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.     

Morphological and photosynthetic variations in the process of spermatia formation from vegetative cells in <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Ueda (Bangiales,Rhodophyta) and their responses to desiccation

Porphyra yezoensis has a macroscopic foliage gametophyte phase with only a single cell layer, and is ideally suited for the study of the sexual differentiation process, from the vegetative cell to the spermatia. Firstly, we compared variations in the responses of the vegetative and male sectors to desiccation. Later, cell tracking experiments were carried out during the formation of spermatia from vegetative cells. The two sectors showed similar tolerance to desiccation, and the formation of spermatia from vegetative cells was independent of the degree of desiccation. Both light and scanning electron microscopy (SEM) observations of the differentiation process showed that the formation of spermatia could be divided into six phases: the one-cell, two-cell, four-cell, eight-cell, pre-release and spermatia phases. Photomicrographs of Fluorescent Brightener staining showed that the released spermatia had no cell walls. Photosynthetic data showed that there was a significant rise in Y(II) in the four-cell phase, indicating an increase in photosynthetic efficiency of PSII during this phase. We propose that this photosynthetic rise may be substantial and provide the increased energy needed for the formation and release of spermatia in P. yezoensis.     

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.     

Gamete recognition during fertilization in a red alga,Antithamnion nipponicum

Summary Fertilization in the marine red algaAntithamnion nipponicum is a highly specific process involving non-motile male gametes, spermatia, and female receptive structures, carpogonia. FITC-lectin and Calcofluor white ST labelling show that the outer cell walls of spermatia differ from vegetative cells in carbohydrate composition. Specific binding of the lectins to spermatial walls was confirmed by lectin-gold labelling on thin sections. Gametic recognition inAntithamnion nipponicum is based on the interaction of a surface carbohydrate on the spermatia with a surface carbohydrate receptor on the trichogynes. Spermatial binding to trichogynes is inhibited by pre-incubation with concanavalin A and trichogyne receptors are blocked by the complementary carbohydrate -D-methyl mannose. The inhibitory effects of concanavalin A to spermatial binding of trichogynes is reversed by preincubation with -D-methyl mannose. The combination of long spermatial appendages and a carbohydrate-carbohydrate receptor-based gamete recognition mechanism make fertilization in this species an efficient process.     

ULTRASTRUCTURE OF SPERMATIAL DEVELOPMENT IN THE PARASITIC RED ALGAE LEVRINGIELLA GARDNERI AND ERYTHROCYSTIS SACCATA12

Morphologically, the development of spermatia in Levringiella gardneri and Erythrocystis saccata is identical, although cytologically several differences are evident. Mature spermatia contain 1 or 2 large spermatial vesicles that contain fibrous material, several small mitochondria, some proplastids, and are surrounded by a wall, either single-layered as in Erythrocystis or triple-layered as in Levringiella. Spermatial vesicles are formed by aggregations of endoplasmic reticulum in Levringiella, whereas concentric membrane bodies and dictyosomes may be involved in Erythrocystis. In addition to being fibrillar, the contents of the vesicle assume a convoluted appearance in Levringiella. Several spermatia are formed per mother cell and are connected by small pit connections which rupture to allow spermatial release from the spermatangial branch.     

Fertilization and the cytoskeleton in the red alga Bostrychia moritziana (Rhodomelaceae,Rhodophyta)

Time-lapse videomicroscopy was used to observe the effects of various cytoskeletal inhibitors on three important fertilization events in Bostrychia moritziana: spermatial mitosis, gamete fusion (formation of a fertilization pore) and nuclear migration along the trichogyne. The microtubule inhibitor oryzalin disrupted spermatial mitosis but had no other effect on fertilization. The actin inhibitors, jasplakinolide, cytochalasin B, latrunculin A and B and mycalolide B inhibited gamete fusion while BDM, a myosin-disrupting drug, inhibited all three major fertilization events. FL-Phallacidin was used to stain actin filaments in spermatia and trichogynes while microtubules were labelled with antibodies at appropriate stages of fertilization. Microtubules were only evident during spermatial nuclear division. Actin filaments were present in both trichogynes and spermatia throughout fertilization; they formed a discrete ring around the fertilization pore and ensheathed male nuclei as the latter migrated into and along the trichogyne. These results suggest that the actin/myosin system plays a role in the events of fertilization.     

SPECIALIZED APPENDAGES ON SPERMATIA FROM THE RED ALGA AGLAOTHAMNION NEGLECTUM (CERAMIALES,CERAMIACEAE) SPECIFICALLY BIND WITH TRICHOGYNES1

Spermatia from Aglaothamnion neglectum Feldmann-Mazoyer specifically bind with trichogynes and hairs of female thalli, One of the functions of hairs on female thalli appears to be the catching of spermatia. Fertilization can occur if a spermatium binds first with a hair and then binds with a trichogyne. The binding of spermatia with trichogynes is not species specific, but only occurs beween closely related species. Spermatia have fimbriate coneshaped appendages projecting from each end that are responsible for initial binding with trichogynes.     

