The genus Coelastrella (Chlorophyceae, Chlorophyta): molecular species delimitation, biotechnological potential, and description of a new species Coelastrella affinis sp. nov., based on an integrative taxonomic approach.

Despite the long research history on the genus Coelastrella, its species diversity and biotechnological potential have not been fully explored. For the first time, cluster analysis of morphological characteristics was done in the representatives of the said genus. The results obtained have shown that morphological similarity does not necessarily indicate a molecular genetic relationship. It the light of it, the taxonomic status of species can reliably be determined using specific DNA region, such as 18S-ITS1-5.8S-ITS2. The V4 and V9 regions of gene 18S rRNA are relatively conservative fragments which are not suitable for species identification. The ITS2 can be used as a "short barcode". Among the advanced machine methods for delimitation species, the most effective algorithm for distinguishing Coelastrella species was the Generalized Mixed Yule Coalescent (GMYC) method. This paper represented for the first time our comprehensive review of the works devoted to the analysis of the biotechnological potential of representatives of the genus Coelastrella and shows that fatty acid composition of the three main chemogroups within the studied genus differs. In the future, this may form the basis for predicting the composition of the fatty acid profile of new strains, which is important while searching for organisms with specified biotechnological properties. In conclusion, an integrative approach was employed to describe Coelastrella affinis sp. nov., a new species of the genus Coelastrella with high biotechnological potential. Also, a new description of C. thermophila var. astaxanthina comb. nov. was proposed.

Green algae (Viridiplantae) in sediments from three lakes on Vega Island, Antarctica, assessed using DNA metabarcoding.

BACKGROUND: Vega Island is located off the eastern tip of the Antarctic Peninsula (Maritime Antarctica), in the Weddell Sea. In this study, we used metabarcoding to investigate green algal DNA sequence diversity present in sediments from three lakes on Vega Island (Esmeralda, Copépodo, and Pan Negro Lakes). METHODS AND RESULTS: Total DNA was extracted and the internal transcribed spacer 2 region of the nuclear ribosomal DNA was used as a DNA barcode for molecular identification. Green algae were represented by sequences representing 78 taxa belonging to Phylum Chlorophyta, of which 32% have not previously been recorded from Antarctica. Sediment from Pan Negro Lake generated the highest number of DNA reads (11,205), followed by Esmeralda (9085) and Copépodo (1595) Lakes. Esmeralda Lake was the richest in terms of number of taxa (59), with Copépodo and Pan Negro Lakes having 30 taxa each. Bray-Curtis dissimilarity among lakes was high (~ 0.80). The Order Chlamydomonadales (Chlorophyceae) gave the highest contribution in terms of numbers of taxa and DNA reads in all lakes. The most abundant taxon was Chlorococcum microstigmatum. CONCLUSIONS: The study confirms the utility of DNA metabarcoding in assessing potential green algal diversity in Antarctic lakes, generating new Antarctic records.

Trophic control of cryptic coralline algal diversity.

Understanding how trophic dynamics drive variation in biodiversity is essential for predicting the outcomes of trophic downgrading across the world's ecosystems. However, assessing the biodiversity of morphologically cryptic lineages can be problematic, yet may be crucial to understanding ecological patterns. Shifts in keystone predation that favor increases in herbivore abundance tend to have negative consequences for the biodiversity of primary producers. However, in nearshore ecosystems, coralline algal cover increases when herbivory is intense, suggesting that corallines may uniquely benefit from trophic downgrading. Because many coralline algal species are morphologically cryptic and their diversity has been globally underestimated, increasing the resolution at which we distinguish species could dramatically alter our conclusions about the consequences of trophic dynamics for this group. In this study, we used DNA barcoding to compare the diversity and composition of cryptic coralline algal assemblages at sites that differ in urchin biomass and keystone predation by sea otters. We show that while coralline cover is greater in urchin-dominated sites (or "barrens"), which are subject to intense grazing, coralline assemblages in these urchin barrens are significantly less diverse than in kelp forests and are dominated by only 1 or 2 species. These findings clarify how food web structure relates to coralline community composition and reconcile patterns of total coralline cover with the widely documented pattern that keystone predation promotes biodiversity. Shifts in coralline diversity and distribution associated with transitions from kelp forests to urchin barrens could have ecosystem-level effects that would be missed by ignoring cryptic species' identities.

