The Chloroplast Genomes of the Green Algae Pyramimonas ...

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 

Research Article 

MBE Advance Access published December 12, 2008 

Monique Turmel,* Marie-Christine Gagnon,* Charley J. O’Kelly,† Christian 

Otis,* and Claude Lemieux* 

*Département de biochimie et de microbiologie, Université Laval, Québec 

(Québec) Canada; and †Botany Department, University of Hawaii, Honolulu 

Running Head: Analysis of 3 Prasinophyte Chloroplast Genomes 

Key Words: prasinophyte green algae, euglenids, chloroplast genome evolution, 

phylogenomics, secondary endosymbiosis, genome reduction, 

horizontal DNA transfers 

Abbreviations: cpDNA – chloroplast DNA, IR – inverted repeat, LSC – large 

single copy region, LSU – large subunit, ML – maximum 

likelihood, ORF – open reading frame, PCR – Polymerase chain 

reaction SC – single copy region, SSC – small single copy region, 

SSU – small subunit 

Corresponding Author: Monique Turmel, Département de biochimie et microbiologie, 

Université Laval, 1030 avenue de la Médecine, Québec (Québec) 

Canada G1V 0A6. 

Tel: (418) 656-2131 ext. 7623; 

FAX: (418) 656-7176; 

Email: monique.turmel@rsvs.ulaval.ca 

© The Author 2008. Published by Oxford University Press on behalf of the Society for Molecular Biology 

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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, 4 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 pravosoli (clade V) 

and the scaly uniflagellate Monomastix (unknown affiliation) were determined, annotated and 

compared to those previously reported for green algae/land plants, including 2 prasinophytes 

(Nephroselmis olivacea, clade III and Ostreococcus tauri, clade II). The chlororachniophyte 

Bigelowiella natans and the euglenid Euglena gracilis, whose chloroplasts originate presumably 

from distinct green algal endosymbionts, were also included in our comparisons. The 3 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 2 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 

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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 2 independent prasinophyte lineages, the Mamiellales and 

the Pycnococcaceae. 

Introduction 

The green plants (Viridiplantae) are divided among two major lineages: the Chlorophyta, 

containing the bulk of the extant green algae, and the Streptophyta, containing the green algae 

belonging to the Charophyceae sensu Mattox and Stewart (1984) and all land plants (Lewis and 

McCourt 2004). It is thought that the first green plants were unicellular green algae bearing 

nonmineralized organic scales on their cell body and/or their flagella (Mattox and Stewart 1984). 

This hypothesis was put forward when it was recognized that flagellated reproductive cells 

(zoospores, gametes) of some taxa in both the Chlorophyta and Streptophyta are covered by a 

layer of square-shaped scales, which also occur as an underlayer in many prasinophytes. Free- 

living scaly flagellates have been ascribed mainly to the Prasinophyceae, a nonmonophyletic 

class representing the earliest divergences of the Chlorophyta (Steinkotter et al. 1994; Nakayama 

et al. 1998; Fawley et al. 2000; Guillou et al. 2004; Proschold and Leliaert 2007). This 

morphologically heterogeneous assemblage of green algae gave rise to the 3 advanced classes 

designated as the Trebouxiophyceae, Ulvophyceae and Chlorophyceae (Lewis and McCourt 

2004). Note that the scaly biflagellate Mesostigma viride, traditionally classified within the 

Prasinophyceae, has been formally excluded from this class and placed in the Streptophyta 

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(Marin and Melkonian 1999; Lemieux et al. 2007; Rodriguez-Ezpeleta et al. 2007). 

Prasinophytes have always fascinated the phycologists because their studies have the potential to 

shed light on the nature of the last common ancestor of all green plants and on the origin of the 

advanced chlorophytes. 

The concept of the class Prasinophyceae has been under constant revision since its formal 

description by Moestrup and Throndsen (1988) (Sym and Pienaar 1993); in the last few years, it 

has profoundly changed with the description of several new taxa and the analysis of 

environmental sequences. Most prasinophytes are found in marine habitats and considerable 

diversity is observed with respect to cell shape and size, flagella number and behavior, mitotic 

and cytokinetic mechanisms, and biochemical features such as accessory photosynthetic 

pigments and storage products (Melkonian 1990; O'Kelly 1992; Sym and Pienaar 1993; Latasa et 

al. 2004). Some species lack flagella, others lack scales, and in some cases, both flagella and 

scales are absent (e.g. Ostreococccus tauri). The small-sized members of the Prasinophyceae, 

particularly those belonging to 3 genera of the Mamiellales (Micromonas, Bathycoccus, and 

Ostreococcus), are prominent in the oceanic picoplankton (comprising organisms less than 3 µm 

in diameter) (Guillou et al. 2004). Included in this category is the smallest-free living eukaryote 

known to date, Ostreococcus tauri (Courties et al. 1994). Phylogenetic studies using molecular 

data, in particular the nuclear-encoded small subunit (SSU) rRNA gene, identified 7 

monophyletic groups of prasinophytes at the base of the Chlorophyta (Steinkotter et al. 1994; 

Nakayama et al. 1998; Fawley et al. 2000; Guillou et al. 2004); however, their order of 

divergence could not be resolved. Despite this uncertainty, it appears that the coccoid form 

evolved more than once in the Prasinophyceae (Fawley et al. 2000; Guillou et al. 2004). Coccoid 

cells are distributed among 4 lineages (clade II, Mamiellales; clade V, Pseudocourfieldiales, 

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Pycnococcaceae; clade VI, Prasinococcales; and clade VII, no order assigned to this clade), two 

of which (clades II and V) exhibit both the coccoid and flagellated cell organizations. 

Comparative analysis of chloroplast genomes has been helpful to resolve problematic 

relationships among green algae and land plants (Wolf et al. 2005; Qiu et al. 2006; Jansen et al. 

2007; Lemieux et al. 2007; Turmel et al. 2008) although the phylogenetic positions of some 

green plant lineages have remained contentious (Pombert et al. 2005; Turmel et al. 2006; 

Lemieux et al. 2007). The only 2 complete chloroplast DNA (cpDNA) sequences currently 

available for prasinophytes, those of the scaly biflagellate Nephroselmis olivacea (clade III, 

Pseudocourfieldiales, Nephroselmidaceae) (Turmel et al. 1999b) and of the tiny coccoid 

Ostreococcus tauri (clade II, Mamiellales) (Robbens et al. 2007), have revealed contrasting 

evolutionary patterns which can be designated as ancestral and reduced-derived, respectively. 

Whereas the 200.8-kb Nephroselmis genome harbors the largest gene repertoire yet reported for 

a chlorophyte (128 different conserved genes compared to about 138 genes for the deepest 

branching streptophyte algae) and has retained many ancestral gene clusters, the nearly 3-fold 

smaller Ostreococcus genome, which is the most compact chlorophyte cpDNA known to date, 

displays a reduced set of 88 genes whose order is highly scrambled. As in most other chloroplast 

genomes, 2 identical copies of a large inverted repeat (IR) are separated by single-copy (SC) 

regions; however, the 2 prasinophyte genomes differ remarkably in their quadripartite 

architectures. The Nephroselmis architectural design closely resembles that found in all 

streptophyte IR-containing cpDNAs: the SC regions are vastly unequal in size, each SC region is 

characterized by a highly conserved set of genes, and the rRNA operon encoded by the IR is 

transcribed toward the small SC (SSC) region. In Ostreococcus, the SC regions have essentially 

the same number of genes; the few genes (just 5) that would be expected to map to the SSC 

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egion in streptophyte cpDNAs are confined to the same SC region, and the rRNA operon is 

transcribed away from the latter SC region (see supplementary fig. 1, Supplementary Material). 

This gene partitioning pattern is reminiscent of that reported for the cpDNAs of the ulvophytes 

Pseudendoclonium akinetum and Oltmannsiellopsis viridis (Pombert et al. 2005; Pombert et al. 

2006). 

To explore the relationships among prasinophyte lineages and to better understand the 

mode of cpDNA evolution in the Prasinophyceae, we sequenced the cpDNAs of the scaly 

quadriflagellate Pyramimonas parkeae (clade I, Pyramimonadales), the coccoid Pycnococcus 

pravosoli (clade V, Pseudocourfieldiales, Pycnococcaceae), and the scaly uniflagellate 

Monomastix (unknown affiliation) and compared these genomes to those previously reported for 

Nephroselmis (Turmel et al. 1999b), Ostreococcus (Robbens et al. 2007), other chlorophytes 

(Wakasugi et al. 1997; Maul et al. 2002; Pombert et al. 2005; Bélanger et al. 2006; de Cambiaire 

et al. 2006; Pombert et al. 2006; de Cambiaire et al. 2007; Brouard et al. 2008), the deep- 

branching streptophytes Mesostigma (Lemieux et al. 2000) and Chlorokybus atmophyticus 

(Lemieux et al. 2007), the euglenid Euglena gracilis (Hallick et al. 1993) and the 

chlororachniophyte Bigelowiella natans (Rogers et al. 2007). The latter photosynthetic 

eukaryotes, which presumably gained their chloroplasts via independent secondary 

endosymbiotic events (Rogers et al. 2007), were included in our comparisons in an attempt to 

gain more detailed information about the green algal donors of their chloroplasts. We found that 

the 3 newly sequenced prasinophyte genomes differ considerably from one another and from 

their previously sequenced homologs at the overall structure, gene content and gene order levels, 

with both the Monomastix and Pycnococcus genomes featuring a reduced pattern of evolution. 

Our phylogenetic analyses of sequence data offered significant insights into the phylogeny and 

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evolution of prasinophytes and provided unequivocal evidence that the euglenid chloroplasts 

were secondarily acquired from a member of the Pyramimonadales. 

Materials and Methods 

Strains and Culture Conditions 

Pyramimonas parkeae (CCMP 726) and Pycnococcus provasolii (CCMP 1203), two 

marine species, were obtained from the Provasoli-Guillard National Center for Culture of Marine 

Phytoplankton (West Boothbay Harbor, Maine) and grown in K medium (Keller et al. 1987) 

under 12 h light/dark cycles. Monomastix sp., a freshwater strain originally collected by H. R. 

Preisig in New Zealand, originates from the personal collection of CJO. This strain, which is 

available upon request to MT, was grown in modified Volvox medium (McCracken et al. 1980) 

under 12 h light/dark cycles. 

Cloning and Sequencing of Chloroplast Genomes 

The complete cpDNA sequences of Pyramimonas, Monomastix and Pycnococcus were 

generated essentially as described previously (Turmel et al. 2005). For each green alga, A + T- 

rich organelle DNA was separated from nuclear DNA by CsCl-bisbenzimide isopycnic 

centrifugation of total cellular DNA (Turmel et al. 1999a). The organelle DNA fraction was 

sheared by nebulization to produce 1500 to 3000-bp fragments that were subsequently cloned 

into a plasmid vector, either pBluescrit II KS+ or pSMART-HCKan (Lucigen Corporation, 

Middleton, WI). After hybridization of the resulting clones with the original DNA used for 

cloning, plasmids from positive clones were purified with the QIAprep 96 Miniprep kit (Qiagen 

Inc., Mississauga, Canada) and sequenced using universal primers. DNA assembly was carried 

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out using AUTOASSEMBLER 2.1.1 (Applied BioSystems, Foster City, CA) or SEQUENCHER 

4.2 (Gene Codes Corporation, Ann Arbor, MI). Distinct contigs of cpDNA origin were ordered 

by polymerase chain reaction (PCR) amplification with primers specific to contig ends. The 

amplified fragments encompassing uncloned regions were sequenced on both strands. 

Chloroplast Genome Analyses 

Genes and all open reading frames (ORFs) larger than 100 codons were identified as 

described previously (Turmel et al. 2006). Secondary structures of group I and group II introns 

were modeled according to Michel et al. (1989) and Michel and Westhof (1990), respectively. 

Short repeats in the Monomastix genome were identified using REPuter 2.74 (Kurtz et al. 2001) 

and the number of copies of each repeat was determined with FINDPATTERNS of the GCG 

package (Accelrys, San Diego, CA). For all 3 newly sequenced prasinophyte genomes, regions 

containing nonoverlapping repeated elements were mapped with RepeatMasker 

(http://www.repeatmasker.org/) running under the WU-BLAST 2.0 search engine 

(http://blast.wustl.edu/), using the repeats ≥ 30 bp identified with REPuter as input sequences. 

