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Thick‐walled rosette‐like snow algae were long thought to be a life stage of various other species of snow algae. Rosette‐like cells have not been cultured, but by manually isolating cells from 38 field samples in southern British Columbia, we assigned a variety of rosette morphologies to DNA sequence. Phylogenetic analysis of Rubisco large‐subunit (rbcL) gene, ribosomal internal transcribed spacer 2 (ITS2) rRNA region, and 18S rRNA gene revealed that the rosette‐like cells form a new clade within the phylogroup Chloromonadinia. Based on these data, we designate a new genus, Rosetta, which comprises five novel species: R. castellata, R. floranivea, R. stellaria, R. rubriterra, and R. papavera. In a survey of 762 snow samples from British Columbia, we observed R. floranivea exclusively on snow overlying high‐elevation glaciers, whereas R. castellata was observed at lower elevations, near the tree line. The other three species were rarely observed. Spherical red cells enveloped in a thin translucent sac were conspecific with Rosetta, possibly a developmental stage. These results highlight the unexplored diversity among snow algae and emphasize the utility of single‐cell isolation to advance the centuries‐old problem of disentangling life stages and cryptic species. [ABSTRACT FROM AUTHOR]

The modern concept of the genus Chloromonas includes a subclade of species found exclusively in snow. We describe Chloromonas (Cr.) fuhrii sp. nov., a new member of this clade, which is closely related to Cr. muramotoi from Japan. The new species is characterised by the absence of a stigma, pyrenoid, and defined papilla, ready loss of flagella in culture, and a single lobed chloroplast. Strains of Cr. fuhrii have been sourced from green, orange, and pink/red snow. The species has been found in Svalbard, Antarctica, and New Zealand, with most genetic variation occurring in Svalbard. The minimal variation present in New Zealand strains suggests a relatively recent arrival. Secondary structure models for ITS2 in the new species demonstrate a separation from Cr. muramotoi due to one CBC. The nine New Zealand strains isolated showed no variation in ITS2 but four variable base positions in ITS1. Inclusion of the single Antarctic and four Svalbard strains resulted in 14 variable positions in ITS2 and 69 in ITS1, with the sequences remaining alignable. The ITS data showed a strong biogeographic signal, both in total in an unrooted tree, and using ITS2 alone with Cr. muramotoi as an outgroup. The absence of a stigma in this species prompted a survey of the literature, showing that stigma absence in Chloromonas is strongly associated with the snow habitat, occurring at about 10 times the rate observed in other habitats, perhaps owing to the difficulty of phototaxis in snow with abundant reflected light. [ABSTRACT FROM AUTHOR]

Snow algae blooms are common occurrences in alpine systems and contribute to increasing snow and glacial ice melt rates. Despite the cosmopolitan distribution of snow algae, little is known about the role their life cycle plays in community composition and activity in snowpack. Mount Baker, in Washington, USA, is a rapidly changing alpine ecosystem. Here we present estimates of cellular biomass, microbial community composition, and primary productivity measurements associated with surficial snow algae blooms and a vertical profile within a snow pit to characterize the microbial ecology of this snowpack system. Chloromonas was the most abundant algae detected in our samples. Sphingobacteriaceae were the most dominant bacteria in samples where fungi were the most abundant eukaryote, whereas Chitinophagaceae dominated when Chlorophyceae were the most abundant eukaryote. In a snow pit vertical profile, we observed more algae in the green-pigmented, flagellated life cycle stage at depth (~30 cm below the surface) than at the surface where the astaxanthin-rich, aplanospore life cycle stage dominated. Higher resolution analyses revealed that Chloromonas-associated photosynthesis transcripts were more abundant at depth than at the surface of the snowpack. These results suggest that there is a photosynthetic niche within alpine snowpack that has unknown effects on the carbon budget and provides potentially undetected primary productivity resulting in a “cryptic” photosynthetic system.

The quadriflagellate genus Chlainomonas frequently dominates red snow globally. It is unusual in several respects, with two separated pairs of flagella, apparent cell division via extrusion of cytoplasmic threads, and being nested phylogenetically within the biflagellate genus Chloromonas. Here, we showed that the austral species Chloromonas (Cr.) rubroleosa, originally described from Antarctic red snow, is a close biflagellate relative of Chlainomonas, challenging the monophyly of Chlainomonas as currently conceived. Sequences of the 18S rRNA gene robustly linked Cr. rubroleosa with near‐identical environmental sequences from Antarctic red snow and Chlainomonas from North America, Japan, and Europe. Furthermore, the 18S rRNA and rbcL gene sequences of Cr. rubroleosa were almost identical to New Zealand and North American collections of Chlainomonas. Cr. rubroleosa and New Zealand Chlainomonas are separated by only a single‐base substitution across the ITS1‐5.8S‐ITS2 rRNA loci (and according to ITS2, the North American collection is the next closest relative). This again raises the possibility that Chlainomonas is a life‐cycle stage of vegetatively biflagellate organisms, although this remains confounded by the scarcity of biflagellates in field populations, the apparent cell division by quadriflagellates, and the absence of Chlainomonas‐type cells in cultures of Cr. rubroleosa. The latter species is broadly similar to Chlainomonas, being poor at swimming, with similar pigment, chloroplast arrangement and ultrastructure, and is relatively large. Increased size is a feature of the wider clade of "Group D" snow algae. A synthesis of field and laboratory investigations may be needed to unravel the life cycle and correct the systematics of this group. [ABSTRACT FROM AUTHOR]