A first view on the unsuspected intragenus diversity of N-glycans in Chlorella microalgae.

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health-promoting food supplements. A mass spectrometry-based survey of N-glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N-glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N-acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan-type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4-O-methyl-N-acetylglucosamine), and substitution of the second N-acetylglucosamine. Analysis of barcode ITS1-5.8S-ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N-glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella-like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N-glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.

Interregulation of CDKA/CDK1 and the Plant-Specific Cyclin-Dependent Kinase CDKB in Control of the Chlamydomonas Cell Cycle.

The cyclin-dependent kinase CDK1 is essential for mitosis in fungi and animals. Plant genomes contain the CDK1 ortholog CDKA and a plant kingdom-specific relative, CDKB. The green alga Chlamydomonas reinhardtii has a long G1 growth period followed by rapid cycles of DNA replication and cell division. We show that null alleles of CDKA extend the growth period prior to the first division cycle and modestly extend the subsequent division cycles, but do not prevent cell division, indicating at most a minor role for the CDK1 ortholog in mitosis in Chlamydomonas. A null allele of cyclin A has a similar though less extreme phenotype. In contrast, both CDKB and cyclin B are essential for mitosis. CDK kinase activity measurements imply that the predominant in vivo complexes are probably cyclin A-CDKA and cyclin B-CDKB. We propose a negative feedback loop: CDKA activates cyclin B-CDKB. Cyclin B-CDKB in turn promotes mitotic entry and inactivates cyclin A-CDKA. Cyclin A-CDKA and cyclin B-CDKB may redundantly promote DNA replication. We show that the anaphase-promoting complex is required for inactivation of both CDKA and CDKB and is essential for anaphase. These results are consistent with findings in Arabidopsis thaliana and may delineate the core of plant kingdom cell cycle control that, compared with the well-studied yeast and animal systems, exhibits deep conservation in some respects and striking divergence in others.

Validation of superior reference genes for qRT-PCR and Western blot analyses in marine Emiliania huxleyi-virus model system.

AIMS: To search for a set of reference genes for reliable gene expression analysis in the globally important marine coccolithophore Emiliania huxleyi-virus model system. METHODS AND RESULTS: Fifteen housekeeping genes (CDKA, CYP15, EFG3, POLAI, RPL30, RPL13, SAMS, COX1, GPB1-2, HSP90, TUA, TUB, UBA1, CAM3 and GAPDH) were evaluated for their stability as potential reference genes for qRT-PCR using ΔCt, geNorm, NormFinder, Bestkeeper and RefFinder software. CDKA, TUA and TUB genes were tested as loading controls for Western blot in the same sample panel. Additionally, target genes associated with cell apoptosis, that is metacaspase genes, were applied to validate the selection of reference genes. The analysis results demonstrated that putative housekeeping genes exhibited significant variations in both mRNA and protein content during virus infection. After a comprehensive analysis with all the algorithms, CDKA and GAPDH were recommended as the most stable reference genes for E huxleyi virus (EhV) infection treatments. For Western blot, significant variation was seen for TUA and TUB, whereas CDKA was stably expressed, consistent with the results of qRT-PCR. CONCLUSIONS: CDKA and GAPDH are the best choice for gene and protein expression analysis than the other candidate reference genes under EhV infection conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The stable internal control genes identified in this work will help to improve the accuracy and reliability of gene expression analysis and gain insight into complex E. huxleyi-EhV interaction regulatory networks.

Combination of ferrocene decorated gold nanoparticles and engineered primers for the direct reagentless determination of isothermally amplified DNA.

