Aurearenophyceae classis nova, a New Class of Heterokontophyta Based on a New Marine Unicellular Alga Aurearena cruciata gen. et sp. nov. Inhabiting Sandy Beaches

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Protist, Vol. 159, 435—457, July 2008 http://www.elsevier.de/protis Published online date 19 March 2008

ORIGINAL PAPER

Aurearenophyceae classis nova, a New Class of Heterokontophyta Based on a New Marine Unicellular Alga Aurearena cruciata gen. et sp. nov. Inhabiting Sandy Beaches Atsushi Kai, Yukie Yoshii2, Takeshi Nakayama, and Isao Inouye1 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan Submitted October 16, 2007; Accepted December 29, 2007 Monitoring Editor: Robert A. Andersen

A new heterokontophyte alga, Aurearena cruciata gen. et sp. nov., was isolated from sandy beaches in Japan. Isolates were characterized by light and electron microscopy, spectroscopy of pigment composition, and molecular phylogenetic analyses using 18S rDNA and rbcL. The alga usually possessed a cell wall but also retained two heterokont flagella beneath the cell wall. Each walled cell first produced only a single flagellate cell that subsequently divided into two flagellate cells. Electronopaque vesicles, possibly associated with cell wall formation, were observed beneath the cell membrane. The chloroplast consisted of two compartments, each enclosed by a chloroplast envelope and the inner membrane of the chloroplast endoplasmic reticulum; these two compartments were surrounded by a common outer membrane of chloroplast endoplasmic reticulum. Molecular phylogenetic trees suggested that this alga was a new and independent member of the clade that included the Phaeophyceae and Xanthophyceae (PX clade). A new class, Aurearenophyceae classis nova was proposed for A. cruciata. & 2008 Elsevier GmbH. All rights reserved. Key words: flagellar apparatus; Xanthophyceae; phylogeny.

Phaeophyceae;

Phaeothamniophyceae;

pigment

composition;

Introduction The heterokontophytes, which include brown algae and diatoms, are major primary producers in the present-day hydrosphere (Falkowski et al. 2004). The brown algae (Phaeophyceae) form large marine forests in coastal regions and provide 1

Corresponding author; fax +81 29 853 4533. e-mail [email protected] (I. Inouye). 2 Present address: Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki, eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan.

& 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2007.12.003

diverse habitats for a variety of animals to grow and reproduce. In addition, they may play a significant role in the global carbon cycle by fixing carbon dioxide and releasing dissolved organic carbon for export to deep water (Itoh et al. 2007; Wada et al. 2007). Brown algae are also economically important as food and as a source of alginic acid and fucoidan (McLachlan 1985; Smit 2004). Ultrastructural observations, biochemical analyses, and molecular phylogenetic approaches have revealed that the greatest phylogenetic

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diversity among photosynthetic organisms lies in the heterokontophyte algae (e.g., Andersen 2004a, b). These algae are classified as the Heterokontophyta and, at present, comprise 15 distinct classes. Of these, eight classes (Bolidophyceae, Chrysomerophyceae, Pelagophyceae, Phaeothamniophyceae, Pinguiophyceae, Schizocladiophyceae, Synchromophyceae, and Synurophyceae) have been recognized in the past two decades (Andersen 1987; Andersen et al. 1993; Bailey et al. 1998; Cavalier-Smith et al. 1995; Guillou et al. 1999; Horn et al. 2007; Kawachi et al. 2002; Kawai et al. 2003). These recent taxonomic developments suggest that our understanding of the true diversity of the heterokont algae is still incomplete. The members of the Heterokontophyta are characterized by formation of flagellate cells that have two unequal heterokont flagella — an anterior flagellum bearing tripartite tubular hairs and a smooth posterior flagellum — and a chloroplast bounded by four membranes (e.g., Andersen 2004a). They are believed to have arisen via so-called secondary endosymbiosis of a red alga (Cavalier-Smith 1993, 1995). Molecular phylogenetic studies, calibrated for evolutionary rates of gene sequences as a molecular clock, suggest that these algae evolved 170—270 Ma (Medlin et al. 1997). They should have evolved much later than the green algae, which acquired a cyanobacterium and converted it to a plastid by primary endosymbiosis. The Heterokontophyta have radiated out rapidly in relatively recent geological periods — the Mesozoic and Cenozoic eras. On the basis of the fossil record, it is well accepted that diatoms appeared 190 Ma in the Jurassic period of the Mesozoic era (Sims et al. 2006). This could be why, despite the morphological distinctness of the classes, it is difficult to elucidate their phylogenetic relationships and branching order based on morphological and biochemical data. Molecular phylogenetic studies of the Heterokontophyta have been based mainly on the sequences of rbcL, a chloroplast-encoded gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO), and the nuclear-encoded small subunit ribosomal RNA gene (18S rDNA) (e.g., Andersen et al. 1993; Bailey et al. 1998; Cavalier-Smith and Chao 2006; Kawai et al. 2003; Van de Peer et al. 1996). These phylogenetic analyses support the monophyly of each class of the Heterokontophyta with confidence, but have failed to determine the relationships among the classes. At present, the global phylogeny of the Heterokontophyta is far

from clear. Despite these undetermined relationships, several higher phylogenetic groups have been recognized. One of these large groups is the clade including the Phaeophyceae and Xanthophyceae (Ariztia et al. 1991; Cavalier-Smith et al. 1995; Van de Peer et al. 1996). O’Kelly and Floyd (1985) suggested that the Chrysomerophyceae and Phaeothamniophyceae be added to this large clade based on ultrastructural studies. Cavalier-Smith et al. (1995) proposed the superclass Fucistia for this large clade. Molecular phylogenetic studies supported the addition of the Chrysomerophyceae, Phaeothamniophyceae, and, subsequently, the Schizocladiophyceae to this clade (Andersen et al. 1998; Bailey et al. 1998; Cavalier-Smith and Chao 2006; Kawai et al. 2003; Saunders et al. 1997). However, the monophyly of this assemblage and the relationship among its members have not been well resolved at the molecular phylogenetic level. The assemblage that includes the Chrysomerophyceae, Phaeophyceae, Phaeothamniophyceae, Schizocladiophyceae, and Xanthophyceae is referred to here as the PX clade, P and X referring to the two major taxa of the clade, the Phaeophyceae and Xanthophyceae. The life forms in the PX clade are very diverse, e.g., unicells, colonies, filaments, coenocytes, or macroscopic thalli. Their habitats are also very diverse, encompassing freshwater, soil, or marine environments. In our opinion, one of the most important evolutionary events to have occurred in the PX clade was the emergence of true multicellularity in the Phaeophyceae. In conjunction with multicellularity, a complex organization of the thallus comparable to the land plants evolved in the Phaeophyceae, including the development of an epidermis and cortex, transfer tissue including sieve plates, sexual organs, and receptacles. The diversification and evolution of brown algal seaweeds have given rise to marine forests that are essential components of present-day coastal ecosystems. All the members of the PX clade possess a cell wall, and one of the characteristic features of this clade is the absence of flagellate cells in the vegetative phase; this is in distinct contrast to the other classes of Heterokontophyta. Approximately 100 years ago, some species in the Xanthophyceae were described as naked forms. However, these species were described based solely on light microscopic features, and there has been no ultrastructural or molecular evidence in support of their presumed taxonomic status. For example, Chlorarachnion reptans, presently recognized as the type species of the Chlorarachniophyta, was

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Aurearena cruciata gen. et sp. nov.

first described as a xanthophyte; however, a detailed ultrastructural study revealed that this alga formed an independent algal phylum (Hibberd and Norris 1984). On the basis of reliable modern data, there is no naked species belonging to the xanthophytes and the PX clade as a whole. At present, therefore, it would be reasonable to exclude these doubtful species from consideration. We discovered a walled unicellular member of the PX clade from sandy beaches in Japan. This alga could not be placed in any of the known classes of the Heterokontophyta. In this paper, we describe the characterization of this novel alga by light and electron microscopy, spectrophotometry, and molecular phylogenetic analyses. Based on the results obtained, we discuss the phylogeny of the PX clade.

Results Taxonomic Treatments Aurearenophyceae Kai, Yoshii, Nakayama et Inouye class. nov. Cellulae, immobiles, parietibus, duis inequalibus flagellis subter parietibus, vel cellulae, nuda, mobilles, duoram flagellorumque. Flagellum longius cum tripartitiones pili flagellares. Chloroplastus lamellarum thylacoidibus tribus appressarum, sine lamella cingulari; DNA chloroplasti nucleoides adspersus formans; chlorophyllis a, b-carotene, diadinoxanthino, diatoxanthino, violaxanthino, antheraxanthino, zeaxanthino, fucoxanthino. Cells, non-motile, surrounded by a cell wall, with two unequal flagella lying inside the cell wall, or cells naked, motile and biflagellate. Longer flagellum with tripartite flagellar hairs. Chloroplast lamellae of three adpressed thylakoids; without girdle lamella; chloroplast DNA scattered; with chlorophyll a, b-carotene, diadinoxanthin, diatoxanthin, violaxanthin, antheraxanthin, zeaxanthin, and fucoxanthin. Aurearenales Kai, Yoshii, Nakayama et Inouye ord. nov. Cum characteristibus classis. With characteristics of the class. Aurearenaceae Kai, Yoshii, Nakayama et Inouye fam. nov. Cum characteristibus classis. With characteristics of the class. Type genus: Aurearena Kai, Yoshii, Nakayama et Inouye

