Comparative plastome and mitogenome analyses indicate that the marine prasinophyte green algae Pycnococcus provasolii and Pseudoscourfieldia marina (Pseudoscourfieldiophyceae class nov., Chlorophyta) represent morphotypes of the same species.

The marine prasinophyte green algae Pycnococcus provasolii and Pseudoscourfieldia marina represent the only extant genera and known species of the Pycnococcaceae. However, their taxonomic status needs to be reassessed, owing to the very close relationship inferred from previous sequence comparisons of individual genes. Although Py. provasolii and Ps. marina are morphologically different, their plastid rbcL and nuclear small subunit rRNA genes were observed to be nearly or entirely identical in sequence, thus leading to the hypothesis that they represent distinct growth forms or alternate life-cycle stages of the same organism. To evaluate this hypothesis, we used organelle genomes as molecular markers. The plastome and mitogenome of Ps. marina UIO 007 were sequenced and compared with those available for two isolates of Py. provasolii (CCMP 1203 and CCAP 190/2). The Ps. marina organelle genomes proved to be almost identical in size and had the same gene content and gene order as their Py. provasolii counterparts. Single nucleotide substitutions and insertions/deletions were localized using genome-scale sequence alignments. Over 99.70% sequence identities were observed in all pairwise comparisons of plastomes and mitogenomes. Alignments of both organelle genomes revealed that Ps. marina UIO 007 is closer to Py. provasolii CCAP 190/2 than are the two Py. provasolii strains to one another. Therefore, our results are not consistent with the placement of Ps. marina and Py. provasolii strains into distinct genera. We propose a taxonomic revision of the Pycnococcaceae and the erection of a new class of Chlorophyta, the Pseudoscourfieldiophyceae.

Voriconazole therapeutic drug monitoring among lung transplant recipients receiving targeted therapy for invasive aspergillosis.

BACKGROUND: Voriconazole is the first line treatment for invasive aspergillosis (IA) Current guidelines suggest performing regular voriconazole therapeutic drug monitoring (TDM) to optimize treatment efficacy. We aimed to determine if TDM was predictive of clinical outcome in LTRs. METHODS: Retrospective chart review was performed for all LTRs with probable or proven IA, treated with voriconazole monotherapy and who underwent TDM during therapy. Clinical outcome and toxicity were measured at 12 weeks. Classification and regression tree (CART) analysis was used to determine the most predictive voriconazole level thresholds for successful outcome. RESULTS: One hundred and eighteen TDM samples from 30 LTRs with IA were analyzed. Three LTRs were excluded due to early treatment discontinuation. The median TDM level was 1.2 µg/ml (range 0.06-7.3). At 12 weeks, 62% (17/27) of patients had a successful outcome, while 37% (10/27) of patients failed therapy. CART analysis determined that the best predictor for successful outcome was a median TDM level >0.72 µg/ml. Seventy percent (14/20) of patients with median TDM above 0.72 µg/ml had a successful outcome, compared to 42.9% (3/7) of patients with a median TDM below 0.72 µg/ml (OR 3.11; 95% CI: 0.53-20.4; P = 0.21). CART analysis determined that a TDM level greater than 2.13 µg/ml was predictive of hepatotoxicity. CONCLUSIONS: Our data suggests that a voriconazole TDM range between 0.72 µg/ml and 2.13 µg/ml may be associated with improved outcomes. Our study is in line with current recommendations on the use of voriconazole TDM in improving outcome and minimizing toxicity in LTR with IA.

Extreme Enlargement of the Inverted Repeat Region in the Plastid Genomes of Diatoms from the Genus Climaconeis.

We sequenced the plastid genomes of three diatoms from the genus Climaconeis, including two strains formerly designated as Climaconeis scalaris. At 208,097 and 216,580 bp, the plastid genomes of the latter strains are the largest ever sequenced among diatoms and their increased size is explained by the massive expansion of the inverted repeat region. Important rearrangements of gene order were identified among the two populations of Climaconeis cf. scalaris. The other sequenced Climaconeis chloroplast genome is 1.5 times smaller compared with those of the Climaconeis cf. scalaris strains and it features an usual quadripartite structure. The extensive structural changes reported here for the genus Climaconeis are compared with those previously observed for other algae and plants displaying large plastid genomes.

Mitochondrial and Plastid Genomes of the Monoraphid Diatom Schizostauron trachyderma.

