A "Total Evidence" Analysis of the Phylogenetic Relationships among the Photosynthetic Stramenopiles

Phylogenetic relationships among the nine major autotrophic stramenopile taxa were inferred in a combined analysis of the rbcL, SSU rDNA, partial LSU rRNA, carotenoid, and ultrastructural data sets. The structure of the shortest combined tree is: (Outgroup, ((((Bacillariophyceae, (Pelagophyceae, Dictyochophyceae)),((Phaeophyceae, Xanthophyceae), Raphidophyceae)), Eustigmatophyceae),(Chrysophyceae, Synurophyceae))). The Synurophyceae/Chrysophyceae is the best supported group followed by the Phaeophyceae/Xanthophyceae and the Pelagophyceae/Dictyochophyceae clades. The monophyletic groups composed of Bacillariophyceae/Pelagophyceae/Dictyochophyceae and Phaeophyceae/Xanthophyceae/Raphidophyceae received the lowest Bremer support values. The optimal combined tree suggests that the diatom frustule is derived from the siliceous "skeleton" in Dictyochophyceae, that the reduced flagellar apparatus arose once in the Bacillariophyceae/Dictyochophyceae/Pelagophyceae clade, and that the specific photoreceptor-eyespot apparatus in Chrysophyceae and the Phaeophyceae/Xantophyceae clade originated independently within the autotrophic stramenopiles. Despite conflicts in tree structure between the most-parsimonious combined phylogeny and the optimal tree(s) of each data partition, it cannot be concluded that extensive incongruence exists between the data sets.     

Supermatrix data highlight the phylogenetic relationships of photosynthetic stramenopiles

Molecular data had consistently recovered monophyletic classes for the heterokont algae, however, the relationships among the classes had remained only partially resolved. Furthermore, earlier studies did not include representatives from all taxonomic classes. We used a five-gene (nuclear encoded SSU rRNA; plastid encoded rbcL, psaA, psbA, psbC) analysis with a subset of 89 taxa representing all 16 heterokont classes to infer a phylogenetic tree. There were three major clades. The Aurearenophyceae, Chrysomerophyceae, Phaeophyceae, Phaeothamniophyceae, Raphidophyceae, Schizocladiophyceae and Xanthophyceae formed the SI clade. The Chrysophyceae, Eustigmatophyceae, Pinguiophyceae, Synchromophyceae and Synurophyceae formed the SII clade. The Bacillariophyceae, Bolidophyceae, Dictyochophyceae and Pelagophyceae formed the SIII clade. These three clades were also found in a ten-gene analysis. The approximately unbiased test rejected alternative hypotheses that forced each class into either of the other two clades. Morphological and biochemical data were not available for all 89 taxa, however, existing data were consistent with the molecular phylogenetic tree, especially for the SIII clade.     

Observations on the ultrastructure of the flagella and periplast in the Cryptophyceae

The flagella in Cryptomonas ovata Ehrenberg and two other un-named strains of Cryptomonas both bear stiff hairs with fine distal filaments of the same type as those found in the Xanthophyceae, the Chrysophyceae sensu stricto, the Phaeophyceae, the Bacillariophyceae, the Eustigmatophyceae and the Oomycetes. On the longer of the two flagella the hairs are 2·5 µm long and in two opposite rows whereas on the shorter flagellum they measure only 1 µm, are arranged in a single row and are more closely spaced. The long flagellum also bears a characteristic lateral swelling with a tuft of hairs of the same type as on the remainder of the flagellum, at approximately the level at which it emerges from the gullet. The hairs on the flagella of Hemiselmis rufescens Parke are distributed in a similar manner to those in Cryptomonas but they are more flexible and the swelling and tuft of hairs appear to be absent from the long flagellum. Hairs are apparently absent from the short flagellum of Chroomonas sp. The periplast in Cryptomonas ovata shows a hexagonal pattern in surface view and in sections of all three Cryptomonas strains appears as a typical plasmalemma underlain by a discontinuous layer of electron-dense material with variable substructure. The distribution of flagellar hairs and the structure of the periplast appear to be characters unique to the Cryptophyceae and these features emphasise the isolated position of this class of algae.     

