Characterization and Phylogenetic Position of the Enigmatic Golden Alga Phaeothamnion Confervicola: Ultrastructure, Pigment Composition and Partial Ssu Rdna Sequence1
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J. Phycol. 34, 286±298 (1998) CHARACTERIZATION AND PHYLOGENETIC POSITION OF THE ENIGMATIC GOLDEN ALGA PHAEOTHAMNION CONFERVICOLA: ULTRASTRUCTURE, PIGMENT COMPOSITION AND PARTIAL SSU RDNA SEQUENCE1 Robert A. Andersen,2 Dan Potter 3 Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine 04575 Robert R. Bidigare, Mikel Latasa 4 Department of Oceanography, 1000 Pope Road, University of Hawaii at Manoa, Honolulu, Hawaii 96822 Kingsley Rowan School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia and Charles J. O'Kelly Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine 04575 ABSTRACT coxanthin, diadinoxanthin, diatoxanthin, heteroxanthin, The morphology, ultrastructure, photosynthetic pig- and b,b-carotene as well as chlorophylls a and c. The ments, and nuclear-encoded small subunit ribosomal DNA complete sequence of the SSU rDNA could not be obtained, (SSU rDNA) were examined for Phaeothamnion con- but a partial sequence (1201 bases) was determined. Par- fervicola Lagerheim strain SAG119.79. The morphology simony and neighbor-joining distance analyses of SSU rDNA from Phaeothamnion and 36 other chromophyte of the vegetative ®laments, as viewed under light micros- È copy, was indistinguishable from the isotype. Light micros- algae (with two Oomycete fungi as the outgroup) indicated copy, including epi¯uorescence microscopy, also revealed that Phaeothamnion was a weakly supported (bootstrap the presence of one to three chloroplasts in both vegetative 5,50%, 52%) sister taxon to the Xanthophyceae rep- cells and zoospores. Vegetative ®laments occasionally trans- resentatives and that this combined clade was in turn a formed to a palmelloid stage in old cultures. An eyespot weakly supported (bootstrap 5,50%, 67%) sister to the was not visible in zoospores when examined with light mi- Phaeophyceae. Based upon ultrastructural observations, croscopy, but small droplets, similar to eyespot droplets, pigment analysis, and SSU rDNA phylogenetic analysis, were apparent beneath the shorter ¯agellum when cells were Phaeothamnion is not a member of the Chrysophyceae viewed with electron microscopy. Zoospores had two ¯agella and should be classi®ed as incertae sedis with af®nities that were laterally inserted in the cell approximately one- to the Xanthophyceae and Phaeophyceae. third of the cell length from the apex. The longer ¯agellum Key index words: 18S rRNA; algae; carotenoids; chloro- was directed anteriorly and the shorter ¯agellum was di- phylls; Chrysophyceae; ¯agellar apparatus; heteroxanthin; rected posteriorly. Electron microscopy revealed the presence microtubular roots; Phaeophyceae; Phaeothamnion; phy- of tubular tripartite ¯agellar hairs on the longer ¯agellum, logeny; Xanthophyceae but no lateral ®laments were found on the tripartite hairs. The general organization of the ¯agellar root system was Abbreviations: bp, boiling point; HPLC, high-perfor- similar to that of zoospores belonging to the Xanthophyceae mance liquid chromatography; SSU rDNA, nuclear-encod- and Phaeophyceae. However, the transitional region of the ed small subunit ribosomal DNA ¯agella contained a transitional helix with four to six gyres. Microtubular root R1 consisted of six microtubules Phaeothamnion is a genus of freshwater ®lamen- at its proximal end and one microtubule at its distal end. tous algae currently classi®ed in the Chrysophyceae Roots R2 and R4 consisted of one microtubule each and (e.g. Bourrelly 1957, Preisig 1995). However, in the root R3 consisted of two microtubules. No rhizoplast was past, Phaeothamnion has been placed in the Phaeo- found. Thin-layer chromatography revealed the presence of phyceae (West 1904) and the Cryptophyceae (Pasch- fucoxanthin, diadinoxanthin, neoxanthin, and hetero- er 1914). Phaeothamnion confervicola Lagerheim is the xanthin as well as chlorophylls a, c1 and c2. High-perfor- type species (Lagerheim 1884, as P. confervicolum), mance liquid chromatography revealed the presence of fu- and it is typi®ed by specimens distributed as no. 608 in the Algae Aquae Dulcis Exsiccatae (Wittrock and 1 Received 26 September 1997. Accepted 31 December 1997. Nordstedt, fasc. 13). There are two described varie- 2 Author for reprint requests; e-mail [email protected]. ties, the nominate variety and P. confervicola var. brit- 3 Present address: Department of Pomology, University of Cal- ifornia, Davis, California 95616. tanica Godward. Phaeothamnion, in turn, is the type 4 Present address: Institut de CieÁncies del Mar, Passeig Joan de genus for the family Phaeothamniaceae and order Borbo s/n, 08039, Barcelona, Spain. Phaeothamniales (Bourrelly 1957) or suborder 286 PHAEOTHAMNION