KARYOLOGY OF BANGIA ATROPURPUREA (RHODOPHYTA,BANGIALES) FROM MEDITERRANEAN AND NORTHEASTERN ATLANTIC POPULATIONS1

We studied the karyology of Bangia atropurpurea (Roth) C. Ag. collected from marine and freshwater populations from the Mediterranean region and some northeastern Atlantic localities. Gametophytic thalli had two haploid karyotypes, n = 3 and n = 4. The n = 4 karyotype was only occasionally present in the Mediterranean and was also found in one Atlantic population, confirming a previous report. We propose that the four-chromosome karyotype is an aneuploid form, n + 1. Chromosomes were frequently observed either in a parallel arrangement or in a circular configuration.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

SEASONAL GROWTH AND PHENOTYPIC VARIATION IN PORPHYRA LINEARIS (RHODOPHYTA) POPULATIONS ON THE WEST COAST OF IRELAND1

The phenology and seasonal growth of Porphyra linearis Grev. were investigated in two morphologically dissimilar populations from the west coast of Ireland. Thallus size and reproductive status of individuals were monitored monthly between June 1997 and June 1998. Both populations exhibited a similar phenology: gametophyte stages appeared on the shore in October, with spermatangial and zygotosporangial sori appearing the following February; the gametophyte stage began to degenerate in April and had disappeared completely by June. However, significant differences in growth and reproduction in the field and in cultures of plants from the two populations were observed. Thallus length and width of individuals from one population were significantly longer throughout the sample period, and reproduction and sporulation occurred 1 month earlier. Also, in situ relative growth rates (RGRs) of plants differed significantly and were correlated with different climatic factors (sunshine, day length, irradiance, rainfall, seawater temperature, and intertidal temperatures), suggesting that plants were affected by two different microhabitats. At one site, blades were more exposed to wave action, sunshine, and extreme minimum temperatures, while at the other site, blades were more protected in winter, spring, and early summer. In culture, RGRs of blades from the second site were higher than RGRs of blades from the first site under short days, corroborating the field results and suggesting a degree of phenotypic differentiation between the two populations. However, there were no sequence divergences of the RUBISCO spacer between strains of the two P. linearis populations.     

MORPHOLOGICAL AND MOLECULAR ANALYSIS OF THE ENDANGERED SPECIES PORPHYRA TENERA (BANGIALES,RHODOPHYTA)1

Porphyra tenera Kjellman, widely cultivated in nori farms before the development of artificial seeding, is currently listed as an endangered species in Japan. To confirm whether a wild‐collected gametophytic blade was P. tenera or the closely related species P. yezoensis Ueda, morphological observations and molecular analyses were made on the pure line HGT‐1 isolated from a wild blade. This pure line was identified as P. tenera based on detailed morphological features. Sequences of the nuclear internal transcribed spacer region 1 and the plastid RUBISCO spacer revealed that P. tenera HGT‐1 was clearly different from P. yezoensis f. narawaensis Miura, the main species cultivated in Japan. PCR‐RFLP analysis of the internal transcribed spacer region was found to be a convenient method for rapid discrimination between P. tenera and cultivated P. yezoensis. The restriction patterns generated by the endonucleases Dra I and Hae III were useful for differentiating between both gametophytic and conchocelis stages of P. tenera HGT‐1 and P. yezoensis f. narawaensis strains. Thus, PCR‐RFLP analysis will serve as a valuable tool for rapid species identification of cultivated Porphyra strains, culture collections of Porphyra strains for breeding material and conservation of biodiversity, and, as codominant cleaved amplified polymorphic sequence markers for interspecific hybridization products between P. tenera and P. yezoensis f. narawaensis. Under the same culture conditions, rate of blade length increase and the blade length‐to‐width ratio were lower in P. tenera HGT‐1 than in P. yezoensis f. narawaensis HG‐4. The HGT‐1 became mature more rapidly than HG‐4 and had thinner blades.     