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.     

ATTACHMENT AND FUSION OF GAMETES DURING FERTILIZATION OF PALMARIA SP. (RHODOPHYTA)1

Fertilization of cultured microscopic female gametophytes by spermatia from field-collected male gametophytes of Palmaria sp. was observed by light and transmission electron microscopy. Liberated spermatia had a prophase-arrested nucleus with a pair of polar rings. The protoplast of spermatia was covered with ca. a 3-μm-thick hyaline covering. After spermatium inoculation, the spermatial covering was attached specifically to the coat surrounding the cell wall of the trichogyne. The spermatial covering was eliminated only at the site of gamete attachment, resulting in direct attachment of the spermatial plasma membrane to the trichogyne within 5 min after spermatium inoculation. This direct attachment was followed by completion of spermatial nuclear division and cell wall formation. The polar rings disappeared before prometaphase. The cytoplasm of the binucleate spermatium invaded the trichogyne cell wall and subsequently fused with the trichogyne cytoplasm. The trichogyne could fuse with many spermatia, and many male nuclei (the derivative nuclei of spermatial nuclear division) could enter the trichogyne cytoplasm.     

PROGRESSION OF SPERMATIAL NUCLEAR DIVISION REQUIRES CALCIUM INFLUX DURING FERTILIZATION OF THE RED ALGA PALMARIA SP.1

During fertilization of the red alga Palmaria sp. (Palmariales), the prophase-arrested nucleus of the uninucleate spermatium resumes its division after direct attachment of the spermatium to the trichogyne of a carpogonium. Treatments that reduce Ca²⁺ influx inhibit the progression of spermatial nuclear division. The ratio of the number of spermatia released from prophase arrest (those in meta-phase to binucleate stages) to the total spermatia attached to trichogynes was significantly reduced by incubating the spermatia in the culture solution having low Ca²⁺ concentration. Similar inhibition was observed by addition of either inorganic (La³⁺ and Co²⁺) or organic (nifedipine and tetramethrin) Ca²⁺ channel inhibitors to the culture solution containing 10 mM Ca²⁺. These results indicate that the prophase/metaphase transition of spermatial nuclear division requires an influx of Ca²⁺ and suggest that Ca²⁺ acts as a second messenger to the mechanical or chemical stimulus that initiates mitotic progression of spermatia in this alga.     

High diversity and widespread occurrence of mitotic spore mats in ectomycorrhizal Pezizales

Fungal mitospores may function as dispersal units and/ or spermatia and thus play a role in distribution and/or mating of species that produce them. Mitospore production in ectomycorrhizal (EcM) Pezizales is rarely reported, but here we document mitospore production by a high diversity of EcM Pezizales on three continents, in both hemispheres. We sequenced the internal transcribed spacer (ITS) and partial large subunit (LSU) nuclear rDNA from 292 spore mats (visible mitospore clumps) collected in Argentina, Chile, China, Mexico and the USA between 2009 and 2012. We collated spore mat ITS sequences with 105 fruit body and 47 EcM root sequences to generate operational taxonomic units (OTUs). Phylogenetic inferences were made through analyses of both molecular data sets. A total of 48 OTUs from spore mats represented six independent EcM Pezizales lineages and included truffles and cup fungi. Three clades of seven OTUs have no known meiospore stage. Mitospores failed to germinate on sterile media, or form ectomycorrhizas on Quercus, Pinus and Populus seedlings, consistent with a hypothesized role of spermatia. The broad geographic range, high frequency and phylogenetic diversity of spore mats produced by EcM Pezizales suggests that a mitospore stage is important for many species in this group in terms of mating, reproduction and/or dispersal.     

Ultrastructural changes in major organelles during spermatial differentiation inBangia (Rhodophyta)

Summary The major organelles within the cells of maleBangia atropurpurea (Roth) C. Ag. filaments undergo a series of ultrastructural transformations during the production of spermatia. Initially, thylakoids within the large axial chloroplast develop a reticulate pattern commencing at the central pyrenoid region. Subsequent changes involve loss of lobes and diminution of volume through division; chloroplasts in final stages contain a few dilated, distorted thylakoids and many plastoglobuli. During differentiation the large nucleolus disappears from the nucleus and four masses of chromatin aggregate near the nuclear envelope. Furrows originating from the nuclear envelope form double membranes around each of the chromatin masses and most of the nucleoplasm is eliminated. Several types of fibrillar vesicles are formed during the process and large floridean starch reserves are utilized. Multilamellar bodies and microbody-like structures occur within the cells during certain phases of spermatiogenesis.     