CONFERVICOLA 287 Phaeothamnineae (Bourrelly 1981, Starmach 1985). MATERIALS AND METHODS Four additional species have been described: P. ar- Phaeothamnion sp. strain SAG119.79 was obtained from the Sam- ticulatum Ettl, P. borzianum Pascher, P. dichrysis Vil- mlung von Algenkulturen at the University of GoÈttingen, Ger- laret, and P. polychrysis (Geitler) Pascher. These spe- many (SchloÈsser 1994). Cells were grown in DYIII (Lehman 1976) cies are separated on the basis of chloroplast num- or DYIV (Andersen et al. 1997) culture media at 12±258 C under ca. 100 mmol´m22´s21 visible light irradiance and either a 14:10 h ber, presence or absence of an eyespot in the zoo- or a 12:12 h LD photocycle. The vegetative features of this strain spore, cell size, basal cell morphology, cell wall were compared with isotype material of P. confervicola obtained thickness, branching patterns of the thalli, and oth- from the Farlow Herbarium of Harvard University (no. 608 in the er morphological features. The type description for Algae Aquae Dulcis Exsiccatae, Wittrock and Nordstedt, fasc. 13). Free-swimming zoospores were obtained by placing ®laments P. confervicola indicates that it has one chloroplast from an old culture (at least 30 days old) into fresh culture me- (Lagerheim 1884), whereas others report more than dium. During the light cycle on the second and third day follow- one chloroplast in at least some cells (Godward ing the culture medium change, zoospore release occurred from 1933, Geitler and Schiman-Czeika 1970). Also, an most ®laments. For electron microscopy, whole mount prepara- erroneous cell measurement has made its way into tions were prepared by collecting zoospores with centrifugation and drying cells directly on grids coated with pioloform (Ted the literature; Starmach (1985) gives the cell length Pella Inc., Redding, California). Cells were negatively stained with for P. confervicola as 20±30 mm even though Lager- a saturated aqueous solution of uranyl acetate. Zoospores pre- heim (1884) clearly reports the cell length to be no pared for electron microscopic thin sectioning were ®xed for 30± more than 12 mm. 60 min at room temperature (ca. 208 C) in a cocktail consisting Several additional golden-colored algae with ®la- of 1 mL 5% glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7, 0.5 mL 4% aqueous osmium tetroxide, and 8.5 mL zoo- mentous or sarcinoid habits have, at one time or spores in DYIII medium. The cells were harvested by gentle ®l- another, been assigned to the Phaeothamniales. tration onto cellulose acetate ®lters (3.0-mm pore size), enrobed Many of these algae, especially those placed in the in 3.0% aqueous ultralow-temperature gelling agarose, dehydrat- Sarcinochrysidales by Gayral and Billard (1986), are ed in an acetone series at 48 C, and embedded in Polybed-Araldite resin (Ted Pella Inc.). no longer assignable to the Chrysophyceae (O'Kelly For pigment analysis by thin layer chromatography (TLC), cells 1989a, Preisig 1995, Saunders et al. 1997). Preisig were concentrated by centrifugation, and pigments were extract- (1995) refers only the genera Chrysoclonium, Phae- ed from cell pellets using a Turrax macerator (Thomas Scienti®c, othamnion, Sphaeridiothrix, and Tetrachrysis to the Swedesboro, New Jersey) and various combinations of acetone, Phaeothamniaceae and places this family in the methanol, acetone:methanol (1:1), and DMSO. The extract was mixed with one-half volume of diethyl ether followed by an excess Chromulinales. Sphaeridiothrix has been considered of 10% aquaeous NaCl (w/v) to concentrate the pigments in the a palmelloid stage of Phaeothamnion by Geitler and epiphase. The epiphase volume was reduced with a stream of Schiman-Czeika (1970), but others have considered nitrogen (N2) gas. The concentrated extract was stored in dark- it to be separate (e.g. Pascher and Vlk 1943, Bour- ness at 2158 C over anhydrous NaCl to remove water. Six TLC relly 1963, Andrews 1970, Dop and Vroman 1976, systems were used. System 1 was a silica gel G/borax plate (Keast and Grant 1976) and a solvent solution of petroleum ether (608± Santos 1976, Cambra 1989). To further complicate 808 C bp):ethyl acetate:diethylamine (58:30:12) (Riley and Wilson matters, the xanthophyte genus Heterodendron, with 1965). System 2 was a silica gel G/borax plate and a solvent so- two species, may also be referable to Phaeothamnion lution of 20% acetone in n-hexane (Chapman 1965). System 3 (Ettl 1959). Species of Heterodendron are otherwise was a silica gel G/borax plate and a solvent solution of 40% ac- nearly indistinguishable from Phaeothamnion except etone in n-hexane (Chapman 1965). System 4 was a silica gel G/borax plate and a solvent solution of 30% acetone in petro- for their yellow-green plastids, and they have been leum ether (608±808 C bp) (Yokahama 1983). System 5 was a assigned to the Xanthophyceae