DIFFERENTIAL LIFE HISTORY PHASE EXPRESSION IN TWO COEXISTING SPECIES OF SCYTOSIPHON (PHAEOPHYCEAE) IN NORTHERN CHILE1

The identity of two phaeophycean taxa that monopolized the middle‐lower rocky intertidal zone of a coastal area chronically exposed to copper mine wastes in northern Chile was unraveled. One of them was preliminarily identified as the gametophytic stage of Scytosiphon lomentaria (Lyngbye) Link. The other, a dark crust, resembled the alternate stage of some Scytosiphon species. Comparative analysis of morphology, life history, and DNA sequences strongly suggests that crusts corresponded to sporophytic S. tenellus Kogame and confirm that erect thalli belonged to S. lomentaria. A clear segregation of erect and crustose thalli was found using internal transcribed spacer region 1 and RUBISCO spacer sequences. Furthermore, whereas crusts always grouped with S. tenellus, erect thalli always grouped with S. lomentaria. Life history studies failed to connect the two entities. First, field‐collected S. tenellus produced progeny that either recycled the crust, which reproduced by unilocular zoidangia, or developed into erect thalli. The latter, unlike typical gametophytic S. lomentaria, developed patchy sori of plurilocular zoidangia. Second, S. lomentaria displayed a direct‐type life cycle, in which progeny from erect individuals only developed into erect thalli and produced only plurilocular zoidangia. This constitutes the first experimental study on Scytosiphon from the Pacific coast of South America and the first report of S. tenellus on this coast. It is also the first report of the crustose stage of Scytosiphon appearing as a perennial and dominant algal species in a temperate rocky intertidal system.     

A taxonomic study of Japanese Scytosiphon (Scytosiphonales, Phaeophyceae), including two new species

Morphological and culture studies were carried out on the genus Scytosiphon (Scytosiphonales, Phaeophyceae) growing in Japan. Morphologies of plurilocular zo-idangia and sporophytic thalli were found to be useful as taxonomic characters. Two types of plurilocular zo-idangial sori were recognized: (i) loosely coherent plurilocular zoidangia without a cuticular layer (loose type); (ii) tightly coherent plurilocular zoidangia with a cuticular layer (coherent type). The sporophytic thallus morphology was different among species in general appearance (tufted or discoid), degree of cohesion of erect filaments, and presence or absence of paraphyses. Two new species are described, Scytosiphon gracilis sp. nov. and Scytosiphon tenellus sp. nov, Scytosiphon gracilis is distinguished from other species by a thin medullary layer, coherent plurilocular zoidangia, no ascocysts associated with plurilocular zoidangia, and Compsonema-like sporophytic thalli. Scytosiphon tenellus is characterized by a thin medullary layer, coherent plurilocular zoidangia, ascocysts among plurilocular zoidangial sori, and Stragularia-like sporophytic thalli. Scytosiphon lo-mentaria (Lyngbye) Link is characterized by a constricted gametophytic thallus, loose plurilocular zoidangia, the presence of ascocysts, and sporophytic thaili identified with Microspongium gelatlnosum Reinke.     

Life history of Chnoospora implexa (Chnoosporaceae, Phaeophyceae) in culture

The life history of the brown alga Chnoospora implexa J. Agardh (Chnoosporaceae, Scytosiphonales) from Japan was studied in laboratory cultures. This species showed a heteromorphic and diphasic life history, alternating between erect gametophytes and discoid sporophytes. The gametophytes were dioecious and produced isogametes. The zygotes developed into sporophytes at 20°C under long‐day conditions, which formed plurilocular zoidangia. Zoids released from the plurilocular zoidangia developed again into sporophytes that always formed plurilocular zoidangia at 20°C and 25°C in long‐day conditions, and mainly unilocular zoidangia at 25°C in short‐day conditions. Zoids released from unilocular zoidangia developed into dioecious gametophytes. At 15°C zygotic erect thalli were formed and were revealed to be diploid by microspectrofluorometric measurements of nuclear DNA contents. The development and reproduction of unfused gametes were similar to those of zygotes. Some strains showed a direct‐type life history; gametophytic thalli were produced, but not via a sporophytic phase.     

