Chloroplast phylogenomic analyses reveal the deepest-branching lineage of the Chlorophyta, Palmophyllophyceae class. nov.

The green plants (Viridiplantae) are an ancient group of eukaryotes comprising two main clades: the Chlorophyta, which includes a wide diversity of green algae, and the Streptophyta, which consists of freshwater green algae and the land plants. The early-diverging lineages of the Viridiplantae comprise unicellular algae, and multicellularity has evolved independently in the two clades. Recent molecular data have revealed an unrecognized early-diverging lineage of green plants, the Palmophyllales, with a unique form of multicellularity, and typically found in deep water. The phylogenetic position of this enigmatic group, however, remained uncertain. Here we elucidate the evolutionary affinity of the Palmophyllales using chloroplast genomic, and nuclear rDNA data. Phylogenetic analyses firmly place the palmophyllalean Verdigellas peltata along with species of Prasinococcales (prasinophyte clade VI) in the deepest-branching clade of the Chlorophyta. The small, compact and intronless chloroplast genome (cpDNA) of V. peltata shows striking similarities in gene content and organization with the cpDNAs of Prasinococcales and the streptophyte Mesostigma viride, indicating that cpDNA architecture has been extremely well conserved in these deep-branching lineages of green plants. The phylogenetic distinctness of the Palmophyllales-Prasinococcales clade, characterized by unique ultrastructural features, warrants recognition of a new class of green plants, Palmophyllophyceae class. nov.

Related papers:

title: Into the deep: New discoveries at the base of the green plant phylogeny
abstract: Recent data have provided evidence for an unrecognised ancient lineage of green plants that persists in marine deep‐water environments. The green plants are a major group of photosynthetic eukaryotes that have played a prominent role in the global ecosystem for millions of years. A schism early in their evolution gave rise to two major lineages, one of which diversified in the world's oceans and gave rise to a large diversity of marine and freshwater green algae (Chlorophyta) while the other gave rise to a diverse array of freshwater green algae and the land plants (Streptophyta). It is generally believed that the earliest‐diverging Chlorophyta were motile planktonic unicellular organisms, but the discovery of an ancient group of deep‐water seaweeds has challenged our understanding of the basal branches of the green plant phylogeny. In this review, we discuss current insights into the origin and diversification of the green plant lineage.

title: Phylogenetic analyses of 18s rdna sequences reveal a new coccoid lineage of the prasinophyceae (Chlorophyta)
abstract: Phylogenetic analyses of 18S rDNA sequences from 25 prasinophytes, including 10 coccoid isolates, reveals that coccoid organisms are found in at least three prasinophyte lineages. The coccoid Ostreococcus tauri is included in the Mamiellales lineage and P ycnococcus provasolii is allied with the flagellate P seudoscourfieldia marina. A previously undescribed prasinophyte lineage is comprised of the coccoid Prasinococcus cf. capsulatus (CCMP 1407) and other isolates tentatively identified as Prasinococcus sp. (CCMP 1202, CCMP 1614, and CCMP 1194), as well as three unnamed coccoids (CCMP 1193, CCMP 1413, and CCMP 1220). No flagellate organisms are known from this lineage. Organisms of this new lineage share some characteristics of both the Pycnococcaceae and the Mamiellales, although relationships among these separate lineages were not supported by bootstrap analyses. An additional unnamed coccoid isolate (CCMP 1205) is separate from all major prasinophyte lineages. The analyses did not resolve the relationships among the major prasinophyte lineages, although they support previous conclusions that the Prasinophyceae are not monophyletic.

title: Diversity of picoplanktonic prasinophytes assessed by direct nuclear SSU rDNA sequencing of environmental samples and novel isolates retrieved from oceanic and coastal marine ecosystems.
abstract: Picoplanktonic prasinophytes are well represented in culture collections and marine samples. In order to better characterize this ecologically important group, we compared the phylogenetic diversity of picoplanktonic prasinophyte strains available at the Roscoff Culture Collection (RCC) and that of nuclear SSU rDNA sequences from environmental clone libraries obtained from oceanic and coastal ecosystems. Among the 570 strains avalaible, 91 belonged to prasinophytes, 65 were partially sequenced, and we obtained the entire SSU rDNA sequence for a selection of 14 strains. Within the 18 available environmental clone libraries, the prasinophytes accounted for 12% of the total number of clones retrieved (142 partial sequences in total), and we selected 9 clones to obtain entire SSU rDNA sequence. Using this approach, we obtained a subsequent genetic database that revealed the presence of seven independent lineages among prasinophytes, including a novel clade (clade VII). This new clade groups the genus Picocystis, two unidentified coccoid strains, and 4 environmental sequences. For each of these seven lineages, at least one representative is available in culture. The three picoplanktonic genera Ostreococcus, Micromonas, and Bathycoccus (order Mamiellales), were the best represented prasinophytes both in cultures and genetic libraries. SSU rDNA phylogenetic analyses suggest that the genus Bathycoccus forms a very homogeneous group. In contrast, the genera Micromonas and Ostreococcus turned out to be quite complex, consisting of three and four independent lineages, respectively. This report of the overall diversity of picoeukaryotic prasinophytes reveals a group of ecologically important and diverse marine microorganims that are well represented by isolated cultures.

