Rampant Horizontal Transfer and Duplication
Eubacteria and Plastids
Charles F. Delwiche’
Department
of Biology,
of Rubisco Genes in
and Jeflrey D. Palmer
Indiana University,
Bloomington
Previous work has shown that molecular phylogenies of plastids, cyanobacteria,
and proteobacteria based on the
rubisco (ribulose-l$bisphosphate
carboxylase/oxygenase)
genes rbcL and rbcS are incongruent with molecular
phylogenies based on other genes and are also incompatible with structural and biochemical information. Although
it has been much speculated that this is the consequence of a single horizontal gene transfer (of a proteobacterial
or mitochondrial rubisco operon into plastids of rhodophytic and chromophytic
algae), neither this hypothesis nor
the alternative hypothesis of ancient gene duplication have been examined in detail. We have conducted phylogenetic
analyses of all available bacterial rbcL sequences, and representative plastid sequences, in order to explore these
alternative hypotheses and fully examine the complexity of rubisco gene evolution. The rbcL phylogeny reveals a
surprising number of gene relationships that are fundamentally incongruent with organismal relationships as inferred
from multiple lines of other molecular evidence. On the order of six horizontal gene transfers are implied by the
form I (L&J rbcL phylogeny, two between cyanobacteria
and proteobacteria,
one between proteobacteria
and
plastids, and three within proteobacteria.
Alternatively, a single ancient duplication of the form I rubisco operon,
followed by repeated and pervasive differential loss of one operon or the other, would account for much of this
inconaruitv.
_ _ In all probability, the rubisco operon has undergone multiple events of both horizontal gene transfer
and gene duplication in different lineages.
Introduction
Plastid
bisphosphate
phylogenies
based on rubisco (ribulose- 1,5carboxylase/oxygenase)
conflict with those
based on other genes (Boczar, Delaney, and Cattolico
1989; Valentin and Zetsche 1989; Martin, Somerville,
and Loiseaux-de
Go& 1992; Douglas 1994; Delwiche,
Kuhsel, and Palmer 1995). Analyses based on 16s and
23s rRNA, t&A, atpB, rpoC1, and HSP60 each indicate
strongly that all plastids are derived from cyanobacterial
ancestors and, possibly, from a single common plastid
ancestor (fig. 1; see also Bhattacharya and Medlin 1995;
Reith 1995; Palenik and Swift 1996). In contrast, analyses based on rbcL (the gene for the large subunit of
rubisco) divide plastids into two groups of widely disparate bacterial affinity. As expected, the rbcL genes of
two of the three primary plastid lineages (chlorophytes,
which include green algae and land plants, and glaucophytes, as exemplified by Cyanophora paradoxa, whose
plastids retain a peptidoglycan
cell wall) group with
those of cyanobacteria.
However, the rbcL genes from
red algae and related secondary plastid lineages (chromophytes, cryptomonads,
etc.) are much more closely related to those of certain proteobacteria
(purple bacteria)
than to cyanobacteria.
The gene for the small subunit of
rubisco, rbcS, shows the same pattern, although it has
not been as widely sampled as rbcL and is too short and
variable to provide enough conserved characters to allow detailed phylogenetic
reconstruction
(Assali et al.
‘Present address: Department of Plant Biology,
Maryland
at College
University
of
Park.
Key words: gene transfer, gene duplication, ribulose- 1,Sbisphosphate carboxylase,
rub&o,
&CL, mosaic evolution, chloroplast phylogeny, molecular phylogeny, cyanobacteria,
proteobacteria.
Address for correspondence
and reprints: Charles Delwiche, Department of Plant Biology, H. J. Patterson Hall, University of Maryland. College Park, Maryland
70742-S 15. E-mail: delwiche@bio.
indiana.edu.
Mr~l. Biol. EL’<>/.13(6):X73-882.
0 1996 by the Soaety
1996
for Molecular Biology
and Evolution.
ISSN: 0737.4038
1991; Morden et al. 1992). No genes other than the rubisco genes have been identified that share this phylogenetic pattern.
The discrepancy
between phylogenies
based on
rbcL and those based on other genes has been noted and
discussed
by several
authors
in the context
of plastid
phylogeny
(e.g., Boczar, Delaney,
and Cattolico
1989;
Valentin and Zetsche
1989; Assali et al. I99 1; Douglas
and Turner 1991; Martin, Somerville,
and Loiseaux-de
Gob 1992; Morden et al. 1992; Delwiche,
Kuhsel, and
Palmer
1995). Although
proposed
mechanisms
have
varied considerably
in their details, most authors have
favored a scenario involving
a single horizontal gene
transfer of a rubisco operon from a proteobacterium
to
a cyanobacterium
or early plastid. An alternative explanation for the discrepancy
is that ancestral proteobacteria and cyanobacteria
possessed two rubisco operons
(Martin, Somerville, and Loiseaux-de Go& 1992). The
different rbcL lineages present in plastids would then
reflect retention of different copies of the gene in the
two main plastid lineages present in the rbcL tree.