A reagent-less DNA sensor has been developed exploiting a combination of gold nanoparticles, modified primers, and isothermal amplification. It is applied to the determination ofKarlodinium armiger, a toxic microalgae, as a model analyte to demonstrate this generic platform. Colloidal gold nanoparticles with an average diameter of 14 ± 0.87 nm were modified with a mixed self-assembled monolayer of thiolated 33-mer DNA probes and (6-mercaptohexyl) ferrocene. Modified primers, exploiting a C3 spacer between the primer-binding site and an engineered single-stranded tail, were used in an isothermal recombinase polymerase amplification reaction to produce an amplicon by two single-stranded tails. These tails were designed to be complementary to a gold electrode tethered capture oligo probe, and an oligo probe immobilized on the gold nanoparticles, respectively. The time required for hybridization of the target tailed DNA with the surface immobilized probe and reporter probe immobilized on AuNPs was optimized and reduced to 10 min, in both cases. Amplification time was further optimized to be 40 min to ensure the maximum signal. Under optimal conditions, the limit of detection was found to be 1.6 fM of target dsDNA. Finally, the developed biosensor was successfully applied to the detection of genomic DNA extracted from a seawater sample that had been spiked with K. armiger cells. The demonstrated generic electrochemical genosensor can be exploited for the detection of any DNA sequence and ongoing work is moving towards an integrated system for use at the point-of-need.

Labyrinthula diatomea n. sp.-A Labyrinthulid Associated with Marine Diatoms.

Labyrinthulomycetes are mostly fungus-like heterotrophic protists that absorb nutrients in an osmotrophic or phagotrophic manner. Members of order Labyrinthulida produce unique membrane-bound ectoplasmic networks for movement and feeding. Among the various types of labyrinthulids' food substrates, diatoms play an important role due to their ubiquitous distribution and abundant biomass. We isolated and cultivated new diatom consuming Labyrinthulida strains from shallow coastal marine sediments. We described Labyrinthula diatomea n. sp. that differs from all known labyrinthulids in both molecular and morphological features. We provided strain delimitation within the genus Labyrinthula based on ITS sequences via haplotype network construction and compared it with previous phylogenetic surveys.

Photosynthetic Sponge-associated Eukaryotes in the Aegean Sea: A Culture-dependent Approach.

Symbioses between sponges and photosynthetic organisms are very diverse regarding the taxonomy and biogeography of both hosts and symbionts; to date, most research has focused on the exploration of bacterial diversity. The present study aims to characterize the culturable diversity of photosynthetic eukaryotes associated with sponges in the Aegean Sea, on which no information exists. Five microalgae strains were isolated from marine sponges; the strains were characterized by morphological features, and the 18S rRNA, 18S-28S Internal Transcribed Spacer, and ribulose-bisphosphate carboxylase large chain (rbcL) sequences. Our polyphasic approach showed that the strains belonged to the green-alga Acrochaete leptochaete, the diatom Nanofrustulum cf. shiloi, the rhodophyte Acrochaetium spongicola, and the chlorachniophyte Lotharella oceanica. A. leptochaete is reported for the first time in sponges, even though green algae are known to be associated with sponges. Nanofrustulum shiloi was found in association with the sponges Agelas oroides and Chondrilla nucula, whereas information existed only for its association with the species Aplysina aerophoba. Acrochaetium spongicola was found for the first time in association with sponges in the eastern Mediterranean. Moreover, we report herein for the first time a sponge-chlorarachniophycean association. Our research revealed new diversity of microalgae associated with sponges and added new records of sponge species, previously unknown for their association with microalgae.

Four New Members of Foliicolous Green Algae Within the Watanabea Clade (Trebouxiophyceae, Chlorophyta) from China.

Members of the Watanabea clade of Trebouxiophyceae are genetically diverse and widely distributed in all kinds of habitats, especially in most terrestrial habitats. Ten new strains of terrestrial algae isolated from the tropical rainforest in China, and four published strains were investigated in this study. Morphological observation and molecular phylogenetic analyses based on the 18S, ITS, rbcL, and tufA genes were used to identify the new strains. Four previously described species were reinvestigated to supplement molecular data and autospores' morphological photographs. The phylogenetic analyses based on 18S only, the concatenated dataset of 18S and ITS, as well as the concatenated dataset of rbcL and tufA, showed the same phylogenetic positions and relationships of these new strains. According to the phylogenetic analysis and morphological comparisons results, we described these 10 strains as four new members within the Watanabea clade, Polulichloris yunnanensis sp. nov., Polulichloris ovale sp. nov., Massjukichlorella orientale sp. nov., and Massjukichlorella minus sp. nov., and two known species, Massjukichlorella epiphytica, and Mysteriochloris nanningensis. Additionally, we provide strong evidence proving that Phyllosiphon, Mysteriochloris, Polulichloris, and Desertella all reproduce through unequal sized autospores.

