horizontal g transfer

HORIZONTAL GENE TRANSFER

BETWEENCHLOROPLASTGENOMES

SENIORSOPHISTORPROJECT

SUPERVISOR: DR KENNETHH. WOLFE

AOIFEMCLYSAGHT 94070091

Senior Sophistor Project 1998 – Aoife McLysaght

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CONTENTS

ABSTRACT..................................................................................................................3

1. INTRODUCTION.................................................................................................4

1.1 HORIZONTAL TRANSFER OFGENETIC INFORMATION.........................................41.2 ORIGINS OFCHLOROPLASTS..............................................................................51.3 UNUSUAL CHLOROPLASTPHYLOGENIES...........................................................71.4 RECOGNITION OFHORIZONTAL TRANSFER........................................................8

2. MATERIALS & METHODS.............................................................................10

3. RESULTS & DISCUSSION...............................................................................15

4. CONCLUSIONS..................................................................................................24

ACKNOWLEDGEMENTS ......................................................................................25

APPENDICES............................................................................................................26

Appendix 1: Neighbour-joining trees from alignments of Euglena , Chlorella,Odontella, and Synechocystis protein sequences..................................................26Appendix 2: Neighbour-joining trees from alignments of Cyanophora, Porphyra,Odontella, and Synechocystis protein sequences..................................................27

REFERENCES...........................................................................................................28


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ABSTRACT

The normal mode of transmission of genetic material is by vertical transfer, i.e.

from parent to offspring. Horizontal transfer is any other kind of transmission,

either within or between species and is thought to have played a significant role

in molecular evolution. The aim of this project was to look for horizontal gene

transfer between chloroplast genomes.

The protein sequences from the complete genomes of twelve plastids and

the cyanobacterium Synechocystis spp. were studied. The phylogenetic

information of proteins common to all of these was assessed for inconruencies

with the species tree. Incongruencies were found which are suggestive of

horizontal gene transfer events in the history of plastid evolution. Such events

probably occurred between the plastids of euglenophytes and the brown algae.


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1. INTRODUCTION

Plastids are cellular organelles found in plants and algae. They have a circular

genome, which is much reduced compared to their cyanobacterial ancestors. The

complete genomes of several plastids and cyanobacteria have been sequenced (see

table 1), which has allowed comparison of the gene content and phylogeny of these

genomes. Particularly important is that among these complete genomes are

representatives of different ‘types’ of

chloroplast i.e. green, red, brown, and

cyanelle.

The normal mode of transmission

of genetic material is by vertical transfer,

i.e. from parent to offspring by sexual or

asexual reproduction. Horizontal (or

lateral) transfer is any other kind of

transfer of genetic material (whole or

partial genes). This project is specifically

concerned with horizontal transfer of genes between distantly related plastids.

1.1 HORIZONTAL TRANSFER OFGENETIC INFORMATION

The uniformity of the genetic code makes it possible for organisms to express foreign

genes (36). This is a feature exploited by viruses for the expression of their genes,

and by scientists in the creation of transgenic organisms. It has been suggested that

horizontal transfer has occurred throughout evolution (14, 35, 37). This has

implications for accepting a single gene phylogeny as a species tree.

Transfer of genetic material might occur between prokaryotes facilitated by

bacteriophages, conjugative plasmids, transformation, or by phagocytosis (31,36).

Possible mechanisms for horizontal transfer also exist in higher organisms in the form

of viruses and transposable elements. The fact that transposable elements can pick up

genes as they transpose means that it is possible for genetic material to be transferred.

Horizontal transfer is postulated by Syvanen to be very important in evolution,

with selection for the capacity for horizontal transfer being responsible for many

GLOSSARY

Paralogues: Genes which diverged after agene duplication event.

Orthologues: Genes which diverged after aspeciation event.

Monophyletic: Derived from a singlecommon ancestor.

Polyphyletic: Derived from two or moreancestors, i.e. having multiple origins.


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‘biological unities’, including the uniformity of the genetic code and similar

embryological development between species (37). Furthermore, the resistance of the

genetic code to drift (e.g. loss of degenerate codons) suggests selective constraints,

and similarly for embryogenesis (37). However, this may be an over-emphasis of the

influence and the incidence of horizontal transfer.

Horozontal gene transfer has the potential to furnish a ‘unique explanation for

a number of possibly conflicting phylogenies and contradictory clocks’(37). It is

suggested that gene conversion events between species would act to counter

mutational frequencies and that the molecular clock would be the sum of these two.

Horizontal transfer would affect the rate of divergence of two sequences but not the

rate of evolution (37).

Some examples of possible horizontal transfer are controversial as it is often

hard to eliminate functional conservation, e.g. mammalianβ-globin genes (35). Other

examples appear more robust, for example, the hypothesised transfer of glucose

phosphate isomerase (gpi) from eukaryotes to prokaryotes (30). Evidence in support

of this is found in the high sequence identity (88%) of thegpi gene inEscherichia coli

with that of Clarkia ungulata(plant). Gpi is absent in someE. coli strains which

suggests loss of the gene in theE. coli lineage, and also absence of selection pressures

to keep this gene which might otherwise explain the conservation (30).