To distinguish between the alternate possibilities of
gene transfer and gene duplication,
we have analyzed
all available eubacterial
rbcL sequences and representative plastid rbcL sequences. We show that the evolutionary history of rubisco is far more complicated than
previously envisioned;
to reconcile it with organismal
phylogeny
requires many more events of lateral gene
transfer and/or gene duplication
and differential
loss
than previously suspected.
Materials
and Methods
Forty-eight
rbcL amino acid sequences (table 1)
were aligned with the GCG (Genetics Computer Group
1991) utility PILEUP The alignment was checked manually and reconciled with alignments based on secondary structural data (Schneider et al. 1990). The total
aligned length was 532 amino acids, of which 497 were
873
874
Delwiche
and Palmer
FIG. I.-Bootstrap
support for relevant plastid and eubacterial
groups from representative
molecular phylogenetic
studies. Included
are those genes for which both red-like and green-like plastid and
representative
eubacterial
sequences have been examined. For each
gene, values in the left column indicate bootstrap support for monophyly of the group in question, while values in the right column indicate the number of taxa represented in that group. The total number
of taxa included in each study is indicated at the head of the column.
Cases that have not been tested (NT) or where bootstrap values are
below 507~ are indicated by shading. (a) Bootstrap support for this
clade was above 50% with only one analytical method, maximum likelihood. (b) The proteobacteria
were not monophyletic,
but bootstrap
support for the observed topology was low (40%). (c) One expected
taxon, Nrisseria gonorrhoeae,
did not fall among the B-proteobacteria.
The studies used to assemble this table were: 16s rRNA, *Bhattacharya and Medlin (1995), :Eisen (1995); 23s rRNA, De Rijk et al.
(1995); tufA, Delwiche, Kuhsel, and Palmer (1995); at@, Douglas and
Murphy (1994);HSP60, Viale et al. (1994); recA. Eisen (1995). Molecular phylogenetic
studies with generally congruent conclusions include: 16s rRNA, Douglas and Turner (1991). DeLong, Frankel, and
Bazylinksi (I 993). Ludwig et al. (1993) Distel and Cavanaugh (1994)
Giovannoni et al. (1988), Olsen, Woese, and Overbeek (1994), Van de
Peer et al. (1994); 23s rRNA. Somerville et al. (1993): r&A. Morden
et al. (1992); atpB, Morden et al. (I 992). Leitsch and Kowallik (1992)
Martin, Jouannic, and Loiseaux-de
Goer (1993); HSP60, Viale and
Arakaki (1994); rrcA, Lloyd and Sharp (1993).
included in the analysis. All sequences were full length
(ca. 470 amino acids) except for those from Prochloron
and Prochlorococcus
(228 amino acids). Percent amino
acid identities of the genes rbcL and rbcS were calculated, excluding gaps, for selected taxa with the GCG
utility DISTANCES.
The alignment is available at http:
//www.bio.indiana.edu/-jpalmer/rubisco-evolution/.
Phylogenetic
analyses were performed with PAUP
v. 3.1.1 (Swofford 1993) and PAUP* v.4.0d31 (Swofford, personal communication).
The most parsimonious
trees were determined with 100 heuristic searches with
random addition
sequences.
Bootstrapping
was performed with 100 replicates, evaluating
each replicate
with 10 heuristic searches with random addition sequences. Neighbor-joining
trees were determined
and
bootstrap analyses were performed using mean character
distances with PAUP* v.4.0d3 1.
Results and Discussion
Phylogenetic
Analysis
Maximum-parsimony
phylogenetic
analysis
of
rbcL amino acid sequences found 24 equally parsimonious trees (fig. 2). Six form II rbcL sequences were
included as outgroups to root the tree (fig. 2). This form
of rubisco, composed of two large subunits but no small
subunits, is restricted to certain proteobacteria
and dinoflagellates
(Gibson and Tabita 1977; Roy and Nier-
zwicki-Bauer
1991; Morse et al. 1995), whereas form I
rubisco (composed of eight large and eight small subunits)-the
subject of this study-is
more widespread,
present in all photosynthetic
plastids (except certain dinoflagellates)
and cyanobacteria,
and widely in proteobacteria. The large subunits of form I and II rubisco,
while readily alignable and clearly homologous (Schneider et al. 1990; Gibson, Falcone, and Tabita 1991), are
so divergent (fig. 3) that one type is customarily used
as outgroup for the other type (e.g., Martin, Somerville,
and Loiseaux-de
Go& 1992). The conclusions
of the
study are not, however, dependent upon rooting of the
tree; in analyses with slightly fewer taxa in which the
form II sequences were excluded, the unrooted tree was
essentially identical to the form I ingroup in the rooted
tree.
The strict consensus of the 24 trees (indicated by
bullets in fig. 2) is consistent with previous analyses of
bacterial and algal form I rbcL sequences, dividing taxa
into two major groups, termed here “green-like”
and
“red-like.”
The green-like group includes two distinct
sets of rbcL sequences, those from cyanobacteria
and
the plastids of Cyanophoru, green algae, plants, and euglenophytes
(these correspond to the “type IB” genes
in the classification
of Tabita [ 1995]), and those from
all examined y-proteobacteria
and one (Y- and one B-proteobacterium (type IA). The red-like group also includes
two distinct sets of sequences, those from red algal,
brown algal (sensu lato), and cryptomonad plastids (type
ID), and those from three (Y- and three B-proteobacterial
sequences (type 1C).