Life histories of Colpomenia sinuosa and Hydroclathrus clathratus (Scytosiphonaceae, Phaeophyceae) in culture

Life-history studies in culture were carried out on Colpomenia sinuosa (Mertens ex Roth) Derbès et Solier and Hydroclathrus clathratus (C. Agardh) Howe (Scytosiphonales, Phaeophyceae) from Japan. These species showed a heteromorphic life-history pattern with an alternation between erect thalli bearing plurilocular zoidangia and prostrate thalli bearing ectocarpoid plurilocular and unilocular zoidangia. Plurizoids released from erect and prostrate thalli developed into prostrate thalli. Unizoids, however, developed into erect thalli. Prostrate thalli produced plurilocular zoidangia in long-day conditions and unilocular zoidangia in short-day conditions at 10-20°C. Prostrate thalli of C. sinuosa formed ascocysts. Germlings of both species did not grow at 5°C.     

Epi-endophytic symbiosis between Laminariocolax aecidioides (Ectocarpales, Phaeophyceae) and Undaria pinnatifida (Laminariales, Phaeophyceae) growing on Argentinian coasts

The present is the first study on epi-endophytic algae on thalli of Undaria pinnatifida growing along Argentinian coasts. The main goal is to describe the nature and the morphology of this symbiosis. Individuals of Laminariocolax aecidioides were detected in both June and December 2004, growing on U. pinnatifida sporophytes. In nature, the epi-endophyte were macroscopically observed as dark zones that partially covered the hosts’ fronds. L. aecidioides vegetative thalli were irregularly branched uniseriate filaments. The life cycle is described from laboratory cultures started from Patagonian populations. Caryology revealed that the sporophytic diploid phase presented 16 chromosomes whereas the gametophytic haploid phase presented 8 chromosomes. Isolates made from thalli growing in the interior of infected hosts developed into filamentous, branched sporophytes that reproduced by both unispores and plurispores that were produced in unilocular and plurilocular sporangia, respectively. The results of this paper also allowed us to conclude that L. aecidioides uses the thalli of U. pinnatifida as a proper substrate. The penetration of endophitic filaments among the host′s cortical cells produced a lateral compression and, finally, their thalli development generated perforations in the host tissues. The effects of the epi-endophytic infection of L. aecidioides on U. pinnatifida are neither severe nor deleterious.     

A NEW BROWN ALGAL ORDER,ISHIGEALES (PHAEOPHYCEAE), ESTABLISHED ON THE BASIS OF PLASTID PROTEIN‐CODING rbcL,psaA, AND psbA REGION COMPARISONS1

The brown algal family Ishigeaceae currently includes a single genus, Ishige Yendo, with two species. The relationship of the family to other brown algal lineages is less studied in terms of their plastid ultrastructure and molecular phylogeny. We determined the sequences of rbcL from four samples of the two Ishige species and nine putative relatives and the psaA and psbA sequences from 37 representatives of the brown algae. Analyses of individual and combined data sets resulted in similar trees; however, the concatenated data gave greater resolution and clade support than each individual gene. In all the phylogenies, the Phaeophyceae was well resolved, the Ectocarpales being placed in a terminal position and the Ishigeaceae ending up in a basal position. From our ultrastructural study, we concluded that the pyrenoid is absent in the Ishigeaceae, despite the presence of a rudimentary pyrenoid in I. okamurae. These results suggest that the Ishigeaceae is an early diverging brown lineage. Our molecular and morphological data, therefore, lead us to exclude the Ishigeaceae from the Ectocarpales s.l., which have an elaborate pyrenoid, and to propose its own order Ishigeales ord. nov. The Ishigeales is distinguished by oligostichous structure of thalli, phaeophycean hairs formed within cryptostomata, unilocular sporangia transformed from terminal cortical cells, and plurilocular sporangia lacking sterile terminal cells. This study is the first to document the utility of the psaA and psbA sequences for brown algae and also the first report on the multigene phylogeny of the Phaeophyceae based on three protein‐coding plastid genes.     

