Chloromonas Species Research Articles

Chloromonas rubrosalmonea sp. nov. (Chlorophyta) causes blooms of salmon-red snow due to high astaxanthin and low chlorophyll content

ABSTRACT Melting snowfields support microbial communities, including blooms of phototrophic psychrophiles. “Watermelon snow” and its flagship species Sanguina nivaloides are mostly found at exposed, alpine or polar sites. Here, we investigated an alga that is morphologically similar but likely less common, thriving in exposed or semi-exposed snowpacks at lower altitudes frequently close to the tree line, and causing characteristic, intensely salmon-red snow surface spots in Central European and Norwegian mountains. Our aim was to illustrate the taxonomic heterogeneity of red snow by performing a cellular and molecular characterization of this species. The phylogenetic analysis placed the alga into the genus Chloromonas, clade “group C”. Single-cell Sanger sequencing enabled us to link the cell morphotype to its genotype. In addition to light microscopy of the snow samples, applying 18S rRNA gene and ITS2 metabarcoding confirmed that this alga dominated the bloom communities. The cysts had multi-layered smooth cell walls, and had a broadly oval to fully spherical shape, in striking contrast to most other Chloromonas species found on snow surfaces. Another remarkable feature was a pronounced astaxanthin accumulation in relation to a low proportion of chlorophylls, occasionally accompanied by a significant reduction of thylakoid membranes. This likely caused the photosynthetic dormancy of mature cysts found in some samples. Based on these data, we describe the snow alga as Chloromonas rubrosalmonea, sp. nov.

Sexual reproduction of the snow alga Chloromonas fukushimae (Volvocales, Chlorophyceae) induced using cultured materials.

Snow algae are microalgae, growing in melting snowpacks, and are thought to act as primary producers in the snow ecosystem. Chloromonas (Volvocales, Chlorophyceae) contains more than 15 snow-inhabiting species. Although vegetative cells and zygotes, or asexual cysts, of snow species of the genus are frequently collected in the field, sexual reproduction and zygote formation in culture have only been induced in C. tughillensis. Here we describe the sexual reproduction of another snow-inhabiting species, C. fukushimae, which was induced using both previously examined and newly established Japanese strains. Mating of isogamous gametes began after mixing two different strains, implying that C. fukushimae is an outcrossing species. Motile and nonmotile zygotes of the species were also described in this report. The nonmotile zygote of C. fukushimae was distinguishable from those of the other snow-inhabiting species of Chloromonas, based on the zygote shape and the presence of several large lipid bodies within the cell. In addition, C. fukushimae carried out sexual reproduction and produced zygotes even under the nitrogen-sufficient condition.

Chloromonas svalbardensis n. sp. with Insights into the Phylogroup Chloromonadinia (Chlorophyceae).

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.

Taxonomic revision of Chloromonas nivalis (Volvocales, Chlorophyceae) strains, with the new description of two snow-inhabiting Chloromonas species.

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.

Comparative ultrastructural analysis of Chloromonas and Gloeomonas (Chlorophyceae): tracing the origin of Gloeomonas-specific basal apparatus traits

: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.

The chemometric approach applied to FTIR spectral data for the analysis of lipid content in microalgae cultivated in different nitrogen sources

Microalgae-based biofuel production has emerged as a potential sector to extend its sustainability. Corresponding to many factors imbibed, nitrogen is considered as an essential element for microalgae cultivation. Among the four studied nitrogen sources, sodium nitrate (Na) and urea (U) showed high lipid content of 35.86 ± 2.3 and 37.35 ± 0.32 %, respectively. In terms of lipid yield, the four investigated nitrogen sources are represented in preferrential order, i.e., urea > sodium nitrate > ammonium nitrate > potassium nitrate. The Fourier transform infrared spectroscopy (FTIR) fingerprint of oil recovered from Chloromonas species ADIITEC-III, cultivated in different nitrogen sources, were analyzed using chemometric techniques. The chemometric techniques, i.e., cluster analysis, multidimensional scaling (MDS), and a multivariate calibration of principal component analysis (PCA), were assessed to analyze the existing spectral signatures specific to the lipid-acyl chain (3000–2800 cm−1) and biomolecular fingerprint region (1800–1000 cm−1) in the FTIR spectrum of the sample. The study of FTIR-coupled chemometric techniques complements the response of the nitrogen sources in lipid yield and confirmed the reliability of the method.