The green-like and red-like rbcL groups are quite
distinct as measured both by branch lengths on phylogenetic trees (fig. 2) and by direct comparisons of amino
acid sequences (fig. 3). Within-group
amino acid sequence identities among the divergent bacterial and eukaryotic taxa examined here are 69%-92%, whereas between-group
identities are much lower, in the range of
53%-60%
(fig. 3, above diagonal). The same general
pattern of sequence relationships also holds for the small
subunit of rubisco; amino acid identities here are consistently higher within the red (52%-70%)
and green
(33%-70%) groups than between the two groups (24%39%; fig. 3, below diagonal).
The major features of the rbcL tree (fig. 2) are
strongly supported, and differences among the 24 shortest trees involve rearrangements
within a few terminal
groups. The critical branches
defining
red-like and
green-like rubisco groups have 100% bootstrap values,
as does the branch separating form I and II rubisco.
There is also strong support for the monophyly of both
green-like and red-like plastids, green-like proteobacterial sequences, and, except for the uncertain position of
the c-Y-proteobacterial “Mn-oxidizing
bacterium,”
the
Tred-like proteobacterial
sequences (fig. 2). Comparable
trees (except for the position of the dinoflagellate
Gonyaulux; see below) and bootstrap support were found in
neighbor-joining
analyses using mean character distances (data not shown).
Lateral Transfer
Table 1
Taxa Used in Phylogenetic
Analysis,
Genbank
Accession
and Duplication
Numbers for &CL, and Classification
Taxon and Strain
of Rubisco
Genes
875
Based on rRNA
rbcL No.
Group
Ml744
U20584
U20585
D90204
M26396
M34536
D28135
D4362 1
D43622
L32182
L22885
D30764
U23 145
Wagner
M64624
X00286
L37437
L42940
MS5061
x70355
Xl7252
Beta”
Beta”
Beta”
Gammah
Gammab
Uncertain’
Gammad
Gammad
Gammad
Alphat
Alpha’
Uncertain’
Alpha”
Alpha”
Alpha”
Alpha”
Betag
Betag
Gamma”
Gamma”
Alpha’
I Proteobacteria
Alcaligenes eutrophus ATCC 17707 (chromosomal)
Alcaligenes eutrophus ATCC 17699 (chromosomal)
Alcaligenes eutrophus ATCC 17699 (plasmid).
Chromatium vinosum ATCC 17899 (rbcL).
Chromatium vinosum ATCC 17899 (rbcA).
Endosymbiont
of Alvinoconcha (Thiobacillus sp.?)
Hydrogenovibrio
marinus MH-110 (form II)
Hydrogenovibrio
murinus MH-110 (form I rbcLI)
Hydrogenovibrio
marks
MH- 1 IO (form I rbcL2)
Mn-oxidizing
bacterium SI85-9al
Nitrobacter vulgaris T3
Pseudomonas hydrogenothermophila
str.TH- 1
Rhodobacter capsulatus ATCC 11166 (form II)
Rhodobacter sphaeroides ATCC 17023 (form II)
Rhodobacter sphaeroides ATCC 17023 (form I)
Rhodospirillum rubrum (form II).
Thiobacillus denitrijicans ATCC 25259 (form II)
Thiobacillus denitrificans ATCC 25259 (form I).
Thiobucillus ferrooxidans Fe1
Thiobacillus ferrooxidans ATCC 19859
Xanthobacter jluvus H4- 14
II. Cyanobacteria
Anabuenu 7120 ATCC 27893
Anacystis nidulans PCC 6301
Prochlorococcus
mnrinus GP2.
Prochloron didrmni (not cultivated)
Prochlorothrix hollandicd
Swechococcus
sp. A 1
Synrchocystis sp. ATCC 27 184
501540
X03220
D21833
D21834
x57359
D13539
X65960
......
......
......
III. Plastids
Ahnfeltia fastigiatd
Antithamnion sp>.
Btypsis
maximd.
Chlumydomonas morwusiiJ.
Chlorelln ellipsoidea C-27
Coleochuete orbicularis var. pondspride
Cryptomonas +
Cynidium
caldarium RK-I
Cyanophora paradoxa 2980 IPP Goettingen
Cylindrotheca sp. N 1
Ectocarpus siliculosud
Euglena gracilis Pringsheim Z.
Gonyuulux p&edru
70 (form II).
Marchantiu polymorphd.
Nicotiuna ucuminatclJ.
Olisthodiscus luteus’
Op:u sntivd
Porphyridium aerugineumj
Pseudotsugu menzie.siiJ
Pyramimonns tetrarhyncus K-0002
A. e~rtrophu\ ATTCC17697
* SSU
rRNA
sequence
for
h SSU
rRNA
sequence
available
L Affiliation
(’ rRNA
mformation
c Based on analysis
F Signature
h Probable
in ribosomal
database
project
tree (RDP;
Redh
Red
Green
Green
Green
Green
Cryptomonad
Red
Glaucophyte
Diatom
Brown
Euglenophyte
Dinoflagellate
Plant
Plant
Diatom
Plant
Red
Plant
Green
http://rdp.life.uiuc.edu).
in RDI?
unknown.
sequence
’ Probable
UO4167
X54532
X55877
Ml5842
D 10997
L13477
X62349
X55524
x53045
M59080
X52503
Ml2109
L41063
X04465
Ml6896
X61918
DO0207
x17597
X52937
L34833
phylogenetic
analysis
phylogenetic
’ Phylogenetic
Y. Igarashi,
16s
placement
personal
rRNA sequence (R.
based on Serwaldt
commumcatmn.