On the sexual reproduction ofDictyosiphon foeniculaceus (Phaeophyceae,Dictyosiphonales)

Dictyosiphon foeniculaceus from Sweden and Newfoundland was studied in laboratory culture. Zoids from unilocular sporangia developed into dioecious microscopic filamentous gametophytes which produced uniseriate plurilocular gametangia in low temperatures (0 to 8 °C). Zygotes and unfused isogametes gave rise to filamentous protonemata on which parenchymatous macroscopic sporophytes were formed. Isolates from Sweden and Newfoundland were interfertile. Although formed in culture, genetically unisexual sporophytes were not detected in nature. Female gametes ofD. foeniculaceus produced a sexual pheromone. It was identified as finavarrene, which is also known as the sperm attractant inAscophyllum nodosum.     

LIFE HISTORY OF COLPOMENIA SINUOSA (SYCTOSIPHONACEAE,PHAEOPHYCEAE) IN THE AZORES1

Colpomenia sinuosa (Mertens ex Roth) Derbès and Solier (Scytosiphonaceae, Phaeophyceae) is a common species on the rocky intertidal shores of the Azores, where reproductive gametophytes occur throughout the year. Life‐history studies of this species were carried out in culture, and both sexual and asexual reproduction were observed. Anisogamous gametes fused to form zygotes. The zygotes gave rise to a filamentous prostrate sporophyte generation bearing unilocular sporangia, under both short‐day and long‐day conditions at 15 and 22°C, and to both unilocular and plurilocular sporangia, under the lower temperature condition. Unispores developed into gametophytes, and plurispores gave rise to filamentous sporophytes. Asexual reproduction was carried out by unfused female gametes and asexual plurispores produced from the same gametophyte. Unfused gametes developed into filamentous prostrate sporophytes producing unilocular sporangia in both culture conditions, and unispores released from the sporangia gave rise to gametophytes. Asexual plurispores from field gametophytes, under both culture conditions, developed directly into new gametophytes. The species exhibited three types of life history: a heteromorphic, diplohaplontic; a heteromorphic, monophasic (both with alternation between the erect and filamentous prostrate thalli); and a monomorphic, monophasic.     

MORPHOLOGY AND LIFE HISTORY OF SCYTOSIPHON LOMENTARIA (SCYTOSIPHONACEAE,PHAEOPHYCEAE) FROM THE AZORES1

Morphological and culture studies of Scytosiphon lomentaria (Lyngbye) Link and Microspongium gelatinosum Reinke (Scytosiphonaceae, Phaeophyceae) were undertaken on material collected on the Island of São Miguel, Azores, where both species were commonly found. Erect thalli of S. lomentaria collected in the field were up to 33 cm long and 2.3 mm wide, tubular, hollow, and commonly constricted at intervals. The plurilocular sporangia were positioned in continuous sori on the thallus surface. Ascocysts were present. In the field, M. gelatinosum formed crustose to slightly pulvinate plants, were spongy in texture, and dark brown to black in color, which were circular or irregularly spreading over several centimeters and firmly attached to the substratum. Sessile unilocular sporangia were located in sori on the crust surface. In culture S. lomentaria plurispores developed into Microspongium‐like crustose prostrate thalli that formed unilocular sporangia. Unispores developed into new erect thalli that formed plurilocular sporangia. Sexual reproduction was not observed. In culture, M. gelatinosum unispores developed into erect thalli identical with S. lomentaria. These results are similar to those reported for other areas and suggest the occurrence in the Azorean plants of a monophasic and heteromorphic life history, involving both entities studied.     

First record of Asteronema rhodochortonoides (Phaeophyceae) for the Pacific Ocean

Morphological observations of a minute, filamentous, branched brown alga epiphytic on Sargassum thun‐bergii (Mertens ex Roth) Kuntze were made on material collected at Tsuyazaki (33°48′N, 130°27′E), Fukuoka Prefecture, southern Japan. This alga was assignable to Asteronema rhodochortonoides (Børgesen) Möller et Parodi in having stellately arranged chloroplasts with several pyrenoids grouped in the center, predominantly apical growth, narrow filaments, and elliptical or broadly elliptical plurilocular zoidangia that are apically or laterally formed on upright filaments. A comparison of partial nuclear small subunit rDNA sequences between the Japanese material and A. rhodochortonoides from the Canary Islands showed only two or three nucleotide differences. This supports our assignment of the Japanese material to this species as a first report for the Pacific Ocean. In laboratory cultures, zoids released from plurilocular zoidangia developed into plants with morphology similar to the field‐collected plants. This cycle repeated without production of unilocular zoidangia in our cultures.     