Progressive and Biased Divergent Evolution Underpins the Origin and Diversification of Peridinin Dinoflagellate Plastids.

Dinoflagellates are algae of tremendous importance to ecosystems and to public health. The cell biology and genome organization of dinoflagellate species is highly unusual. For example, the plastid genomes of peridinin-containing dinoflagellates encode only a minimal number of genes arranged on small elements termed "minicircles". Previous studies of peridinin plastid genes have found evidence for divergent sequence evolution, including extensive substitutions, novel insertions and deletions, and use of alternative translation initiation codons. Understanding the extent of this divergent evolution has been hampered by the lack of characterized peridinin plastid sequences. We have identified over 300 previously unannotated peridinin plastid mRNAs from published transcriptome projects, vastly increasing the number of sequences available. Using these data, we have produced a well-resolved phylogeny of peridinin plastid lineages, which uncovers several novel relationships within the dinoflagellates. This enables us to define changes to plastid sequences that occurred early in dinoflagellate evolution, and that have contributed to the subsequent diversification of individual dinoflagellate clades. We find that the origin of the peridinin dinoflagellates was specifically accompanied by elevations both in the overall number of substitutions that occurred on plastid sequences, and in the Ka/Ks ratio associated with plastid sequences, consistent with changes in selective pressure. These substitutions, alongside other changes, have accumulated progressively in individual peridinin plastid lineages. Throughout our entire dataset, we identify a persistent bias toward non-synonymous substitutions occurring on sequences encoding photosystem I subunits and stromal regions of peridinin plastid proteins, which may have underpinned the evolution of this unusual organelle.

Chloromonas arctica sp. nov., a psychrotolerant alga from snow in the High Arctic (Chlamydomonadales, Chlorophyta).

With the advent of molecular phylogenetic methods, it has become possible to assess the bioversity of snow algae more accurately. In this study, we focused on a morphological, ultrastructural and taxonomic description of a new Chloromonas-like alga isolated from snow in the High Arctic (Svalbard). Light and transmission electron microscopy revealed broad ellipsoidal or ellipsoidal-cylindrical, occasionally spherical cells with a chloroplast without a pyrenoid, an inconspicuous eyespot and a papilla. The size difference and the aforementioned morphological traits clearly distinguished the alga from its closest counterparts within the genus Chloromonas. Moreover, we were able to cultivate the alga at both 5 and 20 °C, revealing the psychrotolerant nature of the strain. Phylogenetic analyses of the plastid rbcL and nuclear 18S rRNA gene showed that the alga is nested within a clade containing a number of psychrotolerant strains within the Chloromonadinia phylogroup (Chlorophyceae). In the rbcL phylogeny, the alga formed an independent lineage, sister to the freshwater species Chloromonas paraserbinowii. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker showed support for a distinct species identity for the new strain. The ITS2 secondary structure of the new isolate differed from the closest matches 'Chlamydomonas' gerloffii and Choloromonas reticulata by three and five compensatory base changes, respectively. Considering the morphological and molecular differences from its closest relatives, a new psychrotolerant species from the Arctic, Choromonas arctica sp. nov., is proposed.

Phylogenic Diversity and Taxonomic Problems of the Dictyosphaerium Morphotype within the Parachlorella Clade (Chlorellaceae, Trebouxiophyceae).

The Parachlorella clade was put forward as a group within the family Chlorellaceae in 2004. Recent molecular analyses have revealed that Dictyosphaerium morphotype algae form several independent lineages within the Parachlorella clade, and new genera and species have been established. In this study, we focus on the diversity of Dictyosphaerium morphotype algae within the Parachlorella clade, based on 42 strains from China. We used combined analyses of morphology and molecular data based on SSU and internal transcribed spacer region (ITS) rDNA sequences to characterize these algae. In addition, the secondary structure of ITS2 was compared to delineate new lineages. Our results revealed high phylogenic diversity of Dictyosphaerium morphotype algae, and we describe five distinct lineages. We examined the morphological features of these five lineages, and morphological differences are difficult to find compared with other Dictyosphaerium morphotype algae. The five distinct lineages were not described as new genera currently. We lastly discuss the taxonomic problems regarding the Dictyosphaerium morphotype within the Parachlorella clade, and possible solutions are considered.

