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) 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.     

NOVEL ICE‐BINDING PROTEINS FROM A PSYCHROPHILIC ANTARCTIC ALGA (CHLAMYDOMONADACEAE,CHLOROPHYCEAE)1

Many cold‐adapted unicellular plants express ice‐active proteins, but at present, only one type of such proteins has been described, and it shows no resemblance to higher plant antifreezes. Here, we describe four isoforms of a second and very active type of extracellular ice‐binding protein (IBP) from a unicellular chlamydomonad alga collected from an Antarctic intertidal location. The alga is a euryhaline psychrophile that, based on sequences of the alpha tubulin gene and an IBP gene, appears to be the same as a snow alga collected on Petrel Island, Antarctica. The IBPs, which do not resemble any known antifreezes, have strong recrystallization inhibition activity and have an ability to slow the drainage of brine from sea ice. These properties, by maintaining liquid environments, may increase survival of the cells in freezing environments. The IBPs have a repeating TXT motif, which has previously been implicated in ice binding in insect antifreezes and a ryegrass antifreeze.     

THE OPTIMUM PH OF FOUR STRAINS OF ACIDOPHILIC SNOW ALGAE IN THE GENUS CHLOROMONAS (CHLOROPHYTA) AND POSSIBLE EFFECTS OF ACID PRECIPITATION1

Four axenic strains of snow algae were examined for optimum pH under laboratory conditions using M-1 growth medium. Growth was measured using cell counts, cell measurements and absorbance readings at 440 nm. Strains C204 and C479A of Chloromonas sp. from the Adirondack Mountains, New York, grew optimally at pH 4.0 to 5.0. Strains C381F and C381G, Chloromonas polyptera (Fritsch) Hoh., Mull. & Roem. from the White Mountains, Arizona, grew optimally at pH 4.5 to 5.0. Growth was significantly higher at pH 4.0 in the northeastern species (Chloromonas sp.), but no significant difference was observed in final growth at pH 4.5, 5.0 and 5.5 between species. It is postulated that the more acidic precipitation in the northeastern United States may be selecting for strains of snow algae with greater tolerance to acidity than in strains from the southwestern United States or that the different pH optima reported are simply species differences. New York strain C204 was also grown in heavily buffered AM medium where it had an optimum pH of 5.0, but cells became irregularly shaped and tended to clump at pH 6.0 to 7.0. Growth of C204 in AM medium was significantly lower than in M-1 medium for snow algae. These findings justify the use of M-1 medium for this type of experimentation.     

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

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.     

拟衣藻系统分类新证据

拟衣藻 (Chloromonas)与衣藻属 (Chlamydomonas)的亲缘关系及分类学位置在藻类学界一直没有定论。其重要原因是单细胞鞭毛类是否具有蛋白核这一特征在系统分类学上具有重要意义 ,而光镜形态与色素体上具 1到多个蛋白核的衣藻极其相似的拟衣藻 ,其不具蛋白核这一特征的稳定性受到许多学者的怀疑。本文观察并报道了中华拟衣藻 (ChloromonasSinica)的超微结构 ,通过对拟衣藻与衣藻超微结构的探究和比对 ,发现除了从孢子时期开始的整个生活史中 ,中华拟衣藻都不具蛋白核外 ,无论显微还是超微结构 ,拟衣藻与衣藻都显示出高度的相似性。从而提出在尚未获得更多资料之前 ,将拟衣藻从衣藻属分离出来成为独立的属较为合适     

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.     

EFFECT OF TEMPERATURE ON TWO ENZYMES FROM A PSYCHROPHILIC CHLOROMONAS (CHLOROPHYTA)1

A psychrophilic green alga belonging to the Chloromonas genus and here named ANT1 was collected in Antarctica. The activities of two enzymes, nitrate reductase and argininosuccinate lyase, were measured at various temperatures and compared to the corresponding enzyme activities in the mesophilic species Chlamydomonas reinhardtii Dangeard. For both enzymes, the temperature for apparent optimal activity was about 20°C lower in ANT1 than in C. reinhardtii. The enzymes were also submitted to various heat treatments before measuring their activities. Both psychrophilic enzymes were more sensitive to heat than the corresponding mesophilic enzymes. It is worth stressing, however, that in both species nitrate reductase was much more sensitive to heat than argininosuccinate lyase, which probably indicates that the peculiar structure of each protein primarily determines its dependence to temperature. Secondary adaptations to low temperatures should then occur to confer the psychrophilic character.     

