ULTRASTRUCTURE AND CYTOCHEMISTRY OF EARLY SPERMATANGIAL DEVELOPMENT IN ANTITHAMNION NIPPONICUM (CERAMIACEAE,RHODOPHYTA)1

Spermatial development and differentiation of wall components were investigated by electron microscopy and cytochemical methods in Antithamnion nipponicum Yamada et Inagaki. The spermatium is composed of two parts, a globular head and two appendages projecting from near the basal portion. The appendages originate form spermatangial vesicles (SVs) and follow a developmental sequence beginning as amorphous material and ending as fully formed fibrous structures compressed with in the SVs. SV formation is due to contributions initially from endoplasmic reticulum and later form dictyosome-derived vesicles. Chemical differentiation of the spermatial wall occurs early in its development. Calcofluor white ST does not label spermatial walls, indicating an absence of cellulose polysaccharides, which are abundant in vegetative cell walls. Labeled lectins show that α-d -methyl manose and / or α-d -glucose as well as N-acetyl-glucosamine, β-d -galactose, and α-l -fucose moieties are present on the spermatial wall but not in the vegetative cell wall. The glyconjugate with α-d -methyl mannose and / or glucose residues, previously reported as a gamete recognition molecule in this species, is distributed along the surface of spermatia as well as in the SV during spermatangial development.     

ONTOGENY,HISTOCHEMISTRY AND FINE STRUCTURE OF CELLULAR INCLUSIONS IN VEGETATIVE CELLS OF ANTITHAMNION DEFECTUM (CERAMIACEAE,RHODOPHYTA)1

The ultrastructure and histochemistry of developing and mature cell inclusions in vegetative cells of Antithamnion defectum Kylin were examined. Those studied were chloroplast inclusions, cytoplasmic crystals and spherical bodies within the vacuole. Chloroplasts of mature vegetative cells contain an interthylakoidal, apparently noncrystalline deposit of undetermined chemical identity. The bodies are parallel to the long axis of the plastid, are square (0.13 μm) in cross-section, and up to 3 μm long. Spherical vacuolar bodies (0.5–1.5 μum diam) are formed during early stages of vacuole formation by accumulation of protein deposits in swelling endoplasmic reticulum (ER) cisternae. Swelling of smooth ER contiguous to the ER containing the deposits results in the vacuole enclosing the spherical bodies. In mature cells, vesicles appear to be secreted into the preformed vacuole. Cytoplasmic proteinaceous crystalloids develop without a bounding membrane and may serve as protein reserves.     

FINE STRUCTURE AND HISTOCHEMISTRY OF VESICLE CELLS OF THE RED ALGA ANTITHAMNION DEFECTUM (CERAMIACEAE)1

The ultrastructure and histochemistry of the refractile, vesiculate cells (“blasenzellen,”“cellules secretrices,”“gland cells”) of Antithamnion defectum Kylin were examined. The refringent vacuolar contents disclosed two components of differing density: an electron opaque, proteinaceous matrix material surrounding cores of irregularly shaped, less opaque material. The cores contain less protein and more unknown material than the matrix. Part or all of the vacuolar material is synthesized by abundant rough endoplasmic reticulum (ER) and deposited in smooth surfaced cisternae that swell to form vesicles. Mitochondria are usually associated with stacks of the swelling cisternae. The vesicles enlarge by continued deposition of synthesized material and coalescence with other vesicles. All vesicles eventually coalesce to form the mature vacuole. A crystalline array of fibrils develops in the cytoplasm during later stages of vacuole enlargement. The crystal contains a sulfated, acidic polysaccharidic material. The chloroplasts, if present, and nucleus degenerate at vacuole maturity. Active release of the vacuolar material does not occur, and organelles for extracellular secretion are not present. Structural evidence suggests a storage, rather than secretory, function for the cells.     

A GLYCOPROTEIN NONCOVALENTLY ASSOCIATED WITH CELL‐WALL POLYSACCHARIDE OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell‐wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS‐PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66‐kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N‐linked terminal mannose. In size‐exclusion chromatography, the 66‐kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66‐kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66‐kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66‐kDa glycoprotein, detected in our in‐house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in‐house expressed sequence tag database of the red microalga Rhodella reticulata. The 66‐kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66‐kDa protein in the biosynthesis of the polysaccharide are discussed.     