Horizontal transfer has been suggested to explain the apparently composite

nature of thePylaiella littoralis plastid genome, alternatively this could be due to

ancient gene duplication and differential loss of paralogues (20). Horizontal transfer

is also believed to have played a role in the appearance of p-elements (transposable

elements) in natural populations ofDrosophila melanogaster(4).

1.2 ORIGINS OFCHLOROPLASTS

The predominant theory on the origin of plastids is by endosymbiosis of a

photosynthetic prokaryote (3, 11). Corollaries to this hypothesis predict secondary

endosymbiotic events where a photosynthetic eukaryotic cell is engulfed by another

eukaryote (9).


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It has been proposed that theEuglena gracilis chloroplast was acquired

through a secondary endosymbiotic event from a green alga (9). This is supported by

the existence of a third membrane around the chloroplast that is not derived from the

endoplasmic reticulum, and is more likely derived from the plasmalemma of the

original symbiont. Strong evidence for secondary endosymbiosis also comes from the

cryptomonad algae where the DNA from the nucleomorph is likely the ‘vestigial

nucleus’ of an algal endosymbiont (6).

The monophyly of all plastids is generally accepted (e.g. 21). Phylogenetic

analysis of plastid and bacterial genes places chloroplasts in a monophyletic group

with cyanobacteria. Theories on the polyphyletic origins of chloroplast were mostly

based on biochemical differences and similarities between chloroplasts (24).

Specifically the differences in pigment composition in the photosynthetic machinery

were thought to reflect the different evolutionary origins of chloroplasts. Green algae

and plants have chlorophylla and b; red algae have chlorophylla and phycobilins;

whereas brown algae use chlorophylla andc. Cyanobacteria use chlorophylla and

phycobilins in their photosynthetic machinery.

The monophyletic theory of chloroplast origins (3) suggests that all

chloroplasts arose from the endosymbiosis of a cyanobacterium with subsequent

divergence giving rise to the different methods of light harvesting. Other theories

propose that the endosymbiotic event occurred several times independently. The

discovery of prochlorophytes (e.g.Prochloron spp. andProchlorothrix hollandica),

photosynthetic bacteria that possess chlorophylla and b, originally appeared to

support the latter theory by providing the missing link from prokaryotes to green

chloroplasts. It was thought that a prochlorophyte was phagocytosed by another cell

to become the chloroplast of the ‘green’ lineage. However, phylogenetic analysis

shows that prochlorophytes fall within the cyanobacterial lineage and are not closely

related to green plastids (22, 41). This suggests that chlorophyllb has either arisen or

been lost several times in the evolution of different plastid lineages, or that horizontal

transfer of the genes for its synthesis has occurred (2). However, it should be noted

that these analyses would not distinguish between actual monophyly, and a

polyphyletic origin involving several closely related cyanobacteria (25).


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Several studies have shown that the Rhodophyta emerged before the lineage

leading to animals, fungi, and green plants (18, 33). This can be reconciled with the

monophyly of chloroplasts if a secondary endosymbiosis event is inferred.

1.3 UNUSUAL CHLOROPLASTPHYLOGENIES

A study on the phylogeny of several chloroplast genes and some of their bacterial

homologues was performed by Mordenet al. (21), who studied five genes:psba,

rbcL, rbcS, tufA, atpB (atpD in bacterialunc operon), and compared them to the

analysis of chloroplast and bacterial SSU (small subunit) rRNA (7). Thepsba, tufA,

atpB, and SSU rRNA trees all show the chloroplasts to form a monophyletic group

with cyanobacteria their closest prokaryote relative. However, therbcL and rbcS

trees show the rhodophytes, phaeophytes, cryptophytes, and chrysophytes grouping

with theβ-proteobacteria (purple bacteria), andβ− andα−proteobacteria respectively.

However the bootstraps (statistical measure of significance) on some of the critical

branches are very low in therbcS tree (21). The authors suggest that this unusual

phylogeny is either due to a horizontal transfer event from the proteobacteria to that

chloroplast lineage (rather than multiple endosymbiotic origins of chloroplasts) or to

transfer from a primitive photosynthetic mitochondrion (since mitochondria are

related toα proteobacteria).

A broad study on the rubisco (ribulose-1,5-bisphosphate

carboxylase/oxygenase) genesrbcSandrbcL of plastids and bacteria confirmed their

unusual phylogeny (5). It is proposed that the best explanation for this is a

combination of ancient duplication followed by differential gene loss, and horizontal

transfer (5).

Another notable incongruency in their trees is that the placement ofEuglena

differs in the SSU rRNA tree from the other trees discussed above (21). It groups

with Ochromonasand Pylaiella in the SSU rRNA tree whereas it groups with the

chlorophytes in the other trees where it was studied (psba, rbcL, rbcS). Unable to

decide on a green-or red-algal origin for theEuglenachloroplast the authors describe

the origins of euglenophytes as ‘perplexing’ (21).


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1.4 RECOGNITION OFHORIZONTAL TRANSFER

The aim of this project was to investigate the occurrence of horizontal gene transfer

between chloroplast genomes. Inherent in this is the need for a mechanism to

recognise such events.