Molecular phylogeny of two unusual brown algae,Phaeostrophion irregulare and Platysiphon glacialis,proposal of the Stschapoviales ord. nov. and Platysiphonaceae fam. nov., and a re‐examination of divergence times for brown algal orders

The molecular phylogeny of brown algae was examined using concatenated DNA sequences of seven chloroplast and mitochondrial genes (atpB, psaA, psaB, psbA, psbC, rbcL, and cox1). The study was carried out mostly from unialgal cultures; we included Phaeostrophion irregulare and Platysiphon glacialis because their ordinal taxonomic positions were unclear. Overall, the molecular phylogeny agreed with previously published studies, however, Platysiphon clustered with Halosiphon and Stschapovia and was paraphyletic with the Tilopteridales. Platysiphon resembled Stschapovia in showing remarkable morphological changes between young and mature thalli. Platysiphon, Halosiphon and Stschapovia also shared parenchymatous, terete, erect thalli with assimilatory filaments in whorls or on the distal end. Based on these results, we proposed a new order Stschapoviales and a new family Platysiphonaceae. We proposed to include Phaeostrophion in the Sphacelariales, and we emended the order to include this foliose member. Finally, using basal taxa not included in earlier studies, the origin and divergence times for brown algae were re‐investigated. Results showed that the Phaeophyceae branched from Schizocladiophyceae ~260 Ma during the Permian Period. The early diverging brown algae had isomorphic life histories, whereas the derived taxa with heteromorphic life histories evolved 155–110 Ma when they branched from the basal taxa. Based on these results, we propose that the development of heteromorphic life histories and their success in the temperate and cold‐water regions was induced by the development of the remarkable seasonality caused by the breakup of Pangaea. Most brown algal orders had diverged by roughly 60 Ma, around the last mass extinction event during the Cretaceous Period, and therefore a drastic climate change might have triggered the divergence of brown algae.     

GLOBAL SYSTEMATIC AND PHYLOGENETIC ANALYSIS OF SARGASSUM IN THE GULF OF MEXICO,CARIBBEAN AND PACIFIC BASIN

Sargassum is one of the most species‐rich genera in the brown algae with over 400 described species worldwide. The bulk of these species occurs in Pacific‐Indian ocean waters with only a small portion found on the Atlantic side of the Isthmus of Panama. Sargassum also has one of the most subdivided and complex taxonomic systems used within the algae. Systematic distinctions within the genus are further complicated by high rates of phenotypic variability in several key morphological characters. Molecular analyses in such systems should allow testing of systematic concepts while providing insights into speciation and evolutionary patterns. Global molecular phylogenetic analyses using both conserved and variable regions of the Rubisco operon (rbcL and rbcL‐IGS‐rbcS) were performed with species from the Gulf of Mexico, Caribbean, and Pacific basin. Results confirm earlier analyses based on rbcL‐IGS‐rbcS from Pacific species at the subgeneric and sectional level while providing additional insights into the systematics and phylogenetics on a global scale. For example, species east of the Isthmus of Panama form a distinct well‐resolved clade within the tropical subgenus. This result in sharp contrast to traditional systematic treatments but provides a window into the evolutionary history of this genus in the Pacific and Atlantic Ocean basins and a possible means to time speciation events.     

