Expressed sequence tag (EST) survey of the highly adapted green algal parasite, Helicosporidium

Helicosporidia are obligate invertebrate pathogens with a unique and highly adapted mode of infection. The evolutionary history of Helicosporidia has been uncertain, but several recent molecular phylogenetic studies have shown an unexpectedly close relationship to green algae, and specifically to the opportunistic pathogen Prototheca. To date, molecular sequences from Helicosporidia are restricted to those genes used for phylogenetic reconstruction and genes related to the existence and function of its cryptic plastid. We have therefore conducted a small expressed sequence tag (EST) project on Helicosporidium sp., yielding about 700 unique sequences. We have examined the functional distribution of known genes, the distribution of EST abundance, and the prevalence of previously unknown gene sequences. To demonstrate the potential utility of large amounts of data, we have used ribosomal proteins to test whether the phylogenetic position of Helicosporidium inferred from a small number of genes is broadly supported by a large number of genes. We conducted phylogenetic analyses on 69 ribosomal proteins and found that 98% supported the green algal origin of Helicosporidia and 80% support a specific relationship with Prototheca. Overall, these data multiply the available molecular information from Helicosporidium 100-fold, which should provide the basis for new insights into these unusual but interesting parasites.     

Detection of Helicosporidium spp. in metagenomic DNA

Distinct isolates of the invertebrate pathogenic alga Helicosporidium sp., collected from different insect hosts and different geographic locations, were processed to sequence the 18S rDNA and β-tubulin genes. The sequences were analyzed to assess genetic variation within the genus Helicosporidium and to design Helicosporidium-specific 18S rDNA primers. The specificity of these primers was demonstrated by testing not only on the Helicosporidium sp. isolates, but also on two trebouxiophyte algae known to be close Helicosporidium relatives, Prototheca wickerhamii and Prototheca zopfii. The genus-specific primers were used to develop a culture-independent assay aimed at detecting the presence of Helicosporidium spp. in environmental waters. The assay was based on the PCR amplification of 18SrDNA gene fragments from metagenomic DNA preparations, and it resulted in the amplification of detectable products for all sampled sites. Phylogenetic analyses that included the environmental sequences demonstrated that all amplification products clustered in a strongly supported, monophyletic Helicosporidium clade, thereby validating the metagenomic approach and the taxonomic origin of the produced environmental sequences. In addition, the phylogenetic analyses established that Helicosporidium spp. isolated from coleopteran hosts are more closely related to each other than they are to the isolate collected from a dipteran host. Finally, the phylogenetic trees depicted intergeneric relationships that supported a Helicosporidium-Prototheca cluster but did not support a Helicosporidium-Coccomyxa grouping, suggesting that pathogenicity to invertebrates evolved at least twice independently within the trebouxiophyte green algae.     

Nucleus-encoded genes for plastid-targeted proteins in Helicosporidium: functional diversity of a cryptic plastid in a parasitic alga

Plastids are the organelles of plants and algae that house photosynthesis and many other biochemical pathways. Plastids contain a small genome, but most of their proteins are encoded in the nucleus and posttranslationally targeted to the organelle. When plants and algae lose photosynthesis, they virtually always retain a highly reduced "cryptic" plastid. Cryptic plastids are known to exist in many organisms, although their metabolic functions are seldom understood. The best-studied example of a cryptic plastid is from the intracellular malaria parasite, Plasmodium, which has retained a plastid for the biosynthesis of fatty acids, isoprenoids, and heme by the use of plastid-targeted enzymes. To study a completely independent transformation of a photosynthetic plastid to a cryptic plastid in another alga-turned-parasite, we conducted an expressed sequence tag (EST) survey of Helicosporidium. This parasite has recently been recognized as a highly derived green alga. Based on phylogenetic relationships to other plastid homologues and the presence of N-terminal transit peptides, we have identified 20 putatively plastid-targeted enzymes that are involved in a wide variety of metabolic pathways. Overall, the metabolic diversity of the Helicosporidium cryptic plastid exceeds that of the Plasmodium plastid, as it includes representatives of most of the pathways known to operate in the Plasmodium plastid as well as many others. In particular, several amino acid biosynthetic pathways have been retained, including the leucine biosynthesis pathway, which was only recently recognized in plant plastids. These two parasites represent different evolutionary trajectories in plastid metabolic adaptation.     

In Vivo and In Vitro Development of the Protist Helicosporidium sp.