Snow algae blooms are common occurrences in alpine systems and contribute to increasing snow and glacial ice melt rates. Despite the cosmopolitan distribution of snow algae, little is known about the role their life cycle plays in community composition and activity in snowpack. Mount Baker, in Washington, USA, is a rapidly changing alpine ecosystem. Here we present estimates of cellular biomass, microbial community composition, and primary productivity measurements associated with surficial snow algae blooms and a vertical profile within a snow pit to characterize the microbial ecology of this snowpack system. Chloromonas was the most abundant algae detected in our samples. Sphingobacteriaceae were the most dominant bacteria in samples where fungi were the most abundant eukaryote, whereas Chitinophagaceae dominated when Chlorophyceae were the most abundant eukaryote. In a snow pit vertical profile, we observed more algae in the green-pigmented, flagellated life cycle stage at depth (~30 cm below the surface) than at the surface where the astaxanthin-rich, aplanospore life cycle stage dominated. Higher resolution analyses revealed that Chloromonas-associated photosynthesis transcripts were more abundant at depth than at the surface of the snowpack. These results suggest that there is a photosynthetic niche within alpine snowpack that has unknown effects on the carbon budget and provides potentially undetected primary productivity resulting in a "cryptic" photosynthetic system. [ABSTRACT FROM AUTHOR]

Snow algae are photosynthetic microbes growing on snowpacks and are commonly observed in mountain areas in Japan. In this study, we report the snow algal blooms observed in Mt. Echigo-Komagatake located in Niigata prefecture, Japan. Field investigation was conducted in late June of 2021 on a snow patch located in an alpine zone at an altitude of 2000 m above sea level. Patches of snow with red, green, or brown color were observed on the snowpack. Microscopic observation showed that the colored snow contained various morpho-types of snow algal cells, which appeared to be similar to those previously reported in Japan. The algal communities of the colored snow were mostly dominated by red spherical cells, which is likely to be Sanguina nivaloides, and consistent with those of the red snow reported in other alpine snowpacks in Japan. However, some samples showed a community consisting of various cell types, including multiple species of genus Chloromonas. The difference in community structure may be due to distance from vegetation around the snow patch, nutrient conditions of the snow, and/or growth period of the algae. The snow algal blooms of Mt. Echigo-Komagatake can be characterized by the appearance of various colors and algal communities on a snowpack. [ABSTRACT FROM AUTHOR]

Abstract Background Pyrenoids are protein microcompartments composed mainly of Rubisco that are localized in the chloroplasts of many photosynthetic organisms. Pyrenoids contribute to the CO2-concentrating mechanism. This organelle has been lost many times during algal/plant evolution, including with the origin of land plants. The molecular basis of the evolutionary loss of pyrenoids is a major topic in evolutionary biology. Recently, it was hypothesized that pyrenoid formation is controlled by the hydrophobicity of the two helices on the surface of the Rubisco small subunit (RBCS), but the relationship between hydrophobicity and pyrenoid loss during the evolution of closely related algal/plant lineages has not been examined. Here, we focused on, the Reticulata group of the unicellular green algal genus Chloromonas, within which pyrenoids are present in some species, although they are absent in the closely related species. Results Based on de novo transcriptome analysis and Sanger sequencing of cloned reverse transcription-polymerase chain reaction products, rbcS sequences were determined from 11 strains of two pyrenoid-lacking and three pyrenoid-containing species of the Reticulata group. We found that the hydrophobicity of the RBCS helices was roughly correlated with the presence or absence of pyrenoids within the Reticulata group and that a decrease in the hydrophobicity of the RBCS helices may have primarily caused pyrenoid loss during the evolution of this group. Conclusions Although we suggest that the observed correlation may only exist for the Reticulata group, this is still an interesting study that provides novel insight into a potential mechanism determining initial evolutionary steps of gain and loss of the pyrenoid.

In algae, it is well established that the pyrenoid, a component of the carbon-concentrating mechanism (CCM), is essential for efficient photosynthesis at low CO2. However, the signal that triggers the formation of the pyrenoid has remained elusive. Here, we show that, in Chlamydomonas reinhardtii, the pyrenoid is strongly induced by hyperoxia, even at high CO2 or bicarbonate levels. These results suggest that the pyrenoid can be induced by a common product of photosynthesis specific to low CO2 or hyperoxia. Consistent with this view, the photorespiratory by-product, H2O2, induced the pyrenoid, suggesting that it acts as a signal. Finally, we show evidence for linkages between genetic variations in hyperoxia tolerance, H2O2 signaling, and pyrenoid morphologies.

Snow algae are photosynthetic microbes that inhabit the melting snow surface in alpine and polar regions. We analyzed the pigment and species composition of colored snow collected on Mt. Tateyama in Japan during the melting seasons of 2015 and 2016. High-performance liquid chromatographic analyses of the pigments extracted from the colored snow showed that their composition varied within the study area and were classified into four types: Type A (astaxanthin-monoester dominant), Type B (medium astaxanthin-monoester content), Type C (abundant primary carotenoids and free-astaxanthin), and Type D (abundant primary carotenoids and astaxanthin diesters). Types A and B were most commonly observed in the study area, whereas Types C and D appeared only at specific sites. Analysis of the 18S ribosomal RNA (18S rRNA) gene revealed six major amplicon sequence variants (ASVs) of snow algae, belonging to the Sanguina, Chloromonas , and Chlainomonas groups. The relative abundance of the algal ASVs showed that Sanguina was dominant (>48%) in both Types A and B, suggesting that the difference in astaxanthin abundance between the two types was caused by the production of pigments in the algal cells. The algal community structures of Types C and D differed from those of Types A and B, indicating that the primary carotenoids and astaxanthin diesters were derived from certain algal species in these types. Therefore, astaxanthin-rich Sanguina algae mostly induced the red snow that appeared widely in this alpine area; however, they were partially dominated by Chloromonas or Chlainomonas algae, causing different pigment compositions. [ABSTRACT FROM AUTHOR]

