Taxonomic studies of nectrioid fungi in Japan. II: The genus <Emphasis Type="Italic">Bionectria</Emphasis>

Based on the result of morphological and the phylogenetic analyses, three Bionectria (Bionectriaceae: Hypocreales) species are added to the mycobiota of Japan. Among them, one found in Okinawa and Kagoshima Prefectures is described as a new species, Bionectria pseudostriatopsis (anamorph: Clonostachys pseudostriatopsis). The other two, B. grammicospora and B. sporodochialis, are new records from Japan. Additional distribution records are given for Bionectria species hitherto known in Japan.     

Characteristics of spore germination and protonemal development in Hypnum pacleseens

The spore germination, protonemal development, and gametophyte differentiation of Hypnum pacleseens were observed in cultivation. Photomicrographs showed that spore germination of Hypnum pacleseens occured within the exospore. Its protonema is massive with filamentous chloronema formed inside. The terminal part of the chloronema differentiated into filamentous caulonema and its rhizoid was derived from the apical cell of the filamentous chloronema. The initial cell of gametophyte differentiated from chloronema and caulonema. Sporeling-type of Hypnum pacleseens is developmentally similar to Glyphmitrium-type.     

Ricerche Embriologiche su Senecio Vulgaris L. var. Thyrrenus Fiori

Abstract

Embryological researches on SENECIO VULGARIS L. var. THYRRENUS Fiori. — Male gametophyte, development of tapetal cells and female gametophyte have been studied in Senecio vulgaris L. var thyrrenus Fiori.

1) The development of male gametophyte is normal. Divisions of the microspore mother cells are of the simultaneous type. The division of the generative nucleus has never been observed till the pollen grain was in the anther.

2) The tapetal cells follow a very simple development. The nucleus of each cell divides only twice starting at the same time with the meiotic divisions of pollen mother cells but ending much earlier; subsequently, as usually happens with the Asteraceae, the ameboid involution of the tapetum begins. Endomitosis or any other process which leads to a polyploidy not due to nuclear fusion, has never been observed.

3) The female gametophyte is eight nucleate of the normal type (Polygonum). At maturity it shows only three antipodal cells whose nucleus undergoes at first, two or three divisions. Only later these new nuclei, always within the antipodal cell, may fuse in a polyvalent one.     

<Emphasis Type="Italic">Calcarisporium xylariicola</Emphasis> sp. nov. and introduction of <Emphasis Type="Italic">Calcarisporiaceae</Emphasis> fam. nov. in Hypocreales

During a survey of fungicolous fungi, a novel taxon from the surface of stroma of an unidentified Xylaria species was collected. Phylogenetic analyses showed that this taxon clustered with Calcarisporium sp. and C. arbuscula isolates, but was resolved as a distinct species. A detailed morphological examination coupled with phylogenetic analysis indicated that the taxon represented a new species. Calcarisporium xylariicola sp. nov. is thus introduced. The new taxon is characterized by short conidiophores with swollen bases and less length/width ratio of conidia that distinguish it from other Calcarisporium species. Calcarisporium is presently placed in Hypocreales genera, incertae sedis genus. Species in the genus are largely fungicolous, or occasionally caulicolous or foliicolous, and have hyaline, erect, verticillate conidiophores and sympodial, polyblastic conidiation. A phylogenetic analysis of combined SSU, ITS, LSU, TEF and RPB2 sequence data from Calcarisporium species and other taxa in Hypocreales indicate that Calcarisporium is a distinct lineage from other families. Therefore, a new family, Calcarisporiaceae, in Hypocreales is introduced.     

