Phylogeny of Ultra-Rapidly Evolving Dinoflagellate Chloroplast Genes: A Possible Common Origin for Sporozoan and Dinoflagellate Plastids

Complete chloroplast 23S rRNA and psbA genes from five peridinin-containing dinoflagellates (Heterocapsa pygmaea, Heterocapsa niei, Heterocapsa rotun-data, Amphidinium carterae, and Protoceratium reticulatum) were amplified by PCR and sequenced; partial sequences were obtained from Thoracosphaera heimii and Scrippsiella trochoidea. Comparison with chloroplast 23S rRNA and psbA genes of other organisms shows that dinoflagellate chloroplast genes are the most divergent and rapidly evolving of all. Quartet puzzling, maximum likelihood, maximum parsimony, neighbor joining, and LogDet trees were constructed. Intersite rate variation and invariant sites were allowed for with quartet puzzling and neighbor joining. All psbA and 23S rRNA trees showed peridinin-containing dinoflagellate chloroplasts as monophyletic. In psbA trees they are related to those of chromists and red algae. In 23S rRNA trees, dinoflagellates are always the sisters of Sporozoa (apicomplexans); maximum likelihood analysis of Heterocapsa triquetra 16S rRNA also groups the dinoflagellate and sporozoan sequences, but the other methods were inconsistent. Thus, dinoflagellate chloroplasts may actually be related to sporozoan plastids, but the possibility of reproducible long-branch artifacts cannot be strongly ruled out. The results for all three genes fit the idea that dinoflagellate chloroplasts originated from red algae by a secondary endosymbiosis, possibly the same one as for chromists and Sporozoa. The marked disagreement between 16S rRNA trees using different phylogenetic algorithms indicates that this is a rather poor molecule for elucidating overall chloroplast phylogeny. We discuss possible reasons why both plastid and mitochondrial genomes of alveolates (Dinozoa, Sporozoa and Ciliophora) have ultra-rapid substitution rates and a proneness to unique genomic rearrangements. Received: 27 December 1999 / Accepted: 24 March 2000     

Sorting wheat from chaff in multi-gene analyses of chlorophyll c-containing plastids

Photosynthetic eukaryotes contain primary, secondary or tertiary plastids, depending on the source of the organelle (a cyanobacterium or a photosynthetic eukaryote). Plastid phylogeny is relatively well investigated, but molecular phylogenies have conflicted as a function of gene choice, taxon-representations, and analytical method. To better understand the influences of these variables, we performed analyses of a multi-gene data set based on 62 plastid-associated genes of 15 taxa representing the major plastid lineages. In an attempt to distinguish phylogenetic signal from non-phylogenetic patterns, we analyzed the data using a wide range of phylogenetic methods and examined the effect of covarion evolution and compositional bias. The data suggest that the chlorophyll c-containing plastids are monophyletic and acquired their plastids from the red algae after the emergence of the Cyanidiales. The relationships among chl c-containing plastids are particularly hard to resolve. This is the largest data set used for this purpose; the analyses show that cryptophyte plastids are sister to other chl c-containing plastids, and haptophyte and peridinin-containing dinoflagellate plastids are closely related.     

Protein import into cyanelles

"Cyanelles" are peptidoglycan-armored plastids of glaucocystophyte algae with close morphological and biochemical resemblance to endosymbiotic cyanobacteria. Genome sequencing and phylogenetic analysis have placed cyanelles on the earliest branch of phototrophic eukaryotes after the singular primary endosymbiotic event, the closest relatives to cyanobacteria among extant plastids. This model is supported by similar mechanisms for the targeting of nucleus-encoded cyanelle and chloroplast precursor proteins whose genes were transferred to the nucleus after the endosymbiotic event. As in chloroplasts, a prokaryote-type Sec preprotein translocase is shown to operate in cyanelle thylakoid membranes.     

