Structure of reproductive apparatus of <Emphasis Type="Italic">Gracilaria/Gracilariopsis lemaneiformis</Emphasis> (Gracilariaceae,Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial development revealed the similar cytodynamic pattern followed by all the Gracilariaceae members developed from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.     

Structure of reproductive apparatus of Gracilaria/ Gracilariopsis lemaneiformis (Gracilariaceae, Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial development revealed the similar cytodynamic pattern followed by all the Gracilariaceae members developed from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released. Supported by the National Natural Sciences Foundation of China (Grant No. 40606034) and the National High-Tech Research and Development Program of China (Grant No. 2006AA10A413)     

COMPARATIVE MORPHOLOGY AND TAXONOMIC STATUS OF GRACILARIOPSIS (GRACILARIALES,RHODOPHYTA)1

The vegetative organization and reproductive development of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Foldvik [including Gracilaria sjoestedtii Kylin] were investigated. Our observations on spermatangial development and post-fertilization features establish that Gracilariopsis Dawson is distinct at the generic level from Gracilaria Greville, and ice propose the resurrection of Gracilariopsis Dawson as a result. Spermatangial parent cells of Gracilariopsis are superficial, initiated in pairs or groups of three by concavo-convex longitudinal and transverse divisions. Each spermatangial parent cell cuts off a single, colorless spermatangium distally by a transverse division. The female reproductive apparatus consists of a supporting cell that bears a two-celled carpogonial branch flanked by two sterile branches, as in Gracilaria. Likewise, up to six sterile cells fuse with the carpogonium after fertilization to produce a primary fusion cell that generates the gonimoblasts; however, a secondary fusion cell is absent. Inner gonimoblast cells unite with cytologically modified cells of the inner pericarp by means of secondary pit-connections. Tubular nutritive cells are absent. The gonimoblast consists of a central sterile tissue interconnected throughout by secondary pit-connections surmounted by a fertile layer composed of carposporangia aligned in straight chains. The distribution of Gracilariopsis is extended to Western Europe.     

STUDIES ON DEVELOPING AND RELEASED SPERMATIA IN THE RED ALGA,TIFFANIELLA SNYDERAE (RHODOPHYTA)1

Developing and released spermatia of the red alga, Tiffaniella snyderae (Farl.) Abb. were studied. Spermatia were observed under hydrodynamically defined conditions and found to be released from the exposed spermatangial heads in a spermatium-plus-strand unit that remained connected to the spermatangial head. Interactions of single-spermatial strands resulted in the formation of multi-spermatial strands as long as 600 μm with as many as 47 spermatia along their length; however, most were 100–200 μm with 8–21 spermatia. Strand length and number of spermatia were correlated. Spermatial strands contracted or extended and rotated as the water velocity past the plant was changed, and in still water the strands retracted into a clump on the spermatangial head surface. Each strand type exhibited a characteristic threshold water velocity at which it reached maximum length, and above which it broke and was carried away. Fluorescence microscopy showed that the strands did not contain nucleic acid (DNA) and could thus be differentiated from filamentous blue-green algal and bacterial epiphytes. Histochemical staining indicated that the strands and spermatial vesicles contained an acidic, sulfated polysaccharide. Chelation of Ca²⁺ with EGTA resulted in strand breakdown suggesting that this divalent cation may be involved in strand integrity. Scanning electron microscopy revealed that release from the spermatangia occurred through tears in the cuticle covering the spermatangial head if it was still present, or from exposed spermatangia. Individual spermatia were attached tangentially to a well-defined strand 0.64 μm in diameter in the contracted state to 0.2 μm in the extended state. Transmission electron microscopy of spermatangial heads showed that immature spermatangia were characterized by a centrally positioned nucleus and abundant ER cisternae filled with a moderately electron dense granular material. Later in development the spermatangia acquire two spermatial vesicles containing highly convoluted fibrillar contents. The cell becomes polarized with the nucleus displaced apically and the spermatial vesicles occupying the basal half of the spermatangium. At maturity one of the vesicles is released basally. Liberated spermatia contain a membrane-bound nucleus and mitochondria and are associated with an oblong accumulation of fibrous material similar in size and position to the strand observed with the SEM. These strands are discussed in relation to red algal fertilization and other phases of the red algal life-history.     