Intuitively, one would imagine that such events would be quite easy to identify

shortly after they occurred by the high sequence identity of the genes in the ‘donor’

and ‘recipient’. However, such relationships become obscured with the passing of

evolutionary time and accumulation of sequence substitutions.

A good method to look for horizontal transfer is by testing phylogenetic

congruency. This involves comparing the phylogenetic tree for a particular protein

from a number of distantly related organisms to that of their known phylogeny (30,

32, 38).

Syvanen sets a couple of criteria for such a type of analysis: a) the sequences

being analysed must contain phylogenetic information; b) the sequences should be

orthologues not paralogues; c) the substitution rate of a particular sequence that shows

incongruency in a particular organism should be comparable to that of the sequence in

the other organisms being analysed (38). He also specifies that the number of genes

being compared should ideally be over ten. Smithet al. adds to this list the criterion

that the trees should be rooted by an outgroup or by duplicated genes and that ideally,

the life histories of the organisms involved should involve some sort of contact (30).

A weakness of this type of analysis is the lack of a statistical test to test the

significance of a particular phylogenetic tree (38). To say that a particular tree differs

from that which is ‘expected’ implies the use of such a test to calculate the

significance of the differnce from the expected. In this project, the tree given by all

the protein alignments together becomes the expected tree and an unexpected tree

topology is one in which an organism (or group of organisms) takes up a radically

different position in the tree supported by a high bootstrap and an otherwise

conventional topology (30).

Crucial to this analysis is the use of homologous sequences from many

distantly related organisms. High sequence similarity alone between two normally

distantly related organisms can only be taken as tentative evidence for horizontal


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transfer. This is because convergent evolution, or some sort of constraint (structural

or functional) may be responsible for the similarity (31).

This analysis will not be sensitive to horizontal transfer between very closely

related species. For example if any horizontal transfer has ever taken place between

rice and maize chloroplast genomes it would not be detected because it would not

change the topology of the tree with the data set being used here.

Differences in GC content of some genes in an organism can sometimes be

used to infer an unusual origin for those genes (38). For exampleSalmonella

typhimuriumandE. coli have similar sized genomes with conserved gene order and

90% of their genes are homologous. However,S. typhimuriumcontains genes

encoding functions not seen inE. coli which accounts for the remaining 10% of the

Salmonellagenome. The GC contents of many of these unique genes is lower than

the genome average of 50%. This is sometimes taken as evidence in support of the

acquisition of these genes by horizontal transfer (38). However, such inferences are

problematic since many factors can influence GC content (38).

Unusual GC content or codon usage cannot be taken as sole evidence for

horizontal transfer since both of these features can be influenced by other factors. For

example, sea urchin retroviral-like elements (SURL elements) have strong codon

usage biases but these were shown not to be due to horizontal transfer (Springer et al.,

1995).

Von Haeseler and Churchill suggest a network model for sequence evolution

and that a network phylogeny could be indicative of horizontal transfer (12).

Another idea for the recognition of horizontal transfer is specific to that by

retroviral vectors. It rests on the assumption that retroviral genes will diverge from

each other faster than host genes because of the error-prone nature of reverse-

transcription (32)

In this project the phylogenetic incongruency test is applied to protein sequences from

plsatid genomes andSynechocystis spp.. Unusual phylogenies were found suggestive

of horizontal transfer events between euglenophytes and brown algae.


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2. MATERIALS & M ETHODS

The complete genomes ofZea mays, Oryza sativa, Nicotiana tabacum, Pinus

thunbergii, Marchantia polymorpha, Chlorella vulgaris, Euglena gracilis, Epifagus

virginiana, Odontella sinensis, Porphyra purpurea, Cyanophora paradoxa, and

Synechocystisspp. were obtained from the EMBL database (see table 1). All protein

sequences for each genome were extracted from the database using the ACNUC

sequence retrieval programme (10) (theNicotiana rpoC1 and rpoC2 sequences were

corrected manually to be in accordance with more recently published sequences (28)).

These were made into a sequence database using the GCG (44) utility TOBLAST.

This protein database was searched for homologues ofEuglena proteins (i.e. the

complete set ofEuglena gracilisproteins was used for the query sequences) using the

BLAST (1) programme BLASTp.

A gene was selected for study if there was an orthologue in at least ten of the

twelve organisms. The parameter E establishes the significance threshold for

reporting high-scoring pairs (HSPs) or groups of HSPs. It can be thought of as the

upper bound on their chance occurrence. The lowest value of E which was accepted

for a gene to be a homologue was E=10-7. However, for most gene groups (31/48) the

lowest value of E was well below that (E=10-15). Table 2 shows a list of the genes

used in this study. Figure 1 shows these genes on theEuglenaplastid chromosome.