Microalgae, the primary producers in aquatic ecosystems, are highly susceptible to heavy metal contamination. In this study, the interactive effects of warming and copper (Cu) toxicity on the physiology (cell density, photosynthetic efficiency, reactive oxygen species (ROS) production, and metal uptake in the biomass) and biochemistry (metabolite) of a freshwater green microalga, Chloromonas augustae (UMACC246), were elucidated. The microalgae were exposed to culture media supplemented with copper (II) sulfate pentahydrate (CuSO4·5H2O) at different concentrations (50, 150, 250 μM) at two temperatures, 25 °C (control) and 30 °C (sub-optimal), for 24 h. The results indicated that C. augustae exhibited a concentration- and temperature-dependent decrease in the cell density. Copper greatly affected the photosynthetic efficiency of C. augustae by reducing the maximum rate of relative electron transport (rETRm), light harvesting efficiency (α), and saturation irradiance (Ek). Warming increased ROS production remarkably in the microalga. Untargeted metabolomics indicated that temperature contributed to the most significant variations in the cultures (p < 0.05) in comparison with Cu toxicity or both factors combined. Compounds such as amino acids and amines were significantly dysregulated in response to warming and Cu toxicity. Pathway analyses showed that the glutathione metabolism, sulfur metabolism, and mechanisms in the amino acid metabolism were regulated, suggesting that the microalga underwent primary metabolism restructuring for survival and adaptation. Overall, the data showed that warming enhanced Cu toxicity in the cultures. This implied that increasing water temperature and metal toxicity due to global warming and anthropogenic activities will probably exacerbate existing threats to the primary producers. [ABSTRACT FROM AUTHOR]

The phylogroup Chloromonadinia (Volvocales, Chlorophyceae) encompasses ecologically versatile green microalgae with a predominantly freshwater and snow lifestyle. Although a few species have been described from soils, the evolutionary history of terrestrial volvocalean flagellates is poorly known due to undersampling. Here we provide new insights into the phylogenetic position of terrestrial flagellates within the Chloromonadinia and investigate six unresolved strains from terrestrial and freshwater habitats in both hemispheres. The terrestrial strains differed in cell morphology, ultrastructure and lifestyle from the freshwater ones, therefore, we expected them to represent distinct evolutionary lineages (species or genera). While the terrestrial strain SAG 25.87 (Chloromonas gracillima comb. & stat. nov.) from Czech mountains clustered within the psychrotolerant core Chloromonas clade, the other terrestrial strains formed a phylogenetically separate lineage which we proposed as Ostravamonas gen. nov. The new genus differs from other closely related Chloromonadinia genera by chloroplast, eyespot, papilla and cell-wall morphology. Two terrestrial strains, CAUP G 1401 (coal spoil heap; Czechia) and SAG 12.72 (acidic soil; New Zealand), were designated as Ostravamonas chlorostellata comb. nov. and represent the first Chloromonadinia microalgae discovered in acidic terrestrial environments. Closely related 'Chlamydomonas' strains SAG 75.81 (ditch; France) and CCAP 11/108 (origin unknown) were reassigned to Ostravamonas meslinii comb. nov. and Ostravamonas tenuiincisa sp. nov., respectively. Our study shows that the traditional 'Chlamydomonas' still hides an overlooked diversity of species with a terrestrial lifestyle that are nested phylogenetically within the Chloromonadinia phylogroup. [ABSTRACT FROM AUTHOR]

Microalgal ice-binding proteins (IBPs) in the polar region are poorly understood at the genome-wide level, although they are important for cold adaptation. Through the transcriptome study with the Arctic green alga Chloromonas sp. KNF0032, we identified six Chloromonas IBP genes (CmIBPs), homologous with the previously reported IBPs from Antarctic snow alga CCMP681 and Antarctic Chloromonas sp. They were organized with multiple exon/intron structures and low-temperature-responsive cis -elements in their promoters and abundantly expressed at low temperature. The biological functions of three representative CmIBPs (CmIBP1, CmIBP2 and CmIBP3) were tested using in vitro analysis and transgenic plant system. CmIBP1 had the most effective ice recrystallization inhibition (IRI) activities in both in vitro and transgenic plants, and CmIBP2 and CmIBP3 had followed. All transgenic plants grown under nonacclimated condition were freezing tolerant, and especially 35S::CmIBP1 plants were most effective. After cold acclimation, only 35S::CmIBP2 plants showed slightly increased freezing tolerance. Structurally, the CmIBPs were predicted to have β-solenoid forms with parallel β-sheets and repeated TXT motifs. The repeated TXT structure of CmIBPs appears similar to the AidA domain-containing adhesin-like proteins from methanogens. We have shown that the AidA domain has IRI activity as CmIBPs and phylogenetic analysis also supported that the AidA domains are monophyletic with ice-binding domain of CmIBPs, and these results suggest that CmIBPs are a type of modified adhesins. [ABSTRACT FROM AUTHOR]

Recent molecular data has strongly suggested that field-collected cysts of snow algae that are morphologically identifiable as the zygotes of Chloromonas nivalis are composed of multiple species. Motile vegetative cells, however, have not been directly obtained from these cysts because of the difficulties involved in inducing their germination. Recently, our comparative molecular analyses, using both field-collected and cultured materials, demonstrated that one Japanese lineage of “C. nivalis zygotes” belongs to C. miwae. Herein, we examined another Japanese lineage of field-collected “C. nivalis zygotes” and a new strain originating from Japan. Our molecular data demonstrated that these two different life cycle stages are conspecific, and that they represent a new species that we herein describe as C. muramotoi sp. nov., based on the vegetative and asexual morphological characteristics of the strain. Multigene phylogenetic analyses showed that this new species was sister to C. miwae. Scanning electron microscopy demonstrated that the cysts of C. muramotoi are different from those of C. miwae, based on the arrangement of the flanges developing on the cell wall. [ABSTRACT FROM AUTHOR]

