Heterotachy and tree building: a case study with plastids and eubacteria

The nature of heterotachy at the center of recent controversy over the relative performance of tree-building methods is different from the form of heterotachy that has been inferred in empirical studies. The latter have suggested that proportions of variable sites (p(var)) vary among orthologues and among paralogues. However, the strength of this inference, describing what may be one of the most important evolutionary properties of sequence data, has remained weak. Consequently, other models of sequence evolution have been proposed to explain some long-branch attraction (LBA) problems that could be attributed to differences in p(var). For an empirical case with plastid and eubacterial RNA polymerase sequences, we confirm using capture-recapture estimates and simulations that p(var) can differ among orthologues in anciently diverged evolutionary lineages. We find that parsimony and a least squares distance method that implements an overly simple model of sequence evolution are susceptible to LBA induced by this form of heterotachy. Although homogeneous maximum likelihood inference was found to be robust to model misspecification in our specific example, we caution against assuming that it will always be so.     

Plastid genome sequence of the cryptophyte alga Rhodomonas salina CCMP1319: lateral transfer of putative DNA replication machinery and a test of chromist plastid phylogeny

Cryptophytes are a group of unicellular algae with chlorophyll c-containing plastids derived from the uptake of a secondary (i.e., eukaryotic) endosymbiont. Biochemical and molecular data indicate that cryptophyte plastids are derived from red algae, yet the question of whether or not cryptophytes acquired their red algal plastids independent of those in heterokont, haptophyte, and dinoflagellate algae is of long-standing debate. To better understand the origin and evolution of the cryptophyte plastid, we have sequenced the plastid genome of Rhodomonas salina CCMP1319: at 135,854 bp, it is the largest secondary plastid genome characterized thus far. It also possesses interesting features not seen in the distantly related cryptophyte Guillardia theta or in other red secondary plastids, including pseudogenes, introns, and a bacterial-derived gene for the tau/gamma subunit of DNA polymerase III (dnaX), the first time putative DNA replication machinery has been found encoded in any plastid genome. Phylogenetic analyses indicate that dnaX was acquired by lateral gene transfer (LGT) in an ancestor of Rhodomonas, most likely from a firmicute bacterium. A phylogenomic survey revealed no additional cases of LGT, beyond a noncyanobacterial type rpl36 gene similar to that recently characterized in other cryptophytes and haptophytes. Rigorous concatenated analysis of 45 proteins encoded in 15 complete plastid genomes produced trees in which the heterokont, haptophyte, and cryptophyte (i.e., chromist) plastids were monophyletic, and heterokonts and haptophytes were each other's closest relatives. However, statistical support for chromist monophyly disappears when amino acids are recoded according to their chemical properties in order to minimize the impact of composition bias, and a significant fraction of the concatenate appears consistent with a sister-group relationship between cryptophyte and haptophyte plastids.     

Covarion structure in plastid genome evolution: a new statistical test

Covarion models of molecular evolution allow the rate of evolution of a site to vary through time. There are few simple and effective tests for covarion evolution, and consequently, little is known about the presence of covarion processes in molecular evolution. We describe two new tests for covarion evolution and demonstrate with simulations that they perform well under a wide range of conditions. A survey of covarion evolution in sequenced plastid genomes found evidence of covarion drift in at least 26 out of 57 genes. Covarion evolution is most evident in first and second codon positions of the plastid genes, and there is no evidence of covarion evolution in third codon positions. Therefore, the significant covarion tests are likely due to changes in the selective constraints of amino acids. The frequency of covarion evolution within the plastid genome suggests that covarion processes of evolution were important in generating the observed patterns of sequence variation among plastid genomes.     

Combined heat shock protein 90 and ribosomal RNA sequence phylogeny supports multiple replacements of dinoflagellate plastids

Dinoflagellates harbour diverse plastids obtained from several algal groups, including haptophytes, diatoms, cryptophytes, and prasinophytes. Their major plastid type with the accessory pigment peridinin is found in the vast majority of photosynthetic species. Some species of dinoflagellates have other aberrantly pigmented plastids. We sequenced the nuclear small subunit (SSU) ribosomal RNA (rRNA) gene of the "green" dinoflagellate Gymnodinium chlorophorum and show that it is sister to Lepidodinium viride, indicating that their common ancestor obtained the prasinophyte (or other green alga) plastid in one event. As the placement of dinoflagellate species that acquired green algal or haptophyte plastids is unclear from small and large subunit (LSU) rRNA trees, we tested the usefulness of the heat shock protein (Hsp) 90 gene for dinoflagellate phylogeny by sequencing it from four species with aberrant plastids (G. chlorophorum, Karlodinium micrum, Karenia brevis, and Karenia mikimotoi) plus Alexandrium tamarense, and constructing phylogenetic trees for Hsp90 and rRNAs, separately and together. Analyses of the Hsp90 and concatenated data suggest an ancestral origin of the peridinin-containing plastid, and two independent replacements of the peridinin plastid soon after the early radiation of the dinoflagellates. Thus, the Hsp90 gene seems to be a promising phylogenetic marker for dinoflagellate phylogeny.     