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Aurearena Kai, Yoshii, Nakayama et Inouye gen. nov. Cellulae dimorphe ut minimae. Cellulae immobiles parietibus. Chloroplastus auratus, stigmate, pyrenoide projecto cum sulco notato. Cellulae mobilles pro parte maxima ovata, flagellis duobus inaequaliibus subapicaliter insertis. Cells having at least two forms. Non-motile cells surrounded by a cell wall. Golden-yellow chloroplast, with stigma, and with projecting pyrenoid having a marked furrow. Motile cells mostly ovate. Two unequal flagella inserted subapically. Type species: A. cruciata Kai, Yoshii, Nakayama et Inouye Etymology: Aurearena ¼ ‘‘golden sand’’ in Latin Aurearena cruciata Kai, Yoshii, Nakayama et Inouye sp. nov. Cellulae immobiles, sphaericae vel subsphaericae 8—15 mm parietibus. Nuculeus unus, duobus corporibus Golgi parabasale. Chloroplastus, centrali pyrenoide cum sulco cruciato, longitudinali ex invaginatione duarum membranarum chloroplasti et reticuli endoplasmatici chloroplasti interioris constanti, latitudinali ex invaginatione duarum membranarum chloroplasti constanti. Cellulae mobiles, plerumque 10—15 mm longae, 5—10 mm latae. Flagellum longius porro directum, mastigonematibus tubularis obsessum, bervius nudum cum basali tumore. Nucleus unus, in parte antica cellulae, prope bina flagella. Chloroplastus pars postica cellulae insidens, cum lobis cum stigmate porpe flagella breve prologato. Non-motile cells, spherical to subspherical, measuring 8—15 mm, including cell wall. A single nucleus, with two parabasal Golgi bodies. Chloroplast with central pyrenoid having cross-shaped furrow; the longitudinal furrow consists of the invagination of two chloroplast membranes and the inner membrane of the chloroplast endoplasmic reticulum, and the transverse furrow consists of the invagination of two chloroplast membranes. Motile cells are mostly 10—15 mm long, 5—10 mm wide. Longer flagellum directed forward, with tubular flagellar hairs; shorter flagellum naked with basal swelling. A single nucleus located in the anterior part of the cell, near the two flagella. Chloroplast located in the posterior part of the cell, with a lobe and with a stigma extended near the short flagellum. Holotype: TNS-AL-53994 deposited in the Herbarium, Tsukuba Botanical Garden, National Science Museum, Tsukuba, Japan (TNS). The strain NIES-1863, used for the holotype, is maintained in the National Institute for Environmental Studies (NIES).

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Type locality: Isonoura, Wakayama, Japan (Fig. 1) Etymology: cruciata ¼ ‘‘cross-shape’’ (Latin) referring to the characteristic invaginations of the chloroplast. The three strains, NIES-1863, NIES-1864, and NIES-1865, were morphologically indistinguishable from one another. Non-motile, spherical and walled cells, measuring 8—15 mm in diameter, including the cell wall (Fig. 2A), dominated in culture. These non-motile cells floated on the water surface or were attached to the wall of culture vessels oriented toward the light. The walled cell possessed a single golden-yellow chloroplast that radiated out from the central pyrenoid (but, see below). A cruciate pattern was visible on the pyrenoid (Fig. 2B). In addition, a yellowish reflective granule, probably homologous to the eyespot of the swimming cell, was often observed in the chloroplast (Fig. 2A). When cultured on a 14:10 h LD cycle, a single free-swimming cell was released from a single walled cell at approximately 11 h after the onset of the light period. The cell formed a pore in the cell wall from which a single swimming cell emerged (Fig. 2C). The swimming cell was ovoid (10—15  5—10 mm) and lacked a cell wall or any other cell covering (Fig. 2D). The cells possessed two subapically inserted flagella, a

long anterior flagellum and a short posterior flagellum. Swimming cells possessed a chloroplast with a yellowish eyespot located near the insertion of the posterior flagellum. Swimming cells were observed for a relatively short period, 2—3 h after they appeared first in the culture. Then, an aggregate of non-motile walled cells was formed at the side of culture vessels oriented toward the light, indicating that the swimming cells were positively phototactic. When observed under the light microscope, swimming cells readily absorbed their flagella into the cell, and became rounded and walled in response to physical stimuli, such as the pressure of cover slip and the contact to sand grains and debris (Fig. 2E). Under normal culture conditions, cell division took place in the swimming cell (Fig. 2F), resulting in two swimming cells that soon transformed into non-motile walled cells. Cells repeated an alternation of a dominant non-motile walled stage and a naked swimming stage within a short period of time. The cell cycle of A. cruciata is shown in Figure 3. The chloroplast nucleoid was of a scattered type (Fig. 4A, B; arrowhead). In old cultures and in the low salinity (18%) condition, large multinucleate cells, up to 40 mm in diameter, sometimes occurred (Fig. 4C, D). These cells possessed a number of eyespot-like granules. When these large cells were transferred into fresh culture medium, they did not release swimming cells but several walled cells. When cells died, the cytoplasm became discolored to dark bluishgreen, whereas the color of the chloroplast remained unchanged (Fig. 5). The cause of this change in cytoplasmic coloration is unknown. Neither sexual reproduction nor the formation of cysts was observed.

Electron Microscopy

Figure 1. The location of sampling sites where Aurearena cruciata was collected. Site 1, Isonoura beach, Wakayama Prefecture, Japan. Site 2, Noma beach, Aichi Prefecture, Japan. Site 3, Hashikui beach, Wakayama Prefecture, Japan.

The configuration of all cellular components, including the flagellar apparatus, was basically identical between the walled and swimming cells. Diagrammatic illustrations of the walled and swimming cells are presented in Figure 6A and B. The cell wall was comprised of fibrous material and the wall was surrounded by mucous material (Figs 7A, 9D, F). Many electron-opaque vesicles were located beneath the plasma membrane (Figs 7A, C, D, 10A). The chloroplast exhibited a complicated architecture when viewed in the electron microscope. It was divided into two compartments (dorsal and ventral in the flagellate cells), each surrounded by three membranes — two membranes of the chloroplast envelope and

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Figure 2. Light micrographs of Aurearena cruciata gen. et sp. nov. A. Walled cell possessing a yellow granule (arrowhead). B. Cruciate pattern visible on the central pyrenoid. C. Single swimming cell emerging from walled cell leaving cell wall behind. D. Swimming cell with eyespot (arrowhead). E. Swimming cell absorbing flagella into the cell (arrowhead). F. Cytokinesis in the swimming cell. Scale bars, 10 mm. l; LF, long flagellum; SF, short flagellum; W, cell wall.

the inner membrane of the chloroplast endoplasmic reticulum (cER) (Fig. 7A—C; arrowhead). Each compartment possessed an invagination of the two membranes of the chloroplast envelope and the associated periplastidal compartment (Fig. 7A—C; double arrowhead). The invagination extended transversely at right angles to the longitudinal boundary of the two compartments and from the center of the pyrenoid complex into dorsal and ventral directions (Fig. 7A, C). This invagination was tubular, or narrow and plate-like, and did not bisect the chloroplast compartment completely (Fig. 7A, B). This architecture resulted in a cruciate pattern of the pyrenoid that was visible under the light microscope (Fig. 2B). The

two compartments of the chloroplast were enveloped in a single common membrane, the outer membrane of the cER (Fig. 7B); therefore, we refer to the chloroplast of A. cruciata as a singular structure. The pyrenoid was situated at the center of the chloroplast and longitudinally bisected by three membranes as mentioned above (Fig. 7A). The chloroplast lamellae consisted of three adpressed thylakoids, and a girdle lamella was absent (Fig. 8A, B). In both walled and swimming cells, a single layer of lipid globules formed an eyespot near the insertion of the posterior flagellum (Fig. 7B, D). The single nucleus was located near the basal bodies, and wedged between two Golgi bodies (Fig. 7D). The outer

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Figure 3. Cell cycle of Aurearena cruciata. A. A flagellate cell inside a cell wall. B. Naked swimming cell emerging through a pore in the cell wall. C. Swimming cell. D. Cytokinesis proceeding in a swimming cell. E. Swimming cell absorbing flagella into the cell. F. Round non-motile cell after absorption of flagella.

Figure 4. Light micrographs of Aurearena cruciata. Nuclear and chloroplast DNA. DIC images (left column: A, C) and fluorescence images of the same cells (right column: B, D). A, B. Nuclear DNA and scattered chloroplast DNA (arrowheads in B). C, D. Multinucleate large cell. Eyespot-like granules are seen (arrowheads in C). Scale bars, 10 mm.

Figure 5. Light micrographs of Aurearena cruciata. The cytoplasm discolored to dark bluish green in dead cell (arrowhead). Scale bar, 10 mm P, Plastid.

nuclear membrane was continuous with the outer membrane of cER in its posterior region and the anterior region of the pyrenoid (Fig. 8C). The ER was distributed throughout the cytoplasm (Fig. 8D). Mitochondria exhibited many tubular cristae (Figs 7A, C, 8D, 10A). Negatively stained material revealed that the long anterior flagellum (LF) possessed tripartite hairs with triple terminal filaments and no lateral filaments (Fig. 9A—C), while the short posterior flagellum (SF) was smooth and possessed a swelling near its base (Figs 7C, 9F, 13F). Two unequal flagella were also present in the walled cell. These lay in a narrow space between the cell wall and the plasma membrane (Fig. 9D, F). The long flagellum possessed tripartite hairs whereas the short flagellum contained a swelling (Fig. 9E, F), indicating that the walled cell had a complete pair of flagella as in the swimming cells. In addition, when the swimming cells became spherical, as shown in Figure 10A and B, the flagellar microtubules were absorbed into the cytoplasm and basal bodies and flagellar roots were retained, suggesting that flagella were decomposed and newly generated in the walled cells. The flagellar apparatus consisted of two basal bodies, four short microtubular roots (R1, R2, R3, and R4, sensu Andersen 1987) and a rhizoplast.

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LF G

LF

W

G

EV

SF

SF

M

C

N

E E

N BB1 C

P

P

EV

M

Figure 6. Diagrammatic illustrations of the walled and swimming cell of Aurearena cruciata, viewed from the lateral side of the cell. A. Walled cell. B. Swimming cell. C, chloroplast; E, eyespot; G, Golgi body; M, mitochondrion; N, nucleus; P, pyrenoid; W, cell wall; EV, electron opaque vesicle; BB, basal body; LF, long anterior flagellum.

A diagrammatic reconstruction of this apparatus is presented in Fig. 11. The transition region of each flagellum consisted of a single transitional plate lying close to the plane of the plasma membrane and a six-gyred transitional helix located distal to the transitional plate (Figs 9D, 12A, B, 13B, F). Two basal bodies were arranged at right angles and located at the subapical ventral face (Fig. 13A—F). The basal bodies of the LF (BB2) and the SF (BB1) were approximately 110 nm in length and contained an electron-dense core (Figs 12A, 13C, 14B). As shown in Figures 13A—C and 14A, the microtubular root R1 originated from the right side of BB2 near its proximal end and extended toward the anterior cell apex. At the proximal end of the root, R1 comprised three microtubules (Fig. 14B). A few secondary cytoskeletal microtubules originated from R1 and extended posteriorly (Fig. 14A, arrowhead). Microtubular root R2, consisting of one to two microtubules, originated between the two basal bodies and extended anteriorly (Fig. 13C). Microtubular root R3, consisting of four microtubules, arose from the proximal end of BB1 and extended between the eyespot and BB1 (Figs 13D, E, 14C). Microtubular root R4, which consisted of a few microtubules, was attached to

the proximal end of BB1 (Fig. 13D, E). The rhizoplast extended from the BB2 and covered the elongated anterior part of the nucleus (Fig. 12B).

