PHYLOGENYOF CHLOROMONAS (CHLOROPHYCEAE): A STUDY OF 185 RIBOSOMAL RNA GENE SEQUENCES1

The unicellular, biflagellate genus Chloromonas differs from its ally, Chlamydomonas, primarily by the absence of pyrenoids in the vegetative stage of the former. As with most green flagellate genera, little is known about phylogenetic affinities within and among Chloromonas species. Phylogenetic analyses of nuclear-encoded small-subunit ribosomal RNA gene sequences demonstrate that a sampling of five Chloromonas taxa, obtained from major culture collections, do not form a monophyletic group. However, only three of these isolates, Chloromonas clathrata, Chloromonas serbinowi, and Chloromonas rosae, are diagnosable morphologically as Chloromonas species by the absence of a pyrenoid in the vegetative stage. The three diagnosable Chloromonas taxa form an alliance with two pyrenoid-bearing chlamydomonads, Chlamydomonas augustae and Chlamydomonas macrostellata. With the exception of Chloromonas serbinowi, which represents the basal lineage within the clade, each of the diagnosable Chloromonas taxa and their pyrenoidbearing Chlamydomonas allies were isolated originally from mountain soils, snow, or cold peat. These observations suggest that habitat, independent of pyrenoid status, may be most closely linked to the natural history of this clade of chlamydomonad flagellates.     

Immunocytochemical Localization of Phosphoribulokinase in Microalgae

Employing immunogold electron microscopy, the subcellular location of the Calvin cycle enzyme phosphoribulokinase (PRK) was determined for two diverse species of microalgae. In both the red alga Porphyridium cruentum and the green alga Chlamydomonas reinhardtii, PRK was distributed throughout the thylakoid-containing chloroplast stroma. In contrast, the next enzyme in the pathway, ribulose 1,5-bisphosphate carboxylase/oxygenase, was predominantly pyrenoid-localized in both species. In Porphyridium, the chloroplast stroma abuts the pyrenoid but in Chlamydomonas and other green algae, the pyrenoid appears encased in a starch sheath. Unique inclusions found in the pyrenoid of Chlamydomonas were immunolabelled by anti-PRK and thus identified as regions of chloroplast stroma. It is postulated that such PRK-containing stromal inclusions in the pyrenoids of Chlamydomonas and perhaps other green algae provide a means for exchange of Calvin cycle metabolites between pyrenoid and stroma.     

COMPARATIVE STUDIES OF PYRENOID ULTRASTRUCTURE IN ALGAE OF THE MONOSTROMA COMPLEX123

The pyrenoid structure in 15 species of the Monostroma complex is very diverse us revealed by a study of the morphology of the pyrenoid matrix, associated starch shell, and pattern of intrapyrenoidalthylakoid bands. From these characteristics 8 types of pyrenoid structure were classified. The variation of pyrenoid structure was shown not only among the species studied, but also between the alternation of generations (M. angicava and M. nitidum). In M. fuscum var. splendens, M. groenlandicum, M. undulatum, and M. zostericola pyrenoid structure is the same throughout the life cycle. The pyrenoid matrix of M. zostericola is surrounded by a double membrane that prevents the direct connection of the pyrenoid matrix with chloroplast thylakoids. The pyrenoid also lacks a starch shell. These findings support the establishment of a new genus Kornmannia by Bliding to include M. zostericola. In addition, similarities in pyrenoid ultrastructure suggest an affinity of Capsosiphon fulvescens with M. groenlandicum.     

Reconsideration of the taxonomy of ellipsoidal species of Chlorella (Trebouxiophyceae, Chlorophyta), with establishment of Watanabea sen. nov.

