J Mol Evol (2004) 58:348–358
DOI: 10.1007/s00239-003-2557-8
New Insights into the Evolutionary History of Type 1 Rhodopsins
Mario X. Ruiz-González,1,2 Ignacio Marı́n1
1
2
Departamento de Genética, Universidad de Valencia, Calle Doctor Moliner 50, Burjassot 46100, Valencia, Spain
Department of Zoology, Trinity College, Dublin, Ireland
Received: 15 May 2003 / Accepted: 9 October 2003
Abstract. Type 1 (archaeal) rhodopsins and related
rhodopsin-like proteins had been described in a few
halophile archaea, c-proteobacteria, a single cyanobacteria, some fungi, and a green alga. In exhaustive
database searches, we detected rhodopsin-related sequences derived not only from additional fungal
species but also from organisms belonging to three
groups in which opsins had hitherto not been described: the a-proteobacterium Magnetospirillum
magnetotacticum, the cryptomonad alga Guillardia
theta, and the dinoflagellate Pyrocystis lunula. Putative plant and human type 1 rhodopsin sequences
found in the databases are demonstrated to be contaminants of fungal origin. However, a highly diverged sequence supposedly from the plant Oryza
sativa was found that is, together with the Pyrocystis
sequence, quite similar to c-proteobacterial rhodopsins. These close relationships suggest that at least
one horizontal gene transfer event involving rhodopsin genes occurred between prokaryotes and
eukaryotes. Alternative hypotheses to explain the
current phylogenetic range of type 1 rhodopsins are
suggested. The broader phylogenetic range found is
compatible with an ancient origin of type 1 rhodopsins, their patchy distribution being caused by losses
in multiple lineages. However, the possibility of ancient horizontal transfer events between distant relatives cannot be dismissed.
Key words: Rhodopsin — Cryptomonad — Dinoflagellate — Horizontal transfer
Correspondence to: Ignacio Marı́n; email: ignacio.marin@uv.es
Introduction
Rhodopsins are members of the seven-transmembrane receptor family, able to react to light by using
retinal as chromophore. There are two types of
rhodopsins, type 1 and type 2 rhodopsins. The bestknown type 2 rhodopsins are the animal visual photoreceptors (reviewed in Yokoyama 2000). Type 1
rhodopsins were discovered in halophile archaea, and
therefore they are often called ‘‘archaeal opsins.’’
Type 1 and type 2 rhodopsins are structurally similar
and perform related functions, but it is unclear
whether they are evolutionary related (reviewed by
Spudich et al. 2000). Archaea contain four classes of
type 1 rhodopsins. Two of them, sensory rhodopsin I
(or simply ‘‘sensory rhodopsin’’; sR) and sensory
rhodopsin II, also known as phoborhodopsin (pR),
function as sensors for phototaxis. The other two are
transport rhodopsins called bacteriorhodopsin (bR),
which functions as a proton-extruding pump, and
halorhodopsin (hR), which is a chloride uptake pump
(reviewed in Ihara et al. 1999; Spudich et al. 2000).
After their discovery in archaea, genes with clear sequence similarities to Type 1 rhodopsins were characterized in other lineages. First, they were found in a
few fungi (Graul and Sadee 1997; Bieszke et al. 1999a;
Idnurm and Howlett 2001; Zhai et al. 2001). Some of
these fungal genes encode proteins with the typical
sequence signatures of light-modulated opsins (Bieszke et al. 1999a), and one has been demonstrated to
bind retinal to form a photoactive pigment with a
photochemical reaction cycle typical of type 1 rhodopsins (Bieszke et al. 1999b). However, despite being
very similar in sequence, some other fungal proteins
349
must be unable to act as photosensors, because they
lack a critical lysine residue involved in retinal binding. This last type of molecules is called opsin-related
proteins (Bieszke et al. 1999a; Spudich et al. 2000).