Chromatographic and Spectrophotometric Analyses of Pigments As shown in Figure 15, high-performance liquid chromatography (HPLC) analysis of pigments extracted from NIES-1863 was performed. The presence of chlorophyll a, fucoxanthin, diadinoxanthin, diatoxanthin, violaxanthin, antheraxanthin, zeaxanthin, and b-carotene was confirmed based on the absorption spectra and specific retention times obtained using the HPLC system. Chlorophyll ( ¼ chlorophyllide) c was not detected.

Molecular Phylogenetic Analyses The sequences of 18S rDNA and rbcL were determined for the three strains of Aurearena cruciata: NIES-1863, NIES-1864, and NIES-1865. The sequences of NIES-1865 were not used for the phylogenetic analyses because these were almost the same as those of the other two strains

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Figure 7. Transmission electron micrographs of Aurearena cruciata. A. Walled cell. Chloroplast divided into two compartments (arrowheads). Each compartment has an invagination (double arrowheads). Electron opaque vesicles are located beneath cell membrane (arrow). Mitochondrial profiles have many tubular cristae. B. Boundary between two compartments (arrowhead) made by the inner cER, and the invagination of the chloroplast envelope and associated periplastidal compartment (double arrowhead). C. Longitudinal section of a swimming cell. Chloroplast has a longitudinal boundary (arrowheads) and a horizontal invagination (double arrowheads). Note the eyespot, which is composed of a single layer of lipid globules; a pear-shaped nucleus; electron opaque vesicles beneath the cell membrane. D. Nucleus wedged between two Golgi bodies. Note the two anterior basal bodies (BB) and the lipid globules within the chloroplast near one basal body (arrowhead). C, chloroplast; E, eyespot; G, Golgi body; M, mitochondrion; N, nucleus; P, pyrenoid; W, cell wall; BB, basal body; LF, long anterior flagellum; Mu, mucous material; SF, short posterior flagellum; ocER, icER, outer and inner chloroplast ER; ocM, icM, outer and inner chloroplast membrane.

of A. cruciata (approximately 10 differences in 18S rDNA sequence positions). In the 18S rDNA tree (Fig. 16), NIES-1863 and NIES-1864 formed a clade (Aurearenophyceae), and this clade clustered with the clade including Phaeothamnion confervicola (the type species of the Phaeothamniophyceae), Stichogloea doederleinii and two undescribed members of the Phaeothamniophyceae (CCMP2289, CCMP2290) (true Phaeothamniophyceae clade ¼ TP clade). In the tree, the monophyly of the Aurearenophyceae and that of the cluster of Aurearenophyceae and the TP clade were supported by high bootstrap (BS) values (100% and 93%, respectively). The monophyly of the TP clade was, however, supported by a relatively weak BS (69%). Pleuro-

chloridella botrydiopsis and Tetrasporopsis fuscescens, previously classified in the Phaeothamniophyceae, based on morphological characters (Bailey et al. 1998), were not positioned in the TP clade. Nevertheless, they robustly formed sister clades to the Xanthophyceae and Chrysomerophyceae, respectively (BS 98% and 100%, respectively). The monophylies of the Xanthophyceae, Chrysomerophyceae, Phaeophyceae, and Phaeophyceae+Schizocladiophyceae were also robustly supported (BS 94%, 100%, 100%, and 100%, respectively). The PX clade, including the Aurearenophyceae, TP clade, Phaeophyceae, Schizocladiophyceae, Xanthophyceae, Pl. botrydiopsis, Chrysomerophyceae, and T. fuscescens, was also supported by a high BS (87%).

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Figure 8. Transmission electron micrographs of Aurearena cruciata. A. Girdle lamella was absent in the chloroplast. B. Chloroplast lamella consisted of three adpressed thylakoids. C. Outer nuclear membrane continuous with the outer cER. D. ER distributed in cytoplasm (arrowheads). C, chloroplast; M, mitochondrion; N, nucleus; ocER, outer chloroplast endoplasmic reticulum.

In the rbcL tree (Fig. 17), the monophylies of both the Aurearenophyceae and the TP clade were well supported (BS 100% and 96%, respectively). Notably, the monophyly of the TP clade was better supported than in the 18S rDNA tree, although the sister relationship between the

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Aurearenophyceae and the TP clade and the monophyly of the PX clade were only weakly supported (BSo50% and 70%, respectively). In the rbcL tree, T. fuscescens and Giraudyopsis sp. (NIES-1862) (Chrysomerophyceae) robustly formed a sister clade (BS 82%). This clade together with Pl. botrydiopsis formed a clade with the Xanthophyceae, though this relationship was not well supported (BSo50%). To obtain further information, phylogenetic analyses of the combined 18S rDNA and rbcL data were performed using concatenate and separate models (18S+rbcL tree). The best tree obtained using ML analysis with the concatenate model (Fig. 18) and the Bayesian tree with the separate model (not shown) gave almost the same topology. In the 18S+rbcL tree, monophyly was well supported for the PX clade, the Aurearenophyceae, and the TP clade by a high BS (99%, 100%, and 100%, respectively) and posterior probability (PP) (1.00, 1.00, and 1.00, respectively). Furthermore, the sister relationship between the Aurearenophyceae and the TP clade was well supported (BS 83% and PP 1.00). The monophyly of each class of the PX clade as well as the Xanthophyceae+Pl. botrydiopsis, the Chrysomerophyceae (Giraudyopsis sp.)+T. fuscescens and Phaeophyceae+Schizocladiophyceae were also supported by 98—100% BS and 1.00 PP. These phylogenetic analyses revealed four subclades within the PX clade: the Phaeophyceae+Schizocladiophyceae clade (PS clade), the Xanthophyceae+Pl. botrydiopsis clade (XP clade), the Chrysomerophyceae+T. fuscescens clade (CT clade), and the A. cruciata+TP clade (AP clade). However, these analyses could not resolve the phylogenetic relationships between these four subclades. In order to evaluate the significance of all the possible topologies of the four subclades (15 ways), the approximately unbiased (AU) test using a separate model was performed for 15 candidate trees in which these subclades were moved to all possible nodes in the best 18S+rbcL tree using the concatenated model (Table 1). With the exception of two alternative trees (6 and 10) the AU test could not reject (po0.05) the selected candidate alternative trees. Therefore, the AU test was unable to resolve the relationships between the subclades in the PX clade. The AU test using the separate model revealed that alternative trees 11, 12, 13, and 14 were more likely than the best 18S+rbcL tree using concatenated model. However, significant differences between the best tree and four alternative trees were rejected, indicating

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Figure 9. Transmission electron micrographs of Aurearena cruciata flagella. A—C. Negatively stained flagella and flagellar hairs. A. Swimming cell has LF possessing tripartite flagellar hairs (arrowhead) and smooth SF. B. Tripartite hairs of the LF showing the absence of lateral filaments along the shaft. C. Triple terminal hairs (arrowhead). D—F. Flagella in the walled cell. D. LF beneath the cell wall and mucous layer. E. Tubular shafts of tripartite hairs inside cell wall. F. Flagellar swelling at the base of the SF. Note the rhizoplast that extends between the basal body and the nucleus. N, nucleus; W, cell wall; BB, basal body; Mu, mucous material. LF, long anterior flagellum; SF, short posterior flagellum; TF, terminal filament; TS, tubular shaft; B, base; Rhz, rhizoplast.

that the AU test did not deny all the possible topologies of the four subclades.

Discussion Aurearena cruciata has typical morphological characters of the Heterokontophyta: (1) it possesses two unequal flagella, a long anterior

flagellum bearing tripartite tubular hairs and a short smooth posterior flagellum; (2) the mitochondrion has tubular cristae; (3) the chloroplast is surrounded by four membranes the outermost of which is continuous with the outer nuclear envelope; and (4) the flagellar apparatus components are comparable with those of other heterokontophytes (but see below). However, A. cruciata is distinct among heterokontophytes in various

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Figure 10. Transmission electron micrographs of Aurearena cruciata. A. Flagellar microtubules absorbed inside the cell (arrowhead). Basal bodies and flagellar root were remained. B. Enlarged view of flagellar microtubules (arrowheads). C, chloroplast; N, nucleus; R, root; BB, basal body.

aspects and cannot be placed in any of the described classes of the Heterokontophyta as discussed below.