title: PIGMENT SUITES AND TAXONOMIC GROUPS IN PRASINOPHYCEAE 1
abstract: Pigment analysis performed on 30 Prasinophyceae strains revealed two main groups: the prasinoxanthin‐containing and prasinoxanthin‐less Prasinophyceae. Prasinoxanthin‐containing Prasinophyceae comprised the orders Mamiellales, Pseudoscourfieldiales (Pycnococcaceae), and Prasinococcales. For this group, classification with pigment composition showed a good agreement with molecular phylogeny. Mamiellales, except Crustomastix stigmatica, accumulated uriolide, micromonal, dihydrolutein, and the pigment Unidentified M1 as characteristic pigments. Prasinococcales and Pseudoscourfieldiales (Pycnococcaceae) lacked micromonal and Unidentified M1. In addition, Pseudoscourfieldiales (Pycnococcaceae) lacked uriolide. A chl c3‐like pigment was present in prasinoxanthin‐containing strains isolated from the deep sea. Common green algae pigments, a loroxanthin derivative, and siphonaxanthin plus derivatives were found in the prasinoxanthin‐less Prasinophyceae, which included strains from Pyramimonadales, Pseudoscourfieldiales (Nephroselmidiaceae), Chlorodendrales, and a new order. Although some associations could be observed, the correspondence between pigments and molecular taxonomy was less clear for this group.

title: The Basal Position of Scaly Green Flagellates among the Green Algae (Chlorophyta) is Revealed by Analyses of Nuclear-Encoded SSU rRNA Sequences.
abstract: The prasinophytes comprise a morphologically heterogeneous assembly of mostly marine flagellates and coccoid taxa, which represent an important component of the nano- and picoplankton, and have previously figured prominently in discussions about the origin and phylogeny of the green plants. To evaluate their putative basal position in the Viridiplantae and to resolve the phylogenetic relationships among the prasinophyte taxa, we determined complete nuclear-encoded SSU rRNA sequences from 13 prasinophyte taxa representing the genera Cymbomonas, Halosphaera, Mamiella, Mantoniella, Micromonas, Pterosperma, Pycnococcus, and Pyramimonas. Phylogenetic analyses of SSU rRNA sequences using distance, parsimony and likelihood methods revealed four independent prasi.nophyte lineages (clades) which constitute the earliest divergences among the Chlorophyta. In order of their divergence these clades are represented by the genera Cymbomonas, Halosphaera, Pterosperma, Pyramimonas (clade I), Mamiella, Mantoniella, Micromonas (clade II), Pseudoscourfieldia (strain CCMP 717), Nephroselmis (clade III), and Tetraselmis, Scherffelia (clade IV). The coccoid Pycnococcus provasolii diverged after clade II, but before clade III. Since no other coccoid prasinophyte taxa were analyzed in this study, the phylogenetic status of this taxon is presently unresolved. Our analyses provide further evidence for the basal phylogenetic position of the scaly green flagellates among the Chlorophyta and raise important questions concerning the class-level classification of the Chlorophyta.

title: Molecular phylogeny and classification of the Mamiellophyceae class. nov. (Chlorophyta) based on sequence comparisons of the nuclear- and plastid-encoded rRNA operons.
abstract: Molecular phylogenetic analyses of the Mamiellophyceae classis nova, a ubiquitous group of largely picoplanktonic green algae comprising scaly and non-scaly prasinophyte unicells, were performed using single and concatenated gene sequence comparisons of the nuclear- and plastid-encoded rRNA operons. The study resolved all major clades within the class, identified molecular signature sequences for most clades through an exhaustive search for non-homoplasious synapomorphies [Marin et al. (2003): Protist 154: 99-145] and incorporated these signatures into the diagnoses of two novel orders, Monomastigales ord nov., Dolichomastigales ord. nov., and four novel families, Monomastigaceae fam. nov., Dolichomastigaceae fam. nov., Crustomastigaceae fam. nov., and Bathycoccaceae fam. nov., within a revised classification of the class. A database search for the presence of environmental rDNA sequences in the Monomastigales and Dolichomastigales identified an unexpectedly large genetic diversity of Monomastigales confined to freshwater, a novel clade (Dolicho_B) in the Dolichomastigaceae from deep sea sediments and a novel freshwater clade in the Crustomastigaceae. The Mamiellophyceae represent one of the ecologically most successful groups of eukaryotic, photosynthetic picoplankters in marine and likely also freshwater environments.

