organelles were lost. The sym-biont starch (S)-making machinery was retained by cryptomonads in the periplastid space (itslocation in the progenitor red alga), relocated to the host cytosol in dinoflagellates (simply by thetransfer of their genes to the host nucleus), and lost by the chromobiotes. The periplastid mem-brane was lost and a triple envelope evolved in the ancestral dinoflagellate, which also evolvedthe carotenoid peridinin. Thylakoids (T) and photosynthesis were lost in the progenitor of Sporozoa. Chlorophylls c1 and c3 and fucoxanthin (f) evolved in the chromobiote ancestor alone,

which evolved thylakoid stacking in threes independently of dinoflagellates. The divergence of chromobiotes into haptophytes and heterokonts (not shown) probably occurred soon after theirorigin, but the divergence of sporozoans from dinoflagellates occurred well after the origin of alveolates. Adapted from Ref. 17.

T r e

n d s i n P l a n t S c i e n c eCryptomonad Chromobiote

Ancestral chromalveolate alga

NucleusRER

RER

Eochromist

Chromista

Protozoa

Nucleomorph –NM, –PB –S

+ f, chl c 1, c 3

Golgi

Sporozoan

Golgi

Dinoflagellate

Non-photosynthetic protozoan

–T

–PPM

–N, –PB

Fusion ofnuclear envelope andperialgal membrane

Starch

Red alga

Host nucleus

Endosymbiontnucleus

Chloroplast

+ chl c 2

Perialgal vacuole

Starch



ancestor lost the red algal plasma membrane

after its divergence from sporozoa, and a triple

plastid envelope was generated analogously

to but independently from euglenoids17. The

number of sporozoan plastid membranes is con-

troversial62 – if, as is sometimes suggested,

some actually have only three, such reductionwould be independent of that in dinoflagellates.

Protein-targeting to alveolate plastids is prob-

ably via the ER and Golgi17,18,23. As in eugle-

noids46, imported dinoflagellate light-harvesting

proteins are made as polyproteins that are later

proteolytically cleaved. Because proteolytic

cleavage is common in Golgi-secreted proteins,

the convergent import via the Golgi in both

groups might have predisposed them to evolve

similar proteolytic processing, differently from

plant and chromist chloroplast proteins.

Dinoflagellate plastid genomes have frag-

mented into single gene circles63, reversing the

concatenation of genes that created the firstchromosomes2. Dinoflagellates appear to have

transferred to the nucleus or lost far more plas-

tid genes than other eukaryotes63, primarily

retaining a handful of hydrophobic thylakoid

protein genes. This possibly facilitated acci-

dental transposition of replicon origins beside

every gene to fragment their genome. As

cyanobacterial RNA polymerase genes have

not been found in dinoflagellate plastid DNA

(Ref. 63), they might (like most mitochondria8)

have lost them and instead use the unigenic

phage-type polymerase that transcribes gene

expression genes in higher plant plastids42.

Tertiary chloroplast replacements

Typical dinoflagellate peridinin-containing

chloroplasts have probably been replaced sym-

biogenetically at least twice by differently pig-

mented chloroplasts from other eukaryotic

algae. As their plastids must lack enough genes

for self-reproduction and neither endosymbiont

nuclei nor mitochondria remain, the host must

have evolved a protein-insertion mechanism,

making them true tertiary organelles, but direct

evidence is lacking. One aberrant dinoflagel-

late, Lepidodinium, has a green plastid with

chlorophyll a and b plus a membrane topology

suggesting a tertiary symbiogenetic incorpora-tion of a green alga. By contrast, a few species,

such as Gymnodinium breve, have hexanoyl-

fucoxanthin and plastid DNA indicative of a

haptophyte endosymbiont7. A third possible

case is Dinophysis: several species have cryp-

tomonad chloroplasts – because Dinophysis

cannot be cultured we cannot rule out the pos-

sibility that they do not multiply and are sim-

ply kleptochloroplasts (‘stolen’ and maintained

temporarily for photosynthesis as many het-

erotrophic protozoa do). The nesting of all three

cases within the peridinin-containing dino-

flagellates on nuclear rRNA trees26 shows that

they are tertiary chloroplast replacementsand not secondary uptakes by a primitively

non-photosynthetic host. By contrast with

primary and secondary symbiogenesis, which

created whole kingdoms, phyla and classes, ter-

tiary symbiogenesis merely replaced existing

chloroplasts and made a few obscure aberrant

species within a single class – the peridinean

dinoflagellates – which is unusually prone toplastid loss and replacement.

We must firmly distinguish symbiogenesis,

which is exceedingly rare, from symbiosis,

which is frequent and evolutionarily easy. In

addition to a non-photosynthetic relic (retained

as an eyespot) of the typical triple-enveloped

plastid, the fucoxanthin-containing dinofla-

gellatesPeridinium balticum and P. foliaceum

contain a photosynthetic diatom cell64 that

is greatly reduced by loss of mitochondria

and frustule. However, the diatom retains a

nucleus and therefore does not need to import

any host proteins – unless it does it would be

an obligate symbiont not a symbiogeneticallyacquired replacement organelle.

The plastid big bang and snowball earth

From the branching patterns of the major groups

of eukaryotic algae on molecular trees6,45,47 it

appears that there was a rapid, almost simulta-

neous, diversification of the three major plant

and three chromist groups, alveolates and cabo-

zoan algae. This explosive radiation was part of

the eukaryotic ‘big bang’, in which single gene

trees cannot confidently resolve the relative

branching order of the major lineages6,45,47, sug-

gesting that the cabozoan and chromalveolate

symbioses occurred shortly after the origin of plastids when host and symbiont were still

closely related. If this is the case, the merger of

two different genomes might have been easier

than if it occurred long after their divergence.

Comparisons with the fossil record suggest that

the origin of plastids might have been in the late

Proterozoic, around 600 My ago shortly after

(perhaps even stimulated by) the melting of the

icecap that enclosed earth for millions of years65.

Protein trees45,47 and the fossil record are consis-

tent with an explosive early radiation of all

eukaryotic algal phyla, implying a relatively

short timescale for the major transitions dis-

cussed here. This interpretation might surprisesome people because it assumes that the scale

of early eukaryotic divergences on 18S rRNA

trees is inflated6,47, and brings into question the

identification of any fossils as eukaryotic before

~700 My ago. Although the new body plans that

distinguish kingdoms and phyla probably all

originated by explosive quantum evolution, its

speed appears less than was theoretically con-

ceivable11. Therefore the view that the big bang

was so rapid that we cannot establish the relative

branching order of kingdoms and phyla is prob-

ably over-pessimistic. Concatenated protein

trees already support monophyly of kingdom

Plantae and their primary chloroplasts33

; in con- junction with other molecular evidence, such as

shared introns and gene order44, such studies

might eventually confirm or refute the monophyly

of cabozoa, Chromista and chromalveolates.

Elucidating the machinery for protein-targeting

into plastids and the division mechanisms

of their extra membranes in each group will

provide even more direct evidence.

Acknowledgements

I thank NSERC (Canada) for a research grant

and the Canadian Institute for Advanced Re-

search for Fellowship support. Space limitations

and this article’s wide scope prevented the cita-

tion of hundreds of important papers. I apolo-

gise to those whose work had to be referred to

obliquely via other reviews rather than directly.

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182


Perspectives


Tom Cavalier-Smith is at the Dept of

Zoology, University of Oxford, South Parks

Road, Oxford, UK OX1 3PS

(tel44 1865 281065;

fax 44 1865 281310;

e-mail [email protected]).

Membrane Heredity and Early Chloroplast Evolution

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