A NEW PHYLOGENY FOR CHROMOPHYTE ALGAE USING 16S-LIKE RRNA SEQUENCES FROM MALLOMONAS PAPILLOSA (SYNUROPHYCEAE) AND TRIBONEMA AEQUALE (XANTHOPHYCEAE)1

The 16S-like ribosomal RNA genes from Mallomonas papillosa Harris et Bradley (Synurophyceae) and Tribonema aequale Pascher (Xanthophyceae) were sequenced and compared to those of other eukaryotes. Mallomonas is closely related to Ochromonas (Chrysophyceae) and supports the general hypothesis of a close phylogenetic relationship between the Synurophyceae and Chrysophyceae. Tribonema is specifically related to Costaria costata (C. A. Agardh) Saunders (Phaeophyceae) demonstrating an unexpected phylogenetic relationship between the Xanthophyceae and Phaeophyceae. Distance and parsimony analysis place these four chromophyte genera in a complex evolutionary assemblage that includes the Bacillariophyceae and Oomycetes but excludes the Dinophyceae. The close relationship between the chromophyte algae and the Öomycete fungi supports the hypothesis that protists with tripartite hairs form a natural assemblage.     

The diversity and phylogeny of the commercially important algal class Eustigmatophyceae,including the new clade Goniochloridales

The Eustigmatophyceae is a class of yellow-green algae allied with the Chrysophyceae and other chlorophyll c possessing stramenopile (heterokont) algae. Some members of the class, especially the marine species of the genus Nannochloropsis, are under intense investigation for their potential for production of biofuels and beneficial fatty acids. The class has generally been thought to comprise a small number of genera and species, and these organisms were considered rare or infrequently encountered. In this study, we examined the phylogeny and diversity of this class by analysis of nuclear 18S rDNA sequence data. Our analysis included sequences from all the named members of the Eustigmatophyceae held in culture collections as well as a number of strains identified in culture collections as Xanthophyceae, new strains with features characteristic of the Eustigmatophyceae, and published data for uncultured DNA clones. The results of these analyses show that the Eustigmatophyceae is far more diverse than generally recognized. Two major lineages are supported in the class, the previously recognized order Eustigmatales and the new clade, Goniochloridales. Additional new lineages were also resolved within each of these major lineages; however, the results of our analyses were considered insufficient for naming these subordinate clades. Several of these lineages comprised only unnamed strains or uncultured DNA clones. Overall, our results indicate that the Eustigmatophyceae is a highly diverse class, with many new species, genera, and families awaiting taxonomic treatment.     

DISTRIBUTION OF THE MITOCHONDRIAL DEVIANT GENETIC CODE AUA FOR METHIONINE IN HETEROKONT ALGAE

The DNA sequence of the cytochrome oxidase subunit I ( COX I) gene (1059 bp), was determined in a number of heterokont algae, including five species of the Phaeophyceae [ Chorda filum (Linnaeus) Stackhouse, Colpomenia bullosa (Saunders) Yamada, Ectocarpus sp., Pseudochorda nagaii (Tokida) Inagaki, Undaria pinnatifida (Harvey) Suringar], and a member of the Raphidophyceae [ Chattonella antiqua (Hada) Ono]. The distribution of a deviant mitochondrial code, the AUA codon for methionine (AUA/Met), which was previously reported in the Xanthophyceae, was inferred from these COX I sequences. Comparative analyses of these sequences revealed that all the algae described above bear the universal genetic code, including the assignment for the AUA codon. A phylogenetic tree was constructed using the obtained sequences along with already-published COX I sequences of various heterokont algae. The clusters of the Xanthophyceae and the Phaeophyceae were resolved as sister groups with high bootstrap support, excluding a bacillariophycean species, a raphidophycean species, and three species of the Eustigmatophyceae. Taking the distribution of the deviant code and the COX I phylogenetic tree together, the genetic code change most probably occurred in an ancestor of the Xanthophyceae after it had branched off from the Phaeophyceae.     