Conserved gene clusters exhibiting identical gene polarities in selected green algal cpDNAs were 

identified using a custom-built program. 

Sequencing of the Monomastix 18S rRNA Gene and Phylogenetic Analysis 

The nuclear-encoded SSU rRNA gene was amplified from total cellular DNA by PCR 

using the specific primers NS1 (White et al. 1990) and 18L (Hamby and Zimmer 1991). The 

resulting PCR product was purified and sequenced directly using these primers and two internal 

primers. The Monomastix nuclear-encoded SSU rDNA sequence was aligned manually against 

8 




the alignment prepared by Guillou et al. (2004) from 83 chlorophytes and 12 streptophytes. A 

data set of 1663 positions was obtained after removing ambiguously aligned regions using 

GBLOCKS 0.91b (Castresana 2000) and the same filtration parameters employed by Guillou et 

al. (2004). Maximum likelihood (ML) trees were inferred using Treefinder (version of April 

2008) (Jobb et al. 2004) with the best model fitting the data [TN + I (proportion of invariable 

sites) + Γ (4 discrete rate categories)] under the Akaike information criterion. Bootstrap values 

were calculated for 100 replications. 

Phylogenetic Inferences from Whole Genome Sequence Data 

An amino acid data set and the corresponding nucleotide data set with first and second 

codon positions were derived from the completely sequenced cpDNAs of Bigelowiella 

(NC_008408), Euglena (NC_001603), and 22 green plants [species names and accession 

numbers, except those for Oedogonium cardiacum (NC_011031) and Leptosira terrestris 

(NC_009681), are provided in table 3 of Lemieux et al. (2007)]. These data sets were allowed to 

contain missing data; however, limitations were imposed to the proportion of missing data by 

selecting for analysis the protein-coding genes that are shared by at least 14 taxa. Seventy genes 

met this criterion: atpA, B, E, F, H, I, ccsA, cemA, chlB, I, L, N, clpP, ftsH, infA, petA, B, D, G, L, 

psaA, B, C, I, J, M, psbA, B, C, D, E, F, H, I, J, K, L, M, N, T, Z, rbcL, rpl2, 5, 14, 16, 20, 23, 32, 

36, rpoA, B, C1, C2, rps2, 3, 4, 7, 8, 9, 11, 12, 14, 18, 19, tufA, ycf1, 3, 4, 12. The amino acid 

data set was prepared as follows. The deduced amino acid sequences from the 70 individual 

genes were aligned using MUSCLE 3.7 (Edgar 2004), the ambiguously aligned regions in each 

alignment were removed using GBLOCKS 0.91b (Castresana 2000) with the –b2 option 

(minimal number of sequences for a flank position) set to 13, and the protein alignments were 

9 




concatenated. To obtain the nucleotide data set, the multiple sequence alignment of each protein 

was converted into a codon alignment, the poorly aligned and divergent regions in each codon 

alignment were excluded using GBLOCKS 0.91b with the options –b2=13 and –t=c (the latter 

specifying that selected sequences are complete codons), the individual codon alignments were 

concatenated, and finally third codon positions were excluded with PAUP* 4.0b10 (Swofford 

2003). Missing characters represented 5.9% and 5.8% of the amino acid and nucleotide data sets, 

respectively. 

Treefinder (version of April 2008) was used to perform the ML analyses and to identify the 

best model fitting the data under the Akaike information criterion. The amino acid data set was 

analyzed using the cpREV + F (observed amino acid frequencies) + Γ (5 categories) model of 

sequence evolution. Trees were inferred from the nucleotide data set using the GTR + Γ (5 

categories) model. Confidence of branch points was estimated by 500 bootstrap replications. 

The Bayesian inference method was conducted using MrBayes 3.1.2 (Ronquist and 

Huelsenbeck 2003). The model selected was cpREV + F + Γ for the inference from the amino 

acid data set and GTR + Γ for the inference of the nucleotide data set. Rates across sites were 

modeled on a discrete gamma distribution with 5 categories. Two independent Markov chain 

Monte Carlo runs, each consisting of 3 heated chains in addition to the cold chain, were carried 

out using the default parameters. For the analysis of the nucleotide data set, the length of each 

run was 3 million generations after a burn-in phase of 500,000 generations; for the amino acid 

data set, it was 1 million generations after a burn-in phase of 150,000 generations. Trees were 

sampled every 100 generations. Convergence of the 2 independent runs was verified according to 

the output of the ‘sump’ command; this output was also used to determine the burn-in phase. 

Posterior probability values were estimated from the trees sampled from both runs using the 

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‘sumt’ command. 

Reconstruction of Ancestral Character States 

A data set of gene content was prepared from the chloroplast genomes of the streptophytes 

Mesostigma and Chlorokybus, the prasinophytes, and Euglena by coding the presence and 

absence of genes as binary characters. Gene order in each of these chloroplast genomes was 

converted to all possible pairs of signed genes (i.e., taking into account gene polarity) and a gene 

order data set was obtained by coding as binary characters the presence/absence of the ancestral 

gene pairs conserved in at least one streptophyte and one prasinophyte. The gene content and 

gene order data sets were merged to produce a data set of combined ancestral characters. Losses 

of these characters on the best tree topology inferred from sequence data were mapped using 

MacClade 4.08 (Maddison and Maddison 2000). The most parsimonious reconstructions of 

ancestral character states were inferred under the Dollo principle of parsimony (Farris 1977). 

Results and Discussion 

Pyramimonas cpDNA Features an Ancestral Quadripartite Structure and a Large Repertoire of 

Genes 

Of the 3 newly sequenced prasinophyte genomes, only that of Pyramimonas displays a 

large IR (table 1). At 101,605 bp, this genome is 2-fold smaller than its Nephroselmis homolog, a 

size difference attributable to a much shorter IR, gene losses, and a more compact gene 

organization. As shown in fig. 1, the 2 copies of the IR sequence, each 13,057 bp in size and 

encoding 11 genes, are separated by SC regions of 10,338 and 65,153 bp comprising 12 and 76 

genes, respectively. On this figure are color-coded the genes whose orthologs are usually found 

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within the IR, the SSC and large SC (LSC) regions in streptophyte cpDNAs. It can be seen that 

the pattern of gene partitioning among the SC regions of the Pyramimonas genome closely 

resembles that observed for streptophytes. Considering that the Pyramimonas IR is about 2-fold 

larger and encodes additional genes relative to that of Mesostigma and that the IR is known to 

contract and expand through gene conversion events (Goulding et al. 1996), the observation that 

the termini of the Pyramimonas IR contain genes characteristic of the adjacent SC regions is not 

surprising. The most important deviation from the highly conserved partitioning pattern 

displayed by streptophytes concerns the locations of chlL and chlN. These 2 genes, which would 

be expected to be present in the SSC region, lie within the IR near the LSC region. 

The Pyramimonas chloroplast genome encodes 110 conserved genes, i.e. genes found in 

several other cpDNAs and usually present in cyanobacteria. The products of these genes consist 

of 81 proteins and 29 RNA species (2 rRNAs and 27 tRNAs) (table 2). The set of 27 tRNAs is 

sufficient to decode all 61 sense codons provided that the tRNA species encoded by trnV(uac), 

trnA(ugc), trnT(ugu), trnS(uga), trnL(uag) and trnP(ugg) recognize all 4 members of their 

respective codon family through superwobble pairing between the first position of the anticodon 

and the third position of the codon (Rogalski et al. 2008). The size of the Pyramimonas 

chloroplast gene complement closely matches those observed for the trebouxiophytes Chlorella 

vulgaris and Leptosira and for the ulvophytes Pseudendoclonium and Oltmansiellopsis (de 

Cambiaire et al. 2007). Although it is significantly reduced compared to its Nephroselmis 

counterpart (table 2), the set of Pyramimonas chloroplast genes includes 6 ndh genes (ndhA and 

ndhD through ndhH) typically present in streptophytes but previously found only in 

Nephroselmis in the Chlorophyta, as well as 2 protein-coding genes reported here for the first 

time in a chlorophyte chloroplast genome, rpl22 and ycf65 (supplementary table 1, 

12 




Supplementary Material). The ycf65 gene is present in both Mesostigma and Chlorokybus but 

missing in the other investigated streptophytes, whereas rpl22 shows a widespread distribution in 

the Streptophyta and also resides in the Euglena chloroplasts. Perhaps not surprisingly, most of 

the 22 chloroplast genes present in Nephroselmis but absent in Pyramimonas are also missing 

from some chlorophytes belonging to the Trebouxiophyceae, Ulvophyceae or Chlorophyceae 

(supplementary table 1, Supplementary Material). Only 5 genes (cemA, petD, petL, psbM, and 

rrf) represent exceptions and interestingly, all 5, except rrf (the 5S rRNA gene), are also lacking 

in the Ostreococcus and Euglena chloroplasts. The analysis of the nuclear genome from both 

Ostreococcus tauri and Ostreococcus lucimarinus revealed that cemA, petD and psbM have been 

transferred to the nucleus (Derelle et al. 2006; Palenik et al. 2007; Robbens et al. 2007). 

Considering that these genes are essential for chloroplast function, they are also likely to be 

nuclear-encoded in Pyramimonas. Since no case of chloroplast to nucleus transfer has been 

documented for rrf, the possibility exists that this conserved gene is present in Pyramimonas 

cpDNA and that its sequence has diverged beyond recognition. 

We found 2 large ORFs that are not associated with any introns, orf454 and orf510. For the 

orf510, present in the LSC region near the IR, our Blast searches against the nonredundant 

protein sequence database of the National Center for Biotechnology Information failed to 

identify any putative function for the potential encoded protein. However, the product of the 

orf454 localized in the IR revealed sequence similarity with the conserved domain of phage 

associated DNA primases (COG3378, E-value = 1e-06). Interestingly, in the course of the 

present study, we have found that the orf389 in the Nephroselmis IR (Turmel et al. 1999b) also 

encodes a putative protein with the conserved domain of phage associated DNA primases 

(COG3378, E-value = 2e-12). Given that viruses have been observed in Pyramimonas (Moestrup 

13 




and Thomsen 1974; Sandaa et al. 2001) and Nephroselmis (Nakayama et al. 2007), it is tempting 

to speculate that the abovementioned orf454 and orf389 originated from horizontal transfer of 

viral genes. There are only a few documented cases of non-standard, free-standing chloroplast 

genes that were acquired via horizontal gene transfer and all these cases involve genes that 

participate in DNA recombination or replication (Khan et al. 2007; Brouard et al. 2008; Cattolico 

et al. 2008). Like the orf454 and orf389, the 2 horizontally transferred genes identified in the 

chlorophycean green alga Oedogonium cardiacum are housed in the IR (Brouard et al. 2008). 

In general, the conserved genes present in Pyramimonas cpDNA are densely packed (table 

1). Prominent exceptions are those in the regions containing the orf454 and orf510 (fig. 1). There 

are 2 cases of overlapping genes (psbC-psbD and ndhC-ndhK); for the remaining genes, 

intergenic spacers vary between 3 and 2517 bp, with an average size of 159 bp. Consistent with 

this high degree of compaction, only a few short repeats, mostly direct repeats, were identified 

(table 2); they are found mainly in the large spacer adjacent to the orf501. 

Like its Ostreococcus and Pycnococcus homologs (see below), the Pyramimonas genome 

features a unique intron, a group II intron in atpB. However, the Pyramimonas atpB intron and 

those of Ostreococcus and Pycnococcus are inserted at different sites and carry distinct ORFs, 

indicating that they arose from separate events of horizontal DNA transfer. It should be pointed 

out here that the currently available chloroplast genome data strongly support the notion that no 

introns were present in the chloroplast of the common ancestor of all green plants (Turmel et al. 

1999b; Lemieux et al. 2000; Lemieux et al. 2007). The orf608 of the Pyramimonas group IIA 

intron is located within domain IV of the intron secondary structure and carries the reverse 

transcriptase (cd01651) and maturase (pfam01348) domains, but not the endonuclease domain, 

of reverse transcriptases encoded by group II introns. The endonuclease domain, which carries 

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out second-strand DNA cleavage during group II intron mobility (Lambowitz and Zimmerly 

2004), was most likely lost after the horizontal transfer of the intron in the Pyramimonas 

chloroplast. The orf608 product shares strong sequence similarity with reverse transcriptases 

encoded by the genomes of firmicute bacteria and by the mitochondrial cox1 genes of fungi, the 

brown alga Pylaiella littoralis, and the cryptophyte Rhodomonas salina. 