Subcultures of SAG 211–9b and 1AM C-211, ultimately derived from CCAP 211/9b, a strain isolated by Pringsheim in 1939 and identified as Chlorella sac-charophila (Kruger) Migula were observed using light and electron microscopy. Their morphology proved to be basically identical. Both have two forms of cells, one (E-form) narrowly to broadly ellipsoidal, the other (S-form) ovoid to spheroidal. The cell wall of both forms is composed of a single smooth layer. The chloroplast of young cells is trough-like or saucer-shaped with a smooth margin, while that of mature cells is band- or cup-shaped with deep incisions. The thylakoid lamellae are loosely stacked and neither form has a pyrenoid. Both types of cells are capable of producing autospores: eight to 16 in E-form cells, two to four in S-form cells. These morphological features are different from those of C. saccharophila, which has a pyrenoid and produces only one form of autospores. In the absence of any existing genus that includes Chlorella-like algae with a simple cell wall, no pyrenoid, and two forms of mature cells and autospores, a new genus, Watanabea, is proposed with the type species W. reniformis.     

SPECIES-SPECIFIC DIFFERENCES OF PYRENOIDS IN CHLORELLA (CHLOROPHYTA)1

The structure of the pyrenoid supports the separation of Chlorella species into two groups based on cell wall chemistry and suggests evolutionary relationships. Chlorella species with a glucan-type wall exhibit quite diverse pyrenoid structures, which may indicate that these species are not closely related. Those species with glucosamine cell walls (C. kessleri, C. sorokiniana, C. vulgaris) are virtually identical in pyrenoid morphology, indicating a closer evolutionary relationship. In the species with glucosamine walls, the thylakoid that penetrates into the pyrenoid matrix, is unijormly double-layered. Pyrenoids in the species with glucan walls show various features: 1) a pyrenoid matrix only, 2) a pyrenoid traversed by a few discs of double thylakoids with many adhering pyrenoglobuli, 3) a pyrenoid penetrated with tubelike structures or 4) a pyrenoid penetrated with many single undulating thylakoids. The pyrenoid structure of the symbiotic Chlorella in Paramecium bursaria resembles those of free-living Chlorella with glucosamine walls.     

Generic and species concepts in Microglena (previously the Chlamydomonas monadina group) revised using an integrative approach

Traditionally the genus Microglena Ehrenberg has been used to contain species that belong to the Chrysophyceae; however, the type species of Microglena, M. monadina, represents a green alga, which was later transferred to the genus Chlamydomonas. The taxonomic status of the genus has therefore remained unclear. We investigated 15 strains previously assigned to C. monadina and two marine species (C. reginae and C. uva-maris) using an integrative approach. Phylogenetic analyses of SSU and ITS rDNA sequences revealed that all strains form a monophyletic lineage within the Chlorophyceae containing species from different habitats. The strains studied showed similar morphology with respect to cell shape and size, but showed differences in chloroplast and pyrenoid structures. Some representatives of this group have the same type of sexual reproduction (homothallic advanced anisogamy). Three different morphotypes could be recognized. Strains belonging to type I have a cup-shaped chloroplast with a massive basal part, in which a large, single, ellipsoidal pyrenoid is located. The members of type II also have a cup-shaped chloroplast, which is partly lobed and has a thinner basal part than type I; here the pyrenoid is half-ring or horseshoe-shaped and occupies different positions in the chloroplast depending on the strain. The strains of type III have multiple pyrenoids, which appear to have developed from the subdivision of a single ring-shaped pyrenoid into several parts. We compared the results of our morphological investigations with the literature and found that 15 strains could be identified with existing species. Two strains did not fit with any described species. As a result of our study, we transfer all strains to the genus Microglena, propose 11 new combinations, and describe two new species. Comparison of the ITS-1 and ITS-2 secondary structures confirmed the species delineations. All species have characteristic compensatory base changes in their ITS secondary structures and are supported by ITS-2 DNA barcodes.     

MOLECULAR PHYLOGENY,ULTRASTRUCTURE, AND TAXONOMIC REVISION OF CHLOROGONIUM (CHLOROPHYTA): EMENDATION OF CHLOROGONIUM AND DESCRIPTION OF GUNGNIR GEN. NOV. AND RUSALKA GEN. NOV.1