Origin and phylogeny of chloroplasts revealed by a simple correlation analysis of complete genomes

The complete sequenced genomes of chloroplast have provided much information on the origin and evolution of this organelle. In this paper we attempt to use these sequences to test a novel approach for phylogenetic analysis of complete genomes based on correlation analysis of compositional vectors. All protein sequences from 21 complete chloroplast genomes are analyzed in comparison with selected archaea, eubacteria, and eukaryotes. The distance-based analysis shows that the chloroplast genomes are most closely related to cyanobacteria, consistent with the endosymbiotic origin of chloroplasts. The chloroplast genomes are separated to two major clades corresponding to chlorophytes (green plants) s.l. and rhodophytes (red algae) s.l. The interrelationships among the chloroplasts are largely in agreement with the current understanding on chloroplast evolution. For instance, the analysis places the chloroplasts of two chromophytes (Guillardia and Odontella) within the rhodophyte lineage, supporting secondary endosymbiosis as the source of these chloroplasts. The relationships among the green algae and land plants in our tree also agree with results from traditional phylogenetic analyses. Thus, this study establishes the value of our simple correlation analysis in elucidating the evolutionary relationships among genomes. It is hoped that this approach will provide insights on comparative genome analysis.     

Phylogenetic analyses indicate that the 19'Hexanoyloxy-fucoxanthin-containing dinoflagellates have tertiary plastids of haptophyte origin

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.     

Dinophyte chloroplasts and phylogeny - A review

Dinophytes acquired chloroplasts obviously early in evolution and later lost them multiple times. Most families and genera contain both photosynthetic and heterotrophic species. Chloroplasts enveloped by three membranes with thylakoids in stacks of three, containing peridinin as the main pigment, are regarded as the original dinophyte plastids. Pyrenoids are generally present. Stigmata, if present, are usually parts of the chloroplast or are modified original plastids. The form II type RUBISCO found in the dinophytes is unique for eukaryotes, otherwise known only in some anaerobic bacteria. It is disputed whether the original dinophyte chloroplasts are derived from a prokaryotic or an eukaryotic endosymbiosis. Various dinoflagellates contain aberrant chloroplasts. Glenodinium foliaceum and Peridinium balticum have a single complete endosymbiont, originally a pcnnate diatom. Podolampas bipes houses several dictyophycean symbiont cells. The “symbionts” of Lepidodiniurn viride and Gymnodinium chlorophorum are highly reduced prasinophyte cells. The chloroplasts of Gymnodinium mikimotoi have aberrant pigments (fucoxanthin derivatives, no peridinin) and fine structure. The dinoflagellate hosts do not seem to contain any parts of the former endosymbiont except the chloroplasts. Photosynthetic Dinophysis species have cryptophycean-like chloroplasts, whereas symbiotic cyanobacteria are found in other members of the Dinophysiales, e.g., Ornithocercus. Various dinophytes, e.g. Gymnodinium aeruginosum, use kleptochloroplasts from ingested cryptophytes transiently for photosynthesis. Original or secondarily acquired chloroplasts can only be used for phylogenetic considerations in exceptionally cases: it seems unlikely that the Prorocentrales have evolved from the Dinophysiales because all Prorocentrales possess original dinoflagellate chloroplasts, whereas no member of the Dinophysiales has such chloroplasts.     

Phylogenetic artifacts can be caused by leucine, serine, and arginine codon usage heterogeneity: dinoflagellate plastid origins as a case study