PURIFICATION OF A SEX‐SPECIFIC LECTIN INVOLVED IN GAMETE BINDING OF AGLAOTHAMNION CALLOPHYLLIDICOLA (RHODOPHYTA)1

Egg and sperm binding and correct recognition is the first stage for successful fertilization. In red algae, spermatial attachment to female trichogynes is mediated by a specific binding between the lectin(s) distributed on the surface of trichogyne and the complementary carbohydrates on the spermatial surface. A female‐specific lectin was isolated from Aglaothamnion callophyllidicola by agarose‐bound fetuin affinity chromatography. Two proteins, 50 and 14 kDa, eluted from the fetuin column were separated using a native‐polyacrylamide gel electrophoresis method and subjected to a gamete binding assay. The 50 kDa protein, which blocked spermatial binding to female trichogynes, was used for further analysis. Internal amino acid sequence of the 50 kDa protein was analyzed using matrix‐assisted laser desorption/ionization‐mass spectrometry and degenerated primers were designed based on the information. A full‐length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR). The cDNA was 1552 bp in length and coded for a protein of 450 amino acids with a deduced molecular mass of 50.7 kDa, which agreed well with the protein data. Real‐time PCR analysis showed that this protein was up‐regulated about 10‐fold in female thalli. As the protein was novel and showed no significant homology to any known proteins, it was designated Rhodobindin.     

PHYSIOLOGICAL ECOTYPES IN THE MARINE ALGA BOSTRYCHIA RADICANS (CERAMIALES,RHODOPHYTA) FROM THE EAST COAST OF THE U.S.A1

The comparative ecophysiology of nine culture isolates of the eulittoral red alga Bostrychia radicans (Montagne) Montague collected at sites from seven states along the east coast of the U.S.A. was investigated. The growth response in relation to different salinity and light conditions as well as photosynthesis-irradiance curves were studied. In addition, the effect of salt treatment on the content of the isomeric polyols d -sorbitol and d -dulcitol was also studied. All isolates grew between salinities of 5.3 and 70 ppt but with quite different optima and maxima. The isolates were all adapted to low light levels, i.e. growth was already recorded at 2.5 μmol photons·m^?2·s^?1, and growth rates peaked between 40 and 60 μmol photons·m^?2·s_-1. These low-light requirements were also reflected by the photosynthesis-irradiance curves: all plants had low light compensation points (2.5–9.7 μmol photons ·m^?2·^?1) and low photon fluence rates for initial saturation of photosynthesis (38.1–84.7 μmol photons·m^?2·s^?1, indicating that these isolates are “shade-adapted.” Isolates from Florida and Georgia synthesized and accumulated both the osmolytes d -sorbitol and d -dulcitol in increasing salinities, whereas only d -sorbitol was present in plants from North Carolina north to Connecticut. d -sorbitol was always strongly involved in osmotic acclimation. In various isolates from the same location in South Carolina, both polyol patterns were found, i.e. d -sorbitol plus d -dulcitol and d -sorbitol only. All data indicate that B. radicans exhibits a broad salinity tolerance and a low-light preference, which explain the successful colonization of this alga on various intertidal and shaded substrates. The data also clearly indicate intraspecific differences among the nine isolates, which is interpreted as development of different physiological ecotypes.     

DIFFERENTIAL LOCALIZATION OF CARRAGEENAN GELLING SEQUENCES IN KAPPAPHYCUS ALVAREZII VAR. TAMBALANG (RHODOPHYTA) WITH FITC-CONJUGATED CARRAGEENAN OLIGOSACCHARIDES1

The ability of kappa (κ) and iota (ι) carrageenans to form gels is dependent upon the regular repeat of disaccharide units along the carbohydrate chain. Short, chemically- and enzymatically-purified fragments of κ and ι carrageenan were conjugated to fluorescein and used as specific hybridization probes for localization of κ and ι carrageenan gelling sequences within the cells walls and intercellular matrices of Kappaphycus alvarezii (Doty) Doty. The probes label cell walls and intercellular matrices under ionic conditions appropriate for gelation of κ and ι carrageenans. The distribution of κ and ι carrageenans in the matrix and cell walls of K. alvarezii was determined with respect to cell type (epidermis, cortex, medulla, and central axis) and age. The κ-probe labels the cell walls of all cell types except epidermal in both young and old tissues. In contrast, the ι-probe labels the cell walls of the epidermis in both young and old tissue and the cell walls of the thylles only in old tissue. Both probes label intercellular matrix material; however, ι-probe labelling is very much weaker than κ-probe labelling. The results indicate that FITC-conjugated carrageenan oligosaccharides are useful tools that provide information on gelling subunit distribution.     