We provide for the first time the complete plastid and mitochondrial genomes of a monoraphid diatom: Schizostauron trachyderma. The mitogenome is 41,957 bp in size and displays two group II introns in the cox1 gene. The 187,029 bp plastid genome features the typical quadripartite architecture of diatom genomes. It contains a group II intron in the petB gene that overlaps the large single-copy and the inverted repeat region. There is also a group IB4 intron encoding a putative LAGLIDADG homing endonuclease in the rnl gene. The multigene phylogenies conducted provide more evidence of the proximity between S. trachyderma and fistula-bearing species of biraphid diatoms.

Screening for Exotic Forest Pathogens to Increase Survey Capacity Using Metagenomics.

Anthropogenic activities have a major impact on the global environment. Canada's natural resources are threatened by the spread of fungal pathogens, which is facilitated by agricultural practices and international trade. Fungi are introduced to new environments and sometimes become established, in which case they can cause disease outbreaks resulting in extensive forest decline. Here, we describe how a nationwide sample collection strategy coupled to next-generation sequencing (NGS) (i.e., metagenomics) can achieve fast and comprehensive screening for exotic invasive species. This methodology can help provide guidance to phytopathology stakeholders such as regulatory agencies. Several regulated invasive species were monitored by processing field samples collected over 3 years (2013 to 2015) near high-risk areas across Canada. Fifteen sequencing runs were required on the Ion Torrent platform to process 398 samples that yielded 45 million reads. High-throughput screening of fungal and oomycete operational taxonomic units using customized fungi-specific ribosomal internal transcribed spacer 1 barcoded primers was performed. Likewise, Phytophthora-specific barcoded primers were used to amplify the adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer. Several Phytophthora spp. were detected by NGS and confirmed by species-specific quantitative polymerase chain reaction (qPCR) assays. The target species Heterobasidion annosum sensu stricto could be detected only through metagenomics. We demonstrated that screening target species using a variety of sampling techniques and NGS-the results of which were validated by qPCR-has the potential to increase survey capacity and detection sensitivity, reduce hands-on time and costs, and assist regulatory agencies to identify ports of entry. Considering that early detection and prevention are the keys in mitigating invasive species damage, our method represents a substantial asset in plant pathology management.

Chloroplast phylogenomic analysis of chlorophyte green algae identifies a novel lineage sister to the Sphaeropleales (Chlorophyceae).

BACKGROUND: The class Chlorophyceae (Chlorophyta) includes morphologically and ecologically diverse green algae. Most of the documented species belong to the clade formed by the Chlamydomonadales (also called Volvocales) and Sphaeropleales. Although studies based on the nuclear 18S rRNA gene or a few combined genes have shed light on the diversity and phylogenetic structure of the Chlamydomonadales, the positions of many of the monophyletic groups identified remain uncertain. Here, we used a chloroplast phylogenomic approach to delineate the relationships among these lineages. RESULTS: To generate the analyzed amino acid and nucleotide data sets, we sequenced the chloroplast DNAs (cpDNAs) of 24 chlorophycean taxa; these included representatives from 16 of the 21 primary clades previously recognized in the Chlamydomonadales, two taxa from a coccoid lineage (Jenufa) that was suspected to be sister to the Golenkiniaceae, and two sphaeroplealeans. Using Bayesian and/or maximum likelihood inference methods, we analyzed an amino acid data set that was assembled from 69 cpDNA-encoded proteins of 73 core chlorophyte (including 33 chlorophyceans), as well as two nucleotide data sets that were generated from the 69 genes coding for these proteins and 29 RNA-coding genes. The protein and gene phylogenies were congruent and robustly resolved the branching order of most of the investigated lineages. Within the Chlamydomonadales, 22 taxa formed an assemblage of five major clades/lineages. The earliest-diverging clade displayed Hafniomonas laevis and the Crucicarteria, and was followed by the Radicarteria and then by the Chloromonadinia. The latter lineage was sister to two superclades, one consisting of the Oogamochlamydinia and Reinhardtinia and the other of the Caudivolvoxa and Xenovolvoxa. To our surprise, the Jenufa species and the two spine-bearing green algae belonging to the Golenkinia and Treubaria genera were recovered in a highly supported monophyletic group that also included three taxa representing distinct families of the Sphaeropleales (Bracteacoccaceae, Mychonastaceae, and Scenedesmaceae). CONCLUSIONS: Our phylogenomic study advances our knowledge regarding the circumscription and internal structure of the Chlamydomonadales, suggesting that a previously unrecognized lineage is sister to the Sphaeropleales. In addition, it offers new insights into the flagellar structures of the founding members of both the Chlamydomonadales and Sphaeropleales.

Chloroplast phylogenomic analysis resolves deep-level relationships within the green algal class Trebouxiophyceae.