title: The chloroplast genomes of the green algae Pyramimonas, Monomastix, and Pycnococcus shed new light on the evolutionary history of prasinophytes and the origin of the secondary chloroplasts of euglenids.
abstract: Because they represent the earliest divergences of the Chlorophyta and include the smallest known eukaryotes (e.g., the coccoid Ostreococcus), the morphologically diverse unicellular green algae making up the Prasinophyceae are central to our understanding of the evolutionary patterns that accompanied the radiation of chlorophytes and the reduction of cell size in some lineages. Seven prasinophyte lineages, four of which exhibit a coccoid cell organization (no flagella nor scales), were uncovered from analysis of nuclear-encoded 18S rDNA data; however, their order of divergence remains unknown. In this study, the chloroplast genome sequences of the scaly quadriflagellate Pyramimonas parkeae (clade I), the coccoid Pycnococcus provasolii (clade V), and the scaly uniflagellate Monomastix (unknown affiliation) were determined, annotated, and compared with those previously reported for green algae/land plants, including two prasinophytes (Nephroselmis olivacea, clade III and Ostreococcus tauri, clade II). The chlorarachniophyte Bigelowiella natans and the euglenid Euglena gracilis, whose chloroplasts originate presumably from distinct green algal endosymbionts, were also included in our comparisons. The three newly sequenced prasinophyte genomes differ considerably from one another and from their homologs in overall structure, gene content, and gene order, with the 80,211-bp Pycnococcus and 114,528-bp Monomastix genomes (98 and 94 conserved genes, respectively) resembling the 71,666-bp Ostreococcus genome (88 genes) in featuring a significantly reduced gene content. The 101,605-bp Pyramimonas genome (110 genes) features two conserved genes (rpl22 and ycf65) and ancestral gene linkages previously unrecognized in chlorophytes as well as a DNA primase gene putatively acquired from a virus. The Pyramimonas and Euglena cpDNAs revealed uniquely shared derived gene clusters. Besides providing unequivocal evidence that the green algal ancestor of the euglenid chloroplasts belonged to the Pyramimonadales, phylogenetic analyses of concatenated chloroplast genes and proteins elucidated the position of Monomastix and showed that the Mamiellales, a clade comprising Ostreococcus and Monomastix, are sister to the Pyramimonadales + Euglena clade. Our results also revealed that major reduction in gene content and restructuring of the chloroplast genome occurred in conjunction with important changes in cell organization in at least two independent prasinophyte lineages, the Mamiellales and the Pycnococcaceae.

title: Phylogeny and Molecular Evolution of the Green Algae
abstract: The green lineage (Viridiplantae) comprises the green algae and their descendants the land plants, and is one of the major groups of oxygenic photosynthetic eukaryotes. Current hypotheses posit the early divergence of two discrete clades from an ancestral green flagellate. One clade, the Chlorophyta, comprises the early diverging prasinophytes, which gave rise to the core chlorophytes. The other clade, the Streptophyta, includes the charophyte green algae from which the land plants evolved. Multi-marker and genome scale phylogenetic studies have greatly improved our understanding of broad-scale relationships of the green lineage, yet many questions persist, including the branching orders of the prasinophyte lineages, the relationships among core chlorophyte clades (Chlorodendrophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae), and the relationships among the streptophytes. Current phylogenetic hypotheses provide an evolutionary framework for molecular evolutionary studies and comparative genomics. This review summarizes our current understanding of organelle genome evolution in the green algae, genomic insights into the ecology of oceanic picoplanktonic prasinophytes, molecular mechanisms underlying the evolution of complexity in volvocine green algae, and the evolution of genetic codes and the translational apparatus in green seaweeds. Finally, we discuss molecular evolution in the streptophyte lineage, emphasizing the genetic facilitation of land plant origins.