Typical type 1 rhodopsin genes were later found in
some uncultured c-proteobacteria (Béjà et al. 2000,
2001; Man et al. 2003). Their product, called proteorhodopsin, is a transport rhodopsin, which works as a
light-driven proton pump (Béjà et al. 2000). Finally,
type 1 rhodopsins have recently been described in the
green alga Chlamydomonas reinhardtii, where they
function as receptors for phototaxis responses
(Sineshchekov et al. 2002) and as light-activated
proton channels when heterologously expressed (Nagel et al. 2002), and in the cyanobacteria Nostoc
(Anabaena) sp., which shows features typical of sensory rhodopsins (Jung et al. 2003).
For type 1 rhodopsins and their relatives, it has
been suggested that vertical transmission plus rare
horizontal gene transfer episodes explains the generation of the patchy evolutionary distribution observed (Bieszke et al. 1999a; Béjà et al. 2000).
Determination of the evolutionary history of these
proteins critically depends on precisely establishing
their evolutionary range and the phylogenetic relationships among rhodopsin genes. We have performed a comprehensive survey of the available type
1 rhodopsin sequences to determine whether horizontal transmission may have occurred. Although
the information is insufficient to determine the evolutionary history of all these genes, at least one
episode of horizontal transfer is suggested by the
close similarity of some eukaryotic and c-proteobacterial rhodopsins. Alternative hypotheses are
proposed to explain the current phylogenetic range
of these genes, so far found in a few, very distant
organisms.
Materials and Methods
Exhaustive TBLASTN searches (Altschul et al. 1997) for type 1
rhodopsins were performed on all the databases available at the
NCBI Web site (http://www.ncbi.nlm.nih.gov/). The database
compiled in the GOLD homepage (found at http://igweb.integratedgenomics.com/GOLD/) were also screened for sequences not
yet present in the NCBI databases. These searches were finished in
March 2003.
Multiple sequence alignments of the protein sequences, conceptually translated from the corresponding nucleotide sequences,
were performed using Clustal X, v. 1.83 (Thompson et al. 1987).
The alignments were corrected manually, editing the sequences
with GeneDoc 2.6 (Nicholas and Nicholas 1997). GeneDoc was
also used to highlight the similarities among sequences shown in
Fig. 1. Rhodopsins are membrane proteins with seven transmembrane helices. The most conserved region, included in our analyses,
spans from the middle of the second helix (helix B) to a few amino
acids beyond the C terminus of the seventh helix (helix G; for a
total of 180 to 200 amino acids). Final phylogenetic trees were
obtained from the protein multiple alignments, after eliminating
the region between helix B and helix C, which cannot be reliable
aligned. For phylogenetic reconstruction, three types of analyses
were performed. First, we used the neighbor-joining (NJ) method
(Saitou and Nei 1987), as implemented in Clustal X, v. 1.83, with
correction for multiple substitutions. One thousand bootstrap
replicates were performed to determine the reliability of tree topology. Second, we utilized maximum parsimony (MP), as implemented in MEGA 2.1 (Kumar et al. 2001). We performed two
types of MP analyses. First, we generated a fast MP analysis from
which a bootstrap consensus tree (Nei and Kumar 2000) was obtained. For this first analysis, parameters were as follow: (1) all sites
included; (2) initial trees obtained by random addition, with 10
replicates; and (3) close-neighbor interchange with search level 3.
The second analysis was much more exhaustive. Parameters were
the same as in the first analysis, except that the number of trees
obtained by random addition was 100,000. This large number of
trees precludes the generation of a bootstrap consensus tree due to
computer power limitations. However, it allows exploration of the
consistency of the topology obtained with the previous, less exhaustive, method. Finally, we performed, as the third approach, a
maximum likelihood (ML) analysis, using PROTML 2.2 (Adachi
and Hasegawa 1992). The version of this program compiled for PC
computers by R. L. Malmberg, which is publicly available at
his Web page (http://dogwood.botany.uga.edu/malmberg/software.html), was used. We employed the random addition option
()q) and the JTT-f amino acid replacement model. The 50 best
trees were evaluated using the resampling of estimated log likelihood method (RELL; Kishino et al. 1990), equivalent to bootstrapping, to obtain the values presented below. Trees presented
below were drawn using TreeView 1.6.6 (Page 1996).
To establish whether other genes have phylogenetic ranges
similar to the one found for type 1 rhodopsin genes, we explored
online the Cluster of Orthologous Groups (COGs) database
(http://www.ncbi.nlm.nih.gov/COG/phylox.html [Tatusov et al.