A test of host specialization by insect vectors as a mechanism for reproductive isolation among entomophilous fungal species

Epichloë species are self incompatible (heterothallic) fungi that must be fertilized by spermatia from individuals of opposite mating type for successful sexual reproduction to occur. Female flies of the genus Botanophila act as vectors of the fungi by ingesting and defecating spermatia (gametes) onto fungal stromata (fruiting bodies) after oviposition. Larvae feed and develop on the stromata and thus maintain a symbiotic relationship with Epichloë fungi. We hypothesized that sole dependence on fertilized stromata as a food source would promote specialization by flies to single compatible host species and that this specialization would promote reproductive isolation among Epichloë species. Analysis of progeny of ascospores from experimental field plots in Zurich, Switzerland, indicated prevalence of specific matings between stromata of the same host, and thus was consistent with the hypothesis that flies are species-specific in their visitation behaviour. Genetic analyses of spermatia contained in the faeces of individual flies also gave some support for this hypothesis. We recovered spermatia of 4 different Epichloë species from fly faeces. Comparison of spermatia found in fly faeces to those available from stromata showed flies avoided Epichloë clarkii and may have preferred Epichloë typhina . Interestingly, these are the only two Epichloë species known to be interfertile with one another. Individual flies tended to carry spermatia predominantly from one fungal species. Thus, flies may adopt a type of "majoring" and "minoring" behaviour when visiting fungi. Yet, Botanophila flies are not monolectic and often visited all hosts that were available within screened cages. In addition to any reproductive isolation flies may provide to some fungal species, differences in competitiveness among spermatia of different species deposited on the same stroma may favor intraspecific matings.     

Three ascomycetes on leaves of evergreen Ilex trees from Japan: Rhytisma ilicis-integrae sp. nov., R. ilicis-latifoliae, and R. ilicis-pedunculosae sp. nov.

Three species belonging to the genus Rhytisma causing tar spot were collected on leaves in evergreen trees of Ilex species from Japan. Rhytisma ilicis-latifoliae, the known species, is found on Ilex latifolia, and R. ilicis-integrae sp. nov. and R. ilicis-pedunculosae sp. nov. are found on I. integra and I. pedunculosa, respectively. Ascomata are formed on the abaxial part of the stromata in all the Rhytisma species studied, and spermogonia are formed on the amphigenous parts in R. ilicis-latifoliae and on the adaxial part in R. ilicis-integrae and R. ilicis-pedunculosae. Shape and size of asci, ascospores, and spermatia are distinctly different among the three species. The morphology of germination tubes from ascospores and appressoria is unique for each Rhytisma species. Yellowish spots arise on the newly developing leaves in mid-May, then abundant spermatia are produced in spermogonia in the three Rhytisma species. In the next year, ascospores are produced in ascomata from early April to late May in R. ilicis-integrae and from early April to early June in R. ilicis-latifoliae and R. ilicis-pedunculosae, and they are considered to be the inocula of disease infection.     

THE ROLE OF F-ACTIN DURING FERTILIZATION IN THE RED ALGA AGLAOTHAMNION OOSUMIENSE (RHODOPHYTA)

The actin cytoskeletons in spermatia and trichogynes of Aglaothamnion oosumiense Itono were studied using fluorescein isothiocyanate (FITC) conjugated phalloidin and the cytoskeletal inhibitors, potassium iodide (KI), cytochalasin-B, and latrunculin-A. Microfilaments were localized to the distal ends of elongated spermatia and trichogynes and were more prominent in the trichogyne before spermatium binding. The actin cytoskeleton in spermatia and trichogynes was disrupted by treatment with 0.6 M KI, 100 μM cytochalasin-B, or 10 μM latrunculin-A. The actin cytoskeleton in trichogynes recovered within 24 h of removal from the inhibitor, but no recovery was observed in spermatia. Spermatial nuclei entered mitosis as soon as spermatia attached to the trichogyne. The greatest percentage (50%– 60%) of spermatia having completed mitosis was obtained at 60 min after spermatial binding to trichogynes. During mitosis, actin accumulated in the center of the spermatium, thereby separating the two daughter nuclei. Cytoskeletal inhibitors did not affect initial binding of spermatia to trichogynes but did block subsequent stages of fertilization, including spermatial mitosis and gamete fusion. The accumulation of cellulose or β-linked polysaccharide on the spermatial surface was also blocked by treatment with actin inhibitors. Exposure of the trichogyne to actin inhibitors after gamete fusion caused spermatial nuclei in trichogynes to stop moving and to condense. These results suggest that the microfilaments involved in nuclear division, cellulose deposition into the spermatial wall, gamete fusion, and migration of spermatial nuclei in trichogynes during fertilization in Aglaothamnion oosumiense.     