Like its Ostreococcus Homolog, Pycnococcus cpDNA has a Reduced Gene Content and is 

Highly Compact 

The Pycnococcus chloroplast genome is the smallest and most compact of the 3 

prasinophyte genomes sequenced during this study (table 1 and fig. 2). It is only 8.6 kb larger 

relative to Ostreococcus cpDNA and contains 10 additional conserved genes, for a total of 98 

genes. In term of size, this gene repertoire, which consists of 65 protein genes and 33 RNA genes 

encoding 2 rRNAs, 30 tRNAs and the RNA component of RNase P (table 2), is similar to that 

observed for chlorophycean green algae (Brouard et al. 2008). The tRNA complement includes 1 

tRNA species not previously documented in any chlorophytes [tRNA Pro (GGG)] but like its 

Ostreococcus homolog, lacks the tRNA species that reads the AUA codon [i.e. the tRNA Ile 

(CAU) where C is modified post-transcriptionally to lysidine]. As in Pyramimonas cpDNA, the 

5S rRNA gene was not detected. Moreover, the Pycnococcus genome is missing the protein- 

coding genes psaJ and rpoB, which are present in all other investigated chlorophytes. Although 

the Pycnococcus, Ostreococcus and Pyramimonas cpDNAs all show a reduced gene content 

compared to the Nephroselmis genome, their sets of genes show substantial differences (table 2). 

No vestigial IR region was identified in Pycnococcus cpDNA. The genes generally found 

in this region are dispersed throughout the genome; in contrast, several genes usually present 

15 




within the SSC region in genomes displaying an ancestral quadripartite structure [chlN, chlL, 

ycf1, cysT and trnP(ggg)] remained clustered together (supplementary fig. 2, Supplementary 

Material). There are 2 cases of overlapping genes (ycf4-rnpB and psbD-psbC); for the other 

coding regions, intergenic spacers were found to vary from 0 to 383 bp, for an average length of 

102 bp. 

The Pycnococcus atpB intron shares with its Ostreococcus counterpart the same insertion 

position and a large ORF in domain IV that features the reverse transcriptase (cd01651), 

maturase (pfam08388), and HNH endonuclease (cd00085) domains of reverse transcriptases 

encoded by group II introns. The Pycnococcus and Ostreococcus intron ORFs share strong 

similarity with one another and with reverse transcriptase genes found in several cyanobacterial 

species as well as in group II introns present in the mitochondrial large subunit (LSU) rRNA 

gene of the red alga Porphyra purpurea (Burger et al. 1999) and the chloroplast psbA genes of 

Chlamydomonas sp. CCMP 1619 (Odom et al. 2004) and Euglena myxocylindracea (Sheveleva 

and Hallick 2004). 

The Monomastix Chloroplast Genome has a Reduced Gene Content but is Loosely Packed with 

Genes 

Compared to its Pycnococcus homolog, the Monomastix chloroplast genome is 34 kb 

larger, has a deficit of four genes, and contains five additional introns (table 1, fig. 3 and 

supplementary fig. 3, Supplementary Material). Its increased size is largely accounted for by the 

expansion of intergenic spacers. The latter vary from 3 to 2566 bp, for an average size of 524 bp, 

and contain a myriad of short repeated sequences rich in G+C. The 94 conserved genes specify 

64 proteins and 30 RNAs (3 rRNAs, 26 tRNAs, and the RNA component of RNase P) (table 2). 

16 




The 26 tRNAs can decode all 61 sense codons assuming that tRNA Arg (ACG), where A is 

modified to inosine, recognizes all 4 codons of the CGX family. The reduced gene content of 

Monomastix is more like the gene complement of Ostreococcus than that of Pycnococcus (table 

2). It features 9 genes that are missing from Ostreococcus and lacks only 3 genes that are present 

in this alga, including psaC, a gene shared by the chloroplasts of all previously investigated 

chlorophytes. Although short dispersed repeats were mapped predominantly to intergenic 

regions, a small fraction was found within the coding regions of 5 genes (ftsH, rpoB, rpoC1, 

rpoC2 and ftsH) and within 2 introns (psbA intron and rrl intron 4) (supplementary fig. 4, 

Supplementary Material). This distribution pattern resembles those reported for other 

chlorophyte cpDNAs rich in short repeats (Maul et al. 2002; Pombert et al. 2005; Bélanger et al. 

2006; de Cambiaire et al. 2006; Pombert et al. 2006; de Cambiaire et al. 2007). Ranging from 19 

and 58 nucleotides, the most abundant short dispersed repeats of Monomastix were classified into 

4 families (A and A1, B and B1, C and D) according to their sequence motifs; moreover, some 

repeats displaying partial sequences characteristic of distinct families were discerned 

(supplementary fig. 5, Supplementary Material). The hybrid nature of the latter dispersed repeats, 

which were assigned to 6 categories (named AB, AC, AD, A1D, A1B and BD), suggests they 

arose through recombination between regions carrying different repeats. 

The Monomastix chloroplast genome contains a single group II intron, located in 

trnK(uuu), and 5 group I introns, 1 of which resides in psbA and 4 in the LSU rRNA gene (rrl) 

(fig. 1). The IIB trnK intron is inserted within the D arm of the tRNA secondary structure 

following G23 and lacks an ORF. All other trnK(uuu) introns that have been identified in 

streptophyte cpDNAs carry an internal ORF with a maturase domain (matK) and are inserted 

within the anticodon loop (Turmel et al. 2006). In view of their ability to encode an homing 

17 




endonuclease, the 5 Monomastix group I introns are likely to be mobile and were probably 

captured via horizontal intracellular and/or intercellular DNA transfer. The IA2 psbA intron, 

found at position 525 relative to the corresponding Mesostigma gene, specifies a potential 

homing endonuclease with the GIY-YIG motif and has chloroplast homologs with the same 

insertion site and highly similar endonuclease genes in the ulvophytes Oltmannsiellopsis and 

Pseudendoclonium and the chlorophycean green algae Oedogonium and Chlamydomonas 

reinhardtii (Brouard et al. 2008). The 4 remaining group I introns encode potential 

LAGLIDADG homing endonucleases (Côté et al. 1993; Lucas et al. 2001) and also share 

identical insertion sites with a large number of chlorophyte (Lucas et al. 2001; Brouard et al. 

2008) and cyanobacterial (Haugen et al. 2007) introns. The first and third LSU rDNA introns, 

whose insertion positions correspond to sites 1931 and 2500 in the E. coli 23S rRNA, fall within 

subgroup IB4, whereas the second and fourth introns inserted at sites 1951 and 2593 belong to 

the IA3 family. Like its Chlamydomonas homolog I-CreI, the Monomastix site-2593 intron- 

encoded homing endonuclease (I-MsoI) has been characterized at the 3-dimensional level in the 

presence of its DNA target site, revealing that the 2 isoschizomers display strikingly different 

protein/DNA contacts (Lucas et al. 2001; Chevalier et al. 2003). Interestingly, at sites 1931, 2500 

and 2593, the Monomastix mitochondrial LSU rRNA gene features introns with similar 

structures and ORFs as those found at identical sites in the chloroplast gene (Lucas et al. 2001) 

(unpublished data of MT, MCG, CO and CL), highlighting the possibility that mobile group I 

introns were exchanged between different organellar compartments in the Monomastix lineage. 

Evidence supporting such intracellular exchanges of group I introns has also been reported for 

the Nephroselmis (Turmel et al. 1999a) and Pseudendoclonium (Pombert et al. 2006) lineages. 

18 




Pyramimonas and Euglena cpDNAs Show Striking Similarities in Gene Order 

Gene orders in the three newly sequenced prasinophyte chloroplast genomes were 

compared with one another and with those in previously examined chlorophytes, the 

streptophytes Mesostigma and Chlorokybus, the euglenid Euglena and the chlorarachniophyte 

Bigelowiella. In all pairwise genome comparisons, except that including Pyramimonas and 

Euglena, the vast majority of the identified syntenic blocks were composed exclusively of gene 

clusters commonly found in streptophytes and chlorophytes. Ancestral clusters of this type 

display substantial variability among the Euglena and prasinophyte genomes (fig. 4). Clearly, the 

gene-rich genome of Nephroselmis exhibits the highest number of genes (94 genes) mapping to 

clusters predating the split of the Chlorophyta and Streptophyta. Breakpoints within ancestral 

clusters proved to be too variable in positions to determine which of the compared genomes are 

the most closely related. Note that our comparisons of the Pyramimonas genome with those of 

Mesostigma and Chlorokybus disclosed ancestral gene linkages that had not been reported in any 

chlorophyte cpDNA (e.g. psbH-petB-petD, R(ccg)-rbcL-atpB-atpE). The ancestral rps2-atpI 

linkage detected in the Euglena genome was also previously unrecognized in chlorophytes. 

Comparison of gene orders in the Pyramimonas and Euglena cpDNAs revealed striking 

similarities between these genomes. Almost 2-thirds of the 87 genes (56 genes) in Euglena 

cpDNA were found to be part of collinear regions, for a total of 16 syntenic blocks. Thirty-five 

of these genes form 8 blocks that exhibit gene linkages unique to Pyramimonas and Euglena (fig. 

5). Four blocks contain exclusively derived linkages, whereas the remaining 4 also include 

ancestral gene linkages present in chlorophytes and streptophytes (the rpl23, rpl32, rps12 and rrs 

clusters). It is interesting to note that in each of the latter 4 blocks, a pair of adjacent genes was 

cleanly excised from the Euglena genome following the formation of the derived linkages. The 

19 




syntenic block containing the triad psbK-ycf12-psaM is not uniquely shared by the Pyramimonas 

and Euglena chloroplasts. Being also present in Chlorella, Pseudendoclonium and 

Oltmansiellopsis but not in streptophytes, this derived cluster must have arisen in prasinophytes 

and have been transmitted by vertical descent to the trebouxiophyte and ulvophyte lineages. 

Monomastix Occupies an Early-Diverging Branch of the Mamiellales in 18S rDNA Trees 

Monomastix has been historically affiliated with the Prasinophyceae; however, the finding 

that its body scales are not typical of those found in prasinophytes but are more like those of the 

chrysophyte Chromulina placentula (Manton 1967) led to the exclusion of this genus from the 

Prasinophyceae (Melkonian 1990; Sym and Pienaar 1993). Very limited molecular information 

has been reported so far for Monomastix, explaining why its phylogenetic status has remained 

enigmatic. In the present study, we determined the sequence of the Monomastix nuclear-encoded 

SSU rRNA gene and compared it with those available for other prasinophytes and some 

representatives of the Trebouxiophyceae, Ulvophyceae and Chlorophyceae. Trees inferred with 

ML unambiguously showed that Monomastix represents an early-diverging lineage of the 

Mamiellales (clade II) (fig. 6). This uniflagellate, which has non-prasinophyte scales, was 

resolved as the first branch of this morphologically diverse clade. An unquestionable affinity 

therefore exists between Ostreococcus and Monomastix even though these 2 taxa belong to 

different lineages of the Mamiellales. The naked Ostreococcus is closely related to the scaly 

Bathycoccus and the clade uniting these nonflagellated genera is sister to that containing the 

flagellated genera Mamiella (2 flagella), Mantoniella (1 flagellum), Micromonas (naked, 1 

flagellum), and the new genus represented by isolate RCC 391 (2 flagella). 

20 




Chloroplast Phylogenomic Analyses Unite the Pyramimonadales with the Mamiellales and 

Identify the Pyramimonadales as the Source of the Euglenid Chloroplasts 

To explore the relationships among prasinophyte lineages (in particular clades I, II, III and 

V) as well as the relationships of chlorophyte chloroplasts with the secondarily acquired 

chloroplasts of Bigelowiella and Euglena, we generated data sets of 70 concatenated proteins and 

genes (first and second codon positions) from completely sequenced chloroplast genomes and 

analyzed them using the ML and Bayesian methods (fig. 7). As expected, both the protein and 

gene trees identified a strongly supported clade uniting the 2 representatives of the Mamiellales, 

Monomastix and Ostreococcus. This clade is sister to a robust monophyletic group clustering the 

Pyramimonas (scaly, 4 or 8 flagella) and Euglena chloroplasts. While this sister relationship 

received 87% bootstrap support in the protein ML tree (fig. 7A), exclusion of the long-branch 

taxa Euglena and Bigelowiella from the analysis resulted in 97% bootstrap support for the 

Pyramimonas + Monomastix + Ostreococcus clade (data not shown). In all analyses, the scaly 

biflagellate Nephroselmis was sister to all chlorophytes analyzed, whereas the position of the 

naked, nonflagellated Pycnococcus remained equivocal. The latter prasinophyte was resolved as 

sister to the core chlorophytes in the protein tree (fig. 7A), but was sister to the Mamiellales, 

Pyramimonadales and euglenids in the gene tree (fig. 7B). The protein and gene trees thus differ 

only in the branching position of the core chlorophytes with respect to the prasinophyte lineages. 