Differentiation of Male Gametes in Gracilaria verrucosa (Rhodophyta, Gracilariales)

The development of male gametes (spermacia) in the red alga Gracilaria verrucosa has been studied using methods of transmission electron microscopy. Early spermatangia located along the wall of the conceptacle show an elongated shape in the thin sections. In the central part of the electron-dense cytoplasm of these cells there is a nucleus; numerous fibrous vesicles are arranged in the periphery. During the process of differentiation, the spermatangia become more rounded in shape and a large spermatangial vesicle is developed. The subsequent development of spermatium is accompanied by polarization of the spermatangium and the subsequent excretion of the spermatangial vesicle. The spermatia are oval cells containing a nucleus and fibrous vesicles. The process of differentiation of male gametes in G. verrucosa does not differ from that in five species of the genus Gracilaria, where it has already been studied. However, any conclusions about the degree of similarity between the spermatia in all the studied species can be made only after a detailed comparative analysis of the ultrastructural characteristics of these gametes.     

Spermatangium formation and sperm discharge in the Japanese pygmy squid Idiosepius paradoxus

In cephalopods, sperm discharge is an important event not only for sperm transfer but also influencing sperm storage capacity of attached spermatangia (everted spermatophores). To investigate sperm discharge from spermatangia and the condition of naturally attached spermatangia in Japanese pygmy squid (Idiosepius paradoxus) we (i) investigated the morphology of spermatophores and spermatangia, and the process of spermatophore evagination and sperm discharge from spermatangia obtained in vitro; (ii) observed spermatangia that were naturally attached to female squids at 6, 12, 18, 24 and 48 h after copulation to investigate alterations in naturally attached spermatangia with time. The spermatophore of I. paradoxus is slender and cylindrical and consists of a sperm mass, a cement body and an ejaculatory apparatus, which is similar to those of loliginid squids. The spermatangium is fishhook-shaped, its distal end being open and narrow. After the spermatangium is formed, the sperm mass gradually moves to the open end of the spermatangium, from where sperm are released. Sperm discharge is a rapid process immediately after the beginning of sperm release, but within 5 min changes to an intermittent release of sperm. Although the volume of residual spermatozoa differed among spermatangia that were naturally attached to a single individual, the probability that spermatangia would be empty increased with time. Most naturally attached spermatangia discharged almost all of their spermatozoa within 24 h after copulation, and no spermatangia were attached to females 48 h after copulation. These results suggest that sperm transfer from the spermatangium to the seminal receptacle must occur within 24 h, and that the spermatangium functions as a transient sperm storage organ in pygmy squids.     

Two species of Dasya (Ceramiales, Rhodophyta) from Bonin Islands, southern Japan, with the description of Dasya boninensis sp. nov.

Two species of Dasya in the Dasyaceae (Ceramiales, Rhodophyta) are reported from Bonin Islands, southern Japan. Dasya murrayana Abbott et Millar, new to Japan, is characterized by the following set of features: the tufted habit (up to 30 erect axes developing from a basal disc), small‐sized (6–10 mm high and 350–500 μm in diameter in the middle region), thinly but completely corticated axes, rigid and incurved pseudolaterals forming corymbose heads at the apices of axes and branches, the absence of adventitious monosiphonous filaments, a large number of tetrasporangial stichidia and spermatangial branches per fertile pseudolateral and slender spermatangial branches (35–45 μm in diameter). Dasya boninensis Masuda, Kurihara et Kogame, sp. nov. is characterized by short but thick (10–30 mm high and 600–1000 μm in diameter at the middle portion), heavily corticated axes, indistinct pericentral cells except for the upper portion in transverse sections, soft, straight pseudolaterals and adventitious monosiphonous filaments densely covering the axis and branches, a small number of tetrasporangial stichidia and spermatangial branches per fertile pseudolateral, thick spermatangial branches (65–90 μm in diameter), and short‐necked cystocarps. A dichotomous key to the taxa found in Japanese waters is given.     