阔叶鳞盖蕨和粗毛鳞盖蕨(碗蕨科)配子体发育的研究

以腐殖土为基质,对阔叶鳞盖蕨Microlepia patyphylla（D．Don）J．Sm．和粗毛鳞盖蕨M.strigosa（Thunb．）Presl进行了孢子繁殖;利用光学显微镜观察和记录了它们的孢子萌发和配子体发育过程。结果表明,两者的孢子及配子体性状极为相似：孢子同型,三裂缝,萌发慢;孢子萌发需要光,萌发方式为书带蕨型Vittaria-type;配子体生长慢,发育类型为铁线蕨型Adiantum—type;原叶体心形或其他形状,无毛状体,多数为雌雄异株。性器类型为薄囊蕨型Leptosporangiate-type。它们既具有大量的原始性状,也具有少数进化性状。与阔叶鳞盖蕨相比,粗毛鳞盖蕨的配子体发育特征更为原始。说明粗己鳞盖蕨的系统位置更加低下。首次观察到阔叶鳞盖蕨中细胞自然死亡时的叶绿体聚集现象。粗毛鳞盖蕨原叶体老化时叶绿体呈现规则的、相互镶嵌的多边形的形状以及在正常光照下粗毛鳞盖蕨某些细胞中的部分叶绿体成链珠状排列等现象。     

EARLY GAMETOPHYTE ONTOGENY OF GLEICHENIA BIFIDA (WILLD.) SPRENG.: PHYLOGENETIC AND ECOLOGICAL IMPLICATIONS

Past investigation of gametophyte ontogeny in Gleichenia bifida indicated that there was considerable plasticity in early developmental stages (spore germination to initiation of two-dimensional growth). Recent examination has shown that this early ontogeny is not so plastic and can be defined by a characteristic series of developmental stages. Cell divisions following germination result in a three-dimensional mass of cells which secondarily initiates the two-dimensional thallus. Early development as a three-dimensional mass correlates with other gametophyte and sporophyte features which indicate Gleichenia to be phylogenetically primitive. This pattern of early ontogeny may also be of adaptive significance in enabling the gametophytes to survive the environmental fluctuations of the exposed, bare-soil habitats which they colonize.     

Practical methodology for gametophyte proliferation and sporophyte production in green penny fern (Lemmaphyllum microphyllum C. Presl) using mechanical fragmentation

Propagation of gametophytes and sporophytes using mechanical fragmentation has been considered a suitable method for mass production of ferns. This study aimed to develop a practical propagation method for Lemmaphyllum microphyllum C. Presl, which is a fern of significant ornamental and medicinal value. Gametophytes were obtained through in vitro spore germination and used for propagation experiments. The gametophyte was mechanically fragmented using a scalpel into small fragments, which were then used to investigate gametophyte proliferation. In addition, the gametophyte was fragmented using a blender and then used to study sporophyte formation. Optimal proliferation conditions of the gametophyte were determined using Murashige and Skoog (MS) basal medium (double-, full-, half-, quarter-strength), Knop medium, and medium components (sucrose, nitrogen sources, activated charcoal), at various concentrations. The fresh weight of the gametophyte was 14-fold higher than that of gametophytes (300 mg) used as culture material, when cultured on double-strength MS. Moreover, 1 g of the gametophyte fragmented in 25 mL of distilled water formed more than 430 sporophytes in a soil mixture in an area of 7.5 cm². The sporophytes were successfully cultivated in the greenhouse after acclimation. A large-scale production method for L. microphyllum that can be easily implemented in a fern production farm is outlined.

     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

GAMETOPHYTES AND YOUNG SPOROPHYTES OF OPHIOGLOSSUM CROTALOPHOROIDES WALT.

The lens-shaped, dorsiventral gametophytes of Ophioglossum crotalophoroides are superficially different from the cylindrical gametophytes of other species of Ophioglossum. However, they have the same features as other Ophioglossum gametophytes (fundamentally axial organization, radially symmetrical apical meristem, radial distribution of gametangia) except that the cylindrical axis is reduced in length. Young sporophyte development is unique in the genus: all primary organs clearly arise from the embryo and develop simultaneously. The length of the life cycle of O. crotalophoroicles is considerably shorter than that of some other species in the Ophioglossaceae. A timetable for gametophyte and young sporophyte development is postulated. Spores germinate soon after they are released in the spring, and mature gametophytes develop by the next growing season. Fertilization occurs approximately one year after spore dispersal, and after two years, the photosynthetic first leaf of the young sporophyte emerges.     