General Cell Morphology The cell wall of A. cruciata is characteristic in that many electron-opaque vesicles are located beneath the plasma membrane. In this respect, A. cruciata is similar to the Phaeothamniophyceae (Andersen et al. 1998; Bailey et al. 1998). However, A. cruciata differs from the Phaeothamniophyceae in terms of chloroplast morphology. The chloroplast of A. cruciata occupies the central part of the cell and has an invagination of the chloroplast envelope and periplastidal compartment into the pyrenoid matrix. In contrast, the chloroplast in members of the Phaeothamniophyceae is peripheral and has no comparable invagination into the

445

Figure 11. Diagrammatic reconstruction of the flagellar apparatus of Aurearena cruciata, viewed from the lateral side of the cell. C, chloroplast; E, eyespot; G, Golgi body; N, nucleus; BB, basal body; CM, cytoskeletal microtubule; LF, long anterior flagellum; R1, root 1; R2, root 2; R3, root 3; R4, root 4; SF, short posterior flagellum; Rhz, rhizoplast.

pyrenoid, or the pyrenoid is entirely absent. A. cruciata and members of the Phaeothamniophyceae also differ with respect to the presence/ absence of a girdle lamella and in the configuration of the plastid nucleoid. A. cruciata has no girdle lamella and the plastid nucleoid is of the scattered type. In contrast, members of the Phaeothamniophyceae have the girdle lamella and a ring-shaped plastid nucleoid (Andersen et al. 1998; Bailey et al. 1998). The membrane topology of the chloroplast of A. cruciata is most distinctive and has not been reported before. A. cruciata has a single chloroplast, because it is enveloped by a single common membrane (the outer membrane of cER), even though the chloroplast is divided into two compartments by the inner membrane of cER, each bounded by three membranes (chloroplast double membranes and the inner membrane of

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Figure 12. Transmission electron micrographs of Aurearena cruciata. A. The flagellar transition region. Six-gyred transitional helix situated distal to the single transitional plate (arrowhead). B. The rhizoplast extended from BB2 and covered the anterior part of the nucleus. BB, basal body; LF, long anterior flagellum; TP, transitional plate; Rhz, rhizoplast.

chloroplast is bounded by a double membrane, and the two chloroplasts are surrounded by two membranes of cER. These chloroplasts have terminal pyrenoids that form an aggregate at the center of the chloroplast complex (Hibberd and Chre`tiennot-Dinet 1979). The nature of the chloroplast in A. cruciata may be comparable to that in S. grande and R. marina, although the membrane topology appears to be more complicated in A. cruciata. Unlike S. grande and R. marina, the outer and inner membranes of the cER do not appear as a pair; there are two compartments bounded by a double membrane of chloroplast envelope and the inner membrane of the cER, and these two compartments are surrounded by the outer membrane of the cER. At present, it is difficult to interpret this membrane topology in terms of the number of chloroplasts. These differences are probability related to the function and metabolism of the chloroplasts or chloroplasts complexes. Therefore, physiological and molecular studies on the chloroplast of A. cruciata will be indispensable to solve this problem. Even so, together with the chloroplast complexes in S. grande and R. marina, the membrane topology of the A cruciata chloroplast is an exception to the universal topology of four-membrane-bounded chloroplasts in extant algae that acquired chloroplasts secondarily from a red algal symbiont(s).

Flagellar Apparatus cER), and two chloroplasts are together surrounded by a single membrane (the outer membrane of cER). This type of chloroplast has never been reported in the Heterokontophyta or in any other algal group that has four chloroplastbounding membranes (Cryptophyta, Haptophyta, and Chlorarachniophyta). This is probably one of the most important morphological features characterizing A. cruciata. Recently, Horn et al. (2007) reported a marine amoeboid heterokontophyte, Synchroma grande that possesses a unique chloroplast complex. S. grande possesses chloroplast aggregates each comprising 6—8 chloroplasts. Each chloroplast has a double membrane and all chloroplasts within an aggregate are surrounded by an additional two membranes (cER). The chloroplasts in the aggregate have terminal pyrenoids that are densely aggregated at the center of the chloroplast aggregate (Horn et al. 2007). Additionally, the chloroplast of Rhizochromulina marina has essentially the same membrane topology as S. grande. R. marina has a chloroplast complex that contains two chloroplasts. Each

The flagellar apparatus of most heterokontophytes has several common characteristics: (1) an anterior flagellum with tripartite tubular hairs and a smooth posterior flagellum; (2) a transitional helix and transitional plate in the flagellar transition region; (3) four microtubular roots, R1 and R2 associated with BB2, and R3 and R4 associated with BB1; and (4) a fibrous structure, the rhizoplast, connecting the basal bodies and nucleus (e.g., Andersen 1991; Preisig 1989). The basic configuration of the microtubules and other components of A. cruciata falls within the range of diversity for the heterokontophyte flagellar apparatuses. However, the flagellar apparatus of A. cruciata is distinct from that of any other group in the Heterokontophyta, including the Phaeothamniophyceae. The R3 of A cruciata is very short and terminates abruptly, as occurs in the Phaeothamniophyceae. However, the R3 of A. cruciata extends laterally between the eyespot and BB1, whereas that of the Phaeothamniophyceae extends posteriorly and to the right of the cell on its ventral surface (Andersen et al. 1998).

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Figure 13. Transmission electron micrographs of Aurearena cruciata. A—F. Consecutive sections of the flagellar apparatus. E, eyespot; BB, basal body; LF, long anterior flagellum; R1, root 1; R2, root 2; R3, root 3; R4, root 4; SF, short posterior flagellum.

Therefore, the flagellar apparatus of A. cruciata is clearly distinguishable from that of the Phaeothamniophyceae.

Flagella in the Non-Motile Walled Stage One of the most unique and important features of A. cruciata is the presence of two unequal flagella, not only in swimming cells but also in non-motile

walled cells, lying beneath the cell wall. This feature has never been reported in previously described heterokontophytes and other eukaryotic algae. In connection with this, it should be noted that only one swimming cell is released from the walled cell, and that cell division occurs only in the swimming stage. However, in old cultures or under conditions of low salinity (18%), non-motile walled cells develop into multinucleate large cells

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possessing a number of eyespot-like granules. This type of cell division indicates that the organization of A. cruciata is essentially flagellate.

Photosynthetic Pigments

Figure 14. Transmission electron micrographs of Aurearena cruciata. Sections of R1 and R4. E, eyespot; BB, basal body; CM, cytoskeletal microtubules; LF, long anterior flagellum; R1, root 1; R3, root 3; SF, short posterior flagellum.

Within the Heterokontophyta, photosynthetic pigments are used as diagnostic characters of the various classes (e.g., Andersen 2004a; Bjørnland and Liaaen-Jensen 1989; Jeffrey 1976). The composition of the photosynthetic pigments of A. cruciata (chlorophyll a, fucoxanthin, diadinoxanthin, diatoxanthin, violaxanthin, antheraxanthin, zeaxanthin, and b-carotene) is different from that of the other classes. The absence of chlorophyll c is a remarkable character, although the Eustigmatophyceae also lack chlorophyll c and the Xanthophyceae have very low concentrations of chlorophyll c (e.g., Antia et al. 1975; Jeffrey 1976). Some xanthophylls not only participate in lightharvesting functions but also protect against photoinhibitory damage (Demmig et al. 1987). In the Heterokontophyta, two xanthophylls cycles are known to play a photoprotective role: the violaxanthin cycle (violaxanthin/antheraxanthin/ zeaxanthin) and the diadinoxanthin cycle (diadinoxanthin/diatoxanthin) (Lanvaud et al. 2004; Uhrmacher et al. 1995). Most heterokontophytes have either one or the other of the violaxanthin or diadinoxanthin photoprotective cycles, and contain little or no pigment of the other cycle (Bjørnland and Liaaen-Jensen 1989 and Table 2). In contrast, A. cruciata appears to possess both violaxanthin and diadinoxanthin cycles since it has high concentrations of both violaxanthin and diadinoxanthin, and also high concentrations of the other xanthophylls comprising these two cycles. This feature of A. cruciata may reflect an adaptation to strong light in habitats such as sandy beaches where A. cruciata was discovered. Indeed, A. cruciata forms a bloom on sand in the very hot and bright conditions of the Japanese mid-summer.

Taxonomy of Aurearena cruciata

Figure 15. HPLC elution profiles of pigments extracted from NIES-1863. Peaks are fucoxanthin (peak 1), violaxanthin (peak 2), diadinoxanthin (peak 3), antheraxanthin (peak 4), diatoxanthin (peak 5), zeaxanthin (peak 6), Chl a (peak 7), b-carotene (peak 8), respectively.

The three strains of A. cruciata examined in this study (NIES-1863, NIES-1864, and NIES-1865) exhibited no morphological differences and consequently should be treated as a single species. The similarities in the 18S rDNA and rbcL sequences further support this taxonomic treatment. There has been no comparable description of coccoid algae possessing flagella beneath the cell wall. Therefore, we proposed a new genus

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Hyphochytrium catenoides (X80344) Outgroup Developayella elegans (U37107) Bolidomonas pacifica (AF167155) 100 Bolidophyceae Bolidomonas mediterranea (AF123596) 99 Phaeodactylum tricornutum (AY485459) 100 Odontella sinensis (Y10570) Bacillariophyceae 100 Thalassiosira pseudonana (AY485452) 92 Detonula confervacea (AF525672) Dictyocha speculum (U14385) 100 Ciliophrys infusionum (L37205) 100 Dictyochophyceae Helicopedinella tricostata (AB097408) 100 Pedinella squamata (AB081517) 52 Aureoumbra lagunensis (ALU40258) 100 Aureococcus anophagefferens (AF117779) Pelagophyceae Pelagococcus subviridis (U14386) 99 Pelagomonas calceolata (U14389) Eustigmatos magna (EMU41051) 100 Eusigmatophyceae Nannochloropsis salina (AB183586) 100 Nannochloropsis gaditana (AB052271) Picophagus flagellatus (AF185051) 100 Chlamydomyxa labyrinthuloides (AJ130893) 91 Synchromophyceae Synchroma grande (DQ788730) Synura uvella (U73222) 100 Synurophyceae Chromulina nebulosa (AF123285) & Chrysocapsa vernalis (AF123283) Chrysophyceae 94 Hibberdia magna (M87331) 96 Polypodochrysis teissieri (AB183614) 50 100 Pinguiochrysis pyriformis (AF438326) Pinguiophyceae Phaeomonas parva (AF438323) Glossomastix chrysoplasta (AF438325) 100 Fibrocapsa japonica (AY788931) 97 Heterosigma akashiwo (AB001287) 100 Raphidophyceae Chattonella marina (AY788928) Chattonella antiqua (AY788922) 99 SchizocladioSchizocladia ischiensis (AB085614) 100 phyceae Scytosiphon lomentaria (L43066) 100 61 Agarum clathratum (AF123576) Phaeophyceae Asteronema rhodochortonoides (AB056156) 75 97 Asterocladon interjectum (AB102865) Tetrasporopsis fuscescens (AB365206) 100 87 Antarctosaccion applanatum (AJ295822) 100 ChrysomeroGiraudyopsis sp. (NEIS-1862)(AB365204) phyceae Giraudyopsis stellifera (U78034) 100 Pleurochloridella botrydiopsis (AB365207) 98 82 Sphaerosorus composita (AJ579333) 55 Chlorellidium pyrenoidosum (AJ579338) Xanthophyceae Botrydium stoloniferum (AF064743) 94 Vaucheria bursata (VBU41646) 89 76 Aurearena cruciata (NEIS-1863)(AB365192) Aureareno100 Aurearena cruciata (NEIS-1864)(AB365194) phyceae Phaeothamnion confervicola (AB365203) 93 Stichogloea doederleinii (AB365201) TP clade 69 98 CCMP2290 (AB365198) 99 CCMP2289 (AB365197) 0.1 0.2

