*Department of Biology, Division of General Genetics,
and
Department of Biology, Division of Limnology, University of Oslo, Oslo, Norway;
and
Department of Cell Biology and Molecular Genetics/Plant Biology, University of Maryland at College Park
Abstract
The three anomalously pigmented dinoflagellates Gymnodinium galatheanum, Gyrodinium aureolum, and Gymnodinium breve have plastids possessing 19'-hexanoyloxy-fucoxanthin as the major carotenoid rather than peridinin, which is characteristic of the majority of the dinoflagellates. Analyses of SSU rDNA from the plastid and the nuclear genome of these dinoflagellate species indicate that they have acquired their plastids via endosymbiosis of a haptophyte. The dinoflagellate plastid sequences appear to have undergone rapid sequence evolution, and there is considerable divergence between the three species. However, distance, parsimony, and maximum-likelihood phylogenetic analyses of plastid SSU rRNA gene sequences place the three species within the haptophyte clade. Pavlova gyrans is the most basal branching haptophyte and is the outgroup to a clade comprising the dinoflagellate sequences and those of other haptophytes. The haptophytes themselves are thought to have plastids of a secondary origin; hence, these dinoflagellates appear to have tertiary plastids. Both molecular and morphological data divide the plastids into two groups, where G. aureolum and G. breve have similar plastid morphology and G. galatheanum has plastids with distinctive features.
Introduction
Chloroplasts (more generally called plastids) are derived from previously free living prokaryotes through endosymbiosis between a cyanobacterium and a eukaryote (for insights and reviews, see Merezhkovsky 1905
; Douglas 1994
; Van De Peer 1996
; Martin et al. 1998
; Palmer and Delwiche 1998
; Delwiche 1999
). The plastids of rhodophytes, chlorophytes, and glaucophytes are surrounded by two membranes and probably directly derived from a cyanobacterial ancestor and are thus called primary plastids. Whether or not these three plastid lineages themselves are the result of one single endosymbiosis event is not known, and there are contradictory data concerning the number of primary endosymbiosis events (Gibbs 1981
; Lockhart et al. 1992
; Palmer 1993
; Palmer and Delwiche 1998
). A number of other protists acquired their plastids via secondary endosymbiosis. Secondary endosymbiosis is a phenomenon whereby one eukaryote engulfs another eukaryote and permanently retains part of its prey as a degenerate endosymbiont (for discussion, see the articles cited above and McFadden et al. 1994
). Most plastids surrounded by more than two membranes are thought to be the result of secondary endosymbiosis events (Gibbs 1981
).
Within the dinoflagellates, there are several different plastid types, and many endosymbiotic events have apparently taken place. The plastids have been acquired from various pigmentation groups (Watanabe et al. 1987
; Farmer and Roberts 1990
; Elbrächter and Schnepf 1996
; Chesnick et al. 1997
). Some species have plastids that are contained within an otherwise independent endosymbiont, and some of these may represent transitional states in which the endosymbiont is consistently present but has apparently not been integrated as a stable organelle. All of these have a secondary nucleus associated with "their" plastids (Dodge 1971
; Tomas and Cox 1973
).
The dinoflagellates with true plastids can roughly be divided into three groups: The most widespread and typical pigmentation pattern is chlorophyll a + c and peridinin. These plastids are typically bound by three membranes (Dodge 1975
) and appear to have a unique organization of the plastid genome ("minicircles"; Zhang, Green, and Cavalier-Smith 1999
). Phylogenetic analyses suggest that they are related to red algal plastids, and they are likely to be of secondary origin (Zhang, Green, and Cavalier-Smith 1999
). A second group, the Dinophysis species, have plastids containing phycobilins and chlorophyll a + c. These plastids resemble those of cryptophytes and are bound by two membranes only (Schnepf and Elbrächter 1988
). Species containing chlorophyll a + c and fucoxanthin derivatives can be said to constitute a third group (Van den Hoek, Mann, and Jahns 1995
). Based on pigmentation data, there appear to be several "subgroups" of the fucoxanthin-containing species (Bjørnland 1990
; Whatley 1993
), and in this work we focused on a group of dinoflagellates that have 19'-hexanoyloxy-fucoxanthin as their main carotenoid (for simplicity, they will be referred to herein as the fucoxanthin-containing dinoflagellates/plastids). These organisms have no girdle lamella in their plastids (Steidinger, Truby, and Dawes 1978
; Kite and Dodge 1985, 1988
), and no extra nuclei have been identified (Kite and Dodge 1988
; Bjørnland 1990
). The three species investigated were Gymnodinium galatheanum Braarud, Gyrodinium aureolum Hulburt, and Gymnodinium breve Davis. They all have pigment composition (Bjørnland 1990
) and plastid ultrastructure (Steidinger, Truby, and Dawes 1978
; Kite and Dodge 1985, 1988
) resembling that of haptophytes.