Organism EMBL EMBL ReferenceAccession SequenceNumber Name

Zea mays maize X86563 CHZMXX Maier et al., (1995)Oryza Sativa rice X15901 CHOSXX Hiratsuka et al., (1989)Nicotiana tabacum tobacco Z00044; S54304 CHNTXX Shinozaki et al., (1986)Pinus thunbergii black pine D17510 CHPTTRPG Tsudzuki et al., (1993)Marchantia polymorpha liverwort X04465; Y00686 CHMPXX Ohyama et al., (1986)Chlorella vulgaris green alga AB001684 CVAB1684 Wakasugi et al.,(1997)Euglena gracilis alga X70810 CLEGCGA Hallick et al., (1993)Epifagus virginiana parasitic plant M81884 CHEVCG Wolfe et al., (1992)Odontella sinensis brwon alga Z67753 OSCHLPLXX Kowallick et al., (1995)Porphyra purpurea red alga Z67753 CHPPPU388 Reith et al., (1995)Cyanophora paradoxa alga U30821 CC30821 Strewalt et al., (1995)Synechocystis cyanobacterium D90899-D90917 Kaneko et al., (1996)

D63999-D64006

Table 1: Reference information on the complete chloroplast and cyanobacterium genomes used.


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Sets of homologous genes were aligned using CLUSTALW (39) and

neighbour-joining trees (27) were drawn ignoring positions with gaps (otherwise gaps

are treated as undetermined nucleotides) and correcting for multiple substitutions.

Bootstrap (8) analyses were performed with 1000 replications using the same

programme package. A bootstrap is a statistical test of how much support there is for

a particular tree topology contained in the sequence being analysed. It is obtained by

reconstruction the alignment by randomly choosing a position in the alignment and

taking those residues, with replacement, until the original number of positions is

reached and constructing a tree for each of these new alignments. The number of

replications is the number of times that this is repeated. The bootstrap value on a

branch is the number of times that branch was given by the randomly constructed

psb

D

psbC

psbA

psbK

ycf4tufArps7

rps12 rpl20 psbI petG

psaApsaB

psbEpsbF psbL

psbJrpl25

rpl2rps19rps3rpl16rpl14rpl5rps8

rpl36rps14

rps2

atpI

atpHa

tpF

atp

A

rps18

ycf9rp

l12rp

s9p

saC

rpl32

rbc

Latp

E

atpBpe

tB

psbNpsb

H

ycf8

(=psb

T)

psbB

rps14rps11

rpoC2

rpoC1

rpoB

chl1

Euglenagracilis

Figure 1: Diagram of Euglena chloroplast chromosome showing the approximateposition of the genes used in this study. Data from Hallicket al. (1993).


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alignment. A high bootstrap is indicative of strong support for a particular branch

from the sequence data.

For the purposes of this project it was necessary to compare the topology of

individual gene trees to an ‘expected’ phylogeny. In order to obtain an overall

phylogeny for these chloroplast genomes the aligned protein sequences of the proteins

that were present in all genomes exceptEpifaguswere concatenated into one large

alignment and a bootstrapped neighbour-joining tree was drawn from it.Epifagusis

excluded because its plastid genome is so drastically reduced compared to other

plastids. Note that the proteins were aligned independently and then the alignments

were concatenated.

To specifically look at the relationship ofEuglenato Chlorella andOdontella

alignments were made of protein sequences from these three organisms and

Synechocystisand neighbour-joining trees drawn from them. Similarly, this was done

for Cyanophora, Porphyra, Odontella, and Synechocystis. For simplicity’s sake, I

will refer to these trees as the ‘reduced’ tree for a particular protein and the trees

involving eleven organisms as the ‘full’ tree.

Aligned chloroplast small subunit (SSU : 16S) rRNA sequences from all

organisms exceptEpifagusandOdontella(unavailable from the RDP) were obtained

from the Ribosome Database Project (RDP)(45) website. Since the relationship of

Euglena to Odontella is particularly interesting, two relatives ofOdontella were

included in the SSU rRNA analysis in its place:Ochromonas danica(golden alga)

andPylaiella littoralis (brown alga).

In order to confirm the relationship betweenOdontella, Ochromonas,

Pylaiella, and Euglena chloroplasts, protein sequences available from the EMBL

database were aligned and trees drawn. The sequences available forPylaiella were

AtpB, RbcL, and AtpE. The only protein sequence available forOchromonaswas

TufA. SynechocystisandChlorella sequences were included to root the tree and show

the position of the ‘green’ lineage respectively.


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Gene Product/FunctionAtpA ATP synthase CF1 subunitαAtpB ATP synthase CF1 subunitβAtpE ATP synthase CF1 subunitεAtpF ATP synthase CF0 subunit I

AtpH ATP synthase CF0 subunit III

AtpI ATP synthase CF0 subunit IV

ChlI* chlorophyll biosynthesisPetB cytochrome b6PetG/E cytochrome b6/f complex, subunit VPsaA PSI, P700 apoprotein A1PsaB PSI, P700 apoprotein A2PsaC PSI, Fe-S polypeptide SU VIIPsaM*# PSI protein MPsbA PSII, D1 reaction-centre proteinPsbB PSII, CP47 chlorophyll apoproteinPsbC PSII, CP43 chlorophyll apoproteinPsbD PSII, D2 reaction-centre proteinPsbE cytochrome b599 α-chain

PsbF cytochrome b599 β-chain

PsbH PSII, phosphoproteinPsbI PSII, protein IPsbJ PSII, protein JPsbK PSII, protein KPsbL PSII, protein LPsbN PSII, protein NPsbT PSII, protein T (ycf8)RbcL Rubisco large subunitRpl12*# 50S ribosomal protein L12Rpl14 50S ribosomal protein L14Rpl16 50S ribosomal protein L16Rpl2 50S ribosomal protein L2Rpl20 50S ribosomal protein L20Rpl22 50S ribosomal protein L22Rpl23 50S ribosomal protein L23Rpl32 50S ribosomal protein L32Rpl36 50S ribosomal protein L36Rpl5*# 50S ribosomal protein L5RpoB RNA polymeraseβ-chainRpoC1 RNA polymeraseβ'-chain

Table 2: Sequences analysed in this project.