1997; Tatusov et al. 2001]) and later we checked the results
obtained by systematic TBLASTN searches against the NCBI
databases, as indicated above.
Results
Table 1 summarizes the information for the 67 type 1
rhodopsins and rhodopsin-like sequences detected.
The multiple alignment of those sequences is shown
in Fig. 1, where it can be observed that 10 sequences
are incomplete. Several organisms for which rhodopsins were not hitherto described appear in Table
1: the a-proteobacterium Magnetospirillum magnetotacticum, the cryptomonad alga Guillardia theta, the
dinoflagellate Pyrocystis lunula, several plant species,
and even a sequence annotated as belonging to our
own species. In Fig. 2, we show a preliminary NJ tree.
It is obvious from Fig. 3 results that all but one of the
putative plant-derived sequences and also the supposedly human-derived sequence are actually most
likely of fungal origin. The single exception is a rice
sequence (Oryza1) that is quite different from any
other rhodopsin sequence described so far and very
different from the fungal proteins.
We then proceeded to eliminate all partial and
obviously contaminant sequences, carefully reanalyzing the remaining 56 full-length sequences. Results
are presented in Fig. 3, where we show the NJ tree,
which is similar to the one shown in Fig. 2, together
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Table 1.
Type 1 rhodopsin sequences detected in our searches
Species and gene name
Eukaryotes (all fungal species, unless indicated)
Aspergillus nidulans
Botrytis cinerea
Candida albicans
Candida albicans
Candida glabrata
Chaetomium globosum
Chlamydomonas reinhardtii (green alga)
Chlamydomonas reinhardtii (green alga)
Coccidioides posadasii
Coriolus versicolor
Cryptococcus neoformas
Gibberella zeae
Guillardia theta (cryptomonad alga)
Histoplasma capsulatum
Homo sapiens (animal)
Leptosphaeria maculans
Magnaporthe grisea
Mycosphaerella graminicola
Mycosphaerella graminicola
Neurospora crassa NOP1
Neurospora crassa YRO2
Oryza sativa (indica) (plant)
Oryza sativa (indica) (plant)
Paracoccidiodes brasiliensis
Prunus persica (plant)
Pyrocystis lunula (dinoflagellate)
Saccharomyces bayanus
Saccharomyces castellii
Saccharomyces cerevisiae YDR033W
Saccharomyces cerevisiae YRO2
Saccharomyces cerevisiae HSP30
Saccharomyces mikatae
Saccharomyces paradoxus
Schizosaccharomyces pombe
Shorgum bicolor (plant)
Triticum aestivum (plant)
Zygosaccharomyces rouxii
Archaea
Haloarcula argentinensis bR
Haloarcula japonica bR
Haloarcula sp. (Andes) bR
Haloarcula vallismortis hR
Haloarcula vallismortis pR
Haloarcula vallismortis sR
Halobacterium salinarum hRp
Halobacterium salinarum hR
Halobacterium salinarum bR
Halobacterium salinarum pR
Halobacterium salinarum sR
Halobacterium sp. AUS bRp
Halobacterium sp. SG1 Hr
Halobacterium sp. SG1 bRp
Halobacterium sp. SG1 sR
Halobacterium sp. NRC-1 bR
Halobacterium sp. (BOP) bR
Halorubrum sodomense hR
Halorubrum sodomense bR
Halorubrum sodomense sR
Haloterrigena sp. st. arg-4 hR
Natromonas pharaonis hR
Natromonas pharaonis pR
Sequence name (Figs. 1 to 3)
Accession no.