We describe the discovery and developmental features of a Helicosporidium sp. isolated from the black fly Simulium jonesi. Morphologically, the helicosporidia are characterized by a distinct cyst stage that encloses three ovoid cells and a single elongate filamentous cell. Bioassays have demonstrated that the cysts of this isolate infect various insect species, including the lepidopterans, Helicoverpa zea, Galleria mellonella, and Manduca sexta, and the dipterans, Musca domestica, Aedes taeniorhynchus, Anopheles albimanus, and An. quadrimaculatus. The cysts attach to the insect peritrophic matrix prior to dehiscence, which releases the filamentous cell and the three ovoid cells. The ovoid cells are short-lived in the insect gut with infection mediated by the penetration of the filamentous cell into the host. Furthermore, these filamentous cells are covered with projections that anchor them to the midgut lining. Unlike most entomopathogenic protozoa, this Helicosporidium sp. can be propagated in simple nutritional media under defined in vitro conditions, providing a system to conduct detailed analysis of the developmental biology of this poorly known taxon. The morphology and development of the in vitro produced cells are similar to that reported for the achorophyllic algae belonging to the genus Prototheca.     

Twenty-fold difference in evolutionary rates between the mitochondrial and plastid genomes of species with secondary red plastids

Within plastid-bearing species, the relative rates of evolution between mitochondrial and plastid genomes are poorly studied, but for the few lineages in which they have been explored, including land plants and green algae, the mitochondrial DNA mutation rate is nearly always estimated to be lower than or equal to that of the plastid DNA. Here, we show that in protists from three distinct lineages with secondary, red algal-derived plastids, the opposite is true: their mitochondrial genomes are evolving 5-30 times faster than their plastid genomes, even when the plastid is nonphotosynthetic. These findings have implications for understanding the origins and evolution of organelle genome architecture and the genes they encode.     

Implications of the Plastid Genome Sequence of Typha (Typhaceae, Poales) for Understanding Genome Evolution in Poaceae

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.     

Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.     

The evolution of the plastid chromosome in land plants: gene content, gene order, gene function

This review bridges functional and evolutionary aspects of plastid chromosome architecture in land plants and their putative ancestors. We provide an overview on the structure and composition of the plastid genome of land plants as well as the functions of its genes in an explicit phylogenetic and evolutionary context. We will discuss the architecture of land plant plastid chromosomes, including gene content and synteny across land plants. Moreover, we will explore the functions and roles of plastid encoded genes in metabolism and their evolutionary importance regarding gene retention and conservation. We suggest that the slow mode at which the plastome typically evolves is likely to be influenced by a combination of different molecular mechanisms. These include the organization of plastid genes in operons, the usually uniparental mode of plastid inheritance, the activity of highly effective repair mechanisms as well as the rarity of plastid fusion. Nevertheless, structurally rearranged plastomes can be found in several unrelated lineages (e.g. ferns, Pinaceae, multiple angiosperm families). Rearrangements and gene losses seem to correlate with an unusual mode of plastid transmission, abundance of repeats, or a heterotrophic lifestyle (parasites or myco-heterotrophs). While only a few functional gene gains and more frequent gene losses have been inferred for land plants, the plastid Ndh complex is one example of multiple independent gene losses and will be discussed in detail. Patterns of ndh-gene loss and functional analyses indicate that these losses are usually found in plant groups with a certain degree of heterotrophy, might rendering plastid encoded Ndh1 subunits dispensable.     

The plastid genome of the red alga Laurencia

We present the 174,935 nt long plastid genome of the red alga Laurencia sp. JFC0032. It is the third plastid genome characterized for the largest order of red algae (Ceramiales). The circular‐mapping plastid genome is small compared to most florideophyte red algae, and our comparisons show a trend toward smaller plastid genome sizes in the family Rhodomelaceae, independent from a similar trend in Cyanidiophyceae. The Laurencia genome is densely packed with 200 annotated protein‐coding genes (188 widely conserved, 3 open reading frames shared with other red algae and 9 hypothetical coding regions). It has 29 tRNAs, a single‐copy ribosomal RNA cistron, a tmRNA, and the RNase P RNA.     