Rubisco genes indicate a close phylogenetic relation between the plastids of Chromophyta and Rhodophyta

The genes for both subunits of Rubisco (rbcL, rbcS) are located on the plastome of the brown alga Ectocarpus siliculosus (Chromophyta, Phaeophyceae). The organization of these genes in the form of an operon was similar to that found in rhodoplasts, cyanobacteria and the plastids of Cryptomonas . Sequence analysis of the complete operon revealed a high degree of homology and great structural similarities to corresponding genes from two red algae. In contrast, sequence homology to Rubisco genes from chloroplasts and cyanobacteria was much lower. This clearly indicated a close phylogenetic relationship between the plastids of Rhodophyta and Chromophyta which seem to have evolved independently from the chloroplasts (polyphyletic origin). Our data suggest that the plastids of Chromophyta and Cryptophyta have originated from endosymbiotic unicellular red algae. Surprisingly, red and brown algal Rubiscos show a significantly higher degree of homology to that from a hydrogen bacterium than to those from cyanobacteria.     

Rapid evolution of the plastid translational apparatus in a nonphotosynthetic plant: Loss or accelerated sequence evolution of tRNA and ribosomal protein genes

Summary The vestigial plastid genome of Epifagus virginiana (beechdrops), a nonphotosynthetic parasitic flowering plant, is functional but lacks six ribosomal protein and 13 tRNA genes found in the chloroplast DNAs of photosynthetic flowering plants. Import of nuclear gene products is hypothesized to compensate for many of these losses. Codon usage and amino acid usage patterns in Epifagus plastic genes have not been affected by the tRNA gene losses, though a small shift in the base composition of the whole genome (toward A + T -richness) is apparent. The ribosomal protein and tRNA genes that remain have had a high rate of molecular evolution, perhaps due to relaxation of constraints on the translational apparatus. Despite the compactness and extensive gene loss, one translational gene (infA, encoding initiation factor 1) that is a pseudogene in tobacco has been maintained intact in Epifagus.Offprint requests to: J.D. Palmer     

Phylogeny of nuclear-encoded plastid-targeted GAPDH gene supports separate origins for the peridinin- and the fucoxanthin derivative-containing plastids of dinoflagellates

Although most photosynthetic dinoflagellates have plastids with peridinin, the three dinoflagellate genera Karenia, Karlodinium, and Takayama possess anomalously pigmented plastids that contain fucoxanthin and its derivatives (19′-hexanoyloxy-fucoxanthin and 19′-butanoyloxy-fucoxanthin) instead of the peridinin. This pigment composition is similar to that of haptophytes. All peridinin-containing dinoflagellates investigated so far have at least two types of glyceraldehyde-3-phosphate dehydrogenase (GAPDH): cytosolic and plastid-targeted forms. In the present study, we cloned and sequenced genes encoding cytosolic and plastid-targeted GAPDH proteins from three species of the fucoxanthin derivative-containing dinoflagellates. Based on the molecular phylogeny, the plastid-targeted GAPDH genes of the fucoxanthin derivative-containing dinoflagellates were closely related to those of haptophyte algae rather than to the peridinin-containing dinoflagellates, while one of several cytosolic versions from the peridinin- and the fucoxanthin derivative-containing dinoflagellates are closely related to each other. Considering a previously reported theory that the plastid-targeted GAPDH from the peridinin-containing dinoflagellates originated by a gene duplication of the cytosolic form before the splitting of the dinoflagellate lineage, it is highly likely that the plastid-targeted GAPDH gene of the peridinin-containing dinoflagellates is original in this algal group and that in the fucoxanthin-containing dinoflagellates, the original plastid-targeted GAPDH was replaced by that of a haptophyte endosymbiont during a tertiary endosymbiosis. The present results strongly support the hypothesis that the plastids of the peridinin- and the fucoxanthin derivative-containing dinoflagellates are of separate origin.     