Aspergillus
Botrytis
Cand albicans HSP30
Cand albicans HSP31
Cand glabrata
Chaetomium
Chlamydomonas CSOA
Chlamydomonas CSOB
Coccidioides
Coriolus
Cryptococcus
Gibberella
Guillardia
Histoplasma
Homo
Leptosphaeria
Magnaporthe
Mycosphaerella1
Mycosphaerella2
Neurospora NOP1
Neurospora YRO2
Oryza1
Oryza2
Paracoccidioides
Prunus
Pyrocystis
Sacch bayanus
Sacch castellii
Sacch cerev YDR033W
Sacch cerev YRO2
Sacch cerev HSP30
Sacch mikatae
Sacch paradoxus
Schizosaccharomyces
Shorgum
Triticum
Zygosaccharomyces
AACD0100055
AL115930
PEDANT ca1507*
PEDANT ca4034*
BZ293756 + BZ297564
BP099207
AV640817
AB058891
AY059409
AB018405
SGTC cneo011005.C1391**
BU059532 + BU067691
AW342219
F_HGC186AR.contig-2909***
AC138524
AF290180
AC098842
AW180117
COGEME mg[0384]****
AF135863
AL356815
AAAA01084480
CA764330
BQ497887
BU041889
AF508258
AACA01000331
AZ926138
Z68196
Z35923
M93123
AABZ01000282
AABY01000119
CAA21219
AW564434
AL821328
AL395409
Ha argentinensis bR
Ha japonica bR
Ha sp bR
Ha vallismortis hR
Ha vallismortis pR
Ha vallismortis sR
Hb salinarum hR
Hb salinarum hR
Hb salinarum bR
Hb salinarum pR
Hb salinarum sR
Hb sp AUS2 bR
Hb sp SG1 hR
Hb sp SG1 bR
Hb sp SG1 sR
Hb sp NRC1 bR
Hb sp bR
Hr sodomense hR
Hr sodomense bR
Hr sodomense sR
Hr sp arg4 hR
N pharaonis hR
N pharaonis pR
D31880
AB029320
S76743
D31881
Z35308
D83748
P16102
D43765
AF306937
AE005080
X51682
S56354
X70292
X70291
X70290
AE004437
AB009620
AB009622
D50848
AB009623
AB009621
J05199
Z35086
(Continued)
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Table 1.
Continued
Species and gene name
Sequence name (Figs. 1 to 3)
Accession no.
Eubacteria
Uncultured c-proteobacterium (MEDA17)
Uncultured c-proteobacterium (MEDA15)
Uncultured c-proteobacterium (PAL B6)
Uncultured c-proteobacterium (BAC 31A8)
Uncultured c-proteobacterium (BAC 40E8)
Magnetospirillum magnetotacticum (a-proteobacterium)
Nostoc sp. (cyanobacterium)
Proteobacterium1
Proteobacterium2
Proteobacterium3
Proteobacterium4
Proteobacterium5
Magnetospirillum
Nostoc
AY250738
AY250740
AF349998
AF279106
AF349976
AAAP01002252
AP003592
Note. Archaeal gene nomenclature follows Ihara et al. (1999) (bR,
bacteriorhodopsin; hR, halorhodopsin; sR, sensory rhodopsin; pR,
phoborhodopsin). All numbers refer to the NCBI database with the
exception of the following: () obtained from the PEDANT database (http://pedant.gsf.de/); () from the Stanford Genome
Technology Center database (http://www-sequence.stanford.edu);
( ) from the Genome Sequencing Center, Washington University School of Medicine database (http://genome.wustl.edu);
and ( ) from the COGEME database (http://cogeme.ex.ac.uk).
with the information for bootstrap/RELL values of
those branches reasonably supported by the three
methods of phylogenetic reconstruction used (see
Materials and Methods). These results fully confirm
previous phylogenetic studies for archaeal proteins
(e.g., Ihara et al. 1999), with the exception of the lack
of support for a monophyletic clade containing all
phoborhodopsins. This may be related to phoborhodopsins evolving at the highest rate among all
archaeal proteins (Ihara et al. 1999). It is also congruent with trees obtained with fewer sequences by
Bieszke et al. (1999a) and Zhai et al. (2001). The
position of proteorhodopsins in our Fig. 3 is different
from the one shown by Béjà et al. (2000), where they
appeared closer to archaeal sensory rhodopsins than
to archaeal halorhodopsins or bacteriorhodopsins.
However, the topology for the most internal branches
in our tree cannot be ascertained with the available
data (e.g., compare Figs. 2 and 3), and therefore, we
conclude that the precise position of proteorhodopsins is ambiguous. Support for all fungal sequences
forming a single monophyletic group increases
slightly when partial sequences are eliminated (see NJ
values in Figs. 2 and 3). Moreover, the low level of
support for this branch is mainly caused by a single
highly divergent sequence, from Cryptococcus neoformans, whose phylogenetic position is ambiguous.