A MOLECULAR PHYLOGENY OF THE HETEROKONT ALGAE BASED ON ANALYSES OF CHLOROPLAST-ENCODED rbcL SEQUENCE DATA1

Nearly complete ribulose-1,5-bisphosphate carboxylase/ oxygenase (rbcL)sequences from 27 taxa of heterokont algae were determined and combined with rbcL sequences obtained from GenBank for four other heterokont algae and three red algae. The phylogeny of the morphologically diverse haterokont algae was inferred from an unambiguously aligned data matrix using the red algae as the root, Significantly higher levels of mutational saturation in third codon positions were found when plotting the pair-wise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In light of this observation, third codon positions were excluded from phylogenetic analyses. Both weighted-parsimony and maximum-likelihood analyses supported with high bootstrap values the monophyly of the nine currently recognized classes of heterokont algae. The Eustigmatophyceae were the most basal group, and the Dictyochophyceae branched off as the second most basal group. The branching pattern for the other classes was well supported in terms of bootstrap values in the weightedparsimony analysis but was weakly supported in the maximum-likelihood analysis (<50%). In the parsimony analysis, the diatoms formed a sister group to the branch containing the Chrysophyceae and Synurophyceae. This clade, charactetized by siliceous structures (frustules, cysts, scales), was the sister group to the Pelagophyceae/Sarcinochrysidales and Phaeo-/Xantho-/ Raphidophyceae clades. In the latter clade, the raphido-phytes were sister to the Phaeophyceae and Xanthophyceae. A relative rate test revealed that the rbcL gene in the Chrysophyceae and Synurophyceae has experienced a significantly different rate of substitutions compared to other classes of heterokont algae. The branch lengths in the maximum-likelihood reconstruction suggest that these two classes have evolved at an accelerated rate. Six major carotenoids were analyzed cladistically to study the usefulness of carotenoid pigmentation as a class-level character in the heterokont algae. In addition, each carotenoid was mapped onto both the rbcL tree and a consensus tree derived from nuclear-encoded small-subunit ribosomal DNA (SSU rDNA) sequences. Carotenoid pigmentation does not provide unambiguous phylogenetic information, whether analyzed cladistically by itself or when mapped onto phylogenetic trees based upon molecular sequence data.     

ALGAE CONTAINING CHLOROPHYLLS a + c ARE PARAPHYLETIC: MOLECULAR EVOLUTIONARY ANALYSIS OF THE CHROMOPHYTA

Sequence comparisons of small subunit ribosomal RNA coding regions from 12 chlorophylls a + c-containing algae were used to infer phylogenetic relationships within the Chromophyta. Three chromophyte lines of descent, delineated by the Bacillariophyceae, the Phaeophyceae/Xanthophyceae, and the Chrysophyceae/Eustigmatophyceae/Synurophyceae are members of a complex evolutionary assemblage, which also includes representatives of the Oomycota (“lower” fungi). Maximum parsimony and distance matrix methods demonstrate a common evolutionary history for these lineages but their relative branching order could not be determined. Other algal species with chlorophylls a + c, including dinoflagellates and prymnesiophytes, are not members of this complex assemblage. Dinoflagellates are specifically related to apicomplexans and ciliates, and the prymnesiophyte, Emiliania huxleyi, represents an independent photosynthetic lineage that separated from other eukaryotes during the nearly simultaneous divergence of plants, animals, fungi, and a number of other protist lineages. The small subunit rRNA phylogenies of chromophytes/oomycetes were compared to those derived from comparisons of ultrastructural characters. Only tubular, tripartite mastigonemes (flagellar hairs) characterized all studied taxa of chromophytes/oomycetes as a monophyletic assemblage.     

CHARACTERIZATION AND PHYLOGENETIC POSITION OF THE ENIGMATIC GOLDEN ALGA PHAEOTHAMNION CONFERVICOLA: ULTRASTRUCTURE, PIGMENT COMPOSITION AND PARTIAL SSU rDNA SEQUENCE

The morphology, ultrastructure, photosynthetic pigments, and nuclear-encoded small subunit ribosomal DNA (SSU rDNA) were examined for Phaeothamnion confervicola Lagerheim strain SAG119.79. The morphology of the vegetative filaments, as viewed under light microscopy, was indistinguishable from the isotype. Light microscopy, including epifluorescence microscopy, also revealed the presence of one to three chloroplasts in both vegetative cells and zoospores. Vegetative filaments occasionally transformed to a palmelloid stage in old cultures. An eyespot was not visible in zoospores when examined with light microscopy, but small droplets, similar to eyespot droplets, were apparent beneath the shorter flagellum when cells were viewed with electron microscopy. Zoospores had two flagella that were laterally inserted in the cell approximately one-third of the cell length from the apex. The longer flagellum was directed anteriorly and the shorter flagellum was directed posteriorly. Electron microscopy revealed the presence of tubular tripartite flagellar hairs on the longer flagellum, but no lateral filaments were found on the tripartite hairs. The general organization of the flagellar root system was similar to that of zoospores belonging to the Xanthophyceae and Phaeophyceae. However, the transitional region of the flagella contained a transitional helix with four to six gyres. Microtubular root R₁ consisted of six microtubules at its proximal end and one microtubule at its distal end. Roots R₂ and R₄ consisted of one microtubule each and root R₃ consisted of two microtubules. No rhizoplast was found. Thin-layer chromatography revealed the presence of fucoxanthin, diadinoxanthin, neoxanthin, and heteroxanthin as well as chlorophylls a, c₁ and c₂. High-performance liquid chromatography revealed the presence of fucoxanthin, diadinoxanthin, diatoxanthin, heteroxanthin, and β,β-carotene as well as chlorophylls a and c. The complete sequence of the SSU rDNA could not be obtained, but a partial sequence (1201 bases) was determined. Parsimony and neighbor-joining distance analyses of SSU rDNA from Phaeothamnion and 36 other chromophyte algae (with two Öomycete fungi as the outgroup) indicated that Phaeothamnion was a weakly supported (bootstrap = <50%, 52%) sister taxon to the Xanthophyceae representatives and that this combined clade was in turn a weakly supported (bootstrap = <50%, 67%) sister to the Phaeophyceae. Based upon ultrastructural observations, pigment analysis, and SSU rDNA phylogenetic analysis, Phaeothamnion is not a member of the Chrysophyceae and should be classified as incertae sedis with affinities to the Xanthophyceae and Phaeophyceae.     