Phylogenetic analyses of first and second codon positions (DNA1 + 2 analysis) and amino acid sequences (protein analysis) are often thought to provide similar estimates of deep-level phylogeny. However, here we report a novel artifact influencing DNA level phylogenetic inference of protein-coding genes introduced by codon usage heterogeneity that causes significant incongruities between DNA1 + 2 and protein analyses. DNA1 + 2 analyses of plastid-encoded psbA genes (encoding of photosystem II D1 proteins) strongly suggest a relationship between haptophyte plastids and typical (peridinin-containing) dinoflagellate plastids. The psbA genes from haptophytes and a subset of the peridinin-type plastids display similar codon usage patterns for Leu, Ser, and Arg, which are each encoded by two separated codon sets that differ at first or first plus second codon positions. Our detailed analyses clearly indicate that these unusual preferences shared by haptophyte and some peridinin-type plastid genes are largely responsible for their strong affinity in DNA analyses. In particular, almost all of the support from DNA level analyses for the monophyly of haptophyte and peridinin-type plastids is lost when the codons corresponding to constant Leu, Ser, and Arg amino acids are excluded, suggesting that this signal comes from rapidly evolving synonymous substitutions, rather than from substitutions that result in amino acid changes. Indeed, protein maximum-likelihood analyses of concatenated PsaA and PsbA amino acid sequences indicate that, although 19' hexanoyloxyfucoxanthin-type (19' HNOF-type) plastids in dinoflagellates group with haptophyte plastids, peridinin-type plastids group weakly with those of stramenopiles. Consequently our results cast doubt on the single origin of peridinin-type and 19' HNOF-type plastids in dinoflagellates previously suggested on the basis of psaA and psbA concatenated gene phylogenetic analyses. We suggest that codon usage heterogeneity could be a more general problem for DNA level analyses of protein-coding genes, even when third codon positions are excluded.     

Translation in chloroplasts

The discovery that chloroplasts have semi-autonomous genetic systems has led to many insights into the biogenesis of these organelles and their evolution from free-living photosynthetic bacteria. Recent developments of our understanding of the molecular mechanisms of translation in chloroplasts suggest selective pressures that have maintained the 100-200 genes of the ancestral endosymbiont in chloroplast genomes. The ability to introduce modified genes into chloroplast genomes by homologous recombination and the recent development of an in vitro chloroplast translation system have been exploited for analyses of the cis-acting requirements for chloroplast translation. Trans-acting translational factors have been identified by genetic and biochemical approaches. Several studies have suggested that chloroplast mRNAs are translated in association with membranes.     

Phylogeny of Dinoflagellate Plastid Genes Recently Transferred to the Nucleus Supports a Common Ancestry with Red Algal Plastid Genes

It is generally accepted that peridinin-containing dinoflagellate plastids are derived from red alga, but whether they are secondary plastids equivalent to plastids of stramenopiles, haptophytes, or cryptophytes, or are tertiary plastids derived from one of the other secondary plastids, has not yet been completely resolved. As secondary plastids, plastid gene phylogeny should mirror that of nuclear genes, while incongruence in the two phylogenies should be anticipated if their origin was as tertiary plastids. We have analyzed the phylogeny of plastid-encoded genes from Lingulodinium as well as that of nuclear-encoded dinoflagellate homologues of plastid-encoded genes conserved in all other plastid genome sequences. Our analyses place the dinoflagellate, stramenopile, haptophyte, and cryptophyte plastids firmly in the red algal lineage, and in particular, the close relationship between stramenopile plastid genes and their dinoflagellate nuclear-encoded homologues is consistent with the hypothesis that red algal-type plastids have arisen only once in evolution.     

Protein synthesis in Petunia hybrida chloroplasts isolated from leaves and cell cultures

Isolation and incubation conditions were established for Petunia hybrida chloroplasts capable of performing in vitro protein and RNA synthesis. Under these conditions, chloroplasts from leaves as well as from the non-photoautotrophic mutant green cell culture AK-2401 are able to incorporate labeled amino acids into polypeptides. Intact chloroplasts can use light as an energy source; photosynthetically-inactive chloroplasts require the addition for ATP for this protein synthesis. Sodium dodecylsulphate polyacrylamide slab gel electrophoresis shows that in isolated leaf chloroplasts at least twenty-five radioactive polypeptide species are synthesized. The three major products synthesized have molecular weights of 52,000, 32,000 and 17,000. Coomassie brilliant-bluestained polypeptide patterns from plastids isolated from the mutant green cell culture AK-2401 differ considerably from those obtained from leaf chloroplasts. The pattern of radioactive polypeptides synthesized in these isolated cell culture plastids also shows differences. These results indicate that the difference in developmental stage observed between plastids from the cell culture AK-2401 and leaves is reflected in an altered expression of the chloroplast DNA.Abbreviations CAP D-threo-chloramphenicol - 2,4-D 2,4-dichlorophenoxyacetic acid - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecylsulphate     

