Department of Molecular and Cellular Biology, Harvard University
Although the endosymbiotic theory of evolution (Margulis 1970
) is widely accepted, the series of events that led to the permanent inclusion of mitochondria and chloroplasts in eukaryotic cells are poorly understood. In particular, the diverse biochemical and morphological properties of chloroplasts have led to suggestions that these organelles have been acquired through multiple primary endosymbiotic events (for a review see Delwiche 1999
). Furthermore, the presence of three and sometimes four (Gibbs 1962
) membranes around certain chloroplasts, some in association with a second nucleus, suggests that secondary endosymbiosisthe incorporation within a eukaryote of a heritable organelle from another eukaryotehas also occurred (Gibbs 1981
). Secondary endosymbioses may also have occurred more than once (Delwiche and Palmer 1997
; Delwiche 1999
); in fact, the possibility that some autotrophic eukaryotes lost their photosynthetic organelles only to regain them in a later endosymbiotic event cannot be excluded. The occurrence of such multiple, sequential endosymbiotic events can only be proven if some remnant of the first endosymbiont was retained by the host cell. Nuclear-encoded genes for chloroplast proteins, for example GAPDH, could provide such evidence.
Although phylogenetic analyses have resolved cytosolic (GapC) and chloroplast (GapA) sequences into two distinct clades (Martin et al. 1993
; Liaud et al. 1997
), recent studies of gapdh isoforms from marine algae have complicated the simple cytosol-chloroplast tree structure. In the dinoflagellate Lingulodinium polyedrum (Stein) Dodge, formerly Gonyaulax polyedra, and in two cryptomonads (Guillardia theta and Pyrenomonas salina) the role of GAPDH in the Calvin cycle is filled by a modified cytosolic isoform (GapC-I), which has a signal sequence at the N-terminal end for intracellular translocation to the chloroplast and, consistent with its anabolic role, amino acid substitutions that allow binding of NADPH (Liaud et al. 1997
; Fagan, Hastings, and Morse 1998
). Phylogenetic analysis places GapC-I firmly in the cytosolic clade (Fagan, Hastings, and Morse 1998
). Homologous isoforms have been identified subsequently in heterokonts and apicomplexans (Liaud et al. 2000
; Fast et al. 2001
).
How the GapC-I isoform was acquired and how pervasive it is among dinoflagellates is unknown. We proposed that it was obtained through lateral transfer (Fagan, Hastings, and Morse 1998
) because dinoflagellates are known to engulf and harbor organelles of other algae (Larsen 1988
; Lewitus, Glasgow, and Burkholder 1999
). In order to check the validity of this hypothesis, we undertook the cloning and analysis of gapdh sequences from other dinoflagellates. Surprisingly, we have found that two species of Pyrocystis (P. lunula and P. noctiluca) possess a GapA isoform, indicating that acquisition of GAPDH genes in the dinophyceae may have occurred more than once and by different mechanisms.
gapdh sequences were amplified by RT-PCR using total RNA from P. lunula as template. After reverse transcription, degenerate oligonucleotide primers coding for the peptide SNASCTT, which is found in the active site of almost all GAPDH isoforms, were used in initial amplifications together with an antisense primer directed to the polyA region of the first strand cDNA. Potential gapdh products, as determined by size, were cloned; sequences identified as homologous to gapdh were used to design nondegenerate primers for subsequent 5' rapid amplification of cDNA ends (RACE) experiments.
A set of overlapping sequences totaling 1,305 bp in length was obtained from P. lunula. The cDNA has a GC content of 59.5% and contains an open reading frame of 1,032 bp, encoding a predicted protein sequence of 344 amino acids (36.6 kDa). A BLAST (Altschul et al. 1990
) search of the protein sequence against the NCBI nonredundant database showed that the protein is homologous to GAPDH, but surprisingly the most statistically significant alignments were with chloroplast GAPDH enzymes (GapA) from Euglena gracilis and the red alga Chondrus crispus. This suggested that the P. lunula sequence is a homolog of the GapA isoforms, corresponding to those found in most other photosynthetic eukaryotes.
Pairwise alignments showed that the predicted protein sequence of this isoform is 37% and 41% similar to the cytosolic (GapC) and chloroplast (GapC-I) isoforms, respectively, from L. polyedrum. This is in striking contrast to the greater similarity (68%) found between the GapC-I isoforms from L. polyedrum and the cryptomonads G. theta and P. salina (Fagan, Hastings, and Morse 1998
). In addition, the P. lunula isoform and the GapA isoform from the green alga E. gracilis show much greater similarity (
72%), supporting the idea that the sequence isolated is a GapA homolog.
In order to confirm that this gene was amplified from P. lunula, Northern blots of total RNA were probed using the P. lunula GapA gene and a P. lunula luciferase (LCF) probe as control; dinoflagellate luciferases are unique to these algae and provide an excellent diagnostic tool to distinguish between dinoflagellate and nondinoflagellate nucleic acids. Under stringent hybridization conditions, GapA and LCF mRNAs are present in almost equal amounts (fig. 1A ), a ratio unlikely for a non-Pyrocystis origin of the GapA gene. A prokaryotic origin can also be dismissed because the gene was isolated using polyA-directed primers and contains a polyA tail.
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We have also obtained a partial sequence of a P. noctiluca GAPDH gene, comprising 760 nucleotides covering the C-terminal end of the coding region (192 amino acids) and the 3'UTR. Although the UTR (184 bp) is only 27% identical to the P. lunula gene 3'UTR (174 bp), the coding sequences are 87% identical, indicating that the two genes are homologous.