Isolation of Pheromone Precursor Genes of Magnaporthe grisea

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.     

AN ULTRASTRUCTURAL STUDY OF SPERMATIUM FORMATION IN THE RUST FUNGUS GYMNOSPORANGIUM JUNIPERI-VIRGINIANAE

Spermatium formation in G. juniperi-virginianae is phialidic. The spermatia are blown out of the tips of spermatiophores which possess a thickened neck region and a distinct, flared collarette. Each spermatium initial is surrounded by a thin wall which is attached to the inner surface of the spermatiophore wall just below the thickened neck region. A spermatium is delimited by a centripetally developing septum and then pushed into the spermogonial cavity by the next spermatium initial. Mature spermatia are ellipsoid with tapered ends and are surrounded by a thin wall. Each contains a single nucleus, many ribosomes, a few small vacuoles, and a number of lipid bodies and mitochondria.     

A new circumscription of the lichen genus <Emphasis Type="Italic">Nephromopsis</Emphasis> (Parmeliaceae,lichenized Ascomycetes)

The phylogeny of the cetrarioid lichens with bifusiform spermatia and dorsiventral thalli which contain usnic acid is reanalysed using three parts of the genome, ITS rDNA, β-tubulin and GAPDH sequences. Molecular data from five cetrarioid species are presented for the first time, and 13 new sequences are combined with sequences from the gene bank to delimit the genus Nephromopsis. A monophyletic clade of Nephromopsis, Tuckneraria, ‘Cetraria’ leucostigma and ‘C.’ melaloma is identified and circumscribed as one genus, Nephromopsis, which now includes 19 species. Four new combinations are presented. A key to the species is provided.     

Association of slugs with the fungal pathogen Epichloë typhina (Ascomycotina: Clavicipitaceae): potential role in stroma fertilisation and disease spread

Epichloë spp. are endophytes of grasses, and form epiphytic external stromata on flowering tillers. E. typhina was first noticed infecting Dactylis glomerata (= orchardgrass, cocksfoot) stands in the Willamette Valley in 1996, and soon became the primary factor limiting the longevity of seed production fields. Several species of slugs are present in these fields, and we investigated their role in E. typhina biology. Pre‐dawn surveys of D. glomerata fields in 2009 and 2010 found Prophysaon andersoni and Arion subfuscus slugs feeding on the fungal stromata. When unfertilised and fertilised immature stromata predominated, approximately 80% of the individuals of these two species that were seen on plants were found on the stromata. As the majority of stromata reached maturity the presence of these species on stromata declined to between 20–40%. The common agricultural slug pest, Deroceras reticulatum, was on stromata only 20% of the time early in the season, and declined to <5% at stromata maturity. Observations of frass from slugs determined that the most common constituent was the food sources upon which the slug species was usually found during these surveys. Typically 100% of the frass from P. andersoni and A. subfuscus contained stroma material, compared to 25% for D. reticulatum. Spermatia, and ascospores later in the season, were commonly seen in the frass of slugs that consumed stromata. Some slugs that had no stroma material in their frass appeared to have consumed spermatia and ascospores from the leaf surface. A multiple‐choice laboratory test confirmed the different proportional preferences of P. andersoni and D. reticulatum for stroma (0.72 vs 0.20) and leaf (0.07 vs 0.38), respectively. Two laboratory multiple‐choice tests, and a field survey, found that P. andersoni preferred unfertilised and immature stroma over mature stroma. D. reticulatum is the most common and abundant slug in Willamette Valley grass seed fields, yet it is the least likely to move spermatia between unfertilised stromata, or ascospores to uninfected plants. P. andersoni and A. subfuscus are mycophagous, frequently transport viable spermatia and ascospores in their frass; yet they are generally confined to field edges. Data and observations suggest the role of slugs in the epidemiology of E. typhina is small compared to other factors.     

Porphyra drewiana, a new species of red algae (Bangiales, Rhodophyta) from Brazil

Porphyra drewiana Coll et Oliveira, sp. nov., is described from plants collected on the south‐east coast of Brazil. The species proposed is monostromatic, monoecious, monoplastidial, without marginal microscopic teeth and does not produce monospores. Both phases, leafy and filamentous, have three chromosomes. Morphologically the most similar species is Porphyra spiralis Oliveira et Coll var. amplifolia Oliveira et Coll, from which it differs by: (i) thallus gross morphology; (ii) scattered pluristromatic areas of vegetative cells; (iii) division of the plastids prior to the nucleus at the first division of the carpospores mother cell; (iv) the number of carpospores and spermatia produced per mother cell; and (v) morphology and behavior of the filamentous phase in cultures. An identification key for the species referred to Brazil is included.