96

Figure 16. Phylogenetic tree based on 18S rDNA sequences and constructed using maximum likelihood method. Bootstrap values larger than 50% are shown at the internal branches. Unambiguously aligned 1593 nucleotide positions from 54 species were used for the analysis.

and species, Aurearena cruciata, for these three strains. Along with ultrastructural similarities, such as cell wall and associated electron-opaque material beneath the cell membrane, the results

of phylogenetic analyses (the best 18S rDNA and 18S+rbcL trees) suggest that A. cruciata is a sister to the Phaeothamniophyceae. However, the unique photosynthetic pigment composition is

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A. Kai et al. Exanthemachrysis gayraliae (AB043701) Pavlova salina (D45847) Pleurochloridella botrydiopsis (AF069499) 82 Tetrasporopsis fuscescens (AF155585) Giraudyopsis sp. (NIES-1862)(AB365205) Botrydium stoloniferum (AF064743) Vaucheria bursata (AF476938) 74 Sphaerosorus composita (AJ579574) 54 64 Chlorellidium pyrenoidosum (AJ579565) 70 Phaeothamnion confervicola (AF064746) 96 Stichogloea globosa (AF155584) Stichogloea doederleinii (AB365202) 100 87 CCMP2289 (AB365199) 100 CCMP2290 (AB365200) 100 Aurearena cruciata NIES-1863 (AB365193) Aurearena cruciata NIES-1864 (AB365195) Schizocladia ischiensis (AB085615) 72 Agarum clathratum (AB035791) 100 Scytosiphon lomentaria (AB022238) Asteronema rhodochortonoides (AJ295825) 97 Asterocladon interjectum (AB102866) 98 Heterosigma akashiwo (AB176660) 100 100 Chattonella marina (DQ273996) Chattonella antiqua (DQ273991) Synchroma grande (DQ788731) 100 Nannochloropsis salina (AB052287) 69 Nannochloropsis gaditana (AB052735) Chromulina nebulosa (AF155876) 100 Synura uvella (AF015586) Chrysocapsa vernalis (AF155877) 93 76 Hibberdia magna (AF015572) 97 Polypodochrysis teissieri (AF438320) 97 Glossomastix chrysoplasta (AF438318) Pinguiochrysis pyriformis (AF438317) 59 Phaeomonas parva (AF438321) Aureoumbra lagunensis (AF117786) 100 Aureococcus anophagefferens (AF117785) Pelagococcus subviridis (AF015580) 97 100 100 Pelagomonas calceolata (U89898) 100 Bolidomonas pacifica (AF333979) Bolidomonas mediterranea (AF333977) 100 Phaeodactylum tricornutum (EF067920) Odontella sinensis (Z67753) 68 Thalassiosira pseudonana (EF067921) 72 Detonula confervacea (AB018006) 100 Dictyocha speculum (AY043280) Ciliophrys infusionum (AB081643) 95 Helicopedinella tricostata (AB097409) 98 0.1 91 Pedinella squamata (AB081639)

Outgroup Chrysomerophyceae Xanthophyceae

TP clade

Aurearenophyceae Schizocladiophyceae Phaeophyceae

Raphidophyceae Synchromophyceae Eustigmatophyceae Synurophyceae & Chrysophyceae

Pinguiophyceae

Pelagophyceae

Bolidophyceae Bacillariophyceae

Dictyochophyceae

Figure 17. Phylogenetic tree based on rbcL sequences and constructed using maximum likelihood method. Bootstrap values larger than 50% are shown at the internal branches. Unambiguously aligned 1380 nucleotide positions from 49 species were used for the analysis.

probably related to two types of photoprotection and many of the ultrastructural characters mentioned above are apparently distinctive. A comparison of selected characters among the members of the PX clade is presented in Table 2. All these data suggest that A. cruciata is a taxon distinct from the Phaeothamniophyceae and other classes of the PX clade. Therefore, we propose a new class, Aurearenophyceae, for A. cruciata in the Heterokontophyta. This class is characterized by the occurrence of flagella beneath the cell wall in the walled non-motile stage and photo-

synthetic pigments probably involved in two photoprotective cycles.

Phylogenetic Position of A. cruciata within the PX Clade The PX clade has been generally recognized by molecular phylogenetic studies. However, the clade has never been satisfactorily supported by statistics (e.g., Bailey et al. 1998; Kawai et al. 2003; Saunders et al. 1997). The PX clade is one

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451

Outgroup (Haptophyta) Chrysomerophyceae

CT clade

Xanthophyceae

XP clade

Pleurochloridella botrydiopsis (AB365207+AF069499) 99

Sphaerosorus composita (AJ579333+AJ579574)

100

Chlorellidium pyrenoidosum (AJ579338+AJ579565) 99

Botrydium stoloniferum (AF064743+AF064743)

100 72

Vaucheria bursata (VBU41646+AF476938) Phaeothamnion confervicola (AB365203+AF064746)

100

Stichogloea doederleinii (AB365201+AB365202)

100

CCMP2289 (AB365197+AB365199) 83

CCMP2290 (AB365198+AB365200) Aurearena cruciata (NIES-1863)(AB365192+AB365193)

100 100

Aurearena cruciata (NIES-1864)(AB365194+AB365195) Schizocladia ischiensis (AB085614+AB085615)

98 100

Agarum clathratum (AF123576+AB035791) Scytosiphon lomentaria (L43066+AB022238) Asteronema rhodochortonoides (AB056156+AJ295825) Asterocladon interjectum (AB102865+AB102866)

100 100

Chattonella marina (AY788928+DQ273996) Chattonella antiqua (AY788922+DQ273991)

PX clade

Schizocladiophyceae

Phaeophyceae

PS clade

Raphidophyceae

Polypodochrysis teissieri (AB183614+AF438320)

100

Pinguiochrysis pyriformis (AF438326+AF438317) Phaeomonas parva (AF438323+AF438321)

Pinguiophyceae

Glossomastix chrysoplasta (AF438325+AF438318)

100 100

Nannochloropsis salina (AB183586+AB052287) Nannochloropsis gaditana (AB052271+AB052735) Synchroma grande (DQ788730+DQ788731)

89 79

Eustigmatophyceae Synchromophyceae

Chromulina nebulosa (AF123285+AF155876)

100

Synura uvella (U73222+AF015586) Chrysocapsa vernalis (AF123283+AF155877)

Synurophyceae & Chrysophyceae

59 Hibberdia magna (M87331+AF015572) 100 Bolidomonas pacifica (AF167155+AF333979)

100

65

AP clade

Aurearenophyceae

94 100 Heterosigma akashiwo (AB001287+AB176660)

57 57

TP clade

Bolidomonas mediterranea (AF123596+AF333977)

100

Bolidophyceae

Phaeodactylum tricornutum (AY485459+EF067920) Odontella sinensis (Y10570+Z67753) 100

100 98

100

Thalassiosira pseudonana (AY485452+EF067920) Detonula confervacea (AF525672+AB018006) Aureoumbra lagunensis (ALU40258+AF117786)

100

Aureococcus anophagefferens (AF117779+AF117785) Pelagococcus subviridis (U14386+AF015580) 100 100 100 0.1 0.2

Bacillariophyceae

Pelagophyceae

Pelagomonas calceolata (U14389+U89898) Dictyocha speculum (U14385+AY043280) Ciliophrys infusionum (L37205+AB081643)

100 100

Helicopedinella tricostata (AB097408+AB097409)

Dictyochophyceae

Pedinella squamata (AB081517+AB081639)

Figure 18. Phylogenetic tree based on 18S rDNA and rbcL sequences using maximum likelihood (concatenated model) and Bayesian methods (separated model). The best tree of ML method is shown. Bootstrap values of ML method larger than 50% are shown. Bayesian posterior probabilities 1.00 are shown in bold. Unambiguously aligned 1597 bp of 18S rDNA and 1380 bp of rbcL from 49 species were used for the analysis.

of the most important lineages in the Heterokontophyta. Recently, based on their phylogenetic analysis, Cavalier-Smith and Chao (2006) recognized the PX clade and treated this clade as the superclass Fucistia. However, their phylogenetic analysis was performed using only the neighbor-

joining (NJ) method and the BS value they obtained was relatively weak (76%). Furthermore, the taxonomic position and definition of Fucistia have not been well established. Cavalier-Smith and Chao defined the Fucistia as a group of heterokontophytes possessing a typical cell wall.

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Based on Andersen (2004a), Andersen et al. (1998), Bailey et al. (1998), Bjørnland and Liaaen-Jensen (1989), Clayton (1989), Coleman (1985), Entwisle and Andersen (1990), Gayral and Billard (1986), Hibberd (1980), Kai (unpublished observation), Kawai et al. (2003), Jeffrey (1976), and O’Kelly (1989). FL ¼ flagellate, CC ¼ coccoid, FI ¼ filamentous, SI ¼ siphonous, MC ¼ multicellular, M ¼ marine, F ¼ freshwater, PO ¼ periplasmic opaque vesicle, CB ¼ chloroplast boundary, GL ¼ girdle lamella, PN ¼ plastid nuceoid (R ¼ ring type, S ¼ scattered type), TF ¼ no. of terminal filaments of mastigoneme, TH ¼ transitional helix, BR ¼ bypassing root, RH ¼ rhizoplast, Fuc ¼ fucoxanthin, Dia ¼ diatoxanthin/diadinoxanthin, Vio ¼ violaxanthin/antheraxanthin/zeaxanthin, Het ¼ heteroxanthin, Vau ¼ vaucheriaxanthin, asterisks mean very small amount, exceptional example in parentheses.