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

Six newly sequenced chloroplast genomes from prasinophyte green algae provide insights into the relationships among prasinophyte lineages and the diversity of streamlined genome architecture in picoplanktonic species.

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

The invasive land flatworm Arthurdendyus triangulatus has repeated sequences in the mitogenome, extra-long cox2 gene and paralogous nuclear rRNA clusters.

Using a combination of short- and long-reads sequencing, we were able to sequence the complete mitochondrial genome of the invasive 'New Zealand flatworm' Arthurdendyus triangulatus (Geoplanidae, Rhynchodeminae, Caenoplanini) and its two complete paralogous nuclear rRNA gene clusters. The mitogenome has a total length of 20,309 bp and contains repetitions that includes two types of tandem-repeats that could not be solved by short-reads sequencing. We also sequenced for the first time the mitogenomes of four species of Caenoplana (Caenoplanini). A maximum likelihood phylogeny associated A. triangulatus with the other Caenoplanini but Parakontikia ventrolineata and Australopacifica atrata were rejected from the Caenoplanini and associated instead with the Rhynchodemini, with Platydemus manokwari. It was found that the mitogenomes of all species of the subfamily Rhynchodeminae share several unusual structural features, including a very long cox2 gene. This is the first time that the complete paralogous rRNA clusters, which differ in length, sequence and seemingly number of copies, were obtained for a Geoplanidae.

Description of Naviculavanseea sp. nov. (Naviculales, Naviculaceae), a new species of diatom from the highly alkaline Lake Van (Republic of Türkiye) with complete characterisation of its organellar genomes and multigene phylogeny.

The current article describes Naviculavanseeasp. nov., a new species of diatom from Lake Van, a highly alkaline lake in Eastern Anatolia (Türkiye). The description is based on light and scanning electron microscopy performed on two monoclonal cultures. The complete nuclear rRNA clusters and plastid genomes have been sequenced for these two strains and the complete mitogenome for one of them. The plastome of both strains shows the probable loss of a functional ycf35 gene. They also exhibit two IB4 group I introns in their rrl, each encoding for a putative LAGLIDADG homing endonuclease, with the first L1917 IB4 intron reported amongst diatoms. The Maximum Likelihood phylogeny inferred from a concatenated alignment of 18S, rbcL and psbC distinguishes N.vanseea sp. nov. from the morphologically similar species Naviculacincta and Naviculamicrodigitoradiata.

The mitochondrial genome of the bioluminescent fish Malacosteusniger Ayres, 1848 (Stomiidae, Actinopterygii) is large and complex, and contains an inverted-repeat structure.

We determined the complete mitogenome sequence of the bioluminescent fish Malacosteusniger using long-read sequencing technologies. The 21,263 bp mitogenome features a complex structure with two copies of a 1198-bp inverted-repeat and a region of 2616-bp containing alternating copies of 16 and 26 bp repeat elements. Whole mitogenome phylogenies inferred from both nucleotide and amino-acid datasets place M.niger among Melanostomiinae. The need for additional complete mitogenome sequences from the subfamily Malacosteinae is discussed.

Mitochondrial genome sequence of the protist Ancyromonas sigmoides Kent, 1881 (Ancyromonadida) from the Sugluk Inlet, Hudson Strait, Nunavik, Québec.

Introduction: There is little information on evolutionarily ancient eukaryotes, which are often referred to as basal eukaryotes, in Arctic waters. Despite earlier studies being conducted in the Russian White Sea, only few have been reported. Methods: Following a shotgun sequence survey of diatom cultures from Sugluk Inlet off the Hudson Strait in Northern Québec, we obtained the complete mitochondrial genome and the operon of nuclear ribosomal RNA genes from a strain that matches that of Ancyromonas sigmoides (Kent, 1881). Results: The sequence of the mitogenome retrieved was 41,889 bp in length and encoded 38 protein-coding genes, 5 non-conserved open-reading frames, and 2 rRNA and 24 tRNA genes. The mitogenome has retained sdh2 and sdh3, two genes of the succinate dehydrogenase complex, which are sometimes found among basal eukaryotes but seemingly missing among the Malawimonadidae, a lineage sister to Ancyromonadida in some phylogenies. The phylogeny inferred from the 18S rRNA gene associated A. sigmoides from Sugluk Inlet with several other strains originating from the Arctic. The study also unveiled the presence of a metagenomic sequence ascribed to bacteria in GenBank, but it was clearly a mitochondrial genome with a gene content highly similar to that of A. sigmoides, including the non-conserved open-reading frames. Discussion: After re-annotation, a phylogeny was inferred from mitochondrial protein sequences, and it strongly associated A. sigmoides with the misidentified organism, with the two being possibly conspecific or sibling species as they are more similar to one another than to species of the genus Malawimonas. Overall our phylogeny showed that the ice associated ancryomonads were clearly distinct from more southerly strains.