Integrated 'Omics', Targeted Metabolite and Single-cell Analyses of Arctic Snow Algae Functionality and Adaptability.

Snow algae are poly-extremophilic microalgae and important primary colonizers and producers on glaciers and snow fields. Depending on their pigmentation they cause green or red mass blooms during the melt season. This decreases surface albedo and thus further enhances snow and ice melting. Although the phenomenon of snow algal blooms has been known for a long time, large aspects of their physiology and ecology sill remain cryptic. This study provides the first in-depth and multi-omics investigation of two very striking adjacent green and red snow fields on a glacier in Svalbard. We have assessed the algal community composition of green and red snow including their associated microbiota, i.e., bacteria and archaea, their metabolic profiles (targeted and non-targeted metabolites) on the bulk and single-cell level, and assessed the feedbacks between the algae and their physico-chemical environment including liquid water content, pH, albedo, and nutrient availability. We demonstrate that green and red snow clearly vary in their physico-chemical environment, their microbial community composition and their metabolic profiles. For the algae this likely reflects both different stages of their life cycles and their adaptation strategies. Green snow represents a wet, carbon and nutrient rich environment and is dominated by the algae Microglena sp. with a metabolic profile that is characterized by key metabolites involved in growth and proliferation. In contrast, the dry and nutrient poor red snow habitat is colonized by various Chloromonas species with a high abundance of storage and reserve metabolites likely to face upcoming severe conditions. Combining a multitude of techniques we demonstrate the power of such complementary approaches in elucidating the function and ecology of extremophiles such as green and red snow algal blooms, which play crucial roles in glacial ecosystems.

A taxonomic study of snow Chloromonas species (Volvocales, Chlorophyceae) based on light and electron microscopy and molecular analysis of cultured material

: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.

Insights into the biology and phylogeny of Chloromonas polyptera (Chlorophyta), an alga causing orange snow in Maritime Antarctica

AbstractIn Antarctica, mass accumulations of psychrophilic algae cause striking phenomena like green, orange, or red snow. This occurs during summer, when coastal snowfields start to melt, become waterlogged and photoautotrophs can thrive. Chloromonas polyptera (Fritsch) Hoham, Mullet & Roemer is a unicellular species that causes orange snow in the vicinity of penguin rockeries. It has been recognized for many decades because of the distinct habitat and the characteristic morphology of cysts with elongated flanges on the outer cell wall. However, closer investigations concerning the ecology or physiology have been sparse so far. Field material was collected from two sites on the Antarctic Peninsula to find out more about metabolic and cellular strategies. The results were compared with a closely related species from high alpine locations, Chloromonas nivalis (Chodat) Hoham & Mullet. Despite the geographical distance, C. polyptera shares several physiological strategies with the alpine relative, such as the formation of cyst stages, saccharose and glycerol as main soluble carbohydrates and the abundant accumulation of the carotenoid astaxanthin. Moreover, photosynthesis is adapted to temperatures of about 1°C. The molecular phylogeny confirmed a close relationship of C. polyptera to other Chloromonas species isolated from snow. Chloromonas polyptera seems to be exclusive to coastal Antarctic ecosystems influenced by animal nutrient input.

Light and electron microscopy and molecular phylogenetic analyses of Chloromonas pseudoplatyrhyncha (Volvocales, Chlorophyceae)

SUMMARY 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.

Re-examination of the snow algal species Chloromonas miwae (Fukushima) Muramoto et al., comb. nov. (Volvocales, Chlorophyceae) from Japan, based on molecular phylogeny and cultured material

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.

EVOLUTIONARY ORIGIN OF GLOEOMONAS (VOLVOCALES, CHLOROPHYCEAE), BASED ON ULTRASTRUCTURE OF CHLOROPLASTS AND MOLECULAR PHYLOGENY1

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) Proschold, B. Marin, U. Schlosser 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.