An ultrastructural study of spore wall development and septal pores in species of the <Emphasis Type="Italic">Pachyphlodes</Emphasis> (Pezizaceae,Pezizales) lineage,with a description of the new species <Emphasis Type="Italic">Pachyphlodes annagardnerae</Emphasis>

Pachyphlodes (Pezizaceae) is a genus of truffle-like fungi that is distributed across the Northern Hemisphere. These fungi form ectomycorrhizae primarily with trees in the Fagaceae family, and occasionally with other host plants. The genus Plicariella (= Scabropezia) is phylogenetically inferred as an ally of, or within, the Pachyphlodes lineage. Despite molecular phylogenetic analyses that show the close relationships of species in these two genera, morphological differences in ascomata shape and color, spore ornamentation, and ascus shape are profound. Here, we studied spore wall development to better understand affinities within the Pachyphlodes–Plicariella lineages. Electron microscopy studies indicate that the initial spore wall development is similar across six Pachyphlodes species and a Plicariella species, despite striking differences in mature spore ornamentation among species. Ultrastructural analyses reveal that differences in spore ornamentation among Pachyphlodes species are due to unique developmental events at the final stages of spore wall deposition. Septal pore ultrastructure in Pachyphlodes species is similar to other Pezizaceae that have been studied. Molecular analyses of the five species studied indicate that four of them have not been previously described. The new species Pachyphlodes annagardnerae is here described, and the ultrastructural features of species of Pachyphlodes, Plicariella, and other Pezizales are compared and discussed.     

Effects of Exogenous Phytohormones on Spore Germination and Morphogenesis of <Emphasis Type="Italic">Polystichum aculeatum</Emphasis> (L.) Roth Gametophyte in vitro Culture

The effects of exogenous phytohormones–gibberellic acid (GA3) and benzyl-aminopurine (BAP)–on the spore germination and morphogenesis of Polystichum aculeatum (L.) Roth gametophyte in vitro culture were studied. In the control, four stages of gametophyte morphogenesis were determined and their periods were established. Spore germination and protonema formation of P. aculeatum occurred according to the Vittaria-type and prothalium development according to the Aspidium-type. The spore germination percentage depended on the storage time duration. It was found that 80–95% of freshly collected spores germinated. Spore viability was within the range of 68–95% after 4–6-month storage under lab conditions and did not exceed 20% after 1.5 year of the storage period. High concentrations of exogenous GA3 (10^–5 and 10^–6 M) and BAP (10–5 M) significantly inhibited spore germination, whereas low concentrations (GA3 10^–7–10^–8 M) had an insignificant stimulating effect or did not affect germination at all (BAP 10^–6, 10^–7, 10^–8 M). All concentrations of exogenous BAP were demonstrated to inhibit the development of P. aculeatum gametophyte at the protonema stage, which might be due to the removal of apical dominance. The inhibiting effect directly depended on BAP concentrations. The formation of abnormal thalli of the P. aculeatum gametophyte in the response to exogenous GA3 treatments occurred as a result of impairment of cell growth by elongation. A direct interrelationship between GA3 concentrations and level of morphological abnormalities and grade of thalli underdevelopment was demonstrated.     

Origin and taxonomic affinities of Thelypteris (subgen. Stegnogramma) burksiorum (Thelypteridaceae)

This paper examines the ecology, frond and spore morphology, gametophytes, and molecular biology ofThelypteris burksiorum to reveal its taxonomic affinities and phytogeographic origin. The species is endemic to Winston County in northern Alabama (U.S.A.) and only occurs in cave-like rockhouse habitats. A related species,T. pilosa, is widespread in central and southern Mexico, Guatemala, and Honduras.Thelypteris pilosa is extremely variable and our data suggest that current recognition of the varietiesmajor andpilosa is not warranted.Thelypteris pilosa is clearly closely related and likely sister toT. burksiorum. Thelypteris burksiorum exhibits the unusual production of gametophytic propagules. This, in combination with current levels of genetic diversity and reproductive biology, suggests thatT. burksiorum is an ancient Tertiary relict and not an example of recent long distance dispersal. Selection in rockhouse habitats has driven species away from their normal life cycle to partial or complete reliance on the gametophyte generation. The combination of a reduced yet fertile sporophyte and a gametophyte that may rely on gametophytic proliferation placesT. burksiorum in an intermediate evolutionary position relative to other rockhouse pteridophytes.     

New hyphomycetes from Restionaceae in fynbos: <Emphasis Type="Italic">Parasarcopodium ceratocaryi</Emphasis> gen. et sp. nov., and <Emphasis Type="Italic">Rhexodenticula elegiae</Emphasis> sp. nov.