Genes in italics were present in all ofZea mays, Oryza, Nicotiana, Pinus, Marchantia, Chlorella,

Euglena , Odontella, Porphyra, Cyanophora, and Synechocystisand were used to make a

concatenated alignment to get a species tree.

* In analysis of Euglena , Chlorella, Odontella, and Synechocystis, but not in analysis of all

organisms

# In analysis ofPorphyra, Cyanophora, Odontella, and Synechocystis, but not in analysis of all

organisms


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RpoC2 RNA polymeraseβ''-chainRps11 30S ribosomal protein S11Rps12 30S ribosomal protein S12Rps14 30S ribosomal protein S14Rps18 30S ribosomal protein S18Rps19 30S ribosomal protein S19Rps2 30S ribosomal protein S2Rps3 30S ribosomal protein S3Rps4 30S ribosomal protein S4Rps7 30S ribosomal protein S7Rps8 30S ribosomal protein S8Rps9*# 30S ribosomal protein S9TufA*# elongation factor TuYcf4Ycf9


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3. RESULTS & D ISCUSSION

The unrooted neighbour-joining tree obtained from all the protein alignments

concatenated together is shown in figure 2. This is interpreted as a species tree for

these organisms and conforms to the widely accepted phylogeny of these species.

The cyanobacteriumSynechocystisis the outgroup. The plants form a monophyletic

group, a feature which is consistently seen in other gene trees (with a few poorly

supported exceptions) and with strong bootstrap support. It appears to be the most

robust, uncontroversial part of the tree.

This tree shows all

‘green’ plastids to be more

closely related to each other

than to those of brown or red

algae or to the cyanelle of

Cyanophora. All branches

of this tree are supported by

strong bootstraps, the lowest

being that on theEuglena -

Chlorella branch of 94.1%.

Given that 95% is generally

accepted as a statistically

significant bootstrap, this

tree appears to be very

robust.

The cyanelle of

Cyanophorais most closely

related to Synechocystis

according to this analysis.

Given the fact that the

cyanelle retains a

peptidoglycan-sensitive cell

wall typical of cyanobacteria

(which is not found on other plastid types) this placement is not surprising.

Synechocystis

Cyanophora

Porphyra

Odontella

Marchantia

Nicotiana

Oryza

Zea Mays

1000

Pinus

1000

1000

Euglena

Chlorella

1000

941

1000

1000

1000

all 0.037

Figure 2: Species tree. Neighbour-joining tree forcombinedprotein alignment.


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Most protein trees drawn either conform exactly to this topology, or any

differences do not have strong support.

As discussed earlier, plastid-encoded RbcL shows interesting phylogeny. The

RbcL tree for this data set is shown in figure 3. This tree shows nothing too unusual

except thatCyanophorais in an unexpected position relative toSynechocystis, but it is

a poorly supported branch (55.1% bootstrap).

The marked difference in branch lengths is interesting. In these neighbour-

joining trees the horizontal branch length is proportional to sequence divergence (and

can be used as an indicator of rate

of divergence if a molecular clock

is assumed). In this tree, all

organisms show a comparable rate

of divergence except the lineage

leading to Odontella and

Porphyra where the rate appears

to be accelerated. Of course, the

preceding statement is assuming

(at least) two things: a)

Synechocystis RbcL is the

outgroup (hence ancestral form) to

all the other RbcLs analysed here;

b) transmission of genetic material

exclusively by vertical transfer.

However, neither of these may be

true.

This tree is unrooted, so

the positions of theSynechocystis

lineage and the Odontella-

Porphyra lineage are

interchangeable without altering the topology of the tree. Redrawing the tree in such

a way shows that it is consistent with the findings of Mordenet al. (21) and of

Delwiche and Palmer (5). That is, that the cyanobacteria RbcL is more closely related

to the green plastid lineage andCyanophorathan are the red and brown plastids. The

Synechocystis.RBCL

Porphyra.RBCL

Odontella.RBCL

Cyanophora.RBCL

Marchantia.RBCL

Nicotiana.RBCL

Oryza.PE32

Zea Mays.RBCL

951

Pinus.RBCL

660

947

Euglena.RBCL

Chlorella.RBCL

963

711

868

1000

551

rbcl 0.057

Figure 3: RbcL protein neighbour-joining tree.


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long branch length is also consistent with their evidence for the red and brown plastid

RbcL being of proteobacterial origin by horizontal transfer, but neither confirms nor

denies this since there were no proteobacteria included in the data set. What this tree

does indicate though, is that the red and brown plastid RbcL is substantially different

from that of other plastids and cyanobacteria.