CLONING AND QUANTITATIVE ANALYSIS OF THE CARBONIC ANHYDRASE GENE FROM PORPHYRA YEZOENSIS1

Carbonic anhydrase (CA), an enzyme that catalyzes the interconversion of CO₂ and HCO₃^?, has a critical role in inorganic carbon acquisition in many kingdoms, including animals, plants, and bacteria. In this study, the full‐length cDNA of the CA gene from Porphyra yezoensis Ueda (denoted as PyCA) was cloned by using an expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE). The nucleotide sequence of PyCA consists of 1,153 bp, including a 5′ untranslated region (UTR) of 177 bp, a 3′ UTR of 151 bp, and an open reading frame (ORF) of 825 bp that can be translated into a 274‐amino‐acid putative peptide with a molecular mass (M) of 29.8 kDa and putative isoelectric point (pI) of 8.51. The predicted polypeptide has significant homology to the β‐CA from bacteria and unicellular algae, such as Porphyridium purpureum. The mRNA in filamentous thalli, leafy thalli, and conchospores was examined, respectively, by real‐time fluorescent quantitative PCR (qPCR), and the levels of PyCA are different at different stages of the life cycle. The lowest level of mRNA was observed in leafy thalli, and the level in filamentous thalli and in the conchospores was 4‐fold higher and 10‐fold higher, respectively.     

New species, observations, and a list of new records of brown algae (Phaeophyceae) from the Hawaiian Islands

Two new species of brown algae (Phaeophyceae), Padina moffittiana Abbott et Huisman, sp. nov. and Cutleria irregularis Abbott et Huisman, sp. nov., are described from the Hawaiian Islands (between 19°04′N, 155°35′W and 28°25′N, 178°20′W). In addition, the new combination Cutleria canariensis is proposed for Aglaozonia canariensis. New observations are presented on Nereia intricata Yamada, a species described 67 years ago and known only from its type specimen. New records of a further 14 species are given for the Hawaiian Islands. These 17 taxa bring the total number of species of brown algae recorded for the Hawaiian Islands to 55, an increase of 28%. Of the 15 new records, two are recent ‘accidental’ introductions: Dictyota flabellata (Collins) Setchell et Gardner and Sargassum muticum (Yendo) Fensholt from California. Six records are notable because of their great distances from previously known collections: Nereia intricata Yamada from the Ryukyu Is., Japan; Discosporangium mesarthrocarpum (Meneghini) Hauck, from the Adriatic, Mediterranean, warm Atlantic and southern Australia; Distromium flabellatum Womersley, Spatoglossum macrodontum J. Agardh, and Sporochnus moorei Harvey, from Australia; Desmarestia ligulata (Lightfoot) Lamouroux from temperate and colder waters in the Pacific and Atlantic. A comparison with some Japanese species of Padina confirms that Padina japonica Yamada should be subsumed with Padina sanctae‐crucis Børgesen, as proposed earlier.     

FIELD MEASUREMENTS OF INORGANIC NITROGEN UPTAKE BY EPIFLORA COMPONENTS OF THE SEAGRASS POSIDONIA OCEANICA (MONOCOTYLEDONS,POSIDONIACEAE)1

Crustose corallines, crustose and erect brown algae, and sessile animals are major components of the epiphytic community of the Mediterranean seagrass Posidonia oceanica (L.) Delile. Production, biomass, and specific composition of this epiphyte–seagrass association are impacted by anthropogenic increase of nutrient load in this oligotrophic area. In this context, nitrogen uptake by P. oceanica and its epiflora was measured using the isotope ¹⁵N at a 10 m depth in the Revellata Bay (Corsica, Mediterranean Sea). Epiflora components showed various seasonal patterns of biomass and abundance. The epiphytic brown algae appeared at the end of spring, later than the crustose corallines, and after the nitrate peak in the bay. Because of their later development in the season, epiphytic brown algae mostly rely on ammonium for their N needs. We hypothesize that the temporal succession of epiphytic organisms plays a crucial role in the N dynamics of this community under natural conditions. The epiphytic brown algae, which have a growth rate one order of magnitude greater than that of crustose corallines, showed lower N‐uptake rates. The greater N‐uptake rates of crustose corallines probably reflect the greater N requirements (i.e., lower C/N ratios) of red algae. We determined that the epiflora incorporated ammonium and nitrate more rapidly than their host. Nevertheless, when biomass was taken into account, P. oceanica was the most important contributor to N uptake from the water column by benthic macrophytes in this seagrass bed.     