Caspi,
personal
et al. (1982)
communication).
and E. Bock.
personal
communicatmn.
of Woese (1987).
’ Probable phylogenetic
1Strain not specified.
groups
tom~Bacillariophyceae;
from
of complete
placement
bahed on Kusano
placement
based on Wiegel
indicated
for plastids
Brown-Phaeophyta;
et al. (1991).
(1992).
are: Red-Rhodophyta;
Dinoflagellate-Pyrrhophyta;
Green-Chlorophyta;
Plant-Embryophyta.
Cryptomonad-Cryptophyta;
Glaucophyte-Glaucocystophyta;
Dia-
876
Delwiche
and Palmer
1a
3
63
I
Rhodobacter sphaeroides II +--,
Proteobacteria
Thiobacillus denitrificans /I +-------------/.
Hydrogenovibrio II +------------1.
1
Dinoflagellate plastid
3 p Proteobacteria
3 y Proteobacteria
3
a Proteobacteria
1p
Form II
Rubisco
-
Proteobacteria
=
100
1
a Proteobacteria
Red type
Form I
Rubisco
I Red and Brown
Plastids
1
1y
3
Cyanobacteria
-
Proteobacteria
-
:
100
q-
50 steps
i
/
/ 3 y Proteobacteria
Hydrogenovibrio Ll +--_j-J
“-wdomonas’
i//us ferrooxidans fe 1’
Thiobacillus ferr. 19859
denitrificans I
Ghr~ytyp?;
nechococcus
labaena
- Anacystis
.rochlorothrix
- Synechocystis
Prochloron
7
Cyanophora
1
3
3
3
n Proteobacteria
y Proteobacteria
8 Proteobacteria
y Proteobacteria
1
Cyanobacteria
r
1
+I~
/! 3
1
3
L
PyFamimonas
721 L_Chlamydomonas
h/ore//a
Bryopsis
sochaete
darchantia
Green type
Form I
Rubisco
Glaucophyte Plastid
Green Plastids
FIG. 2.-Maximum
parsimony tree of rhcL amino acids from all available bacteria and representative
plastids. The tree shown is one of
24 shortest trees of length 2.422, selected arbitrarily. Classification
of taxa based on 16s rRNA and other evidence is indicated to the right.
Bootstrap values above 60% are indicated above the branch, and branches which were not present in the strict consensus of the 24 shortest
trees are indicated by a bullet (0) below the branch. Branch lengths correspond to the inferred number of character state changes on each
branch (see scale bar). Dashed, arrow-headed
lines connect multiple rbcL sequences determined from the same taxon. *Ribosomal RNA sequences are not available for this strain, and proteobacterial
subgroup may be suspect. **Bootstrap analysis placed Chronzcztium L in a clade
with Hwk~~mor~ihrio
L2 and Prochlorococcus
with 60% bootstrap support, but this topology did not occur among the shortest trees.
Incongruence
Phylogeny
with Plastid and Proteobacterial
The division of form I sequences into red-like and
green-like groups is strongly in conflict with all other
studies of chloroplast
phylogeny;
there is little doubt
that all plastids are ultimately cyanobacterial
in origin,
and reasonable evidence that they are monophyletic
in
origin (fig. 1). Furthermore,
the division of proteobacteria into two widely disparate groups is similarly incongruent with current understanding
of bacterial evolution. There is strong evidence from 16s and 23s
rRNA, which is generally supported by data from protein genes (fig. l), that (Y-, p-, and y-proteobacteria
together form a monophyletic
group, with strongly supported subgroups that include the (Y-, p-, and p-/y-proteobacteria (the y-proteobacteria
are split into a paraphyletic group by the P-proteobacteria).
Several of these
monophyletic
groups are divided by the rbcL tree between the red-like and green-like sequence groups. For
example, the rbcL tree places the a-proteobacterium
Nifrobacter
vulgaris
(Strecker et al. 1994) among the
green-type
sequences but Rhodobacter,
Xmthobacter,
and an unnamed Mn-oxidizing
bacterium, also a-proteobacteria, among the red-types. Similarly, rbcL puts
the P-proteobacterium
Alcaligerzes in the red-like group,
but Thionbacillus denitrijcans
(Hernandez et al. 1996)
in the green-like group.
Aspects of the rbcL phylogeny are also incongruent
with eubacterial
gene phylogenies
within the red-like
and green-like groups. In particular, the c1- and p-proteobacterial sequences are intermixed in a manner that
is inconsistent
with the monophyly of the a-proteobacteria, and the cyanobacterium
Proch1orncoccu.s falls solidly among the y-proteobacteria,
contrary to other phylogenetic information.