It appears in some analyses together with nonfungal
sequences (see Fig. 3 legend). Without this sequence,
support for a fungal monophyletic group is much
higher in the three methods of phylogenetic reconstruction (not shown). Two fungal groups are apparent in Figs. 2 and 3 (and, again, they obtain
stronger support when the C. neoformans sequence is
eliminated). The existence of these two classes of type
1 rhodopsin-like proteins in fungi was already suggested by Bieszke et al., (1999a). This dichotomy is
congruent with the known features of these proteins,
because one of the groups contains the opsin-related
sequences in multiple ascomycete species and the
basidiomycete Coriolus versicolor, while the second
branch includes the bona fide Neurospora rhodopsin
gene Nop1 (Bieszke et al. 1999a) and very similar
genes of other ascomycete species. Considering the
sequences for which we have information (see Fig. 1),
we found that only 1 of the 15 genes in the opsinrelated branch contains the critical lysine residue in
the seventh helix, known to be involved in retinal
binding, while all the genes in the Nop1 branch have
it. The already-mentioned problematic sequence of
the basidiomycete C. neoformans also contains that
residue and may thus belong to the Nop1 class. The
latter result suggests that the duplication that gave
rise to the two main groups of fungal genes is older
than the ascomycetes–basidiomycetes split.
Five eukaryotic sequences are excluded from the
fungal group (Figs. 2 and 3). Among them, we found
the two rhodopsin genes recently described in
Chlamydomonas reinhardtii (Nagel et al. 2002; Sineshchekov et al. 2002). Their high similarity suggests
that they derive from a recent duplication. In addition, we detected sequences putatively derived from
the cryptomonad alga Guillardia, the dinoflagellate
Pyrocystis, and the plant Oryza. The phylogenetic
relationships of the algal sequences with other prokaryotic or eukaryotic rhodopsins are unclear (see
Figs. 2 and 3). However, the Pyrocystis and Oryza
sequences are quite similar and appear together with
proteorhodopsins. The sequences of the a-proteobacterium Magnetospirillum magnetotacticum and the
cyanobacterium Nostoc sp. (Jung et al. 2003) are very
different from proteorhodopsins and appear in the
phylogenetic trees as independent branches.
To determine how frequently genes are limited to
four groups as distant as archaea, eukaryotes, proteobacteria, and cyanobacteria, we searched the
COGs database, using the phylogenetic pattern
search option, for genes present in those groups but
absent in other eubacteria. All genes with those
characteristics were then checked individually in
searches against the NCBI sequence databases to
confirm the COGs results. We finally found three
352
Fig. 1a
353
Fig. 1. Multiple-sequence alignments of type 1 rhodopsins and rhodopsin-like proteins. Positions of helices B to G (according to Spudich
et al. 2000) are indicated at the top. Biochemically conserved residues are highlighted with gray (conserved) to black (highly conserved)
shading. Sequences are ordered according to the phylogenetic tree shown in Fig. 2.
clear-cut cases with a similar phylogenetic pattern.
They correspond to (1) the orthologues of the gene
encoding for yeast translation initiation factor SUI1
(COG0023), already described by Kyrpides and
Woese (1998); (2) COG0278, which includes glutaredoxin-like genes of E. coli (ydhD) and S. cerevisiae (GRX3, GRX4, and GRX5) among other
organisms; and (3) orthologues of the E. coli stress
response gene bolA (COG0271). These results show
that the patchy phylogenetic distribution observed
for type 1 rhodopsins and rhodopsin-related sequences is not particularly unusual. It must be considered that these automatic searches are expected to
produce a significant underestimation of the number
of genes with the phylogenetic range that we explored, due to the fact that only a single eukaryote
(Saccharomyces cerevisiae) and a single cyanobacterium (Synechocistis sp.) are included in the COGs
database. In fact, rhodopsins themselves cannot be
detected by such methods, because they are not present in the Synechocistis genome.