DISTRIBUTION,SEASONALITY AND REPRODUCTIVE PHENOLOGY OF BANGIA ATROPURPUREA (RHODOPHYTA) IN RHODE ISLAND,U.S.A.1

Forty-seven sites along the Rhode Island coastline were sampled on a seasonal basis for the presence of the red alga Bangia atropurpurea (Roth) C. Ag. This species was found to be widely distributed, occurring at 94% of the hard, stable substrates sampled. Bangia populations were most, widespread in the winter (71% of sites) and least frequent in the summer (29% of sites). At one location, three high intertidal populations (average height 101 cm) and three low intertidal populations (average height 14 cm) were sampled monthly for two years. The upper intertidal populations appeared in September and persisted until at least May, whereas the lower populations were ephemeral, appearing for only a few months in late winter. The high intertidal populations were present on 85% of the sampling dates white the lower ones were observed on 25% of the sampling dates. Upper intertidal filaments reproduced exclusively by asexual monospores unlike those of the lower populations which were largely sexual. The maximum diameter of asexual and carpogonial filaments was 120 and 250 nm, respectively. Monosporagenesis was strongly correlated to temperature whereas gametogenesis was strongly correlated to photopriod.     

MOLECULAR IDENTIFICATION OF TWO SIBLING SPECIES UNDER THE NAME GRACILARIA CHILENSIS (RHODOPHYTA,GRACILARIALES)1,3

Molecular markers belonging to three different genomes, mitochondrial (cox2‐3 spacer), plastid (RUBISCO spacer), and nuclear (internal transcribed spacer 1), were used to compare Gracilaria chilensis samples collected along the Chilean coast with samples ascribed to G. chilensis from the West Pacific Ocean (New Zealand and Australia). Our data are in agreement with previous studies suggesting two sibling species currently going under the name G. chilensis that co‐occur in New Zealand. One of these, a New Zealand sample previously examined by Bird and others in 1990, is conspecific with G. chilensis from Chile. Finally, our results demonstrate clearly that most of the sequences in GenBank reported as G. chilensis are based on misidentified material.     

AXENIC TISSUE CULTURE AND MORPHOGENESIS IN PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)

To better understand developmental phenomena in macroalgal tissue culture, we examined the morphogenesis of Porphyra yezoensis Ueda (strain TU-1) cultured aseptically in defined synthetic media . Generally, the filamentous thalli (sporophyte; conchocelis phase) of P. yezoensis were densely tufted with uniseriate filaments. The foliose thalli (gametophyte) were monolayered. In this study, axenic filamentous thalli retained their characteristic morphogenesis; there were no obvious differences between morphogenetic traits in unialgal and axenic conditions. However, conchospores, which might have developed into the foliose form under unialgal conditions, germinated into calluslike masses under axenic conditions. Most of the gametophytes gradually lost their typical morphogenesis after the first longitudinal cell division. Some of the calluslike masses developed rhizoidlike structures in several places or along the entire mass. Therefore, we concluded that P. yezoensis, in axenic cultures, loses its typical morphogenesis only during the gametophytic phase. The axenic tissue culture of Porphyra established in this study is a promising assay system for the identification of growth and morphogenetic factors.     

DISTRIBUTION AND SALINITY ADAPTATIONS OF BANGIA ATROPURPUREA (RHODOPHYTA), A PUTATIVE MIGRANT INTO THE LAURENTIAN GREAT LAKES1

In a survey of the distribution of Bangia atropurpurea (Roth) Ag. along the north and east shorelines of the Laurentian Great Lakes, it was observed that this species was concentrated in the lower lakes. Asexual reproduction by monospore formation was the only form of reproduction in these populations. First generation germlings derived from these spores grew in a wide range of salinities from 1 to 26‰, but the rate was inversely proportional to salinity. In addition, acclimation of parent plants to freshwater or marine media prior to sporulation affected growth rates of first generation germlings placed into a series of salinities. By the second and third generation, germlings with a lineage of seawater showed a preference for the higher salinities, 19 and 26‰ Plants collected in Lake Ontario exhibited no decline in photosynthetic rate as salinity was raised by 13‰ for 15 min. At 26‰ the rate was reduced by half and thylakoids were disrupted in the region of the pyrenoid. On the other hand, third generation plants with a history of seawater attained maximum photosynthetic rates at 26‰ and contained a normal thylakoid organization. Three chromosomes were observed in these plants, which agrees with some of the previous findings for marine Bangia. Origins and taxonomic status of Bangia growing In the Great Lakes are discussed in light of these findings.     