The conflict between rbcL and other sources of
phylogenetic
information
is unlikely to reflect methodological error. Although poorly resolved data and weak
analytical methods can create a false appearance of conflict by failing to find the correct tree, and sampling error
(Cao et al. 1994; Cummings, Otto, and Wakeley 1995).
long branch attraction (Felsenstein
1978), and convergent evolution can cause phylogenetic
analyses to be
positively misleading, the robust phylogenetic
tree and
Lateral Transfer
RhodobacterForm
and Duplication
of Rubisco
Genes
877
II
Hydrogenovibrio
7
Rhodobacter Form
70
Xanthobacter
69
71
Alcaligenes
Cryptomonas
Porphyridium
Ectocarpus
Olisthodiscus
Chromatium A/B
Chromatium L/S
Mtrobacter
Prochlorothrci
71
I
Anabaena
33
Cyanophora
32
Euglena
Chlamydomonas
! 36 1 35 I
’ 32 ! 35 /
’
-31131
34 34
39 _::_._.
38 ; 35
38 38 j 32
37
39
34
39
1 38
37
a-_ ..A
36 42
I\!
85
55
’
84
1 84 i
86
83
50
-
83
57 \
’
30_, 30
_-L_ 28
-_s
35
;
27
28
28
30
26.
25
I:
35
41
36
43
38
45
46
:
45
44
35
33
45
i
45;41-53
Form
:
45
SO
I
FIG. 3.-Percent
amino acid identity of rubisco large (above diagonal) and small (below diagonal) subunit sequences from self xted taxa.
Comparisons between the red-like and green-like form I sequence groups are shaded. rbcS is encoded in the nuclear genome of green algae and
plants, but in the chloroplast genome of red-like plastids and Cyanophora.
dramatic differences (figs. 2 and 3) between green-like
and red-like rubiscos make it quite unlikely that these
phenomena explain the rbcL phylogeny.
In some cases, apparently aberrant phylogenies result from inappropriate rooting. But even if one assumes
that form II rubisco is an inappropriate
outgroup, and
rearranges the tree such that it and all form I rubiscos
from proteobacteria
(and red-like plastids and Prochlorococcus) form a monophyletic
group (i.e., by rooting
on cyanobacteria),
only one of the phylogenetic discrepancies is removed, and this at the expense of introducing
considerable
rate heterogeneity
throughout the tree.
The assignment to proteobacterial
subgroups in figure 2 of some strains, particularly those for which 16s
rRNA sequences have not been determined, could be in
error. However, while a few of the incongruencies
noted
here may be attributable to erroneous classification of a
particular strain, there are rbcL sequences from unambiguously
identified bacteria in both the red-like and
green-like sequence groups.
Thus, much or all of the conflict between the rbcL
phylogeny and other information
(fig. 1) must reflect a
discrepancy
between gene and organismal
phylogeny.
The most likely processes underlying
the problematic
rbcL phylogeny are horizontal gene transfer and ancient
gene duplication coupled with differential gene loss. We
will examine these two possibilities in turn, in each case
first focusing on the division of rbcL sequences into redlike and green-like groups, and then on other features
of the rbcL phylogeny.
Horizontal Gene Transfer Between
Red-like Form I Rubiscos
Green-
and
At least four independent
horizontal gene transfers
(fig. 4A) are required to explain the division of plastids
and proteobacteria
into the green-like
and red-like
groups evident in figure 2. To explain plastid phylogeny,
we postulate transfer of a red-like rubisco operon from
a proteobacterium
to a common ancestor of red and
brown plastids. Given that mitochondria probably arose
from within ol-proteobacteria and that this occurred prior
to the origin of plastids (Gray 1992), the donor bacterium should be assignable to a modern proteobacterial
group. Although the donor proteobacterium
is shown in
figure 4A as an o-proteobacterium,
the intermixing
of
(Y- and P-proteobacteria
in the rubisco tree (fig. 2) and
the small number of characterized
bacterial red-like
genes make it difficult to infer the identity of the donor
with confidence.
Within the proteobacteria,
at least three horizontal
gene transfers are implied by the distribution of red- and
green-like genes. A relatively simple scenario (fig. 4A)
involves the transfer of a (cyanobacterial)
green-like
rbcL to an ancestor of y-proteobacteria
early in their
evolution (but after the emergence of the P-proteobacteria from within the y-proteobacteria),
with subsequent
transfers from this green-like y-proteobacterial
lineage
to ancestors of the a-proteobacterium
Nitrobacter vulgaris and the P-proteobacterium
Thiobacillus denitrificans (fig. 4A). A slightly different sequence of transfers
would be postulated if the transfer of a green-type rubisco into the y-proteobacteria
preceded the emergence
of the P-proteobacteria,
or if some of the taxa in figure
2 were not identified correctly.
One potential source of direct evidence for horizontal gene transfer would be differences in codon usage
and base composition between the putatively transferred
gene and other host genes (Groisman, Saier, and Ochman 1992; Aoyama, Haase, and Reeves 1994; Haraya-
878
Delwiche
and Palmer
Rubisco type
Green Red
1
Proteobacteria
Proteobacteria
Proteobacteria
Cyanobacteria
L
Cyanophora
Red and
Brown Algae
Er;rnt21gae
FIG. 4.-Comparison
of gene transfer and gene duplication hypotheses for red VS. green conflicts only. The topology of the two mirrorimage, wide, gray trees shows a simplified “organismal”
phylogeny as inferred from phylogenetic
analyses of 16s rRNA and other genes (fig.