Snow is not only abiotic component of cryosphere but also a true functional ecosystem. In the past were the Vysoké Tatry Mts, the highest mountains of the Carpathians, the most studied mountain area in the Europe in terms of cryoflora. The aim of this study was to investigate the occurrence of snow microorganisms and its environmental conditions also in other high mountains of Slovakia. Due to climatic conditions of high altitudes, snow patches persist in deep concave relief shapes until late of June. Therefore these conditions are suitable for snow microorganisms. Sampling of snow microorganisms was carried out from May to September of 2013 and 2014. In selected mountains were mostly found snow alga cf. Chloromonas nivalis and we found also the presence of individuals of cyanobacteria, microscopic fungi, ciliates, rotifer and tardigrade. Recorded occurrence of thaw-froze cycles during august and high intensity of photosynthetically active radiation (PAR) (max. 2133 μmol.m−2.s−1) could be considered as stress factors this harsh environment. The pH values of taken samples were slightly acidified, in some cases neutral. The conductivity of water from melted snow had high coefficient of variation (values characteristic for distilled water until values 160 μS.cm−1). The chemical analyses determined the higher content of N-NO3 in analysed samples of melted snow in compare with content of N-NH4 and P-PO4. Despite these facts paper confirms potential of Slovak high mountains as suitable place for life of snow microorganisms. [ABSTRACT FROM AUTHOR]

The traditional green algal genus Chloromonas accommodates mesophilic, cold‐tolerant and cold‐adapted microorganisms. In this paper, we studied a new strain isolated from a wet hummock meadow in the High Arctic. We used morphological, ultrastructural and molecular data to assess the taxonomic position and phylogenetic relationships of the new isolate. The observed morphological features generally corresponded to the cold‐tolerant Chloromonas characteristics. However, ellipsoidal or wide ellipsoidal vegetative cells, a massive parietal cup‐shaped chloroplast with a number of continuously connected lobes, a thick cell wall, a prominent hemispherical papilla and the anterior position of an oblong or round eyespot distinguished the alga from all previously described Chloromonas species. Analyses of rbcL and 18S rRNA genes showed that the new strain formed an independent lineage within a clade containing mesophilic and psychrotolerant Chloromonas species. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker supported a separate species identity of the new isolate. Considering the morphological and molecular differences from its relatives, a new psychrotolerant species, Chloromonas svalbardensis, is proposed. Further, our results demonstrated the paraphyletic origin of Chloromonas within Chloromonadinia with genetically, morphologically and ecologically well‐defined clades. We discuss a scenario of a possible Chloromonas split and revision. [ABSTRACT FROM AUTHOR]

Psychrophilic algae blooms can be observed coloring the snow during the melt season in alpine snowfields. These algae are important primary producers on the snow surface environment, supporting the microbial community that coexists with algae, which includes heterotrophic bacteria and fungi. In this study, we analyzed the microbial community of green and red-colored snow containing algae from Mount Asahi, Japan. We found that Chloromonas spp. are the dominant algae in all samples analyzed, and Chlamydomonas is the second-most abundant genus in the red snow. For the bacterial community profile, species belonging to the subphylum Betaproteobacteria were frequently detected in both green and red snow, while members of the phylum Bacteroidetes were also prominent in red snow. Furthermore, multiple independently obtained strains of Chloromonas sp. from inoculates of red snow resulted in the growth of Betaproteobacteria with the alga and the presence of bacteria appears to support growth of the xenic algal cultures under laboratory conditions. The dominance of Betaproteobacteria in algae-containing snow in combination with the detection of Chloromonas sp. with Betaproteobacteria strains suggest that these bacteria can utilize the available carbon source in algae-rich environments and may in turn promote algal growth.

Chloromonas nivalis (Volvocales, Chlorophyceae) is considered a cosmopolitan species of a snow-inhabiting microalga because cysts morphologically identifiable as zygotes of the species are distributed worldwide. However, recent molecular data demonstrated that field-collected cysts identified as the zygotes consist of multiple species. Recently, we demonstrated that species identification of snow-inhabiting Chloromonas species is possible based on light and electron microscopy of asexual life cycles in strains and molecular phylogenetic analyses. Vegetative cells without eyespots and of inverted-teardrop shape have been reported once in North American material of C. nivalis; however, strains with such vegetative cells in snow-inhabiting species of Chloromonas have not been examined taxonomically in detail. Here, we used light and transmission electron microscopy together with molecular analyses of multiple DNA sequences to examine several C. nivalis strains. The morphological data demonstrated that one North American strain could be identified as C. nivalis, whereas three other strains should be re-classified as C. hoshawii sp. nov. and C. remiasii sp. nov. based on vegetative cell morphology, the number of zoospores within the parental cell wall during asexual reproduction, and whether cell aggregates (resulting from repeated divisions of daughter cells retained within a parental cell wall) were observed in the culture. This taxonomic treatment was supported by multigene phylogeny and comparative molecular analyses that included a rapidly evolving DNA region. Our molecular phylogenetic analyses also demonstrated that the North American strain of C. nivalis was phylogenetically separated from the Austrian and Japanese specimens previously identified as C. nivalis based on zygote morphology. [ABSTRACT FROM AUTHOR]

A series of experiments were conducted to obtain an efficient larval rearing protocol for Megabalanus azoricus. The first part of this study investigates the effect of microalgae-based diets on survival and larval development. Mono and mixed-diets were tested at 20 ± 1°C, in a sequence of 11-day feeding experiments. The second part presents a preliminary study on the influence of a biofilm on recruitment and use of oyster spat collectors in a mass rearing system. A photographic record of larval development and a brief reference to the diagnostic features that enable quick larval staging are also presented, along with morphometric measurements. Of the microalgae tested ( Chaetoceros sp., Chloromonas sp., Dunaliella sp., T- Isochrysis sp. and Skeletonema sp.) the mixed-diet Skeletonema sp. with T- Isocrysis sp. showed the highest survival percentages: total survival ranged from 79.7 to 85.7% and 69.7-80.0% of nauplii were in stage VI after 11 days of rearing. Cypris were also present, but only represented 5.3% of the survivors at most. In the mass rearing system juveniles were found settled in the collectors after 25 days, at 20 ± 1°C. However recruitment was less than 1%. Preliminary results showed no settlement preference towards collectors with biofilm. Nevertheless, this study provides the first record of M. azoricus settlement under laboratorial conditions and represent a starting point for future larval rearing studies. [ABSTRACT FROM AUTHOR]