Microbial community changes during a toxic cyanobacterial bloom in an alkaline Hungarian lake.

The Carpathian Basin is a lowland plain located mainly in Hungary. Due to the nature of the bedrock, alluvial deposits, and a bowl shape, many lakes and ponds of the area are characterized by high alkalinity. In this study, we characterized temporal changes in eukaryal and bacterial community dynamics with high throughput sequencing and relate the changes to environmental conditions in Lake Velence located in Fejér county, Hungary. The sampled Lake Velence microbial populations (algal and bacterial) were analyzed to identify potential correlations with other community members and environmental parameters at six timepoints over 6 weeks in the Spring of 2012. Correlations between community members suggest a positive relationship between certain algal and bacterial populations (e.g. Chlamydomondaceae with Actinobacteria and Acidobacteria), while other correlations allude to changes in these relationships over time. During the study, high nitrogen availability may have favored non-nitrogen fixing cyanobacteria, such as the toxin-producing Microcystis aeruginosa, and the eutrophic effect may have been exacerbated by high phosphorus availability as well as the high calcium and magnesium content of the Carpathian Basin bedrock, potentially fostering exopolymer production and cell aggregation. Cyanobacterial bloom formation could have a negative environmental impact on other community members and potentially affect overall water quality as well as recreational activities. To our knowledge, this is the first prediction for relationships between photoautotrophic eukaryotes and bacteria from an alkaline, Hungarian lake.

A Case of Prototheca zopfii Genotype 1 Infection in a Dog (Canis lupus familiaris).

Protothecosis is a rare disease caused by environmental algae of the genus Prototheca. These are saprophytic, non-photosynthetic, aerobic, colorless algae that belong to the Chlorellaceae family. Seven different species have been described. Prototheca zopfii genotype 2 and P. wickerhamii are most commonly involved in pathogenic infections in humans and animals. The objective of this work is to describe, for the first time, a case of protothecosis caused by P. zopfii genotype 1 in a dog. The dog, a 4-year-old mix bred male, was presented to a veterinary clinic in Montevideo, Uruguay, with multiple skin nodules, one of which was excised by surgical biopsy. The sample was examined histologically and processed by PCR, DNA sequencing, and restriction fragments length polymorphisms for the detection and genotyping of P. zopfii. In addition, transmission electron microscopy and scanning electron microscopy were performed. Histology showed severe ulcerative granulomatous dermatitis and panniculitis with myriads of pleomorphic algae. Algal cells were 4-17 µm in size, with an amphophilic, 2-4-µm-thick wall frequently surrounded by a clear halo, contained flocculant material and a deeply basophilic nucleus, and internal septae with daughter cells (endospores) consistent with endosporulation. Ultrastructurally, algal cells/endospores at different stages of development were found within parasitophorous vacuoles in macrophages. Prototheca zopfii genotype 1 was identified by molecular testing, confirming the etiologic diagnosis of protothecosis.

Prototheca blaschkeae subsp. brasiliensis subsp. nov., isolated from cow milk.

A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T).

The First Isolation and Characterisation of the Protist Labyrinthula sp. in Southeastern Australia.

As a result of anthropogenic influences and global climate change, emerging infectious marine diseases are thought to be increasingly more common and more severe than in the past. The aim of our investigation was to confirm the presence of Labyrinthula, the aetiological agent of the seagrass wasting disease, in Southeastern Australia and provide the first isolation and characterisation of this protist, in Australia. Colonies and individual cells were positively identified as Labyrinthula using published descriptions, diagrams, and photographs. Their identity was then confirmed using DNA barcoding of a region of the 18S rRNA gene. Species level identification of isolates was not possible as the taxonomy of the Labyrinthula is still poorly resolved. Still, a diversity of Labyrinthula was isolated from small sections of the southeast coast of Australia. The isolates were grouped into three haplotypes that are biogeographically restricted. These haplotypes are closely related to previously identified saprotrophic clades. The study highlights the need for further investigation into the global distribution of Labyrinthula, including phylogenetic pathogenicity and analysis of host-parasite interactions in response to stressors. Given the results of our analyses, it is prudent to continue research into disease and epidemic agents to better prepare researchers for potential future outbreaks.