We examined the molecular phylogeny and ultrastructure of Chlorogonium and related species to establish the natural taxonomy at the generic level. Phylogenetic analyses of 18S rRNA and RUBISCO LSU (rbcL) gene sequences revealed two separate clades of Chlorogonium from which Chlorogonium (Cg.) fusiforme Matv. was robustly separated. One clade comprised Cg. neglectum Pascher and Cg. kasakii Nozaki, whereas the other clade included the type species Cg. euchlorum (Ehrenb.) Ehrenb., Cg. elongatum (P. A. Dang.) Francé, and Cg. capillatum Nozaki, M. Watanabe et Aizawa. On the basis of unique ultrastructural characteristics, we described Gungnir Nakada gen. nov. comprising three species: G. neglectum (Pascher) Nakada comb. nov., G. mantoniae (H. Ettl) Nakada comb. nov., and G. kasakii (Nozaki) Nakada comb. nov. We also emended Chlorogonium as a monophyletic genus composed of Cg. euchlorum, Cg. elongatum, and Cg. capillatum. Because Cg. fusiforme was distinguished from the redefined Chlorogonium and Gungnir by the structure of its starch plate, which is associated with pyrenoids, we reclassified this species as Rusalka fusiformis (Matv.) Nakada gen. et comb. nov.     

Observations on Cymbella mexicana (Ehrenb.) Cleve var. mexicana (Bacillariophyceae) with special reference to the band structure

Cymbella mexicana (Ehrenb.) Cleve var. mexicana was collected from Tama-gawa (Tama River), Tokyo. The fine structure of the species was examined by scanning electron microscopy (SEM), with special reference to colony formation and band morphology. A mature cingulum of this species is usually composed of four open bands. The correlation between the position of the second band (B2) of the cingulum and the attaching pole of the frustule is discussed.     

Preliminary results of a classification of fifty-one selected northeastern Wisconsin Lakes (USA) using indicator diatom species

Diatom species lists were generated for 51 lakes in northeastern Wisconsin, and then classified by two-way indicator species analysis (TWINSPAN). The lakes were initially divided into two main groupings: Group I lakes which were alkaline lakes of moderate to high productivity, and Group II lakes which were acid lakes of low productivity. Group I lakes were further divided into two subgroupings, and four levels. In total, twelve indicator diatom species were recognized during the TWINSPAN lake classification, these were: Achnanthes exigua Grun., A. lanceolata var. dubia Grun., Amphora ovalis var. Affinis (Kütz.) V.H. ex Det., A. perpusilla (Grun.) Grun., Cymbella cistula (Ehrenb.) Kirch., Diploneis elliptica (Kütz.) Cl., Eunotia pectinalis (O.F. Mull.) Rabh., Fragilaria vaucheriae (Kütz.) Peters., Frustulia rhomboides var. crassinervia (Bréb. ex W. Sm.) Ross, Navicula capitata Ehrenb., N. decussis Østr., and N. scutelloides W.Sm. ex Gregory.     

ELECTRON MICROSCOPY OF CARTERIA AND CHLAMYDOMONAS

Cultures of Chlamydomonas eugametos, Chl. sp., Carteria eugametos, C. crucifera, C. radiosa, and C. sp. were examined with the electron microscope to determine generic differences between Carteria and Chlamydomonas at the ultrastructural level. The ultrastructure of the flagella, mitochondria, dictyosomes, nuclei and ground substance was noted to be similar in all species. The cellular boundary of all species except Chlamydomonas eugametos contains a 250 A intermediate layer of unknown chemical composition between the fibrillar cellulose wall and the outer capsule layer. Four structural features other than the number of flagella distinguish Carteria from Chlamydomonas: the intermediate layer of the cellular boundary, the chloroplast, the pyrenoid and the eyespot. Only in the Carteria species is the intermediate layer traversed by striations or 12-mμ-wide bars. Striations in the cellulose wall surrounding the flagellar channels also appear in Carteria eugametos and C. crucifera. The chloroplast lamellae of the Carteria species are grouped into discrete stacks of invaginated thylakoids termed pseudograna. The chloroplast lamellae of Chlamydomonas are broad and sheet-like and are also invaginated although less frequently than are the pseudograna of Carteria. The phenomenon of infolding of the chloroplast lamellae is suggested as a general developmental process in the formation of new thylakoids. In Carteria, single thylakoids traverse the pyrenoid and there are 2 rows of granules in the eyespot. Favorable micrographs of the eyespot indicate that the granules may be osmiophilic granules of the chloroplast chemically modified for a photoreceptive function.     