On the basis of prior phylogenetic analyses placing Gloeomonas as a lineage within the very diverse genus Chloromonas, it was hypothesized that the morphologically distinct Gloeomonas, bearing widely spaced basal bodies, evolved from ancestral organisms like Chloromonas. In addition, the phylogenetically related Chloromonas ( Cr.) rubrifilum, was expected to possess traits intermediate to the two forms. To test these hypotheses, we performed detailed transmission electron microscope analysis on nine species of these genera. The species were divided into two categories on the basis of ultrastructural features: group 1, consisting of four diverse species of Chloromonas and Ixipapillifera, had a V-shaped basal body arrangement, and group 2, of Cr. rubrifilum and four Gloeomonas species, forming a single clade possessing widely spaced basal bodies. Members of group 1 commonly had a simple, flat plate-like distal connecting fiber (dcf), three or sometimes two microtubules in the sinister root, continuous dexter and sinister striated microtubule-associated fibers (SMAFs), and a coarsely striated proximal fiber (CSPF) at the posterior-most regions of the basal bodies and probasal bodies, but lacked proximal and median proximal connecting fibers. Group 2 inherited these features, but had novel traits including the elongation and modification of the dcf, SMAFs, and CSPF that occurred with separation of basal bodies during growth, a dcf-associated layered structure, as well as protruding flagellar collars. The ultrastructural traits of Gloeomonas are interpreted as being evolutionarily modified from an ancestral Chloromonas morphology. Specific ultrastructural features were determined to be useful in characterizing these genera. Cr. rubrifilum of group 2 was not intermediate morphologically, but possessed the traits of Gloeomonas; thus G. rubrifilum comb. nov. was proposed. [ABSTRACT FROM AUTHOR]

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP’s biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP. [ABSTRACT FROM AUTHOR]

Chloromonas brevispina and C. nivalis (Volvocales, Chlorophyceae) are considered cosmopolitan, as the aplanozygotes assignable to the two species are distributed worldwide. Although recent phylogenetic analyses have indicated the possible polyphyly of aplanozygotes identified as C. nivalis, the analytical resolution was not robust, possibly because only single genes were analysed. Here, we obtained long sequences of multiple DNA regions from 50 identical [on the basis of light microscopy (LM)] aplanozygotes in a field-collected sample, and the phylogenetic positions of these aplanozygotes (identified as C. brevispina and C. nivalis on the basis of LM) were determined with high statistical suppoit using a multigene phylogeny. Comparisons of the present and previously published sequence data indicated that aplanozygotes of C. nivalis originating from Japan and Austria represented at least four different lineages all of which were robustly separated from the North American strain CU563D of C. nivalis. Molecular analyses including the comparison of highly evolving nuclear ribosomal DNA internal transcribed spacer 2 demonstrated that one Japanese lineage of C. nivalis aplanozygotes was conspecific with C. miwae strains. In addition, the present C. brevispina aplanozygote specimens from Japan were assigned to C. krienitzii sp. nov., which was herein delineated on the basis of LM and electron microscopy and the molecular phylogeny of newly established strains. [ABSTRACT FROM AUTHOR]

Variation is essential to ecological and evolutionary dynamics, but genetic variation of quantitative traits may be concentrated in a limited number of dimensions, constraining ecoevolutionary dynamics. We describe high-dimension variation in natural accessions of the model alga, Chlamydomonas reinhardtii, and test the hypothesis that extensive fitness variation across 30 environments is constrained to a small number of axes., We used high-throughput phenotyping to investigate morphological, fitness, and genotype × environment (G × E) variation in 18 natural C. reinhardtii accessions in 30 environments., The organismal phenotypes of cell cycle, cell size, and phototactic behavior exhibited substantial genetic variation between lines, and we found up to 74-fold fitness variation across accessions and environments. Approximately 47% of the extensive G × E variation is accounted for by the first two principal components (PCs) of the G-matrix corresponding to covariation in metals response, nitrogen availability, or salt and nutrient response., The natural variation of C. reinhardtii accessions supports the hypothesis that, despite abundant genetic variation across single environments, the species' adaptive response should be constrained along few major axes of selection. These results highlight the utility of natural accessions for integrating ecoevolutionary and genetic research. [ABSTRACT FROM AUTHOR]

All ice-and snow-related unicellular algae examined so far secrete ice-binding proteins (IBPs) to mitigate freezing damage. Two types of IBP have been identified in chlorophytes. Type 1 IBPs are members of a large family of proteins that share a large domain of unknown function (DUF3494). Previous studies have suggested that the type 1 algal IBP genes were acquired by horizontal gene transfer. To test this hypothesis I sequenced the IBP genes of a snow alga, Chloromonas brevispina. The IBPs were identified by ice affinity purification, de novo sequencing of a tryptic peptide and large-scale sequencing of the transcriptome and genome. C. brevispina has genes for over 20 IBP isoforms, which strongly indicates their importance. The IBPs are all of type 1 and match fungal and bacterial proteins more closely than they match known algal IBPs, providing further evidence that the genes were acquired by horizontal transfer. Modeling of the 3D structures of the IBPs based on the known structure of a homologous protein suggests that the ice-binding site has characteristics that are shared by all DUF3494 proteins. [ABSTRACT FROM AUTHOR]

Although zygote morphology is important in traditional taxonomic systems of snow-inhabiting species of Chloromonas (Volvocales, Chlorophyceae), induction of sexual reproduction or hypnospore formation is difficult in culture. Thus, alternative taxonomic methodologies are required when sexual reproduction does not occur in cultures. Here, we performed a taxonomic study of snow-inhabiting Chloromonas species based on culture strains with elongate or ellipsoidal vegetative cells. Our comparative light and electron microscopy demonstrated that the strains were clearly distinguished into six species based on differences in vegetative cell shape and chloroplast morphology, the number of zoospores within the parental cell and the presence or absence of cell aggregates in old cultures. The six species are Chloromonas chenangoensis, C. fukushimae sp. nov., C. hohamii. C. pichinchae, C. tenuis sp. nov. and C. tughillensis. Although four of the six species formed a small clade based on phylogenetic analysis of multiple genes, their separation was supported by comparison of secondary structures of the internal transcribed spacer 2 of nuclear ribosomal DNA and genetic differences of nuclear- and chloroplast-encoded genes. The present study demonstrated that polyphasic analyses of culture strains of snow Chloromonas species could be used for the identification of natural species [ABSTRACT FROM AUTHOR]