Discussion
Type 1 rhodopsins are present in archaea, eubacteria,
and eukaryotes. However, in each of these three domains, they have been found in just a few species. In
archaea, they seem to be restricted to some related
354
halophilic species. In eubacteria, they have appeared
in c-proteobacteria, a single cyanobacterium, and, as
described above, an a-proteobacterium. In eukaryotes, in addition to the already known rhodopsins in
fungi and green algae, we found that sequences annotated as belonging to a cryptomonad alga (Guillardia theta), a dinoflagellate (Pyrocystis lunula),
several plants, and even humans encoded rhodopsinlike proteins. However, phylogenetic analyses
strongly suggested that all but one of the putative
plant sequences and the human sequence were false
positives of fungal origin. Only one plant sequence,
supposedly from Oryza sativa, had a level of divergence that suggested it was not derived from fungi.
The discovery of these multiple contaminant sequences raises the critical question of whether the
other new eukaryotic sequences found may actually
not belong to the species to which they have been
attributed. This problem must be tackled individually. However, an important preliminary consideration is that it is obvious from our results (Fig. 2) that
rhodopsin sequences provide reasonable phylogenetic
information only for relatively closely related species.
Several data can be used to determine the age of the
splits that separated organisms that appear in supported groups in our trees. It turns out that they are
separated by a few hundreds of millions of years of
independent evolution. Thus, estimates of divergence
time among archaea allowed the origin of all species
containing archaeal type 1 rhodopsins to be traced
back in time to about 700 million years ago (Ihara
et al. 1999). Similarly, rDNA data presented by Suzuki et al. (2001) and our own data on sequence
distances suggest that all characterized proteorhodopsins are present in organisms that diverged at
most a few hundreds of millions of years ago (e.g.,
compare in Figs. 2 and 3 the branch lenghts for
proteorhodopsins with even the slowest-evolving archaeal opsins). Finally, the finding of rhodopsins in
basidiomycetes and ascomycetes determines the origin of these proteins in fungi to be older than 600
million years, when those two groups diverged (Redecker et al. 2000; Redecker 2002). Heckman et al.
(2001) have suggested that the basidiomycetes/ascomycetes split would have occurred much earlier
(perhaps 1.2 billion years ago), but this conclusion
has been critizised by other authors (e.g., RodriguezTrelles et al. 2002). In opposition to those data, organisms that appear in our trees in independent
branches, and whose divergences can be approximately situated in time, split much longer ago. For
example, cyanobacterial and proteobacterial rhodopsins appear in our trees as independent branches.
Feng et al. (1997) estimated that the split of the lineages that generated proteobacteria and cyanobacteria occurred about 2.2 billion years ago. In a recent
combined analysis of paleontological, phenotypic,
and molecular data, Cavalier-Smith (2002) has suggested that the split of the ancestors of cyanobacteria
and proteobacteria may have occurred even earlier,
more than 2.5 billion years ago.
These considerations suggest that the strong similarity of the putative Oryza and Pyrocystis sequences
to c-proteobacterial rhodopsins cannot be explained
by conventional vertical transmission. We should
then postulate a totally unrealistic deacceleration of
the rate of change of those proteins. It is also illogical
to argue that they are of mitochondrial origin. In that
case, the eukaryotic proteins should be more similar
to the a-proteobacterial Magnetospirillum rhodopsin
than to the c-proteobacterial rhodopsins. We are
therefore left with two reasonable alternatives. First,
we can interpret that high similarity as supporting a
relatively recent horizontal transmission. Second,
those sequences may be rhodopsins of proteobacterial origin annotated as belonging to eukaryotic
species. We favor the first option because there is
good preliminary evidence that the Pyrocystis sequence indeed originated from the genome of the
dinoflagellate. The clone that contains this sequence
derives from a cDNA library obtained by Okamoto
et al. (2001). Although the starting RNA derived
from a nonaxenic culture (CCMP731; see ProvasoliGuillard National Center for Culture of Marine
Phytoplankton,
http://ccmp.bigelow.org/SD/dculture.php?strain = CCMP731), the cDNAs were obtained from poly(A) RNA (Okamoto et al. 2001),
thus making the probability of contaminants of
bacterial origin much less likely. Moreover, Okamoto
et al. (2003) have obtained evidence that the expression of this gene follows a circadian rhythm. So far,
this type of rhytmicity has not been described in
proteobacteria (but see Dvornyk et al. 2003). Thus, it
is unlikely that this clone has a proteobacterial contaminant origin, although it is still formally possible
and independent validation is advisable. In any case,
the fact that rhodopsins exist in green algae and
probably also in dinoflagellates suggests that they
may also exist in groups evolutionarily close to those
two. In this context, we think that the Guillardia sequence may be a bona fide cryptomonad rhodopsin.