MOLECULAR PHYLOGENY OF THE HAPTOPHYTA BASED ON THE rbcL GENE AND SEQUENCE VARIATION IN THE SPACER REGION OF THE RUBISCO OPERON

A phylogeny of 21 haptophyte algae was inferred by maximum parsimony, neighbor-joining, and maximum likelihood analyses of sequences of the plastid-encoded gene, rbcL. Sequence variation in the spacer region of the RUBISCO operon was also investigated. In all the rbcL trees constructed, the haptophytes form two distinct clades: one includes the Pavlovales and the other includes the Prymnesiales, Coccosphaerales, and Isochrysidales (all sensu Parke and Green 1976 . This relationship coincides with the recent taxonomic treatment splitting the division into two subclasses, the Prymnesidae and Pavlovidae ( Cavalier-Smith 1989 ) or the Prymnesiophycidae and the Pavlovophycidae using botanical suffixes ( Jordan and Green 1994 ), or into two classes, the Patelliferea and the Pavlovea ( Cavalier-Smith 1993 ). In the Prymnesiophycidae, all the coccolithophorids examined are placed in a single clade, which suggests a single origin of the coccolithophorids and the ability of coccolith formation in the haptophytes. The genus Chrysochromulina is polyphyletic. Species of Chrysochromulina with a very long haptonema and a compressed cell body (typical of species including the type C. parva Lackey) form a clade, including Imantonia, that is often classified in the Isochrysidales in the neighbor-joining tree, whereas some species possessing a nontypical cell body and cell covering form a clade with Prymnesium and Platychrysis in all trees. It is suggested that loss of the haptonema in Imantonia and the reduction in Prymnesium and Platychrysis occurred secondarily and independently in two different lineages. Within the coccolithophorids, four clades are recognized: Pleurochrysis, Calyptrosphaera-Cruciplacolithus-Calcidiscus-Umbilicosphaera, Helicosphaera, and Emiliania-Gephyrocapsa. A non-coccolith-bearing haptophyte, Isochrysis, is an ingroup of the Emiliania-Gephyrocapsa clade, suggesting its secondary loss of the ability to form a coccolith. Sequence comparison of the spacer region of RUBISCO operon supports most results obtained in the analysis of rbcL sequences. Monophyly of the Prymnesiales sensu Parke and Green is still unclear because of low (<50%) bootstrap support for this group.     

COMPARATIVE STUDIES ON THE CELL WALLS OF SEXUAL AND ASEXUAL BANGIA ATROPURPUREA (RHODOPHYTA). I. HISTOCHEMISTRY OF POLYSACCHARIDES1

A comparative histochemical study of the nature and distribution of acidic and neutral cell wall polysaccharides was conducted on marine sexual and asexual filaments of the red alga Bangia atropurpurea (Roth) C. Ag. Outer and inner walls of this species were clearly partitioned according to staining and transmission electron microscopic characteristics. Neutral polysaccharides were detected in the outer coating (cuticle) but were absent from outer and inner walls of all filaments. Acidic polysaccharides were noted in the outer wall material but not in the inner wall layers of any filaments at any developmental stages. The major staining component of vegetative regions of sexual material and all regions of asexual filaments was a highly sulfated polymer. During sexual reproduction only there was a generalized change in the nature of the acidic component, characterized by a decrease in intensity of staining for sulfates in both male and female filaments and the appearance, in female filaments only, of polysaccharides which presumably were carboxylated. Spermatia attached to both male and female filaments in regions where sexual differentiation was initiated and where changes in the outer wall components commenced.     

COMPARATIVE STUDIES ON THE CELL WALLS OF SEXUAL AND ASEXUAL BANGIA ATROPURPUREA (RHODOPHYTA). II ELECTROPHORETIC PATTERNS OF POLYSACCHARIDES1

Cellulose acetate electrophoresis of the hot water soluble polysaccharide extracts from whole filaments, as well as base, mid and tip segments, of marine asexual and sexual Bangia atropurpurea (Roth) C. Ag. Yielded distinctive patterns which demonstrated that changes occur in the outer cell walls during sexual reproduction. Heterogeneity of the sulfated polysaccharide components isolated from outer cell walls was shown to be specifically related to sexual reproduction. Two components (Band I and II) were detected in extracts from tips of sexual filaments, whole only one (Band I) was present in the vegetative segments of all filaments and in asexual reproductive regions. The faster running component (Band II) was detected during the later stages of sexual development, prior to maturation.     