 + + +  ? ?  +    + ? ? + + + + +  ? ?  (+) + +  + + ? + +  + +* +* + ? + + +  + ?  ? ? +    ? + ? ? + + + + ? + ? +  3 1 2 ? 3 ? ? 1—3

Het Vio Dia Fuc Chl.c RH BR TH TF PN

S R R (S) ? R ? R R  + + () ? + + + + +   ?     + +  +  +  + M F F (M) F M F M M (F) FLCC CC, FI CC, FI, SI CC FI CC FI MC Aurearena Phaeothamniophyceae s.s. Xanthophyceae Pleurochloridella Chrysomerophyceae Tetrasporopsis Schizocladiophyceae Phaeophyceae

However, this definition is not entirely sufficient since members of the Eustigmatophyceae also possess a typical cell wall (Okuda et al. 2004). Moreover, they suggested that Ph. confervicola is a sister to Schizocladia ischiensis (BS, 92%), and reclassified this group as the subclass Phaeothamniophycidae of the class Phaeophyceae. Their conclusion was probably arrived at based on the use of an incomplete 18S rDNA sequence from Ph. confervicola (1201 bp) available at Genbank (AF044846) and insufficient taxon sampling for the Phaeothamniophyceae. Our phylogenetic analyses were performed using both ML and Bayesian methods, which are thought to be more robust than the NJ method (Huelsenbeck 1995; Huelsenbeck et al. 2001). Moreover, we used almost complete sequences for four members of the Phaeothamniophyceae and A. cruciata. The ML and Bayesian trees of 18S+rbcL and the 18S ML tree robustly supported the monophyly of the PX clade (18S+rbcL: BS 83%, PP 1.00; 18S: BS 87%). A. cruciata is undoubtedly a new member of the PX clade, and is most closely related to the ‘‘true’’ Phaeothamniophyceae that includes the type species, Ph. confervicola, and other species such as Stichogloea doederleinii.

GL

p-Values of AU tests were estimated by CONSEL (Shimodaira and Hasegawa 2001). Topologies rejected by the *5%. AP, A. cruciata+true Phaeothamniophyceae clade; CT, Chrysomerophyceae+T. fuscescens clade; PS, Phaeophyceae+ Schizocladiophyceae clade; XP, Xanthophyceae+Pl. botrydiopsis clade; Root, outgroup of PX clade.

CF

0.532 0.108 0.134 0.364 0.532 *0.009 0.259 0.301 0.108 *3e067 0.743 0.403 0.411 0.572 0.199

PO

01:(Root,(CT,(PS,(AP,XP)))) 02:(Root,(PS,(CT,(AP,XP)))) 03:(Root,((AP,XP),(PS,CT))) 04:(Root,(XP,(AP,(PS,CT)))) 05:(Root,(AP,(XP,(PS,CT)))) 06:(Root,((AP,CT),(XP,PS))) 07:(Root,(AP,(CT,(XP,PS)))) 08:(Root,(CT,(AP,(XP,PS)))) 09:(Root,(XP,(PS,(AP,CT)))) 10:(Root,(PS,(XP,(AP,CT)))) 11:(Root,((AP,PS),(XP,CT))) 12:(Root,(AP,(PS,(XP,CT)))) 13:(Root,(PS,(AP,(XP,CT)))) 14:(Root,(XP,(CT,(AP,PS)))) 15:(Root,(CT,(XP,(AP,PS))))

Habitat

p-Value (AU)

Table 2. Comparison of selected characters among the members of the PX clade

Candidate trees

Vau

Table 1. The comparison of alternative trees for the relationships among PX clade by the AU test

  +   ? ? 

A. Kai et al.

Organization

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This is the first study in which the monophyly of the PX clade has been highly supported. Bailey et al. (1998) tentatively classified Pleurochloridella botrydiopsis in the Phaeothamniophyceae and, based on morphological characteristics and photosynthetic pigment composition, placed it in its own order, the Pleurochloridellales. Interestingly, our analyses demonstrated that this alga is not a member of the true Phaeothamniophyceae, but is most closely related to the Xanthophyceae. It is known that this alga has both heteroxanthin and fucoxanthin, which is similar to members of the Phaeothamniophyceae; however, the amount of chlorophyll c is very low (Bailey et al. 1998). We observed the zoospore of Pl. botrydiopsis and found that the configuration of the two chloroplasts is similar to that of the Xanthophyceae (unpublished observations). Based on this evidence, a plausible explanation for the observed pigment compositions is that Pl. botrydiopsis emerged from the root of the Xanthophyceae, and that it represents a transition stage associated with the change of photosynthetic pigments to those of the Xanthophyceae; that is, chlorophyll c is decreased but fucoxanthin is retained. Accordingly, Pl. botrydiopsis (Pleurochloridellales) should be placed back into the Xanthophyceae as originally described (Ettl 1956). Bailey et al. (1998) also classified Tetrasporopsis in the Phaeothamniophyceae (Phaeothamniaceae: Phaeothamniales) based on reproductive and ultrastructural characters (Entwisle and Andersen 1990). However, our molecular phylogenetic analyses indicate that T. fuscescens is distantly related to the true Phaeothamniophyceae, but that it is a sister to the Chrysomerophyceae. At present, it is difficult to assess the taxonomic relationship between T. fuscescens and the Chrysomerophyceae because their ultrastructures and photosynthetic pigment compositions have not been investigated in sufficient detail. Previously recognized common characters between Tetrasporopsis and the true Phaeothamniophyceae, such as electron-opaque vesicles at the cell periphery and cell division via eleutheroschisis (Bailey et al. 1998), are obviously not synapomorphic characters (see below). As discussed above, the present study indicates that the Phaeothamniophyceae (and the Phaeothamniaceae) is polyphyletic. This result suggests that the definitive characters of the Phaeothamniophyceae are not synapomorphies but probably symplesiomorphies of the PX clade. The Phaeothamniophyceae should be limited to the clade including Phaeothamnion (true

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Phaeothamniophyceae) and thus needs appropriate taxonomic emendation. However, we prefer to postpone this classification until further information (e.g., ultrastructural, biochemical, and molecular) on the Phaeothamniophyceae and related algae becomes available. Our phylogenetic analyses suggest that the PX clade consists of four subclades: the Phaeophyceae+Schizocladiophyceae clade, the Xanthophyceae+Pl. botrydiopsis clade, the Chrysomerophyceae+T. fuscescens clade, and the A. cruciata+true Phaeothamniophyceae clade. In connection with this, the electron-opaque vesicles observed beneath the plasma membrane in A. cruciata should be considered as an important feature deserving attention. A very similar structure was reported in the true Phaeothamniophyceae, as well as for both Pl. botrydiopsis and T. fuscescens (Bailey et al. 1998; Entwisle and Andersen 1990; unpublished observation). Furthermore, electron-opaque vesicles, referred to as physodes, are known in some members of the Phaeophyceae (Nagasato and and Motomura 2002b; Schoenwaelder and Clayton 2000). Physodes are very similar to the electron-opaque vesicles of A. cruciata and the Phaeothamniophyceae sensu lato, perhaps suggesting that these physodes are associated with cell wall synthesis in brown algae (Nagasato and and Motomura 2002b; Schoenwaelder and Clayton 2000). Because the electron-opaque vesicles or physodes are present in all four PX subclades, one most parsimonous interpretation is that the electron-opaque vesicles are an ancestral character for the PX clade and are related to the acquisition of a cell wall. Basically, the major component of the cell wall is cellulose that is synthesized by a cellulose-synthesizing complex. The complex was observed located on the plasma membrane of the Eustigmatophyceae, Phaeophyceae, Phaeothamniophyceae, Xanthophyceae and various organisms (Okuda, et al. 2004; Peng and Jaffe 1976). On the other hand, it has been suggested that physodes are composed of polyphenols or sulfated polysaccharides (Schoenwaelder 2002). Therefore, the electron-opaque vesicles or physodes might be associated with components of the cell wall other than cellulose, such as mucous material. Moreover, the fact that the electronopaque vesicles or physodes have not been reported in other walled Heterokontophyta (Eustigmatophyceae) and in the oomycetes, a walled group of heterokonts that is closely related to the Heterokontophyta. This fact might imply the electron-opaque vesicles and physodes are

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associated with unique materials of cell wall in the PX clade. In order to test this assumption, the function of the electron-opaque vesicles of A. cruciata and the Phaeothamniophyceae sensu lato should be studied and compared with the physodes of the Phaeophyceae.

Methods Aurearena cruciata strains were isolated from sand samples collected at three sites: Isonoura beach, Wakayama Prefecture, Japan, May 2003; Noma beach, Aichi Prefecture, Japan, June 2004; and Hashikui beach, Wakayama Prefecture, Japan, July 2005 (Fig. 1). Unialgal cultures were established by single-cell isolation using micropipettes. Three strains, NIES-1863 (Isonoura beach), NIES-1864 (Noma beach), and NIES-1865 (Hashikui beach), were established. The cultures were maintained in f/2 medium (Guillard and Ryther 1962) containing 2.67 mg/l NH4Cl, and at a temperature of 25 1C on a 14:10 h LD cycle under white fluorescent tubes at 50 mE/m2/s. For light microscopy, both living cells were observed and photographed using a DMRD microscope (Leica Microsystems, Wetzlar, Germany) equipped with Nomarski differential interference contrast optics. Swimming cells readily lost their flagella and developed a spherical shape in response to physical stimulation such as the pressure of a cover slip; therefore, the specimens fixed with 2.5% glutaraldehyde solution were used for the observation of flagella. For fluorescence microscopy, cells were fixed with 0.5% glutaraldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 7.4) containing 3% NaCl (Nagasato and Motomura 2002a) for 1 h at 4 1C. After rinsing with PHEM, the cells were incubated with 40 -6-diamido-2phenylindole (DAPI; 1 mg ml in PHEM) for 30 min at room temperature in the dark. Samples were observed and photographed using a DMRD microscope. For transmission electron microscopy, both chemical fixation (Figs 5C, 6A, B, 7D, F, 8A, B, 9A, B, 10A—F, and 11A—C) and high-pressure freeze-substitution (Figs 5A, B, D, 6C, and 7E) were adopted. For chemical fixation, 3 ml of cell suspension and an equal volume of a fixative solution containing 2.5% glutaraldehyde, 0.2% osmium tetroxide (OsO4) and 0.25 M sucrose in 0.2 M sodium cacodylate buffer (pH 7.2) were mixed; 1 ml of 4% OsO4 was added 20 s later (final concentration of 0.67%) and cells were then fixed for 1 h at 4 1C. Cells were dehydrated through a 30—100% ethanol series and embedded in Spurr’s resin (Spurr 1969). For the observation of the