There is growing evidence that cryptomonads and
dinoflagellates derive from a common ancestor,
originated after an endosymbiotic event involving a
red alga and a flagellate (reviewed in Archibald and
Keeling 2002). It is attractive then to hypothesize that
rhodopsins were present in the lineage that generated
the closely related green and red algae (e.g., Baldauf
et al. 2000; Moreira et al. 2000; Van de Peer et al.
2000) and that cryptomonads and dinoflagellates
obtained those genes from their red algal ancestor. In
the case of Guillardia, the genome of the red algae has
mostly dissapeared, with fewer than 500 genes in
three small chromosomes found in a structure derived
355
Fig. 2. Preliminary unrooted phylogenetic tree for type 1 rhodopsins and their relatives, obtained using the neighbor-joining
method. Archaeal species are shown in italics, and eubacterial
species in boldface italics. Numbers refer to bootstrap support (as a
percentage). Only those branches with support of at least 40% are
shown. Asterisks refer to rhodopsins assigned to nonfungal species
that are most likely of fungal origin (see text).
from the algal nucleus, called the nucleomorph, remaining (Douglas et al. 2001). We found that the
Guillardia rhodopsin gene does not come from the
fully sequenced chloroplast or nucleomorph genomes. Therefore, it may derive from the mitochondria or the main nucleus. The first option is unlikely,
considering that this gene is also absent from the
mitochondrial genome of another cryptomonad,
Rhodomonas salina (accession number NC_002572).
Thus, it most likely comes from the main (flagellate)
nucleus. Despite this fact, it still may be of red algal
origin, because it may have been transferred, as has
been shown to occur for other genes (e.g., Deane
et al. 2000) from the algal nucleus (now nucleomorph) to the main nucleus. If we thus assume that
the algal and dinoflagellate rhodopsins have an ancient common origin and also that a relatively recent
horizontal transfer event occurred involving a dinoflagellate and a proteobacterium, the transfer must
have been from the eukaryote to the prokaryote. This
356
Fig. 3. Summary of the trees obtained with full-length rhodopsin
sequences. Numbers refer to the bootstrap or RELL values supporting each branch (as percentages), shown as NJ/MP/ML (see
Materials and Methods for details). Only values for those branches
supported by the three methods of phylogenetic reconstruction and
in which at least two of them gave values of support above 40% are
shown. Asterisks refer to branches with support percentages above
95% for each of the three methods of phylogenetic reconstruction.
When the MP value is in parentheses, it means that those branches
were supported by the primary MP analysis but were not present in
the most parsimonious trees obtained in the more exhaustive MP
analysis made by generating 100,000 random trees (see Materials
and Methods). The lack of support for a monophyletic fungal
branch is caused by the Cryptococcus sequence appearing separated
from the other fungi and together with the putative Guillardia
rhodopsin.
is also the expected direction of transfer considering
the relative likelihood of accepting foreign genes in
eukaryotic versus prokaryotic species.
However, we are still left with a second problem:
how species that belong to two evolutionarily very
distant eukaryotic groups, dinoflagellates and plants,
can have proteins that are so similar (Fig. 2). In this
case, we favor the idea that the putative Oryza sequence indeed may have not originated from any
plant genome. This truncated sequence is found in an
isolated 676-bp-long fragment annotated as the
‘‘whole genome shotgun sequence,’’ but it has not
357
been detected in the assembled genomic DNA of either of the two Oryza varieties that have been extensively sequenced. The fully sequenced Arabidopsis
and the other partially sequenced plant genomes also
seem to lack rhodopsins. Moreover, all the other
supposedly plant rhodopsin sequences have been
demonstrated to be of contaminant origin (see
above). The likelihood of this putative Oryza rhodopsin sequence being a false positive (i.e., not a
plant sequence) is thus high. Its origin is mysterious,
however, because it is clearly substantially different
from all the other rhodopsins found so far.