Complete chloroplast genome of the mixotrophic chrysophyte Poterioochromonas malhamensis (Ochromonadales, Synurophyceae) from Van Lake in Eastern Anatolia.

We sequenced the chloroplast genome of Poterioochromonas malhamensis (Pringsheim) R.A.Andersen strain SZCZR2049, which originates from Van Lake in Turkey. This genome is 133,923 bp long, and like those currently available for six phototrophic chrysophytes, it displays a long, gene-rich inverted repeat and a very short single-copy region. Compared to its chrysophyte counterparts, the P. malhamensis inverted repeat differs noticeably in gene content and the whole genome is missing 11 protein-coding genes. The maximum likelihood phylogeny inferred from concatenated protein-coding genes positioned P. malhamensis among the chrysophytes sensu lato as sister to the clade containing the Synurales (Synurophyceae) and Chromulinales (Chrysophyceae).

A gene-rich and compact chloroplast genome of the green alga Nephroselmis pyriformis (N.Carter) Ettl 1982 from the shores of Mersin (Eastern Mediterranean Sea).

We report the complete chloroplast genome of the MED1 strain of Nephroselmis pyriformis from the Eastern Mediterranean Sea. At 111,026 bp, this genome is smaller and more compact than those of Nephroselmis olivacea and Nephroselmis astigmatica, and in contrast to the latter taxa, its inverted repeat contains no complete protein-coding genes. It encodes 3 rRNAs, 33 tRNAs and 94 proteins. Maximum likelihood analysis of a concatenated set of chloroplast genes from green algae belonging to deep-diverging lineages positioned the three Nephroselmis species in a strongly supported clade in which N. pyriformis is sister to N. astigmatica.

Nitzschia anatoliensis sp. nov., a cryptic diatom species from the highly alkaline Van Lake (Turkey).

In this article we describe Nitzschia anatoliensis Górecka, Gastineau & Solak sp. nov., an example of a diatom species inhabiting extreme habitats. The new species has been isolated and successfully grown from the highly alkaline Van Lake in East Turkey. The description is based on morphology (light and scanning electron microscopy), the sequencing of its organellar genomes and several molecular phylogenies. This species could easily be overlooked because of its extreme similarity to Nitzschia aurariae but molecular phylogenies indicate that they are only distantly related. Furthermore, molecular data suggest that N. anatoliensis may occur in several alkaline lakes of Asia Minor and Siberia, but was previously misidentified as Nitzschia communis. It also revealed the very close genetic proximity between N. anatoliensis and the endosymbiont of the dinotom Kryptoperidinium foliaceum, providing additional clues on what might have been the original species of diatoms to enter symbiosis.

Haslea silbo, A Novel Cosmopolitan Species of Blue Diatoms.

Specimens of a new species of blue diatoms from the genus Haslea Simonsen were discovered in geographically distant sampling sites, first in the Canary Archipelago, then North Carolina, Gulf of Naples, the Croatian South Adriatic Sea, and Turkish coast of the Eastern Mediterranean Sea. An exhaustive characterization of these specimens, using a combined morphological and genomic approach led to the conclusion that they belong to a single new to science cosmopolitan species, Haslea silbo sp. nov. A preliminary characterization of its blue pigment shows similarities to marennine produced by Haslea ostrearia, as evidenced by UV-visible spectrophotometry and Raman spectrometry. Life cycle stages including auxosporulation were also observed, providing data on the cardinal points of this species. For the two most geographically distant populations (North Carolina and East Mediterranean), complete mitochondrial and plastid genomes were sequenced. The mitogenomes of both strains share a rare atp6 pseudogene, but the number, nature, and positions of the group II introns inside its cox1 gene differ between the two populations. There are also two pairs of genes fused in single ORFs. The plastid genomes are characterized by large regions of recombination with plasmid DNA, which are in both cases located between the ycf35 and psbA genes, but whose content differs between the strains. The two sequenced strains hosts three plasmids coding for putative serine recombinase protein whose sequences are compared, and four out of six of these plasmids were highly conserved.

The chloroplast genomes of the green algae Pedinomonas minor, Parachlorella kessleri, and Oocystis solitaria reveal a shared ancestry between the Pedinomonadales and Chlorellales.