FACTORS DETERMINING VARIOUS FORMS IN CLADOSIPHON ZOSTERAE (PHAEOPHYCEAE)

The brown alga Cladosiphon zosterae (J. Ag.) Kylin (Chordariaceae) is found to exist in culture in a variety of forms, including filaments, discoid or crustose forms, and two types of erect cylindrical thalli. Asexual spores from plurilocular sporangia of each form can produce any and all forms. Three factors controlling morphological expression in C. zosterae have been identified. These factors include: (1) heteroblasty (formation of filamentous and discoid germlings as a consequence of alternate modes of spore germination), which was independent of all environmental factors tested including temperature, photoperiod, nitrogen source, and general fertility of the growth medium; (2) influence of certain nitrogen sources, notably ammonium, which induce a compact discoid or crustose form; and (3) a morphogenetic factor presumed to be bacterial which determines mode of development and subsequent morphology of the erect Cladosiphon thallus.     

Utility of rbcS gene as a novel target DNA region for brown algal molecular systematics

The usefulness of molecular phylogenetic studies has increased remarkably as the quantity and quality of available DNA sequences has increased. When compared with the progress that has occurred in angiosperms and animals, there have been relatively few target DNA regions identified for use in taxonomic studies of brown algae. Therefore, in this study, we developed a new set of primers to amplify Rubisco small subunit (rbcS) gene sequences and determined the rbcS gene sequences of various species of brown algae including those belonging to Dictyotales, Ectocarpales, Fucales and Sphacelariales. The level of sequence variations in the rbcS gene varied according to the brown algal lineages. When focusing on the relationship of species within the genus Sargassum, the rbcS gene sequences provided useful information regarding the phylogenetic relationship among sections of the subgenus Bactrophycus. Based on the broad applicability and phylogenetic utility of the rbcS gene, we suggest that the sequence be used as a new target region for the molecular systematics of brown algae.     

Molecular and morphologically distinct Scytosiphon species (Scytosiphonales,Phaeophyceae) display similar antioxidant capacities1

The taxonomic status of an alga with complanate thalli, occurring in central Chile and belonging to the genus Scytosiphon, was elucidated. Morphological and molecular features demonstrated that, in addition to the known and widespread constricted S. lomentaria (Lyngb.) Link that occurs along the Chilean coast, there is a Scytosiphon with complanate thalli that occurs only in central Chile—S. gracilis Kogame. Morphological analyses of this previously unreported complanate Scytosiphon showed thalli without constrictions, coherent plurilocular sporangia without ascocysts, and phaeophycean hairs arising from cortical cells. Furthermore, sequences of the internal transcribed spacer (ITS1 and ITS2) regions of the complanate Scytosiphon were 99.8% and 100% identical to those detected in S. gracilis from Korea. ITS‐based comparative analyses showed that complanate Scytosiphon grouped in a different clade than S. lomentaria and S. tenellus Kogame from various parts of the world, including Chilean species. Moreover, molecular analyses suggest the occurrence of two distinct ITS types of S. lomentaria in northern Chile, corresponding to the Korean and Greek types. On the other hand, biochemical analyses of copper‐induced antioxidant responses in S. gracilis and S. lomentaria showed an identical increase in antioxidant enzyme activities. These results suggest that copper tolerance might be a constitutive trait in these species of Scytosiphon.     

Life history of Japanese Stypocaulon durum (Sphacelariales, Phaeophyceae)

Phenology, morphology, life history and responses to different temperature and photoperiod conditions were studied in Japanese Stypocaulon durum (Ruprecht) Okamura. Erect thalli of the species were collected year-round, but the mature thalli forming either uniloc-ular sporangia or two different types of plurilocular structures (evidently gametangia) on separate thalli were found only in winter. ln culture, an isomorphic life history is suggested for the species, alternating between a sporophyte forming unilocular sporangia and gam-etophytes forming plurilocular macro- (female) and micro- (male) gametangia. Contents of unilocular sporangia were not released, but germinated in situ, developing into erect thalli forming plurilocular gametangia. Macrogametangia released aplanogametes (oospores), but male gametangia appeared to be non-functional, although flagellated cells were once formed in the loc-uli. This is the first report of plurilocular gametangia in the species. Although the species grew well and matured under considerably lower temperature conditions than European Stypocaulon scoparium (L.) Sauvageau, its temperature requirements showed similarity to northwestern Atlantic Stypocaulon species. This supports the notion that northwestern Atlantic Stypocaulon is conspecific with S. durum.