Molecular cloning and nucleotide sequence analysis of psbA from the dinoflagellates: Origin of the dinoflagellate plastid

Cloning and sequencing of psbA, the gene encoding D1 protein of photosystem II, from six species of dinoflagellates harboring a peridinin type plastid [Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra Stein, Lingulodinium polyedra (Dodge) Stein, Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech] is reported. Using the polymerase chain reaction technique, the psbA gene was detected in a satellite DNA band isolated from total DNA of A. catenella by CsCl-Hoechst 33258 gradient ultracentrifugation. This finding suggests that in dinoflagellates psbA is encoded in the plastid genome. The deduced amino acid sequences of D1 from the dinoflagellates did not reveal a typical ‘C-terminus extension’, which should be removed by proteolytic cleavage from the D1 precursor. Molecular phylogenetic analysis based on the deduced amino acid sequences of D1 revealed that the six species of dinoflagellates are monophyletic and also showed that dinoflagellates cluster with rhodophytes, a cryptophyte and heterokonts. These results support the hypothesis that the peridinin type plastid in dinoflagellates originated from an engulfed red alga.     

Polyploid evolution and plastid DNA variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in Scandinavia

The Dactylorhiza incarnata/maculata complex (Orchidaceae) was used as a model system to understand genetic differentiation processes in a naturally occurring polyploid complex with much of ongoing diversification and wide distribution in recently glaciated areas in northern Europe. Data were obtained for 12 hypervariable regions in the plastid DNA genome. A total of 166 haplotypes were found in a sample of 1099 plants. Allopolyploid taxa have inherited their plastid genomes from D. maculata s.l. Overall haplotype diversity of the combined group of allopolyploid taxa was comparable to that of maternal D. maculata s.l., but populations of allopolyploids were also more strongly differentiated from each other and contained lower numbers of haplotypes than populations of D. maculata s.l. In addition to haplotypes found in extant D. maculata s.l., the allopolyploids also contained several distinct and widespread haplotypes that were not found in any of the parental lineages. Some of these haplotypes were shared between widespread allopolyploids. Divergent allopolyploids with small distributions did not seem to originate from local polyploidization events, but rather as segregates of already existing allopolyploids. Genetic diversification of allopolyploid Dactylorhiza is the result of repeated polyploid formation, secondary hybridization and introgression between already existing polyploids and extant representatives of parental lineages, hybridization between independently derived polyploid lineages, and phyletic diversification in the group of allopolyploids. Although some polyploid taxa must have evolved after the last glaciation, genetic material from the parental lineages has been transferred continuously for longer periods of time. This combination of processes may explain the taxonomic complexity encountered in Dactylorhiza and other polyploid complexes distributed in previously glaciated parts of Europe.     

Phylogenomic analysis identifies red algal genes of endosymbiotic origin in the chromalveolates

Endosymbiosis has spread photosynthesis to many branches of the eukaryotic tree; however, the history of photosynthetic organelle (plastid) gain and loss remains controversial. Fortuitously, endosymbiosis may leave a genomic footprint through the transfer of endosymbiont genes to the "host" nucleus (endosymbiotic gene transfer, EGT). EGT can be detected through comparison of host genomes to uncover the history of past plastid acquisitions. Here we focus on a lineage of chlorophyll c-containing algae and protists ("chromalveolates") that are postulated to share a common red algal secondary endosymbiont. This plastid is originally of cyanobacterial origin through primary endosymbiosis and is closely related among the Plantae (i.e., red, green, and glaucophyte algae). To test these ideas, an automated phylogenomics pipeline was used with a novel unigene data set of 5,081 expressed sequence tags (ESTs) from the haptophyte alga Emiliania huxleyi and genome or EST data from other chromalveolates, red algae, plants, animals, fungi, and bacteria. We focused on nuclear-encoded proteins that are targeted to the plastid to express their function because this group of genes is expected to have phylogenies that are relatively easy to interpret. A total of 708 genes were identified in E. huxleyi that had a significant Blast hit to at least one other taxon in our data set. Forty-six of the alignments that were derived from the 708 genes contained at least one other chromalveolate (i.e., besides E. huxleyi), red and/or green algae (or land plants), and one or more cyanobacteria, whereas 15 alignments contained E. huxleyi, one or more other chromalveolates, and only cyanobacteria. Detailed phylogenetic analyses of these data sets turned up 19 cases of EGT that did not contain significant paralogy and had strong bootstrap support at the internal nodes, allowing us to confidently identify the source of the plastid-targeted gene in E. huxleyi. A total of 17 genes originated from the red algal lineage, whereas 2 genes were of green algal origin. Our data demonstrate the existence of multiple red algal genes that are shared among different chromalveolates, suggesting that at least a subset of this group may share a common origin.     