Highlights: [•] Lipidomic analysis of snow alga Chloromonas pichinchae was performed by high-resolution mass spectrometry. [•] Triacylglycerols having at least one PUFA were found in C. pichinchae. [•] Silver-LC and NARP-LC/APCI-MS were used for analysis of regioisomers of TAGs. [•] Alga C. pichinchae may be a potential biotechnological source of C16 PUFAs. [ABSTRACT FROM AUTHOR]

Chloromonas kasaiae sp. nov. (Volvocales, Chlorophyceae) was described based on a new culture strain originating from Japan. Based on light microscopy, vegetative cells of this new species were very similar to those of C. reticulata and C. serbinowii. However, C. kasaiae differed from these two species in the ultrastructure of eyespots (number of globule layers). Our phylogenetic analyses, based on a combined data set from nuclear-encoded 18S ribosomal (r) RNA, the ATP synthase beta subunit and P700 chlorophyll α apoprotein A2 genes, robustly resolved that C. kasaiae was separate from C. reticulata. Although these analyses did not resolve the phylogenetic relationship between C. kasaiae and C. serbinowii, separation of the two was supported based on a comparison of the secondary structure of nuclear rDNA internal transcribed spacer 2. [ABSTRACT FROM AUTHOR]

Chloromonas reticulata (Goroschankin) Gobi, the type species for Chloromonas, was taxonomicatly revised using nine strains previously assigned to the species, based on light and transmission electron microscopy and multigene phylogenetic analyses. Based on vegetative cell and papilla shape, eyespot form and position, and the ultrastructure of the eyespot and pyrenoid, the nine strains were classified into four species: C. reticulata; C. rosae (H. & O. Ettl) H. Ettl; C. chlorococcoides (H. Ettl & K. Schwarz) Matsuzaki, Y. Hara & Nozaki comb. nov.; and C. typhlos (Gerloff) Matsuzaki, Y. Hara & Nozaki comb. nov. The taxonomic revision was supported by the robust monophyly of the species resolved in phylogenetic analyses using a combined dataset that included 18S ribosomal (r) RNA, ATP synthase 13-subunit, P700 chlorophyll a-apoprotein Al and A2 genes, and nuclear rDNA internal transcribed spacer 2 (ITS2) sequences. The phylogenetic analyses demonstrated that the pyrenoid-lacking species C. reticulata and the pyrenoid-containing species C. chlorococcoides, which has a dense pyrenoid matrix associated with starch grains, formed a robust monophyletic group to which the pyrenoid-lacking species C. rosae and the pyrenoid-containing species C. typhlos were basal. Comparison of the C. reticulata and C. chlorococcoides nuclear rDNA ITS2 secondary structures revealed two compensatory base changes in the conserved helix III region, suggesting a separation between these sister species. [ABSTRACT FROM AUTHOR]

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Amongst a specialised group of psychrophilic microalgae that have adapted to thrive exclusively in summer snow fields, Chloromonas nivalis has been reported as a species causing green, orange or pink blooms in many alpine and polar regions worldwide. Nevertheless, the cytology, ecophysiology and taxonomy of this species are still unresolved. Intracellular processes during cyst formation, which is the dominant stage on snow fields, were examined with samples from the European Alps to better understand the cellular strategies of a green alga living in this harsh habitat. We show with two different methods, i.e. oxygen optode fluorometry and by chlorophyll fluorescence, that the cysts are photosynthetically highly active, although they do not divide, and that Chloromonas nivalis can cope with low as well as high light conditions. During cyst formation, the chloroplast is fragmented into several smaller parts, enlarging the surface to volume ratio. The pool of xanthophyll-cycle pigments is significantly enlarged, which is different from other snow algae. The cytoplasm is filled with lipid bodies containing astaxanthin, a secondary carotenoid that causes the typical orange colour. The cyst wall surface possesses characteristic elongate flanges, which are assembled extracellulary by accumulation of material in the periplasmatic interspace. Comparison of Chloromonas nivalis samples from different locations (Austrian Alps, Spitsbergen) by molecular methods indicates genetic variations due to spatial isolation, while a North American strain has no close relationship to the taxon. [ABSTRACT FROM AUTHOR]

A strain of Chloromonas pseudoplatyrhyncha (Pascher) P. C. Silva, which has not been studied previously using cultured material, was established from a soil sample collected in Japan and examined by light microscopy, transmission electron microscopy, and molecular phylogenetic analyses. The chloroplasts of this species showed no pyrenoids under light microscopy. However, transmission electron microscopy and the staining methods with carmine after fixation in an acidified hypochlorite solution revealed that Chloromonas pseudoplatyrhyncha actually had multiple, atypical pyrenoids (pyrenoid matrices without associated starch grains) that were angular in shape and distributed in the interior regions of the lobes of the chloroplasts. Although some other species of Chloromonas have atypical pyrenoids in the chloroplast, such angular pyrenoids have not previously been reported within the Volvocales. The present molecular phylogenetic analysis, based on 18S ribosomal RNA, adenosine triphosphate synthase β-subunit, and P700 chlorophyll a-apoprotein A2 gene sequences, demonstrated that Chloromonas pseudoplatyrhyncha belonged to the Chloromonas lineage or Chloromonadinia, in which it occupied a basal position outside a robust, large monophyletic group consisting of 13 species of Chloromonas and Gloeomonas. [ABSTRACT FROM AUTHOR]