title: Chloroplast phylogenomic analysis resolves deep-level relationships within the green algal class Trebouxiophyceae
abstract: BackgroundThe green algae represent one of the most successful groups of photosynthetic eukaryotes, but compared to their land plant relatives, surprisingly little is known about their evolutionary history. This is in great part due to the difficulty of recognizing species diversity behind morphologically similar organisms. The Trebouxiophyceae is a species-rich class of the Chlorophyta that includes symbionts (e.g. lichenized algae) as well as free-living green algae. Members of this group display remarkable ecological variation, occurring in aquatic, terrestrial and aeroterrestrial environments. Because a reliable backbone phylogeny is essential to understand the evolutionary history of the Trebouxiophyceae, we sought to identify the relationships among the major trebouxiophycean lineages that have been previously recognized in nuclear-encoded 18S rRNA phylogenies. To this end, we used a chloroplast phylogenomic approach.ResultsWe determined the sequences of 29 chlorophyte chloroplast genomes and assembled amino acid and nucleotide data sets derived from 79 chloroplast genes of 61 chlorophytes, including 35 trebouxiophyceans. The amino acid- and nucleotide-based phylogenies inferred using maximum likelihood and Bayesian methods and various models of sequence evolution revealed essentially the same relationships for the trebouxiophyceans. Two major groups were identified: a strongly supported clade of 29 taxa (core trebouxiophyceans) that is sister to the Chlorophyceae + Ulvophyceae and a clade comprising the Chlorellales and Pedinophyceae that represents a basal divergence relative to the former group. The core trebouxiophyceans form a grade of strongly supported clades that include a novel lineage represented by the desert crust alga Pleurastrosarcina brevispinosa. The assemblage composed of the Oocystis and Geminella clades is the deepest divergence of the core trebouxiophyceans. Like most of the chlorellaleans, early-diverging core trebouxiophyceans are predominantly planktonic species, whereas core trebouxiophyceans occupying more derived lineages are mostly terrestrial or aeroterrestrial algae.ConclusionsOur phylogenomic study provides a solid foundation for addressing fundamental questions related to the biology and ecology of the Trebouxiophyceae. The inferred trees reveal that this class is not monophyletic; they offer new insights not only into the internal structure of the class but also into the lifestyle of its founding members and subsequent adaptations to changing environments.

title: Ultrastructure of Verdigellas peltata (Palmellaceae, Chlorophyta), a deep-water, palmelloid alga with ferritin and trilaminar sheaths
abstract: The palmelloid green alga Verdigellas peltata D.L. Ballantine et J.N. Norris was collected from depths of 70 to 110 meters in the Bahamas and studied by light microscopy and transmission electron microscopy. Most of the cell was filled by a cup-shaped chloroplast that contained scattered starch grains but no pyrenoid. Some chloroplasts had clusters of granular inclusions that were identified as ferritin based on their particle size, native electron density, and iron content; the last was detected by energy-dispersive X-ray microanalysis. A nucleus, dictyosome, mitochondrion, and large vacuole occupied a pocket mostly surrounded by the chloroplast. Centrioles and basal bodies were absent. The nuclear envelope was continuous with the endoplasmic reticulum, and some cells had one or two spherical, glycoprotein inclusions, c. 1–2 μm in diameter, within the confluent lumen. The Golgi vesicles contributed fibrillar material to the vacuole, but most of the vacuolar contents consisted of appressed sheets...

title: Palmophyllum umbracola sp. nov. (Chlorophyta) from offshore islands of northern New Zealand
abstract: A new green palmelloid alga, Palmophyllum umbracola sp. nov., is described from material collected from the Kermadec and Poor Knights Islands off northern New Zealand. The vivid green, prostrate thalli consist of unconnected, individual cells (6–7 μm diam.) embedded in a firm mucilaginous matrix. Each cell has a single cup-shaped chloroplast with embedded starch grains but without a pyrenoid or eyespot. A single, large organelle, which may be associated with mucilage secretion, is always present at the open end of the cup-shaped chloroplast. The alga possesses the typical chlorophyte pigments chlorophylls a and b, carotene and xanthophylls as well as an unidentified orange pigment. The taxonomic position of Palmophyllum within the Chlorophyta remains uncertain, but provisional placement in the Chlorococcales is suggested.