We investigated the relationship between the P. lunula sequence and those of other GAPDH isoforms further by phylogenetic analyses. Alignments of protein sequences from 36 representative taxa were used, including photosynthetic and nonphotosynthetic eukaryotes and prokaryotes. Phylogenetic trees generated by parsimony and distance analysis gave similar topologies (fig. 2 ).
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Support for this topology comes from phylogenetic analysis of a subset of characters, comprising 167 amino acids of the C-termini of the proteins. This analysis also places the P. noctiluca sequence in the GapA clade, with both Pyrocystis species forming a sister group with Euglena (fig. 2 inset).
If the proteins coded by these two Pyrocystis genes are truly chloroplast GAPDHs, then they should contain N-terminal transit sequences. The 5' end of the P. lunula GapA, though incomplete, does have such characteristics, being rich in hydroxylated residues and long hydrophobic stretches. Indeed, ChloroP (Emanuelsson, Nielsen, and Von Heijne 1999
), an algorithm that has proven to be reliable in predicting transit sequences and their cleavage sites, indicates that the N-terminus of this gene does contain a transit sequence and predicts a cleavage site compatible with the expected start of a mature enzyme (fig. 1C
).
The identification of GapA genes in Pyrocystis species and a GapC-I gene in L. polyedrum raises questions about the origin and distribution of gapdh genes in dinoflagellates. The recent identification of GapC-I in the diatoms Odontella sinensis and Phaeodactylum tricornutum (Liaud et al. 2000
) and in the Apicomplexa (Fast et al. 2001
) indicate that this isoform may be more widely distributed than previously thought. Cryptomonad, diatom, and apicomplexan plastids have been acquired through secondary endosymbiosis, with several lines of evidence, such as ultrastructural, pigmentation, and phylogenetic (Durnford, Aebersold, and Green 1996
), indicating that the plastid donor was a red alga. Although dinoflagellate plastids differ in some respects (e.g., organization of plastid genes on minicircles [Zhang, Green, and Cavalier-Smith 1999
] and lack of phycobilisomes, Jeffrey 1980
), there is phylogenetic evidence from psbA photosystem sequences (Takishita and Uchida 1999
; Zhang, Green, and Cavalier-Smith 2000
) and ribosomal sequences (Zhang, Green, and Cavalier-Smith 1999
) supporting a red algal origin for dinoflagellate plastids also. Thus, the presence of the GapC-I isoform in these phyla could be explained by its acquisition during a single secondary endosymbiotic event in which an ancestor of present-day photosynthetic dinoflagellates, cryptomonads, diatoms, and apicomplexans engulfed a red alga-type eukaryote. As proposed by Fast et al. (2001)
, a duplication of the cytosolic GapC gene in the ancestor, followed by addition of a targeting sequence to one of the copies, could explain the origin of the GapC-I isoform in all these phyla. This theory is supported by phylogenetic analyses, which show GapC-I and GapC clades, comprising taxa from these phyla, as sister groups (Fast et al. 2001
). Indeed, a precedent for such a duplication occurs in the extant gymnosperm Pinus sylvestris (Meyer-Gauen et al. 1994
). On the basis of parsimony alone the single ancestor theory seems highly likely.
But what of the GapA isoform? GapAs are present in red algae, raising the possibility that dinoflagellates acquired it from the red algal endosymbiont in the single ancestor. If there was such an origin, GapA should have been transferred to the nucleus of the single ancestor and then selectively replaced by the duplicated GapC-I in only some dinoflagellates. This seems unlikely. Moreover, the Pyrocystis genes form a clade with Euglena (see fig. 2
) and are thus closely related to the green algal lineage because the position of Euglena in the tree basal to the divergence of the red and green lineages has been shown to be caused by long branch attraction (Figge et al. 1999
). A red algal origin for the Pyrocystis GapA is, therefore, unlikely.
It is possible that these genes arose through lateral transfer from an engulfed photosynthetic prey. But this would surely require that the host, in this case the ancestor to Pyrocystis, be a heterotroph, thus indicating that a second endosymbiotic event has taken place within the dinoflagellates. We propose that the two chloroplast-targeted gapdh isoforms arose from independent endosymbiotic events. It has been suggested that the ancestor to all dinoflagellates was photosynthetic (Cavalier-Smith 1999
), an idea that has received phylogenetic support (Zhang, Green, and Cavalier-Smith 2000
). Extant heterotrophic dinoflagellates, or their ancestors, must therefore have lost their chloroplasts. It is worth noting, however, that phylogenetic analysis of small ribosomal subunit sequences places several heterotrophs (Noctiluca scintillans, Crypthecodinium cohnii) basal to many photosynthetic species (Saunders et al. 1997
), supporting the possibility that a second endosymbiotic event has taken place.
Sequences reported here have been deposited in GenBank and can be retrieved using accession numbers AF406628 (P. lunula) and AF406629 (P. noctiluca).
Acknowledgements
This work was supported by grants from the NIH (MH 46660) and NSF (9982880). We would like to thank Deb Robertson and Thérèse Wilson for their helpful comments and suggestions, and Keith Okamoto for his help in propagating Pyrocystis colonies.
Footnotes
William Martin, Reviewing Editor
Keywords: gapdh
dinoflagellates
endosymbiosis
evolution
Address for correspondence and reprints: J. Woodland Hastings, Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138. hastings{at}fas.harvard.edu
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