Comparative morphology and molecular phylogeny of Apicoporus n. Gen.: a new genus of marine benthic dinoflagellates formerly classified within Amphidinium

The composition of the dinoflagellate genus Amphidinium is currently polyphyletic and includes several species in need of re-evaluation using modern morphological and phylogenetic methods. We investigated a broad range of uncultured morphotypes extracted from marine sediments in the Eastern Pacific Ocean that were similar in morphology to Amphidinium glabrum Hoppenrath and Okolodkov. To determine the number of distinct species associated with this phenotypic diversity, we collected LM, SEM, TEM and small subunit ribosomal DNA sequence information from different morphotypes, including the previously described A. glabrum. Both comparative morphological and molecular phylogenetic data supported the establishment of a new genus, Apicoporus n. gen., including at least two species, A. glaber n. comb., and A. parvidiaboli n. sp. Apicoporus is characterized by having amphiesmal pores and an apical pore covered by a hook-like protrusion; neither of these characters has been observed in other athecate dinoflagellates. The posterior end of Apicoporus parvidiaboli possessed varying degrees of "horn formation", ranging from slight to prominent. By contrast, the posterior end of Apicoporus glaber was distinctively rounded and lacked evidence of horn formation. Although these species were previously interpreted to be obligate heterotrophs, TEM and epifluorescence microscopy demonstrated that some cells of both species had unusually small but otherwise typical dinoflagellate plastids. The number and density of plastids in any particular cell varied significantly in the genus, but the plastids were almost always concentrated at the posterior end of the cells or around the nucleus. The presence of cryptic photosynthetic plastids in these benthic species suggests that photosynthesis might be much more widespread in dinoflagellates than is currently assumed.     

Molecular data and the evolutionary history of dinoflagellates

We have sequenced small-subunit (SSU) ribosomal RNA (rRNA) genes from 16 dinoflagellates, produced phylogenetic trees of the group containing 105 taxa, and combined small- and partial large-subunit (LSU) rRNA data to produce new phylogenetic trees. We compare phylogenetic trees based on dinoflagellate rRNA and protein genes with established hypotheses of dinoflagellate evolution based on morphological data. Protein-gene trees have too few species for meaningful in-group phylogenetic analyses, but provide important insights on the phylogenetic position of dinoflagellates as a whole, on the identity of their close relatives, and on specific questions of evolutionary history. Phylogenetic trees obtained from dinoflagellate SSU rRNA genes are generally poorly resolved, but include by far the most species and some well-supported clades. Combined analyses of SSU and LSU somewhat improve support for several nodes, but are still weakly resolved. All analyses agree on the placement of dinoflagellates with ciliates and apicomplexans (=Sporozoa) in a well-supported clade, the alveolates. The closest relatives to dinokaryotic dinoflagellates appear to be apicomplexans, Perkinsus, Parvilucifera, syndinians and Oxyrrhis. The position of Noctiluca scintillans is unstable, while Blastodiniales as currently circumscribed seems polyphyletic. The same is true for Gymnodiniales: all phylogenetic trees examined (SSU and LSU-based) suggest that thecal plates have been lost repeatedly during dinoflagellate evolution. It is unclear whether any gymnodinialean clades originated before the theca. Peridiniales appear to be a paraphyletic group from which other dinoflagellate orders like Prorocentrales, Dinophysiales, most Gymnodiniales, and possibly also Gonyaulacales originated. Dinophysiales and Suessiales are strongly supported holophyletic groups, as is Gonyaulacales, although with more modest support. Prorocentrales is a monophyletic group only in some LSU-based trees. Within Gonyaulacales, molecular data broadly agree with classificatory schemes based on morphology. Implications of this taxonomic scheme for the evolution of selected dinoflagellate features (the nucleus, mitosis, flagella and photosynthesis) are discussed.     

Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE

The preprotein translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two receptor components, Toc159 and Toc34, and the channel Toc75 form the Toc complex. In this study, we have analyzed the molecular architecture and organization of the Toc complex by blue native PAGE (BN-PAGE), which is a high-resolution method for separating membrane protein complexes under non-denaturing conditions. Pea chloroplasts isolated in the presence of a protease inhibitor cocktail were directly solubilized in detergent solution and analyzed by BN-PAGE and size exclusion chromatography. Subsequent immunoblot analyses indicated that the complex composed of Toc75, Toc159 and Toc34 has a molecular mass of 800-1,000 kDa. Limited proteolysis revealed a core of the Toc complex, which was resistant to proteases and detergent treatments. The stoichiometry of the three Toc proteins was calculated as approximately 1 : 3 : 3 between Toc159 : Toc75 : Toc34. We have also analyzed the Toc complex of etioplasts and root plastids. These plastids were found to have essentially the same sized Toc complex as that of the chloroplast.     

Frequent chloroplast capture among Isodon (Lamiaceae) species in Japan revealed by phylogenies based on variation in chloroplast and nuclear DNA

A recent phylogenetic study based only on chloroplast DNA (cpDNA) variation revealed that populations of an Isodon species are frequently embedded paraphyletically among other Isodon species. This phylogenetic discrepancy between species taxonomy and molecular phylogeny was considered to have resulted from chloroplast DNA captures and/or incomplete lineage sorting. To elucidate which of these factors was mainly responsible for the observed phylogenetic pattern, we performed phylogenetic analyses of multiple populations of Isodon species in Japan using cpDNA variation, three single-copy nuclear genes, and double-digest restriction-site-associated DNA sequencing (ddRAD-seq). Although a species often shared chlorotypes with other species, our phylogenetical analyses based on variation in the three single-copy nuclear genes and the ddRAD-seq data showed that most populations belonging to the same species were monophyletic at the species level, suggesting that chloroplast capture may have frequently occurred between Isodon species. Some populations of an intraspecific taxon were embedded paraphyletically within the species, regardless of the large amount of phylogenetic information in nuclear DNA; this incongruity may have resulted from incomplete lineage sorting.     

Identification of chloroplast signal recognition particle RNA genes

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts. In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome. The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43. An RNA component has not been identified and does not seem to be required for the post-translational cpSRP. The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified. Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible. We have analysed chloroplast genomes from 27 organisms. In higher plant chloroplasts, no SRP RNA genes were identified. However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene. These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP.     

叶绿体基因组研究进展

作为植物细胞器的重要组成部分和光合作用的器官,叶绿体在生物进化的漫长历史中发挥了重要作用.伴随着生物技术的深入发展,人们发现叶绿体基因组结构和序列的信息在揭示物种起源、进化演变及其不同物种之间的亲缘关系等方面具有重要价值.与此同时,比核转化具有明显优势的叶绿体转化技术在遗传改良、生物制剂的生产等方面显示出巨大潜力,而叶绿体基因组结构和序列分析则是叶绿体转化的基石.基于叶绿体的这些重要作用,收集整理了有关的资料,从几个方面归纳了本领域最近的研究进展,希望能使读者对迅速发展的叶绿体基因组研究有更全面的了解,以及对叶绿体基因组在物种的进化、遗传、系统发育关系等方面的作用有更深刻的认识,同时也希望对叶绿体转化技术的研究和广泛应用产生积极作用.     

Evolution of the chloroplast genome

We discuss the suggestion that differences in the nucleotide composition between plastid and nuclear genomes may provide a selective advantage in the transposition of genes from plastid to nucleus. We show that in the adenine, thymine (AT)-rich genome of Borrelia burgdorferi several genes have an AT-content lower than the average for the genome as a whole. However, genes whose plant homologues have moved from plastid to nucleus are no less AT-rich than genes whose plant homologues have remained in the plastid, indicating that both classes of gene are able to support a high AT-content. We describe the anomalous organization of dinoflagellate plastid genes. These are located on small circles of 2-3 kbp, in contrast to the usual plastid genome organization of a single large circle of 100-200 kbp. Most circles contain a single gene. Some circles contain two genes and some contain none. Dinoflagellate plastids have retained far fewer genes than other plastids. We discuss a similarity between the dinoflagellate minicircles and the bacterial integron system.