positively or negatively stained flagellar hairs, approximately 5 ml of a chemically fixed sample (prepared as described above) was dried on Formvar-coated grids at room temperature for 10 min. After rinsing with distilled water, the grids were dried again. A drop of 2% uranyl acetate was placed on the grid and removed after 3 min using filter paper; after drying again, the cells were observed using a transmission electron microscope. For high-pressure freeze-substitution, the cells were centrifuged and the supernatant was discarded. The cells were frozen in a high-pressure freezing apparatus (HPM010; BAL-TEC AG, Balzers, Liechtenstein) that had been cooled with liquid nitrogen (196 1C). The cells were transferred to 2% OsO4 in dry acetone at 80 1C, incubated at 80 1C for 168 h, and then gradually warmed from 80 to 0 1C over a period of 5 h. The cells were held at 0 1C for 2 h, warmed from 0 to 24 1C over a period of 4 h, and then incubated at 24 1C for 1 h (EM AFS; Leica Microsystems). The cells were rinsed three times with dry acetone at room temperature, substituted with a 1—100% Spurr’s series in dry acetone, and then embedded in 100% Spurr’s resin. Sections were cut on a Reichert ULTRACUT S microtome (Leica Microsystems) with a diamond knife, double-stained on drops of 2% of uranyl acetate and lead citrate (Reynolds 1963), and then examined with a JEM-100CXII transmission electron microscope (JEOL, Tokyo, Japan). The NIES-1863 strain was used for the spectroscopic analysis. About 40—50 ml of dense culture were centrifuged and the supernatant was discarded. The residue was homogenized in 100% acetone on ice in the dark by using an ultrasonic homogenizer for pigment extraction. The extract was injected immediately into a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) equipped with a Partisil-5 ODS-3 column (4 mm  250 mm; GL-science, Tokyo, Japan). Pigments were eluted using a linier gradient from 100% solvent A (ion-pairing solution: water:acetone:acetonitrile ¼ 5:25:20:50) to 100% solvent B (acetone:ethyl acetone ¼ 50:50) over a 20min period (Kohata et al. 1991; Yoshii et al. 2002, 2003). The absorption spectra of the pigments were measured at 400—440 nm using a photodiode array detector (SPD-M10A vp; Shimadzu) attached to the Shimadzu HPLC system. DNA was extracted from the three strains of Aurearena cruciata (NIES-1863, NIES-1864, and NIES-1865), Phaeothamnion confervicola Lagerheim (CCMP (The ProvasoliGuillard National Center for Culture of Marine Phytoplankton) 637), Stichogloea doederleinii Wille (CCMP823), Pleurochloridella botrydiopsis Pascher (CCMP1665), Tetrasporopsis fuscescens (Braun et Ku¨tzing) Lemmermann (SAG20.88), Giraudyopsis sp. (NIES-1862) and two undescribed phaeothamniophycean strains (CCMP2289 and CCMP2290). Although SAG20.88 was referred as Chrysocapsa epiphytica Lund, we used this strain as T. fuscescens following the suggestion from Dr. Robert A. Andersen (personal communication)

Table 3. The list of PCR primers Gene

Primer

rbcL

FB1 intRB1 intFB1 intRB2 intFB2 PX_Rend L7Hetero

Modified from Sekiguchi et al. (2002).

50 -AGTGAHCGTTATGAATCWGGTGT-30 50 -ACHACACGWCCRTAGTTTTT-30 50 -AACDTTCCAAGGTCCHGCDAC-30 50 -TAGAADCCTTTAACCATTA-30 50 -TYTGTAARTGGATGCTGCGTATG-30 50 -TCHACGAAATCWGSHGTATC-30 50 -AARSKHCCTTGTGTAAVTCTCA-30

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Aurearena cruciata gen. et sp. nov. and Entwisle and Andersen (1990). For the extraction of T. fuscescens DNA, we reisolated the alga and prepared a unialgal culture as the original culture was contaminated with fungi (information on the SAG culture collection of algae: http://www.epsag.uni-goettingen.de/html/sag.html). Cells were lysed with UNSET buffer (Garriga et al. 1984) or homogenized in liquid nitrogen, and total DNA was extracted from broken cells using a standard phenol/chloroform method followed by precipitation with ethanol (Sambrook et al. 1989). The precipitated DNA was finally purified using a GFXTM Genomic Blood DNA Purification Kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, England). 18S rDNA was amplified by a polymerase chain reaction (PCR) (Saiki et al. 1988) using three sets of oligonucleotide primers: SR1 and SR5, SR4 and SR9, and SR8 and SR12 (Nakayama et al. 1998). The thermal cycle parameters were as follows: denaturation at 95 1C for 30 s (initial denaturation at 95 1C for 5 min), annealing at 50 1C for 1 min, and extension at 72 1C for 1 min. This pattern was repeated for 28 cycles. In order to amplify rbcL, PCR was performed using the following oligonucleotide primer pairs: L2 and LB, L50 and LC, and L6 and L8 (Sekiguchi et al. 2002) or FB1 and intRB1, intFB1 and intRB2, intFB2 and PX_Rend, and intFB2 and L7Hetero. These primers are listed in Table 3. The thermal cycle parameters were as follows: denaturation at 95 1C for 1 min (initial denaturation at 95 1C for 5 min), annealing at 43 or 50 1C for 30 s, and extension at 72 1C for 1 min. This pattern was repeated for 28 cycles. The PCR products were directly sequenced with a DNA autosequencer (ABI Prism 377 and 310; Perkin-Elmer Inc., MA, USA) using the dye-terminator method following the manufacturer’s instructions (Dye-Terminator Cycle Sequencing Core Kit; Perkin-Elmer Inc.). The 18S rDNA and rbcL sequences used for phylogenetic analyses were obtained from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html). The 18S rDNA sequences were aligned manually taking into account both primary and secondary structure information from the European SSU rRNA database (http://www.psb.ugent.be/rRNA/) using BioEdit Sequence Alignment Editor version 4.8.10 (http:// www.mbio.ncsu.edu/BioEdit/bioedit.html). The rbcL sequences were aligned using the profile alignment process of the CLUSTAL X version 1.8 (Thompson et al. 1997; http:// newfish.mbl.edu/Course/Software/ClustalX/) and refined manually using BioEdit Sequence Alignment Editor version 4.8.10. The common gaps of the 18S rDNA sequences were removed to leave 1593 bp. A 1380-bp sequence of rbcL was used for the analysis. For combined analyses of 18S rDNA and rbcL using maximum-likelihood (ML) and Bayesian methods, the 1597- and 1380-bp sequences, respectively, were used. Phylogenetic trees were constructed using the ML method available in PAUP* version 4.0b10 (Swofford 2001). For application of an appropriate nucleotide substitution model to the ML analyses, the likelihood-ratio test in Modeltest version 3.0 (Posada and Crandall 1998) was used. TrN+I+G model for 18S rDNA, the GTR+I+G model for rbcL, and the GTR+I+G model for the concatenated data set of 18S rDNA and rbcL were selected. Under ML analyses, heuristic searches were performed using 10 random-addition replicates with TBR branch swapping. The BS analyses of ML trees were performed with 100 replicates. The Bayesian analysis of the 18S rDNA+rbcL was performed with MRBAYES version 3.1.2 (Huelsenbeck and Ronquist 2001) using a separate GTR+I+G model for each gene partition, with four rate categories. AU tests of 18S rDNA+rbcL data sets using the separate GTR+I+G model were performed with the program CONSEL (Shimodaira and Hasegawa 2001). The

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parameters of the separate GTR+I+G model for each gene were calculated using PAUP* version 4.0b10 and the Modeltest program version 3.0. In order to economize computation time, the 18S rDNA and rbcL sequences of NIES-1865 were excluded from the above analyses; this strain was robustly clustered with NIES-1863 and NIES-1864 in all analyses when it was added.

Acknowledgments We are most grateful to Dr. Robert A. Andersen for the culture information on CCMP2289, CCM P2290, and SAG20.88; Ms. Mayuko Sato (University of Tokyo) and Professor Shigeyuki Kawano (University of Tokyo) for allowing us to use a highpressure freezing apparatus; and Dr. Isamu Wakana (Lake Akan Eco-Museum Center) for use of the HPLC system. We also acknowledge Professor Tetsuo Hashimoto, Professor Yuji Inagaki, Dr. Miako Sakaguchi (University of Tsukuba), and Dr. Takeaki Hanyuda (University of Kobe) for informative suggestions and technical guidance on the molecular phylogenetic analyses. We thank Dr. Shinji Sakaushi (University of Osaka Prefecture) for technical guidance on the staining for fluorescence microscopy. This study was supported by a grant (RFTF00L0162) from the Japan Society for the Promotion of Science (to I. I.).

References Andersen RA (1987) The Synurophyceae classis nov., a new class of algae. Am J Bot 74: 337—353 Andersen RA (1991) The cytoskeleton of chromophyte algae. Protoplasma 164: 143—159 Andersen RA (2004a) Biology and systematics of heterokont and haptophyte algae. Am J Bot 91: 1508—1522 Andersen RA (2004b) A historical review of heterokont phylogeny. Jpn J Phycol 52 (Suppl):153—162 Andersen RA, Potter D, Bidigare RR, Latasa M, Rowan K, O’Kelly CJ (1998) Characterization and phylogenetic position of the enigmatic golden alga Phaeothamnion confervicola: ultrastructure, pigment composition and partial SSU rDNA sequence. J Phycol 34: 286—298 Andersen RA, Saunders GW, Paskind MP, Sexton JP (1993) Ultrastructure and 18S rRNA gene sequence for Pelagomonas calceolata gen. et sp. nov. and the description of a new algal class, the Pelagophyceae classis nov. J Phycol 29: 701—715 Antia NJ, Bisalputra T, Cheng JY, Kalley JP (1975) Pigment and cytological evidence for reclassification of Nannochlorris oculata and Monollantus salina in the Eustigmatophyceae. J Phycol 11: 339—343 Ariztia EV, Anderson RA, Sogin ML (1991) A new phylogeny for chromophyte algae using 16S-like rRNA sequences from