PHYLOGENY OF ATLANTIC SPECIES OF GRACILARIACEAE (RHODOPHYTA) BASED ON SMALL SUBUNIT RIBOSOMAL GENE SEQUENCING

The complete small subunit ribosomal nuclear gene (ssu rDNA) sequence was determined for nine species of Gracilaria and one species of Gracilariopsis which are common on the American Atlantic waters. The sequences were aligned using the secondary structure as reference, including the published sequences of nine other species of Gracilariaceae. A matrix of 1736 sites was constructed with a proportion of 91% invariable sites and very few assumed indels events. All the inferred trees show three main lineages: 1) the strongly divergent lineages of Gracilariopsis; 2) the austral genera Curdiea / Melanthalia; and 3) the lineage of Gracilaria sensu stricto. The later encompasses the following groups: 1) Gracilaria chilensis from the Pacific ocean; 2) a group of cylindrical tropical species with "henriquesiana" spermatangial type; 3) a group of warm temperate cylindrical species with "verrucosa" spermatangial type; and 4) a group of flattened tropical species with mainly "textorii" spermatangial type. The relationship of a species described as Gracilaria pauciramosa from Venezuela was not inequivocally solved. The inferred phylogenetic groups are congruent with morphology and quality of agar.     

Reproductive morphology and taxonomic reappraisal of Exophyllum wentii Weber-van Bosse (Rhodomelaceae, Rhodophyta) from Bali Island, Indonesia

The little‐known and rarely collected alga Exophyllum wentii Weber‐van Bosse is re‐described in detail from the type material, as well as from new collections from Indonesia, which for the first time reveal in detail the structure of cystocarpic and spermatangial plants and the development of tetrasporangial stichidia under culture conditions. New morphological reproductive information confirms placement of the genus Exophyllum within the Rhodomelaceae. Exophyllum is distinguished from other related genera within the Rhodomelaceae by its cartilaginous, non‐trichoblastic decumbent thallus with multiple holdfasts and its discoid spermatangial organs. Some Pacific material earlier attributed to E. wentii was found to be misidentified and re‐assigned to the Dasyaceae.     

布氏轮藻(Chara braunii)生殖器官环境扫描电镜的观察

本文采用环境扫描电镜和光学显微镜,对布氏轮藻(Chara braunii)的藏卵器和藏精器进行了仔细观察。结果表明：藏卵器外的管细胞(包围细胞或螺旋细胞)在卵细胞受精以后,不但没有钙的沉淀、加厚,而且还呈现出逐渐萎缩,最终消失。因此,在古轮藻的研究上,常提到的包围细胞或螺旋细胞,实际上并非是细胞结构,而是受精卵的外壁。     

ELECTRON MICROSCOPIC OBSERVATIONS ON THE DIFFERENTIATION AND RELEASE OF SPERMATIA IN THE MARINE RED ALGA POLYSIPHONIA HENDRYI (CERAMIALES,RHODOMELACEAE)

Spermatial differentiation in Polysiphonia hendryi begins after nonpolar, avacuolate spermatia are cleaved from their mother cells. The spermatia and their mother cells are embedded within the spermatangium, a confluent wall matrix of the male branch. As the young spermatium enlarges and becomes ellipsoid, the wall fibrils of the spermatangium are compressed, forming a separating layer. Spermatia become polar with rough endoplasmic reticulum coalescing to form a large, fibrillar spermatial vacuole that becomes extracytoplasmic in later development. Following spermatial vacuole formation, dictyosomes form and deposit a spermatial wall, severing the spermatial mother-cell pit connection. Enlargement of younger spermatia, which are lateral to the older ones, squeezes the maturing spermatia, pushing them from the male branch, and leaving a scar that compresses and heals. Through this release mechanism, new sites are created for additional spermatial proliferation.     

PROPOSAL OF THE GRACILARIALES ORD. NOV. (RHODOPHYTA) BASED ON AN ANALYSIS OF THE REPRODUCTIVE DEVELOPMENT OF GRACILARIA VERRUCOSA1

The mode of division of vegetative cells, formation of spermatangial parent cells, initiation of the carpogonial branch apparatus, and formation of tetrasporangial initials are homologous developmental processes that are documented for the first time in the type species of the economically important family Gracilariaceae, Gracilaria verrucosa (Hudson) Papenfuss from the British Isles. G. verrucosa is characterized by a supporting cell of intercalary origin that bears a 2-celled carpogonial branch flanked by two sterile branches, direct fusion of cells of sterile branches onto the carpogonium, formation of an extensive carpogonial fusion cell through the incorporation of additional gametophytic cells prior to gonimoblast initiation, gonimoblast initials produced from fusion cell lobes, schizogenous development of the cytocarp cavity, inner gonimoblast cells producing tubular nutritive cells that fuse with cells of the pericarp or floor of the cystocarp, absence of cytologically modified tissue in the floor of the cystocarp, and carposporangial initials produced in clusters or irregular chains. Spermatangial parent cells are generated in flaments from intercalary cortical cells that line an intercellular space forming a ‘pit’ or ‘conceptacle’. Tetrasporangial initials are transformed from terminal cells derived through division of an outer cortical cell. Tetrasporangia are cruciately divided. The Gracilariaceae is removed from Gigartinales and transferred to the new order Gracilariales. Their closest living relatives appear to be agarophytes belonging to the Gelidiales and Ahnfeltiales.     