Accepting that one relatively recent horizontal
transfer may have occurred, the question remains
whether the patchy distribution observed is the consequence of several additional ancient horizontal
transfer events. Until very recently, support for an
ancient origin for type 1 rhodopsins was very weak.
They presented an extremely unusual phylogenetic
distribution, appearing in just a few closely related
species of archaea, proteobacteria, or fungi. These
results were hardly compatible with an ancient origin,
instead favoring explanations based on horizontal
transfer events. However, the demonstration that
rhodopsin genes exist in several other organisms such
as green algae (Nagel et al. 2002; Sineshchekov et al.
2002) and, especially, cyanobacteria (Jung et al.
2003), which are among the most distant relatives of
proteobacteria within the eubacterial domain, weakens the support for horizontal transmission-based
hypotheses. In fact, this broader phylogenetic range is
not unique anymore: We have found, using COGsbased analyses, three other genes with similar phylogenetic distributions. Considering the limitations of
those analyses mentioned in the previous section, it is
likely that more exist. It is therefore more reasonable
now to suggest that type 1 rhodopsins may have
emerged before the splits that gave rise to eubacteria,
eukaryotes, and archaea. Actually, Kyrpides and
Woese (1998) arrived at that conclusion for a case
with a similar phylogenetic distribution. Our description of rhodopsins in additional groups leads to
an even broader phylogenetic range and, thus, increases the likelihood of an extremely ancient origin
for these genes. However, our results do not fully
dismiss the possibility of several ancient horizontal
transfers involving totally unrelated organisms. In
summary, we envisage three main alternatives.
(1) Extremely ancient origin plus a single recent
horizontal transfer: Type 1 rhodopsin origin predates
the splits separating eubacteria, archaea, and eukaryotes, and a single horizontal transfer event occurred between eukaryotes and proteobacteria. In
this case, we must postulate multiple losses in many
independent lineages.
(2) Ancient horizontal transfer to archaea +
symbiosis + recent horizontal transfer: Type 1 rho-
dopsins originated in eubacteria and were later
horizontally transferred to archaea. They were
transferred to eukaryotes by symbiosis, because they
were present in the bacterial ancestors of mitochondria (a-proteobacteria) and chloroplasts (cyanobacteria). However, only a few lineages still conserve
these genes: fungi (derived from mitochondria) and
organisms (green algae, cryptomonads, dinoflagellates) derived from the ancestral cyanobacteria–
eukayote symbiosis that generated chloroplast-containing eukaryotes or that obtained cyanobacterial
genes through secondary symbioses (see Stechmann
and Cavalier-Smith 2002; Yoon et al. 2002).
(3) Two or more ancient horizontal transfers + a
recent horizontal transfer: Rhodopsins originated in
a domain of life (perhaps eubacteria, considering the
very old dichotomy cyanobacteria/proteobacteria)
and they were long ago horizontally transferred once
to archaea and one or more times to eukaryotes.
More recently, an additional horizontal transfer occurred from eukaryotes to eubacteria.
Only additional information, especially characterization of type 1 rhodopsins in more types of organims, may discriminate among these alternatives.
We close with two final notes. First, our phylogenetic reconstructions did not consistently group
together sensory or transport rhodopsins. It is potentially possible to establish whether sensory and
transport rhodopsins are two evolutionarily independent branches, which diverged long ago, or
whether functional shifts between those two types of
proteins have occurred in particular lineages. However, that demonstration will clearly depend on our
ability to determine all the horizontal transfer events
involving these genes that occurred in the past. Second, the available data suggest that type 2 rhodopsins
may be of much more recent origin than type 1
rhodopsins. However, the question of whether they
evolved from type 1 rhodopsins in eukaryotes or
arose independently is still open.
Acknowledgments. We thank J. Woodland Hastings for sharing
unpublished data on the Pyrocystis opsin. This work was supported
by a Generalitat Valenciana grant (BM-011/2002) and a MCYT
grant (GEN2001-4851-C06-02), which provided the funds required
for computer equipment.
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