FIELD AND CULTURE STUDIES ON GROWTH AND REPRODUCTION OF CALLITHAMNION BYSSOIDES (RHODOPHYTA,CERAMIALES) IN NORTH CAROLINA1,2

The life history of Callithamnion byssoides Arnott ex Harv. in Hook. from North Carolina is shown to be of the Polysiphonia-type. Cross-gradient light-temperature culture in 8:16 and 14:10 LD cycles was utilized to investigate the effects of light intensity, temperature and day-length on growth and reproduction. Carposporophyte and tetrasporangium development were controlled by total light energy rather than photoperiod. Results of these studies explain the observed seasonal periodicity of growth and reproduction on the North Carolina coast. Variations between these results and previously published data for this species in Texas are explained in terms of ecotypic differentiation.     

SPECIES OF ANTITHAMNION (RHODOPHYCEAE,CERAMIACEAE) OCCURRING ON THE SOUTHEAST AFRICAN COAST (NATAL)1

Three species of Antithamnion Naegeli were previously known from Natal, all reported since 1984. A more thorough collection of the flora in recent months has revealed that there are nine species in Natal, four of which are newly described. Vegetative characteristics are emphasized in this study of the taxonomy of Antithamnion, the most important being (1) size of axial cells, (2) form of whorl-branchlets, (3) size of whorl-branchlet basal cells, (4) size and shape of whorl-branchlet terminal cells, (5) size and position of gland cells, (6) primary or secondary derivation of indeterminate lateral branches, and (7) the relationship of primary indeterminate branches, if present, to normal arrangement of whorl-branchlets. Two closely related species, A. diminuatum Wollaston and A. eliseae sp. nov. have been grown in laboratory culture allowing for testing of stability and reliability of vegetative characteristics for use in taxonomic studies. Conclusions are that these criteria can be effectively used for these purposes. In addition to A. eliseae, A. nematocladellum, A. pterocladellum and A. adenocladellum also are described as new species. A tropical species previously known from the Caribbean and Red Seas, A. Iherminieri Nasr, a species previously recorded from Japan, A. secundum Itono, a species previously known from California and Japan, A hubbsii Dawson, and two species from Australia, A. diminuatum Wollaston and A. divergens (J. Agardh) J. Agardh, are also described from Natal.     

SPECIES OF ANTITHAMNION (RHODOPHYCEAE,CERAMIACEAE) OCCURRING ON THE SOUTHEAST AFRICAN COAST (NATAL)1

Three species of Antithamnion Naegeli were previously known from Natal, all reported since 1984. A more thorough collection of the flora in recent months has revealed that there are nine species in Natal, four of which are newly described. Vegetative characteristics are emphasized in this study of the taxonomy of Antithamnion, the most important being (1) size of axial cells, (2) form of whorl-branchlets, (3) size of whorl-branchlet basal cells, (4) size and shape of whorl-branchlet terminal cells, (5) size and position of gland cells, (6) primary or secondary derivation of indeterminate lateral branches, and (7) the relationship of primary indeterminate branches, if present, to normal arrangement of whorl-branchlets. Two closely related species, A. diminuatum Wollaston and A. eliseae sp. nov. have been grown in laboratory culture allowing for testing of stability and reliability of vegetative characteristics for use in taxonomic studies. Conclusions are that these criteria can be effectively used for these purposes. In addition to A. eliseae, A. nematocladellum, A. pterocladellum and A. adenocladellum also are described as new species. A tropical species previously known from the Caribbean and Red Seas, A. lherminieri Nasr, a species previously recorded from Japan, A. secundum Itono, a species previously known from California and Japan, A hubbsii Dawson, and two species from Australia, A. diminuatum Wollaston and A. divergens (J. Agardh) J. Agardh, are also described from Natal.     