PERIODICITY OF EPIPELIC UNICELLULAR VOLVOCALES (CHLOROPHYCEAE) IN A SHALLOW ACID POOL1

The occurrence, periodicity and growth of twenty species of unicellular Volvocales on sediments in an acidic pool are described. Minimum populations were recorded in winter, but during the rest of the year standing crops fluctuated rapidly. The greatest species diversity and primary productivity occurred in late spring-early summer and in autumn, when maximum numbers of Chlamydomonas spp. and Chloromonas spp. increased exponentially on the sediments. The chlamydomonads were more numerous in the epipelon than other major algal components such as diatoms, euglenoids, bluegreen algae and desmids. Growth of the chlamydomonad population occurred after the period of maximum diatom standing crop. Evidence shows that rates of primary production were greater in late spring and late summer when species diversity and standing crop or apparent growth rates of unicellular Volvocales were high. Thus these algae which are normally neglected may be more important in primary productivity than previously believed since they grow during periods when larger algae are scarce. Analysis of the data using the multivariate technique of Reciprocal Averaging confirmed seasonal periodicity in this community of epipelic flagellates. It also identified species with distinctive ecological requirements. A relationship between the bicarbonate-alkalinity of the overlying water and the chlamydomonad population was demonstrated by ordination analysis.     

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.     

Role of pyrenoids in the CO2-concentrating mechanism: comparative morphology, physiology and molecular phylogenetic analysis of closely related strains of Chlamydomonas and Chloromonas (Volvocales)

The morphology of the pyrenoid and the physiology of the CO₂-concentrating mechanism (CCM) were investigated in Chlamydomonas (Cd.) mutabilis Gerloff UTEX 578, Cd. radiata Deason et Bold UTEX 966, Cd. augustae Skuja UTEX 1969, Cd. macrostellata Lund SAG 72.81, Cd. bipapillata Bourrelly SAG 11-47, and Chloromonas (Cr.) insignis Gerloff et Ettl NIES-447, all of which are closely related phylogenetically to the pyrenoid-less strains of Chloromonas. In the chloroplasts of Cd. mutabilis UTEX 578, Cd. radiata UTEX 966, Cd. augustae UTEX 1969, and Cd. macrostellata SAG 72.81, a typical, spheroidal, electron-dense pyrenoid matrix surrounded by starch granules was present, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were highly concentrated in the pyrenoid matrix. On the other hand, while the pyrenoid matrix of Cr. insignis NIES-447 was electron-dense that of Cd. bipapillata SAG 11-47 was not, and neither was surrounded by starch granules. The pyrenoid matrices of these two species exhibited a higher concentration of Rubisco molecules than the thylakoid region (thylakoid and stroma) of the chloroplasts; however, the densities of Rubisco molecules in these pyrenoid matrices were low compared with those of the other four Chlamydomonas strains examined in this study and that of Cd. reinhardtii Dangeard. In all six strains examined, the presence of the CCM was indicated by relatively high photosynthetic affinities for CO₂ (low values of K_0.5(CO₂)). However, differences in the inorganic carbon (Ci) pools were recognized in relation to the differences in pyrenoid morphology among the strains. In the typical pyrenoid-containing strains. Cd. mutabilis UTEX 578 and Cd. radiata UTEX 966, the ratio of internal to external inorganic carbon was about 20, while in Cr. insignis NIES-447 and Cd. bipapillata SAG 11-47 the ratio was only 2–3 similar to the two pyrenoid-less, CCM-containing strains of Chloromonas previously examined (E. Morita et al., 1998, Planta 204: 269–276). It is thus speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydomonas/Chloromonas strains and the pyrenoid-less Chloromonas strains previously investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with high bootstrap values. Based on the tree topology resolved, it was inferred that loss of the typical pyrenoids accompanied by a decrease in intracellular Ci pools might have taken place independently in the two groups. Received: 21 August 1998 / Accepted: 30 November 1998