The unrooted

neighbour-joining tree for

the plastid and

cyanobacterium SSU (small

subunit) rRNA alignments is

shown in figure 4. This tree

is notable for its grouping of

Euglenawith the brown and

gold algae. The bootstrap on

the branch groupingEuglena

with Pylaiella littoralis to the

exclusion of Ochromonas

danica is not very high

(51.1%), nor on the branches

separating this lineage from

other non-green plastids and

cyanobacteria (54.1% and

56.2%). However, the

branch that excludesEuglena

from the green lineage is

very strongly supported by a

bootstrap of 99.5%. This

tree is consistent with that of Douglas & Turner which showsEuglenain a lineage

with non-green algae and has cyanobacteria as the outgroup (7). This tree contradicts

the species tree generated from the concatenation of all the protein alignments (figure

2). The SSU rRNA indicates that there is something unusual in the relationship of

euglenophytes to brown algae. The trees drawn to confirm the relationship of

Odontella, Pylaiella, Ochromonas, Chlorella, EuglenaandSynechocystisconsistently

Synechocystis

Cyanophora

Porphyra

Euglena

Pylaiella

Ochromonas

511

Pinus

Zea_mays

Oryza

Nicotiana

1000

415

Marchantia

848

Chlorella

1000

541

995

562

335

SSUrRNA 0.017

Figure 4: Neighbour-joining tree for the chloroplast andcyanobacterium SSU rRNA alignment from the RDP.


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separate theChlorella andEuglenafrom the rest with high bootstraps: AtpB (87.1%);

RbcL (97.9%); TufA (90.3%); AtpE (92.6%). As discussed, other evidence suggests

that RbcL may be a special case, but overall these protein trees confirm that

OchromonasandPylaiella are closely related toOdontella.

The results from the trees drawn forEuglena, Chlorella, Odontella, and

Synechocystisare shown in appendix 1. There are two main advantages to drawing a

reduced tree for clarification

purposes: a) there are fewer

possible tree topologies (for

four operational taxonomic

units [OTUs] there are only

three possible trees); b)

because there are fewer

possible trees the bootstraps

are higher. The topology most

commonly found (37/53 trees)

for these organisms is the one

that groups Euglena with

Chlorella as in the species tree

(figure 2). However, fourteen

genes showEuglenagrouping

with Odontella as it does in

the SSU rRNA tree. Not all of

these show strong bootstraps

but some are supported by

reasonably good bootstraps:

Rpl16 (85.9%); Ycf4 (82.5%);

PsbT (80.3%); Ycf9 (79%); Rps12 (77.7%). The trees drawn for these genes for more

organisms have unusual topologies.

Synechocystis.RPL16

Cyanophora.RPL16

Porphyra.RPL16

Euglena.RPL16

Odontella.RPL16

Chlorella.RPL16

430

225

Marchantia.RPL16

Nicotiana.RPL16

Oryza.RPL16

Zea Mays.RPL16

998

Pinus.RPL16

762

587

249

950

582

rpl16 0.035

Figure 5: Neighbour-joining tree for Rpl16 proteinalignment.


- 19 -

Figure 5 shows the Rpl16 tree. Its topology is very unusual and not easy to

explain. It is impossible to tell if the red and brown algae are among the green

lineages or if the converse is true. However, I think it is noteworthy that in an

otherwise weak tree,Chlorella and Euglenaare excluded from the rest of the green

lineage with a bootstrap of 95%.Chlorella andEuglenaare normally excluded from

the plant lineage, but it this case it might mean that the Rpl16 genes ofEuglenaand/or

Chlorella are more

brown/red alga-like rather

than the converse.

The branches in the

controversial region in the

tree have weak bootstraps.

However, the reduced tree

shows that the Rpl16 protein

of the Euglena plastid is

more closely related to that

of Odontella than that of

Chlorella supported by a

bootstrap of 85.9%. This

indicates that despite the

weak bootstraps in figure 5

the grouping of Euglena

with Odontella rather than

Chlorella is robust.

A phylogenetically

informative site for an

alignment of four sequences is one where there is no gap and where there are at least

two different residues, one shared by more than one organism. For the Rpl16

alignment of Odontella, Euglena , Chlorella, and Synechocystisthere are

approximately 60 informative sites under this definition.

Synechocystis.YCF4

Chlorella.YCF4

Cyanophora.YCF4

Porphyra.YCF4

Odontella.YCF4

546

Marchantia.PE40

Nicotiana.PE38

Oryza.PE37

Zea Mays.ORF185

1000

Pinus.PE58

993

839

Euglena.YCF4

1000

262

354

352

ycf4 0.085

Figure 6: The neighbour-joining tree for Ycf4 protein.


- 20 -

The reduced tree for Ycf4 also groupsOdontellaandEuglenato the exclusion

of Chlorella with a high bootstrap (82.5%), but the number of informative sites in that

alignment to is very low (fewer than 20) and therefore unreliable. The full tree for

this gene (figure 6) has low bootstraps on all branches except those defining the plant

phylogeny.