Gloeomonas is a peculiar unicellular volvocalean genus because it lacks pyrenoids in the chloroplasts under the light microscope and has two flagellar bases that are remote from each other. However, ultrastructural features of chloroplasts are very limited, and no molecular phylogenetic analyses have been carried out in Gloeomonas. In this study, we observed ultrastructural features of chloroplasts of three species of Gloeomonas and Chloromonas rubrifilum (Korshikov ex Pascher) Pröschold, B. Marin, U. Schlösser et Melkonian SAG 3.85, and phylogenetic analyses were carried out based on the combined data set from 18S rRNA, ATP synthase beta-subunit, and P700 chl a–apoprotein A2 gene sequences to deduce the natural phylogenetic positions of the genus Gloeomonas. The present EM demonstrated that the chloroplasts of the three Gloeomonas species and C. rubrifilum SAG 3.85 did not have typical pyrenoids with associated starch grains, but they possessed pyrenoid matrices that protruded interiorly within the stroma regions of the chloroplast. The pyrenoid matrices were large and broad in C. rubrifilum, whereas those of the three Gloeomonas species were recognized in only the small protruded regions of the chloroplast lobes. The present multigene phylogenetic analyses resolved that the three species of Gloeomonas belong to the Chloromonas lineage or Chloromonadinia of the Volvocales, and Chloromonas insignis (Anakhin) Gerloff et H. Ettl NIES-447 and C. rubrifilum SAG 3.85, both of which have pyrenoids without associated starch grains, were positioned basally to the clade composed of the three species of Gloeomonas. Therefore, Gloeomonas might have evolved from such a Chloromonas species through reduction in pyrenoid matrix size within the chloroplast and by separating their two flagellar bases. [ABSTRACT FROM AUTHOR]

Carteria (Ca.) miwae was identified as a volvocalean snow alga distributed in Japan. Although Ca. miwae was recorded from various mountainous areas of Japan in its original description, this species has not been studied in over 40 years. We re-examined Ca. miwae, using culture techniques, electron microscopy and molecular phylogenetic analysis on snow samples collected from Mt. Hakkoda, Aomori Prefecture, and Mt. Gassan, Yamagata Prefecture, Japan. Light and transmission electron microscopy (TEM) of ellipsoidal or elongate-ovoid quadriflagellate cells from field samples revealed the presence of two eyespots and the absence of typical pyrenoids surrounded by starch grains in the chloroplasts, which are both characteristic of Ca. miwae. Single-cell sequencing demonstrated that quadriflagellate and naked biflagellate cells with a single eyespot growing in the same snow sample had identical gene sequences (1128 bp) in the large subunit of Rubisco (rbcL), suggesting that these two cell types represent different stages in the sexual cycle of a single species. Our molecular phylogenetic analysis showed that the quadriflagellate and biflagellate cells, and two new strains of the biflagellate volvocalean genus Chloromonas (Cr.) in the Japanese snow samples, form a small clade within the Chloromonas snow taxa. Thus, quadriflagellate cells can be considered planozygotes within the life cycle of a Chloromonas species growing in snow in Japan, and the new combination Chloromonas miwae (Fukushima) Muramoto et al. is proposed, based on Carteria miwae. The unique morphological characteristics and distinct phylogenetic position of Cr. miwae revealed in the present study suggest that this Japanese species has its own peculiar evolutionary history within the worldwide Chloromonas snow taxa. [ABSTRACT FROM AUTHOR]

The quadriflagellate snow alga Chlainomonas Christen, distributed in New Zealand and North America, has several unusual structural attributes. A process assumed to be cytokinesis involves extrusion of protoplasm from the parent through a narrow canal, C. kolii (J. T. Hardy et Curl) Hoham produces a net-like outer envelope rather than a cell wall, and the flagellar basal apparatus of Chlainomonas consists of two semi-independent pairs of basal bodies. Structural connections between basal body pairs appear minimal, but a connecting system different from that observed in other genera exists within each pair. Phylogenetic analysis using rbcL sequences places Chlainomonas in the Chloromonas clade, other known members of which are all biflagellate. Chlainomonas is split into two robust lineages, with New Zealand collections sharing an origin with northern North American collections. Although the quadriflagellate condition is regarded as ancestral in the Chlorophyceae, we speculate—based on ultrastructural and molecular data presented here—that Chlainomonas represents a derived form that has arisen from fusion of two ancestral biflagellate cells. Other explanations (for example, that Chlainomonas represents a diploid form of a biflagellate species) are remotely possible but are presently at odds with extensive observations of field material. Improvements in techniques for experimental manipulation of these sensitive cryophiles will be required to fully characterize their structure and progress our understanding of their biology. [ABSTRACT FROM AUTHOR]

The optimum temperatures of three species of snow algae were studied using four strains of Chloromonas (Cr.) rosae v. psychrophila, six strains of Cr. tughillensis, and one strain of Cr. chenangoensis. These axenic strains were from Upstate New York except for two of Cr. rosae v. psychrophila from the White Mountains, Arizona. Temperatures tested were from 2.5 to 20°C. The high elevation subalpine Cr. rosae v. psychrophila from New York and Arizona grew from 4 to 20°C and had the greatest cell counts at 4 to 15°C. In contrast, the subalpine to temperate low elevation strains of Cr. tughillensis grew from 2.5 to 10°C and optimally at 2.5 or 5°C, and Cr. chenangoensis grew from 2.5 to 7.5°C and optimally at 2.5 and 5°C. Chloromonas tughillensis and Cr. chenangoensis belong to a genetic subclade with low temperature optima, whereas Cr. rosae v. psychrophila belongs to a subclade with broad temperature optima. In acclimation experiments, there were no significant differences in cell counts when acclimating two Adirondack, New York, strains of Cr. rosae v. psychrophila for two weeks prior to experiments vs. using non-acclimated strains that were moved from 4°C directly to 4, 10, 15, or 20°C. For Cr. tughillensis, four of six strains had significantly higher cell counts when grown at 2.5°C after acclimation at 7.5°C for five months. These are the first reports of temperature optima of snow algae from eastern North America. [ABSTRACT FROM AUTHOR]