Origin and distribution of Calvin cycle fructose and sedoheptulose bisphosphatases in plantae and complex algae: a single secondary origin of complex red plastids and subsequent propagation via tertiary endosymbioses

Sedoheptulose-1,7-bisphosphatase (SBPase) and fructose-1,6-bisphosphatase (FBPase) are essential nuclear-encoded enzymes involved in land plant Calvin cycle and gluconeogenesis. In this study, we cloned seven SBP and seven FBP cDNAs/genes and established sequences from all lineages of photosynthetic eukaryotes, in order to investigate their origin and evolution. Our data are best explained by a single recruitment of plastid-targeted SBP in Plantae after primary endosymbiosis and a further distribution to algae with complex plastids. While SBP is universally found in photosynthetic lineages, its presence in apicomplexa, ciliates, trypanosomes, and ascomycetes is surprising given that no metabolic function beyond the one in the plastid Calvin cycle is described so far. Sequences of haptophytes, cryptophytes, diatoms, and peridinin-containing dinoflagellates (complex red lineage) strongly group together in the SBP tree and the same assemblage is recovered for plastid-targeted FBP sequences, although this is less supported. Both SBP and plastid-targeted FBP are most likely of red algal origin. Including phosphoribulokinase, fructose bisphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase, a total of five independent plastid-related nuclear-encoded markers support a common origin of all complex rhodoplasts via a single secondary endosymbiosis event. However, plastid phylogenies are incongruent with those of the host cell, as illustrated by the cytosolic FBP isoenzyme. These results are discussed in the context of Cavalier-Smith's far-reaching chromalveolate hypothesis. In our opinion, a more plausible evolutionary scenario would be the establishment of a unique secondary rhodoplast and its subsequent spread via tertiary endosymbioses.     

Evolution of the plastid ribosomal RNA operon in a nongreen parasitic plant: Accelerated sequence evolution,altered promoter structure,and tRNA pseudogenes

The nucleotide sequence of a 7.4 kb region containing the entire plastid ribosomal RNA operon of the nongreen parasitic plant Epifagus virginiana has been determined. Analysis of the sequence indicates that all four rRNA genes are intact and almost certainly functional. In contrast, the split genes for tRNA^Ile and tRNA^Ala present in the 16S-23S rRNA spacer region have become pseudogenes, and deletion upstream of the 16S rRNA gene has removed a tRNA^Val gene and most of the promoter region for the rRNA operon. The rate of nucleotide substitution in 16S and 23S rRNAs is several times higher in Epifagus than in tobacco, a related photosynthetic plant. Possible reasons for this, including relaxed translational constraints, are discussed.     

Identification and transcription of transfer RNA genes in dinoflagellate plastid minicircles

The dinoflagellate chloroplast genome is unique in that the genes are found on small circular DNA molecules carrying from one to three genes. In addition, only 14 of the typical chloroplast-located genes have so far been discovered on minicircles, while a number have been transferred to the nucleus. We have sequenced four new minicircles from the dinoflagellate Heterocapsa triquetra, three of which carry a single protein-coding gene (psbD, psbE, petD) and one that appears to be an "empty" circle. Using the tRNA prediction programs ARAGORN and tRNAscan-SE, tRNA-Met was found in the petD circle immediately downstream of the end of petD, while tRNA-Trp and tRNA-Pro were detected in the psbE and petD circles as well as in several chimeric circles of H. triquetra and the psbA minicircles of Heterocapsa pygmaea. RT-PCR showed that the tRNAs were co-transcribed with the protein-coding genes that preceded them, and cleaved from the precursor before a poly(U) tail was added to the mRNA.     

Sequences of 72 plastid genes support the early divergence of Cornales in the asterids

Recent phylogenetic analyses of molecular data have supported different hypotheses of relationships among Cornales,Ericales,and core asterids.Such hypotheses have implications for the evolution of important morphological and embryological features of asterids.In this study we generated plastid genome-scale data of Davidia (Cornales) and Franklinia (Ericales) and combined them with published sequence data of eudicots.Our maximum parsimony,maximum likelihood,and Bayesian analyses generated strongly supported and congruent phylogenetic relationships among the three major lineages of the asterids.Cornales diverges first in asterids; Ericales is sister to the core asterids.Adding two more taxa helps mitigate long branch attraction in parsimony analyses.Sampling 26-28 plastid protein-coding genes may provide satisfactory resolution and support for relationships of eudicots including basal lineages of asterids.     

Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis

Pairwise comparison of whole plastid and draft nuclear genomic sequences of Arabidopsis thaliana and Oryza sativa L. ssp. indica shows that rice nuclear genomic sequences contain homologs of plastid DNA covering about 94 kb (83%) of plastid genome and including one or more full-length intact (without mutations resulting in premature stop codons) homologues of 26 known protein-coding (KPC) plastid genes. By contrast, only about 20 kb (16%) of chloroplast DNA, including a single intact plastid-derived KPC gene, is presented in the nucleus of A. thaliana. Sixteen rice plastid genes have at least one nuclear copy without any mutation or with only synonymous substitutions. Nuclear copies for other ten plastid genes contain both synonymous and non-synonymous substitutions. Multiple ESTs for 25 out of 26 KPC genes were also found, as well as putative promoters for some of them. The study of substitutions pattern shows that some of nuclear homologues of plastid genes may be functional and/or are under the pressure of the positive natural selection. The similar comparative analysis performed on rice chromosome 1 revealed 27 contigs containing plastid-derived sequences, totalling about 84 kb and covering two thirds of chloroplast DNA, with the intact nuclear copies of 26 different KPC genes. One of these contigs, AP003280, includes almost 57 kb (45%) of chloroplast genome with the intact copies of 22 KPC genes. At the same time, we observed that relative locations of homologues in plastid DNA and the nuclear genome are significantly different.     

Phytoplankton distribution patterns in the northwestern Sargasso Sea revealed by small subunit rRNA genes from plastids

Phytoplankton species vary in their physiological properties, and are expected to respond differently to seasonal changes in water column conditions. To assess these varying distribution patterns, we used 412 samples collected monthly over 12 years (1991–2004) at the Bermuda Atlantic Time-Series Study site, located in the northwestern Sargasso Sea. We measured plastid 16S ribosomal RNA gene abundances with a terminal restriction fragment length polymorphism approach and identified distribution patterns for members of the Prymnesiophyceae, Pelagophyceae, Chrysophyceae, Cryptophyceae, Bacillariophyceae and Prasinophyceae. The analysis revealed dynamic bloom patterns by these phytoplankton taxa that begin early in the year, when the mixed layer is deep. Previously, unreported open-ocean prasinophyte blooms dominated the plastid gene signal during convective mixing events. Quantitative PCR confirmed the blooms and transitions of Bathycoccus, Micromonas and Ostreococcus populations. In contrast, taxa belonging to the pelagophytes and chrysophytes, as well as cryptophytes, reached annual peaks during mixed layer shoaling, while Bacillariophyceae (diatoms) were observed only episodically in the 12-year record. Prymnesiophytes dominated the integrated plastid gene signal. They were abundant throughout the water column before mixing events, but persisted in the deep chlorophyll maximum during stratified conditions. Various models have been used to describe mechanisms that drive vernal phytoplankton blooms in temperate seas. The range of taxon-specific bloom patterns observed here indicates that different ‘spring bloom'' models can aptly describe the behavior of different phytoplankton taxa at a single geographical location. These findings provide insight into the subdivision of niche space by phytoplankton and may lead to improved predictions of phytoplankton responses to changes in ocean conditions.     

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.     

The endosymbiotic origin,diversification and fate of plastids

Plastids and mitochondria each arose from a single endosymbiotic event and share many similarities in how they were reduced and integrated with their host. However, the subsequent evolution of the two organelles could hardly be more different: mitochondria are a stable fixture of eukaryotic cells that are neither lost nor shuffled between lineages, whereas plastid evolution has been a complex mix of movement, loss and replacement. Molecular data from the past decade have substantially untangled this complex history, and we now know that plastids are derived from a single endosymbiotic event in the ancestor of glaucophytes, red algae and green algae (including plants). The plastids of both red algae and green algae were subsequently transferred to other lineages by secondary endosymbiosis. Green algal plastids were taken up by euglenids and chlorarachniophytes, as well as one small group of dinoflagellates. Red algae appear to have been taken up only once, giving rise to a diverse group called chromalveolates. Additional layers of complexity come from plastid loss, which has happened at least once and probably many times, and replacement. Plastid loss is difficult to prove, and cryptic, non-photosynthetic plastids are being found in many non-photosynthetic lineages. In other cases, photosynthetic lineages are now understood to have evolved from ancestors with a plastid of different origin, so an ancestral plastid has been replaced with a new one. Such replacement has taken place in several dinoflagellates (by tertiary endosymbiosis with other chromalveolates or serial secondary endosymbiosis with a green alga), and apparently also in two rhizarian lineages: chlorarachniophytes and Paulinella (which appear to have evolved from chromalveolate ancestors). The many twists and turns of plastid evolution each represent major evolutionary transitions, and each offers a glimpse into how genomes evolve and how cells integrate through gene transfers and protein trafficking.