title: Six newly sequenced chloroplast genomes from prasinophyte green algae provide insights into the relationships among prasinophyte lineages and the diversity of streamlined genome architecture in picoplanktonic species
abstract: BackgroundBecause they represent the earliest divergences of the Chlorophyta, the morphologically diverse unicellular green algae making up the prasinophytes hold the key to understanding the nature of the first viridiplants and the evolutionary patterns that accompanied the radiation of chlorophytes. Nuclear-encoded 18S rDNA phylogenies unveiled nine prasinophyte clades (clades I through IX) but their branching order is still uncertain. We present here the newly sequenced chloroplast genomes of Nephroselmis astigmatica (clade III) and of five picoplanktonic species from clade VI (Prasinococcus sp. CCMP 1194, Prasinophyceae sp. MBIC 106222 and Prasinoderma coloniale) and clade VII (Picocystis salinarum and Prasinophyceae sp. CCMP 1205). These chloroplast DNAs (cpDNAs) were compared with those of the six previously sampled prasinophytes (clades I, II, III and V) in order to gain information both on the relationships among prasinophyte lineages and on chloroplast genome evolution.ResultsVarying from 64.3 to 85.6 kb in size and encoding 100 to 115 conserved genes, the cpDNAs of the newly investigated picoplanktonic species are substantially smaller than those observed for larger-size prasinophytes, are economically packed and contain a reduced gene content. Although the Nephroselmis and Picocystis cpDNAs feature a large inverted repeat encoding the rRNA operon, gene partitioning among the single copy regions is remarkably different. Unexpectedly, we found that all three species from clade VI (Prasinococcales) harbor chloroplast genes not previously documented for chlorophytes (ndhJ, rbcR, rpl21, rps15, rps16 and ycf66) and that Picocystis contains a trans-spliced group II intron. The phylogenies inferred from cpDNA-encoded proteins are essentially congruent with 18S rDNA trees, resolving with robust support all six examined prasinophyte lineages, with the exception of the Pycnococcaceae.ConclusionsOur results underscore the high variability in genome architecture among prasinophyte lineages, highlighting the strong pressure to maintain a small and compact chloroplast genome in picoplanktonic species. The unique set of six chloroplast genes found in the Prasinococcales supports the ancestral status of this lineage within the prasinophytes. The widely diverging traits uncovered for the clade-VII members (Picocystis and Prasinophyceae sp. CCMP 1205) are consistent with their resolution as separate lineages in the chloroplast phylogeny.

title: A clade uniting the green algae Mesostigma viride and Chlorokybus atmophyticus represents the deepest branch of the Streptophyta in chloroplast genome-based phylogenies
abstract: BackgroundThe Viridiplantae comprise two major phyla: the Streptophyta, containing the charophycean green algae and all land plants, and the Chlorophyta, containing the remaining green algae. Despite recent progress in unravelling phylogenetic relationships among major green plant lineages, problematic nodes still remain in the green tree of life. One of the major issues concerns the scaly biflagellate Mesostigma viride, which is either regarded as representing the earliest divergence of the Streptophyta or a separate lineage that diverged before the Chlorophyta and Streptophyta. Phylogenies based on chloroplast and mitochondrial genomes support the latter view. Because some green plant lineages are not represented in these phylogenies, sparse taxon sampling has been suspected to yield misleading topologies. Here, we describe the complete chloroplast DNA (cpDNA) sequence of the early-diverging charophycean alga Chlorokybus atmophyticus and present chloroplast genome-based phylogenies with an expanded taxon sampling.ResultsThe 152,254 bp Chlorokybus cpDNA closely resembles its Mesostigma homologue at the gene content and gene order levels. Using various methods of phylogenetic inference, we analyzed amino acid and nucleotide data sets that were derived from 45 protein-coding genes common to the cpDNAs of 37 green algal/land plant taxa and eight non-green algae. Unexpectedly, all best trees recovered a robust clade uniting Chlorokybus and Mesostigma. In protein trees, this clade was sister to all streptophytes and chlorophytes and this placement received moderate support. In contrast, gene trees provided unequivocal support to the notion that the Mesostigma + Chlorokybus clade represents the earliest-diverging branch of the Streptophyta. Independent analyses of structural data (gene content and/or gene order) and of subsets of amino acid data progressively enriched in slow-evolving sites led us to conclude that the latter topology reflects the true organismal relationships.ConclusionIn disclosing a sister relationship between the Mesostigmatales and Chlorokybales, our study resolves the long-standing debate about the nature of the unicellular flagellated ancestors of land plants and alters significantly our concepts regarding the evolution of streptophyte algae. Moreover, in predicting a richer chloroplast gene repertoire than previously inferred for the common ancestor of all streptophytes, our study has contributed to a better understanding of chloroplast genome evolution in the Viridiplantae.

title: New phylogenetic hypotheses for the core Chlorophyta based on chloroplast sequence data
abstract: Phylogenetic relationships in the green algal phylum Chlorophyta have long been subject to debate, especially at higher taxonomic ranks (order, class). The relationships among three traditionally defined and well-studied classes, Chlorophyceae, Trebouxiophyceae, and Ulvophyceae are of particular interest, as these groups are species-rich and ecologically important worldwide. Different phylogenetic hypotheses have been proposed over the past two decades and the monophyly of the individual classes has been disputed on occasion. Our study seeks to test these hypotheses by combining high throughput sequencing data from the chloroplast genome with increased taxon sampling. Our results suggest that while many of the deep relationships are still problematic to resolve, the classes Trebouxiophyceae and Ulvophyceae are likely not monophyletic as currently defined. Our results also support relationships among several trebouxiophycean taxa that were previously unresolved. Finally, we propose that the common term for the grouping of the three classes, “UTC clade,” be replaced with the term “core Chlorophyta” for the well-supported clade containing Chlorophyceae, taxa belonging to Ulvophyceae and Trebouxiophyceae, and the classes Chlorodendrophyceae and Pedinophyceae.