SYNUROPHYCEAE CLASSIS NOV., A NEW CLASS OF ALGAE

The silica-scaled algae (Synuraceae, Chrysophyceae sensu lato) are compared to other Chrysophyceae, Phaeophyceae and Bacillariophyceae with occasional comparisons to other chlorophyll c-containing algae, scaled protozoa and oomycete fungi. The silica-scaled algae have several unique characters which separate them from the above groups and based upon these differences a new order, Synurales ord. nov., and a new class, Synurophyceae class. nov., are described. The major distinguishing characters of the Synurophyceae class. nov. are: they have chlorophylls a and c₁ but lack chlorophyll c₂; their flagellar apparatus includes a microtubular root that loops around two parallel flagella and a flagellar root system which occurs in four absolute orientations; the photoreceptor consists of paired flagellar swellings which are not associated with the cell membrane and chloroplast; no eyespot is present; the nuclear envelope is not or is only weakly associated with the chloroplast endoplasmic reticulum. The Synurophyceae class. nov. are about equally distinct from the Chrysophyceae sensu stricto, Phaeophyceae and Bacillariophyceae when the class level characters are compared. Although the Phaeophyceae have a long history of being placed by themselves in the division Phaeophyta, and the Bacillariophyceae and Chrysophyceae have recently been placed alone in the Bacillariophyta and Chrysophyta, respectively, the similarities found among these classes suggest these algae are not so distinct that they require separate divisions. Tentatively, therefore, the Synurophyceae are placed in the division Phaeophyta along with the Bacillariophyceae and Chrysophyceae sensu stricto.     

α-Carotene and its derivatives have a sole chirality in phototrophic organisms?

Carotenoids in eukaryotic phototrophic organisms can be classified into two groups; β-carotene and its derivatives, and α-carotene and its derivatives. We re-examined distribution of α-carotene and its derivatives among various taxa of aquatic algae (17 classes) and land plants. α-carotene and its derivatives were found from Rhodophyceae (macrophytic type), Cryptophyceae, Euglenophyceae, Chlorarachniophyceae, Prasinophyceae, Chlorophyceae, Ulvophyceae, Charophyceae, and land plants, while they could not be detected from Glaucophyceae, Rhodophyceae (unicellular type), Chryosophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, Xanthophyceae, Eustigmatophyceae, Haptophyceae, and Dinophyceae. We also analyzed the chirality of α-carotene and/or its derivatives, such as lutein and siphonaxanthin, and found all of them had only (6'R)-type, not (6'S)-type.     

PHYLOGENETIC AFFINITIES OF CRYSONEPHELE PALUSTRIS (CHRYSOPHYCEAE) BASED ON INFERRED NUCLEAR SMALL-SUBUNIT RIBOSOMAL RNA SEQUENCE

The nuclear small-subunit ribosomal DNA sequence was determined from the colonial chromophytic alga Chrysonephele palustris Pipes, Tyler, et Leedale using polymerase chain reaction methods. The inferred ribosomal RNA sequence was included in a multiple alignment, containing heterokont chromophytes, and subjected to molecular systematic analyses in order to determine the phylogenetic relationships of this alga . Chrysonephele palustris grouped within the Chrysophyceae and not in an intermediate position between the Chrysophyceae and Eustigmatophyceae, suggesting that Chrysonephele is not a phylogenetic link between these algal classes .     