1; see text). The black and white trees shown within the organismal phylogeny represent form I red-like and green-like rubisco, and indicate
the events that would be implied if the observed distribution of these two kinds of rubisco reflects a history of horizontal gene transfer (A), or
ancient gene duplication and differential loss (B). The type of rubisco present in each group is indicated in the right-center column. The figure
assumes that the rbcL phylogeny and classifications
shown in figure 2 are correct, Conflicts occurring entirely within green-like (i.e., Prochlorococcus) or red-like (i.e., intermixing of o- and P-proteobacterial
sequences) groups are not shown. The diagrams are highly schematic, and
are not intended to indicate the precise timing of the events, Many minor variations of these schemes would also be consistent with the data
(see text).
ma 1994). However, in comparisons
of codon usage in
strains for which multiple genes are available, no cases
were found where rbcL codon usage differed substantially from that of other genes (data not shown). Although codon usage does not provide any evidence in
support of the horizontal transfer hypothesis, this does
not rule out the possibility of gene transfer of sufficient
age that the transferred gene would have fully adjusted
to the recipient cell’s compositional
environment
(highly
expressed genes such as rbcLS undergo relatively rapid
amelioration;
Sharp and Matassi 1994; Ochman and
Lawrence 1995), or of transfer between cells of similar
codon usage. Unfortunately,
too few gene sequences for
reliable comparisons of codon usage are available from
the two taxa-Nitrobacter
vulgaris (two other genes)
and Thiobacillus
denitri$cans
(no other genes)-that
seem to have undergone relatively recent gene transfer
events.
Other Horizontal
Gene Transfers
At least three other cases of horizontal gene transfer must be postulated to account for incongruities
in
the rbcL phylogeny besides those that involve the redgreen-like
split. One of these seems ironclad. Recent
work has shown that, unlike all other algal groups examined, the plastids of several peridinin-containing
dinoflagellates
(such as Gonyaulax) utilize a nucleat-encoded form II rubisco (Morse et al. 1995; Whitney,
Shaw, and Yellowlees 1995; Rowan et al. 1996). Given
the clearly cyanobacterial
nature of the dinoflagellate
chloroplast, it is unlikely that the Gonyaulax rubisco is
of plastid origin; form II rubisco is unknown among
cyanobacteria.
Morse et al. (1995) propose that the Gonyaular rubisco is derived from the mitochondrial
genome, having first undergone gene transfer from the ancestral mitochondrion
to the nuclear genome. Subsequent gene substitution
presumably resulted in the expression of this nuclear-encoded
gene in the chloroplast
in lieu of the putative native form I rubisco. Consistent
with this hypothesis, the Gonyaulax form II sequence
groups within the a-proteobacteria
in this, the first phylogenetic analysis performed with this sequence (fig. 2).
However, neighbor-joining
analysis places the Gonyaulax sequence outside all five diverse proteobacterial
form II sequences with moderately high (78%) bootstrap
support (data not shown). Regardless of whether the sequence is mitochondrial
in origin or the product of transfer from a free-living proteobacterium,
the Gonyaulax
rubisco provides a clear example of successful transfer
and substitution of rubisco genes in nature.
A second very likely horizontal gene transfer involves the rbcL gene of the cyanobacteria
Prochlorococcus and Synechococcus.
The Prochlorococcus
gene
is part of a strongly supported (94%) clade that otherwise consists entirely of green-like proteobacterial
sequences, and is distantly related to the other cyanobacterial sequences (fig. 2), while recent data from G. E
Watson and E R. Tabita (personal communication)
reveal that several marine species of Synechococcus con-
Lateral Transfer
tain rbcL genes that are closely related (>90% amino
acid identity) to the Prochlorococcus
gene. Shimada,
Kanai, and Maruyama (1995) noted the similarity of the
Prochlorococcus
sequence to that of the Chromatium
“cryptic” gene rbcL (Hydrogenovibrio
was not included
in their analysis) and from this inferred a phylogenetic
relationship between Prochlorococcus
and Chromatium.
However, analyses of 16s rRNA (Urbach, Robertson,
and Chisholm 1992) and rpoC1 (Palenik and Haselkorn
1992; Palenik and Swift 1996) indicate that other strains
of Prochlorococcus
are cyanobacterial,
and that several
cyanobacteria
have independently
acquired (or retained)
the chlorophyll
a/b pigment system characteristic
of
“prochlorophytes.”
There is no evidence linking Prochlorococcus or Synechococcus
with the proteobacteria
other than their rbcL sequences. Therefore, these cyanobacteria probably acquired their rubisco genes by lateral
transfer from the green-like proteobacterial
group.