TAXONOMIC STUDY OF TWO NEW GENERA OF FUSIFORM GREEN FLAGELLATES,TABRIS GEN. NOV. AND HAMAKKO GEN. NOV. (VOLVOCALES,CHLOROPHYCEAE)1

On the basis of LM, we isolated strains of two species of fusiform green flagellates that could be assigned to former Chlorogonium (Cg.) Ehrenb. One species, “Cg.”heimii Bourr., lacked a pyrenoid in its vegetative cells and required organic compounds for growth. The other was similar to Cg. elongatum (P. A. Dang.) Francé and “Cg.”acus Nayal, but with slightly smaller vegetative cells. Their molecular phylogeny was also studied based on combined 18S rRNA, RUBISCO LSU (rbcL), and P700 chl a‐apoprotein A2 (psaB) gene sequences. Both species were separated from Chlorogonium emend., Gungnir Nakada and Rusalka Nakada, which were formerly assigned to Chlorogonium. They were accordingly assigned to new genera, Tabris Nakada gen. nov. and Hamakko (Hk.) Nakada gen. nov. as T. heimii (Bourr.) Nakada comb. nov. and Hk. caudatus Nakada sp. nov., respectively. Tabris is differentiated from other genera of fusiform green flagellates by its vegetative cells, which only have two apical contractile vacuoles and lack a pyrenoid in the chloroplast. Hamakko, on the other hand, is distinguishable by the fact that its pyrenoids in vegetative cells are penetrated by flattened thylakoid lamellae.     

中国淡水共球藻纲新记录属种——土佐牧野藻(Makinoella tosaensis Okada)

报道分别从湖北省武汉市内和云南省西双版纳小水池中分离培养的两株绿藻,对其进行了形态和18S r DNA基因序列分析,编号分别为FACHB-1783和FACHB-1784。这两株绿藻具独特的四边形群体形态,通常为4或16个细胞,细胞为宽椭圆形至不规则卵圆形、细胞壁两端无增厚,叶绿体多数、片状,具蛋白核。结合形态和分子系统发育分析,确定这两株绿藻为我国1种淡水共球藻纲新记录属种——土佐牧野藻(Makinoella tosaensis Okada)。基于18S r DNA基因的系统发育研究表明这两株绿藻与分离自韩国的土佐牧野藻基因序列相似度可达99.6%~99.9%,并且以较高的支持值与土佐牧野藻聚在一起。     

Immunocytochemical localization of nitrite reductase in green algae

The distribution of nitrite reductase (EC 1.7.7.1) in the green algae Chlamydomonas reinhardtii, Monoraphidium braunii, Chlorella fusca, and Scenedesmus obliquus was studied by immunoelectron microscopy. The labeling of ultrathin cryosections was performed with anti-nitrite reductase antibodies followed by gold-labeled goat anti-rabbit antibodies. In C. reinhardtii sections, gold label was mainly associated with the pyrenoid, tonoplast, and plasmalemma. Significant labeling was also detected in the thylakoid region. In all other organisms, label density was lower but distributed in the same locations, except that the plasmalemma of S. obliquus was not significantly labeled. From estimates of the relative volume of different cell regions, we found that approximately 80% of the total enzyme is located in the chloroplastic region (thylakoids plus pyrenoid) of C. reinhardtii, M. braunii, and C. fusca, and 97% in the case of S. obliquus.     

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae,Chlamydomonadales)

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.     

CHLOROPLAST DIVISION AND PYRENOID FORMATION IN CHLAMYDOMONAS REINHARDI1

The process of chloroplast division during mitosis in Chlamydomonas reinhardi is followed with the electron microscope. The pyrenoid and the chloroplast reproduce by fission. The dividing chloroplast contains regions of dense material that superficially resemble pyrenoids, but it is concluded that the formation of the dense material is not related to pyrenoid formation in C. reinhardi. The dense material appears to be localized over regions of chloroplast DNA.     