title: The chloroplast genomes of Bryopsis plumosa and Tydemania expeditiones (Bryopsidales, Chlorophyta): compact genomes and genes of bacterial origin
abstract: BackgroundSpecies of Bryopsidales form ecologically important components of seaweed communities worldwide. These siphonous macroalgae are composed of a single giant tubular cell containing millions of nuclei and chloroplasts, and harbor diverse bacterial communities. Little is known about the diversity of chloroplast genomes (cpDNAs) in this group, and about the possible consequences of intracellular bacteria on genome composition of the host. We present the complete cpDNAs of Bryopsis plumosa and Tydemania expeditiones, as well as a re-annotated cpDNA of B. hypnoides, which was shown to contain a higher number of genes than originally published. Chloroplast genomic data were also used to evaluate phylogenetic hypotheses in the Chlorophyta, such as monophyly of the Ulvophyceae (the class in which the order Bryopsidales is currently classified).ResultsBoth DNAs are circular and lack a large inverted repeat. The cpDNA of B. plumosa is 106,859 bp long and contains 115 unique genes. A 13 kb region was identified with several freestanding open reading frames (ORFs) of putative bacterial origin, including a large ORF (>8 kb) closely related to bacterial rhs-family genes. The cpDNA of T. expeditiones is 105,200 bp long and contains 125 unique genes. As in B. plumosa, several regions were identified with ORFs of possible bacterial origin, including genes involved in mobile functions (transposases, integrases, phage/plasmid DNA primases), and ORFs showing close similarity with bacterial DNA methyltransferases. The cpDNA of B. hypnoides differs from that of B. plumosa mainly in the presence of long intergenic spacers, and a large tRNA region. Chloroplast phylogenomic analyses were largely inconclusive with respect to monophyly of the Ulvophyceae, and the relationship of the Bryopsidales within the Chlorophyta.ConclusionsThe cpDNAs of B. plumosa and T. expeditiones are amongst the smallest and most gene dense chloroplast genomes in the core Chlorophyta. The presence of bacterial genes, including genes typically found in mobile elements, suggest that these have been acquired through horizontal gene transfer, which may have been facilitated by the occurrence of obligate intracellular bacteria in these siphonous algae.

title: The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: insights into the architecture of ancestral chloroplast genomes.
abstract: Green plants seem to form two sister lineages: Chlorophyta, comprising the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae, and Streptophyta, comprising the Charophyceae and land plants. We have determined the complete chloroplast DNA (cpDNA) sequence (200,799 bp) of Nephroselmis olivacea, a member of the class (Prasinophyceae) thought to include descendants of the earliest-diverging green algae. The 127 genes identified in this genome represent the largest gene repertoire among the green algal and land plant cpDNAs completely sequenced to date. Of the Nephroselmis genes, 2 (ycf81 and ftsI, a gene involved in peptidoglycan synthesis) have not been identified in any previously investigated cpDNA; 5 genes [ftsW, rnE, ycf62, rnpB, and trnS(cga)] have been found only in cpDNAs of nongreen algae; and 10 others (ndh genes) have been described only in land plant cpDNAs. Nephroselmis and land plant cpDNAs share the same quadripartite structure-which is characterized by the presence of a large rRNA-encoding inverted repeat and two unequal single-copy regions-and very similar sets of genes in corresponding genomic regions. Given that our phylogenetic analyses place Nephroselmis within the Chlorophyta, these structural characteristics were most likely present in the cpDNA of the common ancestor of chlorophytes and streptophytes. Comparative analyses of chloroplast genomes indicate that the typical quadripartite architecture and gene-partitioning pattern of land plant cpDNAs are ancient features that may have been derived from the genome of the cyanobacterial progenitor of chloroplasts. Our phylogenetic data also offer insight into the chlorophyte ancestor of euglenophyte chloroplasts.

title: Analyses of Charophyte Chloroplast Genomes Help Characterize the Ancestral Chloroplast Genome of Land Plants
abstract: Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes.