The reduced tree for

the PsbT protein also showed

the sequences ofOdontella

and Euglena to be more

closely related to each other

than either was toChlorella

supported by a large bootstrap

(80.3%) and the number of

informative sites in this

alignment is more that 120.

This immediately suggests

that this result is more robust

than the previous ones.

Indeed, even the full tree

looks better. The bootstraps

are not strictly speaking

statistically significant

(>95%) but with ten OTUs

the number of possible trees

is very large making it

‘harder’ for any data set to

achieve that level of significance. Given this, and the relatively high number of

informative sites, the bootstraps of 67.6% and 77% that isolate theOdontella-Euglena

lineage from the rest seem respectable. The unusual position ofCyanophorais

worthy of note. However, the PsbT tree including onlyOdontella, Porphyra,

Cyanophora, and Synechocystisplaces Odontella and Porphyra together with a

bootstrap of 54.6%. This contradicts the relationship implied in figure 7 which places

Synechocystisclosest toPorphyrawith a bootstrap of 77%.

Synechocystis.PSBT

Porphyra.PSBT

Euglena.YCF8

Odontella.PSBT

Chlorella.PSBT

Cyanophora.PSBT

Marchantia.PE56

Oryza.PE53

Zea Mays.ORF33

Pinus.PSBT

986

781

930

623

476

676

770

psbt 0.064

Figure 7: Neighbour-joining tree for PsbT


- 21 -

Ycf9 is another protein which groupsOdontellaandEuglenatogether with a

high bootstrap (79%) in the reduced tree. Figure 8 shows the full tree for this protein.

This tree conforms to the

species tree (figure 2) except

for the placement ofEuglena

and Odontella on the same

lineage, supported only by a

weak bootstrap (46.5%).

The tree for Rps4 is

also interesting. Even though

the close relationship between

Euglena and Chlorella is

undisturbed (so that there is

nothing unusual in the reduced

tree) Chlorella and Euglena

form a group withOdontella

to the exclusion of all else

(figure 9). While the branches

of the internal topology of this

lineage are only supported by

quite weak bootstraps, the data

does seem conclusive that

these organisms form a

distinct lineage for this gene. They are isolated from the rest of the tree by bootstraps

of 99.8% and 98.3%, both statistically significant.

The topology of each of the trees for SSU rRNA, Rpl16, Ycf4, PsbT, Ycf9,

and Rps4 could be explained by a horizontal gene transfer event between the

brown/red algae lineage and the green algae lineage. The arrangement of these genes

on the relevant plastid chromosomes is quite interesting.

The genespbsT, ycf4 and ycf9 lie close to each other on theOdontella

chromosome, but are almost equidistant on theEuglenachromosome.

Synechocystis.YCF9

Cyanophora.YCF9

Porphyra.YCF9

Marchantia.ORF62

Pinus.PE120

Oryza.PE9

Zea Mays.ORF62

Nicotiana.PE22

971

980

392

Chlorella.YCF9

899

Euglena.YCF9

Odontella.YCF9

843

465

323

415

ycf9 0.11

Figure 8: Neighbour joining tree for Ycf9.


- 22 -

A more interesting observation concerns therpl16 gene. This gene lies in a

region of conserved linkage between different plastid chromosomes which includes

several genes in this study. Of these, the protein sequences from two genes adjacent

to rpl16 groupedEuglena with Odontella with reasonably high bootstraps in the

reduced tree. These are Rpl14 (64.2%) and Rpl5 (64.3%). Also near these genes in

the Euglenaplastid genome (but

not in Odontella) is the rps14

gene (69.7% bootstrap with

Odontella in reduced tree).

Similarly, ycf4 (82.5%), rps7

(64%), and rps12 (77.7%) are

adjacent in theEuglena plastid

genome but onlyrps7 and rps12

are adjacent in Odontella.

Linkage is intuitively favourable

when suggesting horizontal

transfer for two reasons: a)

homologous recombination is

facilitated; b) it is evidence of

the transfer of genetic material

(especially if the conserved

linkage is not seen in other

organisms). In this case the

linkage is not exclusive to

Euglena and Odontella, but it

would appear to support the

possibility of horizontal transfer.

The reduced trees forCyanophora, Odontella, Porphyra, and Synechocystis

(appendix 2) confirm the relationship between these that is seen in figure 2. As

shown, there are some incongruent trees but it is hard to make any inferences from

these since these four organisms normally have a close relationship.

A problem of interpretation common to the trees in figures 4-9 is deciding on

directionality of any horizontal transfer events, i.e. deciding whether the brown/red

Synechocystis.RPS4

Cyanophora.RPS4

Porphyra.RPS4

Marchantia.RPS4

Nicotiana.RPS4

Oryza.PE26

Zea Mays.RPS4

1000

Pinus.RPS4

848

475

Odontella.RPS4

Euglena.RPS4

Chlorella.RPS4

506

998

609

415

983

rps4 0.06

Figure 9: Neighbour-joining tree for Rps4 protein.


- 23 -

algae have become more green-like or if the converse is true. With this information it

does not seem possible to decide either way.

