GENETIC DIVERGENCE CORRELATES WITH MORPHOLOGICAL AND ECOLOGICAL SUBDIVISION IN THE DEEP-WATER ELK KELP, PELAGOPHYCUS PORRA (PHAEOPHYCEAE)

Pelagophycus porra (Leman) Setchell has a narrow distribution confined to deep water from the Channel Islands off the southern California coast to central Baja California, Mexico. Distinct morphotypes are consistently correlated with distinctive habitats, that is, windward exposures characterized by strong water motion and rocky substrates, and sheltered areas with soft substrates found on the lee sides of the islands. We tested the hypothesis that morphologically and ecologically distinct forms reflect genetically distinct stands. Individuals representing populations from three islands and the mainland were compared using RFLP analyses of the nuclear rDNA internal transcribed spacers (ITS1 and ITS2), chloroplast trn L (UAA) intron sequences, and random amplified polymorphic DNA (RAPDs). No variation was found in a survey of 20 restriction sites of ITS1 (ca. 320 base pair [bp]) and ITS2 (ca. 360 bp) among individuals from six populations. Likewise, comparisons of trn L intron (241 bp) sequences among nine individuals from seven populations were identical with the exception of a CATAGT insert in two adjacent stands. A RAPD analysis of 24 individuals from nine populations (4 windward and 5 leeward) using 16 primers generated 166 bands. Thirty-eight percent of the bands did not vary, 16% were unique to a given individual, and 46% were variable. Neighbor joining analysis produced a well-resolved tree with moderately high bootstrap support in which windward and leeward populations were easily distinguished. The lack of divergence in both the fast evolving nuclear rDNA-ITS and the chloroplast trn L intron does not support the morphotypes as different species. However, the compartmentalized differentiation shown in the RAPD data clearly points to isolation. This, and previous ecological studies that demonstrate habitat specificity suggest that leeward stands probably comprise a species in statu nascendi.     

PHYLOGEOGRAPHY OF CUTLERIA CYLINDRICA (CUTLERIALES,PHAEOPHYCEAE) IN NORTHEASTERN ASIA,AND THE IDENTITY OF AN INTRODUCED POPULATION IN CALIFORNIA1

Cutleria cylindrica Okamura was described from Japan in 1902 and has been reported only from northwestern Asia until its relatively recent discovery in California, USA, and Baja California, Mexico. To clarify the genetic relationships within and among the disjunct populations, we carried out a molecular phylogenetic study, as well as the examination of sex ratio and the life‐history patterns, of populations in Japan, Korea, and California. Based on the DNA sequences of mitochondrial genes cox2, cox3, the open reading frame (ORF) region, and the spacer between cox3 and ORF, a total of 23 haplotypes were detected in the 85 individuals from 20 localities in Japan, Korea, and California. All localities in Japan and Korea included multiple haplotypes, but only a single haplotype was found in California. There was a positive relationship between distance and genetic divergence in Japan and Korea. The single haplotype found in California was the same as one occurring in Japan (Aomori Pref. and Fukuoka Pref.) and Korea (Daedaepo, Pusan). Both male and female gametophytes were distributed in most northeastern Asian populations. Only female gametophytes, developing parthenogenetically from female gametes, were found in California and Aomori Pref., Japan. On the basis of these results, we conclude that the disjunct population of C. cylindrica in California originated from a relatively recent introduction from Japan and shares its origin with the parthenogenetic population in the Tsugaru Strait.     

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system.Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy.Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates.Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks.     

NEUTRAL LIPIDS AS MAJOR STORAGE PRODUCTS IN ZOOSPORES OF THE GIANT KELP MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Neutral lipids, consisting primarily of triacylglycerols, were found to be a major form of carbon reserve in zoospores of the giant kelp Macrocystis pyrifera (L.) C. Ag. The fluorescent stain Nile Red revealed large lipid droplets in the posterior end of the cell, which comprised 20–41% of cellular carbon in newly released spores. Flow cytometric analyses of newly released spores stained with Nile Red revealed considerable variation in the neutral lipid content among spores that was independent of spore size. Lipid droplets were consumed during germination in spores maintained either under constant light or in continual darkness. The availability of light appeared to delay, but did not preclude, lipid use. The rate of lipid use during germination varied considerably among germlings with some cells consuming all of their lipid reserves within 5 h after release. In addition to zoospores, lipid droplets were observed in both male and female gametes. Numerous droplets were observed in eggs, while single lipid droplets were observed in sperm. Neutral lipid droplets were not observed in gametophytes or sporophytes except in developing gametes and spores. Large lipid reserves thus seem to be confined to the microscopic life history stages that presumably have relatively high energy demands. By serving as a supplemental fuel reserve, neutral lipids may be important in extending the effective range of zoospore dispersal.     