Relative rates of evolution among the three genetic compartments of the red alga Porphyra differ from those of green plants and do not correlate with genome architecture

In photosynthetic eukaryotes, relative silent-site nucleotide substitution rates (which can be used to approximate relative mutation rates) among mitochondrial, plastid, and nuclear genomes (mtDNAs, ptDNAs, and nucDNAs) are estimated to be 1:3:10 respectively for seed plants and roughly equal for green algae. These estimates correlate with certain genome characteristics, such as size and coding density, and have therefore been taken to support a relationship between mutation rate and genome architecture. Plants and green algae, however, represent a small fraction of the major eukaryotic plastid-bearing lineages. Here, we investigate relative rates of mutation within the model red algal genus Porphyra. In contrast to plants, we find that the levels of silent-site divergence between the Porphyra purpurea and Porphyra umbilicalis mtDNAs are three times that of their ptDNAs and five times that of their nucDNAs. Moreover, relative mutation rates do not correlate with genome architecture: despite an estimated three-fold difference in their mutation rate, the mitochondrial and plastid genome coding densities are equivalent - an observation that extends to organisms with secondary red algal plastids. These findings are supported by within-species silent-site polymorphism data from P. purpurea.     

The plastid-encoded psbA gene in the dinoflagellate Gonyaulax is not encoded on a minicircle

In all dinoflagellate species studied to date, the plastid genome is highly reduced, with many genes normally found in the plastid genome found instead encoded by the nucleus. Furthermore, those genes still remaining in the plastid are found as primarily single gene minicircles whose size is typically only 2-3 kb. We show here that the plastid genome architecture in the dinoflagellate Gonyaulax polyedra is unusual for this class of organism. In particular, the psbA gene is associated with DNA of roughly 50-150 kb and appears to have an unusually high complexity.     

SecA is plastid-encoded in a red alga: implications for the evolution of plastid genomes and the thylakoid protein import apparatus

Summary Partial sequence analysis of the plastid DNA (ptDNA) from a red alga, Antithamnion sp., revealed the presence of a homologue to the Escherichia coli SecA gene as well as two open reading frames (ORF 510, ORF 179). In addition a sec Y homologue has been detected on the plastid genome by heterologous hybridization. None of these genes has been found in completely sequenced chlorophytic plastid genomes. SecA and secY gene copies were also detected in the ptDNA of a chromophytic alga, indicating that secAY may be ubiquitous in rhodophytes and chromophytes. The significance of these findings for the evolution of plastid genomes and the thylakoid protein import mechanism is discussed.     

REVIEW—CONTRASTING MITOCHONDRIAL GENOME ORGANIZATIONS AND SEQUENCE AFFILIATIONS AMONG GREEN ALGAE: POTENTIAL FACTORS, MECHANISMS, AND EVOLUTIONARY SCENARIOS

The three green algal mitochondrial genomes completely sequenced to date — those of Chlamydomonas reinhardtii Dangeard, Chlamydomonas eugametos Gerloff, and Prototheca wickerhamii Soneda & Tubaki — revealed very different mitochondrial genome organizations and sequence affiliations. The Chlamydomonas genomes resemble the ciliate / fungal / animal counterparts, and the Prototheca genome resembles land plant homologues. This review points out that all the green algal mitochondrial genomes examined to date resemble either the Chlamydomonas or the Prototheca mitochondrial genome; the Chlamydomonas- like mitochondrial genomes are small and have a reduced gene content (no ribosomal protein or 5S rRNA genes and only a few protein-coding and tRNA genes) and fragmented and scrambled rRNA coding regions, whereas the Prototheca- like mitochondrial genomes are larger and have a larger set of protein-coding genes (including ribosomal protein genes), more tRNA genes, and 5S rRNA and conventional continuous small-subunit (SSU) and large-subunit (LSU) rRNA coding regions. It appears, therefore, that the differences previously observed between the mitochondrial genomes of C. reinhardtii and P. wickerhamii extend to the two green algal mitochondrial lineages to which they belong and are significant enough to raise questions about the causes and mechanisms responsible for such contrasting evolutionary strategies among green algae. This review suggests an integrative approach in explaining the occurrence of distinct evolutionary strategies and apparent phylogenetic affiliations among the known green algal mitochondrial lineages. The observed differences could be the result of distinct genetic potentials differentiated during the previous evolutionary history of the flagellate ancestors and / or of subsequent changes in habitat and life history of the more advanced green algal lineages.     