By using capillary techniques to isolate algae for single-cell PCR and testing a large number of different primers, full-length plastid SSU rRNA (16S) gene sequences were determined from the three species. Nuclear SSU rRNA (18S) gene sequences were also determined, and various phylogenetic analyses were performed to study the origin and evolution of the fucoxanthin-containing plastids in the dinoflagellates.
Materials and Methods
Algal Strains, Culture Conditions, and Microscopy
Cell morphology was studied by standard light microscopy techniques. Autofluorescence of the plastids was generated by blue-light excitation and recorded by photography or video image analyses.
Gymnodinium galatheanum and G. aureolum were isolated from the Oslofjord by Karl Tangen and obtained from the division of Marine Botany at Department of Biology, University of Oslo, as was a strain of Pavlova gyrans Butcher. Gymnodinium breve was obtained from CCMP, West Boothbay Harbor, Maine. Chrysochromulina polylepis Manton et Parke was a gift from Bente Edvardsen at the division of Marine Botany (University of Oslo). All dinoflagellate species were cultured in Erd-Schreiber natural seawater medium (Føyn 1934
, modified) under fluorescent illumination (14/10 h L/D cycle).
Cloning and Sequencing
To isolate DNA for our nuclear SSU rDNA (18S) sequences, up to 2 x 106 exponentially growing cells were collected from the cultures by centrifugation (8,000 x g for 5 min). DNA isolation was performed using DNA-binding magnetic beads and ethanol precipitation/washing (Dynabeads DNA DIRECT, Dynal) as described by Rudi et al. (1997)
. Nuclear SSU rDNA was PCR-amplified with primers A and B designed for amplification of haptophytes (Medlin et al. 1988
).
To avoid amplification of bacterial SSU rDNA (16S) for our plastid 16S sequences, single-cell isolations were performed. Cells were capillary isolated from nonaxenic cultures, washed in sterile medium, and then transferred to 200-µl Perkin-Elmer tubes containing sterile distilled water, in which amplification was performed directly.
Primers used to amplify SSU rDNA from the fucoxanthin-containing dinoflagellate plastids were designed with reference to a 16S SSU rDNA alignment including a wide range of both bacterial and plastid sequences (available on request). Both general SSU rDNA and plastid-specific primers were designed (table 1 ) and used in various combinations to amplify overlapping fragments. In addition, some species-specific primers were made to increase the specificity of the reactions and perform primer-walking (table 1 ). All amplifications were done with 5 mM Mg2+, Dynazyme polymerase (Finnzymes), and relatively low annealing temperatures.
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Plastid-specific and general primers (table 1 ) were used to amplify plastid SSU rDNA from Pavlova gyrans (Pavlovophyceae) and Chrysochromulina polylepis (Prymnesiophyceae) from regular DNA isolations (as described for nuclear SSU rDNA).
Phylogenetic Analyses
The framework for our nuclear SSU rDNA alignment was downloaded from the rRNA WWW server (De Rijk, Van de Peer, and De Wachter 1998
), and additional sequences were added using the software SeqPup (Gilbert 1996
). Forty-seven species (table 2
) and 1,709 unambiguously aligned characters were used in the analysis (alignment available on request). An initial topology was determined by neighbor joining (NJ) with LogDet distances using proportion of invariable sites (pinvar) estimated from an NJ tree using Kimura two-parameter distances (K2P). LogDet is thought to perform relatively well on data sets with A+T contents varying between the sequences (Lake 1994
; Lockhart et al. 1994
; Swofford et al. 1996
). Maximum-likelihood distances were determined using a gamma shape parameter (
, four categories)/pinvar/base frequencies/general time reversible (GTR) substitution matrix model, with parameters estimated simultaneously using the NJ tree. These distances were used to calculate a minimum-evolution (ME) tree with 20 heuristic searches using random addition of the sequences and tree bisection-reconnection (TBR) branch swapping. This model was significantly better than simpler nested models when compared according to the likelihood ratio test (Huelsenbeck and Rannala 1997
). The data set was also bootstrapped with 100 replicates, using one heuristic search per replicate (random addition of the sequences) and the same model as the initial searches. All of the analyses were done using PAUP*, version 4.0d64 (test version, D. L. Swofford).