Because phylogenetic analyses based on the whole-genome approach are inherently 

associated with sparse taxon sampling, they can lead to trees robustly supporting an artifactual 

clustering of taxa (Brinkmann and Philippe 2008; Heath et al. 2008). Caution must therefore be 

exercised in the interpretation of the observed topologies. In the case of trees derived from 

complete genome sequences, structural features of these genomes can be used as independent 

21 




data to test topologies (Rokas 2006). In the present study, the strong alliance we uncovered 

between the Pyramimonas and Euglena chloroplasts is strengthened by a number of gene 

linkages that are unique to the cpDNAs of these algae (fig. 5). Based on this finding, we infer 

with confidence that the green algal partner in the secondary endosymbiosis that gave rise to 

euglenids was a member of the Pyramimonadales. Euglenids are unicellular organisms that 

belong to the Excavata, a supergroup of eukaryotes including diverse nonphotosynthetic groups 

like diplomonads, retortamonads, parabasalids, oxymonads and jakobids (Baldauf et al. 2000; 

Keeling et al. 2005; Baldauf 2008). Euglenids are the only photosynthetic excavates and they are 

specifically related to a subgroup containing the kinetoplastids and diplonemids (Triemer and 

Farmer 2007). Prior to our study, published data were consistent with the notion that the euglenid 

chloroplasts evolved from a green algal endosymbiont that was allied to prasinophytes (Ishida et 

al. 1997; Turmel et al. 1999b; Rogers et al. 2007); however, it remained unknown which of the 

monophyletic groups of prasinophytes harbored the closest relative of the euglenid 

endosymbiont. In agreement with our results, the ML tree that Ishida et al. (1997) inferred from 

the amino acid sequences of elongation factor Tu identified a strongly supported clade clustering 

Pyramimonas disomata and the euglenids Euglena gracilis and Astasia longa; however, this 

Pyramimonas species was the only prasinophyte sampled in this single-gene analysis. Likewise, 

considering that Pyramimonas parkeae is the unique representative of the Pyramimonadales in 

our chloroplast phylogenomic study, there remain uncertainties about the exact 

pyramimonadalean lineage that was the source of the euglenid chloroplasts. 

In the eukaryotic tree of life based on nuclear-encoded genes, euglenids and 

chlorarachniophytes fall within distinct branches. Like euglenids, chlorarachniophytes belong to 

a supergroup of eukaryotes that is primarily nonphotosynthetic, the Rhizaria (Keeling et al. 2005; 

22 




Baldauf 2008). By robustly placing Bigelowiella at a separate position from Euglena, our 

chloroplast phylogenomic analyses strongly reinforce the hypothesis that the euglenid and 

chlorarachniophyte chloroplasts trace back to 2 independent secondary endosymbioses (Rogers 

et al. 2007; Takahashi et al. 2007) (fig. 7). Although the chloroplast of Bigelowiella was found to 

be sister to those of the ulvophytes Pseudendoclonium and Oltmansiellopsis in both the protein 

and gene trees, broader sampling of core chlorophytes will be required to pinpoint the closest 

green algal relative of the chlorarachniophyte endosymbiont. 

The most unexpected finding that emerged from our study is the observation that the 

Pyramimonas + Euglena clade is sister to the Monomastix + Ostreococcus clade. While the 

existence of a sister relationship between the Pyramimonadales and Mamiellales has not been 

previously documented, it is compatible with the resemblance that these monophyletic groups 

display at the level of flagellar scale structure (Melkonian 1984; Melkonian 1990; O'Kelly 1992; 

Sym and Pienaar 1993) and with the branching order inferred from 18S rDNA data. Although the 

Pyramimonadales emerge just before the Mamiellales in most 18S rDNA trees (Steinkotter et al. 

1994; Nakayama et al. 1998; Fawley et al. 2000; Guillou et al. 2004), these lineages form a 

weakly supported clade in the ML tree recently reported by Nakayama et al. (2007). No 

similarities were found at the chloroplast gene order level that link the Pyramimonadales and 

Mamiellales to the exclusion of other chlorophyte groups; however, losses of at least 4 genes 

(cemA, cysT, petL and rpl19) could be traced back unambiguously to the common ancestor of the 

Pyramimonadales and Mamiellales (supplementary table 1, Supplementary Material). 

Because the Pyramimonadales and Mamiellales are distinguished by prominent 

morphological differences, the existence of a sister relationship between these lineages has 

important implications for the evolution of prasinophytes. All members of the Pyramimonadales, 

23 




which represent the 5 genera indicated in figure 6 and also probably the Tasmanites (a fossil 

resembling the phycoma stages of Cymbomonas, Pterosperma, and Halosphaera, which has 

been found in Precambrian deposits), share a number of synapomorphic characters and have at 

least 4 flagella and a complex scaly covering consisting of 3 layers of scales on the cell body and 

of 2 layers on the flagella (Melkonian 1984; Melkonian 1990; Sym and Pienaar 1993). The 

intermediate scale layer on the cell body consists of spiderweb-shaped scales in Pterosperma and 

is homologous to the outer scale layer on the flagellum (the limulus scales) and to the spiderweb 

scales of the Mamiellales. The limuloid scales of Cymbomonas are also reminiscent of the 

spiderweb scales of the Mamiellales, particularly during morphogenesis (Moestrup et al. 2003). 

Interestingly, an apparent food-uptake apparatus is present in Cymbomonas, which has been 

interpreted as a character inherited from a phagotrophic ancestor of the green plants and 

subsequently lost during evolution of the green algae (Moestrup et al. 2003). On the other hand, 

the members of the Mamiellales show reduced morphological complexity and are characterized 

by a progressive simplification of cellular structure and a reduction in cell size that occurred 

concomitantly with the loss of scales (Nakayama et al. 1998). They lack an underlayer of square- 

shaped scales (such scales are present in most other prasinophyte lineages and the flagellate 

reproductive cells of streptophytes) and no microtubular flagellar roots are attached to the basal 

body no. 2. A sister relationship between the Pyramimonadales and Mamiellales implies that 

some of the cellular features displayed by the Mamiellales were derived from the more complex 

organization seen in the Pyramimonadales and presumably in the common ancestor of all 

chlorophytes. In this context, it is worth mentioning that the nature of the progenitor of all green 

plants has generated intense debate and is still controversial (Melkonian 1984; O'Kelly 1992; 

24 




Sym and Pienaar 1993). A better understanding of the relationships among prasinophyte lineages 

will be required before one can infer with confidence evolutionary scenarios of cellular changes. 

At present, the identity of the earliest-diverging chlorophyte lineage remains uncertain. 

Intriguingly, the trees inferred from 18S rDNA sequences (Guillou et al. 2004; Nakayama et al. 

2007) are in discordance with the chloroplast phylogenomic trees reported in this study with 

regards to the position of the Nephroselmis genus (clade III). The early-diverging position 

observed for the Nephroselmis representative in chloroplast trees is in agreement with the high 

degree of ancestral features found in the cpDNA of this taxon (see fig. 8) but contrasts sharply 

with the much later divergence observed for the genus in 18S rDNA trees. In the latter trees, the 

branch occupied by Nephroselmis species emerges near the lineage containing Pycnococcus and 

Pseudocourfieldia marina, the clade VII containing only picoplanktonic species, and the clade 

containining the core chlorophytes (Chlorodendrales sensu Melkonian (1990) + 

Trebouxiophyceae + Ulvophyceae + Chlorophyceae). Together, these lineages form a large clade 

that is well supported in ML analysis (fig. 6). Given the close relationship observed on the basis 

of scale structure between Nephroselmis and the genera Tetraselmis and Scherffelia, Nakayama 

et al. (2007) proposed that the common ancestor of the clade containing Nephroselmis and the 

core chlorophytes had 2 layers of small scales on the flagella (squared-shaped scales and rod- 

shaped scales) and cell body (square scales and stellate scales). The abovementioned discrepancy 

between nuclear and chloroplast trees highlights the need for analysis of chloroplast genomes 

from additional prasinophytes. Sampling of chloroplast genomes from all 7 known lineages of 

prasinophytes will be required to determine the exact position of Nephroselmis relative to the 

Pycnococcaceae, Pyramimonadales and Mamiellales. 

25 




Losses of Multiple Ancestral cpDNA Characters in Independent Prasinophyte Lineages are 

Correlated with Major Cellular Remodeling 

To trace some of the evolutionary changes that occurred at the chloroplast genome level 

during the evolution of prasinophytes and euglenids, losses of 62 genes and 75 ancestral gene 

pairs were mapped on the tree topology inferred from sequence data (fig. 8). In this analysis, the 

core chlorophytes were excluded and the streptophytes Mesostigma and Chlorokybus were used 

as outgroup. Although multiple characters were lost in independent lineages, a substantial 

fraction of losses are uniquely shared. In particular, the monophyletic group containing the 

Mamiellales + euglenids + Pyramimonadales and the node linking the latter clade with the 

Pycnococcaceae are supported by several changes that occurred only once. Because the nuclear 

genome of just one prasinophyte genus (Ostreococcus) has been decrypted so far (Derelle et al. 

2006; Palenik et al. 2007), we cannot interpret our results in terms of gene transfers from the 

chloroplast to the nucleus. Most of the genes that vanished from the chloroplast genome 

probably fall into this category; however, some might have disappeared entirely from the cell 

because their requirement is restricted to certain growth and physiological conditions (e.g. the 

chl genes associated with chlorophyll synthesis in the dark, the cys genes involved in sulfate and 

thiosulfate transport, and the ndh genes associated with chlororespiration). 

The chloroplast genome sustained important reduction in gene content in at least 3 separate 

lineages, namely the lineages leading to Euglena, to the mamiellalean genera Monomastix and 

Ostreococcus, and to Pycnococcus (fig. 8). In light of the close affinity of the Pyramimonas and 

Euglena chloroplast genomes, we propose that the secondary endosymbiosis that gave rise to the 

euglenid chloroplasts was accompanied by extensive gene losses. Similar extinction of numerous 

chloroplast genes has been associated with the secondary endosymbiosis that involved the 

26 




capture of a red alga and generated the chloroplasts of heterokonts, cryptophytes and haptophytes 

(Khan et al. 2007; Oudot-Le Secq et al. 2007; Cattolico et al. 2008). With regards to the 

Mamiellales, it appears that the common ancestor of Monomastix and Ostreococcus had already 

experienced multiple chloroplast gene losses (fig. 8), implying that these events might have 

accompanied the simplification of cell organization that presumably coincided with the 

emergence of the Mamiellales. Moreover, as indicated by the higher frequency of genes losses in 

the Ostreococcus lineage compared to the Monomastix lineage, part of the gene losses in the 

former lineage were likely connected with the evolution of the coccoid cell organization and the 

reduction in cell size. Pycnococcus represents an independent coccoid lineage that sustained 

considerable reduction of the chloroplast genome, and as observed for Ostreoccocus, there was 

strong pressure to maintain a compact genome organization. In contrast, the genome of the 

mamiellalean Monomastix followed a divergent evolutionary pathway and became loosely 

packed with genes following proliferation of small dispersed repeats (table 1 and supplementary 

fig. 4, Supplementary Material). 