Gene-rich plastid genomes of two parasitic red algal species,Laurencia australis and L. verruciformis (Rhodomelaceae,Ceramiales), and a taxonomic revision of Janczewskia

Parasitic red algae are an interesting system for investigating the genetic changes that occur in parasites. These parasites have evolved independently multiple times within the red algae. The functional loss of plastid genomes can be investigated in these multiple independent examples, and fine-scale patterns may be discerned. The only plastid genomes from red algal parasites known so far are highly reduced and missing almost all photosynthetic genes. Our study assembled and annotated plastid genomes from the parasites Janczewskia tasmanica and its two Laurencia host species (Laurencia elata and one unidentified Laurencia sp. A25) from Australia and Janczewskia verruciformis, its host species (Laurencia catarinensis), and the closest known free-living relative (Laurencia obtusa) from the Canary Islands (Spain). For the first time we show parasitic red algal plastid genomes that are similar in size and gene content to free-living host species without any gene loss or genome reduction. The only exception was two pseudogenes (moeB and ycf46) found in the plastid genome of both isolates of J. tasmanica, indicating potential for future loss of these genes. Further comparative analyses with the three highly reduced plastid genomes showed possible gene loss patterns, in which photosynthetic gene categories were lost followed by other gene categories. Phylogenetic analyses did not confirm monophyly of Janczewskia, and the genus was subsumed into Laurencia. Further investigations will determine if any convergent small-scale patterns of gene loss exist in parasitic red algae and how these are applicable to other parasitic systems.     

Plastid translation and transcription genes in a non-photosynthetic plant: intact, missing and pseudo genes

The non-photosynthetic, parasitic flowering plant Epifagus virginiana has recently been shown to contain a grossly reduced plastid genome that has lost many photosynthetic and chloro-respiratory genes. We have cloned and sequenced a 3.9 kb domain of plastid DNA from Epifagus to investigate the patterns of evolutionary change in such a reduced genome and to determine which genes are still present and likely to be functional. This 3.9 kb domain is colinear with a 35.4 kb region of tobacco chloroplast DNA, differing from it by a minimum of 11 large deletions varying in length from 354 bp to 11.5 kb, as well as by a number of small deletions and insertions. The nine genes retained in Epifagus encode seven tRNAs and two ribosomal proteins and are coextensive and highly conserved in sequence with homologs in photosynthetic plants. This suggests that these genes are functional in Epifagus and, together with evidence that the Epifagus plastid genome is transcribed, implies that plastid gene products play a role in processes other than photosynthesis and gene expression. Genes that are completely absent include not only photosynthetic genes, but surprisingly, genes encoding three subunits of RNA polymerase, four tRNAs and one ribosomal protein. In addition, only pseudogenes are found for two other tRNAs. Despite these defunct tRNA genes, codon and amino acid usage in Epifagus protein genes is normal. We therefore hypothesize that the expression of plastid genes in Epifagus relies on the import of nuclear encoded tRNAs and RNA polymerase from the cytoplasm.     

Plastid genome sequencing,comparative genomics,and phylogenomics: Current status and prospects

Abstract More than 190 plastid genomes have been completely sequenced during the past two decades due to advances in DNA sequencing technologies. Based on this unprecedented abundance of data, extensive genomic changes have been revealed in the plastid genomes. Inversion is the most common mechanism that leads to gene order changes. Several inversion events have been recognized as informative phylogenetic markers, such as a 30‐kb inversion found in all living vascular plants minus lycopsids and two short inversions putatively shared by all ferns. Gene loss is a common event throughout plastid genome evolution. Many genes were independently lost or transferred to the nuclear genome in multiple plant lineages. The trnR‐CCG gene was lost in some clades of lycophytes, ferns, and seed plants, and all the ndh genes were absent in parasitic plants, gnetophytes, Pinaceae, and the Taiwan moth orchid. Certain parasitic plants have, in particular, lost plastid genes related to photosynthesis because of the relaxation of functional constraint. The dramatic growth of plastid genome sequences has also promoted the use of whole plastid sequences and genomic features to solve phylogenetic problems. Chloroplast phylogenomics has provided additional evidence for deep‐level phylogenetic relationships as well as increased phylogenetic resolutions at low taxonomic levels. However, chloroplast phylogenomics is still in its infant stage and rigorous analysis methodology has yet to be developed.     

Ins and outs of plastid genome evolution

Recent findings have established cracks in the straight-laced image of the plastid genome as a molecule whose sole function is photosynthesis and whose gene content is highly conserved. Genes for numerous non-photosynthetic functions have been identified. Algal plastid genomes contain many genes with no homologs in angiosperms, and the recent transfer of genes from the plastid to the nuclear genome has been described. Wholesale abandonment of genes encoding photosynthetic and gene-expression functions has occurred in the plastid genomes of a non-green plant and alga. The origins of plastid DNA, its use in phylogenetic studies, and the origins of plastid introns are also reviewed.