DIVERSITY OF PLASTID DNA CONFIGURATION AMONG CLASSES OF EUKARYOTE ALGAE1

Information is presented concerning the overall arrangement of plastid DNA (ptDNA) in plastids of approximately 100 spp. of eukaryote algae, representing all classes. The three-dimensional arrangement of the ptDNA was assessed by study of both living and fixed material, stained with the DNA fluorochrome 4′,6-diamidino-2-phenylindole (DAPI), using both phase and fluorescence microscopy. The widespread occurrence of two major types of ptDNA configuration known from prior electron microscopy studies was confirmed. These are (1) DNA densities (nucleoids) of variable size and morphology, scattered throughout the plastid, and (2) a ring nucleoid, beaded or unbeaded, lying just within the girdle lamella. Type 1 is characteristic of Rhodophyta, Dinophyta, Chlorophyta, Cryptophyta, Prymnesiophyceae and Eustigmatophyceae (with one exception). Type 2 is characteristic of Phaeophyceae, Bacillariophyceae, Raphidophyceae, Chrysophyceae (except silicoflagellates and organisms such as Synura and Dinobryon), and Xanthophyceae (with the exception of Vaucheria and three genera known to lack girdle lamellae, Bumilleria, Bumilleriopsis, and Pseudobumilleriopsis). Some of these exceptional forms, as well as Euglenophyta, have configurations of ptDNA not previously recognized. In all the configurations observed, the DNA of a single plastid could be interpreted as being in continuity. This character of plastids appears to be stable under varied conditions of growth and at differing stages of the life cycle, where examined, and has confirmed the reclassification made on other grounds of several taxonomic entities. It has also revealed new questionable classifications. Since DAPI staining is far simpler than serial sectioning for electron microscopy in revealing ptDNA architecture, use of the technique may be valuable for future studies of numerous organisms, both to help in their identification and as an aid to unravelling major taxonomic affinities. In light of the endosymbiont hypothesis, plastid characters may require as great attention as those of the remainder of the cell.     

Green flagellar autofluorescence in brown algal swarmers and their phototactic responses

A flavin-like green autofluorescent substance is noticed to occur in one of the flagella of flagellated cells in the Phaeophyceae, Chrysophyceae, Synurophyceae, Xanthophyceae and Prymnesiophyceae. In the phaeophycean swarmers the autofluorescence occurs in the posterior flagellum throughout its length. It is considered to be involved in the photoreception of phototaxis, since it almost always occurs in the swarmers which have a flagellar swelling and stigma and show phototaxis. In the phaeophycean swarmers, the stigma is shown to act as a concave reflector mirror focusing the reflection light onto the flagellar swelling. In the action spectrum studies, phaeophycean swarmers showed phototaxis between 370 and 520 nm, having two major peaks at 420 or 430 nm and 450 or 460 nm. Their responses were true phototactic and not photophobic. Rotation of the swarmer was shown to be essential in the photoreception ofEctocarpus gametes. Recipient of the Botanical Society Award for Young Scientists, 1991.     

A fine structural study of Olisthodiscus luteus carter

Fine structurally, Olisthodiscus luteus is characterised by possessing a sub-surface layer of electron opaque ‘spheres’ approximately 35 nm in diameter. These ‘spheres’ originate in vesicles surrounding the Golgi apparatus. The flagella are of the heterokont type and are attached, at their bases, by a large complicated root to the nucleus. The mitochondria, besides containing microvilli, contain fine fibrils of material that can be removed by treatment with DNase.

The phyletic affinities of Olisthodiscus are discussed with reference to this fine structural study and recent biochemical work. Although no conclusive evidence is available, it is suggested that Olisthodiscus should be transferred from the Xanthophyceae temporarily to the Chrysophyceae.     

Schizocladia ischiensis: a new filamentous marine chromophyte belonging to a new class,Schizocladiophyceae