The pressure to maintain the ancestral quadripartite architecture became relaxed during the 

evolution of prasinophytes and euglenids. The IR was lost a minimum of 3 times (fig. 8), an 

observation that is not surprising given that independent IR losses have been documented for the 

class Trebouxiophyceae (de Cambiaire et al. 2007) and for land plants (Palmer 1991; Raubeson 

and Jansen 2005). More unexpected was our finding that the 3 examined IR-containing 

prasinophyte cpDNAs differ significantly in the distribution of their genes among the 2 SC 

regions and in the orientation of the IR relative to these regions. While the Nephroselmis genome 

is the most similar to the gene partitioning pattern observed for streptophytes and some non- 

green algae (Turmel et al. 1999b), the reduced genome of Ostreococcus shows a pattern 

27 




(supplementary fig.1, Supplementary Material) more like that observed for the ulvophytes 

Pseudendoclonium and Oltmannsiellopsis (Pombert et al. 2005; Pombert et al. 2006). When the 

latter pattern was identified in Pseudendoclonium, it was hypothesized that it might represent an 

intermediate form between the highly derived pattern found in the chlorophycean green alga 

Chlamydomonas reinhardtii and the ancestral quadripartite structure found in streptophytes, 

Nephroselmis, and probably early-diverging trebouxiophytes, thus lending support to the notion 

that the Ulvophyceae is sister to the Chlorophyceae (Pombert et al. 2005). However, the great 

variability in the quadripartite structure uncovered here for the Prasinophyceae and recently 

reported for the Chlorophyceae (de Cambiaire et al. 2006; Brouard et al. 2008) casts doubt on the 

phylogenetic value of this genomic feature. Clearly, these data indicate that chloroplast genome 

rearrangements led to the exchanges of genes between opposite SC regions on multiple 

occasions during the evolutionary history of chlorophytes. 

Conclusions 

The chloroplast genome of prasinophytes exhibits much more fluidity in gene content and 

arrangement than anticipated from the earlier reports on the Nephroselmis and Ostreococcus 

genomes. Major reduction and restructuring of the chloroplast genome occurred in conjunction 

with changes in cell organization in at least 2 lineages, the Mamiellales and Pycnococcaceae. By 

disclosing the existence of a sister relationship between the Mamiellales and Pyramimonadales, 

our study represents a significant step towards a better understanding of prasinophyte evolution. 

Furthermore, it offers for the first time compelling evidence that the evolutionary history of the 

prasinophytes was directly linked with the acquisition of photosynthesis through secondary 

endosymbiosis by a subgroup of excavates, the euglenids. Two independent lines of evidence, 

28 




trees inferred from sequence data and the presence of uniquely shared derived gene clusters, 

robustly support to the notion that the green algal ancestor of the euglenid chloroplasts belonged 

to the Pyramimonadales. Although sampling of Bigelowiella has not enabled us to pinpoint the 

green algal donor of chlorarachniophytes chloroplasts, the inferred trees strengthen the 

hypothesis that chloroplasts arose independently in chlorarachniophytes and euglenids. 

Considering that pyramimonadaleans are richer in ancestral characters at the chloroplast genome 

level and exhibit a more pronounced level of cell asymmetry and complexity compared to the 

mamiellaleans, it is plausible that cell asymmetry characterized the common ancestor of these 

lineages. Consistent with the hypothesis that the common ancestor of all chlorophytes also 

featured an asymmetrical cell architecture is the observation that Nephroselmis occupies the 

earliest divergence of the Chlorophyta and displays the highest conservation of ancestral 

characters. Future chloroplast genome investigations incorporating the Chlorodendrales, the 2 

picoplanktonic lineages not sampled in the present study, and a broader range of taxa in each 

lineage should resolve further the branching pattern of prasinophyte lineages and clarify the 

number of separate events that gave rise to coccoids and streamlining of the chloroplast genome. 

Supplementary Material 

Supplementary figures 1-5, supplementary table 1, the data sets used in phylogenetic 

analyses, and the data set used to infer the evolutionary scenario of character losses are available 

at Molecular Biology and Evolution online (http://mbe.oxfordjournals.org/). The fully annotated 

chloroplast genome sequences of Monomastix, Pycnococcus and Pyramimonas have been 

deposited in the GenBank database under the accession numbers FJ493497, FJ493498, and 

29 




FJ493499, respectively. The GenBank accession number for the Monomastix 18S rDNA 

sequence determined in this study is FJ493496. 

Acknowledgments 

We thank Mathieu Blais and Bertrand Caillier for their assistance in cloning and 

sequencing the Pyramimonas chloroplast genome. This study was supported by a grant from the 

Natural Sciences and Engineering Research Council of Canada (to M.T. and C.L.). 

Literature Cited 

Baldauf SL. 2008. An overview of the phylogeny and diversity of eucaryotes. J Syst Evol. 

46:263-273. 

Baldauf SL, Roger AJ, Wenk-Siefert I, Doolittle WF. 2000. A kingdom-level phylogeny of 

eukaryotes based on combined protein data. Science. 290:972-977. 

Bélanger A-S, Brouard J-S, Charlebois P, Otis C, Lemieux C, Turmel M. 2006. Distinctive 

architecture of the chloroplast genome in the chlorophycean green alga Stigeoclonium 

helveticum. Mol Gen Genomics. 276:464-477. 

Brinkmann H, Philippe H. 2008. Animal phylogeny and large-scale sequencing: progress and 

pitfalls. J Syst Evol. 46:274-286. 

Brouard J-S, Otis C, Lemieux C, Turmel M. 2008. Chloroplast DNA sequence of the green alga 

Oedogonium cardiacum (Chlorophyceae): Unique genome architecture, derived 

characters shared with the Chaetophorales and novel genes acquired through horizontal 

transfer. BMC Genomics. 9:290. 

30 




Burger G, Saint-Louis D, Gray MW, Lang BF. 1999. Complete sequence of the mitochondrial 

DNA of the red alga Porphyra purpurea. Cyanobacterial introns and shared ancestry of 

red and green algae. Plant Cell. 11:1675-1694. 

Castresana J. 2000. Selection of conserved blocks from multiple alignments for their use in 

phylogenetic analysis. Mol Biol Evol. 17:540-552. 

Cattolico R, Jacobs M, Zhou Y, Chang J, Duplessis M, Lybrand T, McKay J, Ong H, Sims E, 

Rocap G. 2008. Chloroplast genome sequencing analysis of Heterosigma akashiwo 

CCMP452 (West Atlantic) and NIES293 (West Pacific) strains. BMC Genomics. 9:211. 

Chevalier B, Turmel M, Lemieux C, Monnat RJ, Stoddard BL. 2003. Flexible DNA target site 

recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI. J Mol 

Biol. 329:253-269. 

Côté V, Mercier J-P, Lemieux C, Turmel M. 1993. The single group-I intron in the chloroplast 

rrnL gene of Chlamydomonas humicola encodes a site-specific DNA endonuclease (I- 

ChuI). Gene. 129:69-76. 

Courties C, Vaquer A, Troussellier M, Lautier J, Chretiennot-Dinet MJ, Neveux J, Machado C, 

Claustre H. 1994. Smallest eukaryotic organism. Nature. 370:255-255. 

de Cambiaire J-C, Otis C, Lemieux C, Turmel M. 2006. The complete chloroplast genome 

sequence of the chlorophycean green alga Scenedesmus obliquus reveals a compact gene 

organization and a biased distribution of genes on the two DNA strands. BMC Evol Biol. 

6:37. 

de Cambiaire J-C, Otis C, Lemieux C, Turmel M. 2007. The chloroplast genome sequence of the 

green alga Leptosira terrestris: multiple losses of the inverted repeat and extensive 

genome rearrangements within the Trebouxiophyceae. BMC Genomics. 8:213. 

31 




Derelle E, Ferraz C, Rombauts S et al. 2006. Genome analysis of the smallest free-living 

eukaryote Ostreococcus tauri unveils many unique features. Proc Natl Acad Sci USA. 

103:11647-11652. 

Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high 

throughput. Nucleic Acids Res. 32:1792-1797. 

Farris JS. 1977. Phylogenetic analysis under Dollo's Law. Syst Zool. 26:77-88. 

Fawley MW, Yun Y, Qin M. 2000. Phylogenetic analyses of 18S rDNA sequences reveal a new 

coccoid lineage of the Prasinophyceae (Chlorophyta). J Phycol. 36:387-393. 

Goulding SE, Olmstead RG, Morden CW, Wolfe KH. 1996. Ebb and flow of the chloroplast 

inverted repeat. Mol Gen Genet. 252:195-206. 

Guillou L, Eikrem W, Chrétiennot-Dinet M-J, Le Gall F, Massana R, Romari K, Pedrós-Alió C, 

Vaulot D. 2004. Diversity of picoplanktonic prasinophytes assessed by direct nuclear 

SSU rDNA sequencing of environmental samples and novel isolates retrieved from 

oceanic and coastal marine ecosystems. Protist. 155:193-214. 

Hallick RB, Hong L, Drager RG, Favreau MR, Monfort A, Orsat B, Spielmann A, Stutz E. 1993. 

Complete sequence of Euglena gracilis chloroplast DNA. Nucleic Acids Res. 21:3537- 

3544. 

Hamby RK, Zimmer EA. 1991. Ribosomal RNA as a phylogenetic tool in plant systematics. In: 

Soltis P, Soltis D, Doyle J, editors. Molecular Systematics in Plants. New York: 

Routledge, Chapman and Hall. p. 50-91. 

Haugen P, Bhattacharya D, Palmer JD, Turner S, Lewis LA, Pryer KM. 2007. Cyanobacterial 

ribosomal RNA genes with multiple, endonuclease-encoding group I introns. BMC Evol 

Biol. 7:159. 

32 




Heath TA, Hedtke SM, Hillis DM. 2008. Taxon sampling and the accuracy of phylogenetic 

analyses. J Syst Evol. 46:239-257. 

Ishida K, Cao Y, Hasegawa M, Okada N, Hara Y. 1997. The origin of chlorarachniophyte 

plastids, as inferred from phylogenetic comparisons of amino acid sequences of EF-Tu. J 

Mol Evol. 45:682-687. 

Jansen RK, Cai Z, Raubeson LA et al. 2007. Analysis of 81 genes from 64 plastid genomes 

resolves relationships in angiosperms and identifies genome-scale evolutionary patterns. 

Proc Natl Acad Sci USA. 104:19369-19374. 

Jobb G, von Haeseler A, Strimmer K. 2004. TREEFINDER: a powerful graphical analysis 

environment for molecular phylogenetics. BMC Evol Biol. 4:18. 

Keeling PJ, Burger G, Durnford DG, Lang BF, Lee RW, Pearlman RE, Roger AJ, Gray MW. 

2005. The tree of eukaryotes. Trends Ecol Evol. 20:670-676. 

Keller MD, Selvin RC, Claus W, Guillard RRL. 1987. Media for the culture of oceanic 

ultraphytoplankton. J Phycol. 23:633-638. 

Khan H, Parks N, Kozera C, Curtis BA, Parsons BJ, Bowman S, Archibald JM. 2007. Plastid 

genome sequence of the cryptophyte alga Rhodomonas salina CCMP1319: lateral 

transfer of putative DNA replication machinery and a test of chromist plastid phylogeny. 

Mol Biol Evol. 24:1832-1842. 

Kurtz S, Choudhuri JV, Ohlebusch E, Schleiermacher C, Stoye J, Giegerich R. 2001. REPuter: 

the manifold applications of repeat analysis on a genomic scale. Nucleic Acids Res. 

29:4633-4642. 

Lambowitz AM, Zimmerly S. 2004. Mobile group II introns. Annu Rev Genet. 38:1-35. 

33 




Latasa M, Scharek R, Le Gall F, Guillou L. 2004. Pigment suites and taxonomic groups in 

Prasinophyceae. J Phycol. 40:1149-1155. 

Lemieux C, Otis C, Turmel M. 2000. Ancestral chloroplast genome in Mesostigma viride reveals 

an early branch of green plant evolution. Nature. 403:649-652. 

Lemieux C, Otis C, Turmel M. 2007. A clade uniting the green algae Mesostigma viride and 

Chlorokybus atmophyticus represents the deepest branch of the Streptophyta in 

chloroplast genome-based phylogenies. BMC Biology. 5:2. 

Lewis LA, McCourt RM. 2004. Green algae and the origin of land plants. Am J Bot. 91:1535- 

1556. 

Lucas P, Otis C, Mercier J-P, Turmel M, Lemieux C. 2001. Rapid evolution of the DNA-binding 

site in LAGLIDADG homing endonucleases. Nucleic Acids Res. 29:960-969. 

Maddison D, Maddison W. 2000. MacClade 4: analysis of phylogeny and character evolution. 

Sunderland, MA: Sinauer Associates. 

Manton I. 1967. Electron microscopical observations on a clone of Monomastix Scherffel in 

culture. Nova Hedwigia. 14:1-11. 

Marin B, Melkonian M. 1999. Mesostigmatophyceae, a new class of streptophyte green algae 

revealed by SSU rRNA sequence comparisons. Protist. 150:399-417. 