Finally, as previously noted, the three a-proteobacterial sequences within the red-like clade (fig. 2) do not
form the expected (fig. 1) monophyletic group. Assuming
correct identification of bacterial strains, the simplest interpretation of this incongruity
is that one of the three
groups (RhodobacterlXanthobacter,
Alcaligenes,
or the
Mn-oxidizing
bacterium)
acquired its form I rubisco
genes by lateral transfer. Neither Rhodobacter nor Alcaligenes have codon usage patterns that suggest recent
gene transfer (data not shown), indicating that, if transferred, these genes either have already adapted to codon
usage in the host, or were acquired from a bacterium with
similar codon usage. Too few genes are available from
Xanthobacter and the Mn-oxidizing bacterium for reliable
analysis.
Gene Duplication
Form I Rubiscos
and Loss for Green-
and Red-like
If a single duplication
of the rubisco operon occurred prior to the divergence
between cyanobacteria
and proteobacteria,
and if one or the other copy of the
gene were lost (or simply remained undetected) in each
of the lineages that have been examined, then the division of the rbcL phylogeny into green-like and red-like
groups could be fully explained (fig. 4B). Under this
interpretation
the green-like and red-like kinds of rbcL
would be paralogs rather than orthologs (and xenologs),
and the distribution of taxa within the two groups would
reflect which copy had been retained.
The illustrative diagram of figure 4B clearly oversimplifies the complexity of the duplication/loss
hypothesis. For example, if one takes the phylogeny of figure
2 at face value, then four separate gene losses within
the cyanobacteria
are required; red-like rubisco must
have been lost independently
from each lineage that
branched prior to the origin of plastids. Although rRNAbased phylogenies of cyanobacteria
are poorly resolved,
even that study (Nelissen et al. 1995) which postulates
the earliest origin of plastids within the group would
require three independent
losses of red-like rubisco
among cyanobacteria
rather than the one shown in figure
4B. Similarly, the numbers of losses among proteobacteria (fig. 4B) are probably also underestimates
and are
and Duplication
of Rubisco
Genes
879
expected to rise as additional proteobacterial
rbcL sequences are determined.
The duplication hypothesis also implies a long coexistance of green- and red-like rubiscos in several lineages,
as indicated by dual black and white horizontal lines in
figure 4B. The duplicate genes in the cyanobacterial lineages would necessarily have persisted jointly for l-2
billion years, at least until after the origin of plastids and
the separation between the red-like and green-like lineages (Knoll and Golubic 1992; Schopf 1993).
If this is correct, the long persistence of both redgroup and green-group rubisco genes during eubacterial
evolution implies that some extant cyanobacteria
and
proteobacteria
might still contain both types of rubisco
genes. To date, no organism has been identified that has
both red-like and green-like rubisco genes, and indeed,
no cyanobacteria
have yet been found to have multiple
rubisco genes of any type. However, the methods that
have been used to isolate most rubisco genes could have
failed to detect the presence of duplicate genes. Sequence divergence between green-like and red-like rubisco genes is high enough (fig. 3) that heterologous
probes would probably detect only one of the two types
under typical hybridization conditions, and amplification
primers would be expected to be selective for one of the
two types.
Do any plastids contain both green- and red-like
rubisco? The large subunit of rubisco is thought to be
encoded by a single-copy plastid gene in all plants and
algae (except certain dinoflagellates),
but the possibility
that a second, divergent rbcL gene resides in the nucleus
is intrinsically
difficult to rule out.
An additional implication
of the duplication
hypothesis is that the long persistence of these duplicate
genes in eubacterial
evolution would suggest a functional distinction
between green-like and red-like rubiscos. Duplicate genes of identical function are expected
to persist only if there is a selective advantage to having
two copies of the gene. Although there may be some
advantage to having more than one identical copy of a
highly expressed gene such as rbcL, the sequence differences between red-like and green-like
rubisco are
great enough that it is unlikely that both of these types
would persist jointly without each having a functional
role. Substantial kinetic differences between the various
forms of rubisco have been documented (Tabita 1995),
and Rhodobacter is known to control the form of rubisco synthesized
(form I vs. form II) depending upon
the availability of CO, (Gibson 1995). A physiological
distinction
between green-like
and red-like rubiscos,
comparable
to that seen between the two rubiscos in
Rhodobacter
sphaeroides and in Chromatium (see below), would be expected if both types are found in a
living cyanobacterium.
By contrast, horizontal gene transfer postulates that
the differences between green-like and red-like rubiscos
are essentially neutral differences built up over long independent evolutionary
histories. Although many differences between the individual genes certainly reflect
adaptation to the form of photosynthesis
carried out by
the cell, the differences between green-like and red-like
880
Delwiche
and Palmer
rubisco developed
independently,
need not reflect distinct functional
and the two
roles.
types
Other Gene Duplications
The best evidence for duplication of rubisco genes
involves situations other than the red- green-like split,
where a given organism actually contains more than one
rubisco gene. One case involves the coexistence of form
I and II rubiscos in several diverse proteobacteria
(fig.