A new marine filamentous chromophyte Schizocladia ischiensis sp. nov. is described from Naples, Italy, and a new class, Schizocladiophyceae, is proposed to accommodate the species based on morphology, photosynthetic pigment analysis, and rbcL and 18S rRNA gene sequences. The vegetative thallus is composed of branched filaments, 3-7 microm in diameter, containing one to two light brown parietal plastids. Cell walls are composed of layered fibers containing alginates, but lacking cellulose. Plastids are of the typical chromophyte type, containing chlorophylls a and c, and abundant fucoxanthin. Zoospores are formed by direct transformation of vegetative cells or through a process including a multinucleated cell stage. Zoospores are teardrop-shaped with a longer anterior flagellum with tubular mastigonemes and a shorter smooth posterior flagellum with a basal swelling. Flagella have a single basal plate and multi-gyred transitional helix distal to the basal plate. Each zoospore has an eyespot. Phylogenetic analyses using rbcL and 18S rDNA sequences suggest the closest phylogenetic relationship with Phaeophyceae, and then with Xanthophyceae and Phaeothamniophyceae. Nevertheless, Schizocladia differs from Phaeophyceae in some essential features (i.e. cell wall lacking cellulose and plasmodesmata, presence of flagellar transitional helix). Therefore, an independent class Schizocladiophyceae is proposed to accommodate this new taxon.     

Immunogold-labeling analysis of alginate distributions in the cell walls of chromophyte algae

The immunogold electron microscopy technique was employed to detect the presence of alginates in the cell walls of selected chromophyte species. Anti-alginate antiserum labeled the cell walls of Sphacelaria and Scytosiphon (Phaeophyceae), Tribonema, Vaucheria, Botrydium, Botrydiopsis (Xanthophyceae) and an‘un-described filamentous species’ (incertae cedis), but it did not label those of Giraudyopsis, Phaeosaccion (Chrysomeridales), Antithamnion (Rhodophyceae) and Bryopsis (Ulvophyceae). This is the first report of the occurrence of alginates in the chromophyte outside Phaeophyceae. The absence of alginates in Chrysomeridales, which has an unclear phylogenetic position, implies a rather distant phylogenetic relationship of the order Chrysomeridales from Phaeophyceae/Xanthophyceae.     

The ultrastructure and taxonomy of the Chrysophyceae and Prymnesiophyceae (Haptophyceae): a survey with some new observations on the ultrastructure of the Chrysophyceae

Information on the internal structure of the Chrysophyceae is reviewed and some new data are presented. From these a model has been constructed which represents the combination of features constructed to be the basic pattern of cell structure in this group. A brief review is given of modern research, which demonstrates that the choanoflagellates should be removed not only from the Chrysophyccae but also from the plant kingdom. The organization of the Prymnesiophyceae classis nova (= Haptophyceae) is compared with that of the Chrysophyceae, and the relationship of both groups to the Xanthophyccae, Phaeophyceac anti Bacillariophyceae is considered. This leads to the conclusion that the Prymnesiophyceae must be considered as a class separate from the Chrysophyceae. Removal of the Prymnesiophyceae and choanoflagellates from the Chrysophyceae leaves a largely homogeneous and monophyletic group, though the family Pedinellaceae appears to have a unique organization and may be found to be separated from the Chrysophyccae by a phyletic distance at least as great as that between the remaining classes of heterokont algae. It is suggested that this family should occupy a more isolated position in the Chrysophyceae than at present, possibly in a separate order Pedinellales.     

Chloroplast pigments and algal systematics

The chloroplast pigments of one typical representative (Pleurochloris magna) and two potential members (clone BSG Sticho and an isolate called Tunis) of the new class Eustigmatophyceae have been examined by modern methods including mass spectrometry. The three cultures all exhibited the same chloroplast pigments: Chlorophyll a, but no b or c, δ-carotene (I), canthaxanthin (II), violaxanthin (IIIa), and esterified vaucheriaxanthin (IVb) plus some free vaucheriaxanthin (IVa). Furanoid rearrangement of the epoxidic carotenoids complicated the analysis. The unique pigment complement hereby indicated for Eustigmatophyceae is clearly different from the pigment distribution patterns reported for Chlorophyceae and Xanthophyceae.     

PHOSPHOTUNGSTIC ACID-CHROMIC ACID: A SELECTIVE STAIN FOR ALGAL PLASMA MEMBRANES1,2

A phosphotungstic acid-chromic acid mixture selectively stains the plasma membrane of whole cells of selected members of the Chlorophyceae, Charophyceae, Euglenophyceae, Xanthophyceae, Bacillariophyceae, Chrysophyceae, and Rhodophyceae, and the plasma membrane in cell-free fractions of Mougeotia (Chlorophyceae). The procedure is not effective on the plasma membrane of the cyanophycean Scytonema or the cyanophycean endosymbiont of Glaucocystis. Staining of the cell walls of Chlamydomonas, Bangia, and Scytonema and the pellicle and sliding junction of Euglena and Astasia suggest that PTA-CrO₃ reactivity may be associated with glycoproteins in the cell walls and plasma membranes.