Morphological and Genetic Variation in the Cosmopolitan Snow Alga Chloromonas nivalis (Volvocales, Chlorophyta) from Japanese Mountainous Area

Mature zygotes of the cosmopolitan snow alga Chloromonas nivalis (Chodat) Hoham & Mullet [=Scotiella nivalis (Chodat) Fritsch] collected from Mt. Gassan, Yamagata Prefecture, were examined by scanning electron microscopy (SEM) and single-cell sequencing of the large subunit of Rubisco (rbcL) genes. The present SEM demonstrated that Japanese samples have at least two types of flanges on the zygote wall (straight and sigmate). Although these types fell within the morphological variability of C. nivalis as reported previously, at least two types of rbcL (large subunit of Rubisco) gene sequences were detected by using the single-cell sequencing. Although the information of the rbcL genes were limited (340–400 bp), the present phylogenetic analyses revealed that these two Japanese types and C. nivalis originating from North America belong to the lineage composed of Chloromonas snow taxa. Thus, C. nivalis might involve multiple cryptic species of Chloromonas. Further ovservations of other stages of life cycle and more detailed phylogenetic analysis are needed to reveal the correct taxonomic identifications of C. nivalis.

Two new species of green snow algae from Upstate New York, Chloromonas chenangoensis sp. nov. and Chloromonas tughillensis sp. nov. (Volvocales, Chlorophyceae) and the effects of light on their life cycle development

R.W. Hoham, J.D. Berman, H.S. Rogers, J.H. Felio, J.B. Ryba and P.R. Miller. 2006. Two new species of green snow algae from Upstate New York, Chloromonas chenangoensis sp. nov. and Chloromonas tughillensis sp. nov. (Volvocales, Chlorophyceae) and the effects of light on their life cycle development. Phycologia 45: 319–330. DOI: 10.2216/04-103.1Two 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. tughillensis 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. tughillensis 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.

Differences in pyrenoid morphology are correlated with differences in the rbcL genes of members of the Chloromonas lineage (volvocales, chlorophyceae).

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 presumably 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.

A COMBINED 18S rDNA AND rbcL PHYLOGENETIC ANALYSIS OF CHLOROMONAS AND CHLAMYDOMONAS (CHLOROPHYCEAE, VOLVOCALES) EMPHASIZING SNOW AND OTHER COLD‐TEMPERATURE HABITATS1

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.

New records of snow algae for New Zealand, from Mt Philistine, Arthur's Pass National Park

Eight out of nine species of snow algae occurring on Mt Philistine, Arthur's Pass National Park, are new records for New Zealand. Chlainomonas kolii, reported previously in snow beneath tree canopies in the USA, is responsible for blooms which colour the snow red. Chloromonas rubroleosa, which is found in red snow dominated by C. kolii, also occurs in continental Antarctica. Three other species of Chloromonas, including the relatively common bipolar alga previously known as Scotiella antarctica, occur in other snowfields on the mountain. The widely reported Raphidonema nivale was identified in cultures from many snow samples. Two species of chrysophyceans, Chromulina cf. elegans and Chrysophyceae sp. 2 (a cyst, of which the life cycle is not understood) occur in Mt Philistine snow. The few previous investigations of New Zealand snow algae have suffered from lack of culturing and observation of living material. Results presented here show that many more sites require detailed study using these techniques to improve our understanding of the biogeography of New Zealand snow algae, and for comparison with floras elsewhere to be meaningful.

Molecular Phylogeny and Taxonomic Revision of Chlamydomonas (Chlorophyta). I. Emendation of Chlamydomonas Ehrenberg and Chloromonas Gobi, and Description of Oogamochlamys gen. nov. and Lobochlamys gen. nov.

The genus Chlamydomonas (including Chloromonas) is one of the largest green algal genera comprising more than 600 species. To initiate a comprehensive analysis of the phylogeny and systematics of the genus, we determined nuclear-encoded SSU rRNA sequences from 32 strains of Chlamydomonas, Chloromonas and Chlorogonium with emphasis on oogamous taxa and related strains, and incorporated these into global molecular phylogenetic analyses of 132 strains of Chlorophyceae. In addition, we studied the morphology and reproduction of oogamous and related strains by light microscopy. We recognize and designate 18 monophyletic lineages (clades) within the Chlorophyceae, 11 of which are confined to the CW (basal bodies displaced clockwise) subgroup. The majority of clades recognized within the Chlorophyceae do not correspond to any of the traditional classification systems, which are still largely based on the organization level. Strains assigned to Chlamydomonas and Chloromonas were found in seven different clades confirming the polyphyly of the two genera as presently conceived. To initiate the taxonomic revision of Chlamydomonas, C. reinhardtii is proposed as the conserved type of the genus. In consequence, species in clades other than the clade containing C. reinhardtii must be transferred to other genera, a process initiated in this contribution. The oogamous strains studied represent a monophyletic lineage, which is described as Oogamochlamys gen. nov. comprising three species (O. gigantea, O. zimbabwiensis and O. ettlii spec. nov.). The sister clade to Oogamochlamys consists of isogamous strains characterized by chloroplasts with incisions and is described as Lobochlamys gen. nov. with two species (L. culleus and L. segnis). Another clade is characterized by asteroid or perforated, parietal chloroplasts and contains the type species of Chloromonas (C. reticulata). Thus, the polyphyletic Chloromonas (traditionally defined as "Chlamydomonas without pyrenoids") can be legitimized as a monophyletic genus by restriction to this clade and is here emended on the basis of chloroplast characters (the clade contains strains with or without pyrenoids thus rejecting the character "absence of pyrenoids").