Mattox KR, Stewart KD. 1984. Classification of the green algae: a concept based on comparative 

cytology. In: Irvine DEG, John DM, editors. The Systematics of the Green Algae. 

London: Academic Press. p. 29-72. 

Maul JE, Lilly JW, Cui L, dePamphilis CW, Miller W, Harris EH, Stern DB. 2002. The 

Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats. 

Plant Cell. 14:2659-2679. 

34 




McCracken DA, Nadakavukaren MJ, Cain JR. 1980. A biochemical and ultrastructural 

evaluation of the taxonomic position of Glaucosphaera vacuolata Korsch. New Phytol. 

86:39-44. 

Melkonian M. 1984. Flagellar apparatus ultrastructure in relation to green algal classification. In: 

Irvine DEG, John DM, editors. The Systematics of the Green Algae. London: Academic 

Press. p. 73-120. 

Melkonian M. 1990. Phylum Chlorophyta. Class Prasinophyceae. In: Margulis L, Corliss JO, 

Melkonian M, Chapman DJ, editors. Handbook of Protoctista. The Structure, Cultivation, 

Habitats and Life Histories of the Eukaryotic Microorganisms and their Descendants 

Exclusive of Animals, Plants and Fungi. Boston: Jones and Bartlett Publishers. p. 600- 

607. 

Michel F, Umesono K, Ozeki H. 1989. Comparative and functional anatomy of group II catalytic 

introns – a review. Gene. 82:5-30. 

Michel F, Westhof E. 1990. Modelling of the three-dimensional architecture of group I catalytic 

introns based on comparative sequence analysis. J Mol Biol. 216:585-610. 

Moestrup O, Inouye I, Hori T. 2003. Ultrastructural studies on Cymbomonas tetramitiformis 

(Prasinophyceae). I. General structure, scale microstructure, and ontogeny. Can J Bot. 

81:657-671. 

Moestrup O, Thomsen HA. 1974. An ultrastructural study of the flagellate Pyramimonas 

orientalis with particular emphasis on golgi apparatus activity and the flagellar apparatus. 

Protoplasma. 81:247-269. 

35 




Moestrup O, Throndsen J. 1988. Light and electron microscopical studies on pseudoscourfieldia- 

marina a primitive scaly green flagellate prasinophyceae with posterior flagella. Can J 

Bot. 66:1415-1434. 

Nakayama T, Marin B, Kranz HD, Surek B, Huss VAR, Inouye I, Melkonian M. 1998. The basal 

position of scaly green flagellates among the green algae (Chlorophyta) is revealed by 

analyses of nuclear-encoded SSU rRNA sequences. Protist. 149:367-380. 

Nakayama T, Suda S, Kawachi M, Inouye I. 2007. Phylogeny and ultrstructure of Nephroselmis 

and Pseudoscourfieldia (Chlorophyta), including the description of Nephroselmis 

anterostigmatica sp. nov. and a proposal for the Nephroselmidales ord. nov. Phycologia. 

46:680-697. 

O'Kelly CJ. 1992. Flagellar apparatus architecture and the phylogeny of “green algae”: 

chlorophytes, euglenoids, glaucophytes. In: Menzel D, editor. The cytoskeleton of the 

algae. Boca Raton: CRC Press. p. 315-345. 

Odom OW, Shenkenberg DL, Garcia JA, Herrin DL. 2004. A horizontally acquired group II 

intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: in vitro self- 

splicing and genetic evidence for maturase activity. RNA. 10:1097-1107. 

Oudot-Le Secq M-P, Grimwood J, Shapiro H, Armbrust EV, Bowler C, Green BR. 2007. 

Chloroplast genomes of the diatoms Phaeodactylum tricornutum and Thalassiosira 

pseudonana: comparison with other plastid genomes of the red lineage. Mol Genet 

Genomics. 277:427-439. 

Palenik B, Grimwood J, Aerts A et al. 2007. The tiny eukaryote Ostreococcus provides genomic 

insights into the paradox of plankton speciation. Proc Natl Acad Sci USA. 104:7705- 

7710. 

36 




Palmer JD. 1991. Plastid chromosomes: structure and evolution. In: Bogorad L, Vasil K, editors. 

The Molecular Biology of Plastids. San Diego: Academic Press. p. 5-53. 

Pombert J-F, Lemieux C, Turmel M. 2006. The complete chloroplast DNA sequence of the green 

alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the 

chloroplast genome of early diverging ulvophytes. BMC Biology. 4:3. 

Pombert J-F, Otis C, Lemieux C, Turmel M. 2005. The chloroplast genome sequence of the 

green alga Pseudendoclonium akinetum (Ulvophyceae) reveals unusual structural features 

and new insights into the branching order of chlorophyte lineages. Mol Biol Evol. 

22:1903-1918. 

Proschold T, Leliaert F. 2007. Systematics of the green algae: conflict of classic and modern 

approaches. In: Brodie J, Lewis J, editors. Unravelling the Algae: The Past, Present, and 

Future of Algal Systematics. Boca Raton: CRC Press, Taylor & Francis. p. 123-153. 

Qiu YL, Li LB, Wang B et al. 2006. The deepest divergences in land plants inferred from 

phylogenomic evidence. Proc Natl Acad Sci USA. 103:15511-15516. 

Raubeson LA, Jansen RK. 2005. Chloroplast genomes of plants. In: Henry RJ, editor. Plant 

Diversity and Evolution: Genotypic and Phenotypic Variation in Higher Plants. 

Wallingford: CABI Publishing. p. 45-68. 

Robbens S, Derelle E, Ferraz C, Wuyts J, Moreau H, Van de Peer Y. 2007. The complete 

chloroplast and mitochondrial DNA sequence of Ostreococcus tauri: organelle genomes 

of the smallest eukaryote are examples of compaction. Mol Biol Evol. 24:956-968. 

Rodriguez-Ezpeleta N, Philippe H, Brinkmann H, Becker B, Melkonian M. 2007. Phylogenetic 

analyses of nuclear, mitochondrial, and plastid multigene data sets support the placement 

of Mesostigma in the Streptophyta. Mol Biol Evol. 24:723-731. 

37 




Rogalski M, Karcher D, Bock R. 2008. Superwobbling facilitates translation with reduced tRNA 

sets. Nat Struct Mol Biol. 15:192-198. 

Rogers MB, Gilson PR, Su V, McFadden GI, Keeling PJ. 2007. The complete chloroplast 

genome of the chlorarachniophyte Bigelowiella natans: evidence for independent origins 

of chlorarachniophyte and euglenid secondary endosymbionts. Mol Biol Evol. 24:54-62. 

Rokas A. 2006. Genomics and the tree of life. Science. 313:1897-1899. 

Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed 

models. Bioinformatics. 19:1572-1574. 

Sandaa RA, Heldal M, Castberg T, Thyrhaug R, Bratbak G. 2001. Isolation and characterization 

of two viruses with large genome size infecting Chrysochromulina ericina 

(Prymnesiophyceae) and Pyramimonas orientalis (Prasinophyceae). Virology. 290:272- 

280. 

Sheveleva EV, Hallick RB. 2004. Recent horizontal intron transfer to a chloroplast genome. 

Nucleic Acids Res. 32:803-810. 

Steinkotter J, Bhattacharya D, Semmelroth I, Bibeau C, Melkonian M. 1994. Prasinophytes form 

independent lineages within the Chlorophyta: Evidence from ribosomal RNA sequence 

comparisons. J Phycol. 30:340-345. 

Swofford DL. 2003. PAUP*. Phylogenetic analysis using parsimony (*and other methods). 

Version 4. Sunderland, MA: Sinauer Associates. 

Sym SD, Pienaar RN. 1993. The class Prasinophyceae. In: Round FE, Chapman DJ, editors. 

Progress in Phycological Research. Bristol: Biopress Ltd. p. 281-376. 

38 




Takahashi F, Okabe Y, Nakada T, Sekimoto H, Ito M, Kataoka H, Nozaki H. 2007. Origins of 

the secondary plastids of Euglenophyta and Chlorarachniophyta as revealed by an 

analysis of the plastid-targeting, nuclear-encoded gene psbO. J Phycol. 43:1302-1309. 

Triemer R, Farmer M. 2007. A decade of euglenoid molecular phylogenetics. In: Brodie J, Lewis 

J, editors. Unravelling the Algae: The Past, Present, and Future of Algal Systematics. 

Boca Raton: CRC Press, Taylor & Francis. p. 315-330. 

Turmel M, Brouard JS, Gagnon C, Otis C, Lemieux C. 2008. Deep division in the 

Chlorophyceae (Chlorophyta) revealed by chloroplast phylogenomic analyses. J Phycol. 

44:739-750. 

Turmel M, Lemieux C, Burger G, Lang BF, Otis C, Plante I, Gray MW. 1999a. The complete 

mitochondrial DNA sequences of Nephroselmis olivacea and Pedinomonas minor: two 

radically different evolutionary patterns within green algae. Plant Cell. 11:1717-1729. 

Turmel M, Otis C, Lemieux C. 2005. The complete chloroplast DNA sequences of the 

charophycean green algae Staurastrum and Zygnema reveal that the chloroplast genome 

underwent extensive changes during the evolution of the Zygnematales. BMC Biology. 

3:22. 

Turmel M, Otis C, Lemieux C. 1999b. The complete chloroplast DNA sequence of the green 

alga Nephroselmis olivacea: insights into the architecture of ancestral chloroplast 

genomes. Proc Natl Acad Sci USA. 96:10248-10253. 

Turmel M, Otis C, Lemieux C. 2006. The chloroplast genome sequence of Chara vulgaris sheds 

new light into the closest green algal relatives of land plants. Mol Biol Evol. 23:1324- 

1338. 

39 




Wakasugi T, Nagai T, Kapoor M et al. 1997. Complete nucleotide sequence of the chloroplast 

genome from the green alga Chlorella vulgaris: the existence of genes possibly involved 

in chloroplast division. Proc Natl Acad Sci USA. 94:5967-5972. 

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal 

ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White 

TJ, editors. PCR Protocols: A Guide to Methods and Applications. San Diego: Academic 

Press. p. 315-322. 

Wolf PG, Karol KG, Mandoli DF, Kuehl J, Arumuganathan K, Ellis MW, Mishler BD, Kelch 

DG, Olmstead RG, Boore JL. 2005. The first complete chloroplast genome sequence of a 

lycophyte, Huperzia lucidula (Lycopodiaceae). Gene. 350:117-128. 

40 




Table 1 

General Features of Prasinophyte cpDNAs 

Feature Nephroselmis Pyramimonas Pycnococcus Monomastix Ostreococcus 

Size (bp) 

Total 200,799 101,605 80,211 114,528 71,666 

IR 46,137 13,057 – a – a 6,825 

LSC 92,126 65,153 – a – a 35,684 

SSC 16,399 10,338 – a – a 22,332 

A+T (%) 57.9 65.3 60.5 61.0 60.1 

Conserved genes (no.) b 128 110 98 94 88 

Introns 

Fraction of genome (%) 0 2.7 3.3 4.6 5.2 

Group I (no.) 0 0 0 5 0 

Group II (no.) 0 1 1 1 1 

Intergenic sequences c 

Fraction of genome (%) 32.6 19.6 11.6 43.9 15.1 

Average size (bp) 352 159 102 524 115 

Short repeated sequences d 

Fraction of genome (%) 0.5 0.5 0.1 17.6 0 

a 

Because Pycnococcus and Monomastix cpDNAs lack an IR, only the total sizes of theses genomes are given. 

b 

Conserved genes refer to free-standing coding sequences usually present in chloroplast genomes. Genes present in the IR were 

counted only once. 

c 

In addition to conserved genes, all ORFs ≥100 codons were considered as gene sequences. 

d 

Non-overlapping repeat elements were mapped on each genome with RepeatMasker using the repeats ≥30 bp identified with 

REPuter as input sequences. 