Two new hyphomycetous anamorphs were isolated from plant hosts of the Restionaceae in the fynbos of the Cape Floral Kingdom of South Africa. Parasarcopodium ceratocaryi gen. et sp. nov. on Ceratocaryum decipiens produces aseptate, cylindrical conidia with amorphous mucoid appendages at both ends in rows or whorls of phialides on verruculose, mononematous conidiophores. Rhexodenticula elegiae sp. nov. on Elegia capensis produces pale brown, fusiform or broadly fusiform, 3-septate conidia on denticles located in the upper part of smooth, geniculate conidiophores. The morphological characteristics of both taxa are described, illustrated, and contrasted to other morphologically similar fungi. To elucidate the phylogenetic relationships of Parasarcopodium, partial large subunit rRNA (28S) gene sequence was generated from the ex-type strain, which revealed it to be a member of the Bionectriaceae (Hypocreales).     

Morphological and molecular comparison of <Emphasis Type="Italic">Myxobolus procerus</Emphasis> (Kudo, 1934) and <Emphasis Type="Italic">M. intramusculi</Emphasis> n. sp. (Myxozoa) parasitising muscles of the trout-perch <Emphasis Type="Italic">Percopsis omiscomaycus</Emphasis>

Inter- and intracellular cysts from a presumed single species, Myxobolus procerus (Kudo, 1934), parasitising the trout-perch Percopsis omiscomaycus were compared with respect to their spore morphology and small subunit 18S ribosomal DNA. The two cyst types have similar pyriform spores and comparable ranges in spore length and width, size of the polar capsules and number of filament coils. However, fixed spores of the intracellular cysts are significantly shorter (p < 0.05) in mean length than those from intercellular cysts, giving them a broader appearance. Comparing 991 base pairs of 18S rDNA revealed a 20 bp difference between the two cyst types (97.9% similarity), 10 due to base substitutions and 10 due to the presence or absence of a base. The described morphological differences and consistent 2.1% difference in the 18S rDNA reveal that the intracellular form is a separate species, which is described herein as Myxobolus intramusculi n. sp. M. intramusculi n. sp. and M. procerus may be sister species and it is suggested that the two species arose through sympatric speciation involving a switch in site specificity within a single host species.     

Segregation distortion in Arabidopsis <Emphasis Type="Italic">gametophytic factor 1 </Emphasis>(<Emphasis Type="Italic">gfa1</Emphasis>) mutants is caused by a deficiency of an essential RNA splicing factor

The female and male gametophytes are critical components of the angiosperm life cycle and are essential for the reproductive process. The gametophytes share many essential cellular processes with each other and with the sporophyte generation. As a consequence, these processes can only be analyzed genetically in the gametophyte generation. Here, we report the characterization of the gametophytic factor 1 (gfa1) mutant. The gfa1 mutation exhibits reduced transmission through both the female and male gametophytes. Reduced transmission through the female gametophyte is due to an effect on female gametophyte development. By contrast, development of the pollen grain is not affected in gfa1; rather, reduced transmission is likely due to an effect on pollen tube growth. We have identified multiple T-DNA-insertion alleles of gfa1 in a gene encoding a protein with high similarity to Snu114/U5-116 kD proteins from yeast and animals required for normal pre-mRNA splicing. Consistent with its predicted function, the GFA1 gene (At1g06220) is expressed throughout the plant. Together, these data suggest that GFA1 functions in mRNA splicing during the plant life cycle. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

C, N and P nutrition of Lemanea mamillosa Kütz. (Batrachospermales, Rhodophyta) in the Dighty Burn, Angus, U.K.