Reproductive morphology and DNA sequences of the brown alga Platysiphon verticillatus support the new combination Platysiphon glacialis

Platysiphon verticillatus, a brown alga endemic to the Arctic, was described based on vegetative specimens collected at Inglefield Bay, West Greenland. The species is distinctive in having a lanceolate blade‐like thallus terminated by a terete portion, both covered with hair‐like assimilatory filaments. Punctaria glacialis was described from Eastern Greenland, and the species differs from other Punctaria species in lacking hairs and plurilocular zoidangia. Unilocular zoidangia were reported, but instead of zoids being released they formed cell walls in situ developing the appearance of plurilocular zoidangia. However, the fate of the zoids, as well as the walled cells was not traced, and the life history of the alga has remained unclear. By comparing DNA sequences (cox1, cox3, and rDNA ITS2) of specimens morphologically referable to Platysiphon verticillatus and Punctaria glacialis collected at Baffin Island, as well as re‐examining morphology and studying crude cultures, we concluded that they are the same taxonomic entity. Furthermore, their cox3 sequence and vegetative morphology agreed with those of the type specimen of Punctaria glacialis. Consequently, we propose Platysiphon glacialis comb. nov. The life cycle could not be completed in culture, but we hypothesize that in situ germination of the unizoids produces reduced gametophytes housed in peripheral tissue of erect sporophytic thalli.     

A long-term comparison of the benthic algal flora of Clare Island, County Mayo, western Ireland

The marine benthic algal flora of Clare Island, off County Mayo, western shore of Ireland, was investigated; collections of intertidal and subtidal marine algae were made at 16 sites along the eastern and southern shores in the years 1990, 1993 and 2000–2002. The data and observations obtained were compared with the results of a similar survey conducted by Arthur Disbrowe Cotton in 1910–1911. Considering the results of the original survey and the new survey together, the marine algal flora of the island currently totals 293 species; 224 species were recorded by Cotton in the original survey, whereas 223 species were identified in the present study. Most species are common to the original and the new list and the main differences are easily explainable; the new survey used SCUBA diving, which allowed the collection of several subtidal species not collected in 1910, and Cotton reported several microscopic green and brown algae, usually difficult to recognise in the field, which were not rediscovered. The most remarkable differences consist in the current presence of some large intertidal brown algae (Bifurcaria bifurcata, Cystoseira foeniculacea and Cystoseira nodicaulis) that were not reported in the survey of 1910. Two algae, Codium fragile subsp. tomentosoides and Asparagopsis armata, were introduced in Europe after the original survey. At present, the benthic algal assemblages of Clare Island still have basically the same structure and distribution as in 1910 and, if compared with other coastal areas of Europe, the intertidal marine environment of Clare Island appears remarkably well conserved.     

Molecular and biochemical studies of virus infections in two filamentous brown algae

The filamentous marine brown algae Ectocarpus siliculosus and Feldmannia simplex are infected by host specific DNA-viruses. Under Percoll isolation, Ectocarpus siliculosus-virus (EsV)-particles maintained their infective potential.The EsV has a circular genome of dsDNA with a size of 320 kb. A restriction map has been established. The gene of a major coat protein (gp1) was detected in a genomic library and partly sequenced. Using gpl- sequences for polymerase chain reaction (PCR) amplification analysis, EsV specific sequences could be detected in various symptom-free, clonal cultures of Ectocarpus. The PCR was also used to follow the passage of the virus genome during the meiosis of hosts. A monospecific antibody against recombinant gpl was used for immunostaining and infection experiments.The Feldmannia simplex-virus (FlexV-1) has a circular genome with a size of 220 kb and a 43% G+C content. FsV-DNA contains methylated bases. 5-methylcytosine (5 mC) makes up 12% of the total cytosines.     

Host-virus interactions in marine brown algae

Ectocarpus-like marine brown algae are frequently parasitized by polyhedric DNA viruses. Infected hosts have been studied in unialgal and axenic cultures, and the present state of knowledge is summarized in regard to stage-specific virus expression, discharge and survival time of virus particles, infection mechanism, association with host's nuclear genome, passage of the virus genome through mitosis and meiosis of the host, suppression of symptoms and spontaneous recovery of infected plants, host specificity and intergeneric transmission, vitality of infected plants, pandemic occurrence of virus infections, molecular data on Ectocarpus and Feldmannia viruses, and algal DNA-viruses as potential vectors for gene transfer. A scheme for the nomenclature of brown algal viruses is proposed.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.     

Two new farnesylacetone derivatives from the brown alga Sargassum micracanthum

From the brown alga, Sargassum micracanthum, two new farnesylacetone derivatives were isolated and their structures elucidated from spectral analyses and chemical correlation to a known farnesylacetone derivative.