title: Ancestral chloroplast genome in Mesostigma viride reveals an early branch of green plant evolution
abstract: Sequence comparisons suggest that all living green plants belong to one of two major phyla: Streptophyta (land plants and their closest green algal relatives, the charophytes); and Chlorophyta (the rest of green algae). Because no green algae are known that pre-date the Streptophyta/Chlorophyta split, and also because the earliest diverging green algae show considerable morphological variation, the nature of the unicellular flagellate ancestor of the two green plant phyla is unknown. Here we report that the flagellate Mesostigma viride belongs to the earliest diverging green plant lineage discovered to date. We have sequenced the entire chloroplast DNA (118,360 base pairs) of this green alga and have conducted phylogenetic analyses of sequences derived from this genome. Mesostigma represents a lineage that emerged before the divergence of the Streptophyta and Chlorophyta, a position that is supported by several features of its chloroplast DNA. The structure and gene organization of this genome indicate that chloroplast DNA architecture has been extremely well conserved in the line leading to land plants.

title: The complete chloroplast and mitochondrial DNA sequence of Ostreococcus tauri: organelle genomes of the smallest eukaryote are examples of compaction.
abstract: The complete nucleotide sequence of the mt (mitochondrial) and cp (chloroplast) genomes of the unicellular green alga Ostreococcus tauri has been determined. The mt genome assembles as a circle of 44,237 bp and contains 65 genes. With an overall average length of only 42 bp for the intergenic regions, this is the most gene-dense mt genome of all Chlorophyta. Furthermore, it is characterized by a unique segmental duplication, encompassing 22 genes and covering 44% of the genome. Such a duplication has not been observed before in green algae, although it is also present in the mt genomes of higher plants. The quadripartite cp genome forms a circle of 71,666 bp, containing 86 genes divided over a larger and a smaller single-copy region, separated by 2 inverted repeat sequences. Based on genome size and number of genes, the Ostreococcus cp genome is the smallest known among the green algae. Phylogenetic analyses based on a concatenated alignment of cp, mt, and nuclear genes confirm the position of O. tauri within the Prasinophyceae, an early branch of the Chlorophyta.

title: Prasinoderma coloniale gen. et sp. nov., a new pelagic coccoid prasinophyte from the western Pacific Ocean
abstract: The new genus Prasinoderma Hasegawa et Chihara and the new species P. coloniale Hasegawa et Chihara are described from open water of the western Pacific Ocean as the third coccoid prasinophyte of the marine picoplankton. Its main feature is the presence of one or more layers of thick cell wall without any appendages on the cell surface. Asexual reproduction is by bipartition and a new cell wall is formed around each of the two daughter cells. One daughter cell is released while the other retains the parent wall. This type or reproduction is also known in Prasinococcus Miyashita et Chihara, tentatively placed in the family Pycnococcaceae (Mamiellales, Prasinophyceae). During the present study, it was found that Pycnococcus provasolii Guillard reproduces by a type of asexual reproduction essentially similar to that of Prasinococcus and Prasinoderma. The circumscription of the family Pycnococcaceae is emended to adopt this characteristic method of reproduction. Thus, the family currently accommodate...

title: PRASINOCOCCUS CAPSULATUS GEN. ET SP. NOV., A NEW MARINE COCCOID PRASINOPHYTE
abstract: A new genus, Prasinococcus, and a new species, P. capsulatus, are described on the basis of specimen that appeared in enriched cultures inoculated from water samples in the western Pacific Ocean. Cells of this unicellular alga are spherical and surrounded with a large amount of gelatinous matrix. The pigment composition, which includes chlorophylls a and b, prasinoxanthin, Mg 2, 4-diviriylphaeoporphyrin a5 monomethyl ester (Mg 2, 4-D) and 5, 6-epoxy-3, 3′-dihydroxy-5, 6, 7′, 8′-tetrahydro-β-e-caroten-11′, 19-olide (uriolide), is close to that observed in the Mamiellales (Prasinophyceae). The cell is non-flagellate and has a firm cell wall, but no scales on the cell body. The pyrenoid has a characteristic structure in which a mitochondrial lobe and chloroplast outer membranes protrude into the pyrenoid matrix. The cell wall has a projecting appendage like a circle collar surrounded by distinctive holes penetrating the cell wall. Since the cell contains no 3-deoxy-manno-octulosonic acid (KDO), the wall is considered to have a different origin from the cell coverings which consist of fused scales in Tetraselmis species. It has a characteristic way of asexual reproduction in which, after cell division, one cell remains within the parent cell wall while the other is extruded. This suggests that this alga be placed tentatively in the Pycnococcaceae (Order Mamiellales) on the basis of the pigment composition and morphological characteristics, but the taxonomic position of coccoid prasinophytes may need to be reconsidered.