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All of the PAUP*, version 4.0d64, analyses were done at the Center for Information Technology Services (USIT, University of Oslo) using an SGI ONYX MPIS R10000 processor (195 MHz). The fastDNAml/PHYLIP work was done on (six parallel) IBM RS6000 SP POWER2 processors (120 MHz, part of the 32-node IBM cluster at USIT). SeqPup was used on various tabletop Macintosh computers.
Results
Cell Morphology and Nuclear Phylogeny
Gyrodinium aureolum and G. breve shared several distinctive morphological features when compared with G. galatheanum. Gyrodinium aureolum and G. breve both have highly vesiculated cytoplasms (Steidinger, Truby, and Dawes 1978
) and nearly identical plastid morphologies. They have approximately 20 bean-shaped plastids that are easily separated as individual structures on compression of the cell and plastid DNA arranged as beaded bands along the ventral side of each plastid (fig. 1A
; Kite and Dodge 1985
). Gymnodinium galatheanum, on the other hand, has a compact nonvesiculated cytoplasm, the plastid(s) appear to be a single, lobate structure (fig. 1B
; as indicated by Bjørnland and Tangen 1979
; Kite and Dodge 1988
), and the plastid DNA is arranged as scattered nucleoids (fig. 1B
; Kite and Dodge 1988
).
|
Because the origin of the fucoxanthin-containing plastids was uncertain, our analyses included a wide range of photosynthetic taxa, and most major groups of photosynthetic protists can be recognized in the tree (fig. 2 ), with 100% bootstrap support for monophyly of several groups. There is also high support for monophyly of the alveolates (98%), rhodophyta (95%), and chlorophyta (95%; used as an outgroup). The fucoxanthin-containing dinoflagellates are all placed unequivocally within the dinoflagellate group. Gyrodinium aureolum and G. breve group together with 100% support, and they had a high degree of sequence similarity (>99% similarity using JC distances). Gymnodinium galatheanum comes out as a sister species, and our analyses indicate that the three species are closely related and probably belong to the same group within the dinoflagellates (71% bootstrap support).
|
The plastid SSU rDNA sequences from the fucoxanthin-containing dinoflagellates had a relatively low sequence similarity when compared with other homologous sequences present in GenBank. Average JC distances between the fucoxanthin-containing dinoflagellates and the rest of the 16S SSU rDNA sequences in the modified alignment were 23.33 changes per 100 bp (ambiguously aligned characters excluded), whereas the average distance between the rest of the taxa was 16.80. Gyrodinium aureolum and G. breve shared at least two insertions, and G. galatheanum contained at least one insertion with no apparent homolog in any of the GenBank sequences or any of the other two fucoxanthin-containing dinoflagellates. The fucoxanthin-containing dinoflagellate sequences were also quite dissimilar to each other: G. aureolum and G. breve had a nucleotide identity of 85.65% (JC distances), and they were both only ~76% similar to G. galatheanum. Although highly divergent (long branches), the dinoflagellate sequences always grouped together with the haptophyte plastids in phylogenetic analyses using various distance, maximum-likelihood, and parsimony methods (data not shown).