UPDATING THE GENUS DICTYOSPHAERIUM AND DESCRIPTION OF MUCIDOSPHAERIUM GEN. NOV. (TREBOUXIOPHYCEAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA1

Recent molecular analyses of Dictyosphaerium strains revealed a polyphyletic origin of this morphotype within the Chlorellaceae. The type species Dictyosphaerium ehrenbergianum Nägeli formed an independent lineage within the Parachlorella clade, assigning the genus to this clade. Our study focused on three different Dictyosphaerium species to resolve the phylogenetic position of remaining species. We used combined analyses of morphology; molecular data based on SSU and internally transcribed spacer region (ITS) rRNA sequences; and the comparison of the secondary structure of the SSU, ITS‐1, and ITS‐2 for species and generic delineation. The phylogenetic analyses revealed two lineages without generic assignment and two distinct clades of Dictyosphaerium‐like strains within the Parachlorella clade. One clade comprises the lineages with the epitype strain of D. ehrenbergianum Nägeli and two additional lineages that are described as new species (Dictyosphaerium libertatis sp. nov. and Dictyosphaerium lacustre sp. nov.). An emendation of the genus Dictyosphaerium is proposed. The second clade comprises the species Dictyosphaerium sphagnale Hindák and Dictyosphaerium pulchellum H. C. Wood. On the basis of phylogenetic analyses, complementary base changes, and morphology, we describe Mucidosphaerium gen. nov with the four species Mucidosphaerium sphagnale comb. nov., Mucidosphaerium pulchellum comb. nov., Mucidosphaerium palustre sp. nov., and Mucidosphaerium planctonicum sp. nov.     

PHYLOGENETIC POSITION OF CRUSTOMASTIX STIGMATICA SP. NOV. AND DOLICHOMASTIX TENUILEPIS IN RELATION TO THE MAMIELLALES (PRASINOPHYCEAE,CHLOROPHYTA)1

A new marine microalga from the Mediterranean Sea, Crustomastix stigmatica Zingone, is investigated by means of LM, SEM, TEM, and pigment and molecular analyses (nuclear‐encoded small subunit [SSU] rDNA and plastid‐encoded rbcL). Pigment and molecular information is also provided for the related species Dolichomastix tenuilepis Throndsen et Zingone. Crustomastix stigmatica has a bean‐shaped cell body 3–5 μm long and 1.5–2.8 μm wide, with two flagella four to five times the body length. The single chloroplast is pale yellow‐green, cup‐shaped, and lacks a pyrenoid. A small bright yellow stigma is located in the mid‐dorsal part of the cell under the chloroplast membrane. An additional accumulation of osmiophilic globules is at times seen in a chloroplast lobe. Cells lack flat scales, whereas three different types of hair‐like scales are present on the flagella. The main pigments of C. stigmatica are those typical of Mamiellales, though siphonein/siphonaxanthin replaces prasinoxanthin and uriolide is absent. The pigment pool of D. tenuilepis is more similar to that of Micromonas pusilla (Butcher) Manton et Parke and of other Mamiellales. The nuclear SSU rDNA phylogeny shows that the inclusion of C. stigmatica and D. tenuilepis in the Mamiellales retains monophyly for the order. The two species form a distinct clade, which is sister to a clade including all the other Mamiellales. Results of rbcL analyses failed to provide phylogenetic information at both the order and species level. No unique morphological or pigment characteristics circumscribe the mamiellalean clade as a whole nor its two daughter clades.     

Further confirmation of the presence of DNA in the pyrenoid core of the siphonous green algal genus Caulerpa (Caulerpales, Ulvophyceae, Chlorophyta)

We re-examined the distribution of chloroplast DNA (ct-DNA) in the pyrenoid core of Caulerpa okamurae Weber van Bosse and C. lentillifera J. Agardh by fluorescence microscopy after staining the squashes and Technovit sections with DNA fluorochromes such as 4′6-diamidino-2-phenylmdole (DAPI), ethidium bromide, Hoechst 33258 and chromomycin A3. All fluorochromes stained specifically the pyrenoid core on the squashes and Technovit sections. In addition, we present new data on the localization of ct-DNA in the pyrenoid core of two other species of the genus Caulerpa: C. cactoides (Turner) Agardh and C. geminata Harvey.