title: Prasinoderma singularis sp. nov. (Prasinophyceae, Chlorophyta), a solitary coccoid Prasinophyte from the South-East Pacific Ocean.
abstract: During the BIOSOPE cruise in the South-East Pacific Ocean in 2004, several unidentified strains of prasinophytes were isolated into culture. Of these, nine strains composed a group for which the partial 18S rRNA gene sequence was related to Prasinoderma coloniale. The ultrastructure, morphology, division process, pigment composition, genome size and molecular genetic phylogeny of these nine strains were investigated, using P. coloniale as a reference. The 18S rRNA gene sequence of P. singularis sp. nov. shares only 96.9% of identity with that of P. coloniale and contains a conserved insertion of 567bp length not recorded in P. coloniale. When compared to P. coloniale, P. singularis sp. nov. is morphologically characterized by the absence of colonies, smaller cells with a thinner cell wall, and a second cell type with a different cell covering.

title: AN UNRECOGNIZED ANCIENT LINEAGE OF GREEN PLANTS PERSISTS IN DEEP MARINE WATERS 1
abstract: We provide molecular phylogenetic evidence that the obscure genera Palmophyllum Kütz. and Verdigellas D. L. Ballant. et J. N. Norris form a distinct and early diverging lineage of green algae. These palmelloid seaweeds generally persist in deep waters, where grazing pressure and competition for space are reduced. Their distinctness warrants recognition as a new order, the Palmophyllales. Although phylogenetic analyses of both the 18S rRNA gene and two chloroplast genes (atpB and rbcL) are in agreement with a deep‐branching Palmophyllales, the genes are in conflict about its exact phylogenetic placement. Analysis of the nuclear ribosomal DNA allies the Palmophyllales with the prasinophyte genera Prasinococcus and Prasinoderma (Prasinococcales), while the plastid gene phylogeny placed Palmophyllum and Verdigellas as sister clade to all other Chlorophyta.

title: Division of Palmoclathrus stipitatus (Chlorophyta) vegetative cells
abstract: Division of vegetative cells in the morphologically complex palmelloid marine green alga Palmoclathrus stipitatus Womersley, as observed at the light microscope level, involves the division of a uninucleate parent cell into two progeny cells that are initially asymmetrical. This ‘asymmetric binary eleutheroschisis’ occurs primarily in the superficiallayer of thalli partially regenerated from cultured stipe segments. The asymmetry is associated with the segregation of a prominent membrane-bound body into only one of the two progeny. The parental cell wall is discarded and, apparently, eventually incorporated into the colonial matrix. This cell division pattern suggests that Palmoclathrus and the related Palmophyllum are bona fide marine representatives of the predominantly freshwater class Chlorophyceae sensu Mattox & Stewart, and are referrable to the order Chlorococcales; other possibilities, however, cannot be excluded.

title: PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating
abstract: A variety of probabilistic models describing the evolution of DNA or protein sequences have been proposed for phylogenetic reconstruction or for molecular dating. However, there still lacks a common implementation allowing one to freely combine these independent features, so as to test their ability to jointly improve phylogenetic and dating accuracy.



We propose a software package, PhyloBayes 3, which can be used for conducting Bayesian phylogenetic reconstruction and molecular dating analyses, using a large variety of amino acid replacement and nucleotide substitution models, including empirical mixtures or non-parametric models, as well as alternative clock relaxation processes.

title: Detecting and overcoming systematic errors in genome-scale phylogenies.
abstract: Genome-scale data sets result in an enhanced resolution of the phylogenetic inference by reducing stochastic errors. However, there is also an increase of systematic errors due to model violations, which can lead to erroneous phylogenies. Here, we explore the impact of systematic errors on the resolution of the eukaryotic phylogeny using a data set of 143 nuclear-encoded proteins from 37 species. The initial observation was that, despite the impressive amount of data, some branches had no significant statistical support. To demonstrate that this lack of resolution is due to a mutual annihilation of phylogenetic and nonphylogenetic signals, we created a series of data sets with slightly different taxon sampling. As expected, these data sets yielded strongly supported but mutually exclusive trees, thus confirming the presence of conflicting phylogenetic and nonphylogenetic signals in the original data set. To decide on the correct tree, we applied several methods expected to reduce the impact of some kinds of systematic error. Briefly, we show that (i) removing fast-evolving positions, (ii) recoding amino acids into functional categories, and (iii) using a site-heterogeneous mixture model (CAT) are three effective means of increasing the ratio of phylogenetic to nonphylogenetic signal. Finally, our results allow us to formulate guidelines for detecting and overcoming phylogenetic artefacts in genome-scale phylogenetic analyses.