To resolve the exact phylogenetic placement of the fucoxanthin-containing dinoflagellate plastids, an analytical approach similar to that described above was employed. The NJ LogDet tree used for parameter estimation was determined with pinvar set to 0.39, and the topology was generally compatible with other published trees (Van De Peer 1996
; Daugbjerg and Andersen 1997
; tree not shown), although some familiar analytical artifacts were observed (e.g., the euglenophytes grouped together with the heterokonts). This tree was used to estimate the maximum-likelihood value for the pinvar, and this was set to 0.34 for a LogDet analysis using heuristic searches. The best tree was found 4 out of 20 times using this model, and in the tree generated, all of the major groups of plastids can be recognized, albeit with only moderate support for several groups (fig. 3
). The fucoxanthin-containing dinoflagellates always grouped together with the haptophyte plastids, and this clade is supported by an 80% bootstrap value in the distance analyses presented. Consistent with the indels observed in the alignment, G. aureolum and G. breve are held together with 100% bootstrap support, while the position of G. galatheanum has more moderate support (68% bootstrap).
|
Maximum-likelihood analysis with the euglenophytes and chlorarachniophytes excluded provided the strongest support for the basal branching pattern of the haptophytes (including the fucoxanthin-containing dinoflagellates). Both euglenophytes and chlorarachniophytes have proved to be difficult groups in analyses of plastid SSU rDNA (Nelissen et al. 1995
; Van De Peer 1996
), and our analyses were no exception. However, the inclusion or exclusion of representatives of these groups did not affect the placement of the fucoxanthin-containing dinoflagellates with the haptophytes (data not shown). An NJ LogDet analysis (pinvar set to 0.39) was performed with chlorarachniophytes and euglenophytes excluded (tree not shown). The LogDet tree was fully compatible with the ME tree (fig. 3
) and was used to estimate the parameters subsequently used in a maximum-likelihood analysis: transition/transversion ratio, 1.88; base frequenciesA, 0.27; C, 0.21; G, 0.24; T, 0.28. The tree generated by fastDNAml using the F84 model (with no correction for site-to-site rate variation) was fully resolved (fig. 4
). There was moderate (78%) support for monophyly of the haptophyte plastids (including the fucoxanthin-containing dinoflagellates), with P. gyrans as the most basal branching haptophyte species. Monophyly of the haptophyte plastids is consistent with analyses of rbcL (Medlin et al. 1997
; Daugbjerg and Andersen 1997
), and assuming that this is correct, there is 96% bootstrap support for the embedding of the fucoxanthin-containing dinoflagellates within the haptophyte clade.
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The Fucoxanthin-Containing Dinoflagellates Have Plastids of Tertiary Origin
Phylogenetic analyses of plastid SSU rDNA place the plastids of the fucoxanthin-containing dinoflagellates unequivocally among those of haptophytes. The close relationship between the fucoxanthin-containing dinoflagellate plastids and those of haptophytes is also supported by common lack of a girdle lamella and the presence of 19'hexanoyloxy-fucoxanthin in both groups (Steidinger, Truby, and Dawes 1978
; Bjørnland and Tangen 1979
; Kite and Dodge 1985, 1988
). Because the plastids of haptophytes are themselves thought to be secondary in origin (Gibbs 1981
; Cavalier-Smith 1993
; Medlin et al. 1997
), and if the fucoxanthin-containing dinoflagellates acquired their plastids from a haptophyte, then these dinoflagellates are the result of three sequential endosymbiotic events (i.e., they are tertiary plastids; fig. 5
). Our analyses support this conclusion, indicating that the plastids have been sequestered from within the haptophyte group.
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Cryptic Endosymbionts and Tertiary Endosymbiosis
Peridinin has long been recognized as the typical carotenoid for the dinoflagellates. Because the peridinin-pigmented dinoflagellates do not seem to be a monophyletic group (fig. 2
), the distribution of peridinin within the dinoflagellates cannot be explained simply. One possibility is that a plastid with this unique pigment was present in the common ancestor of all dinoflagellate species (Bjørnland and Liaaen-Jensen 1989
; Van den Hoek, Mann, and Jahns 1995
; unpublished data). There seems to be no close relationship between the H. triquetra plastid SSU rDNA and the SSU rDNA sequences from the fucoxanthin-containing dinoflagellates, since no synapomorphic characters are observed between these two plastid types. Because of the very derived nature of the H. triquetra plastid SSU rDNA sequence (sequences with JC distances higher than 100 substitutions per 100 bp cannot be used reliably in distance analyses [Jin and Nei 1990
], and the H. triquetra 16S SSU rDNA was considered too derived even for maximum-likelihood analyses), the exact phylogenetic placement of this sequence remains uncertain, although it seems highly unlikely that the peridinin-type plastid and the fucoxanthin-containing plastids have a common origin. It follows that an ancestor of the fucoxanthin-containing dinoflagellates would have at one time contained a peridinin-type plastid and that this lineage has gone through at least one "switch" in plastid type ("cryptic endosymbiosis"; Henze et al. 1995
), perhaps including a period without plastids. Under this hypothesis, gene-transfers from the cryptic endosymbionts (e.g., peridinin-containing plastids) might have provided the nucleus of the host with enough plastid-derived genes to facilitate such "plastid switches."