There are four possible sources for any plastid gene: a) present since the

original endosymbiotic event (i.e. original plastid gene); b) from the mitochondrial

genome of the same organism; c) from the nuclear genome of the same organism; d)

from another organism, i.e. from horizontal transfer. When a gene tree disagrees with

a species tree it is unlikely that the relationship established after the original

endosymbiotic event has remained undisturbed. The mitochondrial genome is ofα-

proteobacterial origin and the nuclear genome is of archaebacterial origin. Any gene

now encoded by the plastid but which originated in either of these organelles would

be expected to be more distant from this gene in other plastid genomes than is the

cyanobacterial gene, i.e. this gene should not show close relationships to other

plastids.

In the trees in figures 4-9, despite the unusual topology, all ‘unusually-placed’

genes are monophyletic with the other plastids (with the possible exception of

Chlorella in the Ycf4 tree).

Another possible explanation for strange tree topologies is if there was an

ancient gene duplication followed by differential loss of one paralogue. Independent

stochastic loss of one copy of the gene in different lineages opens the possibility that

normally distant lineages retain the same copy of the gene and thus any tree based on

that gene will show them to be more closely related than expected from the species

tree. Such an explanation for these results would require many independent gene

losses along different lineages. It is therefore unlikely to be the sole explanation for

this data, but cannot be eliminated as a contributory factor.


- 24 -

4. CONCLUSIONS

The trees generated from the majority of the protein sequences analysed

conform to the widely accepted species tree for these organisms. However, the

unusual topologies of several trees suggest that there has been more than simply

vertical transfer and speciation involved in the evolution of these genomes. Assuming

that these contradictions are not simply due to errors of sequencing or analysis, there

must be some biological phenomenon to explain these results.

The unusual topology of these trees supported by high bootstraps can most

easily be explained by horizontal transfer, although ancient gene duplication followed

by differential loss of paralogues cannot be eliminated as a contributory factor.

A possibility not investigated here is that horizontal transfer of partial genes is

taking place. There is no reason to assume that entire genes would be transferred (in

fact, it seemly more likely that partial genes should be transferred) unless it is a large

region of chromosome. To investigate this trees would need to be drawn for segments

of genes. If different portions of a gene give conflicting phylogenies then it may be

that horizontal transfer of part of that gene has occurred.

The analysis provides good evidence for horizontal transfer events having

occurred between the chloroplasts of green algae and of red/brown algae. The most

likely candidates for horizontal transfer are the genesrpl16, ycf4 and ycf9 and the

gene for SSU rRNA. In these cases the transfer is consistently hypothesised to be

between the same few organisms so it is possible to infer a single horizontal transfer

event involving several genes. However, the extent and incidence of any transfer

events are undetermined by this study.


- 25 -

ACKNOWLEDGEMENTS

I would like to gratefully acknowledge the help of Dr. Ken Wolfe, Dr.

Andrew Lloyd, Anton Enright, and everyone in the lab. Also, to a friend who proof-

read this manuscript but wished to remain anonymous!


- 26 -

APPENDICES

Appendix 1: Neighbour-joining trees from alignments ofEuglena, Chlorella,Odontella, andSynechocystisprotein sequences.

(((E,C),O),S) (((E,O),C),S) (((O,C),E),S)>80% >80% >80%

Rps4 RbcL Rpl5 (64.3) Rpl16 (85.9) PetB nilPsbF AtpA Rps12 (77.7) Ycf4 (82.5) Rpl20PetG AtpE Rps14 (69.7) PsbT (80.3) PsaMPsbJ AtpI Rps7 (64) Rps18Rpl23 ChlI PsaC (58.4)Rpll36 PsaA Rpl14 (64.2)PsaC PsaB AtpB (56.4)PsbB PsbA AtpH (53.1)PsbC PsbD PsbE (57.7)Rpl2 PsbH Rpl32 (37.8)Rps11 PsbK Ycf9 (79)Rps19 Rpl12Rps4 RpoB

RpoC1RpoC2Rps2Rps3Rps8TufAAtpFPsbIPsbLPsbNRps9

The columns indicate which tree was obtained and whether the bootstrap was greater than or

less than 80%. The values shown in brackets are bootstrap values.


- 27 -

Appendix 2: Neighbour-joining trees from alignments ofCyanophora, Porphyra,Odontella, andSynechocystisprotein sequences.

(((O,P),Cy),S) (((Cy,O),P),S) (((Cy,P),O),S)>80% >80% >80%

RpoC2 RpoC1 Rps4 (57) AtpE (86.6) AtpF Rps8 (99)AtpB ChlI Rps9 (52.7) PetG (97.9) PsaCAtpH PetB TufA (48.4) RpoB (80.6) Rpl2PsbA PsaA Rpl36PsbI PsaB Rps14PsbJ PsaM Rps19PsbK PsbBPsbN PsbCPsbT PsbDRpl12 PsbERpl5 PsbFRps11 PsbHRps12 PsbLRps7 RbcLYcf4 Rpl14Ycf9 Rpl16

Rpl20rps18rps2rps3

The columns indicate which tree was obtained and whether the bootstrap was greater than or

less than 80%. The values shown in brackets are bootstrap values.


- 28 -

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