Abstract. Field measurements of the growth rate of the red freshwater macroalga Lemanca mamillosa Kutz, in the Dighty Burn, together with measurements of water velocity, [CO₂], [NO₃], [NH₃+ NH₄⁺] and [phosphate], have been made between February and July. This period covers the growth of the erect gametophyte and later of the carposporophyte inside the gametophyte. Hydrodynamic studies in the laboratory on benzoic acid models of the gametophyte suggest an average in situ unstirred layer some 12 μm thick. For growth of the gametophyte, this estimated boundary layer thickness, together with the measured inorganic C transport pathway within the plant, suggest that growth is not significantly restricted by CO₂ transport from the bulk phase to the plastids. δ¹³C measurements on source CO₂ and on plant organic C bear this out. Habitat choice (low temperatures: CO₂ enrichment from ground-water input: rapid water flow), plant morphology and anatomy (turbulence-generating ‘knobbles’ on the nodes; plastids close to the outside of the plant), and plant biochemistry (high CO₂ affinity of the RUBISCO carboxylase; quite high carbonic anhydrase activity) are responsible for this lack of limitation by inorganic C transport in the growing gametophyte which lacks HCO₃ transport and a CO₂ concentrating mechanism. Transport through the boundary layer does not significantly restrict acquisition by the plant of N (probably as NH₄⁺, despite the preponderance of NO₃ in the environment) or of P (as orthophosphate) in the field. The membrane transporters, which have high substrate affinities (K½'s about 2 mmol NH₄⁺ m^-3 and < 2 mmol inorganic phosphate m^?3), probably impose the major limitation. The development of the carposporophyte later in the season, and an increase in the thickness of the cortex of the gametophyte, result in an increased (less negative) δ¹³C, suggesting a significant diffusion limitation to CO₂ transport. This conclusion is reinforced by consideration of the opposing effect on Δδ¹³ C of the decreased demand for products of phosphoenolpyruvate carboxylase activity as the N/C ratio decreases late in the growing season.     

Taxonomic studies of nectrioid fungi in Japan. I: The genus Neonectria

Five species of the genus Neonectria (Nectriaceae, Hypocreales) collected from Japan are described with discussion and illustrated. Among them, one found on dead twigs of a broad-leaved tree is described as a new species, Neonectria tokyoensis (anamorph: Cylindrocarpon tokyoense). Neonectria cinnamomea and Neo. discophora are newly added to the Japanese mycobiota. Nectria azureo-ostiolata, recorded in 1977 from Japan, is reexamined as a synonym of Neo. jungneri. Neonectria radicicola, teleomorph of Cy. destructans, a well-known soil-borne plant pathogen in Japan, is newly recorded from Japan. Additional distribution records are provided for the Neonectria species hitherto recorded in Japan.     

Male gametophyte development and two different DNA classes of pollen grains in <Emphasis Type="Italic">Rumex acetosa</Emphasis> L., a plant with an XX/XY<Subscript>1</Subscript>Y<Subscript>2</Subscript> sex chromosome system and a female-biased sex ratio

Female-biased sex ratio is an interesting phenomenon observed in Rumex acetosa, a dioecious plant with an XX/XY₁Y₂ sex chromosome system. Previous authors have suggested that the biased sex ratio in this species is conditioned not only postzygotically (sex-differential sporophytic mortality) but also prezygotically, because the sex ratio of seeds is also female-biased, although to a lesser extent than the sex ratio of flowering plants. The mechanisms underlying female bias in Rumex seeds are only poorly understood. To gain more knowledge of them, we analysed male gametophyte development and used flow cytometry to determine the frequency of female-determining (n = 7, A + X) and male-determining (n = 8, A + Y₁Y₂) pollen grains in anthers. Embryological studies showed a regular course of male gametophyte development in R. acetosa. There were no signs of degeneration of microspores or disturbances in pollen divisions (irregular nuclei, micronuclei, delayed chromosomes and anaphase bridges). The Alexander test revealed only 1.6% nonviable pollen grains within anthers. All mature pollen grains were uniformly equipped with starch granules. The two sexes were shown to substantially differ in nuclear 2C DNA amount in somatic tissues (7.00 pg in 2A + XX females and 7.50 pg in 2A + XY₁Y₂ males), and two clearly different DNA classes of mature pollen grains, with lower and with higher DNA amounts (16.8% difference) were found. Most probably the grains with the lower DNA amount possess seven chromosomes, and grains with the higher DNA amount eight of them. The quantitative ratio of these grains in anthers at anthesis was 1:1.2, very close to the sex ratio of seeds observed by the majority of previous authors. All these observations support the opinion that the sex-ratio bias in Rumex is determined prezygotically to some extent.