Monophyletic or Polyphyletic Origin of the Dinoflagellate Fucoxanthin-Containing Plastids?
Do the endosymbioses represented by G. galatheanum, G. aureolum, and G. breve plastids represent a single event (i.e., uptake of one alga by a single dinoflagellate host) or two or more separate engulfment events of phylogenetically related haptophytes? There is high bootstrap support (100%) for monophyly of the G. aureolum and G. breve plastids; however, association of G. galatheanum with this group finds more moderate support (69% in the maximum-likelihood tree), and the three sequences did not form a monophyletic group under all analytical conditions (e.g., in maximum-likelihood analyses using PAUP*, version 4.0d64, and the reduced data set; data not shown). In the trees in which the fucoxanthin-containing plastids did not form a monophyletic group, G. aureolum and G. breve were monophyletic and the sibling group to all haptophyte plastids except P. gyrans, while G. galatheanum grouped together with Isochrysis sp. and Emiliania huxleyi. Although there was overall low bootstrap support for this arrangement, the morphological differences between G. galatheanum and G. aureolum/G. breve plastids could also argue for two independent endosymbiosis events (see also Tangen and Bjørnland 1981
; Kite and Dodge 1988
). The plastid morphology described for G. galatheanum is not known from any haptophyte, and it is not unreasonable to believe it evolved after the plastids were resident in dinoflagellates. On the sequence level, G. aureolum and G. breve share at least one insertion in their plastid SSU rRNA genes, while G. galatheanum appears to have several unique insertions. Evidence in favor of a monophyletic origin of the fucoxanthin-containing dinoflagellate plastids is the presence of a rare carotenoid, gyroxanthin diester, found only in these species (Bjørnland et al. 1987
).
Unusually High Evolutionary Rates in the Fucoxanthin-Containing Dinoflagellate Plastid SSU rDNA
The establishment of the fucoxanthin-containing plastid appears to be a relatively recent event in dinoflagellate evolution. All dinoflagellates with this pigmentation are closely related as measured by nuclear SSU rDNA (fig. 2
), and they form a well-defined, monophyletic group in various phylogenetic analyses using a large number of nuclear SSU rDNA sequences (unpublished data). Why, then, are their plastids so derived both at the morphological level and at the molecular level (see figs. 1, 3, and 4
)? For example, the G. aureolum and G. breve (99.47% sequence similarity of nuclear SSU rDNA using JC distances) plastid SSU rDNA sequences have a lower degree of sequence similarity than the rhodophyte Porphyra purpurea versus the chlorophyte Chlorella mirabilis plastid SSU rDNA (JC distances). It seems that these plastids have a very high evolutionary rate, but the precise reasons for this apparent acceleration are unknown.
Acknowledgements
We thank Terje Bjørnland, Bente Edvardsen, and Wenche Eikrem for valuable help with taxonomy, morphology, and ecology of the organisms investigated. Andreas Botnen has provided critical help and assistance with the analytical work. This work was supported by a grant from the Norwegian Research Council (grant 118894/431) to K.S.J., Valborg Aschehougs Legat to T.T., and an Alfred P. Sloan Foundation young investigator award for Molecular Studies in Evolution (97-4-3 ME) to C.F.D. T.T. and O.J.D. contributed equally to this work.
Footnotes
Geoffrey McFadden, Reviewing Editor
1 Keywords: dinoflagellates
endosymbiosis
19'hexanoyloxy-fucoxanthin
plastid phylogeny
small subunit ribosomal RNA
1 Present address: Institute of Human Virology, University of Maryland at Baltimore.
2 Present address: GenoVision Cytomics AS, Department of Research, Oslo, Norway.
2 Address for correspondence and reprints: Kjetill S. Jakobsen, Department of Biology, Division of General Genetics, University of Oslo, P.O. Box 1031, Blindern, 0315 Oslo, Norway. E-mail: kjetill.jakobsen{at}bio.uio.no
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