Table 2 

Gene Repertoires of Prasinophyte cpDNAs 

Gene a Nephroselmis Pyramimonas Pycnococcus Monomastix Ostreococcus 

accD + – – – – 

ccsA + + – + – 

cemA + – + – – 

chlB + + – – – 

chlI + + + + – 

chlL + + + – – 

chlN + + + – – 

cysA + – – – – 

cysT + – + – – 

ftsI + – – – – 

ftsW + – – – – 

minD + – – – – 

ndhA + + – – – 

ndhB + + – – – 

ndhC + + – – – 

ndhD + + – – – 

ndhE + + – – – 

ndhF + + – – – 

ndhG + + – – – 

ndhH + + – – – 

ndhI + + – – – 

ndhK + + – – – 

petD + – + + – 

petL + – + – – 

petN + + + + – 

psaC + + + – + 

psaJ + + – + + 

psaM – + – + + 

psbM + – – + – 

rne + – – – – 

rnpB + – + + – 

rpl12 + + – – – 

rpl19 + – + – – 

rpl22 – + – – – 

rpl32 + + – + + 

rpoB + + – + + 

rps9 + + – – + 

rrf + – – + + 

trnG(gcc) + + + + – 

trnI(cau) + + – + – 

trnL(caa) + – – – – 

trnL(gag) + – + – + 




trnP(ggg) – – + – – 

trnR(ccg) + + + – – 

trnS(cga) + – + – – 

trnS(gga) + – + – – 

trnT(ggu) + – – – – 

ycf4 + + + – – 

ycf20 + b + – + – 

ycf47 + – – – – 

ycf62 + – – – – 

ycf65 – + – – – 

ycf81 + – – – – 

a Only the genes that are missing in one or more genomes are indicated. A total of 80 genes are 

shared by all compared cpDNAs: atpA, B, E, F, H, I, clpP, ftsH, infA, petA, B, G, psaA, B, I, 

psbA, B, C, D, E, F,H, I, J, K, L, N, T, Z, rbcL, rpl2, 5, 14, 16, 20, 23, 36, rpoA, C1, C2, rps2, 

3, 4, 7, 8, 11, 12, 14, 18, 19, rrl, rrs, tufA, trnA(ugc), C(gca), D(guc), E(uuc), F(gaa), G(ucc), 

H(gug), I(gau), K(uuu), L(uaa), L(uag), Me(cau), Mf(cau), N(guu), P(ugg), Q(uug), R(acg), 

R(ucu), S(gcu), S(uga), T(ugu), V(uac), W(cca), Y(gua), ycf1, 3, 12. 

b ycf20 is present as a pseudogene in Nephroselmis (unpublished data); it is located downstream 

of ndhE and corresponds to orf111 in the gene map reported by Turmel et al. (1999b). 

43 




FIG. 1.—Gene map of Pyramimonas cpDNA. The 2 copies of the IR sequence are 

represented by thick lines. Genes (filled boxes) on the outside of the map are transcribed in a 

clockwise direction. Coding sequences not commonly found in cpDNA are shown in gray. The 

single intron in atpB is represented by an open box. The color-code denotes the genomic regions 

containing the corresponding genes in the cpDNAs of Nephroselmis and streptophytes: magenta, 

SSC; cyan, LSC; and yellow, IR. Given the variable gene content of the IR in these ancestral- 

type genomes, only the genes invariably present in this region (i.e., those forming the rRNA 

operon) were represented in yellow. tRNA genes are indicated by the 1-letter amino acid code 

(Me, elongator methionine; Mf, initiator methionine) followed by the anticodon in parentheses. 

FIG. 2.—Gene map of Pycnococcus cpDNA. Genes (filled boxes) on the outside of the map 

are transcribed in a clockwise direction. The single intron in atpB is represented by an open box. 

The orf163 and orf175 revealed no detectable similarity with any known gene sequences. The 

genes whose orthologs are found within the IR, SSC and LSC regions in Nephroselmis and 

streptophyte cpDNAs are color-coded in supplementary figure 2, Supplementary Material. 

FIG. 3.—Gene map of Monomastix cpDNA. Genes (filled boxes) on the outside of the map 

are transcribed in a clockwise direction. Introns are represented by open boxes. The orf122 and 

orf125 revealed no detectable similarity with any known gene sequences. The genes whose 

orthologs are found within the IR, SSC and LSC regions in Nephroselmis and streptophyte 

cpDNAs are color-coded in supplementary figure 3, Supplementary Material. 

44 




FIG. 4.—Conservation of ancestral gene clusters in prasinophyte and Euglena cpDNAs. 

Ancestral clusters were defined as those containing genes in the same order and polarity in at 

least 1 streptophyte and 1 prasinophyte. For each genome, the set of genes making up each of the 

identified ancestral clusters is shown as black boxes connected by a horizontal line. Black boxes 

that are contiguous but not linked together indicate that the corresponding genes are not adjacent 

on the genome. Gray boxes denote individual genes that have been relocated elsewhere on the 

chloroplast genome and empty boxes denote missing genes. The relative polarities of the genes 

are not represented in this figure; for this information, consult the maps shown in figures 1-3 or 

that previously reported for the Nephroselmis genome (Turmel et al. 1999b). 

FIG. 5.—Derived gene clusters uniquely shared by the Euglena and Pyramimonas cpDNAs. 

The genes shown as gray boxes represent the derived components of these clusters; those shown 

as black boxes exhibit an ancestral organization. The genes shown as empty boxes are missing in 

Euglena cpDNA. 

FIG. 6.—Phylogenetic position of Monomastix among prasinophytes as inferred from 

nuclear-encoded SSU rDNA sequences. The figure presents the best ML tree. Bootstrap values 

are shown on the corresponding nodes. The names of the taxa whose chloroplast genomes were 

examined in the present study are shown on a black background. Clade numbering follows that 

of Guillou et al. (2004). 

FIG. 7.—Phylogenies inferred from 70 concatenated chloroplast genes (first 2 codon 

positions) and their deduced amino acid sequences. (A) Best ML tree inferred from the amino 

45 




acid data set. (B) Best ML tree inferred from the nucleotide data set. The bootstrap values 

obtained in ML analyses and the posterior probability values obtained in Bayesian analyses are 

shown on the left and right, respectively on the corresponding nodes. 

FIG. 8.—Losses of chloroplast genes and gene pairs during the evolution of prasinophytes 

and euglenids. Unique losses are indicated by squares, whereas convergent losses in 2 or more 

lineages are indicated by triangles. Red and blue symbols refer to losses of genes and gene pairs, 

respectively. Some gene pairs disappeared as a result of gene losses; those that were not 

correlated with any gene losses are denoted by dots. The number below each taxon name 

indicates the total number of conserved genes in the chloroplast genome. Losses of the IR 

occurred in the 3 indicated lineages. 

46 




atpH 

rpl5 

psbK 

T(ugu) 

P(ugg) 

atpF 

psbF 

psbE 

petB 

rpl16 

petG 

R(ccg) 

chlL 

rpl2 

L(uaa) 

rps3 

rps9 

chlB 

rrs 

ndhE 

rpl12 

psbT 

ycf1 

infA 

I(gau) 

rpoB 

psbC 

rpl36 

ndhD 

psaM 

rpoC1 

F(gaa) 

chlI 

rps11 

rps4 

psbL 

rpl23 

rps19 

rpl32 

rpl14 

psaC 

psbJ 

ycf20 

rpl22 

rps8 

rrl 

G(ucc) 

A(ugc) 

W(cca) 

psbH 

rpoC2 

ycf12 

rbcL 

V(uac) 

atpI 

psbB 

ftsH 

rpoA 

ycf3 

psbD 

K(uuu) Y(gua) 

S(uga) 

rps14 

Me(cau) 

atpA 

D(guc) 

chlN 

R(ucu) 

psbA 

orf454 

I(cau) 

ndhI 

rpl20 

ycf65 

ndhF 

petN 

rps7 

C(gca) 

R(acg) 

petA 

chlL 

tufA 

rps12 

ndhH 

clpP 

rps2 

atpB 

rrs 

ndhE 

psaA 

S(gcu) 

I(gau) 

orf510 

atpE 

psbN 

N(guu) 

psaI 

ndhC 

rps18 

psaJ 

ndhA 

Mf(cau) 

G(gcc) 

ndhG 

ndhK 

psaC 

ycf20 

rrl 

L(uag) 

ndhB 

H(gug) 

G(ucc) 

A(ugc) 

W(cca) 

V(uac) 

ccsA 

psbZ 

E(uuc) 

orf608 

psaB 

chlN 

Q(uug) 

psbI 

orf454 

ycf4 

IR A 

IR B 

SSC 

LSC 

Pyramimonas parkeae 

chloroplast DNA 

101,605 bp 

Figure 1 

by guest on July 15, 2013 


Downloaded from

psbK 

rpl20 

T(ugu) 

P(ugg) 

petB 

petG 

R(acg) 

petA 

R(ccg) 

tufA 

clpP 

L(uaa) 

rps2 

L(gag) 

psbT 

F(gaa) 

rpoC1 

rps4 

psaI 

N(guu) 

rps18 

Mf(cau) 

petL 

G(gcc) 

L(uag) 

H(gug) 

A(ugc) 

psbH 

W(cca) 

rpoC2 

ycf12 

rbcL 

V(uac) 

psbB 

ycf3 

cemA 

K(uuu) 

rpl19 

psbZ 

S(uga) 

rps14 

E(uuc) 

petD 

D(guc) 

Q(uug) 

psbI 

rpl5 

atpH 

atpF 

psbF 

petN 

rps7 

psbE 

C(gca) 

rpl16 

chlL 

rps12 

rpl2 

rps3 

atpB 

rrs 

orf583 

psaA 

S(gcu) 

orf175 

infA 

ycf1 

I(gau) 

psbC 

rpl36 

atpE 

chlI 

psbN 

rps11 

psbL 

rpl23 

rps19 

rpl14 

psaC 

psbJ 

rps8 

rrl 

S(cga) 

G(ucc) 

atpI 

rpoA 

ftsH 

orf163 

psbD 

Y(gua) 

rnpB 

Me(cau) 

cysT 

atpA 

S(gga) 

P(ggg) 

psaB 

psbA 

R(ucu) 

chlN 

ycf4 

Pycnococcus provasolii 


80,211 bp 

Figure 2 



Downloaded from

atpH 

psbK 

rpl20 

T(ugu) 

atpF 

rps7 

petG 

R(acg) 

petA 

tufA 

rps12 

clpP 

rps2 

atpB 

psbM 

ycf1 

rpoB 

psbC 

rpl36 

rpoC1 

atpE 

rps11 

rps4 

psaI 

G(gcc) 

ycf20 

H(gug) 

G(ucc) 

psbH 

W(cca) 

orf219 

rpoC2 

ycf12 

V(uac) 

atpI 

ftsH 

rpoA 

ycf3 

ccsA 

psbZ 

rnpB 

rps14 

atpA 

psbA 

R(ucu) 

psbI 

Q(uug) 

I(cau) 

rpl5 

P(ugg) 

psbF 

petN 

psbE 

petB 

rpl16 

C(gca) 

rpl2 

rps3 

L(uaa) 

rrs 

orf170 

psaA 

S(gcu) 

psbT 

infA 

I(gau) 

psaM 

F(gaa) 

chlI 

psbN 

orf161 

N(guu) 

rps18 

psaJ 

psbL 

rrf 

orf122 

Mf(cau) 

rpl23 

rps19 

rpl32 

rpl14 

psbJ 

rps8 

rrl 

L(uag) 

A(ugc) 

rbcL 

psbB 

psbD 

orf248 

K(uuu) 

Y(gua) 

S(uga) 

orf138 

petD 

E(uuc) 

Me(cau) 

D(guc) 

psaB 

orf125 

Monomastix sp. 