Use of Highly Specific Molecular Markers Reveals Positive Correlation between Abundances of Mesodinium cf. major and Its Preferred Prey, Teleaulax amphioxeia, During Red Water Blooms in the Columbia River Estuary.

In a previous study, Teleaulax amphioxeia-the preferred prey of Mesodinium in the Columbia River estuary-were undetectable within intense annual blooms, suggesting blooms are prey-limited or prey are acquired outside of bloom patches. We used a novel molecular approach specifically targeting the prey (i.e., Unique Sequence Element [USE] within the ribosomal RNA 28S D2 regions of T. amphioxeia nucleus and nucleomorph) in estuarine water samples acquired autonomously with an Environmental Sample Processor integrated within a monitoring network (ESP-SATURN). This new approach allowed for both more specific detection of the prey and better constraint of sample variability. A positive correlation was observed between abundances of M. cf. major and T. amphioxeia during bloom periods. The correlation was stronger at depth (> 8.2 m) and weak or nonexistent in the surface, suggesting that predator-prey dynamics become uncoupled when stratification is strong. We confirmed exclusive selectivity for T. amphioxeia by M. cf. major and observed the incorporation of the prey nucleus into a 4-nuclei complex, where it remained functionally active. The specific biomarker for T. amphioxeia was also recovered in M. cf. major samples from a Namibian coastal bloom, suggesting that a specific predator-prey relationship might be widespread between M. cf. major and T. amphioxeia.

Molecular phylogeny and taxonomic revision of the genus Wittrockiella (Pithophoraceae, Cladophorales), including the descriptions of W. australis sp. nov. and W. zosterae sp. nov.

Wittrockiella is a small genus of filamentous green algae that occurs in habitats with reduced or fluctuating salinities. Many aspects of the basic biology of these algae are still unknown and the phylogenetic relationships within the genus have not been fully explored. We provide a phylogeny based on three ribosomal markers (ITS, LSU, and SSU rDNA) of the genus, including broad intraspecific sampling for W. lyallii and W. salina, recommendations for the use of existing names are made, and highlight aspects of their physiology and life cycle. Molecular data indicate that there are five species of Wittrockiella. Two new species, W. australis and W. zosterae, are described, both are endophytes. Although W. lyallii and W. salina can be identified morphologically, there are no diagnostic morphological characters to distinguish between W. amphibia, W. australis, and W. zosterae. A range of low molecular weight carbohydrates were analyzed but proved to not be taxonomically informative. The distribution range of W. salina is extended to the Northern Hemisphere as this species has been found in brackish lakes in Japan. Furthermore, it is shown that there are no grounds to recognize W. salina var. kraftii, which was described as an endemic variety from a freshwater habitat on Lord Howe Island, Australia. Culture experiments indicate that W. australis has a preference for growth in lower salinities over full seawater. For W. amphibia and W. zosterae, sexual reproduction is documented, and the split of these species is possibly attributable to polyploidization.

Complete mitochondrial genomes of prasinophyte algae Pyramimonas parkeae and Cymbomonas tetramitiformis.

Mitochondria are archetypal eukaryotic organelles that were acquired by endosymbiosis of an ancient species of alpha-proteobacteria by the last eukaryotic common ancestor. The genetic information contained within the mitochondrial genome has been an important source of information for resolving relationships among eukaryotic taxa. In this study, we utilized mitochondrial and chloroplast genomes to explore relationships among prasinophytes. Prasinophytes are represented by diverse early-diverging green algae whose physical structures and genomes have the potential to elucidate the traits of the last common ancestor of the Viridiplantae (or Chloroplastida). We constructed de novo mitochondrial genomes for two prasinophyte algal species, Pyramimonas parkeae and Cymbomonas tetramitiformis, representing the prasinophyte clade. Comparisons of genome structure and gene order between these species and to those of other prasinophytes revealed that the mitochondrial genomes of P. parkeae and C. tetramitiformis are more similar to each other than to other prasinophytes, consistent with other molecular inferences of the close relationship between these two species. Phylogenetic analyses using the inferred amino acid sequences of mitochondrial and chloroplast protein-coding genes resolved a clade consisting of P. parkeae and C. tetramitiformis; and this group (representing the prasinophyte clade I) branched with the clade II, consistent with previous studies based on the use of nuclear gene markers.