ARTICLE IN PRESS

456

A. Kai et al.

Mallomonas papillosa (Synurophyceae) and Tribonema aequale (Xanthophyceae). J Phycol 27: 428—436 Bailey JC, Bidigare RR, Christensen SJ, Andersen RA (1998) Phaeothamniophyceae classis nova: a new lineage of chromophytes based upon photosynthetic pigments, rbcL sequence analysis and ultrastructure. Protist 149: 245—263 Bjørnland T, Liaaen-Jensen S (1989) Distribution Patterns of Carotenoids in Relation to Chromophyte Phylogeny and Systematics. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae: Problems and Perspectives, The Systematics Association Special Vol. No. 38. Clarendon Press, Oxford, pp 37—60 Cavalier-Smith T (1993) The Origin, Losses and Gains of Chloroplasts. In Lewin RA (ed) Origins of Plastids. Chapman & Hall, New York, London, pp 291—348 Cavalier-Smith T (1995) Membrane Heredity, Symbiogenesis, and the Multiple Origin of Algae. In Arai R, Kato M, Doi Y (eds) Biodiversity and Evolution. National Science Museum Foundation, Tokyo, pp 75—114 Cavalier-Smith T, Chao EE (2006) Phylogeny and Megasystematics of Phagotrophic Heterokonts (Kingdom Chromista). J Mol Evol 62: 388—420

Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can J Microbiol 8: 229—239 Hibberd DJ (1980) Xanthophyceae. In Cox ER (ed) Phytoflagellates. Elsevier/North-Holland, New York, pp 243—271 Hibberd DJ, Chre`tiennot-Dinet M (1979) The ultrastructure and taxonomy of Rhizochromulina marina gen. et sp. nov., an amoeboid marine chrysophyte. J Mar Biol Ass UK 59: 179—193 Hibberd DJ, Norris RE (1984) Cytology and ultrastructure of Chlorarachnion reptans (Chlorarachniophyta divisio nova, Chlorarachniophyceae classis nova). J Phycol 20: 310—330 Horn S, Ehlers K, Fritzsch G, Gil-Rodrı´guez MC, Wilhelm C, Schnetter R (2007) Synchroma grade spec. nov. (Synchromophyceae class. nov., Heterokontophyta): an amoeboid marine alga with unique plastid complexes. Protist 158: 273—293 Huelsenbeck JP (1995) The robustness of two phylogenetic methods: four-taxon simulations reveal a slight superiority of maximum likelihood over neighbor joining. Mol Biol Evol 12: 843—849 Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754—755

Cavalier-Smith T, Chao EE, Allsopp TEP (1995) Ribosomal RNA evidence for chloroplast loss within Heterokonta: pedinellid relationships and a revised classification of ochristan algae. Arch Protistenkd 145: 209—229

Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP (2001) Evolution-Bayesian inference of phylogeny and its impact on evolutionary biology. Science 294: 2310—2314

Clayton MN (1989) Brown Algae and Chromophyte Phylogeny. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae: Problems and Perspectives, The Systematics Association Special Vol. No. 38. Clarendon Press, Oxford, pp 229—253

Itoh H, Aoki MN, Tsuchiya Y, Sato T, Shinagawa H, Komatsu T, Mikami A, Hama T (2007) Fate of organic matter in faecal pellets egested by epifaunal mesograzers living in a Sargassum forest and its implications for biogeochemical cycling. Mar Ecol Prog Ser 352: 101—112

Coleman AW (1985) Diversity of plastid DNA configuration among classes of eukaryote algae. J Phycol 21: 1—16

Jeffrey SW (1976) The occurrence of chlorophyll c1 and c2 in algae. J Phycol 12: 349—354

Demmig B, Winter K, Kru¨ger A, Czygan F (1987) Photoinhibition and zeaxanthin formation in intact leaves a possible role of the xanthophyll cycle in the dissipation of excess light energy. Plant Physiol 84: 218—224

Kawachi M, Inouye I, Honda D, O’Kelly CJ, Bailey JC, Bidigare RR, Andersen RA (2002) The Pinguiophyceae classis nova, a new class of photosynthetic stramenopiles whose members produce large amounts of omega-3 fatty acids. Phycol Res 50: 31—47

Entwisle TJ, Andersen RA (1990) A re-examination of Tetrasporopsis (Chrysophyceae) and the description of Dermatochrysis gen. nov. (Chrysophyceae): a monostromatic alga lacking cell walls. Phycologia 29: 263—274 Ettl H (1956) Ein Beitrag zur Systematik der Heterkonten. Bot Notiser 109: 411—445 Falkowski PG, Katz ME, Knoll AH, Quigg A, Raven JA, Schofield O, Taylor FJR (2004) The evolution of modern eukaryotic phytoplankton. Science 305: 354—360 Garriga GH, Bertrandt H, Lambowits A (1984) RNA splicing in Neurospora mitochondria: nuclear mutants defective in both splicing and 3’ end synthesis of large rRNA. Cell 36: 623—634 Gayral P, Billard C (1986) A Survey of the Marine Chrysophyceae with Special Reference to the Sarcinochrysidales. In Kristiansen J, Andersen RA (eds) Chrysophytes: Aspects and Problems. Cambridge University Press, New York, pp 37—48 Guillou L, Chre`tiennot-Dinet MJ, Medlin LK, Claustre H, Loiseaux de Goe¨r S, Vaulot D (1999) Bolidomonas: a new genus with two species belonging to a new algal class, the Bolidophycease (Heterokonta). J Phycol 35: 368—381

Kawai H, Maeba S, Sasaki H, Okuda K, Henry EC (2003) Schizocladia ischiensis: a new filamentous marine chromophyte belonging to a new class, Schizocladiophyceae. Protist 154: 211—228 Kohata K, Watanabe M, Yamanaka K (1991) Highly sensitive determination of photosynthetic pigments in marine in situ samples by high-performance liquid chromatography. J Chromatogr 558: 131—140 Lanvaud J, Rousseau B, Etienne A-L (2004) General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae). J Phycol 40: 130—137 McLachlan J (1985) Macroalgae (seaweeds): industrial resources and their utilization. Plant Soil 89: 137—157 Medlin LK, Kooistra WHCF, Potter D, Saunders GW, Andersen RA (1997) Phylogenetic relationships of the ‘golden algae’(haptophytes, heterokont chromophytes) and their plastids. Pl Syst Evol (Suppl) 11: 187—219 Nagasato C, Motomura T (2002a) Influence of the centrosome in cytokinesis of brown algae: polyspermic zygotes of

ARTICLE IN PRESS

Aurearena cruciata gen. et sp. nov. Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae). J Cell Sci 115: 2541—2548 Nagasato C, Motomura T (2002b) Ultrastructural study on mitosis and cytokinesis in Scytosiphon lomentaria zygotes (Scytosiphonales, Phaeophyceae) by freeze-substitution. Protoplasma 219: 140—149 Nakayama T, Marin B, Kranz HD, Surek B, Huss VAR, Inouye I, Melkonian M (1998) The basal position of scaly green flagellates among the green algae (Chlorophyta) is revealed by analyses of nuclear-encoded SSU rRNA sequences. Protist 149: 367—380 O’Kelly CJ (1989) The Evolutionary Origin of the Brown Algae: Information from Studies of Motile Cell Ultrastructure. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae. Clarendon Press, Oxford, pp 255—278

457

Schoenwaelder M, Clayton M (2000) Physode formation in embryos of Phyllospora comosa and Hormosira banksii (Phaeophyceae). Phycologia 39: 1—9 Sekiguchi H, Moriya M, Nakayama T, Inouye I (2002) Vestigial chloroplasts in heterotrophic stramenopiles Pteridomonas danica and Ciliophrys infusionum (Dictyochophyceae). Protist 153: 157—167 Shimodaira H, Hasegawa M (2001) CONSEL: a program for assessing the confidence of phylogenetic tree selection. Bioinformatics 17: 1246—1247 Sims PA, Mann DG, Medlin LK (2006) Evolution of the diatoms: insights from fossil, biological and molecular data. Phycologia 45: 361—402 Smit AJ (2004) Medicinal and pharmaceutical uses of seaweed natural products: a review. J Appl Phycol 16: 245—262

O’Kelly CJ, Floyd GL (1985) Absolute configuration of the flagellar apparatus in Giraudyopsis stellifer (Chrysophyceae, Sarcinochrysidales) zoospores and its siginificance in the evolution of the Phaeophyceae. Phycologia 24: 263—274

Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26: 31—42

Okuda K, Sekida S, Yoshinaga S, Suetomo Y (2004) Cellulose-synthesizing complexes in some chromophyte algae. Cellulose 11: 365—376

Swofford DL ed (2001) PAUP* 4.0b10 for 32-bit Microsoft Windows; Phylogenetic Analysis Using Parsimony (and other Methods), Beta 10. Sinauer Associates, Inc. Publishers, Sunderland, MA

Peng HB, Jaffe LF (1976) Cell wall formation in Pelvetia embryos: a freeze-fracture study. Planta 133: 57—61 Preisig HR (1989) The Flagellar Base Ultrastructure and Phylogeny of Chromophytes. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae. Clarendon Press, Oxford, pp 167—187 Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817—818 Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17: 208—212 Saiki RK, Gelfand DH, Stoffel S, Scharf DJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase. Science 239: 487—491 Sambrook J, Fritch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York Saunders GW, Potter D, Andersen RA (1997) Phylogenetic affinities of the Sarcinochrysidales and Chrysomeridales (Heterokonta) based on analyses of molecular and combined data. J Phycol 33: 310—318 Schoenwaelder M (2002) Phycological reviews 21. The occurrence and cellular significance of physodes in brown algae. Phycologia 41: 125—139

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876—4882 Uhrmacher S, Hanelt D, Nultsch W (1995) Zeaxanthin content and the degree of photoinhibition are linearly correlated in the brown alga Dictyota dichotoma. Mar Biol 123: 159—165 Van de Peer Y, Van der Auwera G, De Wachter R (1996) The evolution of stramenopiles and alveolates as derived by substitution rate calibration of small ribosomal subunit RNA. J Mol Evol 42: 201—221 Wada S, Aoki MN, Tsuchiya Y, Sato T, Shinagawa H, Hama T (2007) Quantitative and qualitative analyses of dissolved organic matter released from Ecklonia cava Kjellman, in Oura Bay, Shimoda, Izu Peninsula, Japan. J Exp Mar Biol Ecol 349: 344—358 Yoshii Y, Takaichi S, Maoka T, Inouye I (2003) Photosynthetic pigment composition in the primitive green alga Mesostigma viride (Prasinophyceae): phylogenetic and evolutionary implications. J Phycol 39: 570—576 Yoshii Y, Takaichi S, Maoka T, Hanada S, Inouye I (2002) Characterization of two unique carotenoid fatty acid esters from Pterosperma cristatum (Prasinophyceae, Chlorophyta). J Phycol 38: 297—303