114,528 bp 

Figure 3 



Downloaded from

C(gca) 

rpoB 

rpoC1 

rpoC2 

rps2 

rrs 

I(gau) 

A(ugc) 

rrl 

R(acg) 

rrf 

minD 

ndhH 

ndhA 

rps15 

ndhI 

ndhE 

ndhG 

ndhF 

ndhD 

psaC 

ycf20 

psbB 

psbT 

psbN 

psbH 

petB 

petD 

clpP 

atpI 

atpH 

atpF 

atpA 

psbE 

psbF 

psbL 

psbJ 

R(ccg) 

rbcL 

atpB 

atpE 

chlN 

chlL 

N(guu) 

ccsA 

ftsW 

rps12 

rps7 

tufA 

rpl19 

cemA 

petL 

ycf4 

petA 

petG 

rpl23 

rpl2 

rps19 

rpl22 

rps3 

rpl14 

rps8 

rpl16 

rpl5 

infA 

rpl36 

rps11 

rpoA 

rps9 

rpl12 

Pycnococcus 

Monomastix 

Ostreococcus 

Pyramimonas 

Euglena 

Nephroselmis 

Pycnococcus 

Monomastix 

Ostreococcus 


psbK 

S(cga) 

accD 

psaI 

Y(gua) 

T(ggu) 

rps14 

Mf(cau) 

chlI 

G(ucc) 

cysA 

E(uuc) 

ftsI 

psbA 

psaJ 

P(ugg) 

W(cca) 

psbZ 

S(uga) 

ftsH 

rpl32 

cysT 

ycf1 

psbD 

psbC 

psaA 

psaB 

rpl20 

D(guc) 

ndhC 

ndhK 

Euglena 

Nephroselmis 

Figure 4 by guest on July 15, 2013 


Downloaded from

Euglena 


Euglena 


rpl23 

rpl2 

rps19 

rpl22 

W(cca) 

chlN 

chlL 

rrs 

rps3 

rpl16 

rpl14 

rpl5 

rps8 

infA 

rpl36 

I(cau) 

rps14 

ycf3 

ycf65 

F(gaa) 

I(gau) 

A(ugc) 

rrl 

psbK 

ycf12 

psaM 

rps4 

rps11 

psaC 

rpl32 

ycf1 

Figure 5 

rpl20 

rps12 

rps7 

tufA 

C(gca) 

rps2 

ycf4 

cemA 

petA 

Q(uug) 

N(guu) 

R(acg) 




79 

50 

54 

100 

98 

94 

98 

90 

98 

51 

93 74 

100 

71 

98 

100 Pycnococcus provasolii CCMP1203 

Pseudoscourfieldia marina K-0017 

Pycnococcus provasolii RCC 244 

Nephroselmis pyriformis CCMP 717 

Nephroselmis pyriformis RCC 499 

Nephroselmis pyriformis MBIC 11099 

Nephroselmis pyriformis MBIC10641 

Nephroselmis olivacea SAG 40.89 

98 Ostreococcus sp. RCC 143 

100 Environmental sequence RA000412.150 

Ostreococcus tauri OTTHO0595 

90 

Ostreococcus sp. RCC 501 

Bathycoccus prasinos BLA77 

100 Bathycoccus prasinos ALMO2 

Environmental sequence RA000412.37 

Micromonas sp. RCC 434 

100 

Environmental sequence BL000921.10 

95 Environmental sequence RA000412.97 

100 Micromonas pusilla CCMP 489 

Micromonas sp. CCMP 490 

Mantoniella squamata CCAP 1965/1 

Mantoniella antarctica 

Unidentified flagellate RCC 391 

85 Mamiella sp. strain: Shizugawa 

Crustomastix sp. 

Dolichomastix tenuilepis 

Monomastix sp. 


Pyramimonas australis 

Pyramimonas parkae strain: Hachijo 

Pyramimonas propulsa NIES251 

Pyramimonas olivacea Strain: Shizugawa 

Pyramimonas disomata Singapore 

Prasinopapilla vacuolata 

100 Pterosperma cristatum NIES 221 

Pterosperma cristatum strain: Yokohama 

Cymbomonas tetramitiformis strain: Shizugawa 

Halosphaera sp. strain: Shizugawa 

Prasinoderma cf coloniale CCMP 1220 

Unidentified coccoid CCMP 1193 


Prasinoderma coloniale MBIC 10720 

Unidentified coccoid MBIC 10622 

Entransia fimbriata UTEX LB 2352 

Picocystis salinarum IM214 

Picocystis salinarum SSFB 

Picocystis salinarum L7 

Klebsormidium subtilissimum UTEX 462 

Marchantia polymorpha 

Zamia pumila 

Coleochaete scutata SAG 100.80 

100 

84 

84 

57 

100 

100 

91 

68 

70 71 

100 

100 

100 

100 

100 

Chlorokybus atmophyticus UTEX LB 2591 

Chlorella minutissima C-1.1.9 

Nanochlorum eucaryotum Mainz 1 

100 

92 

100 

100 

Trebouxia impressa UTEX 892 

Symbiont of radiolarian host: cf. Spongodrymus 331 




Tetraselmis sp. RCC 500 

Tetraselmis sp. MBIC 11125 


Scherffelia dubia 

Tetraselmis sp. RG-07 

Tetraselmis striata PLY 443 

Tetraselmis convolutae 208 



Unidentified coccoid RCC 287 

Environmental sequence OLI11059 



Pseudoscourfieldia marina K-0017 




Ostreococcus sp. MBIC 10636 


Prasinococcus sp. CCMP 1614 

Prasinococcus cf. capsulatus CCMP 1407 



Staurastrum sp. M753 

Genicularia spirotaenia 329 

100 

Chara foetida 

Nitella capillaris 

Chaetosphaeridium globosum M1311 

Mesostigma viride NIES 475 

Neochloris aquatica 

Unidentified coccoid/flagellate CCMP1189 

Chlamydomonas reinhardtii 

100 

Acrosiphonia duriuscula 

Ulothrix zonata SAG 38.86 

Pseudoscourfieldia marina RCC 261 

Pycnococcus provasolii CCMP1199 

Pycnococcus provasolii CCMP1198 

Figure 6 

0.05 

Chlorophyceae 

Ulvophyceae 

Trebouxiophyceae 

Clade IV 

Chlorodendrales 

Clade VII 

Clade V 

Pseudocourfieldiales 

Pycnococcaceae 

Clade III 

Pseudocourfieldiales 

Nephroselmidaceae 

Clade II 

Mamiellales 

Clade I 

Pyramimonadales 

Clade VI 

Prasinococcales 

Streptophyta 




Mesostigma 

Chlorokybus 

Chara 

Zygnema 

Staurastrum 

Chaetosphaeridium 

Physcomitrella 

Marchantia 

Anthoceros 

Nephroselmis 

Euglena 


Monomastix 

Ostreococcus 

Pycnococcus 

Chlorella 

Bigelowiella 

Oltmannsiellopsis 

Pseudendoclonium 

Leptosira 

Oedogonium 

Stigeoclonium 

Scenedesmus 

Chlamydomonas 

0.1 

Mesostigma 

Chlorokybus 

Chara 

Zygnema 

Staurastrum 

Chaetosphaeridium 

Physcomitrella 

Marchantia 

Anthoceros 

Nephroselmis 

Pycnococcus 

Euglena 


Monomastix 

Ostreococcus 

Chlorella 

Leptosira 

Bigelowiella 

Oltmannsiellopsis 

Pseudendoclonium 

Oedogonium 

Stigeoclonium 

Scenedesmus 

Chlamydomonas 

0.05 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

100/100 

95/100 

85/100 

77/100 

100/100 

69/100 

59/100 

45/98 

70/100 

42/50 

44/50 

56/98 

100/100 

100/100 

100/100 

72/100 

93/100 

87/100 

82/100 

100/100 

100/100 

49/83 

99/100 

47/100 

51/96 

57/100 

30/83 

Core chlorophytes 

Prasinophyceae 

Streptophyta 

Core chlorophytes 

Prasinophyceae 

Streptophyta 

A 

B 

Figure 7 



Downloaded from

Monomastix 

114.5 kb 

94 genes 

-IR 

Clade II — Mamiellales 

psaC 

rps9 

L(gag) 

3'rpoA-5'rps9 

3'rps7-5'tufA 

5'C(gca)-5'rpoB 

3'rpoB-5'rpoC1 

3'rpoC1-5'rpoC2 

3'rpoC2-5'rps2 

5'psbH-5'psbN 

3'P(ugg)-5'W(cca) 

3'psbD-5'psbC 

• 

Ostreococcus 

71.7 kb 

88 genes 

chlB 

chlL 

chlN 

ndhA 

ndhB 

ndhC 

ndhD 

ndhE 

ndhF 

ndhG 

ndhH 

ndhI 

ndhK 

rpl12 

rpl22 

ycf4 

ycf65 

L(caa) 

R(ccg) 

3'rps19-5'rpl22 

3'rpl22-5'rps3 

3'infA-5'rpl36 

3'rps9-5'rpl12 

3'rps2-5'atpI 

3'ndhH-5'ndhA 

3'ndhA-5'ndhI 

5'clpP-5'psbB 

3'psbN-3'psbT 

3'psbH-5'petB 

3'chlL-5'chlN 

3'rbcL-5'R(ccg) 

5'atpB-5'rbcL 

5'psaJ-5'P(ugg) 

5'rpl20-5'D(guc) 

3'ndhC-5'ndhK 

Clade III — Nephroselmidaceae 

Nephroselmis 

200.8 kb 

128 genes 

psaM 

rpl22 

ycf20 

ycf65 

P(ggg) 

3'rps19-5'rpl22 


3'rps2-5'atpI 

5'clpP-5'psbB 

3'psbH-5'petB 


5'atpB-5'rbcL 

3'rps14-5'Mf(cau) 

ccsA 

chlI 

petD 

petN 

psbM 

rnpB 

ycf20 

G(gcc) 

I(cau) 

3'petB-5'petD 

Clade I — Pyramimonadales 


• 

• 

• 

• 

• 

• 

101.6 kb 

110 genes 

rrf 

L(caa) 

3’rps2-5’atpI 

3'rrl-5'rrf 


cemA 

cysT 

petL 

rpl19 

P(ggg) 

S(cga) 

S(gga) 

3'rpl36-5'rps11 

3'tufA-5'rpl19 

3'petA-5'petL 

3'petL-5'petG 

3'cysT-5'ycf1 

3'rps14-5'Mf(cau) 

Ancestor 

134 genes 

• 

• 

• 

• 

Euglena 

143.2 kb 

87 genes 

-IR 

petD 

psbM 

rnpB 

L(gag) 


3'petB-5'petD 


accD 

cysA 

ftsI 

ftsW 

minD 

rne 

Figure 8 

ycf47 

ccsA 

chlB 

chlL 

chlN 

clpP 

ftsH 

infA 

ndhA 

ndhB 

ndhC 

ndhD 

ndhE 

ndhF 

ndhG 

ndhH 

ndhI 

ndhK 

petA 

petN 

psaI 

ycf1 

ycf3 

ycf20 

ycf65 

R(ccg) 

3'rps8-5'infA 

3'infA-5'rpl36 

3'rps11-5'rpoA 

3'rpoA-5'rps9 


3'ndhH-5'ndhA 

3'ndhA-5'ndhI 

5'clpP-5'psbB 

3'psbN-3'psbT 

5'psbH-5'psbN 

3'psbH-5'petB 

3'chlL-5'chlN 


5'atpB-5'rbcL 

3'psbD-5'psbC 


3'ndhC-5'ndhK 

• 

• 

ycf62 

ycf81 

T(ggu) 

3'rpl19-5'ycf4 

3'ycf4-5'cemA 

3'cemA-5'petA 

3'psaC-5'ndhD 

3'ndhD-3'ndhF 

3'ndhI-5'ndhG 

3'ndhG-5'ndhE 

3'rrf-5'R(acg) 

3'minD-3'R(acg) 

5'ftsW-5'N(guu) 

3'ccsA-3'N(guu) 

5'ccsA-5'chlL 

3'rpl32-5'cysT 

5'psbZ-5'S(uga) 

3'S(uga)-5'ftsH 

3'ftsI-5'psbA 

5'chlI-5'G(ucc) 

3'psbK-5'S(cga) 

5'cysA-5'E(uuc) 

3'Y(gua)-5'T(ggu) 

5'accD-5'psaI 

Clade V — Pycnococcaceae 

Pycnococcus 

80.2 kb 

98 genes 

• 

• 

• 

• 

• 

• 

• 

• 

-IR 

ccsA 

chlB 

ndhA 

ndhB 

ndhC 

ndhD 

ndhE 

ndhF 

ndhG 

ndhH 

ndhI 

ndhK 

psaJ 

psaM 

psbM 

rpl12 

rpl22 

rpl32 

rpoB 

rps9 

rrf 

ycf20 

ycf65 

I(cau) 

L(caa) 

3'rps19-5'rpl22 


3'rpoA-5'rps9 

3'rps9-5'rpl12 

3'rps7-5'tufA 


3'rpoB-5'rpoC1 


3'rps2-5'atpI 

3'ndhH-5'ndhA 

3'ndhA-5'ndhI 

5'clpP-5'psbB 

3'psbH-5'petB 

3'rrs-5'I(gau) 

3'I(gau)-5'A(ugc) 

3'A(ugc)-5'rrl 

3'rrl-5'rrf 


5'atpB-5'rbcL 




3'ndhC-5'ndhK 

• 

• 

• 

• 

•

The Chloroplast Genomes of the Green Algae Pyramimonas ...

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