2; Akazawa, Takabe, and Kobayashi
1984), which is
thought to reflect a primordial duplication predating diversification
within the extant form I and II types. Because of their simple, homodimeric
(L2) form and low
substrate specificity, form II rubiscos have been widely
regarded as an ancestral form of rubisco (McFadden et
al. 1986; Roy and Nierzwicki-Bauer
1991). However, a
genuinely ancient form of rubisco would be expected to
have a wide phylogenetic
distribution,
whereas form II
rubisco (known only from proteobacteria
and certain dinoflagellates)
is less widespread
than form I rubisco
(present in proteobacteria,
cyanobacteria,
and all other
algal groups). For this reason, and because form II rubiscos appear to be changing faster than form I enzymes
(fig. 3; Rowan et al. 1996), one should not dismiss the
possibility that form II rubisco arose by gene duplication
during proteobacterial
evolution, followed by loss of the
small subunit and acceleration of sequence evolution.
A likely example of more recent gene duplication
is the presence of the two rubisco-encoding
operons
(rbcLS and rbcAB) in the y-proteobacterium
Chromatium vinosum (fig. 2) and comparable
dual operons
(rbcLS1 and rbcLS2) in Hydrogenovibrio
(which also
contains a third, form II gene; Yaguchi et al. 1994). The
Hydrogenovibrio
rbcL1 and Chromatium rbcA group together, to the exclusion of the Hydrogenovibrio
rbcL2
and Chromatium
rbcL (fig. 2). Although the shortest
trees found by parsimony analysis did not place the latter two genes in a monophyletic
group (fig. 2), parsimony bootstrap analysis found 60% support for such a
group (including Prochlorococcus),
and neighbor-joining
analysis found a similar clade with 59% support. Recent
gene transfer is unlikely because codon usage is uniform
among both Chromatium genes (Kobayashi et al. 1991)
and all three Hydrogenovibrio
genes (data not shown) but
is distinct between the two taxa. Consequently,
we believe that a duplication early in y-proteobacterial
evolution, in a common ancestor of Chromatium and Hydrogenovibrio, is the best explanation for these data. The
putatively long period of coexistence of these duplicates
has led to functional differentiation in Chromatium: under
normal culture conditions
the predominant
rubisco is
from the rbcAIB operon, and the rbcWS genes are expressed only at very low levels (if at all), but both genes
encode fully active enzymes with different rate constants
and CO, specificities (Kobayashi et al. 1991).
Finally, Alcaligenes
eutrophus H16 is known to
have two rubisco operons, one located on the bacterial
chromosome,
and the other on a plasmid. A similar situation is known to occur in A. eutrophus ATCC17707
(Anderson and Caton 1987), but, interestingly,
the chromosomal and plasmid rbcL genes from A. eutrophus
H16 are more closely related to each other than either
is to the chromosomal
gene from A. eutrophus
ATCC17707 (fig. 2; unfortunately,
the plasmid-encoded
rbcL sequence from this strain is not available). This
situation could reflect either very recent gene duplication, perhaps complicated by concerted evolution, or lateral transfer between closely related bacteria.
Conclusion
The two hypotheses discussed here, horizontal gene
transfer and gene duplication, need not be mutually exclusive. There is ample reason to believe that both of
these processes have played a role in the evolution of
rubisco: form I and form II rubiscos are probably the
product of an ancient gene duplication, and duplication
is probably also responsible for the dual form I genes
in Alcaligenes H16, and in Chromatium and Hydrogenovibrio, while the Gonyaulax form II gene and Prochlorococcus form I gene probably result from gene transfer. Thus the best explanation
for the overall rubisco
phylogeny will certainly involve some combination
of
these mechanisms.
On the basis of current knowledge,
division
of plastid and proteobacterial
rubiscos into
green-like and red-like groups could reasonably be attributed to either horizontal gene transfer or gene duplication, although on balance we tend to favor horizontal transfer. The phylogenetic
distribution of green-like
and red-like rubiscos is so complex that numerous independent events must be postulated in either case, but
the number of independent
losses necessary under the
duplication hypothesis is potentially huge. Furthermore,
if the duplication hypothesis is correct, it is rather surprising that no bacteria have yet been found that contain
both green- and red-like rubisco, especially given the
implied long periods of coexistence of the genes in various bacterial lineages.
The chaotic nature of the rubisco phylogeny at the
deep level explored in this study is ironic, for rbcL is
by far the most widely sequenced gene for systematic
purposes within algae and plants (e.g., Clegg 1993;
Freshwater et al. 1994; Manhart 1994; Sytsma and Hahn
1994). Fortunately, at these lower taxonomic levels, the
rubisco phylogeny is entirely consistent with other evidence, and phylogenetic
analyses based on rbcL have
been valuable in plant and algal taxonomy. Nor are the
rubisco genes the only genes that show signs of phylogenetic discontinuities
attributable either to gene transfer
(xenology) or duplication
and loss (paralogy). Among
the proteins that seem to have undergone similar events
in bacteria are GAPDH (Martin et al. 1993), HSP60 (Viale et al. 1994), and ATPase (Hilario and Gogarten
1993), and it is reasonable to believe that few, if any,
genes will be entirely free of these complications.
As a
widely sampled and conservative
gene, rbcL provides a
case study of these phenomena,
and it is to be hoped
that an understanding
of the evolution of rubisco will
facilitate interpretation of similar patterns in other genes.
Acknowledgments
We acknowledge Ron Caspi, Jonathon Eisen, Yasuo Igarashi, David Morse, Brian Palenik, Rob Rowan,
Lateral Transfer and Duplication of Rubisco Genes
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