IDENTIFICATION OF CYTOKININS IN SARGASSUM MUTICUM (PHAEOPHYTA) AND PORPHYRA PERFORATA (RHODOPHYTA)1

Combined gas chromatography-mass spectrometry (GCMS) was used to identify and quantify specific cytokinins from Porphyra perforate J. Ag. and Sargassum muticum (Yendo) Fensh. The level of isopentenyladenosine was estimated to be 0.6 μ·kg^?1 fresh weight in Porphyra and 0.9 μ·kg^?1 fresh weight in Sargassum. The level of cis-zeatin riboside was estimated to be 0.2 μ·kg^?1 fresh weight in Sargassum. This is the first definitive identification of a cytokinin from a red alga, and the second report from a brown alga.     

LITHIUM CHLORIDE EXTRACTION OF DNA FROM THE SEAWEED PORPHYRA PERFORATA (RHODOPHYTA)1

Lithium chloride facilitates the softening of cell walls resulting in a simple, quick (2 h) method for DNA extraction from the red seaweed Porphyra perforata J. Agardh. A 5-min treatment of tissues in Lid at 55°C extracts DNA that is relatively free of the viscous polysaccharides and proteins that are usually coextracted in large amounts from cell walls and cytoplasm. This protocol does not require grinding of tissues, hydroxyapatite binding, cetyl trimethyl ammonium bromide treatments, enzymatic treatments, phenol extraction, or CsCl-gradient centrifugation. The resulting DNA is of sufficient quality to be used as a template for polymerase chain reaction amplification.     

KARYOTYPES AND REPRODUCTIVE SEASONALITY OF THE GENUS BANGIA (RHODOPHYTA) IN BRITISH COLUMBIA,CANADA1

Chromosome number and morphology as well as reproductive seasonally were examined in Bangia populations collected from 88 sites along the British Columbia coasts. Sexual filaments were recorded at 35 sites and asexual filaments at 58. Four different types of Bangia were determined. These were characterized by reproductive state and chromosome number, as well as by their seasonally and range of distribution. A summer-fall three-chromosome sexual type occurred from approximately 50° N northward and a winter-spring four-chromosome sexual type had a northerly limit of approximately 54° N, extending to the southernmost areas of British Columbia. Two asexual types occurred, exhibiting little seasonally, six-chromosome populations throughout the coast and three-chromosome populations at three southerly sites only. Karyograms were prepared for the four chromosomal types, assigning tentative centromere locations. Somatic chromosome pairing was reported for the first time in the Rhodophyta.     

NEW INSIGHTS IN THE TAXONOMY OF THE CERAMIUM SINICOLA COMPLEX: RESURRECTION OF CERAMIUM INTERRUPTUM (CERAMIACEAE,RHODOPHYTA)1

Phylogenetic relationships of the Ceramium sinicola complex (C. interruptum and C. sinicola) including C. codicola were studied using nucleotide sequences of rbcL and small subunit rDNA, and the RUBISCO spacer was used for sequence comparison of each species. A reassessment of the taxonomic rank and the evolutionary trend within the complex was inferred from a comparative morphological study and molecular data sets based on 11 samples from eight populations from the Pacific coast of the United States and Mexico. Intraspecific relationships were poorly resolved, but the resurrection of C. interruptum as a distinct species was strongly supported by both morphological and molecular data. Ceramium interruptum is distinguished by the combination of the following features: thalli uncorticated at the first internode above the dichotomy, presence of four corticating filaments, 7–11 segments between branching points, rhizoids digitate, and epiphytic on a variety of hosts. Our molecular analyses show that C. sinicola is the sister group to C. codicola, and C. interruptum is basal to them. These phylogenetic relationships allowed for an assessment of the trend in the evolution of cortication pattern and attachment mode to the host.     