The optimum pH of two species of snow algae from Upstate New York were assessed by studying three axenic strains of Chloromonas tughillensis in a pH range of 3.0-7.0 and three non-axenic strains of Cr. chenangoensis in a pH range of 3.0-8.0. Growth was examined at 0.5 pH intervals. Cell counts at the termination of the experiments differed among strains and among pH intervals in individual strains for both species, and strains of Cr. tughillensis responded differently to changes in pH (p < 0.001) while strains of Cr. chenangoensis did not (p = 0.193). Cell counts and absorbance data for Cr. tughillensis indicated an optimum pH of 4.9-6.1 using regression analysis. Strains of Cr. chenangoensis exhibited higher but insignificantly different counts between pH 7.0 and 8.0 with maxima at pH 7.5, but pH optima were not determined. When the range was expanded to include pH 8.5-9.0, an optimal pH of 7.0-8.0 was determined for strain CU 722B, and this is the first snow alga reported to have an optimum alkaline pH. The highest absorbance values, however, occurred between pH 3.0-4.5 and pH 7.0-8.0. The pH values recorded in the field were 5.0-5.3 for Cr. tughillensis and 6.7-7.6 for Cr. chenangoensis. [ABSTRACT FROM AUTHOR]

Two new species of Volvocalean green snow algae are described from Upstate New York, Chloromonas chenangoensis and Chloromonas tughillensis. According to rbcL sequence analysis, these species belong to a subclade of five snow species of Chloromonas that includes Cr. brevispina, Cr. nivalis and Cr. pichinchae. These species have zoosporangia/gametangia (cell packs) that are propelled by flagella. The life cycles of Cr. chenangoensis and Cr. rughillensis include a change in cell shape from oblong to spherical before the loss of the cell wall. Using 8-h laboratory experiments, a peak in the change of cell shape occurred at 4 h for Cr. chenangoensis and between 2 and 4 h for Cr. rughillensis after the onset of light, and this was followed by a decline in change of cell shape for both species. Spherical cells peaked between 6 and 8 h for Cr. chenangoensis and at 8 h for Cr. tughillensis. Maximum total matings occurred at 4 h for Cr. chenangoensis and 6 h for Cr. tughillensis, which was followed by a peak in quadriflagellate zygotes at 8 h for both species. Mature zygotes (zygospores) were observed only in Cr. tughillensis. [ABSTRACT FROM AUTHOR]

An extensive phylogenetic analysis of the biflagellate genera, Chlamydomonas Ehrenberg and Chloromonas Gobi emend. Wille, was undertaken using 18S rDNA and rbcL gene sequence analysis. Emphasis was placed on 21 cold-tolerant taxa of which 10 are from snow. These taxa occurred in four distinct clades each in the 18S rDNA and rbcL phylogenies, and when taken together suggest at least five distinct origins in cold habitats. Most of these taxa occur in a single clade (A), and all snow species occurred in this clade. In the rbcL and combined rbcL-18S rDNA analyses, the snow taxa fell into three groups. Two groups occurred in subclade 1: Chlamydomonas augustae Skuja CU, Chlamydomonas augustae UTEX, and Chlamydomonas sp.-A and Chloromonas clathrata Korshikov, Chloromonas rosae Ettl CU, and Chloromonas rosae v. psychrophila var. nov. The third snow group, subclade 2, included three species with unique cell divisions, Chloromonas brevispina (Fritsch) Hoham, Roemer et Mullet, Chloromonas pichinchae (Lagerheim) Wille, and Chloromonas sp.-D, and the basal Chloromonas nivalis (Chodat) Hoham et Mullet with normal cell divisions. This suggests that the snow habitat has been colonized at least twice and possibly three times in the history of these biflagellates. In the 18S rDNA tree, one cold-tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa (Pascher et Jahoda) Gerloff et Ettl. In the rbcL tree, three cold-tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa, Chloromonas sp.-ANT1, and Chloromonas sp.-ANT3. These results support previous findings that pyrenoids have been gained and lost several times within this complex. An extensive phylogenetic analysis of the biflagellate genera, Chlamydomonas Ehrenberg and Chloromonas Gobi emend. Wille, was undertaken using 18S rDNA and rbcL gene sequence analysis. Emphasis was placed on 21 cold-tolerant taxa of which 10 are from snow. These taxa occurred in four distinct clades each in the 18S... [ABSTRACT FROM AUTHOR]

Chloromonas is distinguished from Chlamydomonas primarily by the absence of pyrenoids, which are structures that are present in the chloroplasts of most algae and are composed primarily of the CO2-fixing enzyme Rubisco. In this study we compared sequences of the rbcL (Rubisco large subunit-encoding) genes of pyrenoid-less Chloromonas species with those of closely related pyrenoid-containing Chlamydomonas species in the "Chloromonas lineage" and with those of 45 other green algae. We found that the proteins encoded by the rbcL genes had a much higher level of amino acid substitution in members of the Chloromonas lineage than they did in other algae. This kind of elevated substitution rate was not observed, however, in the deduced proteins encoded by two other chloroplast genes that we analyzed: atpB and psaB. The rates of synonymous and nonsynonymous nucleotide substitutions in the rbcL genes indicate that the rapid evolution of these genes in members of the Chloromonas lineage is not due to relaxed selection (as it preasumably is in parasitic land plants). A phylogenetic tree based on rbcL nucleotide sequences nested two Chlamydomonas species as a "pyrenoid-regained" clade within a monophyletic Chloromonas "pyrenoid-lost" clade. Character-state optimization with this tree suggested that the loss and the regain of pyrenoids were accompanied by eight synapomorphic amino acid replacements in the Rubisco large subunit, four of which are positioned in the region involved in its dimerization. However, both the atpB and the psaB sequence data gave robust support for a rather different set of phylogenetic relationships in which neither the "pyrenoid-lost" nor the "pyrenoid-regained" clade was resolved. The appearance of such clades in the rbcL-based tree may be an artifact of convergent evolutionary changes that have occurred in a region of the large subunit that determines whether Rubisco molecules will aggregate to form a visible pyrenoid. [ABSTRACT FROM AUTHOR]