Phylogenetic Analysis Indicates Multiple Origins of Chloroplast Glyceraldehyde-3-Phosphate Dehydrogenase Genes in Dinoflagellates

Thomas F. Fagan and J. Woodland Hastings

Department of Molecular and Cellular Biology, Harvard University

Although the endosymbiotic theory of evolution (Margulis 1970Citation ) 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 1999Citation ). Furthermore, the presence of three and sometimes four (Gibbs 1962Citation ) membranes around certain chloroplasts, some in association with a second nucleus, suggests that secondary endosymbiosis—the incorporation within a eukaryote of a heritable organelle from another eukaryote—has also occurred (Gibbs 1981Citation ). Secondary endosymbioses may also have occurred more than once (Delwiche and Palmer 1997Citation ; Delwiche 1999Citation ); 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. 1993Citation ; Liaud et al. 1997Citation ), 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. 1997Citation ; Fagan, Hastings, and Morse 1998Citation ). Phylogenetic analysis places GapC-I firmly in the cytosolic clade (Fagan, Hastings, and Morse 1998Citation ). Homologous isoforms have been identified subsequently in heterokonts and apicomplexans (Liaud et al. 2000Citation ; Fast et al. 2001Citation ).

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 1998Citation ) because dinoflagellates are known to engulf and harbor organelles of other algae (Larsen 1988Citation ; Lewitus, Glasgow, and Burkholder 1999Citation ). 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. 1990Citation ) 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 1998Citation ). 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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Fig. 1.—(A) Northern blot analysis of total RNA from P. lunula. Equal amounts of RNA were transferred to a nylon support and probed with P32-labeled P. lunula GapA cDNA (lane 1) or P. lunula LCF cDNA (lane 2). (B) PCR of genomic DNA isolated from a single colony of P. lunula. Primer pair products in lanes 2, 3, and 4 matched predicted sizes of 1,113, 913, and 720 bp, respectively. (C) The first 60 N-terminal amino acids of P. lunula GapA. Hydrophobic stretches are underlined, and hydroxylated amino acids are in bold. The ChloroP-predicted cleavage site (arrow) is consistent with the start site of mature enzymes and predicts a final protein of 345 amino acids

 
PCR amplification of this gene from genomic DNA isolated from single colonies of P. lunula grown on kanamycin plates also confirmed its origin. Microscopic examination of these bacteria-free gold-colored colonies showed them to be unialgal. Three primer pairs yielded products of the correct size, as predicted by the cDNA sequence (fig. 1B ). The smallest product was cloned, sequenced, and found to be identical to the same region in the cDNA clone.

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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Fig. 2.—Phylogenetic analysis of GAPDH isoforms from 36 representative taxa. Nodes supported by bootstrap values over 50% are shown. Protein sequences were aligned using Clustal W (Thompson, Higgins, and Gibson 1994Citation ). The equivocal characters were removed manually. Distance trees were generated from 100 bootstrap replicates using software modules of the Phylogeny Inference Package (Protdist, Seqboot, Neighbor, Consense) version 3.572 (Joseph Felsenstein, University of Washington), or Paup* 4.0b8 (Sinauer Associates, Sunderland, Mass.). In the distance tree, support values shown in parenthesis were obtained from an unweighted matrix using Paup*. In the parsimony analysis (Paup*), starting trees were obtained by stepwise addition and the tree-bisection–reconnection (TBR) algorithm employed, and the node labeled by the asterisk collapses to give a polytomy. Pyrocystis positions are shown bold, and the GapC-I clade is boxed. The inset shows a distance analysis of a subset of characters and taxa with the P. noctiluca partial sequence included

 
Using the sequence from the archaea Sulfolobus solfataracus as an out-group, two major clades were resolved by both analytical treatments. The first comprises mostly cytosolic isoforms, including dinoflagellate and cryptophyte GapC sequences. In addition, proteobacterial (Escherichia coli) and cyanobacterial (Anabaena 2) sequences are found in this clade, as are the dinoflagellate and cryptophyte GapC-I sequences, which branch deeply within it; these form a single clade with the GapC-I sequences from heterokonts and the apicomplexan Toxoplasma gondii, previously shown to contain vestigial chloroplasts. The second major clade consists of isoforms that are involved in the Calvin cycle of photosynthetic organisms, cyanobacterial (Anabaena 1) and chloroplast GapA sequences. There is robust support for these two clades in both types of analyses, and the tree topologies are consistent with many published previously (Martin and Cerff 1986Citation ; Liaud et al. 2000Citation ; Fast et al. 2001Citation ). Both parsimony and distance analyses place the P. lunula sequence firmly within the GapA clade. It branches deeply within the clade and forms a sister group with the GapA sequence from E. gracilis.

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 1999Citation ), 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. 2000Citation ) and in the Apicomplexa (Fast et al. 2001Citation ) 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 1996Citation ), 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 1999Citation ] and lack of phycobilisomes, Jeffrey 1980Citation ), there is phylogenetic evidence from psbA photosystem sequences (Takishita and Uchida 1999Citation ; Zhang, Green, and Cavalier-Smith 2000Citation ) and ribosomal sequences (Zhang, Green, and Cavalier-Smith 1999Citation ) 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)Citation , 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. 2001Citation ). Indeed, a precedent for such a duplication occurs in the extant gymnosperm Pinus sylvestris (Meyer-Gauen et al. 1994Citation ). 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. 1999Citation ). 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 1999Citation ), an idea that has received phylogenetic support (Zhang, Green, and Cavalier-Smith 2000Citation ). 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. 1997Citation ), 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 Back

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 Back

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Accepted for publication March 14, 2002.