CRYOPRESERVATION OF THE CONCHOCELIS OF PORPHYRA (RHODOPHYTA) BY APPLYING A SIMPLE PREFREEZING SYSTEM1

The conchocelis cells of four strains of Porphyra yezoensis Udea and four other Porphyra species were cryopreserved in liquid nitrogen (LN) using a programmable freezer or a simple prefreezing system, which consisted of a styrofoam box and a deep-freezer at ?40° C. The cells differed in their freezing tolerance but survived maximally when prefrozen to ?40° C in a cryoprotective solution composed of 10% dimethylsulfoxide and 0.5 M sorbitol in 50% seawater. The cryopreservation was successfully performed by applying the simple prefreezing system as well as by a programmable freezer. Conchocelis cells thawed from the LN temperature formed colonies and retained the ability to form conchospores that grew into gametophytic thalli. This technique using a simple prefreezing system will accelerate the spread of Porphyra cryopreservation.     

CELL DIVISION PATTERNS AND DIURNAL CYCLE OF PORPHYRA ABBOTTAE (RHODOPHYTA,BANGIALES) CARPOGONIAL AND CARPOSPORE DEVELOPMENT1

Post-fertilization development of carpospores in Porphyra is a well-documented phenomenon. Development of the pre-fertilization carpogonial cells from vegetative cells, however, has not been previously described. In Porphyra abbottae Krishn., a rock? intertidal monostromatic species occurring from British Columbia to central California, large cells, designated hue CIS “procarpogonial mother cells” (PMCs), initiated the formation of the carpogonial cells. The PMCs formed during late night mitoses beginning at 0200 h with cytokinesis from 0300-0500 h during short day periods of 10:14 h LD in northern California (38°20′N, 123°03′W and 36°37′N, 121°55′W). The PMC cut off numerous smaller cells which in turn divided equal. Approximately 12 h Inter, at 1500 h (day 1) the Smaller cells were recognizable as carpogonial cells by the presence of trichogynes growing from the cytoplasm out through the cell wall to the thallus surface. In another 24 h (day 2), the fertilized carpogonia had divided into carpospore packets. Spores were released at 1500 h the following day (day 3), their projection creating escape channels through the cell walls.     

LIFE HISTORY AND HYBRIDIZATION STUDIES ON GIGARTINA STELLATA AND PETROCELIS CRUENTA (RHODOPHYTA) IN THE NORTH ATLANTIC1

Fourteen isolates of the crustose marine red alga Petrocelis cruenta J. Agardh from various localities in the British Isles, France (including the type locality), Spain and Portugal gave rise in culture to dioecious foliose plants identifiable as Gigartina stellata (Stackhouse) Batters although two isolates formed only sterile foliose blades. A total of 145 isolates of Gigartina stellata were also grown in culture from various localities in the U.S.A. (Maine), the British Isles, Iceland, Denmark, France, Spain and Portugal using both carpospores and vegetative blade apices. Two basic types of life history were found among these isolates: a direct-type life history involving the formation of further foliose plants from carpospores, some isolates of which also form spermatangia on the same papillae as the cystocarps; and a heteromorphic-type in which only crustose plants resembling Petrocelis cruenta are formed from carpospores. Only heteromorphic-type life histories were found from Spain and Portugal. Both life history types were found in plants from the U.S.A., the British Isles and northern France. Only direct-type life histories were found in plants from Iceland and Denmark. Some Petrocelis-like crusts derived from field collected G. stellata carpospores and Petrocelis crusts of hybrid progeny formed tetrasporangia in 8:16 h LD, 10° C but not in 8:16 h LD, 15° C; 16:8 h LD 10° C or 15° C; and 10:6.5:1: 6.5 h LDLD, 10° C. The spores thus formed were viable and produced normal dioecious male and female gametophytes. Short day and low temperature conditions appear necessary for tetrasporogenesis. The results from crossing experiments with 32 male and 27 female isolates of the heteromorphic-type derived from both G. stellata and P. cruenta showed that two virtually non-interbreeding populations with a high degree of geographical separation exist in the north-eastern Atlantic. Morphological differences between plants from each population are described. On the basis of culture and crossing results, Petrocelis cruenta J. Agardh is placed in synonymy with Gigartina stellata (Stackhouse in Withering) Batters.