The green algae belonging to the Chlorophyta-the lineage sister to that comprising the land plants and their charophycean green algal relatives (Streptophyta)-have been subdivided into four classes (Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae). Yet the Pedinomonadales, an assemblage consisting of tiny, naked uniflagellates with a second basal body, has no clear affiliation with these classes and the branching order of the crown chlorophytes remains unknown. To gain an insight into the phylogenetic position of the Pedinomonadales and the relationships among the recognized chlorophyte classes, we have sequenced the chloroplast genomes of Pedinomonas minor (Pedinomonadales) and of two trebouxiophyceans belonging to the Chlorellales, Parachlorella kessleri (Chlorellaceae) and Oocystis solitaria (Oocystaceae), and compared these genomes with those of previously examined streptophytes and chlorophytes, including Chlorella vulgaris (Chlorellaceae). Unlike their Chlorella homolog, the three newly investigated chloroplast DNAs (cpDNAs) carry a large rRNA-encoding inverted repeat (IR) that divides the genome into large and small single-copy regions. In contrast to the situation found for ulvophycean and chlorophycean cpDNAs, the gene contents of the IR and single-copy regions are strikingly similar to that inferred for the common ancestor of chlorophytes and streptophytes. The intronless 98,340-bp Pedinomonas genome is among the chlorophyte cpDNAs featuring the smallest size and most ancestral gene organization. All 105 conserved genes encoded by this genome are included in the gene repertoires of Oocystis (111 genes) and Chlorella (113 genes), with just trnR(ccg) missing from Parachlorella cpDNA. Trees inferred from 71 cpDNA-encoded genes/proteins of 16 chlorophytes and nine streptophytes showed that Pedinomonas is nested in the Chlorellales, a group of algae lacking flagella. This phylogenetic conclusion is independently supported by uniquely shared gene linkages. We hypothesize that chlorellalean and pedinomonadalean green algae are reduced forms of a distant biflagellate ancestor that might have also given rise to the other known trebouxiophycean lineages. Our structural cpDNA data suggest that the Chlorellales and Pedinomonadales represent a deep branch of core chlorophytes, strengthening the notion that the Trebouxiophyceae emerged before the Ulvophyceae and Chlorophyceae. Our results further emphasize the importance of secondary reduction at both the cellular and genome levels during chlorophyte evolution.

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.

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

A deviant genetic code in the reduced mitochondrial genome of the picoplanktonic green alga Pycnococcus provasolii.

Reduction in size of flagellated chlorophytes occurred multiple times during evolution, providing the opportunity to study the consequences of cell reduction on genome architecture. Recent investigations on the chloroplast genomes of the tiny prasinophyceans Ostreococcus tauri (Mamiellales), Micromonas sp. RCC299 (Mamiellales), and Pycnococcus provasolii (Pseudocourfieldiales) highlighted their extreme compaction and reduced gene repertoires. Genome compaction is also exemplified by the Ostreococcus and Micromonas mitochondrial DNAs (mtDNAs) although they have retained almost all of the about 65 genes presumably present in the mitochondria of ancestral prasinophyceans. In this study, the mitochondrial genome of Pycnococcus was sequenced and compared to those of previously examined chlorophytes. Our results document the first case where cellular reduction of a free-living alga was accompanied by marked reduction in gene content of both the mitochondrial and chloroplast genomes. At 24,321 bp, the intronless Pycnococcus mitochondrial genome falls within the lower size range displayed by green algal mtDNAs. The 36 conserved genes, specifying two rRNAs with conventional structures, 16 tRNAs and 18 proteins, are all encoded on the same DNA strand and represent 88% of the genome. Besides a pronounced codon bias, the protein-coding genes feature a variant genetic code characterized by the use of TGA (normally a stop codon) to code for tryptophan, and the unprecedented use of TTA and TTG (normally leucine codons) as stop codons. We conclude that substantial reduction of the mitochondrial genome occurred in at least three independent chlorophyte lineages and that this process entailed a number of convergent changes in these lineages.

Complete mitogenome of the chlorophyte green alga Marsupiomonas sp. NIES 1824 (Pedinophyceae).

The 25,137-bp mitogenome of the green alga Pedinomonas minor (Pedinomonadales, Pedinophyceae), which belongs to a basal class of the core Chlorophyta, is unusual in displaying a reduced gene content as well as other derived traits. Here, we present the mitogenome of Marsupiomonas sp. NIES 1824 (Marsupiomonadales, Pedinophyceae). Despite its smaller size, this 24,252-bp genome encodes twice as many genes (39) as its P. minor homolog. Besides gradual gene erosion, our comparative analyses revealed that major changes in GC content and codon usage led to the gain of distinct, noncanonical genetic codes during evolution of the mitogenome in the Pedinophyceae.