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.     

Diagnoses and key to the genera of the Gracilariaceae (Gracilariales,Rhodophyta)

A key to the genera of the Gracilariaceae is provided along with a short diagnosis for each genus. Features of the mature cystocarp and spermatangial configurations that separate genera are illustrated.     

Spermatophoric reaction reappraised: Novel insights into the functioning of the loliginid spermatophore based on Doryteuthis plei (Mollusca: Cephalopoda)

During copulation, spermatophores produced by male coleoid cephalopods undergo the spermatophoric reaction, a complex process of evagination that culminates in the attachment of the spermatangium (everted spermatophore containing the sperm mass) on the female's body. To better understand this complicated phenomenon, the present study investigated the functional morphology of the spermatophore of the squid Doryteuthis plei applying in vitro analysis of the reaction, as well as light and electron microscopy investigation of spermatangia obtained either in vitro, or naturally attached on females. Hitherto unnoticed functional features of the loliginid spermatophore require a reappraisal of some important processes involved in the spermatophoric reaction. The most striking findings concern the attachment mechanism, which is not carried out solely by cement adhesive material, as previously believed, but rather by an autonomous, complex process performed by multiple structures during the spermatophoric reaction. During evagination, the ejaculatory apparatus provides anchorage on the targeted tissue, presumably due to the minute stellate particles present in the exposed spiral filament. Consequently, the ejaculatory apparatus maintains the attachment of the tip of the evaginating spermatophore until the cement body is extruded. Subsequently, the cement body passes through a complex structural rearrangement, which leads to the injection of both its viscid contents and pointed oral region onto the targeted tissue. The inner membrane at the oral region of the cement body contains numerous stellate particles attached at its inner side; eversion of this membrane exposes these sharp structures, which presumably adhere to the tissue and augment attachment. Several naturally attached spermatangia were found with their bases implanted at the deposition sites, and the possible mechanisms of perforation are discussed based on present evidence. The function of the complex squid spermatophore and its spermatophoric reaction is revisited in light of these findings. J. Morphol. 2012. © 2011 Wiley Periodicals, Inc.     

New records of Gelidiella pannosa, Pterocladiella caerulescens and Pterocladiella caloglossoides (Rhodophyta, Gelidiales) from Japan

Three gelidialean species, Gelidiella pannosa (Feld‐mann) Feldmann et Hamel, Pterocladiella caerulescens (Kutzing) Santelices et Hommersand and Pterocladiella caloglossoides (Howe) Santelices, are newly reported from Japan, and their characteristic features are described. Monoecious plants of P. caerulescens produce spermatangial sori on: (i) fertile cystocarpic branchlets; (ii) special spermatangial branchlets on a cystocarpic axis; and (iii) branchlets of a special spermatangial axis. The latter two are newly reported in this species. Gelidiella pannosa has numerous unicellular independent points of attachment, whereas P. caerulescens and P. caloglossoides have the peg type of secondary rhizoidal anchorage. In the molecular phylogenetic study using small subunit ribosomal DNA sequences, G. pannosa is included in the Gelidiella clade with 100% bootstrap support in neighbor‐joining (NJ) analysis and 99% in maximum parsimony (MP) analysis. Pterocladiella caerulescens and P. caloglossoides are included in the Pterocladiella clade with 99.7% bootstrap support in NJ analysis and 100% in MP analysis. Each type of secondary rhizoidal attachment is completely consistent with the respective genus clade, which suggests that this morphological characteristic reflects phylogeny within the order Gelidiales.     