PHYLOGENETIC RELATIONSHIPS AMONG LINEAGES OF THE CERAMIACEAE (CERAMIALES,RHODOPHYTA) BASED ON NUCLEAR SMALL SUBUNIT rDNA SEQUENCE DATA1

Phylogenetic relationships among 69 species of the Ceramiales (51 Ceramiaceae, six Dasyaceae, seven Delesseriaceae, and five Rhodomelaceae) were determined based on nuclear SSU rDNA sequence data. We resolved five strongly supported but divergent lineages among the included Ceramiaceae: (i) the genus Inkyuleea, which weakly joins other orders of the Rhodymeniophycidae rather than the Ceramiales in our analyses; (ii) the tribe Spyridieae, which is sister to the remainder of the included ceramialean taxa; (iii) the subfamily Ceramioideae, weakly including the tribe Warrenieae; (iv) the subfamily Callithamnioideae; and (v) the subfamily Compsothamnioideae, which emerges as sister to the Dasyaceae/Delesseriaceae/Rhodomelaceae complex, thus rendering the Ceramiaceae sensu lato unequivocally paraphyletic, as has been argued separately on anatomical grounds by Kylin and Hommersand. Our data support a restricted concept of the Ceramiaceae that includes only one of the five lineages (Ceramioideae) that we have resolved. In addition to failing to ally with the Ceramiales in our molecular analyses, species of Inkyuleea differ substantially from other Ceramiaceae sensu lato in details of pre‐ and postfertilization development. The genus Inkyuleea is here assigned to the Inkyuleeaceae fam. nov., which we provisionally retain in the Ceramiales. Species of Spyridia also differ from the remaining Ceramiaceae in their postfertilization development, and, in light of our molecular data, the genus Spyridia is assigned to the Spyridiaceae. The Callithamnioideae is strongly monophyletic (100% in all analyses), which, in combination with key anatomical differences, supports elevation to family status for this lineage as the Callithamniaceae. Similarly, the Compsothamnioideae is solidly monophyletic in our molecular trees and has a unique suite of defining anatomical characters that supports family status for a complex that we consider to include the tribes Compsothamnieae, Dasyphileae, Griffithsieae, Monosporeae, Ptiloteae, Spermothamnieae, Sphondylothamnieae, Spongoclonieae, and Wrangelieae, for which the reinstated family name Wrangeliaceae is available.     

INDUCTION OF APOMIXIS BY OUTCROSSING BETWEEN GENETICALLY DIVERGENT ENTITIES OF CALOGLOSSA LEPRIEURII (CERAMIALES,RHODOPHYTA) AND EVIDENCE OF HYBRID APOMICTS IN NATURE1

Our previous study revealed that apomixis, recycling of tetrasporophytes, can be generated through outcrossing between genetically divergent entities of Caloglossa monosticha M. Kamiya, though such apomicts have never been found in nature. In the case of C. leprieurii (Mont.) G. Martens, the most widespread species in this genus, many apomictic strains have been isolated worldwide, but it is unknown whether these apomicts evolved through an outcrossing process similar to that in C. monosticha. In this study, heterogeneity of the apomicts and their sexual relatives as well as their evolutionary relationships was examined using the nuclear‐encoded actin gene and plastid‐encoded RUBISCO spacer region. Thirteen out of 18 apomictic strains were heterogeneous and contained divergent actin alleles, whereas only two out of 23 sexual strains were heterogeneous. The five homogeneous apomicts were genetically identical, or quite similar, to the sexual strains isolated from adjacent sites. Furthermore, three of the five homogeneous apomicts frequently produced tetraspores that grew into gametophytes, while all the heterogeneous apomicts never generated gametophytes. Apomictic strains from Florida were allotriploid, and each of the three actin sequences was closely related to those of sexual strains from Florida, Peru, and Mexico/Guatemala. In crossing tests, obligate apomixis was generated through the outcrossing between the male from Madagascar and the female from the northwestern Atlantic. These results suggest that outcrossing between genetically divergent sexual entities is one factor that induces apomixis in C. leprieurii.     