The importance of light and photoperiod in sexual reproduction and geographical distribution in the green snow alga,Chloromonas sp.-D (Chlorophyceae, Volvocales)

The effects of premating light regimes on sexual reproduction and the production of spherical cells in Chloromonas sp.-D, a unicellular green snow alga, were studied using cross-mating strains 582C and 582D isolated from snowpacks associated with mixed hardwood-softwood forests in Whetstone Gulf State Park, Tughill Plateau, NY. Two preacclimation regimes were used, Vita-Lite as controls (530-700 nm peak) and blue light as experimentals (430-460 nm peak) prior to the mating experiments. In blue light, an increase in the number of matings and spherical cells (spheres) produced in the life cycle was observed as the photoperiod increased. This implies that longer photoperiods of blue light are more optimal for sexual reproduction in Chloromonas sp.-D than shorter ones. Under Vita-Lite, there was a significant increase in the number of matings and spheres with the extended 20 : 4 photoperiod compared with the shorter 14 : 10 photoperiod. Under blue light, significantly more matings and spheres occurred than under Vita-Lite using the same irradiance level of 95 μmol photons m -2 s -1 (photosynthetically active radiation [PAR] of 400-700 nm) for the 14: 10 and 20: 4 photoperiods. The results of these experiments suggest that Chloromonas sp.-D. known only from the Tughill Plateau, NY, is not reproducing optimally at this site where it grows and reproduces under an approximate 14: 10 photoperiod in early April. However, in the upper 10 cm of snow in the Tughill Plateau, a blue light irradiance level of 95 μmol photons m 2 s -1 occurs, which is optimal for this species. When these conditions are combined with a 14: 10 photoperiod, the Tughill Plateau appears to be sub-optimal for mating and production of spherical cells. Since Chloromonas sp.-D does not appear to have a dependence on a dark cycle, this would allow it to expand its geographical distribution. It may reproduce more optimally under blue light (95 μmol photons m -2 s -1 ) with an extended photoperiod (>20:4 h, light:dark) in high latitude field sites such as Lake Bienville, Quebec, in eastern North America where other species of Chloromonas are known in snow associated with coniferous forests.

RUBISCO ADAPTATION TO LOW TEMPERATURES: A COMPARATIVE STUDY IN PSYCHROPHILIC AND MESOPHILIC UNICELLULAR ALGAE

Some properties of the ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RUBISCO) from two psychrophilic Chloromonas species have been investigated in relation to their adaptation to cold environments. Contrary to the situation usually encountered with psychrophilic enzymes, the carboxylase activity of both purified “cold” RUBISCO enzymes was lower at low temperatures than that found with the enzyme of the mesophilic alga Chlamydomonas reinhardtii Dangeard. Moreover, the apparent optimal temperature for RUBISCO carboxylase activity was similar for psychrophilic and mesophilic enzymes. Psychrophilic RUBISCOs, however, showed a greater thermosensitivity than the C. reinhardtii enzyme. Genes encoding small and large subunits of RUBISCO from one psychrophilic isolate were sequenced. Comparison of the deduced amino acid sequences to those of higher plants and green algae revealed the substitution of a very highly conserved residue (cysteine247→ serine in the large subunit) that could be responsible, at least in part, for the increased thermosensitivity of the “cold” enzyme. Interestingly, the relative amount of RUBISCO subunits found in the psychrophilic isolates was about twice as high as the amount observed in C. reinhardtii and five other mesophilic algae. The high production of a key enzyme to counterbalance its poor catalytic efficiency at low temperature could constitute a novel type of adaptive mechanism to cold environments.