Taxonomic notes on three species of Herposiphonia (Ceramiales, Rhodophyta) found in Japan

Three species of the red algal genus Herposiphonia (Ceramiales, Rhodomelaceae) found in Japan are described, and taxonomic features of the genus are discussed. Herposiphonia crassa Hollenberg is reported from Japan for the first time and is characterized by thick axes (200–350 µm in diameter) and determinate branches (100–200 µm in diameter), relatively short determinate laterals (400–1200 µm in length) with a large number of periaxial cells (15–19 per segment) and three (occasionally two or four) vigorously developed (1.8–2.5 mm in length by 50–75 µm in diameter basally) trichoblasts on each determinate lateral. Herposiphonia elongata Masuda et Kogame is also reported from Japan for the first time and is characterized by the conspicuous thickening growth of cystocarp‐bearing branches and spermatangial branches with an elongated sterile tip. Some newly found features of Herposiphonia fissidentoides (Holmes) Okamura are presented: the rhizoid production from the central portion of parental periaxial cells in addition to the distal end, virtual absence of vegetative trichoblasts, production of procarpial trichoblasts and spermatangial branches on fertile determinate branches on short indeterminate laterals, cystocarps sometimes with a short spur, and extremely large tetrasporangia.     

TAXONOMY AND PHYLOGENY OF OSMUNDEA (RHODOMELACEAE, RHODOPHYTA) IN ATLANTIC EUROPE

Two species of Osmundea Stackhouse (Rhodomelaceae, Rhodophyta) that occur in Atlantic Europe have been confused under the names Osmundea ramosissima (Oeder) Athanasiadis and Osmundea truncata (Kützing) Nam et Maggs, regarded until now as a synonym of O. ramosissima. An epitype from its type locality (Stavanger, Norway) is selected for Osmundea ramosissima Athanasiadis, recognized here as a valid name for Fucus ramosissimus Oeder, nom. illeg. Details of vegetative and reproductive morphology of O. ramosissima are reported, based on material from France, the British Isles, and Helgoland. Osmundea ramosissima resembles other species of Osmundea in its vegetative axial segments with two pericentral cells and one trichoblast, spermatangial development from apical and epidermal cells (filament type), the formation of five pericentral cells in the procarp‐bearing segment of the female trichoblast, and tetrasporangial production from random epidermal cells. Among the species of Osmundea, O. ramosissima is most similar to O. truncata. Both species have discoid holdfasts, secondary pit connections between epidermal cells, and cup‐shaped spermatangial pits. They differ in that: (a) O. ramosissima lacks lenticular wall thickenings and refractive needle‐like inclusions in medullary cells, both of which are present in O. truncata; (b) O. ramosissima has branched spermatangial filaments that terminate in a cluster of several cells, whereas in O. truncata the unbranched spermatangial filaments have a single large terminal sterile cell; and (c) cystocarps of O. ramosissima lack protuberant ostioles but ostioles are remarkably protuberant in O. truncata. Phylogenetic analyses of rbcL sequences of Laurencia obtusa (Hudson) Lamouroux and all five Atlantic European species of Osmundea, including the type species, strongly support the generic status of Osmundea. Osmundea ramosissima and O. truncata are closely related (5.2% sequence divergence) and form a well‐supported clade sister to a clade consisting of O. pinnatifida (Hudson) Stackhouse, O. osmunda Stackhouse and O. hybrida (A. P. de Candolle) Nam. The formation of secondary pit connections between epidermal cells is a synapomorphy for the O. ramosissima+O. truncata clade. The close relationship between species with cup‐shaped spermatangial pits (Osmundea hybrida) and urn‐shaped pits (Osmundea pinnatifida and Osmundea osmunda) shows that spermatangial pit shape is not an important phylogenetic character. Parsimony analysis of a morphological data set also supports the genus Osmundea but conflicts with the molecular trees in infrageneric relationships, placing O. hybrida basal within the Osmundea clade and grouping O. osmunda and O. pinnatifida but not O. truncata and O. ramosissima. A key to Osmundea species is presented.     

ULTRASTRUCTURE OF SPERMATIUM LIBERATION IN THE MARINE RED ALGA PTILOTA DENSA1

The differentiation of male gametes of the marine red alga Ptilota densa was studied by electron microscopy. Mature primary spermatangia are enveloped by a single cell wall and possess a clearly polar subcellular organization. The nucleus is situated apical to large, striated, fibrous vacuoles which are apparently formed by the repeated fusion of dictyosome vesicles. The transformation and liberation of spermatia from spermatangia involve both the secretion of the fibrous vacuoles at the base of the cell and the subsequent rupturing of the spermatangial cell wall. Liberated spermatia are coated with a thin mucilage layer and contain numerous small vesicles and several mitochondria and dictyosomes. The nucleus is cup-shaped and generally lacks a limiting envelope. These findings are discussed in relation to other light and electron microscopic studies of differentiating spermatangia in red algae.