EFFECTS OF SALINITY ON THE DISTRIBUTION OF CALOGLOSSA LEPRIEURII (RHODOPHYTA) IN THE BRISBANE RIVER,AUSTRALIA1

The role of salinity as a factor determining the distribution of two red algal taxa, Caloglossa leprieurii (Mont.) J. Ag. var. leprieurii and Caloglossa leprieurii var. angusta Jao, along the course of the Brisbane River, Queensland, Australia, was investigated. In the field, C. leprieurii var. angusta tolerated a narrower salinity range (mean salinity = 0.0–18.9) than C. leprieurii var. leprieurii (mean salinity = 2.0–33.8) and occupied areas of lower salinity (salinity expressed according to the Practical Salinity Scale of 1978). Both taxa coexisted for a distance of 23 km along the middle reaches of the river. Cell measurements of specimens of both taxa collected along the river showed an increase in cell sizes upstream from the mouth. Results of a reciprocal transplant experiment and growth responses in a series of laboratory culture studies of the two taxa in a range of salinities are presented. These could be correlated with the field distribution of the algae, demonstrating their euryhalinity and the presence of distinct salinity ecotypes.     

ULTRASTRUCTURE OF EARLY DEVELOPMENT IN THE FEMALE REPRODUCTIVE SYSTEM OF POLYSIPHONIA HARVEYI BAILEY (CERAMIALES,RHODOPHYTA)1

Prefertilization and immediate postfertilization development in the female reproductive branch of Polysiphonia harveyi (Bailey) was studied with the electron microscope. Results pertaining to prefertilization morphology and development are consistent with those established in earlier light microscopic studies, but several unexpected ultrastructural characteristics were discovered. The mature carpogonium was found to have double membrane-bound vacuoles of nuclear origin and the carpogonial nucleus contained a nucleolus with a distinctive crystalline lattice. Trichogynes lacked a nucleus. Of particular interest was the discovery of a highly structured channel of smooth endoplasmic reticulum (SER) which extended uninterrupted, except for pit connections, through the carpogonial branch. It is suggested that the message of fertilization is conducted through the SER channel from the carpogonium to the support cell. Few observations were made on postfertilization branches, but evidence of direct fusion between the carpogonium and auxiliary cell was fairly conclusive. Pit plugs in the female branch were found to be of three morphological types differing by the presence and number of pit membranes. We have designated these plugs Type I–III since differential functioning was not fully ascertained. Our data suggest that pit membranes may serve to stabilize the plug and that those plugs without membranes are more readily dismantled to allow cytoplasmic continuity between cells.     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

LOCAL ADAPTATION OF CERAMIUM TENUICORNE (CERAMIALES,RHODOPHYTA) WITHIN THE BALTIC SEA1

Ceramium tenuicorne (Kützing) Wærn is a red alga that is widely distributed in the brackish Baltic Sea. We studied the growth response of Ceramium to low salinity and nutrient enrichment, using isolades from two regions of the Baltic Sea where the alga approaches its inner distribution limit. Ecotypic differentiation was observed in that differences in growth response among isolates corresponded to salinity conditions in their regions of origin. Isolates from the Gulf of Bothnia (4 psu) had inherently lower growth rates that were not increased when transferred to higher salinity, but were better adapted to very low salinity levels than isolates from the Baltic Proper (7 psu). Further, the results indicate that Ceramium from different regions of the Baltic Sea vary in their responses to nutrient enrichment. The observed differences may be best described as a quantitative difference in the proportion of isolates with hyposaline adaptation. The results indicate that the wide distribution of Ceramium in the Baltic Sea is better explained by the occurrence of locally adapted genotypes than by a generalist life strategy, and provide example of adaptive differentiation in a marine edge environment.     

STRUCTURE AND DEVELOPMENT OF THE CARPOSPOROPHYTE OF NEMALION HELMINTHOIDES (NEMALIONALES,RHODOPHYTA)1

Following fertilization, the carposporophyte of Nemalion helminthoides (Velley in With.) Batters differentiates into four distinct regions: the fusion cell, the sterile gonimoblast cells, the carposporangial mother cells and the carposporangia. The gonimoblast is formed by apically dividing, monopodial filaments of limited growth which may later become pseudodichotomous. Upon differentiation of a terminal carposporangium, a gonimoblast filament may continue to grow sympodially. A single carposporangial mother cell may produce carposporangia in several different directions as well as proliferate successive carposporangia within the sporangial walls that remain after carpospore liberation. As the carposporophyte matures, the gonimoblast initial, the stalk cell, the hypogynous and subhypogynous cells fuse. Except for the fusion cell, all cells of the carposporophyte show organelle polarity and contain a distally located, lobed chloroplast and proximal nucleus.